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Sample records for alpha tc3 cells

  1. Development of a bioartificial pancreas: II. Effects of oxygen on long-term entrapped betaTC3 cell cultures.

    PubMed

    Papas, K K; Long, R C; Sambanis, A; Constantinidis, I

    1999-01-01

    Tissue-engineered pancreatic constructs based on immunoisolated, insulin-secreting cells are promising in providing an effective, relatively inexpensive, long-term treatment for type I (insulin-dependent) diabetes. An in vitro characterization of construct function under conditions mimicking the in vivo environment is essential prior to any extensive animal experimentation. Encapsulated cells may experience hypoxic conditions postimplantation as a result of one or more of the following: the design of the construct; the environment at the implantation site; or the development of fibrosis around the construct. In this work, we studied the effects of 3- and 4-day-long hypoxic episodes on the metabolic and secretory activities and on the levels of intracellular metabolites detectable by phosphorus-31 nuclear magnetic resonance ((31)P NMR) of alginate/poly-L-lysine/alginate entrapped betaTC3 mouse insulinomas continuously perfused with culture medium. Results show that, upon decreasing the oxygen concentration in the surrounding medium, the encapsulated cell system reached a new, lower metabolic and secretory state. Hypoxia drove the cells to a more anaerobic glycolytic metabolism, increased the rates of glucose consumption (GCR) and lactate production (LPR), and reduced the rates of oxygen consumption (OCR) and insulin secretion (ISR). Furthermore, hypoxia reduced the levels of intracellular nucleotide triphosphates (NTP) and phosphorylcholine (PC) and caused a rapid transient increase in inorganic phosphate (P(i)). Upon restoration of the oxygen concentration in the perfusion medium, all parameters returned to their prehypoxic levels within 2 to 3 days following either gradual unidirectional changes (ISR, NTP, PC) or more complicated dynamic patterns (OCR, GCR, LPR). A further increase in oxygen concentration in the perfusion medium drove OCR, ISR, NTP, PC, and P(i) to new, higher levels. It is concluded that (31)P NMR spectroscopy can be used for the prolonged

  2. 99mTc-3P-RGD2 SPECT to monitor early response to bevacizumab therapy in patients with advanced non-small cell lung cancer

    PubMed Central

    Chen, Bin; Zhao, Guoqing; Ma, Qingjie; Ji, Bin; Ji, Tiefeng; Xin, Hua; Gao, Shi

    2015-01-01

    The present study assessed the predictive value of 99mTc-3(poly-(ethylene glycol), PEG) 4-arginine-glycine-aspartic (99mTc-3P-RGD2) single photon emission computed tomography (SPECT) for the early identification of response to antiangiogenic treatment with bevacizumab in patients with advanced non-small cell lung cancer (NSCLC). Patients with advanced NSCLC treated with bevacizumab were prospectively studied with 99mTc-3P-RGD2 SPECT before and after 2 weeks from start of treatment. The tumor response was evaluated with RECIST criteria and related to observed change in the tumor to non-tumor (T/N) ratio for the largest known lesion. Receiver operating characteristic (ROC) curve analysis was used to determine T/N ratio changes with regard to predicting response to bevacizumab therapy. Change in T/N ratio was also related to progression-free survival (PFS) and overall survival (OS). Twenty-six patients were included, and 23 were finally assessable for metabolic response evaluation with 99mTc-3P-RGD2 SPECT. The cut-off value of T/N ratio change defined by ROC analysis was 24.4%. The sensitivity, specificity, and negative predictive value of 99mTc-3P-RGD2 SPECT for predicting tumor response were 81.8%, 91.7%, and 84.6%, respectively. Using the cut-off value defined by ROC analysis on 99mTc-3P-RGD2 SPECT, metabolic non-progressive disease patients (mNP) showed prolonged PFS (5.6 months versus 3.4 months; P < 0.001) and OS (17.1 months versus 8.6 months; P < 0.001) than metabolic progressive disease patients (mP). 99mTc-3P-RGD2 SPECT scan is a promising test to predict tumor response in patients with advanced non-small cell lung cancer early in the course of bevacizumab therapy. PMID:26884883

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  4. The recovery of asteroid 2008 TC3

    NASA Astrophysics Data System (ADS)

    Shaddad, Muawia H.; Jenniskens, Peter; Numan, Diyaa; Kudoda, Ayman M.; Elsir, Saadia; Riyad, Ihab F.; Ali, Awad Elkareem; Alameen, Mohammed; Alameen, Nada M.; Eid, Omer; Osman, Ahmed T.; Abubaker, Mohamed I.; Yousif, Mohamed; Chesley, Steven R.; Chodas, Paul W.; Albers, Jim; Edwards, Wayne N.; Brown, Peter G.; Kuiper, Jacob; Friedrich, Jon M.

    2010-10-01

    On October 7, 2008, asteroid 2008 TC3 impacted Earth and fragmented at 37 km altitude above the Nubian Desert in northern Sudan. The area surrounding the asteroid's approach path was searched, resulting in the first recovery of meteorites from an asteroid observed in space. This was also the first recovery of remains from a fragile "cometary" PE = IIIa/b type fireball. In subsequent searches, over 600 mostly small 0.2-379 g meteorites (named "Almahata Sitta") with a total mass 10.7 kg were recovered from a 30 × 7 km area. Meteorites fell along the track at 1.3 kg km-1, nearly independent of mass between 1 and 400 g, with a total fallen mass of 39 ± 6 kg. The strewn field was shifted nearly 1.8 km south from the calculated approach path. The influence of winds on the distribution of the meteorites, and on the motion of the dust train, is investigated. The majority of meteorites are ureilites with densities around 2.8 g cm-3, some of an anomalous (porous, high in carbon) polymict ureilite variety with densities as low as 1.5 g cm-3. In addition, an estimated 20-30% (in mass) of recovered meteorites were ordinary, enstatite, and carbonaceous chondrites. Their fresh look and matching distribution of fragments in the strewn field imply that they were part of 2008 TC3. For that reason, they are all referred to as "Almahata Sitta." No ureilite meteorites were found that still held foreign clasts, suggesting that the asteroid's clasts were only loosely bound.

  5. 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.

  6. DNA binding activities of the Caenorhabditis elegans Tc3 transposase.

    PubMed Central

    Colloms, S D; van Luenen, H G; Plasterk, R H

    1994-01-01

    Tc3 is a member of the Tc1/mariner family of transposable elements. All these elements have terminal inverted repeats, encode related transposases and insert exclusively into TA dinucleotides. We have studied the DNA binding properties of Tc3 transposase and found that an N-terminal domain of 65 amino acids binds specifically to two regions within the 462 bp Tc3 inverted repeat; one region is located at the end of the inverted repeat, the other is located approximately 180 bp from the end. Methylation interference experiments indicate that this N-terminal DNA binding domain of the Tc3 transposase interacts with nucleotides on one face of the DNA helix over adjacent major and minor grooves. Images PMID:7838706

  7. 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.

  8. Cross-talk between integrins {alpha}1{beta}1 and {alpha}2{beta}1 in renal epithelial cells

    SciTech Connect

    Abair, Tristin D.; Sundaramoorthy, Munirathinam; Chen, Dong; Heino, Jyrki; Ivaska, Johanna; Hudson, Billy G.; Sanders, Charles R.; Pozzi, Ambra; Zent, Roy

    2008-11-15

    The collagen-binding integrins {alpha}1{beta}1 and {alpha}2{beta}1 have profoundly different functions, yet they are often co-expressed in epithelial cells. When both integrins are expressed in the same cell, it has been suggested that {alpha}1{beta}1 negatively regulates integrin {alpha}2{beta}1-dependent functions. In this study we utilized murine ureteric bud (UB) epithelial cells, which express no functionally detectable levels of endogenous integrins {alpha}1{beta}1 and {alpha}2{beta}1, to determine the mechanism whereby this regulation occurs. We demonstrate that UB cells expressing integrin {alpha}2{beta}1, but not {alpha}1{beta}1 adhere, migrate and proliferate on collagen I as well as form cellular cords in 3D collagen I gels. Substitution of the transmembrane domain of the integrin {alpha}2 subunit with that of {alpha}1 results in decreased cell adhesion, migration and cord formation. In contrast, substitution of the integrin {alpha}2 cytoplasmic tail with that of {alpha}1, decreases cell migration and cord formation, but increases proliferation. When integrin {alpha}1 and {alpha}2 subunits are co-expressed in UB cells, the {alpha}1 subunit negatively regulates integrin {alpha}2{beta}1-dependent cord formation, adhesion and migration and this inhibition requires expression of both {alpha}1 and {alpha}2 tails. Thus, we provide evidence that the transmembrane and cytoplasmic domains of the {alpha}2 integrin subunit, as well as the {alpha}1 integrin subunit, regulate integrin {alpha}2{beta}1 cell function.

  9. Methylation of alpha-type embryonic globin gene alpha pi represses transcription in primary erythroid cells.

    PubMed

    Singal, Rakesh; vanWert, Jane M; Ferdinand, Larry

    2002-12-01

    The inverse relationship between expression and methylation of beta-type globin genes is well established. However, little is known about the relationship between expression and methylation of avian alpha-type globin genes. The embryonic alpha(pi)-globin promoter was unmethylated, and alpha(pi)-globin RNA was easily detected in 5-day chicken erythroid cells. A progressive methylation of the CpG dinucleotides in the alpha(pi) promoter associated with loss of expression of alpha(pi)-globin gene was seen during development in primary erythroid cells. A 315-bp alpha(pi)-globin promoter region was cloned in an expression construct (alpha(pi)pGL3E) containing a luciferase reporter gene and SV40 enhancer. The alpha(pi)pGL3E construct was transfected into primary erythroid cells derived from 5-day-old chicken embryos. Methylation of alpha(pi)pGL3E plasmid and alpha(pi)-globin promoter alone resulted in a 20-fold and 7-fold inhibition of expression, respectively. The fully methylated but not the unmethylated 315-bp alpha(pi)-globin gene promoter fragment formed a methyl cytosine-binding protein complex (MeCPC). Chromatin immunoprecipitation assays were combined with quantitative real-time polymerase chain reaction to assess histone acetylation associated with the alpha(pi)-globin gene promoter. Slight hyperacetylation of histone H3 but a marked hyperacetylation of histone H4 was seen in 5-day when compared with 14-day erythroid cells. These results demonstrate that methylation can silence transcription of an avian alpha-type embryonic globin gene in homologous primary erythroid cells, possibly by interacting with an MeCPC and histone deacetylase complex. PMID:12393573

  10. Fibronectin receptor functions in embryonic cells deficient in alpha 5 beta 1 integrin can be replaced by alpha V integrins.

    PubMed Central

    Yang, J T; Hynes, R O

    1996-01-01

    alpha 5 beta 1 integrin mediates cell adhesion to extracellular matrix by interacting with fibronectin (FN). Mouse lines carrying null mutations in genes encoding either the alpha 5 integrin subunit or FN have been generated previously. Both mutations are embryonic lethal with overlapping defects, but the defects of alpha 5-null embryos are less severe. Primary embryonic cells lacking alpha 5 beta 1 are able to adhere to FN, form focal contacts, migrate on FN, and assemble FN matrix. These results suggest the involvement of (an)other FN receptors(s). In this study, we examined functions of alpha 4 beta 1 and alpha V integrins in embryonic cells lacking alpha 5 beta 1. Our analysis of cells lacking both alpha 4 beta 1 and alpha 5 beta 1 showed that alpha 4 beta 1 is also not required for these FN-dependent functions. Using alpha V-specific blocking reagents, we showed that alpha V integrins are required for alpha 5-null cells, but not wild-type cells, to adhere and spread on FN. Our data also showed that, although the expression levels of alpha V integrins on the wild-type and alpha 5-null cells are similar, there is an increase in recruitment of alpha V integrins into focal contacts in alpha 5-null cells plated on FN, indicating that alpha V integrins can compensate functionally for the loss of alpha 5 beta 1 in focal contacts of alpha 5-null cells. Finally, our data suggested possible roles for alpha V integrins in replacing the role of alpha 5 beta 1 in FN matrix assembly in vitro and in FN-dependent embryonic functions in vivo. Images PMID:8930896

  11. Alpha-1 Antitrypsin and Lung Cell Apoptosis.

    PubMed

    Serban, Karina A; Petrache, Irina

    2016-04-01

    Discovery of alpha-1 antitrypsin (A1AT) as the principal circulating inhibitor of neutrophil elastase was critical to the appreciation of protease/antiprotease imbalance involvement in the pathogenesis of emphysema. Additional targets of A1AT have been uncovered, along with their contribution to alveolar wall destruction induced by cigarette smoke exposure. We highlight in this report mechanisms of A1AT antiapoptotic effects on structural lung endothelial cells. This function was largely dependent on uptake of the protein from the circulation via clathrin- and, in part, caveolae-mediated endocytosis and on specific interactions with cysteine proteases such as capsase-3, -6, and -7. Exposures to cigarette smoke diminished A1AT intracellular uptake and its anticaspase action, suggesting that even in A1AT-suficient individuals, cigarette smoke may weaken the serpin's endothelial prosurvival effect. In addition, cigarette smoke exposure or genetic mutations known to induce posttranslational modifications such as oxidation or polymerization may alter A1AT bidirectional intracellular traffic in endothelial cells and thus determine its functional bioavailability in certain lung compartments. Uncovering and harnessing the A1AT canonical and noncanonical mechanisms will advance our understanding of the pathogenesis of emphysema and may provide means to improve the effectiveness of therapies in both A1AT-sufficient and A1AT-deficient individuals. PMID:27115949

  12. 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.

  13. Pancreatic alpha-cell dysfunction in diabetes.

    PubMed

    Burcelin, R; Knauf, C; Cani, P D

    2008-02-01

    A major, yet poorly understood, feature of type 2 diabetes is the excessive hepatic glucose production and the corresponding insulin resistance leading to fasting hyperglycaemia. The tremendous amount of work done to provide the physiological and molecular mechanisms explaining this impairment has led to the emergence of several consensual hypotheses. Among these, is the increased daily and unregulated plasma glucagon concentration in type 2 diabetic patients. Therefore, studies aiming to understand the physiological regulation of glucagon secretion and the corresponding impairment during diabetes are directly relevant to the treatment of type 2 diabetes. Glucagon secretion by alpha-cells is an immediate response to glucopenia. Abnormal secretion of glucagon and other counterregulatory hormones is a hallmark of type 1 and type 2 diabetes and a major limitation to the use of strong hypoglycaemia agents. A few molecular mechanisms of glucose detection triggering counterregulation and in particular inducing glucagon secretion or suppressing it during hyperglycaemic episodes, have been identified. Such mechanisms are related to those of the insulin secreted beta-cell. The glucose transporter GLUT2 and the K-ATP dependent channel, as well as regulatory mechanisms, involved the central nervous system and the gut-brain hormone GLP-1. Over the last years, glucoincretins have provided promising results for the normalization of plasma glucagon concentration of type 2 diabetic patients, which could partly explain the therapeutic benefits of incretin-related therapy. The underlined mechanisms of GLP-1 regulated glucagon secretion are most likely related to the action of the hormone on the activation of the portal and brain glucose sensors. Certainly, strategies aiming to restore glucose-regulated glucagon secretion are important milestones for the treatment of diabetic patient and the prevention of iatrogenic hypoglycaemia. PMID:18640586

  14. Alpha-amanitin resistance: a dominant mutation in CHO cells.

    PubMed

    Lobban, P E; Siminovitch, L

    1975-02-01

    Hybrids of CHO cells were constructed consisting of either a 1:1 or 1:2 ratio of alpha-amanitin-resistant and sensitive cells, respectively. The resistance of such hybrids to killing by the drug was similar but slightly less than that of the resistant parent. The hybrids contained both resistant and wild-type RNA polymerase II, in amounts related to the expected gene dosage. The alpha-amanitin marker therefore is expressed codominantly. PMID:1168545

  15. Nonantioxidant functions of alpha-tocopherol in smooth muscle cells.

    PubMed

    Azzi, A; Breyer, I; Feher, M; Ricciarelli, R; Stocker, A; Zimmer, S; Zingg, J

    2001-02-01

    Most tocopherols and tocotrienols, with the exception of alpha-tocopherol, are not retained by humans. This suggests that alpha-tocopherol is recognized uniquely; therefore, it may exert an exclusive function. alpha-Tocopherol possesses distinct properties that are independent of its prooxidant, antioxidant or radical-scavenging ability. alpha-Tocopherol specifically inhibits protein kinase C, the growth of certain cells and the transcription of the CD36 and collagenase genes. Activation events have also been seen on the protein phosphatase 2A (PP(2)A) and on the expression of other genes (alpha-tropomyosin and connective tissue growth factor). Neither ss-tocopherol nor probucol possessed the same specialty functions as alpha-tocopherol. Recently, we isolated a new ubiquitous cytosolic alpha-tocopherol binding protein (TAP). Its motifs suggest that it is a member of the hydrophobic ligand-binding protein family (CRAL-TRIO). TAP may also be involved in the regulation of cellular alpha-tocopherol concentration and alpha-tocopherol-mediated signaling. PMID:11160565

  16. GM=tc^3 in The Year of Light

    NASA Astrophysics Data System (ADS)

    Riofrio, Louise

    2015-08-01

    2015 is the International Year of Light. With the lack of evidence for primordial gravitational waves, light is an alternative to the darkness of untestable ideas. We may test a hypothesis GM=tc^3, where G is Newton's constant and M, t represent mass and age in the universe. This provides a precise fit to redshifts of Type Ia supernovae and explains the "Faint Young Sun" paradox of astrophysics. A 12-sigma anomaly in laser measurements of lunar orbital evolution can be predicted to 1/10 of a standard deviation, a precise test of theory. Starting in 2016, the Atomic Clock Ensemble in Space (ACES) aboard the International Space Station will search for anisotropies in the speed of light. Growing evidence may indicate a "c change" in physics.

  17. 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

  18. 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. PMID:27190515

  19. Impaired tumour necrosis factor-alpha (TNF-alpha) production and abnormal B cell response to TNF-alpha in patients with systemic lupus erythematosus (SLE).

    PubMed Central

    Mitamura, K; Kang, H; Tomita, Y; Hashimoto, H; Sawada, S; Horie, T

    1991-01-01

    We examined the TNF-alpha activity in culture supernatants of monocytes isolated from the peripheral blood of patients with SLE and of normal individuals. The monocytes from patients with SLE stimulated with silica particles, lipopolysaccharide or Staphylococcus aureus Cowan 1 secreted significantly lower amounts of TNF-alpha than did normal monocytes. A decreased TNF mRNA expression was observed in peripheral blood mononuclear cells stimulated by mitogens from patients with SLE. Furthermore, we examined the effect of recombinant TNF-alpha (rTNF-alpha) on the B cell function in SLE patients. rTNF-alpha inhibited the spontaneous B cell proliferation of SLE, but tended to enhance the normal B cell proliferation. Spontaneous IgM production from SLE B cells was inhibited by rTNF-alpha, but that from normal B cells was not. Spontaneous IgG production was unaffected by rTNF-alpha. Also, rTNF-alpha did not affect the viability of B cells. These findings suggest that an impaired TNF-alpha production and an abnormal B cell response to TNF-alpha play a role in the immunological dysfunction in patients with SLE. Images Fig. 2 PMID:1893618

  20. The interaction of alpha-thalassemia and homozygous sickle-cell disease.

    PubMed

    Higgs, D R; Aldridge, B E; Lamb, J; Clegg, J B; Weatherall, D J; Hayes, R J; Grandison, Y; Lowrie, Y; Mason, K P; Serjeant, B E; Serjeant, G R

    1982-06-17

    Patients with homozygous sickle-cell disease may be homozygous for alpha-thalassemia 2 (alpha-/alpha-), may be heterozygous for alpha-thalassemia 2 (alpha-/alpha alpha), or may have a normal alpha-globin-gene complement (alpha alpha/alpha alpha). We compared the clinical and hematologic features of 44 patients who had sickle-cell disease and homozygous alpha-thalassemia 2 with those of controls with the two hematologic conditions. The patients with homozygous alpha-thalassemia 2 had significantly higher red-cell counts and levels of hemoglobin and hemoglobin A2, as well as significantly lower hemoglobin F, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, reticulocyte counts, irreversibly-sickled cell counts, and serum total bilirubin levels, than those with a normal alpha-globin-gene complement. Heterozygotes (alpha-/alpha alpha) had intermediate values. In the group with homozygous alpha-thalassemia 2, fewer patients had episodes of acute chest syndrome and chronic leg ulceration and more patients had splenomegaly, as compared with patients in other two subgroups. These data confirm previous suggestions that alpha-thalassemia inhibits in vivo sickling in homozygous sickle-cell disease and may be an important genetic determinant of its hematologic severity. PMID:6176865

  1. Alpha-, Delta- and PP-cells

    PubMed Central

    Brereton, Melissa F.; Vergari, Elisa; Zhang, Quan

    2015-01-01

    Islet non-β-cells, the α- δ- and pancreatic polypeptide cells (PP-cells), are important components of islet architecture and intercellular communication. In α-cells, glucagon is found in electron-dense granules; granule exocytosis is calcium-dependent via P/Q-type Ca2+-channels, which may be clustered at designated cell membrane sites. Somatostatin-containing δ-cells are neuron-like, creating a network for intra-islet communication. Somatostatin 1-28 and 1-14 have a short bioactive half-life, suggesting inhibitory action via paracrine signaling. PP-cells are the most infrequent islet cell type. The embryologically separate ventral pancreas anlage contains PP-rich islets that are morphologically diffuse and α-cell deficient. Tissue samples taken from the head region are unlikely to be representative of the whole pancreas. PP has anorexic effects on gastro-intestinal function and alters insulin and glucagon secretion. Islet architecture is disrupted in rodent diabetic models, diabetic primates and human Type 1 and Type 2 diabetes, with an increased α-cell population and relocation of non-β-cells to central areas of the islet. In diabetes, the transdifferentiation of non-β-cells, with changes in hormone content, suggests plasticity of islet cells but cellular function may be compromised. Understanding how diabetes-related disordered islet structure influences intra-islet cellular communication could clarify how non-β-cells contribute to the control of islet function. PMID:26216135

  2. 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

  3. Quantum dot solar cell tolerance to alpha-particle irradiation

    SciTech Connect

    Cress, Cory D.; Hubbard, Seth M.; Landi, Brian J.; Raffaelle, Ryne P.; Wilt, David M.

    2007-10-29

    The effects of alpha-particle irradiation on an InAs quantum dot (QD) array and GaAs-based InAs QD solar cells were investigated. Using photoluminescence (PL) mapping, the PL intensity at 872 and 1120 nm, corresponding to bulk GaAs and InAs QD emissions, respectively, were measured for a five-layer InAs QD array which had a spatially varying total alpha-particle dose. The spectral response and normalized current-voltage parameters of the solar cells, measured as a function of alpha-particle fluence, were used to investigate the change in device performance between GaAs solar cells with and without InAs QDs.

  4. 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. PMID:19325630

  5. Quantitative-Proteomic Comparison of Alpha and Beta Cells to Uncover Novel Targets for Lineage Reprogramming

    PubMed Central

    Mertins, Philipp; Udeshi, Namrata D.; Dančík, Vlado; Fomina-Yadlin, Dina; Kubicek, Stefan; Clemons, Paul A.; Schreiber, Stuart L.; Carr, Steven A.; Wagner, Bridget K.

    2014-01-01

    Type-1 diabetes (T1D) is an autoimmune disease in which insulin-secreting pancreatic beta cells are destroyed by the immune system. An emerging strategy to regenerate beta-cell mass is through transdifferentiation of pancreatic alpha cells to beta cells. We previously reported two small molecules, BRD7389 and GW8510, that induce insulin expression in a mouse alpha cell line and provide a glimpse into potential intermediate cell states in beta-cell reprogramming from alpha cells. These small-molecule studies suggested that inhibition of kinases in particular may induce the expression of several beta-cell markers in alpha cells. To identify potential lineage reprogramming protein targets, we compared the transcriptome, proteome, and phosphoproteome of alpha cells, beta cells, and compound-treated alpha cells. Our phosphoproteomic analysis indicated that two kinases, BRSK1 and CAMKK2, exhibit decreased phosphorylation in beta cells compared to alpha cells, and in compound-treated alpha cells compared to DMSO-treated alpha cells. Knock-down of these kinases in alpha cells resulted in expression of key beta-cell markers. These results provide evidence that perturbation of the kinome may be important for lineage reprogramming of alpha cells to beta cells. PMID:24759943

  6. 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.

  7. Exosomal cell-to-cell transmission of alpha synuclein oligomers

    PubMed Central

    2012-01-01

    Background Aggregation of alpha-synuclein (αsyn) and resulting cytotoxicity is a hallmark of sporadic and familial Parkinson’s disease (PD) as well as dementia with Lewy bodies, with recent evidence implicating oligomeric and pre-fibrillar forms of αsyn as the pathogenic species. Recent in vitro studies support the idea of transcellular spread of extracellular, secreted αsyn across membranes. The aim of this study is to characterize the transcellular spread of αsyn oligomers and determine their extracellular location. Results Using a novel protein fragment complementation assay where αsyn is fused to non-bioluminescent amino-or carboxy-terminus fragments of humanized Gaussia Luciferase we demonstrate here that αsyn oligomers can be found in at least two extracellular fractions: either associated with exosomes or free. Exosome-associated αsyn oligomers are more likely to be taken up by recipient cells and can induce more toxicity compared to free αsyn oligomers. Specifically, we determine that αsyn oligomers are present on both the outside as well as inside of exosomes. Notably, the pathway of secretion of αsyn oligomers is strongly influenced by autophagic activity. Conclusions Our data suggest that αsyn may be secreted via different secretory pathways. We hypothesize that exosome-mediated release of αsyn oligomers is a mechanism whereby cells clear toxic αsyn oligomers when autophagic mechanisms fail to be sufficient. Preventing the early events in αsyn exosomal release and uptake by inducing autophagy may be a novel approach to halt disease spreading in PD and other synucleinopathies. PMID:22920859

  8. Reduced Paneth cell alpha-defensins in ileal Crohn's disease.

    PubMed

    Wehkamp, Jan; Salzman, Nita H; Porter, Edith; Nuding, Sabine; Weichenthal, Michael; Petras, Robert E; Shen, Bo; Schaeffeler, Elke; Schwab, Matthias; Linzmeier, Rose; Feathers, Ryan W; Chu, Hiutung; Lima, Heriberto; Fellermann, Klaus; Ganz, Tomas; Stange, Eduard F; Bevins, Charles L

    2005-12-13

    The pathogenesis of Crohn's disease (CD), an idiopathic inflammatory bowel disease, is attributed, in part, to intestinal bacteria that may initiate and perpetuate mucosal inflammation in genetically susceptible individuals. Paneth cells (PC) are the major source of antimicrobial peptides in the small intestine, including human alpha-defensins HD5 and HD6. We tested the hypothesis that reduced expression of PC alpha-defensins compromises mucosal host defenses and predisposes patients to CD of the ileum. We report that patients with CD of the ileum have reduced antibacterial activity in their intestinal mucosal extracts. These specimens also showed decreased expression of PC alpha-defensins, whereas the expression of eight other PC products either remained unchanged or increased when compared with controls. The specific decrease of alpha-defensins was independent of the degree of inflammation in the specimens and was not observed in either CD of the colon, ulcerative colitis, or pouchitis. The functional consequence of alpha-defensin expression levels was examined by using a transgenic mouse model, where we found changes in HD5 expression levels, comparable to those observed in CD, had a pronounced impact on the luminal microbiota. Thus, the specific deficiency of PC defensins that characterizes ileal CD may compromise innate immune defenses of the ileal mucosa and initiate and/or perpetuate this disease. PMID:16330776

  9. 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.

