Science.gov

Sample records for alpha tc3 cells

  1. Reversal of diabetes by βTC3 cells encapsulated in alginate beads generated by emulsion and internal gelation.

    PubMed

    Hoesli, Corinne A; Kiang, Roger L J; Mocinecová, Dušana; Speck, Madeleine; Mošková, Daniela Jochec; Donald-Hague, Christine; Lacík, Igor; Kieffer, Timothy J; Piret, James M

    2012-05-01

    Encapsulation of insulin-producing cells in alginate beads could improve the treatment of type 1 diabetes by reducing or eliminating the need for immunosuppression. We have recently adapted an emulsion and internal gelation process to β-cell encapsulation. This process has the advantages of being well suited for m(3)/h production rates and allowing the use of increased alginate concentrations. Compared with 1.5% alginate beads generated by a standard extrusion process, 5% alginate emulsion-generated beads demonstrated greater in vitro stability and greater volumetric exclusion of antibody-sized pullulan. When βTC3 cells were transplanted into streptozotocin-induced allogeneic diabetic mice, a significant decrease in the blood glucose levels was seen within 2 days with the 5% emulsion-generated beads but not until >16 days with the 1.5% extrusion-generated beads. This was correlated with higher cell survival and lower graft-specific plasma immunoglobulin levels. These results suggest that higher-concentration alginate beads generated by emulsion and internal gelation have improved graft immunoprotection. The emulsion process is a promising and scalable technology for cellular therapies requiring immune isolation.

  2. Desensitization of glucagon-like peptide 1 receptors in insulin-secreting beta TC3 cells: role of PKA-independent mechanisms.

    PubMed Central

    Gromada, J.; Dissing, S.; Rorsman, P.

    1996-01-01

    1. The cellular processes involved in the desensitization of the glucagon-like peptide 1 receptors were investigated by measurements of the glucagon-like peptide 1(7-36)amide (GLP-1(7-36)amide)-induced increases in intracellular free Ca2+ concentration ([Ca2+]i) in insulin-secreting beta TC3 cells. 2. In the presence of 11.2 mM glucose, stimulation with GLP-1(7-36)amide led to a small membrane depolarization (< 10 mV), induction of electrical activity and a rapid increase in [Ca2+]i. The increase in [Ca2+]i was not observed in the presence of the L-type Ca(2+)-channel antagonist nifedipine. However, nifedipine was ineffective when applied after addition of GLP-1(7-36)amide. 3. The increase in [Ca2+]i evoked by GLP-1-(7-36)amide was transient and even in the continued presence of the agonist, [Ca2+]i returned to the basal value within 4-5 min. The latter process was slowed, but not prevented, by inhibition of protein kinase C (PKC) by staurosporine and Ro31-8220. 4. Short pretreatment of the cells with the phorbol ester, 4-beta-phorbol-12-beta-myristate-13-alpha-acetate (PMA), an activator of PKC, reduced the GLP-1(7-36)amide-evoked increase in [Ca2+]i by 75%. This effect of PMA was fully reversed by staurosporine and Ro31-8220. 5. The ability of GLP-1(7-36)amide to increase [Ca2+]i disappeared upon pre-exposure of the cells to the hormone (desensitization). This process was maximal within 5 min of exposure to the agonist. Following removal of the agonist from the medium, the ability to respond to subsequent stimulation by GLP-1(7-36)amide recovered gradually with time; half and complete recovery requiring > 20 min and 60 min, respectively. The desensitizing action of GLP-1(7-36)amide persisted in the presence of either staurosporine or forskolin and did not require an elevation of [Ca2+]i. 6. Our data suggest that the GLP-1(7-36)amide-evoked increase in [Ca2+]i is initiated by Ca(2+)-influx though voltage-dependent and nifedipine-sensitive L-type Ca2+ channels but

  3. Mechanism of alpha-tocopheryl-phosphate (alpha-TP) transport across the cell membrane

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have reported that alpha-TP is synthesized and hydrolyzed in animal cells and tissues; it modulates also several cell functions (FRBM 39:970, and UBMB Life, 57:23, 2005). While it is similar to alpha-tocopherol (alpha-T), alpha-TP appears to be more potent than alpha-T in inhibiting cell prolifer...

  4. Folate receptor {alpha} regulates cell proliferation in mouse gonadotroph {alpha}T3-1 cells

    SciTech Connect

    Yao, Congjun; Evans, Chheng-Orn; Stevens, Victoria L.; Owens, Timothy R.; Oyesiku, Nelson M.

    2009-11-01

    We have previously found that the mRNA and protein levels of the folate receptor alpha (FR{alpha}) are uniquely over-expressed in clinically human nonfunctional (NF) pituitary adenomas, but the mechanistic role of FR{alpha} has not fully been determined. We investigated the effect of FR{alpha} over-expression in the mouse gonadotroph {alpha}T3-1 cell line as a model for NF pituitary adenomas. We found that the expression and function of FR{alpha} were strongly up-regulated, by Western blotting and folic acid binding assay. Furthermore, we found a higher cell growth rate, an enhanced percentage of cells in S-phase by BrdU assay, and a higher PCNA staining. These observations indicate that over-expression of FR{alpha} promotes cell proliferation. These effects were abrogated in the same {alpha}T3-1 cells when transfected with a mutant FR{alpha} cDNA that confers a dominant-negative phenotype by inhibiting folic acid binding. Finally, by real-time quantitative PCR, we found that mRNA expression of NOTCH3 was up-regulated in FR{alpha} over-expressing cells. In summary, our data suggests that FR{alpha} regulates pituitary tumor cell proliferation and mechanistically may involve the NOTCH pathway. Potentially, this finding could be exploited to develop new, innovative molecular targeted treatment for human NF pituitary adenomas.

  5. Prevalence of -alpha(3.7) and alpha alpha alpha(anti3.7) alleles in sickle cell trait and beta-thalassemia patients in Mexico.

    PubMed

    Nava, María Paulina; Ibarra, Bertha; Magaña, María Teresa; de la Luz Chávez, María; Perea, F Javier

    2006-01-01

    The aim of this study was to determine the frequency of alpha-globin gene mutations in three groups of Mexican unrelated individuals. The first two groups were normal and sickle cell trait individuals from the Costa Chica region, a place with a 12.8% frequency of HbS carriers, and the third group comprised of Mexican mestizo patients with beta-thalassemia. We searched for -alpha(3.7) and -alpha(4.2) alpha(+)-thalassemia deletion alleles, as well as the alpha alpha alpha(anti3.7) triplication through long-gap PCR. The alleles -alpha(3.7) and alpha alpha alpha(anti3.7) were found in the heterozygote state only; 19% of the normal subjects had the -alpha(3.7) allele, and 2% showed the alpha alpha alpha(anti3.7) allele. In individuals with the sickle cell trait, 17% had the -alpha(3.7) deletion, and the alpha alpha alpha(anti3.7) triplication was observed in 3% of these individuals. We revealed that 16% of the subjects with beta-thalassemia showed the -alpha(3.7) deletion and 28% the alpha alpha alpha(anti3.7) triplication. The -alpha(4.2) deletion was not detected in any individual. The frequency of the -alpha(3.7) allele was roughly the same in the three groups studied; this can be explained by the fact that the three groups have common genes from Africa and the Mediterranean, where a high prevalence of alpha(+)-thalassemia has been observed. To our knowledge, the frequency of alpha alpha alpha(anti3.7) triplication observed in the Mexican beta-thalassemia patients is the highest reported. As the -alpha(3.7) and alpha alpha alpha(anti3.7) alleles are very common in our selected populations, we believe that there is a need to investigate systematically the alpha-globin gene mutations in all hemoglobinopathies in the Mexican population.

  6. Alpha1 and Alpha2 Integrins Mediate Invasive Activity of Mouse Mammary Carcinoma Cells through Regulation of Stromelysin-1 Expression

    SciTech Connect

    Lochter, Andre; Navre, Marc; Werb, Zena; Bissell, Mina J

    1998-06-29

    Tumor cell invasion relies on cell migration and extracellular matrix proteolysis. We investigated the contribution of different integrins to the invasive activity of mouse mammary carcinoma cells. Antibodies against integrin subunits {alpha}6 and {beta}1, but not against {alpha}1 and {alpha}2, inhibited cell locomotion on a reconstituted basement membrane in two-dimensional cell migration assays, whereas antibodies against {beta}1, but not against a6 or {alpha}2, interfered with cell adhesion to basement membrane constituents. Blocking antibodies against {alpha}1 integrins impaired only cell adhesion to type IV collagen. Antibodies against {alpha}1, {alpha}2, {alpha}6, and {beta}1, but not {alpha}5, integrin subunits reduced invasion of a reconstituted basement membrane. Integrins {alpha}1 and {alpha}2, which contributed only marginally to motility and adhesion, regulated proteinase production. Antibodies against {alpha}1 and {alpha}2, but not {alpha}6 and {beta}1, integrin subunits inhibited both transcription and protein expression of the matrix metalloproteinase stromelysin-1. Inhibition of tumor cell invasion by antibodies against {alpha}1 and {alpha}2 was reversed by addition of recombinant stromelysin-1. In contrast, stromelysin-1 could not rescue invasion inhibited by anti-{alpha}6 antibodies. Our data indicate that {alpha}1 and {alpha}2 integrins confer invasive behavior by regulating stromelysin-1 expression, whereas {alpha}6 integrins regulate cell motility. These results provide new insights into the specific functions of integrins during tumor cell invasion.

  7. G alpha 16, a G protein alpha subunit specifically expressed in hematopoietic cells.

    PubMed Central

    Amatruda, T T; Steele, D A; Slepak, V Z; Simon, M I

    1991-01-01

    Signal-transduction pathways mediated by guanine nucleotide-binding regulatory proteins (G proteins) determine many of the responses of hematopoietic cells. A recently identified gene encoding a G protein alpha subunit, G alpha 16, is specifically expressed in human cells of the hematopoietic lineage. The G alpha 16 cDNA encodes a protein with predicted Mr of 43,500, which resembles the G q class of alpha subunits and does not include a pertussis toxin ADP-ribosylation site. In comparison with other G protein alpha subunits, the G alpha 16 predicted protein has distinctive amino acid sequences in the amino terminus, the region A guanine nucleotide-binding domain, and in the carboxyl-terminal third of the protein. Cell lines of myelomonocytic and T-cell phenotype express the G alpha 16 gene, but no expression is detectable in two B-cell lines or in nonhematopoietic cell lines. G alpha 16 gene expression is down-regulated in HL-60 cells induced to differentiate to neutrophils with dimethyl sulfoxide. Antisera generated from synthetic peptides that correspond to two regions of G alpha 16 specifically react with a protein of 42- to 43-kDa in bacterial strains that overexpress G alpha 16 and in HL-60 membranes. This protein is decreased in membranes from dimethyl sulfoxide-differentiated HL-60 cells and is not detectable in COS cell membranes. The restricted expression of this gene suggests that G alpha 16 regulates cell-type-specific signal-transduction pathways, which are not inhibited by pertussis toxin. Images PMID:1905813

  8. Evolution and Exploration of Asteroid 2008 TC3

    NASA Astrophysics Data System (ADS)

    Jenniskens, P. M.; Shaddad, M. H.; Almahata Sitta Consortium

    2011-12-01

    We will discuss ongoing work to fully explore the diversity of materials contained in asteroid 2008 TC3, the small 4-m sized asteroid that was observed in space before hitting Earth on October 7, 2008. A small part of the asteroid was recovered in the form of the Almahata Sitta meteorites two months later. The recovered meteorites were mostly ureilites, but very unexpectedly 20-30 percent of other meteorite types were also recovered. Those included enstatite and ordinary chondrites that became part of the Ureilite Parent Body's daughter asteroids during the evolutionary pathway from the terrestrial region to the asteroid belt. An update will be given on ongoing and projected research into the Almahata Sitta strewnfield and the analysis of the individual meteorites by members of the Almahata Sitta consortium.

  9. Asteroid 2008 TC3 Breakup and Meteorite Fractions

    NASA Technical Reports Server (NTRS)

    Goodrich, C.; Jenniskens, P.; Shaddad, M. H.; Zolensky, M. E.; Fioretti, A. M.

    2017-01-01

    The recovery of meteorites from the impact of asteroid 2008 TC3 in the Nubian Desert of Sudan on October 7, 2008, marked the first time meteorites were collected from an asteroid observed in space by astronomical techniques before impacting. Search teams from the University of Khartoum traced the location of the strewn field and collected about 660 meteorites in four expeditions to the fall region, all of which have known fall coordinates. Upon further study, the Almahata Sitta meteorites proved to be a mixed bag of mostly ureilites (course grained, fine grained, and sulfide-metal assemblages), enstatite chondrites (EL3-6, EH3, EH5, breccias) and ordinary chondrites (H5-6, L4-5). One bencubbinite-like carbonaceous chondrite was identified, as well as one unique Rumuruti-like chondrite and an Enstatite achondrite. New analysis: The analysed meteorites so far suggest a high 30-40 percent fraction of non-ureilites among the recovered samples, but that high fraction does not appear to be in agreement with the meteorites in the University of Khartoum (UoK) collection. Ureilites dominate the meteorites that were recovered by the Sudanese teams. To better understand the fraction of recovered materials that fell to Earth, a program has been initiated to type the meteorites in the UoK collection in defined search areas. At this meeting, we will present some preliminary results from that investigation.

  10. The shape and rotation of asteroid 2008 TC3

    NASA Astrophysics Data System (ADS)

    Scheirich, Peter; Durech, Josef; Pravec, Petr; Kozubal, Marek; Dantowitz, Ron; Kaasalainen, Mikko; Betzler, Alberto Silva; Beltrame, Paolo; Muler, Gustavo; Birtwhistle, Peter; Kugel, Francois

    2010-10-01

    On October 6, 2008, a small F-class asteroid 2008 TC3 was discovered by Catalina Sky Survey telescope, and exploded 20 hr later in the Earth's atmosphere at 37 km altitude. We analyzed available photometric data taken from 6 October 21:10 to 7 October 01:46 UT, and created a numerical model of a shape and rotation state of the asteroid. The asteroid was in excited rotational state. We found two approximately mirror solutions of orientation of its angular momentum vector. Rotational and precession periods are 99.20 and 97.00 s (errors of the rotational period for the two solutions are 0.03 and 0.04 s; of the precession period are 0.05 s for both solutions). The volume of the convex model and the length of the longest axis of the dynamically equivalent, equal volume ellipsoid are ? and ?, where pV is surface geometric albedo. Assuming a mean albedo value for F taxonomic class, 0.049 ± 0.010, the volume is (25 ± 10) m3 and the longest axis is (6.7 ± 0.8) m. This volume of the convex model is an upper limit on a real volume of the asteroid, which may be less by up to 20% due to concavities.

  11. The impact and recovery of asteroid 2008 TC(3).

    PubMed

    Jenniskens, P; Shaddad, M H; Numan, D; Elsir, S; Kudoda, A M; Zolensky, M E; Le, L; Robinson, G A; Friedrich, J M; Rumble, D; Steele, A; Chesley, S R; Fitzsimmons, A; Duddy, S; Hsieh, H H; Ramsay, G; Brown, P G; Edwards, W N; Tagliaferri, E; Boslough, M B; Spalding, R E; Dantowitz, R; Kozubal, M; Pravec, P; Borovicka, J; Charvat, Z; Vaubaillon, J; Kuiper, J; Albers, J; Bishop, J L; Mancinelli, R L; Sandford, S A; Milam, S N; Nuevo, M; Worden, S P

    2009-03-26

    In the absence of a firm link between individual meteorites and their asteroidal parent bodies, asteroids are typically characterized only by their light reflection properties, and grouped accordingly into classes. On 6 October 2008, a small asteroid was discovered with a flat reflectance spectrum in the 554-995 nm wavelength range, and designated 2008 TC(3) (refs 4-6). It subsequently hit the Earth. Because it exploded at 37 km altitude, no macroscopic fragments were expected to survive. Here we report that a dedicated search along the approach trajectory recovered 47 meteorites, fragments of a single body named Almahata Sitta, with a total mass of 3.95 kg. Analysis of one of these meteorites shows it to be an achondrite, a polymict ureilite, anomalous in its class: ultra-fine-grained and porous, with large carbonaceous grains. The combined asteroid and meteorite reflectance spectra identify the asteroid as F class, now firmly linked to dark carbon-rich anomalous ureilites, a material so fragile it was not previously represented in meteorite collections.

  12. The impact and recovery of asteroid 2008 TC3

    NASA Astrophysics Data System (ADS)

    Jenniskens, P.; Shaddad, M. H.; Numan, D.; Elsir, S.; Kudoda, A. M.; Zolensky, M. E.; Le, L.; Robinson, G. A.; Friedrich, J. M.; Rumble, D.; Steele, A.; Chesley, S. R.; Fitzsimmons, A.; Duddy, S.; Hsieh, H. H.; Ramsay, G.; Brown, P. G.; Edwards, W. N.; Tagliaferri, E.; Boslough, M. B.; Spalding, R. E.; Dantowitz, R.; Kozubal, M.; Pravec, P.; Borovicka, J.; Charvat, Z.; Vaubaillon, J.; Kuiper, J.; Albers, J.; Bishop, J. L.; Mancinelli, R. L.; Sandford, S. A.; Milam, S. N.; Nuevo, M.; Worden, S. P.

    2009-03-01

    In the absence of a firm link between individual meteorites and their asteroidal parent bodies, asteroids are typically characterized only by their light reflection properties, and grouped accordingly into classes. On 6 October 2008, a small asteroid was discovered with a flat reflectance spectrum in the 554-995nm wavelength range, and designated 2008 TC3 (refs 4-6). It subsequently hit the Earth. Because it exploded at 37km altitude, no macroscopic fragments were expected to survive. Here we report that a dedicated search along the approach trajectory recovered 47 meteorites, fragments of a single body named Almahata Sitta, with a total mass of 3.95kg. Analysis of one of these meteorites shows it to be an achondrite, a polymict ureilite, anomalous in its class: ultra-fine-grained and porous, with large carbonaceous grains. The combined asteroid and meteorite reflectance spectra identify the asteroid as F class, now firmly linked to dark carbon-rich anomalous ureilites, a material so fragile it was not previously represented in meteorite collections.

  13. The Impact Trajectory of Asteroid 2008 TC3

    NASA Astrophysics Data System (ADS)

    Chodas, Paul; Chesley, S.; Yeomans, D.

    2009-09-01

    On October 6, 2008, the small newly discovered near-Earth asteroid 2008 TC3 was found to be on an Earth-impacting trajectory, with impact less than 20 hours away. This was the first ever predicted impact of a near-Earth object. Fortunately, it was immediately clear that the object was only a few meters in size and would almost certainly break up when it entered the Earth's tmosphere. We review the pre-impact orbit computations and predictions, the post-impact reconstructions of the trajectory, and the trajectory geometry. The first prediction of impact was made by the Minor Planet Center (MPC), which quickly made the discovery and subsequent follow-up observations available to the astronomical community and contacted both the NASA/JPL Near-Earth Object Program Office and NASA headquarters. Within an hour of receiving the initial data set, JPL predicted that the atmospheric entry would occur over northern Sudan around 02:46 UT on October 7. As the day progressed and more data arrived from the MPC, the impact prediction accuracy continued to improve. A press release was issued at 21:30 UT announcing the predicted impact later that night. Detections of the actual atmospheric impact event suggested that it was an airburst explosion at an altitude of 37 km with an energy equivalent to about one kiloton of TNT explosives. The airburst occurred at 02:45:45 UT at about 32.2 East longitude and 20.8 North latitude, matching the final impact predictions to within 0.2 s in time and 1.5 km in position.

  14. Trafficking of. cap alpha. -L-fucosidase in lymphoid cells

    SciTech Connect

    DiCioccio, R.A.; Brown, K.S.

    1987-05-01

    The quantity of ..cap alpha..-L-fucosidase in human serum is determined by heredity. The mechanism controlling levels of the enzyme in serum is unknown. To investigate this, lymphoid cell lines derived from individuals with either low, intermediate or high ..cap alpha..-L-fucosidase in serum were established. Steady state levels of extracellular ..cap alpha..-L-fucosidase protein and activity overlapped among the cell lines. Thus, in vivo serum phenotypes of ..cap alpha..-L-fucosidase are not adequately expressed in this system. ..cap alpha..-L-Fucosidase was also metabolically labelled with /sup 35/S-methionine, immunoprecipitated, and examined by SDS-PAGE. Cells pulse-labelled from 0.25-2 h had a major intracellular form of enzyme (Mr = 58,000). Cells pulsed for 1.5 h and chased for 21 h with unlabeled methionine had an intracellular form of Mr = 60,000 and an extracellular form of Mr = 62,000. Cells treated with chloroquine had only the 58,000-form both intra- and extra-cellularly. Moreover, chloroquine did not effect the quantitative distribution of ..cap alpha..-L-fucosidase between cells and medium. In fibroblasts, chloroquine enhanced the secretion of newly made lysosomal enzymes and blocked the processing of intercellular enzyme forms from a higher to a lower molecular mass. Thus, there are trafficking differences between ..cap alpha..-L-fucosidase in lymphoid cells and lysosomal enzymes in fibroblasts. This suggests that alternative targeting mechanisms for lysosomal enzymes exist in these cells.

  15. Airway epithelial cell wound repair mediated by alpha-dystroglycan.

    PubMed

    White, S R; Wojcik, K R; Gruenert, D; Sun, S; Dorscheid, D R

    2001-02-01

    Dystroglycans (DGs) bind laminin matrix proteins in skeletal and cardiac muscle and are expressed in other nonmuscle tissues. However, their expression in airway epithelial cells has not been demonstrated. We examined expression of DGs in the human airway epithelial cell line 1HAEo(-), and in human primary airway epithelial cells. Expression of the common gene for alpha- and beta-DG was demonstrated by reverse transcriptase/ polymerase chain reaction in 1HAEo(-) cells. Protein expression of beta-DG was demonstrated by both Western blot and flow cytometry in cultured cells. Localization of alpha-DG, using both a monoclonal antibody and the alpha-DG binding lectin wheat-germ agglutinin (WGA), was to the cell membrane and nucleus. We then examined the function of DGs in modulating wound repair over laminin matrix. Blocking alpha-DG binding to laminin in 1HAEo(-) monolayers using either glycosyaminoglycans or WGA attenuated cell migration and spreading after mechanical injury. alpha-DG was not expressed in epithelial cells at the wound edge immediately after wound creation, but localized to the cell membrane in these cells within 12 h of injury. These data demonstrate the presence of DGs in airway epithelium. alpha-DG is dynamically expressed and serves as a lectin to bind laminin during airway epithelial cell repair.

  16. The trajectory and impact circumstances of asteroid 2008 TC3

    NASA Astrophysics Data System (ADS)

    Chodas, Paul; Chesley, S.; Yeomans, D.

    2010-05-01

    On October 6, 2008, the small newly discovered near-Earth asteroid 2008 TC3 was found to be on an Earth-impacting trajectory, with impact less than 20 hours away. This was the first ever predicted impact of a near-Earth object. Fortunately, it was immediately clear that the object was only a few meters in size and would therefore almost certainly break up when it entered the Earth's atmosphere. We review the pre-impact orbit computations and predictions, the post-impact reconstructions of the trajectory, and the trajectory geometry. The first prediction of impact was made by the Minor Planet Center (MPC), which quickly made the discovery and subsequent follow-up observations available to the astronomical community and contacted us at the NASA/JPL Near-Earth Object Program Office. Our impact predictions indicated that the atmospheric entry would occur over northern Sudan around 02:46 UT on October 7. Over the course of the day, the number of observations sky-rocketed to several hundred, and the impact prediction uncertainty shrank to ±3 km. Topocentric parallax up to 15 degrees is present in the data and the rates of motion approached 13.4 arcsec/s prior to the object's disappearance into the Earth's umbra. Detections of the actual atmospheric impact event suggested that it was an airburst explosion at an altitude of 37 km with an energy equivalent of about one kiloton of TNT. The airburst occurred at 02:45:45 UT at about 32.2 East longitude and 20.8 North latitude, matching the final impact predictions to within 0.2 s in time and 1.5 km in position. We compute Monte Carlo clones of the final orbit to investigate possible source orbits. Trajectory dispersions remain fairly compact as far back as 1961 , when an Earth close approach at 0.18 ± 0.12 AU scatters the predictions. Only statistical characterizations of the earlier trajectory are possible.

  17. Involvement of the clock gene Rev-erb alpha in the regulation of glucagon secretion in pancreatic alpha-cells.

    PubMed

    Vieira, Elaine; Marroquí, Laura; Figueroa, Ana Lucia C; Merino, Beatriz; Fernandez-Ruiz, Rebeca; Nadal, Angel; Burris, Thomas P; Gomis, Ramon; Quesada, Ivan

    2013-01-01

    Disruption of pancreatic clock genes impairs pancreatic beta-cell function, leading to the onset of diabetes. Despite the importance of pancreatic alpha-cells in the regulation of glucose homeostasis and in diabetes pathophysiology, nothing is known about the role of clock genes in these cells. Here, we identify the clock gene Rev-erb alpha as a new intracellular regulator of glucagon secretion. Rev-erb alpha down-regulation by siRNA (60-70% inhibition) in alphaTC1-9 cells inhibited low-glucose induced glucagon secretion (p<0.05) and led to a decrease in key genes of the exocytotic machinery. The Rev-erb alpha agonist GSK4112 increased glucagon secretion (1.6 fold) and intracellular calcium signals in alphaTC1-9 cells and mouse primary alpha-cells, whereas the Rev-erb alpha antagonist SR8278 produced the opposite effect. At 0.5 mM glucose, alphaTC1-9 cells exhibited intrinsic circadian Rev-erb alpha expression oscillations that were inhibited by 11 mM glucose. In mouse primary alpha-cells, glucose induced similar effects (p<0.001). High glucose inhibited key genes controlled by AMPK such as Nampt, Sirt1 and PGC-1 alpha in alphaTC1-9 cells (p<0.05). AMPK activation by metformin completely reversed the inhibitory effect of glucose on Nampt-Sirt1-PGC-1 alpha and Rev-erb alpha. Nampt inhibition decreased Sirt1, PGC-1 alpha and Rev-erb alpha mRNA expression (p<0.01) and glucagon release (p<0.05). These findings identify Rev-erb alpha as a new intracellular regulator of glucagon secretion via AMPK/Nampt/Sirt1 pathway.

  18. Mast cells express novel functional IL-15 receptor alpha isoforms.

    PubMed

    Bulanova, Elena; Budagian, Vadim; Orinska, Zane; Krause, Hans; Paus, Ralf; Bulfone-Paus, Silvia

    2003-05-15

    Mast cells previously have been reported to be regulated by IL-15 and to express a distinct IL-15R, termed IL-15RX. To further examine IL-15 binding and signaling in mast cells, we have studied the nature of the IL-15R and some of its biological activities in these cells. In this study, we report the existence of three novel isoforms of the IL-15R alpha chain in murine bone marrow-derived mast cells as a result of an alternative exon-splicing mechanism within the IL-15R alpha gene. These correspond to new mRNA transcripts lacking exon 4; exons 3 and 4; or exons 3, 4, and 5 (IL-15R alpha Delta 4, IL-15R alpha Delta 3,4, IL-15R alpha Delta 3,4,5). After transient transfection in COS-7 cells, all IL-15R alpha isoforms associate with the Golgi apparatus, the endoplasmic reticulum, the perinuclear space, and the cell membrane. Analysis of glycosylation pattern demonstrates the usage of a single N-glycosylation site, while no O-glycosylation is observed. Importantly, IL-15 binds with high affinity to, and promotes the survival of, murine BA/F3 cells stably transfected with the IL-15R alpha isoforms. Furthermore, we report that signaling mediated by IL-15 binding to the newly identified IL-15R alpha isoforms involves the phosphorylation of STAT3, STAT5, STAT6, Janus kinase 2, and Syk kinase. Taken together, our data indicate that murine mast cells express novel, fully functional IL-15R alpha isoforms, which can explain the selective regulatory effects of IL-15 on these cells.

  19. Plasmacytoid predendritic cells initiate psoriasis through interferon-alpha production.

    PubMed

    Nestle, Frank O; Conrad, Curdin; Tun-Kyi, Adrian; Homey, Bernhard; Gombert, Michael; Boyman, Onur; Burg, Günter; Liu, Yong-Jun; Gilliet, Michel

    2005-07-04

    Psoriasis is one of the most common T cell-mediated autoimmune diseases in humans. Although a role for the innate immune system in driving the autoimmune T cell cascade has been proposed, its nature remains elusive. We show that plasmacytoid predendritic cells (PDCs), the natural interferon (IFN)-alpha-producing cells, infiltrate the skin of psoriatic patients and become activated to produce IFN-alpha early during disease formation. In a xenograft model of human psoriasis, we demonstrate that blocking IFN-alpha signaling or inhibiting the ability of PDCs to produce IFN-alpha prevented the T cell-dependent development of psoriasis. Furthermore, IFN-alpha reconstitution experiments demonstrated that PDC-derived IFN-alpha is essential to drive the development of psoriasis in vivo. These findings uncover a novel innate immune pathway for triggering a common human autoimmune disease and suggest that PDCs and PDC-derived IFN-alpha represent potential early targets for the treatment of psoriasis.

  20. Effects of TNF-alpha on Endothelial Cell Collective Migration

    NASA Astrophysics Data System (ADS)

    Chen, Desu; Wu, Di; Helim Aranda-Espinoza, Jose; Losert, Wolfgang

    2013-03-01

    Tumor necrosis factor (TNF-alpha) is a small cell-signaling protein usually released by monocytes and macrophages during an inflammatory response. Previous work had shown the effects of TNF-alpha on single cell morphology, migration, and biomechanical properties. However, the effect on collective migrations remains unexplored. In this work, we have created scratches on monolayers of human umbilical endothelial cells (HUVECs) treated with 25ng/mL TNF-alpha on glass substrates. The wound healing like processes were imaged with phase contrast microscopy. Quantitative analysis of the collective migration of cells treated with TNF-alpha indicates that these cells maintain their persistent motion and alignment better than untreated cells. In addition, the collective migration was characterized by measuring the amount of non-affine deformations of the wound healing monolayer. We found a lower mean non-affinity and narrower distribution of non-affinities upon TNF-alpha stimulation. These results suggest that TNF-alpha introduces a higher degree of organized cell collective migration.

  1. Engineered secreted T-cell receptor alpha beta heterodimers.

    PubMed Central

    Grégoire, C; Rebaï, N; Schweisguth, F; Necker, A; Mazza, G; Auphan, N; Millward, A; Schmitt-Verhulst, A M; Malissen, B

    1991-01-01

    We have produced a soluble form of a mouse alpha beta T-cell antigen receptor (TCR) by shuffling its variable (V) and constant (C) domains to the C region of an immunoglobulin kappa light chain. These chimeric molecules composed of V alpha C alpha C kappa and V beta C beta C kappa chains were efficiently secreted (up to 1 micrograms/ml) by transfected myeloma cells as noncovalent heterodimers of about 95-kDa molecular mass. In the absence of direct binding measurement, we have refined the epitopic analysis of the soluble V alpha C alpha C kappa-V beta C beta C kappa dimers and shown that they react with an anti-clonotypic antibody and two antibodies directed to the C domain of the TCR alpha and beta chains. Conversely, we have raised three distinct monoclonal antibodies against the soluble TCR heterodimers and shown that they recognize surface-expressed TCRs. Two of these antibodies were found to react specifically with the products of the V alpha 2 (V delta 8) and V beta 2 gene segments, respectively. When considered together, these data suggest that these soluble TCR molecules are folded in a conformation indistinguishable from that which they assume at the cell surface. Images PMID:1716770

  2. Tetrahydro-iso-alpha Acids Antagonize Estrogen Receptor Alpha Activity in MCF-7 Breast Cancer Cells.

    PubMed

    Lempereur, Maëlle; Majewska, Claire; Brunquers, Amandine; Wongpramud, Sumalee; Valet, Bénédicte; Janssens, Philippe; Dillemans, Monique; Van Nedervelde, Laurence; Gallo, Dominique

    2016-01-01

    Tetrahydro-iso-alpha acids commonly called THIAA or Tetra are modified hop acids extracted from hop (Humulus lupulus L.) which are frequently used in brewing industry mainly in order to provide beer bitterness and foam stability. Interestingly, molecular structure of tetrahydro-iso-alpha acids is close to a new type of estrogen receptor alpha (ERα) antagonists aimed at disrupting the binding of coactivators containing an LxxLL motif (NR-box). In this work we show that THIAA decreases estradiol-stimulated proliferation of MCF-7 (ERα-positive breast cancer cells). Besides, we show that it inhibits ERα transcriptional activity. Interestingly, this extract fails to compete with estradiol for ERα binding and does not significantly impact the receptor turnover rate in MCF-7 cells, suggesting that it does not act like classical antiestrogens. Hence, we demonstrate that THIAA is able to antagonize ERα estradiol-induced recruitment of the LxxLL binding motif.

  3. Tetrahydro-iso-alpha Acids Antagonize Estrogen Receptor Alpha Activity in MCF-7 Breast Cancer Cells

    PubMed Central

    Lempereur, Maëlle; Majewska, Claire; Brunquers, Amandine; Wongpramud, Sumalee; Valet, Bénédicte; Janssens, Philippe; Dillemans, Monique; Van Nedervelde, Laurence; Gallo, Dominique

    2016-01-01

    Tetrahydro-iso-alpha acids commonly called THIAA or Tetra are modified hop acids extracted from hop (Humulus lupulus L.) which are frequently used in brewing industry mainly in order to provide beer bitterness and foam stability. Interestingly, molecular structure of tetrahydro-iso-alpha acids is close to a new type of estrogen receptor alpha (ERα) antagonists aimed at disrupting the binding of coactivators containing an LxxLL motif (NR-box). In this work we show that THIAA decreases estradiol-stimulated proliferation of MCF-7 (ERα-positive breast cancer cells). Besides, we show that it inhibits ERα transcriptional activity. Interestingly, this extract fails to compete with estradiol for ERα binding and does not significantly impact the receptor turnover rate in MCF-7 cells, suggesting that it does not act like classical antiestrogens. Hence, we demonstrate that THIAA is able to antagonize ERα estradiol-induced recruitment of the LxxLL binding motif. PMID:27190515

  4. p38 MAPK mediates renal tubular cell TNF-alpha production and TNF-alpha-dependent apoptosis during simulated ischemia.

    PubMed

    Meldrum, K K; Meldrum, D R; Hile, K L; Yerkes, E B; Ayala, A; Cain, M P; Rink, R C; Casale, A J; Kaefer, M A

    2001-08-01

    Ischemia causes renal tubular cell loss through apoptosis; however, the mechanisms of this process remain unclear. Using the renal tubular epithelial cell line LLC-PK(1), we developed a model of simulated ischemia (SI) to investigate the role of p38 MAPK (mitogen-activated protein kinase) in renal cell tumor necrosis factor-alpha (TNF-alpha) mRNA production, protein bioactivity, and apoptosis. Results demonstrate that 60 min of SI induced maximal TNF-alpha mRNA production and bioactivity. Furthermore, 60 min of ischemia induced renal tubular cell apoptosis at all substrate replacement time points examined, with peak apoptotic cell death occurring after either 24 or 48 h. p38 MAPK inhibition abolished TNF-alpha mRNA production and TNF-alpha bioactivity, and both p38 MAPK inhibition and TNF-alpha neutralization (anti-porcine TNF-alpha antibody) prevented apoptosis after 60 min of SI. These results constitute the initial demonstration that 1) renal tubular cells produce TNF-alpha mRNA and biologically active TNF-alpha and undergo apoptosis in response to SI, and 2) p38 MAPK mediates renal tubular cell TNF-alpha production and TNF-alpha-dependent apoptosis after SI.

  5. Almahata Sitta (=asteroid 2008 TC3) and the search for the ureilite parent body

    NASA Astrophysics Data System (ADS)

    Jenniskens, Peter; Vaubaillon, Jérémie; Binzel, Richard P.; DeMeo, Francesca E.; Nesvorný, David; Bottke, William F.; Fitzsimmons, Alan; Hiroi, Takahiro; Marchis, Franck; Bishop, Janice L.; Vernazza, Pierre; Zolensky, Michael E.; Herrin, Jason S.; Welten, Kees C.; Meier, Matthias M. M.; Shaddad, Muawia H.

    2010-10-01

    This article explores what the recovery of 2008 TC3 in the form of the Almahata Sitta meteorites may tell us about the source region of ureilites in the main asteroid belt. An investigation is made into what is known about asteroids with roughly the same spectroscopic signature as 2008 TC3. A population of low-inclination near-Earth asteroids is identified with spectra similar to 2008 TC3. Five asteroid families in the Main Belt, as well as a population of ungrouped asteroids scattered in the inner and central belts, are identified as possible source regions for this near-Earth population and 2008 TC3. Three of the families are ruled out on dynamical and spectroscopic grounds. New near-infrared spectra of 142 Polana and 1726 Hoffmeister, lead objects in the two other families, also show a poor match to Almahata Sitta. Thus, there are no Main Belt spectral analogs to Almahata Sitta currently known. Space weathering effects on ureilitic materials have not been investigated, so that it is unclear how the spectrum of the Main Belt progenitor may look different from the spectra of 2008 TC3 and the Almahata Sitta meteorites. Dynamical arguments are discussed, as well as ureilite petrogenesis and parent body evolution models, but these considerations do not conclusively point to a source region either, other than that 2008 TC3 probably originated in the inner asteroid belt.

  6. Monitoring the antiviral effect of alpha interferon on individual cells.

    PubMed

    Kim, Chon Saeng; Jung, Jong Ha; Wakita, Takaji; Yoon, Seung Kew; Jang, Sung Key

    2007-08-01

    An infectious hepatitis C virus (HCV) cDNA clone (JFH1) was generated recently. However, quantitative analysis of HCV infection and observation of infected cells have proved to be difficult because the yield of HCV in cell cultures is fairly low. We generated infectious HCV clones containing the convenient reporters green fluorescent protein (GFP) and Renilla luciferase in the NS5a-coding sequence. The new viruses responded to antiviral agents in a dose-dependent manner. Responses of individual cells containing HCV to alpha interferon (IFN-alpha) were monitored using GFP-tagged HCV and time-lapse confocal microscopy. Marked variations in the response to IFN-alpha were observed among HCV-containing cells.

  7. Persistent induction of HIF-1alpha and -2alpha in cardiomyocytes and stromal cells of ischemic myocardium.

    PubMed

    Jürgensen, Jan Steffen; Rosenberger, Christian; Wiesener, Michael S; Warnecke, Christina; Hörstrup, Jan H; Gräfe, Michael; Philipp, Sebastian; Griethe, Wanja; Maxwell, Patrick H; Frei, Ulrich; Bachmann, Sebastian; Willenbrock, Roland; Eckardt, Kai-Uwe

    2004-09-01

    Hypoxia-inducible factor (HIF)-1alpha and -2alpha are key regulators of the transcriptional response to hypoxia and pivotal in mediating the consequences of many disease states. In the present work, we define their temporo-spatial accumulation after myocardial infarction and systemic hypoxia. Rats were exposed to hypoxia or underwent coronary artery ligation. Immunohistochemistry was used for detection of HIF-1alpha and -2alpha proteins and target genes, and mRNA levels were determined by RNase protection. Marked nuclear accumulation of HIF-1alpha and -2alpha occurred after both systemic hypoxia and coronary ligation in cardiomyocytes as well as interstitial and endothelial cells (EC) without pronounced changes in HIF mRNA levels. While systemic hypoxia led to widespread induction of HIF, expression after coronary occlusion occurred primarily at the border of infarcted tissue. This expression persisted for 4 wk, included infiltrating macrophages, and colocalized with target gene expression. Subsets of cells simultaneously expressed both HIF-alpha subunits, but EC more frequently induced HIF-2alpha. A progressive increase of HIF-2alpha but not HIF-1alpha occurred in areas remote from the infarct, including the interventricular septum. Cardiomyocytes and cardiac stromal cells exhibit a marked potential for a prolonged transcriptional response to ischemia mediated by HIF. The induction of HIF-1alpha and -2alpha appears to be complementary rather than solely redundant.

  8. Nicotine inhibits Fc epsilon RI-induced cysteinyl leukotrienes and cytokine production without affecting mast cell degranulation through alpha 7/alpha 9/alpha 10-nicotinic receptors.

    PubMed

    Mishra, Neerad C; Rir-sima-ah, Jules; Boyd, R Thomas; Singh, Shashi P; Gundavarapu, Sravanthi; Langley, Raymond J; Razani-Boroujerdi, Seddigheh; Sopori, Mohan L

    2010-07-01

    Smokers are less likely to develop some inflammatory and allergic diseases. In Brown-Norway rats, nicotine inhibits several parameters of allergic asthma, including the production of Th2 cytokines and the cysteinyl leukotriene LTC(4). Cysteinyl leukotrienes are primarily produced by mast cells, and these cells play a central role in allergic asthma. Mast cells express a high-affinity receptor for IgE (FcepsilonRI). Following its cross-linking, cells degranulate and release preformed inflammatory mediators (early phase) and synthesize and secrete cytokines/chemokines and leukotrienes (late phase). The mechanism by which nicotine modulates mast cell activation is unclear. Using alpha-bungarotoxin binding and quantitative PCR and PCR product sequencing, we showed that the rat mast/basophil cell line RBL-2H3 expresses nicotinic acetylcholine receptors (nAChRs) alpha7, alpha9, and alpha10; exposure to exceedingly low concentrations of nicotine (nanomolar), but not the biologically inactive metabolite cotinine, for > or = 8 h suppressed the late phase (leukotriene/cytokine production) but not degranulation (histamine and hexosaminidase release). These effects were unrelated to those of nicotine on intracellular free calcium concentration but were causally associated with the inhibition of cytosolic phospholipase A(2) activity and the PI3K/ERK/NF-kappaB pathway, including phosphorylation of Akt and ERK and nuclear translocation of NF-kappaB. The suppressive effect of nicotine on the late-phase response was blocked by the alpha7/alpha9-nAChR antagonists methyllycaconitine and alpha-bungarotoxin, as well as by small interfering RNA knockdown of alpha7-, alpha9-, or alpha10-nAChRs, suggesting a functional interaction between alpha7-, alpha9-, and alpha10-nAChRs that might explain the response of RBL cells to nanomolar concentrations of nicotine. This "hybrid" receptor might serve as a target for novel antiallergic/antiasthmatic therapies.

  9. Targeting Prostate Cancer Stem Cells with Alpha-Particle Therapy.

    PubMed

    Ceder, Jens; Elgqvist, Jörgen

    2016-01-01

    Modern molecular and radiopharmaceutical development has brought the promise of tumor-selective delivery of antibody-drug conjugates to tumor cells for the diagnosis and treatment of primary and disseminated tumor disease. The classical mode of discourse regarding targeted therapy has been that the antigen targeted must be highly and homogenously expressed in the tumor cell population, and at the same time exhibit low expression in healthy tissue. However, there is increasing evidence that the reason cancer patients are not cured by current protocols is that there exist subpopulations of cancer cells that are resistant to conventional therapy including radioresistance and that these cells express other target antigens than the bulk of the tumor cells. These types of cells are often referred to as cancer stem cells (CSCs). The CSCs are tumorigenic and have the ability to give rise to all types of cells found in a cancerous disease through the processes of self-renewal and differentiation. If the CSCs are not eradicated, the cancer is likely to recur after therapy. Due to some of the characteristics of alpha particles, such as short path length and high density of energy depositions per distance traveled in tissue, they are especially well suited for use in targeted therapies against microscopic cancerous disease. The characteristics of alpha particles further make it possible to minimize the irradiation of non-targeted surrounding healthy tissue, but most importantly, make it possible to deliver high-absorbed doses locally and therefore eradicating small tumor cell clusters on the submillimeter level, or even single tumor cells. When alpha particles pass through a cell, they cause severe damage to the cell membrane, cytoplasm, and nucleus, including double-strand breaks of DNA that are very difficult to repair for the cell. This means that very few hits to a cell by alpha particles are needed in order to cause cell death, enabling killing of cells, such as CSCs

  10. Targeting Prostate Cancer Stem Cells with Alpha-Particle Therapy

    PubMed Central

    Ceder, Jens; Elgqvist, Jörgen

    2017-01-01

    Modern molecular and radiopharmaceutical development has brought the promise of tumor-selective delivery of antibody–drug conjugates to tumor cells for the diagnosis and treatment of primary and disseminated tumor disease. The classical mode of discourse regarding targeted therapy has been that the antigen targeted must be highly and homogenously expressed in the tumor cell population, and at the same time exhibit low expression in healthy tissue. However, there is increasing evidence that the reason cancer patients are not cured by current protocols is that there exist subpopulations of cancer cells that are resistant to conventional therapy including radioresistance and that these cells express other target antigens than the bulk of the tumor cells. These types of cells are often referred to as cancer stem cells (CSCs). The CSCs are tumorigenic and have the ability to give rise to all types of cells found in a cancerous disease through the processes of self-renewal and differentiation. If the CSCs are not eradicated, the cancer is likely to recur after therapy. Due to some of the characteristics of alpha particles, such as short path length and high density of energy depositions per distance traveled in tissue, they are especially well suited for use in targeted therapies against microscopic cancerous disease. The characteristics of alpha particles further make it possible to minimize the irradiation of non-targeted surrounding healthy tissue, but most importantly, make it possible to deliver high-absorbed doses locally and therefore eradicating small tumor cell clusters on the submillimeter level, or even single tumor cells. When alpha particles pass through a cell, they cause severe damage to the cell membrane, cytoplasm, and nucleus, including double-strand breaks of DNA that are very difficult to repair for the cell. This means that very few hits to a cell by alpha particles are needed in order to cause cell death, enabling killing of cells, such as CSCs

  11. Bax alpha perturbs T cell development and affects cell cycle entry of T cells.

    PubMed Central

    Brady, H J; Gil-Gómez, G; Kirberg, J; Berns, A J

    1996-01-01

    Bax alpha can heterodimerize with Bcl-2 and Bcl-X(L), countering their effects, as well as promoting apoptosis on overexpression. We show that bax alpha transgenic mice have greatly reduced numbers of mature T cells, which results from an impaired positive selection in the thymus. This perturbation in positive selection is accompanied by an increase in the number of cycling thymocytes. Further to this, mature T cells overexpressing Bax alpha have lower levels of p27Kip1 and enter S phase more rapidly in response to interleukin-2 stimulation than do control T cells, while the converse is true of bcl-2 transgenic T cells. These data indicate that apoptotic regulatory proteins can modulate the level of cell cycle-controlling proteins and thereby directly impact on the cell cycle. Images PMID:9003775

  12. Laminin isoforms and their integrin receptors in glioma cell migration and invasiveness: Evidence for a role of alpha5-laminin(s) and alpha3beta1 integrin.

    PubMed

    Kawataki, Tomoyuki; Yamane, Tetsu; Naganuma, Hirofumi; Rousselle, Patricia; Andurén, Ingegerd; Tryggvason, Karl; Patarroyo, Manuel

    2007-11-01

    Glioma cell infiltration of brain tissue often occurs along the basement membrane (BM) of blood vessels. In the present study we have investigated the role of laminins, major structural components of BMs and strong promoters of cell migration. Immunohistochemical studies of glioma tumor tissue demonstrated expression of alpha2-, alpha3-, alpha4- and alpha5-, but not alpha1-, laminins by the tumor vasculature. In functional assays, alpha3 (Lm-332/laminin-5)- and alpha5 (Lm-511/laminin-10)-laminins strongly promoted migration of all glioma cell lines tested. alpha1-Laminin (Lm-111/laminin-1) displayed lower activity, whereas alpha2 (Lm-211/laminin-2)- and alpha4 (Lm-411/laminin-8)-laminins were practically inactive. Global integrin phenotyping identified alpha3beta1 as the most abundant integrin in all the glioma cell lines, and this laminin-binding integrin exclusively or largely mediate the cell migration. Moreover, pretreatment of U251 glioma cells with blocking antibodies to alpha3beta1 integrin followed by intracerebral injection into nude mice inhibited invasion of the tumor cells into the brain tissue. The cell lines secreted Lm-211, Lm-411 and Lm-511, at different ratios. The results indicate that glioma cells secrete alpha2-, alpha4- and alpha5-laminins and that alpha3- and alpha5-laminins, found in brain vasculature, selectively promote glioma cell migration. They identify alpha3beta1 as the predominant integrin and laminin receptor in glioma cells, and as a brain invasion-mediating integrin.

  13. Secretion of alpha 1-antitrypsin by alveolar epithelial cells.

    PubMed

    Venembre, P; Boutten, A; Seta, N; Dehoux, M S; Crestani, B; Aubier, M; Durand, G

    1994-06-13

    We have investigated the ability of alveolar epithelial cells (human A549 cell line and rat type-II pneumocytes) to produce alpha 1-antitrypsin (AAT). Northern blot analysis demonstrated the presence of an AAT-specific mRNA transcript in A549 cells. Unstimulated A549 cells secreted immunoreactive AAT at a rate of 0.51 +/- 0.04 ng/10(6) cells/h, with a modified glycosylation compared to serum AAT. AAT formed a complex with neutrophil elastase. Rat type-II pneumocytes secreted immunoreactive AAT. Our results suggest that alveolar epithelial cells could participate in antiprotease defense within the lung through local AAT production.

  14. Taraxacum officinale induces cytotoxicity through TNF-alpha and IL-1alpha secretion in Hep G2 cells.

    PubMed

    Koo, Hyun-Na; Hong, Seung-Heon; Song, Bong-Keun; Kim, Cheorl-Ho; Yoo, Young-Hyun; Kim, Hyung-Min

    2004-01-16

    Taraxacum officinale (TO) has been frequently used as a remedy for women's disease (e.g. breast and uterus cancer) and disorders of the liver and gallbladder. Several earlier studies have indicated that TO exhibits anti-tumor properties, but its mechanism remains to be elucidated. In this study, we investigated the effect of TO on the cytotoxicity and production of cytokines in human hepatoma cell line, Hep G2. Our results show that TO decreased the cell viability by 26%, and significantly increased the tumor necrosis factor (TNF)-alpha and interleukin (IL)-1alpha production compared with media control (about 1.6-fold for TNF-alpha, and 2.4-fold for IL-1alpha, P < 0.05). Also, TO strongly induced apoptosis of Hep G2 cells as determined by flow cytometry. Increased amounts of TNF-alpha and IL-1alpha contributed to TO-induced apoptosis. Anti-TNF-alpha and IL-1alpha antibodies almost abolished it. These results suggest that TO induces cytotoxicity through TNF-alpha and IL-1alpha secretion in Hep G2 cells.

  15. T-cell receptor V alpha and C alpha alleles associated with multiple and myasthenia gravis.

    PubMed Central

    Oksenberg, J R; Sherritt, M; Begovich, A B; Erlich, H A; Bernard, C C; Cavalli-Sforza, L L; Steinman, L

    1989-01-01

    Polymorphic markers in genes encoding that alpha chain of the human T-cell receptor (TcR) have been detected by Southern blot analysis in Pss I digests. Polymorphic bands were observed at 6.3 and 2.0 kilobases (kb) with frequencies of 0.30 and 0.44, respectively, in the general population. Using the polymerase chain reaction (PCR) method, we amplified selected sequences derived from the full-length TcR alpha cDNA probe. These PCR products were used as specific probes to demonstrate that the 6.3-kb polymorphic fragment hybridizes to the variable (V)-region probe and the 2.0-kb fragment hybridizes to the constant (C)-region probe. Segregation of the polymorphic bands was analyzed in family studies. To look for associations between these markers and autoimmune diseases, we have studied the restriction fragment length polymorphism distribution of the Pss I markers in patients with multiple sclerosis, myasthenia gravis, and Graves disease. Significant differences in the frequency of the polymorphic V alpha and C alpha markers were identified between patients and healthy individuals. Images PMID:2915992

  16. Human fat cell alpha-2 adrenoceptors. I. Functional exploration and pharmacological definition with selected alpha-2 agonists and antagonists

    SciTech Connect

    Galitzky, J.; Mauriege, P.; Berlan, M.; Lafontan, M.

    1989-05-01

    This study was undertaken to investigate more fully the pharmacological characteristics of the human fat cell alpha-2 adrenoceptor. Biological assays were performed on intact isolated fat cells while radioligand binding studies were carried out with (/sup 3/H)yohimbine in membranes. These pharmacological studies brought: (1) a critical definition of the limits of the experimental conditions required for the exploration of alpha-2 adrenergic responsiveness on human fat cells and membranes; (2) an improvement in the pharmacological definition of the human fat cell postsynaptic alpha-2 adrenoceptor. Among alpha-2 agonists, UK-14,304 was the most potent and the relative order of potency was: UK-14,304 greater than p-aminoclonidine greater than clonidine = B-HT 920 greater than rilmenidine. For alpha-2 antagonists, the potency order was: yohimbine greater than idazoxan greater than SK F-86,466 much greater than benextramine; (3) a description of the impact of benextramine (irreversible alpha-1/alpha-2 antagonist) on human fat cell alpha-2 adrenergic receptors and on human fat cell function; the drug inactivates the alpha-2 adrenergic receptors with a minor impact on beta adrenergic receptors and without noticeable alterations of fat cell function as assessed by preservation of beta adrenergic and Al-adenosine receptor-mediated lipolytic responses; and (4) a definition of the relationship existing between alpha-2 adrenergic receptor occupancy, inhibition of adenylate cyclase activity and antilipolysis with full and partial agonists. The existence of a receptor reserve must be taken into account when evaluating alpha-2 adrenergic receptor distribution and regulation of human fat cells.

  17. Role of alpha 5 beta 1 integrin in determining malignant properties of colon carcinoma cells.

    PubMed

    Gong, J; Wang, D; Sun, L; Zborowska, E; Willson, J K; Brattain, M G

    1997-01-01

    We characterized the expression of alpha 5 beta 1 integrin in two distinct phenotypes of colon carcinoma cell lines. Highly invasive colon cell lines (designated Group I cell lines) expressed higher levels of integrin alpha 5 beta 1 mRNA and protein than did poorly invasive colon cell lines (designated Group III cell lines). The relatively high expression of integrin alpha 5 beta 1 in Group I cell lines resulted in strong enhancement of cell adhesion to fibronectin (FN) tissue culture plates, whereas Group III cell lines showed little or no enhancement of cell adhesion by coating. There was no significant difference between Group I and Group III cell lines with respect to cell adhesion to laminin and collagen IV. Cell adhesion to FN in Group I cells was mainly mediated by integrin alpha 5 beta 1 because a monoclonal anti-alpha 5 subunit antibody could block cell adhesion to FN, whereas anti-alpha 2 and anti-alpha 3 antibodies had no effect on cell adhesion to FN. The divergence of alpha 5 beta 1 expression in these two distinct colon carcinoma phenotypes suggested that high expression of alpha 5 beta 1 might contribute to malignant progression in this model system. To test this hypothesis, GEO cells, a Group III cell line that did not express alpha 5 integrin, were transfected with the alpha 5 subunit. Stable transfection of alpha 5 sense cDNA into a typical GEO-limiting dilution clone led to the expression of alpha 5 subunit mRNA and cell surface alpha 5 beta 1 protein. The alpha 5 sense transfectants showed enhanced attachment to FN-coated plates and were more tumorigenic when the cells were injected into athymic nude mice. These results indicate that inappropriately high alpha 5 beta 1 integrin expression contributes to malignant progression in colon carcinoma.

  18. Radon measurement of natural gas using alpha scintillation cells.

    PubMed

    Kitto, Michael E; Torres, Miguel A; Haines, Douglas K; Semkow, Thomas M

    2014-12-01

    Due to their sensitivity and ease of use, alpha-scintillation cells are being increasingly utilized for measurements of radon ((222)Rn) in natural gas. Laboratory studies showed an average increase of 7.3% in the measurement efficiency of alpha-scintillation cells when filled with less-dense natural gas rather than regular air. A theoretical calculation comparing the atomic weight and density of air to that of natural gas suggests a 6-7% increase in the detection efficiency when measuring radon in the cells. A correction is also applicable when the sampling location and measurement laboratory are at different elevations. These corrections to the measurement efficiency need to be considered in order to derive accurate concentrations of radon in natural gas.

  19. Inhibition of THP-1 cell adhesion to endothelial cells by alpha-tocopherol and alpha-tocotrienol is dependent on intracellular concentration of the antioxidants.

    PubMed

    Noguchi, Noriko; Hanyu, Ryuhei; Nonaka, Aya; Okimoto, Yuko; Kodama, Tatsuhiko

    2003-06-15

    Vitamin E analogs such as alpha-tocopherol and alpha-tocotrienol have been shown to reduce endothelial expression of adhesion molecules. The reactivity of alpha-tocopherol and alpha-tocotrienol in inhibiting lipid peroxidation in vitro was essentially identical but the inhibition of adhesion of THP-1 cells, a monocytic-"like" cell line, to endothelial cells differs substantially. To determine the mechanism underlying this response, human umbilical vein endothelial cells (HUVECs) were assessed for their ability to accumulate vitamin E analogs. alpha-Tocotrienol accumulated in HUVECs to levels approximately 10-fold greater than that of alpha-tocopherol. The decrease in expression of vascular cell adhesion molecule-1 (VCAM-1) and the adhesion of THP-1 cells to HUVECs by alpha-tocopherol and alpha-tocotrienol was also determined. Both alpha-tocopherol and alpha-tocotrienol suppressed VCAM-1 expression and adhesion of THP-1 cells to HUVECs in a concentration-dependent manner. The efficacy of tocotrienol for reduction of VCAM-1 expression and adhesion of THP-1 cells to HUVECs was also 10-fold higher than that of tocopherol. The inhibitory effects of vitamin E analogs on the adhesiveness of endothelial cells clearly correlated with their intracellular concentrations. The data demonstrated that, in assessing the biological responses of antioxidants, intracellular accumulation and metabolism were additional important factors that must be considered.

  20. glucagon is essential for alpha cell transdifferentiation and beta cell neogenesis

    PubMed Central

    Ye, Lihua; Robertson, Morgan A.; Hesselson, Daniel; Stainier, Didier Y. R.; Anderson, Ryan M.

    2015-01-01

    The interconversion of cell lineages via transdifferentiation is an adaptive mode of tissue regeneration and an appealing therapeutic target. However, its clinical exploitation is contingent upon the discovery of contextual regulators of cell fate acquisition and maintenance. In murine models of diabetes, glucagon-secreting alpha cells transdifferentiate into insulin-secreting beta cells following targeted beta cell depletion, regenerating the form and function of the pancreatic islet. However, the molecular triggers of this mode of regeneration are unknown. Here, using lineage-tracing assays in a transgenic zebrafish model of beta cell ablation, we demonstrate conserved plasticity of alpha cells during islet regeneration. In addition, we show that glucagon expression is upregulated after injury. Through gene knockdown and rescue approaches, we also find that peptides derived from the glucagon gene are necessary for alpha-to-beta cell fate switching. Importantly, whereas beta cell neogenesis was stimulated by glucose, alpha-to-beta cell conversion was not, suggesting that transdifferentiation is not mediated by glucagon/GLP-1 control of hepatic glucose production. Overall, this study supports the hypothesis that alpha cells are an endogenous reservoir of potential new beta cells. It further reveals that glucagon plays an important role in maintaining endocrine cell homeostasis through feedback mechanisms that govern cell fate stability. PMID:25852199

  1. A T-cell specific transcriptional enhancer element 3 prime of C sub. alpha. in the human T-cell receptor. alpha. locus

    SciTech Connect

    Ho, Icheng; Yang, Lihsuan; Morle, G.; Leiden, J.M. )

    1989-09-01

    A transcriptional enhancer element has been identified 4.5 kilobases 3{prime} of C{sub {alpha}} (constant region {alpha} chain) in the human T-cell receptor (TCR) {alpha}-chain locus. This enhancer is active on both a TCR V{sub {alpha}} (variable region {alpha} chain) promoter and the minimal simian virus 40 promoter in TCR {alpha}/{beta} Jurkat and EL4 cells but is inactive on a V{sub {alpha}} promoter TCR {gamma}/{delta} PEER and Molt-13 cells, clone 13 B cells, and HeLa fibroblasts. The enhancer has been localized to a 116-base-pair BstXI/Dra I restriction enzyme fragment, which lacks immunoglobulin octamer and {kappa}B enhancer motifs but does contain a consensus cAMP-response element (CRE). DNase I footprint analyses demonstrated that the minimal enhancer contains two binding sites for Jurkat nuclear proteins. One of these sites corresponds to the CRE, while the other does not correspond to a known transcriptional enhancer motif. These data support a model in which TCR {alpha} gene transcription is regulated by a unique set of cis-acting sequences and trans-acting factors, which are differentially active in cells of the TCR {alpha}/{beta} lineage. In addition, the TCR {alpha} enhancer may play a role in activating oncogene expression in T-lymphoblastoid tumors that have previously been shown to display chromosomal translocations into the human TCR {alpha} locus.

  2. Recovery of Saccharomyces cerevisiae mating-type a cells from G1 arrest by alpha factor.

    PubMed Central

    Chan, R K

    1977-01-01

    Mating-type a cells of the yeast Saccharomyces cerevisiae that had been specifically arrested in the G1 phase of the cell cycle by alpha factor, an oligopeptide pheromone made by alpha cells, recovered and resumed cell division after a period of inhibition which was dependent on the concentration of alpha factor used. These treated a cells were more resistant to alpha factor than untreated a cells, but lost their resistance upon further cell division. However, cells arrested for 6 h were no more resistant to alpha factor than cells arrested for only 2.5 h. Mating-type a strains could inactivate or remove alpha factor from the culture fluid, but two a sterile (nonmating) mutants and an a/alpha diploid strain could not. These results suggest that a cells have a mechanism, which may involve uptake or inactivation of alpha factor, for recovering from alpha factor arrest. However, the results do not distinguish between a recovery mechanism which is constitutive and one which is induced by alpha factor. The loss of alpha factor activity during recovery appeared to be primarily cell contact mediated, although an extracellular, diffusible inhibitor of alpha factor that is labile or that functions stoichiometrically could not be ruled out. PMID:400792

  3. Interferon-alpha and dexamethasone inhibit adhesion of T cells to endothelial cells and synovial cells

    PubMed Central

    Eguchi, K.; Kawakami, A.; Nakashima, M.; Ida, H.; Sakito, S.; Matsuoka, N.; Terada, K.; Sakai, M.; Kawabe, Y.; Fukuda, T.; Ishimaru, T.; Kurouji, K.; Fujita, N.; Aoyagi, T.; Maeda, K.; Nagataki, S.

    1992-01-01

    We investigated whether interferon-gamma (IFN-γ), interferon-alpha (IFN-α) and glucocorticoids affected the adhesion of T cells to human umbilical endothelial cells or human synovial cells. About 30% of peripheral blood T cells could bind to unstimulated endothelial cells, but only a few T cells could bind to unstimulated synovial cells. When both endothelial cells and synovial cells were cultured with recombinant IFN-γ (rIFN-γ), the percentage of T cell binding to both types of cells increased in a dose-dependent manner. rIFN-α and dexamethasone blocked the T cell binding to unstimulated endothelial cells. Furthermore, rIFN-α and dexamethasone suppressed T cell binding to both endothelial cells and synovial cells stimulated by IFN-γ, and also inhibited intercellular adhesion molecule-1 (ICAM-1) expression on both endothelial cells and synovial cells stimulated by IFN-γ. These results suggest that IFN-α and glucocorticoids may inhibit T cell binding to endothelial cells or synovial cells by modulating adhesion molecule expression on these cells. PMID:1606729

  4. Concomitant T-cell receptor alpha and delta gene rearrangements in individual T-cell precursors.

    PubMed Central

    Thompson, S D; Pelkonen, J; Hurwitz, J L

    1990-01-01

    A debate has recently surfaced concerning the degree of precommitment attained by alpha beta and gamma delta T-cell precursors prior to T-cell receptor (TCR) gene rearrangement. It has been suggested that precursors may be precommitted to rearrange either alpha or delta genes, but not both, thus giving rise to alpha beta- and gamma delta-producing T cells, respectively. Alternatively, the precursors may be flexible with regard to potential TCR gene rearrangements. To address this controversy, the gene rearrangements among a group of T-cell hybridomas from fetal, newborn, and early postnatal mouse thymi were examined. Six probes spanning the delta and alpha loci were used in Southern blot analyses to characterize the rearrangements which occurred on homologous chromosomes in each cell. Although homologous chromosomes often rearranged in synchrony within the alpha locus, a number of hybridomas were found which had retained a delta rearrangement on one chromosome and an alpha rearrangement on the second. Results show that a precommitment by T cells to rearrange delta or alpha genes in a mutually exclusive manner is not an absolute feature of mouse thymocyte development. Images PMID:2164690

  5. Alpha1-antitrypsin protects beta-cells from apoptosis.

    PubMed

    Zhang, Bin; Lu, Yuanqing; Campbell-Thompson, Martha; Spencer, Terry; Wasserfall, Clive; Atkinson, Mark; Song, Sihong

    2007-05-01

    Beta-cell apoptosis appears to represent a key event in the pathogenesis of type 1 diabetes. Previous studies have demonstrated that administration of the serine proteinase inhibitor alpha1-antitrypsin (AAT) prevents type 1 diabetes development in NOD mice and prolongs islet allograft survival in rodents; yet the mechanisms underlying this therapeutic benefit remain largely unclear. Herein we describe novel findings indicating that AAT significantly reduces cytokine- and streptozotocin (STZ)-induced beta-cell apoptosis. Specifically, strong antiapoptotic activities for AAT (Prolastin, human) were observed when murine insulinoma cells (MIN6) were exposed to tumor necrosis factor-alpha. In a second model system involving STZ-induced beta-cell apoptosis, treatment of MIN6 cells with AAT similarly induced a significant increase in cellular viability and a reduction in apoptosis. Importantly, in both model systems, treatment with AAT completely abolished induced caspase-3 activity. In terms of its activities in vivo, treatment of C57BL/6 mice with AAT prevented STZ-induced diabetes and, in agreement with the in vitro analyses, supported the concept of a mechanism involving the disruption of beta-cell apoptosis. These results propose a novel biological function for this molecule and suggest it may represent an effective candidate for attempts seeking to prevent or reverse type 1 diabetes.

  6. Integrin {alpha}6 cleavage: A novel modification to modulate cell migration

    SciTech Connect

    Pawar, Sangita C.; Demetriou, Manolis C.; Nagle, Raymond B.; Bowden, G. Tim; Cress, Anne E. . E-mail: acress@azcc.arizona.edu

    2007-04-01

    Integrins play a major role in cell adhesion and migration. Previous work reported that a cleaved form of integrin {alpha}6 ({alpha}6p) was detected in invasive human prostate cancer tissue, absent in normal prostate tissue and was produced by urokinase-type Plasminogen Activator (uPA) in a plasmin-independent manner. Using site-directed mutagenesis we identified amino acid residues R594 and R595, located in the 'stalk' region of integrin {alpha}6, as essential for cleavage. The cleavage site is located on the extracellular region of the protein between the {beta}-barrel domain and the thigh domain. Prostate cancer cells (PC3N) were stably transfected to overexpress the cleavable, wild-type (PC3N-{alpha}6-WT) or the non-cleavable form of integrin {alpha}6 (PC3N-{alpha}6-RR). The number of cells invading laminin 111- and laminin 332-coated filters by PC3N-{alpha}6-WT cells increased by threefold as compared to PC3N-{alpha}6-RR cells. Plasminogen activator inhibitor-1 (PAI-1) reduced the invasion of PC3N-{alpha}6-WT cells by approximately 42% through laminin 332-coated filters and plasmin inhibitor aprotinin had no significant effect. Linear cell migration increased production of integrin {alpha}6p in the PC3N-{alpha}6-WT cells and not in the PC3N-{alpha}6-RR cells and 32% of the PC3N-{alpha}6-WT cells migrated on laminin 111 in the linear migration assay as compared to the 5% PC3N-{alpha}6-RR cells. These data taken together suggest that the uPA-mediated cell surface cleavage of the {alpha}6 integrin extracellular domain is involved in tumor cell invasion and migration on laminin.

  7. Alpha-adrenergic blocker mediated osteoblastic stem cell differentiation

    SciTech Connect

    Choi, Yoon Jung; Lee, Jue Yeon; Lee, Seung Jin; Chung, Chong-Pyoung; Park, Yoon Jeong

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Doxazocin directly up-regulated bone metabolism at a low dose. Black-Right-Pointing-Pointer Doxazocin induced osteoblastic stem cell differentiation without affecting cell proliferation. Black-Right-Pointing-Pointer This osteogenic stem cell differentiation is mediated by ERK-signal dependent pathway. -- Abstract: Recent researches have indicated a role for antihypertensive drugs including alpha- or beta-blockers in the prevention of bone loss. Some epidemiological studies reported the protective effects of those agents on fracture risk. However, there is limited information on the association with those agents especially at the mechanism of action. In the present study, we investigated the effects of doxazosin, an alpha-blocker that is clinically used for the treatment of benign prostatic hyperplasia (BPH) along with antihypertensive medication, on the osteogenic stem cell differentiation. We found that doxazosin increased osteogenic differentiation of human mesenchymal stem cells, detected by Alizarin red S staining and calcein. Doxazosin not only induced expression of alkaline phosphatase, type I collagen, osteopontin, and osteocalcin, it also resulted in increased phosphorylation of extracellular signal-regulated kinase (ERK1/2), a MAP kinase involved in osteoblastic differentiation. Treatment with U0126, a MAP kinase inhibitor, significantly blocked doxazosin-induced osteoblastic differentiation. Unrelated to activation of osteogenic differentiation by doxazosin, we found that there were no significant changes in adipogenic differentiation or in the expression of adipose-specific genes, including peroxisome proliferator-activated receptor {gamma}, aP2, or LPL. In this report, we suggest that doxazosin has the ability to increase osteogenic cell differentiation via ERK1/2 activation in osteogenic differentiation of adult stem cells, which supports the protective effects of antihypertensive drug on fracture risk and

  8. The crystal structure of a TL/CD8{alpha}{alpha} complex at 2.1 {angstrom} resolution : implications for modulation of T cell activation and memory.

    SciTech Connect

    Liu, Y.; Xiong, Y.; Naidenko, O. V.; Liu, J.-H.; Zhang, R.; Joachimiak, A.; Kronenberg, M.; Cheroutre, H.; Reinherz, E. L.; Wang, J.-H.; Biosciences Division; Dana-Farber Cancer Inst.; Harvard Medical School; La Jolla Inst. of Allergy and Immunology

    2003-02-01

    TL is a nonclassical MHC class I molecule that modulates T cell activation through relatively high-affinity interaction with CD8{alpha}{alpha}. To investigate how the TL/CD8{alpha}{alpha} interaction influences TCR signaling, we characterized the structure of the TL/CD8{alpha}{alpha} complex using X-ray crystallography. Unlike antigen-presenting molecules, the TL antigen-binding groove is occluded by specific conformational changes. This feature eliminates antigen presentation, severely hampers direct TCR recognition, and prevents TL from participating in the TCR activation complex. At the same time, the TL/CD8{alpha}{alpha} interaction is strengthened through subtle structure changes in the TL {alpha}3 domain. Thus, TL functions to sequester and redirect CD8{alpha}{alpha} away from the TCR, modifying lck-dependent signaling.

  9. Cell wall alpha1-3glucans induce the aggregation of germinating conidia of Aspergillus fumigatus.

    PubMed

    Fontaine, Thierry; Beauvais, Anne; Loussert, Céline; Thevenard, Benoît; Fulgsang, Claus C; Ohno, Naohito; Clavaud, Cécile; Prevost, Marie-Christine; Latgé, Jean-Paul

    2010-08-01

    The germination of Aspergillus fumigatus conidia can be divided into four stages: breaking of dormancy, isotropic swelling, establishment of cell polarity, and formation of a germ tube. Swelling of conidia is associated in liquid medium with a multi-cellular aggregation that produced large clumps of conidia. Conidial aggregation can be specifically prevented by the addition of alpha1-3glucanase. Swollen conidia specifically adhere to insoluble alpha1-3glucan chains. Electron microscopy studies showed that cell wall alpha1-3glucan chains became exposed at the cell surface during the swelling. These results demonstrate that cell wall alpha1-3glucans play an essential role in the aggregation between swollen conidia. Experiments with alpha1-3glucan coated latex beads show that alpha1-3glucan chains interacted between them without the requirement of any other cell wall component suggesting that biophysical properties of alpha1-3glucans are solely responsible for conidial aggregation.

  10. Molecular mechanism of {alpha}-tocopheryl-phosphate transport across the cell membrane

    SciTech Connect

    Negis, Yesim; Meydani, Mohsen; Zingg, Jean-Marc; Azzi, Angelo . E-mail: angelo.azzi@tufts.edu

    2007-07-27

    {alpha}-Tocopheryl-phosphate ({alpha}-TP) is synthesized and hydrolyzed in animal cells and tissues where it modulates several functions. {alpha}-TP is more potent than {alpha}-T in inhibiting cell proliferation, down-regulating CD36 transcription, inhibiting atherosclerotic plaque formation. Administration of {alpha}-TP to cells or animals requires its transfer through membranes, via a transporter. We show here that {alpha}-TP is passing the plasma membrane via a system that is inhibited by glibenclamide and probenecid, inhibitors of a number of transporters. Glibenclamide and probenecid prevent dose-dependently {alpha}-TP inhibition of cell proliferation. The two inhibitors act on ATP binding cassette (ABC) and organic anion transporters (OAT). Since ABC transporters function to export solutes and {alpha}-TP is transported into cells, it may be concluded that {alpha}-TP transport may occur via an OAT family member. Due to the protection by glibenclamide and probenecid on the {alpha}-TP induced cell growth inhibition it appears that {alpha}-TP acts after its uptake inside cells.

  11. ERR{alpha} regulates osteoblastic and adipogenic differentiation of mouse bone marrow mesenchymal stem cells

    SciTech Connect

    Rajalin, Ann-Marie; Pollock, Hanna; Aarnisalo, Piia

    2010-05-28

    The orphan nuclear receptor estrogen-related receptor-{alpha} (ERR{alpha}) has been reported to have both a positive and a negative regulatory role in osteoblastic and adipocytic differentiation. We have studied the role of ERR{alpha} in osteoblastic and adipogenic differentiation of mesenchymal stem cells. Bone marrow mesenchymal stem cells were isolated from ERR{alpha} deficient mice and their differentiation capacities were compared to that of the wild-type cells. ERR{alpha} deficient cultures displayed reduced cellular proliferation, osteoblastic differentiation, and mineralization. In the complementary experiment, overexpression of ERR{alpha} in MC3T3-E1 cells increased the expression of osteoblastic markers and mineralization. Alterations in the expression of bone sialoprotein (BSP) may at least partially explain the effects on mineralization as BSP expression was reduced in ERR{alpha} deficient MSCs and enhanced upon ERR{alpha} overexpression in MC3T3-E1 cells. Furthermore, a luciferase reporter construct driven by the BSP promoter was efficiently transactivated by ERR{alpha}. Under adipogenic conditions, ERR{alpha} deficient cultures displayed reduced adipocytic differentiation. Our data thus propose a positive role for ERR{alpha} in osteoblastic and adipocytic differentiation. The variability in the results yielded in the different studies implies that ERR{alpha} may play different roles in bone under different physiological conditions.

  12. AKAP 18 alpha and gamma have opposing effects on insulin release in INS-1E cells.

    PubMed

    Josefsen, Knud; Lee, Ying C; Thams, Peter; Efendic, Suad; Nielsen, Jens H

    2010-01-04

    A-kinase anchoring proteins (AKAPs) are known to compartmentalise protein kinase(s) to discrete cellular locations. Here we show that silencing of AKAP 18 alpha or gamma expression results in decreased or increased glucose-stimulated insulin secretion in INS-1E cells. Glucose stimulates AKAP 18 alpha and inhibits AKAP 18 gamma mRNA expressions while palmitate markedly reduces AKAP 18 alpha expression. Human growth hormone (GH) stimulates AKAP 18 alpha expression and attenuates palmitate-induced suppression of AKAP 18 alpha mRNA level. The roles of AKAP 18 alpha and gamma in mediating insulin release are consistent with their respective regulations by glucose.

  13. Cell cycle regulation and p53 activation by protein phosphatase 2C alpha.

    PubMed

    Ofek, Paula; Ben-Meir, Daniella; Kariv-Inbal, Zehavit; Oren, Moshe; Lavi, Sara

    2003-04-18

    Protein phosphatase 2C (PP2C) dephosphorylates a broad range of substrates, regulating stress response and growth-related pathways in both prokaryotes and eukaryotes. We now demonstrate that PP2C alpha, a major mammalian isoform, inhibits cell growth and activates the p53 pathway. In 293 cell clones, in which PP2C alpha expression is regulated by a tetracycline-inducible promoter, PP2C alpha overexpression led to G(2)/M cell cycle arrest and apoptosis. Furthermore, PP2C alpha induced the expression of endogenous p53 and the p53-responsive gene p21. Activation of the p53 pathway by PP2C alpha took place both in cells harboring endogenous p53, as well as in p53-null cells transfected with exogenous p53. Induction of PP2C alpha resulted in an increase in the overall levels of p53 protein as well as an augmentation of p53 transcription activity. The dephosphorylation activity of PP2C alpha is essential to the described phenomena, as none of these effects was detected when an enzymatically inactive PP2C alpha mutant was overexpressed. p53 plays an important role in PP2C alpha-directed cell cycle arrest and apoptosis because perturbation of p53 expression in human 293 cells by human papillomavirus E6 led to a significant increase in cell survival. The role of PP2C alpha in p53 activation is discussed.

  14. Thermophysical properties of Almahata Sitta meteorites (asteroid 2008 TC3) for high-fidelity entry modeling

    NASA Astrophysics Data System (ADS)

    Loehle, Stefan; Jenniskens, Peter; Böhrk, Hannah; Bauer, Thomas; Elsäßer, Henning; Sears, Derek W.; Zolensky, Michael E.; Shaddad, Muawia H.

