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Sample records for alpha tc3 cells

  1. In Vitro Phenotypic, Genomic and Proteomic Characterization of a Cytokine-Resistant Murine β-TC3 Cell Line

    PubMed Central

    Coppola, Antonina; Tomasello, Laura; Pizzolanti, Giuseppe; Pucci-Minafra, Ida; Albanese, Nadia; Di Cara, Gianluca; Cancemi, Patrizia; Pitrone, Maria; Bommarito, Alessandra; Carissimi, Elvira; Zito, Giovanni; Criscimanna, Angela; Galluzzo, Aldo; Giordano, Carla

    2012-01-01

    Type 1 diabetes mellitus (T1DM) is caused by the selective destruction of insulin-producing β-cells. This process is mediated by cells of the immune system through release of nitric oxide, free radicals and pro-inflammatory cytokines, which induce a complex network of intracellular signalling cascades, eventually affecting the expression of genes involved in β-cell survival. The aim of our study was to investigate possible mechanisms of resistance to cytokine-induced β-cell death. To this purpose, we created a cytokine-resistant β-cell line (β-TC3R) by chronically treating the β-TC3 murine insulinoma cell line with IL-1β + IFN-γ. β-TC3R cells exhibited higher proliferation rate and resistance to cytokine-mediated cell death in comparison to the parental line. Interestingly, they maintained expression of β-cell specific markers, such as PDX1, NKX6.1, GLUT2 and insulin. The analysis of the secretory function showed that β-TC3R cells have impaired glucose-induced c-peptide release, which however was only moderately reduced after incubation with KCl and tolbutamide. Gene expression analysis showed that β-TC3R cells were characterized by downregulation of IL-1β and IFN-γ receptors and upregulation of SOCS3, the classical negative regulator of cytokines signaling. Comparative proteomic analysis showed specific upregulation of 35 proteins, mainly involved in cell death, stress response and folding. Among them, SUMO4, a negative feedback regulator in NF-kB and JAK/STAT signaling pathways, resulted hyper-expressed. Silencing of SUMO4 was able to restore sensitivity to cytokine-induced cell death in β-TC3R cells, suggesting it may play a key role in acquired cytokine resistance by blocking JAK/STAT and NF-kB lethal signaling. In conclusion, our study represents the first extensive proteomic characterization of a murine cytokine-resistant β-cell line, which might represent a useful tool for studying the mechanisms involved in resistance to cytokine-mediated β-cell

  2. 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.

  3. 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

  4. Overall sulfation of heparan sulfate from pancreatic islet β-TC3 cells increases maximal fibril formation but does not determine binding to the amyloidogenic peptide islet amyloid polypeptide.

    PubMed

    Hull, Rebecca L; Peters, Michael J; Perigo, Susan Potter; Chan, Christina K; Wight, Thomas N; Kinsella, Michael G

    2012-10-26

    Islet amyloid, a pathologic feature of type 2 diabetes, contains the islet β-cell peptide islet amyloid polypeptide (IAPP) as its unique amyloidogenic component. Islet amyloid also contains heparan sulfate proteoglycans (HSPGs) that may contribute to amyloid formation by binding IAPP via their heparan sulfate (HS) chains. We hypothesized that β-cells produce HS that bind IAPP via regions of highly sulfated disaccharides. Unexpectedly, HS from the β-cell line β-TC3 contained fewer regions of highly sulfated disaccharides compared with control normal murine mammary gland (NMuMG) cells. The proportion of HS that bound IAPP was similar in both cell lines (∼65%). The sulfation pattern of IAPP-bound versus non-bound HS from β-TC3 cells was similar. In contrast, IAPP-bound HS from NMuMG cells contained frequent highly sulfated regions, whereas the non-bound material demonstrated fewer sulfated regions. Fibril formation from IAPP was stimulated equally by IAPP-bound β-TC3 HS, non-bound β-TC3 HS, and non-bound NMuMG HS but was stimulated to a greater extent by the highly sulfated IAPP-bound NMuMG HS. Desulfation of HS decreased the ability of both β-TC3 and NMuMG HS to stimulate IAPP maximal fibril formation, but desulfated HS from both cell types still accelerated fibril formation relative to IAPP alone. In summary, neither binding to nor acceleration of fibril formation from the amyloidogenic peptide IAPP is dependent on overall sulfation in HS synthesized by β-TC3 cells. This information will be important in determining approaches to reduce HS-IAPP interactions and ultimately prevent islet amyloid formation and its toxic effects in type 2 diabetes.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  6. 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.

  7. The impact trajectory of asteroid 2008 TC3

    NASA Astrophysics Data System (ADS)

    Farnocchia, Davide; Jenniskens, Peter; Robertson, Darrel K.; Chesley, Steven R.; Dimare, Linda; Chodas, Paul W.

    2017-09-01

    The impact of asteroid 2008 TC3 was an unprecedented event-the first ever predicted impact of a near-Earth object. When it was first detected about 20 h before impact, 2008 TC3 was still farther away than the Moon. Once it was recognized as an impactor and announced as such, 2008 TC3 began to receive considerable attention from astronomical observers. Using the unprecedented dataset of nearly 900 astrometric observations and the latest observation debiasing and weighting techniques, we estimate the precise trajectory of 2008 TC3 and its impact ground track. At the entry point into the atmosphere, the 3-σ formal uncertainty in predicted position is an ellipse only 1.4 km × 0.15 km in size. The locations of the many meteorites recovered from the desert floor mark the asteroid's actual ground track and provide a unique opportunity to validate trajectory models. We find that the second-order zonal harmonics of the Earth gravity field moves the ground track by more than 1 km and the location along the ground track by more than 2 km, while non-zonal and higher order harmonics change the impact prediction by less than 20 m. The contribution of atmospheric drag to the trajectory of 2008 TC3 is similar to the numerical integration error level, a few meters, down to an altitude of 50 km. Integrating forward to lower altitudes and ignoring the break-up of 2008 TC3, atmospheric drag causes an along-track deviation that can be as large as a few kilometers at sea level.

  8. 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.

  9. Evolving Function and Potential of Pancreatic Alpha Cells

    PubMed Central

    Stanojevic, Violeta; Habener, Joel F.

    2015-01-01

    The alpha cells that co-occupy the islets in association with beta cells have been long recognized as the source of glucagon, a hyperglycemia-producing and diabetogenic hormone. Although the mechanisms that control the functions of alpha cells, glucagon secretion, and the role of glucagon in diabetes have remained somewhat enigmatic over the fifty years since their discovery, seminal findings during the past few years have moved alpha cells into the spotlight of scientific discovery. These findings obtained largely from studies in mice are: Alpha cells have the capacity to trans-differentiate into insulin-producing beta cells. Alpha cells contain a GLP-1 generating system that produces GLP-1 locally for paracrine actions within the islets that likely promotes beta cell growth and survival and maintains beta cell mass. Impairment of glucagon signaling both prevents the occurrence of diabetes in conditions of the near absence of insulin and expands alpha cell mass. Alpha cells appear to serve as helper cells or guardians of beta cells to ensure their health and well-being. Of potential relevance to the possibility of promoting the transformation of alpha to beta cells is the observation that impairment of glucagon signaling leads to a marked increase in alpha cell mass in the islets. Such alpha cell hyperplasia provides an increased supply of alpha cells for their transdifferentiation into new beta cells. In this review we discuss these recent discoveries from the perspective of their potential relevance to the treatment of diabetes. PMID:26696515

  10. 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.

  11. 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.

  12. 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

  13. Signalling functions of alpha-tocopherol in smooth muscle cells.

    PubMed

    Azzi, A; Boscoboinik, D; Clément, S; Ozer, N K; Ricciarelli, R; Stocker, A; Tasinato, A; Sirikçi, O

    1997-01-01

    alpha-Tocopherol but not beta-tocopherol, activates protein phosphatase 2A, decreases protein kinase C activity and attenuates smooth muscle cell proliferation at physiological concentrations. beta-Tocopherol prevents the effects of alpha-tocopherol. Inhibition of protein kinase C alpha, but not of the other isoforms, by the inhibitor Gö6976 prevents the effect of alpha-tocopherol. Protein kinase C alpha, immunoprecipitated from alpha-tocopherol treated cells, is less phosphorylated and inactive. It is proposed that the specific activation of protein phosphatase 2A by alpha-tocopherol results in dephosphorylation and inactivation of protein kinase C alpha. Finally, this cascade of events leads to smooth muscle cell proliferation inhibition.

  14. d-alpha-tocopherol control of cell proliferation.

    PubMed

    Azzi, A; Boscoboinik, D; Chatelain, E; Ozer, N K; Stäuble, B

    1993-01-01

    Uncontrolled cell growth is at the basis of neoplastic proliferation and arteriosclerotic lesions. In vitro proliferation of vascular smooth muscle cells, Balb c/3T3 fibroblasts, retinal neuroepithelial cells and neuroblastoma cells is inhibited by d-alpha-tocopherol. On the contrary Chinese hamster ovary cells, osteosarcoma cells and macrophages are not sensitive. PDGF-BB activated proliferation is highly d-alpha-tocopherol sensitive while lysophosphatidic acid induced growth is poorly inhibited. d-beta-Tocopherol, an analogue of d-alpha-tocopherol, with similar antioxidant properties, does not inhibit proliferation. Protein kinase C activity is inhibited by d-alpha-tocopherol but not by d-beta-tocopherol, suggesting a central role of this enzyme in the control of cell proliferation by d-alpha-tocopherol. Activation of the transcription activation complex AP-1 (but not NFKB) is prevented by d-alpha-tocopherol and not by d-beta-tocopherol.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. Alpha-Synuclein to the Rescue: Immune Cell Recruitment by Alpha-Synuclein during Gastrointestinal Infection.

    PubMed

    Labrie, Viviane; Brundin, Patrik

    2017-09-02

    Intraneuronal accumulation of misfolded alpha-synuclein in the central and peripheral nervous systems is strongly linked to Parkinson disease (PD) and other related synucleinopathies. In rare inherited forms of PD, point mutations or gene multiplications mediate the formation of alpha-synuclein protein aggregates. However, in most PD cases it is presumed that the combined effects of ageing and environmental factors drive the formation of alpha-synuclein aggregates. Despite advances regarding alpha-synuclein pathobiology, the normal functions of this protein and factors that regulate its expression are not well understood. We discuss a recent study reporting that viral infection induces alpha-synuclein expression in neurons of the gastrointestinal tract. Alpha-synuclein levels increased during norovirus infection in the duodenum of children. In an in vitro paradigm, monomeric and oligomeric alpha-synuclein acted as chemoattractants for neutrophils and monocytes, and promoted the maturation of dendritic cells. This suggests that alpha-synuclein facilitates immune responses to infection. We explore the possibility that intestinal infections, and associated inflammation, place individuals at increased risk of PD by increasing alpha-synuclein levels and promoting the formation of alpha-synuclein aggregates that propagate in a prion-like fashion via the vagal nerve to the brainstem. © 2017 S. Karger AG, Basel.

  20. 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.

  1. Alpha thalassemia changes erythrocyte heterogeneity in sickle cell disease.

    PubMed Central

    Noguchi, C T; Dover, G J; Rodgers, G P; Serjeant, G R; Antonarakis, S E; Anagnou, N P; Higgs, D R; Weatherall, D J; Schechter, A N

    1985-01-01

    Homozygous alpha-thalassemia has the beneficial effect in sickle cell anemia of reducing the hemolytic severity while changing several other hematological parameters. We examined in detail the cellular basis of some of these hematologic alterations. We find that the broad distribution in erythrocyte density and the large proportion of dense cells associated with sickle cell anemia are both reduced with coexisting alpha-thalassemia. Measurements of glycosylated hemoglobin levels as a function of cell density indicate that the accelerated increase in cell density, beyond normal cell aging, in sickle cell anemia is also reduced with alpha-thalassemia. The patients with homozygous alpha-thalassemia and sickle cell disease have slightly lower levels of hemoglobin F than the nonthalassemic sickle cell patients. Examination of hemoglobin F production revealed that the proportion of hemoglobin F containing reticulocytes remained unchanged, as did the proportion of hemoglobin F in cells containing hemoglobin F (F cells). Preferential survival of F cells occurs in sickle cell anemia, with or without alpha-thalassemia, and the slight difference in hemoglobin F levels appear to reflect differences in numbers of circulating F cells. Thus, in sickle cell disease with coexisting alpha-thalassemia, the change in the erythrocyte density profile, possibly due to inhibition of polymerization-related increases in cell density, explains the hematological improvement. PMID:2581999

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. Formation of recombinant triple-helical [alpha 1(IV)]2 alpha 2(IV) collagen molecules in CHO cells.

    PubMed

    Fukuda, K; Hori, H; Utani, A; Burbelo, P D; Yamada, Y

    1997-02-03

    Collagen IV molecules represent a major structural component of basement membranes providing a network of support for the supramolecular structure. Like other collagens, collagen IV forms a triple-helical molecule composed of three alpha chains. Six different alpha chains exist for collagen IV, although the most common isoform consists of two alpha 1(IV) and one alpha 2(IV) chain. To understand the molecular mechanism of triple-helical formation of collagen IV, we expressed recombinant alpha 1(IV) and alpha 2(IV) mouse collagen chains in Chinese hamster ovary (CHO) cells. An expression vector containing the full length cDNA for the mouse alpha 1(IV) chain was stably transfected into CHO cells and a cell line, A222, which expressed recombinant alpha 1(IV) chains was selected. These A222 cells were then infected with a retroviral expression vector containing the mouse alpha 2(IV) chain and a cell line, A222-A2, stably expressing both recombinant alpha 1(IV) and alpha 2(IV) chains was obtained. Immunoprecipitation of A222 cell lysates revealed a high level of alpha 1(IV) chain monomer, which was unable to form a homotrimer. Analysis of A222-A2 cell lysates revealed the presence of both monomeric alpha 2(IV) and alpha 1(IV) chains as well as a higher molecular weight collagen IV species. Second dimensional SDS-PAGE analysis demonstrated that the high molecular weight species was a heterotrimer consisting of two alpha 1(IV) and one alpha 2(IV) chain. This heterotrimer collagen IV species was pepsin-resistant indicating the formation of a stable triple-helical structure. Pulse-chase experiments showed that the monomer alpha 1(IV) chain was secreted, but at a much slower rate than the heterotrimer. Together these results demonstrate that the alpha 1(IV) chain is not capable of forming homotrimers and suggest that the coexpression with the alpha 2(IV) chain is necessary to form a triple-helical structure.

  8. Mutagenicity of alpha particles in Ehrlich ascites tumor cells

    SciTech Connect

    Iliakis, G.

    1984-07-01

    Cell killing and the induction of mutation to thioguanine resistance were measured after exposure of Ehrlich ascites tumor cells to 150-kV X rays and /sup 241/Am ..cap alpha.. particles. The curve describing the induction of mutations was almost linear after exposure to ..cap alpha.. particles but upward bending after exposure to X rays, apparently reaching a final slope similar to that obtained after exposure to ..cap alpha.. particles. The number of mutants induced per viable cell by ..cap alpha.. particles at a given level of cell killing was similar to that induced by X rays. The RBE values obtained for cell killing and the induction of mutations are compared with each other, and the possible involvement of repair processes in determining the RBE is discussed.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. Origin and Dynamical Evolution of Ureilites and 2008TC3/Almahata Sitta.

    NASA Astrophysics Data System (ADS)

    Goodrich, Cyrena

    In October, 2008, a small asteroid with an F-type reflectance spectrum, 2008 TC3, exploded over the Nubian desert and deposited material in a strewn field in Northern Sudan. Searches to-date have led to collection of >600 stones thought to be derived from 2008 TC3. These materials are named Almahata Sitta (AHS), and represent the first recovered meteorite from a spectrally observed asteroid. The discovery, tracking, spectral analysis and recovery of material from 2008 TC3/AHS was a remarkable series of events, and a testament to the benefits of cross-disciplinary and international cooperation. But the list of remarkable things about this object does not end there. The first AHS samples studied showed an anomalous type of material belonging to the class of ureilite meterorites. Ureilites are one of the least understood classes of primitive achondrites, and arguably one of the most important for understanding early solar system processes that bridge the chondrite-to-achondrite transition. Thus, the advent of a new type of ureilite, potentially providing new information in the form of fresh, unweathered material, is certainly welcome. Ensuing studies of more samples showed a great diversity of ureilite materials, leading to classification of AHS as an anomalous polymict ureilite. Even more remarkably, however, ~20-30% of the recovered clasts were found to be non- ureilitic, instead representing a variety of different chondrite types. Thus, 2008 TC3/AHS appears to be a new type of breccia, unlike any meteorite known to date. Nevertheless, there are strong indications that 2008 TC3/AHS, "normal" polymict ureilites, and main group ureilites share a common lineage. We hypothesize that the immediate parent of 2008 TC3/AHS was also the immediate parent of all ureilitic material that has been sampled in recent times by meteorites on Earth, and that using 2008 TC3/AHS as a starting point, we can trace the history of this material back through many stages of Solar System

  6. 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

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

    PubMed

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

    1989-02-01

    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.

  8. Integrin alpha 6 regulates glioblastoma stem cells

    PubMed Central

    Lathia, Justin D.; Gallagher, Joseph; Heddleston, John M.; Wang, Jialiang; Eyler, Christine E.; MacSwords, Jennifer; Wu, Qiulian; Vasanji, Amit; McLendon, Roger E.; Hjelmeland, Anita B.; Rich, Jeremy N.

    2010-01-01

    Summary Cancer stem cells (CSCs) are a subpopulation of tumor cells suggested to be critical for tumor maintenance, metastasis, and therapeutic resistance. Prospective identification and targeting of CSCs are therefore priorities for the development of novel therapeutic paradigms. While CSC enrichment has been achieved with cell surface proteins including CD133 (Prominin-1), the roles of current CSC markers in tumor maintenance remain unclear. We examined the glioblastoma stem cell (GSC) perivascular microenvironment in patient specimens to identify enrichment markers with a functional significance and identified integrin α6 as a candidate. Integrin α6 is co-expressed with conventional GSC markers and enriches for GSCs. Targeting integrin α6 in GSCs inhibits self-renewal, proliferation, and tumor formation capacity. Our results provide evidence that GSCs express high levels of integrin α6, which can not only serve as an enrichment marker but also as a promising anti-glioblastoma therapy. PMID:20452317

  9. 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.

  10. 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.

  11. Control of yeast cell type by the mating type locus: positive regulation of the alpha-specific STE3 gene by the MAT alpha 1 product.

    PubMed

    Sprague, G F; Jensen, R; Herskowitz, I

    1983-02-01

    The mating type locus (MAT) determines the three yeast cell types, a, alpha, and a/alpha. It has been proposed that alleles of this locus, MATa and MAT alpha, encode regulators that control expression of unlinked genes necessary for mating and sporulation. Specifically, the alpha 1 product of MAT alpha is proposed to be a positive regulator of alpha-specific genes. To test this view, we have assayed RNA production from the alpha-specific STE3 gene in the three cell types and in mutants defective in MAT alpha. The STE3 gene was cloned by screening a yeast genomic clone bank for plasmids that complement the mating defect of ste3 mutants. Using the cloned STE3 gene as a probe, we find that alpha cells produce STE3 RNA, whereas a and a/alpha cells do not. Furthermore, mat alpha 1 mutants do not produce STE3 RNA, whereas mat alpha 2 mutants do. These results show that the STE3 gene, required for mating only by alpha cells, is expressed only in alpha cells. They show also that production of RNA from the STE3 gene requires that alpha 1 product of MAT alpha. Thus alpha 1 positively regulates at least one alpha-specific gene by increasing the level of that gene's RNA product.

  12. 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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. [Effect of dominant negative HIF-1alpha (dn HIF-1alpha) on biological characteristics of uterine cervix cancer cells].

    PubMed

    Tang, Bin-Zhi; Zhao, Feng-Yan; Wei, Ting; Mu, De-Zhi; Mao, Meng; Fu, Qiang; Zhang, Lin; Qu, Yia

    2008-05-01

    To explore the effect of dominant negative HIF-1alpha (dn HIF-1alpha) on biological characteristics of uterine cervix cancer cell SiHa and elucidate the related mechanism. pcDNA3. 1-dn HIF-1alpha was transfected into SiHa cells. The expression of HIF-1alpha and VEGF protein were detected by immunocytochemical method and Western Blotting. The growth proliferation of cells was surveyed by the MTT assay and cell apoptosis was detected through TUNEL after treated with CoCl2, meanwhile the results were compared with the group transfected with mock plasmid and untransfected group. After successfully transfected with relevant plasmid, there's no obvious difference of expression of HIF-1alpha among dn HIF-1alpha group, pcDNA3. 1 group, and untransfected group, however the expression of VEGF of dn HIF-1alpha group was significantly lower than that of the others (P < 0. 05). The proliferation ability of dn HIF-1alpha group was obviously lower than that of the other two (P < 0.05), whether it was under normoxia or chemical hypoxia induced by CoCl2. The characteristic apoptotic morphology was most significantly apparent in dn HIF-1alpha group among these three (P < 0.05). Domain negative HIF-1alpha can inhibit the proliferation of uterine cervix cancer cell and accelerate its apoptosis under hypoxia induced by CoCl2, as well as decrease the expression of VEGF protein. The implications of all this were that the domain negative HIF-1alpha may play an important role in the therapy of uterine cervix cancer.

  14. Adult pancreatic alpha-cells: a new source of cells for beta-cell regeneration.

    PubMed

    Chung, Cheng-Ho; Levine, Fred

    2010-01-01

    Beta-cell deficit is the major pathological feature in type 1 and type 2 diabetes patients, and plays a key role in disease progression. In principle, beta-cell regeneration can occur by replication of pre-existing beta-cells, or by beta-cell neogenesis from stem/progenitors. Unfortunately, beta-cell replication is limited by the almost complete absence of beta-cells in patients with type 1 diabetes, and the increasing recognition that the beta-cell replicative capacity declines severely with age. Therefore, beta-cell neogenesis has received increasing interest. Many different cell types within the pancreas have been suggested as potential beta-cell stem/progenitor cells, but the data have been conflicting. In some cases, this may be due to different regeneration models. On the other hand, different results have been obtained with similar regeneration models, leading to confusion about the nature and existence of beta-cell neogenesis in adult animals. Here, we review the major candidates for adult regeneration pathways, and focus on the recent discovery that alpha-cells can function as a novel beta-cell progenitor. Of note, this is a pathway that appears to be unique to beta-cell neogenesis in the adult, as the embryonic pathway of beta-cell neogenesis does not proceed through a glucagon-positive intermediate. We conclude that beta-cell neogenesis from alpha-cells is a new pathway of potential therapeutic significance, making it of high importance to elucidate the molecular events in alpha- to beta-cell conversion.

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. alpha-thalassemia in Bantu population from Congo-Brazzaville: its interaction with sickle cell anemia.

    PubMed

    Mouélé, R; Pambou, O; Feingold, J; Galactéros, F

    2000-01-01

    Deletional alpha(+)-thalassemia (-alpha(3.7)) was investigated in four groups of unrelated individuals from the Bantu population (newborns, normal adults, sickle cells trait carriers, sickle cell anemia patients) of Brazzaville, Congo. The frequency of the (-alpha(3.7)) chromosome was similar between newborns (f = 0.40) and adult subjects (f = 0.36), and between sicklers and nonsickler subjects. The frequency of the (-alpha(3.7)) chromosome in sickle cell anemia patients (SS patients) did not change when age was stratified. The hematological characteristics of SS patients with (-alpha/alphaalpha, -alpha/-alpha) and without (alphaalpha/alphaalpha) alpha(+)-thalassemia were similar to those reported in Jamaican and US sickle cell anemia patients. alpha(+)-Thalassemia had an effect on the percentage of hemoglobin S in sickle cell trait carriers. Thus, the high frequency of alpha(+)-thalassemia in the Congolese population presumably results from this disorder having a selective advantage favoring survival. However, the frequency of alpha(+)-thalassemia was not affected by age. Although in this selective tropical environment, alpha(+)-thalassemia as elsewhere markedly affects the hematological characteristics of sickle cell anemia patients, however our data provide no evidence that alpha(+)-thalassemia increases survival of SS patients. Copyright 1999 S. Karger AG, Basel

  4. Morphology and Tracer Coupling Pattern of Alpha Ganglion Cells in the Mouse Retina

    PubMed Central

    VÖLGYI, BÉLA; ABRAMS, JOSEPH; PAUL, DAVID L.; BLOOMFIELD, STEWART A.

    2010-01-01

    Alpha cells are a type of ganglion cell whose morphology appears to be conserved across a number of mammalian retinas. In particular, alpha cells display the largest somata and dendritic arbors at a given eccentricity and tile the retina as independent on- (ON) and off-center (OFF) subtypes. Mammalian alpha cells also express a variable tracer coupling pattern, which often includes homologous (same cell type) coupling to a few neighboring alpha cells and extensive heterologous (different cell type) coupling to two to three amacrine cell types. Here, we use the gap junction-permeant tracer Neurobiotin to determine the architecture and coupling pattern of alpha cells in the mouse retina. We find that alpha cells show the same somatic and dendritic architecture described previously in the mammal. However, alpha cells show varied tracer coupling patterns related to their ON and OFF physiologies. ON alpha cells show no evidence of homologous tracer coupling but are coupled heterologously to at least two types of amacrine cell whose somata lie within the ganglion cell layer. In contrast, OFF alpha cells are coupled to one another in circumscribed arrays as well as to two to three types of amacrine cell with somata occupying the inner nuclear layer. We find that homologous coupling between OFF alpha cells is unaltered in the connexin36 (Cx36) knockout (KO) mouse retina, indicating that it is not dependent on Cx36. However, a subset of the heterologous coupling of ON alpha cells and all the heterologous coupling of OFF alpha cells are eliminated in the KO retina, suggesting that Cx36 comprises most of the junctions made with amacrine cells. PMID:16175559

  5. 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

  6. The human T cell receptor alpha variable (TRAV) genes.

    PubMed

    Scaviner, D; Lefranc, M P

    2000-01-01

    'Human T Cell Receptor Alpha Variable (TRAV) Genes', the eighth report of the 'IMGT Locus in Focus' section, comprises four tables: (1) 'Number of human germline TRAV genes at 14q11 and potential repertoire'; (2) 'Human germline TRAV genes at 14q11'; (3) 'Human TRAV allele table', and (4) 'Correspondence between the different human TRAV gene nomenclatures'. These tables are available at the IMGT Marie-Paule page of IMGT, the international ImMunoGeneTics database (http://imgt.cines.fr:8104) created by Marie-Paule Lefranc, Université Montpellier II, CNRS, France. Copyright 2000 S. Karger AG, Basel

  7. 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.

  8. 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.

  9. Correlation between human vascular smooth muscle cell proliferation and protein kinase C alpha-expression: effect of d-alpha-tocopherol.

    PubMed

    Marilley, D; Mosieniak, G; Boscoboinik, D; Azzi, A

    1996-11-01

    Population doublings of four different human aortic vascular smooth muscle cell strains correlate with the amount of protein kinase C alpha present in these cells. d-alpha-Tocopherol inhibits, at different extents, protein kinase C activity in all cells studied. Furthermore, the extent of inhibition positively correlates with amount of protein kinase C alpha expression and not with that of the other isoforms. It is suggested that, in human aortic smooth muscle cells, protein kinase C alpha modulates cell proliferation and serves as a target for d-alpha-tocopherol inhibition.