  10. THE CELL-BOUND ALPHA-AMYLASES OF STREPTOCOCCUS BOVIS.

    PubMed

    WALKER, G J

    1965-02-01

    1. The cell-bound alpha-amylase of Streptococcus bovis has been isolated from other carbohydrases in the cell extract by chromatography on DEAE-cellulose. The enzyme has been compared with the extracellular alpha-amylase produced by this organism. 2. The two amylases had similar action patterns on amylose, the main product being maltotriose with smaller amounts of maltose and a little glucose. 3. The cell-bound amylase hydrolysed maltopentaose and maltohexaose at a similar rate to the hydrolysis of amylose. Maltotetraose was hydrolysed six times more slowly, and maltotriose 280 times more slowly, than amylose. 4. Studies with end-labelled maltodextrins revealed that the cell-bound alpha-amylase preferentially hydrolysed the third linkage from the non-reducing end, liberating maltotriose. The linkage at the reducing end of maltotriose was more easily hydrolysed than the other. 5. Egg-white lysozyme and the extracellular enzymes of Streptomyces albus lysed the cell walls of Streptococcus bovis, releasing amylase into the medium. In the presence of 0.6 m-sucrose 10% of the maximal amylase activity was released by lysozyme. Suspension of the spheroplasts in dilute buffer caused the rupture of the cytoplasmic membrane and the liberation of amylase. 6. A sensitive method for determining the ability of amylases to degrade starch granules is described. PMID:14346085

  11. Quinazoline-derived alpha1-adrenoceptor antagonists induce prostate cancer cell apoptosis via an alpha1-adrenoceptor-independent action.

    PubMed

    Benning, Cynthia M; Kyprianou, Natasha

    2002-01-15

    Recent evidence suggests that the quinazoline-based alpha1-adrenoceptor antagonists, doxazosin and terazosin, exhibit a potent apoptotic effect against prostate tumor epithelial cells, whereas tamsulosin, a sulfonamide-based alpha1-adrenoceptor antagonist, was ineffective in inducing a similar apoptotic effect against prostate cells (Cancer Res., 60: 4550-4555, 2000). In this study, to identify the precise molecular mechanism underlying this apoptosis induction, we examined whether doxazosin and terazosin (both piperazinyl quinazolines) affect prostate growth via an alpha1-adrenoceptor-independent action. Transfection-mediated overexpression of alpha1-adrenoceptor in human prostate cancer cells, DU-145 (that lack alpha1-adrenoceptor), did not alter the ability of prostate cancer cells to undergo apoptosis in response to quinazolines. Significantly enough, there was no modification of the apoptotic threshold of the androgen-sensitive prostate cancer cells, LNCaP, to either quinazoline-based alpha1-agonist by androgens. Furthermore, human normal prostate epithelial cells exhibited a very low sensitivity to the apoptotic effects of doxazosin compared with that observed for the malignant prostate cells. These findings provide the first evidence that the apoptotic activity of the quinazoline-based alpha1-adrenoceptor antagonists (doxazosin and terazosin) against prostate cancer cells is independent of: (a) their capacity to antagonize alpha1-adrenoceptors; and (b) the hormone sensitivity status of the cells. This may have potential therapeutic significance in the use of quinazoline-based alpha1-adrenoceptor antagonists (already in clinical use for the treatment of hypertension and benign prostate hyperplasia) for the treatment of androgen-independent human prostate cancer. PMID:11809715

  12. 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

  13. HIF-1alpha and HIF-2alpha Are Differentially Activated in Distinct Cell Populations in Retinal Ischaemia

    PubMed Central

    Mowat, Freya M.; Luhmann, Ulrich F. O.; Smith, Alexander J.; Lange, Clemens; Duran, Yanai; Harten, Sarah; Shukla, Deepa; Maxwell, Patrick H.; Ali, Robin R.; Bainbridge, James W. B.

    2010-01-01

    Background Hypoxia plays a key role in ischaemic and neovascular disorders of the retina. Cellular responses to oxygen are mediated by hypoxia-inducible transcription factors (HIFs) that are stabilised in hypoxia and induce the expression of a diverse range of genes. The purpose of this study was to define the cellular specificities of HIF-1alpha and HIF-2alpha in retinal ischaemia, and to determine their correlation with the pattern of retinal hypoxia and the expression profiles of induced molecular mediators. Methodology/Principal Findings We investigated the tissue distribution of retinal hypoxia during oxygen-induced retinopathy (OIR) in mice using the bio-reductive drug pimonidazole. We measured the levels of HIF-1alpha and HIF-2alpha proteins by Western blotting and determined their cellular distribution by immunohistochemistry during the development of OIR. We measured the temporal expression profiles of two downstream mediators, vascular endothelial growth factor (VEGF) and erythropoietin (Epo) by ELISA. Pimonidazole labelling was evident specifically in the inner retina. Labelling peaked at 2 hours after the onset of hypoxia and gradually declined thereafter. Marked binding to Müller glia was evident during the early hypoxic stages of OIR. Both HIF-1alpha and HIF-2alpha protein levels were significantly increased during retinal hypoxia but were evident in distinct cellular distributions; HIF-1alpha stabilisation was evident in neuronal cells throughout the inner retinal layers whereas HIF-2alpha was restricted to Müller glia and astrocytes. Hypoxia and HIF-alpha stabilisation in the retina were closely followed by upregulated expression of the downstream mediators VEGF and EPO. Conclusions/Significance Both HIF-1alpha and HIF-2alpha are activated in close correlation with retinal hypoxia but have contrasting cell specificities, consistent with differential roles in retinal ischaemia. Our findings suggest that HIF-2alpha activation plays a key role in

  14. 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.

  15. Altered enteroendocrine cell expression in T cell receptor alpha chain knock-out mice.

    PubMed

    Rubin, D C; Zhang, H; Qian, P; Lorenz, R G; Hutton, K; Peters, M G

    2000-10-15

    Mice lacking T cell receptor alpha chain (TCRalpha(-/-)) develop inflammation of the colon. We have examined the effect of this inflammation on the colonic epithelium by studying markers of epithelial cuff, enteroendocrine, and immune cell differentiation. Using immunohistochemical techniques, colons were compared in normal C57/BL6 and murine TCR alpha(-/-) mice aged 2 and 3 weeks and 3-11 months. TCR alpha(-/-) mice aged 3-11 months had histologic evidence of inflammation with increased expression of CD45, CD4+, CD8+, and B220+ cells and a decrease in expression of IgA+ cells. There was a decrease in the number of cholecystokinin, serotonin, and neurotensin enteroendocrine expressing cells in the colon of TCR alpha(-/-) mice. These changes were not present in 2-3-week-old suckling/weaning mice. In contrast, peptide tyrosine tyrosine (PYY), glucagon-like peptide-1, and gastrin expression did not change and small intestinal enteroendocrine cells remained unaltered. The change in colonic enteroendocrine cell expression appears to be a specific response, since only a subset of these cells was altered, and the epithelium was intact by histologic analysis. The absence of functional T cells in TCR alpha(-/-) colon has a marked effect on differentiation of a specific subpopulation of enteroendocrine cells, prior to loss of integrity of the epithelium. PMID:11054861

  16. Hepatocyte nuclear factor-3 alpha (HNF-3{alpha}) negatively regulates androgen receptor transactivation in prostate cancer cells

    SciTech Connect

    Lee, Hyun Joo; Hwang, Miok; Chattopadhyay, Soma; Choi, Hueng-Sik; Lee, Keesook

    2008-03-07

    The androgen receptor (AR) is involved in the development and progression of prostate cancers. However, the mechanisms by which this occurs remain incompletely understood. In previous reports, hepatocyte nuclear factor-3{alpha} (HNF-3{alpha}) has been shown to be expressed in the epithelia of the prostate gland, and has been determined to regulate the transcription of prostate-specific genes. In this study, we report that HNF-3{alpha} functions as a novel corepressor of AR in prostatic cells. HNF-3{alpha} represses AR transactivation on target promoters containing the androgen response element (ARE) in a dose-dependent manner. HNF-3{alpha} interacts physically with AR, and negatively regulates AR transactivation via competition with AR coactivators, including GRIP1. Furthermore, HNF-3{alpha} overexpression reduces the androgen-induced expression of prostate-specific antigen (PSA) in LNCaP cells. Taken together, our findings indicate that HNF-3{alpha} is a novel corepressor of AR, and predict its effects on the proliferation of prostate cancer cells.

  17. Alpha-tocopherol inhibits agonist-induced monocytic cell adhesion to cultured human endothelial cells.

    PubMed Central

    Faruqi, R; de la Motte, C; DiCorleto, P E

    1994-01-01

    Antioxidants have been proposed to be anti-atherosclerotic agents; however, the mechanisms underlying their beneficial effects are poorly understood. We have examined the effect of alpha-tocopherol (alpha-tcp) on one cellular event in atherosclerotic plaque development, monocyte adhesion to stimulated endothelial cells (ECs). Human umbilical vein ECs were pretreated with alpha-tcp before stimulation with known agonists of monocyte adhesion: IL-1 (10 ng/ml), LPS (10 ng/ml), thrombin (30 U/ml), or PMA (10 nM). Agonist-induced monocytic cell adhesion, but not basal adhesion, was inhibited in a time- and concentration-dependent manner by alpha-tcp. The IC50 of alpha-tcp on an IL-1-induced response was 45 microM. The inhibition correlated with a decrease in steady state levels of E-selectin mRNA and cell surface expression of E-selectin which is consistent with the ability of a monoclonal antibody to E-selectin to inhibit monocytic cell adhesion in this system. Probucol (50 microM) and N-acetylcysteine (20 mM) also inhibited agonist-induced monocytic cell adhesion; whereas, several other antioxidants had no significant effect. Protein kinase C (PKC) does not appear to play a role in the alpha-tcp effect since no suppression of phosphorylation of PKC substrates was observed. Activation of the transcription factor NF-kappa B is reported to be necessary but not sufficient for E-selectin expression in EC. Electrophoretic mobility shift assays failed to show an alpha-tcp-induced decrease in activation of this transcription factor after cytokine stimulation. It has been hypothesized that alpha-tcp acts as an anti-atherosclerotic molecule by inhibiting generation of oxidized LDL--a putative triggering molecule in the atherosclerotic process. Our results point to a novel alternative mechanism of action of alpha-tcp. Images PMID:7518838

  18. Cultured Ito cells of rat liver express the alpha 2-macroglobulin gene.

    PubMed

    Andus, T; Ramadori, G; Heinrich, P C; Knittel, T; Meyer zum Büschenfelde, K H

    1987-11-01

    Ito cells were isolated from rat liver and kept in culture for up to 13 days. The capability of the Ito cells to synthesize alpha 2-macroglobulin was analyzed at different times after isolation and by pulse-chase experiments. Newly synthesized alpha 2-macroglobulin was determined by immunoprecipitation followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography. alpha 2-Macroglobulin synthesis was hardly detectable in Ito cells and their media 3 days after plating. However, 5-11 days after the isolation of the cells, increasing amounts of alpha 2-macroglobulin were synthesized. The results of pulse-chase experiments performed on day 7 showed that radioactively labeled alpha 2-macroglobulin decreased in the intracellular compartment and increased in the culture medium. alpha 2-Macroglobulin was identified by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non-reducing conditions. Furthermore, when unlabeled alpha 2-macroglobulin was added during the immunoprecipitation, a competition was observed. Incubation of pancreatic elastase with culture medium of rat Ito cells or rat hepatocytes led to the same cleavage products as found with alpha 2-macroglobulin. alpha 2-Macroglobulin-specific mRNA could be demonstrated by Northern blot analysis of total RNA extracted from rat Ito cells. Under the conditions where alpha 2-macroglobulin was synthesized in Ito cells, no synthesis of alpha 1-macroglobulin, alpha 1-inhibitor 3, alpha 1-proteinase inhibitor, alpha 1-acid glycoprotein, alpha 1-acute-phase globulin (T-kininogen) and albumin could be demonstrated. It is concluded that alpha 2-macroglobulin is a true secretory protein of rat Ito cells in culture. This could be of importance for collagen metabolism in liver diseases. PMID:2444437

  19. 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

  20. The structure of cell wall alpha-glucan from fission yeast.

    PubMed

    Grün, Christian H; Hochstenbach, Frans; Humbel, Bruno M; Verkleij, Arie J; Sietsma, J Hans; Klis, Frans M; Kamerling, Johannis P; Vliegenthart, Johannes F G

    2005-03-01

    Morphology and structural integrity of fungal cells depend on cell wall polysaccharides. The chemical structure and biosynthesis of two types of these polysaccharides, chitin and (1-->3)-beta-glucan, have been studied extensively, whereas little is known about alpha-glucan. Here we describe the chemical structure of alpha-glucan isolated from wild-type and mutant cell walls of the fission yeast Schizosaccharomyces pombe. Wild-type alpha-glucan was found to consist of a single population of linear glucose polymers, approximately 260 residues in length. These glucose polymers were composed of two interconnected linear chains, each consisting of approximately 120 (1-->3)-linked alpha-d-glucose residues and some (1-->4)-linked alpha-D-glucose residues at the reducing end. By contrast, alpha-glucan of an alpha-glucan synthase mutant with an aberrant cell morphology and reduced alpha-glucan levels consisted of a single chain only. We propose that alpha-glucan biosynthesis involves an ordered series of events, whereby two alpha-glucan chains are coupled to create mature cell wall alpha-glucan. This mature form of cell wall alpha-glucan is essential for fission-yeast morphogenesis. PMID:15470229

  1. Functional analysis of the cytoplasmic domain of the integrin {alpha}1 subunit in endothelial cells.

    PubMed

    Abair, Tristin D; Bulus, Nada; Borza, Corina; Sundaramoorthy, Munirathinam; Zent, Roy; Pozzi, Ambra

    2008-10-15

    Integrin alpha1beta1, the major collagen type IV receptor, is expressed by endothelial cells and plays a role in both physiologic and pathologic angiogenesis. Because the molecular mechanisms whereby this collagen IV receptor mediates endothelial cell functions are poorly understood, truncation and point mutants of the integrin alpha1 subunit cytoplasmic tail (amino acids 1137-1151) were generated and expressed into alpha1-null endothelial cells. We show that alpha1-null endothelial cells expressing the alpha1 subunit, which lacks the entire cytoplasmic tail (mutant alpha1-1136) or expresses all the amino acids up to the highly conserved GFFKR motif (mutant alpha1-1143), have a similar phenotype to parental alpha1-null cells. Pro(1144) and Leu(1145) were shown to be necessary for alpha1beta1-mediated endothelial cell proliferation; Lys(1146) for adhesion, migration, and tubulogenesis and Lys(1147) for tubulogenesis. Integrin alpha1beta1-dependent endothelial cell proliferation is primarily mediated by ERK activation, whereas migration and tubulogenesis require both p38 MAPK and PI3K/Akt activation. Thus, distinct amino acids distal to the GFFKR motif of the alpha1 integrin cytoplasmic tail mediate activation of selective downstream signaling pathways and specific endothelial cell functions. PMID:18647959

  2. 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.

  3. 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.

  4. 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.

  5. Endogenous catecholamine enhances the dysfunction of unfolded protein response and alpha-synuclein oligomerization in PC12 cells overexpressing human alpha-synuclein.

    PubMed

    Ito, Satoru; Nakaso, Kazuhiro; Imamura, Keiko; Takeshima, Takao; Nakashima, Kenji

    2010-01-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by the selective loss of dopaminergic neurons and the presence of Lewy bodies. alpha-Synuclein is a major component of Lewy bodies. Recently, many studies have focused on the interaction between alpha-synuclein and catecholamine in the pathogenesis of PD. However, no detailed relationship between cathecholamine and alpha-synuclein cytotoxicity has been elucidated. Therefore, this study established PC12 cell lines which overexpress human alpha-synuclein in a tetracycline-inducible manner. The overexpression of human alpha-synuclein increased the number of apoptotic cells in a long-term culture. Moreover, human alpha-synuclein expressing PC12 cells demonstrated an increased vulnerability to several stressors in a short culture period. Thapsigargin increased the SDS soluble oligomers of alpha-synuclein associated with catecholamine-quinone. The unfolded protein response (UPR) study showed that thapsigargin increased eIF2alpha phosphorylation and nuclear GADD153/CHOP induction under alpha-synuclein overexpressed conditions. The activities of the ATF6alpha and IRE1alpha pathways decreased. These findings suggest that an overexpression of alpha-synuclein partly inactivates the UPR. alpha-Methyltyrosine inhibited the dysfunction of the UPR caused by an overexpression of human alpha-synuclein. Therefore, these findings suggest that the coexistence of human alpha-synuclein with catecholamine enhances the endoplasmic reticulum stress-related toxicity in PD pathogenesis. PMID:19835916

  6. Alpha-naphthylisothiocyanate cytotoxicity in hepatocytes and other cultured cells

    SciTech Connect

    Bailie, M.B.; Roth, R.A. )

    1991-03-11

    Alpha-naphthylisothiocyanate (ANIT) is a model hepatotoxicant that causes injury to liver parenchymal and bile ductular cells in vivo. In this study, toxicity to various cells in culture was evaluated. In short term cultures of rat hepatocytes (HCs), a 4hr exposure to ANIT caused a concentration dependent increase in cytotoxicity as measured by lactate dehydrogenase (LDH) release. HCs cultured for 24hr or longer demonstrated a delay in ANIT-induced LDH release when compared to 2.5hr cultures. In addition, the magnitude of the cytotoxic response was greater in longer term cultures. The threshold for ANIT-induced cytotoxicity in HCs was between 20 and 63uM. In porcine endothelial cell cultures, ANIT cytotoxicity was similar to that seen in HCs. In two transformed cells lines, the Swiss 3T3 fibroblast and WB cell, a 24hr exposure to ANTI caused a concentration dependent increase in LDH release. Like the HCs, the threshold concentration was between 20 and 63uM. These results indicate that ANIT is directly cytotoxic to various cells in culture. Since endothelium and fibroblasts are deficient in cytochrome P-450 mixed function oxidase activity, ANIT toxicity in culture may be largely independent of this xenobiotic metabolizing system.

  7. DNA polymorphisms and mutations of the tumor necrosis factor-alpha (TNF-alpha) promoter in Langerhans cell histiocytosis (LCH).

    PubMed

    Wu, W S; McClain, K L

    1997-10-01

    Langerhans cell histiocytosis (LCH) is a clonal proliferation of dendritic histiocytes expressing elevated levels of tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 (IL-1), and leukemia inhibitory factor (LIF). The cause of the increased cytokine levels is unknown, but DNA sequence changes in promoters could alter expression. The TNF-alpha and IFN-gamma promoter DNA sequences of 12 LCH patients were studied and compared with normal individuals by dideoxy fingerprinting and DNA sequencing. Functional consequences of polymorphic or mutated sequences were assessed by cloning altered and control promoter sequences into a luciferase reporter gene vector. Electrophoretic mobility shifts (EMSA) after binding of nuclear extracts from a macrophage cell line (U-937) by mutated promoters were compared with controls. Five of 12 LCH patients had alterations in the TNF-alpha promoter DNA sequence. None were found in the IFN-gamma gene promoter. Of the 5 with TNF-alpha DNA alterations, 2 were at position -308, which has been described as a G-A polymorphism associated with upregulation of TNF-alpha in some patients with infections or immune-mediated diseases. The polymorphism at -308 but not the other TNF-alpha promoter mutations caused a 3-fold to 7-fold increased production of the luciferase reporter gene. EMSA showed that the -308 mutant promoters bound fewer nuclear proteins than normals. Polymorphisms of the TNF-alpha promoter in LCH patients could increase the production of that cytokine. PMID:9355965

  8. 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.

  9. Selective production of interferon-alpha subtypes by cultured peripheral blood mononuclear cells and lymphoblastoid cell lines.

    PubMed Central

    Greenway, A L; Overall, M L; Sattayasai, N; Rowley, M J; Hertzog, P J; McMullen, G L; Cheetham, B F; Marzuki, S

    1992-01-01

    The biological significance of the existence of multiple interferon-alpha (IFN-alpha) subtypes is unknown but may represent a finely tuned mechanism whereby different subtypes are produced in response to different stimuli. To investigate the expression of individual IFN-alpha subtypes, polyclonal antipeptide antisera designed to react with all IFN-alpha subtypes, or with a particular subtype, IFN-alpha 2 or IFN-alpha 4, have been produced. In this study we demonstrate the utility of these antisera for the detection, using indirect immunofluorescence staining, of intracellular IFN-alpha produced by human peripheral blood mononuclear cells (PBMC) and lymphoblastoid cells. Secreted IFN-alpha was also investigated by bioassay and a sandwich radioimmunoassay (RIA), using two monoclonal antibodies (mAb) and specific for IFN-alpha 4. The PBMC were shown to produce IFN reactive with all three polyclonal antisera, after stimulation with Sendai virus. The lymphoblastoid cells also produced IFN, including IFN-alpha 2, but IFN-alpha 4 was not detected either intracellularly, by immunofluorescence, or in the medium, by sandwich RIA. The immunofluorescence studies also demonstrate that in the absence of viral stimulation IFN-alpha is found in the cytoplasm of PBMC and lymphoblastoid cells but not secreted in detectable levels. The finding that two lymphoblastoid cell lines do not produce the subtype IFN-alpha 4 raises important questions as to whether other cell lines and cell types produce IFN-alpha subtypes selectively, and whether individual IFN-alpha subtypes have different roles in human physiology and pathology. Images Figure 1 Figure 2 Figure 3 PMID:1537595

  10. Serum-alpha-hydroxybutyrate dehydrogenase levels in sickle-cell disease and sickle-cell crisis.

    PubMed

    White, J M; Billimoria, F; Muller, M A; Davis, L R; Stroud, C E

    1978-03-11

    In patients with sickle-cell disease (Hb S/S disease) who are not in crisis, the serum-level of alpha-hydroxybutyrate dehydrogenase (alpha-H.B.D.) is significantly above normal. The degree of elevation correlates with the severity of the disease. During infarctive crises the level increases still further by a factor of 2 to 3, and it returns to the patient's normal level on recovery. The level of alpha-H.B.D. thus provides an index of the severity of the disease. Also, it allows distinction between true infarctive crisis, infection, and fake symptoms. In 5 patients a rise in the level of alpha-H.B.D. above the patient's normal level was detected several days before the onset of symptoms, which suggests that there may be a "prodromal" phase. PMID:76073

  11. 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. PMID:8140855

  12. Neuronal-type alpha-bungarotoxin receptors and the alpha 5-nicotinic receptor subunit gene are expressed in neuronal and nonneuronal human cell lines.

    PubMed Central

    Chini, B; Clementi, F; Hukovic, N; Sher, E

    1992-01-01

    alpha-Bungarotoxin (alpha Bgtx) is a toxin known to interact with muscle nicotinic receptors and with some neuronal nicotinic receptors. We show that alpha Bgtx binding sites are also expressed in nonmuscle and nonneuronal human cells, including small cell lung carcinoma and several epithelial cell lines. These receptors are immunologically related to the alpha Bgtx receptors of unknown function described in the nervous system and in the IMR32 neuroblastoma cell line and are distinct from muscle nicotinic receptors. We have also cloned from IMR32 cells the human alpha 5-nicotinic receptor subunit, which is supposed to participate in the formation of alpha Bgtx receptors. Transcripts corresponding to the alpha 5-subunit gene were found not only in neuroblastoma cells but also in all the cell lines expressing alpha Bgtx receptors, with the exception of the TE671 cell line, whose nicotinic receptor subunits are of the muscle type. We conclude that both alpha Bgtx receptors and the alpha 5-nicotinic subunit gene are not neuron-specific, as previously thought, but are expressed in a number of human cell lines of various origin. Images PMID:1542648

  13. Stem cell factor induces HIF-1{alpha} at normoxia in hematopoietic cells

    SciTech Connect

    Pedersen, Malin; Loefstedt, Tobias; Sun Jianmin; Holmquist-Mengelbier, Linda; Pahlman, Sven; Roennstrand, Lars

    2008-12-05

    Signaling by the receptor for stem cell factor (SCF), c-Kit, is of major importance for hematopoiesis, melanogenesis and reproduction, and the biological responses are commonly proliferation and cell survival. Thus, constitutive activation due to c-Kit mutations is involved in the pathogenesis of several forms of cancer, e.g. leukemias, gastrointestinal stromal tumors and testicular tumors. Tumor survival requires oxygen supply through induced neovascularization, a process largely mediated by the vascular endothelial growth factor (VEGF), a prominent target of the transcription factors hypoxia-inducible factor-1 (HIF-1) and HIF-2. Using Affymetrix microarrays we have identified genes that are upregulated following SCF stimulation. Interestingly, many of the genes induced were found to be related to a hypoxic response. These findings were corroborated by our observation that SCF stimulation of the hematopoietic cell lines M-07e induces HIF-1{alpha} and HIF-2{alpha} protein accumulation at normoxia. In addition, SCF-induced HIF-1{alpha} was transcriptionally active, and transcribed HIF-1 target genes such as VEGF, BNIP3, GLUT1 and DEC1, an effect that could be reversed by siRNA against HIF-1{alpha}. We also show that SCF-induced accumulation of HIF-1{alpha} is dependent on both the PI-3-kinase and Ras/MEK/Erk pathways. Our data suggest a novel mechanism of SCF/c-Kit signaling in angiogenesis and tumor progression.

  14. Non random usage of T cell receptor alpha gene expression in atopy using anchored PCR.

    PubMed

    Mansur, A H; Gelder, C M; Holland, D; Campell, D A; Griffin, A; Cunliffe, W; Markham, A F; Morrison, J F

    1996-01-01

    The T cell receptor (TCR) alpha beta heterodimer recognises antigenic peptide fragments presented by Class II MHC. This interaction initiates T cell activation and cytokine release with subsequent recruitment of inflammatory cells. Previous work from our group suggests a qualitative difference in variable alpha gene expression in atopy as compared to non atopic controls. In this study we examine TCR alpha repertoire using anchored PCR to provide a quantitative assessment of the V alpha and J alpha repertoire. One atopic (DRB1*0701,DRB1*15: DRB4*0101, DRB5*01: DQB1* 0303, DQB1*601/2) and one non-atopic (DRB1*0701,DRB1*03011/2: DRB4*01, DRB3*0x: DQB1* 0303, DQB1*0201/2) control were studied. Variable gene usage was markedly limited in the atopic individual. V alpha 1, 3, 8 accounted for 60% and J alpha 12, 31 30% of the gene usage. There was evidence of preferential V alpha-J alpha gene pairing and clonal expansion. We conclude that there is a marked non random TCR alpha gene distribution in atopy using both V alpha family and anchored PCR. This may be due in part to antigen driven clonal expansion. PMID:9095269

  15. Target site choice of the related transposable elements Tc1 and Tc3 of Caenorhabditis elegans.

    PubMed Central

    van Luenen, H G; Plasterk, R H

    1994-01-01

    We have investigated the target choice of the related transposable elements Tc1 and Tc3 of the nematode C. elegans. The exact locations of 204 independent Tc1 insertions and 166 Tc3 insertions in an 1 kbp region of the genome were determined. There was no phenotypic selection for the insertions. All insertions were into the sequence TA. Both elements have a strong preference for certain positions in the 1 kbp region. Hot sites for integration are not clustered or regularly spaced. The orientation of the integrated transposon has no effect on the distribution pattern. We tested several explanations for the target site preference. If simple structural features of the DNA (e.g. bends) would mark hot sites, we would expect the patterns of the two related transposons Tc1 and Tc3 to be similar; however we found them to be completely different. Furthermore we found that the sequence at the donor site has no effect on the choice of the new insertion site, because the insertion pattern of a transposon that jumps from a transgenic donor site is identical to the insertion pattern of transposons jumping from endogenous genomic donor sites. The most likely explanation for the target choice is therefore that the primary sequence of the target site is recognized by the transposase. However, alignment of the Tc1 and Tc3 integration sites does not reveal a strong consensus sequence for either transposon. PMID:8127662

  16. 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.