    2017-02-01

    Asteroid 2008 TC3 was characterized in a unique manner prior to impacting Earth's atmosphere, making its October 7, 2008, impact a suitable field test for or validating the application of high-fidelity re-entry modeling to asteroid entry. The accurate modeling of the behavior of 2008 TC3 during its entry in Earth's atmosphere requires detailed information about the thermophysical properties of the asteroid's meteoritic materials at temperatures ranging from room temperature up to the point of ablation (T 1400 K). Here, we present measurements of the thermophysical properties up to these temperatures (in a 1 atm. pressure of argon) for two samples of the Almahata Sitta meteorites from asteroid 2008 TC3: a thick flat-faced ureilite suitably shaped for emissivity measurements and a thin flat-faced EL6 enstatite chondrite suitable for diffusivity measurements. Heat capacity was determined from the elemental composition and density from a 3-D laser scan of the sample. We find that the thermal conductivity of the enstatite chondrite material decreases more gradually as a function of temperature than expected, while the emissivity of the ureilitic material decreases at a rate of 9.5 × 10-5 K-1 above 770 K. The entry scenario is the result of the actual flight path being the boundary to the load the meteorite will be affected with when entering. An accurate heat load prediction depends on the thermophysical properties. Finally, based on these data, the breakup can be calculated accurately leading to a risk assessment for ground damage.

  15. (99)mTc-3PRGD2 scintimammography in palpable and nonpalpable breast lesions.

    PubMed

    Liu, Lin; Song, Yan; Gao, Shi; Ji, Tiefeng; Zhang, Haishan; Ji, Bin; Chen, Ben; Jia, Bing; Wang, Fan; Xu, Zheli; Ma, Qingjie

    2014-01-01

    The aim of this study was to explore the diagnostic performance of 99mTc-3(poly-(ethylene glycol),PEG)4-RGD2 (99mTc-3PRGD2) scintimammography (SMM) in patients with either palpable or nonpalpable breast lesions and compare SMM to mammography to assess the possible incremental value of SMM in breast cancer detection. We also investigated the αvβ3 expression in malignant and benign breast lesions. Ninety-four patients with 110 lesions were included in this study. Mammograms were evaluated according to the Breast Imaging Reporting and Data System (BI-RADS) by a specialized imaging radiologist. Prone SMM was performed 1 hour after injection of 99mTc-3PRGD2. Scintigraphic images were interpreted independently by two experienced nuclear medicine physicians using a three-point system, and the kappa value was calculated to determine the interreader agreement. The McNemar test was used to compare SMM and mammography with respect to sensitivity, specificity, and accuracy. Diagnostic values for breast cancer detection were evaluated for each lesion. Immunohistochemistry was performed to evaluate integrin αvβ3 expression. Histopathology revealed 46 malignant lesions and 64 benign lesions. The overall sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of SMM were 83%, 73%, 77%, 69%, and 85%, respectively. The kappa value between the two reviewers was 0.63. The diagnostic values of SMM were higher than those of mammography in evaluating overall breast lesions. A sensitivity of 91% was achieved when SMM and mammography results were combined with 60% of all false-negative mammography findings classified as true-positive results by SMM. Integrin αvβ3 expression was positively identified using SMM imaging. SMM is a promising tool to avoid unnecessary biopsies when used in addition to mammography and can be used to image αvβ3 expression in breast cancer with good image quality.

  16. Evasion mechanisms to tumor necrosis factor alpha (TNF-alpha) of small cell lung carcinoma and non-small cell lung carcinoma cell lines: comparison with the erythroleukaemia K-562 cell line.

    PubMed

    López-González, J S; Hernández García, A; Noyola, M I; Cázares, D A; Mandoki, J J; Morales, F M; Mendieta, I C; Caloca, J V

    2000-03-01

    The tumour necrosis factor alpha (TNF-alpha) is produced by mononuclear phagocytes as a defence mechanism against malignant cells. However, these cells can evade destruction by TNF-alpha. The present study evaluates in three lung cancer cell lines (small cell carcinoma NCI-H69, adenocarcinoma A-427, squamous carcinoma SK-MES-1) and one erythroleukaemia (K-562) cell line the following evasion mechanisms: (1) inhibition of TNF-alpha production, in indirect and direct co-cultures with monocytes; (2) the expression of type I and type II receptors for TNF-alpha (TNFRI and TNFRII) by tumour cell lines, using indirect immunofluorescence and flow cytometry; (3) the sensitivity of tumour cell lines to the toxic action of recombinant human TNF-alpha (rhTNF-alpha). With the exception of cell line NCI-H69, the other tumour cell lines liberated soluble factors that inhibited TNF-alpha production in monocytes. This effect occurred even after membrane contact with the A-427 and SK-MES-1 cell lines. Erythroleukaemia K-562 cells expressed both types of receptors for TNF-alpha, whereas the NCI-H69 cells expressed only TNFRI, and the A-427 and SK-MES-1 cells expressed no receptors. Lines NCI-H69, A-427 and K-562 were insensitive to the cytotoxic action of rhTNF-alpha. In conclusion, different lung cancer cell lines may evade destruction by TNF-alpha by various mechanisms that range from blocking TNF-alpha production by monocytes to blocking the cytotoxic action of this molecule. For selecting the most effective immunotherapy, knowledge of the evasion mechanisms would be useful.

  17. Tumor necrosis factor-{alpha} enhances IL-15-induced natural killer cell differentiation

    SciTech Connect

    Lee, Jiwon; Lee, Suk Hyung; Shin, Nara; Jeong, Mira; Kim, Mi Sun; Kim, Mi Jeong; Yoon, Suk Ran; Chung, Jin Woong; Kim, Tae-Don; Choi, Inpyo

    2009-09-04

    The differentiation of natural killer (NK) cells is regulated by various factors including soluble growth factors and transcription factors. Here, we have demonstrated that tumor necrosis factor-{alpha} (TNF-{alpha}) is a positive regulator of NK cell differentiation. TNF-{alpha} augmented the IL-15-induced expression of NK1.1 and CD122 in mature NK cells, and TNF-{alpha} alone also induced NK cell maturation as well as IL-15. TNF-{alpha} also increased IFN-{gamma} production in NK cells in the presence of IL-15. Meanwhile, mRNA expression of several transcription factors, including T-bet and GATA-3, was increased by the addition of TNF-{alpha} and IL-15. In addition, TNF-{alpha} increased nuclear factor-kappa B (NF-{kappa}B) activity in NK cells and inhibition of NF-{kappa}B impeded TNF-{alpha}-enhanced NK cell maturation. Overall, these data suggest that TNF-{alpha} significantly increased IL-15-driven NK cell differentiation by increasing the expression of transcription factors that play crucial roles in NK cell maturation and inducing the NF-{kappa}B activity.

  18. Cell surface alpha 2,6 sialylation affects adhesion of breast carcinoma cells.

    PubMed

    Lin, Shaoqiang; Kemmner, Wolfgang; Grigull, Sabine; Schlag, Peter M

    2002-05-15

    Tumor-associated alterations of cell surface glycosylation play a crucial role in the adhesion and metastasis of carcinoma cells. The aim of this study was to examine the effect of alpha 2,6-sialylation on the adhesion properties of breast carcinoma cells. To this end mammary carcinoma cells, MDA-MB-435, were sense-transfected with sialyltransferase ST6Gal-I cDNA or antisense-transfected with a part of the ST6Gal-I sequence. Sense transfectants showed an enhanced ST6Gal-I mRNA expression and enzyme activity and an increased binding of the lectin Sambucus nigra agglutinin (SNA), specific for alpha 2,6-linked sialic acid. Transfection with ST6Gal-I in the antisense direction resulted in less enzyme activity and SNA reactivity. A sense-transfected clone carrying increased amounts of alpha 2,6-linked sialic acid adhered preferentially to collagen IV and showed reduced cell-cell adhesion and enhanced invasion capacity. In contrast, antisense transfection led to less collagen IV adhesion but enhanced homotypic cell-cell adhesion. In another approach, inhibition of ST6Gal-I enzyme activity by application of soluble antisense-oligodeoxynucleotides was studied. Antisense treatment resulted in reduced ST6 mRNA expression and cell surface 2,6-sialylation and significantly decreased collagen IV adhesion. Our results suggest that cell surface alpha 2,6-sialylation contributes to cell-cell and cell-extracellular matrix adhesion of tumor cells. Inhibition of sialytransferase ST6Gal-I by antisense-oligodeoxynucleotides might be a way to reduce the metastatic capacity of carcinoma cells.

  19. Cell cycle expression of two replicative DNA polymerases alpha and delta from Schizosaccharomyces pombe.

    PubMed Central

    Park, H; Francesconi, S; Wang, T S

    1993-01-01

    We have investigated the expression of two Schizosaccharomyces pombe replicative DNA polymerases alpha and delta during the cell cycle. The pol alpha+ and pol delta+ genes encoding DNA polymerases alpha and delta were isolated from S. pombe. Both pol alpha+ and pol delta+ genes are single copy genes in haploid cells and are essential for cell viability. In contrast to Saccharomyces cerevisiae homologs, the steady-state transcripts of both S. pombe pol alpha+ and pol delta+ genes were present throughout the cell cycle. Sequence analysis of the pol alpha+ and pol delta+ genes did not reveal the Mlu I motifs in their upstream sequences that are involved in cell cycle-dependent transcription of S. cerevisiae DNA synthesis genes as well as the S. pombe cdc22+ gene at the G1/S boundary. However, five near-match Mlu I motifs were found in the upstream region of the pol alpha+ gene. S. pombe DNA polymerases alpha and delta proteins were also expressed constantly throughout the cell cycle. In addition, the enzymatic activity of the S. pombe DNA polymerase alpha measured by in vitro assay was detected at all stages of the cell cycle. Thus, these S. pombe replicative DNA polymerases, like that of S. pombe cdc17+ gene, are expressed throughout the cell cycle at the transcriptional and protein level. These results indicate that S. pombe has at least two regulatory modes for the expression of genes involved in DNA replication and DNA precursor synthesis. Images PMID:8443413

  20. Oxidative inactivation of alpha 1-proteinase inhibitor by alveolar epithelial type II cells.

    PubMed

    Wallaert, B; Aerts, C; Gressier, B; Gosset, P; Voisin, C

    1993-12-01

    The aim of this work was to evaluate the ability of guinea pig alveolar epithelial type II cells to generate significant amounts of reactive oxygen species to inactivate alpha 1-proteinase inhibitor (alpha 1-PI). Inactivation of alpha 1-PI was evaluated by its inhibitory activity against porcine pancreatic elastase and was expressed as a percentage. The same experiments were performed in parallel with alveolar macrophages (AM) obtained from the same animals and with MRC-5 fibroblasts. Both type II cells and AM released significant amounts of hydrogen peroxide and superoxide, whereas the fibroblasts did not. Unstimulated type II cells (0.5 +/- 2%), AM (1.2 +/- 1.5%), and fibroblasts (0.5 +/- 0.5%) were unable to inactivate alpha 1-PI. Addition of phorbol myristate acetate did not increase their ability to inactivate alpha 1-PI. In contrast, type II cells (79.7 +/- 7%) and AM (80.1 +/- 8%) dramatically inactivated alpha 1-PI in the presence of myeloperoxidase (25 mU/ml), whereas fibroblasts did not. Addition of catalase to the reaction significantly prevented the inactivation of alpha 1-PI. Western blot analysis of alpha 1-PI did not reveal a significant proteolysis of alpha 1-PI, which supports the hypothesis that, in the presence of neutrophil-derived myeloperoxidase, type II cells may oxidatively inactivate alpha 1-PI.

  1. Integrin alpha 8 beta 1 promotes attachment, cell spreading, and neurite outgrowth on fibronectin.

    PubMed Central

    Müller, U; Bossy, B; Venstrom, K; Reichardt, L F

    1995-01-01

    The integrin alpha 8 subunit, isolated by low stringency hybridization, is a novel integrin subunit that associates with beta 1. To identify ligands, we have prepared a function-blocking antiserum to the extracellular domain of alpha 8, and we have established by transfection K562 cell lines that stably express alpha 8 beta 1 heterodimers on the cell surface. We demonstrate here by cell adhesion and neurite outgrowth assays that alpha 8 beta 1 is a fibronectin receptor. Studies on fibronectin fragments using RGD peptides as inhibitors show that alpha 8 beta 1 binds to the RGD site of fibronectin. In contrast to the endogenous alpha 5 beta 1 fibronectin receptor in K562 cells, alpha 8 beta 1 not only promotes cell attachment but also extensive cell spreading, suggesting functional differences between the two receptors. In chick embryo fibroblasts, alpha 8 beta 1 is localized to focal adhesions. We conclude that alpha 8 beta 1 is a receptor for fibronectin and can promote attachment, cell spreading, and neurite outgrowth on fibronectin. Images PMID:7626807

  2. Effects of alpha-crystallin on lens cell function and cataract pathology.

    PubMed

    Andley, Usha P

    2009-09-01

    The development of cataracts is a debilitating eye condition which is common in elderly patients and afflicts millions worldwide. Cataracts result from the deposition of aggregated proteins in the eye which causes clouding of the lens, light scattering, and obstruction of vision. Non-syndromic, hereditary human cataract development is linked to point mutations in the CRYAA and CRYAB genes which encode alphaA and alphaB-crystallin. The alpha-crystallins are small heat shock proteins which play central roles in maintaining lens transparency and refractive properties. The discovery in 1992 that these proteins possess chaperone-like activity has led most researchers to focus on the ability of alpha-crystallins to prevent protein aggregation in vitro. While the ability of alpha-crystallins to efficiently trap aggregation-prone denatured proteins in vitro is thought to delay the development of age-related cataracts in vivo, alpha-crystallins have additional functions which may also contribute to cataract pathology. In addition to chaperone activity, alpha-crystallins are known to protect cells from stress-induced apoptosis, regulate cell growth, and enhance genomic stability. They also physically and functionally interact with both the cell membrane and cytoskeleton. Functional changes in alpha-crystallin have been shown to modify membrane and cell-cell interactions and lead to lens cell pathology in vivo. This article focuses on the multiple diverse roles of alphaA-crystallin in the maintenance of lens function and cataract development in vivo.

  3. Targeted depletion of lymphotoxin-alpha-expressing TH1 and TH17 cells inhibits autoimmune disease.

    PubMed

    Chiang, Eugene Y; Kolumam, Ganesh A; Yu, Xin; Francesco, Michelle; Ivelja, Sinisa; Peng, Ivan; Gribling, Peter; Shu, Jean; Lee, Wyne P; Refino, Canio J; Balazs, Mercedesz; Paler-Martinez, Andres; Nguyen, Allen; Young, Judy; Barck, Kai H; Carano, Richard A D; Ferrando, Ron; Diehl, Lauri; Chatterjea, Devavani; Grogan, Jane L

    2009-07-01

    Uncontrolled T helper type 1 (T(H)1) and T(H)17 cells are associated with autoimmune responses. We identify surface lymphotoxin-alpha (LT-alpha) as common to T(H)0, T(H)1 and T(H)17 cells and employ a unique strategy to target these subsets using a depleting monoclonal antibody (mAb) directed to surface LT-alpha. Depleting LT-alpha-specific mAb inhibited T cell-mediated models of delayed-type hypersensitivity and experimental autoimmune encephalomyelitis. In collagen-induced arthritis (CIA), preventive and therapeutic administration of LT-alpha-specific mAb inhibited disease, and immunoablated T cells expressing interleukin-17 (IL-17), interferon-gamma and tumor necrosis factor-alpha (TNF-alpha), whereas decoy lymphotoxin-beta receptor (LT-betaR) fusion protein had no effect. A mutation in the Fc tail, rendering the antibody incapable of Fcgamma receptor binding and antibody-dependent cellular cytotoxicity activity, abolished all in vivo effects. Efficacy in CIA was preceded by a loss of rheumatoid-associated cytokines IL-6, IL-1beta and TNF-alpha within joints. These data indicate that depleting LT-alpha-expressing lymphocytes with LT-alpha-specific mAb may be beneficial in the treatment of autoimmune disease.

  4. Biologically active monoiodinated alpha-MSH derivatives for receptor binding studies using human melanoma cells

    SciTech Connect

    Eberle, A.N.; Verin, V.J.; Solca, F.; Siegrist, W.; Kueenlin, C.B.; Bagutti, C.; Stutz, S.; Girard, J. , University Hospital, Basel )

    1991-01-01

    Three different monoiodinated radioligands of alpha-MSH (alpha-melanocyte-stimulating hormone) were compared in a binding assay with human D10 melanoma cells: (Tyr(125I)2)-alpha-MSH, (Tyr(125I)2,NIe4)-alpha-MSH, and (Tyr(125I)2,NIe4,D-Phe7)-alpha-MSH. They were prepared either by the classical chloramine T method or by the Enzymobead method. A simple and rapid purification scheme was developed consisting of a primary separation on reversed-phase C18 silica cartridges immediately after the iodination, followed by HPLC purification before each binding experiment. Biological testing of the three radioligands showed that they all retained high melanotropic activity in the B16 melanin assay and the Anolis melanophore assay. However, in human D10 melanoma cells, (Tyr(125I)2,NIe4)-alpha-MSH led to a high degree of non-specific binding to the cells which could not be displaced by excess alpha-MSH and only partially by (NIe4)-alpha-MSH. The (Tyr(125I)2,NIe4,D-Phe7)-alpha-MSH tracer gave similar results but with a much lower proportion of non-specific binding. On the other hand, (Tyr(125I)2)-alpha-MSH proved to be an excellent radioligand whose non-specific binding to the D10 cells was not higher than 20% of the total binding.

  5. Targeted Therapies: Bevacizumab and interferon-alpha in metastatic renal-cell carcinoma.

    PubMed

    Bukowski, Ronald M

    2009-05-01

    Rini and colleagues provide additional data on bevacizumab and interferon-alpha in clear-cell carcinoma of the kidney; a comparison of these results with the findings from contemporary trials suggests that bevacizumab and interferon-alpha is another clinically useful treatment option for patients with metastatic renal-cell carcinoma.

  6. Role of integrin alpha 4 beta 7/alpha 4 beta P in lymphocyte adherence to fibronectin and VCAM-1 and in homotypic cell clustering

    PubMed Central

    1992-01-01

    Integrins are heterodimeric cell surface proteins that mediate both cell-cell and cell-extracellular matrix interactions. We and others recently identified cDNAs encoding a novel integrin beta subunit, beta 7, in lymphocytes. We have now detected beta 7 mRNA in mouse TK-1 T lymphoma cells, which are known to express the putative Peyer's patch homing receptor alpha 4 beta P. We used an anti-peptide antiserum and a novel mAb against the beta 7 subunit to show that TK-1 cells express beta 7 as the only subunit associated with alpha 4. We conclude that beta 7 and beta P are identical. We also show that activated peripheral blood T cells express alpha 4 beta 7. We studied the function of alpha 4 beta 7/alpha 4 beta P in TK-1 cells, which do not express very late antigen (VLA)-4 (alpha 4 beta 1). Cells adhered to intact fibronectin and to a fibronectin fragment containing the CS-1 region, but not to a fragment containing the RGD sequence. Adhesion to fibronectin was inhibited by antibodies to alpha 4, suggesting that alpha 4 beta 7 is a fibronectin receptor. We confirmed that alpha 4 beta 7 binds to the CS- 1 region of fibronectin using affinity chromatography. TK-1 cell adhesion to the vascular cell adhesion molecule VCAM-1 was also inhibited by antibodies to alpha 4, implying that alpha 4 beta 7 also plays a role in the adherence of lymphocytes to endothelial cells. TK-1 cell binding to fibronectin and VCAM-1 is markedly increased by brief PMA stimulation. We also found that mAbs against alpha 4 and beta 7 induce homotypic clustering of TK-1 cells. Taken together these results suggest that alpha 4 beta 7/alpha 4 beta P recognizes some or all of the same widely distributed ligands recognized by VLA-4 (alpha 4 beta 1) and that the role of alpha 4 beta 7/alpha 4 beta P may not be restricted to lymphocyte homing. PMID:1372909

  7. The p53 inhibitor, pifithrin-{alpha}, suppresses self-renewal of embryonic stem cells

    SciTech Connect

    Abdelalim, Essam Mohamed; Tooyama, Ikuo

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer We determine the role of p53 in ES cells under unstressful conditions. Black-Right-Pointing-Pointer PFT-{alpha} suppresses ES cell proliferation. Black-Right-Pointing-Pointer PFT-{alpha} induces ES cell cycle arrest. Black-Right-Pointing-Pointer PFT-{alpha} downregulates Nanog and cyclin D1. -- Abstract: Recent studies have reported the role of p53 in suppressing the pluripotency of embryonic stem (ES) cells after DNA damage and blocking the reprogramming of somatic cells into induced pluripotent stem (iPS) cells. However, to date no evidence has been presented to support the function of p53 in unstressed ES cells. In this study, we investigated the effect of pifithrin (PFT)-{alpha}, an inhibitor of p53-dependent transcriptional activation, on self-renewal of ES cells. Our results revealed that treatment of ES cells with PFT-{alpha} resulted in the inhibition of ES cell propagation in a dose-dependent manner, as indicated by a marked reduction in the cell number and colony size. Also, PFT-{alpha} caused a cell cycle arrest and significant reduction in DNA synthesis. In addition, inhibition of p53 activity reduced the expression levels of cyclin D1 and Nanog. These findings indicate that p53 pathway in ES cells rather than acting as an inactive gene, is required for ES cell proliferation and self-renewal under unstressful conditions.

  8. Functional analysis of alpha 1 beta 1 integrin in human natural killer cells.

    PubMed

    Pérez-Villar, J J; Melero, I; Gismondi, A; Santoni, A; López-Botet, M

    1996-09-01

    Upon activation with interleukin (IL)-2 human natural killer (NK) cells acquire on their surface the alpha 1 beta 1 and alpha 2 beta 1 integrins and down-regulate the expression of alpha 6 beta 1. By employing alpha 1 beta 1-specific monoclonal antibody (mAb) HP-2B6, characterized in our laboratory, we examined the functional role of the alpha 1 beta 1 integrin in NK cells. Treatment with HP-2B6 mAb partially interfered with attachment of cultured NK cells to type I collagen, and combined with an anti-alpha 2 beta 1 (TEA 1/41) mAb, it completely abrogated cell adhesion to this extracelular matrix protein. In contrast, NK cell attachment to laminin was completely blocked by the anti-beta 1 LIA 1/2 mAb, but was unaffected by alpha 1 and alpha 2-specific mAb; as alpha 3 beta 1 and alpha 6 beta 1 were undetectable, the data indicate that the alpha 1 beta 1 integrin binding sites for type I collagen and laminin are different. Incubation with anti-alpha 1 HP-2B6 or its F(ab')2 fragments specifically induced a rapid homotypic aggregation of NK cells that was dependent on active metabolism, an intact cytoskeleton and the presence of divalent cations (Ca2+ and Mg2+); homotypic cell adhesion was selectively blocked by anti-CD18, CD11a or CD54 mAb. In addition, stimulation of cultured NK cells with the anti-alpha 1 HP-2B6 enhanced TNF-alpha production and induced tyrosine phosphorylation of a 110-kDa protein. Pretreatment with specific inhibitors of protein tyrosine kinase (PTK) activity (tyrphostin 25 and herbimycin A) completely abrogated the functional effects induced by the anti-alpha 1 HP-2B6 mAb. Our data show that ligation of the alpha 1 beta 1 integrin positively modulates IL-2-activated NK cell function via a PTK-dependent pathway.

  9. (alpha)IIb Integrin, a novel marker for hemopoietic progenitor cells.

    PubMed

    Corbel, Catherine; Vaigot, Pierre; Salaün, Josselyne

    2005-01-01

    Integrin (alpha)IIb(beta)3 (abbreviated as (alpha)IIb), also known as GPIIb-IIIa or CD41/CD61, is a cell adhesion molecule expressed on cells belonging to the megakaryocytic lineage. Aiming to identify new markers of hemopoietic progenitor cells (HPC), we undertook a developmental study of this molecule since it remains controversial if this integrin is expressed by various progenitors. We reported the expression pattern of two integrins, in both of which the beta3 chain is present, respectively associated with alphaV and alpha IIb in the chick embryo. While at E3.5, the earliest time at which these integrins can be detected, (alpha)V(beta)3 becomes expressed by endothelial cells in the aorta (and only in the aorta), (alpha)IIb(beta)3 becomes detected in the well-defined intra-aortic clusters made up of HPC. The latter were found to be multilineage progenitors when sorted for (alpha)IIb expression and analyzed by means of clonogenic assays. In mice also, (alpha)IIb is expressed in the intra-embryonic site of HPC generation, the intra-arterial clusters in the embryo proper, as well as in sites where HPC migrate. Finally we provided the first evidence in two species that multipotent HPC expressing (alpha)IIb are able to differentiate not only into cells of the erythroid and myeloid lineages but also into lymphocytes. These cell populations actually coexpress (alpha)IIb and c-Kit. These data establish (alpha)IIb as a novel marker for HPC, which appears at very early stages in the embryo. Capitalizing on this finding, other investigators confirmed it and suggested that (alpha)IIb plays a role in regulating hematopoietic development.

  10. Suppression of estrogen receptor-alpha transactivation by thyroid transcription factor-2 in breast cancer cells

    SciTech Connect

    Park, Eunsook; Gong, Eun-Yeung; Romanelli, Maria Grazia; Lee, Keesook

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer TTF-2 was expressed in mammary glands and breast cancer cells. Black-Right-Pointing-Pointer TTF-2 repressed ER{alpha} transactivation. Black-Right-Pointing-Pointer TTF-2 inhibited the proliferation of breast cancer cells. -- Abstract: Estrogen receptors (ERs), which mediate estrogen actions, regulate cell growth and differentiation of a variety of normal tissues and hormone-responsive tumors through interaction with cellular factors. In this study, we show that thyroid transcription factor-2 (TTF-2) is expressed in mammary gland and acts as ER{alpha} co-repressor. TTF-2 inhibited ER{alpha} transactivation in a dose-dependent manner in MCF-7 breast cancer cells. In addition, TTF-2 directly bound to and formed a complex with ER{alpha}, colocalizing with ER{alpha} in the nucleus. In MCF-7/TTF-2 stable cell lines, TTF-2 repressed the expression of endogenous ER{alpha} target genes such as pS2 and cyclin D1 by interrupting ER{alpha} binding to target promoters and also significantly decreased cell proliferation. Taken together, these data suggest that TTF-2 may modulate the function of ER{alpha} as a corepressor and play a role in ER-dependent proliferation of mammary cells.

  11. Conservation of mouse alpha A-crystallin promoter activity in chicken lens epithelial cells.

    PubMed

    Donovan, D M; Sax, C M; Klement, J F; Li, X; Chepelinsky, A B; Piatigorsky, J

    1992-10-01

    Previous transfection experiments have shown that 162 base pairs (bp) of the 5' flanking sequence of the chicken alpha A-crystallin gene are required for promoter activity in primary chicken lens epithelial cells (PLE), while only 111 bp of the 5' flanking sequence are needed for activity of the mouse alpha A-crystallin promoter in transfected chicken PLE cells or in a SV40 T-antigen-transformed transfected mouse lens epithelial cell line (alpha TN4-1). The effect of site-directed mutations covering positions -111 to -34 of the mouse alpha A-crystallin promoter fused to the bacterial chloramphenicol acetyltransferase (CAT) gene was compared in transfected chicken PLE cells and mouse alpha TN4-1 cells; selected mutations were also examined in a nontransformed rabbit lens epithelial cell line (N/N1003A). In general, the same mutations reduced promoter activity in the transfected lens cells from all three species, although differences were noted. The mutations severely affected regions -111/-106 and -69/-40 regions in all the transfected cells examined; by contrast, mutations at positions -105/-99 and -87/-70 had a somewhat greater effect in the chicken PLE than the mouse alpha TN4-1 cells, while mutations of the -93/-88 sequence reduced expression in the alpha TN4-1 but not the PLE cells. A partial cDNA with sequence similarity to alpha A-CRYPB1 of the mouse has been isolated from a chicken lens library; mouse alpha A-CRYBP1 is a putative transcription factor which binds to the -66/-55 sequence of the mouse alpha A-crystallin promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Differential regulation of phosphoinositide metabolism by alphaVbeta3 and alphaVbeta5 integrins upon smooth muscle cell migration.

    PubMed

    Paulhe, F; Racaud-Sultan, C; Ragab, A; Albiges-Rizo, C; Chap, H; Iberg, N; Morand, O; Perret, B

    2001-11-09

    Smooth muscle cell migration is a key step of atherosclerosis and angiogenesis. We demonstrate that alpha(V)beta(3) and alpha(V)beta(5) integrins synergistically regulate smooth muscle cell migration onto vitronectin. Using an original haptotactic cell migration assay, we measured a strong stimulation of phosphoinositide metabolism in migrating vascular smooth muscle cells. Phosphatidic acid production and phosphoinositide 3-kinase IA activation were triggered only upon alpha(V)beta(3) engagement. Blockade of alpha(V)beta(3) engagement or phospholipase C activity resulted in a strong inhibition of smooth muscle cell spreading on vitronectin. By contrast, blockade of alpha(V)beta(5) reinforced elongation and polarization of cell shape. Moreover, Pyk2-associated tyrosine kinase and phosphoinositide 4-kinase activities measured in Pyk2 immunoprecipitates were stimulated upon cell migration. Blockade of either alpha(V)beta(3) or alpha(V)beta(5) function, as well as inhibition of phospholipase C activity, decreased both Pyk2-associated activities. We demonstrated that the Pyk2-associated phosphoinositide 4-kinase corresponded to the beta isoform. Our data point to the metabolism of phosphoinositides as a regulatory pathway for the differential roles played by alpha(V)beta(3) and alpha(V)beta(5) upon cell migration and identify the Pyk2-associated phosphoinositide 4-kinase beta as a common target for both integrins.

  13. Failure of isolated rat tibial periosteal cells to 5 alpha reduce testosterone to 5 alpha-dihydrotestosterone

    SciTech Connect

    Turner, R.T.; Bleiberg, B.; Colvard, D.S.; Keeting, P.E.; Evans, G.; Spelsberg, T.C. )

    1990-07-01

    Periosteal cells were isolated from tibiae of adult male rats after collagenase treatment. Northern blot analysis of total cytoplasmic RNA extracted from the isolated periosteal cells was positive for expression of genes encoding the osteoblast marker proteins osteocalcin (BGP) and pre-pro-alpha 2(I) chain of type 1 precollagen. The isolated periosteal cells were incubated with 1 nM (3H)testosterone (({sup 3}H)T) for up to 240 minutes and the reaction products separated by high-performance liquid chromatography. ({sup 3}H)5 alpha-dihydrotestosterone (({sup 3}H)DHT) was not detected in extracts of periosteal cell incubations. In contrast, ({sup 3}H)DHT was produced in a time-dependent manner by cells from seminal vesicles. These results suggest that testosterone 5 alpha-reductase activity is not expressed by osteoblasts in rat tibial periosteum and that the anabolic effects of androgens in this tissue are not mediated by locally produced DHT.

  14. Identification of novel targets for PGC-1{alpha} and histone deacetylase inhibitors in neuroblastoma cells

    SciTech Connect

    Cowell, Rita M. Talati, Pratik; Blake, Kathryn R.; Meador-Woodruff, James H.; Russell, James W.

    2009-02-06

    Recent evidence suggests that the transcriptional coactivator peroxisome proliferator activated receptor {gamma} coactivator 1{alpha} (PGC-1{alpha}) is involved in the pathology of Huntington's Disease (HD). While animals lacking PGC-1{alpha} express lower levels of genes involved in antioxidant defense and oxidative phosphorylation in the brain, little is known about other targets for PGC-1{alpha} in neuronal cells and whether there are ways to pharmacologically target PGC-1{alpha} in neurons. Here, PGC-1{alpha} overexpression in SH-SY5Y neuroblastoma cells upregulated expression of genes involved in mitochondrial function, glucose transport, fatty acid metabolism, and synaptic function. Overexpression also decreased vulnerability to hydrogen peroxide-induced cell death and caspase 3 activation. Treatment of cells with the histone deacetylase inhibitors (HDACi's) trichostatin A and valproic acid upregulated PGC-1{alpha} and glucose transporter 4 (GLUT4). These results suggest that PGC-1{alpha} regulates multiple pathways in neurons and that HDACi's may be good candidates to target PGC-1{alpha} and GLUT4 in HD and other neurological disorders.

  15. Transforming growth factor alpha induces collagen degradation and cell migration in differentiating human epidermal raft cultures.

    PubMed Central

    Turksen, K; Choi, Y; Fuchs, E

    1991-01-01

    When cultured on plastic and treated with transforming growth factor alpha (TGF alpha), human keratinocytes exhibit an increase in proliferation at the colony periphery, apparently as a consequence of enhanced cell migration (Barrandon and Green, 1987). To investigate the effects of TGF alpha on a differentiating stratified squamous epithelium and to begin to examine the molecular basis mediating this influence, we cultured human epidermal cells on a gelled lattice of collagen and fibroblasts, floating on the air-liquid interface. Under these conditions, raft cultures differentiate and exhibit morphological and biochemical features of human skin in vivo (Asselineau et al., 1986; Kopan et al., 1987). When 3-wk-old raft cultures were treated with TGF alpha, basal cells showed a marked increase in cell proliferation. At elevated concentrations of TGF alpha, the organization of cells within the artificial tissue changed and islands of basal cells entered the collagen matrix. Biochemical analysis of the response revealed that type I collagenase and gelatinase were induced by keratinocytes within 12 h after TGF alpha treatment. In contrast, invasion of basal cells into the collagen matrix was not significant until 48-72 h post-treatment, suggesting that collagenase and gelatinase production may be a prerequisite to this phenomenon. These results have important implications for the possible role of TGF alpha in squamous cell carcinoma and tumor invasion. Images PMID:1663788

  16. EF-1[alpha] Is Associated with a Cytoskeletal Network Surrounding Protein Bodies in Maize Endosperm Cells.

    PubMed Central

    Clore, A. M.; Dannenhoffer, J. M.; Larkins, B. A.

    1996-01-01

    By using indirect immunofluorescence and confocal microscopy, we documented changes in the distribution of elongation factor-1[alpha] (EF-1[alpha]), actin, and microtubules during the development of maize endosperm cells. In older interphase cells actively forming starch grains and protein bodies, the protein bodies are enmeshed in EF-1[alpha] and actin and are found juxtaposed with a multidirectional array of microtubules. Actin and EF-1[alpha] appear to exist in a complex, because we observed that the two are colocalized, and treatment with cytochalasin D resulted in the redistribution of EF-1[alpa]. These data suggest that EF-1[alpha] and actin are associated in maize endosperm cells and may help to explain the basis of the correlation we found between the concentration of EF-1[alpha] and lysine content. The data also support the hypothesis that the cytoskeleton plays a role in storage protein deposition. The distributions of EF-1[alpha] actin, and microtubules change during development. We observed that in young cells before the accumulation of starch and storage protein, EF-1[alpha], actin, and microtubules are found mainly in the cell cortex or in association with nuclei. PMID:12239373

  17. Comparable susceptibilities of human 293 cells and insect Tn-5B1-4 cells to photoactivated alpha-terthienyl.

    PubMed

    Huang, Qingchun; Liu, Yang; Zhan, Taisong; Deng, Yunfei; He, Yuan

    2010-03-10

    The hope is that photoactive compounds acting as potential insecticides will have reduced environmental risk, but that is not necessarily the case. In an attempt to elucidate the risk by which photoactivated alpha-terthienyl (alpha-T) affects human health, the effects of exposure of human 293 cells and insect Tn-5B1-4 cells to photoactivated alpha-T at certain doses were characterized. Photoactivated alpha-T exhibited dose dependence of toxicity and time kinetics of phototoxic activation on the growth of 293 cells (EC(50) = 6.23 microg/mL) and Tn-5B1-4 cells (EC(50) = 3.36 microg/mL). 293 cells appeared to be anchorage-independent, inflated, and broken; Tn-5B1-4 cells showed significant necrosis. ROS productions and lipid peroxidation of 293 cells were always lower than that of Tn-5B1-4 cells in the treatments of alpha-T at the same dose. Moreover, photoactivated alpha-T caused nonselective DNA damage in 293 and Tn-5B1-4 cells at a 10 microg/mL dose and induced cell-cycle progression of 293 cells to increase apoptosis of cells and G1 arrest and decrease in S phase cell population, whereas Tn-5B1-4 cells showed S arrest accompanied by a dose-dependent decrease in G1 and G2 phase cells at a 5 microg/mL dose. These observations suggest that Tn-5B1-4 cells are more susceptible to the action of photoactivated alpha-T than 293 cells, but photoactivated alpha-T as an efficient insecticide might be a potential factor in human mutagenic progression.

  18. Detection of pregnancy associated alpha 2-glycoprotein (alpha 2-PAG), an immunosuppressive agent, in IgA producing plasma cells and in body secretions.

    PubMed Central

    Horne, C H; Armstrong, S S; Thomson, A W; Thompson, W D

    1983-01-01

    Using a combined indirect immunoperoxidase (PAP)/direct immunofluorescence (IF) staining technique, pregnancy associated alpha 2-glycoprotein (alpha 2-PAG) has been shown to be present solely in the IgA producing plasma cells. alpha 2-PAG was undetectable in plasma cells synthesizing any of the other immunoglobulin isotypes (IgG, IgM, IgD and IgE). Furthermore, we have observed increased numbers of alpha 2-PAG positive cells in sites recognized for their local IgA production e.g. lactating breast, salivary gland, respiratory and gastrointestinal mucosae. In support of this cellular association with IgA, alpha 2-PAG was measured by enzyme immunoassay in colostrum, breast milk, saliva, jejunal secretions, bile and urine. The relevance of this novel and preferential association of immunosuppressive alpha 2-PAG with IgA is discussed with regard to the regulation of gut immunoresponsiveness. Images Fig. 1 Fig. 2 Fig. 3 PMID:6342887

  19. Thrombopoietin-induced Dami cells as a model for alpha-granule biogenesis.

    PubMed

    Briquet-Laugier, Véronique; El Golli, Nargès; Nurden, Paquita; Lavenu-Bombled, Cécile; Dubart-Kupperschmitt, Anne; Nurden, Alan; Rosa, Jean-Philippe

    2004-09-01

    Megakaryocytic alpha-granules contain secretory proteins relevant to megakaryocyte and platelet functions. Understanding alpha-granule biogenesis is hampered because human primary megakaryocytes are difficult to manipulate. Existing promegakaryocytic cell lines do not spontaneously exhibit mature alpha-granules. Dami cells, transfected with the megakaryocytic platelet factor 4, fused to GFP (PF4-GFP), were induced in the presence of thrombopoietin (TPO), a megakaryocyte cytokine and PMA. Using confocal microscopy, PF4-GFP colocalized with von Willebrand Factor (vWF) in newly formed storage granules. Immunoelectron microscopy demonstrated alpha-granule-like features, including a dense core or parallel tubules and colocalization of PF4-GFP and vWF. Hence, TPO-treated Dami cells are a suitable model to study alpha-granules and their biogenesis.

  20. Dehydrocostus lactone enhances tumor necrosis factor-alpha-induced apoptosis of human leukemia HL-60 cells.

    PubMed

    Oh, G S; Pae, H O; Chung, H T; Kwon, J W; Lee, J H; Kwon, T O; Kwon, S Y; Chon, B H; Yun, Young Gab

    2004-05-01

    Sesquiterpene lactones have raised considerable interest because of their ability to block the activation of nuclear transcription factor-kappaB (NF-kappaB). NF-kappaB plays an important role in the resistance of cancer cells to the induction of apoptosis by anticancer drugs and tumor necrosis factor-alpha (TNF-alpha). Pharmacological inhibition of NF-kappaB offers the promise of enhancing the efficacy of anticancer therapies. Here, we demonstrate that dehydrocostus lactone (DL), the major sesquiterpene lactone isolated from the roots of Saussurea lappa, inhibits NF-kappaB activation by preventing TNF-alpha-induced degradation and phosphorylation of its inhibitory protein I-kappaB alpha in human leukemia HL-60 cells and that DL renders HL-60 cells susceptible to TNF-alpha-induced apoptosis by enhancing caspase-8 and caspase-3 activities.

  1. T-cell receptor V sub. alpha. and C sub. alpha. alleles associated with multiple sclerosis and myasthenia gravis

    SciTech Connect

    Oksenberg, J.R.; Cavalli-Sforza, L.L.; Steinman, L. ); Sherritt, M.; Bernard, C.C. ); Begovich, A.B.; Erlich, H.A. )

    1989-02-01

    Polymorphic markers in genes encoding the {alpha} chain of the human T-cell receptor (TcR) have been detected by Southern blot analysis in Pss I digests. Polymorphic bands were observed at 6.3 and 2.0 kilobases (kb) with frequencies of 0.30 and 0.44, respectively, in the general population. Using the polymerase chain reaction (PCR) method, the authors amplified selected sequences derived from the full-length TcR {alpha} cDNA probe. These PcR products were used as specific probes to demonstrate that the 6.3-kb polymorphic fragment hybridizes to the variable (V)-region probe and the 2.0-kb fragment hybridizes to the constant (C)-region probe. Segregation of the polymorphic bands was analyzed in family studies. To look for associations between these markers and autoimmune diseases, the authors have studied the restriction fragment length polymorphism distribution of the Pss I markers in patients with multiple sclerosis, myasthenia gravis, and Graves disease. Significant differences in the frequency of the polymorphic V{sub {alpha}} and C{sub {alpha}} markers were identified between patients and healthy individuals.

  2. Analysis of T cell antigen receptor (TCR) expression by human peripheral blood CD4-8- alpha/beta T cells demonstrates preferential use of several V beta genes and an invariant TCR alpha chain

    PubMed Central

    1993-01-01

    CD4-CD8- (double negative [DN]) alpha/beta T cells are a largely uncharacterized subpopulation of unknown function. To investigate whether these cells are selected to recognize particular antigens or antigen-presenting molecules, DN alpha/beta T cells were purified from the peripheral blood of five normal donors and their T cell receptor (TCR) alpha and beta chains were examined. Random cloning of TCR alpha chains by single-sided polymerase chain reaction (PCR) amplification identified an invariant rearrangement between V alpha 24 and J alpha Q, with no N region diversity, which was expressed preferentially by DN alpha/beta T cells from all donors. Random cloning also identified a precise V alpha 7.2-J alpha (IGRJa14) rearrangement, with two variable amino acids encoded in the V-J junction, which was enriched in the DN alpha/beta T cell preparations from some, but not all, donors. Analysis of TCR beta chains by quantitative PCR amplification demonstrated that the expression of four V beta gene families, V beta 2, 8, 11, and 13, was markedly increased in these DN alpha/beta T cell preparations. The expression of particular TCRs by DN alpha/beta T cells from multiple donors indicates that these cells, or at least a subpopulation of cells with this phenotype, recognize a limited spectrum of antigens and suggests that they may use nonpolymorphic antigen-presenting molecules. PMID:8391057

  3. Interaction between HP1{alpha} and replication proteins in mammalian cells

    SciTech Connect

    Auth, Tanja . E-mail: tauth@uni-bonn.de; Kunkel, Elisabeth; Grummt, Friedrich . E-mail: grummt@biozentrum.uni-wuerzburg.de

    2006-10-15

    HP1 is an essential heterochromatin-associated protein known to play an important role in the organization of heterochromatin as well as in the transcriptional regulation of heterochromatic and euchromatic genes both in repression and activation. Using the yeast two-hybrid system and immunoprecipitation, we report here that murine HP1{alpha} interacts with the preRC proteins ORC1, ORC2 and CDC6. Immunofluorescence staining and EGFP/DsRed fusion proteins revealed a colocalization of HP1{alpha} with ORC1, ORC2 and CDC6 in heterochromatin, supporting the notion that ORC and probably CDC6 play an important role in murine HP1{alpha} function. Besides that, we also observed a colocalization of HP1{alpha} with {gamma}-tubulin suggesting a centrosomal localization of HP1{alpha} in murine cells. To gain insight into HP1{alpha} function, we applied the RNAi technique. Depletion of HP1{alpha} leads to a slow down of cell proliferation, an aberrant cell cycle progression as well as to multinucleated cells with insufficiently organized microtubule. These results together indicate that HP1{alpha} exerts functions in mitosis and cytokinesis.

  4. Production of alpha-terpineol from Escherichia coli cells expressing thermostable limonene hydratase.

    PubMed

    Savithiry, N; Cheong, T K; Oriel, P

    1997-01-01

    The genes encoding a thermostable limonene hydratase have been located on a cloned fragment in Escherichia coli conferring growth on limonene and production of the monoterpenes perillyl alcohol and alpha-terpineol. Whole cell bioconversion studies at elevated temperature employing both an aqueous phase and neat limonene phase demonstrated significant production of alpha-terpineol with additional production of carvone.

  5. A karyopherin alpha2 nuclear transport pathway is regulated by glucose in hepatic and pancreatic cells.

    PubMed

    Cassany, Aurélia; Guillemain, Ghislaine; Klein, Christophe; Dalet, Véronique; Brot-Laroche, Edith; Leturque, Armelle

    2004-01-01

    We studied the role of the karyopherin alpha2 nuclear import carrier (also known as importin alpha2) in glucose signaling. In mhAT3F hepatoma cells, GFP-karyopherin alpha2 accumulated massively in the cytoplasm within minutes of glucose extracellular addition and returned to the nucleus after glucose removal. In contrast, GFP-karyopherin alpha1 distribution was unaffected regardless of glucose concentration. Glucose increased GFP-karyopherin alpha2 nuclear efflux by a factor 80 and its shuttling by a factor 4. These glucose-induced movements were not due to glycolytic ATP production. The mechanism involved was leptomycin B-insensitive, but phosphatase- and energy-dependent. HepG2 and COS-7 cells displayed no glucose-induced GFP-karyopherin alpha2 movements. In pancreatic MIN-6 cells, the glucose-induced movements of karyopherin alpha2 and the stimulation of glucose-induced gene transcription were simultaneously lost between passages 28 and 33. Thus, extracellular glucose regulates a nuclear transport pathway by increasing the nuclear efflux and shuttling of karyopherin alpha2 in cells in which glucose can stimulate the transcription of sugar-responsive genes.

  6. Alpha-amylase production is induced by sulfuric acid in rice aleurone cells.

    PubMed

    Mitsunaga, Shin-ichiro; Kobayashi, Midori; Fukui, Satoe; Fukuoka, Kayoko; Kawakami, Osamu; Yamaguchi, Junji; Ohshima, Masahiro; Mitsui, Toshiaki

    2007-12-01

    The hydrolytic enzyme alpha-amylase (EC 3.2.1.1) is produced mainly in aleurone cells of germinating cereals, and the phytohormone gibberellin (GA) is essential for its induction. However, in rice (Oryza sativa L.), sulfuric acid (H(2)SO(4)) induces alpha-amylase production in aleurone tissue even in the absence of GA. Here, the pre-treatment of rice aleurone cells with H(2)SO(4) and incubation in water induced alpha-amylase activity, as if the cells had been incubated in GA solution.

  7. Quantitation of alpha-factor internalization and response during the Saccharomyces cerevisiae cell cycle.

    PubMed Central

    Zanolari, B; Riezman, H

    1991-01-01

    The alpha-factor pheromone binds to specific cell surface receptors on Saccharomyces cerevisiae a cells. The pheromone is then internalized, and cell surface receptors are down-regulated. At the same time, a signal is transmitted that causes changes in gene expression and cell cycle arrest. We show that the ability of cells to internalize alpha-factor is constant throughout the cell cycle, a cells are also able to respond to pheromone throughout the cycle even though there is cell cycle modulation of the expression of two pheromone-inducible genes, FUS1 and STE2. Both of these genes are expressed less efficiently near or just after the START point of the cell cycle in response to alpha-factor. For STE2, the basal level of expression is modulated in the same manner. Images PMID:1656226

  8. Interferon-resistant Daudi cells are deficient in interferon-alpha-induced ISGF3 alpha activation, but remain sensitive to the interferon-alpha-induced increase in ISGF3 gamma content.

    PubMed

    Dron, M; Tovey, M G

    1993-10-01

    Low levels of the transcription factor ISGF3 alpha were detected in the cytoplasm and nucleus of untreated Daudi cells, which increased markedly following interferon (IFN) treatment. In contrast no ISGF3 alpha was detected in an IFN-resistant clone of Daudi cells, DIF8, and only low levels were detected in these cells after IFN-alpha treatment. High levels of ISGF3 were produced in vitro, however, by the addition of ISGF3 alpha to extracts of IFN-treated DIF8 cells, indicating that IFN is unable to produce substantial amounts of functional ISGF3 alpha in DIF8 cells. A second clone of IFN-resistant Daudi cells, DIF3, also exhibited defective ISGF3 alpha production, which was restored to normal in the subclone DIF3REV5 that had reverted to high IFN sensitivity. Thus, the antiproliferative effect of IFN on Daudi cells and derived clones is closely related to the level of ISGF3 present in the nucleus of these cells. IFN-alpha, however, also enhances the content of ISGF3 gamma in IFN-resistant cells as well as certain proteins of unknown function, raising the possibility that a second pathway of IFN-alpha signal transduction, distinct from the ISGF3 pathway, remains functional in both DIF8 and DIF3 cells.

  9. Distinctive selection mechanisms govern the T cell receptor repertoire of peripheral CD4-CD8- alpha/beta T cells

    PubMed Central

    1992-01-01

    The T cell receptor (TCR) repertoire of CD4+ and CD8+ alpha/beta T cells is heavily influenced by positive and negative selection events that occur during T cell development in the thymus. The coreceptors CD4 and CD8 appear to be essential for this selection to occur. To gain insight into whether T cells that express TCR alpha/beta but lack either coreceptor (CD4- CD8- TCR alpha/beta or alpha/beta double- negative [DN] cells) are also subject to positive and negative selection, and whether selection can occur in the absence of coreceptors, we have performed an extensive immunogenetic analysis of the TCR V beta repertoire of alpha/beta DN cells in lymph nodes of normal mice. Our results show that alpha/beta DN cells appear to be unaffected by clonal deletion of V beta 5 and V beta 11 in I-E- expressing mice, and do not undergo deletion of V beta 6- and V beta 8.1-expressing T cells in Mls-1a-positive mice. They are also unaffected by positive selection of V beta 17a+ T cells in the context of I-Aq. The results suggest that most selection events require the participation of CD4 and CD8, while alpha/beta DN cells are unselected. This argues that most alpha/beta DN cells probably have never expressed CD4 or CD8. However, a unique form of repertoire selection occurs: enrichment of V beta 17a+ alpha/beta DN cells in I-E+ mice. This could be an instance of coreceptor-independent selection. PMID:1512537

  10. Thiazolidinediones inhibit REG I{alpha} gene transcription in gastrointestinal cancer cells

    SciTech Connect

    Yamauchi, Akiyo; Takahashi, Iwao; Takasawa, Shin; Nata, Koji; Noguchi, Naoya; Ikeda, Takayuki; Yoshikawa, Takeo; Shervani, Nausheen J.; Suzuki, Iwao; Uruno, Akira; Unno, Michiaki; Okamoto, Hiroshi Sugawara, Akira

    2009-02-13

    REG (Regenerating gene) I{alpha} protein functions as a growth factor for gastrointestinal cancer cells, and its mRNA expression is strongly associated with a poor prognosis in gastrointestinal cancer patients. We here demonstrated that PPAR{gamma}-agonist thiazolidinediones (TZDs) inhibited cell proliferation and REG I{alpha} protein/mRNA expression in gastrointestinal cancer cells. TZDs inhibited the REG I{alpha} gene promoter activity, via its cis-acting element which lacked PPAR response element and could not bind to PPAR{gamma}, in PPAR{gamma}-expressing gastrointestinal cancer cells. The inhibition was reversed by co-treatment with a specific PPAR{gamma}-antagonist GW9662. Although TZDs did not inhibit the REG I{alpha} gene promoter activity in PPAR{gamma}-non-expressing cells, PPAR{gamma} overexpression in the cells recovered their inhibitory effect. Taken together, TZDs inhibit REG I{alpha} gene transcription through a PPAR{gamma}-dependent pathway. The TZD-induced REG I{alpha} mRNA reduction was abolished by cycloheximide, indicating the necessity of novel protein(s) synthesis. TZDs may therefore be a candidate for novel anti-cancer drugs for patients with gastrointestinal cancer expressing both REG I{alpha} and PPAR{gamma}.

  11. PKC{alpha} expression regulated by Elk-1 and MZF-1 in human HCC cells

    SciTech Connect

    Hsieh, Y.-H.; Wu, T.-T.; Tsai, J.-H.; Huang, C.-Y.; Hsieh, Y.-S.; Liu, J.-Y. . E-mail: jyl@csmu.edu.tw

    2006-01-06

    Our previous study found that PKC{alpha} was highly expressed in the poor-differentiated human HCC cells and associated with cell migration and invasion. In this study, we further investigated the gene regulation of this enzyme. We showed that PKC{alpha} expression enhancement in the poor-differentiated human HCC cells was found neither by DNA amplification nor by increasing mRNA stability using differential PCR and mRNA decay assays. After screening seven transcription factors in the putative cis-acting regulatory elements of human PKC{alpha} promoters, only Elk-1 and MZF-1 antisense oligonucleotide showed a significant reduction in the PKC{alpha} mRNA level. They also reduced cell proliferation, cell migratory and invasive capabilities, and DNA binding activities in the PKC{alpha} promoter region. Over-expression assay confirmed that the PKC{alpha} expression may be modulated by these two factors at the transcriptional level. Therefore, these results may provide a novel mechanism for PKC{alpha} expression regulation in human HCC cells.

  12. In vitro reprogramming of pancreatic alpha cells towards a beta cell phenotype following ectopic HNF4α expression.

    PubMed

    Sangan, Caroline B; Jover, Ramiro; Heimberg, Harry; Tosh, David

    2015-01-05

    There is currently a shortage of organ donors available for pancreatic beta cell transplantation into diabetic patients. An alternative source of beta cells is pre-existing pancreatic cells. While we know that beta cells can arise directly from alpha cells during pancreatic regeneration we do not understand the molecular basis for the switch in phenotype. The aim of the present study was to investigate if hepatocyte nuclear factor 4 alpha (HNF4α), a transcription factor essential for a normal beta cell phenotype, could induce the reprogramming of alpha cells towards potential beta cells. We utilised an in vitro model of pancreatic alpha cells, the murine αTC1-9 cell line. We initially characterised the αTC1-9 cell line before and following adenovirus-mediated ectopic expression of HNF4α. We analysed the phenotype at transcript and protein level and assessed its glucose-responsiveness. Ectopic HNF4α expression in the αTC1-9 cell line induced a change in morphology (1.7-fold increase in size), suppressed glucagon expression, induced key beta cell-specific markers (insulin, C-peptide, glucokinase, GLUT2 and Pax4) and pancreatic polypeptide (PP) and enabled the cells to secrete insulin in a glucose-regulated manner. In conclusion, HNF4α reprograms alpha cells to beta-like cells.

  13. alpha-Lactalbumin species variation, HAMLET formation, and tumor cell death.

    PubMed

    Pettersson, Jenny; Mossberg, Ann-Kristin; Svanborg, Catharina

    2006-06-23

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a tumoricidal complex of apo alpha-lactalbumin and oleic acid, formed in casein after low pH treatment of human milk. This study examined if HAMLET-like complexes are present in casein from different species and if isolated alpha-lactalbumin from those species can form such complexes with oleic acid. Casein from human, bovine, equine, and porcine milk was separated by ion exchange chromatography and active complexes were only found in human casein. This was not explained by alpha-lactalbumin sequence variation, as purified bovine, equine, porcine, and caprine alpha-lactalbumins formed complexes with oleic acid with biological activity similar to HAMLET. We conclude that structural variation of alpha-lactalbumins does not preclude the formation of HAMLET-like complexes and that natural HAMLET formation in casein was unique to human milk, which also showed the highest oleic acid content.

  14. Effect of transforming growth factor-alpha on inositol phospholipid metabolism in human epidermoid carcinoma cells

    SciTech Connect

    Kato, M.; Takenawa, T.; Twardzik, D.R.

    1988-08-01

    Transforming growth factor-alpha (TGF-alpha) stimulates (in a dose-dependent manner) the incorporation of (/sup 32/P)Pi into phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), and phosphatidic acid (PA) in the human epidermoid carcinoma cell line (A431). The effect of TGF-alpha on the incorporation was found to be similar to that of EGF. On the other hand, a striking difference in the activation of diacylglycerol (DG) kinase activity was seen between TGF-alpha and EGF. At least 100 times more TGF-alpha was required to achieve maximal stimulation of DG kinase activity relative to EGF. These results suggest that the activation of DG kinase by TGF-alpha may involve a mechanism independent from or subsequent to activation of the EGF receptor.

  15. Quantitative analysis of topoisomerase II{alpha} to rapidly evaluate cell proliferation in brain tumors

    SciTech Connect

    Oda, Masashi; Arakawa, Yoshiki; Kano, Hideyuki; Kawabata, Yasuhiro; Katsuki, Takahisa; Shirahata, Mitsuaki; Ono, Makoto; Yamana, Norikazu; Hashimoto, Nobuo; Takahashi, Jun A. . E-mail: jat@kuhp.kyoto-u.ac.jp

    2005-06-17

    Immunohistochemical cell proliferation analyses have come into wide use for evaluation of tumor malignancy. Topoisomerase II{alpha} (topo II{alpha}), an essential nuclear enzyme, has been known to have cell cycle coupled expression. We here show the usefulness of quantitative analysis of topo II{alpha} mRNA to rapidly evaluate cell proliferation in brain tumors. A protocol to quantify topo II{alpha} mRNA was developed with a real-time RT-PCR. It took only 3 h to quantify from a specimen. A total of 28 brain tumors were analyzed, and the level of topo II{alpha} mRNA was significantly correlated with its immuno-staining index (p < 0.0001, r = 0.9077). Furthermore, it sharply detected that topo II{alpha} mRNA decreased in growth-inhibited glioma cell. These results support that topo II{alpha} mRNA may be a good and rapid indicator to evaluate cell proliferate potential in brain tumors.

  16. T-cell receptor alpha chain plays a critical role in antigen-specific suppressor cell function.

    PubMed Central

    Kuchroo, V K; Byrne, M C; Atsumi, Y; Greenfield, E; Connolly, J B; Whitters, M J; O'Hara, R M; Collins, M; Dorf, M E

    1991-01-01

    Antigen-specific suppressor T-cell hybridomas release soluble suppressor factors (TsF) in the supernatant that modulate both in vivo delayed-type hypersensitivity and in vitro plaque-forming cell responses in an antigen-specific manner. To study the relationship between the T-cell receptor (TcR) and TsF, we developed a series of TcR alpha- or TcR beta- expression variants from suppressor T-cell hybridomas that expressed the CD3-TcR alpha/beta complex. We demonstrate that loss of TcR alpha but not TcR beta mRNA was accompanied by the concomitant loss of suppressor bioactivity. Homologous transfection of TcR alpha cDNA into a TcR alpha- beta+ clone reconstituted both CD3-TcR expression and suppressor function. Furthermore, suppressor activity from TcR beta- variants was specifically absorbed by antigen and anti-TcR alpha antibodies, but not by anti-CD3 or anti-TcR beta affinity columns. These data directly establish a role for the TcR alpha chain in suppressor T-cell function and suggest that the TcR alpha chain is part of the antigen-specific TsF molecule. Images PMID:1833764

  17. Spatial Characterization of Polycyclic Aromatic Hydrocarbons in 2008 TC3 Samples

    NASA Astrophysics Data System (ADS)

    Sabbah, Hassan; Morrow, A.; Zare, R. N.; Jenniskens, P.

    2009-09-01

    Hassan Sabbah1, Amy L. Morrow1, Richard N. Zare1 and Petrus Jenniskens2 1Stanford University, Stanford, California 94305, 2 SETI Institute, Carl Sagan Center, 515 North Whisman Road, Mountain View, California 94043, USA. In October 2006 a small asteroid (2-3 meters) was observed in outer space. On October 7, 2008, it entered the Earth's atmosphere creating a fireball over Northern Sudan. Some 280 meteorites were collected by the University of Khartoum. In order to explore the existence of organic materials, specifically polycyclic aromatic hydrocarbons (PAHs), we applied two-step laser desorption laser ionization mass spectrometry (L2MS) to some selected fragments. This technique consists of desorbing with a pulsed infrared laser beam the solid materials into a gaseous phase with no fragmentation followed by resonance enhanced multiphoton ionization to analyze the PAH content. L2MS was already applied to an array of extraterrestrial objects including interplanetary dust particles IDPs, carbonaceous chondrites and comet coma particles. Moreover, spatial resolution of PAHs in 2008 TC3 samples was achieved to explore the heterogeneity within individual fragments. The results of these studies and their contribution to understanding the formation of this asteroid will be discussed.

  18. Lysosomal alpha-glucosidase: cell-specific processing and altered maturation in HT-29 colon cancer cells.

    PubMed Central

    Francí, C; Egea, G; Arribas, R; Reuser, A J; Real, F X

    1996-01-01

    We have previously described the abnormal localization of resident Golgi proteins and O-glycans in the rough endoplasmic reticulum of mucin-secreting HT-29 M6 colon cancer cells, suggesting altered protein trafficking in these cells [Egea, Francí, Gambús, Lesuffleur, Zweibaum and Real (1993) J. Cell Sci. 105, 819-830]. In the present work, we have chosen lysosomal alpha-glucosidase as a reporter to examine the intracellular traffic of glycoproteins in M6 cells. We have compared the synthesis and processing of alpha-glucosidase in mucin-secreting M6 cells and in Caco-2 colon cancer cells, the latter resembling normal absorptive intestinal epithelium. Our results show that alpha-glucosidase processing and secretion is markedly delayed in M6 cells as compared to Caco-2 cells or normal fibroblasts, and this delay is caused by an accumulation of alpha-glucosidase precursor form in the trans-Golgi network. Furthermore, treatment in Caco-2 cells with brefeldin A led to changes in alpha-glucosidase maturation similar to those observed in untreated M6 cells. To determine whether altered processing occurs in other cultured cells, a panel of cancer cell lines and cultures from normal exocrine pancreas were examined. In pancreas-derived cultures, alpha-glucosidase showed a processing pattern different from that described until now. Only HT-29 cells and HT-29-derived subpopulations displayed a defect in alpha-glucosidase maturation. In conclusion, alpha-glucosidase processing is more diverse than has previously been described; this finding may have tissue-specific functional implications. PMID:8660303

  19. Hydroxyl-HIF2-alpha is potential therapeutic target for renal cell carcinomas

    PubMed Central

    Isono, Takahiro; Chano, Tokuhiro; Yoshida, Tetsuya; Kageyama, Susumu; Kawauchi, Akihiro; Suzaki, Masafumi; Yuasa, Takeshi

    2016-01-01

    Dormant cancer cells are deprivation-resistant, and cause a number of problems for therapeutic approaches for cancers. Renal cell carcinomas (RCCs) include deprivation-resistant cells that are resistant to various treatments. In this study, the specific characteristics of deprivation-resistant cells were transcriptionally identified by next generation sequencing. The hypoxia-inducible factors (HIF) transcription factor network was significantly enhanced in deprivation-resistant RCCs compared to the sensitive RCCs. Deprivation-resistant RCCs, that had lost Von Hippel-Lindau tumor suppressor expression, expressed hydroxyl-HIF2-alpha in the nucleus, but not sensitive-RCCs. Hydroxyl-HIF-alpha was also expressed in nuclei of RCC tissue samples. Knockdown for HIF2-alpha, but not HIF1-alpha, induced cell death related to a reduction in HIF-related gene expression in deprivation-resistant RCC cells. Chetomin, a nuclear HIF-inhibitor, induced marked level of cytotoxicity in deprivation-resistant cells, similar to the knockdown of HIF2-alpha. Therefore, hydroxyl-HIF2-alpha might be a potential therapeutic target for RCCs. PMID:27822416

  20. Hydroxyl-HIF2-alpha is potential therapeutic target for renal cell carcinomas.

    PubMed

    Isono, Takahiro; Chano, Tokuhiro; Yoshida, Tetsuya; Kageyama, Susumu; Kawauchi, Akihiro; Suzaki, Masafumi; Yuasa, Takeshi

    2016-01-01

    Dormant cancer cells are deprivation-resistant, and cause a number of problems for therapeutic approaches for cancers. Renal cell carcinomas (RCCs) include deprivation-resistant cells that are resistant to various treatments. In this study, the specific characteristics of deprivation-resistant cells were transcriptionally identified by next generation sequencing. The hypoxia-inducible factors (HIF) transcription factor network was significantly enhanced in deprivation-resistant RCCs compared to the sensitive RCCs. Deprivation-resistant RCCs, that had lost Von Hippel-Lindau tumor suppressor expression, expressed hydroxyl-HIF2-alpha in the nucleus, but not sensitive-RCCs. Hydroxyl-HIF-alpha was also expressed in nuclei of RCC tissue samples. Knockdown for HIF2-alpha, but not HIF1-alpha, induced cell death related to a reduction in HIF-related gene expression in deprivation-resistant RCC cells. Chetomin, a nuclear HIF-inhibitor, induced marked level of cytotoxicity in deprivation-resistant cells, similar to the knockdown of HIF2-alpha. Therefore, hydroxyl-HIF2-alpha might be a potential therapeutic target for RCCs.

  1. Antigenic modulation of metastatic breast and ovary carcinoma cells by intracavitary injection of IFN-alpha.

    PubMed Central

    Giacomini, P.; Mottolese, M.; Fraioli, R.; Benevolo, M.; Venturo, I.; Natali, P. G.

    1992-01-01

    Antigenic modulation of major histocompatibility and tumour associated antigens was observed in neoplastic cells obtained from patients with pleural and abdominal effusions of breast and ovary carcinomas following a single intracavitary dose of 18 x 10(6) U recombinant IFN-alpha. This regimen resulted in antigenic modulation in seven out of 11 tested cases, suggesting a potential, although limited, responsiveness of at least a fraction of breast and ovary carcinoma cells to in situ biomodification with IFN-alpha. PMID:1503908

  2. Tumor necrosis factor-alpha is expressed by glomerular visceral epithelial cells in human membranous nephropathy.

    PubMed Central

    Neale, T. J.; Rüger, B. M.; Macaulay, H.; Dunbar, P. R.; Hasan, Q.; Bourke, A.; Murray-McIntosh, R. P.; Kitching, A. R.

    1995-01-01

    The role of tumor necrosis factor alpha (TNF-alpha) was examined in biopsy-proven glomerulonephritis by immunohistochemistry, in situ hybridization, immunogold electron microscopy, immunoassay in serum and urine, and urinary immunoblot. Striking glomerular capillary wall and visceral glomerular epithelial cell TNF-alpha protein staining was observed in all cases of membranous nephropathy and membranous lupus nephropathy. Staining was less frequently observed in crescentic glomerulonephritis and in isolated cases of other histological subtypes of glomerulonephritis, usually in association with glomerular macrophages. By immunogold electron microscopy TNF-alpha was localized in membranous nephropathy within the visceral glomerular epithelial cells, and also in the glomerular basement membrane, especially in relation to immune deposits. In situ hybridization localized TNF-alpha mRNA exclusively to glomerular epithelial cells in all biopsies with membranous morphology but not in other histological subtypes. Concentrations of TNF-alpha were significantly increased compared with normal controls in the urine of patients with membranous nephropathy and with crescentic glomerulonephritis. The expression of TNF-alpha by glomerular epithelial cells exclusively and universally in biopsies showing a membranous morphology strongly suggests this cytokine has a role in the pathogenesis of membranous nephropathy. Images Figure 1 Figure 2 Figure 3 Figure 5 PMID:7778683

  3. The human alpha 2-macroglobulin receptor: identification of a 420-kD cell surface glycoprotein specific for the activated conformation of alpha 2-macroglobulin

    PubMed Central

    1990-01-01

    Ligand affinity chromatography was used to purify a cell surface alpha 2-macroglobulin (alpha 2M) receptor. Detergent extracts of human placenta were applied to an affinity matrix consisting of alpha 2M, previously reacted with methylamine, coupled to Sepharose. Elution with EDTA specifically released polypeptides with apparent molecular masses of 420 and 39 kD. In some preparations, small amounts of a 90-kD polypeptide were observed. The 420- and 39-kD polypeptides appear specific for the forms of alpha 2M activated by reaction with proteinases or methylamine and do not bind to an affinity matrix consisting of native alpha 2M coupled to Sepharose. Separation of these two polypeptides was accomplished by anion exchange chromatography, and binding activity was exclusively associated with the 420-kD polypeptide. The purified 420-kD protein binds to the conformationally altered forms of alpha 2M that are known to specifically interact with alpha 2M receptors and does not bind to native alpha 2M. Binding of the 420-kD polypeptide to immobilized wheat germ agglutinin indicates that this polypeptide is a glycoprotein. The cell surface localization of the 420-kD glycoprotein was confirmed by affinity chromatography of extracts from surface radioiodinated fibroblasts. These properties suggest that the 420-kD polypeptide is a cell surface receptor for the activated forms of alpha 2M. PMID:1691187

  4. Expression and hypoxic regulation of hif1alpha and hif2alpha during early blood and endothelial cell differentiation in chick.

    PubMed

    Ota, Kanako; Nagai, Hiroki; Sheng, Guojun

    2007-08-01

    HIF1 and HIF2 are major mediators for hypoxia sensing and response. Their roles in early differentiation of two key cell types involved in oxygen supply in amniotes, the primitive blood cells and endothelial cells, are unclear. We show that, in pre-circulation avian embryos, hif1alpha and hif2alpha are expressed in embryonic and extraembryonic tissues, respectively. hif2alpha, first identified as epas1, is not present in endothelial cells at any pre-circulation stage under either normoxia or hypoxia conditions. Differentiating blood cells express low levels of hif2alpha under normoxia, but show a strong and rapid upregulation under hypoxia. Blood cell differentiation, however, is not affected under either hypoxia or hyperoxia conditions.

  5. PDE7 inhibitor TC3.6 ameliorates symptomatology in a model of primary progressive multiple sclerosis

    PubMed Central

    Mestre, L; Redondo, M; Carrillo-Salinas, F J; Morales-García, J A; Alonso-Gil, S; Pérez-Castillo, A; Gil, C; Martínez, A; Guaza, C

    2015-01-01

    Background and Purpose cAMP plays an important role in the transduction of signalling pathways involved in neuroprotection and immune regulation. Control of the levels of this nucleotide by inhibition of cAMP-specific PDEs such as PDE7 may affect the pathological processes of neuroinflammatory diseases like multiple sclerosis (MS). In the present study, we evaluated the therapeutic potential of the selective PDE7 inhibitor, TC3.6, in a model of primary progressive multiple sclerosis (PPMS), a rare and severe variant of MS. Experimental Approach Theiler’s murine encephalomyelitis virus-induced demyelinated disease (TMEV-IDD) is one of the models used to validate the therapeutic efficacy of new drugs in MS. As recent studies have analysed the effect of PDE7 inhibitors in the EAE model of MS, here the TMEV-IDD model was used to test their efficacy in a progressive variant of MS. Mice were subjected to two protocols of TC3.6 administration: on the pre-symptomatic phase and once the disease was established. Key Results Treatment with TC3.6 ameliorated the disease course and improved motor deficits of infected mice. This was associated with down-regulation of microglial activation and reduced cellular infiltrates. Decreased expression of pro-inflammatory mediators such as COX-2 and the cytokines, IL-1β, TNF-α, IFN-γ and IL-6 in the spinal cord of TMEV-infected mice was also observed after TC3.6 administration. Conclusion These findings support the importance of PDE7 inhibitors, and specifically TC3.6, as a novel class of agents with therapeutic potential for PPMS. Preclinical studies are needed to determine whether their effects translate into durable clinical benefits. PMID:25994655

  6. Mechanisms for the proliferation of eosinophilic leukemia cells by FIP1L1-PDGFR{alpha}

    SciTech Connect

    Ishihara, Kenji; Kitamura, Hajime; Hiraizumi, Kenji; Kaneko, Motoko; Takahashi, Aki; Zee, OkPyo; Seyama, Toshio; Hong, JangJa; Ohuchi, Kazuo; Hirasawa, Noriyasu

    2008-02-22

    The constitutively activated tyrosine kinase Fip1-like 1 (FIP1L1)-platelet-derived growth factor receptor {alpha} (PDGFR{alpha}) causes eosinophilic leukemia EoL-1 cells to proliferate. Recently, we demonstrated that histone deacetylase inhibitors suppressed this proliferation and induced the differentiation of EoL-1 cells into eosinophils in parallel with a decrease in the level of FIP1L1-PDGFR{alpha}. In this study, we analyzed the mechanism by which FIP1L1-PDGFR{alpha} induces the proliferation and whether the suppression of cell proliferation triggers the differentiation into eosinophils. The FIP1L1-PDGFR{alpha} inhibitor imatinib inhibited the proliferation of EoL-1 cells and decreased the level of the oncoprotein c-Myc as well as the phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase (JNK). The proliferation of EoL-1 cells and expression of c-Myc were also inhibited by the MEK inhibitor U0126 and JNK inhibitor SP600125. The expression of the eosinophilic differentiation marker CCR3 was not induced by imatinib. These findings suggest that FIP1L1-PDGFR{alpha} induces the proliferation of EoL-1 cells through the induction of c-Myc expression via ERK and JNK signaling pathways, but is not involved in the inhibition of differentiation toward mature eosinophils.

  7. Melanocyte stimulating hormone peptides inhibit TNF-alpha signaling in human dermal fibroblast cells.

    PubMed

    Hill, R P; MacNeil, S; Haycock, J W

    2006-02-01

    Alpha-melanocyte stimulating hormone (alpha-MSH) has been identified as a potent anti-inflammatory in various tissues including the skin. It has previously been shown in skin cell keratinocytes and melanocytes/melanoma cells that MSH peptides inhibit TNF-alpha stimulated NF-kappaB activity and intercellular adhesion molecule-1 (ICAM-1) upregulation. However, the precise anti-inflammatory role of MSH peptides in dermal fibroblasts is unclear. Some studies report on pro-inflammatory responses, while others on anti-inflammatory responses. The present study confirms MC1R expression in cultured human dermal fibroblasts and reports that the MSH peptides alpha-MSH and KP(-D-)V inhibit TNF-alpha stimulated NF-kappaB activity and ICAM-1 upregulation, consistent with an anti-inflammatory role. However, involvement of IkappaB-alpha regulation by either peptide was not confirmed, supporting a mechanism independent of the NF-kappaB inhibitor. In conclusion, alpha-MSH and KP(-D-)V peptides have an anti-inflammatory action on dermal fibroblast signaling by inhibiting the pro-inflammatory activity of TNF-alpha in vitro.

  8. Pseudomonas aeruginosa and tumor necrosis factor-alpha attenuate Clara cell secretory protein promoter function.

    PubMed

    Harrod, Kevin S; Jaramillo, Richard J

    2002-02-01

    The Clara cell secretory protein (CCSP, also CC-10/uterglobin) is a 16-kD homodimeric protein abundantly expressed in the airways of mammals. Although the molecular function is unknown, gene-targeting studies indicate CCSP as a regulator of lung inflammation following acute respiratory infection or injury. CCSP is decreased in the lungs of mice following acute Pseudomonas aeruginosa (P.a.) infection. In the present study, the role of decreased promoter function in the regulation of CCSP by P.a. was assessed using an in vitro co-culture system and in vivo studies of transgenic mice. CCSP promoter activity in lung epithelial cells was markedly decreased by P.a. or tumor necrosis factor-alpha (TNF-alpha) in a dose-dependent manner. Regulation of CCSP promoter function by either P.a. or TNF-alpha was localized to the proximal 166 bp flanking region of the CCSP promoter activity. Decreased regulation of the CCSP promoter by P.a. or TNF-alpha was specific to CCSP, as human surfactant protein D (SP-D) promoter activity was unaffected or increased by P.a. or TNF-alpha, respectively. A neutralizing antibody against human TNF-alpha was able to reverse both the TNF-alpha- mediated as well as P.a.-mediated decrease in CCSP promoter function in lung epithelial cells. TNF-alpha secretion by lung epithelial cells coincided with the decrease in CCSP promoter function following P.a. administration. Using a transgenic mouse model, P.a. administration to the lung markedly attenuated CCSP promoter-conferred gene expression in vivo. The attenuation of CCSP promoter activity in lung epithelial cells by P.a. involves, in part, autocrine/paracrine secretion of TNF-alpha, which in turn regulates CCSP transcription through cis-active elements in the proximal promoter region.

  9. Functional effect of point mutations in the alpha-folate receptor gene of CABA I ovarian carcinoma cells.

    PubMed

    Mangiarotti, F; Miotti, S; Galmozzi, E; Mazzi, M; Sforzini, S; Canevari, S; Tomassetti, A

    2001-01-01

    The alpha-folate receptor (alpha FR) is overexpressed in 90% of nonmucinous ovarian carcinomas. In addition to the known role of alpha FR binding and mediating the internalization of folates, functional interaction of alpha FR with signaling molecules was recently shown. To identify a model to study the role of alpha FR in ovarian carcinoma, we characterized the alpha FR gene in the ovarian carcinoma cell line CABA I in comparison to a reference line, IGROV1. In CABA I cells, Northern blot analysis revealed an alpha FR transcript of the expected length and FACS analysis indicated receptor expression on the cell membrane; however, RNase protection assay revealed no specific signals. Southern blot and genomic PCR analysis suggested the presence of a rearrangement(s) involving the 5' region of the gene in CABA I cells as compared to IGROV1 cells. Cloning and sequencing of CABA I alpha FR cDNA revealed several point mutations. The partitioning of alpha FR in membrane microdomains from CABA I cells and its association with regulatory molecules was comparable to that of IGROV1 cells. By contrast, the alpha FR expressed on the CABA I cell membrane bound folic acid with lower affinity, and ectopic expression of the corresponding cDNA in CHO cells confirmed impaired folic acid binding. Thus, CABA I cells may provide a tool to delineate functional domains of the alpha FR.