  10. T cell receptor V[alpha] gene segment with alternate splicing in the junctional region

    SciTech Connect

    Marche, P.N.; Six, A.; Gahery, H.; Gris-Liebe, C.; Cazenave, P.A.; Jouvin-Marche, E. )

    1993-11-15

    The locus encoding mouse TCR-[alpha] chain includes approximately 100 V[alpha] gene segments that can be organized in about 20 structural subfamilies. Southern blot analysis of a T cell line derived from the BALB/c strain, M5T, has indicated that both [alpha] loci were rearranged, as assessed by the deletion of the [delta] locus, and that the V[alpha] gene-segment involved in one of the rearrangements did not belong to any of the V[alpha] subfamilies already described. Transcripts of TCR-[alpha] chains from the M5T line were cloned after cDNA synthesis and anchored-polymerase chain reaction, revealing a V[alpha] gene segment of an as yet unidentified subfamily, V[alpha]5T. Molecular cloning of germ-line V[alpha]5T gene segments has shown that this subfamily contained two members, one of them being a pseudogene. The two members were located to each extremity of the [alpha] locus associated with a member of the V[alpha]13 and V[alpha]BWB subfamilies. Analysis of transcripts bearing the V[alpha]5T gene segment in the M5T line as well as in thymocytes has revealed that J[alpha] are frequently absent. This is due to an alternate donor splice site generated at the V[alpha]5T-J[alpha] junction that leads to a splicing from the end of V[alpha]5T to C[alpha] instead of the J[alpha] to C[alpha] conventional splicing. The impact of J[alpha] spliced-out transcripts on the allelic exclusion process is discussed. 41 refs., 5 figs.

  11. 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.

  12. 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.

  13. 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.

  14. (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.

  15. Extinction of alpha1-antitrypsin expression in cell hybrids is independent of HNF1alpha and HNF4 and involves both promoter and internal DNA sequences.

    PubMed Central

    Bulla, G A

    1999-01-01

    In rat hepatoma x fibroblast somatic cell hybrids, extinction of rat alpha1-antitrypsin (alpha1AT) gene expression is accompanied by the loss of liver-enriched transcription factors hepatocyte nuclear factor 1 (HNF1alpha) and hepatocyte nuclear factor 4 (HNF4). Previous analysis showed that forced expression of functional HNF1alpha failed to prevent extinction of the rat alpha1AT locus in cell hybrids. Here I show that ectopic co-expression of HNF1alpha plus HNF4 fails to prevent extinction of either rat or human alpha1AT genes in cell hybrids. A 40 kb human alpha1AT minilocus integrated into the rat genome is fully silenced in cell hybrids in the presence of transacting factors. The integrated alpha1AT promoter, but not a viral or ubiquitously active promoter, is repressed 35-fold in the cell hybrids. In addition, position effects also contributed to extinction of many integrated transgenes in a cell type-dependent manner. Finally, internal DNA sequences within the human alpha1AT gene contributed dramatically to the extinction phenotype, resulting in a further 10- to 30-fold reduction in alpha1AT gene expression in cell hybrids. Thus, multiple mechanisms contribute to silencing of tissue-specific gene expression of the alpha1AT gene in cell hybrids. PMID:9927755

  16. Inhibition of cell proliferation by alpha-tocopherol. Role of protein kinase C.

    PubMed

    Boscoboinik, D; Szewczyk, A; Hensey, C; Azzi, A

    1991-04-05

    The effect of alpha-tocopherol (vitamin E) on the proliferation of vascular smooth muscle cells (A7r5), human osteosarcoma cells (Saos-2), fibroblasts (Balb/3T3), and neuroblastoma cells (NB2A) has been studied. The proliferation of vascular smooth muscle cells was inhibited by physiologically relevant concentrations of alpha-tocopherol, neuroblastoma cells were only sensitive to higher alpha-tocopherol concentrations, and proliferation of the other cell lines was not inhibited. The inhibition of smooth muscle cell proliferation was specific for alpha-tocopherol. Trolox, phytol, and alpha-tocopherol esters had no effect. Proliferation of smooth muscle cells stimulated by platelet-derived growth factor or endothelin was completely sensitive to alpha-tocopherol. If smooth muscle cells were stimulated by fetal calf serum, proliferation was 50% inhibited by alpha-tocopherol. No effect of alpha-tocopherol was observed when proliferation of smooth muscle cells was stimulated by bombesin and lysophosphatidic acid. The possibility of an involvement of protein kinase C in the cell response to alpha-tocopherol was suggested by experiments with the isolated enzyme and supported by the 2- to 3-fold stimulation of phorbol ester binding induced by alpha-tocopherol in sensitive cells. Moreover, alpha-tocopherol also caused inhibition of protein kinase C translocation induced by phorbol esters and inhibition of the phosphorylation of its 80-kDa protein substrate in smooth muscle cells. A model is discussed by which alpha-tocopherol inhibits cell proliferation by interacting with the cytosolic protein kinase C, thus preventing its membrane translocation and activation.

  17. 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.

  18. Alpha thalassemia among sickle cell anaemia patients in Kampala, Uganda.

    PubMed

    Lubega, Irene; Ndugwa, Christopher M; Mworozi, Edison A; Tumwine, James K

    2015-06-01

    Sickle cell anaemia is prevalent in sub Saharan Africa. While α+-thalassaemia is known to modulate sickle cell anaemia, its magnitude and significance in Uganda have hitherto not been described. To determine the prevalence of α+thalassaemia among sickle cell anaemia patients in Mulago Hospital and to describe the clinical and laboratory findings in these patients. A cross sectional study was carried out on patients with sickle cell anaemia in Kampala. Dried blood spots were used to analyze for the deletional α+ thalassaemia using multiplex polymerase chain reaction. Of the 142 patients with sickle cell anaemia, 110 (77.5%) had the αα+thalassaemia deletion. The gene frequency of (-α) was 0.425. Ninety one percent (100/110) of those with α+thalassaemia were heterozygous (αα/α-). Amongst the patients older than 60 months, 15 (83.3%) of those without αα+thalassaemia had significant hepatomegaly of greater than 4 cm compared to 36 (45.6%) of those with α+thalassaemia (p=0.003). The gene frequency of (-α) of 0.425 noted in this study is higher than that reported from many places in Africa. Concurrent alpha thalassemia might be a protective trait against significant hepatomegaly in sickle cell anaemia patients more than 60 months of age at Mulago hospital.

  19. 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.

  20. Loss of LAP2 alpha delays satellite cell differentiation and affects postnatal fiber-type determination.

    PubMed

    Gotic, Ivana; Schmidt, Wolfgang M; Biadasiewicz, Katarzyna; Leschnik, Michael; Spilka, Rita; Braun, Juliane; Stewart, Colin L; Foisner, Roland

    2010-03-31

    Lamina-associated polypeptide 2 alpha (LAP2 alpha) is a nucleoplasmic protein implicated in cell cycle regulation through its interaction with A-type lamins and the retinoblastoma protein. Mutations in lamin A/C and LAP2 alpha cause late onset striated muscle diseases, but the molecular mechanisms are poorly understood. To study the role of LAP2 alpha in skeletal muscle function and postnatal tissue homeostasis, we generated complete and muscle-specific LAP2 alpha knockout mice. Whereas overall muscle morphology, function, and regeneration were not detectably affected, the myofiber-associated muscle stem cell pool was increased in complete LAP2 alpha knockout animals. At molecular level, the absence of LAP2 alpha preserved the stem cell-like phenotype of Lap2 alpha(-/-) primary myoblasts and delayed their in vitro differentiation. In addition, loss of LAP2 alpha shifted the myofiber-type ratios of adult slow muscles toward fast fiber types. Conditional Cre-mediated late muscle-specific ablation of LAP2 alpha affected early stages of in vitro myoblast differentiation, and also fiber-type determination, but did not change myofiber-associated stem cell numbers in vivo. Our data demonstrate multiple and distinct functions of LAP2 alpha in muscle stem cell maintenance, early phases of myogenic differentiation, and muscle remodeling.

  1. 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.

  2. Survival of human lung epithelial cells following in vitro alpha-particle irradiation with absolute determination of the number of alpha-particle traversals of individual cells.

    PubMed

    Søyland, C; Hassfjell, S P

    2000-10-01

    To throw light on human exposure to domestic radon and radon progeny, the effects of low doses of alpha-particle irradiation on normal human lung epithelial cells has been studied. At such low exposure levels the concept of dose is inadequate due to the stochastic variation in the number of alpha-particle traversals per cell. The objective of the current study was to establish an accurate survival curve for human lung epithelial cells with absolute determination of the exact number of alpha-particle traversals of individual cells. Irradiation of L132 cells growing in tracketch detector-based cell dishes, was performed using a collimated alpha-particle beam from a 210Po source. The number of alpha-particle traversals through each individual cell was scored by using a technique of retrospective track-etch dosimetry. This technique is based upon image matching and mapping of corresponding cell and alpha-particle track images. The spatial resolution of the hit determination procedure was +/-0.9/microm. Surviving fractions of cells (SF) showed strict dependence on the number of nuclear traversals (n), with SF(n)= a exp(-bn), a=0.957 (+/- 0.046), b = 0.587 (+/- 0.059), R2 =98.8%. No significant dependence on the number of nuclear membrane traversals (m) or the number of cytoplasm traversals (c) was observed.

  3. Comparison of pancreatic beta cells and alpha cells under hyperglycemia: Inverse coupling in pAkt-FoxO1.

    PubMed

    Kim, Mi-Kyung; Shin, Hyun Mi; Jung, HyeSook; Lee, EunJu; Kim, Tae Kyoon; Kim, Tae Nyun; Kwon, Min Jeong; Lee, Soon Hee; Rhee, Byoung Doo; Park, Jeong Hyun

    2017-09-01

    Type 2 diabetes manifests beta cell deficiencies and alpha cell expansion which is consistent with relative insulin deficiency and glucagon oversecretion. The effects of hyperglycemia on alpha cells are not as understood in comparison to beta cells. Hyperglycemia increases oxidative stress, which induces Akt activation or FoxO activation, depending on cell type. Several studies independently reported that FoxO1 translocations in alpha cells and beta cells were opposite. We compared the responses of pancreatic alpha cells and beta cells against hyperglycemia. Alpha TC-1 cells and Beta TC-6 cells were incubated with control (5mM Glucose) or high glucose (33mM Glucose) with or without PI3K inhibitor or FoxO1 inhibitor. We assessed PI3K, pAkt and phosphorylated FoxO1 (pFoxO1) in both cell lines. Immunostaining of BrdU and FoxO1 was detected by green fluorescence microscopy and confocal microscopy. Hyperglycemia and H2O2 decreased PI3K and pAKT in beta cells, but increased them in alpha cells. FoxO1 localizations and pFoxO1 expressions between alpha cells and beta cells were opposite. Proliferation of beta cells was decreased, but alpha cell proliferation was increased under hyperglycemia. Antioxidant enzymes including superoxide dismutase (SOD) and catalase were increased in beta cells and they were reversed with FoxO1 inhibitor treatment. Increased proliferation in alpha cells under hyperglycemia was attenuated with PI3K inhibitor. In conclusion, hyperglycemia increased alpha cell proliferation and glucagon contents which are opposite to beta cells. These differences may be related to contrasting PI3K/pAkt changes in both cells and subsequent FoxO1 modulation. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. 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.

  5. Integrin alpha(3)-subunit expression modulates alveolar epithelial cell monolayer formation.

    PubMed

    Lubman, R L; Zhang, X L; Zheng, J; Ocampo, L; Lopez, M Z; Veeraraghavan, S; Zabski, S M; Danto, S I; Borok, Z

    2000-07-01

    We investigated expression of the alpha(3)-integrin subunit by rat alveolar epithelial cells (AECs) grown in primary culture as well as the effects of monoclonal antibodies with blocking activity against the alpha(3)-integrin subunit on AEC monolayer formation. alpha(3)-Integrin subunit mRNA and protein were detectable in AECs on day 1 and increased with time in culture. alpha(3)- and beta(1)-integrin subunits coprecipitated in immunoprecipitation experiments with alpha(3)- and beta(1)-subunit-specific antibodies, consistent with their association as the alpha(3)beta(1)-integrin receptor at the cell membrane. Treatment with blocking anti-alpha(3) monoclonal antibody from day 0 delayed development of transepithelial resistance, reduced transepithelial resistance through day 5 compared with that in untreated AECs, and resulted in large subconfluent patches in monolayers viewed by scanning electron microscopy on day 3. These data indicate that alpha(3)- and beta(1)-integrin subunits are expressed in AEC monolayers where they form the heterodimeric alpha(3)beta(1)-integrin receptor at the cell membrane. Blockade of the alpha(3)-integrin subunit inhibits formation of confluent AEC monolayers. We conclude that the alpha(3)-integrin subunit modulates formation of AEC monolayers by virtue of the key role of the alpha(3)beta(1)-integrin receptor in AEC adhesion.

  6. Molecular basis of alpha-tocopherol control of smooth muscle cell proliferation.

    PubMed

    Azzi, A; Aratri, E; Boscoboinik, D; Clément, S; Ozer, N K; Ricciarelli, R; Spycher, S

    1998-01-01

    Rat and human vascular smooth muscle cell proliferation is specifically sensitive to alpha-tocopherol, but not beta-tocopherol. The former, but not the latter, is capable of limiting proliferation and inhibiting protein kinase C activity in a dose-dependent manner. The phenomenon occurs at concentrations in the range 10-50 microM. beta-tocopherol addition together with alpha-tocopherol, prevents both cell growth and protein kinase C inhibition. alpha-tocopherol increases de novo synthesis of protein kinase C molecules. The enzyme specific activity, however, is diminished, due to a decreased phosphorylation of protein kinase C, occurring in the presence of alpha-tocopherol. Experiments with protein kinase C isoform-specific inhibitors and precipitating antibodies show that the only isoform affected by alpha-tocopherol is protein kinase C-alpha. The effect of alpha-tocopherol is prevented by okadaic acid indicating a phosphatase of the PP2A type as responsible for protein kinase C-alpha dephosphorylation produced in the presence of alpha-tocopherol. At a gene level alpha-tocopherol but not beta-tocopherol induces a transient activation of alpha-tropomyosin gene transcription and protein expression. It is proposed that, by inhibiting protein kinase C activity via an activation of a phosphatase PP2A, alpha-tocopherol controls smooth muscle cell proliferation through changes in gene expression.

  7. 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.

  8. Nicotine promotes cell migration through alpha7 nicotinic acetylcholine receptor in gastric cancer cells.

    PubMed

    Lien, Yung-Chang; Wang, Weu; Kuo, Li-Jen; Liu, Jun-Jen; Wei, Po-Li; Ho, Yuan-Soon; Ting, Wen-Chien; Wu, Chih-Hsiung; Chang, Yu-Jia

    2011-09-01

    The objective was to study the mechanism of nicotine-enhanced migration of gastric cancer cells. Long-term cigarette smoking increases the risk of gastric cancer mortality. Tobacco-specific mitogen, nicotine, was reported to correlate with cancer progression on gastric cancer. Since metastasis is the major cause of cancer death, the influence of nicotine on the migration of gastric cancer cells remains to be determined. The influence of nicotine on migration of gastric cancer cells was evaluated by transwell assay and wound-healing migration assay. Receptor-mediated migration was studied by both inhibitor and small interfering RNA. Alpha7 nicotinic acetylcholine receptor, alpha7-nAChR, was identified in gastric cancer cell lines, AGS cells. Nicotine enhanced AGS cell migration in transwell assay and wound-healing migration assay in a dose-dependent manner. We used inhibitor and siRNA to demonstrate that alpha7-nAChR mediated nicotine-enhanced gastric cancer cell migration through downregulation E-cadherin and upregulation ZEB-1 and snail. Tobacco-specific mitogen, nicotine, enhanced gastric cancer metastasis through alpha7-nAChR and suppression of E-cadherin level-one of the hallmarks of epithelial to mesenchymal transition. Therefore, patients with gastric cancer should avoid smoking.

  9. Levels of tumor necrosis factor alpha/cachectin (TNF alpha) in sera from patients with sickle cell disease.

    PubMed

    Malavé, I; Perdomo, Y; Escalona, E; Rodriguez, E; Anchustegui, M; Malavé, H; Arends, T

    1993-01-01

    Serum levels of tumor necrosis factor alpha/cachectin (TNF alpha) were studied in a group of adult patients with sickle cell disease (SCD), which include 31 patients with homozygous SS hemoglobinopathy and 10 patients bearing double heterozygous SC hemoglobinopathy and in their matched normal controls. All patients tested did not show any form of crisis for at least 4 weeks prior to the extraction of the sample. The amount of TNF alpha in serum was quantitated by means of an immunoenzymatic assay with a lower limit of detection of 25 pg/ml. The percentage of sera with detectable levels of TNF alpha was significantly increased in SCD patients as compared with the normal controls. Mean TNF alpha values in individuals with detectable levels of the cytokine were also significantly higher in the whole group of SCD patients and in patients bearing either SS or SC hemoglobinopathies than in the control group. An inverse correlation was observed between the percentages of Hb F and the levels of TNF alpha found in the sera from the patients.

  10. 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

  11. alpha-Synuclein stimulates differentiation of osteosarcoma cells: relevance to down-regulation of proteasome activity.

    PubMed

    Fujita, Masayo; Sugama, Shuei; Nakai, Masaaki; Takenouchi, Takato; Wei, Jianshe; Urano, Tomohiko; Inoue, Satoshi; Hashimoto, Makoto

    2007-02-23

    Because a limited study previously showed that alpha-synuclein (alpha-syn), the major pathogenic protein for Parkinson disease, was expressed in differentiating brain tumors as well as various peripheral cancers, the main objective of the present study was to determine whether alpha-syn might be involved in the regulation of tumor differentiation. For this purpose, alpha-syn and its non-amyloidogenic homologue beta-syn were stably transfected to human osteosarcoma MG63 cell line. Compared with beta-syn-overexpressing and vector-transfected cells, alpha-syn-overexpressing cells exhibited distinct features of differentiated osteoblastic phenotype, as shown by up-regulation of alkaline phosphatase and osteocalcin as well as inductive matrix mineralization. Further studies revealed that proteasome activity was significantly decreased in alpha-syn-overexpressing cells compared with other cell types, consistent with the fact that proteasome inhibitors stimulate differentiation of various osteoblastic cells. In alpha-syn-overexpressing cells, protein kinase C (PKC) activity was significantly decreased, and reactivation of PKC by phorbol ester significantly restored the proteasome activity and abrogated cellular differentiation. Moreover, activity of lysosome was up-regulated in alpha-syn-overexpressing cells, and treatment of these cells with autophagy-lysosomal inhibitors resulted in a decrease of proteasome activity associated with up-regulation of alpha-syn expression, leading to enhance cellular differentiation. Taken together, these results suggest that the stimulatory effect of alpha-syn on tumor differentiation may be attributed to down-regulation of proteasome, which is further modulated by alterations of various factors, such as protein kinase C signaling pathway and a autophagy-lysosomal degradation system. Thus, the mechanism of alpha-syn regulation of tumor differentiation and neuropathological effects of alpha-syn may considerably overlap with each other.

  12. 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.

  13. Detection of a T cell receptor delta chain with an anti-TCR alpha chain serum.

    PubMed

    Leca, G; Bories, J C; Davi, F; Bensussan, A

    1990-04-01

    Two types of T cell antigen-specific receptors have been described. Most peripheral blood T lymphocytes express, at their surface, an antigen receptor consisting of alpha and beta subunits, while a small subset of thymocytes and a minority of mature T lymphocytes express a heterodimeric receptor termed gamma delta. Whereas the gene segments localization corresponding to the TCR gamma and beta chains are separate, genes encoding the joining and the constant regions of TCR delta chain are located between the TCR V alpha region and the J alpha-C alpha gene cluster. To determine whether V alpha gene segments are used by delta chains, immunoprecipitations from human TCR gamma delta expressing cell clones were performed with an anti-alpha serum. The results show that a rabbit antiserum raised against the purified REX TCR alpha subunit immunoprecipitates a TCR delta chain from the cell surface of only one human T cell clone termed SO1. However, since no SO1 RNA hybridization is observed with REX TCR V alpha probe and SO1 cloned cells do react with an anti-V delta 2 monoclonal antibody, we conclude that TCR delta and alpha chains expressed a limited structural homology and that REX TCR V alpha gene do not seem to be frequently used in a functional delta chain.

  14. 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

  15. Regulation of apoptotic and growth inhibitory activities of C/EBP{alpha} in different cell lines

    SciTech Connect

    Wang Guoli; Shi Xiurong; Salisbury, Elizabeth; Timchenko, Nikolai A.

    2008-04-15

    C/EBP{alpha} is expressed in many tissues and inhibits cell growth. In this paper, we have examined mechanisms which regulate activities of C/EBP{alpha} in cell lines derived from different tissues. We found that C/EBP{alpha} possesses strong pro-apoptotic activity in NIH3T3 cells, while this activity is not detected in 3T3-L1, Hep3B2 and HEK293 cells. Micro-array data show that C/EBP{alpha} activates many genes of apoptosis signaling in NIH3T3 cells. One of these genes, ARL6IP5, is a direct target of C/EBP{alpha} and is a key mediator of the apoptosis. Using C/EBP{alpha} mutants which do not cause cell death; we have found that C/EBP{alpha} does not arrest proliferation of NIH3T3 cells. The lack of growth arrest in NIH3T3 cells correlates with the inhibition of p16INK4 and with low levels of cyclin D3. The limited growth inhibitory activity of C/EBP{alpha} is also observed in Hep3B2 cells which express low levels of cyclin D3. Elevation of cyclin D3 restores growth inhibitory activity of C/EBP{alpha} in NIH3T3 and in Hep3B2 cells. These data show that apoptotic and growth inhibitory activities of C/EBP{alpha} are differentially regulated in different cells and that cooperation of cyclin D3 and C/EBP{alpha} is required for the inhibition of proliferation.

  16. Chloroquine modulates HIV-1-induced plasmacytoid dendritic cell alpha interferon: implication for T-cell activation.

    PubMed

    Martinson, Jeffrey A; Montoya, Carlos J; Usuga, Xiomara; Ronquillo, Rollie; Landay, Alan L; Desai, Seema N

    2010-02-01

    Plasmacytoid dendritic cells (pDC) contribute to antiviral immunity mainly through recognition of microbial products and viruses via intracellular Toll-like receptor 7 (TLR7) or TLR9, resulting in the production of type I interferons (IFNs). Although interferons reduce the viral burden in the acute phase of infection, their role in the chronic phase is unclear. The presence of elevated plasma IFN-alpha levels in advanced HIV disease and its association with microbial translocation in chronic HIV infection lead us to hypothesize that IFN-alpha could contribute to immune activation. Blocking of IFN-alpha production using chloroquine, an endosomal inhibitor, was tested in a novel in vitro model system with the aim of characterizing the effects of chloroquine on HIV-1-mediated TLR signaling, IFN-alpha production, and T-cell activation. Our results indicate that chloroquine blocks TLR-mediated activation of pDC and MyD88 signaling, as shown by decreases in the levels of the downstream signaling molecules IRAK-4 and IRF-7 and by inhibition of IFN-alpha synthesis. Chloroquine decreased CD8 T-cell activation induced by aldrithiol-2-treated HIV-1 in peripheral blood mononuclear cell cultures. In addition to blocking pDC activation, chloroquine also blocked negative modulators of the T-cell response, such as indoleamine 2,3-dioxygenase (IDO) and programmed death ligand 1 (PDL-1). Our results indicate that TLR stimulation and production of IFN-alpha by pDC contribute to immune activation and that blocking of these pathways using chloroquine may interfere with events contributing to HIV pathogenesis. Our results suggests that a safe, well-tolerated drug such as chloroquine can be proposed as an adjuvant therapeutic candidate along with highly active antiretroviral therapy to control immune activation in HIV-1 infection.

  17. [Detection of Clostridium novyi type B alpha toxin using cell culture systems

    PubMed

    Borrmann, Erika; Schulze, Frank

    1998-01-01

    The aim of our study was to investigate if a cell culture assay can replace the toxin neutralisation test in mice for the potency testing of alpha toxoid containing clostridial vaccines for veterinary use. The basis for the development of our cell culture assay was the detection of the cytotoxic/cytopathic action of alpha toxin on cells in culture. Nine permanent cell lines were examined for their reaction to the alpha toxin. The action of the toxin was determined after three days by microscopic examination and MTT assay. The alpha toxin exhibited the strongest effect on the ESH-L cells. We were able to show that the cytopathic effect was neutralised by the international standard for gas gangrene antitoxin (C.novyi) but never by heterologous antisera. Our results showed that the ESH-L cell line was a suitable indicator for the detection of the cytotoxic effect of alpha toxin.

  18. 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.

  19. 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.

  20. Transforming growth factor-alpha precursors in human colon carcinoma cells.

    PubMed

    Asbert, M; Montaner, B; Pérez-Tomás, R

    2001-06-01

    Among the proteins of the epidermal growth factor family, transforming growth factor-alpha (TGF-alpha) may be an especially reliable indicator of metastasis or prognosis in human colorectal carcinomas. Moreover, anomalous forms of TGF-alpha have been detected in several tissues of cancer origin, suggesting a role of these forms in the development of the disease. This study was designed to identify the presence of TGF-alpha precursors in different colon cancer cell lines by mean of immunocytochemistry and western blotting techniques. Pro-TGF-alpha was detected in all cell lines tested. Staining for pro-TGF-alpha was observed in cytoplasm. Monoclonal antibody to TGF-alpha detected two bands of 20 and 21 kDa. Polyclonal antibody to pro-TGF-alpha revealed five bands ranging from 15 to 24 kDa. All these proteins were also detected in nonmalignant cells expressing a transfected rat pro-TGF-alpha gene. In conclusions, transformation in these human colon carcinoma cells is not due to the presence of anomalous forms of TGF-alpha precursors.

  1. 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.

  2. Tumor necrosis factor alpha selectively sensitizes human immunodeficiency virus-infected cells to heat and radiation

    SciTech Connect

    Wong, G.H.; McHugh, T.; Weber, R.; Goeddel, D.V. )

    1991-05-15

    We report here that infection of the human T-cell line HUT-78 with human immunodeficiency virus (HIV) increases its sensitivity to heat and radiation toxicity. A possible explanation for this result may be the reduced expression of manganous superoxide dismutase (MnSOD) in HIV-infected cells compared to uninfected cells. Tumor necrosis factor alpha (TNF-alpha) further sensitizes HIV-infected cells but not uninfected cells to heat and radiation. This is consistent with the ability of TNF-alpha to induce the expression of MnSOD in uninfected but not in HIV-infected cells. HIV-infected HUT-78 cell lines engineered to overexpress MnSOD are more resistant to heat and radiation than HIV-infected cells that do not overexpress MnSOD. However, treatment with TNF-alpha still sensitizes these cells to heat and radiation.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. (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.