  17. Interferon-alpha stimulates production of interleukin-10 in activated CD4+ T cells and monocytes.

    PubMed

    Aman, M J; Tretter, T; Eisenbeis, I; Bug, G; Decker, T; Aulitzky, W E; Tilg, H; Huber, C; Peschel, C

    1996-06-01

    In the present study, we investigated the effect of interferon-alpha (IFN-alpha) on the expression of interleukin-10 (IL-10) mRNA and protein synthesis in human monocytes and CD4+ T cells. In mononuclear cells, IFN-alpha induced expression of IL-10 mRNA and further enhanced lipopolysaccharide (LPS)-stimulated IL-10 expression. In purified monocytes, a strong expression of IL-10 mRNA induced by LPS was not further enhanced by IFN-alpha. In highly purified CD4+ T cells, IFN-alpha upregulated IL-10 mRNA upon activation with phytohemagglutinin and phorbol myristate acetate. In purified monocytes, an effect of IFN-alpha on IL-10 protein synthesis was dependent on costimulation with LPS. Maximal stimulation of IL-10 protein by IFN-alpha was seen after prolonged incubation periods of 48 to 96 hours, whereas IFN-gamma reduced IL-10 production in the early incubation period. Similar effects of IFN-alpha were observed in CD4+ T cells activated with CD3 and CD28 monoclonal antibodies. Addition of IFN-alpha caused an increase of IL-10 in culture supernatants of activated T-helper cells of more than 100% after 96 hours of incubation. In contrast, other cytokines, including IFN-gamma and IL-4, had no influence on IL-10 secretion stimulated by CD3 and CD28 in CD4+ T cells. In serum samples of IFN-alpha-treated individuals, we failed to detect an influence of cytokine treatment on IL-10 serum levels, confirming the requirement of additional activating signals for IFN-alpha-mediated effects on IL-10 synthesis. In conclusion, IFN-alpha enhances the late induction of IL-10, which physiologically occurs upon stimulation of monocytes and T cells. Biologically, this effect might enhance the negative-feedback mechanism ascribed to IL-10, which limits inflammatory reactions. PMID:8639843

  18. 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.

  19. The mutual clonal origin of the lymphoplasmocytic and lymphoma cell in alpha-heavy chain disease.

    PubMed Central

    Ramot, B; Levanon, M; Hahn, Y; Lahat, N; Moroz, C

    1977-01-01

    Biosynthetic studies in alpha-heavy chain disease were performed on the gut tumour which was composed mainly of lymphoplasmocytic cells and on the mesenteric lymph node tumour composed mainly of immunoblasts. The gut tumour cells synthesised alpha-heavy chains and secreted them during 2-5 hr culture, whereas the lymph node tumour cells synthesized alpha-heavy chains which were shed into the culture medium only after 20 hr. These chains were shown to be present on the surface of the immunoblastic tumour cells by enzymatic radioiodination. Both the surface and the secreted alpha-heavy chain of the lymph node and gut tumour were found to be smaller than the alpha-heavy chain of myeloma proteins. These results suggest that the lymphoblasmocytic and the immunoblastic tumour cells originate from the same defective clone. PMID:405165

  20. 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.

  1. Integrins alpha(6A)beta 1 and alpha(6B)beta 1 promote different stages of chondrogenic cell differentiation.

    PubMed

    Segat, Daniela; Comai, Riccardo; Di Marco, Eddi; Strangio, Antonella; Cancedda, Ranieri; Franzi, Adriano T; Tacchetti, Carlo

    2002-08-30

    The differentiation of chondrocytes and of several other cell types is associated with a switch from the alpha(6B) to the alpha(6A) isoform of the laminin alpha(6)beta(1) integrin receptor. To define whether this event plays a functional role in cell differentiation, we used an in vitro model system that allows chick chondrogenic cells to remain undifferentiated when cultured in monolayer and to differentiate into chondrocytes when grown in suspension culture. We report that: (i) upon over-expression of the human alpha(6B), adherent chondrogenic cells differentiate to stage I chondrocytes (i.e. increased type II collagen, reduced type I collagen, fibronectin, alpha(5)beta(1) and growth rate, loss of fibroblast morphology); (ii) the expression of type II collagen requires the activation of p38 MAP kinase; (iii) the over-expression of alpha(6A) induces an incomplete differentiation to stage I chondrocytes, whereas no differentiation was observed in alpha(5) and mock-transfected control cells; (iv) a prevalence of the alpha(6A) subunit is necessary to stabilize the differentiated phenotype when cells are transferred to suspension culture. Altogether, these results indicate a functional role for the alpha(6B) to alpha(6A) switch in chondrocyte differentiation; the former promotes chondrocyte differentiation, and the latter is necessary in stabilizing the differentiated phenotype. PMID:12077132

  2. 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.

  3. 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.

  4. Cell surface expression and turnover of the alpha-subunit of the epithelial sodium channel.

    PubMed

    Kleyman, T R; Zuckerman, J B; Middleton, P; McNulty, K A; Hu, B; Su, X; An, B; Eaton, D C; Smith, P R

    2001-08-01

    The renal epithelial cell line A6, derived from Xenopus laevis, expresses epithelial Na(+) channels (ENaCs) and serves as a model system to study hormonal regulation and turnover of ENaCs. Our previous studies suggest that the alpha-subunit of Xenopus ENaC (alpha-xENaC) is detectable as 150- and 180-kDa polypeptides, putative immature and mature alpha-subunit heterodimers. The 150- and 180-kDa alpha-xENaC were present in distinct fractions after sedimentation of A6 cell lysate through a sucrose density gradient. Two anti-alpha-xENaC antibodies directed against distinct domains demonstrated that only 180-kDa alpha-xENaC was expressed at the apical cell surface. The half-life of cell surface-expressed alpha-xENaC was 24-30 h, suggesting that once ENaC matures and is expressed at the plasma membrane, its turnover is similar to that reported for mature cystic fibrosis transmembrane conductance regulator. No significant changes in apical surface expression of alpha-xENaC were observed after treatment of A6 cells with aldosterone for 24 h, despite a 5.3-fold increase in short-circuit current. This lack of change in surface expression is consistent with previous observations in A6 cells and suggests that aldosterone regulates ENaC gating and increases channel open probability. PMID:11457713

  5. Differential expression of alpha-subunits of G-proteins in human neuroblastoma-derived cell clones.

    PubMed

    Klinz, F J; Yu, V C; Sadée, W; Costa, T

    1987-11-16

    The distribution of alpha- and beta-subunits of G-proteins was analyzed in membranes of three cell clones which are derived from the human neuroblastoma cell line SK-N-SH. The neuroblast-like clone SH-SY5Y shows a pattern of G-proteins very similar to that of human brain cortex with high levels of Gi alpha and Go alpha but low levels of G40 alpha. The intermediate clone SH-IN contains high levels of Go alpha and Gi alpha and moderate levels of G40 alpha. The non-neuronal clone SH-EP shows high levels of G40 alpha but lacks Go alpha. Differentiation of the neuroblast-like clone SH-SY5Y by retinoic acid or nerve growth factor does not change the amount of Gi alpha or Go alpha in the membrane. PMID:3119368

  6. 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.

  7. Widespread histologic distribution of the alpha 2 beta 1 integrin cell-surface collagen receptor.

    PubMed Central

    Zutter, M. M.; Santoro, S. A.

    1990-01-01

    The alpha 2 beta 1 integrin (platelet membrane glycoprotein Ia-IIa, VLA-2, ECMR-II) functions as a cell surface receptor for collagen. The authors have determined the histologic distribution of the alpha 2 beta 1 receptor in normal tissues by immunohistochemical technique. The studies revealed that the alpha 2 beta 1 receptor was expressed on fibroblasts, endothelial cells, and epithelial cells from multiple sites including skin, tonsil, breast, sweat gland, gastrointestinal tract, lung, bladder, cervix, and prostate. Follicular dendritic cells of the lymph node, tonsil, and spleen and dendritic cells of the thymus also expressed the alpha 2 beta 1 receptor. The receptor also was present on Schwann cells of ganglia and on neuroglia. Greatly enhanced expression of the receptor in regions of proliferating epithelium suggests that enhanced expression of alpha 2 beta 1 is associated with orderly, regulated cell proliferation. The circumferential staining pattern of the alpha 2 beta 1 integrin within many epithelia is virtually identical to that observed for other adhesive receptors, such as the cadherins, which have been implicated in cell-cell adhesion. Images Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 16 Figure 17 PMID:2164774

  8. 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.

  9. 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.

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

    SciTech Connect

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

    2009-02-13

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

  11. The mouse dead-end gene isoform alpha is necessary for germ cell and embryonic viability.

    PubMed

    Bhattacharya, Chitralekha; Aggarwal, Sita; Zhu, Rui; Kumar, Madhu; Zhao, Ming; Meistrich, Marvin L; Matin, Angabin

    2007-03-30

    Inactivation of the dead-end (Dnd1) gene in the Ter mouse strain results in depletion of primordial germ cells (PGCs) so that mice become sterile. However, on the 129 mouse strain background, loss of Dnd1 also increases testicular germ cell tumor incidence in parallel to PGC depletion. We report that inactivation of Dnd1 also affects embryonic viability in the 129 strain. Mouse Dnd1 encodes two protein isoforms, DND1-isoform alpha (DND1-alpha) and DND1-isoform beta (DND1-beta). Using isoform-specific antibodies, we determined DND1-alpha is expressed in embryos and embryonic gonads whereas DND1-beta expression is restricted to germ cells of the adult testis. Our data implicate DND1-alpha isoform to be necessary for germ cell viability and therefore its loss in Ter mice results in PGC depletion, germ cell tumor development and partial embryonic lethality in the 129 strain. PMID:17291453

  12. Fish oils lower rat plasma and hepatic, but not immune cell alpha-tocopherol concentration.

    PubMed

    Alexander, D W; McGuire, S O; Cassity, N A; Fritsche, K L

    1995-10-01

    These studies were designed to measure the impact of different fish oil sources of dietary (n-3) polyunsaturated fatty acid on the alpha-tocopherol content of rat immune cells. In the first experiment, rats were fed diets containing either lard, corn oil, menhaden fish oil or cod liver oil. In the second study, sardine fish oil replaced corn oil. Dietary fat source did not significantly influence body weights or the yield of immune cells in either study. In both studies, plasma and liver alpha-tocopherol concentrations were significantly lower in (n-3) polyunsaturated fatty acid-fed rats than in rats fed lard. In the first study, immune cell alpha-tocopherol concentrations followed those observed in the plasma and liver. These concentrations closely paralleled the amount of RRR-alpha-tocopheryl acetate added to diets and not the total vitamin E present, which was the same for all treatment groups. However, in the second study, alpha-tocopherol concentration of immune cells was not significantly different among rats fed lard, menhaden fish oil, and sardine fish oil. In that study both the amount and form of vitamin E were carefully balanced across dietary treatment groups. In conclusion, despite having similar amounts of (n-3) polyunsaturated fatty acids, two out of three fish oils tested did not lower immune cell alpha-tocopherol concentration even in the face of significantly reduced plasma and liver alpha-tocopherol concentrations. PMID:7562101

  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. Mouse pre-immunocytes as non-proliferating multipotent precursors of macrophages, interferon-producing cells, CD8alpha(+) and CD8alpha(-) dendritic cells.

    PubMed

    Bruno, L; Seidl, T; Lanzavecchia, A

    2001-11-01

    In this study we characterize in mouse bone marrow and peripheral blood a homogeneous cell subset expressing Ly6C, CD31 and CD11c, that can give rise to multiple cell types involved in the immune response. Under the aegis of M-CSF or GM-CSF these cells rapidly differentiate without division to either macrophages or immature dendritic cells, which can be further induced to mature by LPS stimulation. In fetal thymic organ cultures the same cells generate both CD8alpha(+) and CD8alpha(-) dendritic cells in comparable proportion as found in normal thymus. The Ly6C(+), CD31(+) and CD11c(+) cells express not only TLR2 and TLR4, which are characteristic of myeloid dendritic cells, but also TLR7 and TLR9, which conversely are characteristic of human interferon-producing cells. Moreover, following stimulation with influenza virus, they rapidly express high levels of IFN-alpha mRNA. Finally these precursors are increased in bone marrow and peripheral blood during systemic inflammation. These cells are defined as "pre-immunocytes" to underline the fact that they serve in a flexible fashion multiple, and often divergent, functions required for the immune response to pathogens. PMID:11745359

  15. Biotransformation of alpha- and 6beta-santonin by fungus and plant cell cultures.

    PubMed

    Yang, L; Dai, J; Sakai, J-I; Ando, M

    2006-06-01

    One fungus, Abisidia coerulea IFO 4011, and suspended cell cultures of one plant, Asparagus officinalis, were employed to bioconvert alpha- and 6beta-santonin. Incubation of alpha-santonin with the cell cultures of the fungus afforded two products, 11beta-hydroxy-alpha-santonin (1, in 76.5% yield) and 8alpha-hydroxy-alpha-santonin (2, in 2.0% yield). And from 6beta-santonin, four major products (3, 4, 5 and 6) and four minor products (7, 8, 9 and 10) were obtained, including 8alpha-hydroxylated products in trace yields. Very interestingly, a skeletal rearrangement occurred and a guaiane product (13) formed in a very low yield when alpha-santonin incubating with A.officinalis cell cultures, while not in the case of 6beta-santonin as substrate. Among the obtained 15 products, 2, 7, 8, 9, 10 and 12 are new compounds. The fact of 8alpha hydroxylation of santonin enables the formation of 8,12-eudesmanolide instead of 6,12-eudesmanolide and some useful modification at C-8 position. In addition, these reactions would provide evidence for the biogenesis between different types of eudesmane and/or guaiane compounds in the plants in nature. PMID:16864442

  16. Conversion of adult pancreatic alpha-cells to beta-cells after extreme beta-cell loss.

    PubMed

    Thorel, Fabrizio; Népote, Virginie; Avril, Isabelle; Kohno, Kenji; Desgraz, Renaud; Chera, Simona; Herrera, Pedro L

    2010-04-22

    Pancreatic insulin-producing beta-cells have a long lifespan, such that in healthy conditions they replicate little during a lifetime. Nevertheless, they show increased self-duplication after increased metabolic demand or after injury (that is, beta-cell loss). It is not known whether adult mammals can differentiate (regenerate) new beta-cells after extreme, total beta-cell loss, as in diabetes. This would indicate differentiation from precursors or another heterologous (non-beta-cell) source. Here we show beta-cell regeneration in a transgenic model of diphtheria-toxin-induced acute selective near-total beta-cell ablation. If given insulin, the mice survived and showed beta-cell mass augmentation with time. Lineage-tracing to label the glucagon-producing alpha-cells before beta-cell ablation tracked large fractions of regenerated beta-cells as deriving from alpha-cells, revealing a previously disregarded degree of pancreatic cell plasticity. Such inter-endocrine spontaneous adult cell conversion could be harnessed towards methods of producing beta-cells for diabetes therapies, either in differentiation settings in vitro or in induced regeneration. PMID:20364121

  17. [Cloning the alpha-amylase gene of Streptococcus bovis and its expression in Bacillus subtilis cells].

    PubMed

    Iakorski, P; Kuntsova, M M; Loseva, E F; Khasanov, F K

    1991-06-01

    The gene coding for alpha-amylase from the ruminant bacterium Streptococcus bovis was cloned on the plasmid pMX39 in Bacillus subtilis cells. An alpha-amylase positive colony was isolated in the initial screening of 3900 colonies on the medium containing insoluble starch. The size of the insert was approximately 2.8 kb. The recombinant plasmid was stably maintained in Bacillus subtilis cells under the nonselective conditions. PMID:1944323

  18. Bimatoprost and prostaglandin F(2 alpha) selectively stimulate intracellular calcium signaling in different cat iris sphincter cells.

    PubMed

    Spada, Clayton S; Krauss, Achim H-P; Woodward, David F; Chen, June; Protzman, Charles E; Nieves, Amelia L; Wheeler, Larry A; Scott, David F; Sachs, George

    2005-01-01

    Bimatoprost is a synthetic analog of prostaglandin F(2 alpha) ethanolamide (prostamide F(2 alpha)), and shares a pharmacological profile consistent with that of the prostamides. Like prostaglandin F(2 alpha) carboxylic acid, bimatoprost potently lowers intraocular pressure in dogs, primates and humans. In order to distinguish its mechanism of action from prostaglandin F(2 alpha), fluorescence confocal microscopy was used to examine the effects of bimatoprost, prostaglandin F(2 alpha) and 17-phenyl prostaglandin F(2 alpha) on calcium signaling in resident cells of digested cat iris sphincter, a tissue which exhibits contractile responses to both agonists. Constant superfusion conditions obviated effective conversion of bimatoprost. Serial challenge with 100 nM bimatoprost and prostaglandin F(2 alpha) consistently evoked responses in different cells within the same tissue preparation, whereas prostaglandin F(2 alpha) and 17-phenyl prostaglandin F(2 alpha) elicited signaling responses in the same cells. Bimatoprost-sensitive cells were consistently re-stimulated with bimatoprost only, and prostaglandin F(2 alpha) sensitive cells could only be re-stimulated with prostaglandin F(2 alpha). The selective stimulation of different cells in the same cat iris sphincter preparation by bimatoprost and prostaglandin F(2 alpha), along with the complete absence of observed instances in which the same cells respond to both agonists, strongly suggests the involvement of distinct receptors for prostaglandin F(2 alpha) and bimatoprost. Further, prostaglandin F(2 alpha) but not bimatoprost potently stimulated calcium signaling in isolated human embryonic kidney cells stably transfected with the feline- and human-prostaglandin F(2 alpha) FP-receptor and in human dermal fibroblast cells, and only prostaglandin F(2 alpha) competed with radioligand binding in HEK-feFP cells. These studies provide further evidence for the existence of a bimatoprost-sensitive receptor that is distinct from

  19. 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

  20. Fish oil source differentially affects rat immune cell alpha-tocopherol concentration.

    PubMed

    McGuire, S O; Alexander, D W; Fritsche, K L

    1997-07-01

    We have previously reported that both the source of dietary fish oil and the chemical form of vitamin E supplied in the diet affect the vitamin E status of immune cells in rats. The purpose of this study was to investigate further the effect of fish oil source on immune cell vitamin E status using free alpha-tocopherol (alpha-T) at the AIN recommended level as the sole source of vitamin E. Sixty weanling female rats were fed semipurified, high fat (20 g/100 g) diets containing either tocopherol-stripped lard (LRD), menhaden fish oil (MFO), sardine fish oil (SRD) or cod liver oil (CLO) as the primary lipid source. Endogenous alpha-T concentration was measured and equalized to 150 mg/kg oil by addition of free RRR-alpha-T to each lipid source, allowing for a final concentration of alpha-T in the mixed diet of 30 mg/kg. An additional group of rats was fed LRD without supplemental vitamin E (LRD-) as a negative control. After feeding experimental diets for 5 or 10 wk, tissues were collected for alpha-T analysis by HPLC. After 5 wk, plasma and liver alpha-T (micromol alpha-T/g lipid) were significantly lower in SRD- and CLO-fed rats compared with LRD-fed rats. At 10 wk, only plasma alpha-T in CLO-fed rats remained significantly depressed. Plasma and liver alpha-T concentrations (micromol alpha-T/g lipid) were not significantly lower in MFO-fed rats than LRD-fed rats at either time point. Compared with LRD, feeding MFO to rats for 5 or 10 wk resulted in significantly greater alpha-T content of immune cells. In similar fashion, SRD-fed rats, compared with LRD-fed rats, also had significantly greater alpha-T content in splenocytes at both time points and greater thymocyte alpha-T at 10 wk. In all instances, the alpha-T status of rats fed CLO was indistinguishable from that of rats fed the vitamin E-free diet (LRD-). These data further demonstrate the complexity of the relationship between vitamin E status and dietary (n-3) polyunsaturated fatty acids (PUFA). PMID:9202096

  1. Differences in properties between human alphaA- and alphaB-crystallin proteins expressed in Escherichia coli cells in response to cold and extreme pH.

    PubMed Central

    Takeuchi, Satoru; Mandai, Yumi; Otsu, Akiko; Shirakawa, Taro; Masuda, Katsuyoshi; Chinami, Masanobu

    2003-01-01

    It has been reported that alphaA-crystallin has greater protective effects against apoptosis in lens epithelial cells than alphaB-crystallin [Andley, Song, Wawrousek, Fleming and Bassnett (2000) J. Biol. Chem. 275, 36823-36831]. Because the alphaA-crystallin proteins are specifically expressed in the vertebrate lens, we examine the non-specific properties of both alphaA- and alphaB-crystallins in an Escherichia coli system. E. coli cells were transformed with the inducible protein expression vector pET-11a, harbouring the gene for either human alphaA- or alphaB-crystallin, and two other control plasmids, pET-1la vector alone or pGEX-2T vector encoding GST (glutathione S-transferase). These cells were exposed to various stress conditions, such as cold-shock at 4 degrees C or extremely low or high pH environments (pH 4.7 or pH 8.0) for 6 h, and survival of the host cells and the solubility of the expressed target proteins in the cytosol were examined. Under these stress conditions, the cells expressing alphaB-crystallin protein demonstrated significantly improved survival when compared with the other cells, and the expressed protein in the cytosol was almost soluble, in contrast with the alphaA-crystallin protein. Differences in the amino acid sequence between the proteins in a phenylalanine-rich region next to the N-terminal consensus alpha-crystallin domain was considered to be responsible for chaperone activity and cell survival. PMID:12826011

  2. Protective effect of alpha-mangostin against oxidative stress induced-retinal cell death

    PubMed Central

    Fang, Yuan; Su, Tu; Qiu, Xiaorong; Mao, Pingan; Xu, Yidan; Hu, Zizhong; Zhang, Yi; Zheng, Xinhua; Xie, Ping; Liu, Qinghuai

    2016-01-01

    It is known that oxidative stress plays a pivotal role in age-related macular degeneration (AMD) pathogenesis. Alpha-mangostin is the main xanthone purified from mangosteen known as anti-oxidative properties. The aim of the study was to test the protective effect of alpha-mangostin against oxidative stress both in retina of light-damaged mice model and in hydrogen peroxide (H2O2)-stressed RPE cells. We observed that alpha-mangostin significantly inhibited light-induced degeneration of photoreceptors and 200 μM H2O2-induced apoptosis of RPE cells. 200 μM H2O2-induced generation of reactive oxygen species (ROS) and light-induced generation of malondialdehyde (MDA) were suppressed by alpha-mangostin. Alpha-mangostin stimulation resulted in an increase of superoxide dismutase (SOD) activity, glutathione peroxidase (GPX) activity and glutathione (GSH) content both in vivo and vitro. Furthermore, the mechanism of retinal protection against oxidative stress by alpha-mangostin involves accumulation and the nuclear translocation of the NF-E2-related factor (Nrf2) along with up-regulation the expression of heme oxygenas-1 (HO-1). Meanwhile, alpha-mangostin can activate the expression of PKC-δ and down-regulate the expression of mitogen-activated protein kinases (MAPKs), including ERK1/2, JNK, P38. The results suggest that alpha-mangostin could be a new approach to suspend the onset and development of AMD. PMID:26888416

  3. Protective effect of alpha-mangostin against oxidative stress induced-retinal cell death.

    PubMed

    Fang, Yuan; Su, Tu; Qiu, Xiaorong; Mao, Pingan; Xu, Yidan; Hu, Zizhong; Zhang, Yi; Zheng, Xinhua; Xie, Ping; Liu, Qinghuai

    2016-01-01

    It is known that oxidative stress plays a pivotal role in age-related macular degeneration (AMD) pathogenesis. Alpha-mangostin is the main xanthone purified from mangosteen known as anti-oxidative properties. The aim of the study was to test the protective effect of alpha-mangostin against oxidative stress both in retina of light-damaged mice model and in hydrogen peroxide (H2O2)-stressed RPE cells. We observed that alpha-mangostin significantly inhibited light-induced degeneration of photoreceptors and 200 μM H2O2-induced apoptosis of RPE cells. 200 μM H2O2-induced generation of reactive oxygen species (ROS) and light-induced generation of malondialdehyde (MDA) were suppressed by alpha-mangostin. Alpha-mangostin stimulation resulted in an increase of superoxide dismutase (SOD) activity, glutathione peroxidase (GPX) activity and glutathione (GSH) content both in vivo and vitro. Furthermore, the mechanism of retinal protection against oxidative stress by alpha-mangostin involves accumulation and the nuclear translocation of the NF-E2-related factor (Nrf2) along with up-regulation the expression of heme oxygenas-1 (HO-1). Meanwhile, alpha-mangostin can activate the expression of PKC-δ and down-regulate the expression of mitogen-activated protein kinases (MAPKs), including ERK1/2, JNK, P38. The results suggest that alpha-mangostin could be a new approach to suspend the onset and development of AMD. PMID:26888416

  4. Retinoic acid modulates RAR alpha and RAR beta receptors in human glioma cell lines.

    PubMed

    Carpentier, A F; Leonard, N; Lacombe, J; Zassadowski, F; Padua, R A; Degos, L; Daumas-Duport, C; Chomienne, C

    1999-01-01

    To identify retinoic acid (RA) signalling pathways involved in growth and differentiation in cells of the glial lineage, two human glioma ceh lines were studied. The three RA receptors (RARs) mRNAs were constitutively expressed, and of the three RXRs, RXR beta appeared predominant. Western blotting analysis confirmed the constitutive expression of RAR alpha and RAR beta. Treatment with all-trans-RA induced morphological changes in the two cell lines, which progressed from their normal pattern of randomly oriented spindle-shaped cells to fibroblast-like glial cells. RA up-regulated RAR alpha and RAR beta mRNAs in both cell lines. Interestingly, RA treatment up-regulated RAR beta proteins but not RAR alpha proteins, suggesting post-transcriptional regulations of RAR transcripts in glioma cells. PMID:10652610

  5. 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.

  6. Activity of DL-alpha-Difluoromethylarginine and Polyamine Analogues against Cryptosporidium parvum Infection in a T-Cell Receptor Alpha-Deficient Mouse Model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The in vivo effectiveness of a series of conformationally restricted polyamine analogs alone and in combination with DL-alpha-difluoromethylarginine (DFMA) towards a T-cell receptor-alpha deficient mouse model infection of Cryptosporidium parvum was tested. Polyamine analogues were constructed from ...

  7. AMPA receptors serum-dependently mediate GABAA receptor alpha1 and alpha6 subunit down-regulation in cultured mouse cerebellar granule cells.