  10. Differential effects of pentoxifylline on the production of tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) by monocytes and T cells.

    PubMed Central

    Schandené, L; Vandenbussche, P; Crusiaux, A; Alègre, M L; Abramowicz, D; Dupont, E; Content, J; Goldman, M

    1992-01-01

    Pentoxifylline (PTX) is a methylxanthine compound known to inhibit the production of tumour necrosis factor-alpha (TNF-alpha) by monocytic cells. In this study, we found that PTX differentially regulates the production of TNF-alpha and interleukin-6 (IL-6). Indeed, PTX at high concentrations triggers the production of IL-6 but not of TNF-alpha by peripheral blood mononuclear cells (PBMC). Further experiments indicated that monocytes are responsible for this PTX-induced IL-6 production. When PBMC were stimulated with LPS, PTX was found to inhibit the secretion of TNF-alpha as well as the accumulation of TNF-alpha messenger RNA (mRNA). In contrast, no inhibitory effect was observed on the induction of IL-6. Similar results were obtained when PBMC were stimulated with OKT3 monoclonal antibody (mAb). In addition, the in vivo administration of PTX in transplant patients receiving the first dose of OKT3 allowed to decrease the systemic release of TNF-alpha but not of IL-6. Since monocytes represent a major source of TNF-alpha and IL-6 in these settings, additional experiments were performed in vitro on purified T cells stimulated with the CLB-T3/3, an anti-CD3 mAb which does not require the presence of accessory cells to activate T cells. In this system, PTX was found to inhibit the secretion of both TNF-alpha and IL-6 by T cells. We suggest that cAMP could be involved in these differential effects of PTX on production of TNF-alpha and of IL-6. Images Figure 3 PMID:1385797

  11. Rapid capping in alpha-spectrin-deficient MEL cells from mice afflicted with hereditary hemolytic anemia

    PubMed Central

    1994-01-01

    A spectrin-based membrane skeleton is important for the stability and organization of the erythrocyte. To study the role of spectrin in cells that possess complex cytoskeletons, we have generated alpha-spectrin- deficient erythroleukemia cell lines from sph/sph mice. These cells contain beta-spectrin, but lack alpha-spectrin as determined by immunoblot and Northern blot analyses. The effects of alpha-spectrin deficiency are apparent in the cells' irregular shape and fragility in culture. Capping of membrane glycoproteins by fluorescent lectin or antibodies occurs more rapidly in sph/sph than in wild-type erythroleukemia cells, and the caps appear more concentrated. The data support the idea that spectrin plays an important role in organizing membrane structure and limiting the lateral mobility of integral membrane glycoproteins in cells other than mature erythrocytes. PMID:8195289

  12. Effects of alpha-particles on survival and chromosomal aberrations in human mammary epithelial cells

    NASA Technical Reports Server (NTRS)

    Durante, M.; Grossi, G. F.; Gialanella, G.; Pugliese, M.; Nappo, M.; Yang, T. C.

    1995-01-01

    We have studied the radiation responses of a human mammary epithelial cell line, H184B5 F5-1 M/10. This cell line was derived from primary mammary cells after treatment with chemicals and heavy ions. The F5-1 M/10 cells are immortal, density-inhibited in growth, and non-tumorigenic in athymic nude mice and represent an in vitro model of the human epithelium for radiation studies. Because epithelial cells are the target of alpha-particles emitted from radon daughters, we concentrated our studies on the efficiency of alpha-particles. Confluent cultures of M/10 cells were exposed to accelerated alpha-particles [beam energy incident at the cell monolayer = 3.85 MeV, incident linear energy transfer (LET) in cell = 109 keV/microns] and, for comparison, to 80 kVp x-rays. The following endpoints were studied: (1) survival, (2) chromosome aberrations at the first postirradiation mitosis, and (3) chromosome alterations at later passages following irradiation. The survival curve was exponential for alpha-particles (D0 = 0.73 +/- 0.04 Gy), while a shoulder was observed for x-rays (alpha/beta = 2.9 Gy; D0 = 2.5 Gy, extrapolation number 1.6). The relative biological effectiveness (RBE) of high-LET alpha-particles for human epithelial cell killing was 3.3 at 37% survival. Dose-response curves for the induction of chromosome aberrations were linear for alpha-particles and linearquadratic for x-rays. The RBE for the induction of chromosome aberrations varied with the type of aberration scored and was high (about 5) for chromosome breaks and low (about 2) for chromosome exchanges.(ABSTRACT TRUNCATED AT 250 WORDS).

  13. Involvement of Mst1 in tumor necrosis factor-{alpha}-induced apoptosis of endothelial cells

    SciTech Connect

    Ohtsubo, Hideki; Ichiki, Toshihiro Imayama, Ikuyo; Ono, Hiroki; Fukuyama, Kae; Hashiguchi, Yasuko; Sadoshima, Junichi; Sunagawa, Kenji

    2008-03-07

    Mammalian sterile 20-kinase 1 (Mst1), a member of the sterile-20 family protein kinase, plays an important role in the induction of apoptosis. However, little is know about the physiological activator of Mst1 and the role of Mst1 in endothelial cells (ECs). We examined whether Mst1 is involved in the tumor necrosis factor (TNF)-{alpha}-induced apoptosis of ECs. Western blot analysis revealed that TNF-{alpha} induced activation of caspase 3 and Mst1 in a time- and dose-dependent manner. TNF-{alpha}-induced Mst1 activation is almost completely prevented by pretreatment with Z-DEVD-FMK, a caspase 3 inhibitor. Nuclear staining with Hoechst 33258 and fluorescence-activated cell sorting of propidium iodide-stained cells showed that TNF-{alpha} induced apoptosis of EC. Diphenyleneiodonium, an inhibitor of NADPH oxidase, and N-acetylcysteine, a potent antioxidant, also inhibited TNF-{alpha}-induced activation of Mst1 and caspase 3, as well as apoptosis. Knockdown of Mst1 expression by short interfering RNA attenuated TNF-{alpha}-induced apoptosis but not cleavage of caspase 3. These results suggest that Mst1 plays an important role in the induction of TNF-{alpha}-induced apoptosis of EC. However, positive feedback mechanism between Mst1 and caspase 3, which was shown in the previous studies, was not observed. Inhibition of Mst1 function may be beneficial for maintaining the endothelial integrity and inhibition of atherogenesis.

  14. Epidermal growth factor controls smooth muscle alpha-isoactin expression in BC3H1 cells

    PubMed Central

    1988-01-01

    We have examined the effects of epidermal growth factor (EGF), platelet- derived growth factor, and insulin on the differentiation of a mouse vascular smooth muscle-like cell line, the BC3H1 cells. On the basis of cell morphology and smooth muscle alpha-isoactin synthesis, we demonstrate that EGF at physiological concentrations prevents the differentiation of these cells, whereas platelet-derived growth factor has no apparent effect. The induction of alpha-isoactin synthesis by serum deprivation is inhibited by EGF in a dose-dependent manner with a half-maximal effect at 3-5 ng/ml and a maximal inhibition at approximately 30 ng/ml. Northern analysis also shows that EGF blocks the accumulation of alpha-isoactin mRNA normally observed during cell differentiation. Addition of EGF to differentiated cells results in a repression of alpha-isoactin synthesis, a stimulation of beta- and gamma-isoactin synthesis, and the stabilization of the nonmuscle isoactins. The synthesis of creatine phosphokinase, a muscle-specific noncontractile protein, is also regulated by EGF in a similar fashion. Modulation by EGF of alpha-isoactin expression is not affected by aphidicolin and is therefore independent of its mitogenic effect on these cells. Insulin is not required for observation of the EGF- dependent effects but instead seems to promote differentiation. Our results show that EGF can replace serum in controlling the differentiation of BC3H1 cells. PMID:3279054

  15. Tumor necrosis factor alpha leads to increased cell surface expression of CXCR4 in SK-N-MC cells.

    PubMed

    Rostasy, Kevin; Gorgun, Gullue; Kleyner, Yelena; Garcia, Anthony; Kramer, Michael; Melanson, Suzanne M; Mathys, Jean Marie; Yiannoutsos, Constantin; Skolnik, Paul R; Navia, Bradford A

    2005-07-01

    Both host and viral factors play an important role in the pathogenesis of human immunodeficiency virus (HIV)-associated bran injury. In this study, the authors examined the interactions between tumor necrosis factor (TNF)-alpha, CXCR4, the alpha chemokine receptor, and three HIV isolates, including the T-tropic viruses, HIV-1(MN) and HIV-1(IIIB), and the dual tropic virus, HIV-1(89.6). The authors show by flow cytometry that treatment of differentiated SK-N-MC cells with TNF-alpha induces a significant increase in the cell surface expression of CXCR4 in a time- and dose-dependent manner. The effect is partly regulated at the level of transcription. To assess the biological significance of this finding, we show that TNF-alpha potentiates the ability of the above mentioned HIV isolates to induce neuronal apoptosis and that the effect is significantly reduced by pretreating cells with monoclonal antibodies to either CXCR4 and TNF-alpha. Together these results suggest that TNF-alpha may render neuronal cells vulnerable to the apoptotic effects of HIV by increasing the cell surface expression of CXCR4 and thus identify another mechanism by which TNF-alpha contributes to the pathogenesis of HIV-associated brain injury.

  16. Alpha-2 adrenergic and serotonin-1B receptors in the OK cell, an opossum kidney cell line

    SciTech Connect

    Murphy, T.J.

    1988-01-01

    Alpha-2 adrenergic and serotonin-1B (5HT{sub 1B}) receptors, both negatively-coupled to adenylyl cyclase, were characterized in the OK cell line, a renal proximal tubule epithelial cell line derived from the kidney of a North American opossum. In membrane saturation radioligand binding experiments, ({sup 3}H)yohimbine and ({sup 3}H)rauwolscine labeled an equivalent number of binding sites. Detailed pharmacological analysis of OK cell alpha-2 adrenergic receptors in competition binding assays indicate this receptor is neither an alpha-2A nor an alpha-2B adrenergic receptor subtype, although the alpha-2B receptor subtype-selective drugs prazosin, ARC-239 and chlorpromazine have affinities for OK cell alpha-2 adrenergic receptors similar to those at the alpha-2B receptor subtype. Determinations of agonist potency for inhibition of PTH-stimulated cyclic AMP production and radioligand binding analysis using ({sup 125}I)({minus})-cyanopindolol indicate that a 5HT{sub 1B} receptor is expressed in the OK cell line. A biochemical effector system coupled to this receptor subtype has not been previously described. Several compounds appear to be potent agonists at the 5TH{sub 1B} receptor including the beta adrenergic antagonists cyanopindolol, pindolol, propranolol and alprenolol.

  17. Deficient repair of chemical adducts in alpha DNA of monkey cells

    SciTech Connect

    Zolan, M.E.; Cortopassi, G.A.; Smith, C.A.; Hanawalt, P.C.

    1982-03-01

    Researchers have examined excision repair of DNA damage in the highly repeated alpha DNA sequence of cultured African green monkey cells. Irradiation of cells with 254 nm ultraviolet light resulted in the same frequency of pyrimidine dimers in alpha DNA and the bulk of the DNA. The rate and extent of pyrimidine dimer removal, as judged by measurement of repair synthesis, was also similar for alpha DNA and bulk DNA. In cells treated with furocoumarins and long-wave-length ultraviolet light, however, repair synthesis in alpha DNA was only 30% of that in bulk DNA, although it followed the same time course. Researchers found that this reduced repair was not caused by different initial amounts of furocoumarin damage or by different sizes of repair patches, as researchers found these to be similar in the two DNA species. Direct quantification demonstrated that fewer furocoumarin adducts were removed from alpha DNA than from bulk DNA. In cells treated with another chemical DNA-damaging agent, N-acetoxy-2-acetylaminofluorene, repair synthesis in alpha DNA was 60% of that in bulk DNA. These results show that the repair of different kinds of DNA damage can be affected to different extents by some property of this tandemly repeated heterochromatic DNA. To our knowledge, this is the first demonstration in primate cells of differential repair of cellular DNA sequences.

  18. The Alpha Stem Cell Clinic: A Model for Evaluating and Delivering Stem Cell-Based Therapies

    PubMed Central

    DeWitt, Natalie D.; Feigal, Ellen G.

    2012-01-01

    Summary Cellular therapies require the careful preparation, expansion, characterization, and delivery of cells in a clinical environment. There are major challenges associated with the delivery of cell therapies and high costs that will limit the companies available to fully evaluate their merit in clinical trials, and will handicap their application at the present financial environment. Cells will be manufactured in good manufacturing practice or near-equivalent facilities with prerequisite safety practices in place, and cell delivery systems will be specialized and require well-trained medical and nursing staff, technicians or nurses trained to handle cells once delivered, patient counselors, as well as statisticians and database managers who will oversee the monitoring of patients in relatively long-term follow-up studies. The model proposed for Alpha Stem Cell Clinics will initially use the capacities and infrastructure that exist in the most advanced tertiary medical clinics for delivery of established bone marrow stem cell therapies. As the research evolves, they will incorporate improved procedures and cell preparations. This model enables commercialization of medical devices, reagents, and other products required for cell therapies. A carefully constructed cell therapy clinical infrastructure with the requisite scientific, technical, and medical expertise and operational efficiencies will have the capabilities to address three fundamental and critical functions: 1) fostering clinical trials; 2) evaluating and establishing safe and effective therapies, and 3) developing and maintaining the delivery of therapies approved by the Food and Drug Administration, or other regulatory agencies. PMID:23197634

  19. Synthesis of IL-1 alpha and IL-1 beta by arterial cells in atherosclerosis.

    PubMed Central

    Moyer, C. F.; Sajuthi, D.; Tulli, H.; Williams, J. K.

    1991-01-01

    Interleukin-1 (IL-1) has been implicated as a regulatory protein in the development and clinical sequelae of atherosclerosis. To determine which cells in the atherosclerotic plaque synthesize IL-1 in situ, the authors evaluated histologic sections of iliac arteries from cynomolgus monkeys using probes for IL-1 alpha and beta. A polyclonal antibody to IL-1 alpha and beta was used to determine if proteins were concomitantly produced. The predominant cells expressing IL-1 alpha and beta mRNA were foam cells in the intima. Adherent leukocytes and vascular smooth muscle cells (VSMCs) expressed mRNA for IL-1 alpha. Microvascular endothelium expressed mRNA for both IL-1 alpha and beta. IL-1 proteins were located frequently in cells expressing IL-1 mRNA. These results indicate that endothelium and VSMCs, in conjunction with macrophages, serve as localized sources of IL-1 protein synthesis. These findings suggest that vascular cells may contribute directly to the pathogenesis of atherosclerotic vascular disease by actively secreting potent biologic mediators that modify vascular and immune cell function. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:2012178

  20. Alpha-particle-induced p53 protein expression in a rat lung epithelial cell strain.

    PubMed

    Hickman, A W; Jaramillo, R J; Lechner, J F; Johnson, N F

    1994-11-15

    Other investigators have shown that both sparsely ionizing and UV radiation cause cell cycle arrest that is associated with increased expression of wild-type p53 protein. The effect of exposure to alpha-particles from 238Pu on the induction of the p53 protein has now been examined in cultured lung epithelial cells derived from male F344 rats. The number of cells having increased levels of p53 protein was determined by flow cytometry after the cells had been stained with a monoclonal antibody to p53. alpha-Particle irradiation caused a dose-dependent increase in p53 protein levels detectable at doses as low as 0.6 cGy, with no evidence of a threshold. An increase in p53 protein also occurred in X-irradiated cells. However, no increase was seen in cells exposed to less than 10 cGy of X-rays, indicating the existence of a relatively higher DNA damage threshold for sparsely ionizing radiation. In addition, more cells exposed to low doses of alpha radiation had increased p53 protein levels than would be predicted based on the number of nuclei expected to be traversed by an alpha-particle, suggesting that alpha-particles cause genetic damage by mechanisms in addition to direct interactions with DNA.

  1. PPAR{alpha} agonists up-regulate organic cation transporters in rat liver cells

    SciTech Connect

    Luci, Sebastian; Geissler, Stefanie; Koenig, Bettina; Koch, Alexander; Stangl, Gabriele I.; Hirche, Frank; Eder, Klaus . E-mail: klaus.eder@landw.uni-halle.de

    2006-11-24

    It has been shown that clofibrate treatment increases the carnitine concentration in the liver of rats. However, the molecular mechanism is still unknown. In this study, we observed for the first time that treatment of rats with the peroxisome proliferator activated receptor (PPAR)-{alpha} agonist clofibrate increases hepatic mRNA concentrations of organic cation transporters (OCTNs)-1 and -2 which act as transporters of carnitine into the cell. In rat hepatoma (Fao) cells, treatment with WY-14,643 also increased the mRNA concentration of OCTN-2. mRNA concentrations of enzymes involved in carnitine biosynthesis were not altered by treatment with the PPAR{alpha} agonists in livers of rats and in Fao cells. We conclude that PPAR{alpha} agonists increase carnitine concentrations in livers of rats and cells by an increased uptake of carnitine into the cell but not by an increased carnitine biosynthesis.

  2. Posttranscriptional regulation of collagen alpha1(I) mRNA in hepatic stellate cells.

    PubMed Central

    Stefanovic, B; Hellerbrand, C; Holcik, M; Briendl, M; Aliebhaber, S; Brenner, D A

    1997-01-01

    The hepatic stellate cell (HSC) is the primary cell responsible for the dramatic increase in the synthesis of type I collagen in the cirrhotic liver. Quiescent HSCs contain a low level of collagen alpha1(I) mRNA, while activated HSCs contain about 60- to 70-fold more of this mRNA. The transcription rate of the collagen alpha1(I) gene is only two fold higher in activated HSCs than in quiescent HSCs. In assays using actinomycin D or 5,6-dichlorobenzimidazole riboside collagen alpha1(I) mRNA has estimated half-lives of 1.5 h in quiescent HSCs and 24 h in activated HSCs. Thus, this 16-fold change in mRNA stability is primarily responsible for the increase in collagen alpha1(I) mRNA steady-state level in activated HSCs. We have identified a novel RNA-protein interaction targeted to the C-rich sequence in the collagen alpha1(I) mRNA 3' untranslated region (UTR). This sequence is localized 24 nucleotides 3' to the stop codon. In transient transfection experiments, mutation of this sequence diminished accumulation of an mRNA transcribed from a collagen alpha1(I) minigene and in stable transfections decreased the half-life of collagen alpha1(I) minigene mRNA. Binding to the collagen alpha1(I) 3' UTR is present in cytoplasmic extracts of activated but not quiescent HSCs. It contains as a subunit alphaCP, which is also found in the complex involved in stabilization of alpha-globin mRNA. The auxiliary factors necessary to promote binding of alphaCP to the collagen 3' UTR are distinct from the factors necessary for binding to the alpha-globin sequence. Since alphaCP is expressed in both quiescent and activated HSCs, these auxiliary factors are responsible for the differentially expressed RNA-protein interaction at the collagen alpha1(I) mRNA 3' UTR. PMID:9271398

  3. The coffee diterpene kahweol inhibits tumor necrosis factor-{alpha}-induced expression of cell adhesion molecules in human endothelial cells

    SciTech Connect

    Kim, Hyung Gyun; Kim, Ji Young; Hwang, Yong Pil; Lee, Kyung Jin; Lee, Kwang Youl; Kim, Dong Hee; Kim, Dong Hyun; Jeong, Hye Gwang . E-mail: hgjeong@chosun.ac.kr

    2006-12-15

    Endothelial cells produce adhesion molecules after being stimulated with various inflammatory cytokines. These adhesion molecules play an important role in the development of atherogenesis. Recent studies have highlighted the chemoprotective and anti-inflammatory effects of kahweol, a coffee-specific diterpene. This study examined the effects of kahweol on the cytokine-induced monocyte/human endothelial cell interaction, which is a crucial early event in atherogenesis. Kahweol inhibited the adhesion of TNF{alpha}-induced monocytes to endothelial cells and suppressed the TNF{alpha}-induced protein and mRNA expression of the cell adhesion molecules, VCAM-1 and ICAM-1. Furthermore, kahweol inhibited the TNF{alpha}-induced JAK2-PI3K/Akt-NF-{kappa}B activation pathway in these cells. Overall, kahweol has anti-inflammatory and anti-atherosclerotic activities, which occurs partly by down-regulating the pathway that affects the expression and interaction of the cell adhesion molecules on endothelial cells.

  4. Hypertonic saline attenuates TNF-alpha-induced NF-kappaB activation in pulmonary epithelial cells.

    PubMed

    Nydam, Trevor L; Moore, Ernest E; McIntyre, Robert C; Wright, Franklin L; Gamboni-Robertson, Fabia; Eckels, Phillip C; Banerjee, Anirban

    2009-05-01

    Resuscitation with hypertonic saline (HTS) attenuates acute lung injury (ALI) and modulates postinjury hyperinflammation. TNF-alpha-stimulated pulmonary epithelium is a major contributor to hemorrhage-induced ALI. We hypothesized that HTS would inhibit TNF-alpha-induced nuclear factor (NF)-kappaB proinflammatory signaling in pulmonary epithelial cells. Therefore, we pretreated human pulmonary epithelial cells (A549) with hypertonic medium (180 mM NaCl) for 30 min, followed by TNF-alpha stimulation (10 ng/mL). Key regulatory steps and protein concentrations in this pathway were assessed for significant alterations. Hypertonic saline significantly reduced TNF-alpha-induced intercellular adhesion molecule 1 levels and NF-kappaB nuclear localization. The mechanism is attenuated phosphorylation and delayed degradation of IkappaB alpha. Hypertonic saline did not alter TNF-alpha-induced p38 mitogen-activated protein kinase phosphorylation or constitutive vascular endothelial growth factor expression, suggesting that the observed inhibition is not a generalized suppression of protein phosphorylation or cellular function. These results show that HTS inhibits TNF-alpha-induced NF-kappaB activation in the pulmonary epithelium and, further, our understanding of its beneficial effects in hemorrhage-induced ALI.

  5. Erythroid cell-specific alpha-globin gene regulation by the CP2 transcription factor family.

    PubMed

    Kang, Ho Chul; Chae, Ji Hyung; Lee, Yeon Ho; Park, Mi-Ae; Shin, June Ho; Kim, Sung-Hyun; Ye, Sang-Kyu; Cho, Yoon Shin; Fiering, Steven; Kim, Chul Geun

    2005-07-01

    We previously demonstrated that ubiquitously expressed CP2c exerts potent erythroid-specific transactivation of alpha-globin through an unknown mechanism. This mechanism is reported here to involve specific CP2 splice variants and protein inhibitor of activated STAT1 (PIAS1). We identify a novel murine splice isoform of CP2, CP2b, which is identical to CP2a except that it has an additional 36 amino acids encoded by an extra exon. CP2b has an erythroid cell-specific transcriptional activation domain, which requires the extra exon and can form heteromeric complexes with other CP2 isoforms, but lacks the DNA binding activity found in CP2a and CP2c. Transcriptional activation of alpha-globin occurred following dimerization between CP2b and CP2c in erythroid K562 and MEL cells, but this dimerization did not activate the alpha-globin promoter in nonerythroid 293T cells, indicating that an additional erythroid factor is missing in 293T cells. PIAS1 was confirmed as a CP2 binding protein by the yeast two-hybrid screen, and expression of CP2b, CP2c, and PIAS1 in 293T cell induced alpha-globin promoter activation. These results show that ubiquitously expressed CP2b exerts potent erythroid cell-specific alpha-globin gene expression by complexing with CP2c and PIAS1.

  6. Stromal cell-derived factor-1{alpha} (SDF-1{alpha}/CXCL12) stimulates ovarian cancer cell growth through the EGF receptor transactivation

    SciTech Connect

    Porcile, Carola; Bajetto, Adriana . E-mail: bajetto@cba.unige.it; Barbieri, Federica; Barbero, Simone; Bonavia, Rudy; Biglieri, Marianna; Pirani, Paolo; Florio, Tullio . E-mail: florio@cba.unige.it; Schettini, Gennaro

    2005-08-15

    Ovarian cancer (OC) is the leading cause of death in gynecologic diseases in which there is evidence for a complex chemokine network. Chemokines are a family of proteins that play an important role in tumor progression influencing cell proliferation, angiogenic/angiostatic processes, cell migration and metastasis, and, finally, regulating the immune cells recruitment into the tumor mass. We previously demonstrated that astrocytes and glioblastoma cells express both the chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1), and that SDF-1{alpha} treatment induced cell proliferation, supporting the hypothesis that chemokines may play an important role in tumor cells' growth in vitro. In the present study, we report that CXCR4 and SDF-1 are expressed in OC cell lines. We demonstrate that SDF-1{alpha} induces a dose-dependent proliferation in OC cells, by the specific interaction with CXCR4 and a biphasic activation of ERK1/2 and Akt kinases. Our results further indicate that CXCR4 activation induces EGF receptor (EGFR) phosphorylation that in turn was linked to the downstream intracellular kinases activation, ERK1/2 and Akt. In addition, we provide evidence for cytoplasmic tyrosine kinase (c-Src) involvement in the SDF-1/CXCR4-EGFR transactivation. These results suggest a possible important 'cross-talk' between SDF-1/CXCR4 and EGFR intracellular pathways that may link signals of cell proliferation in ovarian cancer.

  7. HAMLET (human alpha-lactalbumin made lethal to tumor cells) triggers autophagic tumor cell death.

    PubMed

    Aits, Sonja; Gustafsson, Lotta; Hallgren, Oskar; Brest, Patrick; Gustafsson, Mattias; Trulsson, Maria; Mossberg, Ann-Kristin; Simon, Hans-Uwe; Mograbi, Baharia; Svanborg, Catharina

    2009-03-01

    HAMLET, a complex of partially unfolded alpha-lactalbumin and oleic acid, kills a wide range of tumor cells. Here we propose that HAMLET causes macroautophagy in tumor cells and that this contributes to their death. Cell death was accompanied by mitochondrial damage and a reduction in the level of active mTOR and HAMLET triggered extensive cytoplasmic vacuolization and the formation of double-membrane-enclosed vesicles typical of macroautophagy. In addition, HAMLET caused a change from uniform (LC3-I) to granular (LC3-II) staining in LC3-GFP-transfected cells reflecting LC3 translocation during macroautophagy, and this was blocked by the macroautophagy inhibitor 3-methyladenine. HAMLET also caused accumulation of LC3-II detected by Western blot when lysosomal degradation was inhibited suggesting that HAMLET caused an increase in autophagic flux. To determine if macroautophagy contributed to cell death, we used RNA interference against Beclin-1 and Atg5. Suppression of Beclin-1 and Atg5 improved the survival of HAMLET-treated tumor cells and inhibited the increase in granular LC3-GFP staining. The results show that HAMLET triggers macroautophagy in tumor cells and suggest that macroautophagy contributes to HAMLET-induced tumor cell death.

  8. Ultraviolet irradiation of monkey cells enhances the repair of DNA adducts in alpha DNA

    SciTech Connect

    Leadon, S.A.; Hanawalt, P.C.

    1984-11-01

    Excision repair of bulky adducts in alpha DNA of African green monkey cells has previously been shown to be deficient relative to that in the overall genome. We have found that u.v. irradiation of these cells results in the enhanced removal of both aflatoxin B1 (AFB1) and acetylaminofluorene (AAF) adducts from the alpha DNA sequences without affecting repair in the bulk of the DNA. The degree of enhanced removal of AFB1 is dependent upon the u.v. dose and the time interval between irradiation and AFB1 treatment. The u.v. enhancement is not inhibited by cycloheximide. Exposure of the cells to dimethylsulfate or gamma-rays does not affect AFB1 adduct repair. The formation and removal of N-acetoxy-2-acetylaminofluorene (NA-AAF) adducts from alpha and bulk DNA was studied in detail. A higher initial level of the acetylated C8 adduct of guanine was found in alpha DNA than in bulk DNA. Although both the acetylated and deacetylated C8 adducts were removed from the two DNA species, the level of repair was significantly greater in the bulk DNA. Irradiation of cells with u.v. prior to treatment with NA-AAF enhanced the removal of both adducts from alpha DNA with little or no effect on repair in bulk DNA. We conclude that the presence of u.v. photoproducts or some intermediate in their processing alters the chromatin structure of alpha DNA thereby rendering bulky adducts accessible to repair enzymes. In addition, the differential formation and repair of AAF adducts in alpha DNA compared with that in the bulk of the genome supports the hypothesis of an altered chromatin structure for alpha domains.

  9. Bovine somatotropin attenuates phorbol ester-induced prostaglandin F2alpha production in bovine endometrial cells.

    PubMed

    Badinga, L; Guzeloglu, A; Thatcher, W W

    2002-03-01

    The recent observation that bovine somatotropin (bST) treatment at a timed insemination improves pregnancy rates in lactating dairy cows raises the possibility that growth hormone (GH) may modulate the endocrine and biochemical cross talk between the conceptus and maternal uterus at the time of pregnancy establishment in cattle. The objective of this study was to characterize the cellular and molecular mechanisms by which exogenous GH affects phorbol ester-induced prostaglandin F2alpha (PGF2alpha) production in cultured bovine endometrial (BEND) cells. Serum-deprived BEND cells were incubated with or without recombinant bovine GH (rbGH), insulin-like growth factor (IGF)-I, recombinant bovine interferon (rbIFN)-tau or a combination of rbGH + rbIFN-tau for 3 h and then treated with phorbol 12,13-dibutyrate (PDBu) for an additional 6 h. Exogenous PDBu increased PGF2alpha secretion and steady-state levels of COX-2 mRNA within 3 h. Priming of BEND cells with rbGH reduced PGF2alpha response to PDBu, whereas cotreatment with IGF-I amplified PDBu induction of PGF2alpha. Preincubation of cell monolayers with rbIFN-tau suppressed PGF2alpha and COX-2 mRNA responses to PDBu. Inhibitory effects of rbGH and rbIFN-tau on PDBu-induced PGF2alpha production were additive. Results provide the first direct evidence that supplemental bST may interact with conceptus-secreted IFN-tau to modulate PGF2alpha secretion at the critical time of maternal recognition of pregnancy.

  10. Characterization of G-protein alpha subunits in the Gq class: expression in murine tissues and in stromal and hematopoietic cell lines.

    PubMed Central

    Wilkie, T M; Scherle, P A; Strathmann, M P; Slepak, V Z; Simon, M I

    1991-01-01

    Murine G alpha 14 and G alpha 15 cDNAs encode distinct alpha subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins). These alpha subunits are related to members of the Gq class and share certain sequence characteristics with G alpha q, G alpha 11, and G alpha 16, such as the absence of a pertussis toxin ADP-ribosylation site. G alpha 11 and G alpha q are ubiquitously expressed among murine tissues but G alpha 14 is predominantly expressed in spleen, lung, kidney, and testis whereas G alpha 15 is primarily restricted to hematopoietic lineages. Among hematopoietic cell lines, G alpha 11 mRNA is found in all cell lines tested, G alpha q is expressed widely but is not found in most T-cell lines, G alpha 15 is predominantly expressed in myeloid and B-cell lineages, and G alpha 14 is expressed in bone marrow adherent (stromal) cells, certain early myeloid cells, and progenitor B cells. Polyclonal antisera produced from synthetic peptides that correspond to two regions of G alpha 15 react with a protein of 42 kDa expressed in B-cell membranes and in Escherichia coli transformed with G alpha 15 cDNA. The expression patterns that were observed in mouse tissues and cell lines indicate that each of the alpha subunits in the Gq class may be involved in pertussis toxin-insensitive signal-transduction pathways that are fundamental to hematopoietic cell differentiation and function. Images PMID:1946421

  11. ATP Release from Human Airway Epithelial Cells Exposed to Staphylococcus aureus Alpha-Toxin

    PubMed Central

    Baaske, Romina; Richter, Mandy; Möller, Nils; Ziesemer, Sabine; Eiffler, Ina; Müller, Christian; Hildebrandt, Jan-Peter

    2016-01-01

    Airway epithelial cells reduce cytosolic ATP content in response to treatment with S. aureus alpha-toxin (hemolysin A, Hla). This study was undertaken to investigate whether this is due to attenuated ATP generation or to release of ATP from the cytosol and extracellular ATP degradation by ecto-enzymes. Exposure of cells to rHla did result in mitochondrial calcium uptake and a moderate decline in mitochondrial membrane potential, indicating that ATP regeneration may have been attenuated. In addition, ATP may have left the cells through transmembrane pores formed by the toxin or through endogenous release channels (e.g., pannexins) activated by cellular stress imposed on the cells by toxin exposure. Exposure of cells to an alpha-toxin mutant (H35L), which attaches to the host cell membrane but does not form transmembrane pores, did not induce ATP release from the cells. The Hla-mediated ATP-release was completely blocked by IB201, a cyclodextrin-inhibitor of the alpha-toxin pore, but was not at all affected by inhibitors of pannexin channels. These results indicate that, while exposure of cells to rHla may somewhat reduce ATP production and cellular ATP content, a portion of the remaining ATP is released to the extracellular space and degraded by ecto-enzymes. The release of ATP from the cells may occur directly through the transmembrane pores formed by alpha-toxin. PMID:27929417

  12. Interleukin-7 receptor alpha is essential for the development of gamma delta + T cells, but not natural killer cells

    PubMed Central

    1996-01-01

    Mice that lack a functional gamma c subunit of the receptors for interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15 display profound defects in lymphoid development. The IL-7/IL-7R system represents a critical interaction for conventional T and B cell development. In this report, the role of IL-7R alpha in the development of lymphoid lineages other than conventional T and B cells was examined. We demonstrate that gamma delta + T cells were absent in IL-7R alpha-deficient mice, whereas the development and function of natural killer cells were normal. Thus, IL-7R alpha function is required for the development of gamma delta + T cells but not natural killer cells. PMID:8691145

  13. Involvement of DNA polymerase alpha in host cell reactivation of UV-irradiated herpes simplex virus

    SciTech Connect

    Nishiyama, Y.; Yoshida, S.; Maeno, K.

    1984-02-01

    Aphidicolin is a potent inhibitor of both host cell DNA polymerase alpha and herpes simplex virus (HSV)-induced DNA polymerase but has no effect on DNA polymerases beta and gamma of host cells. By using an aphidicolin-resistant mutant (Aphr) of HSV, a possible involvement of DNA polymerase alpha in host cell reactivation of UV-damaged HSV was studied. Plaque formation by UV-irradiated Aphr was markedly inhibited by 1 microgram of aphidicolin per ml, which did not affect the plating efficiency of nonirradiated Aphr. Aphidicolin added before 12 h postinfection inhibited plaque formation by irradiated Aphr, which became aphidicolin insensitive after 36 h postinfection. The results strongly suggest that host cell DNA polymerase alpha is involved in the repair of UV-irradiated HSV DNA.

  14. Karyopherin alpha2: a control step of glucose-sensitive gene expression in hepatic cells.

    PubMed Central

    Guillemain, Ghislaine; Muñoz-Alonso, Maria J; Cassany, Aurélia; Loizeau, Martine; Faussat, Anne-Marie; Burnol, Anne-Françoise; Leturque, Armelle

    2002-01-01

    Glucose is required for an efficient expression of the glucose transporter GLUT2 and other genes. We have shown previously that the intracytoplasmic loop of GLUT2 can divert a signal, resulting in the stimulation of glucose-sensitive gene transcription. In the present study, by interaction with the GLUT2 loop, we have cloned the rat karyopherin alpha2, a receptor involved in nuclear import. The specificity of the binding was restricted to GLUT2, and not GLUT1 or GLUT4, and to karyopherin alpha2, not alpha1. When rendered irreversible by a cross-linking agent, this transitory interaction was detected in vivo in hepatocytes. A role for karyopherin alpha2 in the transcription of two glucose-sensitive genes was investigated by transfection of native and inactive green fluorescent protein-karyopherin alpha2 in GLUT2-expressing hepatoma cells. The amount of inactive karyopherin alpha2 receptor reduced, in a dose-dependent manner, the GLUT2 and liver pyruvate kinase mRNA levels by competition with endogenous active receptor. In contrast, the overexpression of karyopherin alpha2 did not significantly stimulate GLUT2 and liver pyruvate kinase mRNA accumulation in green fluorescent protein-sorted cells. The present study suggests that, in concert with glucose metabolism, karyopherin alpha2 transmits a signal to the nucleus to regulate glucose-sensitive gene expression. The transitory tethering of karyopherin alpha2 to GLUT2 at the plasma membrane might indicate that the receptor can load the cargo to be imported locally. PMID:11988093

  15. Karyopherin alpha2: a control step of glucose-sensitive gene expression in hepatic cells.

    PubMed

    Guillemain, Ghislaine; Muñoz-Alonso, Maria J; Cassany, Aurélia; Loizeau, Martine; Faussat, Anne-Marie; Burnol, Anne-Françoise; Leturque, Armelle

    2002-05-15

    Glucose is required for an efficient expression of the glucose transporter GLUT2 and other genes. We have shown previously that the intracytoplasmic loop of GLUT2 can divert a signal, resulting in the stimulation of glucose-sensitive gene transcription. In the present study, by interaction with the GLUT2 loop, we have cloned the rat karyopherin alpha2, a receptor involved in nuclear import. The specificity of the binding was restricted to GLUT2, and not GLUT1 or GLUT4, and to karyopherin alpha2, not alpha1. When rendered irreversible by a cross-linking agent, this transitory interaction was detected in vivo in hepatocytes. A role for karyopherin alpha2 in the transcription of two glucose-sensitive genes was investigated by transfection of native and inactive green fluorescent protein-karyopherin alpha2 in GLUT2-expressing hepatoma cells. The amount of inactive karyopherin alpha2 receptor reduced, in a dose-dependent manner, the GLUT2 and liver pyruvate kinase mRNA levels by competition with endogenous active receptor. In contrast, the overexpression of karyopherin alpha2 did not significantly stimulate GLUT2 and liver pyruvate kinase mRNA accumulation in green fluorescent protein-sorted cells. The present study suggests that, in concert with glucose metabolism, karyopherin alpha2 transmits a signal to the nucleus to regulate glucose-sensitive gene expression. The transitory tethering of karyopherin alpha2 to GLUT2 at the plasma membrane might indicate that the receptor can load the cargo to be imported locally.

  16. Overexpression of {alpha}-catenin increases osteoblastic differentiation in mouse mesenchymal C3H10T1/2 cells

    SciTech Connect

    Kim, Dohee; Yang, Jae-Yeon; Shin, Chan Soo

    2009-05-15

    {alpha}- and {beta}-Catenin link cadherins to the actin-based cytoskeleton at adherens junctions and regulate cell-cell adhesion. Although roles of cadherins and canonical Wnt-/{beta}-catenin-signaling in osteoblastic differentiation have been extensively studied, the role of {alpha}-catenin is not known. Murine embryonic mesenchymal stem cells, C3H10T1/2 cells, were transduced with retrovirus encoding {alpha}-catenin (MSCV-{alpha}-catenin-HA-GFP). In the presence of Wnt-3A conditioned medium or osteogenic medium ({beta}-glycerol phosphate and ascorbic acid), cells overexpressing {alpha}-catenin showed enhanced osteoblastic differentiation as measured by alkaline phosphatase (ALP) staining and ALP activity assay compared to cells transduced with empty virus (MSCV-GFP). In addition, mRNA expression of osteocalcin and Runx2 was significantly increased compared to control. Cell aggregation assay revealed that {alpha}-catenin overexpression has significantly increased cell-cell aggregation. However, cellular {beta}-catenin levels (total, cytoplasmic-nuclear ratio) and {beta}-catenin-TCF/LEF transcriptional activity did not change by overexpression of {alpha}-catenin. Knock-down of {alpha}-catenin using siRNA decreased osteoblastic differentiation as measured by ALP assay. These results suggest that {alpha}-catenin overexpression increases osteoblastic differentiation by increasing cell-cell adhesion rather than Wnt-/{beta}-catenin-signaling.

  17. Alpha-lactalbumin unfolding is not sufficient to cause apoptosis, but is required for the conversion to HAMLET (human alpha-lactalbumin made lethal to tumor cells).

    PubMed

    Svensson, Malin; Fast, Jonas; Mossberg, Ann-Kristin; Düringer, Caroline; Gustafsson, Lotta; Hallgren, Oskar; Brooks, Charles L; Berliner, Lawrence; Linse, Sara; Svanborg, Catharina

    2003-12-01

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a complex of human alpha-lactalbumin and oleic acid (C18:1:9 cis) that kills tumor cells by an apoptosis-like mechanism. Previous studies have shown that a conformational change is required to form HAMLET from alpha-lactalbumin, and that a partially unfolded conformation is maintained in the HAMLET complex. This study examined if unfolding of alpha-lactalbumin is sufficient to induce cell death. We used the bovine alpha-lactalbumin Ca(2+) site mutant D87A, which is unable to bind Ca(2+), and thus remains partially unfolded regardless of solvent conditions. The D87A mutant protein was found to be inactive in the apoptosis assay, but could readily be converted to a HAMLET-like complex in the presence of oleic acid. BAMLET (bovine alpha-lactalbumin made lethal to tumor cells) and D87A-BAMLET complexes were both able to kill tumor cells. This activity was independent of the Ca(2+)site, as HAMLET maintained a high affinity for Ca(2+) but D87A-BAMLET was active with no Ca(2+) bound. We conclude that partial unfolding of alpha-lactalbumin is necessary but not sufficient to trigger cell death, and that the activity of HAMLET is defined both by the protein and the lipid cofactor. Furthermore, a functional Ca(2+)-binding site is not required for conversion of alpha-lactalbumin to the active complex or to cause cell death. This suggests that the lipid cofactor stabilizes the altered fold without interfering with the Ca(2+)site.

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

    SciTech Connect

    Song, Xiaomin; Luo, Yongzhang

    2010-07-16

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

  19. Identification of cDNA encoding an additional. alpha. subunit of a human GTP-binding protein: Expression of three. alpha. sub i subtypes in human tissues and cell lines

    SciTech Connect

    Kim, S.; Ang, S.L.; Bloch, D.B.; Bloch, K.D.; Kawahara, Y.; Tolman, C.; Lee, R.; Seidman, J.G.; Neer, E.J. )

    1988-06-01

    The guanine nucleotide-binding proteins (G proteins), which mediate hormonal regulation of many membrane functions, are composed of {alpha}, {beta}, and {gamma} subunits. The authors have cloned and characterized cDNA from a human T-cell library encoding a form of {alpha}{sub i} that is different from the human {alpha}{sub i} subtypes previously reported. {alpha}{sub i} is the {alpha} subunit of a class of G proteins that inhibits adenylate cyclase and regulates other enzymes and ion channels. This cDNA encodes a polypeptide of 354 amino acids and is assigned to encode the {alpha}{sub i-3} subtype of G proteins on the basis of its similarity to other {alpha}{sub i}-like cDNAs and the presence of a predicted site for ADP ribosylation by pertussis toxin. They have determined the expression of mRNA for this and two other subtypes of human {alpha}{sub i} ({alpha}{sub i-1} and {alpha}{sub i-2}) in a variety of human fetal tissues and in human cell lines. All three {alpha}{sub i} subtypes were present in the tissues tested. However, analysis of individual cell types reveals specificity of {alpha}{sub i-1} expression. mRNA for {alpha}{i-1} is absent in T cells, B cells, and monocytes but is present in other cell lines. The finding of differential expression of {alpha}{sub i-1} genes may permit characterization of distinct physiological roles for this {alpha}{sub i} subunit. mRNA for {alpha}{sub i-2} and {alpha}{sub i-3} was found in all the primary and transformed cell lines tested. Thus, some cells contain all three {alpha}{sub i} subtypes. This observation raises the question of how cells prevent cross talk among receptors that are coupled to effectors through such similar {alpha} proteins.

  20. Endothelial Lu/BCAM glycoproteins are novel ligands for red blood cell alpha4beta1 integrin: role in adhesion of sickle red blood cells to endothelial cells.

    PubMed

    El Nemer, Wassim; Wautier, Marie-Paule; Rahuel, Cécile; Gane, Pierre; Hermand, Patricia; Galactéros, Frédéric; Wautier, Jean-Luc; Cartron, Jean-Pierre; Colin, Yves; Le Van Kim, Caroline

    2007-04-15

    The Lutheran (Lu) blood group and basal cell adhesion molecule (BCAM) antigens are both carried by 2 glycoprotein isoforms of the immunoglobulin superfamily representing receptors for the laminin alpha(5) chain. In addition to red blood cells, Lu/BCAM proteins are highly expressed in endothelial cells. Abnormal adhesion of red blood cells to the endothelium could potentially contribute to the vaso-occlusive episodes in sickle cell disease. Considering the presence of integrin consensus-binding sites in Lu/BCAM proteins, we investigated their potential interaction with integrin alpha(4)beta(1), the unique integrin expressed on immature circulating sickle red cells. Using cell adhesion assays under static and flow conditions, we demonstrated that integrin alpha(4)beta(1) expressed on transfected cells bound to chimeric Lu-Fc protein. We showed that epinephrine-stimulated sickle cells, but not control red cells, adhered to Lu-Fc via integrin alpha(4)beta(1) under flow conditions. Antibody-mediated activation of integrin alpha(4)beta(1) induced adhesion of sickle red cells to primary human umbilical vein endothelial cells; this adhesion was inhibited by soluble Lu-Fc and vascular cell adhesion molecule-1 (VCAM-1)-Fc proteins. This novel interaction between integrin alpha(4)beta(1) in sickle red cells and endothelial Lu/BCAM proteins could participate in sickle cell adhesion to endothelium and potentially play a role in vaso-occlusive episodes.

  1. Activation of phospholipase C by the alpha subunits of the Gq and G11 proteins in transfected Cos-7 cells.

    PubMed

    Wu, D Q; Lee, C H; Rhee, S G; Simon, M I

    1992-01-25

    High efficiency transient transfection was used to introduce cDNA corresponding to various G protein alpha subunits into Cos-7 cells. The proteins that were subsequently synthesized were detected with specific G protein alpha subunit antipeptide antiserum and were localized in the membrane fraction of the cell. Cells that were prelabeled with the [3H]inositol and transfected with G alpha q and G alpha 11 cDNA showed marked increases in formation of [3H]inositol phosphates after stimulation with aluminum fluoride. Co-transfection with cDNAs corresponding to phosphoinositide specific phospholipase C beta 1 (PI-PLC beta 1) and to G alpha q or G alpha 11 resulted in even higher levels of inositol phosphate formation. The introduction of mutations that convert residue glutamine 209 to leucine in G alpha q and G alpha 11 resulted in persistent activation of PI-PLC and high steady state levels of inositol phosphates. On the other hand, transfection with a variety of other G alpha subunit cDNAs, i.e. G alpha Z, G alpha OA, G alpha OB, transducin, and the glutamine 205 to leucine mutants of G alpha Z and of G alpha OA did not increase inositol phosphate formation. To further test the specificity of G protein activation of PI-PLC, a cell-free system was prepared by using washed membranes of transiently transfected cells and purified PI-PLC beta 1. Membranes derived from G alpha q and G alpha 11, but not G alpha OA transfected cells, showed guanosine 5-O-thiotriphosphate (GTP gamma S)-stimulated PIP2 hydrolysis. The activity seen in the system reconstituted with membranes derived from G alpha 11-transfected cells was blocked by preincubation with specific G alpha 11 antipeptide antibodies. All of these results are consistent with the conclusion that G alpha q and G alpha 11 cDNA encode proteins that in the presence of GTP gamma S specifically activate PI-PLC.

  2. Targeted inhibition of {alpha}v{beta}3 integrin with an RNA aptamer impairs endothelial cell growth and survival

    SciTech Connect

    Mi Jing; Zhang Xiuwu; Giangrande, Paloma H.; McNamara, James O.; Nimjee, Shahid M.; Sarraf-Yazdi, Shiva; Sullenger, Bruce A.; Clary, Bryan M. . E-mail: mi001@duke.edu

    2005-12-16

    {alpha}v{beta}3 integrin is a crucial factor involved in a variety of physiological processes, such as cell growth and migration, tumor invasion and metastasis, angiogenesis, and wound healing. {alpha}v{beta}3 integrin exerts its effect by regulating endothelial cell (EC) migration, proliferation, and survival. Inhibiting the function of {alpha}v{beta}3 integrin, therefore, represents a potential anti-cancer, anti-thrombotic, and anti-inflammatory strategy. In this study, we tested an RNA aptamer, Apt-{alpha}v{beta}3 that binds recombinant {alpha}v{beta}3 integrin, for its ability to bind endogenous {alpha}v{beta}3 integrin on the surface of cells in culture and to subsequently affect cellular response. Our data illustrate that Apt-{alpha}v{beta}3 binds {alpha}v{beta}3 integrin expressed on the surface of live HUVECs. This interaction significantly decreases both basal and PDGF-induced cell proliferation as well as inhibition of cell adhesion. Apt-{alpha}v{beta}3 can also reduce PDGF-stimulated tube formation and increase HUVEC apoptosis through inhibition of FAK phosphorylation pathway. Our results demonstrate that by binding to its target, Apt-{alpha}v{beta}3 can efficiently inhibit human EC proliferation and survival, resulting in reduced angiogenesis. It predicts that Apt-{alpha}v{beta}3 could become useful in both tumor imaging and the treatment of tumor growth, atherosclerosis, thrombosis, and inflammation.

  3. Identification of alpha 2-adrenergic receptor sites in human retinoblastoma (Y-79) and neuroblastoma (SH-SY5Y) cells

    SciTech Connect

    Kazmi, S.M.; Mishra, R.K.

    1989-02-15

    The existence of specific alpha 2-adrenergic receptor sites has been shown in human retinoblastoma (Y-79) and neuroblastoma (SH-SH5Y) cells using direct radioligand binding. (/sup 3/H)Rauwolscine, a selective alpha 2-adrenergic receptor antagonist, exhibited high affinity, saturable binding to both Y-79 and SH-SY5Y cell membranes. The binding of alpha 1 specific antagonist, (/sup 3/H)Prazocine, was not detectable in either cell type. Competition studies with antagonists yielded pharmacological characteristics typical of alpha 2-adrenergic receptors: rauwolscine greater than yohimbine greater than phentolamine greater than prazocine. Based on the affinity constants of prazocine and oxymetazoline, it appears that Y-79 cells contain alpha 2A receptor, whereas SH-SY5Y cells probably represent a mixture of alpha 2A and alpha 2B receptors. alpha 2-agonists clonidine and (-)epinephrine inhibition curves yielded high and low affinity states of the receptor in SH-SY5Y cells. Gpp(NH)p and sodium ions reduced the proportion of high affinity sites of alpha 2 receptors. These two neuronal cell lines of human origin would prove useful in elucidating the action and regulation of human alpha 2-adrenergic receptors and their interaction with other receptor systems.

  4. Production of transforming growth factor. cap alpha. in human pancreatic cancer cells: evidence for a superagonist autocrine cycle

    SciTech Connect

    Smith, J.J.; Derynck, R.; Korc, M.

    1987-11-01

    Previous work showed that cultured human pancreatic cancer cells overexpress the epidermal growth factor (EGF) receptor. In the present study, the authors sought to determine whether some of these cell lines produce transforming growth factor ..cap alpha.. (TGF-..cap alpha..). Utilizing a radiolabeled TGF-..cap alpha.. cDNA in hybridization experiments, they determined that ASPC-1, T/sub 3/M/sub 4/, PANC-1, COLO-357, and MIA PaCa-2 cell lines expressed TGF-..cap alpha.. mRNA. Serum-free medium conditioned by T/sub 3/M/sub 4/ and ASPC-1 cells contained significant amounts of TGF-..cap alpha.. protein. Although unlabeled TGF-..cap alpha.. readily competed with /sup 125/I-labeled EGF for binding, each cell line exhibited lower surface binding and internalization of /sup 125/I-labeled TGF-..cap alpha.. as compared to /sup 125/I-labeled EGF. Both TGF-..cap alpha.. and EGF significantly enhanced the anchorage-independent growth of PANC-1, T/sub 3/M/sub 4/, and ASPC-1 cells. However, TGF-..cap alpha.. was 10- to 100-fold more potent than EGF. These findings suggest that the concomitant overexpression of EGF receptors and production of TGF-..cap alpha.. may represent an efficient mechanism for certain cancer cells to obtain a growth advantage.

  5. PDGFBB promotes PDGFR{alpha}-positive cell migration into artificial bone in vivo

    SciTech Connect

    Yoshida, Shigeyuki; Iwasaki, Ryotaro; Kawana, Hiromasa; Miyauchi, Yoshiteru; Hoshi, Hiroko; Miyamoto, Hiroya; Mori, Tomoaki; Kanagawa, Hiroya; Katsuyama, Eri; Fujie, Atsuhiro; Hao, Wu; and others

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer We examined effects of PDGFBB in PDGFR{alpha} positive cell migration in artificial bones. Black-Right-Pointing-Pointer PDGFBB was not expressed in osteoblastic cells but was expressed in peripheral blood cells. Black-Right-Pointing-Pointer PDGFBB promoted PDGFR{alpha} positive cell migration into artificial bones but not osteoblast proliferation. Black-Right-Pointing-Pointer PDGFBB did not inhibit osteoblastogenesis. -- Abstract: Bone defects caused by traumatic bone loss or tumor dissection are now treated with auto- or allo-bone graft, and also occasionally by artificial bone transplantation, particularly in the case of large bone defects. However, artificial bones often exhibit poor affinity to host bones followed by bony union failure. Thus therapies combining artificial bones with growth factors have been sought. Here we report that platelet derived growth factor bb (PDGFBB) promotes a significant increase in migration of PDGF receptor {alpha} (PDGFR{alpha})-positive mesenchymal stem cells/pre-osteoblastic cells into artificial bone in vivo. Growth factors such as transforming growth factor beta (TGF{beta}) and hepatocyte growth factor (HGF) reportedly inhibit osteoblast differentiation; however, PDGFBB did not exhibit such inhibitory effects and in fact stimulated osteoblast differentiation in vitro, suggesting that combining artificial bones with PDGFBB treatment could promote host cell migration into artificial bones without inhibiting osteoblastogenesis.

  6. Association of alpha interferon production with natural killer cell lysis of U937 cells infected with human immunodeficiency virus.

    PubMed Central

    Rappocciolo, G; Toso, J F; Torpey, D J; Gupta, P; Rinaldo, C R

    1989-01-01

    Mononuclear leukocytes from human immunodeficiency virus (HIV)-seronegative and -seropositive homosexual men lysed HIV-infected U937 cells to a significantly greater degree than uninfected U937 cells. Depletion of cell subsets with monoclonal antibodies and complement indicated that the effector cells were primarily of the CD16+ phenotype. Acid-stable alpha interferon (IFN-alpha) production induced by the HIV-infected cells correlated with, although was not an absolute requisite for, preferential lysis of the infected targets. The activity of these CD16+, natural killer (NK) cells decreased in relation to the duration of HIV infection and the presence of acquired immunodeficiency syndrome. Pretreatment of peripheral blood mononuclear cells from HIV-seronegative subjects, but not HIV-seropositive men, with IFN-alpha or recombinant interleukin-2 enhanced lysis of both uninfected and HIV-infected U937 cells. These results suggest that IFN-alpha-associated, NK-like mechanisms are active in the cytotoxic response against HIV-infected cells and that HIV infection results in an early and progressive depression of such responses. Prospective investigations may be useful in determining the role of this NK cell response in the natural history and pathogenesis of HIV infection and the efficacy of therapeutic modalities. PMID:2913035

  7. Polarization of yeast cells in spatial gradients of alpha mating factor.

    PubMed Central

    Segall, J E

    1993-01-01

    The process of cell fusion during mating of the yeast Saccharomyces cerevisiae is mediated by factors secreted by the mating partners. Spatial gradients of one of these mating factors, alpha-factor, polarized the growth of projections by MATa cells. The site of previous budding did not affect the direction of polarization, and subsequent budding was also polarized if mating factor was removed. Orientation occurred in the presence of nocodazole, suggesting that microtubules were not critical. At extremely low concentrations of alpha-factor, sst2-mutants (which in genetic studies do not discriminate between partners producing different amounts of alpha-factor) were able to polarize their projections. The sensitivity of this spatial sensing mechanism in wild-type cells is such that differences in receptor occupancy estimated to be about 1% are sufficient for orientation. Images Fig. 1 Fig. 2 Fig. 3 PMID:8397402

  8. The outline structure of the T-cell alpha beta receptor.

    PubMed Central

    Chothia, C; Boswell, D R; Lesk, A M

    1988-01-01

    From an analysis of the immunoglobulins of known structure we derive a list of 40 sites crucial for the conserved structure of the variable domains. We show that, with marginal exceptions, the sequences of the T-cell alpha beta receptors contain, at sites homologous to these 40, the same or very similar residues. Thus the V alpha-V beta dimer has a framework structure very close to that of the immunoglobulins. Further comparisons show that parts of the surface of the V alpha-V beta framework are hypervariable. They also show that the loops that form the antigen-binding site are similar in size to those commonly found in the immunoglobulins but have different conformations. Only limited sequence variations occur in the first loop of the antigen-binding site in both V alpha and V beta. This, and their geometrical arrangement, suggest that they mainly interact with the MHC proteins. PMID:3208747

  9. Human epidermal T cells predominantly belong to the lineage expressing alpha/beta T cell receptor

    PubMed Central

    1990-01-01

    The epidermis of clinically normal-appearing human skin harbors a phenotypically heterogeneous population of T lymphocytes (TCs), the majority of which are CD2+/CD3+/CD5+ "memory" cells, but in an unactivated state, and express the TCR-alpha/beta. In contrast to murine skin, only a very minor subpopulation of CD3+ cells in the human epidermis bears the TCR-gamma/delta. Epidermal TCs primarily are distributed along the rete ridges in the basal keratinocyte layer and are often in close apposition to Langerhans cells (LCs). These TCs were propagated from epidermal cell suspensions after stimulation with TC activating agents (Con A, rIL-1, rIL-2), then evaluated for phenotypic features and TCR diversity. Similar to the in situ situation, most were CD4-/CD8+/TCR-alpha/beta+. In addition, two cultures contained TCR- gamma/delta+ cells; one of these determined to be an adherent CD4-/CD8+ population. Epidermal TCs were significantly (p less than 0.0001) more abundant in the sole than in the other body regions examined (i.e., 40 vs. 7 CD3+ cells/linear centimeter of epidermis) and seemed to have a particular affinity for the acrosyringial epithelium of eccrine sweat ducts. Moreover, the sole usually contained a greater number of CD8+ relative to CD4+ TCs, whereas the epidermal CD4/CD8 ratio in the trunk and extremities was quite variable, although the trend also was towards a slightly larger percentage of CD8+ cells. Collectively, our data suggest that the volar epidermis has a unique microenvironment which is responsible for both the higher density of TCs, preferentially CD8+, and lower number of LCs. This study has not only provided evidence for significant regional variability in the human epidermal TC population of normal skin, but also strengthens the concept for skin-associated lymphoid tissues (SALT), whereby memory TCs recirculate back to the epidermis and interact with resident antigen-presenting cells (i.e., LC). PMID:2182763

  10. Inhibition of TNF-alpha induced cell death in human umbilical vein endothelial cells and Jurkat cells by protocatechuic acid.

    PubMed

    Zhou-Stache, J; Buettner, R; Artmann, G; Mittermayer, C; Bosserhoff, A K

    2002-11-01

    The Chinese herb radix Salviae miltiorrhizae (RSM) is used in traditional Chinese medicine as a treatment for cardiovascular and cerebrovascular diseases. Several components of the plant extract from salvia mitorrhiza bunge have been determined previously, one of which is protocatechuic acid (PAC). It has been found, in the study, that PAC inhibited TNF-alpha-induced cell death of human umbilical vein endothelial cells (HUVECs) and Jurkat cells in a concentration of 100 microM when applied 2 h prior to TNF-alpha exposure. Molecular studies revealed that PAC activated NF-kappaB with a maximum effect after 30 min of treatment. Inhibition of NF-kappaB action by MG132 and NF-kappaB inhibitory peptide suppressed the cell-protective effect of PAC. Further, degradation of IkBalpha occurred in response to PAC treatment. The results provide evidence that activation of NF-kappaB plays an important role in mediating the cell-protecting effect of PAC on HUVECs and Jurkat cells. Further studies are required to test whether PAC, a component of radix salviae miltiorrhizae, could be useful in preventing in vivo cell death resulting from cardiovascular or cerebrovascular diseases.

  11. Cell shape and interaction defects in alpha-spectrin mutants of Drosophila melanogaster

    PubMed Central

    1993-01-01

    We show that the alpha-spectrin gene is essential for larval survival and development by characterizing several alpha-spectrin mutations in Drosophila. P-element minigene rescue and sequence analysis were used to identify the alpha-spectrin gene as the l(3)dre3 complementation group of the Dras-Roughened-ecdysoneless region of chromosome 3 (Sliter et al., 1988). Germ line transformants carrying an alpha-spectrin cDNA, whose expression is driven by the ubiquitin promoter, fully rescued the first to second instar lethality characteristic of the l(3)dre3 alleles. The molecular defects in two gamma-ray-induced alleles were identified. One of these mutations, which resulted in second instar lethality, contained a 73-bp deletion in alpha-spectrin segment 22 (starting at amino acid residue 2312), producing a premature stop codon between the two EF hands found in this segment. The second mutation, which resulted in first instar lethality, contained a 20 base pair deletion in the middle of segment 1 (at amino acid residue 92), resulting in a premature stop codon. Examination of the spectrin- deficient larvae revealed a loss of contact between epithelial cells of the gut and disruption of cell-substratum interactions. The most pronounced morphological change was seen in tissues of complex cellular architecture such as the middle midgut where a loss of cell contact between cup-shaped cuprophilic cells and neighboring interstitial cells was accompanied by disorganization of the cuprophilic cell brush borders. Our examination of spectrin deficient larvae suggests that an important role of non-erythroid spectrin is to stabilize cell to cell interactions that are critical for the maintenance of cell shape and subcellular organization within tissues. PMID:8276898

  12. Fibroblast activation protein-alpha and dipeptidyl peptidase IV (CD26): cell-surface proteases that activate cell signaling and are potential targets for cancer therapy.

    PubMed

    Kelly, Thomas

    2005-01-01

    Fibroblast activation protein-alpha (FAP-alpha) and dipeptidyl peptidase IV (DPPIV) are serine proteases with post-prolyl peptidase activities that can modify tumor cell behavior. FAP-alpha and DPPIV can form heteromeric complexes with each other and may function coordinately to modulate the growth, differentiation, adhesion, and metastasis of tumor cells. This review is focused on FAP-alpha and summarizes a series of studies showing that elevated expression of FAP-alpha results in profound changes in growth and malignant behavior of tumor cells. Depending on the model system investigated, FAP-alpha expression causes dramatic promotion or suppression of tumor growth. In the case of tumor promotion, FAP-alpha expression can drive tumor growth by increasing angiogenesis and by decreasing the anti-tumor response of the immune system. In the case of tumor suppression, FAP-alpha can decrease tumorigenicity of mouse melanoma cells and restore contact inhibition and growth factor dependence even when it is catalytically inactive, implying that protein-protein interactions mediate these effects. Understanding how FAP-alpha activates cell signaling is critical to determining how FAP-alpha mediates growth promotion versus growth suppression in the different model systems and ultimately in human cancer patients. In particular, the roles of FAP-alpha protease activity and FAP-alpha complex formation with DPPIV and other surface molecules in activating cell signaling need to be elucidated since these represent potential targets for therapeutic intervention.

  13. Alpha-irradiation-induced G2 delay: a period of cell recovery

    SciTech Connect

    Lucke-Huhle, C.

    1982-02-01

    Exponentially growing Chinese hamster V79 cells were delayed in G2 very efficiently by 3.4-MeV ..cap alpha.. particles. In comparison with the effect caused by sparsely ionizing /sup 60/Co ..gamma.. rays, G2 delay after ..cap alpha.. irradiation was greater by a factor of 6.7 and 4.2 for doses <0.5 Gy and >0.5 Gy, respectively, if the slopes of the dose-effect curves are compared. While at low doses (0.03-0.5 Gy) G2 arrest was reversible within 10 hr, increasing doses (0.5-4.38 Gy) of ..cap alpha.. irradiation blocked increasing fractions of cells for more than 16 hr, as determined by flow cytometry, and only some of these were able to complete mitosis. Addition of caffeine, however, reduced G2 arrest considerably if given directly after irradiation and reversed G2 arrest if added 8 hr after 4.38 Gy of ..cap alpha.. particles, a time when most of the cells already had accumulated in G2, caffeine treatment during G2 decreased survival after ..cap alpha.. irradiation by factors of 1.3 and 1.7 for 1 and 2 mM caffeine, respectively.

  14. Dehydroepiandrosterone inhibits the TNF-alpha-induced inflammatory response in human umbilical vein endothelial cells.

    PubMed

    Gutiérrez, Gisela; Mendoza, Criselda; Zapata, Estrella; Montiel, Angélica; Reyes, Elba; Montaño, Luis Felipe; López-Marure, Rebeca

    2007-01-01

    Dehydroepiandrosterone (DHEA) has a protective role against atherosclerosis. We determined the effect of pharmacological doses of DHEA upon the adhesion of monocytic U937 cells to human umbilical vein endothelial cells (HUVEC), as well as the expression of adhesion and chemoattractant molecules, the translocation of NF-kappaB, the degradation of IkappaB-alpha and the production of reactive oxygen species (ROS) in HUVEC. Adhesion of U937 cells to DHEA-treated HUVEC was evaluated by co-culture experiments using [(3)H]-thymidine-labeled U937 cells. The expression of adhesion and chemoattractant molecules was evaluated by flow cytometry and RT-PCR, respectively; NF-kappaB translocation was determined by Electrophoretic Mobility Shift Assay (EMSA) and IkappaB-alpha degradation by Western blot. ROS production was determined by the reduction of fluorescent DCFDA. TNF-alpha was used to induce inflammatory responses in HUVEC. One hundred micromolar of DHEA-treatment inhibited the TNF-alpha-induced expression of ICAM-1, E-selectin, ROS production and U937 cells adhesion to HUVEC, and interfered with NF-kappaB translocation and IkappaB-alpha degradation. DHEA at the above mention concentration also inhibited the mRNA expression of MCP-1 and IL-8 in basal conditions but not in TNF-alpha-stimulated conditions. Our results suggest that DHEA inhibits the expression of molecules involved in the inflammatory process, therefore it could be used as an alternative in the treatment of chronic inflammatory diseases such as atherosclerosis.

  15. Prediction of Lung Cells Oncogenic Transformation for Induced Radon Progeny Alpha Particles Using Sugarscape Cellular Automata

    PubMed Central

    Baradaran, Samaneh; Maleknasr, Niaz; Setayeshi, Saeed; Akbari, Mohammad Esmaeil

    2014-01-01

    Background Alpha particle irradiation from radon progeny is one of the major natural sources of effective dose in the public population. Oncogenic transformation is a biological effectiveness of radon progeny alpha particle hits. The biological effects which has caused by exposure to radon, were the main result of a complex series of physical, chemical, biological and physiological interactions. The cellular and molecular mechanisms for radon-induced carcinogenesis have not been clear yet. Methods Various biological models, including cultured cells and animals, have been found useful for studying the carcinogenesis effects of radon progeny alpha particles. In this paper, sugars cape cellular automata have been presented for computational study of complex biological effect of radon progeny alpha particles in lung bronchial airways. The model has included mechanism of DNA damage, which has been induced alpha particles hits, and then formation of transformation in the lung cells. Biomarkers were an objective measure or evaluation of normal or abnormal biological processes. In the model, the metabolism rate of infected cell has been induced alpha particles traversals, as a biomarker, has been followed to reach oncogenic transformation. Results The model results have successfully validated in comparison with “in vitro oncogenic transformation data” for C3H 10T1/2 cells. This model has provided an opportunity to study the cellular and molecular changes, at the various stages in radiation carcinogenesis, involving human cells. Conclusion It has become well known that simulation could be used to investigate complex biomedical systems, in situations where traditional methodologies were difficult or too costly to employ. PMID:25250147

  16. Omentin inhibits TNF-{alpha}-induced expression of adhesion molecules in endothelial cells via ERK/NF-{kappa}B pathway

    SciTech Connect

    Zhong, Xia; Li, Xiaonan; Liu, Fuli; Tan, Hui; Shang, Deya

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer Omentin inhibited TNF-{alpha}-induced adhesion of THP-1 cells to HUVECs. Black-Right-Pointing-Pointer Omentin reduces expression of ICAM-1 and VCAM-1 induced by TNF-{alpha} in HUVECs. Black-Right-Pointing-Pointer Omentin inhibits TNF-{alpha}-induced ERK and NF-{kappa}B activation in HUVECs. Black-Right-Pointing-Pointer Omentin supreeses TNF-{alpha}-induced expression of ICAM-1 and VCAM-1 via ERK/NF-{kappa}B pathway. -- Abstract: In the present study, we investigated whether omentin affected the expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in tumor necrosis factor-{alpha} (TNF-{alpha}) induced human umbilical vein endothelial cells (HUVECs). Our data showed that omentin decreased TNF-{alpha}-induced expression of ICAM-1 and VCAM-1 in HUVECs. In addition, omentin inhibited TNF-{alpha}-induced adhesion of THP-1 cells to HUVECs. Further, we found that omentin inhibited TNF-{alpha}-activated signal pathway of nuclear factor-{kappa}B (NF-{kappa}B) by preventing NF-{kappa}B inhibitory protein (I{kappa}B{alpha}) degradation and NF-{kappa}B/DNA binding activity. Omentin pretreatment significantly inhibited TNF-{alpha}-induced ERK activity and ERK phosphorylation in HUVECs. Pretreatment with PD98059 suppressed TNF-{alpha}-induced NF-{kappa}B activity. Omentin, NF-kB inhibitor (BAY11-7082) and ERK inhibitor (PD98059) reduced the up-regulation of ICAM-1 and VCAM-1 induced by TNF-{alpha}. These results suggest that omentin may inhibit TNF-{alpha}-induced expression of adhesion molecules in endothelial cells via blocking ERK/NF-{kappa}B pathway.

  17. Inhibition of cell surface mediated plasminogen activation by a monoclonal antibody against alpha-Enolase.

    PubMed

    López-Alemany, Roser; Longstaff, Colin; Hawley, Stephen; Mirshahi, Massoud; Fábregas, Pere; Jardí, Merce; Merton, Elizabeth; Miles, Lindsey A; Félez, Jordi

    2003-04-01

    Localization of plasmin activity on leukocyte surfaces plays a critical role in fibrinolysis as well as in pathological and physiological processes in which cells must degrade the extracellular matrix in order to migrate. The binding of plasminogen to leukocytic cell lines induces a 30- to 80-fold increase in the rate of plasminogen activation by tissue-type (tPA) and urokinase-type (uPA) plasminogen activators. In the present study we have examined the role of alpha-enolase in plasminogen activation on the cell surface. We produced and characterized a monoclonal antibody (MAb) 11G1 against purified alpha-enolase, which abrogated about 90% of cell-dependent plasminogen activation by either uPA or tPA on leukocytoid cell lines of different lineages: B-lymphocytic, T-lymphocytic, granulocytic, and monocytic cells. In addition, MAb 11G1 also blocked enhancement of plasmin formation by peripheral blood neutrophils and monocytes. In contrast, MAb 11G1 did not affect plasmin generation in the presence of fibrin, indicating that this antibody did not interact with fibrinolytic components in the absence of cells. These data suggest that, although leukocytic cells display several molecules that bind plasminogen, alpha-enolase is responsible for the majority of the promotion of plasminogen activation on the surfaces of leukocytic cells.

  18. Genetic modification of alphaGal expression in xenogeneic endothelial cells yields a complex immunological response.

    PubMed

    Fischbeck, J A; Baier, J M; Akella, R; Hern-Anderson, D; Schmidt, C E

    2001-12-01

    The source of cells for tissue engineering applications remains a hurdle, predominantly for procedures in which there is insufficient time to harvest a patient's own cells. Animal cells are readily available, but undergo immune rejection. Rejection of animal (i.e., xenogeneic) tissue involves practically every component of the immune system. The initial phase, hyperacute rejection (HAR), involves natural xenoreactive antibodies and the complement system, and leads to endothelial cell lysis and rapid tissue destruction. The cell-surface epitope, galactose-alpha(1,3)-galactose (alphaGal), is presumed to play a key role in HAR. The later stage of immune response (delayed xenograft rejection or DXR), is mediated by immune cells such as monocytes. Carbohydrates are likely also involved in DXR, but their role in this phase of the immune response is less clear. A better understanding of all stages of xenogeneic immune rejection may make it feasible to create cell lines that are immune tolerant. In these studies, we have genetically modified bovine endothelial cells to study the roles of carbohydrates in immune rejection. Our studies suggest that one or more epitopes other than alphaGal may influence complement-mediated lysis. Furthermore, antibodies, as instigators in the complement response, and monocytes appear to recognize different cell surface epitopes.

  19. Integrin alpha chains exhibit distinct temporal and spatial localization patterns in epithelial cells of the Drosophila ovary.

    PubMed

    Dinkins, Michael B; Fratto, Victoria M; Lemosy, Ellen K

    2008-12-01

    Integrins are heterodimeric transmembrane receptors that modulate cell adhesion, migration, and signaling. Multiple integrin chains contribute to development and morphogenesis of a given tissue. Here, we analyze the expression of Drosophila integrin alpha chains in the ovarian follicular epithelium, a model for tissue morphogenesis and cell migration. We find expression throughout development of the beta chain, betaPS. Alpha chains, however, exhibit both spatial and temporal expression differences. alphaPS1 and alphaPS2 integrins are detected during early and mid-oogenesis on apical, lateral, and basal membranes with the betaPS chain, whereas alphaPS3-family integrins (alphaPS3, alphaPS4, alphaPS5) are expressed in anterior cells late in oogenesis. Surprisingly, we find that alphaPS3-family integrins are dispensable for dorsal appendage morphogenesis but play a role in the final length of the egg, suggesting redundant functions of integrins in a simple tissue. We also demonstrate roles for alphaPS3betaPS integrin in border cell migration and in stretch cells.

  20. Tumor necrosis factor-{alpha} enhanced fusions between oral squamous cell carcinoma cells and endothelial cells via VCAM-1/VLA-4 pathway

    SciTech Connect

    Song, Kai; Zhu, Fei; Zhang, Han-zhong; Shang, Zheng-jun

    2012-08-15

    Fusion between cancer cells and host cells, including endothelial cells, may strongly modulate the biological behavior of tumors. However, no one is sure about the driving factors and underlying mechanism involved in such fusion. We hypothesized in this study that inflammation, one of the main characteristics in tumor microenvironment, serves as a prominent catalyst for fusion events. Our results showed that oral cancer cells can fuse spontaneously with endothelial cells in co-culture and inflammatory cytokine tumor necrosis factor-{alpha} (TNF-{alpha}) increased fusion of human umbilical vein endothelium cells and oral cancer cells by up to 3-fold in vitro. Additionally, human oral squamous cell carcinoma cell lines and 35 out of 50 (70%) oral squamous carcinoma specimens express VLA-4, an integrin, previously implicated in fusions between human peripheral blood CD34-positive cells and murine cardiomyocytes. Expression of VCAM-1, a ligand for VLA-4, was evident on vascular endothelium of oral squamous cell carcinoma. Moreover, immunocytochemistry and flow cytometry analysis revealed that expression of VCAM-1 increased obviously in TNF-{alpha}-stimulated endothelial cells. Anti-VLA-4 or anti-VCAM-1 treatment can decrease significantly cancer-endothelial adhesion and block such fusion. Collectively, our results suggested that TNF-{alpha} could enhance cancer-endothelial cell adhesion and fusion through VCAM-1/VLA-4 pathway. This study provides insights into regulatory mechanism of cancer-endothelial cell fusion, and has important implications for the development of novel therapeutic strategies for prevention of metastasis. -- Highlights: Black-Right-Pointing-Pointer Spontaneous oral cancer-endothelial cell fusion. Black-Right-Pointing-Pointer TNF-{alpha} enhanced cell fusions. Black-Right-Pointing-Pointer VCAM-1/VLA-4 expressed in oral cancer. Black-Right-Pointing-Pointer TNF-{alpha} increased expression of VCAM-1 on endothelial cells. Black

  1. Differential use of the regulatory elements of the alpha B-crystallin enhancer in cultured murine lung (MLg), lens (alpha TN4-1) and muscle (C2C12) cells.

    PubMed

    Haynes, J I; Gopal-Srivastava, R; Frederikse, P H; Piatigorsky, J

    1995-04-03

    The mouse alpha B-crystallin-encoding gene (alpha B-cry) is highly expressed in the lens and expressed to lesser extents in other tissues. Here, we investigated alpha B-cry expression in mouse-lung-derived MLg cells. Two sizes of MLg alpha B-cry transcripts comigrated with alpha B-cry transcripts contained in total and poly(A)+RNA from mouse lung, with preference for the larger species in the MLg cells. Expression of both alpha B-cry promoter/cat reporter gene constructs and alpha B-cry enhancer (nt -427/-259)/herpes simplex virus (HSV) thymidine kinase promoter (ptk)/human growth hormone reporter gene (hGH) constructs was studied in transfected MLg cells and the results compared with those obtained from alpha TN4-1 lens and C2C12 muscle cells. The alpha B-cry enhancer increased activity of the endogenous and tk promoters approx. 2-fold in the MLg cells, in contrast to its 3-7-fold effect in alpha TN4-1 cells and 17-20-fold effect in C2C12 myotubes. Site-specific mutagenesis of the previously identified enhancer control elements, alpha B-E-1 (nt -407 to -397), alpha BE-2 (-360 to -327) and MRF (-300 to -288), decreased enhancer strength in transfected MLg cells. DNase I footprinting showed that MLg nuclear proteins occupy only alpha BE-1 and alpha BE-2. Previous data have shown that lens cells use alpha BE-1, alpha BE-2 and alpha BE-3, while muscle cells use, in addition, the muscle regulatory factor-binding site (MRF). Thus, the present experiments correlate tissue-specific enhancer strength and the number of control elements utilized.