  8. Effect of alpha-tocopherol and alpha-tocopheryl quinone on the radiosensitivity of thiol-depleted mammalian cells

    SciTech Connect

    Hodgkiss, R.J.; Stratford, M.R.; Watfa, R.R.

    1989-05-01

    The effect of hypoxic cell radiosensitizers is increased when mammalian cells are depleted of endogenous glutathione by buthionine sulphoximine pre-treatment in vitro; a similar gain has not been observed in tumors in vivo despite evidence of glutathione depletion in vivo following buthionine sulphoximine treatment. However, concentrations of biological reducing agents other than glutathione were not measured in the in vivo experiments. Other reducing agents found in tumors include alpha-tocopherol, which reduces the sensitizing efficiency of nitro-aromatic sensitizers in thiol-depleted mammalian cells. These data suggest that the failure to observe large gains in misonidazole sensitizing efficiency in thiol-depleted tumors in vivo may be due, in part, to the presence of biological reducing agents such as alpha-tocopherol.

  9. 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.

  10. 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.

  11. Identification of potential target genes of ROR-alpha in THP1 and HUVEC cell lines.

    PubMed

    Gulec, Cagri; Coban, Neslihan; Ozsait-Selcuk, Bilge; Sirma-Ekmekci, Sema; Yildirim, Ozlem; Erginel-Unaltuna, Nihan

    2017-04-01

    ROR-alpha is a nuclear receptor, activity of which can be modulated by natural or synthetic ligands. Due to its possible involvement in, and potential therapeutic target for atherosclerosis, we aimed to identify ROR-alpha target genes in monocytic and endothelial cell lines. We performed chromatin immunoprecipitation (ChIP) followed by tiling array (ChIP-on-chip) for ROR-alpha in monocytic cell line THP1 and endothelial cell line HUVEC. Following bioinformatic analysis of the array data, we tested four candidate genes in terms of dependence of their expression level on ligand-mediated ROR-alpha activity, and two of them in terms of promoter occupancy by ROR-alpha. Bioinformatic analyses of ChIP-on-chip data suggested that ROR-alpha binds to genomic regions near the transcription start site (TSS) of more than 3000 genes in THP1 and HUVEC. Potential ROR-alpha target genes in both cell types seem to be involved mainly in membrane receptor activity, signal transduction and ion transport. While SPP1 and IKBKA were shown to be direct target genes of ROR-alpha in THP1 monocytes, inflammation related gene HMOX1 and heat shock protein gene HSPA8 were shown to be potential target genes of ROR-alpha. Our results suggest that ROR-alpha may regulate signaling receptor activity, and transmembrane transport activity through its potential target genes. ROR-alpha seems also to play role in cellular sensitivity to environmental substances like arsenite and chloroprene. Although, the expression analyses have shown that synthetic ROR-alpha ligands can modulate some of potential ROR-alpha target genes, functional significance of ligand-dependent modulation of gene expression needs to be confirmed with further analyses. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. 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)

  13. 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.

  14. RRR-alpha-tocopherol regulation of gene transcription in response to the cell oxidant status.

    PubMed

    Azzi, A; Boscoboinik, D; Fazzio, A; Marilley, D; Maroni, P; Ozer, N K; Spycher, S; Tasinato, A

    1998-01-01

    RRR-alpha-Tocopherol, but not RRR-beta-tocopherol, negative regulates proliferation of vascular smooth muscle cells at physiological concentrations. At the same concentrations RRR-alpha-tocopherol inhibits protein kinase C activity, whereas RRR-beta-tocopherol is ineffective. Furthermore, RRR-beta-tocopherol prevents the inhibition of cell growth and of protein kinase C activity caused by RRR-alpha-tocopherol. The negative regulation by RRR-alpha-tocopherol of protein kinase C activity appears to be the cause of smooth muscle cell growth inhibition. RRR-alpha-Tocopherol does not act by binding to protein kinase C directly but presumably by preventing protein kinase C activation. A second RRR-alpha-tocopherol effect has been found at the level of AP 1, the latter becoming activated by RRR-alpha-tocopherol under condition of protein kinase C inhibition or down regulation. AP-1 inhibition by RRR-alpha-tocopherol is seen, however, under condition of protein kinase C stimulation. Compositional changes of AP-1 have been found to be at the basis of the RRR-alpha-tocopherol effects. RRR-beta-tocopherol, provided with similar antioxidant properties, not only it does not affect AP 1 but it prevents the effects of RRR-alpha-tocopherol. Moreover, it has been observed that RRR-alpha-tocopherol is able to affect TRE regulated gene transcription. It is concluded that RRR-alpha-tocopherol acts specifically in vascular smooth muscle cells, by controlling a signal transduction pathway leading to cell proliferation by a non-antioxidant mechanism.

  15. 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.

  16. 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

  17. Lymphoblastoid interferon-alpha inhibits T cell proliferation and expression of eosinophil-activating cytokines.

    PubMed

    Krishnaswamy, G; Smith, J K; Srikanth, S; Chi, D S; Kalbfleisch, J H; Huang, S K

    1996-10-01

    T cell-derived cytokines, such as interleukin-5 (IL-5) and granulocyte-macrophage colony-stimulating factor (GM-CSF) activate eosinophils, whereas other cytokines, such as tumor necrosis factor (TNF)-alpha and IL-13, determine eosinophil recruitment. Interferon-alpha (IFN-alpha), a leukocyte-derived cytokine, has been shown to have beneficial effects in eosinophil-mediated disorders, such as the hypereosinophilic syndrome and a murine model of allergic asthma, where it inhibited eosinophil recruitment. We tested the hypothesis that IFN-alpha acted in eosinophil-mediated disorders by modulating T cell cytokine expression. Peripheral blood mononuclear cells (PBMC) or human ragweed-specific TH1 (2B8) and TH2 (2D2) T cell clones were cultured in the presence of 5 micrograms/ml of phytohemagglutinin (PHA) or 25 micrograms/ml of antigen Amb a 1 (short ragweed allergen), respectively, and lymphoblastoid IFN-alpha (varying from 0 to 10,000 U/ml). We assessed T cell proliferation by [3H]thymidine incorporation and production of IL-5 and GM-CSF by ELISA. Expression of cytokine transcripts was analyzed by the reverse transcription-polymerase chain reaction technique (RT-PCR). IFN-alpha induced a dose-dependent suppression of T cell proliferation of both PBMC (p < 0.001) and the T cell clones (p < 0.001). IFN-alpha inhibited gene expression of IL-5, GM-CSF, TNF-alpha, and IL-13 in PBMC. Furthermore, IFN-alpha significantly inhibited mitogen-induced and antigen-induced production of IL-5 and GM-CSF. IFN-alpha may benefit eosinophil-mediated disorders by inhibiting T cell function and production of cytokines active on human eosinophils.

  18. Alpha-tocopherol (vitamin E) regulates vascular smooth muscle cell proliferation and protein kinase C activity.

    PubMed

    Boscoboinik, D; Szewczyk, A; Azzi, A

    1991-04-01

    Alpha-Tocopherol (vitamin E) protects against free radical damage, which has been implicated in aging, cancer initiation, and atherosclerosis. We have found that physiological concentrations of alpha-tocopherol specifically inhibited aorta smooth muscle cell (VSMC, line A7r5) proliferation and protein kinase C (PKC) activity. Other water and lipid soluble antioxidants were inactive. alpha-Tocopherol inhibition of PKC and of VSMC proliferation may represent a physiological mechanism, relevant to the onset of diseased states such as atherosclerosis.

  19. 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

  20. Alpha-tocopherol as a modulator of smooth muscle cell proliferation.

    PubMed

    Azzi, A; Boscoboinik, D; Clément, S; Marilley, D; Ozer, N K; Ricciarelli, R; Tasinato, A

    1997-10-01

    The effects of alpha-tocopherol and beta-tocopherol have been studied in rat and human aortic smooth muscle cells. Alpha-tocopherol, but not beta-tocopherol, inhibited smooth muscle cell proliferation and protein kinase C in a dose-dependent manner, at concentrations ranging from 10 to 50 microM. Beta-tocopherol added simultaneously with alpha-tocopherol prevented both proliferation and protein kinase C inhibition. Protein kinase C inhibition was cell cycle-dependent and it was prevented by okadaic acid, a protein phosphatase inhibitor. Protein kinase C activity measured from aortas of cholesterol-fed rabbits was also inhibited by alpha-tocopherol. By using protein kinase C (PKC) isoform-specific inhibitors and immunoprecipitation reactions it was found that PKC-alpha was selectively inhibited by alpha-tocopherol. Further, an activation of protein phosphatase 2A by alpha-tocopherol was found, which caused PKC-alpha dephosphorylation and inhibition. Ultimately, this cascade of events at the level of cell signal transduction leads to the inhibition of smooth muscle cell proliferation.

  1. Alpha-, gamma- and delta-tocopherols reduce inflammatory angiogenesis in human microvascular endothelial cells.

    PubMed

    Wells, Shannon R; Jennings, Merilyn H; Rome, Courtney; Hadjivassiliou, Vicky; Papas, Konstantinos A; Alexander, Jonathon S

    2010-07-01

    Vitamin E, a micronutrient (comprising alpha-, beta-, gamma- and delta-tocopherols, alpha-, beta-, gamma- and delta-tocotrienols), has documented antioxidant and non-antioxidant effects, some of which inhibit inflammation and angiogenesis. We compared the abilities of alpha-, gamma- and delta-tocopherols to regulate human blood cytotoxicity (BEC) and lymphatic endothelial cytotoxicity (LEC), proliferation, invasiveness, permeability, capillary formation and suppression of TNF-alpha-induced VCAM-1 as in vitro models of inflammatory angiogenesis. alpha-, gamma- and delta-tocopherols were not toxic to either cell type up to 40 microM. In BEC, confluent cell density was decreased by all concentrations of delta- and gamma-tocopherol (10-40 microM) but not by alpha-tocopherol. LEC showed no change in cell density in response to tocopherols. delta-Tocopherol (40 microM), but not other isomers, decreased BEC invasiveness. In LEC, all doses of gamma-tocopherol, as well as the highest dose of alpha-tocopherol (40 microM), decreased cell invasiveness. delta-Tocopherol had no effect on LEC invasiveness at any molarity. delta-Tocopherol dose dependently increased cell permeability at 48 h in BEC and LEC; alpha- and gamma-tocopherols showed slight effects. Capillary tube formation was decreased by high dose (40 microM) concentrations of alpha-, gamma- and delta-tocopherol, but showed no effects with smaller doses (10-20 microM) in BEC. gamma-Tocopherol (10-20 microM) and alpha-tocopherol (10 microM), but not delta-tocopherol, increased LEC capillary tube formation. Lastly, in BEC, alpha-, gamma- and delta-tocopherol each dose-dependently reduced TNF-alpha-induced expression of VCAM-1. In LEC, there was no significant change to TNF-alpha-induced VCAM-1 expression with any concentration of alpha-, gamma- or delta-tocopherol. These data demonstrate that physiological levels (0-40 microM) of alpha-, gamma- and delta-tocopherols are nontoxic and dietary tocopherols, especially delta

  2. Osmotic stress sensitizes naturally resistant cells to TNF-alpha-induced apoptosis.

    PubMed

    Franco, D L; Nojek, I M; Molinero, L; Coso, O A; Costas, M A

    2002-10-01

    Most cells are naturally resistant to TNF-alpha-induced cell death and become sensitized when NF-kappaB transactivation is blocked or in the presence of protein synthesis inhibitors that prevent the expression of anti-apoptotic genes. In this report we analyzed the role of osmotic stress on TNF-alpha-induced cell death. We found that it sensitizes the naturally resistant HeLa cells to TNF-alpha-induced apoptosis, with the involvement of an increase in the activity of several kinases, the inhibition of Bcl-2 expression, and a late increase on NF-kappaB activation. Cell death occurs regardless of the enhanced NF-kappaB activity, whose inhibition produces an increase in apoptosis. The inhibition of p38 kinase, also involved in NF-kappaB activation, significantly increases the effect of osmotic stress on TNF-alpha-induced cell death.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. Isolation and characterization of a new mutant human cell line unresponsive to alpha and beta interferons.

    PubMed

    John, J; McKendry, R; Pellegrini, S; Flavell, D; Kerr, I M; Stark, G R

    1991-08-01

    Previously we described human cell line 2fTGH, in which expression of guanine phosphoribosyltransferase is tightly controlled by the upstream region of interferon (IFN)-stimulated human gene 6-16. After mutagenesis of 2fTGH and selection with 6-thioguanine and IFN-alpha, we isolated 11.1, a recessive mutant that does not respond to IFN-alpha. We now describe U2, a second recessive mutant, selected similarly, that complements 11.1. U2 had no response to IFN-alpha or IFN-beta, and its response to IFN-gamma was partially defective. Although many genes did respond to IFN-gamma in U2, the 9-27 gene did not and the antiviral response of U2 cells to IFN-gamma was greatly reduced. Band shift assays showed that none of the transcription factors normally induced in 2fTGH cells by IFN-alpha (E and M) or IFN-gamma (G) were induced in U2. However, extracts of untreated U2 cells gave rise to a novel band that was increased by treatment with IFN-gamma but not IFN-alpha. Band shift complementation assays revealed that untreated and IFN-gamma-treated U2 cells lack the functional E gamma subunit of transcription factor E and that IFN-alpha-treated U2 cells do contain the functional E alpha subunit.

  17. 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.

  18. 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}.

  19. Angiotensin II receptor blocker inhibits tumour necrosis factor-alpha-induced cell damage in human renal proximal tubular epithelial cells.

    PubMed

    Kagawa, Toru; Takao, Toshihiro; Horino, Taro; Matsumoto, Reiko; Inoue, Kousuke; Morita, Tatsuhito; Hashimoto, Kozo

    2008-06-01

    We investigated the effect of angiotensin II (AII) type 1 (AT1) and angiotensin II type 2 (AT2) receptor blockers on tumour necrosis factor alpha (TNF-alpha)-induced cell damage in human renal proximal tubular epithelial cells (RPTEC). The lactate dehydrogenase (LDH) and N-acetyl-beta-glucosaminidase (NAG) release into the medium after TNF-alpha treatment in RPTEC were determined using modified commercial procedures. In addition, the levels of caspase 3/7 activity in RPTEC were measured after TNF-alpha treatment with AlphaTau1 or AT2 receptor blockers. Finally we investigated the change of p22phox protein levels after TNF-alpha with AlphaTau1 or AT2 receptor blockers in RPTEC. Tumour necrosis factor alpha (10(-8) mol/L) significantly increased LDH and NAG release into the medium from RPTEC. AlphaTau1 receptor blockers, olmesartan and valsartan (10(-9)-10(-6) mol/L) showed a significant reduction on TNF-alpha-induced LDH and NAG release in RPTEC. AT2 receptor blocker, PD123319 (10(-7)-10(-5) mol/L) also decreased TNF-alpha-induced LDH and NAG release in RPTEC. Blockade of both AlphaTau1 and AT2 receptor indicated additional reduction on TNF-alpha-induced LDH and NAG release. TNF-alpha (10(-8) mol/L) treatment showed small but significant increases of caspase 3/7 activity in RPTEC, and AT1 and AT2 receptor blockers (10(-8) mol/L) comparably decreased TNF-alpha-induced caspase 3/7 activity. Significant increases of p22phox protein levels were observed in TNF-alpha-treated group in RPTEC. However, only AlphaTau1 (10(-8) mol/L) but not AT2 (10(-5) mol/L) receptor blocker significantly decreased TNF-alpha-induced p22phox protein levels. The present study demonstrates that TNF-alpha induces renal tubular cell damage in RPTEC and AT1/AT2 receptor blockers showed cytoprotective effects probably via at least partly different mechanism.

  20. 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

  1. Oncogenic Transformation of Mammalian Cells by Ultrasoft X-Rays and Alpha Particles

    NASA Astrophysics Data System (ADS)

    Yang, T. C.; Craise, L. M.; Raju, M. R.

    For a better understanding of oncogenic cell transformation by ionizing radiation, we conducted experiments with ultrasoft X rays and low energy alpha particles. Confluent C3HlOTl/2 cells were irradiated by Al-K (1.5 keV) X rays or alpha particles from plutonium through a thin mylar sheet, on which the cells attached and grew. Our results indicated that Al-K X rays were more effective in causing cell inactivation and oncogenic transformation than 60Co gamma rays but less effective than 1.0 and 3.7 MeV alpha particles. There was no significant difference between 1.0 and 3.7 MeV alpha particles in transforming cells although the latter were slightly more effective than the former in producing lethal effect. These results indicated that track structure is important in causing biological effects by ionizing radiation

  2. Molecular basis of alpha-tocopherol inhibition of smooth muscle cell proliferation in vitro.

    PubMed

    Boscoboinik, D; Chatelain, E; Bartoli, G M; Azzi, A

    1992-01-01

    The molecular events responsible for the inhibition of cell proliferation by alpha-tocopherol have been investigated. Smooth muscle cells in vitro have been shown to be specifically inhibited by alpha-tocopherol with a concomitant inhibition of protein kinase C activity. beta-Tocopherol was inactive, despite its similar radical scavenging activity. The point of inhibition of alpha-tocopherol relative to the cell cycle was localized in the late G1 phase. A second effect of alpha-tocopherol observed with smooth muscle cells was the stimulation of protein kinase C biosynthesis in both the S and G2 phases of the cell cycle. The implications of these findings for the onset of arteriosclerosis are discussed.

  3. Pancreatic-derived factor (FAM3B), a novel islet cytokine, induces apoptosis of insulin-secreting beta-cells.

    PubMed

    Cao, Xiaopei; Gao, Zhiyong; Robert, Claudia E; Greene, Scott; Xu, Gang; Xu, Weizhen; Bell, Ewan; Campbell, Don; Zhu, Yuan; Young, Robert; Trucco, Matteo; Markmann, James F; Naji, Ali; Wolf, Bryan A

    2003-09-01

    PANDER (PANcreatic DERived factor, FAM3B), a newly discovered secreted cytokine, is specifically expressed at high levels in the islets of Langerhans of the endocrine pancreas. To evaluate the role of PANDER in beta-cell function, we investigated the effects of PANDER on rat, mouse, and human pancreatic islets; the beta-TC3 cell line; and the alpha-TC cell line. PANDER protein was present in alpha- and beta-cells of pancreatic islets, insulin-secreting beta-TC3 cells, and glucagon-secreting alpha-TC cells. PANDER induced islet cell death in rat and human islets. Culture of beta-TC3 cells with recombinant PANDER had a dose-dependent inhibitory effect on cell viability. This effect was also time-dependent. PANDER caused apoptosis of beta-cells as assessed by electron microscopy, annexin V fluorescent staining, and flow-cytometric terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. PANDER did not affect cytosolic Ca(2+) levels or nitric oxide levels. However, PANDER activated caspase-3. Hence, PANDER may have a role in the process of pancreatic beta-cell apoptosis.

  4. Genomic organization of the mouse T cell receptor V alpha family.

    PubMed Central

    Jouvin-Marche, E; Hue, I; Marche, P N; Liebe-Gris, C; Marolleau, J P; Malissen, B; Cazenave, P A; Malissen, M

    1990-01-01

    Based on the analysis of V alpha gene segment deletions in a panel of T lymphomas, we have constructed a map of the mouse T cell receptor alpha/delta region and assigned the relative position of 72 distinct V gene segments. Three major observations have emerged from such studies. First, members of a given V alpha subfamily are not organized in discrete units along the chromosome but largely interspersed with members of other V alpha subfamilies. Second, analysis of the deletion map suggests the existence of repetitive patterns (V alpha clusters) in the chromosomal distribution of the V alpha gene segments. Third, the present-day organization of the V alpha/delta region may be readily explained by a series of sequential duplications involving three ancestral V alpha clusters. Direct evidence for the existence of these unique structural features has been gained by cloning approximately 370 kb of DNA and positioning 26 distinct V alpha gene segments belonging to six different subfamilies. Finally, the relationships existing between the V alpha/delta gene segment organization and usage are discussed in terms of position-dependent models. Images Fig. 1. Fig. 2. Fig. 4. PMID:2357962

  5. 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.

  6. 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.

  7. Nano-XRF Study of Earliest Solar Condensates in EL-3 Fragments from the Almahata Sitta TC3 Asteroid

    NASA Astrophysics Data System (ADS)

    Simionovici, A. S.; Lemelle, L.; Boyet, M.; Gillet, Ph.; Rivard, C.; El Goresy, A.

    2014-09-01

    A nano-XRF study of enstatite chondritic fragments of the Almahata Sitta TC3 asteroid was performed at the ID21 beamline of the ESRF synchrotron in Grenoble, France. We report on the identification and composition of idiomorphic sinoite crystals.

  8. Dual Energy Nano-XRF Quantification in EL-3 Fragments of the Almahata Sitta TC3 Asteroid

    NASA Astrophysics Data System (ADS)

    Simionovici, A. S.; David, G.; Lemelle, L.; Boyet, M.; Gillet, Ph.; Rivard, C.; El Goresy, A.

    2015-07-01

    We studied idiomorphic sinoite crystals in MS-17/177 fragments of A-S TC3 asteroid by dual energy XRF nano-imaging at ESRF (Grenoble, France), down to O/N and confirm previous findings favoring the CaS-Si2N2O condensation sequence scheme.

  9. 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.

  10. Alpha 2-macroglobulin used to isolate intracellular endopeptidases from mammalian cells in culture.

    PubMed Central

    Slot, L A; Hendil, K B

    1988-01-01

    Extracts of cell cultures labelled with [3H]leucine were incubated with human alpha 2-macroglobulin (alpha 2M), a plasma proteinase inhibitor. The proteinase-alpha 2M complexes were then precipitated with immobilized monoclonal antibodies to alpha 2M and analysed by SDS/polyacrylamide-gel electrophoresis. Parallel experiments were done with methylamine-inactivated alpha 2M to check for unspecific binding of cell proteins to alpha 2M. Several 3H-labelled cell proteins bound to active, but not to inactivated, alpha 2M. Such proteins are likely to be proteinases. Putative endopeptidases of subunit Mr 112000, 78,000, 53,000, and in some experiments 88,000 and 16,000, were trapped by alpha 2M in supernatant fractions from IMR90 human fibroblasts, EBTr bovine fibroblasts and HeLa human carcinoma cells. No additional proteins were trapped in the presence of ATP. The Mr-78,000 endopeptidase was identified as calpain II by immunoblotting. At pH 5.3 putative endopeptidases of subunit Mr 80,000, 53,000 and 28,000-32,000 were trapped from IMR90-fibroblast extracts. Immunoblotting showed that both cathepsin B and cathepsin D were present in the Mr-28,000-32,000 electrophoretic bands. The use of alpha 2M and immobilized antibody to alpha 2M thus allows a rapid enrichment of endopeptidases from cell extracts. Some potentials and limitations of the method are discussed. Images Fig. 4. Fig. 5. PMID:2462415

  11. Differential molecular and cellular responses of GLP-1 secreting L-cells and pancreatic alpha cells to glucotoxicity and lipotoxicity.

    PubMed

    Vasu, Srividya; Moffett, R Charlotte; McClenaghan, Neville H; Flatt, Peter R

    2015-08-01

    Knowledge of the effects of glucotoxic and lipotoxic environments on proglucagon producing intestinal L cells and pancreatic alpha cells is limited compared with pancreatic beta cells. This study compares the in vitro responses of these cell types to hyperglycaemia and hyperlipidaemia. Glucose (30 mM) and palmitate (0.5mM) reduced GLUTag and MIN6 cell viability while alpha TC1 cells were sensitive only to lipotoxicity. Consistent with this, Cat mRNA expression was substantially higher in GLUTag and alpha TC1 cells compared to MIN6 cells. Glucose and palmitate reduced GLUTag cell secretory function while hypersecretion of glucagon was apparent from alpha TC1 cells. Glucose exposure increased transcription of Cat and Sod2 in MIN6 and GLUTag cells respectively while it decreased transcription of Cat and Gpx1 in alpha TC1 cells. Palmitate increased transcription of Cat and Sod2 in all three cell lines. Upregulation of antioxidant enzyme expression by palmitate was accompanied by an increase in Nfkb1 transcription, indicative of activation of defence pathways. Lipotoxicity activated ER stress response, evident from increased Hspa4 mRNA level in GLUTag and MIN6 cells. Glucose and palmitate-induced DNA damage and apoptosis, with substantially smaller effects in alpha TC1 cells. Thus alpha cells are resistant to gluco- and lipotoxicity, partly reflecting higher expression of genes involved in antioxidant defence. In contrast, intestinal L cells, like beta cells, are prone to gluco- and lipotoxicity, possibly contributing to abnormalities of GLP-1 secretion in type 2 diabetes. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Detection of Clostridium novyi type B alpha toxin by cell culture systems.

    PubMed

    Borrmann, E; Schulze, F

    1999-07-01

    Ten permanent cell lines were examined for their reaction to the Clostridium novyi alpha toxin. The action of the toxin was determined after 3 days by microscopic examination and the MTT assay. The alpha toxin exhibited the strongest effect on ESH-L cells rather than other cell lines. Vero and SFT-R cells reacted in a comparable way, but less sensitively. We were able to show that the cytopathic effect on the three types of cells was neutralised by the international standard for gas gangrene antitoxin (C. novyi) but in no case by heterologous antisera. Our results have shown that the three cell lines were specific indicators for the detection of the cytopathic effect of alpha toxin. The cytopathic effect can be measured reproducibly by the cell culture assay used. These results are suitable as the starting point for the development of the neutralisation test using cell cultures.

  13. 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.

  14. 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

  15. 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

  16. 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

  17. Glucose suppression of glucagon secretion: metabolic and calcium responses from alpha-cells in intact mouse pancreatic islets.

    PubMed

    Le Marchand, Sylvain J; Piston, David W

    2010-05-07

    Glucagon is released from alpha-cells present in intact pancreatic islets at glucose concentrations below 4 mm, whereas higher glucose levels inhibit its secretion. The mechanisms underlying the suppression of alpha-cell secretory activity are poorly understood, but two general types of models have been proposed as follows: direct inhibition by glucose or paracrine inhibition from non-alpha-cells within the islet of Langerhans. To identify alpha-cells for analysis, we utilized transgenic mice expressing fluorescent proteins targeted specifically to these cells. Measurements of glucagon secretion from pure populations of flow-sorted alpha-cells show that contrary to its effect on intact islets, glucose does stimulate glucagon secretion from isolated alpha-cells. This observation argues against a direct inhibition of glucagon secretion by glucose and supports the paracrine inhibition model. Imaging of cellular metabolism by two-photon excitation of NAD(P)H autofluorescence indicates that glucose is metabolized in alpha-cells and that glucokinase is the likely rate-limiting step in this process. Imaging calcium dynamics of alpha-cells in intact islets reveals that inhibiting concentrations of glucose increase the intracellular calcium concentration and the frequency of alpha-cell calcium oscillations. Application of candidate paracrine inhibitors leads to reduced glucagon secretion but did not decrease the alpha-cell calcium activity. Taken together, the data suggest that suppression occurs downstream from alpha-cell calcium signaling, presumably at the level of vesicle trafficking or exocytotic machinery.