    PubMed

    Uusi-Oukari, Mikko; Kontturi, Leena-Stiina; Kallinen, Sampsa A; Salonen, Virpi

    2010-04-01

    Depolarization of cultured mouse cerebellar granule cells with potassium or kainate results in developmentally arrested state that includes down-regulation of GABA(A) receptor alpha1, alpha6 and beta2 subunit expression. These subunits are normally strongly expressed in cerebellar granule cells from second postnatal week throughout the adulthood. In the present study we demonstrate that selective activation of AMPA subtype of glutamate receptors down-regulates alpha1 and alpha6 subunit mRNA expression. Removal of AMPA agonist from culture medium restores expression of these subunits indicating reversibility of the down-regulation. In serum-free culture medium AMPA receptor activation did not down-regulate alpha1 or alpha6 subunit expression. Furthermore, the down-regulation was strongly attenuated when the cells were cultured in the presence of dialysed fetal calf serum. The results indicate that down-regulation of GABA(A) receptor alpha1 and alpha6 subunits by AMPA receptor activation is dependent on the presence of low molecular weight compounds present in fetal calf serum. In order to study mouse cerebellar granule cell maturation and/or regulation of GABA(A) receptor subunit expression in culture, the experiments should be performed in the absence of fetal calf serum. PMID:20170697

  8. G Protein Coupled Receptors in Embryonic Stem Cells: A Role for Gs-Alpha Signaling

    PubMed Central

    Layden, Brian T.; Newman, Marsha; Chen, Fei; Fisher, Amanda; Lowe, William L.

    2010-01-01

    Background Identification of receptor mediated signaling pathways in embryonic stem (ES) cells is needed to facilitate strategies for cell replacement using ES cells. One large receptor family, largely uninvestigated in ES cells, is G protein coupled receptors (GPCRs). An important role for these receptors in embryonic development has been described, but little is known about GPCR expression in ES cells. Methodology/Principal Findings We have examined the expression profile of 343 different GPCRs in mouse ES cells demonstrating for the first time that a large number of GPCRs are expressed in undifferentiated and differentiating ES cells, and in many cases at high levels. To begin to define a role for GPCR signaling in ES cells, the impact of activating Gs-alpha, one of the major alpha subunits that couples to GPCRs, was investigated. Gs-alpha activation resulted in larger embryoid bodies (EBs), due, in part, to increased cell proliferation and prevented the time-related decline in expression of transcription factors important for maintaining ES cell pluripotency. Significance/Conclusions These studies suggest that Gs-alpha signaling contributes to ES cell proliferation and pluripotency and provide a framework for further investigation of GPCRs in ES cells. PMID:20161705

  9. 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).

  10. Effect of 5alpha-dihydrotestosterone and testosterone on apoptosis in human dermal papilla cells.

    PubMed

    Winiarska, A; Mandt, N; Kamp, H; Hossini, A; Seltmann, H; Zouboulis, C C; Blume-Peytavi, U

    2006-01-01

    Pathogenetic mechanisms in androgenetic alopecia are not yet fully understood; however, it is commonly accepted that androgens like testosterone (T) and 5alpha-dihydrotestosterone (5alpha-DHT) inhibit hair follicle activity with early induction of the catagen. Thus, we investigated the influence of T and 5alpha-DHT on proliferation, cell death and bcl-2/bax expression in cultured dermal papilla cells (DPC) from nonbalding scalp regions of healthy volunteers. T and 5alpha-DHT induced apoptosis in DPC in a dose-dependent and time-related manner; in addition a necrotic effect due to T at 10(-5) M was found. Interestingly, bcl-2 protein expression was decreased in T- and 5alpha-DHT-treated cells, leading to an increase in the bax/bcl-2 ratio. In addition, T and 5alpha-DHT induced proteolytic cleavage of caspase 8 and inhibited proliferation of DPC at 10(-5) M. High concentrations of T and 5alpha-DHT were needed to induce apoptotic effects in DPC. These data suggest that DPC from nonbalding scalp regions do have the capacity to undergo apoptosis, but need a high androgen stimulus. The present study provides an interesting new pathogenetic approach in androgenetic alopecia. PMID:16931898

  11. Transient functional expression of alphaVbeta 3 on vascular cells during wound repair.

    PubMed Central

    Clark, R. A.; Tonnesen, M. G.; Gailit, J.; Cheresh, D. A.

    1996-01-01

    During early granulation tissue formation of wound repair, new capillaries invade the fibrin clot, a process that undoubtedly requires an interaction of vascular cells with the wound provisional matrix composed mainly of fibrin, fibronectin, and vitronectin. Integrin alphaVbeta3 is the vascular cell receptor for these wound-associated adhesive proteins. Therefore, we investigated the expression of this receptor on new capillaries of healing full-thickness cutaneous porcine wounds. During granulation tissue formation, alphaVbeta3 was expressed specifically on capillary sprouts invading the central fibrin clot whereas the closely related integrin alphaVbeta5 failed to localize to these cells. Cyclic peptides or antibody antagonists of alphaVbeta3 specifically inhibited granulation tissue formation in a transient manner during the period of invasive angiogenesis. Immunolocalization studies revealed that alphaVbeta3 became aggregated and lost from sprouting vessels after treatment with a peptide antagonist. In contrast, beta 1 integrins were not modulated by this treatment. Once granulation tissue filled the wound and invasive angiogenesis terminated, the alphaVbeta3 showed little or no expression in the granulation tissue microvasculature. These data demonstrate that integrin alphaVbeta3 plays a fundamental, but transient, role during invasive angiogenesis and granulation tissue formation in a healing wound. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 8 Figure 9 Figure 10 PMID:8623913

  12. The Alpha Stem Cell Clinic: a model for evaluating and delivering stem cell-based therapies.

    PubMed

    Trounson, Alan; DeWitt, Natalie D; Feigal, Ellen G

    2012-01-01

    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

  13. 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

  14. In vitro antitumor cytotoxic T lymphocyte response induced by dendritic cells transduced with DeltaNp73alpha recombinant adenovirus.

    PubMed

    Hu, Yijie; He, Yong; Srivenugopal, Kalkunte S; Fan, Shizhi; Jiang, Yaoguang

    2007-11-01

    DeltaNp73alpha, the N-terminal truncated form of p73alpha is a candidate tumor antigen because of its selective expression in many human cancers and lack of expression in normal tissues. Therefore, we investigated the effects of dendritic cells infected with adenoviral DeltaNp73alpha (DNp73alpha) on breaking immune tolerance and induction of immunity against DNp73alpha-expressing (A549 lung cancer, K-562 leukemia) and non-expressing (MCF-7 breast cancer) cell lines. Immature dendritic cells generated in the presence of interleukin-4 and granulocyte/macrophage colony-stimulating factor from a human umbilical cord blood were transduced with a recombinant adenoviral (Ad) vector encoding full-length human DNp73alpha cDNA (Ad-DNp73alpha) or a control vector Ad-EGFP, using the centrifugal force method. Induction of DNp73alpha-specific CTL response was evaluated by a cytotoxic assay against the three human tumor cell lines with different DNp73alpha expression levels. The viability and activation status of transduced dendritic cells were assessed by flow cytometry. The dendrocyte/Ad-DNp73alpha-activated cytotoxic T lymphocytes showed significantly higher cytotoxicity against the cell lines A549/DNp73alpha, K-562 that expressed DNp73alpha than the DNp73alpha-null MCF-7 cells. The DCs/Ad-DNp73alpha showed higher survival rates than the DCs/Ad-EGFP or untransduced DCs, presumably due to the inhibition of cell death. These findings, with potential applications for immunotherapy, demonstrate that dendrocytes transduced with Ad-DNp73alpha can induce specific and sustained T cell responses against tumors expressing this variant p53-related gene. PMID:17914557

  15. 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.

  16. Possible Role of Non-Muscle Alpha-Actinins in Muscle Cell Mechanosensitivity

    PubMed Central

    Ogneva, Irina V.; Biryukov, Nikolay S.; Leinsoo, Toomas A.; Larina, Irina M.

    2014-01-01

    The main hypothesis suggested that changes in the external mechanical load would lead to different deformations of the submembranous cytoskeleton and, as a result, dissociation of different proteins from its structure (induced by increased/decreased mechanical stress). The study subjects were fibers of the soleus muscle and cardiomyocytes of Wistar rats. Changes in external mechanical conditions were reconstructed by means of antiorthostatic suspension of the animals by their tails for 6, 12, 18, 24 and 72 hours. Transversal stiffness was measured by atomic force microscopy imaging; beta-, gamma-actin, alpha-actinin 1 and alpha-actinin 4 levels in membranous and cytoplasmic fractions were quantified by Western blot analysis; expression rates of the corresponding genes were studied using RT-PCR. Results: In 6 hours, alpha-actinin 1 and alpha-actinin 4 levels decreased in the membranous fraction of proteins of cardiomyocytes and soleus muscle fibers, respectively, but increased in the cytoplasmic fraction of the abovementioned cells. After 6–12 hours of suspension, the expression rates of beta-, gamma-actin, alpha-actinin 1 and alpha-actinin 4 were elevated in the soleus muscle fibers, but the alpha-actinin 1 expression rate returned to the reference level in 72 hours. After 18–24 hours, the expression rates of beta-actin and alpha-actinin 4 increased in cardiomyocytes, while the alpha-actinin 1 expression rate decreased in soleus muscle fibers. After 12 hours, the beta- and gamma-actin content dropped in the membranous fraction and increased in the cytoplasmic protein fractions from both cardiomyocytes and soleus muscle fibers. The stiffness of both cell types decreased after the same period of time. Further, during the unloading period the concentration of nonmuscle actin and different isoforms of alpha-actinins increased in the membranous fraction from cardiomyocytes. At the same time, the concentration of the abovementioned proteins decreased in the soleus

  17. CCL20/macrophage inflammatory protein 3alpha and tumor necrosis factor alpha production by primary uterine epithelial cells in response to treatment with lipopolysaccharide or Pam3Cys.

    PubMed

    Crane-Godreau, Mardi A; Wira, Charles R

    2005-01-01

    Having previously shown that CCL20/macrophage inflammatory protein 3alpha and tumor necrosis factor alpha (TNF-alpha) are released by polarized primary rat uterine epithelial cells (UEC) in response to Escherichia coli but not to Lactobacillus rhamnosus, we sought to determine if epithelial cells are responsive to pathogen-associated molecular patterns (PAMP), including lipopolysaccharide (LPS), lipoteichoic acid (LTA), and Pam(3)Cys, a bacterial lipoprotein analog. Epithelial cells were grown to confluence on Nunc cell culture inserts prior to apical treatment with PAMPs. In response to LPS, LTA, and Pam(3)Cys (EMC Microcollection GmbH, Tubingen, Germany), CCL20 levels increased (4- to 10-fold) while PAMPs caused increased TNF-alpha (1- to 4-fold) in the medium collected after 24 h of incubation. Both apical and basolateral secretion of CCL20 and TNF-alpha increased in response to PAMPs, but treatments had no effect on cell viability and integrity, as measured by transepithelial resistance. Time course studies of CCL20 and TNF-alpha release in response to Pam(3)Cys and LPS indicated that CCL20 release peaked between 2 and 4 h after treatment, whereas TNF-alpha release was gradual over the length of the incubation. Freeze-thaw and cell lysis experiments, along with actinomycin D studies, suggested that CCL20 and TNF-alpha are synthesized in response to PAMP stimulation. Taken together, these studies demonstrate that E. coli and selected PAMPs have direct effects on the production of CCL20 and TNF-alpha without affecting cell integrity. Since CCL20 is known to be both chemotactic and antimicrobial, the increase in apical and basolateral release by UEC in response to PAMPs suggests a new mechanism of innate immune protection in the female reproductive tract. PMID:15618187

  18. 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.

  19. Tumor necrosis factor alpha induces expression of human immunodeficiency virus in a chronically infected T-cell clone.

    PubMed Central

    Folks, T M; Clouse, K A; Justement, J; Rabson, A; Duh, E; Kehrl, J H; Fauci, A S

    1989-01-01

    Tumor necrosis factor alpha (TNF-alpha), also known as cachectin, was demonstrated to induce the expression of human immunodeficiency virus (HIV) in a chronically infected T-cell clone (ACH-2). Concentrations of recombinant TNF-alpha as low as 50 pg/ml induced a significant increase over background of HIV expression in the ACH-2 cells as determined by supernatant reverse transcriptase activity. The HIV-inducing effects of TNF-alpha could not be explained by toxic effects on the cells. In addition, both the uninfected parental cell line (A3.01) and the infected ACH-2 cells were shown to have high-affinity receptors for TNF-alpha. Transient-transfection experiments demonstrated that the inductive effects of TNF-alpha were due to specific activation of the HIV long terminal repeat. These studies provide evidence that TNF-alpha may play a role in the mechanisms of pathogenesis of HIV infection. Images PMID:2784570

  20. 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

  1. 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.

  2. Protein kinase C-alpha and -beta play antagonistic roles in the differentiation process of THP-1 cells.

    PubMed

    Dieter, P; Schwende, H

    2000-05-01

    The roles of protein kinase C (PKC) isoenzymes in the differentiation process of THP-1 cells are investigated. Inhibition of PKC by RO 31-8220 reduces the phagocytosis of latex particles and the release of superoxide, prostaglandin E(2) (PGE(2)), and tumour necrosis factor (TNF)-alpha. The proliferation of THP-1 cells is slightly enhanced by RO 31-8220. Stable transfection of THP-1 cells with asPKC-alpha, and incubation of THP-1 cells with antisense (as) PKC-alpha oligodeoxynucleotides reduces PKC-alpha levels and PKC activity. asPKC-alpha-transfected THP-1 cells show a decreased phagocytosis and a decreased release of superoxide, PGE(2) and TNF-alpha. The proliferation of asPKC-alpha-transfected THP-1 cells is enhanced. Stable transfection of THP-1 cells with asPKC-beta, and incubation of THP-1 cells with asPKC-beta oligodeoxynucleotides, reduces PKC-beta levels and PKC activity. asPKC-beta-transfected THP-1 cells show a decreased phagocytosis, a decreased TNF-alpha release, and a decreased proliferation. However, no difference is measured in the release of superoxide and PGE(2). These results suggest that: (1) PKC-alpha but not PKC-beta is involved in the release of superoxide and PGE(2); (2) TNF-alpha release and the phagocytosis of latex particles are mediated by PKC-alpha, PKC-beta, and other PKC isoenzymes; and (3) PKC-alpha and PKC-beta play antagonistic roles in the differentiation process of THP-1 cells. PKC-alpha promotes the differentiation process of THP-1 cells, PKC-beta retards the differentiation of THP-1 cells into macrophage-like cells. PMID:10822170

  3. Infection of human fallopian tube epithelial cells with Neisseria gonorrhoeae protects cells from tumor necrosis factor alpha-induced apoptosis.

    PubMed

    Morales, Priscilla; Reyes, Paz; Vargas, Macarena; Rios, Miguel; Imarai, Mónica; Cardenas, Hugo; Croxatto, Horacio; Orihuela, Pedro; Vargas, Renato; Fuhrer, Juan; Heckels, John E; Christodoulides, Myron; Velasquez, Luis

    2006-06-01

    Following infection with Neisseria gonorrhoeae, bacteria may ascend into the Fallopian tubes (FT) and induce salpingitis, a major cause of infertility. In the FT, interactions between mucosal epithelial cells and gonococci are pivotal events in the pathogen's infection cycle and the inflammatory response. In the current study, primary FT epithelial cells were infected in vitro with different multiplicities of infection (MOI) of Pil+ Opa+ gonococci. Bacteria showed a dose-dependent association with cells and induced the secretion of tumor necrosis factor alpha (TNF-alpha). A significant finding was that gonococcal infection (MOI = 1) induced apoptosis in approximately 30% of cells, whereas increasing numbers of bacteria (MOI = 10 to 100) did not induce apoptosis. Apoptosis was observed in only 11% of cells with associated bacteria, whereas >84% of cells with no adherent bacteria were apoptotic. TNF-alpha was a key contributor to apoptosis, since (i) culture supernatants from cells infected with gonococci (MOI = 1) induced apoptosis in naïve cultures, suggesting that a soluble factor was responsible; (ii) gonococcal infection-induced apoptosis was inhibited with anti-TNF-alpha antibodies; and (iii) the addition of exogenous TNF-alpha induced apoptosis, which was inhibited by the presence of increasing numbers of bacteria (MOI = 10 to 100). These data suggest that TNF-alpha-mediated apoptosis of FT epithelial cells is likely a primary host defense mechanism to prevent pathogen colonization. However, epithelial cell-associated gonococci have evolved a mechanism to protect the cells from undergoing TNF-alpha-mediated apoptosis, and this modulation of the host innate response may contribute to establishment of infection. Understanding the antiapoptotic mechanisms used by Neisseria gonorrhoeae will inform the pathogenesis of salpingitis and could suggest new intervention strategies for prevention and treatment of the disease. PMID:16714596

  4. One-step purification of functional human and rat pancreatic alpha cells.

    PubMed

    Köhler, Martin; Daré, Elisabetta; Ali, Muhammed Yusuf; Rajasekaran, Subu Surendran; Moede, Tilo; Leibiger, Barbara; Leibiger, Ingo B; Tibell, Annika; Juntti-Berggren, Lisa; Berggren, Per-Olof

    2012-02-01

    Pancreatic alpha cells contribute to glucose homeostasis by the regulated secretion of glucagon, which increases glycogenolysis and hepatic gluconeogenesis in response to hypoglycemia. Alterations of glucagon secretion are observed in diabetic patients and exacerbate the disease. The restricted availability of purified primary alpha cells has limited our understanding of their function in health and disease. This study was designed to establish convenient protocols for the purification of viable alpha cells from rat and human pancreatic islets by FACS, using intrinsic cellular properties. Islets were isolated from the pancreata of Wistar rats or deceased human organ donors. Dispersed islet cells were separated by FACS based on light scatter and autofluorescence. Purity of sorted cells was evaluated by immunocytochemistry using hormone specific antibodies. Relative hormone expression was further determined by quantitative RT-PCR. Viability was determined by Annexin V and propidium iodide staining and function was assessed by monitoring cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) using Fura-2/AM. We developed species-specific FACS gating strategies that resulted in populations consisting mainly of alpha cells (96.6 ± 1.4%, n = 3 for rat; 95.4 ± 1.7%, n = 4 for human, mean ± SEM). These cell fractions showed ~5-fold and ~4-fold enrichment (rat and human, respectively) of glucagon mRNA expression compared to total ungated islet cells. Most of the sorted cells were viable and functional, as they responded with an increase in [Ca(2+)](i) upon stimulation with L-arginine (10 mM). The majority of the sorted human alpha cells responded also to stimulation with kainate (100 μM), whereas this response was infrequent in rat alpha cells. Using the same sample preparation, but a different gating strategy, we were also able to sort rat and human populations enriched in beta cells. In conclusion, we have simplified and optimized a method for the purification of rat

  5. Human dermal mast cells contain and release tumor necrosis factor alpha, which induces endothelial leukocyte adhesion molecule 1.

    PubMed Central

    Walsh, L J; Trinchieri, G; Waldorf, H A; Whitaker, D; Murphy, G F

    1991-01-01

    Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine that mediates endothelial leukocyte interactions by inducing expression of adhesion molecules. In this report, we demonstrate that human dermal mast cells contain sizeable stores of immunoreactive and biologically active TNF-alpha within granules, which can be released rapidly into the extracellular space upon degranulation. Among normal human dermal cells, mast cells are the predominant cell type that expresses both TNF-alpha protein and TNF-alpha mRNA. Moreover, induction of endothelial leukocyte adhesion molecule 1 expression is a direct consequence of release of mast cell-derived TNF-alpha. These findings establish a role for human mast cells as "gatekeepers" of the dermal microvasculature and indicate that mast cell products other than vasoactive amines influence endothelium in a proinflammatory fashion. Images PMID:1709737

  6. Effects of alpha-chlorohydrin and gonadectomy on the adenohypophysial cells of male rats and gerbils.

    PubMed

    Dixit, V P; Lohiya, N K; Jain, H C

    1974-05-01

    The cytological changes occurring in the gonadotropic cells of the anterior lobe of the pituitary in 30 male rats and 30 male gerbils after administration of alpha-chlorohydrin were compared with those occurring following bilateral gonadectomy. A decline in the weights of testes and accessory sex glands and inhibited spermatogenesis were noted in rats and gerbils following alpha-chlorohydrin administration. Alpha-cholorohydrin changed the appearance of the delta-basilphils, cell cytoplasm showed fine granulation and vacuolization, and castration vacuoles appeared in many delta-basiphils of the anteroir lobe of the pituitary. The compound brought about transient changes resembling those of castration in the anterior lobe of the pituitary of male rats and gerbils. In gerbils, there was also a marked decrease in the number of acidophils. The results show that alpha-chlorohydrin exerts its effects on androgen-dependent structures, e.g., seminal vesicles, ventral prostrate, epididymides, and perineal complex. PMID:4841391

  7. 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.

  8. GW8510 Increases Insulin Expression in Pancreatic Alpha Cells through Activation of p53 Transcriptional Activity

    PubMed Central

    Fomina-Yadlin, Dina; Kubicek, Stefan; Vetere, Amedeo; He, Kaihui Hu; Schreiber, Stuart L.; Wagner, Bridget K.

    2012-01-01

    Background Expression of insulin in terminally differentiated non-beta cell types in the pancreas could be important to treating type-1 diabetes. Previous findings led us to hypothesize involvement of kinase inhibition in induction of insulin expression in pancreatic alpha cells. Methodology/Principal Findings Alpha (αTC1.6) cells and human islets were treated with GW8510 and other small-molecule inhibitors for up to 5 days. Alpha cells were assessed for gene- and protein-expression levels, cell-cycle status, promoter occupancy status by chromatin immunoprecipitation (ChIP), and p53-dependent transcriptional activity. GW8510, a putative CDK2 inhibitor, up-regulated insulin expression in mouse alpha cells and enhanced insulin secretion in dissociated human islets. Gene-expression profiling and gene-set enrichment analysis of GW8510-treated alpha cells suggested up-regulation of the p53 pathway. Accordingly, the compound increased p53 transcriptional activity and expression levels of p53 transcriptional targets. A predicted p53 response element in the promoter region of the mouse Ins2 gene was verified by chromatin immunoprecipitation (ChIP). Further, inhibition of Jun N-terminal kinase (JNK) and p38 kinase activities suppressed insulin induction by GW8510. Conclusions/Significance The induction of Ins2 by GW8510 occurred through p53 in a JNK- and p38-dependent manner. These results implicate p53 activity in modulation of Ins2 expression levels in pancreatic alpha cells, and point to a potential approach toward using small molecules to generate insulin in an alternative cell type. PMID:22242153

  9. Complete sequence of three alpha-tubulin cDNAs in Chinese hamster ovary cells: each encodes a distinct alpha-tubulin isoprotein.

    PubMed Central

    Elliott, E M; Henderson, G; Sarangi, F; Ling, V

    1986-01-01

    The genome of Chinese hamster ovary (CHO) cells contains a complex family of approximately 16 alpha-tubulin genes, many of which may be pseudogenes. We present here the complete cDNA sequences of three expressed alpha-tubulin genes; one of these genes has been identified only in CHO cells. The noncoding regions of these three CHO alpha-tubulin genes differed significantly, but their coding regions were highly conserved. Nevertheless, we observed differences in the predicted amino acid sequences for the three genes. A comparison of the CHO alpha-tubulin sequences with all of the sequences available for mammals allowed assignment of the alpha-tubulin genes to three classes. The proteins encoded by the members of two of these classes showed no class-specific amino acids among the mammalian species examined. The gene belonging to the third class encoded an isoprotein which was clearly distinct, and members of this class may play a unique role in vivo. Sequencing of the three alpha-tubulin genes was also undertaken in CMR795, a colcemid-resistant clonal CHO cell line which has previously been shown to have structural and functional alterations in its tubulin proteins. We found differences in the tubulin nucleotide sequence compared with the parental line; however, no differences in the alpha-tubulin proteins encoded in the two cell lines were observed. PMID:3773896

  10. Inhibition of Calpain Prevents Manganese-Induced Cell Injury and Alpha-Synuclein Oligomerization in Organotypic Brain Slice Cultures

    PubMed Central

    Xu, Bin; Liu, Wei; Deng, Yu; Yang, Tian-Yao; Feng, Shu; Xu, Zhao-Fa

    2015-01-01

    Overexposure to manganese has been known to promote alpha-synuclein oligomerization and enhance cellular toxicity. However, the exact mechanism of Mn-induced alpha-synuclein oligomerization is unclear. To explore whether alpha-synuclein oligomerization was associated with the cleavage of alpha-synuclein by calpain, we made a rat brain slice model of manganism and pretreated slices with calpain inhibitor II, a cell-permeable peptide that restricts the activity of calpain. After slices were treated with 400 μM Mn for 24 h, there were significant increases in the percentage of apoptotic cells, lactate dehydrogenase release, intracellular [Ca2+]i, calpain activity, and the mRNA and protein expression of calpain 1 and alpha-synuclein. Moreover, the number of C- and N-terminal fragments of alpha-synuclein and the amount of alpha-synuclein oligomerization also increased. These results also showed that calpain inhibitor II pretreatment could reduce Mn-induced nerve cell injury and alpha-synuclein oligomerization. Additionally, there was a significant decrease in the number of C- and N-terminal fragments of alpha-synuclein in calpain inhibitor II-pretreated slices. These findings revealed that Mn induced the cleavage of alpha-synuclein protein via overactivation of calpain and subsequent alpha-synuclein oligomerization in cultured slices. Moreover, the cleavage of alpha-synuclein by calpain 1 is an important signaling event in Mn-induced alpha-synuclein oligomerization. PMID:25756858

  11. 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.

  12. 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.

  13. The Atlantic Salmon MHC class II alpha and beta promoters are active in mammalian cell lines.

    PubMed

    Vestrheim, O; Lundin, M; Syed, M

    2007-01-01

    The major histocompatibility complex class II (MHCII) genes are only constitutively expressed in certain immune response cells such as B cells, macrophages, dendritic cells and other antigen presenting cells. This cell specific expression pattern and the presence of conserved regions such as the X-, X2-, Y-, and W-boxes make the MHCII promoters especially interesting as vector constructs. We tested whether the Atlantic salmon (Salmo salar L.) MHCII promoters can function in cell lines from other organisms. We found that the salmon MHCII alpha and MHCII beta promoters could drive expression of a LacZ reporter gene in adherent lymphoblast cell lines from dog (DH82) and rabbit (HybL-L). This paper shows that the promoters of Atlantic salmon MHCII alpha and beta genes can function in mammalian cell lines. PMID:17934904

  14. Mutagenesis and repair by low doses of alpha radiation in mammalian cells.

    PubMed

    Puck, Theodore T; Johnson, Robert; Webb, Patricia; Cui, Helen; Valdez, Joseph G; Crissman, Harry

    2002-09-17

    Low doses of alpha radiation in basements have been causally implicated in lung cancer. Previous studies have concentrated on high dose effects, for which no significant repair was found. In the present study, the methodology for measuring mutation by quantitating mitotic breaks and gaps was found to be applicable to G2-phase Chinese hamster ovary cells irradiated with 10-50 cGy of alpha radiation. The mutation yield in such cells closely resembles that of gamma irradiation. Caffeine, which inhibits repair, produces the same straight line increase of alpha and gamma mutation yields plotted against the dose. In the absence of caffeine, the repair of alpha radiation lesions is almost twice as great as for gamma radiation. Mitotic index changes substantiate these interpretations. It is proposed that the higher ion density associated with alpha radiation may result in fewer lesions being missed by the repair processes. The quantitation of chromosomal lesions for G2 cells exposed to low doses of alpha radiation, gamma radiation, or chemical mutagens in the presence and absence of caffeine is a rapid and reproducible methodology. Protection from mutational disease in a fashion similar to the use of sanitation for infectious disease appears practical. PMID:12198179

  15. Sphingolipid signalling: molecular basis and role in TNF-alpha-induced cell death.

    PubMed

    Malagarie-Cazenave, Sophie; Andrieu-Abadie, Nathalie; Ségui, Bruno; Gouazé, Valérie; Tardy, Claudine; Cuvillier, Olivier; Levade, Thierry

    2002-12-01

    Various lipidic molecules serve as second messengers for transducing signals from the cell surface to the cell interior and trigger specific cellular responses. Sphingolipids represent a complex group of lipids that have recently emerged as new transducers in eukaryotic cells. Several sphingolipid molecules are able to modulate cell growth, differentiation and death. This review summarises current knowledge of the signalling functions of sphingolipids, especially in the regulation of tumour necrosis factor [alpha] (TNF-[alpha])-mediated cytotoxic effects. TNF-[alpha] is a multifaceted cytokine that controls a wide range of immune responses in mammals, including induction of programmed cell death (also called apoptosis). On the basis of recent observations, a working model is proposed for the molecular mechanisms underlying regulation of sphingolipid generation following TNF-[alpha] receptor 1 activation. The implications of these findings for the development of future pharmacological strategies to prevent the cytotoxic TNF-[alpha] response and subsequent cellular dysfunctions (as seen in various human diseases) are discussed. PMID:14987386

  16. Lability of DNA polymerase alpha correlated with decreased DNA synthesis and increased age in human cells

    SciTech Connect

    Busbee, D.; Sylvia, V.; Stec, J.; Cernosek, Z.; Norman, J.