  2. Genomic organization of the human T-cell antigen-receptor alpha/delta locus.

    PubMed

    Satyanarayana, K; Hata, S; Devlin, P; Roncarolo, M G; De Vries, J E; Spits, H; Strominger, J L; Krangel, M S

    1988-11-01

    Two clusters of overlapping cosmid clones comprising about 100 kilobases (kb) at the human T-cell antigen-receptor alpha/delta locus were isolated from a genomic library. The structure of the germ-line V delta 1 variable gene segment was determined. V delta 1 is located 8.5 kb downstream of the V alpha 13.1 gene segment, and both V segments are arranged in the same transcriptional orientation. The V alpha 17.1 segment is located between V delta 1 and the D delta, J delta, C delta region (containing the diversity, joining, and constant gene segments). Thus, V delta and V alpha segments are interspersed along the chromosome. The germ-line organization of the D delta 2, J delta 1, and J delta 2 segments was determined. Linkage of C delta to the J alpha region was established by identification of J alpha segments within 20 kb downstream of C delta. The organization of the locus was also analyzed by field-inversion gel electrophoresis. The unrearranged V delta 1 and D delta, J delta, C delta regions are quite distant from each other, apparently separated by a minimum of 175-180 kb.

  3. Increased antiviral activity of microscale-purified HuIFN alpha 8 (human interferon alpha 8) over HuIFN alpha 2b in Hep-2 cells challenged with Mengo virus.

    PubMed

    García, Julio César Sánchez; Ariza, Alejandro Miranda; Lasa, Alexis Musacchio; González, Luis Javier; Perez, Vladimir Besada

    2007-11-01

    Human proteins are not routinely expressed at high levels in Escherichia coli for, among other reasons, different codon usage. Several purification procedures have been applied to recover recombinant proteins for further biological characterization. However, the vast majority involve costly chromatography procedures. In the present study, both (Hu)IFN(alpha 2b) (human interferon alpha 2b) and (Hu)IFN(alpha 8) were expressed efficiently in E. coli BL21-codonplus-RIL. Subsequently, both recombinant proteins were purified to homogeneity by passive elution from reverse-stained SDS/PAGE gels, a cost-effective purification procedure. After purification, both recovered proteins were biologically active. The (Hu)IFN(alpha 8) subtype induced 1.46-fold more antiviral activity than (Hu)IFN(alpha 2b) using Hep-2 human laryngeal carcinoma cell challenged with Mengo virus.

  4. TNF-{alpha} promotes cell survival through stimulation of K{sup +} channel and NF{kappa}B activity in corneal epithelial cells

    SciTech Connect

    Wang Ling; Reinach, Peter; Lu, Luo . E-mail: lluou@ucla.edu

    2005-11-15

    Tumor necrosis factor (TNF-{alpha}) in various cell types induces either cell death or mitogenesis through different signaling pathways. In the present study, we determined in human corneal epithelial cells how TNF-{alpha} also promotes cell survival. Human corneal epithelial (HCE) cells were cultured in DMEM/F-12 medium containing 10% FBS. TNF-{alpha} stimulation induced activation of a voltage-gated K{sup +} channel detected by measuring single channel activity using patch clamp techniques. The effect of TNF-{alpha} on downstream events included NF{kappa}B nuclear translocation and increases in DNA binding activities, but did not elicit ERK, JNK, or p38 limb signaling activation. TNF-{alpha} induced increases in p21 expression resulting in partial cell cycle attenuation in the G{sub 1} phase. Cell cycle progression was also mapped by flow cytometer analysis. Blockade of TNF-{alpha}-induced K{sup +} channel activity effectively prevented NF{kappa}B nuclear translocation and binding to DNA, diminishing the cell-survival protective effect of TNF-{alpha}. In conclusion, TNF-{alpha} promotes survival of HCE cells through sequential stimulation of K{sup +} channel and NF{kappa}B activities. This response to TNF-{alpha} is dependent on stimulating K{sup +} channel activity because following suppression of K{sup +} channel activity TNF-{alpha} failed to activate NF{kappa}B nuclear translocation and binding to nuclear DNA.

  5. Different patterns of 5{alpha}-reductase expression, cellular distribution, and testosterone metabolism in human follicular dermal papilla cells

    SciTech Connect

    Liu, Shicheng Yamauchi, Hitoshi

    2008-04-18

    Androgens regulate hair growth, and 5{alpha}-reductase (5{alpha}R) plays a pivotal role in the action of androgens on target organs. To clarify the molecular mechanisms responsible for controlling hair growth, the present study presents evidence that the human follicular dermal papilla cells (DPCs) from either beard (bDPCs) or scalp hair (sDPCs) possess endogenous 5{alpha}R activity. Real-time RT-PCR revealed that the highest level of 5{alpha}R1 mRNA was found in bDPCs, followed by sDPCs, and a low but detectable level of 5{alpha}R1 mRNA was observed in fibroblasts. Minimally detectable levels of 5{alpha}R2 mRNA were found in all three cell types. A weak band at 26 kDa corresponding to the human 5{alpha}R1 protein was detected by Western blot in both DPCs, but not in fibroblasts. Immuonofluorescence analysis confirmed that 5{alpha}R1 was localized to the cytoplasm rather than in the nuclei in both DPCs Furthermore, a 5{alpha}R assay using [{sup 14}C]testosterone labeling in intact cells revealed that testosterone was transformed primarily into androstenedione, and in small amounts, into DHT. Our results demonstrate that the 5{alpha}R activities of either bDPCs or sDPCs are stronger than that of dermal fibroblasts, despite the fact that the major steroidogenic activity is attributed to 17{beta}-HSD rather than 5{alpha}R among the three cell types. The 5{alpha}R1 inhibitor MK386 exhibited a more potent inhibitory effect on 5{alpha}R activity than finasteride (5{alpha}R2 inhibitor) in bDPCs.

  6. Telomere attrition in beta and alpha cells with age.

    PubMed

    Tamura, Yoshiaki; Izumiyama-Shimomura, Naotaka; Kimbara, Yoshiyuki; Nakamura, Ken-Ichi; Ishikawa, Naoshi; Aida, Junko; Chiba, Yuko; Matsuda, Yoko; Mori, Seijiro; Arai, Tomio; Fujiwara, Mutsunori; Poon, Steven S S; Ishizaki, Tatsuro; Araki, Atsushi; Takubo, Kaiyo; Ito, Hideki

    2016-06-01

    We have reported telomere attrition in β and α cells of the pancreas in elderly patients with type 2 diabetes, but it has not been explored how the telomere lengths of these islet cells change according to age in normal subjects. To examine the telomere lengths of β and α cells in individuals without diabetes across a wide range of ages, we conducted measurement of the telomere lengths of human pancreatic β and α cells obtained from 104 autopsied subjects without diabetes ranging in age from 0 to 100 years. As an index of telomere lengths, the normalized telomere-centromere ratio (NTCR) was determined for β (NTCRβ) and α (NTCRα) cells by quantitative fluorescence in situ hybridization (Q-FISH). We found NTCRβ and NTCRα showed almost the same levels and both decreased according to age (p < 0.001 for both). NTCRs decreased more rapidly with age and were more widely distributed (p = 0.036 for NTCRβ, p < 0.001 for NTCRα) in subjects under 18 years of age than in subjects over 18 years. There was a positive correlation between NTCRβ and NTCRα only among adult subjects (p < 0.001). In conclusion, the telomeres of β and α cells become shortened with normal aging process.

  7. The primary structure of the alpha 4 subunit of VLA-4: homology to other integrins and a possible cell-cell adhesion function.

    PubMed Central

    Takada, Y; Elices, M J; Crouse, C; Hemler, M E

    1989-01-01

    VLA-4 is a cell surface heterodimer in the integrin superfamily of adhesion receptors. Anti-VLA-4 antibodies inhibited cytolytic T cell activity, with inhibitory activity directed against the effector T cells rather than their targets. Thus, whereas other VLA receptors appear to mediate cell--matrix interactions, VLA-4 may have a cell--cell adhesion function. To facilitate comparative studies of VLA-4 and other integrins, cDNA clones for the human alpha 4 subunit of VLA-4 were selected and then sequenced. The 3805 bp sequence encoded for 999 amino acids, with an N-terminus identical to that previously obtained from direct sequencing of purified alpha 4 protein. The alpha 4 amino acid sequence was 17-24% similar to other integrin alpha chains with known sequences. Parts of the alpha 4 sequence most conserved in other alpha chains include (i) the positions of 19/24 cysteine residues, (ii) three potential divalent cation binding sites of the general structure DXDXDGXXD and (iii) the transmembrane region. However, alpha 4 stands apart from all other known integrin alpha subunit sequences because (i) alpha 4 has neither an inserted I-domain, nor a disulfide-linked C-terminal fragment, (ii) its sequence is the most unique and (iii) only alpha 4 has a potential protease cleavage site, near the middle of the coding region, which appears responsible for the characteristic 80,000 and 70,000 Mr fragments of alpha 4. Images PMID:2788572

  8. Erythropoietin protects myocardin-expressing cardiac stem cells against cytotoxicity of tumor necrosis factor-{alpha}

    SciTech Connect

    Madonna, Rosalinda; Shelat, Harnath; Xue, Qun; Willerson, James T.; De Caterina, Raffaele; Geng, Yong-Jian

    2009-10-15

    Cardiac stem cells are vulnerable to inflammation caused by infarction or ischemic injury. The growth factor, erythropoietin (Epo), ameliorates the inflammatory response of the myocardium to ischemic injury. This study was designed to assess the role of Epo in regulation of expression and activation of the cell death-associated intracellular signaling components in cardiac myoblasts stimulated with the proinflammatory cytokine tumor necrosis factor (TNF)-{alpha}. Cardiac myoblasts isolated from canine embryonic hearts characterized by expression of myocardin A, a promyogenic transcription factor for cardiovascular muscle development were pretreated with Epo and then exposed to TNF-{alpha}. Compared to untreated cells, the Epo-treated cardiac myoblasts exhibited better morphology and viability. Immunoblotting revealed lower levels of active caspase-3 and reductions in iNOS expression and NO production in Epo-treated cells. Furthermore, Epo pretreatment reduced nuclear translocation of NF-{kappa}B and inhibited phosphorylation of inhibitor of kappa B (I{kappa}B) in TNF-{alpha}-stimulated cardiac myoblasts. Thus, Epo protects cardiac myocyte progenitors or myoblasts against the cytotoxic effects of TNF-{alpha} by inhibiting NF-{kappa}B-mediated iNOS expression and NO production and by preventing caspase-3 activation.

  9. Alpha2 adrenoceptors regulate proliferation of human intestinal epithelial cells

    PubMed Central

    Schaak, S; Cussac, D; Cayla, C; Devedjian, J; Guyot, R; Paris, H; Denis, C

    2000-01-01

    BACKGROUND AND AIMS—Previous studies on rodents have suggested that catecholamines stimulate proliferation of the intestinal epithelium through activation of α2 adrenoceptors located on crypt cells. The occurrence of this effect awaits demonstration in humans and the molecular mechanisms involved have not yet been elucidated. Here, we examined the effect of α2 agonists on a clone of Caco2 cells expressing the human α2A adrenoceptor.
METHODS—Cells were transfected with a bicistronic plasmid containing the α2C10 and neomycin phosphotransferase genes. G418 resistant clones were assayed for receptor expression using radioligand binding. Receptor functionality was assessed by testing its ability to couple Gi proteins and to inhibit cAMP production. Mitogen activated protein kinase (MAPK) phosphorylation was followed by western blot, and cell proliferation was estimated by measuring protein and DNA content.
RESULTS—Permanent transfection of Caco2 cells allowed us to obtain a clone (Caco2-3B) expressing α2A adrenoceptors at a density similar to that found in normal human intestinal epithelium. Caco2-3B retained morphological features and brush border enzyme expression characteristic of enterocytic differentiation. The receptor was coupled to Gi2/Gi3 proteins and its stimulation caused marked diminution of forskolin induced cAMP production. Treatment of Caco2-3B with UK14304 (α2 agonist) induced a rapid increase in the phosphorylation state of MAPK, extracellular regulated protein kinase 1 (Erk1), and 2 (Erk2). This event was totally abolished in pertussis toxin treated cells and in the presence of kinase inhibitors (genistein or PD98059). It was unaffected by protein kinase C downregulation but correlated with a transient increase in Shc tyrosine phosphorylation. Finally, sustained exposure of Caco2-3B to UK14304 resulted in modest but significant acceleration of cell proliferation. None of these effects was observed in the parental cell line Caco2.

  10. Vitronectin enhances internalization of crocidolite asbestos by rabbit pleural mesothelial cells via the integrin alpha v beta 5.

    PubMed Central

    Boylan, A M; Sanan, D A; Sheppard, D; Broaddus, V C

    1995-01-01

    The mechanism by which pleural mesothelial cells, the likely progenitor cells of asbestos-induced mesothelioma, recognize and internalize crocidolite asbestos is unknown. Because incubation of asbestos fibers with serum increases their association with cells, we asked whether a protein coat on asbestos increased internalization of fibers via specific cellular receptors. Coating crocidolite with citronectin, but not with fibronectin or other proteins, increased fiber internalization by rabbit pleural mesothelial cells, as measured by a new technique using fluorescence confocal microscopy. Receptors for vitronectin, alpha v beta 3 and alpha v beta 5, were identified on mesothelial cells. Inhibiting vitronectin receptors by plating cells on a vitronectin substrate or incubating cells with excess soluble vitronectin reduced internalization of vitronectin-coated crocidolite. Inhibition of alpha v beta 5, but not alpha v beta 3, with blocking antibodies similarly reduced internalization. In addition, alpha v beta 5, but not alpha v beta 3, showed immunocytochemical colocalization with fibers. Of biologic relevance, coating crocidolite with serum also increased internalization via alpha v beta 5, an effect dependent on the vitronectin in serum. We conclude that pleural mesothelial cells recognize and internalize vitronectin- and serum-coated asbestos via the integrin alpha v beta 5. Since integrins initiate some of the same signaling pathways as does asbestos, our findings may provide insights into the mechanisms of asbestos-induced biologic effects. Images PMID:7560092

  11. TRIM32 promotes retinoic acid receptor {alpha}-mediated differentiation in human promyelogenous leukemic cell line HL60

    SciTech Connect

    Sato, Tomonobu; Okumura, Fumihiko; Iguchi, Akihiro; Ariga, Tadashi; Hatakeyama, Shigetsugu

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer TRIM32 enhanced RAR{alpha}-mediated transcriptional activity even in the absence of RA. Black-Right-Pointing-Pointer TRIM32 stabilized RAR{alpha} in the human promyelogenous leukemic cell line HL60. Black-Right-Pointing-Pointer Overexpression of TRIM32 in HL60 cells induced granulocytic differentiation. Black-Right-Pointing-Pointer TRIM32 may function as a coactivator for RAR{alpha}-mediated transcription in APL cells. -- Abstract: Ubiquitination, one of the posttranslational modifications, appears to be involved in the transcriptional activity of nuclear receptors including retinoic acid receptor {alpha} (RAR{alpha}). We previously reported that an E3 ubiquitin ligase, TRIM32, interacts with several important proteins including RAR{alpha} and enhances transcriptional activity of RAR{alpha} in mouse neuroblastoma cells and embryonal carcinoma cells. Retinoic acid (RA), which acts as a ligand to nuclear receptors including RAR{alpha}, plays crucial roles in development, differentiation, cell cycles and apoptosis. In this study, we found that TRIM32 enhances RAR{alpha}-mediated transcriptional activity even in the absence of RA and stabilizes RAR{alpha} in the human promyelogenous leukemic cell line HL60. Moreover, we found that overexpression of TRIM32 in HL60 cells suppresses cellular proliferation and induces granulocytic differentiation even in the absence of RA. These findings suggest that TRIM32 functions as one of the coactivators for RAR{alpha}-mediated transcription in acute promyelogenous leukemia (APL) cells, and thus TRIM32 may become a potentially therapeutic target for APL.

  12. Reduction of cell viability induced by IFN-alpha generates impaired data on antiviral assay using Hep-2C cells.

    PubMed

    de Oliveira, Edson R A; Lima, Bruna M M P; de Moura, Wlamir C; Nogueira, Ana Cristina M de A

    2013-12-31

    Type I interferons (IFNs) exert an array of important biological functions on the innate immune response and has become a useful tool in the treatment of various diseases. An increasing demand in the usage of recombinant IFNs, mainly due to the treatment of chronic hepatitis C infection, augmented the need of quality control for this biopharmaceutical. A traditional bioassay for IFN potency assessment is the cytopathic effect reduction antiviral assay where a given cell line is preserved by IFN from a lytic virus activity using the cell viability as a frequent measure of end point. However, type I IFNs induce other biological effects such as cell-cycle arrest and apoptosis that can influence directly on viability of many cell lines. Here, we standardized a cytopathic effect reduction antiviral assay using Hep-2C cell/mengovirus combination and studied a possible impact of cell viability variations caused by IFN-alpha 2b on responses generated on the antiviral assay. Using the four-parameter logistic model, we observed less correlation and less linearity on antiviral assay when responses from IFN-alpha 2b 1000 IU/ml were considered in the analysis. Cell viability tests with MTT revealed a clear cell growth inhibition of Hep-2C cells under stimulation with IFN-alpha 2b. Flow cytometric cell-cycle analysis and apoptosis assessment showed an increase of S+G2 phase and higher levels of apoptotic cells after treatment with IFN-alpha 2b 1000 IU/ml under our standardized antiviral assay procedure. Considering our studied dose range, we also observed strong STAT1 activation on Hep-2C cells after stimulation with the higher doses of IFN-alpha 2b. Our findings showed that the reduction of cell viability driven by IFN-alpha can cause a negative impact on antiviral assays. We assume that the cell death induction and the cell growth inhibition effect of IFNs should also be considered while employing antiviral assay protocols in a quality control routine and emphasizes the

  13. Curcumin inhibits interferon-{alpha} induced NF-{kappa}B and COX-2 in human A549 non-small cell lung cancer cells

    SciTech Connect

    Lee, Jeeyun |; Im, Young-Hyuck | E-mail: imyh@smc.samsung.co.kr; Jung, Hae Hyun; Kim, Joo Hyun; Park, Joon Oh |; Kim, Kihyun |; Kim, Won Seog |; Ahn, Jin Seok

    2005-08-26

    The A549 cells, non-small cell lung cancer cell line from human, were resistant to interferon (IFN)-{alpha} treatment. The IFN-{alpha}-treated A549 cells showed increase in protein expression levels of NF-{kappa}B and COX-2. IFN-{alpha} induced NF-{kappa}B binding activity within 30 min and this increased binding activity was markedly suppressed with inclusion of curcumin. Curcumin also inhibited IFN-{alpha}-induced COX-2 expression in A549 cells. Within 10 min, IFN-{alpha} rapidly induced the binding activity of a {gamma}-{sup 32}P-labeled consensus GAS oligonucleotide probe, which was profoundly reversed by curcumin. Taken together, IFN-{alpha}-induced activations of NF-{kappa}B and COX-2 were inhibited by the addition of curcumin in A549 cells.

  14. Human neural cell adhesion molecule L1 and rat homologue NILE are ligands for integrin alpha v beta 3

    PubMed Central

    1996-01-01

    Integrin alpha v beta 3 is distinct in its capacity to recognize the sequence Arg-Gly-Asp (RGD) in many extra-cellular matrix (ECM) components. Here, we demonstrate that in addition to the recognition of ECM components, alpha v beta 3 can interact with the neural cell adhesion molecule L1-CAM; a member of the immunoglobulin superfamily (IgSF). M21 melanoma cells displayed significant Ca(++)-dependent adhesion and spreading on immunopurified rat L1 (NILE). This adhesion was found to be dependent on the expression of the alpha v-integrin subunit and could be significantly inhibited by an antibody to the alpha v beta 3 heterodimer. M21 cells also displayed some alpha v beta 3-dependent adhesion and spreading on immunopurified human L1. Ligation between this ligand and alpha v beta 3 was also observed to promote significant haptotactic cell migration. To map the site of alpha v beta 3 ligation we used recombinant L1 fragments comprising the entire extracellular domain of human L1. Significant alpha v beta 3-dependent adhesion and spreading was evident on a L1 fragment containing Ig-like domains 4, 5, and 6. Importantly, mutation of an RGD sequence present in the sixth Ig-like domain of L1 abrogated M21 cell adhesion. We conclude that alpha v beta 3-dependent recognition of human L1 is dependent on ligation of this RGD site. Despite high levels of L1 expression the M21 melanoma cells did not display significant adhesion via a homophilic L1-L1 interaction. These data suggest that M21 melanoma cells recognize and adhere to L1 through a mechanism that is primarily heterophilic and integrin dependent. Finally, we present evidence that melanoma cells can shed and deposit L1 in occluding ECM. In this regard, alpha v beta 3 may recognize L1 in a cell-cell or cell- substrate interaction. PMID:8636223

  15. Two distinct T-cell receptor alpha-chain transcripts in a rabbit T-cell line: implications for allelic exclusion in T cells.

    PubMed Central

    Marche, P N; Kindt, T J

    1986-01-01

    Information relevant to allelic exclusion in T cells has been obtained by a study of cDNA clones corresponding to alpha-chain genes of the T-cell receptor in the rabbit T-cell line RL-5. One clone contains a variable-joining-constant (VJC) sequence encoding a complete alpha chain of the T-cell receptor. A second has an identical constant region and includes a distinct variable-joining (VJ) sequence. However, a single-base deletion in the variable region places the remainder of the second transcript out-of-phase and appears to be the product of a rearrangement involving a variable region of the T-cell receptor alpha-chain pseudogene. Presence of two variable-joining-constant (VJC) transcripts in the same cell line indicates that alpha-chain gene rearrangement is not affected by transcription of a complete alpha-chain mRNA and suggests that steps after mRNA synthesis are involved in the allelic exclusion process for alpha-chain genes. Comparison of rabbit alpha-chain sequences with those of man and mouse revealed interspecies conservation in constant and variable regions. Genomic Southern blot analyses using a rabbit constant region of the T-cell receptor alpha-chain probe revealed the presence of a single constant region gene. Hybridization with variable region probes defined two distinct multigenic subfamilies. Homology between certain rabbit and murine variable regions of the T-cell receptor alpha-chain sequences suggests that the existence of subfamilies predated divergence of these species. Images PMID:3485798

  16. Inhibition of tumour necrosis factor-alpha (TNF-alpha) release from mast cells by the anti-inflammatory drugs, sodium cromoglycate and nedocromil sodium.

    PubMed

    Bissonnette, E Y; Enciso, J A; Befus, A D

    1995-10-01

    TNF-alpha is a cytokine thought to be involved in the pathogenesis of asthma and in several other inflammatory conditions. Given recent evidence that mast cells (MC) are an important source of TNF-alpha, we investigated the effects of two anti-inflammatory drugs, nedocromil sodium (NED) and sodium cromoglycate (SCG), on rat MC-derived TNF-alpha. We established that at least 2 h pretreatment with NED or SCG followed by washing was required to inhibit TNF-alpha-dependent cytotoxicity by rat peritoneal MC (PMC). A maximum inhibition of TNF-alpha occurred after 6 h treatment. The inhibitory effect of NED and SCG (10(-5)-10(-3)M) was concentration-dependent (20-37% for NED and 16-37% for SCG). The time-course analysis and the use of cycloheximide, an inhibitor of protein synthesis, provided strong evidence that new protein synthesis by the MC is required for this inhibitory effect. Furthermore, 24 h treatment with 1 mM NED inhibited the levels of mRNA for TNF-alpha by 59-83%. In addition to the effect on TNF-alpha-dependent cytotoxicity by MC, 20 min pretreatment with 10(-4) M NED and SCG inhibited antigen-stimulated TNF-alpha release (6h) by 42% and 48%, respectively. Interestingly, the functionally distinct intestinal mucosal MC (IMMC) is unresponsive to these drugs with regard to histamine secretion. However, as with PMC, 2h pretreatment with NED or SCG inhibited TNF-alpha-dependent cytotoxicity by IMMC. These effects may be important in the action of these drugs in vivo in the late phase reaction in asthma or other inflammatory conditions.

  17. Inhibition of tumour necrosis factor-alpha (TNF-alpha) release from mast cells by the anti-inflammatory drugs, sodium cromoglycate and nedocromil sodium.

    PubMed Central

    Bissonnette, E Y; Enciso, J A; Befus, A D

    1995-01-01

    TNF-alpha is a cytokine thought to be involved in the pathogenesis of asthma and in several other inflammatory conditions. Given recent evidence that mast cells (MC) are an important source of TNF-alpha, we investigated the effects of two anti-inflammatory drugs, nedocromil sodium (NED) and sodium cromoglycate (SCG), on rat MC-derived TNF-alpha. We established that at least 2 h pretreatment with NED or SCG followed by washing was required to inhibit TNF-alpha-dependent cytotoxicity by rat peritoneal MC (PMC). A maximum inhibition of TNF-alpha occurred after 6 h treatment. The inhibitory effect of NED and SCG (10(-5)-10(-3)M) was concentration-dependent (20-37% for NED and 16-37% for SCG). The time-course analysis and the use of cycloheximide, an inhibitor of protein synthesis, provided strong evidence that new protein synthesis by the MC is required for this inhibitory effect. Furthermore, 24 h treatment with 1 mM NED inhibited the levels of mRNA for TNF-alpha by 59-83%. In addition to the effect on TNF-alpha-dependent cytotoxicity by MC, 20 min pretreatment with 10(-4) M NED and SCG inhibited antigen-stimulated TNF-alpha release (6h) by 42% and 48%, respectively. Interestingly, the functionally distinct intestinal mucosal MC (IMMC) is unresponsive to these drugs with regard to histamine secretion. However, as with PMC, 2h pretreatment with NED or SCG inhibited TNF-alpha-dependent cytotoxicity by IMMC. These effects may be important in the action of these drugs in vivo in the late phase reaction in asthma or other inflammatory conditions. Images Fig. 6 PMID:7554404

  18. Staging of alpha-synuclein in the olfactory bulb in a model of Parkinson's disease: cell types involved.

    PubMed

    Ubeda-Bañon, Isabel; Saiz-Sanchez, Daniel; de la Rosa-Prieto, Carlos; Mohedano-Moriano, Alicia; Fradejas, Noelia; Calvo, Soledad; Argandoña-Palacios, Lucia; Garcia-Muñozguren, Susana; Martinez-Marcos, Alino

    2010-08-15

    Impaired olfaction is an early symptom of Parkinson's disease. The underlying neuropathology likely includes alpha-synucleinopathy in the olfactory bulb at an earlier stage (Braak's stage1) than pathology in the substantia nigra, which is not observed until stage 3. In this report, we investigated the distribution and cell types affected by alpha-synuclein in the olfactory bulb of transgenic mice (2-8 months of age) expressing the human A53T variant of alpha-synuclein. alpha-Synuclein immunostaining progressively affects interneurons and mitral cells. Double labeling studies demonstrate that dopaminergic cells are hardly involved, whereas glutamatergic- and calcium binding protein-positive cells are severely affected. This temporal evolution and the cell types expressing alpha-synuclein are reminiscent of idiopathic Parkinson's disease and support the usefulness of this model to address specific topics in the premotor phase of the disease.

  19. Increase in gene dosage is a mechanism of HIF-1alpha constitutive expression in head and neck squamous cell carcinomas.

    PubMed

    Secades, Pablo; Rodrigo, Juan Pablo; Hermsen, Mario; Alvarez, Cesar; Suarez, Carlos; Chiara, María-Dolores

    2009-05-01

    The HIF-1alpha protein plays a key role in the cellular response to hypoxia via transcriptional regulation of genes involved in erythropoiesis, angiogenesis, and metabolism. Overexpression of HIF-1alpha is commonly found in solid tumors in significant association with increased patient mortality and resistance to therapy. The predominant mode of HIF-1alpha regulation by hypoxia occurs at the level of protein stability. In addition to hypoxia, HIF-1alpha protein stability and synthesis is regulated by nonhypoxic signals such as inactivation of tumor suppressors and activation of oncogenes. Here, we show that an increase in gene dosage may contribute to HIF-1alpha mRNA and protein overexpression in a nonhypoxic environment in head and neck squamous cell carcinomas (HNSCC). Increased HIF-1alpha gene dosage was found in one out of five HNSCC-derived cell lines and three out of 27 HNSCC primary tumors. Significantly, increased gene dosage in those samples was associated with high HIF-1alpha mRNA and protein levels. Normoxic overexpression of HIF-1alpha protein in HNSCC-derived cell lines was also paralleled by higher expression levels of HIF-1alpha target genes. Array CGH analysis confirmed the copy number increase of HIF-1alpha gene and revealed that the gene is contained within a region of amplification at 14q23-q24.2 both in the cell line and primary tumors. In addition, FISH analysis revealed the presence of 11-13 copies on a tetraploid background in SCC2 cells. These data suggest that increased HIF-1alpha gene dosage is a mechanism of HIF-1alpha protein overexpression in HNSCC that possibly prepares the cells for a higher activity in an intratumoral hypoxic environment.

  20. Grepafloxacin inhibits tumor necrosis factor-alpha-induced interleukin-8 expression in human airway epithelial cells.

    PubMed

    Hashimoto, S; Matsumoto, K; Gon, Y; Maruoka, S; Hayashi, S; Asai, Y; Machino, T; Horie, T

    2000-01-01

    We examined the effect of grepafloxacin (GPFX), a new fluoroquinolone antimicrobial agent, on interleukin-8 (IL-8) expression in tumor necrosis factor-alpha (TNF-alpha)-stimulated human airway epithelial cells (AEC). GPFX inhibited IL-8 protein production as well as mRNA expression in a concentration-dependent manner (2.5 - 25 micro g/ml), but the inhibition of IL-8 expression by corresponding concentrations of GPFX to serum and airway lining fluids was not complete. We discuss the modulatory effect of GPFX on IL-8 production in the context of its efficacy on controlling chronic airway inflammatory diseases.

  1. Tumor necrosis factor-alpha modulates survival, proliferation, and neuronal differentiation in neonatal subventricular zone cell cultures.

    PubMed

    Bernardino, Liliana; Agasse, Fabienne; Silva, Bruno; Ferreira, Raquel; Grade, Sofia; Malva, João O

    2008-09-01

    Tumor necrosis factor (TNF)-alpha has been reported to modulate brain injury, but remarkably, little is known about its effects on neurogenesis. We report that TNF-alpha strongly influences survival, proliferation, and neuronal differentiation in cultured subventricular zone (SVZ) neural stem/progenitor cells derived from the neonatal P1-3 C57BL/6 mice. By using single-cell calcium imaging, we developed a method, based on cellular response to KCl and/or histamine, that allows the functional evaluation of neuronal differentiation. Exposure of SVZ cultures to 1 and 10 ng/ml mouse or 1 ng/ml human recombinant TNF-alpha resulted in increased differentiation of cells displaying a neuronal-like profile of [Ca2+](i) responses, compared with the predominant profile of immature cells observed in control, nontreated cultures. Moreover, by using neutralizing antibodies for each TNF-alpha receptor, we found that the proneurogenic effect of 1 ng/ml TNF-alpha is mediated via tumor necrosis factor receptor 1 activation. Accordingly, the percentage of neuronal nuclear protein-positive neurons was increased following exposure to mouse TNF-alpha. Interestingly, exposure of SVZ cultures to 1 ng/ml TNF-alpha induced cell proliferation, whereas 10 and 100 ng/ml TNF-alpha induced apoptotic cell death. Moreover, we found that exposure of SVZ cells to TNF-alpha for 15 minutes or 6 hours caused an increase in the phospho-stress-activated protein kinase/c-Jun N-terminal kinase immunoreactivity initially in the nucleus and then in growing axons, colocalizing with tau, consistent with axonogenesis. Taken together, these results show that TNF-alpha induces neurogenesis in neonatal SVZ cell cultures of mice. TNF-alpha, a proinflammatory cytokine and a proneurogenic factor, may play a central role in promoting neurogenesis and brain repair in response to brain injury and infection.

  2. Tumor cell alpha-N-acetylgalactosaminidase activity and its involvement in GcMAF-related macrophage activation.

    PubMed

    Mohamad, Saharuddin B; Nagasawa, Hideko; Uto, Yoshihiro; Hori, Hitoshi

    2002-05-01

    Alpha-N-acetyl galactosaminidase (alpha-NaGalase) has been reported to accumulate in serum of cancer patients and be responsible for deglycosylation of Gc protein, which is a precursor of GcMAF-mediated macrophage activation cascade, finally leading to immunosuppression in advanced cancer patients. We studied the biochemical characterization of alpha-NaGalase from several human tumor cell lines. We also examined its effect on the potency of GcMAF to activate mouse peritoneal macrophage to produce superoxide in GcMAF-mediated macrophage activation cascade. The specific activity of alpha-NaGalases from human colon tumor cell line HCT116, human hepatoma cell line HepG2, and normal human liver cells (Chang liver cell line) were evaluated using two types of substrates; GalNAc-alpha-PNP (exo-type substrate) and Gal-beta-GalNAc-alpha-PNP (endo-type substrate). Tumor-derived alpha-NaGalase having higher activity than normal alpha-NaGalase, had higher substrate specificity to the exo-type substrate than to the endo-type substrate, and still maintained its activity at pH 7. GcMAF enhance superoxide production in mouse macrophage, and pre-treatment of GcMAF with tumor cell lysate reduce the activity. We conclude that tumor-derived alpha-NaGalase is different in biochemical characterization compared to normal alpha-NaGalase from normal Chang liver cells. In addition, tumor cell-derived alpha-NaGalase decreases the potency of GcMAF on macrophage activation.

  3. Thrombopoietin enhances the alpha IIb beta 3-dependent adhesion of megakaryocytic cells to fibrinogen or fibronectin through PI 3 kinase.

    PubMed

    Zauli, G; Bassini, A; Vitale, M; Gibellini, D; Celeghini, C; Caramelli, E; Pierpaoli, S; Guidotti, L; Capitani, S

    1997-02-01

    The effect of thrombopoietin (TPO) on the functional activity of surface alpha IIb beta 3 (GPIIbIIIa) was investigated in both primary human megakaryocytic cells, derived from peripheral blood CD34+ cells, and HEL hematopoietic cell line. TPO (100 ng/mL) induced a sixfold to ninefold enhancement of adhesion of both primary megakaryocytic and HEL cells to plates coated with either fibrinogen or fibronectin and a parallel increase of immunoreactivity to the PAC1 monoclonal antibody (MoAb) and fluorescein isothiocyanate-fibrinogen, both of which recognize an activated state of alpha IIb beta 3. The enhanced adhesion to fibrinogen or fibronectin was mediated by the Arg-Gly-Asp (RGD) recognition sequence of alpha IIb beta 3, as it was abolished by pretreatment of cells with saturating concentrations of RGDS peptide. A MoAb specific for the alpha IIb beta subunit of alpha IIb beta 3 also inhibited cell attachment to fibrinogen or fibronectin, while MoAb to anti-alpha v beta 3 or anti-alpha 5 integrins were completely ineffective, clearly indicating that alpha IIb beta 3 participates in this association. A role for PI 3 kinase (PI 3-K) in the TPO-mediated increase in alpha IIb beta 3 function in megakaryocytic cells was suggested by the ability of the PI 3-K inhibitor wortmannin (100 nmol/L) and antisense oligonucleotides directed against the p85 regulatory subunit of PI 3-K to completely block the TPO-induced increase in alpha IIb beta 3 integrin activity upon TPO stimulation. The modulation of adhesiveness to extracellular matrix proteins containing the RGD motif mediated by TPO likely plays a physiologic role in megakaryocytopoiesis, as pretreatment of CD34+ cells with RGDS or anti-alpha IIb MoAb significantly reduced the number of megakaryocytic colonies obtained in a fibrinclot semisolid assay.

  4. Alpha/sub 2/-adrenergic receptors on a platelet precursor cell line, HEL

    SciTech Connect

    McKernan, R.M.; Motulsky, H.J.; Rozansky, D.; Insel, P.A.

    1986-03-01

    The authors have identified ..cap alpha../sub 2/-adrenergic receptors on human erythroleukemia HEL cells, a suspension-growing, bone-marrow-derived cell line related to human platelets. Intact HEL cells were studied using radioligand binding and cAMP accumulation assays. The authors identified saturable specific binding of the ..cap alpha../sub 2/-antagonist (/sup 3/H)yohimbine (yoh) in cells incubated at 37/sup 0/C for 1 hr (B/sub max/ 5900 +/- 2100 sites/cell, K/sub d/ 3.6 +/- 0.9 nM, n = 7). Competition for (/sup 3/H)yoh binding sites with antagonists confirmed that these sites were similar to human ..cap alpha../sub 2/-adrenoceptors from platelets and other resources, as typified by their high affinity for WY-26392, yohimbine and idazoxan, and very low affinity for prazosin. Studies at 37/sup 0/C revealed a low affinity of these sites for catecholamines (K/sub i/ for (-)-epinephrine, 21 ..mu..M; (-)-norepinephrine, 45 ..mu..M, (+)-epinephrine, 80 ..mu..M). When experiments were conducted at 4 /sup 0/C, (-)-epinephrine was able to compete for only 50-60% of the sites specifically labelled by (/sup 3/H)yoh at 37/sup 0/, but (-)-epinephrine had an approximately 10-fold greater affinity for these sites (K/sub i/ at 4 /sup 0/C = 2.4 ..mu..M). In addition, epinephrine inhibited cAMP accumulation stimulated by forskolin and PGE/sub 1/ in HEL cells; this response was inhibited by pertussis toxin. The authors conclude that HEL cells possess ..cap alpha../sub 2/-adrenergic receptors linked to G/sub i/ and thus should serve as a useful model to explore metabolism and regulation of these receptors in human cells.

  5. Transforming growth factor-alpha in vivo stimulates epithelial cell proliferation in digestive tissues of suckling rats.

    PubMed Central

    Hormi, K; Lehy, T

    1996-01-01

    BACKGROUND: The role that exogenous transforming growth factor-alpha (TGF-alpha) may exert on cell proliferation in vivo is poorly understood. AIM: To investigate the effect of rat TGF-alpha on epithelial cell proliferation in all suckling rat digestive tissues and to compare it with that of rat epidermal growth factor (EGF). ANIMAL AND METHODS: TGF-alpha and EGF were given three times daily either subcutaneously (10 or 20 micrograms/kg) or intraperitoneally (100 micrograms/kg) to rats from the ninth postnatal day. Cell proliferation was assessed through 5-bromo- 2-deoxyuridine incorporation and estimation of labelling indices. RESULTS: For both growth factors, the highest dose given for only two days significantly increased stomach and intestinal weights compared with controls (p < 0.05 to p < 0.001). The proliferative responded depended on the dose given, colonic mucosa being the most sensitive whereas oxyntic mucosa remained unresponsive. TGF-alpha was as potent as EGF in stimulating epithelial cell proliferation in antral, duodenal, and colonic mucosae. However, EGF was more active on oesophageal and jejunal cell proliferation whereas TGF-alpha was more active on pancreatic exocrine cell proliferation and the differences between the two growth factor treated groups were significant. CONCLUSIONS: These results prove for the first time the stimulating effect in vivo of exogenous rat TGF-alpha on epithelial cell proliferation in rat digestive tissues during the developmental period and support a functional role for TGF-alpha at that time. PMID:8944561

  6. Alpha1-antichymotrypsin induces TNF-alpha production and NF-kappaB activation in the murine N9 microglial cell line.

    PubMed

    Braghin, Elisa; Galimberti, Daniela; Scarpini, Elio; Bresolin, Nereo; Baron, Pierluigi

    2009-12-18

    Microglia are known to accumulate in senile plaques of Alzheimer's disease (AD) together with a set of proteins including alpha(1)-antichymotrypsin (ACT). To investigate the biological effects of the interaction between ACT and microglia, we examined cytokine production by the murine N9 microglial cell line after ACT treatment. Real-time PCR analysis and specific immunoassays demonstrate that ACT triggers mRNA expression and release of TNF-alpha by N9 microglial cells. Furthermore, we show that ACT induces a significant increase in NF-kappaB nuclear translocation. Taken together, these data demonstrate that ACT might contribute to the inflammatory mechanisms present in AD senile plaques.

  7. Alpha tumor necrosis factor contributes to CD8{sup +} T cell survival in the transition phase

    SciTech Connect

    Shi, Meiqing; Ye, Zhenmin; Umeshappa, Keshav Sokke; Moyana, Terence; Xiang, Jim . E-mail: jxiang@scf.sk.ca

    2007-08-31

    Cytokine and costimulation signals determine CD8{sup +} T cell responses in proliferation phase. In this study, we assessed the potential effect of cytokines and costimulations to CD8{sup +} T cell survival in transition phase by transferring in vitro ovalbumin (OVA)-pulsed dendritic cell-activated CD8{sup +} T cells derived from OVA-specific T cell receptor transgenic OT I mice into wild-type C57BL/6 mice or mice with designated gene knockout. We found that deficiency of IL-10, IL-12, IFN-{gamma}, CD28, CD40, CD80, CD40L, and 41BBL in recipients did not affect CD8{sup +} T cell survival after adoptive transfer. In contrast, TNF-{alpha} deficiency in both recipients and donor CD8{sup +} effector T cells significantly reduced CD8{sup +} T cell survival. Therefore, our data demonstrate that the host- and T cell-derived TNF-{alpha} signaling contributes to CD8{sup +} effector T cell survival and their transition to memory T cells in the transition phase, and may be useful information when designing vaccination.

  8. Polycystin-1 promotes PKC{alpha}-mediated NF-{kappa}B activation in kidney cells

    SciTech Connect

    Banzi, Manuela; Aguiari, Gianluca; Trimi, Viky; Mangolini, Alessandra; Pinton, Paolo; Witzgall, Ralph; Rizzuto, Rosario; Senno, Laura del . E-mail: sen@unife.it

    2006-11-17

    Polycystin-1 (PC1), the PKD1 gene product, is a membrane receptor which regulates many cell functions, including cell proliferation and apoptosis, both typically increased in cyst lining cells in autosomal dominant polycystic kidney disease. Here we show that PC1 upregulates the NF-{kappa}B signalling pathway in kidney cells to prevent cell death. Human embryonic kidney cell lines (HEK293{sup CTT}), stably expressing a PC1 cytoplasmic terminal tail (CTT), presented increased NF-{kappa}B nuclear levels and NF-{kappa}B-mediated luciferase promoter activity. This, consistently, was reduced in HEK293 cells in which the endogenous PC1 was depleted by RNA interference. CTT-dependent NF-{kappa}B promoter activation was mediated by PKC{alpha} because it was blocked by its specific inhibitor Ro-320432. Furthermore, it was observed that apoptosis, which was increased in PC1-depleted cells, was reduced in HEK293{sup CTT} cells and in porcine kidney LtTA cells expressing a doxycycline-regulated CTT. Staurosporine, a PKC inhibitor, and parthenolide, a NF-{kappa}B inhibitor, significantly reduced the CTT-dependent antiapoptotic effect. These data reveal, therefore, a novel pathway by which polycystin-1 activates a PKC{alpha}-mediated NF-{kappa}B signalling and cell survival.

  9. Sclareol protects Staphylococcus aureus-induced lung cell injury via inhibiting alpha-hemolysin expression.

    PubMed

    Ouyang, Ping; Sun, Mao; He, Xuewen; Wang, Kaiyu; Yin, Zhongqiong; Fu, Hualin; Li, Yinglun; Geng, Yi; Shu, Gang; He, Changliang; Liang, Xiaoxia; Lai, Weiming; Li, Lixia; Zou, Yuanfeng; Song, Xu; Yin, Lizi

    2016-09-23

    Staphylococcus aureus (S. aureus) is a common Gram-positive bacterium that causes serious infections in human and animals. With the continuous emergence of the methicillin-resistant S. aureus (MRSA) strains, antibiotics have limited efficacy in treating MRSA infections. Accordingly, novel agents that act on new targets are desperately needed to combat these infections. S. aureus alpha-hemolysin plays an indispensable role in its pathogenicity. In this study, we demonstrate that sclareol, a fragrant chemical compound found in clary sage, can prominently decrease alpha-hemolysin secretion in S. aureus strain USA300 at sub-inhibitory concentrations. Hemolysis assays, western-blotting and RT-PCR were used to detect the production of alpha-hemolysin in the culture supernatant. When USA300 was co-cultured with and A549 epithelial cells, sclareol could protect A549 cells at a final concentration of 8 µg/ml. The protective capability of sclareol against the USA300-mediated injury of A549 cells was further shown by cytotoxicity assays and live/dead analysis. In conclusion, sclareol was shown to inhibit the production of S. aureus alpha-hemolysin. Sclareol has potential for development as a new agent to treat S. aureus infections.

  10. The HPV-16 E7 oncogene sensitizes malignant cells to IFN-alpha-induced apoptosis

    SciTech Connect

    Wang, Yisong

    2005-10-01

    Interferons (IFNs) exert antitumor effects in several human malignancies, but their mechanism of action is unclear. There is a great variability in sensitivity to IFN treatment depending on both tumor type and the individual patient. The reason for this variable sensitivity is not known. The fact that several IFN-induced anticellular effects are exerted through modulation of proto-oncogenes and tumor suppressor genes may indicate that the malignant genotype may be decisive in the cell's sensitivity to IFN. To determine if a deregulated oncogene could alter the cellular response to IFN, a mouse lymphoma cell line (J3D) was stably transfected with the viral human papillomavirus-16 (HPV-16) E7 oncogene. The E7-transfected cells and their respective mock-transfected sister clones were treated with IFN-{alpha} and examined for possible IFN-induced anticellular effects. We found that the E7-transfected clones were greatly sensitized to IFN-{alpha}-induced apoptosis compared with their mock-transfected counterparts. Induction of apoptosis in the transfected cells correlated with the ability of IFN to activate parts of the proapoptotic machinery specifically in these cells, including activation of caspases and the proapoptotic protein Bak. In summary, our data suggest that transfection of malignant cells with the E7 oncogene can sensitize them to IFN-{alpha}-induced apoptosis. This demonstrates that an oncogenic event may alter the cellular sensitivity to IFN and might also have implications for treatment of HPV related diseases with IFN.

  11. Method of using alpha-1 acid glycoprotein on T-cells as a marker for alzheimer's disease

    SciTech Connect

    Fudenberg, H.H.

    1989-01-31

    A method is described of diagnosing a dementia of the Alzheimer's type characterized by a change in the percentage of T-cells bearing surface membrane alpha-1 acid glycoprotein which comprises providing T-cells from a subject, determining the percentage of those T cells which bear surface membrane alpha-1 acid glycoprotein, and comparing that percentage of the percentage of T cells which bear the glycoprotein in a control, whereby the dementia is diagnosed.

  12. An in-cell alpha detection system for radioisotope component assembly operations

    SciTech Connect

    Carteret, B.A. ); Goles, R.W. )

    1991-09-01

    A remotely operated alpha detection system is being developed for use at the Radioisotope Power Systems Facility at the US Department of Energy's Hanford Site. It will be used in hot cells being constructed to assemble components of Radioisotope Thermoelectric Generators for space power applications. The in-cell detection equipment will survey radiological swipe samples to determine smearable surface contamination levels on radioisotope fuel, fueled components, and hot-cell work areas. This system is potentially adaptable to other hot cell and glovebox applications where radiation dose rates and contamination levels are expected to be low. 2 figs.

  13. Integrin alpha 3 beta 1 participates in the phagocytosis of extracellular matrix molecules by human breast cancer cells.

    PubMed Central

    Coopman, P J; Thomas, D M; Gehlsen, K R; Mueller, S C

    1996-01-01

    The mechanisms and receptors involved in phagocytosis by nonhematopoietic cells are not well understood. The involvement of the alpha 3 beta 1 integrin in phagocytosis of the extracellular matrix by human breast cancer cells was studied. The possible role of this integrin was suggested since alpha 3 and beta 1 but not alpha 2 subunits are concentrated at membrane sites where local degradation of fluorescently labeled gelatin occurs. Strikingly, anti-alpha 3 integrin monoclonal antibodies (mAbs) stimulate the phagocytosis of fluorescently labeled gelatin films, gelatin beads, and Matrigel films in a quantitative phagocytosis assay. Stimulation of the gelatin uptake by the anti-alpha 3 mAb is dose responsive, saturable, and time dependent. Antibodies against other integrin subunits have a lower stimulatory effect (anti-beta 1) or no significant effect (anti-alpha 2, -alpha 5, -alpha 6, and -alpha v) on gelatin phagocytosis. The synthetic HGD-6 human laminin peptide that binds specifically the alpha 3 beta 1 integrin, but not the scrambled HSGD-6 control peptide, also markedly stimulates gelatin uptake in a dose-responsive way. Furthermore, the stimulatory effects of the HGD-6 peptide and the anti-alpha 3 mAb are additive, suggesting that they might promote phagocytosis in different ways. Other laminin (YIGSR, IKVAV) and fibronectin (GRGDS) peptides have no effect on gelatin phagocytosis. Immunofluorescence shows that the alpha 3 and the beta 1, but not the alpha 2 integrin subunit, concentrate into patches on the cell surface after treatment with their respective mAbs. And, both gelatin and the alpha 3 beta 1 but not the alpha 2 beta 1 integrin are cointernalized and routed to acidic vesicles such as lysosomes. In conclusion, we demonstrate that human breast cancer cells locally degrade and phagocytose the extracellular matrix and show for the first time that the alpha 3 beta 1 integrin participates in this phagocytosis. We hypothesize that the anti-alpha 3

  14. Effect of Zanthoxylum schinifolium on TNF-alpha-induced vascular inflammation in human umbilical vein endothelial cells.

    PubMed

    Cao, Li Hua; Lee, Yun Jung; Kang, Dae Gill; Kim, Jin Sook; Lee, Ho Sub

    2009-01-01

    Pro-inflammatory cytokines induce the injury of endothelial cells in response to increases of adhesion molecules, leading to vascular inflammation and the development of atherosclerosis. In this study, we evaluated an ethanol extract of Zanthoxylum schinifolium (EZS) to determine if it inhibits the expressions of cellular adhesion molecules in human umbilical vein endothelial cells (HUVEC). When pretreatment of HUVEC with EZS, EZS suppressed the expression levels of cell adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-), and E-selectin induced by TNF-alpha. The adhesion of HL-60 cells to TNF-alpha-induced endothelial cells was decreased significantly in a concentration-dependent manner. Furthermore, TNF-alpha-induced MCP-1 and IL-8 mRNA expression levels were also attenuated by pretreatment with EZS. In addition, EZS suppressed TNF-alpha-induced production of reactive oxygen species (ROS). EZS inhibited NF-kappaB activation and IkappaB-alpha phosphorylation induced by TNF-alpha, subsequent degradation of IkappaB-alpha. Finally, EZS inhibited TNF-alpha-induced p38 MAPK and c-Jun N-terminal kinase (JNK) phosphorylation. Taken together, these results demonstrate that EZS suppresses vascular inflammatory process, which may be closely related to the inhibition of ROS, JNK, p38 MAPK and NF-kappaB activation in HUVEC.

  15. The nectin-1{alpha} transmembrane domain, but not the cytoplasmic tail, influences cell fusion induced by HSV-1 glycoproteins

    SciTech Connect

    Subramanian, Ravi P.; Dunn, Jennifer E.; Geraghty, Robert J. . E-mail: rgeragh@uky.edu

    2005-09-01

    Nectin-1 is a receptor for herpes simplex virus (HSV), a member of the immunoglobulin superfamily, and a cellular adhesion molecule. To study domains of nectin-1{alpha} involved in cell fusion, we measured the ability of nectin-1{alpha}/nectin-2{alpha} chimeras, nectin-1{alpha}/CD4 chimeras, and transmembrane domain and cytoplasmic tail mutants of nectin-1{alpha} to promote cell fusion induced by HSV-1 glycoproteins. Our results demonstrate that only chimeras and mutants containing the entire V-like domain and a link to the plasma membrane conferred cell-fusion activity. The transmembrane domain and cytoplasmic tail of nectin-1 were not required for any viral receptor or cell adhesion function tested. Cellular cytoplasmic factors that bind to the nectin-1{alpha} cytoplasmic tail, therefore, did not influence virus entry or cell fusion. Interestingly, the efficiency of cell fusion was reduced when membrane-spanning domains of nectin-1{alpha} and gD were replaced by glycosylphosphatidylinositol tethers, indicating that transmembrane domains may play a modulatory role in the gD/nectin-1{alpha} interaction in fusion.

  16. In vitro cell irradiation systems based on 210Po alpha source: construction and characterisation

    NASA Technical Reports Server (NTRS)

    Szabo, J.; Feher, I.; Palfalvi, J.; Balashazy, I.; Dam, A. M.; Polonyi, I.; Bogdandi, E. N.

    2002-01-01

    One way of studying the risk to human health of low-level radiation exposure is to make biological experiments on living cell cultures. Two 210Po alpha-particle emitting devices, with 0.5 and 100 MBq activity, were designed and constructed to perform such experiments irradiating monolayers of cells. Estimates of dose rate at the cell surface were obtained from measurements by a PIPS alpha-particle spectrometer and from calculations by the SRIM 2000, Monte Carlo charged particle transport code. Particle fluence area distributions were measured by solid state nuclear track detectors. The design and dosimetric characterisation of the devices are discussed. c2002 Elsevier Science Ltd. All rights reserved.

  17. Hepatocyte nuclear factor 4alpha orchestrates expression of cell adhesion proteins during the epithelial transformation of the developing liver.

    PubMed

    Battle, Michele A; Konopka, Genevieve; Parviz, Fereshteh; Gaggl, Alexandra Lerch; Yang, Chuhu; Sladek, Frances M; Duncan, Stephen A

    2006-05-30

    Epithelial formation is a central facet of organogenesis that relies on intercellular junction assembly to create functionally distinct apical and basal cell surfaces. How this process is regulated during embryonic development remains obscure. Previous studies using conditional knockout mice have shown that loss of hepatocyte nuclear factor 4alpha (HNF4alpha) blocks the epithelial transformation of the fetal liver, suggesting that HNF4alpha is a central regulator of epithelial morphogenesis. Although HNF4alpha-null hepatocytes do not express E-cadherin (also called CDH1), we show here that E-cadherin is dispensable for liver development, implying that HNF4alpha regulates additional aspects of epithelial formation. Microarray and molecular analyses reveal that HNF4alpha regulates the developmental expression of a myriad of proteins required for cell junction assembly and adhesion. Our findings define a fundamental mechanism through which generation of tissue epithelia during development is coordinated with the onset of organ function.

  18. The PPFLMLLKGSTR motif in globular domain 3 of the human laminin-5 {alpha}3 chain is crucial for integrin {alpha}3{beta}1 binding and cell adhesion

    SciTech Connect

    Kim, Jin-Man; Park, Won Ho; Min, Byung-Moo . E-mail: bmmin@snu.ac.kr

    2005-03-10

    Laminin-5 regulates various cellular functions, including cell adhesion, spreading, and motility. Here, we expressed the five human laminin {alpha}3 chain globular (LG) domains as monomeric, soluble fusion proteins, and examined their biological functions and signaling. Recombinant LG3 (rLG3) protein, unlike rLG1, rLG2, rLG4, and rLG5, played roles in cell adhesion, spreading, and integrin {alpha}3{beta}1 binding. More significantly, we identified a novel motif (PPFLMLLKGSTR) in the LG3 domain that is crucial for these responses. Studies with the synthetic peptides delineated the PPFLMLLKGSTR peptide within LG3 domain as a major site for both integrin {alpha}3{beta}1 binding and cell adhesion. Substitution mutation experiments suggest that the Arg residue is important for these activities. rLG3 protein- and PPFLMLLKGSTR peptide-induced keratinocyte adhesion triggered cell signaling through FAK phosphorylation at tyrosine-397 and -577. To our knowledge, this is the first report demonstrating that the PPFLMLLKGSTR peptide within the LG3 domain is a novel motif that is capable of supporting integrin {alpha}3{beta}1-dependent cell adhesion and spreading.

  19. Traversal of cells by radiation and absorbed fraction estimates for electrons and alpha particles

    SciTech Connect

    Eckerman, K.F.; Ryman, J.C.; Taner, A.C.; Kerr, G.D.

    1985-01-01

    Consideration of the pathlength which radiation traverses in a cell is central to algorithms for estimating energy deposition on a cellular level. Distinct pathlength distributions occur for radionuclides: (1) uniformly distributed in space about the cell (referred to as -randomness); (2) uniformly distributed on the surface of the cell (S-randomness); and (3) uniformly distributed within the cell volume (I-randomness). For a spherical cell of diameter d, the mean pathlengths are 2/3d, 1/2d, and 3/4d, respectively, for these distributions. Algorithms for simulating the path of radiation through a cell are presented and the absorbed fraction in the cell and its nucleus are tabulated for low energy electrons and alpha particles emitted on the surface of spherical cells. The algorithms and absorbed fraction data should be of interest to those concerned with the dosimetry of radionuclide-labeled monoclonal antibodies. 8 refs., 3 figs., 2 tabs.

  20. Sequestration of mutated alpha1-antitrypsin into inclusion bodies is a cell-protective mechanism to maintain endoplasmic reticulum function.

    PubMed

    Granell, Susana; Baldini, Giovanna; Mohammad, Sameer; Nicolin, Vanessa; Narducci, Paola; Storrie, Brian; Baldini, Giulia

    2008-02-01

    A variant alpha1-antitrypsin with E342K mutation has a high tendency to form intracellular polymers, and it is associated with liver disease. In the hepatocytes of individuals carrying the mutation, alpha1-antitrypsin localizes both to the endoplasmic reticulum (ER) and to membrane-surrounded inclusion bodies (IBs). It is unclear whether the IBs contribute to cell toxicity or whether they are protective to the cell. We found that in hepatoma cells, mutated alpha1-antitrypsin exited the ER and accumulated in IBs that were negative for autophagosomal and lysosomal markers, and contained several ER components, but not calnexin. Mutated alpha1-antitrypsin induced IBs also in neuroendocrine cells, showing that formation of these organelles is not cell type specific. In the presence of IBs, ER function was largely maintained. Increased levels of calnexin, but not of protein disulfide isomerase, inhibited formation of IBs and lead to retention of mutated alpha1-antitrypsin in the ER. In hepatoma cells, shift of mutated alpha1-antitrypsin localization to the ER by calnexin overexpression lead to cell shrinkage, ER stress, and impairment of the secretory pathway at the ER level. We conclude that segregation of mutated alpha1-antitrypsin from the ER to the IBs is a protective cell response to maintain a functional secretory pathway.

  1. Aggregated Alpha-Synuclein Transfer Efficiently between Cultured Human Neuron-Like Cells and Localize to Lysosomes

    PubMed Central

    Severinsson, Emelie; Agholme, Lotta; Bergström, Joakim; Ingelsson, Martin

    2016-01-01

    Parkinson’s disease and other alpha-synucleinopathies are progressive neurodegenerative diseases characterized by aggregates of misfolded alpha-synuclein spreading throughout the brain. Recent evidence suggests that the pathological progression is likely due to neuron-to-neuron transfer of these aggregates between neuroanatomically connected areas of the brain. As the impact of this pathological spreading mechanism is currently debated, we aimed to investigate the transfer and subcellular location of alpha-synuclein species in a novel 3D co-culture human cell model based on highly differentiated SH-SY5Y cells. Fluorescently-labeled monomeric, oligomeric and fibrillar species of alpha-synuclein were introduced into a donor cell population and co-cultured with an EGFP-expressing acceptor-cell population of differentiated neuron-like cells. Subsequent transfer and colocalization of the different species were determined with confocal microscopy. We could confirm cell-to-cell transfer of all three alpha-synuclein species investigated. Interestingly the level of transferred oligomers and fibrils and oligomers were significantly higher than monomers, which could affect the probability of seeding and pathology in the recipient cells. Most alpha-synuclein colocalized with the lysosomal/endosomal system, both pre- and postsynaptically, suggesting its importance in the processing and spreading of alpha-synuclein. PMID:28030591

  2. Induction of a bystander mutagenic effect of alpha particles in mammalian cells

    NASA Technical Reports Server (NTRS)

    Zhou, H.; Randers-Pehrson, G.; Waldren, C. A.; Vannais, D.; Hall, E. J.; Hei, T. K.; Chatterjee, A. (Principal Investigator)

    2000-01-01

    Ever since the discovery of X-rays was made by Rontgen more than a hundred years ago, it has always been accepted that the deleterious effects of ionizing radiation such as mutation and carcinogenesis are attributable mainly to direct damage to DNA. Although evidence based on microdosimetric estimation in support of a bystander effect appears to be consistent, direct proof of such extranuclear/extracellular effects are limited. Using a precision charged particle microbeam, we show here that irradiation of 20% of randomly selected A(L) cells with 20 alpha particles each results in a mutant fraction that is 3-fold higher than expected, assuming no bystander modulation effect. Furthermore, analysis by multiplex PCR shows that the types of mutants induced are significantly different from those of spontaneous origin. Pretreatment of cells with the radical scavenger DMSO had no effect on the mutagenic incidence. In contrast, cells pretreated with a 40 microM dose of lindane, which inhibits cell-cell communication, significantly decreased the mutant yield. The doses of DMSO and lindane used in these experiments are nontoxic and nonmutagenic. We further examined the mutagenic yield when 5-10% of randomly selected cells were irradiated with 20 alpha particles each. Results showed, likewise, a higher mutant yield than expected assuming no bystander effects. Our studies provide clear evidence that irradiated cells can induce a bystander mutagenic response in neighboring cells not directly traversed by alpha particles and that cell-cell communication process play a critical role in mediating the bystander phenomenon.

  3. Identification of sequence elements that confer cell-type-specific control of MF alpha 1 expression in Saccharomyces cerevisiae.

    PubMed Central

    Inokuchi, K; Nakayama, A; Hishinuma, F

    1987-01-01

    The MF alpha 1 gene of Saccharomyces cerevisiae, a major structural gene for mating pheromone alpha factor, is an alpha-specific gene whose expression is regulated by the mating-type locus. To study the role of sequences upstream of MF alpha 1 in its expression and regulation, we generated two sets of promoter deletions: upstream deletions and internal deletions. By analyzing these deletions, we have identified a TATA box and two closely related, tandemly arranged upstream activation sites as necessary elements for MF alpha 1 expression. Two upstream activation sites were located ca. 300 and 250 base pairs upstream of the MF alpha 1 transcription start points, which were also determined in this study. Each site contained a homologous 22-base-pair sequence, and both sites were required for maximum transcription level. The distance between the upstream activation sites and the transcription start points could be altered without causing loss of transcription efficiency, and the sites were active in either orientation with respect to the coding region. These elements conferred cell type-specific expression on a heterologous promoter. Analysis with host mating-type locus mutants indicates that these sequences are the sites through which the MAT alpha 1 product exerts its action to activate the MF alpha 1 gene. Homologous sequences with these elements were found in other alpha-specific genes, MF alpha 2 and STE3, and may mediate activation of this set of genes by MAT alpha 1. Images PMID:2959859

  4. Early detection of rheumatoid arthritis in rats and humans with 99mTc-3PRGD2 scintigraphy: imaging synovial neoangiogenesis

    PubMed Central

    Wang, Xiangcheng; Zhao, Zhenfang; Wang, Tao; Wang, Xuemei; Li, Xiao-Feng

    2017-01-01

    Objectives: To validate 99mTc-labeled arginylglycylaspartic acid (99mTc-3PRGD2) scintigraphy as a means to image synovial neoangiogenesis in joints afflicted by rheumatoid arthritis and to investigate its potential in the early detection and management of rheumatoid arthritis. Methods: Rheumatoid arthritis and osteoarthritis were generated in Sprague Dawley rats by type II collagen immunization and papain injection, respectively. Rats were imaged with 99mTc-3PRGD2 and 99mTc- methyl diphosphonate (99mTc MDP). X-ray images were also obtained and assessed by a radiologist. Immunohistochemistry of αvβ3 and CD31confirmed the onset of synovial neoangiogenesis. The effect of bevacizumab on rheumatoid arthritis was followed with 99mTc-3PRGD2 scintigraphy. A patient with rheumatoid arthritis and a healthy volunteer were scanned with 99mTc-3PRGD2. Results: Two weeks after immunization, a significant increase in 99mTc-3PRGD2 was observed in the joints of the rheumatoid arthritis model though uptake in osteoarthritis model and untreated controls was low. 99mTc-MDP whole body scans failed to distinguish early rheumatoid arthritis joints from healthy controls. The expression of αvβ3 and CD31was significantly higher in the joints of rheumatoid arthritis rats compared to normal controls. In serial 99mTc-3PRGD2 scintigraphy studies, 99mTc-3PRGD2 uptake increased in parallel with disease progression. Bevacizumab anti-angiogenetic therapy both improved the symptoms of the rheumatoid arthritis rats and significantly decreased 99mTc-3PRGD2 uptake. Significantly higher 99mTc-3PRGD2 accumulation was also observed in rheumatoid arthritis joints in the patient. Conclusions: Our findings indicate that 99mTc-3PRGD2 scintigraphy could detect early rheumatoid arthritis by imaging the associated synovial neoangiogenesis, and may be useful in disease management. PMID:27992368

  5. Dual requirement for the Ig alpha immunoreceptor tyrosine-based activation motif (ITAM) and a conserved non-Ig alpha ITAM tyrosine in supporting Ig alpha beta-mediated B cell development.

    PubMed

    Pike, Kelly A; Ratcliffe, Michael J H

    2005-02-15

    Surface Ig (sIg) expression is a critical checkpoint during avian B cell development. Only cells that express sIg colonize bursal follicles, clonally expand, and undergo Ig diversification by gene conversion. Expression of a heterodimer, in which the extracellular and transmembrane domains of murine CD8alpha or CD8beta are fused to the cytoplasmic domains of chicken Igalpha (chIgalpha) or Igbeta, respectively (murine CD8alpha (mCD8alpha):chIgalpha + mCD8beta:chIgbeta), or an mCD8alpha:chIgalpha homodimer supported bursal B cell development as efficiently as endogenous sIg. In this study we demonstrate that B cell development, in the absence of chIgbeta, requires both the Igalpha ITAM and a conserved non-ITAM Igalpha tyrosine (Y3) that has been associated with binding to B cell linker protein (BLNK). When associated with the cytoplasmic domain of Igbeta, the Igalpha ITAM is not required for the induction of strong calcium mobilization or BLNK phosphorylation, but is still necessary to support B cell development. In contrast, mutation of the Igalpha Y3 severely compromised calcium mobilization when expressed as either a homodimer or a heterodimer with the cytoplasmic domain of Igbeta. However, coexpression of the cytoplasmic domain of Igbeta partially complemented the Igalpha Y3 mutation, rescuing higher levels of BLNK phosphorylation and, more strikingly, supporting B cell development.

  6. The neuro-steroid, 3beta androstene 17alpha diol exhibits potent cytotoxic effects on human malignant glioma and lymphoma cells through different programmed cell death pathways.

    PubMed

    Graf, M R; Jia, W; Loria, R M

    2007-09-03

    The neuro-steroids 3beta-androstene-17alpha-diol (17alpha-AED), 3beta-androstene-17beta-diol (17beta-AED), 3beta-androstene-7alpha,-17beta-triol (7alpha-AET) and 3beta-androstene-7beta,-17beta-triol (7beta-AET) are metabolites of dehydroepiandrosterone and are produced in neuro-ectodermal tissue. Both epimers of androstenediols (17alpha-AED and 17beta-AED) and androstenetriols (7alpha-AET and 7beta-AET) have markedly different biological functions of their chemical analogue. We investigated the cytotoxic activity of these neuro-steroids on human T98G and U251MG glioblastoma and U937 lymphoma cells. Proliferation studies showed that 17alpha-AED is the most potent inhibitor, with an IC(50) approximately 15 microM. For T98G glioma, 90% inhibition was achieved with 25 muM of 17alpha-AED. Other neuro-steroids tested only marginally suppressed cell proliferation. Reduced cell adherence and viability could be detected after 18 h of 17alpha-AED exposure. Treatment with 17alpha-AED induced a significant level of apoptosis in U937 lymphoma cells, but not in the glioma cells. Cytopathology of 17alpha-AED-treated T98G cells revealed the presence of multiple cytoplasmic vacuoles. Acridine orange staining demonstrated the formation of acidic vesicular organelles in 17alpha-AED-treated T98G and U251MG, which was inhibited by bafilomycin A1. These findings indicate that 17alpha-AED bears the most potent cytotoxic activity of the neuro-steroids tested, and the effectiveness may depend on the number of hydroxyls and their position on the androstene molecule. These cytotoxic effects may utilize a non-apoptotic pathway in malignant glioma cells.

  7. Synchronization of HeLa cell cultures by inhibition of DNA polymerase alpha with aphidicolin.

    PubMed Central

    Pedrali-Noy, G; Spadari, S; Miller-Faurès, A; Miller, A O; Kruppa, J; Koch, G

    1980-01-01

    Both the inhibitory effect of aphidicolin on the replicative alpha-polymerase and the reversibility of its action in vivo (Pedrali-Noy & Spadari, 1979, Biochem. Biophys. Res. Commun. 88, 1194-2002) allow the synchronization of cells in culture. Aphidicolin prevents G1 cells from entering the DNA synthetic period, blocks cells in "S" phase, allows G2, M and G1 cells to continue the cell cycle and to accumulate at the G1/S border. Aphidicolin is a more useful reagent than hydroxyurea and thymidine because it does not affect cell viability or "S" phase duration and does not interfere with the synthesis of dNTPs or DNA polymerases. In fact cells exposed to the drug continue to synthesize all three DNA polymerases alpha, beta and gamma as well as all dNTPs which, when the block is removed, are present at levels optimal for DNA initiation and replication. The technique is simple and can be applied to cells growing in suspension or monolayers and allows one to harvest large quantities of synchronized cells. PMID:6775308

  8. The involvement of hypoxia-inducible factor-1alpha in the susceptibility to gamma-rays and chemotherapeutic drugs of oral squamous cell carcinoma cells.

    PubMed

    Sasabe, Eri; Zhou, Xuan; Li, Dechao; Oku, Naohisa; Yamamoto, Tetsuya; Osaki, Tokio

    2007-01-15

    The transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha) is the key regulator that controls the hypoxic response of mammalian cells. The overexpression of HIF-1alpha has been demonstrated in many human tumors. However, the role of HIF-1alpha in the therapeutic efficacy of chemotherapy and radiotherapy in cancer cells is poorly understood. In this study, we investigated the influence of HIF-1alpha expression on the susceptibility of oral squamous cell carcinoma (OSCC) cells to chemotherapeutic drugs (cis-diamminedichloroplatinum and 5-fluorouracil) and gamma-rays. Treatment with chemotherapeutic drugs and gamma-rays enhanced the expression and nuclear translocation of HIF-1alpha, and the susceptibility of OSCC cells to the drugs and gamma-rays was negatively correlated with the expression level of HIF-1alpha protein. The overexpression of HIF-1alpha induced OSCC cells to become more resistant to the anticancer agents, and down-regulation of HIF-1alpha expression by small interfering RNA enhanced the susceptibility of OSCC cells to them. In the HIF-1alpha-knockdown OSCC cells, the expression of P-glycoprotein, heme oxygenase-1, manganese-superoxide dismutase and ceruloplasmin were downregulated and the intracellular levels of chemotherapeutic drugs and reactive oxygen species were sustained at higher levels after the treatment with the anticancer agents. These results suggest that enhanced HIF-1alpha expression is related to the resistance of tumor cells to chemo- and radio-therapy and that HIF-1alpha is an effective therapeutic target for cancer treatment.

  9. Inhibition of mucin glycosylation by aryl-N-acetyl-alpha-galactosaminides in human colon cancer cells

    SciTech Connect

    Kuan, S.F.; Byrd, J.C.; Basbaum, C.; Kim, Y.S. )

    1989-11-15

    Specific inhibitors of the glycosylation of O-glycosidically linked glycoproteins have not previously been described. When tested for their effects on mucin glycosylation in a mucin-producing colon cancer cell line, LS174T, benzyl-, phenyl-, and p-nitrophenyl-N-acetyl-alpha-galactosaminide inhibited the formation of fully glycosylated mucin in a dose-dependent manner. Free aryl-oligosaccharides were found in the medium of treated cells labeled with ({sup 3}H)glucosamine, ({sup 3}H)galactose, ({sup 3}H)fucose, ({sup 3}H)mannosamine, or phenyl-alpha-(6-{sup 3}H) N-acetylgalactosamine. UDP-Gal:GalNAc-beta 1,3-galactosyltransferase was inhibited by aryl-N-acetyl-alpha-galactosaminides but not by a number of other aryl-glycosides. Treatment with these inhibitors also causes reversible morphologic changes including formation of intercellular cysts. Aryl-N-acetyl-alpha-galactosaminides can be useful for the structural and functional studies of mucin macromolecules and other O-linked glycoproteins.

  10. Mushroom acidic glycosphingolipid induction of cytokine secretion from murine T cells and proliferation of NK1.1 {alpha}/{beta} TCR-double positive cells in vitro

    SciTech Connect

    Nozaki, Hirofumi; Itonori, Saki; Sugita, Mutsumi; Nakamura, Kimihide; Ohba, Kiyoshi; Suzuki, Akemi; Kushi, Yasunori

    2008-08-29

    Interferon (IFN)-{gamma} and interleukin (IL)-4 regulate many types of immune responses. Here we report that acidic glycosphingolipids (AGLs) of Hypsizigus marmoreus and Pleurotus eryngii induced secretion of IFN- {gamma} and IL-4 from T cells in a CD11c-positive cell-dependent manner similar to that of {alpha}-galactosylceramide ({alpha}-GalCer) and isoglobotriaosylceramide (iGb3), although activated T cells by AGLs showed less secretion of cytokine than those activated by {alpha}-GalCer. In addition, stimulation of these mushroom AGLs induced proliferation of NK1.1 {alpha}/{beta} TCR-double positive cells in splenocytes. Administration of a mixture of {alpha}-GalCer and AGLs affected the stimulation of {alpha}-GalCer and generally induced a subtle Th1 bias for splenocytes but induced an extreme Th2 bias for thymocytes. These results suggested that edible mushroom AGLs contribute to immunomodulation.

  11. Proliferation of Estrogen Receptor alpha Positive Mammary Epithelial Cells is Restrained by TGFbeta1 in Adult Mice

    SciTech Connect

    Ewan, Kenneth B.R.; Oketch-Rabah, Hellen A.; Ravani, Shraddha A.; Shyamala, G.; Moses, Harold L.; Barcellos-Hoff, Mary Helen

    2005-03-03

    Transforming growth factor {beta}1 (TGF{beta}1) is a potent inhibitor of mammary epithelial proliferation. In human breast, estrogen receptor {alpha} (ER{alpha}) cells rarely co-localize with markers of proliferation, but their increased frequency correlates with breast cancer risk. To determine whether TGF{beta}1 is necessary for the quiescence of ER{alpha}-positive population, we examined mouse mammary epithelial gland at estrus. Approximately 35% of cells showed TGF{beta}1 activation, which co-localized with nuclear receptor-phosphorylated Smad 2/3, indicating that TGF{beta} signaling is autocrine. Furthermore, nuclear Smad co-localized with nuclear ER{alpha}. To test whether TGF{beta} was functional, we examined genetically engineered mice with different levels of TGF{beta}1. ER{alpha} co-localization with markers of proliferation (i.e. Ki-67 or BrdU) at estrus was significantly increased in the mammary glands of Tgf{beta}1 C57/bl/129SV heterozygote mice. This relationship was maintained following pregnancy, but was absent at puberty. Conversely, mammary epithelial expression of constitutively active TGF{beta}1 via the MMTV promoter suppressed proliferation of ER{alpha} positive cells. Thus, TGF{beta}1 activation functionally restrains ER{alpha} positive cells from proliferating in adult mammary gland. Accordingly, we propose that TGF{beta}1 dysregulation may promote proliferation of ER{alpha} positive cells associated with breast cancer risk in humans.

  12. Uncoupling of stem cell inhibition from monocyte chemoattraction in MIP-1alpha by mutagenesis of the proteoglycan binding site.

    PubMed

    Graham, G J; Wilkinson, P C; Nibbs, R J; Lowe, S; Kolset, S O; Parker, A; Freshney, M G; Tsang, M L; Pragnell, I B

    1996-12-02

    We have studied the role of proteoglycans in the function of Macrophage Inflammatory Protein-1 alpha (MIP-1alpha), a member of the proteoglycan binding chemokine family. Sequence and peptide analysis has identified a basic region within MIP-1alpha which appears to be the major determinant of proteoglycan binding and we have now produced a mutant of MIP-1alpha lacking the basic charges on two of the amino acids within this proteoglycan binding site. This mutant (Hep Mut) appears to have lost the ability to bind to proteoglycans. Bioassay of Hep Mut indicates that it has retained stem cell inhibitory properties but has a compromised activity as a monocyte chemoattractant, thus suggesting uncoupling of these two properties of MIP-1alpha. Receptor studies have indicated that the inactivity of Hep Mut on human monocytes correlates with its inability to bind to CCR1, a cloned human MIP-1alpha receptor. In addition, studies using proteoglycan deficient cells transfected with CCR1 have indicated that the proteoglycan binding site in MIP-1alpha is a site that is also involved in the docking of MIP-1alpha to the monocyte receptor. The site for interaction with the stem cell receptor must therefore be distinct, suggesting that MIP-1alpha utilizes different receptors for these two different biological processes.

  13. Uncoupling of stem cell inhibition from monocyte chemoattraction in MIP-1alpha by mutagenesis of the proteoglycan binding site.