  18. Changes in erythrocytes following supplementation with alpha-tocopherol in children suffering from sickle cell anaemia.

    PubMed

    Jaja, S I; Aigbe, P E; Gbenebitse, S; Temiye, E O

    2005-06-01

    Low plasma vitamin E level in children with sickle cell anaemia has been related to elevated level of irreversibly sickled cells which has been implicated in the pathogenesis of painful crisis. In adult patients supplementation with alpha-tocopherol has been shown to reduce irreversibly sickled cell count. In-addition, vitamin E supplementation increases resting forearm blood flow and reduces forearm vascular resistance in adult sickle cell anaemia sufferers. The aim of the study was to examine the effects of supplementation with alpha-tocopherol on arterial blood pressure, some haematological parameters and osmotic fragility in children suffering from sickle cell anaemia. The effects of supplementation with alpha-tocopherol (100 mg. per day for 6 weeks) on packed cell volume (PCV) haemoglobin concentration ([Hb]), mean corpuscular haemoglobin concentration (MCHC), per cent foetal haemoglobin (% HbF), per cent irreversibly sickled cells (%ISC), erythrocyte osmotic fragility and blood pressure have been assessed in ten (10) children suffering from sickle cell anaemia. Their ages ranged from 4.0 to 10.0 years. Supplementation with alpha-tocopherol significantly increased packed cell volume (p<0.001), haemoglobin concentration (p<0.01) and per cent foetal haemoglobin (p<0.001), but significantly reduced mean corpuscular haemoglobin concentration (p<0.01) and per cent irreversibly sickled cells (p<0.001). alpha-Tocopherol also increased the resistance of the cells to lysis. Supplementation with alpha-tocopherol had little or no effect on arterial blood pressure. This study shows that short-term supplementation with alpha-tocopherol may be beneficial to the paediatric sickle cell sufferer.

  19. 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.

  20. 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

  1. [Apoptosis of mesenchymal cell line MBA-1 induced by core binding factor alpha 1].

    PubMed

    Liu, Min; Zhou, Hou-de; He, Yu-lin; Xie, Hui; Liao, Er-yuan

    2006-02-01

    To investigate the effect of two core binding factors alpha 1 (Cbfa1) isfroms (Cbfa1/P56 and Cbfa1/P57) on the apoptosis of mesenchymal cell line MBA-1. The two Cbfal isfroms were transiently transfected into MBA-1 cells, then the changes of apoptosis rate were observed by flow cytometer. The protein expressions of Cbfa1, Bax, Bcl-2, caspase-3, and caspase-9, cytochrome-C and TNF-alpha were determined by Western immunoblot. After the transient transfection with the two isforms of Cbfa1, MBA-1, the cells apoptotic rates increased, and the ratio of Bax/Bcl-2, the expressions of cytochrome-C, caspase-3, caspase-9, and TNF-alpha were significantly increased. Cbfa1 can promote the apoptosis in mesenchymal cell line MBA-1. Bax/Bcl-2, cytochrome-C, caspase-9, caspase-3, and TNF-alpha are also involved in the apoptosis pathway.

  2. 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

  3. Glycosidases of Ehrlich ascites tumor cells and ascitic fluid--purification and substrate specificity of alpha-N-acetylgalactosaminidase and alpha-galactosidase: comparison with coffee bean alpha-galactosidase.

    PubMed

    Yagi, F; Eckhardt, A E; Goldstein, I J

    1990-07-01

    Ehrlich ascites tumor cells and ascitic fluid were assayed for glycosidase activity. alpha-Galactosidase and beta-galactosidase, alpha- and beta-mannosidase, alpha-N-acetylgalactosaminidase, and beta-N-acetylglucosaminidase activities were detected using p-nitrophenyl glycosides as substrates. alpha-Galactosidase and alpha-N-acetylgalactosaminidase were isolated from Ehrlich ascites tumor cells on epsilon-aminocaproylgalactosylamine-Sepharose. alpha-Galactosidase was purified 160,000-fold and was free of other glycosidase activities. alpha-N-Acetylgalactosaminidase was also purified 160,000-fold but exhibited a weak alpha-galactosidase activity which appears to be inherent in this enzyme. Substrate specificity of the alpha-galactosidase was investigated with 12 substrates and compared with that of the corresponding coffee bean enzyme. The pH optimum of the Ehrlich cell alpha-galactosidase centered near 4.5, irrespective of substrate, whereas the pH optimum of the coffee bean enzyme for PNP-alpha-Gal was 6.0, which is 1.5 pH units higher than that for other substrates of the coffee bean enzyme. The reverse was found for alpha-N-acetylgalactosaminidase: the pH optimum for the hydrolysis of PNP-alpha-GalNAc was 3.6, lower than the pH 4.5 required for the hydrolysis of GalNAc alpha 1,3Gal. Coffee bean alpha-galactosidase showed a relatively broad substrate specificity, suggesting that it is suited for cleaving many kinds of terminal alpha-galactosyl linkages. On the other hand, the substrate specificity of Ehrlich alpha-galactosidase appears to be quite narrow. This enzyme was highly active toward the terminal alpha-galactosyl linkages of Ehrlich glycoproteins and laminin, both of which possess Gal alpha 1, 3Gal beta 1,4GlcNAc beta-trisaccharide sequences. The alpha-N-acetylgalactosaminidase was found to be active toward the blood group type A disaccharide, and trisaccharide, and glycoproteins with type A-active carbohydrate chains.

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. 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).

  14. 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).

  15. 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.

  16. 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

  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. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. Regulation of cell cycle and cyclins by 16alpha-hydroxyestrone in MCF-7 breast cancer cells.

    PubMed

    Lewis, J S; Thomas, T J; Klinge, C M; Gallo, M A; Thomas, T

    2001-12-01

    It has been suggested that alterations in estradiol (E(2)) metabolism, resulting in increased production of 16alpha-hydroxyestrone (16alpha-OHE(1)), is associated with an increased risk of breast cancer. In the present study, we examined the effects of 16alpha-OHE(1)on DNA synthesis, cell cycle progression, and the expression of cell cycle regulatory genes in MCF-7 breast cancer cells. G(1) synchronized cells were treated with 1 to 25 nM 16alpha-OHE(1) for 24 and 48 h. [(3)H]Thymidine incorporation assay showed that 16alpha-OHE(1) caused an 8-fold increase in DNA synthesis compared with that of control cells, whereas E(2) caused a 4-fold increase. Flow cytometric analysis of cell cycle progression also demonstrated the potency of 16alpha-OHE(1) in stimulating cell growth. When G(1) synchronized cells were treated with 10 nM 16alpha-OHE(1) for 24 h, 62+/-3% of cells were in S phase compared with 14+/-3% and 52+/-2% of cells in the control and E(2)-treated groups respectively. In order to explore the role of 16alpha-OHE(1) in cell cycle regulation, we examined its effects on cyclins (D1, E, A, B1), cyclin dependent kinases (Cdk4, Cdk2), and retinoblastoma protein (pRB) using Western and Northern blot analysis. Treatment of cells with 10 nM 16alpha-OHE(1) resulted in 4- and 3-fold increases in cyclin D1 and cyclin A, respectively, at the protein level. There was also a significant increase in pRB phosphorylation and Cdk2 activation. In addition, transient transfection assay using an estrogen response element-driven luciferase reporter vector showed a 15-fold increase in estrogen receptor-mediated transactivation compared with control. These results show that 16alpha-OHE(1) is a potent estrogen capable of accelerating cell cycle kinetics and stimulating the expression of cell cycle regulatory proteins.

  4. 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.

  5. 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

  6. Oxidative stress induces nuclear translocation of C-terminus of {alpha}-synuclein in dopaminergic cells

    SciTech Connect

    Xu Shengli; Zhou Ming; Yu Shun; Cai Yanning; Zhang Alex; Ueda, Kenji; Chan Piu . E-mail: pbchan@bjsap.org

    2006-03-31

    Growing evidence suggests that oxidative stress is involved in the neuronal degeneration and can promote the aggregation of {alpha}-synuclein. However, the role of {alpha}-synuclein under physiological and pathological conditions remains poorly understood. In the present study, we examined the possible interaction between the {alpha}-synuclein and oxidative stress. In a dopaminergic cell line MES23.5, we have found that the 200 {mu}M H{sub 2}O{sub 2} treatment induced the translocation of {alpha}-synuclein from cytoplasm to nuclei at 30 min post-treatment. The immunoactivity of {alpha}-synuclein became highly intensive in the nuclei after 2 h treatment. The protein translocated to nucleus was a 10 kDa fragment of C-terminus region of {alpha}-synuclein, while full-length {alpha}-synuclein remained in cytoplasm. Thioflavine-S staining suggested that the C-terminal fragment in the nuclei has no {beta}-sheet structures. Our present results indicated that 200 {mu}M H{sub 2}O{sub 2} treatment induces the intranuclear accumulation of the C-terminal fragment of {alpha}-synuclein in dopaminergic neurons, whose role remains to be investigated.

  7. 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.

  8. [Effect of HIF-1alpha on sex hormone levels and germ cell apoptosis of mice].

    PubMed

    Chen, Xiang-Mei; Xiong, Yan-Lei; Gong, Hui; Xu, Cheng-Li

    2013-07-01

    To study the effect of hypoxia on hypothalamus-adenohypophysis-testis axis hormone levels, germ cell apoptosis and hypoxia-inducible factor-1alpha (HIF-1alpha) expression in testis of adolescent mice, and explore HIF-1alpha regulation on the reproductive function of male mice. Eighty SPF grade adolescent C57BL/6 mice were randomly divided into normoxia group, hypoxia 3, 7, 14 and 28 d groups. The level of serum testosterone (T), free testosterone (FT), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) was analyzed by ELISA. Detected the sperm count, motility rate and abnormal sperm rate of epididymal sperm suspension. The apoptosis cells in testis were determined using TUNEL method. The expression of HIF-1alpha was analyzed using Western blot. Compared with corresponding normoxia group, serum T, FT, FSH and LH concentrations in hypoxia 3 d group were significantly higher (P < 0.05); T and LH concentrations in hypoxia 14 d group were significantly lower (P < 0.05). Sperm count and motility rate in hypoxia 7 and 14 d groups significantly declined (P < 0.05); abnormal sperm rate in all hypoxia groups significantly increased (P < 0.05). The apoptosis index (AI) of germ cells in hypoxia 7, 14 and 28 d groups significantly increased (P < 0.05), and the levels of HIF-1alpha protein expression were significantly higher (P < 0.05). HIF-1alpha protein highly expressed in mice testis could induce germ cell apoptosis increased in chronic hypoxia environment.

  9. Identification of the T-cell receptor alpha variable (TRAV) gene(s) in T-cell malignancies.

    PubMed

    Hinz, T; Kabelitz, D

    2000-12-01

    Due to the lack of a complete range of monoclonal antibodies (mAb) it is often impossible to rapidly identify by flow cytometry the T-cell receptor variable genes in patients suffering from T-cell malignancies. This applies especially to the alpha variable genes (TRAV), since only very few anti-TcR variable alpha mAb are available. We describe a very rapid method for inverse PCR amplification of the TcR alpha chain without prior purification of the double-stranded cDNA, provide the sequences for appropriate oligonucleotides, and describe a buffer system that dramatically enhances the amplification efficiency as compared to standard conditions.

  10. 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.

  11. Interferon alpha-2a as alternative treatment for conjunctival squamous cell carcinoma.

    PubMed

    Cruzado-Sánchez, D; Salas-Diaz, M; Tellez, W A; Alvarez-Matos, S E; Serpa-Frías, S

    2017-05-15

    A 35 year-old male patient with a history of HIV infection characterized by progressive tumour growth in bulbar conjunctiva of the left eye, corresponding to conjunctival squamous cell carcinoma that responded to treatment with interferon alpha-2a. Interferon alpha-2b has been used at conjunctival level as a topical immunomodulator treatment, with complete remission of epithelial neoplasms being observed. However, there have not been any previous publications on the use of interferon alpha-2a, which differs from interferon alpha-2b in a single amino acid, for the treatment of conjunctival squamous cell carcinoma. Copyright © 2017 Sociedad Española de Oftalmología. Publicado por Elsevier España, S.L.U. All rights reserved.

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

    PubMed

    Wu, Yu; Zhang, Guojian; Wang, Xiangcheng; Zhao, Zhenfang; Wang, Tao; Wang, Xuemei; Li, Xiao-Feng

    2017-01-24

    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. 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. 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. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. The glucose sensor protein glucokinase is expressed in glucagon-producing alpha-cells.

    PubMed Central

    Heimberg, H; De Vos, A; Moens, K; Quartier, E; Bouwens, L; Pipeleers, D; Van Schaftingen, E; Madsen, O; Schuit, F

    1996-01-01

    Expression of glucokinase in hepatocytes and pancreatic 6-cells is of major physiologic importance to mammalian glucose homeostasis. Liver glucokinase catalyzes the first committed step in the disposal of glucose, and beta-cell glucokinase catalyzes a rate-limiting step required for glucose-regulated insulin release. The present study reports the expression of glucokinase in rat glucagon-producing alpha-cells, which are negatively regulated by glucose. Purified rat alpha-cells express glucokinase mRNA and protein with the same transcript length, nucleotide sequence, and immunoreactivity as the beta-cell isoform. Glucokinase activity accounts for more than 50% of glucose phosphorylation in extracts of alpha-cells and for more than 90% of glucose utilization in intact cells. The glucagon-producing tumor MSL-G-AN also contained glucokinase mRNA, protein, and enzymatic activity. These data indicate that glucokinase may serve as a metabolic glucose sensor in pancreatic alpha-cells and, hence, mediate a mechanism for direct regulation of glucagon release by extracellular glucose. Since these cells do not express Glut2, we suggest that glucose sensing does not necessarily require the coexpression of Glut2 and glucokinase. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8692940

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. Induction of endothelial cell activation by a triple helical alpha2beta integrin ligand, derived from type I collagen alpha1(I)496-507.

    PubMed

    Baronas-Lowell, Diane; Lauer-Fields, Janelle L; Fields, Gregg B

    2004-01-09

    Endothelial cell activation involves the elevated expression of cell adhesion molecules, chemoattractants, chemokines, and cytokines. These expression profiles may be regulated by integrin-mediated cell signaling pathways. In the current study, an alpha2beta1 integrin triple helical peptide ligand derived from type I collagen residues alpha1(I)496-507 was examined for induction of human aortic endothelial cell (HAEC) activation. In addition, a "miniextracellular matrix" composed of a mixture of the alpha1(I)496-507 ligand and a second, alpha-helical ligand incorporating the endothelial cell proliferating region of SPARC (secreted protein acidic and rich in cysteine) was studied for induction of HAEC activation. Following HAEC adhesion to alpha1(I)496-507, mRNA expression of E-selectin-1, vascular and intercellular cell adhesion molecules-1, and monocytic chemoattractant protein-1 was stimulated, whereas that of endothelin-1 was inhibited. Enzyme-linked immunosorbent assay analysis demonstrated that E-selectin-1 and monocytic chemoattractant protein-1 expression was also stimulated, whereas endothelin-1 protein expression diminished. Engagement of the alpha2beta1 integrin initiated a HAEC response similar to that of tumor necrosis factor-alpha-induced HAECs but was not sufficient to induce an inflammatory response. Addition of the SPARC119-122 region had only a slight effect on HAEC activation. Other cell-extracellular matrix interactions appear to be required to elicit an inflammatory response. The alpha2beta1 integrin specific triple helical peptide ligand described herein represents a more general in vitro model system by which gene expression and protein production profiles induced by binding to a single cellular receptor type can be quantified.

  7. 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.

  8. Role of alpha7-nicotinic acetylcholine receptor in human non-small cell lung cancer proliferation.

    PubMed

    Paleari, L; Catassi, A; Ciarlo, M; Cavalieri, Z; Bruzzo, C; Servent, D; Cesario, A; Chessa, L; Cilli, M; Piccardi, F; Granone, P; Russo, P

    2008-12-01

    Lung cancer is the most common cause of cancer death in the world. Cigarette smoking represents the major risk factor. Nicotine, an active component of cigarettes, can induce cell proliferation, angiogenesis and apoptosis resistance. All these events are mediated through the nicotinic acetylcholine receptor (nAChR) expressed on lung cancer cells. We speculate that new insights into the pathophysiological roles of nAChR may lead to new therapeutic avenues to reduce non-small cell lung cancer (NSCLC) tumour growth. Human samples of NSCLC, cell lines and mouse models were utilized in Western blotting, reverse transcriptase polymerase chain reaction and apoptosis studies. Human NSCLC tissues expressed alpha7-nAChR. This expression was higher in smoking patients with squamous carcinomas than those with adenocarcinomas and in male smoking patients than in females. All the data support the hypothesis that major expression of alpha7-nAChR is related to major activation of the Rb-Raf-1/phospho-ERK/phospho-p90RSK pathway. alpha7-nAChR antagonists, via mitochondria associated apoptosis, inhibited proliferation of human NSCLC primary and established cells. Nicotine stimulates tumour growth in a murine model, A549 cells orthotopically grafted. The effects of nicotine were associated with increases in phospho-ERK in tumours. Proliferation effects of nicotine could be blocked by inhibition of alpha7-nAChR by the high affinity ligand alpha-cobratoxin. These results showed that alpha7-nAChR plays an important role in NSCLC cell growth and tumour progression as well as in cell death.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. Stevioside counteracts the alpha-cell hypersecretion caused by long-term palmitate exposure.

    PubMed

    Hong, J; Chen, L; Jeppesen, P B; Nordentoft, I; Hermansen, K

    2006-03-01

    Long-term exposure to fatty acids impairs beta-cell function in type 2 diabetes, but little is known about the chronic effects of fatty acids on alpha-cells. We therefore studied the prolonged impact of palmitate on alpha-cell function and on the expression of genes related to fuel metabolism. We also investigated whether the antihyperglycemic agent stevioside was able to counteract these effects of palmitate. Clonal alpha-TC1-6 cells were cultured with palmitate in the presence or absence of stevioside. After 72 h, we evaluated glucagon secretion, glucagon content, triglyceride (TG) content, and changes in gene expression. Glucagon secretion was dose-dependently increased after 72-h culture, with palmitate at concentrations >or=0.25 mM (P< 0.05). Palmitate (0.5 mM) enhanced TG content of alpha-cells by 73% (P< 0.01). Interestingly, stevioside (10(-8) and 10(-6) M) reduced palmitate-stimulated glucagon release by 22 and 45%, respectively (P< 0.01). There was no significant change in glucagon content after 72-h culture with palmitate and/or stevioside. Palmitate increased carnitine palmitoyltransferase I (CPT I) mRNA level, whereas stevioside enhanced CPT I, peroxisome proliferator-activated receptor-gamma, and stearoyl-CoA desaturase gene expressions in the presence of palmitate (P<0.05). In conclusion, long-term exposure to elevated fatty acids leads to a hypersecretion of glucagon and an accumulation of TG content in clonal alpha-TC1-6 cells. Stevioside was able to counteract the alpha-cell hypersecretion caused by palmitate and enhanced the expression of genes involved in fatty acid metabolism. This indicates that stevioside may be a promising antidiabetic agent in treatment of type 2 diabetes.

  14. 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.

  15. The role of hydrogen peroxide and RRR-alpha-tocopherol in smooth muscle cell proliferation.

    PubMed

    Azzi, A; Cantoni, O; Ozer, N; Boscoboinik, D; Spycher, S

    1996-01-01

    Oxidants can be considered early growth signals, since they have been shown to activate a number of pathways that are also stimulated by growth factors. In particular, H(2)O(2) activates the protein kinase C signal transduction pathway in smooth muscle cells. These events certainly play a role in the activation of the DNA synthesis machinery although it is still unclear whether they can also regulate the lethal response. Evidence exists of an oxidant-mediated increase in tyrosine protein phosphorylation as an early event in the signal transduction cascade of growth factor receptors, leading to augmentation of cell proliferation. Oxidants can also induce transcription of enzymes, such as ornithine decarboxylase and the phosphatase CL-100. CL-100 is the first example of a new class of protein phosphatases responsible for modulating the activation of MAP kinase following exposure of quiescent cells to growth factors and further implicates MAP kinase activation/deactivation in the cellular response to hydrogen peroxide. Moreover H(2)O(2) activates the MAP kinase cascade by stimulating the tyrosine kinase and protein kinase C pathways. JNK1, a relative of the MAP kinase group, is activated by dual phosphorylation at Thr and Tyr during the UV response. RRR-alpha-tocopherol and RRR-beta-tocopherol have different and competing effects on smooth muscle cell proliferation, indicating that they do not act as antioxidants. The earliest event brought by RRR-alpha-tocopherol in the signal transduction cascade contolling receptor mediated cell growth is the inhibition of the transcription factor AP-1, activated by phorbol esters. RRR-beta-tocopherol alone is without effect but in combination with RRR-alpha-tocopherol prevents the AP-1-inhibiting effect of the latter. Protein kinase C is inhibited by RRR-alpha-tocopherol and not by RRR-beta-tocopherol, which also in this case prevented the effect of RRR-alpha-tocopherol. The inhibition of RRR-alpha-tocopherol of protein kinase C

  16. 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.

  17. 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.

  18. 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.

  19. T-Type calcium channel alpha1G and alpha1H subunits in human retinoblastoma cells and their loss after differentiation.

    PubMed

    Hirooka, Kazuyuki; Bertolesi, Gabriel E; Kelly, Melanie E M; Denovan-Wright, Eileen M; Sun, Xiaolu; Hamid, Jawed; Zamponi, Gerald W; Juhasz, Alexander E; Haynes, Lawrence W; Barnes, Steven

    2002-07-01

    Human retinoblastoma cells are multipotent retinal precursor cells capable of differentiating into photoreceptors, neurons, and glia. The current-voltage relation of the undifferentiated cells is dominated by a transient inward current that disappears shortly after differentiation. In 20 mM Ba(2+)-containing bath solutions, the current has an activation midpoint near -25 mV and appears to be fully inactivated at -20 mV. Sr(2+) and Ca(2+) are preferred charge carriers relative to Ba(2+), and the current vanishes in the absence of these divalent cations. Cd(2+) blocks the current with an IC(50) of 160 microM, and Ni(2+) blocks in a biphasic manner with IC(50)s of 22 and 352 microM. The current is unaffected when sodium is replaced with other monovalent cations, and it is insensitive to nifedipine, omega-conotoxin GVIA, omega-agatoxin IVA, and omega-conotoxin MVIIC. RT-PCR revealed the presence of alpha 1G and alpha 1H mRNA in undifferentiated cells, but following differentiation, a striking reduction of both alpha 1G and alpha 1H mRNA was found, and this was paralleled by the loss of T-type Ca channel currents. alpha 1I subunit mRNA levels were low in undifferentiated and differentiated cells. These results suggest that T-type Ca channels could play a role in undifferentiated retinoblastoma cell physiology since alpha 1G and alpha 1H Ca channel subunit expression is reduced in cells that have differentiated and exited the cell cycle.

  20. Interferon-alpha down-regulates the interleukin-6 receptor in a human multiple myeloma cell line, U266.

    PubMed Central

    Anthes, J C; Zhan, Z; Gilchrest, H; Egan, R W; Siegel, M I; Billah, M M

    1995-01-01

    The effects of interferon-alpha (IFN-alpha) on the interleukin-6 (IL-6) receptor in a multiple myeloma cell line, U266, have been examined. IFN-alpha inhibits [3H]thymidine incorporation in U266 cells in a time- and dose-dependent manner. Furthermore, IFN-alpha inhibits the ability of IL-6 to induce increases in [3H]thymidine incorporation. While IFN-alpha suppresses the ability of 125I-IL-6 to bind to the IL-6 receptor on U266 cells, this effect is not due to competition of IFN-alpha with IL-6 for the IL-6 receptor. Although IFN-alpha induces IL-6 synthesis in the U266 cell, inhibition of IL-6 binding occurs when IL-6 synthesis is minimal. Furthermore, after pretreatment of U266 cells with neutralizing anti-IL-6 antibodies, IFN-alpha still inhibits 125I-IL-6 binding. These data suggest that IFN-alpha inhibition of 125I-IL-6 binding does not involve IL-6 synthesis. IFN-alpha reduces 125I-IL-6 binding without affecting its affinity, suggesting that IFN-alpha inhibits IL-6 receptor expression. Although pretreatment with cycloheximide inhibits 125I-IL-6 binding, IFN-alpha does not cause a selective decrease in the levels of gp130 or IL-6 receptor mRNA at times when 125I-IL-6 binding is inhibited. These observations indicate that IFN-alpha lowers IL-6 receptor density on U266 cells by mechanisms other than competitive binding or lowering IL-6 receptor mRNA production. Receptor down-regulation may be a mechanism of IFN-alpha-induced inhibition of growth in U266 cells. Images Figure 9 PMID:7619053

  1. 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

  2. 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

  3. 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

  4. Hepatocyte differentiation of WIF-B cells includes a high capacity of interleukin-6-mediated induction of alpha 1-acid glycoprotein and alpha 2-macroglobulin.

    PubMed

    Guillonneau, F; Drechou, A; Poüs, C; Chevalier, S; Lardeux, B; Cassio, D; Durand, G

    1999-01-11

    Responsiveness to cytokine-mediated acute inflammatory stimuli of the highly differentiated and polarized WIF-B hybrid cell line was studied by measuring the induction of alpha 1-acid glycoprotein and alpha 2-macroglobulin mRNAs after interleukin-1, interleukin-6 and tumor necrosis factor-alpha treatments in the presence of dexamethasone. Compared with their Fao parent, WIF-B cells were 10 times more responsive to 24-h interleukin-6 induction regarding alpha 2-macroglobulin induction. At variance from the response measured in Fao cells, the late effects of interleukin-6 treatment confirmed the higher sensitivity of WIF-B cells to this cytokine as a 72-h treatment as 10 times more effective than a 24-h treatment at inducting alpha 1-acid glycoprotein mRNA. These findings highlight the hepatocyte differentiation of WIF-B cells compared with other hepatoma cell lines, with respect to the regulation of acute-phase protein gene expression. They also make WIF-B cells a convenient model to study the molecular effects of interleukin-6 in terms of transduction and/or transcription, and the many cross-talks that occur during the regulation of acute-phase protein gene expression.

  5. 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

  6. 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

  7. 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.

  8. Ablation of phosphoinositide-3-kinase class II alpha suppresses hepatoma cell proliferation

    SciTech Connect

    Ng, Stanley K.L.; Neo, Soek-Ying; Yap, Yann-Wan; Karuturi, R. Krishna Murthy; Loh, Evelyn S.L.; Liau, Kui-Hin; Ren, Ee-Chee

    2009-09-18

    Cancer such as hepatocellular carcinoma (HCC) is characterized by complex perturbations in multiple signaling pathways, including the phosphoinositide-3-kinase (PI3K/AKT) pathways. Herein we investigated the role of PI3K catalytic isoforms, particularly class II isoforms in HCC proliferation. Among the siRNAs tested against the eight known catalytic PI3K isoforms, specific ablation of class II PI3K alpha (PIK3C2{alpha}) was the most effective in impairing cell growth and this was accompanied by concomitant decrease in PIK3C2{alpha} mRNA and protein levels. Colony formation ability of cells deficient for PIK3C2{alpha} was markedly reduced and growth arrest was associated with increased caspase 3 levels. A small but significant difference in gene dosage and expression levels was detected between tumor and non-tumor tissues in a cohort of 19 HCC patients. Taken together, these data suggest for the first time that in addition to class I PI3Ks in cancer, class II PIK3C2{alpha} can modulate HCC cell growth.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. Prostaglandin F2 alpha stimulates progesterone secretion by porcine luteal cells in vitro throughout the estrous cycle.