    1987-12-01

    DNA excision repair and mitogen-initiated blastogenesis in human cells declined in efficiency as an apparent function of decreased DNA polymerase alpha specific activity with increased age of the cell donor. DNA polymerase alpha isolated from fetal cells contained a single, high-specific-activity enzyme form that could not be further activated and that was stable with regard to enzyme activity and affinity for DNA template-primer. DNA polymerase alpha isolated from adult-derived cells contained both low-specific-activity and high-specific-activity forms. The low-activity enzyme form, which showed low affinity of binding to DNA template-primer, was activated by treatment with phosphatidylinositol, /sup 32/P-ATP, and phosphatidylinositol kinase, resulting in a /sup 32/P-labeled enzyme that exhibited high affinity of binding to DNA template-primer. The activated enzyme was unstable, exhibiting a loss of /sup 32/P-label correlated with the loss of both specific activity and high affinity of binding to DNA template-primer. The data suggest that DNA polymerase alpha isolated from adult-derived human cells has low-activity and high-activity forms. Decreased specific activity of DNA polymerase alpha correlated with increased age of the donor appears to be a function of loss of an enzyme activator molecule resulting in diminished ability of the enzyme to bind DNA template-primer.

  17. Claudin 1 mediates tumor necrosis factor alpha-induced cell migration in human gastric cancer cells

    PubMed Central

    Shiozaki, Atsushi; Shimizu, Hiroki; Ichikawa, Daisuke; Konishi, Hirotaka; Komatsu, Shuhei; Kubota, Takeshi; Fujiwara, Hitoshi; Okamoto, Kazuma; Iitaka, Daisuke; Nakashima, Shingo; Nako, Yoshito; Liu, Mingyao; Otsuji, Eigo

    2014-01-01

    AIM: To investigate the role of claudin 1 in the regulation of genes involved in cell migration and tumor necrosis factor alpha (TNF-α)-induced gene expression in human gastric adenocarcinoma cells. METHODS: Knockdown experiments were conducted with claudin 1 small interfering RNA (siRNA), and the effects on the cell cycle, apoptosis, migration and invasion were analyzed in human gastric adenocarcinoma MKN28 cells. The gene expression profiles of cells were analyzed by microarray and bioinformatics. RESULTS: The knockdown of claudin 1 significantly inhibited cell proliferation, migration and invasion, and increased apoptosis. Microarray analysis identified 245 genes whose expression levels were altered by the knockdown of claudin 1. Pathway analysis showed that the top-ranked molecular and cellular function was the cellular movement related pathway, which involved MMP7, TNF-SF10, TGFBR1, and CCL2. Furthermore, TNF- and nuclear frctor-κB were the top-ranked upstream regulators related to claudin 1. TNF-α treatment increased claudin 1 expression and cell migration in MKN28 cells. Microarray analysis indicated that the depletion of claudin 1 inhibited 80% of the TNF-α-induced mRNA expression changes. Further, TNF-α did not enhance cell migration in the claudin 1 siRNA transfected cells. CONCLUSION: These results suggest that claudin 1 is an important messenger that regulates TNF-α-induced gene expression and migration in gastric cancer cells. A deeper understanding of these cellular processes may be helpful in establishing new therapeutic strategies for gastric cancer. PMID:25548484

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

    SciTech Connect

    Song, Xiaomin; Luo, Yongzhang

    2010-07-16

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

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

    SciTech Connect

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

    1988-06-01

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

  20. Tumor necrosis factor-alpha (TNF-alpha) concentrations from whole blood cultures correlate with isolated peripheral blood mononuclear cell cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many cellular immune assays are impractical because they require labor-intensive isolation of cells from their natural environment. The objectives of this study were to determine the relationship between cell culture supernatant TNF-alpha from isolated peripheral blood mononuclear cells (PBMC) and w...

  1. 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.

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

    SciTech Connect

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

    2005-12-16

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

  3. 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

  4. 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.

  5. Severe osteoporosis due to systemic mast cell disease: successful treatment with interferon alpha-2B.

    PubMed

    Lehmann, T; Beyeler, C; Lämmle, B; Hunziker, T; Vock, P; Olah, A J; Dahinden, C; Gerber, N J

    1996-09-01

    We describe a 33-year old man suffering from severe vertebral osteoporosis and urticaria pigmentosa due to systemic mast cell disease (SMCD). Because i.v. clodronate therapy could not prevent further vertebral fractures, an additional treatment with interferon alpha-2b was initiated. During 24 months of treatment, our patient had no further pain episodes, no new vertebral fractures were discovered, trabecular bone mineral density (BMD) increased significantly and urticarial symptoms improved. Nevertheless, the extent of skin lesions remained unchanged. On histological examination, a remarkable decrease of mast cells was observed in the bone marrow, but not in the skin. Five months after discontinuation of interferon alpha-2b, trabecular BMD decreased and urticarial symptoms deteriorated. These findings illustrate a beneficial effect of interferon alpha-2b on SMCD-induced osteoporosis as well as urticarial symptoms, and raise the question whether this treatment may have a diverse impact on mast cell population in different tissues. PMID:8810675

  6. Tumour necrosis factor-alpha stimulates invasiveness of T-cell hybridomas and cytotoxic T-cell clones by a pertussis toxin-insensitive mechanism.

    PubMed Central

    La Rivière, G; Klein Gebbinck, J W; Schipper, C A; Roos, E

    1992-01-01

    Tumour necrosis factor-alpha (TNF-alpha) stimulated invasion by mouse T-cell hybridomas and cytotoxic T-lymphocyte clones into rat embryo fibroblast monolayers. The effect on these highly invasive cells was limited: invasion was stimulated maximally to 130% of controls. However, when cells were pretreated with pertussis toxin (PT), which inhibits invasion to +/- 20% of controls, a clearcut effect was observed: 400 U TNF-alpha per ml stimulated invasion usually two- to threefold, and sometimes even up to 10-fold. Therefore, experiments were done with PT-pretreated cells. Stimulation was dose dependent and maximal at 200-400 U TNF-alpha per ml. An anti-TNF-alpha monoclonal antibody completely abolished TNF-alpha-induced invasion. The effect was maximal 30 min after addition of cells and TNF-alpha to the monolayer and then declined. TNF-alpha preincubation of T-cell hybridoma cells, but not of fibroblasts, had a similar stimulatory effect, which was also maximal after 30 min. This shows that TNF-alpha acts directly on the T-cell hybridoma cells. Invasive T-cell hybridomas colonize many tissues from the blood similarly as normal T cells. Our data thus suggest that TNF-alpha can stimulate migration of normal T lymphocytes into inflamed tissues and can promote metastasis of malignant T lymphomas. The signals involved are transmitted via a pertussis toxin-insensitive pathway. PMID:1551690

  7. Effects of warm acclimation on Na+,K+-ATPase alpha-subunit expression in chloride cells of Antarctic fish.

    PubMed

    Brauer, Philip R; Sanmann, Jennifer N; Petzel, David H

    2005-07-01

    The teleosts Trematomus bernacchii thrive in southern oceanic waters with temperatures below 0 degrees C. These fish have serum osmolalities almost double those found in fish of temperate waters, thereby lowering their serum's freezing point and the energy needed for ionic homeostasis. Upon warm acclimation to 4 degrees C, T. bernacchii decrease their serum osmolality and increase the Na+,K+-ATPase activity in their gills. Na+,K+-ATPase alpha1-, alpha2-, and alpha3-subunit isoforms are expressed in the gills of T. bernacchii and it is thought that Na+,K+-ATPase subunit composition in chloride cells changes with warm acclimation. Using immunohistochemistry, we compared the number of chloride cells expressing various alpha-isoforms of the Na+,K+-ATPase in the gills of cold- and warm-acclimated T. bernacchii. We found no change in the number of alpha2- or alpha3-immunopositive cells in warm-acclimated fish gills or in the number of cells immunopositive for the Na+,K+,2Cl- cotransporter. However, the number of pan-alpha-immunopositive (recognizing all three alpha-isoforms) and alpha1-immunopositive cells both increased in warm-acclimated fish. This suggests that changes in the number of alpha1-isoform-expressing chloride cells could contribute to the increased Na+,K+-ATPase activity that occurs with warm-acclimation. PMID:15912523

  8. Tumor suppressor p53 and its homologue p73alpha affect cell migration.

    PubMed

    Sablina, Anna A; Chumakov, Peter M; Kopnin, Boris P

    2003-07-25

    The p53 tumor suppressor plays a central role in the negative control of growth and survival of abnormal cells. Previously we demonstrated that in addition to these functions, p53 expression affects cell morphology and lamellar activity of the cell edge (Alexandrova, A., Ivanov, A., Chumakov, P. M., Kopnin, P. B., and Vasiliev, J. M. (2000) Oncogene 19, 5826-5830). In the present work we studied the effects of p53 and its homologue p73alpha on cell migration. We found that loss of p53 function correlated with decreased cell migration that was analyzed by in vitro wound closure test and Boyden chamber assay. The decreased motility of p53-deficient cells was observed in different cell contexts: human foreskin fibroblasts (BJ), human colon and lung carcinoma cell lines (HCT116 and H1299, respectively), as well as mouse normal fibroblasts from lung and spleen, peritoneal macrophages, and keratinocytes. On the other hand, overexpression of the p53 family member p73alpha stimulated cell migration. Changes in cell migration correlated directly with transcription activation induced by p53 or p73alpha. Noteworthy, p53 modulated cell motility in the absence of stress. The effect of p53 and p73alpha on cell migration was mediated through the activity of the phosphatidylinositol 3-kinase/Rac1 pathway. This p53/p73 function was mainly associated with some modulation of intracellular signaling rather than with stimulation of production of secreted motogenic factors. The identified novel activity of the p53 family members might be involved in regulation of embryogenesis, wound healing, or inflammatory response. PMID:12750388

  9. 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

  10. 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.

  11. 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.

  12. WS6 induces both alpha and beta cell proliferation without affecting differentiation or viability

    PubMed Central

    Boerner, Brian P.; George, Nicholas M.; Mir, Shakeel U.R.; Sarvetnick, Nora E.

    2016-01-01

    Agents that stimulate human pancreatic beta cell proliferation are needed to improve diabetes mellitus treatment. Recently, a small molecule, WS6, was observed to stimulate human beta cell proliferation. However, little is known about its other effects on human islets. To better understand the role of WS6 as a possible beta cell regenerative therapy, we carried out in-depth phenotypic analysis of WS6-treated human islets, exploring its effects on non-beta cell proliferation, beta cell differentiation, and islet cell viability. WS6 not only stimulated beta cell proliferation in cultured human islets (in agreement with previous reports), but also human alpha cell proliferation, indicating that WS6 is not a beta cell-specific mitogen. WS6 did not change the proportion of insulin-positive beta cells or the expression of beta cell-specific transcription factors, suggesting that WS6 does not alter beta cell differentiation, and WS6 had no effect on human islet cell apoptosis or viability. In conclusion, WS6 stimulates proliferation of both human beta and alpha cells while maintaining cellular viability and the beta cell differentiated phenotype. These findings expand the literature on WS6 and support the suggestion that WS6 may help increase human islet mass needed for successful treatment of diabetes. PMID:25739404

  13. 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.

  14. Inhibition of protein synthesis by imexon reduces HIF-1alpha expression in normoxic and hypoxic pancreatic cancer cells.

    PubMed

    Samulitis, Betty K; Landowski, Terry H; Dorr, Robert T

    2009-02-01

    Hypoxia-inducing factor-1 alpha (HIF-1alpha), is a major survival factor for tumor cells growing in a low oxygen environment. The anti-cancer agent imexon binds thiols and causes accumulation of reactive oxygen species (ROS) in pancreatic cancer cells. Unlike many cytotoxic agents, imexon is equi-cytotoxic in human MiaPaCa-2 and Panc-1 cells grown in normoxic (21% O(2)) and hypoxic (1% O(2)) conditions. Western blot analyses of imexon-treated cells demonstrated that imexon reduces HIF-1alpha protein levels in both normoxic and hypoxic conditions in a time- and concentration-dependant fashion. Gemcitabine did not similarly affect HIF-1alpha levels. Imexon did not reduce transcription of new HIF-1alpha mRNA, but did reduce the synthesis of new proteins, including HIF-1alpha, measured by (35)S methionine/cysteine (Met/Cys) incorporation. Concurrently, the half-life of existing HIF-1alpha protein was increased by imexon, in association with a marked inhibition of chymotryptic activity in the 20S proteasome. The inhibition of HIF-1alpha translation was not specific, rather it was part of a general decrease in protein translation caused by imexon. This inhibitory effect on translation did not involve phosphorylation of eukaryotic initiation factor-2alpha (eIF-2alpha) and was not closely correlated to cell growth inhibition by imexon, suggesting that mechanisms other than protein synthesis inhibition contribute to the drug's cytotoxic effects. In summary, imexon blocks the translation of new proteins, including HIF-1alpha, and this effect overcomes an increase in the stability of preformed HIF-1alpha due to proteasome inhibition by imexon. Because net HIF-1alpha levels are reduced by imexon, combination studies with other drugs affected by HIF-1alpha survival signaling are warranted. PMID:18607542

  15. Transactivation of human immunodeficiency virus type 1 long terminal repeats by cell surface tumor necrosis factor alpha.

    PubMed Central

    Tadmori, W; Mondal, D; Tadmori, I; Prakash, O

    1991-01-01

    Tumor necrosis factor alpha (TNF-alpha) is expressed in secreted and cell surface (csTNF-alpha) forms by activated monocytic and T cells. In this report, we demonstrate that csTNF-alpha may predominantly regulate the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) activation in the promonocytic cell line U937 and in the Epstein-Barr virus-transformed B-cell line BH1. Anti-TNF-alpha antibody suppressed both the constitutive expression of the HIV-1 LTR in BH1 cells and the expression induced by phorbol 12-myristate 13-acetate in U937 cells. This suppression was found to be mediated via csTNF-alpha. No correlation between the HIV-1 LTR activation and the secretion of TNF-alpha was evident in these cell lines. Suppression of TNF-alpha secretion by cyclosporin A or by a serine protease inhibitor did not suppress the HIV-1 LTR activation. These observations suggest a novel biological role for csTNF-alpha in the immunopathogenesis of AIDS. PMID:1942242

  16. 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.

  17. The RNA polymerase II of an alpha-amanitin-resistant Chinese hamster ovary cell line.

    PubMed

    Lobban, P E; Siminovitch, L; Ingles, C J

    1976-05-01

    Amal, an alpha-amanitin-resistant mutant of the Chinese hamster ovary cell line, contains an RNA polymerase activity which elutes from DEAE-Sephadex at a salt concentration characteristic of an RNA polymerase II, but which is not sensitive to alpha-amanitin at levels where the polymerase II of wild-type cells is strongly inhibited. This result suggests that Amal owes its amanitin-resistant phenotype to a mutation affecting one of its genes for RNA polymerase II. To test this hypothesis, we purified the enzyme from Amal and then compared its properties with those of the wild-type enzyme. The mutant enzyme is indeed a polymerase II, and is over 600 times less sensitive to alpha-amanitin and more thermolabile than the wild-type enzyme. PMID:954093

  18. Isolation and characterisation of Kasumi-1 human myeloid leukaemia cell line resistant to tumour necrosis factor alpha-induced apoptosis.

    PubMed Central

    Ido, M.; Hayashi, K.; Kato, S.; Ogawa, H.; Komada, Y.; Zhau, Y. W.; Zhang, X. L.; Sakurai, M.; Suzuki, K.

    1996-01-01

    Tumour necrosis factor (TNF)-alpha induces apoptosis in a human acute myeloid leukaemia cell line, Kasumi-1. To examine the role of protein phosphorylation in signal transduction of TNF-alpha-induced apoptosis, a variant cell line resistant to TNF-alpha was established by an intermittent challenge of Kasumi-1 cells with increasing concentrations of TNF-alpha for 6 months. The mechanism of resistance to TNF-alpha appears to be in the post-receptor pathway because expression of p55 TNF receptor in the variant cells is increased compared with that of the parental Kasumi-1 cells. In renaturation assays, TNF-alpha induced a rapid activation of different protein kinases of different molecular weights, including the 50 kDa protein kinase (PK50) followed by the 35 kDa protein kinase (PK35), in the parental Kasumi-1 cells. The dose-response of TNF-alpha required to activate PK50 and PK35 was closely related to concentrations of TNF-alpha that induced apoptosis. Treatment of Kasumi-1 cells with ceramide also activated PK35. In TNF-resistant variant cells, activation of PK35 in response to TNF-alpha or ceramide was practically nil. These findings suggest that activation of PK35 through the ceramide pathway may play an important role in signal transduction of TNF-alpha in the Kasumi-1 cell line, while the decreased activation of PK35 may explain the insensitivity of the variant cells towards TNF-alpha. Images Figure 3 Figure 4 Figure 5 Figure 6 PMID:8562342

  19. 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.

  20. Study on CXCR4/SDF-1alpha axis in lymph node metastasis of cervical squamous cell carcinoma.

    PubMed

    Zhang, J-P; Lu, W-G; Ye, F; Chen, H-Z; Zhou, C-Y; Xie, X

    2007-01-01

    CXCR4/stromal-cell-derived factor-1alpha (SDF-1alpha) is involved in many cancer metastatic mechanisms. Cervical squamous cell cancer (SCC) tissues (n=35), normal cervical tissues (n=10), metastatic (n=10) and nonmetastatic lymph nodes (n=50), and Hela cells were stained immunohistochemically with CXCR4 monoclonal antibody (mAb). Meanwhile, lymph nodes were stained immunohistochemically with rabbit anti-SDF-1alpha. In vitro invasion of Hela cells was evaluated using Transwell Permeable Supports (Corning, NY), in which Hela cells with/without CXCR4 mAb preincubation were seeded in the upper chambers and medium containing 0-100 ng/mL SDF-1alpha was added to the lower compartments. For evaluating the effect of CXCR4/SDF-1alpha on proliferation of cervical cancer cells, Hela cells were cultured for 72 h exposed to SDF-1alpha with and without CXCR4 mAb. We found that CXCR4 was expressed on SCC cells in all cervical cancer, metastatic lymph node, and Hela cells but not in normal cervix. SDF-1alpha was expressed on lymph cells in all lymph nodes. SDF-1alpha induced the directed migration of Hela cells with a concentration-dependent model, which was inhibited by CXCR4 mAb (P<0.05). SDF-1alpha also stimulated the proliferation of Hela cells mediated by CXCR4 (P<0.05). CXCR4/SDF-1alpha axis probably participates in the metastasis toward lymph nodes in cervical cancer. PMID:17362322

  1. Cell-cell interaction promotes rat marrow stromal cell differentiation into endothelial cell via activation of TACE/TNF-alpha signaling

    PubMed Central

    Xu, Jian; Liu, Xinfeng; Chen, Jieli; Zacharek, Alex; Cui, Xu; Savant-Bhonsale, Smita; Chopp, Michael; Liu, Zhenguo

    2010-01-01

    Objective Marrow stromal cells (MSCs) are capable of differentiating into various cell types including endothelial cells. Microenvironment is important in cell fate determination. Tumor necrosis factor alpha converting enzyme (TACE), a well characterized “sheddase”, participates in the differentiation process of multiple lineages by the proteolytic release of membrane-bound proteins such as tumor necrosis factor alpha (TNF-alpha). Methods and Results We investigated the endothelial differentiation of MSCs under two co-culture conditions: 1) direct MSCs-rat brain microvascular endothelial cells (rBMECs) contact co-culture; and 2) indirect co-culture of MSCs and rBMECs. Also, we examined the role of TACE/TNF-alpha signaling in the process of differentiation under direct co-culture condition. We found that endothelial differentiation of MSCs was substantially enhanced in MSCs-rBMECs direct contact co-culture, but not in indirect transwell co-culture condition. Transcript levels of TACE and TNF-alpha as well as TACE protein expression were significantly upregulated in direct, but not in indirect co-culture condition. Addition of human recombinant TACE promoted gene expression of endothelial specific markers including vWF, CD31, VE-cadherin, Flk-1 and Flt-1 in the differentiating MSCs. Furthermore, inhibition of TACE with TAPI-2 or inhibition of TNF-alpha with Etanercept attenuated endothelial differentiation of MSCs in the direct co-culture condition. Conclusions We demonstrated for the first time that direct MSCs-rBMECs interaction stimulated the endothelial differentiation of MSCs via TACE/TNF alpha signaling. PMID:19796498

  2. 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.

  3. Binding of human interferon alpha to cells of different sensitivities: studies with internally radiolabeled interferon retaining full biological activity.

    PubMed Central

    Yonehara, S; Yonehara-Takahashi, M; Ishii, A

    1983-01-01

    The characteristics of interferon binding to various cells with different interferon sensitivity were studied by using [3H]leucine-labeled, pure human interferon alpha from Namalwa cells. Scatchard analysis of the binding data on cells sensitive to interferon alpha (human FL and fibroblasts and bovine MDBK) indicated the presence of two kinds of binding sites with high and low affinities. The binding constants of the high-affinity sites in these cells were similar (4 X 10(10) to 11 X 10(10) M-1). Cells insensitive to human interferon alpha (human HEC-1 and mouse L cells) were shown to have only low-affinity sites, suggesting that high-affinity binding sites are indispensable for interferon sensitivity and represent interferon receptors. However, the number of sites in three human diploid fibroblast strains and one strain trisomic for chromosome 21 were not proportionally correlated to the interferon sensitivity of the cells. The high-affinity binding to human cells was completely inhibited by both nonradioactive human interferons alpha and beta in a similar manner, but binding to bovine MDBK cells, on which human interferon beta is practically inactive, was inhibited effectively only by interferon alpha and not by beta. These results suggest that the receptor for human interferon alpha is common to human interferon beta in human cells, whereas the receptor on bovine cells binds only human interferon alpha. PMID:6300453

  4. 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.

  5. Alpha-defensins 1-3 release by dendritic cells is reduced by estrogen

    PubMed Central

    2011-01-01

    Background During pregnancy the immune system of the mother must protect any activation that may negatively affect the fetus. Changes in susceptibility to infection as well as resolution of some autoimmune disorders represent empirical evidence for pregnancy related alterations in immunity. Sex hormones reach extremely high levels during pregnancy and have been shown to have direct effects on many immune functions including the antiviral response of dendritic cells. Among the immunologically active proteins secreted by monocyte derived DCs (MDDC) are the alpha-defensins 1-3. This family of cationic antimicrobial peptides has a broad spectrum of microbicidal activity and has also been shown to link innate to adaptive immunity by attracting T cells and immature DCs, which are essential for initiating and polarizing the immune response. Methods We compare culture-generated monocyte derived DCs (MDDCs) with directly isolated myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) and measure their alpha-defensins 1-3 secretion by ELISA both, in basal situations and after hormone (E2 or PG) treatments. Moreover, using a cohort of pregnant women we isolated mDCs from blood and also measure the levels of these anti-microbial peptides along pregnancy. Results We show that mDCs and pDCs constitutively produce alpha-defensins 1-3 and at much higher levels than MDDCs. Alpha-defensins 1-3 production from mDCs and MDDCs but not pDCs is inhibited by E2. PG does not affect alpha-defensins 1-3 in any of the populations. Moreover, alpha-defensins 1-3 production by mDCs was reduced in the later stages of pregnancy in 40% of the patients. Conclusions Here, we demonstrate that mDCs and pDCs secrete alpha-defensins 1-3 and present a novel effect of E2 on the secretion of alpha-defensins 1-3 by dendritic cells. PMID:21861873

  6. 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.

  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 Ss; 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. PMID:27216158

  8. 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

  9. Alpha2 adrenoceptors regulate proliferation of human intestinal epithelial cells

    PubMed Central

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

    2000-01-01

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

  10. Cell-cell contact between marrow stromal cells and myeloma cells via VCAM-1 and alpha(4)beta(1)-integrin enhances production of osteoclast-stimulating activity.

    PubMed

    Michigami, T; Shimizu, N; Williams, P J; Niewolna, M; Dallas, S L; Mundy, G R; Yoneda, T

    2000-09-01

    Myeloma is a unique hematologic malignancy that exclusively homes in the bone marrow and induces massive osteoclastic bone destruction presumably by producing cytokines that promote the differentiation of the hematopoietic progenitors to osteoclasts (osteoclastogenesis). It is recognized that neighboring bone marrow stromal cells influence the expression of the malignant phenotype in myeloma cells. This study examined the role of the interactions between myeloma cells and neighboring stromal cells in the production of osteoclastogenic factors to elucidate the mechanism underlying extensive osteoclastic bone destruction. A murine myeloma cell line 5TGM1, which causes severe osteolysis, expresses alpha(4)beta(1)-integrin and tightly adheres to the mouse marrow stromal cell line ST2, which expresses the vascular cell adhesion molecule-1 (VCAM-1), a ligand for alpha(4)beta(1)-integrin. Co-cultures of 5TGM1 with primary bone marrow cells generated tartrate-resistant acid phosphatase-positive multinucleated bone-resorbing osteoclasts. Co-cultures of 5TGM1 with ST2 showed increased production of bone-resorbing activity and neutralizing antibodies against VCAM-1 or alpha(4)beta(1)-integrin inhibited this. The 5TGM1 cells contacting recombinant VCAM-1 produced increased osteoclastogenic and bone-resorbing activity. The activity was not blocked by the neutralizing antibody to known osteoclastogenic cytokines including interleukin (IL)-1, IL-6, tumor necrosis factor, or parathyroid hormone-related peptide. These data suggest that myeloma cells are responsible for producing osteoclastogenic activity and that establishment of direct contact with marrow stromal cells via alpha(4)beta(1)-integrin/VCAM-1 increases the production of this activity by myeloma cells. They also suggest that the presence of stromal cells may provide a microenvironment that allows exclusive colonization of myeloma cells in the bone marrow. (Blood. 2000;96:1953-1960) PMID:10961900

  11. 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. PMID:12062184

  12. 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.