    PubMed Central

    Graham, G J; Wilkinson, P C; Nibbs, R J; Lowe, S; Kolset, S O; Parker, A; Freshney, M G; Tsang, M L; Pragnell, I B

    1996-01-01

    We have studied the role of proteoglycans in the function of Macrophage Inflammatory Protein-1 alpha (MIP-1alpha), a member of the proteoglycan binding chemokine family. Sequence and peptide analysis has identified a basic region within MIP-1alpha which appears to be the major determinant of proteoglycan binding and we have now produced a mutant of MIP-1alpha lacking the basic charges on two of the amino acids within this proteoglycan binding site. This mutant (Hep Mut) appears to have lost the ability to bind to proteoglycans. Bioassay of Hep Mut indicates that it has retained stem cell inhibitory properties but has a compromised activity as a monocyte chemoattractant, thus suggesting uncoupling of these two properties of MIP-1alpha. Receptor studies have indicated that the inactivity of Hep Mut on human monocytes correlates with its inability to bind to CCR1, a cloned human MIP-1alpha receptor. In addition, studies using proteoglycan deficient cells transfected with CCR1 have indicated that the proteoglycan binding site in MIP-1alpha is a site that is also involved in the docking of MIP-1alpha to the monocyte receptor. The site for interaction with the stem cell receptor must therefore be distinct, suggesting that MIP-1alpha utilizes different receptors for these two different biological processes. Images PMID:8978677

  14. Fibronectin type III5 repeat contains a novel cell adhesion sequence, KLDAPT, which binds activated alpha4beta1 and alpha4beta7 integrins.

    PubMed

    Moyano, J V; Carnemolla, B; Domínguez-Jiménez, C; García-Gila, M; Albar, J P; Sánchez-Aparicio, P; Leprini, A; Querzé, G; Zardi, L; Garcia-Pardo, A

    1997-10-03

    The region of fibronectin encompassing type III repeats 4-6 contains a low affinity heparin binding domain, but its physiological significance is not clear. We have studied whether this domain is able to interact with cells as already shown for other heparin binding domains of fibronectin. A computer search based on homologies with known active sites in fibronectin revealed the sequence KLDAPT located in FN-III5. A synthetic peptide containing this sequence induced lymphoid cell adhesion upon treatment with the activating anti-beta1 monoclonal antibody (mAb) TS2/16 or with Mn2+, indicating that KLDAPT was binding to an integrin. A recombinant fragment containing repeat III5 (FN-III5) also mediated adhesion of TS2/16/Mn2+-treated cells while the FN-III6 fragment did not. Soluble KLDAPT peptide inhibited cell adhesion to FN-III5 as well as to a 38-kDa fibronectin fragment and VCAM-1, two previously known ligands for alpha4beta1 integrin. KLDAPT also competed with the binding of soluble alkaline phosphatase-coupled VCAM-Ig to Mn2+-treated alpha4beta1. Furthermore, mAbs anti-alpha4 and anti-alpha4beta7, but not mAbs to other integrins, inhibited cell adhesion to FN-III5 and KLDAPT. These results therefore establish a cell adhesive function for the FN-III5 repeat and show that KLDAPT is a novel fibronectin ligand for activated alpha4 integrins.

  15. Adenovirus-mediated transfer of a recombinant human alpha 1-antitrypsin cDNA to human endothelial cells.

    PubMed Central

    Lemarchand, P; Jaffe, H A; Danel, C; Cid, M C; Kleinman, H K; Stratford-Perricaudet, L D; Perricaudet, M; Pavirani, A; Lecocq, J P; Crystal, R G

    1992-01-01

    To evaluate the feasibility of using a replication-deficient recombinant adenovirus to transfer human genes to the human endothelium, human umbilical vein endothelial cells were infected in vitro with adenovirus vectors containing the lacZ gene or a human alpha 1-antitrypsin (alpha 1AT) cDNA. After in vitro infection with the lacZ adenovirus vector, cultured endothelial cells expressed beta-galactosidase. In parallel studies with the alpha 1AT adenovirus vector, infected cells expressed human alpha 1AT transcripts, as evidenced by in situ hybridization and Northern analysis, and de novo synthesized and secreted glycosylated, functional alpha 1AT within 6 hr of infection, as shown by [35S]methionine labeling and immunoprecipitation. Quantification of the culture supernatants demonstrated 0.3-0.6 micrograms of human alpha 1AT secreted per 10(6) cells in 24 hr, for at least 14 days after adenovirus vector infection. To demonstrate the feasibility of direct transfer of genes into endothelial cells in human blood vessels, lacZ or alpha 1AT adenovirus vectors were placed in the lumen of intact human umbilical veins ex vivo. Histologic evaluation of the veins after 24 hr demonstrated transfer and expression of the lacZ gene specifically to the endothelium. alpha 1AT adenovirus infection resulted both in expression of alpha 1AT transcripts in the endothelium and in de novo synthesis and secretion of alpha 1AT. Quantification of alpha 1AT in the vein perfusates showed average levels of 13 micrograms/ml after 24 hr. These observations strongly support the feasibility of in vivo human gene transfer to the endothelium mediated by replication-deficient adenovirus vectors. Images PMID:1631146

  16. Oxygen-dependent expression of hypoxia-inducible factor-1alpha in renal medullary cells of rats.

    PubMed

    Zou, A P; Yang, Z Z; Li, P L; Cowley AW, J R

    2001-08-28

    Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcription factor that regulates the oxygen-dependent expression of a number of genes. This transcription factor may contribute to the abundant expression of many genes in renal medullary cells that function normally under hypoxic conditions. The present study was designed to determine the characteristics of HIF-1alpha cDNA cloned from the rat kidney and the expression profile of HIF-1alpha in different kidney regions and to explore the mechanism activating or regulating HIF-1alpha expression in renal medullary cells. A 3,718-bp HIF-1alpha cDNA from the rat kidney was first cloned and sequenced using RT-PCR and TA cloning technique. It was found that 823 amino acids deduced from this renal HIF-1alpha cDNA had 99%, 96%, and 90% identity with rat, mouse, or human HIF-1alpha deposited in GenBank, respectively. The 3'-untranslated region of HIF-1alpha mRNA from the rat kidney contained seven AUUUA instability elements, five of which were found to be conserved among rat, mouse, and human HIF-1alpha. Northern blot analyses demonstrated a corticomedullary gradient of HIF-1alpha mRNA expression in the kidney, with the greatest abundance in the renal inner medulla. Western blot analyses also detected a higher HIF-1alpha protein level in the nuclear extracts from the renal medulla than the renal cortex. A classic loop diuretic, furosemide (10 mg/kg ip), markedly increased renal medullary Po(2) levels from 22.5 to 52.2 mmHg, which was accompanied by a significant reduction of HIF-1alpha transcripts in renal medullary tissue. In in vitro experiments, low Po(2), but not elevated osmolarity, was found to significantly increase HIF-1alpha mRNA in renal medullary interstitial cells and inner medullary collecting duct cells. These results indicate that HIF-1alpha is more abundantly expressed in the renal medulla compared with the renal cortex. Increased abundance of HIF-1alpha mRNA in the renal medulla may represent an adaptive

  17. Modulation of hematopoiesis via alpha 1-adrenergic receptors on bone marrow cells.

    PubMed

    Maestroni, G J; Conti, A

    1994-03-01

    We have recently demonstrated that adrenergic agents can affect hematopoiesis after syngeneic bone marrow transplantation in mice. In particular, chemical sympathectomy by 6-hydroxydopamine (6-OHDA) and/or administration of the alpha 1-adrenergic antagonist prazosin were shown to increase the concentration of blood granulocytes, platelets, and bone marrow colony-forming units-granulocyte/macrophage (CFU-GM), and to induce a granulocytic hyperplasia of the spleen. Here we show that prazosin can also enhance myelopoiesis and platelet formation in normal mice. Furthermore, noradrenaline and the alpha 1-adrenergic agonist methoxamine could directly inhibit the in vitro growth of GM-CFU. The effect of noradrenaline was counteracted by prazosin and by other alpha-adrenergic antagonists such as phentolamine and yohimbine, in the following order of potency: prazosin > phentolamine > yohimbine. In line with these results, we were able to demonstrate that 3H-prazosin binds specifically to both bone marrow cell membranes and intact bone marrow cells. Scatchard analysis of the binding to intact cells revealed the presence of two binding sites. A kd of 0.98 +/- 0.32 nM and a B max of 5 +/- 2.9 fM/2 x 10(6) cells characterized the higher affinity site, while the lower affinity site displayed a kd of 55.9 +/- 8.2 nM and a B max of 44 +/- 7.7 fM/mg protein. These saturation studies, together with competition experiments to evaluate the ability of various adrenergic compounds to displace 3H-prazosin binding, classified the higher affinity site as an alpha 1-adrenergic receptor. The remaining low affinity binding site remains to be characterized. Furthermore, separation of bone marrow cells by counterflow centrifugal elutriation (CCE) showed that the high-affinity binding is due to a lymphoid/stem cell fraction with no blasts and no GM-CFU progenitors. The low-affinity site was apparent on the rotor-off fraction, which was enriched with GM-CFU progenitor cells. These findings

  18. Functional analysis of {alpha}1,3/4-fucosyltransferase VI in human hepatocellular carcinoma cells

    SciTech Connect

    Guo, Qiya; Guo, Bin; Wang, Yingming; Wu, Jun; Jiang, Wenjun; Zhao, Shenan; Qiao, Shouyi; Wu, Yanhua

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Human FUT6 is up-regulated in HCC tissues. Black-Right-Pointing-Pointer Expression of FUT6 promotes G0/G1-S transition and cell growth. Black-Right-Pointing-Pointer FUT6 confers a growth advantage in vivo. Black-Right-Pointing-Pointer FUT6 suppresses p21 expression through modulating PI3K/Akt signaling. -- Abstract: The {alpha}1,3/4-fucosyltransferases (FUT) subfamily are key enzymes in cell surface antigen synthesis during various biological processes. A novel role of FUTs in tumorigenesis has been discovered recently, however, the underlying mechanism remains largely unknown. Here, we characterized FUT6, a member of {alpha}1,3/4-FUT subfamily, in human hepatocellular carcinoma (HCC). In HCC tissues, the expression levels of FUT6 and its catalytic product SLe{sup x} were significantly up-regulated. Overexpression of FUT6 in HCC cells enhanced S-phase cell population, promoted cell growth and colony formation ability. Moreover, subcutaneously injection of FUT6-overexpressing cells in nude mice promoted cell growth in vivo. In addition, elevating FUT6 expression markedly induced intracellular Akt phosphorylation, and suppressed the expression of the cyclin-dependent kinases inhibitor p21. Bath application of the PI3K inhibitor blocked FUT6-induced Akt phosphorylation, p21 suppression and cell proliferation. Our results suggest that FUT6 plays an important role in HCC growth by regulating the PI3K/Akt signaling pathway.

  19. NeuAc alpha 2,3gal-glycoconjugate expression determines cell susceptibility to the porcine rubulavirus LPMV.

    PubMed

    Reyes-Leyva, J; Espinosa, B; Hernandez, J; Zenteno, R; Vallejo, V; Hernández-Jáuregui, P; Zenteno, E

    1997-10-01

    Relevance of membrane sialoglycoconjugates as receptors for infection by the porcine rubulavirus has been determined in vitro by sugar and lectin competition assays and by inhibition of glycosylation. Our results show that NeuAc alpha 2,3Gal but not NeuAc alpha 2,6Gal inhibits the virus infectivity of Vero cells, and the virus was effectively blocked with the lectin Maackia amurensis, specific for NeuAc alpha 2,3Gal. Inhibition of the cellular glycosylation with tunicamycin, deoxinojirimycin as well as neuraminidase treatment diminishes the viral capacity to bind and infect this cell line. Dexamethasone, which promotes the activity of sialyl alpha 2,6 glycosyltransferase, also diminishes the cell susceptibility for infection. This is the first report confirming that NeuAc alpha-2,3Gal recognition is determinant in the pathogenesis of the porcine rubulavirus.

  20. Tumor necrosis factor alpha gene expression in human monocytic THP-1 cells exposed to beryllium.

    PubMed

    Galbraith, G M; Pandey, J P; Schmidt, M G; Arnaud, P; Goust, J M

    1996-01-01

    Chronic beryllium disease, which results from occupational exposure to particulate beryllium, is characterized by the development of lung granulomas and progressive pulmonary fibrosis. Increased production of proinflammatory cytokines (e.g., tumor necrosis factor alpha and interleukin-1 beta) by pulmonary alveolar macrophages occurs in many chronic fibrotic lung diseases and is thought to contribute to the disease process. The purpose of the present study was to investigate cytokine production by human monocytic cells exposed to beryllium in vitro. The results indicated that such cells respond to beryllium ions in the presence of fluoride by accumulation of messenger ribonucleic acid for both tumor necrosis factor alpha and interleukin-1 beta. These findings suggest that inhaled beryllium may directly stimulate the production of these cytokines by alveolar macrophages in vitro.

  1. alpha2beta1 integrin controls association of Rac with the membrane and triggers quiescence of endothelial cells.

    PubMed

    Cailleteau, Laurence; Estrach, Soline; Thyss, Raphael; Boyer, Laurent; Doye, Anne; Domange, Barbara; Johnsson, Nils; Rubinstein, Eric; Boucheix, Claude; Ebrahimian, Teni; Silvestre, Jean-Sebastien; Lemichez, Emmanuel; Meneguzzi, Guerrino; Mettouchi, Amel

    2010-07-15

    Integrin receptors and their extracellular matrix ligands provide cues to cell proliferation, survival, differentiation and migration. Here, we show that alpha2beta1 integrin, when ligated to the basement membrane component laminin-1, triggers a proliferation arrest in primary endothelial cells. Indeed, in the presence of strong growth signals supplied by growth factors and fibronectin, alpha2beta1 engagement alters assembly of mature focal adhesions by alpha5beta1 and leads to impairment of downstream signaling and cell-cycle arrest in the G1 phase. Although the capacity of alpha5beta1 to signal for GTP loading of Rac is preserved, the joint engagement of alpha2beta1 interferes with membrane anchorage of Rac. Adapting the 'split-ubiquitin' sensor to screen for membrane-proximal alpha2 integrin partners, we identified the CD9 tetraspanin and further establish its requirement for destabilization of focal adhesions, control of Rac subcellular localization and growth arrest induced by alpha2beta1 integrin. Altogether, our data establish that alpha2beta1 integrin controls endothelial cell commitment towards quiescence by triggering a CD9-dependent dominant signaling.

  2. The role of transforming growth factor alpha production and ErbB-2 overexpression in induction of tumorigenicity of lung epithelial cells.

    PubMed Central

    Hamburger, A. W.; Fernandes, A.; Murakami, M.; Gerwin, B. I.

    1998-01-01

    Over-expression of erbB-2 is associated with shortened survival of patients with lung adenocarcinomas. We demonstrated that human lung epithelial cells, overexpressing erbB-2, formed tumours in nude mice only when high levels of transforming growth factor (TGF)-alpha were produced (E6T cells). To define the role that TGF-alpha production played in induction of tumorigenicity, a non-tumorigenic TGF-alpha-negative clone of ErbB-2 overexpressing cells (E2 cells) was transfected with an expression vector for TGF-alpha (E2alpha cells). Transfected clones produced TGF-alpha at 11-25% of the level produced by the E6T cell line. Tumorigenic E6T cells transfected with a TGF-alpha antisense vector (E6TA cells) expressed only 6% of the TGF-alpha level of the parental cells. Clones of E6T, E6TA, E2 and E2alpha were inoculated into athymic nude mice to measure tumorigenic potential. E6T cells formed tumours with a 70% efficiency. E2, E6TA and E2alpha cells failed to form tumours. The levels of EGFR were similar in non-tumorigenic E2 and tumorigenic E6T cells but higher in E2alpha and E6TA cells, and ErbB-2 were greatly overexpressed in an E2alpha clone. In vitro, ErbB-2 co-immunoprecipitated with EGFR in lysates of unstimulated E6T and E2alpha TGF-alpha-producing cells, indicating that the lower TGF-alpha levels were sufficient to induce in vitro heterodimerization. These studies suggest that induction of the tumorigenic phenotype depends on achieving a threshold level of TGF-alpha sufficient to activate downstream signalling by ErbB-2 containing active heterodimers. Images Figure 1 Figure 2 Figure 3 PMID:9569041

  3. Tumor suppressor KAI1 affects integrin {alpha}v{beta}3-mediated ovarian cancer cell adhesion, motility, and proliferation

    SciTech Connect

    Ruseva, Zlatna; Geiger, Pamina Xenia Charlotte; Hutzler, Peter; Kotzsch, Matthias; Luber, Birgit; Schmitt, Manfred; Gross, Eva; Reuning, Ute

    2009-06-10

    The tetraspanin KAI1 had been described as a metastasis suppressor in many different cancer types, a function for which associations of KAI1 with adhesion and signaling receptors of the integrin superfamily likely play a role. In ovarian cancer, integrin {alpha}v{beta}3 correlates with tumor progression and its elevation in vitro provoked enhanced cell adhesion accompanied by significant increases in cell motility and proliferation in the presence of its major ligand vitronectin. In the present study, we characterized integrin {alpha}v{beta}3-mediated tumor biological effects as a function of cellular KAI1 restoration and proved for the first time that KAI1, besides its already known physical crosstalk with {beta}1-integrins, also colocalizes with integrin {alpha}v{beta}3. Functionally, elevated KAI1 levels drastically increased integrin {alpha}v{beta}3/vitronectin-dependent ovarian cancer cell adhesion. Since an intermediate level of cell adhesive strength is required for optimal cell migration, we next studied ovarian cancer cell motility as a function of KAI1 restoration. By time lapse video microscopy, we found impaired integrin {alpha}v{beta}3/vitronectin-mediated cell migration most probably due to strongly enhanced cellular immobilization onto the adhesion-supporting matrix. Moreover, KAI1 reexpression significantly diminished cell proliferation. These data strongly indicate that KAI1 may suppress ovarian cancer progression by inhibiting integrin {alpha}v{beta}3/vitronectin-provoked tumor cell motility and proliferation as important hallmarks of the oncogenic process.

  4. The humoral response in TCR alpha-/- mice. Can gammadelta-T cells support the humoral immune response?

    PubMed

    Lindroth, K; Troye-Blomberg, M; Singh, M; Dieli, F; Ivanyi, J; Fernández, C

    2002-03-01

    An optimal humoral response requires T-cell help; however, it has been questioned if this help comes exclusively from alphabeta-T cells or whether gammadelta-T cells also contribute. We have attempted to answer this question by studying the humoral response in T-cell receptor alpha-chain knockout (alpha-/-) mice, which lack the alphabetaT cell subset. Two model antigens were used to characterize the response: the thymus-independent (TI) antigen native dextran B512 (Dx), and the thymus-dependent (TD) antigen heat shock protein (HSP65) from Mycobacterium tuberculosis. When challenged with Dx, the alpha-/- mice elicited a strong antibody response and formed rudimentary germinal centres (GCs), a T-cell dependent reaction. In contrast, the humoral response to HSP65 was poor. However, alpha-/- mice became primed when challenged with HSP65, because when supplemented with wild-type thymocytes, the antigen-primed animals were able to mount a stronger response than the nonprimed ones when challenged with HSP65. A crucial step seems to be the collaboration between gammadeltaT cells and antigen presenting cells (APCs), as splenocytes from alpha-/- mice were able to respond to HSP65 in an environment containing primed-APCs. Based on these results, we propose a model for B-cell activation in the alpha-/- mice.

  5. GRO-alpha in normal and pathological thyroid tissues and its regulation in thyroid-derived cells.

    PubMed

    Aust, G; Steinert, M; Boltze, C; Kiessling, S; Simchen, C

    2001-09-01

    Thyroid glands affected by Graves' disease (GD) show striking leukocytic infiltration, mainly by T-cells. The mechanisms by which the various leukocytes are maintained in the thyroid are unknown. Growth-regulated oncogene-alpha (GRO-alpha) in interaction with its receptor CXCR2 is a chemoattractant for both T-cells and neutrophils and may be one of the chemokines involved in the cell maintenance. GRO-alpha and CD18 mRNA as a marker of leukocytic infiltration were quantified in thyroid tissue using competitive RT-PCR. We found very high GRO-alpha mRNA levels in all thyroid tissues. In GD patients (n=16), the GRO-alpha mRNA did not correlate with the CD18 mRNA level or thyroid peroxidase and TSH-receptor antibodies in patients' sera. In thyroid autonomy (n=10), the GRO-alpha mRNA levels were significantly lower in autonomous single adenomas compared with the corresponding normal tissue. In order to define the cellular source of GRO-alpha mRNA and protein, we examined various thyroid-derived cells. Thyrocytes, thyroid-derived leukocytes and fibroblasts showed basal GRO-alpha mRNA and protein expression, which was remarkably upregulated by different stimuli in vitro. The expression of GRO-alpha by thyroid carcinoma cell lines confirms that thyrocytes may actually produce GRO-alpha. As shown by flow cytometry and immunohistology, CD68+ monocytes/macrophages are the only cell population strongly expressing CXCR2 in the thyroid.

  6. Disruption by interferon-alpha of an autocrine interleukin-6 growth loop in IL-6-dependent U266 myeloma cells by homologous and heterologous down-regulation of the IL-6 receptor alpha- and beta-chains.

    PubMed Central

    Schwabe, M; Brini, A T; Bosco, M C; Rubboli, F; Egawa, M; Zhao, J; Princler, G L; Kung, H F

    1994-01-01

    IL-6 is an autocrine growth factor for U266 myeloma cells and their growth is inhibited by IFN-alpha or IL-6 mAb. We asked, therefore, whether IFN-alpha-induced growth inhibition involved IL-6. IFN-alpha and mAb against IL-6, the IL-6R alpha-(gp80) or beta-chain (gp130) potently inhibited U266 cells. Remarkably, this effect occurred despite IFN-alpha-augmented secretion of endogenous IL-6. However, examining the IL-6R revealed that IFN-alpha drastically curtailed expression of the IL-6R alpha- and beta-chain. This effect occurred on two different levels (protein and mRNA) and by two different mechanisms (directly and indirectly through IL-6). First, IFN-alpha, but not IL-6, greatly decreased gp80 and, to a lesser extent, gp130 mRNA levels which resulted in a loss of IL-6 binding sites. Second, IFN-alpha-induced IL-6 predominantly down-regulated membrane-bound gp130. IFN-alpha-mediated decrease of gp80 levels was not detected on IL-6-independent myeloma (RPMI 8226) or myeloid cells (U937). We conclude that IFN-alpha inhibited IL-6-dependent myeloma cell growth by depriving U266 cells of an essential component of their autocrine growth loop, a functional IL-6R. Images PMID:7989587

  7. Resolution and purification of free primase activity from the DNA primase-polymerase alpha complex of HeLa cells.

    PubMed Central

    Vishwanatha, J K; Baril, E F

    1986-01-01

    DNA primase activity has been resolved from a purified DNA primase-polymerase alpha complex of HeLa cells by hydrophobic affinity chromatography on phenylSepharose followed by chromatography on hexylagarose. This procedure provides a good yield (55%) of DNA primase that is free from polymerase alpha. The free DNA primase activity was purified to near homogeneity and its properties characterized. Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of the purified free DNA primase showed a major protein staining band of Mr 70,000. The native enzyme in velocity sedimentation has an S20'W of 5. DNA primase synthesizes RNA oligomers with single-stranded M-13 DNA, poly(dT) and poly(dC) templates that are elongated by the DNA polymerase alpha in a manner that has already been described for several purified eukaryotic DNA primase-polymerase alpha complexes. The purified free DNA primase activity is resistant to neutralizing anti-human DNA polymerase alpha antibodies, BuPdGTP and aphidicolin that specifically inhibit the free DNA polymerase alpha and also DNA polymerase alpha complexed with the primase. The free primase activity is more sensitive to monovalent salt concentrations and is more labile than polymerase alpha. Taken together these results indicate that the DNA primase and polymerase alpha activities of the DNA primase-polymerase alpha complex reside on separate polypeptides that associate tightly through hydrophobic interactions. Images PMID:3786132

  8. In utero transplantation of monocytic cells in cats with alpha-mannosidosis

    PubMed Central

    Abkowitz, Janis L.; Sabo, Kathleen M.; Yang, Zhantao; Vite, Charles H.; Shields, Laurence E.; Haskins, Mark E.

    2009-01-01

    Background Lysosomal storage diseases are devastating illnesses, in large part because of their neurological consequences. Since significant morbidity occurs prenatally, in utero therapy is an attractive therapeutic approach. Methods We studied the feasibility and efficacy of in utero (IU) injections of monocytic cells (derived from normal marrow) in feline alpha-mannosidosis. Heterozygous cats were interbred to produce affected (homozygous) and control (heterozygous and wild-type) offspring. Thirty-seven pregnancies were studied in which fetuses were transplanted intraperitoneally (1 × 108 cells/kg recipient) at gestational day 27–33 and then each week for two weeks (term=63 days). After birth, affected kittens were evaluated clinically and pathologically, tissue alpha-mannosidase (MANB) levels were assayed, and in many studies, the numbers of MANB-containing cells were enumerated. When male donor cells were transplanted into female recipients, engraftment was also quantified using PCR to amplify a Y-chromosome-specific sequence. Results We establish methods to transplant cats intraperitoneally while IU using ultrasound guidance, thus, describing a new large animal model for prenatal therapy. We show that the donor monocytic cells engraft and persist (for up to 125 days) in brain, liver, and spleen, albeit at levels below those needed to alter the clinical or pathological progression of the alpha-mannosidosis. Conclusions This is the first study of monocyte transplantation in a large animal model of a lysosomal storage disorder and demonstrates its feasibility, safety, and promise. Delivering cells IU may be a useful strategy to prevent morbidities before a definitive therapy, such as hematopoietic stem cell transplantation, can be administered after birth. PMID:19667933

  9. Alpha 1,3 fucosyltransferases are master regulators of prostate cancer cell trafficking.

    PubMed

    Barthel, Steven R; Wiese, Georg K; Cho, Jaehyung; Opperman, Matthew J; Hays, Danielle L; Siddiqui, Javed; Pienta, Kenneth J; Furie, Bruce; Dimitroff, Charles J

    2009-11-17

    How cancer cells bind to vascular surfaces and extravasate into target organs is an underappreciated, yet essential step in metastasis. We postulate that the metastatic process involves discrete adhesive interactions between circulating cancer cells and microvascular endothelial cells. Sialyl Lewis X (sLe(X)) on prostate cancer (PCa) cells is thought to promote metastasis by mediating PCa cell binding to microvascular endothelial (E)-selectin. Yet, regulation of sLe(X) and related E-selectin ligand expression in PCa cells is a poorly understood factor in PCa metastasis. Here, we describe a glycobiological mechanism regulating E-selectin-mediated adhesion and metastatic potential of PCa cells. We demonstrate that alpha1,3 fucosyltransferases (FT) 3, 6, and 7 are markedly elevated in bone- and liver-metastatic PCa and dictate synthesis of sLe(X) and E-selectin ligands on metastatic PCa cells. Upregulated FT3, FT6, or FT7 expression induced robust PCa PC-3 cell adhesion to bone marrow (BM) endothelium and to inflamed postcapillary venules in an E-selectin-dependent manner. Membrane proteins, CD44, carcinoembryonic antigen (CEA), podocalyxin-like protein (PCLP), and melanoma cell adhesion molecule (MCAM) were major scaffolds presenting E-selectin-binding determinants on FT-upregulated PC-3 cells. Furthermore, elevated FT7 expression promoted PC-3 cell trafficking to and retention in BM through an E-selectin dependent event. These results indicate that alpha1,3 FTs could enhance metastatic efficiency of PCa by triggering an E-selectin-dependent trafficking mechanism.

  10. CD90-positive cells, an additional cell population, produce laminin {alpha}2 upon transplantation to dy{sup 3k}/dy{sup 3k} mice

    SciTech Connect

    Fukada, So-ichiro Yamamoto, Yukiko; Segawa, Masashi; Sakamoto, Kenta; Nakajima, Mari; Sato, Masaki; Morikawa, Daisuke; Uezumi, Akiyoshi; Miyagoe-Suzuki, Yuko; Takeda, Shin'ichi; Tsujikawa, Kazutake; Yamamoto, Hiroshi

    2008-01-01

    Laminin {alpha}2 is a component of skeletal and cardiac muscle basal lamina. A defect of the laminin {alpha}2 chain leads to severe congenital muscular dystrophy (MDC1A) in humans and dy/dy mice. Myogenic cells including myoblasts, myotubes, and myofibers in skeletal muscle are a possible source of the laminin {alpha}2 chain, and myogenic cells are thus proposed as a cell source for congenital muscular dystrophy therapy. However, we observed production of laminin {alpha}2 in non-myogenic cells of normal mice, and we could enrich these laminin {alpha}2-producing cells in CD90{sup +} cell fractions. Intriguingly, the number of CD90{sup +} cells increased dramatically during skeletal muscle regeneration in mice. This fraction did not include myogenic cells but exhibited a fibroblast-like phenotype. Moreover, these cells were resident in skeletal muscle, not derived from bone marrow. Finally, the production of laminin {alpha}2 in CD90{sup +} cells was not dependent on fusion with myogenic cells. Thus, CD90{sup +} cells are a newly identified additional cell fraction that increased during skeletal muscle regeneration in vivo and could be another cell source for therapy for lama2-deficient muscular dystrophy.

  11. Radiation-induced increase in expression of the alpha IIb beta 3 integrin in melanoma cells: effects on metastatic potential.

    PubMed

    Onoda, J M; Piechocki, M P; Honn, K V

    1992-06-01

    We investigated the effects of nonlethal gamma radiation on the metastatic potential of the murine tumor cell line, B16 melanoma. The ability of B16 cells to adhere to fibronectin, which is in part mediated by the alpha IIb beta 3 integrin receptor, is predictive of metastatic potential. We determined that exposure to 0.25-2.5 Gy gamma radiation significantly enhanced B16 cell adhesion to fibronectin. The radiation-enhanced adhesion was dependent on enhanced expression of the alpha IIb beta 3 integrin. We observed that 15 min after 0.5 Gy radiation, 99% of irradiated B16 tumor cells were positively labeled with monoclonal antibodies directed against alpha IIb beta 3 compared to 22% of sham-irradiated cells. Radiation-enhanced expression of the alpha IIb beta 3 receptor is reversible and down-regulation begins within 2-4 h postirradiation. Finally, we found that irradiation significantly enhanced the ability of B16 cells to form metastases in a lung colony assay. It is concluded that a relationship exists between radiation effects on the B16 tumor cells, alpha IIb beta 3 receptor expression, adhesion in vitro, and metastasis in vivo. We suggest that low-dose radiation, at levels comparable to those used in fractionated or hyperfractionated radiotherapy, may alter the metastatic phenotype and potential of surviving tumor cells via a rapid alteration in their surface expression of alpha IIb beta 3 integrin receptors.

  12. Alpha1beta1 integrin is crucial for accumulation of epidermal T cells and the development of psoriasis.

    PubMed

    Conrad, Curdin; Boyman, Onur; Tonel, Giulia; Tun-Kyi, Adrian; Laggner, Ute; de Fougerolles, Antonin; Kotelianski, Victor; Gardner, Humphrey; Nestle, Frank O

    2007-07-01

    Psoriasis is a common T cell-mediated autoimmune inflammatory disease. We show that blocking the interaction of alpha1beta1 integrin (VLA-1) with collagen prevented accumulation of epidermal T cells and immunopathology of psoriasis. Alpha1beta1 integrin, a major collagen-binding surface receptor, was exclusively expressed by epidermal but not dermal T cells. Alpha1beta1-positive T cells showed characteristic surface markers of effector memory cells and contained high levels of interferon-gamma but not interleukin-4. Blockade of alpha1beta1 inhibited migration of T cells into the epidermis in a clinically relevant xenotransplantation model. This was paralleled by a complete inhibition of psoriasis development, comparable to that caused by tumor necrosis factor-alpha blockers. These results define a crucial role for alpha1beta1 in controlling the accumulation of epidermal type 1 polarized effector memory T cells in a common human immunopathology and provide the basis for new strategies in psoriasis treatment focusing on T cell-extracellular matrix interactions.

  13. Respiratory epithelial cell expression of human transforming growth factor-alpha induces lung fibrosis in transgenic mice.

    PubMed Central

    Korfhagen, T R; Swantz, R J; Wert, S E; McCarty, J M; Kerlakian, C B; Glasser, S W; Whitsett, J A

    1994-01-01

    Increased production of EGF or TGF-alpha by the respiratory epithelial cells has been associated with the pathogenesis of various forms of lung injury. Growth factors and cytokines are thought to act locally, via paracrine and autocrine mechanisms, to stimulate cell proliferation and matrix deposition by interstitial lung cells resulting in pulmonary fibrosis. To test whether TGF-alpha mediates pulmonary fibrotic responses, we have generated transgenic mice expressing human TGF-alpha under control of regulatory regions of the human surfactant protein C (SP-C) gene. Human TGF-alpha mRNA was expressed in pulmonary epithelial cells in the lungs of the transgenic mice. Adult mice bearing the SP-C-TGF-alpha transgene developed severe pulmonary fibrosis. Fibrotic lesions were observed in peribronchial, peribronchiolar, and perivascular regions, as well as subjacent to pleural surfaces. Lesions consisted of fibrous tissue that included groups of epithelial cells expressing endogenous SP-C mRNA, consistent with their identification as distal respiratory epithelial cells. Peripheral fibrotic regions consisted of thickened pleura associated with extensive collagen deposition. Alveolar architecture was disrupted in the transgenic mice with loss of alveoli in the lung parenchyma. Pulmonary epithelial cell expression of TGF-alpha in transgenic mice disrupts alveolar morphogenesis and produces fibrotic lesions mediated by paracrine signaling between respiratory epithelial and interstitial cells of the lung. Images PMID:8163670

  14. Apoptosis and tumor cell death in response to HAMLET (human alpha-lactalbumin made lethal to tumor cells).

    PubMed

    Hallgren, Oskar; Aits, Sonja; Brest, Patrick; Gustafsson, Lotta; Mossberg, Ann-Kristin; Wullt, Björn; Svanborg, Catharina

    2008-01-01

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a molecular complex derived from human milk that kills tumor cells by a process resembling programmed cell death. The complex consists of partially unfolded alpha-lactalbumin and oleic acid, and both the protein and the fatty acid are required for cell death. HAMLET has broad antitumor activity in vitro, and its therapeutic effect has been confirmed in vivo in a human glioblastoma rat xenograft model, in patients with skin papillomas and in patients with bladder cancer. The mechanisms of tumor cell death remain unclear, however. Immediately after the encounter with tumor cells, HAMLET invades the cells and causes mitochondrial membrane depolarization, cytochrome c release, phosphatidyl serine exposure, and a low caspase response. A fraction of the cells undergoes morphological changes characteristic of apoptosis, but caspase inhibition does not rescue the cells and Bcl-2 overexpression or altered p53 status does not influence the sensitivity of tumor cells to HAMLET. HAMLET also creates a state of unfolded protein overload and activates 20S proteasomes, which contributes to cell death. In parallel, HAMLET translocates to tumor cell nuclei, where high-affinity interactions with histones cause chromatin disruption, loss of transcription, and nuclear condensation. The dying cells also show morphological changes compatible with macroautophagy, and recent studies indicate that macroautophagy is involved in the cell death response to HAMLET. The results suggest that HAMLET, like a hydra with many heads, may interact with several crucial cellular organelles, thereby activating several forms of cell death, in parallel. This complexity might underlie the rapid death response of tumor cells and the broad antitumor activity of HAMLET.

  15. Interleukin-6 enhances whereas tumor necrosis factor alpha and interferons inhibit integrin expression and adhesion of human mast cells to extracellular matrix proteins.

    PubMed

    Schoeler, Dagmar; Grützkau, Andreas; Henz, Beate M; Küchler, Jens; Krüger-Krasagakis, Sabine

    2003-05-01

    Integrins are expressed on mast cells and constitute an essential prerequisite for the accumulation of the cells at sites of inflammation. In order to clarify a potential contribution of inflammatory cytokines to this process, we have studied the modulation of integrin expression and adhesion of immature human mast cells (HMC-1) to extracellular matrix proteins by interleukin-6, tumor necrosis factor alpha, interferon-alpha and interferon-gamma. Corticosteroids were used for comparison. On fluorescence-activated cell sorter analysis, preincubation of cells for 48 h with different concentrations of interleukin-6 induced a significant, up to 40%, increase of alpha v alpha 5, CD49b (alpha 2), CD49e (alpha 5), CD49f (alpha 6), and CD51 (alpha v). In contrast, different concentrations of tumor necrosis factor alpha, interferon-alpha, interferon-gamma, and dexamethasone (10-8-10-10 M) inhibited expression of adhesion receptors by up to 60%, reaching significance for some but not all integrins. On semiquantitative polymerase chain reaction analysis, interleukin-6, the other cytokines, and corticosteroids significantly modulated expression of alpha1, alpha v and alpha 5 integrin chains at mRNA level. Functional significance of these findings was proven in adhesion assays using fibronectin, laminin, and vitronectin, with interleukin-6 causing significant enhancement of adhesion in all cases, tumor necrosis factor alpha and dexamethasone inducing significant reduction of adhesion to fibronectin and laminin, and interferon-gamma significantly inhibiting adhesion to fibronectin only. Specificity of interleukin-6-induced changes was demonstrated using antibodies against alpha1 and alpha 5 integrins in unstimulated and interleukin-6-prestimulated cells. These data show that interleukin-6 stimulates mast cell adhesion to extracellular matrix and thus allows for the accumulation of the cells at tissue sites by enhancing integrin expression, whereas tumor necrosis factor alpha

  16. Human cord blood T-cell receptor alpha beta cell responses to protein antigens of Paracoccidioides brasiliensis yeast forms.

    PubMed Central

    Munk, M E; Kaufmann, S H

    1995-01-01

    Paracoccidioides brasiliensis causes a chronic granulomatous mycosis, prevalent in South America, and cell-mediated immunity represents the principal mode of protection against this fungal infection. We investigated the response of naive cord blood T cells to P. brasiliensis lysates. Our results show: (1) P. brasiliensis stimulates T-cell expansion, interleukin-2 (IL-2) production and differentiation into cytotoxic T cells; (2) T-cell stimulation depends on P. brasiliensis processing and major histocompatibility complex (MHC) class II expression; (3) the responsive T-cell population expresses alpha beta T-cell receptors (TCR) with different V beta gene products, CD4 and CD45RO; (4) the P. brasiliensis components involved in T-cell expansion primarily reside in a high molecular weight (100,000 MW) and a low molecular weight (< 1000 MW) protein fraction. These results indicate that protein antigens of P. brasiliensis stimulate cord blood CD4 alpha beta T cells, independent from in vivo presensitization, and thus question direct correlation of positive in vitro responses with protective immunity in vivo. PMID:7890308

  17. Lung carcinomas decrease the number of monocytes/macrophages (CD14+ cells) that produce TNF-alpha.

    PubMed

    Lopez-Gonzalez, Jose Sullivan; Avila-Moreno, Federico; Prado-Garcia, Heriberto; Aguilar-Cazares, Dolores; Mandoki, Juan Jose; Meneses-Flores, Manuel

    2007-03-01

    The role that inflammation plays in cancer is puzzling. In peripheral blood, TNF-alpha-producing monocytes (CD14+ cells) were compared among patients with lung cancer, patients with tuberculosis and healthy donors; also, in pleural effusion TNF-alpha-producing CD14+ cells were compared between tuberculous patients and lung cancer patients. To analyze the level of the cellular alteration in TNF-alpha production, an experimental model was set up. TNF-alpha-producing CD14+ cells in peripheral blood from lung cancer patients were significantly lower than those from healthy donors. In pleural effusion, TNF-alpha-producing CD14+ cells were significantly lower in lung cancer patients than in tuberculous patients. Based on the results obtained from an experimental model, we suggest that this phenomenon was attributed to a reduced expression of TNF-alpha transcript. These findings provide evidence that lung carcinomas reduce TNF-alpha production by macrophages, possibly by inducing in these cells an M2 phenotype, which favor tumor progression.

  18. Virus-cell interactions regulating induction of tumor necrosis factor alpha production in macrophages infected with herpes simplex virus.

    PubMed

    Paludan, S R; Mogensen, S C

    2001-11-01

    Macrophages respond to virus infections by rapidly secreting proinflammatory cytokines, which play an important role in the first line of defense. Tumor necrosis factor alpha (TNF-alpha) is one of the major macrophage-produced cytokines. In this study we have investigated the virus-cell interactions responsible for induction of TNF-alpha expression in herpes simplex virus (HSV)-infected macrophages. Both HSV type 1 (HSV-1) and HSV-2 induced TNF-alpha expression in macrophages activated with gamma interferon (IFN-gamma). This induction was to some extent sensitive to UV treatment of the virus. Virus particles unable to enter the cells displayed reduced capacity to stimulate TNF-alpha expression but retained a significant portion which was abolished by HSV-specific antibodies. Recombinant HSV-1 glycoprotein D was able to trigger TNF-alpha secretion in concert with IFN-gamma. Sugar moieties of HSV glycoproteins have been reported to be involved in induction of IFN-alpha but did not contribute to TNF-alpha expression in macrophages. Moreover, the entry-dependent portion of the TNF-alpha induction was investigated with HSV-1 mutants and found to be independent of the tegument proteins VP16 and UL13 and partly dependent on nuclear translocation of the viral DNA. Finally, we found that macrophages expressing an inactive mutant of the double-stranded RNA (dsRNA)-activated protein kinase (PKR) produced less TNF-alpha in response to infectious HSV infection than the empty-vector control cell line but displayed the same responsiveness to UV-inactivated virus. These results indicate that HSV induces TNF-alpha expression in macrophages through mechanisms involving (i) viral glycoproteins, (ii) early postentry events occurring prior to nuclear translocation of viral DNA, and (iii) viral dsRNA-PKR.

  19. Formation of triads without the dihydropyridine receptor alpha subunits in cell lines from dysgenic skeletal muscle

    PubMed Central

    1996-01-01

    Muscular dysgenesis (mdg/mdg), a mutation of the skeletal muscle dihydropyridine receptor (DHPR) alpha 1 subunit, has served as a model to study the functions of the DHPR in excitation-contraction coupling and its role in triad formation. We have investigated the question of whether the lack of the DHPR in dysgenic skeletal muscle results in a failure of triad formation, using cell lines (GLT and NLT) derived from dysgenic (mdg/mdg) and normal (+/+) muscle, respectively. The lines were generated by transfection of myoblasts with a plasmid encoding a Large T antigen. Both cell lines express muscle-specific proteins and begin organization of sarcomeres as demonstrated by immunocytochemistry. Similar to primary cultures, dysgenic (GLT) myoblasts show a higher incidence of cell fusion than their normal counterparts (NLT). NLT myotubes develop spontaneous contractile activity, and fluorescent Ca2+ recordings show Ca2+ release in response to depolarization. In contrast, GLTs show neither spontaneous nor depolarization-induced Ca2+ transients, but do release Ca2+ from the sarcoplasmic reticulum (SR) in response to caffeine. Despite normal transverse tubule (T-tubule) formation, GLT myotubes lack the alpha 1 subunit of the skeletal muscle DHPR, and the alpha 2 subunit is mistargeted. Nevertheless, the ryanodine receptor (RyR) frequently develops its normal, clustered organization in the absence of both DHPR alpha subunits in the T-tubules. In EM, these RyR clusters correspond to T-tubule/SR junctions with regularly spaced feet. These findings provide conclusive evidence that interactions between the DHPR and RyR are not involved in the formation of triad junctions or in the normal organization of the RyR in the junctional SR. PMID:8707823

  20. Phosphorylation of eIF2 alpha in Sf9 cells: a stress, survival and suicidal signal.

    PubMed

    Aarti, Iyer; Rajesh, Kamindla; Ramaiah, Kolluru V A

    2010-06-01

    An analysis of the stress-induced phosphorylation of the alpha-subunit of eukaryotic initiation factor (eIF2alpha) involved in translation regulation, in the ovarian cells of Spodoptera frugiperda (Sf9) for its role in cell survival and death reveals that it stimulates casapase activation and cell death in the absence of BiP, a chaperone and stress marker of the endoplasmic reticulum (ER). While Phospho-JNK and GADD-153 levels are elevated in non-ER stress-induced eIF2alpha phosphorylation-mediated cell death, ATF4 levels are elevated both in response to ER and non-ER stress-induced eIF2alpha phosphorylation. Infection of Sf9 cells by wt and a mutant Deltapk2 baculovirus that harbor the anti-apoptotic p35 gene induces BiP expression. However, UV-induced eIF2alpha phosphorylation and caspase activation are mitigated more efficiently by wt, but not by Deltapk2 baculovirus that lacks pk2, an inhibitor of eIF2alpha kinase. z-VAD-fmk, a caspase inhibitor reduces the late stages, but not the initial stages of non-ER stress-induced eIF2alpha phosphorylation, thereby suggesting that eIF2alpha phosphorylation is a cause and consequence of caspase activation. The importance of BiP affecting the delicate balance between eIF2alpha phosphorylation-mediated cell survival and death is further supported by the findings that tunicamycin-treated cells expressing BiP resist eIF2alpha phosphorylation-mediated cell death and addition of a purified recombinant mutant phosphomimetic form, but not wt eIF2alpha, stimulates caspase activation in cell extracts devoid of BiP. These findings therefore suggest that eIF2alpha phosphorylation is primarily a stress signal and evokes adaptive or apoptotic responses depending on its cellular location, changes in gene expression, coincident signaling activities, and inter-protein interactions.

  1. Follicular dendritic cell tumor of the mediastinum: expression of fractalkine and SDF-1alpha as mast cell chemoattractants.

    PubMed

    Guettier, Catherine; Validire, Pierre; Emilie, Dominique; Tricottet, Viviane; Sebagh, Mylène; Anjo, Aurora; Misset, Jean-Louis; Reynes, Michel

    2006-02-01

    Follicular dendritic cell tumor (FDCT) is a rare tumor mainly located in laterocervical lymph nodes. We report one case of mediastinal FDCT associated with a history of bullous skin disease and clinically obvious immunosuppression. This tumor was characterized by heavy mast cell infiltration. Mast cells were in close relationship with tumor cells as demonstrated by ultrastructural examination and their presence are probably related with the strong expression of mast cell chemoattractants as fraktalkine and stromal cell-derived factor-1alpha by tumor cells. The long follow-up period of more than 17 years allowed to us assess the relatively indolent evolution of this tumor characterized by three slowly growing local recurrences without metastasis.

  2. Investigating the role of protein folding and assembly in cell-type dependent expression of alpha7 nicotinic receptors using a green fluorescent protein chimera.

    PubMed

    Lee, H K; Gwalani, L; Mishra, V; Anandjiwala, P; Sala, F; Sala, S; Ballesta, J J; O'Malley, D; Criado, M; Loring, R H

    2009-03-09

    To test the hypothesis that cell-dependent expression of alpha7 receptors is due to differences in protein folding or assembly, we constructed a chimeric rat alpha7 subunit with green fluorescent protein (GFP) at the receptor C-terminal. Expression of alpha7-GFP in Xenopus oocytes resulted in currents that were indistinguishable from wild type receptors but were only 33% of control. (125)I-alpha-bungarotoxin (alphaBGT) binding at the oocyte surface was reduced to 23% of wild type. Transfection of alpha7-GFP into GH4C1 cells produced fluorescence that was less intense than GFP alone, but showed significant alpha-BGT binding compared to transfection with GFP. In contrast, alpha7-GFP transfection in SH-EP1, HEK293 and CHO-CAR cells produced fluorescence without alphaBGT binding. Flow cytometry of cells transfected with alpha7-GFP indicated fluorescence in both SH-EP1 and GH4C1 cells, but surface toxin binding sites and sites immunoprecipitated using anti-GFP antibodies were undetectable in SH-EP1 cells, suggesting a problem in folding/assembly rather than trafficking. Surprisingly, integrated fluorescence intensities in GH4C1 cells transfected with alpha7-GFP did not correlate with amounts of cell surface or immunoprecipitable alphaBGT binding. Therefore, GFP folding at the C-terminal of the alpha7-GFP chimera is cell-line independent, but toxin binding is highly cell-line dependent, suggesting that if altered protein folding is involved in the cell-type dependence of alpha7 receptor expression, the phenomenon is restricted to specific protein domains. Further, C-terminal GFP-labeled alpha7 receptors decreased the efficiency of folding/assembly not only of chimeric subunits, but also wild-type subunits, suggesting that the C-terminal is an important domain for alpha7 receptor assembly.

  3. alpha-Linolenic acid protects renal cells against palmitic acid lipotoxicity via inhibition of endoplasmic reticulum stress.

    PubMed

    Katsoulieris, Elias; Mabley, Jon G; Samai, Mohamed; Green, Irene C; Chatterjee, Prabal K

    2009-11-25

    Unsaturated fatty acids may counteract the lipotoxicity associated with saturated fatty acids. Palmitic acid induced endoplasmic reticulum (ER) stress and caused apoptotic and necrotic cell death in the renal proximal tubular cell line, NRK-52E. We investigated whether alpha-linolenic acid, an unsaturated fatty acid, protected against ER stress and cell death induced by palmitic acid or by other non-nutrient ER stress generators. Incubation of NRK-52E cells for 24h with palmitic acid produced a significant increase in apoptosis and necrosis. Palmitic acid also increased levels of three indicators of ER stress - the phosphorylated form of the eukaryotic initiation factor 2alpha (eIF2alpha), C/EBP homologous protein (CHOP), and glucose regulated protein 78 (GRP78). alpha-Linolenic acid dramatically reduced cell death and levels of all three indicators of ER stress brought about by palmitic acid. Tunicamycin, which induces ER stress by glycosylation of proteins, produced similar effects to those obtained using palmitic acid; its effects were partially reversed by alpha-linolenic acid. Salubrinal (a phosphatase inhibitor) causes increased levels of the phosphorylated form of eIF2alpha - this effect was partially reversed by alpha-linolenic acid. Palmitoleate, a monosaturated fatty acid, had similar effects to those of alpha-linolenic acid. These results suggest that part of the mechanism of protection of the kidney by unsaturated fatty acids is through inhibition of ER stress, eIF2alpha phosphorylation and consequential reduction of CHOP protein expression and apoptotic renal cell death.

  4. Neuropeptide Y inhibits spontaneous alpha-melanocyte-stimulating hormone (alpha-MSH) release via a Y(5) receptor and suppresses thyrotropin-releasing hormone-induced alpha-MSH secretion via a Y(1) receptor in frog melanotrope cells.

    PubMed

    Galas, Ludovic; Tonon, Marie-Christine; Beaujean, Delphine; Fredriksson, Robert; Larhammar, Dan; Lihrmann, Isabelle; Jegou, Sylvie; Fournier, Alain; Chartrel, Nicolas; Vaudry, Hubert

    2002-05-01

    In amphibians, the secretion of alpha-MSH by melanotrope cells is stimulated by TRH and inhibited by NPY. We have previously shown that NPY abrogates the stimulatory effect of TRH on alpha-MSH secretion. The aim of the present study was to characterize the receptor subtypes mediating the action of NPY and to investigate the intracellular mechanisms involved in the inhibitory effect of NPY on basal and TRH-induced alpha-MSH secretion. Y(1) and Y(5) receptor mRNAs were detected by RT-PCR and visualized by in situ hybridization histochemistry in the intermediate lobe of the pituitary. Various NPY analogs inhibited in a dose-dependent manner the spontaneous secretion of alpha-MSH from perifused frog neurointermediate lobes with the following order of potency porcine peptide YY (pPYY) > frog NPY (fNPY) > porcine NPY (pNPY)-2-36) > pNPY-(13-36) > [D-Trp(32)]pNPY > [Leu(31),Pro(34)]pNPY. The stimulatory effect of TRH (10(-8)6 M) on alpha-MSH release was inhibited by fNPY, pPYY, and [Leu(31),Pro(34)]pNPY, but not by pNPY-(13-36) and [D-Trp(32)]pNPY. These data indicate that the inhibitory effect of fNPY on spontaneous alpha-MSH release is preferentially mediated through Y(5) receptors, whereas the suppression of TRH-induced alpha-MSH secretion by fNPY probably involves Y(1) receptors. Pretreatment of neurointermediate lobes with pertussis toxin (PTX; 1 microg/ml; 12 h) did not abolish the inhibitory effect of fNPY on cAMP formation and spontaneous alpha-MSH release, but restored the stimulatory effect of TRH on alpha-MSH secretion, indicating that the adenylyl cyclase pathway is not involved in the action of fNPY on TRH-evoked alpha-MSH secretion. In the majority of melanotrope cells, TRH induces a sustained and biphasic increase in cytosolic Ca(2+) concentration. Preincubation of cultured cells with fNPY (10(-7) M) or omega-conotoxin GVIA (10(-7) M) suppressed the plateau phase of the Ca(2+) response induced by TRH. However, although fNPY abrogated TRH-evoked alpha

  5. EORTC (30885) randomised phase III study with recombinant interferon alpha and recombinant interferon alpha and gamma in patients with advanced renal cell carcinoma. The EORTC Genitourinary Group.

    PubMed Central

    De Mulder, P. H.; Oosterhof, G.; Bouffioux, C.; van Oosterom, A. T.; Vermeylen, K.; Sylvester, R.

    1995-01-01

    In the treatment of renal cell carcinoma both complete (CRs) and partial remissions (PRs) have been obtained using recombinant (r) interferon alpha (IFN-alpha), with response rates ranging from 0 to 31% (mean 16%). rIFN-gamma is a potent immunostimulating agent, but the clinical experience of its use is limited and results are conflicting. In a phase II study with the combination of rIFN-alpha 2c (Boehringer Ingelheim) and rIFN-gamma (Genentech, supplied by Boehringer Ingelheim) in 31 eligible patients, a response rate of 25% was recorded. Based on this observation a randomised phase III study was initiated to investigate the possible advantage of the addition rIFN-gamma to rIFN-alpha 2c treatment. Treatment consisted of rIFN-alpha 2c 30 micrograms m-2 = 10 x 10(6) IU m-2 s.c. twice weekly in arm A and the same dose of rIFN-alpha combined with rIFN-gamma 100 micrograms m-2 = 2 x 10(6) IU m-2 in arm B. Eligibility criteria included documented progression of disease; patients with bone lesions only and overt central nervous system metastases were excluded. Between November 1988 and September 1990, 102 patients were entered into the study. An interim analysis showed a response in 7/53 (13%) patients (two CRs and five PRs) in the rIFN-alpha 2c monotherapy arm and in 2/45 (4%) (one CR and one PR) patients in the combination arm. This difference was not statistically significant (P = 0.17). The probability of missing an eventual 10% advantage for the combination is 0.001. The numbers are insufficient to rule out a negative effect of the addition of rIFN-gamma. The dose intensity of IFN-alpha 2c for the two treatment arms was the same. The addition of rIFN-gamma does not improve the response rate of rIFN-alpha 2c monotherapy. A possible detrimental effect cannot be excluded. PMID:7841054

  6. Stress-induced apoptosis in Spodoptera frugiperda (Sf9) cells: baculovirus p35 mitigates eIF2 alpha phosphorylation.

    PubMed

    Aparna, Gunda; Bhuyan, Abani K; Sahdev, Sudhir; Hasnain, Seyed E; Kaufman, Randal J; Ramaiah, Kolluru V A

    2003-12-30

    Spodoptera frugiperda (Sf9) ovarian cells, natural hosts for baculovirus, are good model systems to study apoptosis and also heterologous gene expression. We report that uninfected Sf9 cells readily undergo apoptosis and show increased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) in the presence of agents such as UVB light, etoposide, high concentrations of cycloheximide, and EGTA. In contrast, tunicamycin, A23187, and low concentrations of cycloheximide promoted eIF2alpha phosphorylation in Sf9 cells but without apoptosis. These findings therefore suggest that increased eIF2alpha phosphorylation does not always necessarily lead to apoptosis, but it is a characteristic hallmark of stressed cells and also of cells undergoing apoptosis. Cell death induced by the above agents was abrogated by infection of Sf9 cells with wild-type (wt) AcNPV. In contrast, Sf9 cells when infected with vAcdelta35, a virus carrying deletion of the antiapoptotic p35 gene, showed increased apoptosis and enhanced eIF2alpha phosphorylation. Further, a recombinant wt virus vAcS51D expressing human S51D, a phosphomimetic form of eIF2alpha, induced apoptosis in UVB pretreated Sf9 cells. However, infection with vAcS51A expressing a nonphosphorylatable form (S51A) of human eIF2alpha partially reduced apoptosis. Consistent with these findings, it has been observed here that caspase activation has led to increased eIF2alpha phosphorylation, while caspase inhibition by z-VAD-fmk reduced eIF2alpha phosphorylation selectively in cells exposed to proapoptotic agents. These findings therefore suggest that the stress signaling pathway determines apoptosis, and caspase activation is a prerequisite for increased eIF2alpha phosphorylation in Sf9 cells undergoing apoptosis. The findings also reinforce the conclusion for the first time that the "pancaspase inhibitor" baculovirus p35 mitigates eIF2alpha phosphorylation.

  7. Alpha-ketoglutarate Curbs Differentiation and Induces Cell Death in Mesenchymal Stromal Precursors with Mitochondrial Dysfunction.

    PubMed

    Singh, Karmveer; Krug, Linda; Basu, Abhijit; Meyer, Patrick; Treiber, Nicolai; Vander Beken, Seppe; Wlaschek, Meinhard; Kochanek, Stefan; Bloch, Wilhelm; Geiger, Hartmut; Maity, Pallab; Scharffetter-Kochanek, Karin

    2017-04-11

    Increased concentrations of reactive oxygen species (ROS) originating from dysfunctional mitochondria contribute to diverse aging-related degenerative disorders. But so far little is known about the impact of distinct ROS on metabolism and fate of stromal precursor cells. We here demonstrate that an increase in superoxide anion radicals due to superoxide dismutase 2 (Sod2) deficiency in stromal precursor cells suppress osteogenic and adipogenic differentiation through fundamental changes in the global metabolite landscape. Our data identify impairment of the pyruvate and L-glutamine metabolism causing toxic accumulation of alpha-ketoglutarate in the Sod2 deficient and intrinsically aged stromal precursor cells as a major cause for their reduced lineage differentiation. Alpha-ketoglutarate accumulation led to enhanced nucleocytoplasmic vacuolation and chromatin condensation-mediated cell death in Sod2 deficient stromal precursor cells as a consequence of DNA damage, Hif-1α instability and reduced histone H3 (Lys27) acetylation. These findings hold promise for prevention and treatment of mitochondrial disorders commonly associated with aged individuals. This article is protected by copyright. All rights reserved.

  8. Mutagenic effects of a single and an exact number of alpha particles in mammalian cells

    NASA Technical Reports Server (NTRS)

    Hei, T. K.; Wu, L. J.; Liu, S. X.; Vannais, D.; Waldren, C. A.; Randers-Pehrson, G.

    1997-01-01

    One of the main uncertainties in risk estimation for environmental radon exposure using lung cancer data from underground miners is the extrapolation from high- to low-dose exposure where multiple traversal is extremely rare. The biological effects of a single alpha particle are currently unknown. Using the recently available microbeam source at the Radiological Research Accelerator Facility at Columbia University, we examined the frequencies and molecular spectrum of S1- mutants induced in human-hamster hybrid (A(L)) cells by either a single or an exact number of alpha particles. Exponentially growing cells were stained briefly with a nontoxic concentration of Hoechst dye for image analysis, and the location of individual cells was computer-monitored. The nucleus of each cell was irradiated with either 1,2,4, or 8 alpha particles at a linear energy transfer of 90 keV/microm consistent with the energy spectrum of domestic radon exposure. Although single-particle traversal was only slightly cytotoxic to A(L) cells (survival fraction approximately 0.82), it was highly mutagenic, and the induced mutant fraction averaged 110 mutants per 10(5) survivors. In addition, both toxicity and mutant induction were dose-dependent. Multiplex PCR analysis of mutant DNA showed that the proportion of mutants with multilocus deletions increased with the number of particle traversals. These data provide direct evidence that a single a particle traversing a nucleus will have a high probability of resulting in a mutation and highlight the need for radiation protection at low doses.

  9. Mutagenic effects of a single and an exact number of alpha particles in mammalian cells.

    PubMed

    Hei, T K; Wu, L J; Liu, S X; Vannais, D; Waldren, C A; Randers-Pehrson, G

    1997-04-15

    One of the main uncertainties in risk estimation for environmental radon exposure using lung cancer data from underground miners is the extrapolation from high- to low-dose exposure where multiple traversal is extremely rare. The biological effects of a single alpha particle are currently unknown. Using the recently available microbeam source at the Radiological Research Accelerator Facility at Columbia University, we examined the frequencies and molecular spectrum of S1- mutants induced in human-hamster hybrid (A(L)) cells by either a single or an exact number of alpha particles. Exponentially growing cells were stained briefly with a nontoxic concentration of Hoechst dye for image analysis, and the location of individual cells was computer-monitored. The nucleus of each cell was irradiated with either 1,2,4, or 8 alpha particles at a linear energy transfer of 90 keV/microm consistent with the energy spectrum of domestic radon exposure. Although single-particle traversal was only slightly cytotoxic to A(L) cells (survival fraction approximately 0.82), it was highly mutagenic, and the induced mutant fraction averaged 110 mutants per 10(5) survivors. In addition, both toxicity and mutant induction were dose-dependent. Multiplex PCR analysis of mutant DNA showed that the proportion of mutants with multilocus deletions increased with the number of particle traversals. These data provide direct evidence that a single a particle traversing a nucleus will have a high probability of resulting in a mutation and highlight the need for radiation protection at low doses.

  10. Interferon beta 2/interleukin 6 modulates synthesis of alpha 1-antitrypsin in human mononuclear phagocytes and in human hepatoma cells.

    PubMed Central

    Perlmutter, D H; May, L T; Sehgal, P B

    1989-01-01

    The cytokine IFN beta 2/IL-6 has recently been shown to regulate the expression of genes encoding hepatic acute phase plasma proteins. INF beta 2/IL-6 has also been shown to be identical to MGI-2, a protein that induces differentiation of bone marrow precursor cells toward mature granulocytes and monocytes. Accordingly, we have examined the effect of IFN beta 2/IL-6 on expression of the IL-1- and tumor necrosis factor-unresponsive acute phase protein alpha 1-antitrypsin (alpha 1 AT) in human hepatoma-derived hepatocytes and in human mononuclear phagocytes. Purified human fibroblast and recombinant IFN beta 2/IL-6 each mediate a specific increase in steady-state levels of alpha 1 AT mRNA and a corresponding increase in net synthesis of alpha 1 AT in primary cultures of human peripheral blood monocytes as well as in HepG2 and Hep3B cells. Thus, the effect of IFN beta 2/IL-6 on alpha 1 AT gene expression in these cells is primarily due to an increase in accumulation of alpha 1 AT mRNA and can be distinguished from the direct, predominantly translational effect of bacterial lipopolysaccharide on expression of this gene in monocytes and macrophages. The results indicate that IFN beta 2/IL-6 regulates acute phase gene expression, specifically alpha 1 AT gene expression, in extrahepatic as well as hepatic cell types. Images PMID:2472425

  11. Prostaglandin F{sub 2{alpha}} regulates cytokine responses of mast cells through the receptors for prostaglandin E

    SciTech Connect

    Kaneko, Izumi; Hishinuma, Takanori; Suzuki, Kaori; Owada, Yuji; Kitanaka, Noriko; Kondo, Hisatake; Goto, Junichi; Furukawa, Hiroshi; Ono, Masao

    2008-03-14

    There is an increasing body of evidence that prostanoids modulate mast cell functions and contribute to the development of allergic inflammation. The present study aimed to identify an undetermined function of prostaglandin (PG) F{sub 2{alpha}} in mast cell activation and the signaling mechanism involved in it. Simultaneous quantification of prostanoids by liquid chromatography/tandem mass spectrometry revealed the constitutive release of PGF{sub 2{alpha}}, thromboxane B{sub 2}, and 6-keto-PGF{sub 1{alpha}} from bone marrow-derived mast cells (BMMCs). Upon activation of BMMCs by lipopolysaccharide, the cytokine production in BMMCs was enhanced when the culture was supplemented with PGF{sub 2{alpha}}. However, F prostanoid receptor-a selective receptor for PGF{sub 2{alpha}}-was not detected in BMMCs. Further investigations performed using prostanoid receptor antagonists revealed an alternative mechanism wherein the receptors for PGE species-E prostanoid receptors-mediated the PGF{sub 2{alpha}} signal in BMMCs. The present study provides an insight into a novel function of PGF{sub 2{alpha}}, i.e., an autocrine accelerator for mast cell activation.

  12. Number and Brightness analysis of alpha-synuclein oligomerization and the associated mitochondrial morphology alterations in live cells

    PubMed Central

    Plotegher, N.; Gratton, E.; Bubacco, L.

    2014-01-01

    Background Alpha-synuclein oligomerization is associated to Parkinson's disease etiopathogenesis. The study of alpha-synuclein oligomerization properties in live cell and the definition of their effects on cellular viability are among fields expected to provide the knowledge required to unravel the mechanism(s) of toxicity that lead to the disease. Methods We used Number and Brightness method, which is a method based on fluorescence fluctuation analysis, to monitor alpha-synuclein tagged with EGFP aggregation in living SH-SY5Y cells. The presence of alpha-synuclein oligomers detected with this method was associated with intracellular structure conditions, evaluated by fluorescence confocal imaging. Results Cells overexpressing alpha-synuclein-EGFP present a heterogeneous ensemble of oligomers constituted by less than 10 monomers, when the protein approaches a threshold concentration value of about 90 nM in the cell cytoplasm. We show that the oligomeric species are partially sequestered by lysosomes and that the mitochondria morphology is altered in cells presenting oligomers, suggesting that these mitochondria may be dysfunctional. Conclusions We showed that alpha-synuclein overexpression in SH-SY5Y causes the formation of alpha-synuclein oligomeric species, whose presence is associated with mitochondrial fragmentation and autophagic-lysosomal pathway activation in live cells. General significance The unique capability provided by the Number and Brightness analysis to study alpha-synuclein oligomers distribution and properties, and the study their association to intracellular components in single live cells is important to forward our understanding of the molecular mechanisms Parkinson’s disease and it may be of general significance when applied to the study of other aggregating proteins in cellular models. PMID:24561157

  13. Retrograde inflammatory signaling from neutrophils to endothelial cells by soluble interleukin-6 receptor alpha.

    PubMed Central

    Modur, V; Li, Y; Zimmerman, G A; Prescott, S M; McIntyre, T M

    1997-01-01

    Endothelial cells initiate the inflammatory response by recruiting and activating leukocytes. IL-6 is not an agonist for this, but we found soluble IL-6 receptor alpha-subunit (IL-6Ralpha), with their constitutive IL-6 synthesis, stimulated endothelial cells to synthesize E-selectin, intracellular adhesion molecule-1, vascular cellular adhesion molecule-1, IL-6, and IL-8, and to bind neutrophils. Neutrophils express significant amounts of IL-6Ralpha and upon stimulation shed it: this material activates endothelial cells through a newly constituted IL-6 receptor. Retrograde signaling from PMN activated in the extravascular compartment to surrounding endothelial cells will recruit more and a wider variety of leukocytes. The limiting signal is a soluble receptor, not a cytokine. PMID:9389739

  14. Actin capping protein alpha maintains vestigial-expressing cells within the Drosophila wing disc epithelium.

    PubMed

    Janody, Florence; Treisman, Jessica E

    2006-09-01

    Tissue patterning must be translated into morphogenesis through cell shape changes mediated by remodeling of the actin cytoskeleton. We have found that Capping protein alpha (Cpa) and Capping protein beta (Cpb), which prevent extension of the barbed ends of actin filaments, are specifically required in the wing blade primordium of the Drosophila wing disc. cpa or cpb mutant cells in this region, but not in the remainder of the wing disc, are extruded from the epithelium and undergo apoptosis. Excessive actin filament polymerization is not sufficient to explain this phenotype, as loss of Cofilin or Cyclase-associated protein does not cause cell extrusion or death. Misexpression of Vestigial, the transcription factor that specifies the wing blade, both increases cpa transcription and makes cells dependent on cpa for their maintenance in the epithelium. Our results suggest that Vestigial specifies the cytoskeletal changes that lead to morphogenesis of the adult wing.

  15. Functional response to SDF1 alpha through over-expression of CXCR4 on adult subventricular zone progenitor cells.

    PubMed

    Liu, Xian Shuang; Chopp, Michael; Santra, Manoranjan; Hozeska-Solgot, Ann; Zhang, Rui Lan; Wang, Lei; Teng, Hua; Lu, Mei; Zhang, Zheng Gang

    2008-08-21

    The chemokine receptor CXCR4 and its ligand, stromal cell derived factor-1 alpha (SDF1 alpha) regulate neuroblast migration towards the ischemic boundary after stroke. Using loss- and gain-function, we investigated the biological effect of CXCR4/SDF1 alpha on neural progenitor cells. Neural progenitor cells, from the subventricular zone (SVZ) of the adult rat, were transfected with rat CXCR4-pLEGFP-C1 and pSIREN-RetroQ-CXCR4-siRNA retroviral vectors. Migration assay analysis showed that inhibition of CXCR4 by siRNA significantly reduced cell migration compared to the empty vector, indicating that CXCR4 mediated neural progenitor cell motility. When neural progenitor cells were cultured in growth medium containing bFGF (20 ng/ml), over-expression of CXCR4 significantly reduced the cell proliferation as measured by the number of bromodeoxyuridine+ (BrdU+) cells (26.4%) compared with the number in the control group (54.0%). Addition of a high concentration of SDF1 alpha (500 ng/ml) into the progenitor cells with over-expression of CXCR4 reversed the cell proliferation back to the control levels (57.6%). Immunostaining analysis showed that neither over-expression nor inhibition of CXCR4 altered the population of neurons and astrocytes, when neural progenitor cells were cultured in differentiation medium. These in vitro results suggest that CXCR4/SDF1 alpha primarily regulates adult neural progenitor cell motility but not differentiation, while over-expression of CXCR4 in the absence of SDF1 alpha decreases neural progenitor cell proliferation.

  16. Apoptosis induced by tumor necrosis factor-alpha in rat hepatocyte cell lines expressing hepatitis B virus.

    PubMed Central

    Guilhot, S.; Miller, T.; Cornman, G.; Isom, H. C.

    1996-01-01

    Three well differentiated SV40-immortalized rat hepatocyte cell lines, CWSV1, CWSV2, and CWSV14, and Hepatitis B Virus (HBV)-producing cell lines derived from them were examined for sensitivity to tumor necrosis factor (TNF)-alpha. CWSV1, CWSV2, and CWSV14 cells were co-transfected with a DNA construct containing a dimer of the HBV genome and the neo gene and selected in G418 to generate stable cell lines. Characterization of these cell lines indicated that they contain integrated HBV DNA, contain low molecular weight HBV DNA compatible with the presence of HBV replication intermediates, express HBV transcripts, and produce HBV proteins. The viability of CWSV1, CWSV2, and CWSV2 cells was not significantly altered when they were treated with TNF-alpha at concentrations as high as 20,000 U/ml. The HBV-expressing CWSV1 cell line, SV1di36, and the HBV-expressing CWSV14 cell line, SV14di208, were also not killed when treated with TNF-alpha. However, the HBV-expressing CWSV2 cell line, SV2di366, was extensively killed when treated with TNF-alpha at concentrations ranging from 200 to 20,000 U/ml. Analysis of several different HBV-producing CWSV2 cell lines indicated that TNF-alpha killing depended upon the level of HBV expression. The TNF-alpha-induced cell killing in high HBV-producing CWSV2 cell lines was accompanied by the presence of an oligonucleosomal DNA ladder characteristic of apoptosis. Images Figure 2 Figure 3 Figure 4 Figure 6 Figure 9 Figure 10 Figure 11 PMID:8774135

  17. Curative and beta cell regenerative effects of alpha1-antitrypsin treatment in autoimmune diabetic NOD mice.

    PubMed

    Koulmanda, Maria; Bhasin, Manoj; Hoffman, Lauren; Fan, Zhigang; Qipo, Andi; Shi, Hang; Bonner-Weir, Susan; Putheti, Prabhakar; Degauque, Nicolas; Libermann, Towia A; Auchincloss, Hugh; Flier, Jeffrey S; Strom, Terry B

    2008-10-21

    Invasive insulitis is a destructive T cell-dependent autoimmune process directed against insulin-producing beta cells that is central to the pathogenesis of type 1 diabetes mellitus (T1DM) in humans and the clinically relevant nonobese diabetic (NOD) mouse model. Few therapies have succeeded in restoring long-term, drug-free euglycemia and immune tolerance to beta cells in overtly diabetic NOD mice, and none have demonstrably enabled enlargement of the functional beta cell mass. Recent studies have emphasized the impact of inflammatory cytokines on the commitment of antigen-activated T cells to various effector or regulatory T cell phenotypes and insulin resistance and defective insulin signaling. Hence, we tested the hypothesis that inflammatory mechanisms trigger insulitis, insulin resistance, faulty insulin signaling, and the loss of immune tolerance to islets. We demonstrate that treatment with alpha1-antitrypsin (AAT), an agent that dampens inflammation, does not directly inhibit T cell activation, ablates invasive insulitis, and restores euglycemia, immune tolerance to beta cells, normal insulin signaling, and insulin responsiveness in NOD mice with recent-onset T1DM through favorable changes in the inflammation milieu. Indeed, the functional mass of beta cells expands in AAT-treated diabetic NOD mice.

  18. Lipidomic profile of GM95 cell death induced by Clostridium perfringens alpha-toxin.

    PubMed

    Manni, Marco M; Valero, Juan G; Pérez-Cormenzana, Miriam; Cano, Ainara; Alonso, Cristina; Goñi, Félix M

    2017-03-01

    Clostridium perfringens alpha-toxin (ATX) is considered as a prototype of cytotoxic bacterial phospholipases C, and is the major virulence factor in C. perfringens-induced gas gangrene. It is known that, depending on the dose, ATX causes membrane disruption and cytolysis or only limited hydrolysis of its substrates. In the latter case, toxin activity leads to the unregulated generation of bioactive lipids that can ultimately induce cell death. We have characterized apoptosis and necrosis in highly ATX-sensitive, ganglioside-deficient cells exposed to different concentrations of ATX and we have studied the lipidomic profile of cells treated with ATX as compared to native cells to detect the main changes in the lipidomic profile and the possible involvement of lipid signals in cell death. ATX causes both apoptosis and necrosis, depending on dose and time. ATX activates cell death, stimulating the release of cytochrome C from mitochondria and the consequent activation of caspases-3. Moreover GM95 cells treated with ATX showed important lipidomic alterations, among them we detected a general decrease in several phospholipid species and important changes in lipids involved in programmed cell death e.g. ceramide. The data suggest two different mechanisms of cell death caused by ATX, one leading to (mainly saturated) glycerophospholipid hydrolysis related to an increase in diacylglycerols and associated to membrane damage and necrosis, and a second mechanism involving chiefly sphingomyelin hydrolysis and generation of proapoptotic lipidic mediators such as ceramide, N-acylethanolamine and saturated non-esterified fatty acids.

  19. Regulation of the human SLC25A20 expression by peroxisome proliferator-activated receptor alpha in human hepatoblastoma cells

    SciTech Connect

    Tachibana, Keisuke; Takeuchi, Kentaro; Inada, Hirohiko; Yamasaki, Daisuke; Ishimoto, Kenji; Tanaka, Toshiya; Hamakubo, Takao; Sakai, Juro; Kodama, Tatsuhiko; Doi, Takefumi

    2009-11-20

    Solute carrier family 25, member 20 (SLC25A20) is a key molecule that transfers acylcarnitine esters in exchange for free carnitine across the mitochondrial membrane in the mitochondrial {beta}-oxidation. The peroxisome proliferator-activated receptor alpha (PPAR{alpha}) is a ligand-activated transcription factor that plays an important role in the regulation of {beta}-oxidation. We previously established tetracycline-regulated human cell line that can be induced to express PPAR{alpha} and found that PPAR{alpha} induces the SLC25A20 expression. In this study, we analyzed the promoter region of the human slc25a20 gene and showed that PPAR{alpha} regulates the expression of human SLC25A20 via the peroxisome proliferator responsive element.

  20. Co-receptor choice by V alpha14i NKT cells is driven by Th-POK expression rather than avoidance of CD8-mediated negative selection.

    PubMed

    Engel, Isaac; Hammond, Kirsten; Sullivan, Barbara A; He, Xi; Taniuchi, Ichiro; Kappes, Dietmar; Kronenberg, Mitchell

    2010-05-10

    Mouse natural killer T (NKT) cells with an invariant V alpha14-J alpha18 rearrangement (V alpha14 invariant [V alpha14i] NKT cells) are either CD4(+)CD8(-) or CD4(-)CD8(-). Because transgenic mice with forced CD8 expression in all T cells exhibited a profound NKT cell deficit, the absence of CD8 has been attributed to negative selection. We now present evidence that CD8 does not serve as a coreceptor for CD1d recognition and that the defect in development in CD8 transgene homozygous mice is the result of a reduction in secondary T cell receptor alpha rearrangements. Thymocytes from mice hemizygous for the CD8 transgene have a less severe rearrangement defect and have functional CD8(+) V alpha14i NKT cells. Furthermore, we demonstrate that the transcription factor Th, Poxviruses and Zinc finger, and Krüppel family (Th-POK) is expressed by V alpha14i NKT cells throughout their differentiation and is necessary both to silence CD8 expression and for the functional maturity of V alpha14i NKT cells. We therefore suggest that Th-POK expression is required for the normal development of V alpha14i NKT cells and that the absence of CD8 expression by these cells is a by-product of such expression, as opposed to the result of negative selection of CD8-expressing V alpha14i NKT cells.

  1. Irradiation of Mesenchymal Stromal Cells with Low and High Doses of Alpha Particles Induces Senescence and/or Apoptosis.