    PubMed

    Gadsby, J E; Earnest, K L

    1994-08-01

    In this study we examined the stimulatory effects of PGF2 alpha on progesterone secretion by porcine luteal cells on different days of the estrous cycle, and the effects of PGF2 alpha, A23187 and PMA on progesterone secretion by isolated large and small luteal cells, in vitro. Corpora lutea were obtained from cycling pigs (days 6-16), collagenase dispersed and luteal cells incubated in medium 199 in the absence or presence of increasing doses of PGF2 alpha, A23187, and PMA. Progesterone concentrations in spent media were measured by RIA. PGF2 alpha stimulation of progesterone secretion by mixed luteal cells did not vary significantly throughout the estrous cycle. Progesterone secretion by large, but not small, luteal cells was increased (p < 0.05) in a dose-dependent fashion by PGF2 alpha. A23187 also caused a dose-dependent increase in progesterone secretion by large luteal cells but inhibited small luteal cells. Progesterone secretion by both large and small luteal cells was significantly increased by increasing doses of PMA. We conclude that the stimulatory response of luteal cells to PGF2 alpha in vitro did not correlate with PGF2 alpha receptor concentrations (not measured in this study), and we speculate that calcium/protein kinase C may be involved in mediating the stimulatory action of PGF2 alpha on luteal cell progesterone secretion.

  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. 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.

  18. Central tolerance regulates B cells reactive with Goodpasture antigen alpha3(IV)NC1 collagen

    PubMed Central

    Zhang, Ying; Su, Susan C.; Hecox, Douglas B.; Brady, Graham F.; Mackin, Katherine M.; Clark, Amy G.; Foster, Mary H.

    2008-01-01

    Patients and rodents with Goodpasture’s syndrome (GPS) develop severe autoimmune crescentic glomerulonephritis, kidney failure, and lung hemorrhage due to binding of pathogenic autoantibodies to the NC1 domain of the alpha3 chain of type IV collagen. Target epitopes are cryptic, normally hidden from circulating antibodies by protein-protein interactions and the highly tissue-restricted expression of the alpha3(IV) collagen chain. Based on this limited antigen exposure, it has been suggested that target epitopes are not available as B cell tolerogens. To determine how pathogenic anti-GPS autoantibody responses are regulated, we generated an immunoglobulin (Ig) transgenic (Tg) mouse model that expresses an Ig that binds alpha3(IV)NC1 collagen epitopes recognized by serum IgG of patients with GPS. Phenotypic analysis reveals B cell depletion and light chain editing in Tg mice. To determine the default tolerance phenotype in the absence of receptor editing and endogenous lymphocyte populations, we crossed Tg mice two generations with mice deficient in recombinase activating gene (Rag). Resulting Tg Rag-deficient mice have central B cell deletion. Thus development of Tg anti-alpha3(IV)NC1 collagen B cells is halted in the bone marrow, at which point the cells are deleted unless rescued by a Rag enzyme-dependent process, such as editing. The central tolerance phenotype implies that tolerizing self antigen is expressed in bone marrow. PMID:18941198

  19. Central tolerance regulates B cells reactive with Goodpasture antigen alpha3(IV)NC1 collagen.

    PubMed

    Zhang, Ying; Su, Susan C; Hecox, Douglas B; Brady, Graham F; Mackin, Katherine M; Clark, Amy G; Foster, Mary H

    2008-11-01

    Patients and rodents with Goodpasture's syndrome (GPS) develop severe autoimmune crescentic glomerulonephritis, kidney failure, and lung hemorrhage due to binding of pathogenic autoantibodies to the NC1 domain of the alpha3 chain of type IV collagen. Target epitopes are cryptic, normally hidden from circulating Abs by protein-protein interactions and the highly tissue-restricted expression of the alpha3(IV) collagen chain. Based on this limited Ag exposure, it has been suggested that target epitopes are not available as B cell tolerogens. To determine how pathogenic anti-GPS autoantibody responses are regulated, we generated an Ig transgenic (Tg) mouse model that expresses an Ig that binds alpha3(IV)NC1 collagen epitopes recognized by serum IgG of patients with GPS. Phenotypic analysis reveals B cell depletion and L chain editing in Tg mice. To determine the default tolerance phenotype in the absence of receptor editing and endogenous lymphocyte populations, we crossed Tg mice two generations with mice deficient in Rag. Resulting Tg Rag-deficient mice have central B cell deletion. Thus, development of Tg anti-alpha3(IV)NC1 collagen B cells is halted in the bone marrow, at which point the cells are deleted unless rescued by a Rag enzyme-dependent process, such as editing. The central tolerance phenotype implies that tolerizing self-Ag is expressed in bone marrow.

  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. [Preparation of transfusable human universal red blood cell with recombinant alpha-galactosidase].

    PubMed

    Gong, Feng; Lü, Qiu-Shuang; You, Ying; Gao, Hong-Wei; Bao, Guo-Qiang; Gao, Xin; Li, Su-Bo; Li, Li-Li; Wang, Ying-Li; Tian, Shu-Guang; Zhang, Zhi-Xin; Zhang, Ping; Zhang, Yang-Pei

    2005-04-01

    In order to meet the demand for safe transfusion in special conditions and to utilize the donated blood supply efficiently, technology has been developed to convert erythrocytes from type A, B, or AB to "universal donor" blood. Conversion of blood type B to O was performed by means of recombinant alpha-galactosidase digestion. The results showed that blood type B to O was converted successfully, 1 transfusion unit of red cells of group B (100 ml totally) could converted to universal blood cells in the optimal conditions including pH 5.6, 26 degrees C, 2 hours, obturation and sterilization. It is concluded that the universal red blood cells converted from group B to group O are conformed to demand of identification rules of biological products, no harmful effects of alpha-galactosidase on cell structure and function are observed. The converted red cells can stored in 4 degrees C for 21 days.

  5. Individual Amino Acid Supplementation Can Improve Energy Metabolism and Decrease ROS Production in Neuronal Cells Overexpressing Alpha-Synuclein.

    PubMed

    Delic, Vedad; Griffin, Jeddidiah W D; Zivkovic, Sandra; Zhang, Yumeng; Phan, Tam-Anh; Gong, Henry; Chaput, Dale; Reynes, Christian; Dinh, Vinh B; Cruz, Josean; Cvitkovic, Eni; Placides, Devon; Frederic, Ernide; Mirzaei, Hamed; Stevens, Stanley M; Jinwal, Umesh; Lee, Daniel C; Bradshaw, Patrick C

    2017-06-15

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein accumulation and loss of dopaminergic neurons in the substantia nigra (SN) region of the brain. Increased levels of alpha-synuclein have been shown to result in loss of mitochondrial electron transport chain complex I activity leading to increased reactive oxygen species (ROS) production. WT alpha-synuclein was stably overexpressed in human BE(2)-M17 neuroblastoma cells resulting in increased levels of an alpha-synuclein multimer, but no increase in alpha-synuclein monomer levels. Oxygen consumption was decreased by alpha-synuclein overexpression, but ATP levels did not decrease and ROS levels did not increase. Treatment with ferrous sulfate, a ROS generator, resulted in decreased oxygen consumption in both control and alpha-synuclein overexpressing cells. However, this treatment only decreased ATP levels and increased ROS production in the cells overexpressing alpha-synuclein. Similarly, paraquat, another ROS generator, decreased ATP levels in the alpha-synuclein overexpressing cells, but not in the control cells, further demonstrating how alpha-synuclein sensitized the cells to oxidative insult. Proteomic analysis yielded molecular insights into the cellular adaptations to alpha-synuclein overexpression, such as the increased abundance of many mitochondrial proteins. Many amino acids and citric acid cycle intermediates and their ester forms were individually supplemented to the cells with L-serine, L-proline, L-aspartate, or L-glutamine decreasing ROS production in oxidatively stressed alpha-synuclein overexpressing cells, while diethyl oxaloacetate or L-valine supplementation increased ATP levels. These results suggest that dietary supplementation with individual metabolites could yield bioenergetic improvements in PD patients to delay loss of dopaminergic neurons.

  6. 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.

  7. 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.

  8. Over-expression of CCL3 MIP-1alpha in a blastoid mantle cell lymphoma with hypercalcemia.

    PubMed

    Hattori, Norimichi; Nakamaki, Tsuyoshi; Ariizumi, Hirotsugu; Homma, Mayumi; Yamochi-Onizuka, Toshiko; Ota, Hidekazu; Tomoyasu, Shigeru

    2010-05-01

    We analyzed a case with the blastoid variant of mantle cell lymphoma (MCL-BV), a rare subtype of B-cell lymphoma, presenting with marked hypercalcemia at diagnosis. Enzyme-linked immunosorbent assay (ELISA) showed elevated serum levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-1alpha (MIP-1alpha), and type I collagen telopeptide, but not parathyroid hormone, calcitriol or parathyroid hormone-related peptide at diagnosis, suggesting local osteoclastic hypercalcemia in this case. By reverse transcription polymerase chain reaction (RT-PCR) analysis, we found predominant expression of mRNA for MIP-1alpha in addition to those for receptor-activator of nuclear-factor kappa B ligand (RANKL), TNF-alpha, and IL-6 in lymphoma cells obtained from the patient. Furthermore, recombinant MIP-1alpha significantly stimulated (3)H-thymidine uptake by isolated MCL cells in vitro. Treatment with intravenous fluids, bisphosphonate, and methylprednisolone followed by combination chemotherapy promptly corrects the hypercalcemia and successfully induced complete remission, which was accompanied by a decrease of these cytokines in the serum, including MIP-1alpha. In the present case, MIP-1alpha, an osteoclast-activating factor produced by mantle lymphoma cells, may contribute to the development of hypercalcemia. It likely acts through RANKL expression in tumor cells and/or stroma cells, as indicated in multiple myeloma (MM) and adult T-cell leukemia/lymphoma (ATLL). Furthermore, MIP-1alpha is also involved in the development of an aggressive phenotype on MCL by stimulating proliferation of these lymphoma cells. In summary, the present study demonstrated that MIP-1alpha is an important factor in the development of both hypercalcemia and an aggressive phenotype in some types of B-cell lymphoma.

  9. 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.

  10. 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.

  11. 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.

  12. Laminin alpha 2 enables glioblastoma stem cell growth

    PubMed Central

    Lathia, Justin D.; Li, Meizhang; Hall, Peter E.; Gallagher, Joseph; Hale, James S.; Wu, Qiulian; Venere, Monica; Levy, Emily; Rani, MR Sandhya; Huang, Ping; Bae, Eunnyung; Selfridge, Julia; Cheng, Lin; Guvenc, Hacer; McLendon, Roger E.; Nakano, Ichiro; Sloan, Andrew E.; Phillips, Heidi S.; Lai, Albert; Gladson, Candece; Bredel, Markus; Bao, Shideng; Hjelmeland, Anita B.; Rich, Jeremy N.

    2013-01-01

    Objective Glioblastomas (GBMs) are lethal cancers that display cellular hierarchies parallel to normal brain. At the apex are GBM stem cells (GSCs), which are relatively resistant to conventional therapy. An important driver of malignancy and self-renewal in GSCs are interactions with the adjacent perivascular niche. Extracellular matrix (ECM) cues instruct neural stem/progenitor cell-niche interactions and the objective of our study was to elucidate its composition and contribution to GSC maintenance in the perivascular niche. Methods We interrogated human tumor tissue for immunofluorescence analysis and derived GSC from tumor tissues for functional studies. Bioinformatics analyses were conducted by mining publicly available databases. Results We find that laminin ECM proteins are localized to the perivascular GBM niche and inform negative patient prognosis. To identify the source of laminins, we characterized cellular elements within the niche and found that laminin α chains were expressed by non-stem tumor cells and tumor associated endothelial cells (ECs). RNA interference targeting laminin α2 inhibited GSC growth and self-renewal. In co-culture studies of GSCs and ECs, laminin α2 knockdown in ECs resulted in decreased tumor growth. Interpretation Our studies highlight the contribution of non-stem tumor cell-derived laminin juxtracrine signaling. As laminin α2 has recently been identified as a molecular marker of aggressive ependymoma, we propose that the brain vascular ECM promotes tumor malignancy through maintenance of the GSC compartment providing not only a molecular fingerprint but also a possible therapeutic target. PMID:23280793

  13. Arsenite enhances tumor necrosis factor-{alpha}-induced expression of vascular cell adhesion molecule-1

    SciTech Connect

    Tsou, T.-C. . E-mail: tctsou@nhri.org.tw; Yeh, Szu Ching; Tsai, E.-M.; Tsai, F.-Y.; Chao, H.-R.; Chang, Louis W.

    2005-11-15

    Epidemiological studies demonstrated a high association of vascular diseases with arsenite exposure. We hypothesize that arsenite potentiates the effect of proinflammatory cytokines on vascular endothelial cells, and hence contributes to atherosclerosis. In this study, we investigated the effect of arsenite and its induction of glutathione (GSH) on vascular cell adhesion molecule-1 (VCAM-1) protein expression in human umbilical vein endothelial cells (HUVECs) in response to tumor necrosis factor-{alpha} (TNF-{alpha}), a typical proinflammatory cytokine. Our study demonstrated that arsenite pretreatment potentiated the TNF-{alpha}-induced VCAM-1 expression with up-regulations of both activator protein-1 (AP-1) and nuclear factor-{kappa}B (NF-{kappa}B). To elucidate the role of GSH in regulation of AP-1, NF-{kappa}B, and VCAM-1 expression, we employed L-buthionine (S,R)-sulfoximine (BSO), a specific {gamma}-glutamylcysteine synthetase ({gamma}-GCS) inhibitor, to block intracellular GSH synthesis. Our investigation revealed that, by depleting GSH, arsenite attenuated the TNF-{alpha}-induced VCAM-1 expression as well as a potentiation of AP-1 and an attenuation of NF-{kappa}B activations by TNF-{alpha}. Moreover, we found that depletion of GSH would also attenuate the TNF-{alpha}-induced VCAM-1 expression with a down-regulation of the TNF-{alpha}-induced NF-{kappa}B activation and without significant effect on AP-1. On the other hand, the TNF-{alpha}-induced VCAM-1 expression could be completely abolished by inhibition of AP-1 or NF-{kappa}B activity, suggesting that activation of both AP-1 and NF-{kappa}B was necessary for VCAM-1 expression. In summary, we demonstrate that arsenite enhances the TNF-{alpha}-induced VCAM-1 expression in HUVECs via regulation of AP-1 and NF-{kappa}B activities in a GSH-sensitive manner. Our present study suggested a potential mechanism for arsenite in the induction of vascular inflammation and vascular diseases via modulating the actions

  14. 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

  15. 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

  16. 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.

  17. Overexpression of hypoxia-inducible factor 1 alpha improves immunomodulation by dental mesenchymal stem cells.

    PubMed

    Martinez, Victor G; Ontoria-Oviedo, Imelda; Ricardo, Carolina P; Harding, Sian E; Sacedon, Rosa; Varas, Alberto; Zapata, Agustin; Sepulveda, Pilar; Vicente, Angeles

    2017-09-29

    Human dental mesenchymal stem cells (MSCs) are considered as highly accessible and attractive MSCs for use in regenerative medicine, yet some of their features are not as well characterized as other MSCs. Hypoxia-preconditioning and hypoxia-inducible factor 1 (HIF-1) alpha overexpression significantly improves MSC therapeutics, but the mechanisms involved are not fully understood. In the present study, we characterize immunomodulatory properties of dental MSCs and determine changes in their ability to modulate adaptive and innate immune populations after HIF-1 alpha overexpression. Human dental MSCs were stably transduced with green fluorescent protein (GFP-MSCs) or GFP-HIF-1 alpha lentivirus vectors (HIF-MSCs). A hypoxic-like metabolic profile was confirmed by mitochondrial and glycolysis stress test. Capacity of HIF-MSCs to modulate T-cell activation, dendritic cell differentiation, monocyte migration, and polarizations towards macrophages and natural killer (NK) cell lytic activity was assessed by a number of functional assays in co-cultures. The expression of relevant factors were determined by polymerase chain reaction (PCR) analysis and enzyme-linked immunosorbent assay (ELISA). While HIF-1 alpha overexpression did not modify the inhibition of T-cell activation by MSCs, HIF-MSCs impaired dendritic cell differentiation more efficiently. In addition, HIF-MSCs showed a tendency to induce higher attraction of monocytes, which differentiate into suppressor macrophages, and exhibited enhanced resistance to NK cell-mediated lysis, which supports the improved therapeutic capacity of HIF-MSCs. HIF-MSCs also displayed a pro-angiogenic profile characterized by increased expression of CXCL12/SDF1 and CCL5/RANTES and complete loss of CXCL10/IP10 transcription. Immunomodulation and expression of trophic factors by dental MSCs make them perfect candidates for cell therapy. Overexpression of HIF-1 alpha enhances these features and increases their resistance to allogenic NK

  18. 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

  19. alpha-Lipoic acid and ascorbate prevent LDL oxidation and oxidant stress in endothelial cells.

    PubMed

    Sabharwal, Anup K; May, James M

    2008-02-01

    Both alpha-lipoic acid (LA) and ascorbic acid (vitamin C) have been shown to improve endothelial dysfunction, a precursor of atherosclerosis. Since oxidant stress can cause endothelial dysfunction, we tested the interaction and efficacy of these antioxidants in preventing oxidant damage to lipids due to both intra- and extracellular oxidant stresses in EA.hy926 endothelial cells. LA spared intracellular ascorbate in culture and in response to an intracellular oxidant stress induced by the redox cycling agent menadione. Extracellular oxidant stress generated by incubating cells for 2 h in with 0.2 mg/ml LDL and 5 muM Cu2+ caused a time-dependent increase of the lipid peroxidation product malondialdehyde in both cells and LDL, preceded by rapid disappearance of; alpha-tocopherol in LDL. alpha-Lipoic acid at concentrations of 40-80 microM blunted these effects. Similarly, intracellular ascorbate concentrations of 1-2 mM also prevented Cu2+-induced lipid peroxidation in LDL and cells. Cu2+-dependent oxidation of LDL in the presence of ascorbate-loaded cells decreased intracellular ascorbate by 20%, but this decrease was not reversed by LA. Both LA and ascorbate protect endothelial cells and LDL from either intra- or extracellular oxidant stress, but that LA does not spare ascorbate in oxidatively stressed cells.

  20. Differential expression of hypoxia inducible factor-1 alpha and tumor cell proliferation between squamous cell carcinomas and adenocarcinomas among operable non-small cell lung carcinomas.

    PubMed Central

    Lee, Chang Hun; Lee, Min Ki; Kang, Chi Duk; Kim, Young Dae; Park, Do Youn; Kim, Jee Yeon; Sol, Mee Young; Suh, Kang Suek

    2003-01-01

    This study aimed to evaluate whether the elevated level of hypoxia-inducible factor-1alpha (HIF-1alpha) correlated with histologic types, angiogenesis, tumor cell proliferation, and clinical parameters in common non-small cell lung carcinomas (NSCLCs). We performed immunohistochemical stains using paraffin-embedded tissue blocks from 84 cases of operable NSCLC [No. of squamous cell carcinoma (SCC), 45; No. of adenocarcinoma (AC), 39]. HIF-1alpha expression was related with histologic types (66.7% in SCCs vs 20.5% in ACs, p<0.001), but not with lymph node status, tumor stage, vascular endothelial growth factor expression, microvessel density (MVD), and proliferating cell nuclear antigen (PCNA) index (p>0.05, respectively). As for the histologic types, MVD and PCNA index were significantly higher in SCCs than in ACs (p=0.009 and p=0.016, respectively). Among HIF-1alpha positive carcinomas, MVD was significantly higher in HIF-1alpha positive SCCs than in HIF-1alpha positive ACs (p=0.023). The overall survival curves were not associated with HIF-1alpha expression or any other histologic parameters (p>0.05). These findings suggest that HIF-1alpha expression in NSCLCs may play a differential role according to histologic types, but its prognostic significance is indeterminate. PMID:12692416

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. TGF-alpha can act as morphogen and/or mitogen in a colon-cancer cell line.

    PubMed

    Liu, D; Gagliardi, G; Nasim, M M; Alison, M R; Oates, T; Lalani, E N; Stamp, G W; Pignatelli, M

    1994-02-15

    Transforming growth factor alpha (TGF-alpha) has multifunctional biological effects on a variety of mesenchymal and epithelial cells. It is a potent mitogen for a number of normal and transformed cell types, regulates extracellular matrix (ECM) production and promotes breast, kidney and lung morphogenesis. To clarify the role of ECM proteins in the morphogenetic and mitogenic effects of TGF-alpha, we have used a human colon carcinoma cell line (SW1222) which expresses EGF receptor. Here we show that TGF-alpha at 1 ng/ml increases the proliferation of SW1222 cells, but only when they are cultured on plastic rather than collagen-coated plates. Higher concentrations of TGF-alpha (10 ng/ml) did not increase cell proliferation but significantly enhanced the crypt-like glandular differentiation when cells were grown in 3-dimensional collagen gel (p = 0.027). These effects were accompanied by increased expression of alpha 2 beta 1 and alpha 3 beta 1 integrin molecules, which are receptors for extracellular matrix proteins, and by a statistically significant increase in binding of SW1222 cells to type-1 collagen. The effects of TGF-alpha both on binding to type-1 collagen and on morphological differentiation in 3-dimensional collagen gel were inhibited by monoclonal antibodies recognizing the alpha 2 beta 1 integrin. These data indicate that the morphogenetic or mitogenic activities of TGF-alpha are critically dependent on cellular interactions with extracellular matrix proteins and are primarily mediated by the alpha 2 beta 1 integrin receptor. Inappropriate expression of this growth factor, seen in tumours whose cell-matrix interactions are greatly impaired, could have deleterious effects on the maintenance of normal tissue architecture and growth control.

  12. 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.

  13. 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.

  14. Ability of cell-sized beads bearing tumor cell membrane proteins to stimulate LAK cells to secrete interferon-gamma and tumor necrosis factor-alpha.

    PubMed

    Chong, A S; Pinkard, J K; Lam, K S; Scuderi, P; Hersh, E M; Grimes, W J

    1991-04-15

    We recently reported that lymphokine activated killer (LAK) cells were stimulated to release both interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) when stimulated by a variety of tumor cells. We proposed then that the released cytokines may play a role in mediating tumor cell regression in vivo. In this paper, we provide further information on the nature of the signals, provided by the tumor cells (K562 erythroleukemia), that stimulate LAK cells to secrete IFN-gamma and TNF-alpha. Using a previously published protocol for coating tumor-membrane molecules onto cell-sized hydrophobic beads (also called pseudocytes), we demonstrate that the signal provided by the tumor cell is membrane associated. Beads coated with K562 membranes stimulated LAK cells to release IFN-gamma and TNF-alpha. The pretreatment of these beads with trypsin and sodium periodate eliminated the ability of these pseudocytes to stimulate cytokine release in LAK cells. The glycoproteins that stimulate LAK cells to secrete IFN-gamma and TNF-alpha were further enriched by their ability to bind concanavalin A (Con A, Jack Bean). To determine if the tumor-associated molecules that stimulate LAK cells to release IFN-gamma and TNF-alpha are also the molecules involved in mediating tumor cell lysis, we tested the ability of the Con A binding and nonbinding proteins to inhibit the LAK cell-mediated lysis of K562 cells. Our results demonstrate that molecules that inhibited LAK cell-mediated cytotoxicity were not enriched by Con A. These results are therefore consistent with the conclusion that different sets of tumor-associated molecules are involved in the stimulation of LAK cells to secrete cytokine and in the induction of LAK cells to mediate tumor cell cytolysis.

  15. Inhibition of protein kinase C activity and vascular smooth muscle cell growth by d-alpha-tocopherol.

    PubMed

    Boscoboinik, D O; Chatelain, E; Bartoli, G M; Stäuble, B; Azzi, A

    1994-12-30

    The inhibition by d-alpha-tocopherol of protein kinase C activity has been studied in synchronised A7r5 rat smooth muscle cells during the cell cycle. Cell protein kinase C activity has been found to oscillate, with a minimum in the G0 phase, a maximum in the late G1 phase and a new minimum in the S phase. An inhibition of protein kinase C activity by d-alpha-tocopherol appears to be at the basis of cell growth inhibition. Nevertheless, the amount of the different protein kinase C isoenzymes present in smooth muscle cells, measured by their specific antibodies, does not change during the cell cycle in both untreated and d-alpha-tocopherol-treated cells. The possible mechanisms of protein kinase C modulation during the cell cycle and of its inhibition by d-alpha-tocopherol are discussed.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. Melatonin inhibits maneb-induced aggregation of alpha-synuclein in rat pheochromocytoma cells.

    PubMed

    Ishido, Masami

    2007-03-01

    Melatonin, a secretory product of the pineal gland, is involved in the regulation of circadian and seasonal rhythms, in oncostasis, and in inducing osteoblast differentiation. Furthermore, melatonin is a scavenger of a number of reactive oxygen and reactive nitrogen species both in vitro and in vivo. In this study, the antioxidant nature of melatonin was shown to prevent cultured neural cells from apoptosis induced by endocrine-disrupting chemical, maneb. The neurotoxicity of the fungicide, maneb (1 microg/mL), on the PC12 cells was elicited through apoptotic cell death, concomitant with aggregation of alpha-synuclein, a feature of Parkinson's disease. Activation of caspase-3/7 was associated with this process. A fluorescence rationing technique using a mitochondrial dye revealed that maneb altered the mitochondrial membrane potential of the neural cells. However, melatonin (1 nm) largely prevented the neural cells from the neural toxicant by inhibition of both caspase-3/7 activation and disruption of the mitochondrial transmembrane potential. Furthermore, aggregation of alpha-synuclein by maneb was also inhibited by melatonin. Thus, melatonin prevents maneb-induced neurodegeneration at a nighttime physiological blood concentration, most likely by inhibiting the aggregation of alpha-synuclein as well as preventing mitochondrial dysfunction in PC 12 cells.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. Lycopene in association with alpha-tocopherol inhibits at physiological concentrations proliferation of prostate carcinoma cells.

    PubMed

    Pastori, M; Pfander, H; Boscoboinik, D; Azzi, A

    1998-09-29

    The effect of lycopene alone or in association with other antioxidants was studied on the growth of two different human prostate carcinoma cell lines (the androgen insensitive DU-145 and PC-3). It was found that lycopene alone was not a potent inhibitor of prostate carcinoma cell proliferation. However, the simultaneous addition of lycopene together with alpha-tocopherol, at physiological concentrations (less than 1 microM and 50 microM, respectively), resulted in a strong inhibitory effect of prostate carcinoma cell proliferation, which reached values close to 90 %. The effect of lycopene with alpha-tocopherol was synergistic and was not shared by beta-tocopherol, ascorbic acid and probucol. Copyright 1998 Academic Press.