  13. 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

  14. Identification of a putative alpha-glucan synthase essential for cell wall construction and morphogenesis in fission yeast

    PubMed Central

    Hochstenbach, Frans; Klis, Frans M.; van den Ende, Herman; van Donselaar, Elly; Peters, Peter J.; Klausner, Richard D.

    1998-01-01

    The cell wall protects fungi against lysis and determines their cell shape. Alpha-glucan is a major carbohydrate component of the fungal cell wall, but its function is unknown and its synthase has remained elusive. Here, we describe a fission yeast gene, ags1+, which encodes a putative alpha-glucan synthase. In contrast to the structure of other carbohydrate polymer synthases, the predicted Ags1 protein consists of two probable catalytic domains for alpha-glucan assembly, namely an intracellular domain for alpha-glucan synthesis and an extracellular domain speculated to cross-link or remodel alpha-glucan. In addition, the predicted Ags1 protein contains a multipass transmembrane domain that might contribute to transport of alpha-glucan across the membrane. Loss of Ags1p function in a temperature-sensitive mutant results in cell lysis, whereas mutant cells grown at the semipermissive temperature contain decreased levels of cell wall alpha-glucan and fail to maintain rod shapes, causing rounding of the cells. These findings demonstrate that alpha-glucan is essential for fission yeast morphogenesis. PMID:9689051

  15. Direct visualisation of peptide hormones in cultured pancreatic islet alpha- and beta-cells by intact-cell mass spectrometry.

    PubMed

    Buchanan, Christina M; Malik, Arpita S; Cooper, Garth J S

    2007-01-01

    The application of intact-cell mass spectrometry (ICM) by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry to achieve direct protein-profiling of bacterial species is now well established. However, this methodology has not to our knowledge been applied to the analysis of mammalian cells in routine culture. Here, we describe a novel application of ICM by which we have identified proteins in intact cells from two lines representative of pancreatic islet alpha- and beta-cells. Adherent alphaTC1 clone 9 and betaTC6 F7 cells were harvested into phosphate-buffered saline (PBS) using enzyme-free dissociation buffer before 1 microL of cell suspension was spotted onto MALDI plates. Cells were overlaid with sinapinic acid then washed with pure water before application of a final coat of sinapinic acid. Data in the 2000-20,000 m/z range were acquired in linear mode on a Voyager DE-Pro mass spectrometer. The proteins which ionised were composed in large part of peptide hormones (e.g. insulin and glucagon) known to be packaged into the secretory granules of the beta- and alpha-cells respectively. However, in addition to visualising the peptides expected to be associated with these cells, a mass consistent with oxyntomodulin was identified in the cultured alpha-cells, a finding not previously reported to our knowledge. In summary, this paper describes, for the first time, a rapid and direct method useful for identifying secretory products in intact endocrine cells. PMID:17918213

  16. 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.

  17. 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.

  18. Cell, matrix changes and alpha-smooth muscle actin expression in repair of the canine meniscus.

    PubMed

    Kambic, H E; Futani, H; McDevitt, C A

    2000-01-01

    Processes in the repair of a crevice in the knee joint meniscus were investigated in 10 dogs. Two 2-mm cylindrical plugs from each medial meniscus were removed, rendered acellular by freezing and thawing, and then reinserted into the meniscus. Dogs were euthanized at intervals of 3-52 weeks after surgery. The crevice between the plug and meniscus at 3 weeks after surgery was filled with a tissue containing alpha-smooth muscle actin-positive cells. One year after surgery, the plug had remodeled and was populated with spindle-shaped and fibrochondrocyte-like cells. The plug had an appearance intermediate between that of hyaline and fibrocartilage at this time, with a seamless integration in sites between the remodeled plug and the surrounding meniscus. alpha-smooth muscle actin-positive cells were concentrated at the interface of the remodeled plug and adjacent meniscus and at the surface of the plug. Therefore, remodeling of both the plug and meniscal tissue and the participation of alpha-smooth muscle actin-positive cells appear essential for integration of the plug into the adjacent meniscal tissue. Cells in the superficial zone of the meniscus seem to be active in the repair process. A change in both the phenotype of the cells and the quality of the matrix toward a more hyaline state appears to be an integral part of the remodeling process in the meniscus. PMID:11208183

  19. 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.

  20. 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.

  1. TNF-Alpha Represses Transcription of Human Bone Morphogenetic Protein-4 in Lung Epithelial Cells

    PubMed Central

    Zhu, Nian-Ling; Li, Changgong; Huang, Hao Hao; Sebald, Matthew; Londhe, Vedang A.; Heisterkamp, Nora; Warburton, David; Bellusci, Saverio; Minoo, Parviz

    2007-01-01

    Bone Morphogenetic Proteins are key signaling molecules in vertebrate development. Little is known about Bmp gene regulation in any organ. In Drosophila, the Bmp gene, dpp is regulated by Dorsal, the invertebrate homologue of Rel-NFkB. In this study we examined whether TNF-alpha, which activates NF-kB, can regulate Bmp4 gene expression. TNF-alpha reduced Bmp4 mRNA in lung adenocarcinoma A549 cells and repressed transcriptional activity of the human Bmp4 promoter in a dose-dependent manner. Similar repression was observed when the Bmp4 promoter was co-transfected with a p65 (RelA) expression vector in the absence of TNF-alpha treatment, suggesting that RelA mediates the effect of TNF-alpha. In support of this finding, the repressor effect of TNF-alpha on Bmp4 was abrogated by a co-transfected dominant negative mutant of IkB (S32A/S36A). The human Bmp4 promoter contains 3 putative consensus binding sites for NF-kB. Surprisingly, only one of the latter binding sites was capable of binding NFkB. Repressor effect of NFkB was not dependent on any of the three binding sites, but localized to a 122 bp fragment which bound both RelA and SP1. SP1stimulated transcription, whereas increasing doses of RelA opposed this effect. In vivo, TNF-alpha inhibited branching morphogenesis and LacZ gene expression in Bmp4-lacz transgenic lungs. These data support a model in which TNF-alpha-induced RelA interacts with SP1 to bring about transcriptional repression of Bmp4 gene. These findings provide a mechanistic paradigm for interactions between mediators of inflammation and morphogenesis with relevant implications for normal lung development and pathogenesis of disease. PMID:17350185

  2. [Treatment of advanced renal cell carcinoma with a combination of interferon alpha and gamma].

    PubMed

    Naito, S; Yasumasu, T; Kumazawa, J; Hiratsuka, Y; Sakamoto, K; Iguchi, A; Masaki, Z; Hasui, Y; Osada, Y; Kurozumi, T

    1995-08-01

    A total of 29 patients with advanced renal cell carcinoma entered a pilot study of combination therapy with interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma). IFN-alpha (HLBI: 3 x 10(6) IU, BALL 1:5 x 10(6) IU, IFN-alpha-2a: 9 x 10(6) IU or IFN-alpha-2b: 6 x 10(6) IU) was given intramuscularly every day and IFN-gamma (IFN-gamma-1a: 3 x 10(6) JRU) was given intravenously by drip infusion 3 times a week (every 2-3 days). The treatment was continued for 3 months as the induction therapy, and then the tumor response was evaluated. Of the 22 evaluable patients, 4 achieved a partial response (PR), 10 showed no change (NC), and in 8 the disease had progressed (PD) during the therapy. Thus, the overall response rate was 18.2% [95% confidence interval (CI) 2.1-34.3%]. A favorable response tended to be obtained in patients with good performance status or small pulmonary metastases, or in those who had no prior therapy with IFN-alpha, who received this treatment immediately subsequent to radical nephrectomy, or who received IFN-gamma as much as possible according to this regimen. Toxicity was evaluated in 28 patients: fever, general fatigue, anorexia, leukocytopenia and impaired liver function were frequently noted, and 3 patients were withdrawn from the study because of such adverse effects. In patients who had a PR or NC, the same dosage of IFN-alpha was continued to be given intramuscularly 2-3 times a week (every 2-4 days) as the maintenance therapy.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7474618

  3. Cloning and sequencing of the gene for alpha antigen from Mycobacterium avium and mapping of B-cell epitopes.

    PubMed Central

    Ohara, N; Matsuo, K; Yamaguchi, R; Yamazaki, A; Tasaka, H; Yamada, T

    1993-01-01

    The complete nucleotide sequence of alpha antigen secreted from Mycobacterium avium (A-alpha) was determined. The gene encodes 330 amino acids, including 40 amino acids for the signal peptide, followed by 290 amino acids for the mature protein with a molecular mass of 30,811 Da. This is the first sequence of A-alpha. Comparisons between A-alpha and alpha antigens of Mycobacterium leprae, Mycobacterium bovis BCG, and Mycobacterium kansasii showed highly homologous regions which suggested a conserved functional domain and two less-homologous regions. Serological analysis of recombinant A-alpha, expressed by a series of deletion constructs, indicated the possibility that A-alpha carries at least six B-cell epitopes. The three antigenic determinants were common to Mycobacterium tuberculosis, M. kansasii, and M. avium. The results also suggested the possibility that there are three species-specific epitopes. Images PMID:7681039

  4. A selective alpha1D-adrenoreceptor antagonist inhibits human prostate cancer cell proliferation and motility "in vitro".

    PubMed

    Colciago, Alessandra; Mornati, Ornella; Ferri, Nicola; Castelnovo, Luca Franco; Fumagalli, Laura; Bolchi, Cristiano; Pallavicini, Marco; Valoti, Ermanno; Negri-Cesi, Paola

    2016-01-01

    The progression of prostate cancer (PC) to a metastatic hormone refractory disease is the major contributor to the overall cancer mortality in men, mainly because the conventional therapies are generally ineffective at this stage. Thus, other therapeutic options are needed as alternatives or in addition to the classic approaches to prevent or delay tumor progression. Catecholamines participate to the control of prostate cell functions by the activation of alpha1-adrenoreceptors (alpha1-AR) and increased sympathetic activity has been linked to PC development and evolution. Molecular and pharmacological studies identified three alpha1-AR subtypes (A, B and D), which differ in tissue distribution, cell signaling, pharmacology and physiological role. Within the prostate, alpha1A-ARs mainly control stromal cell functions, while alpha1B- and alpha1D- subtypes seem to modulate glandular epithelial cell growth. The possible direct contribution of alpha1D-ARs in tumor biology is supported by their overexpression in PC. The studies here presented investigate the "in vitro" antitumor action of A175, a selective alpha1D-AR antagonist we have recently obtained by modifying the potent, but not subtype-selective alpha1-AR antagonist (S)-WB4101, in the hormone-refractory PC3 and DU145 PC cell lines. The results indicate that A175 has an alpha1D-AR-mediated significant and dose-dependent antiproliferative action that possibly involves the induction of G0/G1 cell cycle arrest, but not apoptosis. In addition, A175 reduces cell migration and adhesiveness to culture plates. In conclusion, our work clarified some cellular aspects promoted by alpha1D-AR activity modulation and supports a further pharmacological approach in the cure of hormone-refractory PC, by targeting specifically this AR subtype. PMID:26621245

  5. 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.

  6. 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.

  7. 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.

  8. The alpha 2-adrenoceptors of the human retinoblastoma cell line (Y79) may represent an additional example of the alpha 2C-adrenoceptor.

    PubMed Central

    Gleason, M. M.; Hieble, J. P.

    1992-01-01

    1. In agreement with the literature, correlation of the ability of a series of agonists and antagonists to displace [3H]-rauwolscine binding shows the alpha 2-adrenoceptors of HT29 cells, NG108-15 cells, OK cells and homogenates of rat sublingual gland to represent four distinct subtypes. 2. [3H]-rauwolscine also bound with high affinity (KD = 0.30 +/- 0.10 mM) to a human retinoblastoma cell line (Y79). Specific binding represents 73% of total binding, and a Bmax of 38 +/- 1 fmol mg-1 protein was determined. 3. Correlation of antagonist affinities against [3H]-rauwolscine with corresponding values in the other four tissue sources showed the Y79 cells to resemble most closely the OK cells, the prototype example of an alpha 2C-adrenoceptor, with a correlation coefficient of 0.90 and a regression slope of 1.01 being obtained for 10 antagonists in these two systems. 4. Comparison of KD values for [3H]-rauwolscine also showed a similarity between the OK cells (0.19 +/- 0.07 nM) and Y79 cells. 5. These data suggest that the human retinoblastoma cell line may represent an additional example of the alpha 2C-adrenoceptor subtype. PMID:1358385

  9. TC3: Convective and radiative heating of MSRO for simplest kinetic models

    NASA Astrophysics Data System (ADS)

    Surzhikov, Sergey T.

    2005-05-01

    Numerical simulation results for radiative heating of whole surface of space vehicle Mars Sampler Return Orbiter (MSRO) are presented and analyzed. Two calculation cases are considered: non-catalytic and pseudo-catalytic surface. The calculation data were obtained by the radiative gas dynamic code RGDSV-2 developed in Institute for Problems in Mechanics Russian Academy of Sciences (IPMech RAS), which is intended for study of aerothermodynamics of space vehicles entering planetary atmospheres and for diagnostics of trajectory parameters by measuring spectrum of heat radiation from high-temperature gas around space vehicles. Distinguishing feature of the code is proper prediction of directional spectral emissive power of a space vehicle at its braking trajectory in an atmosphere and also prediction of radiative heating of whole surface of space vehicles. Simplest kinetic models were used for obtaining calculation data presented in the paper in accordance with the Test Case 3 (TC-3) problem statement [1]. Vibrational excitations of diatomic and three atomic molecules were neglected.

  10. 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.

  11. 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.

  12. Activation of two new alpha(1,3)fucosyltransferase activities in Chinese hamster ovary cells by 5-azacytidine.

    PubMed Central

    Potvin, B; Stanley, P

    1991-01-01

    Several mammalian alpha(1,3)fucosyltransferases (alpha[1,3]Fuc-T) that synthesize carbohydrates containing alpha(1,3)fucosylated lactosamine units have been identified. Although Chinese hamster ovary (CHO) cells do not express alpha(1,3)Fuc-T activity, the rare mutants LEC11 and LEC12, isolated after mutagenesis or DNA transfection, each express an alpha(1,3)Fuc-T that may be distinguished by several criteria. Two new CHO mutants possessing alpha(1,3)Fuc-T activity (LEC29 and LEC30) have now been isolated after treatment of a CHO cell population with 5-azacytidine (5-AzaC), ethylnitrosourea (ENU), or 5-AzaC followed by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Like LEC12, both mutants possess an N-ethylmaleimide-resistant alpha(1,3)Fuc-T activity that can utilize a variety of acceptors and both express the Lewis X (Lex) determinant (Gal beta[1,4](Fuc alpha[1,3])GlcNAc beta 1)) but not the sialyl alpha(2,3)Lex determinant on cell-surface carbohydrates. However, LEC29 and LEC30 may be distinguished from LEC11 and LEC12, as well as from each other, on the basis of their unique patterns of lectin resistance and their abilities to bind the VIM-2 monoclonal antibody that recognizes carbohydrates terminating in NeuNAc alpha(2,3)Gal beta(1,4)GlcNAc beta(1,3)Gal beta(1,4)(Fuc alpha[1,3])GlcNAc beta and also by the different in vitro substrate specificities and kinetic properties of their respective alpha(1,3)Fuc-T activities. The combined data provide good evidence that the LEC29 and LEC30 alpha(1,3)Fuc-Ts are novel transferases encoded by distinct gene products. PMID:1724918

  13. 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.

  14. 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.

  15. 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.

  16. 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

    2016-01-01

    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 . PMID:27460926

  17. 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.

  18. Filamentous nerve cell inclusions in neurodegenerative diseases: tauopathies and alpha-synucleinopathies.

    PubMed Central

    Goedert, M

    1999-01-01

    Alzheimer's disease and Parkinson's disease are the most common neurodegenerative diseases. They are characterized by the degeneration of selected populations of nerve cells that develop filamentous inclusions before degeneration. The neuronal inclusions of Alzheimer's disease are made of the microtubule-associated protein tau, in a hyperphosphorylated state. Recent work has shown that the filamentous inclusions of Parkinson's disease are made of the protein alpha-synuclein and that rare, familial forms of Parkinson's disease are caused by missense mutations in the alpha-synuclein gene. Besides Parkinson's disease, the filamentous inclusions of two additional neurodegenerative diseases, namely dementia with Lewy bodies and multiple system atrophy, have also been found to be made of alpha-synuclein. Abundant filamentous tau inclusions are not limited to Alzheimer's disease. They are the defining neuropathological characteristic of frontotemporal dementias such as Pick's disease, and of progressive supranuclear palsy and corticobasal degeneration. The recent discovery of mutations in the tau gene in familial forms of frontotemporal dementia has provided a direct link between tau dysfunction and dementing disease. The new work has established that tauopathies and alpha-synucleinopathies account for most late-onset neurodegenerative diseases in man. The formation of intracellular filamentous inclusions might be the gain of toxic function that leads to the demise of affected brain cells. PMID:10434313

  19. 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.

  20. dl-. cap alpha. -tocopheryl succinate enhances the effect of. gamma. -irradiation on neuroblastoma cells in culture

    SciTech Connect

    Sarri, A.; Prasad, K.N.

    1984-01-01

    The effect of dl-..cap alpha..-tocopheryl (vitamin E) succinate in modifying the radiation response of mouse neuroblastoma (NBP/sub 2/) and mouse fibroblast (L-cells) cells in culture was studied on the criterion of growth inhibition (due to cell death and inhibition of cell division). Results show that vitamin E succinate markedly enhanced the effect of /sub 60/CO-..gamma..-irradiation on NB cells, but it did not significantly modify the effect of irradiation on mouse fibroblasts. Sodium succinate plus ethanol (0.25% final concentration) did not modify the radiation response of NB cells or fibroblasts. Butylated hydroxyanisole, a lipid soluble antioxidant, also enhanced the effect of irradiation on NB cells, indicating that the effect of vitamin E in modifying the radiation response may be mediated, in part, by antioxidation mechanisms.

  1. Cyclic mechanical stretching and interleukin-1alpha synergistically up-regulate prostacyclin secretion in cultured human uterine myometrial cells.

    PubMed

    Korita, D; Itoh, H; Sagawa, N; Yura, S; Yoshida, M; Kakui, K; Takemura, M; Nuamah, M A; Fujii, S

    2004-03-01

    Prostacyclin (PGI2), a potent uterine smooth muscle relaxant, is postulated to be a major prostaglandin (PG) secreted from the human myometrium. PGI2 metabolite concentrations in the maternal plasma were reported to be elevated during pregnancy, especially during labor. Recently, we developed cultured human myometrial cells from pregnant women and reported that cyclic mechanical stretching mimicking labor increased PGI2 secretion from these cells by up-regulating PGI2 synthase promoter activities. Since elevation of cervical/vaginal interleukin-1alpha (IL-1alpha) concentrations is also a characteristic feature of delivery, and IL-1alpha is a known stimulator of PG synthesis, we investigated a possible synergistic effect of cyclic mechanical stretching and IL-1alpha on PGI2 production in cultured human myometrial cells. Treatment with IL-1alpha (10 ng/ml) significantly augmented (4- to 60-fold) the secretion of PGI2, prostaglandin E2 (PGE2), prostaglandin F2alpha (PGF2alpha) and thromboxane A2 (TXA2) from cultured human myometrial cells obtained from non-pregnant and pregnant women as well as in cultured human umbilical artery and cultured human coronary artery smooth muscle cells (p < 0.05 for all comparisons). However, labor-like cyclic mechanical stretching up-regulated IL-1alpha-augmented PGI2 secretion from myometrial cells obtained from non-pregnant and pregnant women 2.1- to 2.8-fold (p < 0.05 for all comparisons), but not PGE2, PGF2alpha nor TXA2. Moreover, such an augumentation of PGI2 secretion by cyclic mechanical stretching was not observed in cultured human umbilical artery nor in cultured human coronary artery smooth muscle cells. These results suggest that cyclic mechanical stretching by labor, in concert with IL-1alpha stimulation, contributes to the increase in myometrial PGI2 secretion during delivery. PMID:15255281

  2. Negative feedback between prostaglandin and alpha- and beta-chemokine synthesis in human microglial cells and astrocytes.

    PubMed

    Janabi, N; Hau, I; Tardieu, M

    1999-02-01

    The understanding of immune surveillance and inflammation regulation in cerebral tissue is essential in the therapy of neuroimmunological disorders. We demonstrate here that primary human glial cells were able to produce alpha- and beta-chemokines (IL-8 > growth related protein alpha (GROalpha) > RANTES > microphage inflammatory protein (MIP)-1alpha and MIP-1beta) in parallel to PGs (PGE2 and PGF2alpha) after proinflammatory cytokine stimulation: TNF-alpha + IL-1beta induced all except RANTES, which was induced by TNF-alpha + IFN-gamma. Purified cultures of astrocytes and microglia were also induced by the same combination of cytokines, to produce all these mediators except MIP-1alpha and MIP-1beta, which were produced predominantly by astrocytes. The inhibition of PG production by indomethacin led to a 37-60% increase in RANTES, MIP-1alpha, and MIP-1beta but not in GROalpha and IL-8 secretion. In contrast, inhibition of IL-8 and GRO activities using neutralizing Abs resulted in a specific 6-fold increase in PGE2 but not in PGF2alpha production by stimulated microglial cells and astrocytes, whereas Abs to beta-chemokines had no effect. Thus, the production of PGs in human glial cells down-regulates their beta-chemokine secretion, whereas alpha-chemokine production in these cells controls PG secretion level. These data suggest that under inflammatory conditions, the intraparenchymal production of PGs could control chemotactic gradient of beta-chemokines for an appropriate effector cell recruitment or activation. Conversely, the elevated intracerebral alpha-chemokine levels could reduce PG secretion, preventing the exacerbation of inflammation and neurotoxicity. PMID:9973432

  3. 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.

  4. The effects of 1alpha,24(S)-dihydroxyvitamin D(2) analog on cancer cell proliferation and cytokine expression.

    PubMed

    Shany, S; Levy, Y; Lahav-Cohen, M

    2001-01-01

    It is well established that 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), the active metabolite of vitamin D, plays a role in regulating proliferation and differentiation of cells, in addition to its classic function in mineral homeostasis. Recent studies have also provided evidence for the involvement of 1alpha,25(OH)(2)D(3) in regulating the immune system. However, therapeutic application of 1alpha,25(OH)(2)D(3) to hyperproliferative diseases such as cancer, or for immunologic purposes, is thwarted by its hypercalcemic activity. In order to overcome this obstacle, analogs of 1alpha,25(OH)(2)D(3) have been produced that exhibit decreased hypercalcemic activity while retaining the growth and immunologic regulating properties. In the present study, the efficacy of 1alpha,24(S)-dihydroxyvitamin D(2) (1alpha,24(S)(OH)(2)D(2)), a vitamin D(2) analog, in restraining cell proliferation was compared to that of 1alpha,25(OH)(2)D(3). In parallel studies, cancer cell lines were grown in increased concentrations (10(-10)-10(-7) M) of each compound for various incubation periods (1-4 days). Growth was assessed by measuring [(3)H]thymidine incorporation. The results revealed that 1alpha,24(S)(OH)(2)D(2) significantly inhibits proliferation to an extent similar to that observed for 1alpha,25(OH)(2)D(3). Moreover, incubating the human leukemia cell line, HL-60, with 1alpha,24(S)(OH)(2)D(2) resulted in an induction of differentiation of these promyelomonocyte cells into monocyte-macrophage-like cells, in a manner similar to that observed with 1alpha,25(OH)(2)D(3). Using a Western procedure, it was also shown that 1alpha,24(S)(OH)(2)D(2) like 1alpha,25(OH)(2)D(3) enhances the expression of vitamin D receptors (VDR) in the rat osteosarcoma cell line, ROS 17/2.8. The expression of tumor necrosis factor (TNF) alpha (TNF-alpha) in human peritoneal macrophages (HPM) obtained from uremic patients treated with continuous ambulatory peritoneal dialysis (CAPD) was found to be

  5. Establishment of type II 5alpha-reductase over-expressing cell line as an inhibitor screening model.

    PubMed

    Jang, Sunhyae; Lee, Young; Hwang, Seong-Lok; Lee, Min-Ho; Park, Su Jin; Lee, In Ho; Kang, Sangjin; Roh, Seok-Seon; Seo, Young-Joon; Park, Jang-Kyu; Lee, Jeung-Hoon; Kim, Chang Deok

    2007-01-01

    Dihydrotestosterone (DHT) is the most potent male hormone that causes androgenetic alopecia. The type II 5alpha-reductase is an enzyme that catalyzes the conversion of testosterone (T) to DHT, therefore it can be expected that specific inhibitors for type II 5alpha-reductase may improve the pathophysiologic status of androgenetic alopecia. In this study, we attempted to establish the reliable and convenient screening model for type II 5alpha-reductase inhibitors. After transfection of human cDNA for type II 5alpha-reductase into HEK293 cells, the type II 5alpha-reductase over-expressing stable cells were selected by G418 treatment. RT-PCR and Western blot analyses confirmed that type II 5alpha-reductase gene was expressed in the stable cells. In in vitro enzymatic assay, 10 microg of stable cell extract completely converted 1 microCi (approximately 0.015 nmol) of T into DHT. The type II 5alpha-reductase activity was inhibited by finasteride in a dose-dependent manner, confirming the reliability of screening system. In cell culture condition, 2 x 10(5) of stable cells completely converted all the input T (approximately 0.03 nmol) into DHT by 4h incubation, demonstrating that the stable cell line can be used as a cell-based assay system. Using this system, we selected the extracts of Curcumae longae rhizoma and Mori ramulus as the potential inhibitors for type II 5alpha-reductase. These results demonstrate that the type II 5alpha-reductase over-expressing stable cell line is a convenient and reliable model for screening and evaluation of inhibitors. PMID:17646096

  6. Quinazoline-based alpha 1-adrenoceptor antagonists induce prostate cancer cell apoptosis via TGF-beta signalling and I kappa B alpha induction.

    PubMed

    Partin, J V; Anglin, I E; Kyprianou, N

    2003-05-19

    Previous studies documented the ability of quinazoline-based alpha1-adrenoceptor antagonists to induce apoptosis in prostate cancer cells via an alpha 1-adrenoceptor-independent mechanism. In this study we investigated the molecular events initiating this apoptotic effect. Since transforming growth factor-beta 1 (TGF-beta 1) mediates prostate epithelial cell apoptosis, we hypothesised that the activation of the TGF-beta 1 pathway underlies the quinazoline-based apoptotic effect in prostate cancer cells. Treatment of the androgen-independent human prostate cancer cells PC-3 with doxazosin resulted in a strong caspase-3 activation within 24 h, whereas tamsulosin, a sulphonamide-based alpha 1-adrenoceptor antagonist, had no significant apoptotic effect against prostate cancer cells. To identify the molecular components involved in this quinazoline-mediated apoptosis, cDNA microarray analysis of PC-3 prostate cancer cells treated with doxazosin (3 h) was performed. Induced expression of several genes was observed including p21(WAF-1) and I kappa B alpha (inhibitor of NF-kappa B alpha). Relative quantitative reverse transcription-polymerase chain reaction analysis revealed induction of several TGF-beta1 signalling effectors: Induction of mRNA for Smad4 and the TGF-beta1-regulated apoptosis-inducing transcription factor TGF-beta1-inducible early gene (TIEG1) was detected within the first 6 h of doxazosin treatment. Upregulation of I kappa B alpha at both the mRNA and protein level was also detected after 6 h of treatment. Furthermore, doxazosin resulted in a considerable elevation in Smad4 and TIEG protein expression (6 h). A 'latent' increase in TGF-beta mRNA expression was detected after 48 h of treatment. These findings suggest that the quinazoline-based doxazosin mediates prostate cancer apoptosis by initially inducing the expression of TGF-beta1 signalling effectors and subsequently I kappa B alpha. The present study provides an initial insight into the molecular

  7. 5-Azacytidine treatment of HA-A melanoma cells induces Sp1 activity and concomitant transforming growth factor alpha expression.