    PubMed

    Alessio, Nicola; Esposito, Giuseppe; Galano, Giovanni; De Rosa, Roberto; Anello, Pasquale; Peluso, Gianfranco; Tabocchini, Maria Antonella; Galderisi, Umberto

    2017-03-02

    The use of high-linear energy transfer charged particles is gaining attention as a medical tool because of the emission of radiations with an efficient cell-killing ability. Considerable interest has developed in the use of targeted alpha-particle therapy for the treatment of micrometastases. Moreover, the use of helium beams is gaining momentum, especially for treating pediatric tumors. We analyzed the effects of alpha particles on bone marrow mesenchymal stromal cells (MSCs), which have a subpopulation of stem cells capable of generating adipocytes, chondrocytes, and osteocytes. Further, these cells contribute toward maintenance of homeostasis in the body. MSCs were irradiated with low and high doses of alpha particles or X-rays and a comparative biological analysis was performed. At a low dose (40 mGy), alpha particles exhibited a limited negative effect on the biology of MSCs compared with X-rays. No significant perturbation of cell cycle was observed, and a minimal increase in apoptosis or senescence was detected. Self-renewal was preserved as revealed by the CFU assay. On the contrary, with 2000 mGy alpha particles we observed adverse effects on the vitality, functionality, and stemness of MSCs. These results are the consequence of different proportion of cells targeted by alpha particles or X-rays and the quality of induced DNA damage. The present study suggests that radiotherapy with alpha particles may spare healthy stem cells more efficaciously than X-ray treatments, an observation that should be taken into consideration by physicians while planning irradiation of tumor areas close to stem cell niches, such as bone marrow. This article is protected by copyright. All rights reserved.

  2. The effect of interferon-{alpha} on the expression of cytochrome P450 3A4 in human hepatoma cells

    SciTech Connect

    Flaman, Anathea S.; Gravel, Caroline; Hashem, Anwar M.; Tocchi, Monika; Li Xuguang

    2011-06-01

    Interferon {alpha} (IFN{alpha}) is used to treat malignancies and chronic viral infections. It has been found to decrease the rate of drug metabolism by acting on cytochrome P450 enzymes, but no studies have investigated the consequences of IFN{alpha} treatment on the CYP3A4 isoform, responsible for the metabolism of a majority of drugs. In this study, we have examined the effect of IFN{alpha} on CYP3A4 catalytic activity and expression in human hepatoma cells. We found that IFN{alpha} inhibits CYP3A4 activity and rapidly down-regulates the expression of CYP3A4, independent of de novo protein synthesis. Pharmacologic inhibitors and a dominant-negative mutant expression plasmid were used to dissect the molecular pathway required for CYP3A4 suppression, revealing roles for Jak1 and Stat1 and eliminating the involvement of the p38 mitogen-activated and extracellular regulated kinases. Treatment of hepatoma cells with IFN{alpha} did not affect the nuclear localization or relative abundance of Sp1 and Sp3 transcription factors, suggesting that the suppression of CYP3A4 by IFN{alpha} does not result from inhibitory Sp3 out-competing Sp1. To our knowledge, this is the first report that IFN{alpha} down-regulates CYP3A4 expression largely through the JAK-STAT pathway. Since IFN{alpha} suppresses CYP3A4 expression, caution is warranted when IFN{alpha} is administered in combination with CYP3A4 substrates to avoid the occurrence of adverse drug interactions.

  3. Analysis of SM22alpha-deficient mice reveals unanticipated insights into smooth muscle cell differentiation and function.

    PubMed

    Zhang, J C; Kim, S; Helmke, B P; Yu, W W; Du, K L; Lu, M M; Strobeck, M; Yu, Q; Parmacek, M S

    2001-02-01

    SM22alpha is a 22-kDa smooth muscle cell (SMC) lineage-restricted protein that physically associates with cytoskeletal actin filament bundles in contractile SMCs. To examine the function of SM22alpha, gene targeting was used to generate SM22alpha-deficient (SM22(-/-LacZ)) mice. The gene targeting strategy employed resulted in insertion of the bacterial lacZ reporter gene at the SM22alpha initiation codon, permitting precise analysis of the temporal and spatial pattern of SM22alpha transcriptional activation in the developing mouse. Northern and Western blot analyses confirmed that the gene targeting strategy resulted in a null mutation. Histological analysis of SM22(+/-LacZ) embryos revealed detectable beta-galactosidase activity in the unturned embryonic day 8.0 embryo in the layer of cells surrounding the paired dorsal aortae concomitant with its expression in the primitive heart tube, cephalic mesenchyme, and yolk sac vasculature. Subsequently, during postnatal development, beta-galactosidase activity was observed exclusively in arterial, venous, and visceral SMCs. SM22alpha-deficient mice are viable and fertile. Their blood pressure and heart rate do not differ significantly from their control SM22alpha(+/-) and SM22alpha(+/+) littermates. The vasculature and SMC-containing tissues of SM22alpha-deficient mice develop normally and appear to be histologically and ultrastructurally similar to those of their control littermates. Taken together, these data demonstrate that SM22alpha is not required for basal homeostatic functions mediated by vascular and visceral SMCs in the developing mouse. These data also suggest that signaling pathways that regulate SMC specification and differentiation from local mesenchyme are activated earlier in the angiogenic program than previously recognized.

  4. Inhibition of the glucose transporter SGLT2 with dapagliflozin in pancreatic alpha cells triggers glucagon secretion.

    PubMed

    Bonner, Caroline; Kerr-Conte, Julie; Gmyr, Valéry; Queniat, Gurvan; Moerman, Ericka; Thévenet, Julien; Beaucamps, Cédric; Delalleau, Nathalie; Popescu, Iuliana; Malaisse, Willy J; Sener, Abdullah; Deprez, Benoit; Abderrahmani, Amar; Staels, Bart; Pattou, François

    2015-05-01

    Type 2 diabetes (T2D) is characterized by chronic hyperglycemia resulting from a deficiency in insulin signaling, because of insulin resistance and/or defects in insulin secretion; it is also associated with increases in glucagon and endogenous glucose production (EGP). Gliflozins, including dapagliflozin, are a new class of approved oral antidiabetic agents that specifically inhibit sodium-glucose co-transporter 2 (SGLT2) function in the kidney, thus preventing renal glucose reabsorption and increasing glycosuria in diabetic individuals while reducing hyperglycemia. However, gliflozin treatment in subjects with T2D increases both plasma glucagon and EGP by unknown mechanisms. In spite of the rise in EGP, T2D patients treated with gliflozin have lower blood glucose levels than those receiving placebo, possibly because of increased glycosuria; however, the resulting increase in plasma glucagon levels represents a possible concerning side effect, especially in a patient population already affected by hyperglucagonemia. Here we demonstrate that SGLT2 is expressed in glucagon-secreting alpha cells of the pancreatic islets. We further found that expression of SLC5A2 (which encodes SGLT2) was lower and glucagon (GCG) gene expression was higher in islets from T2D individuals and in normal islets exposed to chronic hyperglycemia than in islets from non-diabetics. Moreover, hepatocyte nuclear factor 4-α (HNF4A) is specifically expressed in human alpha cells, in which it controls SLC5A2 expression, and its expression is downregulated by hyperglycemia. In addition, inhibition of either SLC5A2 via siRNA-induced gene silencing or SGLT2 via dapagliflozin treatment in human islets triggered glucagon secretion through KATP channel activation. Finally, we found that dapagliflozin treatment further promotes glucagon secretion and hepatic gluconeogenesis in healthy mice, thereby limiting the decrease of plasma glucose induced by fasting. Collectively, these results identify a

  5. CPT1{alpha} over-expression increases long-chain fatty acid oxidation and reduces cell viability with incremental palmitic acid concentration in 293T cells

    SciTech Connect

    Jambor de Sousa, Ulrike L.; Koss, Michael D.; Fillies, Marion; Gahl, Anja; Scheeder, Martin R.L.; Cardoso, M. Cristina; Leonhardt, Heinrich; Geary, Nori; Langhans, Wolfgang; Leonhardt, Monika . E-mail: monika.leonhardt@inw.agrl.ethz.ch

    2005-12-16

    To test the cellular response to an increased fatty acid oxidation, we generated a vector for an inducible expression of the rate-limiting enzyme carnitine palmitoyl-transferase 1{alpha} (CPT1{alpha}). Human embryonic 293T kidney cells were transiently transfected and expression of the CPT1{alpha} transgene in the tet-on vector was activated with doxycycline. Fatty acid oxidation was measured by determining the conversion of supplemented, synthetic cis-10-heptadecenoic acid (C17:1n-7) to C15:ln-7. CPT1{alpha} over-expression increased mitochondrial long-chain fatty acid oxidation about 6-fold. Addition of palmitic acid (PA) decreased viability of CPT1{alpha} over-expressing cells in a concentration-dependent manner. Both, PA and CPT1{alpha} over-expression increased cell death. Interestingly, PA reduced total cell number only in cells over-expressing CPT1{alpha}, suggesting an effect on cell proliferation that requires PA translocation across the mitochondrial inner membrane. This inducible expression system should be well suited to study the roles of CPT1 and fatty acid oxidation in lipotoxicity and metabolism in vivo.

  6. Induction of cytotoxicity in human lung adenocarcinoma cells by 6-O-carboxypropyl-alpha-tocotrienol, a redox-silent derivative of alpha-tocotrienol.

    PubMed

    Yano, Yoshihisa; Satoh, Haruna; Fukumoto, Keiko; Kumadaki, Itsumaro; Ichikawa, Tomio; Yamada, Kazuhiko; Hagiwara, Kiyokazu; Yano, Tomohiro

    2005-07-10

    Tocotrienols are one of the most potent anticancer agents of all natural compounds and the anticancer property may be related to the inactivation of Ras family molecules. The anticancer potential of tocotrienols, however, is weakened due to its short elimination half life in vivo. To overcome the disadvantage and reinforce the anticancer activity in tocotrienols, we synthesized a redox-silent analogue of alpha-tocotrienol (T3), 6-O-carboxypropyl-alpha-tocotrienol (T3E). We estimated the possibility of T3E as a new anticancer agent against lung adenocarcinoma showing poor prognosis based on the mutation of ras gene. T3E showed cytotoxicity against A549 cells, a human lung adenocarcinoma cell line with a ras gene mutation, in a dose-dependent manner (0-40 microM), whereas T3 and a redox-silent analogue of alpha-tocopherol (T), 6-O-carboxypropyl-alpha-tocopherol (TE), showed much less cytotoxicity in cells within 40 microM. T3E cytotoxicity was based on the accumulation of cells in the G1-phase of the cell-cycle and the subsequent induction of apoptosis. Similar to this event, 24-hr treatment of A549 cells with 40 microM T3E caused the inhibition of Ras farnesylation, and a marked decrease in the levels of cyclin D required for G1/S progression in the cell-cycle and Bcl-xL, a key anti-apoptotic molecule. Moreover, the T3E-dependent inhibition of RhoA geranyl-geranylation is an inducing factor for the occurrence of apoptosis in A549 cells. Our results suggest that T3E suppresses Ras and RhoA prenylation, leading to negative growth control against A549 cells. In conclusion, a redox-silent analogue of T3, T3E may be a new candidate as an anticancer agent against lung adenocarcinoma showing poor prognosis based on the mutation of ras genes.

  7. Feasibility study on the use of polyallyldiglycol-carbonate cell dishes in TUNEL assay for alpha particle radiobiological experiments

    NASA Astrophysics Data System (ADS)

    Chan, K. F.; Yum, E. H. W.; Wan, C. K.; Fong, W. F.; Yu, K. N.

    2007-08-01

    In the present work, we have studied the feasibility of a method based on polyallyldiglycol-carbonate (PADC) films to investigate the effects of alpha particles on HeLa cervix cancer cells. Thin PADC films with thickness of about 20 μm were prepared from commercially available CR-39 films by chemical etching to fabricate custom-made petri dishes for cell culture, which could accurately record alpha particle hit positions. A special method involving "base tracks" for aligning the images of cell nuclei and alpha particle hits has been proposed, so that alpha particle transversals of cell nuclei can be visually counted. Radiobiological experiments were carried out to induce DNA damages, with the TdT-mediated d UTP Nick- End Labeling (TUNEL) fluorescence method employed to detect DNA strand breaks. The staining results were investigated by flow cytometer. The preliminary results showed that more strand breaks occurred in cells hit by alpha particles with lower energies. Moreover, large TUNEL positive signals were obtained even with small percentages of cells irradiated and TUNEL signals were also obtained from non-targeted cells. These provided evidence for the bystander effect.

  8. Bacterial cell wall products increases stabilization of HIF-1 alpha in an oligodendrocyte cell line preconditioned by cobalt chloride or desferrioxamine.

    PubMed

    Yao, Song-yi; Soutto, Mohammed; Sriram, Subramaniam

    2008-08-30

    We examined the effect of lipopolysaccharide (LPS) or lipotechoic acid (LTA) on the regulation of hypoxia inducible factor (HIF-1) alpha on the MO3.13 cells, a human oligodendroglial cell line. Our study shows that MO3.13 cells express the toll like receptors (TLR's) but do not increase cellular levels of HIF-1 alpha following exposure to bacterial cell wall products. When MO3.13 cells were preconditioned by desferrioxamine (DFO) or cobalt chloride (CoCl(2)) and then treated with either LPS or LTA, HIF-1 alpha levels were higher than that induced by DFO or CoCl(2) alone. The increase in HIF-1 alpha was due to increased protein stability that was mediated by activation of the ERK-MAP kinase pathway.

  9. Characterization of the cytokine pattern of porcine bone marrow-derived cells treated with 1alpha,25(OH)D.

    PubMed

    Sipos, W; Duvigneau, J C; Schmoll, F; Exel, B; Hofbauer, G; Baravalle, G; Hartl, R T; Dobretsberger, M; Pietschmann, P

    2005-10-01

    The biologically active form of vitamine D(3) [1alpha,25(OH)(2)D(3)] has recently been described not only to influence bone metabolism but also to exert immunomodulating activities, which may have an impact on bone formation/resorption as well. In this study, we analysed the effects of 1alpha,25(OH)(2)D(3) on the cytokine pattern of porcine bone marrow-derived cells from piglets aged 1-3 weeks. After culture for 1 week, the number of osteoclasts was determined, with tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated cells being considered osteoclasts. Cultured bone marrow cell-derived mRNA was subjected to semiquantitative RT-PCR specific for a panel of porcine cytokines (IL-1alpha, IL-6, IL-8, IL-10, and TNF-alpha). In addition, an immunofluorescence analysis using anti-porcine mAbs specific for IL-1beta, IL-2, IL-4, IL-6, IL-12, TNF-alpha, and IFN-gamma was performed. In order to prove the existence of a porcine homologue of the receptor activator of NF-kappaB ligand (RANKL) bone marrow cell- as well as porcine white blood cell-derived mRNA was investigated by RT-PCR using primer pairs specific for murine RANKL. Cell culture supernatant was analysed for soluble RANKL by means of an ELISA designed for quantification of human RANKL. By means of RT-PCR, expression of IL-1alpha, IL-6, IL-8, IL-10 and TNF-alpha mRNA could be found in cells cultured with and without 1alpha,25(OH)(2)D(3). Immunofluorescence analysis revealed that IL-1, IL-6, and TNF-alpha were produced by both stromal cells and osteoclasts. Besides its known osteoclastogenic effects, 1alpha,25(OH)(2)D(3) tended to downregulate the respective cytokines, but significantly upregulated RANKL expression. The homology between the porcine RANKL-specific sequence and the corresponding human RANKL sequence was 79%. The data found support the idea that porcine bone marrow cell cultures may provide a suitable alternative to murine systems in human osteological research.

  10. The intermediate filament protein vimentin binds specifically to a recombinant integrin {alpha}2/{beta}1 cytoplasmic tail complex and co-localizes with native {alpha}2/{beta}1 in endothelial cell focal adhesions

    SciTech Connect

    Kreis, Stephanie; Schoenfeld, Hans-Joachim; Melchior, Chantal; Steiner, Beat; Kieffer, Nelly . E-mail: kieffer@cu.lu

    2005-04-15

    Integrin receptors are crucial players in cell adhesion and migration. Identification and characterization of cellular proteins that interact with their short {alpha} and {beta} cytoplasmic tails will help to elucidate the molecular mechanisms by which integrins mediate bi-directional signaling across the plasma membrane. Integrin {alpha}2{beta}1 is a major collagen receptor but to date, only few proteins have been shown to interact with the {alpha}2 cytoplasmic tail or with the {alpha}2{beta}1 complex. In order to identify novel binding partners of a {alpha}2{beta}1cytoplasmic domain complex, we have generated recombinant GST-fusion proteins, incorporating the leucine zipper heterodimerization cassettes of Jun and Fos. To ascertain proper functionality of the recombinant proteins, interaction with natural binding partners was tested. GST-{alpha}2 and GST-Jun {alpha}2 bound His-tagged calreticulin while GST-{beta}1 and GST-Fos {beta}1 proteins bound talin. In screening assays for novel binding partners, the immobilized GST-Jun {alpha}2/GST-Fos {beta}1 heterodimeric complex, but not the single subunits, interacted specifically with endothelial cell-derived vimentin. Vimentin, an abundant intermediate filament protein, has previously been shown to co-localize with {alpha}v{beta}3-positive focal contacts. Here, we provide evidence that this interaction also occurs with {alpha}2{beta}1-enriched focal adhesions and we further show that this association is lost after prolonged adhesion of endothelial cells to collagen.

  11. Targeted Cytoplasmic Irradiation with Alpha Particles Induces Mutations in Mammalian Cells

    NASA Astrophysics Data System (ADS)

    Wu, Li-Jun; Randers-Pehrson, Gerhard; Xu, An; Waldren, Charles A.; Geard, Charles R.; Yu, Zengliang; Hei, Tom K.

    1999-04-01

    Ever since x-rays were shown to induce mutation in Drosophila more than 70 years ago, prevailing dogma considered the genotoxic effects of ionizing radiation, such as mutations and carcinogenesis, as being due mostly to direct damage to the nucleus. Although there was indication that alpha particle traversal through cellular cytoplasm was innocuous, the full impact remained unknown. The availability of the microbeam at the Radiological Research Accelerator Facility of Columbia University made it possible to target and irradiate the cytoplasm of individual cells in a highly localized spatial region. By using dual fluorochrome dyes (Hoechst and Nile Red) to locate nucleus and cellular cytoplasm, respectively, thereby avoiding inadvertent traversal of nuclei, we show here that cytoplasmic irradiation is mutagenic at the CD59 (S1) locus of human-hamster hybrid (AL) cells, while inflicting minimal cytotoxicity. The principal class of mutations induced are similar to those of spontaneous origin and are entirely different from those of nuclear irradiation. Furthermore, experiments with radical scavenger and inhibitor of intracellular glutathione indicated that the mutagenicity of cytoplasmic irradiation depends on generation of reactive oxygen species. These findings suggest that cytoplasm is an important target for genotoxic effects of ionizing radiation, particularly radon, the second leading cause of lung cancer in the United States. In addition, cytoplasmic traversal by alpha particles may be more dangerous than nuclear traversal, because the mutagenicity is accomplished by little or no killing of the target cells.

  12. PPARγ regulates the mitochondrial dysfunction in human neural stem cells with tumor necrosis factor alpha.

    PubMed

    Chiang, M-C; Cheng, Y-C; Lin, K-H; Yen, C-H

    2013-01-15

    Peroxisome proliferator-activated receptor gamma (PPARγ) belongs to a family of ligand-activated transcription factors, and its ligands are known to control many physiological and pathological conditions. The hypothesis of our study was that the PPARγ agonist (rosiglitazone) could mediate tumor necrosis factor alpha (TNFα) related to the regulation of human neural stem cells (hNSCs), by which TNFα possibly fulfills important roles in neuronal impairment. The results show that PPARγ mediates the cell viability of hNSCs via the downregulation of the activity of caspase 3, indicating that this rescue effect of PPARγ could improve the reduced levels of two mitochondrial regulators, adenosine monophosphate-activated protein kinase (AMPK) and Sirtuin 1 (SIRT1) in the hNSCs with TNFα. The stimulation of mitochondrial function by PPARγ was associated with activation of the PPAR coactivator1 alpha (PGC1α) pathway by up-regulation of oxidative defense and mitochondrial systems. The above protective effects appeared to be exerted by a direct activation of the rosiglitazone, because it protected hNSCs from TNFα-evoked oxidative stress and mitochondrial deficiency. Here we show that the rosiglitazone protects hNSCs against Aβ-induced apoptosis and promotes cell survival. These findings extend our understanding of the central role of PPARγ in TNFα-related neuronal impairment, which probably increases risks of neurodegenerative diseases. The anti-inflammatory effects of PPARγ in the hNSCs with TNFα, and the involved mechanisms were also characterized.

  13. Autophagy-associated alpha-arrestin signaling is required for conidiogenous cell development in Magnaporthe oryzae

    PubMed Central

    Dong, Bo; Xu, Xiaojin; Chen, Guoqing; Zhang, Dandan; Tang, Mingzhi; Xu, Fei; Liu, Xiaohong; Wang, Hua; Zhou, Bo

    2016-01-01

    Conidiation patterning is evolutionarily complex and mechanism concerning conidiogenous cell differentiation remains largely unknown. Magnaporthe oryzae conidiates in a sympodial way and uses its conidia to infect host and disseminate blast disease. Arrestins are multifunctional proteins that modulate receptor down-regulation and scaffold components of intracellular trafficking routes. We here report an alpha-arrestin that regulates patterns of conidiation and contributes to pathogenicity in M. oryzae. We show that disruption of ARRDC1 generates mutants which produce conidia in an acropetal array and ARRDC1 significantly affects expression profile of CCA1, a virulence-related transcription factor required for conidiogenous cell differentiation. Although germ tubes normally develop appressoria, penetration peg formation is dramatically impaired and Δarrdc1 mutants are mostly nonpathogenic. Fluorescent analysis indicates that EGFP-ARRDC1 puncta are well colocalized with DsRed2-Atg8, and this distribution profile could not be altered in Δatg9 mutants, suggesting ARRDC1 enters into autophagic flux before autophagosome maturation. We propose that M. oryzae employs ARRDC1 to regulate specific receptors in response to conidiation-related signals for conidiogenous cell differentiation and utilize autophagosomes for desensitization of conidiogenous receptor, which transmits extracellular signal to the downstream elements of transcription factors. Our investigation extends novel significance of autophagy-associated alpha-arrestin signaling to fungal parasites. PMID:27498554

  14. Human DNA polymerase alpha gene: sequences controlling expression in cycling and serum-stimulated cells.

    PubMed Central

    Pearson, B E; Nasheuer, H P; Wang, T S

    1991-01-01

    We have investigated the DNA polymerase alpha promoter sequence requirements for the expression of a heterologous gene in actively cycling cells and following serum addition to serum-deprived cells. An 11.4-kb genomic clone that spans the 5' end of this gene and includes 1.62 kb of sequence upstream from the translation start site was isolated. The transcription start site was mapped at 46 +/- 1 nucleotides upstream from the translation start site. The upstream sequence is GC rich and lacks a TATA sequence but has a CCAAT sequence on the opposite strand. Analysis of a set of deletion constructs in transient transfection assays demonstrated that efficient expression of the reporter in cycling cells requires 248 bp of sequence upstream from the cap site. Clustered within these 248 nucleotides are sequences similar to consensus sequences for Sp1-, Ap1-, Ap2-, and E2F-binding sites. The CCAAT sequence and the potential E2F- and Ap1-binding sites are shown to be protected from DNase I digestion by partially purified nuclear proteins. The DNA polymerase alpha promoter can confer upon the reporter an appropriate, late response to serum addition. No single sequence element could be shown to confer serum inducibility. Rather, multiple sequence elements appear to mediate the full serum response. Images PMID:2005899

  15. Prothymosin alpha is a chromatin-remodelling protein in mammalian cells.

    PubMed Central

    Gomez-Marquez, J; Rodríguez, P

    1998-01-01

    Prothymosin alpha (ProTalpha) is an abundant mammalian acidic nuclear protein whose expression is related to cell proliferation. Here we report that in HL-60 cells overexpressing ProTalpha, the accessibility of micrococcal nuclease to chromatin is strongly increased. In the DNA ladder generated by the nuclease activity, the sizes of the mononucleosome (146 bp, the DNA fragment that is bound to the histone octamer) and its multimers correspond to nucleosomes lacking histone H1. The percentage of histone-H1-depleted chromatin (active chromatin) is also higher in the cells overexpressing ProTalpha. On the basis of these and previous findings, we propose a biological role for ProTalpha in the remodelling of chromatin fibres through its interaction with histone H1. PMID:9639554

  16. Eicosapentaenoic acid inhibits TNF-{alpha}-induced matrix metalloproteinase-9 expression in human keratinocytes, HaCaT cells

    SciTech Connect

    Kim, Hyeon Ho; Lee, Youngae; Eun, Hee Chul Chung, Jin Ho

    2008-04-04

    Eicosapentaenoic acid (EPA) is an omega-3 ({omega}-3) polyunsaturated fatty acid (PUFA), which has anti-inflammatory and anti-cancer properties. Some reports have demonstrated that EPA inhibits NF-{kappa}B activation induced by tumor necrosis factor (TNF)-{alpha} or lipopolysaccharide (LPS) in various cells. However, its detailed mode of action is unclear. In this report, we investigated whether EPA inhibits the expression of TNF-{alpha}-induced matrix metalloproteinases (MMP)-9 in human immortalized keratinocytes (HaCaT). TNF-{alpha} induced MMP-9 expression by NF-{kappa}B-dependent pathway. Pretreatment of EPA inhibited TNF-{alpha}-induced MMP-9 expression and p65 phosphorylation. However, EPA could not affect I{kappa}B-{alpha} phosphorylation, nuclear translocation of p65, and DNA binding activity of NF-{kappa}B. EPA inhibited TNF-{alpha}-induced p65 phosphorylation through p38 and Akt inhibition and this inhibition was IKK{alpha}-dependent event. Taken together, we demonstrate that EPA inhibits TNF-{alpha}-induced MMP-9 expression through inhibition of p38 and Akt activation.

  17. Overexpression of prothymosin alpha accelerates proliferation and retards differentiation in HL-60 cells.

    PubMed

    Rodríguez, P; Viñuela, J E; Alvarez-Fernández, L; Buceta, M; Vidal, A; Domínguez, F; Gómez-Márquez, J

    1998-05-01

    Prothymosin alpha (ProTalpha) is an acidic nuclear protein the expression of which is related to the proliferation and differentiation processes in mammalian cells. In the present study we have stably transfected HL-60 cells, a biological system that allows the study of both proliferation and differentiation, with recombinant vectors encoding sense and antisense ProTalpha mRNA. In the HL-60 cell clones overexpressing ProTalpha we observed an acceleration in the growth rate, whereas expression of the antisense orientation showed the opposite effect. Moreover, cell-cycle analysis demonstrated that the G1-phase was shortened in the cells expressing the sense construct. Before studying how ProTalpha affects differentiation, we showed that the down-regulation of ProTalpha gene during differentiation occurs in all mammalian cell lines (HL-60, K562, U937, MEL C88, N2A and PC12) analysed. The biological effect evoked by the induction of the ProTalpha sense vector was the retardation of cell differentiation, although expression of the antisense construct showed no effect on differentiation. In conclusion, our findings provide evidence that ProTalpha is directly implicated in cellular proliferation and that the maintenance of high levels of ProTalpha inside HL-60 cells is incompatible with their ability to differentiate.

  18. Overexpression of prothymosin alpha accelerates proliferation and retards differentiation in HL-60 cells.

    PubMed Central

    Rodríguez, P; Viñuela, J E; Alvarez-Fernández, L; Buceta, M; Vidal, A; Domínguez, F; Gómez-Márquez, J

    1998-01-01

    Prothymosin alpha (ProTalpha) is an acidic nuclear protein the expression of which is related to the proliferation and differentiation processes in mammalian cells. In the present study we have stably transfected HL-60 cells, a biological system that allows the study of both proliferation and differentiation, with recombinant vectors encoding sense and antisense ProTalpha mRNA. In the HL-60 cell clones overexpressing ProTalpha we observed an acceleration in the growth rate, whereas expression of the antisense orientation showed the opposite effect. Moreover, cell-cycle analysis demonstrated that the G1-phase was shortened in the cells expressing the sense construct. Before studying how ProTalpha affects differentiation, we showed that the down-regulation of ProTalpha gene during differentiation occurs in all mammalian cell lines (HL-60, K562, U937, MEL C88, N2A and PC12) analysed. The biological effect evoked by the induction of the ProTalpha sense vector was the retardation of cell differentiation, although expression of the antisense construct showed no effect on differentiation. In conclusion, our findings provide evidence that ProTalpha is directly implicated in cellular proliferation and that the maintenance of high levels of ProTalpha inside HL-60 cells is incompatible with their ability to differentiate. PMID:9560301

  19. Karyopherin Alpha 1 Regulates Satellite Cell Proliferation and Survival by Modulating Nuclear Import.

    PubMed

    Choo, Hyo-Jung; Cutler, Alicia; Pavlath, Grace K

    2016-07-19

    Satellite cells are stem cells with an essential role in skeletal muscle repair. Precise regulation of gene expression is critical for proper satellite cell quiescence, proliferation, differentiation and self-renewal. Nuclear proteins required for gene expression are dependent on the nucleocytoplasmic transport machinery to access to nucleus, however little is known about regulation of nuclear transport in satellite cells. The best characterized nuclear import pathway is classical nuclear import which depends on a classical nuclear localization signal (cNLS) in a cargo protein and the heterodimeric import receptors, karyopherin alpha (KPNA) and beta (KPNB). Multiple KPNA1 paralogs exist and can differ in importing specific cNLS proteins required for cell differentiation and function. We show that transcripts for six Kpna paralogs underwent distinct changes in mouse satellite cells during muscle regeneration accompanied by changes in cNLS proteins in nuclei. Depletion of KPNA1, the most dramatically altered KPNA, caused satellite cells in uninjured muscle to prematurely activate, proliferate and undergo apoptosis leading to satellite cell exhaustion with age. Increased proliferation of satellite cells led to enhanced muscle regeneration at early stages of regeneration. In addition, we observed impaired nuclear localization of two key KPNA1 cargo proteins: p27, a cyclin-dependent kinase inhibitor associated with cell cycle control and lymphoid enhancer factor 1, a critical cotranscription factor for β-catenin. These results indicate that regulated nuclear import of proteins by KPNA1 is critical for satellite cell proliferation and survival and establish classical nuclear import as a novel regulatory mechanism for controlling satellite cell fate. Stem Cells 2016.

  20. Homeobox A7 stimulates breast cancer cell proliferation by up-regulating estrogen receptor-alpha

    SciTech Connect

    Zhang, Yu; Cheng, Jung-Chien; Huang, He-Feng; Leung, Peter C.K.

    2013-11-01

    Highlights: •HOXA7 regulates MCF7 cell proliferation. •HOXA7 up-regulates ERα expression. •HOXA7 mediates estrogen-induced MCF7 cell proliferation. -- Abstract: Breast cancer is the most common hormone-dependent malignancy in women. Homeobox (HOX) transcription factors regulate many cellular functions, including cell migration, proliferation and differentiation. The aberrant expression of HOX genes has been reported to be associated with human reproductive cancers. Estradiol (E2) and its nuclear receptors, estrogen receptor (ER)-alpha and ER-beta, are known to play critical roles in the regulation of breast cancer cell growth. However, an understanding of the potential relationship between HOXA7 and ER in breast cancer cells is limited. In this study, our results demonstrate that knockdown of HOXA7 in MCF7 cells significantly decreased cell proliferation and ERα expression. In addition, HOXA7 knockdown attenuated E2-induced cell proliferation as well as progesterone receptor (PR) expression. The stimulatory effects of E2 on cell proliferation and PR expression were abolished by co-treatment with ICI 182780, a selective ERα antagonist. In contrast, overexpression of HOXA7 significantly stimulated cell proliferation and ERα expression. Moreover, E2-induced cell proliferation, as well as PR expression, was enhanced by the overexpression of HOXA7. Neither knockdown nor overexpression of HOXA7 affected the ER-beta levels. Our results demonstrate a novel mechanistic role for HOXA7 in modulating breast cancer cell proliferation via regulation of ERα expression. This finding contributes to our understanding of the role HOXA7 plays in regulating the proliferation of ER-positive cancer cells.

  1. Developmentally regulated enzymes and cyclic AMP-binding sites in Dictyostelium discoideum cells blocked during development by alpha-chymotrypsin.

    PubMed Central

    Schmidt, J A; Stirling, J L

    1982-01-01

    When cells of the slime mould Dictyostelium discoideum are allowed to starve in the presence of alpha-chymotrypsin, they are blocked in development at the stage where tight aggregates form tips. Analysis of developmentally regulated enzymes has shown that alpha-mannosidase, beta-N-acetylglucosaminidase, threonine deaminase, tyrosine aminotransferase, beta-glucosidase and the carbohydrate-binding protein discoidin are unaffected, but enzymes that show an increase in specific activity during post-aggregative development, namely glycogen phosphorylase, UDP-glucose pyrophosphorylase, UDP-galactose 4-epimerase, UDP-galactose polysaccharide transferase and alkaline phosphatase, did not show the characteristic increase when development was blocked by alpha-chymotrypsin. Recovery of cells from the effects of alpha-chymotrypsin was accompanied by the formation of fruiting bodies and a concomitant increase in the specific activity of UDP-glucose pyrophosphorylase. Uptake or efflux of 45Ca2+ was not altered in the presence of alpha-chymotrypsin. Cells allowed to develop in alpha-chymotrypsin, or treated with the enzyme for 15 min, had a markedly reduced ability to bind cyclic AMP with low affinity; high-affinity binding was unaffected. Pronase had a similar effect on cyclic AMP binding, but trypsin, which does not alter developmental processes, has no effect on cyclic AMP binding to D. discoideum cells. PMID:7150239

  2. Modulation of alpha smooth muscle actin and desmin expression in perisinusoidal cells of normal and diseased human livers.

    PubMed Central

    Schmitt-Gräff, A.; Krüger, S.; Bochard, F.; Gabbiani, G.; Denk, H.

    1991-01-01

    It has been suggested that perisinusoidal liver cells (PSC) play a pivotal role in the pathogenesis of fibrocontractive changes. Using light and electron microscopic immunolocalization techniques, a series of 207 normal and pathologic human liver specimens were evaluated for the expression of alpha smooth muscle (SM) actin and desmin in this and other nonparenchymal cell types. In normal adult liver tissue, PSCs were practically devoid of desmin and exceptionally stained for alpha-SM actin, whereas this actin isoform frequently was encountered in PSCs from the embryonic to the adolescent period. A broad spectrum of pathologic conditions was accompanied by the presence of alpha-SM actin containing PSCs; these were detected preferentially in periportal or perivenular zones according to the predominant location of the underlying hepatocellular damage. The occurrence of this PSC phenotype generally was associated with fibrogenesis and was in some cases detected earlier than overt collagen accumulation. Fibrous bands subdividing liver tissue in cirrhosis and focal nodular hyperplasia, as well as desmoplastic reaction to malignant tumors, contained alpha-SM actin-rich cells admixed with variable proportions of cells coexpressing desmin. In end stages, this population was less numerous than in active fibrotic or cirrhotic processes. Using immunogold electron microscopy, alpha-SM actin was localized in microfilament bundles of typical PSCs. Our results are compatible with the assumption that the appearance of alpha-SM actin and desmin-expressing myofibroblasts results at least in part from a phenotypic modulation of PSCs. Images Figure 1 Figure 2 PMID:2024709

  3. Diallyl trisulfide, a chemopreventive agent from Allium vegetables, inhibits alpha-secretases in breast cancer cells.

    PubMed

    Kiesel, Violet A; Stan, Silvia D

    2017-03-18

    Breast cancer affects one in eight women throughout the course of their lifetime creating a demand for novel prevention strategies against this disease. The Notch signaling pathway is often aberrantly activated in human malignancies including breast cancer. Alpha secretases, including ADAM (A Disintegrin and Metalloprotease) -10 and -17, are proteases that play a key role in the cleavage of cell surface molecules and subsequent ligand-mediated activation of Notch signaling pathway. High expression levels of ADAM10 and 17 have been clinically associated with a lower disease-free survival in breast cancer patients. This study was undertaken to determine the effect of diallyl trisulfide (DATS), a bioactive organosulfide found in garlic and other Allium vegetables, on alpha secretases in breast cancer cells. Here we report for the first time that DATS inhibits the expression of ADAM10 and ADAM17 in estrogen-independent MDA-MB-231 and estrogen-dependent MCF-7 breast cancer cells, and in Harvey-ras (H-Ras) transformed MCF10A-H-Ras breast epithelial cells. We also show that DATS induces a dose-dependent reduction in colony formation ability of MDA-MB-231 and MCF-7 cells, suggesting a long-term effect of DATS on growth inhibition of breast cancer cells. Furthermore, we show that DATS inhibits the Notch ligands Jagged-1 and Jagged-2 involved in activation of Notch signaling pathway. Collectively, these findings show that DATS targets Notch pathway components overexpressed in breast cancer tumors and may serve as a functionally relevant bioactive for breast cancer prevention.

  4. In vitro study of alpha 2-adrenoceptor turnover and metabolism using the adenocarcinoma cell line HT29

    SciTech Connect

    Paris, H.; Taouis, M.; Galitzky, J.

    1987-11-01

    The biosynthesis rate of the receptor was studied in postconfluent HT29 cells, when its density expressed as fmol/mg of cell membrane protein is constant, by following the recovery of the receptor binding capacity after blockade with the non-reversible alpha-adrenergic antagonist benextramine. Study of the inhibition of (/sup 3/H)yohimbine and (/sup 3/H)UK-14,304 binding showed that benextramine was a more potent antagonist at alpha 2-adrenoceptor than phenoxybenzamine. The incubation of intact HT29 cells for 30 min in the presence of 10(-5) M benextramine irreversibly blocked more than 95% of the alpha 2-adrenoceptors and totally suppressed the inhibitory effect of UK-14,304 on cyclic AMP production. The blockade appeared specific, since benextramine effects were prevented by alpha 2-adrenergic agents. Moreover, neither vasoactive intestinal polypeptide responsiveness nor other tested aspects of the regulation of the adenylate cyclase was altered by the treatment. Study of the time course of receptor recovery after irreversible blockade indicated that alpha 2-adrenoceptors reappeared in the cells with a monoexponential kinetic. The linearization of the repopulation curve obtained with the labeled antagonist (/sup 3/H)yohimbine allowed the determination of the rate constant for receptor degradation (k = 0.0268 +/- 0.0025 hr-1) and the rate of receptor synthesis (6.91 +/- 0.64 fmol/mg of cell membrane protein/hr) corresponding to the synthesis of about 500 receptors/cell/hr. The alpha 2-adrenoceptor half-life was 26 +/- 3 hr. Measurement of the biological effects associated to the alpha-adrenoceptor stimulation during the course of receptor recovery indicated a relationship between the number of cell receptors and the percentage of inhibition of the cyclic AMP accumulation induced by forskolin.

  5. Suppression of human prostate cancer cell growth by alpha1-adrenoceptor antagonists doxazosin and terazosin via induction of apoptosis.

    PubMed

    Kyprianou, N; Benning, C M

    2000-08-15

    Recent evidence from our laboratory has demonstrated that alpha1-adrenoceptor antagonists doxazosin and terazosin induced apoptosis in prostate epithelial and smooth muscle cells in patients with benign prostatic hypertrophy (BPH; J. Urol., 159: 1810-1815, 1998; J. Urol., 161: 2002-2007, 1999). In this study, we investigated the biological action of three alpha1-adrenoceptor antagonists, doxazosin, terazosin, and tamsulosin, against prostate cancer cell growth. The antigrowth effect of the three alpha1-adrenoceptor antagonists was examined in two human prostate cancer cell lines, PC-3 and DU-145, and a prostate smooth muscle cell primary culture, SMC-1, on the basis of: (a) cell viability assay; (b) rate of DNA synthesis; and (c) induction of apoptosis. Our results indicate that treatment of prostate cancer cells with doxazosin or terazosin results in a significant loss of cell viability, via induction of apoptosis in a dose-dependent manner, whereas tamsulosin had no effect on prostate cell growth. Neither doxazosin nor terazosin exerted a significant effect on the rate of cell proliferation in prostate cancer cells. Exposure to phenoxybenzamine, an irreversible inhibitor of alpha1-adrenoceptors, does not abrogate the apoptotic effect of doxazosin or terazosin against human prostate cancer or smooth muscle cells. This suggests that the apoptotic activity of doxazosin and terazosin against prostate cells is independent of their capacity to antagonize alpha1-adrenoceptors. Furthermore, an in vivo efficacy trial demonstrated that doxazosin administration (at tolerated pharmacologically relevant doses) in SCID mice bearing PC-3 prostate cancer xenografts resulted in a significant inhibition of tumor growth. These findings demonstrate the ability of doxazosin and terazosin (but not tamsulosin) to suppress prostate cancer cell growth in vitro and in vivo by inducing apoptosis without affecting cell proliferation. This evidence provides the rationale for targeting both

  6. The mucin epiglycanin on TA3/Ha carcinoma cells prevents alpha 6 beta 4- mediated adhesion to laminin and kalinin and E-cadherin-mediated cell- cell interaction

    PubMed Central

    1994-01-01

    TA3/Ha murine mammary carcinoma cells grow in suspension, do not adhere to extracellular matrix molecules, but do adhere to hepatocytes and form liver metastases upon intraportal injection. Recently we showed that the integrin alpha 6 beta 4 on the TA3/Ha cells is involved in adhesion to hepatocytes. However, despite high cell surface levels of alpha 6 beta 4, TA3/Ha cells do not adhere to the alpha 6 beta 4 ligands laminin and kalinin. Here we show that this is due to the mucin epiglycanin that is highly expressed on TA3/Ha cells. Some monoclonal antibodies generated against epiglycanin induced capping of most of the epiglycanin molecules. TA3/Ha cells treated with these mAb did adhere to laminin and kalinin, and an epithelial monolayer was formed on kalinin, with alpha 6 beta 4 localized in HD1-containing hemidesmosome- like structures and E-cadherin at the cell-cell contact sites. Similar results were obtained after treatment of TA3/Ha cells with O- sialoglycoprotein endopeptidase which removes all epiglycanin. In addition, the enzyme induced E-cadherin-mediated cell-cell aggregation. Both treatments also enhanced the adhesion to hepatocytes, but given the potent antiadhesive effect of epiglycanin it is remarkable that nontreated TA3/Ha cells adhere to hepatocytes at all. We found that during this interaction, epiglycanin was redistributed. We conclude that epiglycanin can completely prevent both intercellular and matrix adhesion, but that this effect can be overcome in certain intercellular interactions because of the induced redistribution of the mucin. PMID:7528749

  7. Effect of salivary gland adenocarcinoma cell-derived alpha-N-acetylgalactosaminidase on the bioactivity of macrophage activating factor.

    PubMed

    Matsuura, Takashi; Uematsu, Takashi; Yamaoka, Minoru; Furusawa, Kiyofumi

    2004-03-01

    The aim of this study was to clarify the effects of alpha-N-acetylgalactosaminidase (alpha-NaGalase) produced by human salivary gland adenocarcinoma (SGA) cells on the bioactivity of macrophage-activating factor (GcMAF). High exo-alpha-NaGalase activity was detected in the SGA cell line HSG. HSG alpha-NaGalase had both exo- and endo-enzyme activities, cleaving the Gal-GalNAc and GalNAc residues linked to Thr/Ser but not releasing the [NeuAc2-6]GalNac residue. Furthermore, GcMAF enzymatically prepared from the Gc protein enhanced the superoxide-generation capacity and phagocytic activity of monocytes/macrophages. However, GcMAF treated with purified alpha-NaGalase did not exhibit these effects. Thus, HSG possesses the capacity to produce larger quantities of alpha-NaGalase, which inactivates GcMAF produced from Gc protein, resulting in reduced phagocytic activity and superoxide-generation capacity of monocytes/macrophages. The present data strongly suggest that HSG alpha-NaGalase acts as an immunodeficiency factor in cancer patients.

  8. The Role of Plasmacytoid Dendritic Cells in Innate and Adaptive Immune Responses against Alpha Herpes Virus Infections

    PubMed Central

    Schuster, Philipp; Boscheinen, Jan Bernardin; Tennert, Karin; Schmidt, Barbara

    2011-01-01

    In 1999, two independent groups identified plasmacytoid dendritic cells (PDC) as major type I interferon- (IFN-) producing cells in the blood. Since then, evidence is accumulating that PDC are a multifunctional cell population effectively coordinating innate and adaptive immune responses. This paper focuses on the role of different immune cells and their interactions in the surveillance of alpha herpes virus infections, summarizes current knowledge on PDC surface receptors and their role in direct cell-cell contacts, and develops a risk factor model for the clinical implications of herpes simplex and varicella zoster virus reactivation. Data from studies involving knockout mice and cell-depletion experiments as well as human studies converge into a “spider web”, in which the direct and indirect crosstalk between many cell populations tightly controls acute, latent, and recurrent alpha herpes virus infections. Notably, cells involved in innate immune regulations appear to shape adaptive immune responses more extensively than previously thought. PMID:22312349

  9. Tomatidine and lycotetraose, hydrolysis products of alpha-tomatine by Fusarium oxysporum tomatinase, suppress induced defense responses in tomato cells.

    PubMed

    Ito, Shin-ichi; Eto, Tomomi; Tanaka, Shuhei; Yamauchi, Naoki; Takahara, Hiroyuki; Ikeda, Tsuyoshi

    2004-07-30

    Many fungal pathogens of tomato produce extracellular enzymes, collectively known as tomatinases, that detoxify the preformed antifungal steroidal glycoalkaloid alpha-tomatine. Tomatinase from the vascular wilt pathogen of tomato Fusarium oxysporum f. sp. lycopersici cleaves alpha-tomatine into the aglycon tomatidine (Td) and the tetrasaccharide lycotetraose (Lt). Although modes of action of alpha-tomatine have been extensively studied, those of Td and Lt are poorly understood. Here, we show that both Td and Lt inhibit the oxidative burst and hypersensitive cell death in suspension-cultured tomato cells. A tomatinase-negative F. oxysporum strain inherently non-pathogenic on tomato was able to infect tomato cuttings when either Td or Lt was present. These results suggest that tomatinase from F. oxysporum is required not only for detoxification of alpha-tomatine but also for suppression of induced defense responses of host.

  10. Secreted phosphoprotein 1 (SPP1, osteopontin) binds to integrin alpha v beta 6 on porcine trophectoderm cells and integrin alpha v beta 3 on uterine luminal epithelial cells, and promotes trophectoderm cell adhesion and migration.

    PubMed

    Erikson, David W; Burghardt, Robert C; Bayless, Kayla J; Johnson, Greg A

    2009-11-01

    Conceptus implantation involves pregnancy-specific alterations in extracellular matrix at the conceptus-maternal interface. Secreted phosphoprotein 1 (SPP1, osteopontin) is induced just before implantation and is present at the conceptus-maternal interface in mammals. In the present study, we investigated mechanisms by which SPP1 facilitates porcine conceptus and uterine luminal epithelial cell attachment. Native bovine milk and wild-type rat recombinant SPP1 stimulated trophectoderm cell migration. Bovine milk SPP1, ovine uterine SPP1, and recombinant wild-type, but not mutated, rat SPP1 promoted dose- and cation-dependent attachment of porcine trophectoderm and uterine luminal epithelial cells, which was markedly reduced in the presence of a linear Arg-Gly-Asp integrin-blocking peptide. Affinity chromatography and immunoprecipitation experiments revealed direct binding of alpha v beta 6 trophectoderm and alpha v beta 3 uterine epithelial cell integrins to SPP1. Immunofluorescence microscopy using SPP1-coated microspheres revealed colocalization of the alpha v integrin subunit and talin at focal adhesions as well as at the apical domain of trophectoderm cells. Similarly, immunofluorescence staining of implantation sites in frozen gravid uterine cross sections localized SPP1 and alpha v integrin to the apical surfaces of trophectoderm and luminal epithelium and beta 3 integrin to the apical surface of luminal epithelium. To our knowledge, the present study is the first to demonstrate functionally that SPP1 directly binds specific integrins to promote trophectoderm cell migration and attachment to luminal epithelium that may be critical to conceptus elongation and implantation.

  11. Modulation of the expression of integrins on glial cells during experimental autoimmune encephalomyelitis. A central role for TNF-alpha.

    PubMed Central

    Previtali, S. C.; Archelos, J. J.; Hartung, H. P.

    1997-01-01

    Integrins comprise a group of adhesion receptors involved in cell-cell and cell-extracellular matrix interactions. Evidence is accumulating that integrins expressed on mononuclear cells play a central role in the induction of autoimmune diseases of the central nervous system. The effects of integrins on glial cell behavior, myelination, and angiogenesis suggest that they may also have a role in resolving inflammation in the nervous system and in promoting tissue repair. We investigated the temporospatial expression of integrins in the rat central nervous system during the course of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. A higher expression of alpha v- and beta 4-integrin subunits in astrocytes and alpha 2 integrin in oligodendrocytes was observed in active lesions of experimental autoimmune encephalomyelitis, in comparison with controls. Proinflammatory cytokines, primarily TNF-alpha, also enhanced alpha v, beta 4, and alpha 2 expression in purified glial cells ex vivo. Furthermore, we observed that the expression of some integrin subunits was modulated in the cerebral vasculature during inflammation. Our results suggest an active role for glial and vascular integrins in the regulation of autoimmune diseases of the central nervous system, opening an avenue for new potential immunotherapies. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 9 PMID:9358769

  12. Megakaryocytic cells synthesize and platelets secrete alpha5-laminins, and the endothelial laminin isoform laminin 10 (alpha5beta1gamma1) strongly promotes adhesion but not activation of platelets.

    PubMed

    Nigatu, Ayele; Sime, Wondossen; Gorfu, Gezahegn; Geberhiwot, Tarekegn; Andurén, Ingegerd; Ingerpuu, Sulev; Doi, Masayuki; Tryggvason, Karl; Hjemdahl, Paul; Patarroyo, Manuel

    2006-01-01

    Following vascular injury, basement membrane (BM) components of the blood vessels are exposed to circulating cells and may contribute to hemostasis and/or thrombosis. Laminins 8 (LN-8) (alpha4beta1gamma1) and 10 (LN-10) (alpha5beta1gamma1) are major laminin isoforms of the endothelial BM, and LN-8 is also secreted by activated platelets. In the present study, we demonstrate synthesis of alpha5-laminins LN-10 and LN-11 (alpha5beta2gamma1) by megakaryocytic cells, and intracellular expression of these laminin isoforms in blood platelets. In contrast to platelet LN alpha4 chain that had an apparent molecular weight of 180 kDa and associated mostly to LNbeta1 chain, platelet LNalpha5 consisted of 300/350 kDa polypeptides and associated mainly to LNbeta2. Both alpha4- and alpha5-laminins were secreted by platelets following stimulation. When compared to recombinant human (rh) LN-8, rhLN-10 was much more adhesive to platelets, though adhesion to both proteins was largely mediated via alpha6beta1 integrin. In spite of their adhesive properties, rhLN-8 and rhLN-10 induced neither P-selectin expression nor cell aggregation, two signs of platelet activation. This study demonstrates synthesis/expression of heterotrimeric alpha5-laminins in hematopoietic/blood cells, and provides evidence for the adhesive, but not activating, role of endothelial laminin isoforms in platelet biology.

  13. NK T cell-induced protection against diabetes in V alpha 14-J alpha 281 transgenic nonobese diabetic mice is associated with a Th2 shift circumscribed regionally to the islets and functionally to islet autoantigen.

    PubMed

    Laloux, V; Beaudoin, L; Jeske, D; Carnaud, C; Lehuen, A

    2001-03-15

    The onset of autoimmune diabetes is related to defective immune regulation. Recent studies have shown that NK T cells are deficient in number and function in both diabetic patients and nonobese diabetic (NOD) mice. NK T cells, which are CD1d restricted, express a TCR with an invariant V alpha 14-J alpha 281 chain and rapidly produce large amounts of cytokines. V alpha 14-J alpha 281 transgenic NOD mice have increased numbers of NK T cells and are protected against diabetes onset. In this study we analyzed where and how NK T cells interfere with the development of the anti-islet autoimmune response. NK T cells, which are usually rare in lymph nodes, are abundant in pancreatic lymph nodes and are also present in islets. IL-4 mRNA levels are increased and IFN-gamma mRNA levels decreased in islets from diabetes-free V alpha 14-J alpha 281 transgenic NOD mice; the IgG1/IgG2c ratio of autoantibodies against glutamic acid decarboxylase is also increased in these mice. Treatment with IL-12 (a pro-Th1 cytokine) or anti-IL-4 Ab abolishes the diabetes protection in V alpha 14-J alpha 281 NOD mice. The protection from diabetes conferred by NK T cells is thus associated with a Th2 shift within islets directed against autoantigen such as glutamic acid decarboxylase. Our findings also demonstrate the key role of IL-4.

  14. Low doses of alpha particles do not induce sister chromatid exchanges in bystander Chinese hamster cells defective in homologous recombination

    SciTech Connect

    Nagasawa, H; Wilson, P F; Chen, D J; Thompson, L H; Bedford, J S; Little, J B

    2007-10-26

    We reported previously that the homologous recombinational repair (HRR)-deficient Chinese hamster mutant cell line irs3 (deficient in the Rad51 paralog Rad51C) showed only a 50% spontaneous frequency of sister chromatid exchange (SCE) as compared to parental wild-type V79 cells. Furthermore, when irradiated with very low doses of alpha particles, SCEs were not induced in irs3 cells, as compared to a prominent bystander effect observed in V79 cells (Nagasawa et al., Radiat. Res. 164, 141-147, 2005). In the present study, we examined additional Chinese hamster cell lines deficient in the Rad51 paralogs Rad51C, Rad51D, Xrcc2, and Xrcc3 as well as another essential HRR protein, Brca2. Spontaneous SCE frequencies in non-irradiated wild-type cell lines CHO, AA8 and V79 were 0.33 SCE/chromosome, whereas two Rad51C-deficient cell lines showed only 0.16 SCE/chromosome. Spontaneous SCE frequencies in cell lines defective in Rad51D, Xrcc2, Xrcc3, and Brca2 ranged from 0.23-0.33 SCE/chromosome, 0-30% lower than wild-type cells. SCEs were induced significantly 20-50% above spontaneous levels in wild-type cells exposed to a mean dose of 1.3 mGy of alpha particles (<1% of nuclei traversed by an alpha particle). However, induction of SCEs above spontaneous levels was minimal or absent after {alpha}-particle irradiation in all of the HRR-deficient cell lines. These data suggest that Brca2 and the Rad51 paralogs contribute to DNA damage repair processes induced in bystander cells (presumably oxidative damage repair in S-phase cells) following irradiation with very low doses of alpha particles.

  15. Effect of Interleukin-1beta and Tumor Necrosis Factor-alpha on Gene Expression in Human Endothelial Cells

    DTIC Science & Technology

    2003-06-01

    bela 3 (phosphatidylinositoJ.specific) AA846573 CD47 antigen (Rh-related antigen, integrin-associated signal transducer) AA455448 proteasome ( prosome ...macropain) activator subunit 1 (PA28 alpha) T47815 proteasome ( prosome , macropain) activator subunH 2 (PA28 beta) H65395 stimulated trans-acting...class I, C AA464246 natural kiter cell transcript 4 AA458965 diubiqu~in N33920 proteasome ( prosome , macropain) subunit, alpha type, 6 AA047338 matrix

  16. Mating type-specific cell-cell recognition of Saccharomyces cerevisiae: cell wall attachment and active sites of a- and alpha-agglutinin.

    PubMed Central

    Cappellaro, C; Baldermann, C; Rachel, R; Tanner, W

    1994-01-01

    Mating type-specific agglutination of Saccharomyces cerevisiae a and alpha cells depends on the heterophilic interaction of two cell surface glycoproteins, the gene products of AG alpha 1 and AGA2. Evidence is presented with immunogold labelling that the alpha-agglutinin is part of the outer fimbrial cell wall coat. The a-agglutinin is bound via two S-S bridges (Cys7 and Cys50) to a cell wall component, most probably the gene product of AGA1. His273 of alpha-agglutinin has previously been shown to be essential for a- and alpha-agglutinin interaction and a model based on two opposing ion-pairs had been proposed. By site-directed mutagenesis this possibility has now been excluded. With the help of various peptides, either chemically synthesized, obtained by proteolysis of intact glycosylated a-agglutinin or prepared from a fusion protein expressed in Escherichia coli, the biologically active region of a-agglutinin was located at the C-terminus of the molecule. A peptide consisting of the C-terminal 10 amino acids (GSPIN-TQYVF) was active in nanomolar concentrations. Saccharide moieties, therefore, are not essential for the mating type-specific cell-cell interaction; glycosylated peptides are, however, four to five times more active than non-glycosylated ones. Comparisons of the recognition sequences of the S. cerevisiae agglutinins with that of the Dictyostelium contact site A glycoprotein (gp80), as well as with those of the various families of cell adhesion molecules of higher eucaryotes, have been made and are discussed. Images PMID:7957044

  17. Recombinant interferon-alpha selectively inhibits the production of interleukin-5 by human CD4+ T cells.

    PubMed Central

    Schandené, L; Del Prete, G F; Cogan, E; Stordeur, P; Crusiaux, A; Kennes, B; Romagnani, S; Goldman, M

    1996-01-01

    The effects of recombinant IFN-alpha on the production of IL-5 by human CD4+ T cells were first analyzed on resting CD4+ T cells purified from normal PBMC and stimulated either with a combination of PMA and anti-CD28 mAb or anti-CD3 mAb cross-linked on B7-1/CD32-transfected mouse fibroblasts. We found that IFN-alpha profoundly inhibited in a dose-dependent manner IL-5 production by resting CD4+ T cells whereas IL-10 was upregulated in both systems. The addition of a neutralizing anti-IL-10 mAb to PMA and anti-CD28 mAb upregulated IL-5 production by resting CD4+ T cells but did not prevent IFN-alpha-induced IL-5 inhibition. We then analyzed the effect of IFN-alpha on the production of cytokines by differentiated type 2 helper (Th2) CD4+CD3- cells isolated from peripheral blood of two patients with the hypereosinophilic syndrome. In both cases, IFN-alpha markedly inhibited IL-5 production while it induced mild upregulation of IL-4 and IL-10. Finally, the inhibitory effect of IFN-alpha on IL-5 production was confirmed on a panel of Th2 and Th0 clones generated in vitro. In 2 out of 6 clones, IL-5 inhibition was associated with upregulation of IL-4 and IL-10. We conclude that IFN-alpha selectively downregulates IL-5 synthesis by human CD4+ T cells. PMID:8567949

  18. The IL-15R alpha chain signals through association with Syk in human B cells.

    PubMed

    Bulanova, E; Budagian, V; Pohl, T; Krause, H; Dürkop, H; Paus, R; Bulfone-Paus, S

    2001-12-01

    The alpha-chain of the IL-15R (IL-15Ralpha) serves as the specific, high-affinity receptor for IL-15. It is expressed by lymphoid and nonlymphoid cells, including B cell lymphoma lines. In this study, we have further explored IL-15Ralpha-mediated signaling in activated primary B cells and in Raji cells, a human B-lymphoblastoid cell line which expresses the IL-15Ralpha and IL-2Rgamma chains, but lacks the IL-2Rbeta chain. Stimulation of Raji cells with IL-15 induces their proliferation and rescues them from C2-ceramide-induced apoptosis. By immunoprecipitation and Western blotting, we show that treatment of Raji cells and activated primary B cells with IL-15 induces coprecipitation of Syk kinase with the IL-15Ralpha chain. Upon association, the activated Syk kinase phosphorylates the IL-15Ralpha chain as well as phospholipase Cgamma, which coprecipitates with Syk. Furthermore, transfection of Raji cells with stem-loop Syk antisense oligonucleotides prevents IL-15Ralpha and phospholipase Cgamma phosphorylation as well as the inhibition of apoptosis by IL-15. Mutation of a defined region of the intracellular signaling portion of IL-15Ralpha (Tyr227) abrogates both the IL-15Ralpha/Syk association and IL-15Ralpha phosphorylation. Taken together, this suggests that Syk kinase physically and functionally associates with the IL-15Ralpha chain in B cells and that Syk plays a key role in mediating IL-15-induced signal transduction, thus accounting for the distinct functional consequences of IL-15 vs IL-2 binding to B cells.

  19. T cell responses to HLA-A*0201-restricted peptides derived from human alpha fetoprotein.

    PubMed

    Butterfield, L H; Meng, W S; Koh, A; Vollmer, C M; Ribas, A; Dissette, V B; Faull, K; Glaspy, J A; McBride, W H; Economou, J S

    2001-04-15

    alpha fetoprotein (AFP)-derived peptide epitopes can be recognized by human T cells in the context of MHC class I. We determined the identity of AFP-derived peptides, presented in the context of HLA-A*0201, that could be recognized by the human (h) T cell repertoire. We screened 74 peptides and identified 3 new AFP epitopes, hAFP(137-145), hAFP(158-166), and hAFP(325-334), in addition to the previously reported hAFP(542-550.) Each possesses two anchor residues and stabilized HLA-A*0201 on T2 cells in a concentration-dependent class I binding assay. The peptides were stable for 2-4 h in an off-kinetics assay. Each peptide induced peptide-specific T cells in vitro from several normal HLA-A*0201 donors. Importantly, these hAFP peptide-specific T cells also were capable of recognizing HLA-A*0201(+)/AFP(+) tumor cells in both cytotoxicity assays and IFN-gamma enzyme-linked immunospot assays. The immunogenicity of each peptide was tested in vivo with HLA-A*0201/K(b)-transgenic mice. After immunization with each peptide emulsified in CFA, draining lymph node cells produced IFN-gamma on recognition of cells stably transfected with hAFP. Furthermore, AFP peptide-specific T cells could be identified in the spleens of mice immunized with dendritic cells transduced with an AFP-expressing adenovirus (AdVhAFP). Three of four AFP peptides could be identified by mass spectrometric analysis of surface peptides from an HLA-A*0201 human hepatocellular carcinoma (HCC) cell line. Thus, compelling immunological and physiochemical evidence is presented that at least four hAFP-derived epitopes are naturally processed and presented in the context of class I, are immunogenic, and represent potential targets for hepatocellular carcinoma immunotherapy.

  20. Induction of the cholesterol biosynthesis pathway in differentiated Caco-2 cells by the potato glycoalkaloid alpha-chaconine.

    PubMed

    Mandimika, Tafadzwa; Baykus, Hakan; Poortman, Jenneke; Garza, Cutberto; Kuiper, Harry; Peijnenburg, Ad

    2007-10-01

    Glycoalkaloids are naturally occurring toxins in potatoes, which at high levels may induce toxic effects in humans, mainly on the gastrointestinal tract by cell membrane disruption. In order to better understand the molecular mechanisms underlying glycoalkaloid toxicity, we examined the effects of alpha-chaconine on gene expression in the Caco-2 intestinal epithelial cell line using DNA microarrays. Caco-2 cells were exposed for 6h to 10 microM alpha-chaconine in three independent experiments (randomized block design). The most prominent finding from our gene expression and pathway analyses was the upregulation of expression of several genes involved in cholesterol biosynthesis. This to some extent is in line with the literature-described mechanism of cell membrane disruption by glycoalkaloids. In addition, various growth factor signaling pathways were found to be significantly upregulated. This study is useful in understanding the mechanism(s) of alpha-chaconine toxicity, which may be extended to other potato glycoalkaloids more generally.

  1. The synergistic necrohemorrhagic action of Clostridium perfringens perfringolysin and alpha toxin in the bovine intestine and against bovine endothelial cells

    PubMed Central

    2013-01-01

    Bovine necrohemorrhagic enteritis is a major cause of mortality in veal calves. Clostridium perfringens is considered as the causative agent, but there has been controversy on the toxins responsible for the disease. Recently, it has been demonstrated that a variety of C. perfringens type A strains can induce necrohemorrhagic lesions in a calf intestinal loop assay. These results put forward alpha toxin and perfringolysin as potential causative toxins, since both are produced by all C. perfringens type A strains. The importance of perfringolysin in the pathogenesis of bovine necrohemorrhagic enteritis has not been studied before. Therefore, the objective of the current study was to evaluate the role of perfringolysin in the development of necrohemorrhagic enteritis lesions in calves and its synergism with alpha toxin. A perfringolysin-deficient mutant, an alpha toxin-deficient mutant and a perfringolysin alpha toxin double mutant were less able to induce necrosis in a calf intestinal loop assay as compared to the wild-type strain. Only complementation with both toxins could restore the activity to that of the wild-type. In addition, perfringolysin and alpha toxin had a synergistic cytotoxic effect on bovine endothelial cells. This endothelial cell damage potentially explains why capillary hemorrhages are an initial step in the development of bovine necrohemorrhagic enteritis. Taken together, our results show that perfringolysin acts synergistically with alpha toxin in the development of necrohemorrhagic enteritis in a calf intestinal loop model and we hypothesize that both toxins act by targeting the endothelial cells. PMID:23782465

  2. Purification and characterization of the glycoprotein hormone. cap alpha. -subunit-like material secreted by HeLa cells

    SciTech Connect

    Cox, G.S.; Rimerman, R.A.

    1988-08-23

    The protein secreted by HeLa cells that cross-reacts with antiserum developed against the ..cap alpha..-subunit of human chorionic gonadotropin (hCG) has been purified approximately 30,000-fold from concentrated culture medium by organic solvent fractionation followed by ion exchange, gel filtration, and lectin affinity chromatography. The final preparation had a specific activity (by RIA) of 6.8 x 10/sup 5/ ng of ..cap alpha../mg of protein and appeared homogeneous by electrophoresis on reducing/denaturing polyacrylamide gels (SDS-PAGE). Amino acid analysis indicated that HeLa-..cap alpha.. had a composition very similar to that of the urinary hCG ..cap alpha..-subunit. However, comparison of hCG-..cap alpha.. and HeLa-..cap alpha.. demonstrated that the tumor-associated subunit was not identical with its normal counterpart. The purified tumor protein had an apparent molecular weight greater than that of the urinary ..cap alpha..-subunit when analyzed by SDS-PAGE, and this difference was even greater when a partially purified preparation was examined by an immunoblot technique (Western). Isoelectric focusing of the HeLa and hCG subunits demonstrated that the tumor protein had a lower pI. Immunoprecipitation and electrophoresis of ..cap alpha..-subunit from HeLa cultures labeled with (/sup 3/H)fucose indicated that the tumor subunit was fucosylated, whereas analysis of hCG-..cap alpha.. hydrosylates by HPLC confirmed previous reports that the placental subunit does not contain fucose. The results indicate that, regardless of whether or not a single ..cap alpha..-subunit gene is being expressed in both normal and neoplastic tissues, posttranslational modifications lead to a highly altered subunit in the tumor. The differences observed may be useful in diagnosing neoplastic vs hyperplastic conditions and may lend insight into the mechanism of ectopic hormone production by tumors.

  3. BACE2 functions as an alternative alpha-secretase in cells.

    PubMed

    Yan, R; Munzner, J B; Shuck, M E; Bienkowski, M J

    2001-09-07

    BACE1 and BACE2 define a new subfamily of membrane-anchored aspartyl proteases. Both endoproteases share similar structural organization including a prodomain, a catalytic domain formed via DTG and DSG active site motifs, a single transmembrane domain, and a short C-terminal tail. BACE1 has been identified as the Alzheimer's beta-secretase, whereas BACE2 was mapped to the Down's critical region of human chromosome 21. Herein we show that purified BACE2 can be autoactivated in vitro. Purified BACE2 cleaves human amyloid precursor protein (APP) sequences at the beta-secretase site, and near the alpha-secretase site, mainly at A beta-Phe(20)--Ala(21) and also at A beta-Phe(19)--Phe(20). Alternatively, in cells BACE2 has a limited effect on the beta-secretase site but efficiently cleaves the sequences near the alpha-secretase site. The in vitro specificity of APP processing by BACE2 is distinct from that observed in cells. BACE2 localizes in the endoplasmic reticulum, Golgi, trans-Golgi network, endosomes, and plasma membrane, and its cellular localization patterns depend on the presence of its transmembrane domain. BACE2 chimeras that increase localization of BACE2 in the trans-Golgi network do not change its APP processing patterns. Thus, BACE2 can be distinguished from BACE1 on the basis of autoprocessing of the prosegment, APP processing specificity, and subcellular localization patterns.

  4. Dynamic elastic behavior of alpha-satellite DNA domains visualized in situ in living human cells

    PubMed Central

    1996-01-01

    We have constructed a fluorescent alpha-satellite DNA-binding protein to explore the motile and mechanical properties of human centromeres. A fusion protein consisting of human CENP-B coupled to the green fluorescent protein (GFP) of A. victoria specifically targets to centromeres when expressed in human cells. Morphometric analysis revealed that the alpha-satellite DNA domain bound by CENPB-GFP becomes elongated in mitosis in a microtubule-dependent fashion. Time lapse confocal microscopy in live mitotic cells revealed apparent elastic deformations of the central domain of the centromere that occurred during metaphase chromosome oscillations. These observations demonstrate that the interior region of the centromere behaves as an elastic element that could play a role in the mechanoregulatory mechanisms recently identified at centromeres. Fluorescent labeling of centromeres revealed that they disperse throughout the nucleus in a nearly isometric expansion during chromosome decondensation in telophase and early G1. During interphase, centromeres were primarily stationary, although motility of individual or small groups of centromeres was occasionally observed at very slow rates of 7-10 microns/h. PMID:8909532

  5. Fluorescence lifetime images of green fluorescent protein in HeLa cells during TNF-alpha induced apoptosis.

    PubMed

    Ito, Toshiyuki; Oshita, Shugo; Nakabayashi, Takakazu; Sun, Fan; Kinjo, Masataka; Ohta, Nobuhiro

    2009-06-01

    Fluorescence lifetime images of HeLa cells expressing enhanced green fluorescent protein (EGFP) have been measured as apoptosis is induced by tumor necrosis factor-alpha (TNF-alpha) in combination with cycloheximide. The fluorescence lifetime of EGFP is found to decrease after the induction of apoptosis, indicating that the change in environment occurs around the chromophore of EGFP with the apoptosis process. The fluorescence lifetime imaging technique can be used to perform in vivo observation of cell death processes. Fluorescence lifetime measurements are useful to examine the induction of the apoptosis process, even when a morphological change of each cell cannot be observed because of a low spatial resolution.

  6. PTH-related protein upregulates integrin {alpha}6{beta}4 expression and activates Akt in breast cancer cells

    SciTech Connect

    Shen Xiaoli; Falzon, Miriam . E-mail: mfalzon@utmb.edu

    2006-11-15

    Breast cancer is the most common carcinoma that metastasizes to bone. Tumor-produced parathyroid hormone-related protein (PTHrP), a known stimulator of osteoclastic bone resorption, is a major mediator of the osteolytic process in breast cancer. We have previously shown that PTHrP increases breast cancer cell proliferation, survival, migration, and pro-invasive integrin {alpha}6{beta}4 expression. To determine the role of integrin {alpha}6{beta}4 in these PTHrP-mediated effects, we utilized two strategies to modulate expression of the {alpha}6 and {beta}4 subunits in parental and PTHrP-overexpressing MDA-MB-231 and MCF-7 cells: overexpression of {alpha}6{beta}4 by transfection with constructs encoding the {alpha}6 and {beta}4 subunits, and suppression of endogenous {alpha}6{beta}4 expression by transfection with siRNAs targeting these subunits. We now show that the effects of PTHrP are mediated via upregulation of integrin {alpha}6{beta}4 expression. We also show that integrin {alpha}6{beta}4 expression is modulated at the mRNA level, indicating a transcriptional and/or post-transcriptional mechanism of action for PTHrP. PTHrP expression also increased the levels of phosphorylated Akt, with a consequent increase in the levels of phosphorylated (inactive) glycogen synthase kinase-3 (GSK-3). The role of PTHrP in breast cancer growth and metastasis may thus be mediated via upregulation of integrin {alpha}6{beta}4 expression and Akt activation, with consequent inactivation of GSK-3.

  7. Interleukin-2 enhances the production of tumor necrosis factor-alpha in activated B-type chronic lymphocytic leukemia (B-CLL) cells.

    PubMed

    Larsson, L G; Carlsson, M; Schena, M; Lantz, M; Caligaris-Cappio, F; Nilsson, K

    1993-02-01

    Tumor necrosis factor-alpha (TNF-alpha) has recently been implicated as a regulator growth and differentiation of normal and malignant B cells. We utilized a selected clone (I-83) of primary resting B-type chronic lymphocytic leukemia (B-CLL) cells, inducible to activation, growth and differentiation in vitro, as a model system to study the possible role of TNF-alpha as an autocrine growth factor for such cells. Our results show that unstimulated I-83 B-CLL cells produced a low level of TNF-alpha mRNA, as shown by Northern blot analysis, and cytoplasmic TNF-alpha, determined in individual cells by immunocytochemistry. Secreted TNF-alpha could, however, not be detected in the medium by ELISA. TNF-alpha synthesis and secretion was, however, induced to high levels by stimulation of the B-CLL cells with interleukin-2 (IL-2) after activation by 12-O-tetradecanoylphorbol-13-acetate (TPA) or Staphylococcus aureus Cowan strain I (SAC) and B-cell stimulatory factor-MP6 (thioredoxin). A moderate increase in TNF-alpha secretion was also induced by TPA or IL-2 alone. IL-4 did not have any major effects on the production of TNF-alpha in activated cells, but inhibited the IL-2-induced production of TNF-alpha in SAC-activated cells. The cell surface expression of TNF-alpha receptors (TNF-R), as determined by binding assay using 125I-labelled recombinant TNF-alpha (rTNF-alpha), was also induced after SAC or TPA activation, but shed receptors (TNF-binding proteins) were only observed after TPA activation. Exogenously added rTNF-alpha in combination with TPA or SAC induced a high level of DNA synthesis in I-83 B-CLL cells. The increased endogenous production and secretion of TNF-alpha during induced growth stimulation, the induced expression of TNF-R, and the mitogenic effect of TNF-alpha on activated B-CLL cells raise the question whether TNF-alpha may function as an autocrine co-stimulator of B-CLL cell growth as recently suggested. anti-TNF-alpha and anti-TNF-R antibodies

  8. Self-renewal and pluripotency is maintained in human embryonic stem cells by co-culture with human fetal liver stromal cells expressing hypoxia inducible factor 1alpha.

    PubMed

    Ji, Lei; Liu, Yu-xiao; Yang, Chao; Yue, Wen; Shi, Shuang-shuang; Bai, Ci-xian; Xi, Jia-fei; Nan, Xue; Pei, Xue-Tao

    2009-10-01

    Human embryonic stem (hES) cells are typically maintained on mouse embryonic fibroblast (MEF) feeders or with MEF-conditioned medium. However, these xenosupport systems greatly limit the therapeutic applications of hES cells because of the risk of cross-transfer of animal pathogens. The stem cell niche is a unique tissue microenvironment that regulates the self-renewal and differentiation of stem cells. Recent evidence suggests that stem cells are localized in the microenvironment of low oxygen. We hypothesized that hypoxia could maintain the undifferentiated phenotype of embryonic stem cells. We have co-cultured a human embryonic cell line with human fetal liver stromal cells (hFLSCs) feeder cells stably expressing hypoxia-inducible factor-1 alpha (HIF-1alpha), which is known as the key transcription factor in hypoxia. The results suggested HIF-1alpha was critical for preventing differentiation of hES cells in culture. Consistent with this observation, hypoxia upregulated the expression of Nanog and Oct-4, the key factors expressed in undifferentiated stem cells. We further demonstrated that HIF-1alpha could upregulate the expression of some soluble factors including bFGF and SDF-1alpha, which are released into the microenvironment to maintain the undifferentiated status of hES cells. This suggests that the targets of HIF-1alpha are secreted soluble factors rather than a cell-cell contact mechanism, and defines an important mechanism for the inhibition of hESCs differentiation by hypoxia. Our findings developed a transgene feeder co-culture system and will provide a more reliable alternative for future therapeutic applications of hES cells.

  9. Pax6-induced alteration of cell fate: shape changes, expression of neuronal alpha tubulin, postmitotic phenotype, and cell migration.

    PubMed

    Cartier, Laetitia; Laforge, Terese; Feki, Anis; Arnaudeau, Serge; Dubois-Dauphin, Michel; Krause, Karl-Heinz

    2006-04-01

    The transcription factor Pax6 plays an important role in the development of the central nervous system. To understand its mechanism of action, we transduced HeLa cells with a Pax6-expressing lentiviral vector. Upon transduction, HeLa cells markedly changed shape and formed neuritelike extensions. Pax6-transduced HeLa cells expressed high levels of neuronal alpha3 tubulin, demonstrating a partial transdifferentiation towards a neuronal phenotype. Neurons are postmitotic cells. Pax6-transduced HeLa cells became postmitotic through mechanisms involving up-regulation of p53 and cyclin-dependent kinase inhibitor p21. One of the most striking effects of Pax6 was observed by time-lapse videomicroscopy: cells started to dissociate from cell clusters and displayed intense migratory activity. Migration was accompanied by dynamic and reversible shape changes. Our results identified three elements of Pax6 action: (i) expression of neuron-specific genes; (ii) establishment of a postmitotic phenotype; and (iii) involvement in the regulation of cell shape and cell migration.

  10. Selective blockade and recovery of cell surface alpha 2-adrenergic receptors in human erythroleukemia (HEL) cells. Studies with the irreversible antagonist benextramine

    SciTech Connect

    McKernan, R.M.; Strickland, W.R.; Insel, P.A.

    1988-01-01

    alpha 2-Adrenergic receptors are present on human erythroleukemia (HEL) cells, both on the cell surface and in a sequestered compartment. In the current study we show that benextramine, a hydrophilic irreversible antagonist, can be used to investigate alpha 2-adrenergic receptor compartmentation in these cells. In membranes prepared from HEL cells, benextramine competed for all alpha 2-adrenergic receptors ( (/sup 3/H)yohimbine sites). In intact cells, at 4 degrees, benextramine exhibited a biphasic competition curve for alpha 2-adrenergic receptors, with EC50 values of approximately 10 microM and greater than 1 mM for the high and low affinity components, respectively. We propose that the alpha 2-adrenergic receptors preferentially blocked by benextramine are those on the surface of the cell, whereas those with low affinity are sequestered receptors because: 1) only epinephrine-accessible sites are removed by prior treatment of cells with benextramine, 2) a preparation enriched with surface membranes is also enriched in receptors with a high affinity for benextramine; and 3) after blockade of cell surface receptors (54 +/- 6% of total sites, n = 7) by benextramine, the ability of the alpha 2-adrenergic agonists epinephrine and UK-14,304 to inhibit forskolin-stimulated cAMP accumulation is lost. The latter result implies that only cell surface and not sequestered receptors are functionally coupled to adenylate cyclase. The return of receptors from the sequestered compartment to the cell surface and the recovery of alpha 2-adrenergic receptor function were measured after HEL cells were treated with benextramine (50 microM for 1 hr at 4 degrees). The recovery of receptor binding (t1/2 = 25 min) was somewhat slower than the recovery of function (t1/2 approximately 8 min).