  12. 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.

  13. 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.

  14. Alpha-particle-induced charge collection in scaled dram cells with advanced structures

    NASA Astrophysics Data System (ADS)

    Takeuchi, K.; Aoki, M.; Watanabe, Y.; Itoh, K.

    1990-11-01

    Alpha-particle-induced charge collection in scaled DRAM cells with advanced structures are experimentally examined. It is concluded that the storage charge necessary to avoid the soft-errors, QC, is proportional to the diagonal length of the depletion region in most types of scaled DRAM cells including PMOS cells. For some types of DRAM cells, however, additional effects on the QC should be considered: (a) for DRAM cells with LOCOS isolation, the collected charge enhancement caused by the charge multiplication at the LOCOS junction edge becomes prominent with scaling, (b) for substrate-plate SPC (Sheat Plate Capacitor) trench cells, the potential slope towards the surface caused by the p+ plate regions enhance the charge collection, (c) for PMOS cells, the collected charge enhancement through the charge multiplication can be weaker than that in NMOS cells.

  15. [Expression of interferon alpha family gene of Chinese marmot in eukaryotic and prokaryotic cells].

    PubMed

    Lu, Yin-ping; Wang, Bao-ju; Huang, Hong-ping; Tian, Yong-jun; Yang, Yan; Dong, Ji-hua; Lu, Meng-ji; Yang, Dong-liang

    2006-02-01

    To investigate the function of interferon alpha (IFNalpha) in a Chinese marmot model of hepatitis B, we expressed the Chinese marmot (Marmota himalayana) IFNalpha family gene (IFNA) in eukaryotic cells and prokaryotic cells. Eukaryotic and prokaryotic expression plasmids harboring Chinese marmot interferon alpha gene with different genotypes were generated using molecular cloning technology. We detected the biological activity of all expression products by viral protection assay, and analyzed their differences and species restriction of the biological activity. The Chinese marmot functional genotype IFNalpha was expressed in the baby hamster kidney (BHK) cell line, and these products protected WH12/6 cells challenged by encephalomyocarditis virus (EMCV). The Chinese marmot IFN-alpha5 also expressed in E. Coli induced by IPTG, and purified fusion protein had antiviral biological activity. The biologic activity displayed differences among different subtype IFNalpha, and it had strict species restriction. The IFNalpha family gene of the Chinese marmot can be expressed in both eukaryotic and prokaryotic cells, and the expression products show antiviral activity in a protection assay. This study provides, for the first time, evidence that IFNalpha from the Chinese marmot has an antiviral function in vitro and can be used to improve the efficacy of current therapies for HBV infection in our Chinese marmot model.

  16. 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.

  17. Induction of interferon-gamma from natural killer cells by immunostimulatory CpG DNA is mediated through plasmacytoid-dendritic-cell-produced interferon-alpha and tumour necrosis factor-alpha.

    PubMed

    Marshall, Jason D; Heeke, Darren S; Abbate, Christi; Yee, Priscilla; Van Nest, Gary

    2006-01-01

    Immunostimulatory sequences (ISS) that contain CpG motifs have been demonstrated to exert antipathogen and antitumour immunity in animal models through several mechanisms, including the activation of natural killer (NK) cells to secrete interferon-gamma (IFN-gamma) and to exert lytic activity. Since NK cells lack the ISS receptor TLR9, the exact pathway by which NK cells are activated by ISS is unclear. We determined that ISS-induced IFN-gamma from NK cells is primarily dependent upon IFN-alpha release from plasmacytoid dendritic cells (PDCs), which directly activates the NK cell. However, further analysis indicated that other PDC-released soluble factor(s) may contribute to IFN-gamma induction. Indeed, tumour necrosis factor-alpha (TNF-alpha) was identified as a significant contributor to ISS-mediated activation of NK cells and was observed to act in an additive fashion with IFN-alpha in the induction of IFN-gamma from NK cells and to up-regulate CD69 expression on NK cells. This activity of TNF-alpha, however, was dependent upon the presence of PDC-derived factors such as type I interferon. These results illustrate an important function for type I interferon in innate immunity, which is not only to activate effectors like NK cells directly, but also to prime them for enhanced activation by other factors such as TNF-alpha.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  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. Menthol Enhances an Antiproliferative Activity of 1alpha,25-Dihydroxyvitamin D(3) in LNCaP Cells.

    PubMed

    Park, Eun-Jung; Kim, Su-Hwa; Kim, Byung-Joo; Kim, Sung-Young; So, Insuk; Jeon, Ju-Hong

    2009-03-01

    1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], the most active form of vitamin D(3), and its analogues have therapeutic benefits for prostate cancer treatment. However, the development of hypercalcemia is an obstacle to clinical applications of 1alpha,25(OH)(2)D(3) for cancer therapy. In this study, we provide evidence that menthol, a key component of peppermint oil, increases an anti-proliferation activity of 1alpha,25(OH)(2)D(3) in LNCaP prostate cancer cells. We found that menthol per se does not exhibit antiproliferative activity, but it is able to enhance 1alpha,25(OH)(2)D(3)-mediated growth inhibition in LNCaP cells. Fluorometric assays using Fura-2 showed that 1alpha,25(OH)(2)D(3) does not induce acute Ca(2+) response, whereas menthol evokes an increase in [Ca(2+)](i), which suggests that cross-talks of menthol-induced Ca(2+) signaling with 1alpha,25(OH)(2)D(3)-mediated growth inhibition pathways. In addition, Western blot analysis revealed that 1alpha,25(OH)(2)D(3) and menthol cooperatively modulate the expression of bcl-2 and p21 which provides the insight into the molecular mechanisms underlying the enhanced 1alpha,25(OH)(2)D(3)-mediated growth inhibition by menthol. Thus, our findings suggest that menthol may be a useful natural compound to enhance therapeutic effects of 1alpha,25(OH)(2)D(3).

  5. 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

  6. 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.

  7. Genomic organization of the human T-cell receptor variable {alpha} (TCRAV) gene cluster

    SciTech Connect

    Ibberson, M.R.; Copier, J.P.; So, A.K.

    1995-07-20

    A long-range physical map of the human T-cell receptor variable {alpha} (TCRAV) locus was produced using 23 V{alpha} subgroup-specific probes. Linkage disequilibrium across the locus was also studied using polymorphic TCRAV markers. Pulsed-field gel electrophoresis was used to map V{alpha} gene segments onto one SfiI fragment of 500 kb and two of 200 kb using DNA from peripheral blood neutrophils. PCR and conventional Southern techniques on Jurkat, CEM, and H9 T-cell lines were used to establish the 5{prime} to 3{prime} order of the gene segments and the relative positions of V{alpha} gene segments on the SfiI fragments. The linkage disequilibrium study used single-stranded conformation polymorphism analysis to genotype 100 normal caucasoid subjects for TCRAV5S1, V6S1, V8S1, V17S1, and V21S1 polymorphisms. Strong linkage disequilibrium was detected between V5S1 and V8S1, in concordance with the physical map. This new information will be useful for future studies of genetic variation at the TCRAV locus, its role in the shaping of the TCR repertoire, and its possible contribution to autoimmune diseases. 46 refs., 3 figs., 5 tabs.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. Overexpression of estrogen receptor-alpha in the endometrial carcinoma cell line Ishikawa: inhibition of growth and angiogenic factors.

    PubMed

    Ali, Syed Hamid; O'Donnell, Amy L; Mohamed, Seema; Mousa, Shaker; Dandona, Paresh

    2004-12-01

    A high level of estrogen receptor-alpha (ER-alpha) is believed to be favorable in the prognosis and treatment of endometrial, ovarian, and breast cancer. High levels of ER-alpha have been shown to inhibit the growth and invasive, metastatic potential of breast cancer cell lines. To bring about these inhibitory effects, ER-alpha probably acts through other cellular factors involved in the regulation of cell growth. To investigate the role of high levels ER-alpha in growth inhibition of endometrial cancer cells. A human ER-alpha cDNA was stably overexpressed in an endometrial cancer cell line, namely, Ishikawa. ER-alpha-overexpressing, parent, and control Ishikawa cells were grown in vitro and their growth rates were compared by cell count. ER-alpha-overexpressing and parent Ishikawa cells were also grown in vitro as tumors in a chicken chorioallantoic membrane (CAM) model, and tumor growth and angiogenesis was measured. Finally, levels of angiogenesis-modulating factors, nitric oxide synthase (NOS), and vascular endothelial growth factor (VEGF) were examined in relation to ER overexpression. The growth of Ishikawa cells was found inhibited in culture as well as in the CAM model. Angiogenesis of CAM tumors was also found inhibited in ER-overexpressing cells. Angiogenic factor VEGF was inhibited whereas the activity of NOS was found elevated following ER overexpression. Our work on the Ishikawa cell line indicates that high levels of ER-alpha in endometrial cancer may inhibit cancer growth by modulating angiogenic factors, thereby limiting the blood supply to the growing tumor. Our results support the earlier data from other groups that have shown a positive correlation between high ER content and better prognosis of endometrial cancers.

  13. 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.

  14. 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

  15. 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.

  16. A novel Gs alpha mutant in a patient with Albright hereditary osteodystrophy uncouples cell surface receptors from adenylyl cyclase.

    PubMed

    Schwindinger, W F; Miric, A; Zimmerman, D; Levine, M A

    1994-10-14

    Albright hereditary osteodystrophy (AHO) is an autosomal-dominant disorder characterized by decreased expression of Gs alpha and widespread tissue resistance to hormones that activate adenylyl cyclase. We identified a single mutation, R385H, in the Gs alpha gene of a subject with AHO who had evidence for a dysfunctional Gs alpha protein. The R385H substitution is near the carboxyl terminus of the Gs alpha protein and is located five amino acids upstream of the R389P mutation that uncouples Gs alpha from cell surface receptors in the unc clone of S49 murine lymphoma. To test the biological activity of the R385H mutant, we transiently expressed wild type, R385H, and R389P Gs alpha cDNAs in COS-1 cells. Neither of the mutant Gs alpha proteins stimulated adenylyl cyclase in response to l-isoproterenol (1 to 30 microM). By contrast, both mutant Gs alpha proteins showed activation of adenylyl cyclase in response to forskolin (10 microM) and fluoroaluminate (10 mM). We propose that the R385H mutation produces a Gs alpha molecule that is unable to interact with hormone receptors and results in uncoupling of adenylyl cyclase from cell surface receptors. This uncoupling mutation represents a new type of molecular defect that can result in AHO.

  17. Teaching tactical combat casualty care using the TC3 sim game-based simulation: a study to measure training effectiveness.

    PubMed

    Sotomayor, Teresita M

    2010-01-01

    The effectiveness of games as instructional tools has been debated over the past several decades. This is due to the lack of empirical data to support such claims. The US ARMY developed a game-based simulation to support Tactical Combat Casualty Care (TCCC) Training. The TC3 Game based Simulation is a first person game that allows a Soldier to play the role of a combat medic during an infantry squad mission in an urban environment. This research documents results from a training effectiveness evaluation conducted at the Department of Combat Medic Training (Ft Sam Houston) in an effort to explore the capability of the game based simulation as a potential tool to support the TCCC program of instruction. Reaction to training, as well as, acquisition of knowledge and transfer of skills were explored using Kirkpatrick's Model of Training Effectiveness Evaluation. Results from the evaluation are discussed.

  18. Entorhinal cortex pre-alpha cell clusters in schizophrenia: quantitative evidence of a developmental abnormality.

    PubMed

    Falkai, P; Schneider-Axmann, T; Honer, W G

    2000-06-01

    Previous studies using semiquantitative or qualitative techniques demonstrated abnormalities of positioning of clusters of neurons (pre-alpha cells) in the entorhinal cortex in schizophrenia, suggesting a developmental mechanism could contribute to the illness. Recent quantitative studies of laminar thickness and laminar cell counts have been less consistent, and several failed to replicate the finding. However, none of the quantitative studies focused on the position of the pre-alpha cell clusters. To study pre-alpha cell position in detail, we examined the entorhinal cortex in serial sections from 21 control and 19 schizophrenic brains. Cluster position relative to the gray-white matter junction and cluster size were measured. Quantitative assessment of 1991 clusters indicated clusters were positioned relatively closer to the gray-white matter junction in the anterior half of schizophrenic entorhinal cortices. In addition, the size of clusters in males with schizophrenia was reduced. These results support the model of schizophrenia as an illness in which brain development is impaired. The findings in males with schizophrenia may indicate the presence of more severe pathology, or an additional pathogenic mechanism.

  19. Single-cell TCRseq: paired recovery of entire T-cell alpha and beta chain transcripts in T-cell receptors from single-cell RNAseq.

    PubMed

    Redmond, David; Poran, Asaf; Elemento, Olivier

    2016-07-27

    Accurate characterization of the repertoire of the T-cell receptor (TCR) alpha and beta chains is critical to understanding adaptive immunity. Such characterization has many applications across such fields as vaccine development and response, clone-tracking in cancer, and immunotherapy. Here we present a new methodology called single-cell TCRseq (scTCRseq) for the identification and assembly of full-length rearranged V(D)J T-cell receptor sequences from paired-end single-cell RNA sequencing reads. The method allows accurate identification of the V(D)J rearrangements for each individual T-cell and has the novel ability to recover paired alpha and beta segments. Source code is available at https://github.com/ElementoLab/scTCRseq .

  20. Isolation of hybrid cell clones that contain deletion and non-deletion defects of alpha-thalassemia in man.

    PubMed

    Deisseroth, A; Bode, U; Lebo, R; Dozy, A; Kan, Y W

    1980-06-01

    We have succeeded in isolating hybrid mouse erythroleukemia cell clones from a patient with hemoglobin H disease, which exhibit either deletion or nondeletion mutations of the human alpha-globin genes. Analysis of one of these hybrid clones that had retained a human chromosome 16 from the patient's cells showed that both human alpha-globin had been deleted. Several clones of another hybrid cell had retained a human chromsome 16 from the patient's cells, which contained both human alpha-globin genes on an EcoRI fragment of 23 kilobases (kb). These latter hybrid clones showed the presence of human alpha-globin chains at detectable but low levels. These studies show that there are two different types of human chromosome 16 in this patient and that the nondeletion mutation of human alpha-globin genes leading to hemoglobin H diseases in this patient acts in cis to the two alpha-globin genes remaining in his cells. The close correlation between the pattern of human alpha-globin gene expression in the patient and in the hybrid cells suggests that this method of transfer of human globin genes to rodent cells will be a useful one for study of mutations affecting the expression of differentiated genes that lead to disease in man.

  1. CD1d-mediated interaction between activated T cells and B cells is essential to B-cell proliferation and anti-alpha-Gal antibody production.

    PubMed

    Liu, S; Kandeva, T; Tchervenkov, J

    2009-01-01

    Antibody-mediated rejection is central to ABO-incompatible transplantation as well as to xenotransplantation. The carbohydrate structure of xenoantigen alpha-Gal is highly analogous to the human blood group antigens. Both require memory B-cell activation for antibody production. We hypothesized that B cells, reactive to the alpha-Gal xenoantigen, required the presence of fully activated T cells to survive and proliferate in vitro. This hypothesis was contrary to the traditional theory that the response of B cells to carbohydrate antigens is T cell independent (Wong and Arsequell: Immunobiology of Carbohydrates. New York: Kluwer; 2003). When we compared the capacity of B cells to proliferate, we observed that activated T cells were necessary for B-cell proliferation. However, this proliferation was independent of the presence of antigen. A relevant question was also to investigate the role of the specific class of T cells: the CD1d-restricted iNKT (iNKT) cells in the activation of alpha-Gal-reactive B cells. The iNKT cells are reactive to glycolipids and capable of producing both Th1 and Th2 cytokine responses. We therefore wanted to determine the role of the iNKT cells as mediators of a Th2-type response when B cells were exposed to a glycolipid antigen extracted from pig red blood cells, which express blockade of the alpha-Gal epitope. We observed that the interaction between B cells and iNKT cells prevents B-cell proliferation and anti-alpha-Gal antibody production.

  2. Ablation of AMP-activated protein kinase alpha1 and alpha2 from mouse pancreatic beta cells and RIP2.Cre neurons suppresses insulin release in vivo.

    PubMed

    Sun, G; Tarasov, A I; McGinty, J; McDonald, A; da Silva Xavier, G; Gorman, T; Marley, A; French, P M; Parker, H; Gribble, F; Reimann, F; Prendiville, O; Carzaniga, R; Viollet, B; Leclerc, I; Rutter, G A

    2010-05-01

    AMP-activated protein kinase (AMPK) is an evolutionarily conserved enzyme and a target of glucose-lowering agents, including metformin. However, the precise role or roles of the enzyme in controlling insulin secretion remain uncertain. The catalytic alpha1 and alpha2 subunits of AMPK were ablated selectively in mouse pancreatic beta cells and hypothalamic neurons by breeding Ampkalpha1 [also known as Prkaa1]-knockout mice, bearing floxed Ampkalpha2 [also known as Prkaa2] alleles (Ampkalpha1 ( -/- ),alpha2( fl/fl ),), with mice expressing Cre recombinase under the rat insulin promoter (RIP2). RIP2 was used to express constitutively activated AMPK selectively in beta cells in transgenic mice. Food intake, body weight and urinary catecholamines were measured using metabolic cages. Glucose and insulin tolerance were determined after intraperitoneal injection. Beta cell mass and morphology were analysed by optical projection tomography and confocal immunofluorescence microscopy, respectively. Granule docking, insulin secretion, membrane potential and intracellular free Ca(2+) were measured with standard techniques. Trigenic Ampkalpha1 ( -/- ),alpha2( fl/fl ) expressing Cre recombinase and lacking both AMPKalpha subunits in the beta cell, displayed normal body weight and increased insulin sensitivity, but were profoundly insulin-deficient. Secreted catecholamine levels were unchanged. Total beta cell mass was unaltered, while mean islet and beta cell volume were reduced. AMPK-deficient beta cells displayed normal glucose-induced changes in membrane potential and intracellular free Ca(2+), while granule docking and insulin secretion were enhanced. Conversely, betaAMPK transgenic mice were glucose-intolerant and displayed defective insulin secretion. Inhibition of AMPK activity within the beta cell is necessary, but not sufficient for stimulation of insulin secretion by glucose to occur. AMPK activation in extrapancreatic RIP2.Cre-expressing cells might also influence

  3. 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

  4. 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.

  5. 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

  6. Radiosensitization by inhibition of IkappaB-alpha phosphorylation in human glioma cells.

    PubMed

    Ding, Gui-Rong; Honda, Naoko; Nakahara, Takehisa; Tian, Furong; Yoshida, Masami; Hirose, Hideki; Miyakoshi, Junji

    2003-08-01

    To assess the role of nuclear factor kappaB (NFKB) in cellular radiosensitivity, three different IkappaB-alpha (also known as NFKBIA) expression plasmids, i.e., S-IkappaB (mutations at (32, 36)Ser), Y-IkappaB (a mutation at (42)Tyr), and SY-IkappaB, were constructed and introduced into human brain tumor M054 cells. The clones were named as M054-S8, M054-Y2 and M054-SY4, respectively. Compared to the parental cell line, M054-S8 and M054-Y2 cells were more sensitive to X rays while M054-SY4 cells exhibited the greatest sensitivity. After treatment with N-acetyl-Leu-Leu-norleucinal, a proteasome inhibitor, the X-ray sensitivity of M054-S8 and M054-SY4 cells did not change, while that of M054-Y2 cells and the parental cells was enhanced. An increase in X-ray sensitivity accompanied by a decrease in translocation of NFKB to the nucleus in parental cells was observed after treatment with pervanadate, an inhibitor of tyrosine phosphatase, as well as in M054-S8 and M054-SY4 cells. Repair of potentially lethal damage (PLD) was observed in the parental cells but not in the clones. Four hours after irradiation (8 Gy), the expression of TP53 and phospho-p53 ((15)Ser) was induced in the parental cells but not in M054-S8, M054-Y2 or M054-SY4 cells. Our data suggest that inhibition of IkappaB-alpha phosphorylation at serine or tyrosine acts independently in sensitizing cells to X rays. NFKB may play a role in determining radiosensitivity and PLD repair in malignant glioma cells; TP53 may also be involved.

  7. 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.

  8. Induction of alpha and beta chemokines by intestinal epithelial cells stimulated with Campylobacter jejuni.

    PubMed

    Bakhiet, Moiz; Al-Salloom, Fajer Subah; Qareiballa, Ahmed; Bindayna, Khalid; Farid, Iman; Botta, Giuseppe A

    2004-04-01

    To investigate the production of dynamic alpha and beta chemokines represented by interleukin-8 (IL-8) as alpha chemokine and CCL2 (monocyte-chemoattractant protein-1, CCR2 ligand), CCL4 (macrophage-inflammatory protein-1beta, CCR5 ligand), CCL3 (macrophage-inflammatory protein-1alpha, CCR1/5 ligand), (CCL5, regulated upon activation, normal T-cell expressed and secreted (RANTES, CCR5 ligand) as beta chemokines by the human intestinal cell line INT407 stimulated with factors produced by living Campylobacter jejuni (C. jejuni) and those present within sonicated and filtrated bacteria. We used immunohistochemical technique modified to detect intracellular production of cytokines protein and RT-PCR to read RNA messages for evaluation of de novo cytokine synthesis. Living bacteria induced increased numbers of IL-8, CCL4 and CCL2 but not CCL3 or CCL5 producing cells. Low numbers of IL-8, CCL4 and CCL2 producing cells were detected with filtrated supernatant compared to living and sonicated bacteria. A non-significant low number of chemokine producing cells was noted when comparing numbers of chemokine producing cells stimulated with living C. jejuni to those stimulated with sonicated bacteria, indicating that the triggering factors involved in stimulation with living bacteria were still active after sonication, but they were largely lost upon filtration. The mRNA signals for IL-8 were noted in conformity with its protein levels as increased IL-8 mRNA signals were registered after stimulation with living and sonicated bacteria but not with filtrated supernatant. Preferential production of chemokines probably induced by membrane associate factors of C. jejuni acting on intestinal epithelial cells is presented. These chemokines are suggested to be part of an inflammatory network affecting cell types that contribute to initiation and/or resolution of the infection.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. Characterization of synthesis and storage of TGF-alpha in rat parotid acinar and intercalated duct cells.

    PubMed

    Login, G R; Yang, J; Bryan, K P; Digenis, E C; McBride, J; Elovic, A; Quissell, D O; Dvorak, A M; Wong, D T

    1997-03-01

    Although the expression and biological role of transforming growth factor-alpha (TGF-alpha) have been explored in a variety of normal cells in mammalian species, little is known about the storage of TGF-alpha in secretory cells of exocrine organs. Parotid glands from four rats were homogenized for RNA isolation followed by reverse transcription-polymerase chain reaction to determine the presence of TGF-alpha message. In situ hybridization using a hamster-specific TGF-alpha riboprobe was done on paraffin sections. Parotid gland and isolated acinar cells were processed for transmission electron microscopy (TEM) and postembedding immunogold labeled for TGF-alpha. Gold particles were counted on approximately 200 granules in 10 acinar cells and in 10 intercalated duct cells. Labeling density was calculated as the number of gold particles per square micrometer +/- SD. Statistical significance was calculated using one-way analysis of variance. Using multiple technologies, we have established that rat parotid acinar and intercalated duct cells synthesize TGF-alpha and store the precursor form of this cytokine in their secretory granules.

  16. 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

  17. Overexpression of Reg3alpha increases cell growth and the levels of cyclin D1 and CDK4 in insulinoma cells.

    PubMed

    Cui, Wei; De Jesus, Kristine; Zhao, Hong; Takasawa, Shin; Shi, Bingyin; Srikant, Coimbatore B; Liu, Jun-Li

    2009-06-01

    Regenerating gene (Reg) family protein Reg3alpha is normally expressed in pancreatic acinar and endocrine cells. In order to explore its effect on islet beta-cell replication, insulinoma MIN6 cells were stably transfected with murine Reg3alpha cDNA. Determined using real-time PCR and Western blots, the levels of Reg3alpha mRNA and protein in Reg3alpha-transfected clones were increased 10- and 6-fold, respectively. Western blots also revealed that the protein was released into the culture medium, consistent with an endocrine effect. In MTT cell proliferation assay, Reg3alpha-overexpressing cells exhibited a 2-fold increase in the rate of cell growth. In order to investigate the intracellular mechanism, we studied cell cycle regulatory proteins. In Reg3alpha-expressing cells, we detected 2.2- and 2.5-fold increased levels of cyclin D1 and CDK4, respectively, which paralleled a 1.8-fold increase in the rate of Akt phosphorylation. It is established that beta-cell replication is associated with increased cyclin D1 and CDK4 levels; deficiency in CDK4 or cyclin D2 results in reduced beta-cell mass and diabetes. Our results suggest that Reg3alpha stimulates beta-cell replication, by activating Akt kinase and increasing the levels of cyclin D1/CDK4.

  18. Alpha 7 subunit of nAChR regulates migration of human mesenchymal stem cells.

    PubMed

    Schraufstatter, Ingrid U; DiScipio, Richard G; Khaldoyanidi, Sophia K

    2009-01-01

    The efficient migration of mesenchymal stem cells (MSCs) to diseased tissues is required for the fulfillment of their regenerative potential. Recruitment of circulating cells into the damaged tissues is regulated by a complex network, which includes the non-neural cholinergic system. We found that human MSCs (hMSCs) express nicotinic acetylcholine receptor subunits alpha 7, beta 2 and beta 4. The receptor agonist nicotine caused calcium (Ca(++)) influx into hMSCs suggesting that the calcium ion channel alpha 7 homopolymer mediated this response. While high concentrations of nicotine (10(5)M) induced hMSC apoptosis, physiological concentrations (10(7)M) did not interfere with cell survival. At non-toxic concentrations, nicotine increased spontaneous migration of hMSCs, whereas chemotaxis of hMSCs toward C3a and bFGF in vitro and migration of intravenously infusion hMSCs into bone marrow and spleen in vivo were inhibited. The antagonist for the alpha 7 homopolymer, bungarotoxin, blocked the inhibitory effect of nicotine on chemotactic factor-induced migration of hMSCs. These findings reveal an involvement of the non-neural cholinergic system in regulation of hMSC migration.

  19. Alpha 7 Subunit of nAChR Regulates Migration of Human Mesenchymal Stem Cells

    PubMed Central

    Schraufstatter, Ingrid U.; DiScipio, Richard G.; Khaldoyanidi, Sophia K.