    PubMed Central

    Shin, T H; Paterson, A J; Grant, J H; Meluch, A A; Kudlow, J E

    1992-01-01

    Evidence indicates DNA methylation as a part of the regulatory machinery controlling mammalian gene expression. The human melanoma cell line HA-A expresses low levels of transforming growth factor alpha (TGF-alpha). TGF-alpha mRNA accumulated, however, in response to DNA demethylation induced by a nucleoside analog, 5-azacytidine (5-azaC). The importance of DNA methylation in the TGF-alpha promoter region was examined by a transient transfection assay with luciferase reporter plasmids containing a portion of the TGF-alpha promoter. 5-azaC treatment of HA-A cells before the transfection caused a significant increase in the luciferase activity. Since input plasmids were confirmed to remain unmethylated, DNA demethylation of the TGF-alpha promoter itself does not account for the observed increase in TGF-alpha mRNA. Using an electrophoretic mobility shift assay, enhanced formation of protein-TGF-alpha promoter complex was detected in response to 5-azaC treatment. This 5-azaC-induced complex was shown to contain the transcription factor Sp1 by the following criteria: the protein-DNA complex formed on the TGF-alpha promoter contained immunoreactive Sp1; the mobility of the complex in an electrophoretic mobility shift assay was similar to that formed by recombinant Sp1; and DNase I footprinting analysis demonstrated that the 5-azaC-induced complex produced a footprint on the TGF-alpha promoter identical to that of authentic Sp1. These observations suggest that 5-azaC induces TGF-alpha expression by augmenting the Sp1 activity. However, neither the Sp1 mRNA nor its protein was induced by 5-azaC. These results suggest that in HA-A cells, TGF-alpha expression is down-modulated by DNA methylation. In addition, this process may involve the specific regulation of Sp1 activity without altering the amount of the transcription factor. Images PMID:1380648

  8. CD20-targeted tetrameric interferon-alpha, a novel and potent immunocytokine for the therapy of B-cell lymphomas.

    PubMed

    Rossi, Edmund A; Goldenberg, David M; Cardillo, Thomas M; Stein, Rhona; Chang, Chien-Hsing

    2009-10-29

    Interferon-alpha (IFN-alpha) has direct inhibitory effects on some tumors and is a potent stimulator of both the innate and adaptive immune systems. A tumor-targeting antibody-IFN-alpha conjugate (mAb-IFN-alpha) could kill by direct actions of the monoclonal antibody (mAb) and IFN-alpha on tumor cells and also potentiate a tumor-directed immune response. The modular Dock-and-Lock method (DNL) was used to generate 20-2b, the first immunocytokine having 4 cytokine (IFN-alpha2b) groups that are fused to the humanized anti-CD20 mAb, veltuzumab. Additional mAb-IFN-alpha constructs, each retaining potent IFN-alpha2b biologic activity, also were produced by DNL. The 20-2b shows enhanced antibody-dependent cellular cytotoxicity compared with veltuzumab but lacks complement-dependent cytotoxicity. The 20-2b inhibits in vitro proliferation of lymphoma cells and depletes them from whole human blood more potently than the combination of veltuzumab and a nontargeting, irrelevant, mAb-IFN-alpha. The 20-2b demonstrated superior therapeutic efficacy compared with veltuzumab or nontargeting mAb-IFN-alpha in 3 human lymphoma xenograft models, even though mouse immune cells respond poorly to human IFN-alpha2b. Targeting IFN-alpha with an anti-CD20 mAb makes the immunocytokine more potent than either agent alone. These findings suggest that 20-2b merits clinical evaluation as a new candidate antilymphoma therapeutic. PMID:19710501

  9. Effect of alpha Ni3S2 on arachidonic acid metabolites in cultured human lung cells (L132 cell line).

    PubMed

    Shirali, P; Teissier, E; Marez, T; Hildebrand, H F; Haguenoer, J M

    1994-04-01

    Our previous investigations have shown evidence of an interaction between alpha Ni3S2 and membranous and cellular lipids of lung cells with a significant increase in the linoleic, linolenic and arachidonic acid pool. The present work is designed to follow the metabolic fate of arachidonic acid in alpha Ni3S2-exposed human embryonic pulmonary epithelial cells (L132) in culture (50 microM alpha Ni3S2 for 3 days). The metabolites of arachidonic acid were assessed by HPLC determination coupled with UV or electrochemical detection. We determined malondialdehyde (MDA), hydroxyeicosatetraenoic acid (HETE), leukotrienes (LT) and reduced glutathione (GSH). In exposed cells we observed a significant increase of MDA, which is a breakdown product of lipid peroxidation. In addition, we noted significant increases of 5-HETE and 15-HETE in L132 cells resulting from the enzymatic reduction of 5-HPETE and 15-HPETE respectively. There was also a simultaneous decrease of GSH--confirmed by a strong decrease of GSH in exposed cells with respect to controls. 5-HPETE is furthermore converted to epoxides such as leukotriene A4 and we also quantified in exposed cells a significant increase of its subsequent catabolites LTB4, LTC4 and LTE4. These investigations show clearly that exposure of L132 cells to alpha Ni3S2 enhances lipid peroxidation based upon direct measurements of MDA and other metabolites of arachidonic acid. This lipid peroxidation is an autocatalytic free-radical process and could be responsible for DNA damage. PMID:8149492

  10. Endogenous interleukin 1 alpha must be transported to the nucleus to exert its activity in human endothelial cells.

    PubMed Central

    Maier, J A; Statuto, M; Ragnotti, G

    1994-01-01

    We have previously shown that the signal peptideless cytokine interleukin 1 alpha (IL-1 alpha) may play a role as an intracellular regulator of human endothelial cell senescence (J. A. M. Maier, P. Voulalas, D. Roeder, and T. Maciag, Science 249:1570-1574, 1990). To investigate the potential intracellular function of IL-1 alpha, transformed endothelial cells were transfected with the human cDNAs that code for the two forms of IL-1 alpha, the precursor molecule IL-1(1-271) and the mature protein IL-1(113-271). The subcellular localization of the two different polypeptides was investigated directly or by using chimeric genes constructed by fusion of different fragments of the IL-1 alpha gene and the beta-galactosidase open reading frames. The IL-1(113-271) protein was cytoplasmic, while IL-1(1-271) was nuclear. The basic cluster at the NH2 terminus of IL-1, KVLKKRR, has been shown to mediate IL-1 alpha nuclear targeting. Moreover, nuclear localization of IL-1 alpha correlates with impaired cell growth and expression of some IL-1 alpha-inducible genes. These results suggest that transport of endogenous IL-1(1-271) into the nucleus is required for it to modulate endothelial cell function. Images PMID:8114717

  11. 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.

  12. 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. PMID:8629860

  13. Tumour necrosis factor-alpha up-regulates decay-accelerating factor gene expression in human intestinal epithelial cells.

    PubMed Central

    Andoh, A; Fujiyama, Y; Sumiyoshi, K; Sakumoto, H; Okabe, H; Bamba, T

    1997-01-01

    The increased expression of decay-accelerating factor (DAF) has been detected in intestinal epithelial cells at the inflamed mucosa. In this study, we examined the effects of tumour necrosis factor (TNF)-alpha on DAF expression in three intestinal epithelial cell lines. DAF mRNA expression was evaluated by Northern blot analysis, and DAF protein expression was analysed by biotin labelling and immunoprecipitation. TNF-alpha induced a marked increase in DAF mRNA and protein expression in HT-29, T84 and Caco-2 cells. In HT-29 cells, the effects of TNF-a on DAF mRNA accumulation were observed in a dose-dependent manner; DAF mRNA accumulation reached a maximum at 3-6 hr, and then gradually decreased. These effects of TNF-alpha required de novo protein synthesis. Messenger RNA stability studies suggested that TNF-alpha partially regulated DAF gene expression by a posttranscriptional mechanism. Moreover, the combination of TNF-alpha and interleukin (IL)-4 induced an additive increase in DAF mRNA accumulation in HT-29 and T84 cells. In human intestinal epithelial cells, TNF-alpha acts as a potent inducer of DAF mRNA expression, indicating an important role for TNF-alpha in the regulation of DAF expression at the inflamed mucosa. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 6 Figure 7 Figure 8 PMID:9155641

  14. A pathway for cell wall anchorage of Saccharomyces cerevisiae alpha-agglutinin.

    PubMed Central

    Lu, C F; Kurjan, J; Lipke, P N

    1994-01-01

    Saccharomyces cerevisiae alpha-agglutinin is a cell wall-anchored adhesion glycoprotein. The previously identified 140-kDa form, which contains a glycosyl-phosphatidylinositol (GPI) anchor (D. Wojciechowicz, C.-F. Lu, J. Kurjan, and P. N. Lipke, Mol. Cell. Biol. 13:2554-2563, 1993), and additional forms of 80, 150, 250 to 300, and > 300 kDa had the properties of intermediates in a transport and cell wall anchorage pathway. N glycosylation and additional modifications resulted in successive increases in size during transport. The 150- and 250- to 300-kDa forms were membrane associated and are likely to be intermediates between the 140-kDa form and a cell surface GPI-anchored form of > 300 kDa. A soluble form of > 300 kDa that lacked the GPI anchor had properties of a periplasmic intermediate between the plasma membrane form and the > 300-kDa cell wall-anchored form. These results constitute experimental support for the hypothesis that GPI anchors act to localize alpha-agglutinin to the plasma membrane and that cell wall anchorage involves release from the GPI anchor to produce a periplasmic intermediate followed by linkage to the cell wall. Images PMID:8007981

  15. HIF-1alpha Deficiency Attenuates the Cardiomyogenesis of Mouse Embryonic Stem Cells

    PubMed Central

    Kudová, Jana; Procházková, Jiřina; Vašiček, Ondřej; Perečko, Tomáš; Sedláčková, Miroslava; Pešl, Martin; Pacherník, Jiří

    2016-01-01

    Cardiac cell formation, cardiomyogenesis, is critically dependent on oxygen availability. It is known that hypoxia, a reduced oxygen level, modulates the in vitro differentiation of pluripotent cells into cardiomyocytes via hypoxia inducible factor-1alpha (HIF-1α)-dependent mechanisms. However, the direct impact of HIF-1α deficiency on the formation and maturation of cardiac-like cells derived from mouse embryonic stem cells (mESC) in vitro remains to be elucidated. In the present study, we demonstrated that HIF-1α deficiency significantly altered the quality and quantity of mESC-derived cardiomyocytes. It was accompanied with lower mRNA and protein levels of cardiac cell specific markers (myosin heavy chains 6 and 7) and with a decreasing percentage of myosin heavy chain α and β, and cardiac troponin T-positive cells. As to structural aspects of the differentiated cardiomyocytes, the localization of contractile proteins (cardiac troponin T, myosin heavy chain α and β) and the organization of myofibrils were also different. Simultaneously, HIF-1α deficiency was associated with a lower percentage of beating embryoid bodies. Interestingly, an observed alteration in the in vitro differentiation scheme of HIF-1α deficient cells was accompanied with significantly lower expression of the endodermal marker (hepatic nuclear factor 4 alpha). These findings thus suggest that HIF-1α deficiency attenuates spontaneous cardiomyogenesis through the negative regulation of endoderm development in mESC differentiating in vitro. PMID:27355368

  16. 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.

  17. 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

  18. Mannosidase 2, alpha 1 deficiency is associated with ricin resistance in embryonic stem (ES) cells.

    PubMed

    Wang, Wei; Hale, Christine; Goulding, Dave; Haslam, Stuart M; Tissot, Bérangère; Lindsay, Christopher; Michell, Stephen; Titball, Rick; Yu, Jun; Toribio, Ana Luisa; Rossi, Raffaella; Dell, Anne; Bradley, Allan; Dougan, Gordon

    2011-01-01

    Host gene products required for mediating the action of toxins are potential targets for reversing or controlling their pathogenic impact following exposure. To identify such targets libraries of insertional gene-trap mutations generated with a PiggyBac transposon in Blm-deficient embryonic stem cells were exposed to the plant toxin, ricin. Resistant clones were isolated and genetically characterised and one was found to be a homozygous mutant of the mannosidase 2, alpha 1 (Man2α1) locus with a matching defect in the homologous allele. The causality of the molecular lesion was confirmed by removal of the transposon following expression of PB-transposase. Comparative glycomic and lectin binding analysis of the Man2α1 (-/-) ricin resistant cells revealed an increase in the levels of hybrid glycan structures and a reduction in terminal β-galactose moieties, potential target receptors for ricin. Furthermore, naïve ES cells treated with inhibitors of the N-linked glycosylation pathway at the mannosidase 2, alpha 1 step exhibited either full or partial resistance to ricin. Therefore, we conclusively identified mannosidase 2, alpha 1 deficiency to be associated with ricin resistance. PMID:21886775

  19. 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

  20. 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

  1. HIF-1alpha Expression Profile in Intratumoral and Peritumoral Inflammatory Cells as a Prognostic Marker for Squamous Cell Carcinoma of the Oral Cavity

    PubMed Central

    Mendes, Suzanny Oliveira; dos Santos, Marcelo; Peterle, Gabriela Tonini; Maia, Lucas de Lima; Stur, Elaine; Agostini, Lidiane Pignaton; de Carvalho, Marcos Brasilino; Tajara, Eloiza Helena; Louro, Iúri Drumond; Trivilin, Leonardo Oliveira; da Silva-Conforti, Adriana Madeira Álvares

    2014-01-01

    The HIF-1 transcriptional complex is responsible for controlling transcription of over 100 genes involved in cell hypoxia response. HIF-1alpha subunit is stabilized in hypoxia conditions, creating the HIF-1 nuclear transcription factor. In inflammatory cells, high HIF-1alpha expression induces lymphocytic immunosuppression, decreasing tumoral antigen recognition, which promotes tumor growth. The present work investigated the relationship between HIF-1alpha expression in lymphocytes populating the intratumoral and peritumoral region of 56 patients with oral cancer. Our data indicates a prognostic value for this expression. High HIF-1alpha expression in peritumoral inflammatory cells is significantly related to worse patient outcome, whereas high expression in the intratumoral lymphoid cells correlates with a better prognosis. A risk profile indicating the chance of disease relapse and death was designed based on HIF-1alpha expression in tumoral inflammatory cells, defining low, intermediate and high risks. This risk profile was able to determine that high HIF-1alpha expression in peritumoral cells correlates with worse prognosis, independently of intratumoral expression. Low HIF-1alpha in tumor margins and high expression in the tumor was considered a low risk profile, showing no cases of disease relapse and disease related death. Intermediate risk was associated with low expression in tumor and tumor margins. Our results suggest that HIF-1alpha expression in tumor and peritumoral inflammatory cells may play an important role as prognostic tumor marker. PMID:24416312

  2. 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.

  3. Increased tumor necrosis factor alpha (TNF-alpha) and natural killer cell (NK) function using an integrative approach in late stage cancers.

    PubMed

    See, Darryl; Mason, Stephanie; Roshan, Ramesh

    2002-05-01

    Natural products may increase cytotoxic activity of Natural Killer Cells (NK) Tumor Necrosis Factor alpha (TNF-alpha) while decreasing DNA damage in patients with late-stage cancer. Pilot studies have suggested that a combination of Nutraceuticals can raise NK cell function and TNF-alpha alpha activity and result in improved clinical outcomes in patients with late stage cancer. The objective of the study is to determine if Nutraceuticals can significantly raise NK function and TNF levels in patients with late stage cancer. After informed consent was obtained, 20 patients with stage IV, end-stage cancer were evaluated (one bladder, five breast, two prostate, one neuroblastoma, two non-small cell lung, three colon, 1 mesothelioma, two lymphoma, one ovarian, one gastric, one osteosarcoma). Transfer Factor Plus (TFP+, 3 tablets 3 times per day), IMUPlus (non denatured milk whey protein, 40 gm/day); Intravenous (50 to 100 gm/day) and oral (1-2 gm/day) ascorbic acid; Agaricus Blazeii Murill teas (10 gm/day); Immune Modulator Mix (a combination of vitamin, minerals, antioxidants and immune-enhancing natural products); nitrogenated soy extract (high levels of genistein and dadzein) and Andrographis Paniculata (500 mg twice, daily) were used. Baseline NK function by standard 4 h 51Cr release assay and TNF alpha and receptor levels were measured by ELISA from resting and phytohemagglutinin (PHA) stimulated adherent and non-adherent Peripheral Blood Mononuclear Cell (PBMC). Total mercaptans and glutathione in plasma were taken and compared to levels measured 6 months later. Complete blood counts and chemistry panels were routinely monitored. As of a mean of 6 months, 16/20 patients were still alive. The 16 survivors had significantly higher NK function than baseline (p < .01 for each) and TNF-alpha levels in all four cell populations studied (p < .01 for each). Total mercaptans (p < .01) and TNF-alpha receptor levels were significantly reduced (p < .01). It was also observed

  4. Monte-Carlo dosimetry on a realistic cell monolayer geometry exposed to alpha particles

    NASA Astrophysics Data System (ADS)

    Barberet, Ph; Vianna, F.; Karamitros, M.; Brun, T.; Gordillo, N.; Moretto, Ph; Incerti, S.; Seznec, H.

    2012-04-01

    The energy and specific energy absorbed in the main cell compartments (nucleus and cytoplasm) in typical radiobiology experiments are usually estimated by calculations as they are not accessible for a direct measurement. In most of the work, the cell geometry is modelled using the combination of simple mathematical volumes. We propose a method based on high resolution confocal imaging and ion beam analysis (IBA) in order to import realistic cell nuclei geometries in Monte-Carlo simulations and thus take into account the variety of different geometries encountered in a typical cell population. Seventy-six cell nuclei have been imaged using confocal microscopy and their chemical composition has been measured using IBA. A cellular phantom was created from these data using the ImageJ image analysis software and imported in the Geant4 Monte-Carlo simulation toolkit. Total energy and specific energy distributions in the 76 cell nuclei have been calculated for two types of irradiation protocols: a 3 MeV alpha particle microbeam used for targeted irradiation and a 239Pu alpha source used for large angle random irradiation. Qualitative images of the energy deposited along the particle tracks have been produced and show good agreement with images of DNA double strand break signalling proteins obtained experimentally. The methodology presented in this paper provides microdosimetric quantities calculated from realistic cellular volumes. It is based on open-source oriented software that is publicly available.

  5. Monte-Carlo dosimetry on a realistic cell monolayer geometry exposed to alpha particles.

    PubMed

    Barberet, Ph; Vianna, F; Karamitros, M; Brun, T; Gordillo, N; Moretto, Ph; Incerti, S; Seznec, H

    2012-04-21

    The energy and specific energy absorbed in the main cell compartments (nucleus and cytoplasm) in typical radiobiology experiments are usually estimated by calculations as they are not accessible for a direct measurement. In most of the work, the cell geometry is modelled using the combination of simple mathematical volumes. We propose a method based on high resolution confocal imaging and ion beam analysis (IBA) in order to import realistic cell nuclei geometries in Monte-Carlo simulations and thus take into account the variety of different geometries encountered in a typical cell population. Seventy-six cell nuclei have been imaged using confocal microscopy and their chemical composition has been measured using IBA. A cellular phantom was created from these data using the ImageJ image analysis software and imported in the Geant4 Monte-Carlo simulation toolkit. Total energy and specific energy distributions in the 76 cell nuclei have been calculated for two types of irradiation protocols: a 3 MeV alpha particle microbeam used for targeted irradiation and a ²³⁹Pu alpha source used for large angle random irradiation. Qualitative images of the energy deposited along the particle tracks have been produced and show good agreement with images of DNA double strand break signalling proteins obtained experimentally. The methodology presented in this paper provides microdosimetric quantities calculated from realistic cellular volumes. It is based on open-source oriented software that is publicly available. PMID:22456322

  6. Effect of coacervated alpha-elastin on proliferation of vascular smooth muscle and endothelial cells.

    PubMed

    Ito, S; Ishimaru, S; Wilson, S E

    1998-04-01

    The arterial wall injury associated with arterial graft implantation causes smooth muscle cells (SMCs) in the media to migrate and proliferate in the intima at the graft-artery junction resulting in anastomotic intimal hyperplasia (AIH). An important step in developing a small-diameter prosthesis may be to stimulate endothelialization and thereby inhibit AIH. In this study, we investigated the effect of coacervated and crosslinked alpha-elastin on proliferation of SMCs and endothelial cells (ECs) in vitro. Coacervation is an important step in the conversion of proelastin to make an elastin fiber in vivo. SMCs and ECs were prepared from porcine aortic media and endothelium, respectively. SMCs and ECs (three to five passages, 4 x 10[4] cells/well) were seeded onto 12 well plates, coated and crosslinked with 0 or 10 mg/mL of coacervated alpha-elastin. After the 1st, 2nd, or 3rd day of cultivation, proliferation was assayed by scintillation counting of [3H]-thymidine incorporation. For the 4th day only, 0, 0.1, 1, 10 mg/mL concentration of coacervated alpha-elastin was coated and crosslinked. SMC proliferation (1st, 2nd day: p<0.005; 3rd, 4th day: p<0.0001) was significantly inhibited over time and dose dependently, eg, 0.1 mg/mL (45.7+/-2.3%: % of control p<0.005), 1 mg/mL (5.9+/-0.7%, p<0.0005), 10 mg/mL (2.8+/-0.4%, p<0.0005). EC proliferation was inhibited over time by 10 mg/mL of coacervated alpha-elastin (2nd, 3rd day: p<0.005; 4th day: p<0.0001), but proliferation (132.8+/-9.9%: % of control p=NS) was stimulated by 0.1 mg/mL of coacervated alpha-elastin. These results suggest that coating and crosslinking a coacervated alpha-elastin into the structure of arterial prosthesis may inhibit AIH and stimulate endothelialization. PMID:9555932

  7. Costimulation-dependent modulation of experimental autoimmune encephalomyelitis by ligand stimulation of V alpha 14 NK T cells.

    PubMed

    Pál, E; Tabira, T; Kawano, T; Taniguchi, M; Miyake, S; Yamamura, T

    2001-01-01

    Experimental autoimmune encephalomyelitis (EAE) is a Th1 cell-mediated autoimmune disease that can be protected against by stimulating regulatory cells. Here we examined whether EAE can be purposefully modulated by stimulating Valpha14 NK T cells with the CD1d-restricted ligand alpha-galactosylceramide (alpha-GC). EAE induced in wild-type C57BL/6 (B6) mice was not appreciably altered by injection of alpha-GC. However, EAE induced in IL-4 knockout mice and IFN-gamma knockout mice was enhanced or suppressed by alpha-GC, respectively. This indicates that the IL-4 and IFN-gamma triggered by alpha-GC may play an inhibitory or enhancing role in the regulation of EAE. We next studied whether NK T cells of wild-type mice may switch their Th0-like phenotype toward Th1 or Th2. Notably, in the presence of blocking B7.2 (CD86) mAb, alpha-GC stimulation could bias the cytokine profile of NK T cells toward Th2, whereas presentation of alpha-GC by CD40-activated APC induced a Th1 shift of NK T cells. Furthermore, transfer of the alpha-GC-pulsed APC preparations suppressed or enhanced EAE according to their ability to polarize NK T cells toward Th2 or Th1 in vitro. These results have important implications for understanding the role of NK T cells in autoimmunity and for designing a therapeutic strategy targeting NK T cells. PMID:11123351

  8. Binding of RANTES, MCP-1, MCP-3, and MIP-1alpha to cells in human skin.

    PubMed Central

    Hub, E.; Rot, A.

    1998-01-01

    Based on their ability to induce leukocyte chemotaxis and adhesion to endothelial cells (ECs), chemokines have been implicated in driving inflammatory leukocyte emigration. Recently, it was suggested that chemokines can accomplish their pro-emigratory role more effectively while being bound to the luminal surface of the ECs. Previously, such binding was demonstrated in situ in human skin for the prototype alpha-chemokine interleukin (IL)-8. Here we used an in situ binding assay to investigate the binding characteristics of several beta-chemokines in intact human skin. RANTES, MCP-1, and MCP-3 bound, similar to IL-8, in a specific saturable manner to the ECs of venules and small veins but not arteries or capillaries. RANTES inhibited MCP-1 and MCP-3 binding and vice versa, indicating that the EC binding sites are shared among these beta-chemokines; moreover, IL-8 and RANTES cross-competed for each other's binding, suggesting that the same chemokine binding sites are used by members of alpha- and beta-chemokine subfamilies. Conversely, MIP-1alpha did not bind to the ECs and did not compete for binding of RANTES. Analogous to IL-8, all of the tested beta-chemokines bound to the resident dermal cells. As a novel aspect of chemokine interaction with cells in normal skin, we observed specific, saturable binding of RANTES, MCP-1, and MCP-3 but not MIP-1alpha or IL-8 to the ECs of dermal afferent lymphatic vessels. RANTES, MCP-1, and MCP-3 also cross-competed for each other's binding to lymphatics, suggesting a common binding site with a novel chemokine binding profile. We suggest that the chemokine binding to the ECs of lymphatics may be involved in the process of leukocyte entry into the afferent lymphatic vessels. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:9502417

  9. Rotavirus spike protein VP5* binds alpha2beta1 integrin on the cell surface and competes with virus for cell binding and infectivity.