  11. cap alpha. -chain locus of the T-cell antigen receptor is involved in the t(10; 14) chromosome translocation of T-cell acute lymphocytic leukemia

    SciTech Connect

    Kagan, J.; Finan, J.; Letofsky, J.; Besa, E.C.; Nowell, P.C.; Croce, C.M.

    1987-07-01

    Human leukemic T cells carrying a t(10;14)(q24;q11) chromosome translocation were fused with mouse leukemic T cells, and the hybrids were examined for genetic markers of human chromosomes 10 and 14. Hybrids containing the human 10q+ chromosome had the human genes for terminal deoxynucleotidyltransferase that has been mapped at 10q23-q25 and for C/sub ..cap alpha../ (the constant region of TCRA (the ..cap alpha..-chain locus of the T-cell antigen receptor gene)), but not for V/sub ..cap alpha../ (the variable region of TCRA). Hybrids containing the human 14q- chromosome retained the V/sub ..cap alpha../genes. Thus the 14q11 breakpoint in the t(10;14) chromosome translocation directly involves TCRA, splitting the locus in a region between the V/sub ..cap alpha../ and the C/sub ..cap alpha../ genes. These results suggest that the translocation of the C/sub ..cap alpha../ locus to a putative cellular protooncogene located proximal to the breakpoint at 10q24, for which the authors propose the name TCL3, results in its deregulation, leading to T-cell leukemia. Since hybrids with the 10q+ chromosome also retained the human terminal deoxynucleotidyltransferase gene, it is further concluded that the terminal deoxynucleotidyltransferase locus is proximal to the TCL3 gene, at band 10q23-q24.

  12. Immune protective effect of human alpha-1-antitrypsin gene during β cell transplantation in diabetic mice.

    PubMed

    Yang, Lu; Liao, Yu-Ting; Yang, Xiao-Fei; Reng, Li-Wei; Qi, Hui; Li, Fu-Rong

    2015-05-01

    Type 1 diabetes (T1D) is a chronic autoimmune disease in which β cells are destroyed. Islet transplantation is the most promising therapeutic treatment for T1D patients. However, allograft rejection and autoimmune reaction have been recognized as primary causes of graft loss after transplantation. Alpha-1-antitrypsin (AAT) is an important serine protease inhibitor in serum. AAT is characterized by anti-inflammation, anti-apoptosis, and induction-specific immunological tolerance. In this study, we successfully established NIT-hAAT cell lines, which are murine islet β cell lines with stable expression of human AAT (hAAT) gene. These NIT-hAAT cells were transplanted under the left kidney capsule of BALB/c diabetic mice. Interestingly, the sustained expression of hAAT in vivo can block the inflammatory cell infiltration and reduce the production of proinflammatory cytokines to effectively prevent nonspecific inflammation. Results showed that hAAT can inhibit the proliferation of lymphocytes, shift the balance between Th17 and Treg, and suppress the maturation of dendritic cells. Therefore, hAAT can serve as a beneficial immunomodulator that limits immune rejection to prolong islet allograft survival and achieve long-term successful transplant outcomes.

  13. Integrin-Alpha IIb Identifies Murine Lymph Node Lymphatic Endothelial Cells Responsive to RANKL

    PubMed Central

    Cordeiro, Olga G.; Chypre, Mélanie; Brouard, Nathalie; Rauber, Simon; Alloush, Farouk; Romera-Hernandez, Monica; Bénézech, Cécile; Li, Zhi; Eckly, Anita; Coles, Mark C.; Rot, Antal; Yagita, Hideo; Léon, Catherine; Ludewig, Burkhard; Cupedo, Tom; Lanza, François; Mueller, Christopher G.

    2016-01-01

    Microenvironment and activation signals likely imprint heterogeneity in the lymphatic endothelial cell (LEC) population. Particularly LECs of secondary lymphoid organs are exposed to different cell types and immune stimuli. However, our understanding of the nature of LEC activation signals and their cell source within the secondary lymphoid organ in the steady state remains incomplete. Here we show that integrin alpha 2b (ITGA2b), known to be carried by platelets, megakaryocytes and hematopoietic progenitors, is expressed by a lymph node subset of LECs, residing in medullary, cortical and subcapsular sinuses. In the subcapsular sinus, the floor but not the ceiling layer expresses the integrin, being excluded from ACKR4+ LECs but overlapping with MAdCAM-1 expression. ITGA2b expression increases in response to immunization, raising the possibility that heterogeneous ITGA2b levels reflect variation in exposure to activation signals. We show that alterations of the level of receptor activator of NF-κB ligand (RANKL), by overexpression, neutralization or deletion from stromal marginal reticular cells, affected the proportion of ITGA2b+ LECs. Lymph node LECs but not peripheral LECs express RANK. In addition, we found that lymphotoxin-β receptor signaling likewise regulated the proportion of ITGA2b+ LECs. These findings demonstrate that stromal reticular cells activate LECs via RANKL and support the action of hematopoietic cell-derived lymphotoxin. PMID:27010197

  14. Estrogen related receptor alpha in castration-resistant prostate cancer cells promotes tumor progression in bone

    PubMed Central

    Delliaux, Carine; Gervais, Manon; Kan, Casina; Benetollo, Claire; Pantano, Francesco; Vargas, Geoffrey; Bouazza, Lamia; Croset, Martine; Bala, Yohann; Leroy, Xavier; Rosol, Thomas J; Rieusset, Jennifer; Bellahcène, Akeila; Castronovo, Vincent; Aubin, Jane E; Clézardin, Philippe; Duterque-Coquillaud, Martine; Bonnelye, Edith

    2016-01-01

    Bone metastases are one of the main complications of prostate cancer and they are incurable. We investigated whether and how estrogen receptor-related receptor alpha (ERRα) is involved in bone tumor progression associated with advanced prostate cancer. By meta-analysis, we first found that ERRα expression is correlated with castration-resistant prostate cancer (CRPC), the hallmark of progressive disease. We then analyzed tumor cell progression and the associated signaling pathways in gain-of-function/loss-of-function CRPC models in vivo and in vitro. Increased levels of ERRα in tumor cells led to rapid tumor progression, with both bone destruction and formation, and direct impacts on osteoclasts and osteoblasts. VEGF-A, WNT5A and TGFβ1 were upregulated by ERRα in tumor cells and all of these factors also significantly and positively correlated with ERRα expression in CRPC patient specimens. Finally, high levels of ERRα in tumor cells stimulated the pro-metastatic factor periostin expression in the stroma, suggesting that ERRα regulates the tumor stromal cell microenvironment to enhance tumor progression. Taken together, our data demonstrate that ERRα is a regulator of CRPC cell progression in bone. Therefore, inhibiting ERRα may constitute a new therapeutic strategy for prostate cancer skeletal-related events. PMID:27776343

  15. Acetyl-CoA carboxylase alpha is essential to breast cancer cell survival.

    PubMed

    Chajès, Véronique; Cambot, Marie; Moreau, Karen; Lenoir, Gilbert M; Joulin, Virginie

    2006-05-15

    Activation of de novo fatty acid synthesis is a characteristic feature of cancer cells. We have recently described an interaction between acetyl-CoA carboxylase alpha (ACCalpha), a key enzyme in fatty acid synthesis, and BRCA1, which indicates a possible connection between lipid synthesis and genetic factors involved in susceptibility to breast and ovarian cancers. For this reason, we explored the role of ACCalpha in breast cancer cell survival using an RNA interference (RNAi) approach. We show that specific silencing of either the ACCalpha or the fatty acid synthase (FAS) genes in cancer cells results in a major decrease in palmitic acid synthesis. Depletion of the cellular pool of palmitic acid is associated with induction of apoptosis concomitant with the formation of reactive oxygen species (ROS) and mitochondrial impairment. Expression of a small interfering RNA (siRNA)-resistant form of ACCalpha mRNA prevented the effect of ACCalpha-RNAi but failed to prevent the effect of FAS gene silencing. Furthermore, supplementation of the culture medium with palmitate or with the antioxidant vitamin E resulted in the complete rescue of cells from both ACCalpha and FAS siRNA-induced apoptosis. Finally, human mammary epithelial cells are resistant to RNAi against either ACCalpha or FAS. These data confirm the importance of lipogenesis in cancer cell survival and indicate that this pathway represents a key target for antineoplastic therapy that, however, might require specific dietary recommendation for full efficacy.

  16. Alpha fetoprotein antagonises benzyl isothiocyanate inhibition of the malignant behaviors of hepatocellular carcinoma cells

    PubMed Central

    Zhu, Mingyue; Li, Wei; Guo, Junli; Lu, Yan; Dong, Xu; Lin, Bo; Chen, Yi; Zhang, Xueer; Li, Mengsen

    2016-01-01

    Benzyl isothiocyanate (BITC) is a dietary isothiocyanate derived from cruciferous vegetables. Recent studies showed that BITC inhibited the growth of many cancer cells, including hepatocellular carcinoma (HCC) cells. Alpha-fetoprotein (AFP) is a important molecule for promoting progression of HCC, in the present investigation, we explore the influence of AFP on the role of BITC in the malignant behaviours of HCC cells, and the potential underlying mechanisms. We found thatBITC inhibited viability, migration, invasion and induced apoptosis of human liver cancer cell lines, Bel 7402(AFP producer) and HLE(non-AFP producer) cells in vitro. The role of BITC involve in promoting actived-caspase-3 and PARP-1 expression, and enhancing caspase-3 activity but decreasing MMP-2/9, survivin and CXCR4 expression. AFP antagonized the effect of BITC. This study suggests that BITC induced significant reductions in the viability of HCC cell lines. BITC may activate caspase-3 signal and inhibit the expression of growth- and metastasis-related proteins; AFP is an pivotal molecule for the HCC chemo-resistance of BITC. PMID:27716619

  17. In vitro expression of the alpha-smooth muscle actin isoform by rat lung mesenchymal cells: regulation by culture condition and transforming growth factor-beta.

    PubMed

    Mitchell, J J; Woodcock-Mitchell, J L; Perry, L; Zhao, J; Low, R B; Baldor, L; Absher, P M

    1993-07-01

    alpha-Smooth muscle actin (alpha SM actin)-containing cells recently have been demonstrated in intraalveolar lesions in both rat and human tissues following lung injury. In order to develop model systems for the study of such cells, we examined cultured lung cell lines for this phenotype. The adult rat lung fibroblast-like "RL" cell lines were found to express alpha SM actin mRNA and protein and to organize this actin into stress fiber-like structures. Immunocytochemical staining of subclones of the RL87 line demonstrated the presence in the cultures of at least four cell phenotypes, one that fails to express alpha SM actin and three distinct morphologic types that do express alpha SM actin. The proportion of cellular actin that is the alpha-isoform was modulated by the culture conditions. RL cells growing at low density expressed minimal alpha SM actin. On reaching confluent densities, however, alpha SM actin increased to at least 20% of the total actin content. This effect, combined with the observation that the most immunoreactive cells were those that displayed overlapping cell processes in culture, suggests that cell-cell contact may be involved in actin isoform regulation in these cells. Similar to the response of some smooth muscle cell lines, alpha SM actin expression in RL cells also was promoted by conditions, e.g., maintenance in low serum medium, which minimize cell division. alpha SM actin expression was modulated in RL cells by the growth factor transforming growth factor-beta. Addition of this cytokine to growing cells substantially elevated the proportion of alpha SM actin protein.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Calsyntenins are secretory granule proteins in anterior pituitary gland and pancreatic islet alpha cells.

    PubMed

    Rindler, Michael J; Xu, Chong-Feng; Gumper, Iwona; Cen, Chuan; Sonderegger, Peter; Neubert, Thomas A

    2008-04-01

    Calsyntenins are members of the cadherin superfamily of cell adhesion molecules. They are present in postsynaptic membranes of excitatory neurons and in vesicles in transit to neuronal growth cones. In the current study, calsyntenin-1 (CST-1) and calsyntenin-3 (CST-3) were identified by mass spectrometric analysis (LC-MS/MS) of integral membrane proteins from highly enriched secretory granule preparations from bovine anterior pituitary gland. Immunofluorescence microscopy on thin frozen sections of rat pituitary revealed that CST-1 was present only in gonadotropes where it colocalized with follicle-stimulating hormone in secretory granules. In contrast, CST-3 was present not only in gonadotrope secretory granules but also in those of somatotropes and thyrotropes. Neither protein was detected in mammatropes. In addition, CST-1 was also localized to the glucagon-containing secretory granules of alpha cells in the pancreatic islets of Langerhans. Results indicate that calsyntenins function outside the nervous system and potentially are modulators of endocrine function.

  19. Isorhamnetin Attenuates Staphylococcus aureus-Induced Lung Cell Injury by Inhibiting Alpha-Hemolysin Expression.

    PubMed

    Jiang, Lanxiang; Li, Hongen; Wang, Laiying; Song, Zexin; Shi, Lei; Li, Wenhua; Deng, Xuming; Wang, Jianfeng

    2016-03-01

    Staphylococcus aureus, like other gram-positive pathogens, has evolved a large repertoire of virulence factors as a powerful weapon to subvert the host immune system, among which alpha-hemolysin (Hla), a secreted pore-forming cytotoxin, plays a preeminent role. We observed a concentration-dependent reduction in Hla production by S. aureus in the presence of sub-inhibitory concentrations of isorhamnetin, a flavonoid from the fruits of Hippophae rhamnoides L., which has little antibacterial activity. We further evaluate the effect of isorhamnetin on the transcription of the Hla-encoding gene hla and RNAIII, an effector molecule in the agr system. Isorhamnetin significantly down-regulated RNAIII expression and subsequently inhibited hla transcription. In a co-culture of S. aureus and lung cells, topical isorhamnetin treatment protected against S. aureus-induced cell injury. Isorhamnetin may represent a leading compound for the development of anti-virulence drugs against S. aureus infections.

  20. Effects of TNF-alpha inhibitors on the number of epidermal Langerhans cells in uninvolved skin of psoriatic patients: a pilot study.

    PubMed

    Piaserico, Stefano; Zattra, Edoardo; Michelotto, Anna; Alaibac, Mauro

    2013-09-01

    Only limited data are available on the effects of TNF-alpha inhibitors on dendritic cells. However, TNF-alpha plays a central role in the biology of dendritic cells, both with regard to their maturity process and mobilization to secondary lymphoid organs. In particular, the effects of TNF-alpha inhibitors on Langerhans cells in healthy skin have never been investigated. In this pilot study, we aimed to assess the change of the density of Langerhans cells within the normal, not photo-exposed, skin of 17 psoriatic patients, before and after 16 weeks of treatment with TNF-alpha inhibitors. Most of the patients (88%) showed an increase or a similar density of Langerhans cells after 16 weeks of therapy with TNF-alpha inhibitors compared with baseline values. Only 2 patients (12%) showed a reduction of these cells following therapy with TNF-alpha inhibitors.

  1. Alpha-actinin-1 phosphorylation modulates pressure-induced colon cancer cell adhesion through regulation of focal adhesion kinase-Src interaction.

    PubMed

    Craig, David H; Haimovich, Beatrice; Basson, Marc D

    2007-12-01

    Physical forces including pressure, strain, and shear can be converted into intracellular signals that regulate diverse aspects of cell biology. Exposure to increased extracellular pressure stimulates colon cancer cell adhesion by a beta(1)-integrin-dependent mechanism that requires an intact cytoskeleton and activation of focal adhesion kinase (FAK) and Src. alpha-Actinin facilitates focal adhesion formation and physically links integrin-associated focal adhesion complexes with the cytoskeleton. We therefore hypothesized that alpha-actinin may be necessary for the mechanical response pathway that mediates pressure-stimulated cell adhesion. We reduced alpha-actinin-1 and alpha-actinin-4 expression with isoform-specific small interfering (si)RNA. Silencing of alpha-actinin-1, but not alpha-actinin-4, blocked pressure-stimulated cell adhesion in human SW620, HT-29, and Caco-2 colon cancer cell lines. Cell exposure to increased extracellular pressure stimulated alpha-actinin-1 tyrosine phosphorylation and alpha-actinin-1 interaction with FAK and/or Src, and enhanced FAK phosphorylation at residues Y397 and Y576. The requirement for alpha-actinin-1 phosphorylation in the pressure response was investigated by expressing the alpha-actinin-1 tyrosine phosphorylation mutant Y12F in the colon cancer cells. Expression of Y12F blocked pressure-mediated adhesion and inhibited the pressure-induced association of alpha-actinin-1 with FAK and Src, as well as FAK activation. Furthermore, siRNA-mediated reduction of alpha-actinin-1 eliminated the pressure-induced association of alpha-actinin-1 and Src with beta(1)-integrin receptor, as well as FAK-Src complex formation. These results suggest that alpha-actinin-1 phosphorylation at Y12 plays a crucial role in pressure-activated cell adhesion and mechanotransduction by facilitating Src recruitment to beta(1)-integrin, and consequently the association of FAK with Src, to enhance FAK phosphorylation.

  2. Estrogen receptor alpha is cell cycle-regulated and regulates the cell cycle in a ligand-dependent fashion.

    PubMed

    JavanMoghadam, Sonia; Weihua, Zhang; Hunt, Kelly K; Keyomarsi, Khandan

    2016-06-17

    Estrogen receptor alpha (ERα) has been implicated in several cell cycle regulatory events and is an important predictive marker of disease outcome in breast cancer patients. Here, we aimed to elucidate the mechanism through which ERα influences proliferation in breast cancer cells. Our results show that ERα protein is cell cycle-regulated in human breast cancer cells and that the presence of 17-β-estradiol (E2) in the culture medium shortened the cell cycle significantly (by 4.5 hours, P < 0.05) compared with unliganded conditions. The alterations in cell cycle duration were observed in the S and G2/M phases, whereas the G1 phase was indistinguishable under liganded and unliganded conditions. In addition, ERα knockdown in MCF-7 cells accelerated mitotic exit, whereas transfection of ERα-negative MDA-MB-231 cells with exogenous ERα significantly shortened the S and G2/M phases (by 9.1 hours, P < 0.05) compared with parental cells. Finally, treatment of MCF-7 cells with antiestrogens revealed that tamoxifen yields a slower cell cycle progression through the S and G2/M phases than fulvestrant does, presumably because of the destabilizing effect of fulvestrant on ERα protein. Together, these results show that ERα modulates breast cancer cell proliferation by regulating events during the S and G2/M phases of the cell cycle in a ligand-dependent fashion. These results provide the rationale for an effective treatment strategy that includes a cell cycle inhibitor in combination with a drug that lowers estrogen levels, such as an aromatase inhibitor, and an antiestrogen that does not result in the degradation of ERα, such as tamoxifen.

  3. Pretranslational regulation of ectopic hCG alpha production in ChaGo lung cancer cells by sodium butyrate.

    PubMed

    Nagelberg, S B; Burnside, J; Maniatis, A; Lippman, S S; Weintraub, B D

    1985-12-31

    Ectopic production of hCG and its free alpha subunit by ChaGo lung cancer cells is stimulated by sodium butyrate. To investigate pretranslational regulation in this system, we examined the response of the hCG alpha and beta subunit mRNAs in ChaGo-K1 cells, a clone that produces free hCG alpha but no hCG or hCG beta in the basal state. When a Northern blot of total RNA from ChaGo cells was hybridized to a [32P]- labeled hCG alpha cDNA probe, a single band was detected that was identical in size (approximately 850 bases) to placental hCG alpha mRNA. RNA from butyrate-stimulated (5 mM, 24 h) ChaGo cells contained 7.7 times as much hCG alpha mRNA as RNA from control ChaGo cells. This increase appeared to be relatively selective since no difference in total polyA-containing mRNA levels was detected between butyrate-treated and control cells by [32P]oligo(dT) hybridization. In addition, no hCG beta mRNA was detected when Northern and dot blots were hybridized to an hCG beta cDNA probe. In a time course experiment, hCG alpha mRNA accumulation in butyrate-treated cells increased significantly by 8 h with a maximum increase of 6.1-fold at 24 h compared to control values. Major differences in immunoactive hCG alpha accumulation were not apparent, however, until after 24 h. These studies show that stimulation of ChaGo hCG alpha production by butyrate can be completely accounted for by pretranslational events and that failure to detect hCG or free hCG beta production by these cells is not due to poorly translatable RNA or post-translational protein degradation. Thus, exclusive ectopic production of only one of the hCG subunits is likely to be due to selective genomic expression.

  4. Suppressive effect of Elf-1 on FcepsilonRI alpha-chain expression in primary mast cells.

    PubMed

    Wang, Qing-Hui; Nishiyama, Chiharu; Nakano, Nobuhiro; Shimokawa, Naomi; Hara, Mutsuko; Kanada, Shunsuke; Ogawa, Hideoki; Okumura, Ko

    2008-10-01

    The high-affinity receptor for immunoglobulin E (IgE), FcepsilonRI, is specifically expressed in mast cells and basophils and plays a key role in IgE-mediated allergic reactions. The transcription factor Elf-1 has been previously identified to bind to the promoter of the human FcepsilonRI alpha-chain, which is essential for the function and expression of FcepsilonRI. In the present study, Elf-1 siRNA was conducted to evaluate the effects of Elf-1 on FcepsilonRI alpha-chain expression in the primary mouse mast cells, bone marrow-derived mast cells (BMMC). Introduction of Elf-1 siRNA effectively reduced expression levels of Elf-1 mRNA and protein in BMMC. Transient reporter assay showed that the knockdown of Elf-1 by siRNA resulted in increased FcepsilonRI alpha-chain promoter activity, while overexpression of Elf-1 suppressed alpha-chain promoter activity in BMMC. Elf-1 siRNA-treated BMMC exhibited marked upregulation of FcepsilonRI alpha-chain transcription, whereas beta-chain mRNA was not affected by Elf-1 siRNA. Chromatin immunoprecipitation assay showed that the amount of transcription factor PU.1, recognizing the cis-element close to the Elf-1-site on the FcepsilonRI alpha-chain promoter, was significantly increased by introduction of Elf-1 siRNA. These results indicate that Elf-1 negatively regulates FcepsilonRI alpha-chain expression by suppressing PU.1-mediated transcription of the alpha-chain in BMMC.

  5. Cell-associated alpha-amylases of butyrate-producing Firmicute bacteria from the human colon.

    PubMed

    Ramsay, Alan G; Scott, Karen P; Martin, Jenny C; Rincon, Marco T; Flint, Harry J

    2006-11-01

    Selected butyrate-producing bacteria from the human colon that are related to Roseburia spp. and Butyrivibrio fibrisolvens showed a good ability to utilize a variety of starches for growth when compared with the Gram-negative amylolytic anaerobe Bacteroides thetaiotaomicron. A major cell-associated amylase of high molecular mass (140-210 kDa) was detected in each strain by SDS-PAGE zymogram analysis, and genes corresponding to these enzymes were analysed for two representative strains. Amy13B from But. fibrisolvens 16/4 is a multi-domain enzyme of 144.6 kDa that includes a family 13 glycoside hydrolase domain, and duplicated family 26 carbohydrate-binding modules. Amy13A (182.4 kDa), from Roseburia inulinivorans A2-194, also includes a family 13 domain, which is preceded by two repeat units of approximately 116 aa rich in aromatic residues, an isoamylase N-terminal domain, a pullulanase-associated domain, and an additional unidentified domain. Both Amy13A and Amy13B have N-terminal signal peptides and C-terminal cell-wall sorting signals, including a modified LPXTG motif similar to that involved in interactions with the cell surface in other Gram-positive bacteria, a hydrophobic transmembrane segment, and a basic C terminus. The overexpressed family 13 domains showed an absolute requirement for Mg2+ or Ca2+ for activity, and functioned as 1,4-alpha-glucanohydrolases (alpha-amylases; EC 3.2.1.1). These major starch-degrading enzymes thus appear to be anchored to the cell wall in this important group of human gut bacteria.

  6. Andrographolide down-regulates hypoxia-inducible factor-1{alpha} in human non-small cell lung cancer A549 cells

    SciTech Connect

    Lin, Hui-Hsuan; Tsai, Chia-Wen; Chou, Fen-Pi; Wang, Chau-Jong; Hsuan, Shu-Wen; Wang, Cheng-Kun; Chen, Jing-Hsien

    2011-02-01

    Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess multiple pharmacological activities. In our previous study, Andro had been shown to inhibit non-small cell lung cancer (NSCLC) A549 cell migration and invasion via down-regulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Here we demonstrated that Andro inhibited the expression of hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) in A549 cells. HIF-1{alpha} plays an important role in tumor growth, angiogenesis and lymph node metastasis of NSCLC. The Andro-induced decrease of cellular protein level of HIF-1{alpha} was correlated with a rapid ubiquitin-dependent degradation of HIF-1{alpha}, and was accompanied by increased expressions of hydroxyl-HIF-1{alpha} and prolyl hydroxylase (PHD2), and a later decrease of vascular endothelial growth factor (VEGF) upon the treatment of Andro. The Andro-inhibited VEGF expression appeared to be a consequence of HIF-1{alpha} inactivation, because its DNA binding activity was suppressed by Andro. Molecular data showed that all these effects of Andro might be mediated via TGF{beta}1/PHD2/HIF-1{alpha} pathway, as demonstrated by the transfection of TGF{beta}1 overexpression vector and PHD2 siRNA, and the usage of a pharmacological MG132 inhibitor. Furthermore, we elucidated the involvement of Andro in HIF-1{alpha} transduced VEGF expression in A549 cells and other NSCLC cell lines. In conclusion, these results highlighted the potential effects of Andro, which may be developed as a chemotherapeutic or an anti-angiogenesis agent for NSCLC in the future.

  7. 17{alpha}-Estradiol arrests cell cycle progression at G{sub 2}/M and induces apoptotic cell death in human acute leukemia Jurkat T cells

    SciTech Connect

    Jun, Do Youn; Park, Hae Sun; Kim, Jun Seok; Kim, Jong Sik; Park, Wan; Song, Bang Ho; Kim, Hee-Sook; Taub, Dennis; Kim, Young Ho

    2008-09-15

    A pharmacological dose (2.5-10 {mu}M) of 17{alpha}-estradiol (17{alpha}-E{sub 2}) exerted a cytotoxic effect on human leukemias Jurkat T and U937 cells, which was not suppressed by the estrogen receptor (ER) antagonist ICI 182,780. Along with cytotoxicity in Jurkat T cells, several apoptotic events including mitochondrial cytochrome c release, activation of caspase-9, -3, and -8, PARP degradation, and DNA fragmentation were induced. The cytotoxicity of 17{alpha}-E{sub 2} was not blocked by the anti-Fas neutralizing antibody ZB-4. While undergoing apoptosis, there was a remarkable accumulation of G{sub 2}/M cells with the upregulatoin of cdc2 kinase activity, which was reflected in the Thr56 phosphorylation of Bcl-2. Dephosphorylation at Tyr15 and phosphorylation at Thr161 of cdc2, and significant increase in the cyclin B1 level were underlying factors for the cdc2 kinase activation. Whereas the 17{alpha}-E{sub 2}-induced apoptosis was completely abrogated by overexpression of Bcl-2 or by pretreatment with the pan-caspase inhibitor z-VAD-fmk, the accumulation of G{sub 2}/M cells significantly increased. The caspase-8 inhibitor z-IETD-fmk failed to influence 17{alpha}-E{sub 2}-mediated caspase-9 activation, but it markedly reduced caspase-3 activation and PARP degradation with the suppression of apoptosis, indicating the contribution of caspase-8; not as an upstream event of the mitochondrial cytochrome c release, but to caspase-3 activation. In the presence of hydroxyurea, which blocked the cell cycle progression at the G{sub 1}/S boundary, 17{alpha}-E{sub 2} failed to induce the G{sub 2}/M arrest as well as apoptosis. These results demonstrate that the cytotoxicity of 17{alpha}-E{sub 2} toward Jurkat T cells is attributable to apoptosis mainly induced in G{sub 2}/M-arrested cells, in an ER-independent manner, via a mitochondria-dependent caspase pathway regulated by Bcl-2.

  8. Interaction between Ca/sup + +/-channel antagonists and. cap alpha. /sub 2/-adrenergic receptors in rabbit ileal cell membrane

    SciTech Connect

    Homeidan, F.R.; Wicks, J.; Cusolito, S.; El-Sabban, M.E.; Sharp, G.W.G.; Donowitz, M.

    1986-03-05

    An interaction between Ca/sup + +/-channel antagonists and the ..cap alpha../sub 2/-adrenergic receptor on active electrolyte transport was demonstrated in rabbit ileum. Clonidine, an ..cap alpha../sub 2/-agonist, stimulated NaCl absorption apparently by Ca/sup + +/-channel antagonism since it inhibited /sup 45/Ca/sup + +/ uptake across the basolateral membrane and decreased total ileal calcium content. This stimulation was inhibited by the Ca/sup + +/-channel antagonists dl- and l-verapamil and cadmium but not by nifedipine. The binding of /sup 3/H-yohimbine, a specific ..cap alpha../sub 2/-adrenergic antagonist, was studied on purified ileal cell membranes using a rapid filtration technique. dl-Verapamil and Cd/sup + +/ inhibited the specific binding of /sup 3/H-yohimbine over the same concentration range in which they affected transport. In contrast, nifedipine had no effect on binding, just as it had no effect on clonidine-stimulated NaCl absorption. These data demonstrate that there is an interaction between Ca/sup + +/-channels and ..cap alpha../sub 2/-adrenergic receptors in ileal basolateral membranes. Some Ca/sup + +/-channel antagonists alter ..cap alpha../sub 2/-adrenergic binding to the receptor and ..cap alpha../sub 2/-agonist binding leads to changes in Ca/sup + +/ entry. A close spatial relationship between the Ca/sup + +/-channel and the ..cap alpha../sub 2/-receptor could explain the data.

  9. Cell growth kinetics of the human cell line Colo-205 irradiated with photons and astatine-211 alpha-particles.

    PubMed

    Palm, S; Andersson, H; Bäck, T; Claesson, I; Delle, U; Hultborn, R; Jacobsson, L; Köpf, I; Lindegren, S

    2000-01-01

    Cell growth kinetics following Astatine-211 (211At, alpha-particle emitter) and photon irradiation were studied for the human colorectal cell line Colo-205. A growth assay using 96-well plates was chosen. The growth kinetics could be simulated by assuming certain fractions of cells with various proliferative capacities, i.e. from none up to 5 cell doublings, in addition to the defined survivors with remaining unlimited clonogenic capacity. No significant difference in cell growth characteristics was seen between 211At and photon irradiation. The cell doubling time, as calculated from the increment in optical density, was compared with the results from BrdU experiments in the early phases of growth (Tpot = 18.5 +/- 0.6 h for LDR (low dose rate) photon irradiated and 20.3 +/- 0.8 hours for sham-irradiated cells 40-45 hours post-irradiation) confirming the transient accelerated growth of irradiated cells. No statistically significant difference in growth was found between LDR, MDR (medium dose rate) and HDR (high dose rate) photon irradiation.

  10. Multimeric complexes of the PML-retinoic acid receptor alpha fusion protein in acute promyelocytic leukemia cells and interference with retinoid and peroxisome-proliferator signaling pathways.

    PubMed Central

    Jansen, J H; Mahfoudi, A; Rambaud, S; Lavau, C; Wahli, W; Dejean, A

    1995-01-01

    The t(15;17) chromosomal translocation, specific for acute promyelocytic leukemia (APL), fuses the PML gene to the retinoic acid receptor alpha (RAR alpha) gene, resulting in expression of a PML-RAR alpha hybrid protein. In this report, we analyzed the nature of PML-RAR alpha-containing complexes in nuclear protein extracts of t(15;17)-positive cells. We show that endogenous PML-RAR alpha can bind to DNA as a homodimer, in contrast to RAR alpha that requires the retinoid X receptor (RXR) dimerization partner. In addition, these cells contain oligomeric complexes of PML-RAR alpha and endogenous RXR. Treatment with retinoic acid results in a decrease of PML-RAR alpha protein levels and, as a consequence, of DNA binding by the different complexes. Using responsive elements from various hormone signaling pathways, we show that PML-RAR alpha homodimers have altered DNA-binding characteristics when compared to RAR alpha-RXR alpha heterodimers. In transfected Drosophila SL-3 cells that are devoid of endogenous retinoid receptors PML-RAR alpha inhibits transactivation by RAR alpha-RXR alpha heterodimers in a dominant fashion. In addition, we show that both normal retinoid receptors and the PML-RAR alpha hybrid bind and activate the peroxisome proliferator-activated receptor responsive element from the Acyl-CoA oxidase gene, indicating that retinoids and peroxisome proliferator receptors may share common target genes. These properties of PML-RAR alpha may contribute to the transformed phenotype of APL cells. Images Fig. 1 Fig. 2 Fig. 3 PMID:7638205

  11. DOPAL is Transmissible to and Oligomerizes Alpha-Synuclein in Human Glial Cells

    PubMed Central

    Jinsmaa, Yunden; Sullivan, Patricia; Sharabi, Yehonatan; Goldstein, David S.

    2016-01-01

    Introduction Glial cytoplasmic inclusions (GCIs) containing alpha-synuclein (AS) are a neuropathologic hallmark of multiple system atrophy (MSA). Oligomerized AS is thought to be the pathogenic form of the protein. Glial cells normally express little AS, but they can take up AS from the extracellular fluid. 3,4-dihydroxyphenylacetaldehyde (DOPAL), an obligate intermediate in the intra-neuronal metabolism of dopamine (DA), potently oligomerizes AS. In this study we tested whether DOPAL is taken up by human glial cells and augments intracellular oligomerization of AS. Methods DOPAL (exogenous or endogenous from co-incubation with PC12 cells) and AS (native or A53T mutant form) were added to the incubation medium of glial cells (glioblastoma or MO3.13 oligodendrocytes). Glial cellular contents of DOPAL and its intracellular metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were measured at up to 180 minutes of incubation. Glial cellular AS oligomers were quantified by Western blotting. Results Neither glioblastoma nor MO3.13 cells contained endogenous catecholamines or AS. Co-incubation of the cells with DA-producing PC12 cells produced time-related increases in DOPAL and DOPAC contents. Similarly, glial cellular DOPAL and DOPAC contents increased rapidly after addition of DOPAL to the medium. After addition of native or A53T-AS, intracellular AS also increased. Incubation of glial cells with both DOPAL and AS enhanced the intracellular oligomerization of native and A53T-AS. Conclusions DOPAL is transmissible to glial cells and enhances intracellular oligomerization of AS. An interaction of DOPAL with AS might help explain the formation of CGIs in MSA. PMID:26777075

  12. Hypoxia Inducible Factor 1 Alpha Is Expressed in Germ Cells throughout the Murine Life Cycle.

    PubMed

    Takahashi, Natsumi; Davy, Philip M C; Gardner, Lauren H; Mathews, Juanita; Yamazaki, Yuki; Allsopp, Richard C

    2016-01-01

    Pluripotent stem cells of the early embryo, and germ line cells, are essential to ensure uncompromised development to adulthood as well as species propagation, respectively. Recently, the transcription factor hypoxia inducible factor 1 alpha (Hif1α) has been shown to have important roles in embryonic stem cells; in particular, regulation of conversion to glycolytic metabolism and, as we have shown, maintenance of functional levels of telomerase. In the present study, we sought to assess whether Hif1α was also expressed in the primitive cells of the murine embryo. We observed expression of Hif1α in pre-implantation embryos, specifically the 2-cell stage, morula, and blastocyst. Robust Hif1α expression was also observed in male and female primordial germ cells. We subsequently assessed whether Hif1α was expressed in adult male and female germ cells. In the testis, Hif1α was robustly expressed in spermatogonial cells, in both juvenile (6-week old) and adult (3-month old) males. In the ovaries, Hif1α was expressed in mature oocytes from adult females, as assessed both in situ and in individual oocytes flushed from super-ovulated females. Analysis of Hif1α transcript levels indicates a mechanism of regulation during early development that involves stockpiling of Hif1α protein in mature oocytes, presumably to provide protection from hypoxic stress until the gene is re-activated at the blastocyst stage. Together, these observations show that Hif1α is expressed throughout the life-cycle, including both the male and female germ line, and point to an important role for Hif1α in early progenitor cells.

  13. Hypoxia Inducible Factor 1 Alpha Is Expressed in Germ Cells throughout the Murine Life Cycle

    PubMed Central

    Gardner, Lauren H.; Mathews, Juanita; Yamazaki, Yuki; Allsopp, Richard C.

    2016-01-01

    Pluripotent stem cells of the early embryo, and germ line cells, are essential to ensure uncompromised development to adulthood as well as species propagation, respectively. Recently, the transcription factor hypoxia inducible factor 1 alpha (Hif1α) has been shown to have important roles in embryonic stem cells; in particular, regulation of conversion to glycolytic metabolism and, as we have shown, maintenance of functional levels of telomerase. In the present study, we sought to assess whether Hif1α was also expressed in the primitive cells of the murine embryo. We observed expression of Hif1α in pre-implantation embryos, specifically the 2-cell stage, morula, and blastocyst. Robust Hif1α expression was also observed in male and female primordial germ cells. We subsequently assessed whether Hif1α was expressed in adult male and female germ cells. In the testis, Hif1α was robustly expressed in spermatogonial cells, in both juvenile (6-week old) and adult (3-month old) males. In the ovaries, Hif1α was expressed in mature oocytes from adult females, as assessed both in situ and in individual oocytes flushed from super-ovulated females. Analysis of Hif1α transcript levels indicates a mechanism of regulation during early development that involves stockpiling of Hif1α protein in mature oocytes, presumably to provide protection from hypoxic stress until the gene is re-activated at the blastocyst stage. Together, these observations show that Hif1α is expressed throughout the life-cycle, including both the male and female germ line, and point to an important role for Hif1α in early progenitor cells. PMID:27148974

  14. Expression and secretion of immunoglobulin alpha heavy chain with diverse VDJ recombinations by human epithelial cancer cells.

    PubMed

    Zheng, Hui; Li, Ming; Ren, Wei; Zeng, Liang; Liu, Hai-dan; Hu, Duosha; Deng, Xiyun; Tang, Min; Shi, Ying; Gong, Jianping; Cao, Ya

    2007-03-01

    Generally, only B lymphocytes express immunoglobulin. Recently, we found the expression of Ig alpha heavy chain in human epithelial cancer cells unexpectedly. We first detected Ig VDJ-Calpha and Ialpha-Calpha transcripts in multiple cancer cell lines. Further, the configuration of the Ig heavy chain genomic locus was analyzed in human cancer cells. We found that cancer cells have the recombination VDJ region, but bear Ig Salpha region in germline configuration, which is different from Ig expression pattern in B cells. And human epithelial cancers possess the essential effectors including RAG-1 and RAG-2, but not activation induced cytidine deaminase (AID) protein. These provide further proofs for Ig alpha expression. In addition, we found that human cancer cells not only express the protein of Ig alpha chain, but also secrete the protein in secretory IgA (SIgA) pattern. Importantly, diverse CDR3 recombinations were found in human cancer cells of different epithelial origin. Since IgA is the key immunoglobulin which contributes to local immunity of mucous membrane, the aberrant expression of Ig alpha heavy chain might increase our further comprehension to development and immunity of cancers.

  15. Identification of gene-based responses in human blood cells exposed to alpha particle radiation

    PubMed Central

    2014-01-01

    Background The threat of a terrorist-precipitated nuclear event places humans at danger for radiological exposures. Isotopes which emit alpha (α)-particle radiation pose the highest risk. Currently, gene expression signatures are being developed for radiation biodosimetry and triage with respect to ionizing photon radiation. This study was designed to determine if similar gene expression profiles are obtained after exposures involving α-particles. Methods Peripheral blood mononuclear cells (PBMCs) were used to identify sensitive and robust gene-based biomarkers of α-particle radiation exposure. Cells were isolated from healthy individuals and were irradiated at doses ranging from 0-1.5 Gy. Microarray technology was employed to identify transcripts that were differentially expressed relative to unirradiated cells 24 hours post-exposure. Statistical analysis identified modulated genes at each of the individual doses. Results Twenty-nine genes were common to all doses with expression levels ranging from 2-10 fold relative to control treatment group. This subset of genes was further assessed in independent complete white blood cell (WBC) populations exposed to either α-particles or X-rays using quantitative real-time PCR. This 29 gene panel was responsive in the α-particle exposed WBCs and was shown to exhibit differential fold-changes compared to X-irradiated cells, though no α-particle specific transcripts were identified. Conclusion Current gene panels for photon radiation may also be applicable for use in α-particle radiation biodosimetry. PMID:25017500

  16. Alpha-Dystrobrevin and its associated proteins in human promyelocytic leukemia cells induced to apoptosis.

    PubMed

    Navakauskienė, Rūta; Treigytė, Gražina; Borutinskaitė, Veronika-Viktorija; Matuzevičius, Dalius; Navakauskas, Dalius; Magnusson, Karl-Eric

    2012-06-18

    Dystrobrevin is a dystrophin-related component of the dystrophin-associated protein complex (DAPC). Using alpha-dystrobrevin as indicator, we aimed to elucidate the interaction network of the DAPC with other proteins during apoptosis of promyelocytic HL-60 cells. The precise role(s) of DBs are not known, but we and others have shown that they play a role in intracellular signal transduction and cellular organization. Apoptosis was induced with etoposide in the absence or presence of Z-VAD to block caspase activity, and we then followed the cellular distribution of α-DB and its association with other proteins, using confocal imaging and cell fractions analyses after immune-precipitation with anti-α-DB and mass spectrometry. Confocal imaging revealed distinct spatial relocalizations of α-DB between the cell membrane, cytosol and nucleus after induction of apoptosis. The expression levels of the identified proteins were evaluated with computer-assisted image analysis of the gels. We thus identified associations with structural and transport proteins (tropomyosin, myosin), membrane (ADAM21, syntrophin), ER-Golgi (TGN51, eIF38) and nuclear (Lamins, ribonucleoprotein C1/C2) proteins. These results suggest that apoptosis-induction in HL-60 cells involves not only classical markers of apoptosis but also a network α-DB-associated proteins at the cell membrane, the cytoplasm and nucleus, affecting key cellular transport processes and cellular structure.

  17. Stromal Cell-Derived Factor-1 Alpha is Cardioprotective After Myocardial Infarction

    PubMed Central

    Saxena, Ankur; Fish, Jason E.; White, Michael D.; Yu, Sangho; Smyth, James WP; Shaw, Robin M.; DiMaio, J. Michael; Srivastava, Deepak

    2009-01-01

    Background Heart disease is a leading cause of mortality throughout the world. Tissue damage from vascular occlusive events results in the replacement of contractile myocardium by nonfunctional scar tissue. The potential of new technologies to regenerate damaged myocardium is significant, although cell-based therapies must overcome several technical barriers. One possible cell-independent alternative is the direct administration of small proteins to damaged myocardium. Methods and Results Here we show that the secreted signaling protein stromal cell-derived factor-1 alpha (SDF-1α), which activates the cell-survival factor protein kinase B (PKB/Akt) via the G-protein-coupled receptor CXCR4, protected tissue after an acute ischemic event in mice and activated Akt within endothelial cells and myocytes of the heart. Significantly better cardiac function than in control mice was evident as early as 24 hours post-infarction as well as at 3, 14 and 28 days post-infarction. Prolonged survival of hypoxic myocardium was followed by an increase in levels of vascular endothelial growth factor (VEGF) protein and neo-angiogenesis. Consistent with improved cardiac function, mice exposed to SDF-1α demonstrated significantly decreased scar formation than control mice. Conclusions These findings suggest that SDF-1α may serve a tissue-protective and regenerative role for solid organs suffering a hypoxic insult. PMID:18427137

  18. SIP1/NHERF2 enhances estrogen receptor alpha transactivation in breast cancer cells

    PubMed Central

    Meneses-Morales, Ivan; Tecalco-Cruz, Angeles C.; Barrios-García, Tonatiuh; Gómez-Romero, Vania; Trujillo-González, Isis; Reyes-Carmona, Sandra; García-Zepeda, Eduardo; Méndez-Enríquez, Erika; Cervantes-Roldán, Rafael; Pérez-Sánchez, Víctor; Recillas-Targa, Félix; Mohar-Betancourt, Alejandro; León-Del-Río, Alfonso

    2014-01-01

    The estrogen receptor alpha (ERα) is a ligand-activated transcription factor that possesses two activating domains designated AF-1 and AF-2 that mediate its transcriptional activity. The role of AF-2 is to recruit coregulator protein complexes capable of modifying chromatin condensation status. In contrast, the mechanism responsible for the ligand-independent AF-1 activity and for its synergistic functional interaction with AF-2 is unclear. In this study, we have identified the protein Na+/H+ Exchanger RegulatoryFactor 2 (NHERF2) as an ERα-associated coactivator that interacts predominantly with the AF-1 domain of the nuclear receptor. Overexpression of NHERF2 in breast cancer MCF7 cells produced an increase in ERα transactivation. Interestingly, the presence of SRC-1 in NHERF2 stably overexpressing MCF7 cells produced a synergistic increase in ERα activity. We show further that NHERF2 interacts with ERα and SRC-1 in the promoter region of ERα target genes. The binding of NHERF2 to ERα in MCF7 cells increased cell proliferation and the ability of MCF7 cells to form tumors in a mouse model. We analyzed the expression of NHERF2 in breast cancer tumors finding a 2- to 17-fold increase in its mRNA levels in 50% of the tumor samples compared to normal breast tissue. These results indicate that NHERF2 is a coactivator of ERα that may participate in the development of estrogen-dependent breast cancer tumors. PMID:24771346

  19. Interaction of the human cytomegalovirus particle with the host cell induces hypoxia-inducible factor 1 alpha

    SciTech Connect

    McFarlane, Steven; Nicholl, Mary Jane; Sutherland, Jane S.; Preston, Chris M.

    2011-05-25

    The cellular protein hypoxia-inducible factor 1 alpha (HIF-1{alpha}) was induced after infection of human fibroblasts with human cytomegalovirus (HCMV). HCMV irradiated with ultraviolet light (uv-HCMV) also elicited the effect, demonstrating that the response was provoked by interaction of the infecting virion with the cell and that viral gene expression was not required. Although induction of HIF-1{alpha} was initiated by an early event, accumulation of the protein was not detected until 9 hours post infection, with levels increasing thereafter. Infection with uv-HCMV resulted in increased abundance of HIF-1{alpha}-specific RNA, indicating stimulation of transcription. In addition, greater phosphorylation of the protein kinase Akt was observed, and the activity of this enzyme was required for induction of HIF-1{alpha} to occur. HIF-1{alpha} controls the expression of many cellular gene products; therefore the findings reveal new ways in which interaction of the HCMV particle with the host cell may cause significant alterations to cellular physiology.

  20. Pro-gliogenic effect of IL-1alpha in the differentiation of embryonic neural precursor cells in vitro.

    PubMed

    Ajmone-Cat, Maria Antonietta; Cacci, Emanuele; Ragazzoni, Ylenia; Minghetti, Luisa; Biagioni, Stefano

    2010-05-01

    Inflammation is regarded as a main obstacle to brain regeneration. Major detrimental effects are attributed to microglial/macrophagic products, such as TNF-alpha and interleukin (IL)-6. The role of cytokines of the IL-1 family, particularly of IL-1alpha, in the modulation of neural precursor cell (NPC) properties is less characterized. IL-1alpha is one of the most abundant cytokines released upon acute stimulation of microglia with lipopolysaccharide and is down-regulated upon chronic stimulation. As we recently demonstrated, acutely activated microglia reduces NPC survival, prevent neuronal differentiation and promote glial differentiation. Chronically activated microglia are instead permissive to NPC survival and neuronal differentiation, and less effective in promoting astrocytic differentiation. We thus investigated whether IL-1alpha could contribute to the effects of acutely activated microglia on NPC. We found that NPC express functional IL-1 receptors and that exposure to recombinant IL-1alpha strongly enhances NPC differentiation into astrocytes, without affecting cell viability and neuronal differentiation. In the same conditions, recombinant IL-1beta has pro-gliogenic effects at concentrations 10-fold higher than those found in activated microglial conditioned media. Interestingly, immunodepletion of IL-1alpha in activated microglial conditioned media fails to revert microglial pro-gliogenic action and slightly enhances neuronal differentiation, revealing that other microglial-derived factors contribute to the modulation of NPC properties.

  1. The adhesive and migratory effects of osteopontin are mediated via distinct cell surface integrins. Role of alpha v beta 3 in smooth muscle cell migration to osteopontin in vitro.

    PubMed Central

    Liaw, L; Skinner, M P; Raines, E W; Ross, R; Cheresh, D A; Schwartz, S M; Giachelli, C M

    1995-01-01

    Osteopontin is an arginine-glycine-aspartate containing acidic glycoprotein postulated to mediate adhesion, migration, and biomineralization in diverse tissues. The mechanisms explaining this multifunctionality are not well understood, although it is known that one osteopontin receptor is the alpha v beta 3 integrin. In this work, we studied human smooth muscle cells varying in alpha v beta 3 levels to identify additional osteopontin receptors. We report that, in addition to alpha v beta 3, both alpha v beta 5 and alpha v beta 1 are osteopontin receptors. Moreover, the presence or absence of alpha v beta 3 on the cell surface altered the adhesive and migratory responses of smooth muscle cells to osteopontin. Adhesion of alpha v beta 3-deficient cell populations to osteopontin was only half that of cells containing alpha v beta 3, and migration toward an osteopontin gradient in the Boyden chamber was dependent on cell surface alpha v beta 3. Although alpha v beta 3-deficient smooth muscle cells were unable to migrate to osteopontin, they did migrate significantly in response to vitronectin and fibronectin. These findings represent the first description of alpha v beta 5 and alpha v beta 1 as osteopontin receptors and suggest that, while adhesion to osteopontin is supported by integrins containing beta 1, beta 3, and beta 5, migration in response to osteopontin appears to depend on alpha v beta 3. Thus, interaction with distinct receptors is one mechanism by which osteopontin may initiate multiple functions. Images PMID:7532190

  2. Sesamin attenuates intercellular cell adhesion molecule-1 expression in vitro in TNF-alpha-treated human aortic endothelial cells and in vivo in apolipoprotein-E-deficient mice.

    PubMed

    Wu, Wen-Huey; Wang, Shu-Huei; Kuan, I-I; Kao, Ya-Shi; Wu, Pei-Jhen; Liang, Chan-Jung; Chien, Hsiung-Fei; Kao, Chiu-Hua; Huang, Ching-Jang; Chen, Yuh-Lien

    2010-09-01

    Sesame lignans have antioxidative and anti-inflammatory properties. We focused on the effects of the lignans sesamin and sesamol on the expression of endothelial-leukocyte adhesion molecules in tumor necrosis factor-alpha (TNF-alpha)-treated human aortic endothelial cells (HAECs). When HAECs were pretreated with sesamin (10 or 100 microM), the TNF-alpha-induced expression of intercellular cell adhesion molecule-1 (ICAM-1) was significantly reduced (35 or 70% decrease, respectively) by Western blotting. Sesamol was less effective at inhibiting ICAM-1 expression (30% decrease at 100 microM). Sesamin and sesamol reduced the marked TNF-alpha-induced increase in human antigen R (HuR) translocation and the interaction between HuR and the 3'UTR of ICAM-1 mRNA. Both significantly reduced the binding of monocytes to TNF-alpha-stimulated HAECs. Sesamin significantly attenuated TNF-alpha-induced ICAM-1 expression and cell adhesion by downregulation of extracellular signal-regulated kinase 1/2 and p38. Furthermore, in vivo, sesamin attenuated intimal thickening and ICAM-1 expression seen in aortas of apolipoprotein-E-deficient mice. Taken together, these data suggest that sesamin inhibits TNF-alpha-induced extracellular signal-regulated kinase/p38 phosphorylation, nuclear translocation of NF-kappaB p65, cytoplasmic translocalization of HuR and thereby suppresses ICAM-1 expression, resulting in reduced adhesion of leukocytes. These results also suggest that sesamin may prevent the development of atherosclerosis and inflammatory responses.

  3. Tumor necrosis factor alpha promotes the expression of immunosuppressive proteins and enhances the cell growth in a human bone marrow-derived stem cell culture

    SciTech Connect

    Miettinen, Johanna A.; Pietilae, Mika; Salonen, Riikka J.; Ohlmeier, Steffen; Ylitalo, Kari; Huikuri, Heikki V.; Lehenkari, Petri

    2011-04-01

    Mesenchymal stem cells (MSCs) are widely used in experimental treatments for various conditions that involve normal tissue regeneration via inflammatory repair. It is known that MSCs can secrete multiple soluble factors and suppress inflammation. Even though the effect of MSCs on inflammation has been extensively studied, the effect of inflammation on MSCs is poorly understood. One of the major cytokines released at the site of inflammation is tumor necrosis factor alpha (TNF-{alpha}) which is known to induce MSC invasion and proliferation. Therefore, we wanted to test the effects of TNF-{alpha} exposure on MSCs derived from human bone marrow. We found, as expected, that cell proliferation was significantly enhanced during TNF-{alpha} exposure. However, according to the cell surface marker analysis, the intensity of several antigens in the minimum criteria panel for MSCs proposed by International Society of Cellular Therapy (ISCT) was decreased dramatically, and in certain cases, the criteria for MSCs were not fulfilled. In addition, TNF-{alpha} exposure resulted in a significant but transient increase in human leukocyte antigen and CD54 expression. Additional proteomic analysis by two-dimensional difference gel electrophoresis and mass spectrometry revealed three proteins whose expression levels decreased and 8 proteins whose expression levels increased significantly during TNF-{alpha} exposure. The majority of these proteins could be linked to immunosuppressive and signalling pathways. These results strongly support reactive and immunosuppressive activation of MSCs during TNF-{alpha} exposure, which might influence MSC differentiation stage and capacity.

  4. Cytotoxicity of alpha-particle-emitting m-[211At]astatobenzylguanidine on human neuroblastoma cells.

    PubMed

    Strickland, D K; Vaidyanathan, G; Zalutsky, M R

    1994-10-15

    Radioiodinated m-iodobenzylguanidine (MIBG) has been used with only limited success for the treatment of neural crest tumors including neuroblastoma. Use of an MIBG analogue labeled with 211At could be advantageous because of the shorter range and higher linear energy transfer of its alpha-particle emissions compared with the beta-particles emitted by 131I. The potential utility of m-[211At]astatobenzylguanidine for the treatment of neuroblastoma was investigated in vitro using 3 human neuroblastoma cell lines known to take up MIBG [SK-N-SH, SK-N-BE(2C), and SK-SY5Y] and a control line lacking MIBG uptake (SK-N-MC). Maximum binding of m-[211At]astatobenzylguanidine ([211At] MABG) to 5 x 10(5) cells after a 2-h incubation ranged from 61% for SK-N-SH to 1% for SK-N-MC. Using a limiting dilution clonogenic assay, the cytotoxicity for SK-N-SH cells of [211At]MABG was compared with [211At]astatide and no-carrier-added [131I]MIBG. A D0 of 5.8 nCi/ml was calculated for [211At]MABG compared with 482 nCi/ml for [211At] astatide, indicating a more than 80-fold enhanced cytotoxicity for the specifically targeted alpha-particles of [211At]MABG. For [211At]MABG, the D0 corresponded to only 6.4 211At atoms bound/cell compared with 9000 atoms/cell for no-carrier-added [131I]MIBG. The D0 values measured for [211At]MABG treatment of SK-SY5Y, SK-N-BE(2C), and SK-N-MC cells were 50, 5.8, and 11,043 nCi/ml, respectively, corresponding to 7.04, 6.46, and 171.79 211At atoms bound/cell. In conclusion, these results have demonstrated that [211At]MABG is considerably more cytotoxic than [131I]MIBG and that [211At]MABG could have great potential as a radiotherapeutic agent for the treatment of neuroblastoma.

  5. In vitro protective effects of two extracts from bergamot peels on human endothelial cells exposed to tumor necrosis factor-alpha (TNF-alpha).

    PubMed

    Trombetta, Domenico; Cimino, Francesco; Cristani, Mariateresa; Mandalari, Giuseppina; Saija, Antonella; Ginestra, Giovanna; Speciale, Antonio; Chirafisi, Joselita; Bisignano, Giuseppe; Waldron, Keith; Narbad, Arjan; Faulds, Craig B

    2010-07-28

    Bergamot ( Citrus bergamia Risso) is a less commercialized Citrus fruit, mainly used for its essential oil extracted from the peel. Bergamot peel (BP) represents about 60% of the processed fruits and is regarded as primary waste. However, it contains good amounts of useful compounds, such as pectins and flavonoids. Many of the bioactivities of Citrus flavonoids appear to impact vascular endothelial cells. Herein, we report the protective effect of two flavonoid-rich extracts from BP (endowed with radical-scavenging properties and lacking genotoxic activity) against alterations in cell modifications induced by the pleiotropic inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) on human umbilical vein endothelial cells (HUVECs), as demonstrated by monitoring intracellular levels of malondialdehyde/4-hydroxynonenal, reduced and oxidized glutathione and superoxide dismutase activity, and the activation status of nuclear factor-kappaB (NF-kappaB). Thus, BP appears to be a potential source of natural antioxidant/anti-inflammatory phytocomplexes to be employed as ingredients of nutraceutical products or functional foods.

  6. Minimal requirements for exocytosis. A study using PC 12 cells permeabilized with staphylococcal alpha-toxin

    SciTech Connect

    Ahnert-Hilger, G.; Bhakdi, S.; Gratzl, M.

    1985-10-15

    The membrane-permeabilizing effects of streptolysin O, staphylococcal alpha-toxin, and digitonin on cultured rat pheochromocytoma cells were studied. All three agents perturbed the plasma membrane, causing release of intracellular YWRb and uptake of trypan blue. In addition, streptolysin O and digitonin also damaged the membranes of secretory vesicles, including a parallel release of dopamine. In contrast, the effects of alpha-toxin appeared to be strictly confined to the plasma membrane, and no dopamine release was observed with this agent. The exocytotic machinery, however, remained intact and could be triggered by subsequent introduction of micromolar concentrations of Ca2+ into the medium. Dopamine release was entirely Ca2+ specific and occurred independent of the presence or absence of other cations or anions including K+ glutamate, K+ acetate, or Na+ chloride. Ca2+-induced exocytosis did not require the presence of Mg2+-ATP in the medium. The process was insensitive to pH alterations in the range pH 6.6-7.2, and appeared optimal at an osmolarity of 300 mosm/kg. Toxin permeabilization seems to be an excellent method for studying the minimal requirements for exocytosis.

  7. The stromal cell-derived factor-1alpha dependent migration of human cord blood CD34 haematopoietic stem and progenitor cells switches from protein kinase C (PKC)-alpha dependence to PKC-alpha independence upon prolonged culture in the presence of Flt3-ligand and interleukin-6.

    PubMed

    Kasenda, Benjamin; Kassmer, Susannah H; Niggemann, Bernd; Schiermeier, Sven; Hatzmann, Wolfgang; Zänker, Kurt S; Dittmar, Thomas

    2008-09-01

    Addition of the inflammatory cytokine interleukin (IL)-6 to the culture medium of human cord blood haematopoietic stem and progenitor cells (HSPCs) has been shown to lead to an altered stromal cell-derived factor-1alpha-dependent migratory phenotype. This study investigated whether this effect was attributed to a differential engagement of protein kinase C (PKC) isotypes. The migratory activity of both Flt3-ligand and Flt3-ligand/IL-6 cultured cord blood HSPCs was PKC-alpha dependent on day 1, but PKC-alpha independent after 5 d of cultivation. PKC-alpha expression was not down-regulated in cells cultured for 5 d indicating a switch of signalling molecules directing cell migration.

  8. The IL-6/sIL-6R treatment of a malignant melanoma cell line enhances susceptibility to TNF-{alpha}-induced apoptosis

    SciTech Connect

    Wagley, Yadav; Yoo, Yung-Choon; Seo, Han Geuk; Rhee, Man Hee; Kim, Tae-Hyoung; Kang, Keon Wook; Nah, Seung-Yeol; Oh, Jae-Wook . E-mail: ohjw@mail.chosun.ac.kr

    2007-03-23

    Melanoma is an intractable tumor that has shown very impressive and promising response to local administration of high dose recombinant TNF-{alpha} in combination with IFN-{gamma} in clinical studies. In this study, we investigated the effect of IL-6/sIL-6R on TNF-{alpha}-resistant B16/F10.9 melanoma cells. A low dose of TNF-{alpha} or IL-6/sIL-6R had minimal affect on the cell growth. However, the highly active fusion protein of sIL-6R and IL-6 (IL6RIL6), covalently linked by a flexible peptide, sensitized TNF-{alpha}-resistant F10.9 melanoma cells to TNF-{alpha}-induced apoptosis. Stimulation of the cells with IL6RIL6 plus TNF-{alpha} resulted in both the activation of caspase-3 and the reduction of bcl-2 expression. Flow cytometry analysis showed that IL6RIL6-upregulated TNF-R55 and TNF-R75 expression, suggesting an increase in TNF-{alpha} responsiveness by IL6RIL6 resulting from the induction of TNF receptors. Moreover, exposure of F10.9 cells to neutralizing antibody to TNF-R55 significantly inhibited IL6RIL6/TNF-{alpha}-induced cytotoxicity. These results suggest that the IL6/sIL6R/gp130 system, which sensitizes TNF-{alpha}-resistant melanoma cells to TNF-{alpha}-induced apoptosis, may provide a new target for immunotherapy.

  9. TNF-alpha-induced apoptosis is prevented by erythropoietin treatment on SH-SY5Y cells

    SciTech Connect

    Pregi, Nicolas Wenker, Shirley; Vittori, Daniela; Leiros, Claudia Perez; Nesse, Alcira

    2009-02-01

    The growth factor erythropoietin (Epo) has shown neuronal protective action in addition to its well known proerythroid activity. Furthermore, Epo has dealt with cellular inflammation by inhibiting the expression of several proinflammatory cytokines, such as IL-1 and TNF-{alpha}. The action of TNF can have both apoptotic and antiapoptotic consequences due to altered balance between different cell signalling pathways. This work has focused on the apoptotic effects of this cytokine and the potential protective action of Epo. The model we used was neuroblastoma SH-SY5Y cells cultured in the presence of 25 ng/ml TNF-{alpha} or pretreated with 25 U/ml Epo for 12 h before the addition of TNF-{alpha}. Apoptosis was evaluated by differential cell count after Hoechst staining, analysis of DNA ladder pattern, and measurement of caspase activity. Despite its ability to induce NF-{kappa}B nuclear translocation, TNF-{alpha} induced cell death, which was found to be associated to upregulation of TNF Receptor 1 expression. On the other hand, cells activated by Epo became resistant to cell death. Prevention of death receptor upregulation and caspase activation may explain this antiapoptotic effect of Epo, which may be also favoured by the induction of a higher expression of protective factors, such as Bcl-2 and NF-{kappa}B, through mechanisms involving Jak/STAT and PI3K signalling pathways.

  10. Interferon-alpha production by swine dendritic cells is inhibited during acute infection with foot-and-mouth disease virus.

    PubMed

    Nfon, Charles K; Ferman, Geoffrey S; Toka, Felix N; Gregg, Douglas A; Golde, William T

    2008-03-01

    Viruses have evolved multiple mechanisms to evade the innate immune response, particularly the actions of interferons (IFNs). We have previously reported that exposure of dendritic cells (DCs) to foot-and-mouth disease virus (FMDV) in vitro yields no infection and induces a strong type I IFN (IFN-alpha and IFN-beta) response, indicating that DCs may play a critical role in the innate response to the virus. In vivo, FMDV induces lymphopenia and reduced T-cell proliferative responses to mitogen, viral effects that may contribute to evasion of early immune responses. In this study we analyzed the in vivo effects of FMDV infection on the IFN-alpha response of two populations of dendritic cells. During the acute phase of infection of swine, production of IFN-alpha from monocyte-derived DCs (MoDCs) and skin-derived DCs (skin DCs) is inhibited. This effect occurs concurrently with rising viral titers in the blood; however, these cells are not productively infected. Interestingly, there are no changes in the capability of these DCs to take up particles and process antigens, indicating that antigen-presenting cell function is normal. These data indicate that inhibition of the IFN-alpha response of dendritic cell populations from blood and skin by FMDV enhances viral pathogenesis in infected animals.

  11. Survivin-2B promotes autophagy by accumulating IKK alpha in the nucleus of selenite-treated NB4 cells.

    PubMed

    Shi, K; An, J; Shan, L; Jiang, Q; Li, F; Ci, Y; Wu, P; Duan, J; Hui, K; Yang, Y; Xu, C

    2014-02-20

    Survivin-2B, a known splice variant of survivin, has been reported to promote cell death in some cancer cells, although it keeps prosurvival function in others, and the mechanisms are unclear. In this report, we discovered that selenite, an antitumor agent, switched protective autophagy to apoptosis in NB4 cells. In this process, the level of survivin-2B was decreased and the interaction between IKK alpha and survivin-2B in the nucleus was attenuated, which further led to the decrease of nuclear IKK alpha. As a result, P73, a known transcript factor of UVRAG, was downregulated. Therefore, the expression of UVRAG, one of the initiators of autophagy, was inhibited. The regulatory status of survivin-2B was also proved in NB4 cells after different chemicals' exposure and in other tumor cell lines (Jurkat, HCT116). Finally, experiments in vivo confirmed that the alterations of survivin-2B, IKK alpha, P73 and UVRAG were the same as that in vitro. Taken together, survivin-2B promoted autophagy and further regulated cell death by accumulating and stabilizing IKK alpha in the nucleus.

  12. Molecular basis of cross-reactivity among allergen-specific human T cells: T-cell receptor V alpha gene usage and epitope structure.

    PubMed Central

    Mohapatra, S S; Mohapatra, S; Yang, M; Ansari, A A; Parronchi, P; Maggi, E; Romagnani, S

    1994-01-01

    Cross-reactivities between the major grass pollen allergens, at the level of T-cell recognition was examined employing several Lolium perenne I (Lol p I)-specific human T-cell clones. Nine of these Lol p I-specific T-cell clones exhibited cross-recognition of the recombinant Poa pratensis IX (Poa p IX) allergen, rKBG7.2, indicating that these two major antigens of a grass pollen share T-cell epitopes. Furthermore, proliferative responses of two other T-cell clones demonstrated that individual allergens of diverse grass pollens also possess common T-cell epitopes. Examination of the T-cell receptor (TcR) V alpha genes of these T-cell clones indicated that these cloned cells utilized distinct J alpha genes and that nine out of 10 clones possessed V alpha 13 gene. Furthermore, sequence comparisons of several allergenic molecules indicated that this cross-reactivity may be due to the presence of epitope(s) with structure(s) similar to the major T-cell epitope of Poa p IX allergens. Taken together, these results suggest for the first time that the major grass pollen allergens share cross-reacting T-cell epitope(s), and that this cross-reactivity is due to the structural homologies among allergens and restricted usage of TcR V alpha genes. PMID:7510663

  13. Approaches to maximizing stable expression of alpha 1-antitrypsin in transformed CHO cells.

    PubMed

    Paterson, T; Innes, J; Moore, S

    1994-01-01

    A variety of approaches to maximizing the production of recombinant human alpha 1-antitrypsin (AAT) in Chinese hamster ovary (CHO) cells have been investigated. The highly active and inducible human cytomegalovirus immediate early (IE) promoter/enhancer was used to drive transcription of a recombinant AAT gene in transiently transfected and stably transformed CHO cells. The AAT gene was modified to incorporate highly efficient 3'RNA processing signals from the herpes simplex virus type 2 IE gene 5, and optimal translational initiation signals were created by site-directed mutagenesis. The effect of flanking the recombinant gene with matrix attachment regions was investigated. Combinations of these modifications allowed secretion of up to 44 micrograms AAT/ml per day by cell lines growing in serum-rich medium. This could be increased to up to 100 micrograms AAT/ml per day upon chemical induction of expression by propionate, butyrate or hexamethylene bisacetamide. Cell lines adapted to grow in protein-free medium produced less AAT but still responded to chemical induction to secrete up to 14 micrograms/ml per day of readily purified AAT.

  14. Alpha-1-antitrypsin for the improvement of autoimmunity and allograft rejection in beta cell transplantation.

    PubMed

    Ye, Jian; Liao, Yu-Ting; Jian, You-Qiang; Zhang, Xiao-Dan; Wei, Pei; Qi, Hui; Deng, Chun-Yan; Li, Fu-Rong

    2013-02-01

    Islet transplantation offers hope for patients with type 1 diabetes, which is an autoimmune disease. However, islet transplant recipients must overcome two obstacles in both allograft rejection and autoimmune reaction. Alpha-1-antitrypsin (a1-proteinase inhibitor, AAT) possesses anti-inflammatory properties, reduces cytokine-mediated islet damage, and induces specific immune tolerance. In this study, an insulinoma cell line, NIT-1, was transfected with human AAT (hAAT), named NIT-hAAT, and was transplanted to the left renal subcapsular spaces of 7-week-old female non-obese diabetic (NOD) mice (n=22). Cyclophosphamide(CY) was administered to synchronize and accelerate the development of diabetes. Thus, the immunosuppressive and cytoprotective activity of hAAT in β-cell transplantation was investigated. NIT-hAAT has immunomodulatory properties, which delay the onset of autoimmune diabetes, reduce diabetes incidence, inhibit insulitis and β-cell apoptosis, and dampen transplant site inflammation. We propose that NIT-hAAT has a dual function by improving islet autoimmunity and protecting transplanted β-cells from allograft rejection. However, the low expression of hAAT in vivo results in the inability of NIT-hAAT to induce long-term specific immune tolerance and to completely block allograft rejection.

  15. [Alpha-1 Antitrypsin Affects U0126-Induced Cytotoxicity in Colon Cancer Cell Line (HCT116)].

    PubMed

    Ljujic, M; Mijatovic, S; Bulatovic, M Z; Mojic, M; Maksimovic-Ivanic, D; Radojkovic, D; Topic, A

    2016-01-01

    Alpha-1-antitrypsin (AAT), an acute phase protein, is the principal circulatory anti-protease. This multifunctional protein is encoded by the SERPINA1 gene. Although AAT was recognised as a potential tumour marker, its role in cancer biology remains unknown. Given that it has been demonstrated that AAT has an anti-apoptotic property against non-malignant cells, we aimed to investigate whether AAT affects apoptosis in a colon cancer cell line (HCT116). The presence of AAT in the HCT116 cell culture antagonized cytotoxicity of blockers of MEK1/2, PI3K/Akt pathways as well as NF-κB. The dominantly recovered cell viability was observed in the co-treatment with MEK1/2 inhibitor U0126. In addition, it was revealed that AAT almost completely abolished U0126-induced apoptosis through maintenance of the autophagy process. Our study revealed for the first time that the observed cyto-protection triggered by AAT was accompanied by sustained autophagy which opposed apoptosis. These results may contribute to understanding of the role of AAT in cancer development and evaluation of efficacy of cancer therapy.

  16. Ubiquitination of tissue transglutaminase is modulated by interferon alpha in human lung cancer cells.

    PubMed Central

    Esposito, Carla; Marra, Monica; Giuberti, Gaia; D'Alessandro, Anna Maria; Porta, Raffaele; Cozzolino, Anna; Caraglia, Michele; Abbruzzese, Alberto

    2003-01-01

    The addition of 2500 i.u./ml interferon alpha (IFNalpha) for 48 h induced apoptosis, and caused an approx. 4-fold increase in the activity and expression of tissue transglutaminase (tTG), in human lung cancer H1355 cells. However, the increase in mRNA levels for tTG was just 1.6-fold. On the basis of these data, we investigated whether tTG levels may be regulated through regulation of its degradation via ubiquitination. It was found that 2500 i.u./ml IFNalpha induced a time-dependent decrease in tTG ubiquitination. On the other hand, addition of the proteasome inhibitor lactacystin led to accumulation of the ubiquitinated form of the enzyme and to a consequent increase in its expression. Treatment of the cells with the two agents combined antagonized the accumulation of the ubiquitinated isoforms of tTG induced by lactacystin and caused a potentiation of tTG expression. Moreover, the tTG inducer retinoic acid was also able to cause increased expression and ubiquitination of tTG in H1355 cells. The addition of monodansylcadaverine (a tTG inhibitor) to IFNalpha-treated H1355 cells completely antagonized growth inhibition and apoptosis induced by the cytokine. In conclusion, we demonstrate for the first time that tTG is ubiquitinated and degraded by a proteasome-dependent pathway. Moreover, IFNalpha can, at least in part, induce apoptosis through the modulation of this pathway. PMID:12401132

  17. Human mast cells transmigrate through human umbilical vein endothelial monolayers and selectively produce IL-8 in response to stromal cell-derived factor-1 alpha.

    PubMed

    Lin, T J; Issekutz, T B; Marshall, J S

    2000-07-01

    Mature mast cells are generally considered to be less mobile cells residing within tissue sites. However, mast cell numbers are known to increase in the context of inflammation, and mast cells are recognized to be important in regulating local neutrophil infiltration. CXC chemokines may play a critical role in this process. In this study two human mast cell-like lines, HMC-1 and KU812, and human cord blood-derived primary cultured mast cells were employed to examine role of stromal cell-derived factor-1 (SDF-1) in regulating mast cell migration and mediator production. It was demonstrated that human mast cells constitutively express mRNA and protein for CXCR4. Stimulation of human mast cells with SDF-1, the only known ligand for CXCR4, induced a significant increase in intracellular calcium levels. In vitro, SDF-1 alpha mediated dose-dependent migration of human cord blood-derived mast cells and HMC-1 cells across HUVEC monolayers. Although SDF-1 alpha did not induce mast cell degranulation, it selectively stimulated production of the neutrophil chemoattractant IL-8 without affecting TNF-alpha, IL-1beta, IL-6, GM-CSF, IFN-gamma, or RANTES production, providing further evidence of the selective modulation of mast cell function by this chemokine. These findings provide a novel, SDF-1-dependent mechanism for mast cell transendothelial migration and functional regulation, which may have important implications for the local regulation of mast cells in disease.

  18. Interferon alpha impairs insulin production in human beta cells via endoplasmic reticulum stress.

    PubMed

    Lombardi, Angela; Tomer, Yaron

    2017-02-23

    Despite substantial advances in the research exploring the pathogenesis of Type 1 Diabetes (T1D), the pathophysiological mechanisms involved remain unknown. We hypothesized in this study that interferon alpha (IFNα) participates in the early stages of T1D development by triggering endoplasmic reticulum (ER) stress. To verify our hypothesis, human islets and human EndoC-βH1 cells were exposed to IFNα and tested for ER stress markers, glucose stimulated insulin secretion (GSIS) and insulin content. IFNα treatment induced upregulation of ER stress markers including Binding immunoglobulin Protein, phospho-eukaryotic translation initiation factor 2α, spliced- X-box binding protein-1, C/EBP homologous protein and activating transcription factor 4. Intriguingly, IFNα treatment did not impair GSIS but significantly decreased insulin production in both human islets and EndoC-βH1 cells. Furthermore, IFNα decreased the expression of both proinsulin convertase 1 and proinsulin convertase 2, suggesting an altered functional state of the beta cells characterized by a slower proinsulin-insulin conversion. Pretreatment of both human islets and EndoC-βH1 cells with chemical chaperones 4-phenylbutyric acid and tauroursodeoxycholic acid completely prevented IFNα effects, indicating an ER stress-mediated impairment of insulin production. We demonstrated for the first time that IFNα elicits ER stress in human beta cells providing a novel mechanistic role for this virus-induced cytokine in the development of T1D. Compounds targeting molecular processes altered in ER-stressed beta cells could represent a potential therapeutic strategy to prevent IFNα-induced beta cell dysfunction in the early onset of T1D.

  19. [beta]-hexosaminidase isozymes from cells cotransfected with [alpha] and [beta] cDNA constructs: Analysis of the [alpha]-subunit missense mutation associated with the adult form of Tay-Sachs disease

    SciTech Connect

    Brown, C.A.; Mahuran, D.J. )

    1993-08-01

    In vitro mutagenesis and transient expression in COS cells has been used to associate a missense mutation with a clinical or biochemical phenotype. Mutations affecting the [alpha]-subunit of [beta]-hexosaminidase A ([alpha][beta]) (E.C.3.2.1.52) result in Tay-Sachs disease. Because hexosaminidase A is heterodimeric, analysis of [alpha]-chain mutations is not straightforward. The authors examine three approaches utilizing previously identified mutations affecting [alpha]-chain folding. These involve transfection of (1) the [alpha] cDNA alone; (2) a [beta] cDNA construct encoding a [beta]-subunit substituted at a position homologous to that of the [alpha]-subunit, and (3) both [alpha] and [beta] cDNAs. The latter two procedures amplified residual activity levels over that of patient samples, an effect not previously found with mutations affecting an [open quotes]active[close quotes] [alpha]Arg residue. This effect may help to discriminate between protein-folding and active-site mutations. The authors conclude that, with proper controls, the latter method of cotransfection can be used to evaluate the effects and perhaps to predict the clinical course of some [alpha]-chain mutations. Using this technique, they demonstrate that the adult-onset Tay-Sachs mutation, [alpha]Gly[yields]Ser[sup 269], does not directly affect [alpha][beta] dimerization but exerts an indirect effect on the dimer through destabilizing the folded [alpha]-subunit at physiological temperatures. Two other [alpha] mutations linked to more severe phenotypes appear to inhibit the initial folding of the subunit. 36 refs., 2 figs., 5 tabs.