    2010-01-01

    The efficient migration of mesenchymal stem cells (MSCs) to diseased tissues is required for the fulfillment of their regenerative potential. Recruitment of circulating cells into the damaged tissues is regulated by a complex network, which includes the non-neural cholinergic system. We found that human MSCs (hMSCs) express nicotinic acetylcholine receptor subunits alpha 7, beta 2 and beta 4. The receptor agonist nicotine caused calcium (Ca++) influx into hMSCs suggesting that the calcium ion channel alpha 7 homopolymer mediated this response. While high concentrations of nicotine (10−5M) induced hMSC apoptosis, physiological concentrations (10−7M) did not interfere with cell survival. At non-toxic concentrations, nicotine increased spontaneous migration of hMSCs, whereas chemotaxis of hMSCs toward C3a and bFGF in vitro and migration of intravenously infusion hMSCs into bone marrow and spleen in vivo were inhibited. The antagonist for the alpha 7 homopolymer, bungarotoxin, blocked the inhibitory effect of nicotine on chemotactic factor-induced migration of hMSCs. These findings reveal an involvement of the non-neural cholinergic system in regulation of hMSC migration. PMID:20720594

  20. 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

  1. 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

  2. Interaction between the Helicobacter pylori CagA and alpha-Pix in gastric epithelial AGS cells.

    PubMed

    Baek, Hye Yeon; Lim, Joo Weon; Kim, Hyeyoung

    2007-01-01

    The gastric pathogen Helicobacter pylori (H. pylori) translocates the CagA protein into epithelial cells by a type IV secretion process. Upon translocation into the host cell cytosol, CagA undergoes tyrosine phosphorylation. Phosphorylation of CagA occurs within the C terminus of the protein and is mediated by members of the Src family of tyrosine kinases. Phosphorylation of CagA induces the dephosphorylation of as yet unidentified cellular proteins, rearrangements of the host cell actin cytoskeleton, and cell scattering. This article aims to determine the cellular protein that interacts with CagA. Gastric epithelial AGS cells were stimulated with CagA-positive H. pylori (NCTC11637, at a bacteria/cell ratio of 500:1) and cultured in antibiotic-free medium. Proteins were isolated from the cells with or without H. pylori infection. CagA-interactive protein was determined by immunoprecipitation using anti-CagA antibody and proteomic analysis. We found that alpha-Pix interacts with CagA and alpha-Pix was constitutively expressed in AGS cells. Upon H. pylori stimulation, CagA was translocated into the cells and the expression of alpha-Pix (PAK-interactive exchange factor) was increased in AGS cells time dependently. The interaction of alpha-Pix with CagA was increased by H. pylori infection in AGS cells. Phosphorylation of CagA induces the dephosphorylation of alpha-Pix in AGS cells. alpha-Pix is a family of PAK-binding proteins that strongly activates PAK (p21-activated tyrosine kinase). PAK regulates changes in gene expression and mediates actin cytoskeletal and cell morphological changes. The novel finding of this study is that phosphorylation of CagA induces the dephosphorylation of alpha-Pix, which may modulate cytoskeletal changes of gastric epithelial cells through PAK.

  3. D-Glucosamine down-regulates HIF-1{alpha} through inhibition of protein translation in DU145 prostate cancer cells

    SciTech Connect

    Park, Jee-Young; Park, Jong-Wook; Suh, Seong-Il; Baek, Won-Ki

    2009-04-24

    D-Glucosamine has been reported to inhibit proliferation of cancer cells in culture and in vivo. In this study we report a novel response to D-glucosamine involving the translation regulation of hypoxia inducible factor (HIF)-1{alpha} expression. D-Glucosamine caused a decreased expression of HIF-1{alpha} under normoxic and hypoxic conditions without affecting HIF-1{alpha} mRNA expression in DU145 prostate cancer cells. D-Glucosamine inhibited HIF-1{alpha} accumulation induced by proteasome inhibitor MG132 and prolyl hydroxylase inhibitor DMOG suggesting D-glucosamine reduces HIF-1{alpha} protein expression through proteasome-independent pathway. Metabolic labeling assays indicated that D-glucosamine inhibits translation of HIF-1{alpha} protein. In addition, D-glucosamine inhibited HIF-1{alpha} expression induced by serum stimulation in parallel with inhibition of p70S6K suggesting D-glucosamine inhibits growth factor-induced HIF-1{alpha} expression, at least in part, through p70S6K inhibition. Taken together, these results suggest that D-glucosamine inhibits HIF-1{alpha} expression through inhibiting protein translation and provide new insight into a potential mechanism of the anticancer properties of D-glucosamine.

  4. 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.

  5. 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.

  6. TNF-alpha modulates the differentiation induced by butyrate in the HT-29 human colon adenocarcinoma cell line.

    PubMed

    Kovaríková, M; Pacherník, J; Hofmanová, J; Zadák, Z; Kozubík, A

    2000-09-01

    The aim of this study was to determine whether and how tumour necrosis factor alpha (TNF-alpha) modulates butyrate effects. After the treatment of human colon adenocarcinoma HT-29 cells with sodium butyrate (NaBt), TNF-alpha or with their combinations we detected cell cycle (flow cytometry), cell proliferation (amidoblack and MTT assays), the amount of dead (floating) and apoptotic cells (flow cytometry and fluorescence microscopy), and the level of differentiation by alkaline phosphatase (ALP) activity (spectrophotometry), relative F-actin content (confocal laser scanning microscopy analysis) and E-cadherin expression (Western blot analysis). Both TNF-alpha and NaBt decreased cell growth in a dose-dependent manner. After combined treatment of the cells with both agents used, either none or additive effects were observed as compared with NaBt treatment alone. The level of dead and apoptotic cells was dose-dependently increased after this combined treatment. In contrast, TNF-alpha suppressed ALP activity and F-actin accumulation induced by NaBt. The results suggest that TNF-alpha does not influence significantly the antiproliferative effects of NaBt but, contrary to its potentiation of apoptosis, it markedly reduces NaBt-induced differentiation of HT-29 colon adenocarcinoma cells.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. Do HLA genes play a prominent role in determining T cell receptor V{alpha} segment usage in humans?

    SciTech Connect

    Gulwani-Akolkar, B.; Shi, B.; Akolkar, P.N.

    1995-04-15

    Previous studies in humans have demonstrated that HLA genes can profoundly influence the TCR V{beta} repertoire. To similarly assess the influence of HLA genes on the TCR V{alpha} segment repertoire, the V{alpha} repertoires of 12 individuals from three unrelated families were determined by quantitative PCR. Each family contained at least one pair of HLA-identical and -nonidentical siblings. Repertoire analysis was performed on purified CD4{sup +} and CD8{sup +} cells by using V{alpha}-specific primers. We were unable to demonstrate more similar V{alpha} repertoires between HLA-identical siblings than between HLA-nonidentical siblings. In contrast, when a similar analysis was performed on the same individuals for the V{beta} repertoire, HLA-identical siblings were found to have significantly more similar repertoires than HLA-nonidentical siblings. Furthermore, both the V{alpha} and V{beta} repertoires of monozygotic twins showed striking similarity. Despite our inability to shown an influence of HLA genes on the V{alpha} repertoire, we did observe a very strong skewing in terms of preferential expression on CD4{sup +} or CD8{sup +} cells of several V{alpha} segments, notably TCRAV1, -2, -5, -6, -7, -11, -12, and -13. These studies suggest that HLA genes play less of a role in determining V{alpha} segment usage than V{beta}. Nevertheless, the pronounced skewing of V{alpha} segment expression in the CD4{sup +} or CD8{sup +} populations suggests some role for HLA genes in determining the V{alpha} TCR repertoire. Furthermore, the striking similarity of V{alpha} repertoires of identical twins suggests a major role for non-HLA genes in determining the V{alpha} repertoire. 35 refs., 8 figs., 3 tabs.

  12. 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

  13. Toxicities of trichosanthin and alpha-momorcharin, abortifacient proteins from Chinese medicinal plants, on cultured tumor cell lines.

    PubMed

    Tsao, S W; Ng, T B; Yeung, H W

    1990-01-01

    Trichosanthin and alpha-momorcharin are abortifacient proteins extracted from Chinese medicinal herbs. Study of their in vitro cytotoxicities showed that the two proteins selectively injured choriocarcinoma and melanoma cells. Hepatoma cells represented the most resistant cell line among the various cell lines investigated. Cytotoxicity profiles of trichosanthin and alpha-momorcharin differed from those of anti-cancer drugs which interfere with DNA metabolism such as cisplatin, methotrexate and 5-fluorouracil. Radioactive precursor incorporation studies suggested that the two abortifacient proteins inhibited cellular protein synthesis. The marked decrease in secretion of human chorionic gonadotrophin and progesterone by choriocarcinoma cells after treatment with the proteins could be attributed mainly to loss of cells.

  14. 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.

  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. 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

  17. Sensitization of RPE cells by alphaB-crystallin siRNA to SAHA-induced stage 1 apoptosis through abolishing the association of alphaB-crystallin with HDAC1 in SC35 speckles.

    PubMed

    Noh, Seung Jin; Jeong, Woo Jin; Rho, Jee Hyun; Shin, Dong Min; Ahn, Hee Bae; Park, Woo Chan; Rho, Sae Heun; Soung, Young Hwa; Kim, Tae Hyun; Park, Bong Soo; Yoo, Young Hyun

    2008-11-01

    To better understand the mechanism underlying the anti-apoptotic activity of alphaB-crystallin in RPE cells. Cells of the human retinal pigment epithelial line ARPE-19 were treated with a histone deacetylase inhibitor (HDACI), suberoylanilide hydroxamic acid (SAHA), with or without alphaB-crystallin siRNA. To examine the mechanism underlying the cell death induced in ARPE-19 cells, nuclear staining, flow cytometry, DNA electrophoresis, pulse field gel electrophoresis, Western blot analysis, confocal microscopy, and coimmunoprecipitation assay were undertaken. The present study demonstrated that an HDACI, SAHA, at the usual doses or the silencing of alphaB-crystallin by siRNA alone did not effectively induce apoptosis in ARPE-19 cells. Silencing of alphaB-crystallin likely abolishes the anti-apoptotic activity of alphaB-crystallin. The data indicated that silencing of alphaB-crystallin sensitizes ARPE19 cells to SAHA-induced apoptosis and leads them to stage 1 apoptosis. alphaB-Crystallin associates with HDAC1 on SC35 speckles, and silencing of alphaB-crystallin abolishes this association, resulting in the induction of apoptosis. The data indicated that the association between alphaB-crystallin and HDAC1 on SC35 speckles plays a pivotal role in anti-apoptotic activity. Knockout of alphaB-crystallin may be a promising new approach to enhance therapeutic potency for proliferative vitreoretinopathy without compromising efficacy.

  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. Hypoxia-inducible factor 1-alpha release after intracoronary versus intramyocardial stem cell therapy in myocardial infarction.

    PubMed

    Gyöngyösi, Mariann; Hemetsberger, Rayyan; Posa, Aniko; Charwat, Silvia; Pavo, Noemi; Petnehazy, Ors; Petrasi, Zsolt; Pavo, Imre J; Hemetsberger, Hani; Benedek, Imre; Benedek, Teodora; Benedek, Istvan; Kovacs, Istvan; Kaun, Christoph; Maurer, Gerald

    2010-04-01

    We have investigated the effect of stem cell delivery on the release of hypoxia-inducible factor 1 alpha (HIF-1alpha) in peripheral circulation and myocardium in experimental myocardial ischemia. Closed-chest, reperfused myocardial infarction (MI) was created in domestic pigs. Porcine mesenchymal stem cells (MSCs) were cultured and delivered (9.8 +/- 1.2 x 10(6)) either percutaneously NOGA-guided transendocardially (Group IM) or intracoronary (Group IC) 22 +/- 4 days post-MI. Pigs without MSC delivery served as sham control (Group S). Plasma HIF-1alpha was measured at baseline, immediately post- and at follow-up (FUP; 2 h or 24 h) post-MSC delivery by ELISA kit. Myocardial HIF-1alpha expression of infarcted, normal myocardium, or border zone was determined by Western blot. Plasma level of HIF-1alpha increased immediately post-MI (from 278 +/- 127 to 631 +/- 375 pg/ml, p < 0.05). Cardiac delivery of MSCs elevated the plasma levels of HIF-1alpha significantly (p < 0.05) in groups IC and IM immediately post-MSC delivery, and returned to baseline level at FUP, without difference between the groups IC and IM. The myocardial tissue HIF-1alpha expression in the infarcted area was higher in Group IM than in Group IC or S (1,963 +/- 586 vs. 1,307 +/- 392 vs. 271 +/- 110 activity per square millimeter, respectively, p < 0.05), while the border zone contained similarly lower level of HIF-1alpha, but still significantly higher as compared with Group S. Trend towards increase in myocardial expression of HIF-1alpha was measured in Group IM at 24 h, in contrast to Group IC. In conclusion, both stem cell delivery modes increase the systemic and myocardial level of HIF-1alpha. Intramyocardial delivery of MSC seems to trigger the release of angiogenic HIF-1alpha more effectively than does intracoronary delivery.

  20. Dieldrin induces ubiquitin-proteasome dysfunction in alpha-synuclein overexpressing dopaminergic neuronal cells and enhances susceptibility to apoptotic cell death.

    PubMed

    Sun, Faneng; Anantharam, Vellareddy; Latchoumycandane, Calivarathan; Kanthasamy, Arthi; Kanthasamy, Anumantha G

    2005-10-01

    Exposure to pesticides is implicated in the etiopathogenesis of Parkinson's disease (PD). The organochlorine pesticide dieldrin is one of the environmental chemicals potentially linked to PD. Because recent evidence indicates that abnormal accumulation and aggregation of alpha-synuclein and ubiquitin-proteasome system dysfunction can contribute to the degenerative processes of PD, in the present study we examined whether the environmental pesticide dieldrin impairs proteasomal function and subsequently promotes apoptotic cell death in rat mesencephalic dopaminergic neuronal cells overexpressing human alpha-synuclein. Overexpression of wild-type alpha-synuclein significantly reduced the proteasomal activity. Dieldrin exposure dose-dependently (0-70 microM) decreased proteasomal activity, and 30 microM dieldrin inhibited activity by more than 60% in alpha-synuclein cells. Confocal microscopic analysis of dieldrin-treated alpha-synuclein cells revealed that alpha-synuclein-positive protein aggregates colocalized with ubiquitin protein. Further characterization of the aggregates with the autophagosomal marker mondansyl cadaverine and the lysosomal marker and dot-blot analysis revealed that these protein oligomeric aggregates were distinct from autophagosomes and lysosomes. The dieldrin-induced proteasomal dysfunction in alpha-synuclein cells was also confirmed by significant accumulation of ubiquitin protein conjugates in the detergent-insoluble fraction. We found that proteasomal inhibition preceded cell death after dieldrin treatment and that alpha-synuclein cells were more sensitive than vector cells to the toxicity. Furthermore, measurement of caspase-3 and DNA fragmentation confirmed the enhanced sensitivity of alpha-synuclein cells to dieldrin-induced apoptosis. Together, our results suggest that increased expression of alpha-synuclein predisposes dopaminergic cells to proteasomal dysfunction, which can be further exacerbated by environmental exposure to certain

  1. Enzymatic removal of alpha-galactosyl epitopes from porcine endothelial cells diminishes the cytotoxic effect of natural antibodies.

    PubMed

    LaVecchio, J A; Dunne, A D; Edge, A S

    1995-10-27

    Xenotransplantation of tissues between discordant species such as pig into human is not yet feasible due to the problem of hyperacute rejection. This rapid response to xenogeneic tissue is mediated by natural antibodies that react with antigens on the xenograft. A number of xenoantigens consist of carbohydrate residues, and a terminal galactose in alpha linkage has been shown to be involved in hyperacute rejection of pig-to-human xenografts. We show that alpha-linked galactose on porcine endothelial cells is a major epitope recognized by IgG and IgM antibodies present in monkey and human sera. Endothelial cells that had been treated with alpha-galactosidase did not react with fluorescein-labeled Griffonia simplicifolia I B4 (GS-IB4), a lectin that detects the alpha-galactosyl epitope on intact cells. The reactivity of both human and cynomolgus monkey serum with endothelial cells was decreased by 59% to 90% after treatment with coffee bean alpha-galactosidase. Using a colorimetric assay for cell viability, we show that natural antibodies present in the sera of cynomolgus monkey and humans are cytotoxic to porcine endothelial cells in the presence of exogenously added complement. When the terminal alpha-galactosy residues were removed by enzymatic digestion, the cytotoxic effect of natural antibodies on porcine endothelial cells was diminished by > 80%. Evaluation of the time course of reappearance of the alpha-galactosyl epitope at the cell surface revealed that 48 hr after alpha-galactosidase treatment, binding of GS-IB4 was diminished by 60%. These results suggest that glycosidase treatment of cells to be transplanted could prevent hyperacute rejection mediated by natural antibodies.

  2. 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.

  3. 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.

  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. 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.

  7. 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.

  8. Genetic signatures of differentiation induced by 1alpha,25-dihydroxyvitamin D3 in human colon cancer cells.

    PubMed

    Pálmer, Héctor G; Sánchez-Carbayo, Marta; Ordóñez-Morán, Paloma; Larriba, María Jesús; Cordón-Cardó, Carlos; Muñoz, Alberto

    2003-11-15

    Epidemiological and preclinical data indicate that vitamin D and its most active metabolite 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] have anticancer activity. Accordingly, clinical trials are under way using several nonhypercalcemic 1alpha,25(OH)(2)D(3) analogues against various neoplasms including colon cancer. 1alpha,25(OH)(2)D(3) induces proliferation arrest and epithelial differentiation of human SW480-ADH colon cancer cells. We examined the gene expression profiles associated with 1alpha,25(OH)(2)D(3) exposure using oligonucleotide microarrays. 1alpha,25(OH)(2)D(3) changed the expression levels of numerous previously unreported genes, including many involved in transcription, cell adhesion, DNA synthesis, apoptosis, redox status, and intracellular signaling. Most genes were up-regulated, and only a small fraction were down-regulated. Fourteen of 17 candidate genes studied were validated as 1alpha,25(OH)(2)D(3) target genes by Northern and Western blotting or immunocytochemistry. They included c-JUN, JUNB, JUND, FREAC-1/FoxF1, ZNF-44/KOX7, plectin, filamin, keratin-13, G(0)S2, and the putative tumor suppressors NES-1 and protease M. There was little overlap between genes regulated after short (4 h) or long (48 h) exposure. Gene regulatory effects of 1alpha,25(OH)(2)D(3) in SW480-ADH cells differed from those in LS-174T cells, which lack E-cadherin and do not differentiate in response to 1alpha,25(OH)(2)D(3). Data from this study reveal that 1alpha,25(OH)(2)D(3) causes a profound change in gene expression profiles and provide a mechanistic basis to the ongoing clinical studies using nonhypercalcemic vitamin D(3) derivatives for colon cancer prevention and treatment.

  9. Immunity to the alpha(1,3)galactosyl epitope provides protection in mice challenged with colon cancer cells expressing alpha(1,3)galactosyl-transferase: a novel suicide gene for cancer gene therapy.

    PubMed

    Unfer, Robert C; Hellrung, Daniel; Link, Charles J

    2003-03-01

    Human immunity to alpha(1,3)Galactosyl epitopes (alpha Gal) may provide the means for a successful cancer gene therapy that uses the immune system to identify and to destroy tumor cells expressing the suicide gene alpha(1,3)Galactosyltransferase (alpha GT). Innate antibody specific for cell surface alpha Gal constitutes a high percentage of circulating IgG and IgM immunoglobulins in humans and is the basis for complement-mediated hyperacute xenograft rejection and antibody-dependent cell-mediated cytotoxicity. In humans, the gene for alpha GT is mutated, and cells do not express the alpha Gal moiety. We hypothesized that human tumor cells induced to express the alpha Gal epitope would be killed by the hosts' innate immunity. Previous in vitro work by our group has demonstrated complement-mediated lysis of alpha Gal-transduced human tumor cells in culture by human serum. To induce antibodies to alpha Gal in this in vivo study, alpha GT knockout mice were used to determine whether immunization with alpha Gal could provide protection from challenge with alpha Gal-expressing murine MC38 colon cancer cells. Knockout mice were immunized either a single time, or twice, with rabbit RBC. Antibody titers to alpha Gal measured by indirect ELISA were significantly higher in mice immunized twice and approached the titers observed in human serum. Anti-alpha Gal antibodies were predominantly of the IgG1 and IgG3 subtype. Immunized knockout mice were challenged i.p. with varying doses of alpha Gal(+) MC38 colon carcinoma cells. Nonimmunized control groups consisting of alpha GT knockout mice, and wild-type C57BL/6 mice were challenged as well with MC38 cells. Immunized mice survived and exhibited slower tumor development in comparison to nonimmunized knockout and control mice. This study demonstrates, in vivo, the protective benefit of an immune response to the alpha Gal epitope. Our results provide a basis to pursue additional development of this cancer gene therapy strategy.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. Introduction of the human pro. cap alpha. 1(I) collagen gene into pro. cap alpha. 1(I)-deficient Mov-13 mouse cells leads to formation of functional mouse-human hybrid type I collagen

    SciTech Connect

    Schnieke, A.; Dziadek, M.; Bateman, J.; Mascara, T.; Harbers, K.; Gelinas, R.; Jaenisch, R.

    1987-02-01

    The Mov-13 mouse strain carries a retroviral insertion in the pro..cap alpha..1(I) collagen gene that prevents transcription of the gene. Cell lines derived from homozygous embryos do not express type I collagen although normal amounts of pro..cap alpha..2 mRNA are synthesized. The authors have introduced genomic clones of either the human or mouse pro..cap alpha..1(I) collagen gene into homozygous cell lines to assess whether the human or mouse pro..cap alpha..1(I) chains can associate with the endogenous mouse pro..cap alpha..2(I) chain to form stable type I collagen. The human gene under control of the simian virus 40 promoter was efficiently transcribed in the transfected cells. Protein analyses revealed that stable heterotrimers consisting of two human ..cap alpha..1 chains and one mouse ..cap alpha..2 chain were formed and that type I collagen was secreted by the transfected cells at normal rates. However, the electrophoretic migration of both ..cap alpha..1(I) and ..cap alpha..2(I) chains in the human-mouse hybrid molecules were retarded, compared to the ..cap alpha..(I) chains in control mouse cells. Inhibition of the posttranslational hydroxylation of lysine and proline resulted in comigration of human and mouse ..cap alpha..1 and ..cap alpha..2 chains, suggesting that increased posttranslational modification caused the altered electrophoretic migration in the human-mouse hybrid molecules. Amino acid sequence differences between the mouse and human ..cap alpha.. chains may interfere with the normal rate of helix formation and increase the degree of posttranslational modifications similar to those observed in patients with lethal perinatal osteogenesis imperfecta. The Mov-13 mouse system should allow the authors to study the effect specific mutations introduced in transfected pro..cap alpha..1(I) genes have on the synthesis, assembly, and function of collagen I.

  16. Introduction of the human pro alpha 1(I) collagen gene into pro alpha 1(I)-deficient Mov-13 mouse cells leads to formation of functional mouse-human hybrid type I collagen.

    PubMed Central

    Schnieke, A; Dziadek, M; Bateman, J; Mascara, T; Harbers, K; Gelinas, R; Jaenisch, R

    1987-01-01

    The Mov-13 mouse strain carries a retroviral insertion in the pro alpha 1(I) collagen gene that prevents transcription of the gene. Cell lines derived from homozygous embryos do not express type I collagen although normal amounts of pro alpha 2 mRNA are synthesized. We have introduced genomic clones of either the human or mouse pro alpha 1(I) collagen gene into homozygous cell lines to assess whether the human or mouse pro alpha 1(I) chains can associate with the endogenous mouse pro alpha 2(I) chain to form stable type I collagen. The human gene under control of the simian virus 40 promoter was efficiently transcribed in the transfected cells. Protein analyses revealed that stable heterotrimers consisting of two human alpha 1 chains and one mouse alpha 2 chain were formed and that type I collagen was secreted by the transfected cells at normal rates. However, the electrophoretic migration of both alpha 1(I) and alpha 2(I) chains in the human-mouse hybrid molecules were retarded, compared to the alpha (I) chains in control mouse cells. Inhibition of the posttranslational hydroxylation of lysine and proline resulted in comigration of human and mouse alpha 1 and alpha 2 chains, suggesting that increased posttranslational modification caused the altered electrophoretic migration in the human-mouse hybrid molecules. Amino acid sequence differences between the mouse and human alpha chains may interfere with the normal rate of helix formation and increase the degree of posttranslational modifications similar to those observed in patients with lethal perinatal osteogenesis imperfecta. The Mov-13 mouse system should allow us to study the effect specific mutations introduced in transfected pro alpha 1(I) genes have on the synthesis, assembly, and function of collagen I. Images PMID:3468512

  17. Retinoic acid receptor alpha mediates growth inhibition by retinoids in rat pancreatic carcinoma DSL-6A/C1 cells.

    PubMed Central

    Brembeck, F. H.; Kaiser, A.; Detjen, K.; Hotz, H.; Foitzik, T.; Buhr, H. J.; Riecken, E. O.; Rosewicz, S.

    1998-01-01

    During carcinogenesis, pancreatic acinar cells can dedifferentiate into ductal adenocarcinoma of the pancreas. DSL-6A/C1 cells represent an in vitro model of this carcinogenic sequence. This study was designed to examine the effects of retinoids on cell growth in DSL-6A/C1 cells and to characterize further the molecular mechanisms underlying the antiproliferative actions of retinoids. Treatment of DSL-6A/C1 cells with retinoids results in a time- and dose-dependent inhibition of cell growth, paralleled by a retinoid-mediated transactivation of a pTK::betaRAREx2-luciferase reporter construct transiently transfected into DSL-6A/C1 cells. Retinoid receptor expression was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) using subtype-specific primers and demonstrated expression of retinoic acid receptor alpha (RAR-alpha), RAR-beta and retinoid X receptor alpha (RXR-alpha). Using a panel of receptor subtype-specific agonists, the RAR-alpha specific agonist Ro 40-6055 was the most potent retinoid in terms of growth inhibition. Furthermore, all-trans-retinoic acid-mediated growth inhibition and transactivation was completely blocked by the RAR-alpha-specific antagonist Ro 41-5253. In summary, the RAR-alpha subtype predominantly mediates the antiproliferative effects of retinoids in DSL-6A/C1 cells. Furthermore, this cell system provides a feasible tool to study the molecular mechanisms underlying the growth inhibitory effects of retinoids in ductal pancreatic carcinoma cells derived from a primary acinar cell phenotype. Images Figure 1 Figure 5 PMID:9823968

  18. Retinoic acid receptor alpha mediates growth inhibition by retinoids in rat pancreatic carcinoma DSL-6A/C1 cells.

    PubMed

    Brembeck, F H; Kaiser, A; Detjen, K; Hotz, H; Foitzik, T; Buhr, H J; Riecken, E O; Rosewicz, S

    1998-11-01

    During carcinogenesis, pancreatic acinar cells can dedifferentiate into ductal adenocarcinoma of the pancreas. DSL-6A/C1 cells represent an in vitro model of this carcinogenic sequence. This study was designed to examine the effects of retinoids on cell growth in DSL-6A/C1 cells and to characterize further the molecular mechanisms underlying the antiproliferative actions of retinoids. Treatment of DSL-6A/C1 cells with retinoids results in a time- and dose-dependent inhibition of cell growth, paralleled by a retinoid-mediated transactivation of a pTK::betaRAREx2-luciferase reporter construct transiently transfected into DSL-6A/C1 cells. Retinoid receptor expression was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) using subtype-specific primers and demonstrated expression of retinoic acid receptor alpha (RAR-alpha), RAR-beta and retinoid X receptor alpha (RXR-alpha). Using a panel of receptor subtype-specific agonists, the RAR-alpha specific agonist Ro 40-6055 was the most potent retinoid in terms of growth inhibition. Furthermore, all-trans-retinoic acid-mediated growth inhibition and transactivation was completely blocked by the RAR-alpha-specific antagonist Ro 41-5253. In summary, the RAR-alpha subtype predominantly mediates the antiproliferative effects of retinoids in DSL-6A/C1 cells. Furthermore, this cell system provides a feasible tool to study the molecular mechanisms underlying the growth inhibitory effects of retinoids in ductal pancreatic carcinoma cells derived from a primary acinar cell phenotype.