  20. Involvement of phosphatidylinositol 3-kinase in stromal cell-derived factor-1 alpha-induced lymphocyte polarization and chemotaxis.

    PubMed

    Vicente-Manzanares, M; Rey, M; Jones, D R; Sancho, D; Mellado, M; Rodriguez-Frade, J M; del Pozo, M A; Yáñez-Mó, M; de Ana, A M; Martínez-A, C; Mérida, I; Sánchez-Madrid, F

    1999-10-01

    The role of phosphatidylinositol 3-kinase (PI3-kinase), an important enzyme involved in signal transduction events, has been studied in the polarization and chemotaxis of lymphocytes induced by the chemokine stromal cell-derived factor-1 alpha (SDF-1 alpha). This chemokine was able to directly activate p85/p110 PI3-kinase in whole human PBL and to induce the association of PI3-kinase to the SDF-1 alpha receptor, CXCR4, in a pertussis toxin-sensitive manner. Two unrelated chemical inhibitors of PI3-kinase, wortmannin and Ly294002, prevented ICAM-3 and ERM protein moesin polarization as well as the chemotaxis of PBL in response to SDF-1 alpha. However, they did not interfere with the reorganization of either tubulin or the actin cytoskeleton. Moreover, the transient expression of a dominant negative form of the PI3-kinase 85-kDa regulatory subunit in the constitutively polarized Peer T cell line inhibited ICAM-3 polarization and markedly reduced SDF-1 alpha-induced chemotaxis. Conversely, overexpression of a constitutively activated mutant of the PI3-kinase 110-kDa catalytic subunit in the round-shaped PM-1 T cell line induced ICAM-3 polarization. These results underline the role of PI3-kinase in the regulation of lymphocyte polarization and motility and indicate that PI3-kinase plays a selective role in the regulation of adhesion and ERM proteins redistribution in the plasma membrane of lymphocytes.

  1. The role of hypoxia inducible factor 1alpha in cobalt chloride induced cell death in mouse embryonic fibroblasts.

    PubMed

    Vengellur, A; LaPres, J J

    2004-12-01

    Cobalt has been widely used in the treatment of anemia and as a hypoxia mimic in cell culture and it is known to activate hypoxic signaling by stabilizing the hypoxia inducible transcription factor 1alpha (HIF1alpha). However, cobalt exposure can lead to tissue and cellular toxicity. These studies were conducted to determine the role of HIF1alpha in mediating cobalt-induced toxicity. Mouse embryonic fibroblasts (MEFs) that were null for the HIF1alpha protein were used to show that HIF1alpha protein plays a major role in mediating cobalt-induced cytotoxicity. Previous work from our lab and others has shown that two BH3 domain containing cell death genes, BNip3 and NIX, are targets of hypoxia signaling. These experiments document that BNip3 and NIX expression is HIF1alpha-dependent, and cobalt induces their expression in a time and dose dependent manner. In addition, their expression is correlated with an increase in BNIP3 and NIX protein. Characteristically, the elevated level of BNIP3 was correlated with an increased presence of chromatin condensation, one marker for cell injury. Interestingly, this increased chromosomal condensation was not coupled to caspase-3 activation as usually seen in a typical apoptotic response. These results show that HIF1alpha is playing a major role in mediating cobalt-induced toxicity in mouse embryonic fibroblasts and may offer a possible mechanism for the underlying pathology of injuries seen in workers exposed to environmental contaminants that can influence the hypoxia signaling system, such as cobalt.

  2. Ribozyme modulation of lipopolysaccharide-induced tumor necrosis factor-alpha production by peritoneal cells in vitro and in vivo.

    PubMed

    Sioud, M

    1996-05-01

    We have utilized synthetic ribozymes to modulate the lipopolysaccharide (LPS)-induced production of tumor necrosis factor-alpha (TNF-alpha) by peritoneal cells. Two hammerhead ribozymes (mRz1 and mRz2) were prepared by transcription in vitro and their activities in vitro and in vivo were investigated. Both ribozymes cleaved their RNA target with an apparent turnover number (kcat) of 2 min(-1), and inhibited TNF-alpha gene expression in vitro by 50% and 70%, respectively. When mRz1 and mRz2, entrapped in liposomes, were delivered into mice by intraperitoneal injection, they inhibited LPS-induced TNF-alpha gene expression in vivo with mRz2 being the most effective. This enhanced activity could result from the facilitation of catalysis by cellular endogenous proteins, since they specifically bind to mRz2 as compared to mRz1. Furthermore, a significant mRz2 activity can be recovered from peritoneal cells 2 days post-administration in vivo. The anti-TNF-alpha ribozyme treatment in vivo resulted in a more significant reduction of LPS-induced IFN-gamma protein secretion compared to IL-10. In contrast to this pleiotropic effect, the anti-TNF-alpha ribozyme treatment did not affect the heterogenous expression of Fas ligand by peritoneal cells, indicating the specificity of the treatment. Taken together, the present data indicate that the biological effects of TNF-alpha can be modulated by ribozymes. In addition, the data suggest that ribozymes can be administered in a drug-like manner, and therefore indicate their potential in clinical applications.

  3. An approach for producing transgenic cloned cows by nuclear transfer of cells transfected with human alpha 1-antitrypsin gene.

    PubMed

    Jang, Goo; Bhuiyan, M M U; Jeon, Hyun Yong; Ko, Kyeong Hee; Park, Hee Jung; Kim, Min Kyu; Kim, Joung Ju; Kang, Sung Keun; Lee, Byeong Chun; Hwang, Woo Suk

    2006-06-01

    In an attempt to produce transgenic cloned cows secreting alpha 1-antitrypsin (alpha1-AT) protein into milk, bovine cumulus cells were transfected with a plasmid containing an alpha1-AT gene and green fluorescent protein (GFP) reporter gene using Fugene 6 as a lipid carrier. The GFP-expressing cells were selected and transferred into enucleated bovine oocytes. Couplets were fused, chemically activated and cultured. Developmental competence was monitored and the number of inner cell mass (ICM) and trophectoderm (TE) cells in blastocysts were counted after differential staining. The percentage of blastocysts was lower (P < 0.05) in transgenic cloned embryos compared to non-transgenic cloned embryos (23% versus 35%). No difference in the numbers of ICM and TE cells between the two groups of embryos was observed. One or two GFP-expressing blastocysts were transferred into the uterus of each recipient cow. Out of 49 recipient cows, three pregnancies were detected by non-return estrus and rectal palpation. However, the pregnancies failed to maintain to term; two fetuses were aborted at Day 60 and 150, respectively, and one fetus at Day 240. The genomic DNA from the aborted fetus was amplified by polymerase chain reaction (PCR) to investigate integration of the transgene in the fetus. The expected PCR product was sequenced and was identical to the sequence of alpha1-AT transgene. In conclusion, the present study demonstrated that developmental competence of cloned embryos derived from transgenic donor cells was lower than embryos derived from non-transfected donor cells. Although we failed to obtain a viable transgenic cloned calf, integration of alpha1-AT gene into the fetus presents the possibility of producing transgenic cloned cows by somatic cell nuclear transfer.

  4. High glucose concentrations attenuate hypoxia-inducible factor-1{alpha} expression and signaling in non-tumor cells

    SciTech Connect

    Dehne, Nathalie; Bruene, Bernhard

    2010-04-15

    Hypoxia-inducible factor (HIF) is the major transcription factor mediating adaption to hypoxia e.g. by enhancing glycolysis. In tumor cells, high glucose concentrations are known to increase HIF-1{alpha} expression even under normoxia, presumably by enhancing the concentration of tricarboxylic acid cycle intermediates, while reactions of non-tumor cells are not well defined. Therefore, we analyzed cellular responses to different glucose concentrations in respect to HIF activation comparing tumor to non-tumor cells. Using cells derived from non-tumor origin, we show that HIF-1{alpha} accumulation was higher under low compared to high glucose concentrations. Low glucose allowed mRNA expression of HIF-1 target genes like adrenomedullin. Transfection of C{sub 2}C{sub 12} cells with a HIF-1{alpha} oxygen-dependent degradation domaine-GFP fusion protein revealed that prolyl hydroxylase (PHD) activity is impaired at low glucose concentrations, thus stabilizing the fusion protein. Mechanistic considerations suggested that neither O{sub 2} redistribution nor an altered redox state explains impaired PHD activity in the absence of glucose. In order to affect PHD activity, glucose needs to be metabolized. Amino acids present in the medium also diminished HIF-1{alpha} expression, while the addition of fatty acids did not. This suggests that glucose or amino acid metabolism increases oxoglutarate concentrations, which enhances PHD activity in non-tumor cells. Tumor cells deprived of glutamine showed HIF-1{alpha} accumulation in the absence of glucose, proposing that enhanced glutaminolysis observed in many tumors enables these cells to compensate reduced oxoglutarate production in the absence of glucose.

  5. Association of myosin I alpha with endosomes and lysosomes in mammalian cells.

    PubMed

    Raposo, G; Cordonnier, M N; Tenza, D; Menichi, B; Dürrbach, A; Louvard, D; Coudrier, E

    1999-05-01

    Myosin Is, which constitute a ubiquitous monomeric subclass of myosins with actin-based motor properties, are associated with plasma membrane and intracellular vesicles. Myosin Is have been proposed as key players for membrane trafficking in endocytosis or exocytosis. In the present paper we provide biochemical and immunoelectron microscopic evidence indicating that a pool of myosin I alpha (MMIalpha) is associated with endosomes and lysosomes. We show that the overproduction of MMIalpha or the production of nonfunctional truncated MMIalpha affects the distribution of the endocytic compartments. We also show that truncated brush border myosin I proteins, myosin Is that share 78% homology with MMIalpha, promote the dissociation of MMIalpha from vesicular membranes derived from endocytic compartments. The analysis at the ultrastructural level of cells producing these brush border myosin I truncated proteins shows that the delivery of the fluid phase markers from endosomes to lysosomes is impaired. MMIalpha might therefore be involved in membrane trafficking occurring between endosomes and lysosomes.

  6. Association of Myosin I Alpha with Endosomes and Lysosomes in Mammalian Cells

    PubMed Central

    Raposo, Graça; Cordonnier, Marie-Neige; Tenza, Danièle; Menichi, Bernadette; Dürrbach, Antoine; Louvard, Daniel; Coudrier, Evelyne

    1999-01-01

    Myosin Is, which constitute a ubiquitous monomeric subclass of myosins with actin-based motor properties, are associated with plasma membrane and intracellular vesicles. Myosin Is have been proposed as key players for membrane trafficking in endocytosis or exocytosis. In the present paper we provide biochemical and immunoelectron microscopic evidence indicating that a pool of myosin I alpha (MMIα) is associated with endosomes and lysosomes. We show that the overproduction of MMIα or the production of nonfunctional truncated MMIα affects the distribution of the endocytic compartments. We also show that truncated brush border myosin I proteins, myosin Is that share 78% homology with MMIα, promote the dissociation of MMIα from vesicular membranes derived from endocytic compartments. The analysis at the ultrastructural level of cells producing these brush border myosin I truncated proteins shows that the delivery of the fluid phase markers from endosomes to lysosomes is impaired. MMIα might therefore be involved in membrane trafficking occurring between endosomes and lysosomes. PMID:10233157

  7. Infection of Human Fallopian Tube Epithelial Cells with Neisseria gonorrhoeae Protects Cells from Tumor Necrosis Factor Alpha-Induced Apoptosis

    PubMed Central

    Morales, Priscilla; Reyes, Paz; Vargas, Macarena; Rios, Miguel; Imarai, Mónica; Cardenas, Hugo; Croxatto, Horacio; Orihuela, Pedro; Vargas, Renato; Fuhrer, Juan; Heckels, John E.; Christodoulides, Myron; Velasquez, Luis

    2006-01-01

    Following infection with Neisseria gonorrhoeae, bacteria may ascend into the Fallopian tubes (FT) and induce salpingitis, a major cause of infertility. In the FT, interactions between mucosal epithelial cells and gonococci are pivotal events in the pathogen's infection cycle and the inflammatory response. In the current study, primary FT epithelial cells were infected in vitro with different multiplicities of infection (MOI) of Pil+ Opa+ gonococci. Bacteria showed a dose-dependent association with cells and induced the secretion of tumor necrosis factor alpha (TNF-α). A significant finding was that gonococcal infection (MOI = 1) induced apoptosis in approximately 30% of cells, whereas increasing numbers of bacteria (MOI = 10 to 100) did not induce apoptosis. Apoptosis was observed in only 11% of cells with associated bacteria, whereas >84% of cells with no adherent bacteria were apoptotic. TNF-α was a key contributor to apoptosis, since (i) culture supernatants from cells infected with gonococci (MOI = 1) induced apoptosis in naïve cultures, suggesting that a soluble factor was responsible; (ii) gonococcal infection-induced apoptosis was inhibited with anti-TNF-α antibodies; and (iii) the addition of exogenous TNF-α induced apoptosis, which was inhibited by the presence of increasing numbers of bacteria (MOI = 10 to 100). These data suggest that TNF-α-mediated apoptosis of FT epithelial cells is likely a primary host defense mechanism to prevent pathogen colonization. However, epithelial cell-associated gonococci have evolved a mechanism to protect the cells from undergoing TNF-α-mediated apoptosis, and this modulation of the host innate response may contribute to establishment of infection. Understanding the antiapoptotic mechanisms used by Neisseria gonorrhoeae will inform the pathogenesis of salpingitis and could suggest new intervention strategies for prevention and treatment of the disease. PMID:16714596

  8. Pancreatic alpha-cells from female mice undergo morphofunctional changes during compensatory adaptations of the endocrine pancreas to diet-induced obesity.

    PubMed

    Merino, Beatriz; Alonso-Magdalena, Paloma; Lluesma, Mónica; Ñeco, Patricia; Gonzalez, Alejandro; Marroquí, Laura; García-Arévalo, Marta; Nadal, Angel; Quesada, Ivan

    2015-06-25

    Obesity is frequently associated with insulin resistance. To compensate for this situation and maintain normoglycaemia, pancreatic beta-cells undergo several morphofunctional adaptations, which result in insulin hypersecretion and hyperinsulinaemia. However, no information exists about pancreatic alpha-cells during this compensatory stage of obesity. Here, we studied alpha-cells in mice fed a high-fat diet (HFD) for 12 weeks. These animals exhibited hyperinsulinaemia and normoglycaemia compared with control animals in addition to hypoglucagonaemia. While the in vivo response of glucagon to hypoglycaemia was preserved in the obese mice, the suppression of glucagon secretion during hyperglycaemia was impaired. Additionally, in vitro glucagon release at low glucose levels and glucagon content in isolated islets were decreased, while alpha-cell exocytosis remained unchanged. Assessment of morphological parameters revealed that alpha-cell area was reduced in the pancreas of the obese mice in association with alpha-cell hypotrophy, increased apoptosis and decreased proliferation. HFD feeding for 24 weeks led to significant deterioration in beta-cell function and glucose homeostasis. Under these conditions, the majority of alpha-cell changes were reversed and became comparable to controls. These findings indicate that pancreatic compensatory adaptations during obesity may also involve pancreatic alpha-cells. Additionally, defects in alpha-cell function during obesity may be implicated in progression to diabetes.

  9. Alpha-chaconine, a potato glycoalkaloid, induces apoptosis of HT-29 human colon cancer cells through caspase-3 activation and inhibition of ERK 1/2 phosphorylation.

    PubMed

    Yang, Seun-Ah; Paek, Seung-Hwan; Kozukue, Nobuyuki; Lee, Kap-Rang; Kim, Jung-Ae

    2006-06-01

    Although alpha-chaconine, one of the two major potato trisaccharide glycoalkaloids, have shown cytotoxic effects on human cancer cells, the exact mechanism of this action of alpha-chaconine is not completely understood. In this study, we found that alpha-chaconine induced apoptosis of HT-29 cells in a time- and concentration-dependent manner by using flow cytometric analysis. We also found that caspase-3 activity and the active form of caspase-3 were increased 12 h after alpha-chaconine treatment. Caspase inhibitors, N-Ac-DEVD-CHO and Z-VAD-fmk, prevented alpha-chaconine-induced apoptosis, whereas alpha-chaconine-induced apoptosis was potentiated by PD98059, an extracellular signal-regulated kinase (ERK) inhibitor. However, pretreatment of the cells with LY294002 and SB203580, inhibitors of PI3K and p38, respectively, BAPTA-AM, an intracellular Ca(2+) chelator, and antioxidants such as N-acetylcysteine (NAC) and Trolox had no effect on the alpha-chaconine-induced cell death. In addition, phosphorylation of ERK was reduced by the treatment with alpha-chaconine. Moreover, alpha-chaconine-induced caspase-3 activity was further increased by the pretreatment with PD98059. Thus, the results indicate that alpha-chaconine induces apoptosis of HT-29 cells through inhibition of ERK and, in turn, activation of caspase-3.

  10. Integrin {alpha}{sub v}{beta}{sub 3} antagonist Cilengitide enhances efficacy of radiotherapy in endothelial cell and non-small-cell lung cancer models

    SciTech Connect

    Albert, Jeffrey M.; Cao, Carolyn; Geng, Ling; Leavitt, Lauren; Hallahan, Dennis E.; Lu Bo . E-mail: bo.lu@vanderbilt.edu

    2006-08-01

    Purpose: Integrins {alpha}{sub v}{beta}{sub 3} and {alpha}{sub v}{beta}{sub 5} are important in tumor growth and angiogenesis and have been recently explored as targets for cancer therapy. Radiotherapy also inhibits tumor growth and affects vasculature. We explored the combination of integrin antagonist Cilengitide (EMD 121974) and ionizing radiation. Methods and Materials: Levels of {alpha}{sub v}{beta}{sub 3} were determined for human umbilical vein endothelial cells (HUVEC), as well as H157 and H460 human non-small-cell lung cancer cells, using FACS analysis and immunofluorescence imaging. Clonogenic assays, Western immunoblots probed for cleaved caspase 3, and Annexin-V probing were used to evaluate cell survival and apoptosis. A cell detachment assay and matrigel assay were used to further examine the effects of treatment. Results: Human umbilical vein endothelial cells had the highest {alpha}{sub v}{beta}{sub 3} level, followed by H157, and H460. Interestingly, we found that 5 Gy irradiation induced expression of {alpha}{sub v}{beta}{sub 3} in all cell lines. Clonogenic assays showed a radiosensitizing effect with Cilengitide, and calculation of the dose enhancement ratio showed that the effect was highest in HUVECs (1.38), followed by H157 (1.19), and H460 (1.10), corresponding to the levels of target expression. There was an increase in apoptotic cells after combination treatment with Cilengitide and radiation, and there was an increase in detached cells after treatment with Cilengitide. Additionally, there was decreased endothelial tubule formation after combination treatment. Conclusions: We conclude that radiation induces expression of {alpha}{sub v}{beta}{sub 3} integrin in endothelial and non-small-cell lung cancer models, and that integrin antagonist Cilengitide is a radiosensitizer in proportion to the levels of target integrin expression.

  11. The human transforming growth factor type alpha coding sequence is not a direct-acting oncogene when overexpressed in NIH 3T3 cells.

    PubMed Central

    Finzi, E; Fleming, T; Segatto, O; Pennington, C Y; Bringman, T S; Derynck, R; Aaronson, S A

    1987-01-01

    A peptide secreted by some tumor cells in vitro imparts anchorage-independent growth to normal rat kidney (NRK) cells and has been termed transforming growth factor type alpha (TGF-alpha). To directly investigate the transforming properties of this factor, the human sequence coding for TGF-alpha was placed under the control of either a metallothionein promoter or a retroviral long terminal repeat. These constructs failed to induce morphological transformation upon transfection of NIH 3T3 cells, whereas viral oncogenes encoding a truncated form of its cognate receptor, the EGF receptor, or another growth factor, sis/platelet-derived growth factor 2, efficiently induced transformed foci. When NIH 3T3 clonal sublines were selected by transfection of TGF-alpha expression vectors in the presence of a dominant selectable marker, they were shown to secrete large amounts of TGF-alpha into the medium, to have downregulated EGF receptors, and to be inhibited in growth by TGF-alpha monoclonal antibody. These results indicated that secreted TGF-alpha interacts with its receptor at a cell surface location. Single cell-derived TGF-alpha-expressing sublines grew to high saturation density in culture. However, when plated as single cells on contact-inhibited monolayers of NIH 3T3 cells, they failed to form colonies, whereas v-sis- and v-erbB-transfected cells formed transformed colonies under the same conditions. Moreover, TGF-alpha-expressing sublines were not tumorigenic in nude mice. These and other results imply that TGF-alpha exerts a growth-promoting effect on the entire NIH 3T3 cell population after secretion into the medium but little, if any, effect on the individual cell synthesizing this factor. It is concluded that the normal coding sequence for TGF-alpha is not a direct-acting oncogene when overexpressed in NIH 3T3 cells. Images PMID:3035551

  12. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) induces initiation factor 2 alpha phosphorylation and translation inhibition in PC12 cells.

    PubMed

    Muñoz, F; Martín, M E; Salinas, M; Fando, J L

    2001-03-09

    We have investigated the effect of the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) on protein synthesis rate and initiation factor 2 (eIF2) phosphorylation in PC12 cells differentiated with nerve growth factor. FCCP treatment induced a very rapid 2-fold increase in intracellular Ca(2+) concentration that was accompanied by a strong protein synthesis rate inhibition (68%). The translation inhibition correlated with an increased phosphorylation of the alpha subunit of eIF2 (eIF2 alpha) (25% vs. 7%, for FCCP-treated and control cells, respectively) and a 1.7-fold increase in the double-stranded RNA-dependent protein kinase activity. No changes in the PKR endoplasmic reticulum-related kinase or eIF2 alpha phosphatase were found. Translational regulation may play a significant role in the process triggered by mitochondrial calcium mobilization.

  13. A Stress-Induced Small RNA Modulates Alpha-Rhizobial Cell Cycle Progression

    PubMed Central

    Robledo, Marta; Frage, Benjamin; Wright, Patrick R.; Becker, Anke

    2015-01-01

    Mechanisms adjusting replication initiation and cell cycle progression in response to environmental conditions are crucial for microbial survival. Functional characterization of the trans-encoded small non-coding RNA (trans-sRNA) EcpR1 in the plant-symbiotic alpha-proteobacterium Sinorhizobium meliloti revealed a role of this class of riboregulators in modulation of cell cycle regulation. EcpR1 is broadly conserved in at least five families of the Rhizobiales and is predicted to form a stable structure with two defined stem-loop domains. In S. meliloti, this trans-sRNA is encoded downstream of the divK-pleD operon. ecpR1 belongs to the stringent response regulon, and its expression was induced by various stress factors and in stationary phase. Induced EcpR1 overproduction led to cell elongation and increased DNA content, while deletion of ecpR1 resulted in reduced competitiveness. Computationally predicted EcpR1 targets were enriched with cell cycle-related mRNAs. Post-transcriptional repression of the cell cycle key regulatory genes gcrA and dnaA mediated by mRNA base-pairing with the strongly conserved loop 1 of EcpR1 was experimentally confirmed by two-plasmid differential gene expression assays and compensatory changes in sRNA and mRNA. Evidence is presented for EcpR1 promoting RNase E-dependent degradation of the dnaA mRNA. We propose that EcpR1 contributes to modulation of cell cycle regulation under detrimental conditions. PMID:25923724

  14. Nicotine promotes cell proliferation via {alpha}7-nicotinic acetylcholine receptor and catecholamine-synthesizing enzymes-mediated pathway in human colon adenocarcinoma HT-29 cells

    SciTech Connect

    Wong, Helen Pui Shan; Yu Le; Lam, Emily Kai Yee; Tai, Emily Kin Ki; Wu, William Ka Kei; Cho, Chi Hin . E-mail: chcho@cuhk.edu.hk

    2007-06-15

    Cigarette smoking has been implicated in colon cancer. Nicotine is a major alkaloid in cigarette smoke. In the present study, we showed that nicotine stimulated HT-29 cell proliferation and adrenaline production in a dose-dependent manner. The stimulatory action of nicotine was reversed by atenolol and ICI 118,551, a {beta}{sub 1}- and {beta}{sub 2}-selective antagonist, respectively, suggesting the role of {beta}-adrenoceptors in mediating the action. Nicotine also significantly upregulated the expression of the catecholamine-synthesizing enzymes [tyrosine hydroxylase (TH), dopamine-{beta}-hydroxylase (D{beta}H) and phenylethanolamine N-methyltransferase]. Inhibitor of TH, a rate-limiting enzyme in the catecholamine-biosynthesis pathway, reduced the actions of nicotine on cell proliferation and adrenaline production. Expression of {alpha}7-nicotinic acetylcholine receptor ({alpha}7-nAChR) was demonstrated in HT-29 cells. Methyllycaconitine, an {alpha}7-nAChR antagonist, reversed the stimulatory actions of nicotine on cell proliferation, TH and D{beta}H expression as well as adrenaline production. Taken together, through the action on {alpha}7-nAChR nicotine stimulates HT-29 cell proliferation via the upregulation of the catecholamine-synthesis pathway and ultimately adrenaline production and {beta}-adrenergic activation. These data reveal the contributory role {alpha}7-nAChR and {beta}-adrenoceptors in the tumorigenesis of colon cancer cells and partly elucidate the carcinogenic action of cigarette smoke on colon cancer.

  15. Inhibition of MAP kinase promotes the recruitment of corepressor SMRT by tamoxifen-bound estrogen receptor alpha and potentiates tamoxifen action in MCF-7 cells

    SciTech Connect

    Hong, Wei; Chen, Linfeng; Li, Juan; Yao, Zhi

    2010-05-28

    Estrogen receptor alpha (ER{alpha}), a ligand controlled transcription factor, plays an important role in breast cancer growth and endocrine therapy. Tamoxifen (TAM) antagonizes ER{alpha} activity and has been applied in breast cancer treatment. TAM-bound ER{alpha} associates with nuclear receptor-corepressors. Mitogen-activated protein kinase (MAPK) has been elucidated to result in cross-talk between growth factor and ER{alpha} mediated signaling. We show that activated MAPK represses interaction of TAM-bound ER{alpha} with silencing mediator for retinoid and thyroid hormone receptors (SMRT) and inhibits the recruitment of SMRT by ER{alpha} to certain estrogen target genes. Blockade of MAPK signaling cascade with MEK inhibitor U0126 promotes the interaction and subsequently inhibits ER{alpha} activity via enhanced recruitment of SMRT, leading to reduced expression of ER{alpha} target genes. The growth rate of MCF-7 cells was decelerated when treated with both TAM and U0126. Moreover, the growth of MCF-7 cells stably expressing SMRT showed a robust repression in the presence of TAM and U0126. These results suggest that activated MAPK signaling cascade attenuates antagonist-induced recruitment of SMRT to ER{alpha}, suggesting corepressor mediates inhibition of ER{alpha} transactivation and breast cancer cell growth by antagonist. Taken together, our finding indicates combination of antagonist and MAPK inhibitor could be a helpful approach for breast cancer therapy.

  16. Brucella suis prevents human dendritic cell maturation and antigen presentation through regulation of tumor necrosis factor alpha secretion.

    PubMed

    Billard, Elisabeth; Dornand, Jacques; Gross, Antoine

    2007-10-01

    Brucella is a facultative intracellular pathogen and the etiological agent of brucellosis. In some cases, human brucellosis results in a persistent infection that may reactivate years after the initial exposure. The mechanisms by which the parasite evades clearance by the immune response to chronically infect its host are unknown. We recently demonstrated that dendritic cells (DCs), which are critical components of adaptive immunity, are highly susceptible to Brucella infection and are a preferential niche for the development of the bacteria. Here, we report that in contrast to several intracellular bacteria, Brucella prevented the infected DCs from engaging in their maturation process and impaired their capacities to present antigen to naïve T cells and to secrete interleukin-12. Moreover, Brucella-infected DCs failed to release tumor necrosis factor alpha (TNF-alpha), a defect involving the bacterial protein Omp25. Exogenous TNF-alpha addition to Brucella-infected DCs restored cell maturation and allowed them to present antigens. Two avirulent mutants of B. suis, B. suis bvrR and B. suis omp25 mutants, which do not express the Omp25 protein, triggered TNF-alpha production upon DC invasion. Cells infected with these mutants subsequently matured and acquired the ability to present antigens, two properties which were dramatically impaired by addition of anti-TNF-alpha antibodies. In light of these data, we propose a model in which virulent Brucella alters the maturation and functions of DCs through Omp25-dependent control of TNF-alpha production. This model defines a specific evasion strategy of the bacteria by which they can escape the immune response to chronically infect their host.

  17. Sustained expression of alpha1-antitrypsin after transplantation of manipulated hematopoietic stem cells.

    PubMed

    Wilson, Andrew A; Kwok, Letty W; Hovav, Avi-Hai; Ohle, Sarah J; Little, Frederic F; Fine, Alan; Kotton, Darrell N

    2008-08-01

    Inherited mutations in the human alpha(1)-antitrypsin (AAT) gene lead to deficient circulating levels of AAT protein and a predisposition to developing emphysema. Gene therapy for individuals deficient in AAT is an attractive goal, because transfer of a normal AAT gene into any cell type able to secrete AAT should reverse deficient AAT levels and attenuate progression of lung disease. Here we present an approach for AAT gene transfer based on the transplantation of lentivirally transduced hematopoietic stem cells (HSCs). We develop a novel dual-promoter lentiviral system to transfer normal human AAT cDNA as well as a fluorescent tracking "reporter gene" into murine HSCs. After transplantation of 3,000 transduced HSCs into irradiated mouse recipients, we demonstrate simultaneous and sustained systemic expression of both genes in vivo for at least 31 weeks. The stem cells transduced with this protocol maintain multipotency, self-renewal potential, and the ability to reconstitute the hematopoietic systems of both primary and secondary recipients. This lentiviral-based system may be useful for investigations requiring the systemic secretion of anti-proteases or cytokines relevant to the pathogenesis of a variety of lung diseases.

  18. Overexpression of Rho GDP-dissociation inhibitor alpha predicts poor survival in oral squamous cell carcinoma.

    PubMed

    Chiang, Wei-Fan; Ho, Hsu-Chueh; Chang, Hua-Yuan; Chiu, Chien-Chih; Chen, Yuh-Ling; Hour, Tzyh-Chyuan; Chuang, Shu-Ju; Wu, Yu-Jen; Chen, Hau-Ren; Chen, Jen-Hao; Liu, Shyun-Yeu; Lu, Chin-Li; Chen, Jeff Yi-Fu

    2011-06-01

    Oral cancer has emerged as one of the fastest growing malignancies in Taiwan. However, biomarkers that reliably predict clinical outcomes have yet to be identified. This study was aimed to identify tumor-associated proteins that could be prognostic biomarkers for oral cancer. We compared the protein expression between oral squamous cell carcinoma (OSCC) tissues and adjacent non-cancerous matched tissues (NCMTs) by proteomics. We found that Rho GDP-dissociation inhibitor alpha (RhoGDIα) was differentially expressed in frozen cancerous samples and OSCC cell lines but not in NCMTs. Furthermore, our results indicated that RhoGDIα was selectively upregulated in 78 OSCC tissue sections (p<0.001), and this high expression was significantly correlated with increased tumor size (p<0.05) and poor overall survival (p<0.01). There was a trend that RhoGDIα expression was localized in the cytoplasm of cancer cells but was localized in the plasma membrane of NCMTs. Finally, expression of RhoGDIα was validated to be an independent prognostic indicator for overall survival (p<0.01). These results have identified a novel biomarker that may be useful for prediction of poor prognosis in OSCC patients.

  19. Alpha-smooth muscle actin expression and structure integrity in chondrogenesis of human mesenchymal stem cells.

    PubMed

    Hung, Shih-Chieh; Kuo, Pei-Yin; Chang, Ching-Fang; Chen, Tain-Hsiung; Ho, Larry Low-Tone

    2006-06-01

    The expression of alpha-smooth muscle actin (SMA) by human mesenchymal stem cells (hMSCs) during chondrogenesis was investigated by the use of pellet culture. Undifferentiated hMSCs expressed low but detectable amounts of SMA and the addition of transforming growth factor beta1 (TGF-beta1) to the culture medium increased SMA expression in a dose-dependent manner. Differentiation in pellet culture was rapidly induced in the presence of TGF-beta1 and was accompanied by the development of annular layers at the surface of the pellet. These peripheral layers lacked expression of glycosaminoglycan and type II collagen during early differentiation. Progress in differentiation increased the synthesis of glycosaminoglycan and type II collagen and the expression of SMA in these layers. Double-staining for type II collagen and SMA by immunofluorescence demonstrated the differentiation of hMSCs into cells positive for these two proteins. The addition of cytochalasin D, a potent inhibitor of the polymerization of actin microfilaments, caused damage to the structural integrity and surface smoothness of the chondrogenic pellets. The SMA-positive cells in the peripheral layers of the chondrogenic pellets mimic those within the superficial layer of articular cartilage and are speculated to play a major role in cartilage development and maintenance.

  20. Proteomic Studies of Nitrated Alpha-Synuclein Microglia Regulation by CD4+CD25+ T Cells

    PubMed Central

    Reynolds, Ashley D.; Stone, David K.; Mosley, R. Lee; Gendelman, Howard E.

    2009-01-01

    Microglial inflammatory responses affect Parkinson's disease (PD) associated nigrostriatal degeneration. This is triggered, in measure, by misfolded, nitrated alpha-synuclein (N-α-syn) contained within Lewy bodies that are released from dying or dead dopaminergic neurons into the extravascular space. N-α-syn-stimulated microglial immunity is regulated by CD4+ T cells. Indeed, CD4+CD25+regulatory T cells (Treg) induce neuroprotective immune responses. This is seen in rodent models of stroke, amyotrophic lateral sclerosis, human immunodeficiency virus associated dementia, and PD. To elucidate the mechanism for Treg-mediated microglial responses, we used a proteomic platform integrating difference gel electrophoresis and tandem mass spectrometry peptide sequencing. These tests served to determine the consequences of Treg on the N-α-syn stimulated microglia. The data demonstrated that Treg substantially alter the microglial proteome in response to N-α-syn. This is seen through Treg's abilities to suppress microglial neurotoxic proteins linked to cell metabolism, migration, protein transport and degradation, redox biology, cytoskeletal, and bioenergetic activities. We conclude that Treg modulate the N-α-syn microglial proteome and, in this way, can slow the tempo and course of PD. PMID:19432400

  1. Metabolism of 17alpha-ethynylestradiol by intact liver parenchymal cells isolated from mouse and rat.

    PubMed

    Helton, E D; Casciano, D A; Althaus, Z R; Plant, H D

    1977-12-01

    Liver parenchymal cells isolated by perfusion from female C3H/HeN-MTV+Nctr mice and Sprague-Dawley rats were incubated with [6,7-3H] 17alpha-ethynylestradiol (EE2). The incubates were individually fractionated into free steroid (organic phase), steroid conjugates (aqueous), and bound steroids (macromolecular pellet). The rat had significantly less total free radioactive steroid but significantly more total conjugated and irreversibly bound radioactivity than the mouse. However, when the metabolic conversion of EE2 was compared in the rat and the mouse on a cellular basis (metabolic clearance per 10(6) cells), the rat was found to be less efficient than the mouse. The two species were essentially equivalent in their covalent binding when expressed on a per 10(6) cell basis. Purification of the free radiolabeled steriods on LH-20 demonstrated the mouse to have the parent compound and on identifiable 2-OH-EE2 fraction. The rat had EE2 and an identifiable 2-methoxy-EE2 fraction. A major metabolite fraction for both species was very nonpolar and, although not identified, was found to be ethynylated as demonstrated by silver-sulfoethylcellulose chromatography. The conjugate fractions of the mouse were indicative of glucuronide conjugation, whereas the rat had additional conjugate fractions suggestive of sulfoconjugation.

  2. In liver fibrosis, dendritic cells govern hepatic inflammation in mice via TNF-alpha.

    PubMed

    Connolly, Michael K; Bedrosian, Andrea S; Mallen-St Clair, Jon; Mitchell, Aaron P; Ibrahim, Junaid; Stroud, Andrea; Pachter, H Leon; Bar-Sagi, Dafna; Frey, Alan B; Miller, George

    2009-11-01

    Hepatic fibrosis occurs during most chronic liver diseases and is driven by inflammatory responses to injured tissue. Because DCs are central to modulating liver immunity, we postulated that altered DC function contributes to immunologic changes in hepatic fibrosis and affects the pathologic inflammatory milieu within the fibrotic liver. Using mouse models, we determined the contribution of DCs to altered hepatic immunity in fibrosis and investigated the role of DCs in modulating the inflammatory environment within the fibrotic liver. We found that DC depletion completely abrogated the elevated levels of many inflammatory mediators that are produced in the fibrotic liver. DCs represented approximately 25% of the fibrotic hepatic leukocytes and showed an elevated CD11b+CD8- fraction, a lower B220+ plasmacytoid fraction, and increased expression of MHC II and CD40. Moreover, after liver injury, DCs gained a marked capacity to induce hepatic stellate cells, NK cells, and T cells to mediate inflammation, proliferation, and production of potent immune responses. The proinflammatory and immunogenic effects of fibrotic DCs were contingent on their production of TNF-alpha. Therefore, modulating DC function may be an attractive approach to experimental therapeutics in fibro-inflammatory liver disease.

  3. Galectin-1 suppresses alpha2(I) collagen through Smad3 in renal epithelial cells.

    PubMed

    Okano, K; Uchida, K; Nitta, K; Hayashida, T

    2008-10-01

    Transforming growth factor (TGF-beta1) promotes renal fibrogenesis through activation of Smads. Galectin-1 is reported to prevent experimental glomerulonephritis. Here we investigated the fact that transfected galectin-1 significantly suppressed the transcription of alpha2(I) collagen (COL1A2) in TGF-beta1- activated human renal epithelial cells. Conversely, galectin-1 silencing RNA reduced secretion of type I collagen by HKC cells. Galectin-1 significantly decreased activation of a TGF-beta1-responsive reporter construct and of a minimal reporter construct that contains four repeats of the Smad binding element (SBE). Galectin-1 had no effect on phosphorylation of Smad3 at the linker region and C-terminus, whereas it decreased affinity of Smad3 to the SBE. Additionally, the inhibitory effect of galectin-1 disappeared using a mutated reporter construct, 376 m-LUC, in which a potential Smad recognition site within the promoter is mutated. Taken together, the results suggest that galectin-1 decreases Smad3-complex from binding to the SBE, down-regulating transcription of COL1A2 in TGF-beta1-stimulated renal epithelial cells.

  4. Analysis of the activation state of alpha4beta1 integrin in human B cell lines derived from myeloma, leukemia or lymphoma.

    PubMed

    García-Gila, M; Cabañas, C; García-Pardo, A

    1997-12-01

    Myeloma cells specifically localize in the bone marrow and rarely circulate in blood. To study whether this immobilization could be partially explained by the presence of constitutively activated integrins, particularly alpha4beta1, we used the activation reporter HUTS-21 anti-beta1 mAb. These analyses showed that beta1 integrins on myeloma cells were moderately active and could be upregulated similarly to integrins on lymphoma or leukemia cells. Myeloma cells were also tested for their ability to attach to RGD-containing fibronectin fragments, a property of activated (but not resting) alpha4beta1. Two cell lines adhered to these fragments and this was inhibited by anti-alpha5 but not by anti-alpha4 mAbs. These results show that myeloma cells bear low/moderately active alpha4beta1 and support the notion that multiple interactions contribute to their localization in the bone marrow.

  5. Hepatitis B Virus X Protein Driven Alpha Fetoprotein Expression to Promote Malignant Behaviors of Normal Liver Cells and Hepatoma Cells

    PubMed Central

    Zhu, Mingyue; Lu, Yan; Li, Wei; Guo, Junli; Dong, Xu; Lin, Bo; Chen, Yi; Xie, Xieju; Li, Mengsen

    2016-01-01

    Background: The infection of Hepatitis B virus (HBV) is closely associated with the development of hepatocellular carcinoma(HCC), HBV-X protein(HBx) is able to induce expression of alpha-fetoprotein(AFP) in normal liver cells, and AFP harbors a function to promote malignant transformation of normal liver cells, but the role AFP playing in malignant behaviors of HCC cells is still unclear. Methods: Fifty-six liver tissue samples were collected from the clinical patients through hepatectomy(include normal liver tissues, HBV-related hepatitis liver tissues and HBV-related HCC tissues), and diagnosis of these tissues by pathology section, expression of AFP, Ras and CXCR4 were evidenced by immunohisochemical staining and Western blotting; The proliferation of human normal liver cells line L-02 cells and human hepatoma cells line, HLE cells(non AFP-producing) were performed by MTT method; Repaired capacity of L-02 and HLE cells were compared by wound healing assay; Migration and invasion of these cells were analyzed by Transwell chamber assay; HBx expressed vectors(pcDNA3.1-HBx) were constructed and transfected into L-02 and HLE cells, effects of pcDNA3.1-HBx on the malignant behaviors were also detected by MTT, Transwell chamber assay and the expression of AFP, Ras and CXCR4 were evidenced by Western blotting. Results: we found that expression of AFP, Ras and CXCR4 in HBV-related HCC and lymph nodes metastasis tissues were significantly elevated compared with HBV-related HCC, non metastasis tissues and HBV-related hepatitis tissues; Expression of AFP, Ras and CXCR4 in HBV-related hepatitis tissues were significantly enhanced compared with normal liver tissues; The growth ratio, migratory and invasive ability, expression of AFP, Ras and CXCR4 of the cells were outstanding promoted while L-02 and HLE cells were transfected with pcDNA3.1-HBx vectors. The proliferation ratio, migration and invasion ability, and expression of Ras and CXCR4 were significantly inhibited while

  6. alpha-Melanocyte-stimulating hormone, MSH 11-13 KPV and adrenocorticotropic hormone signalling in human keratinocyte cells.

    PubMed

    Elliott, Richard J; Szabo, Marika; Wagner, Mark J; Kemp, E Helen; MacNeil, Sheila; Haycock, John W

    2004-04-01

    alpha-MSH signals by binding to the melanocortin-1 receptor (MC-1R) and elevating cyclic AMP in several different cells. The anti-inflammatory properties of this peptide are also believed to be cyclic AMP dependent. The carboxyl terminal tripeptides of alpha-MSH (KPV / KP-D-V) are the smallest minimal sequences reported to prevent inflammation but it is not known if they operate via MC-1R or cyclic AMP. The aim of this study was to examine the intracellular signalling of key MSH and ACTH peptides in human keratinotocytes. No elevation in cyclic AMP was detected in either HaCaT or normal human keratinocytes in response to alpha-MSH, KPV or ACTH peptides. Rapid and acute intracellular calcium, however, were observed in HaCaT keratinocytes in response to alpha-MSH (10(-15)-10(-7) M), KPV (10(-15)-10(-7) M), KP-D-V (10(-15)-10(-7) M) and ACTH (10(-15)-10(-7) M), but only in the presence of PIA, an adenosine agonist that inhibits the cyclic AMP pathway. Normal keratinocytes responded to all the above peptides but in addition responded to ACTH 1-17 (10(-13)-10(-7) M) in contrast to the HaCaT keratinocytes. Stable transfection of Chinese hamster ovary cells with the MC-1 receptor showed that alpha-MSH and the KPV peptides elevated intracellular calcium.

  7. RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND THE HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY

    EPA Science Inventory

    RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY.
    MC Cardon, PC Hartig,LE Gray, Jr. and VS Wilson.
    U.S. EPA, ORD, NHEERL, RTD, Research Triangle Park, NC, USA.
    Typically, in vitro hazard assessments for ...

  8. RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND THE HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY

    EPA Science Inventory

    Rainbow Trout Androgen Receptor Alpha And Human Androgen Receptor: Comparisons in the COS Whole Cell Binding Assay
    Mary C. Cardon, L. Earl Gray, Jr. and Vickie S. Wilson
    U.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology Division, Research Triangle...

  9. 5'-nitro-indirubinoxime inhibits inflammatory response in TNF-alpha stimulated human umbilical vein endothelial cells.

    PubMed

    Kim, Eun-Jung; Park, Won-Hwan; Ahn, Sang-Gun; Yoon, Jung-Hoon; Kim, Si-Wouk; Kim, Soo-A

    2010-07-01

    Inflammation plays a critical role in the development of atherosclerosis and TNF-alpha, a major inflammatory cytokine, induces inflammatory responses by enhancing the expression of adhesion molecules and the secretion of inflammatory mediators. Indirubin is an active compound of Polygonum tinctorium Lour (P. tinctorium) that has the ability to suppress inflammation. Previously, we described the novel indirubin derivative, 5'-nitro-indirubinoxime (5'-NIO), and demonstrated that it has potent anti-proliferative activity against various human cancer cells. In this study, we examined the effect of 5'-NIO on the TNF-alpha induced inflammatory conditions of human umbilical vein endothelial cells (HUVECs). We found that 5'-NIO inhibited TNF-alpha induced MCP-1 and IL-8 expression at the RNA and protein levels in HUVECs. Specifically, 5'-NIO significantly inhibited the TNF-alpha stimulated release of MCP-1 and IL-8, with levels that were only 19.8% and 30.9% of those of untreated control cells, respectively. Furthermore, 5'-NIO largely inhibited the adhesion of U937 cells to HUVECs by decreasing the expression level of ICAM-1 and VCAM-1. Overall, these observations suggest that 5'-NIO has the potential for use as an anti-atherosclerotic agent.

  10. BINDING OF STEROIDS AND ENVIRONMENTAL CHEMICALS TO THE RAINBOW TROUT ANDROGEN RECEPTOR ALPHA EXPRESSED IN COS CELLS

    EPA Science Inventory

    Binding of Steroids and Environmental Chemicals to the Rainbow Trout Androgen Receptor Alpha Expressed in COS Cells.

    Mary C. Cardon, L. Earl Gray. Jr., Phillip C. Hartig and Vickie S. Wilson
    U.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology...

  11. Crossreactive T Cells Spotlight the Germline Rules for [alpha beta] T Cell-Receptor Interactions with MHC Molecules

    SciTech Connect

    Dai, Shaodong; Huseby, Eric S.; Rubtsova, Kira; Scott-Browne, James; Crawford, Frances; Macdonald, Whitney A.; Marrack, Philippa; Kappler, John W.

    2008-10-31

    To test whether highly crossreactive {alpha}{beta} T cell receptors (TCRs) produced during limited negative selection best illustrate evolutionarily conserved interactions between TCR and major histocompatibility complex (MHC) molecules, we solved the structures of three TCRs bound to the same MHC II peptide (IA{sup b}-3K). The TCRs had similar affinities for IA{sup b}-3K but varied from noncrossreactive to extremely crossreactive with other peptides and MHCs. Crossreactivity correlated with a shrinking, increasingly hydrophobic TCR-ligand interface, involving fewer TCR amino acids. A few CDR1 and CDR2 amino acids dominated the most crossreactive TCR interface with MHC, including V{beta}8 48Y and 54E and V{alpha}4 29Y, arranged to impose the familiar diagonal orientation of TCR on MHC. These interactions contribute to MHC binding by other TCRs using related V regions, but not usually so dominantly. These data show that crossreactive TCRs can spotlight the evolutionarily conserved features of TCR-MHC interactions and that these interactions impose the diagonal docking of TCRs on MHC.

  12. Reduced interferon-alpha production by Epstein-Barr virus transformed B-lymphoblastoid cell lines and lectin-stimulated lymphocytes in congenital dyserythropoietic anaemia type I.

    PubMed

    Wickramasinghe, S N; Hasan, R; Smythe, J

    1997-08-01

    The concentrations of interferon-alpha (IFN-alpha) in supernatants from cultures of Epstein-Barr virus (EBV) transformed B-lymphoblastoid cell lines derived from seven patients with congenital dyserythropoietic anaemia (CDA) type I were below the 95% confidence limits for those derived from six healthy subjects. In contrast, the concentrations of IFN-alpha in supernatants from cultures of EBV-transformed lymphoblastoid cell lines derived from four patients with other types of CDA and four patients with hereditary sideroblastic anaemia were normal. Supernatants from cultures of peripheral blood lymphocytes stimulated with phytohaemagglutinin or pokeweed mitogen contained less IFN-alpha when the cells were derived from patients with CDA type I than when derived from healthy subjects. Since patients with CDA type I show a substantial haematological response to treatment with IFN-alpha, the data suggest that impaired IFN-alpha production may be an important pathogenetic mechanism in CDA type I.

  13. The CD8alpha(+) dendritic cell is responsible for inducing peripheral self-tolerance to tissue-associated antigens.

    PubMed

    Belz, Gabrielle T; Behrens, Georg M N; Smith, Chris M; Miller, Jacques F A P; Jones, Claerwen; Lejon, Kristina; Fathman, C Garrison; Mueller, Scott N; Shortman, Ken; Carbone, Francis R; Heath, William R

    2002-10-21

    We previously described a mechanism for the maintenance of peripheral self-tolerance. This involves the cross-presentation of tissue-associated antigens by a bone marrow-derived cell type that stimulates the proliferation and ultimate deletion of self-reactive CD8 T cells. This process has been referred to as cross-tolerance. Here, we characterize the elusive cell type responsible for inducing cross-tolerance as a CD8alpha(+) dendritic cell (DC). To achieve this aim, transgenic mice were generated expressing yellow fluorescent protein (YFP) linked to CTL epitopes for ovalbumin and glycoprotein B (gB) of herpes simplex virus under the rat insulin promoter (RIP). Although tracking of YFP was inconclusive, the use of a highly sensitive gB-specific hybridoma that produced beta-galactosidase on encounter with antigen, enabled detection of antigen presentation by cells isolated from the pancreatic lymph node. This showed that a CD11c(+)CD8alpha(+) cell was responsible for cross-tolerance, the same DC subset as previously implicated in cross-priming. These data indicate that CD8alpha(+) DCs play a critical role in both tolerance and immunity to cell-associated antigens, providing a potential mechanism by which cytotoxic T lymphocyte can be immunized to viral antigens while maintaining tolerance to self.

  14. Effect of recombinant human interferon-alpha A/D on in vivo murine tumor cell growth.

    PubMed

    Uno, K; Shimizu, S; Inaba, K; Kitaura, M; Nakahira, K; Kato, T; Yamaguchi, Y; Muramatsu, S

    1988-05-01

    We investigated the effect of human recombinant interferon-alpha A/D A/D-IFN), which is known to delay the growth of murine tumor cells, on the growth of S1 and R1 subline cells of murine Meth A fibrosarcoma in the peritoneal cavity of mice. In vitro growth of S1 cells was sensitive to, and that of R1 cells was resistant to, the direct effect of A/D-IFN, as with murine natural IFN-alpha/beta, which was used originally to isolate these sublines. In vivo, however, the growth of not only S1 cells but also R1 cells was suppressed by the administration of A/D-IFN, and the survival time of tumor-bearing mice was prolonged. Although A/D-IFN had a direct effect on S1 cells in vivo, R1 cells were susceptible only to the indirect effect via the host cells. Macrophages (M phi) harvested from the peritoneal cavity of A/D-IFN-treated mice bearing ascitic R1 cells were very effective in suppressing the in vitro growth of R1 cells; those from non-R1-bearing A/D-IFN-treated mice were less effective. The results of in vitro experiments indicate that M phi are very probably activated by the synergism of A/D-IFN and M phi diameter-activating factor(s) produced by lymphoid cells in tumor-bearing mice.

  15. Alpha-bungarotoxin binding to target cell in a developing visual system by carboxylated nanodiamond

    NASA Astrophysics Data System (ADS)

    Liu, Kuang-Kai; Chen, Mei-Fang; Chen, Po-Yi; Lee, Tony J. F.; Cheng, Chia-Liang; Chang, Chia-Ching; Ho, Yen-Peng; Chao, Jui-I.

    2008-05-01

    Biological molecules conjugating with nanoparticles are valuable for applications including bio-imaging, bio-detection, and bio-sensing. Nanometer-sized diamond particles have excellent electronic and chemical properties for bio-conjugation. In this study, we manipulated the carboxyl group produced on the surface of nanodiamond (carboxylated nanodiamond, cND) for conjugating with alpha-bungarotoxin (α-BTX), a neurotoxin derived from Bungarus multicinctus with specific blockade of alpha7-nicotinic acetylcholine receptor (α7-nAChR). The electrostatic binding of cND-α-BTX was mediated by the negative charge of the cND and the positive charge of the α-BTX in physiological pH conditions. Sodium dodecyl sulfate-polyacrylamide gel analysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS) spectra displayed that α-BTX proteins were conjugated with cND particles via non-covalent bindings. The green fluorescence of the cND particles combining with the red fluorescence of tetramethylrhodamine-labeled α-BTX presented a yellow color at the same location, which indicated that α-BTX proteins were conjugated with cND particles. Xenopus laevis's oocytes expressed the human α7-nAChR proteins by microinjection with α7-nAChR mRNA. The cND-α-BTX complexes were bound to α7-nAChR locating on the cell membrane of oocytes and human lung A549 cancer cells analyzed by laser scanning confocal microscopy. The choline-evoked α7-nAChR-mediated inward currents of the oocytes were blocked by cND-α-BTX complexes in a concentration-dependent manner using two-electrode voltage-clamp recording. Furthermore, the fluorescence intensity of cND-α-BTX binding on A549 cells could be quantified by flow cytometry. These results indicate that cND-conjugated α-BTX still preserves its biological activity in blocking the function of α7-nAChR, and provide a visual system showing the binding of α-BTX to α7-nAChR.

  16. Alpha-bungarotoxin binding to target cell in a developing visual system by carboxylated nanodiamond.

    PubMed

    Liu, Kuang-Kai; Chen, Mei-Fang; Chen, Po-Yi; Lee, Tony J F; Cheng, Chia-Liang; Chang, Chia-Ching; Ho, Yen-Peng; Chao, Jui-I

    2008-05-21

    Biological molecules conjugating with nanoparticles are valuable for applications including bio-imaging, bio-detection, and bio-sensing. Nanometer-sized diamond particles have excellent electronic and chemical properties for bio-conjugation. In this study, we manipulated the carboxyl group produced on the surface of nanodiamond (carboxylated nanodiamond, cND) for conjugating with alpha-bungarotoxin (α-BTX), a neurotoxin derived from Bungarus multicinctus with specific blockade of alpha7-nicotinic acetylcholine receptor (α7-nAChR). The electrostatic binding of cND-α-BTX was mediated by the negative charge of the cND and the positive charge of the α-BTX in physiological pH conditions. Sodium dodecyl sulfate-polyacrylamide gel analysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS) spectra displayed that α-BTX proteins were conjugated with cND particles via non-covalent bindings. The green fluorescence of the cND particles combining with the red fluorescence of tetramethylrhodamine-labeled α-BTX presented a yellow color at the same location, which indicated that α-BTX proteins were conjugated with cND particles. Xenopus laevis's oocytes expressed the human α7-nAChR proteins by microinjection with α7-nAChR mRNA. The cND-α-BTX complexes were bound to α7-nAChR locating on the cell membrane of oocytes and human lung A549 cancer cells analyzed by laser scanning confocal microscopy. The choline-evoked α7-nAChR-mediated inward currents of the oocytes were blocked by cND-α-BTX complexes in a concentration-dependent manner using two-electrode voltage-clamp recording. Furthermore, the fluorescence intensity of cND-α-BTX binding on A549 cells could be quantified by flow cytometry. These results indicate that cND-conjugated α-BTX still preserves its biological activity in blocking the function of α7-nAChR, and provide a visual system showing the binding of α-BTX to α7-nAChR.

  17. Molecular interactions between T cells and fibroblast-like synoviocytes: role of membrane tumor necrosis factor-alpha on cytokine-activated T cells.

    PubMed

    Tran, Chinh N; Lundy, Steven K; White, Peter T; Endres, Judith L; Motyl, Christopher D; Gupta, Raj; Wilke, Cailin M; Shelden, Eric A; Chung, Kevin C; Urquhart, Andrew G; Fox, David A

    2007-11-01

    The mechanism of fibroblast-like synoviocyte (FLS) transformation into an inflammatory phenotype in rheumatoid arthritis (RA) is not fully understood. FLS interactions with invading leukocytes, particularly T cells, are thought to be a critical component of this pathological process. Resting T cells and T cells activated through the T-cell receptor have previously been shown to induce inflammatory cytokine production by FLS. More recently, a distinct population of T cells has been identified in RA synovium that phenotypically resembles cytokine-activated T (Tck) cells. Using time lapse microscopy, the interactions of resting, superantigen-activated, and cytokine-activated T cells with FLS were visualized. Rapid and robust adhesion of Tck and superantigen-activated T cells to FLS was observed that resulted in flattening of the T cells and a crawling movement on the FLS surface. Tck also readily activated FLS to produce interleukin IL-6 and IL-8 in a cell contact-dependent manner that was enhanced by exogenous IL-17. Although LFA-1 and ICAM-1 co-localized at the Tck-FLS synapse, blocking the LFA-1/ICAM-1 interaction did not substantially inhibit Tck effector function. However, antibody blocking of membrane tumor necrosis factor (TNF)-alpha on the Tck surface did inhibit FLS cytokine production, thus illustrating a novel mechanism for involvement of TNF-alpha in cell-cell interactions in RA synovium and for the effectiveness of TNF-alpha blockade in the treatment of RA.

  18. Chemokine gene expression in the murine renal cell carcinoma, RENCA, following treatment in vivo with interferon-alpha and interleukin-2.

    PubMed Central

    Sonouchi, K.; Hamilton, T. A.; Tannenbaum, C. S.; Tubbs, R. R.; Bukowski, R.; Finke, J. H.

    1994-01-01

    The expression of three chemoattractant cytokine (chemokine) messenger (m)RNAs in the murine renal cell carcinoma (RENCA) from mice treated with a combination of interferon-alpha (IFN-alpha) and interleukin-2 was examined and related to tumor infiltration by inflammatory leukocytes. Using a semi-quantitative reverse transcriptase polymerase chain reaction assay, mRNAs encoding the KC, JE, and IP-10 genes were all elevated in tumor tissue from mice treated systemically with IFN-alpha/interleukin-2 for 4 days. Similarly, the mRNA for tumor necrosis factor-alpha (TNF-alpha) was also increased in tumors from treated as compared to control animals. The same tumors showed a significant increase in Mac-1+ leukocytes, which correlated well with the increase in chemokine and TNF-alpha gene expression. The renal cell carcinoma tumor itself may be responsible for the expression of chemokine genes in the tumor bed following cytokine therapy. Cultures of freshly explanted RENCA cells expressed significant levels of chemokine mRNAs when stimulated in vitro with IFN alpha, IFN gamma, and/or interleukin-2, demonstrating that this tumor cell has potential for expression of these genes in vivo. In contrast, TNF-alpha expression was not detected in cultured tumor cells. Thus TNF-alpha may be expressed by infiltrating monocytes following exposure to recombinant cytokine therapy. Images Figure 1 Figure 2 Figure 4 PMID:8160774

  19. Alpha-chaconine-reduced metastasis involves a PI3K/Akt signaling pathway with downregulation of NF-kappaB in human lung adenocarcinoma A549 cells.

    PubMed

    Shih, Yuan-Wei; Chen, Pin-Shern; Wu, Cheng-Hsun; Jeng, Ya-Fang; Wang, Chau-Jong

    2007-12-26

    Alpha-chaconine, isolated from Solanum tuberosum Linn., is a naturally occurring steroidal glycoalkaloid in potato sprouts. Some reports demonstrated that alpha-chaconine had various anticarcinogenic properties. The aim of this study is to investigate the inhibitory effect of alpha-chaconine on lung adenocarcinoma cell metastasis in vitro. We chose the highly metastatic A549 cells, which were treated with various concentrations of alpha-chaconine to clarify the potential of inhibiting A549 cells invasion and migration. Data showed that alpha-chaconine inhibited A549 cell invasion/migration according to wound healing assay and Boyden chamber assay. Our results also showed that alpha-chaconine could inhibit phosphorylation of c-Jun N-terminal kinase (JNK) and Akt, whereas it did not affected phosphorylation of extracellular signal regulating kinase (ERK) and p38. In addition, alpha-chaconine significantly decreased the nuclear level of nuclear factor kappa B (NF-kappaB) and the binding ability of NF-kappaB. These results suggested that alpha-chaconine inhibited A549 cell metastasis by a reduction of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) activities involving suppression of phosphoinositide 3-kinase/Akt/NF-kappaB (PI3K/Akt/NF-kappaB) signaling pathway. Inhibiting metastasis by alpha-chaconine might offer a pivotal mechanism for its effective chemotherapeutic action.

  20. Beta3 subunits promote expression and nicotine-induced up-regulation of human nicotinic alpha6* nicotinic acetylcholine receptors expressed in transfected cell lines.

    PubMed

    Tumkosit, Prem; Kuryatov, Alexander; Luo, Jie; Lindstrom, Jon

    2006-10-01

    Nicotinic acetylcholine receptors (AChRs) containing alpha6 subunits are typically found at aminergic nerve endings where they play important roles in nicotine addiction and Parkinson's disease. alpha6* AChRs usually contain beta3 subunits. beta3 subunits are presumed to assemble only in the accessory subunit position within AChRs where they do not participate in forming acetylcholine binding sites. Assembly of subunits in the accessory position may be a critical final step in assembly of mature AChRs. Human alpha6 AChRs subtypes were permanently transfected into human tsA201 human embryonic kidney (HEK) cell lines. alpha6beta2beta3 and alpha6beta4beta3 cell lines were found to express much larger amounts of AChRs and were more sensitive to nicotine-induced increase in the amount of AChRs than were alpha6beta2 or alpha6beta4 cell lines. The increased sensitivity to nicotine-induced up-regulation was due not to a beta3-induced increase in affinity for nicotine but probably to a direct effect on assembly of AChR subunits. HEK cells express only a small amount of mature alpha6beta2 AChRs, but many of these subunits are on the cell surface. This contrasts with Xenopus laevis oocytes, which express a large amount of incorrectly assembled alpha6beta2 subunits that bind cholinergic ligands but form large amorphous intracellular aggregates. Monoclonal antibodies (mAbs) were made to the alpha6 and beta3 subunits to aid in the characterization of these AChRs. The alpha6 mAbs bind to epitopes C-terminal of the extracellular domain. These data demonstrate that both cell type and the accessory subunit beta3 can play important roles in alpha6* AChR expression, stability, and up-regulation by nicotine.

  1. beta-Naphthoflavone protects from peritonitis by reducing TNF-alpha-induced endothelial cell activation.

    PubMed

    Hsu, Sheng-Yao; Liou, Je-Wen; Cheng, Tsung-Lin; Peng, Shih-Yi; Lin, Chi-Chen; Chu, Yuan-Yuan; Luo, Wei-Cheng; Huang, Zheng-Kai; Jiang, Shinn-Jong

    2015-12-01

    β-Naphthoflavone (β-NF), a ligand of the aryl hydrocarbon receptor, has been shown to possess anti-oxidative properties. We investigated the anti-oxidative and anti-inflammatory potential of β-NF in human microvascular endothelial cells treated with tumor necrosis factor-alpha (TNF-α). Pretreatment with β-NF significantly inhibited TNF-α-induced intracellular reactive oxygen species, translocation of p67(phox), and TNF-α-induced monocyte binding and transmigration. In addition, β-NF significantly inhibited TNF-α-induced ICAM-1 and VCAM-1 expression. The mRNA expression levels of the inflammatory cytokines TNF-α and IL-6 were reduced by β-NF, as was the infiltration of white blood cells, in a peritonitis model. The inhibition of adhesion molecules was associated with suppressed nuclear translocation of NF-κB p65 and Akt, and suppressed phosphorylation of ERK1/2 and p38. The translocation of Egr-1, a downstream transcription factor involved in the MEK-ERK signaling pathway, was suppressed by β-NF treatment. Our findings show that β-NF inhibits TNF-α-induced NF-kB and ERK1/2 activation and ROS generation, thereby suppressing the expression of adhesion molecules. This results in reduced adhesion and transmigration of leukocytes in vitro and prevents the infiltration of leukocytes in a peritonitis model. Our findings also suggest that β-NF might prevent TNF-α-induced inflammation.

  2. Fluorescent Protein–Labeled Glucocorticoid Receptor alpha Isoform Trafficking in Cultured Human Trabecular Meshwork Cells

    PubMed Central

    Dibas, Adnan; Jiang, Ming; Fudala, Rafal; Gryczynski, Ignacy; Gryczynski, Zygmunt; Clark, Abbot F.; Yorio, Thomas

    2012-01-01

    Purpose. To characterize the roles of the cytoskeleton and heat shock protein 90 (HSP90) in steroid-induced glucocorticoid receptor alpha (GRα) translocation in cultured human trabecular meshwork cells. Methods. Stably transfected red fluorescent protein (RFP)-GRα NTM5 cell lines were developed. Nuclear localization of RFP-GRα in NTM5 cells treated with vehicle (ethanol), dexamethasone (DEX), or RU486 was measured in cytosolic and nuclear fractions by western blotting and laser confocal microscopy. Cytochalasin D, colchicine, and 17-demethoxygeldanamycin (17AAG, an HSP90 inhibitor), were tested for their abilities to affect GRα trafficking. Nuclear export of RFP-GRα was studied using confocal microscopy following DEX or RU486 removal. Results. NTM5 cells transfected with RFP-GRα showed a clear cytosolic localization of receptor that underwent nuclear localization after DEX treatment. RFP-GRα translocation was temperature sensitive, occurring at 37°C but not at room temperature. Neither cytochalasin D nor colchicine blocked DEX-induced or RU486-induced RFP-GRα nuclear translocation; however, 17AAG prevented DEX-induced RFP-GRα nuclear translocation. Both nuclear import and export of DEX-induced RFP-GRα were faster than RU-486–induced nuclear shuttling. Conclusions. RFP-GRα receptor behaves similarly to the wild-type GRα with its cytosolic localization and shuttling to nucleus after DEX or RU486 treatment. HSP90 is required for nuclear translocation, but the disruption of cytoskeleton had no effect on nuclear translocation of RFP-GRα. PMID:22447868

  3. Pronounced differences between the native K+ channels and KAT1 and KST1 alpha-subunit homomers of guard cells.

    PubMed

    Brüggemann, L; Dietrich, P; Dreyer, I; Hedrich, R

    1999-01-01

    Stomatal opening is the result of K(+)-salt accumulation in guard cells. Potassium uptake in these motor cells is mediated by voltage-dependent, K(+)-selective ion channels. Here we compare the invitro properties of two guard-cell K(+)-channel alpha-subunits from Arabidopsis thaliana (L.) Heynh. (KAT1) and Solanum tuberosum L. (KST1) after heterologous expression with the respective K(+)-transport characteristics in their mother cell. The KAT1 and KST1 subunits when expressed in Xenopus oocytes shared the basic features of the K(+)-uptake channels in the corresponding guard cells, including voltage dependence and single-channel conductance. Besides these similarities, the electrophysiological comparison of K+ channels in the homologous and the heterologous expression systems revealed pronounced differences with respect to modulation and block by extracellular cations. In the presence of 1 mM Cs+, 50% of the guard-cell K(+)-uptake channels (GCKClin) in A. thaliana and S. tuberosum, were inhibited upon hyperpolarization to -90 mV. For a similar effect on KAT1 and KST1 in oocytes, voltages as negative as -155 mV were required. In contrast, compared to the K+ channels in vivo the functional alpha-subunit homomers almost lacked a voltage-dependent block by extracellular Ca2+. Similar to the block by Cs+ and Ca2+, the acid activation of the alpha-homomers was less pronounced in oocytes. Upon acidification the voltage-dependence shifted by 82 and 90 mV for GCKCLin in A. thaliana and S. tuberosum, respectively, but only by 25 mV for KAT1 and KST1. From the differences in K(+)-channel modulation in vivo and after heterologous expression we conclude that the properties of functional guard-cell K(+)-uptake channels result either from the heterometric assembly of different alpha-subunits or evolve from cell-type specific posttranslational modification.

  4. The integrin alpha 9 beta 1 mediates cell attachment to a non-RGD site in the third fibronectin type III repeat of tenascin.

    PubMed

    Yokosaki, Y; Palmer, E L; Prieto, A L; Crossin, K L; Bourdon, M A; Pytela, R; Sheppard, D

    1994-10-28

    We have previously reported the sequence of the integrin alpha 9 subunit, a partner of the beta 1 subunit that is expressed in basal keratinocytes, hepatocytes, airway epithelial cells, and smooth and skeletal muscle. In the present study, we have stably expressed alpha 9 beta 1 on the surface of the human embryonic kidney cell line 293 and the human colon carcinoma cell line SW480 and used these transfected cells lines to identify ligand(s) for this integrin. Transfected cells did not appear to utilize alpha 9 beta 1 for attachment to the extracellular matrix proteins fibronectin, laminin, vitronectin, fibrinogen, thrombospondin, or type I or IV collagen. However, in contrast to mock transfectants, both 293 cells and SW480 cells expressing alpha 9 beta 1 adhered to intact chicken tenascin. By utilizing a variety of recombinant fragments of tenascin, we were able to localize the binding site for alpha 9 beta 1 to the third type III repeat. This repeat contains the arginine-glycine-aspartic acid (RGD) tripeptide that has been shown to serve as a binding site in tenascin for alpha v-integrins. However, the RGD site does not appear to be the binding site for alpha 9 beta 1, as the attachment of alpha 9 transfectants to this fragment was not inhibited by RGD peptide, nor by changing the RGD site to RAD or RAA.

  5. Fibronectin and alpha5 integrin regulate keratinocyte cell cycling. A mechanism for increased fibronectin potentiation of T cell lymphokine-driven keratinocyte hyperproliferation in psoriasis.

    PubMed Central

    Bata-Csorgo, Z; Cooper, K D; Ting, K M; Voorhees, J J; Hammerberg, C

    1998-01-01

    In addition to being T lymphocyte-driven, psoriasis may be due in part to abnormal integrin expression. Normal-appearing (uninvolved) skin from psoriatic patients was examined to determine whether altered fibronectin or its receptor expression is detectable before development of psoriatic lesions. In contrast to skin from normal subjects, we detect by immunofluorescence the abnormal presence of plasma fibronectin in the basal cell layer of the epidermis of psoriatic uninvolved skin. Furthermore, increased fibronectin exposure superinduces the in vitro cell cycle induction and expansion of psoriatic nonlesional keratinocytes in response to a cocktail of T cell lymphokines. Fibronectin alone also appeared to increase cell cycle entry among uninvolved but not normal keratinocytes. Concordantly, the alpha5 integrin fibronectin receptor, but not alpha2 or alpha3, is overexpressed in the in vivo nonlesional psoriatic epidermis. The involvement of alpha5beta1 in the early outgrowth of clonogenic keratinocytes in the ex vivo culture was demonstrated by the ability of anti-alpha5 mAb to inhibit keratinocyte growth on fibronectin. Thus, the fibronectin receptor appears to be one of the components required for the development of the hyperresponsiveness of psoriatic keratinocytes to signals for proliferation provided by lymphokines produced by intralesional T lymphocytes in psoriasis. PMID:9525994

  6. Adrenergic activation of steroid 5alpha-reductase gene expression in rat C6 glioma cells: involvement of cyclic amp/protein kinase A-mediated signaling pathway.

    PubMed

    Morita, Kyoji; Arimochi, Hideki; Tsuruo, Yoshihiro

    2004-01-01

    Steroid 5alpha-reductase (5alpha-R) is well known as the enzyme converting progesterone and other steroid hormones to their 5alpha-reduced metabolites and has been reported to be localized in both neuronal and glial cells in the brain. Previously, the enzyme activity in glial cells has been shown to be enhanced either by coculturing with neuronal cells or by adding the conditioned medium of neuronal cells, suggesting a possible implication of neuro-glial interactions in the regulation of neurosteroid metabolism in the brain. In the present studies, the effects of adrenergic agonists on 5alpha-R mRNA and protein levels in rat C6 glioma cells were examined as one of the model experiments for investigating the influence of neuronal activity on the expression of 5alpha-R gene in the glial cell. The direct challenge of beta-adrenergic agonists to glioma cells resulted in the rapid and transient elevation of 5alpha-R mRNA levels through the activation of the cyclic AMP (cAMP)/protein kinase A-mediated signaling pathway. Further studies showed that cAMP-induced 5alpha-R mRNA expression was completely abolished by pretreatment of cells with actinomycin D and also indicated that the elevation of 5alpha-R mRNA levels was accompanied by an increase in enzyme protein in the cells. These findings provide strong evidence that the stimulation of beta-adrenergic receptors might induce the transcriptional activation of 5alpha-R gene expression in glial cells, proposing the possibility that neuronal activity might be involved in the production of neuroactive 5alpha-reduced steroids in the brain.

  7. Axonal regulation of Schwann cell integrin expression suggests a role for alpha 6 beta 4 in myelination

    PubMed Central

    1993-01-01

    Ensheathment and myelination of axons by Schwann cells in the peripheral nervous system requires contact with a basal lamina. The molecular mechanism(s) by which the basal lamina promotes myelination is not known but is likely to reflect the activity of integrins expressed by Schwann cells. To initiate studies on the role of integrins during myelination, we characterized the expression of two integrin subunits, beta 1 and beta 4, in an in vitro myelination system and compared their expression to that of the glial adhesion molecule, the myelin-associated glycoprotein (MAG). In the absence of neurons, Schwann cells express significant levels of beta 1 but virtually no beta 4 or MAG. When Schwann cells are cocultured with dorsal root ganglia neurons under conditions promoting myelination, expression of beta 4 and MAG increased dramatically in myelinating cells, whereas beta 1 levels remained essentially unchanged. (In general agreement with these findings, during peripheral nerve development in vivo, beta 4 levels also increase during the period of myelination in sharp contrast to beta 1 levels which show a striking decrease.) In cocultures of neurons and Schwann cells, beta 4 and MAG appear to colocalize in nascent myelin sheaths but have distinct distributions in mature sheaths, with beta 4 concentrated in the outer plasma membrane of the Schwann cell and MAG localized to the inner (periaxonal) membrane. Surprisingly, beta 4 is also present at high levels with MAG in Schmidt-Lanterman incisures. Immunoprecipitation studies demonstrated that primary Schwann cells express beta 1 in association with the alpha 1 and alpha 6 subunits, while myelinating Schwann cells express alpha 6 beta 4 and possibly alpha 1 beta 1. beta 4 is also downregulated during Wallerian degeneration in vitro, indicating that its expression requires continuous Schwann cell contact with the axon. These results indicate that axonal contact induces the expression of beta 4 during Schwann cell

  8. Human melanoma cells derived from lymphatic metastases use integrin alpha v beta 3 to adhere to lymph node vitronectin.

    PubMed Central

    Nip, J; Shibata, H; Loskutoff, D J; Cheresh, D A; Brodt, P

    1992-01-01

    Human melanoma is a highly metastatic cancer and the regional lymph nodes are generally the first site of metastasis. Adhesion to cryostat sections of human lymph nodes was therefore studied using two human melanoma models established from lymph node metastases, namely, MeWo cell lines of diverse metastatic potentials and a highly metastatic cell line of recent origin designated MIM/8. We found a good correlation between the metastatic potentials of the melanoma cells as measured in nude mice and their ability to adhere to cryostat sections of human lymph nodes. When adhesion to immobilized extracellular matrix proteins was measured, a significant increase in adhesion, which correlated with increased metastasis, was seen mainly on vitronectin and to a lesser extent on fibronectin. The adhesion to vitronectin and to the frozen sections were specifically blocked by an RGD-containing peptide, mAb 661 to vitronectin and mAb LM609 to integrin alpha v beta 3. FACS analysis revealed a significant and specific increase in cell surface expression of alpha v beta 3 on the metastatic cells as compared to the parent line. Together these results suggest that the adhesion of melanoma cells to lymph node vitronectin via the alpha v beta 3 receptor plays a role in the process of lymphatic dissemination. Images PMID:1383272

  9. Bench-level characterization of a CMOS standard-cell D-latch using alpha-particle sensitive test circuits

    NASA Technical Reports Server (NTRS)

    Blaes, B. R.; Soli, G. A.; Buehler, M. G.

    1991-01-01

    A methodology is described for predicting the SEU susceptibility of a standard-cell D-latch using an alpha-particle sensitive SRAM, SPICE critical charge simulation results, and alpha-particle interaction physics. Measurements were made on a 1.6-micron n-well CMOS 4-kb test SRAM irradiated with an Am-241 alpha-particle source. A collection depth of 6.09 micron was determined using these results and TRIM computer code. Using this collection depth and SPICE derived critical charge results on the latch design, an LET threshold of 34 MeV sq cm/mg was predicted. Heavy ion tests were then performed on the latch and an LET threshold of 41 MeV sq cm/mg was determined.

  10. COUP-TF II homodimers are formed in preference to heterodimers with RXR alpha or TR beta in intact cells.

    PubMed Central

    Butler, A J; Parker, M G

    1995-01-01

    Chicken ovalbumin upstream promoter-transcription factor (COUP-TF) represses the transcriptional activity of a number of nuclear receptors, including that of retinoid receptors (RAR and RXR) and thyroid hormone receptors (TR). Since COUP-TF is capable of binding to DNA in vitro either as a homodimer or as a heterodimer with RXR or TR, it has not been possible to distinguish between competitive DNA binding and heterodimer formation as a mechanism to account for the repression. Using a two-hybrid system we have investigated the dimerisation properties of COUP-TF II in intact cells. In conditions where COUP-TF II homodimers and RXR alpha-RAR alpha heterodimers were formed we were unable to detect the formation of heterodimers between COUP-TF II and RXR alpha. Moreover, we were unable to detect an interaction between COUP-TF II and RXR alpha on DNA. Similarly COUP-TF II homodimers and RXR alpha-TR beta heterodimers are favoured over COUP-TF II-TR beta heterodimers. We conclude that the formation of functionally inactive heterodimers is unlikely to represent a general mechanism by which COUP-TF represses the transcriptional activity of nuclear receptors and favour a model in which repression is mediated by COUP-TF homodimers competing for binding to DNA. Images PMID:7479078

  11. Hypoxia reduces constitutive and TNF-{alpha}-induced