  19. HNF-1alpha participates in glucose regulation of sucrase-isomaltase gene expression in epithelial intestinal cells.

    PubMed

    Gu, Ning; Adachi, Tetsuya; Matsunaga, Tetsuro; Tsujimoto, Gozoh; Ishihara, Akihiko; Yasuda, Koichiro; Tsuda, Kinsuke

    2007-02-16

    Sucrase-isomaltase (SI) gene expression is negatively regulated by glucose, but its molecular mechanism is not completely clear. The purpose of this study is to investigate whether HNF-1alpha and HNF-1beta contribute to glucose regulation of SI gene expression. To explore this question, we examined the association of gene expressions between SI and HNF-1alpha and HNF-1beta in Caco-2 cells cultured in medium containing 2.0 and 16.7 mM glucose. We found that gene expression of HNF-1alpha but not HNF-1beta exhibits a positive correlation with that of SI regulated by glucose. Moreover, to elucidate whether glucose regulation of SI gene expression is changed when HNF-1alpha and HNF-1beta are inhibited, we produced three stable cell lines, in which dominant-negative mutant HNF-1alphaT539fsdelC, mutant HNF-1betaR177X, and empty vector (as a control), respectively, were stably expressed. We found that the glucose regulation of SI gene expression was significantly attenuated in HNF-1alphaT539fsdelC cells, but it was well maintained in empty vector and HNF-1betaR177X cells. These results suggest that HNF-1alpha participates in glucose regulation of SI gene expression.

  20. 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.

  1. Isolation of a taxol-resistant Chinese hamster ovary cell mutant that has an alteration in alpha-tubulin.

    PubMed Central

    Cabral, F; Abraham, I; Gottesman, M M

    1981-01-01

    Taxol is a plant alkaloid that has antimitotic activity and appears to stabilize microtubules [Schiff, P. B., Fant, J. & Horwitz, S. B. (1979) Nature (London) 277, 665-667]. Taxol-resistant cells were selected from a population of UV-mutagen-treated Chinese hamster ovary cells by a single-step procedure. These mutants have normal morphologies and growth rates but are 2- to 3-fold more resistant to the toxic effects of the drug than the wild-type parent. One out of 20 mutants screened by two-dimensional electrophoresis for chemical alterations in tubulin had an "extra" spot with a more acidic isoelectric point that alpha-tubulin. This extra spot was shown to be an electrophoretic variant alpha-tubulin by its copurification with tubulin in crude microtubule-containing preparations and by one-dimensional peptide mapping. The alpha-tubulin mutant was found to be temperature sensitive for growth, and this property was used as the basis for the selection of revertants. Seventeen temperature-resistant revertants of the alpha-tubulin mutant were selected for their ability to grow at 40 degrees C and three of these revertants were found to have simultaneously lost their taxol resistance and the electrophoretic variant alpha-tubulin. These results provide evidence that an alteration in alpha-tubulin can confer taxon resistance on a mammalian cell line and suggest that alpha-tubulin is essential for cell viability. Images PMID:6117076

  2. Paxillin binding to the alpha 4 integrin subunit stimulates LFA-1 (integrin alpha L beta 2)-dependent T cell migration by augmenting the activation of focal adhesion kinase/proline-rich tyrosine kinase-2.

    PubMed

    Rose, David M; Liu, Shouchun; Woodside, Darren G; Han, Jaewon; Schlaepfer, David D; Ginsberg, Mark H

    2003-06-15

    Engagement of very late Ag-4 (integrin alpha(4)beta(1)) by ligands such as VCAM-1 markedly stimulates leukocyte migration mediated by LFA-1 (integrin alpha(L)beta(2)). This form of integrin trans-regulation in T cells requires the binding of paxillin to the alpha(4) integrin cytoplasmic domain. This conclusion is based on the abolition of trans-regulation in Jurkat T cells by an alpha(4) mutation (alpha(4)(Y991A)) that disrupts paxillin binding. Furthermore, cellular expression of an alpha(4)-binding fragment of paxillin that blocks the alpha(4)-paxillin interaction, selectively blocked VCAM-1 stimulation of alpha(L)beta(2)-dependent cell migration. The alpha(4)-paxillin association mediates trans-regulation by enhancing the activation of tyrosine kinases, focal adhesion kinase (FAK) and/or proline-rich tyrosine kinase-2 (Pyk2), based on two lines of evidence. First, disruption of the paxillin-binding site in the alpha(4) tail resulted in much less alpha(4)beta(1)-mediated phosphorylation of Pyk2 and FAK. Second, transfection with cDNAs encoding C-terminal fragments of Pyk2 and FAK, which block the function of the intact kinases, blocked alpha(4)beta(1) stimulation of alpha(L)beta(2)-dependent migration. These results define a proximal protein-protein interaction of an integrin cytoplasmic domain required for trans-regulation between integrins, and establish that augmented activation of Pyk2 and/or FAK is an immediate signaling event required for the trans-regulation of integrin alpha(L)beta(2) by alpha(4)beta(1).

  3. Dexamethasone protection from TNF-alpha-induced cell death in MCF-7 cells requires NF-kappaB and is independent from AKT

    PubMed Central

    Machuca, Catalina; Mendoza-Milla, Criselda; Córdova, Emilio; Mejía, Salvador; Covarrubias, Luis; Ventura, José; Zentella, Alejandro

    2006-01-01

    Background The biochemical bases for hormone dependence in breast cancer have been recognized as an important element in tumor resistance, proliferation and metastasis. On this respect, dexamethasone (Dex) dependent protection against TNF-alpha-mediated cell death in the MCF-7 cell line has been demonstrated to be a useful model for the study of this type of cancer. Recently, cytoplasmic signaling induced by steroid receptors has been described, such as the activation of the PI3K/Akt and NF-kappaB pathways. We evaluated their possible participation in the Dex-dependent protection against TNF-alpha-mediated cell death. Results Cellular cultures of the MCF-7 cell line were exposed to either, TNF-alpha or TNF-alpha and Dex, and cell viability was evaluated. Next, negative dominants of PI3K and IkappaB-alpha, designed to block the PI3K/Akt and NF-kappaB pathways, respectively, were transfected and selection and evaluation of several clones overexpressing the mutants were examined. Also, correlation with inhibitor of apoptosis proteins (IAPs) expression was examined. Independent inhibition of these two pathways allowed us to test their participation in Dex-dependent protection against TNF-alpha-cytotoxicity in MCF-7 cells. Expression of the PI3K dominant negative mutant did not alter the protection conferred by Dex against TNF-alpha mediated cell death. Contrariwise, clones expressing the IkappaB-alpha dominant negative mutant lost the Dex-conferred protection against TNF-alpha. In these clones degradation of c-IAP was accelerated, while that of XIAP was remained unaffected. Conclusion NF-kappaB, but not PI3K/Akt activation, is required for the Dex protective effect against TNF-alpha-mediated cell death, and correlates with lack of degradation of the anti-apoptotic protein c-IAP1. PMID:16504042

  4. 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.

  5. 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

  6. 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

  7. Cell type-specific transcriptional regulation of the gene encoding importin-{alpha}1

    SciTech Connect

    Kamikawa, Yasunao; Yasuhara, Noriko; Yoneda, Yoshihiro

    2011-08-15

    Importin-{alpha}1 belongs to a receptor family that recognizes classical nuclear localization signals. Encoded by Kpna2, this receptor subtype is highly expressed in mouse embryonic stem (ES) cells. In this study, we identified a critical promoter region in Kpna2 and showed that the expression of this gene is differentially regulated in ES cells and NIH3T3 cells. Conserved CCAAT boxes are required for Kpna2 promoter activity in both ES and NIH3T3 cells. Interestingly, deletion of the region from nucleotide position - 251 to - 179 bp resulted in a drastic reduction in Kpna2 transcriptional activity only in ES cells. This region contains Krueppel-like factor (Klf) binding sequences and is responsible for transactivation of the gene by Klf2 and Klf4. Accordingly, endogenous Kpna2 mRNA levels decreased in response to depletion of Klf2 and Klf4 in ES cells. Our results suggest that Klf2 and Klf4 function redundantly to drive high level of Kpna2 expression in ES cells. -- Research Highlights: {yields} We showed the cell type-specific transcriptional regulation of Kpna2 encoding importin-al. {yields} NF-Y binds the CCAAT boxes to activate Kpna2 transcription in NIH3T3 cells. {yields} Klf2 and Klf4 redundantly activate the expression of Kpna2 in ES cells.

  8. Transport of alpha-amylase across the basolateral membrane of the pancreatic acinar cell.

    PubMed Central

    Isenman, L D; Rothman, S S

    1977-01-01

    The flux of alpha-amylase (1,4-alpha-D-glucan glucanohydrolase; EC 3.2.1.1) across the basolateral membrane of the acinar cell was measured in the cell-to-bath direction using the whole rabbit pancreas in organ culture. This in vitro preparation is polarized so that apical and basolateral secretions can be collected separately. The unstimulated amylase flux from cell to bath was substantial at the initial rate (approximately three times the concurrent apical flux). With time, bath amylase approached a steady-state concentration, suggesting an equilbrating process. During the same time interval, ductal amylase secretion remained constant. At the steady state, the amylase concentration in the bath was at least an order of magnitude less than its ductal concentration. Hourly replacement of bathing medium reproduced the initial rate of amylase release into the bath for five consecutive hours. Pancreozymin (cholecystokinin), a peptide hormone, did not alter the steady-state bath amylase content, although it greatly augmented ductal amylase secretion. In contrast, a cholinergic agonist greatly increased both the flux from the cell to bath and the ductal secretion of amylase. Taken together, these results indicate a natural bidirectional permeability of the basolateral membrane to digestive enzyme and support evidence previously obtained suggesting that such a permeability might exist. PMID:302947

  9. 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.

  10. Effects of alpha-thalassemia and sickle polymerization tendency on the urine-concentrating defect of individuals with sickle cell trait.

    PubMed Central

    Gupta, A K; Kirchner, K A; Nicholson, R; Adams, J G; Schechter, A N; Noguchi, C T; Steinberg, M H

    1991-01-01

    A defect in urine concentrating ability occurs in individuals with sickle cell trait (HbAS). This may result from intracellular polymerization of sickle hemoglobin (HbS) in erythrocytes, leading to microvascular occlusion, in the vasa recta of the renal medulla. To test the hypothesis that the severity of the concentrating defect is related to the percentage of sickle hemoglobin present in erythrocytes, urinary concentrating ability was examined after overnight water deprivation, and intranasal desmopressin acetate (dDAVP) in 27 individuals with HbAS. The HbAS individuals were separated into those who had a normal alpha-globin genotype (alpha alpha/alpha alpha), and those who were either heterozygous (-alpha/alpha alpha) or homozygous (-alpha/-alpha) for gene-deletion alpha-thalassemia, because alpha-thalassemia modulates the HbS concentration in HbAS. The urinary concentrating ability was less in the alpha alpha/alpha alpha genotype than in the -alpha/alpha alpha or -alpha/-alpha genotypes (P less than 0.05). After dDAVP, the urine osmolality was greater in patients with the -alpha/-alpha genotype than with the -alpha/alpha alpha genotype (882 +/- 37 vs. 672 +/- 38 mOsm/kg H2O) (P less than 0.05); patients with the -alpha/alpha alpha genotype had greater concentrating ability than individuals with a normal alpha-globin gene arrangement. There was an inverse linear correlation between urinary osmolality after dDAVP and the percentage HbS in all patients studied (r = -0.654; P less than 0.05). A linear correlation also existed for urine concentrating ability and the calculated polymerization tendencies for an oxygen saturation of 0.4 and O (r = -0.62 and 0.69, respectively). We conclude that the severity of hyposthenuria in HbAS is heterogeneous. It is determined by the amount of HbS polymer, that in turn is dependent upon the percentage HbS, which is itself related to the alpha-globin genotype. Images PMID:1752955

  11. Patterns of alpha-L-fucosidase in acute myeloid leukemia cells. Comparison with promyelocytic HL-60 cell line.

    PubMed

    Dosbaa, I; Bernard, M; Foglietti, M J; Percheron, F; Emiliani, C

    1992-12-15

    Changes were observed in alpha-L-fucosidase forms in cells from acute myelocytic leukemias (AML). Total alpha-L-fucosidase activity was not significantly different for normal granulocytes and leukemic cells, but enzymic profiles obtained by chromatofocusing are quite different. In granulocyte profile, two main peaks are present (B and more acidic A) which were eluted at pH 5.2 with a shoulder at pH 4.6. In AMLs the B form is present but weakly expressed, whereas the more acidic forms are the major ones. This pattern may be related either to the malignancy character or to the stage at which the differentiation is stopped. Experiments on an HL-60 cell line (promyelocytic cells corresponding to the AML 3 type) showed that differentiation induced by dimethyl sulfoxide leads to the appearance of the B form present in normal mature cells. Thus the repartition of the enzyme forms seems to be related to the stage of differentiation of the myelocytic cells.

  12. Potential protective effects of alpha-pinene against cytotoxicity caused by aspirin in the IEC-6 cells.

    PubMed

    Bouzenna, Hafsia; Hfaiedh, Najla; Giroux-Metges, Marie-Agnès; Elfeki, Abdelfattah; Talarmin, Hélène

    2017-09-01

    Alpha-pinene is a key compound of the essential oils extracted from many species of coniferous trees. It is known for its biological activities. The aim of the present study was to determine the preventive effect of alpha-pinene on aspirin-induced toxicity in vitro, using IEC-6 cells, and to investigate its antioxidant activities. The antioxidant activities were determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP). The cytotoxicity and oxidative stress were detected by cell viability, antioxidant enzyme activity, malondialdehyde (MDA) and GSH production, and the activation of MAPK pathways. The results indicated that alpha-pinene revealed an important antioxidant activity. It was evaluated by DPPH test (EC50=310±10μg/mL) and FRAP test (EC50=238±18.92μg/mL). The co-exposure of alpha-pinene with aspirin on cells significantly increased the survival of cells and the level of GSH, and decreased the levels of MDA and total SOD and the activity of Mn-SOD. In addition, the activation of p38 and JNK was blocked by alpha-pinene. Therefore, these findings suggest that alpha-pinene can protect IEC-6 cells against aspirin-induced oxidative stress. Copyright © 2017. Published by Elsevier Masson SAS.

  13. The Retinoic Acid Receptor-alpha mediates human T-cell activation and Th2 cytokine and chemokine production.

    PubMed

    Dawson, Harry D; Collins, Gary; Pyle, Robert; Key, Michael; Taub, Dennis D

    2008-04-16

    We have recently demonstrated that all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (9-cis RA) promote IL-4, IL-5 and IL-13 synthesis, while decreasing IFN-gamma and TNF-alpha expression by activated human T cells and reduces the synthesis of IL-12p70 from accessory cells. Here, we have demonstrated that the observed effects using ATRA and 9-cis RA are shared with the clinically useful RAR ligand, 13-cis retinoic acid (13-cis RA), and the retinoic acid receptor-alpha (RAR-alpha)-selective agonist, AM580 but not with the RAR-beta/gamma ligand, 4-hydroxyphenylretinamide (4-HPR). The increase in type 2 cytokine production by these retinoids correlated with the expression of the T cell activation markers, CD69 and CD38. The RAR-alpha-selective agonist, AM580 recapitulated all of the T cell activation and type 2 cytokine-inducing effects of ATRA and 9-cis-RA, while the RAR-alpha-selective antagonist, RO 41-5253, inhibited these effects. These results strongly support a role for RAR-alpha engagement in the regulation of genes and proteins involved with human T cell activation and type 2 cytokine production.

  14. 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.

  15. 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.

  16. Initiation of remote hepatic injury in the rat: interactions between Kupffer cells, tumor necrosis factor-alpha, and microvascular perfusion.

    PubMed

    Brock, R W; Lawlor, D K; Harris, K A; Potter, R F

    1999-07-01

    Severe trauma may initiate a systemic inflammatory response, which in turn may result in remote organ injury. After limb ischemia/reperfusion (I/R), intravital fluorescence microscopy was applied to the livers of normotensive rats to investigate the initiation of remote injury to the liver. Additionally, we determined whether Kupffer cell activation and tumor necrosis factor-alpha (TNF-alpha) were involved, via perfusion deficits, in such injury. TNF-alpha, measured by immunoassay, peaked at 30 minutes of reperfusion, but returned to baseline within 60 minutes. Limb I/R resulted in significant increases to global hepatocellular injury measured by alanine transaminase (ALT) and lethal hepatocyte injury as seen with intravital fluorescence microscopy. Although the number of perfused sinusoids went unchanged, a significantly augmented perfusion heterogeneity was measured. After 1.5 hours of reperfusion, both TNF-alpha and Kupffer cells were shown to contribute to global hepatocellular injury (e.g., ALT). After 3 hours, TNF-alpha was no longer essential for this injury, suggesting that some other mechanism(s) activated Kupffer cells and initiated hepatocellular injury. Using propidium iodide and fluorescence microscopy, we found that both TNF-alpha and Kupffer cell activation were necessary to drive hepatocytes toward lethal injury. No additional benefits were observed with a combination of TNF-alpha inhibition and Kupffer cell suppression. These results not only implicate both Kupffer cells and TNF-alpha in the initiation of remote hepatic injury, but suggest that sinusoidal perfusion deficits are not essential for the initiation of such injury. Other mechanism(s) are likely involved in the pathogenesis of remote hepatic parenchymal injury.

  17. 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

  18. Retinoic acid-induced expression of CD38 antigen in myeloid cells is mediated through retinoic acid receptor-alpha.

    PubMed

    Drach, J; McQueen, T; Engel, H; Andreeff, M; Robertson, K A; Collins, S J; Malavasi, F; Mehta, K

    1994-04-01

    CD38 is a leukocyte differentiation antigen that has been thought to be a phenotypic marker of different subpopulations of T- and B-lymphocytes. In myeloid cells, CD38 is expressed during early stages of differentiation. Virtually no information is available on regulation and functions of CD38. Recently we reported that all-trans-retinoic acid (ATRA) is a potent and highly specific inducer of CD38 expression in human promyelocytic leukemia cells. Here we report that ATRA-induced expression of CD38 antigen in myeloid cells is mediated through retinoic acid-alpha receptor (RAR alpha). ATRA failed to induce CD38 expression in a mutant subclone of the HL-60 myeloid leukemia cell line (designated HL-60R) that is relatively resistant to ATRA-induced granulocytic differentiation. Retroviral vector-mediated transduction of RA receptor (RAR alpha) into this HL-60R subclone completely restored the sensitivity of these cells to ATRA in terms of their ability to express CD38. In contrast, CD38 expression was not inducible by ATRA in HL-60R cells, transfected with a functional RAR beta, RAR gamma, or RXR alpha receptor. Induction of CD38 in acute promyelocytic and acute myeloblastic leukemia cells was independent of ATRA-induced cytodifferentiation. Following culture with ATRA, increased CD38 protein levels were also observed in normal CD34+ bone marrow cells, but not on normal circulating granulocytes. From these results, we conclude that CD38 is ATRA inducible in myeloid leukemia cells and normal CD34+ bone marrow cells. This effect is independent of differentiation and is mediated by RAR alpha in HL-60 cells, suggesting a similar role for RAR alpha in CD38 expression in other hematopoietic cells.

  19. Clinical usefulness of alpha-crystallin antibodies in non-small cell lung cancer patients.

    PubMed

    Cherneva, Radostina; Petrov, Danail; Georgiev, Ognian; Trifonova, Nedka

    2010-01-01

    The non-invasive approach of finding biomarkers in peripheral blood of cancer patients makes it useful for clinical application and cancer screening. The aim of the study was to explore the clinical utility of alpha-crystallin antibodies as markers for diagnosis of non-small cell lung cancer (NSCLC) and screening among high-risk groups. Alpha-crystallin antibodies were detected with enzyme-linked immunosorbent assay (ELISA) in 51 NSCLC patients, 38 high-risk chronic obstructive pulmonary disease (COPD) patients and 52 age and sex matched healthy volunteers. Alpha-crystallin IgG antibodies differed significantly between the groups of cancer patients and the healthy volunteers (P<0.001). A cut-off value of 0.317 discerned NSCLC patients with sensitivity 62%, and specificity 72% among the control group. The assay was effective in distinguishing the patients with and without lymphogenic metastatic spread of the disease (P=0.045): sensitivity 60%, and specificity 70%. The clinical significance of this marker has a modest implication in lung cancer diagnosis and screening in high-risk groups. Its importance as a prognostic marker or a marker of disease recurrence and lymph node micrometastasis should be further explored.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. Asynchronous Crystal Cell Expansion during Lithiation of K+-Stabilized {alpha}-MnO2

    SciTech Connect

    Yuan, Yifei; Nie, Anmin; Odegard, Gregory M.; Xu, Rui; Zhou, Dehua; Sunand, Santhanagopalan; He, Kun; Asayesh-Ardakani, Hasti; Meng, Dennis Desheng; Klie, Robert F.; Johnson, Christopher; Lu, Jun; Shahbazian-Yassar, Reza

    2015-05-01

    alpha-MnO2 is a promising material for Li-ion batteries and has unique tunneled structure that facilitates the diffusion of Li+. The overall electrochemical performance of a-MnO2 is determined by the tunneled structure stability during its interaction with Li+, the mechanism of which is, however, poorly understood. In this paper, a novel tetragonal-orthorhombic-tetragonal symmetric transition during lithiation of K+-stabilized a-MnO2 is observed using in situ transmission electron microscopy. Atomic resolution imaging indicated that 1 x 1 and 2 x 2 tunnels exist along c ([001]) direction of the nanowire. The morphology of a partially lithiated nanowire observed in the ?100? projection is largely dependent on crystallographic orientation ([100] or [010]), indicating the existence of asynchronous expansion of alpha-MnO(2)s tetragonal unit cell along a and b lattice directions, which results in a tetragonal-orthorhombic-tetragonal (TOT) symmetric transition upon lithiation. Such a TOT transition is confirmed by diffraction analysis and Mn valence quantification. Density functional theory (DFT) confirms that Wyckoff 8h sites inside 2 x 2 tunnels are the preferred sites for Li+ occupancy. The sequential Li+ filling at 8h sites leads to asynchronous expansion and symmetry degradation of the host lattice as well as tunnel instability upon lithiation. These findings provide fundamental understanding for appearance of stepwise potential variation during the discharge of Li/alpha-MnO2 batteries as well as the origin for low practical capacity and fast capacity fading of alpha-MnO2 as an intercalated electrode.

  6. Lithium increases PGC-1alpha expression and mitochondrial biogenesis in primary bovine aortic endothelial cells.

    PubMed

    Struewing, Ian T; Barnett, Corey D; Tang, Tao; Mao, Catherine D

    2007-06-01

    Lithium is a therapeutic agent commonly used to treat bipolar disorder and its beneficial effects are thought to be due to a combination of activation of the Wnt/beta-catenin pathway via inhibition of glycogen synthase kinase-3beta and depletion of the inositol pool via inhibition of the inositol monophosphatase-1. We demonstrated that lithium in primary endothelial cells induced an increase in mitochondrial mass leading to an increase in ATP production without any significant change in mitochondrial efficiency. This increase in mitochondrial mass was associated with an increase in the mRNA levels of mitochondrial biogenesis transcription factors: nuclear respiratory factor-1 and -2beta, as well as mitochondrial transcription factors A and B2, which lead to the coordinated upregulation of oxidative phosphorylation components encoded by either the nuclear or mitochondrial genome. These effects of lithium on mitochondrial biogenesis were independent of the inhibition of glycogen synthase kinase-3beta and independent of inositol depletion. Also, expression of the coactivator PGC-1alpha was increased, whereas expression of the coactivator PRC was not affected. Lithium treatment rapidly induced a decrease in activating Akt-Ser473 phosphorylation and inhibitory Forkhead box class O (FOXO1)-Thr24 phosphorylation, as well as an increase in activating c-AMP responsive element binding (CREB)-Ser133 phosphorylation, two mechanisms known to control PGC-1alpha expression. Together, our results show that lithium induces mitochondrial biogenesis via CREB/PGC-1alpha and FOXO1/PGC-1alpha cascades, which highlight the pleiotropic effects of lithium and reveal also novel beneficial effects via preservation of mitochondrial functions.

  7. Structure-function studies of PANDER, an islet specific cytokine inducing cell death of insulin-secreting beta cells.

    PubMed

    Yang, Jichun; Gao, Zhiyong; Robert, Claudia E; Burkhardt, Brant R; Gaweska, Helena; Wagner, Amary; Wu, Jianmei; Greene, Scott R; Young, Robert A; Wolf, Bryan A

    2005-08-30

    PANDER (pancreatic derived factor, FAM3B) is a novel cytokine, present in insulin secretory granules, that induces apoptosis of alpha and beta cells of mouse, rat, and human islets in a dose- and time-dependent manner, and may be implicated in diabetes. PANDER has the predicted secondary structure of 4 alpha-helical bundles with an up-up-down-down topology, and two disulfide bonds. Eleven mutated PANDERs were constructed and expressed in beta-TC3 cells to identify the essential region of PANDER involved in beta-cell death. Beta-cell function was assessed by assays of cell viability and insulin secretion. Based on quantitative real-time RT-PCR all mutant PANDERs had similar mRNA expression levels in beta-TC3 cells. Immunoblotting showed that ten of eleven mutant PANDER proteins were synthesized and detected in beta-TC3 cells. A mutant PANDER with no signal peptide, however, was not expressed. Truncation of helix D alone caused a 40-50% decrease in PANDER's activity, while truncation of both helices C and D resulted in a 75% loss of activity. In contrast, truncation of the N-terminus of PANDER (helix A, the loop between helices A and B, and the first two cysteines) had no effect on PANDER-induced beta-cell death. The third and fourth cysteines of PANDER, C91 and C229, were shown to form one disulfide bond and be functionally important. Finally, the region between Cys91 and Phe152 constitutes the active part of PANDER, based on the demonstration that mutants with truncation of helix B or C caused decreased beta-cell death and did not inhibit insulin secretion, as compared to wild-type PANDER. Hence, helices B and C and the second disulfide bond of PANDER are essential for PANDER-induced beta-cell death.

  8. 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.

  9. 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 inve