    PubMed

    Graham, Kate L; Takada, Yoshikazu; Coulson, Barbara S

    2006-05-01

    Rotaviruses recognize several cell-surface molecules, including the alpha2beta1 integrin, and the processes of rotavirus cell attachment and entry appear to be multifactorial. The VP5* subunit of the rotavirus spike protein VP4 contains the alpha2beta1 ligand sequence Asp-Gly-Glu at residues 308-310. Binding to alpha2beta1 and infectivity of monkey rotavirus strain RRV and human rotavirus strain Wa, but not porcine rotavirus strain CRW-8, are inhibited by peptides containing Asp-Gly-Glu. Asp308 and Gly309 are necessary for the binding of RRV VP5* (aa 248-474) to expressed I domain of the alpha2 integrin subunit. Here, the ability of RRV VP5* to bind cells and affect rotavirus-integrin interactions was determined. Interestingly, VP5* bound to cells at 4 and 37 degrees C, both via alpha2beta1 and independently of this integrin. Prior VP5* binding at 37 degrees C eliminated RRV binding to cellular alpha2beta1 and reduced RRV and Wa infectivity in MA104 cells by 38-46 %. VP5* binding did not affect the infectivity of CRW-8. VP5* binding at 4 degrees C did not affect permissive-cell infection by RRV, indicating an energy requirement for VP5* competition with virus for infectivity. Mutagenesis of VP5* Asp308 and Gly309 eliminated VP5* binding to alpha2beta1 and the VP5* inhibition of rotavirus cell binding and infection, but not alpha2beta1-independent cell binding by VP5*. These studies show for the first time that expressed VP5* binds cell-surface alpha2beta1 using Asp308 and Gly309 and inhibits the infection of homologous and heterologous rotaviruses that use alpha2beta1 as a receptor. PMID:16603530

  10. Role of alpha 5 beta 1 integrin in TGF-beta 1-costimulated CD8+ T cell growth and apoptosis.

    PubMed

    Rich, S; Van Nood, N; Lee, H M

    1996-10-01

    TGF-beta 1 regulates cell growth, differentiation, and adhesion and is a potent immunosuppressant, in part through its well-recognized growth-inhibitory effects. However, certain T cell subsets, particularly of naive phenotype, can instead be costimulated to proliferate by TGF-beta 1. We have previously demonstrated that naive murine CD8+ T cells, TCR activated by platebound anti-CD3 Ab or SEB superantigen, are growth stimulated by TGF-beta 1, acquire a memory phenotype, express elevated IL-10 and TGF-beta 1, and cause T cell growth inhibition as effector CD8+ T cells. TGF-beta 1 causes growth among certain nonlymphoid cells in part by inducing or mimicking integrin activation. The present studies thus addressed mediation of TGF-beta 1-dependent growth and survival of anti-CD3-triggered CD8+ T cells via beta 1 integrins. TGF-beta 1 reduced anti-CD3-activated alpha 4 beta 1 integrin expression and constitutive adhesion to fibronectin, while initial alpha 5 beta 1 expression was heightened and adhesive function sustained. Fibronectin-based RGD peptides that bind alpha 5 beta 1 integrins and alpha 5 or beta 1 integrin chain-specific Abs blocked TGF-beta 1-dependent proliferation, while connecting segment-1 peptide that binds alpha 4 beta 1 integrin and alpha 4 chain-specific Abs had no effect. Cross-linked alpha 5- but not alpha 4-specific Ab mimicked TGF-beta 1 function by costimulating CD8+ T cell growth. TGF-beta 1 also caused RGD peptide-sensitive CD8+ T cell aggregation. Additionally, TGF-beta 1-costimulated proliferation correlated with TGF-beta 1 protection of CD8+ T cells from anti-CD3-induced apoptosis. RGD peptides and alpha 5 integrin-specific Ab abolished TGF-beta 1 prevention of activation-induced apoptosis. Therefore, TGF-beta 1 costimulates CD8+ T cell growth via activation of the alpha 5 beta 1 integrin and/or its ligand and supports sustained growth at least in part by alpha 5 beta 1-mediated protection from activation-induced apoptosis. PMID:8816397

  11. Spontaneous expression of a low affinity Fc receptor for IgA (Fc alpha R) on human B cell lines.

    PubMed Central

    Millet, I; Briere, F; Vincent, C; Rousset, F; Andreoni, C; De Vries, J E; Revillard, J P

    1989-01-01

    Expression of receptors for IgA (Fc alpha Rs) was investigated on a panel of 35 human B cell lines by labelling with human secretory IgA (0.5 mg/ml) and flow cytometry analysis after staining with fluoresceinated goat anti-human secretory component and/or anti-alpha chain F(ab')2 fragments. Receptors for IgA could be demonstrated on one out of nine Burkitt's lymphoma cell lines, three out of five myeloma cell lines and five out of 21 lymphoblastoid cell lines. The percentage of Fc alpha R-positive cells within the same B cell line varied upon repeated examination. Human dimeric IgA1 lambda myeloma protein revealed the same number of IgA receptor positive cells as did secretory IgA, whereas monomeric IgA did not bind to Fc alpha R. Detection of Fc alpha R was not inhibited when the tests were carried out in the presence of human dimeric IgG, IgM, asialo-orosomucoid, and secretory component but it was abrogated by pre-treatment of the cells with trypsin. The binding characteristics of Fc alpha Rs were studied on the myeloma cell line Esteve, using 125I-labelled human dimeric IgA and secretory IgA. The binding was dose-dependent with rapid kinetics and specific inhibition by unlabelled secretory IgA. Scatchard plot analysis resulted in an equilibrium constant K ranging from 3.2 to 4.7 x 10(6) M/l. No correlation was observed between Fc alpha R expression and differentiation stage, monoclonality, polyclonality of the cell lines, or Ig class produced by the B cells. PMID:2788048

  12. Retroviral interleukin 1alpha gene transfer in bone marrow stromal cells in a primate model: induction of myelopoiesis stimulation.

    PubMed

    de Revel, Thierry; Becard, Nicolas; Sorg, Tania; Rousseau, Sandrine; Spano, Jean Philippe; Thiebot, Hugues; Methali, Magid; Gras, Gabriel; Le Grand, Roger; Dormont, Dominique

    2002-09-01

    Effects of interleukin 1-alpha (IL-1alpha), a proinflammatory cytokine with pleiotropic activity, in the myelopoietic setting, is mainly linked to its ability to increase haematopoietic growth factor production by bone marrow stromal cells. In order to minimize systemic effects of IL-1alpha therapy, we proposed a model of retroviral IL-1alpha gene transfer within bone marrow stromal cells in the macaque cynomolgus. Invitro, 10-15% of bone marrow stromal cells was effectively transduced by retroviral vector (murine Moloney leukaemia virus-derived) expressing IL-1alpha/LacZ, or LacZ alone as control marker, as assessed by betaGal staining. IL-1alpha gene expression was upregulated [semiquantitative reverse transcription polymerase chain reaction (RT-PCR)] within the transduced cells and the cell supernatant showed an increased production of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage (GM)-CSF (enzyme-linked immunosorbent assay) and an increased clonogenic activity (colony-forming cell assay). Ex vivo autologous expanded IL-1alpha/LacZ transduced bone marrow stromal cells were reinfused in two macaques (and two control animals for LacZ alone as controls), without clinical systemic toxicity; LacZ expression by RT-PCR was detected in one animal of each group between d 4 and 9. A slight increase of the peripheral blood leucocyte counts (both polymorphonuclear cells and monocytes) of the two animals transduced with IL-1alpha/LacZ was observed within 10 d, indicating stimulation of myelopoiesis. PMID:12181061

  13. Halogenated benzimidazole carboxamides target integrin alpha4 beta1 on T and B cell lymphomas3456

    PubMed Central

    Carpenter, Richard D.; Natarajan, Arutselvan; Lau, Edmond Y.; Andrei, Mirela; Solano, Danielle M.; Lightstone, Felice C.; DeNardo, Sally J.; Lam, Kit S.; Kurth, Mark J.

    2011-01-01

    Integrin alpha-4 beta-1 (alpha4 beta1, alpha(4)beta(1), α4β1) is an attractive but poorly understood target for selective diagnosis and treatment of T and B cell lymphomas. This report focuses on the rapid microwave preparation, structure-activity relationships and biological evaluation of medicinally pertinent benzimidazole heterocycles as integrin α4β1 antagonists. We documented tumor uptake of derivatives labelled with I-125 in xenograft murine models of B-cell lymphoma. Molecular homology models of integrin α4β1 predicted that docked halobenzimidazole carboxamides have the halogen atom in a suitable orientation for halogen-hydrogen bonding. The high affinity halogenated ligands identified offer attractive tools for medicinal and biological use, including fluoro and iodo derivatives with potential radiodiagnostic (18F) or radiotherapeutic (131I) applications, or chloro and bromo analogues that could provide structural insights into integrin-ligand interactions through photoaffinity, cross-linking/mass spectroscopy, and X-ray crystallographic studies. PMID:20530664

  14. DNA binding of Pd(TC3), a conformable cationic porphyrin with a long-lived triplet state.

    PubMed

    Ghimire, Srijana; Bork, Matthew A; Zhang, Hanyu; Fanwick, Phillip E; Zeller, Matthias; Choi, Jong Hyun; McMillin, David R

    2016-09-28

    The goal of this work has been to synthesize and investigate Pd(TC3), an intercalating porphyrin that has conformable substituents capable of groove binding to B-form DNA. (TC3 denotes the doubly deprotonated form of 5,10,15,20-tetra[3-(3'-methylimidazolium-1'-yl)prop-1-yl]porphyrin.) Palladium(ii) is an apt choice for the central metal ion because it remains strictly four-coordinate and provides for a luminescent triplet excited state with a long lifetime. The DNA hosts are hairpin-forming sequences programmed to differ in base composition. Luminescence, absorbance, and circular dichroism results are consistent with the idea that congruent structural reorganization takes place at the host and ligand during uptake. Photoexcitation of DNA-bound Pd(TC3) generates a comparatively modest steady state concentration of singlet oxygen, due to a relatively slow reaction with molecular oxygen in solution. The sheer size of the substituent groups disfavors quenching, but groove-binding interactions compound the problem by inhibiting mobility. The results show how ligand design affects adduct structure as well as function. PMID:27534907

  15. Isolation of human alpha 2HS-glycoprotein synthesized by Sf9 cells.

    PubMed

    Lörincz, Z; Kalabay, L; Cseh, S; Závodszky, P; Arnaud, P; Jakab, L

    1998-01-01

    The human alpha 2HS-glycoprotein is a negative acute phase protein synthesized by hepatocytes. Because of its fragility and the difficulty of its purification, we used recombinant DNA techniques to produce the protein in order to investigate its biological effects. The cDNA coding for the whole alpha 2HS-glycoprotein from human liver library had been cloned into the baculovirus expression vector system using the pVL 1392 transfer vector and Sf9 cells. The recombinant protein was synthesized in the Sf9 cells and was isolated on a hydroxiapatite column from the culture medium. Western blot analysis indicated that the cells synthesized large quantities of the recombinant human protein. The molecular mass of the recombinant AHSG was the same as that of the protein in human plasma but was slightly lower than that of AHSG in the cell culture supernatants of HepG2 and higher than that of AHSG from Hep3B cell cultures, respectively. PMID:9873947

  16. Detailed 8-transistor SRAM cell analysis for improved alpha particle radiation hardening in nanometer technologies

    NASA Astrophysics Data System (ADS)

    Bota, Sebastià A.; Torrens, Gabriel; Verd, Jaume; Segura, Jaume

    2015-09-01

    Eight-transistor (8T) cells were introduced to improve variability tolerance, cell stability and low-voltage operation in high-speed SRAM caches by decoupling the read and write design requirements. Altogether, 8T-SRAM can be designed without significant area penalty over 6T-SRAM. Ionizing radiation effects are nowadays a major concern for reliability and dependability of emerging electronic SRAM devices, even for sea-level applications. In this paper we demonstrate from experimental results that the 8T-SRAM also exhibits an enhanced overall intrinsic tolerance to alpha particle radiation even though its critical charge values are smaller than conventional 6T cells. We have experimentally found that the soft error rate measured in accelerated experiments with alpha particles in SRAM devices implemented in a 65 nm CMOS is 56% better for 8T cells with respect to standard 6T-cells. Even more, we show that this value can be increased up to a 200% through transistor sizing optimization.

  17. 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.

  18. Neuroprotective Effects of Alpha-Mangostin on MPP+-Induced Apoptotic Cell Death in Neuroblastoma SH-SY5Y Cells

    PubMed Central

    Janhom, Prachya; Dharmasaroja, Permphan

    2015-01-01

    In vitro studies have shown that extracts from mangosteen (Garcinia mangostana Linn.) act as antioxidants and cytoprotective agents against oxidative damage. The protective effect of alpha-mangostin, the major xanthone found in the pericarp of the mangosteen, in cellular models of Parkinson's disease (PD), has not been investigated. This study aims to investigate whether alpha-mangostin could protect SH-SY5Y neuroblastoma cells from MPP+-induced apoptosis. The effects of alpha-mangostin on MPP+-induced cell death were evaluated with a cell viability assay, staining for nuclear DNA morphology, flow cytometry for apoptotic cells and reactive oxygen species (ROS) production, quantitative real-time PCR for the expression of p53, Bax, and Bcl-2, and western blot analysis for cleaved caspase-3. Concomitant treatment with alpha-mangostin attenuated the effect of MPP+ on cell viability and apoptotic cell death. Alpha-mangostin reduced ROS formation induced by MPP+. Bax/Bcl-2 expression ratio and expression of p53 were significantly lower in cells cocultured with alpha-mangostin and MPP+. The cotreated cells showed a significant decrease in activated caspase-3 compared with MPP+ treatment alone. Our data suggest that cytoprotection of alpha-mangostin against MPP+-induced apoptosis may be associated with the reduction of ROS production, modulating the balance of pro- and antiapoptotic genes, and suppression of caspase-3 activation. PMID:26357513

  19. Neuroprotective Effects of Alpha-Mangostin on MPP(+)-Induced Apoptotic Cell Death in Neuroblastoma SH-SY5Y Cells.

    PubMed

    Janhom, Prachya; Dharmasaroja, Permphan

    2015-01-01

    In vitro studies have shown that extracts from mangosteen (Garcinia mangostana Linn.) act as antioxidants and cytoprotective agents against oxidative damage. The protective effect of alpha-mangostin, the major xanthone found in the pericarp of the mangosteen, in cellular models of Parkinson's disease (PD), has not been investigated. This study aims to investigate whether alpha-mangostin could protect SH-SY5Y neuroblastoma cells from MPP(+)-induced apoptosis. The effects of alpha-mangostin on MPP(+)-induced cell death were evaluated with a cell viability assay, staining for nuclear DNA morphology, flow cytometry for apoptotic cells and reactive oxygen species (ROS) production, quantitative real-time PCR for the expression of p53, Bax, and Bcl-2, and western blot analysis for cleaved caspase-3. Concomitant treatment with alpha-mangostin attenuated the effect of MPP(+) on cell viability and apoptotic cell death. Alpha-mangostin reduced ROS formation induced by MPP(+). Bax/Bcl-2 expression ratio and expression of p53 were significantly lower in cells cocultured with alpha-mangostin and MPP(+). The cotreated cells showed a significant decrease in activated caspase-3 compared with MPP(+) treatment alone. Our data suggest that cytoprotection of alpha-mangostin against MPP(+)-induced apoptosis may be associated with the reduction of ROS production, modulating the balance of pro- and antiapoptotic genes, and suppression of caspase-3 activation. PMID:26357513

  20. 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. PMID:9108052

  1. 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.

  2. Alpha fetoprotein plays a critical role in promoting metastasis of hepatocellular carcinoma cells.

    PubMed

    Lu, Yan; Zhu, Mingyue; Li, Wei; Lin, Bo; Dong, Xu; Chen, Yi; Xie, Xieju; Guo, Junli; Li, Mengsen

    2016-03-01

    A high level of serum alpha fetoprotein (AFP) is positively associated with human hepatocellular carcinoma (HCC) carcinogenesis and metastasis; however, the function of AFP in HCC metastasis is unknown. This study has explored the effects of AFP on regulating metastatic and invasive capacity of human HCC cells. Forty-seven clinical patients' liver samples were collected and diagnosed; HCC cells line, Bel 7402 cells (AFP-producing) and liver cancer cell line cells (non-AFP-producing) were selected to analyse the role of AFP in the metastasis of HCC cells. The results indicated that high serum concentration of AFP was positively correlated with HCC intrahepatic, lymph nodes and lung metastasis. Repressed expression of AFP significantly inhibited the capability of migration and invasion of Bel 7402 cells, expression of keratin 19 (K19), epithelial cell adhesion molecule (EpCAM), matrix metalloproteinase 2/9 (MMP2/9) and CXC chemokine receptor 4 (CXCR4) were also down-regulated in Bel 7402 cells; migration and invasion, expression of K19, EpCAM, MMP2/9 and CXCR4 were significantly enhanced when HLE cells were transfected with AFP-expressed vector. The results demonstrated that AFP plays a critical role in promoting metastasis of HCC; AFP promoted HCC cell invasion and metastasis via up-regulating expression of metastasis-related proteins. Thus, AFP may be used as a novel therapeutic target for treating HCC patients. PMID:26756858

  3. A delta T-cell receptor deleting element transgenic reporter construct is rearranged in alpha beta but not gamma delta T-cell lineages.

    PubMed Central

    Shutter, J; Cain, J A; Ledbetter, S; Rogers, M D; Hockett, R D

    1995-01-01

    T cells can be divided into two groups on the basis of the expression of either alpha beta or gamma delta T-cell receptors (TCRs). Because the TCR delta chain locus lies within the larger TCR alpha chain locus, control of the utilization of these two receptors is important in T-cell development, specifically for determination of T-cell type: rearrangement of the alpha locus results in deletion of the delta coding segments and commitment to the alpha beta lineage. In the developing thymus, a relative site-specific recombination occurs by which the TCR delta chain gene segments are deleted. This deletion removes all D delta, J delta, and C delta genes and occurs on both alleles. This delta deletional mechanism is evolutionarily conserved between mice and humans. Transgenic mice which contain the human delta deleting elements and as much internal TCR delta chain coding sequence as possible without allowing the formation of a complete delta chain gene were developed. Several transgenic lines showing recombinations between deleting elements within the transgene were developed. These lines demonstrate that utilization of the delta deleting elements occurs in alpha beta T cells of the spleen and thymus. These recombinations are rare in the gamma delta population, indicating that the machinery for utilization of delta deleting elements is functional in alpha beta T cells but absent in gamma delta T cells. Furthermore, a discrete population of early thymocytes containing delta deleting element recombinations but not V alpha-to-J alpha rearrangements has been identified. These data are consistent with a model in which delta deletion contributes to the implementation of a signal by which the TCR alpha chain locus is rearranged and expressed and thus becomes an alpha beta T cell. PMID:8524269

  4. New pterocarpanquinones: synthesis, antineoplasic activity on cultured human malignant cell lines and TNF-alpha modulation in human PBMC cells.

    PubMed

    Netto, Chaquip D; da Silva, Alcides J M; Salustiano, Eduardo J S; Bacelar, Thiago S; Riça, Ingred G; Cavalcante, Moises C M; Rumjanek, Vivian M; Costa, Paulo R R

    2010-02-15

    A new pterocarpanquinone (5a) was synthesized through a palladium catalyzed oxyarylation reaction and was transformed, through electrophilic substitution reaction, into derivatives 5b-d. These compounds showed to be active against human leukemic cell lines and human lung cancer cell lines. Even multidrug resistant cells were sensitive to 5a, which presented low toxicity toward peripheral blood mononuclear cells (PBMC) cells and decreased the production of TNF-alpha by these cells. In the laboratory these pterocarpanquinones were reduced by sodium dithionite in the presence of thiophenol at physiological pH, as NAD(P)H quinone oxidoredutase-1 (NQO1) catalyzed two-electron reduction, and the resulting hydroquinone undergo structural rearrangements, leading to the formation of Michael acceptors, which were intercepted as adducts of thiophenol. These results suggest that these compounds could be activated by bioreduction. PMID:20117936

  5. Two novel, putatively cell wall-associated and glycosylphosphatidylinositol-anchored alpha-glucanotransferase enzymes of Aspergillus niger.

    PubMed

    van der Kaaij, R M; Yuan, X-L; Franken, A; Ram, A F J; Punt, P J; van der Maarel, M J E C; Dijkhuizen, L

    2007-07-01

    In the genome sequence of Aspergillus niger CBS 513.88, three genes were identified with high similarity to fungal alpha-amylases. The protein sequences derived from these genes were different in two ways from all described fungal alpha-amylases: they were predicted to be glycosylphosphatidylinositol anchored, and some highly conserved amino acids of enzymes in the alpha-amylase family were absent. We expressed two of these enzymes in a suitable A. niger strain and characterized the purified proteins. Both enzymes showed transglycosylation activity on donor substrates with alpha-(1,4)-glycosidic bonds and at least five anhydroglucose units. The enzymes, designated AgtA and AgtB, produced new alpha-(1,4)-glycosidic bonds and therefore belong to the group of the 4-alpha-glucanotransferases (EC 2.4.1.25). Their reaction products reached a degree of polymerization of at least 30. Maltose and larger maltooligosaccharides were the most efficient acceptor substrates, although AgtA also used small nigerooligosaccharides containing alpha-(1,3)-glycosidic bonds as acceptor substrate. An agtA knockout of A. niger showed an increased susceptibility towards the cell wall-disrupting compound calcofluor white, indicating a cell wall integrity defect in this strain. Homologues of AgtA and AgtB are present in other fungal species with alpha-glucans in their cell walls, but not in yeast species lacking cell wall alpha-glucan. Possible roles for these enzymes in the synthesis and/or maintenance of the fungal cell wall are discussed. PMID:17496125

  6. 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). PMID:12794117

  7. Alpha fetoprotein antagonizes apoptosis induced by paclitaxel in hepatoma cells in vitro.

    PubMed

    Zhu, Mingyue; Li, Wei; Lu, Yan; Dong, Xu; Chen, Yi; Lin, Bo; Xie, Xieju; Guo, Junli; Li, Mengsen

    2016-01-01

    Hepatocellular carcinoma (HCC) cell resistance to the effects of paclitaxel has not been adequately addressed. In this study, we found that paclitaxel significantly inhibited the viability of HLE, Bel 7402 and L-02 cells in a dose- and time-dependent manner. HLE cells and L-02 cells resisted the cytotoxicity of paclitaxel when transfected with pcDNA3.1-afp vectors. However, Bel 7402 cell sensitivity to paclitaxel was increased when transfected with alpha fetoprotein (AFP)-siRNA. Bel 7402 cell resistance to paclitaxel was associated with the expression of the "stemness" markers CD44 and CD133. Paclitaxel significantly inhibited growth and promoted apoptosis in HLE cells and L-02 cells by inducing fragmentation of caspase-3 and inhibiting the expression of Ras and Survivin, but pcDNA3.1-afp vectors prevented these effects. However, paclitaxel could not significantly promote the cleavage of caspase-3 or suppress the expression of Ras and Survivin in Bel 7402 cells. Silenced expression of AFP may be synergistic with paclitaxel to restrain proliferation and induce apoptosis, enhance cleavage of caspase-3, and suppress the expression of Ras and Survivin. Taken together, AFP may be an important molecule acting against paclitaxel-inhibited proliferation and induced apoptosis in HCC cells via repressing the activity of caspase-3 and stimulating the expression of Ras and Survivin. Targeted inhibition of AFP expression after treatment with paclitaxel is an available strategy for the therapy of patients with HCC. PMID:27255186

  8. Alpha fetoprotein antagonizes apoptosis induced by paclitaxel in hepatoma cells in vitro

    PubMed Central

    Zhu, Mingyue; Li, Wei; Lu, Yan; Dong, Xu; Chen, Yi; Lin, Bo; Xie, Xieju; Guo, Junli; Li, Mengsen

    2016-01-01

    Hepatocellular carcinoma (HCC) cell resistance to the effects of paclitaxel has not been adequately addressed. In this study, we found that paclitaxel significantly inhibited the viability of HLE, Bel 7402 and L-02 cells in a dose- and time-dependent manner. HLE cells and L-02 cells resisted the cytotoxicity of paclitaxel when transfected with pcDNA3.1-afp vectors. However, Bel 7402 cell sensitivity to paclitaxel was increased when transfected with alpha fetoprotein (AFP)-siRNA. Bel 7402 cell resistance to paclitaxel was associated with the expression of the “stemness” markers CD44 and CD133. Paclitaxel significantly inhibited growth and promoted apoptosis in HLE cells and L-02 cells by inducing fragmentation of caspase-3 and inhibiting the expression of Ras and Survivin, but pcDNA3.1-afp vectors prevented these effects. However, paclitaxel could not significantly promote the cleavage of caspase-3 or suppress the expression of Ras and Survivin in Bel 7402 cells. Silenced expression of AFP may be synergistic with paclitaxel to restrain proliferation and induce apoptosis, enhance cleavage of caspase-3, and suppress the expression of Ras and Survivin. Taken together, AFP may be an important molecule acting against paclitaxel-inhibited proliferation and induced apoptosis in HCC cells via repressing the activity of caspase-3 and stimulating the expression of Ras and Survivin. Targeted inhibition of AFP expression after treatment with paclitaxel is an available strategy for the therapy of patients with HCC. PMID:27255186

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

    SciTech Connect

    Kamikawa, Yasunao; Yasuhara, Noriko; Yoneda, Yoshihiro; Department of Biochemistry, Graduate School of Medicine, Osaka University; JST, CREST, Graduate School of Frontier Biosciences, Osaka University, Yamada-oka, Suita, Osaka 565-0871

    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.

  10. The endocytic protein alpha-Adaptin is required for numb-mediated asymmetric cell division in Drosophila.

    PubMed

    Berdnik, Daniela; Török, Tibor; González-Gaitán, Marcos; Knoblich, Juergen A

    2002-08-01

    During asymmetric cell division in Drosophila sensory organ precursor cells, the Numb protein localizes asymmetrically and segregates into one daughter cell, where it influences cell fate by repressing signal transduction via the Notch receptor. We show here that Numb acts by polarizing the distribution of alpha-Adaptin, a protein involved in receptor-mediated endocytosis. alpha-Adaptin binds to Numb and localizes asymmetrically in a Numb-dependent fashion. Mutant forms of alpha-Adaptin that no longer bind to Numb fail to localize asymmetrically and cause numb-like defects in asymmetric cell division. Our results suggest a model in which Numb influences cell fate by downregulating Notch through polarized receptor-mediated endocytosis, since Numb also binds to the intracellular domain of Notch. PMID:12194853

  11. Tat-APE1/ref-1 protein inhibits TNF-{alpha}-induced endothelial cell activation

    SciTech Connect

    Song, Yun Jeong; Lee, Ji Young; Joo, Hee Kyoung; Kim, Hyo Shin; Lee, Sang Ki; Lee, Kwon Ho; Cho, Chung-Hyun; Park, Jin Bong; Jeon, Byeong Hwa

    2008-03-28

    Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/ref-1) is a multifunctional protein involved both in DNA base excision repair and redox regulation. In this study we evaluated the protective role of Tat-mediated APE1/ref-1 transduction on the tumor necrosis factor (TNF)-{alpha}-activated endothelial activation in cultured human umbilical vein endothelial cells. To construct Tat-APE1/ref-1 fusion protein, human full length of APE1/ref-1 was fused with Tat-protein transduction domain. Purified Tat-APE1/ref-1 fusion protein efficiently transduced cultured endothelial cells in a dose-dependent manner and reached maximum expression at 1 h after incubation. Transduced Tat-APE1/ref-1 showed inhibitory activity on the TNF-{alpha}-induced monocyte adhesion and vascular cell adhesion molecule-1 expression in cultured endothelial cells. These results suggest Tat-APE1/ref-1 might be useful to reduce vascular endothelial activation or vascular inflammatory disorders.

  12. 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

  13. Integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha ) interacts directly with the metastasis suppressor nm23-H2, and both proteins are targeted to newly formed cell adhesion sites upon integrin engagement.

    PubMed

    Fournier, Henri-Noël; Dupé-Manet, Sandra; Bouvard, Daniel; Lacombe, Marie-Lise; Marie, Christiane; Block, Marc R; Albiges-Rizo, Corinne

    2002-06-01

    Cell adhesion-dependent signaling implicates cytoplasmic proteins interacting with the intracellular tails of integrins. Among those, the integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha) has been shown to interact specifically with the beta(1) integrin cytoplasmic domain. Although it is likely that this protein plays an important role in controlling cell adhesion and migration, little is known about its actual function. To search for potential ICAP-1alpha-binding proteins, we used a yeast two-hybrid screen and identified the human metastatic suppressor protein nm23-H2 as a new partner of ICAP-1alpha. This direct interaction was confirmed in vitro, using purified recombinant ICAP-1alpha and nm23-H2, and by co-immunoprecipitation from CHO cell lysates over-expressing ICAP-1alpha. The physiological relevance of this interaction is provided by confocal fluorescence microscopy, which shows that ICAP-1alpha and nm23-H2 are co-localized in lamellipodia during the early stages of cell spreading. These adhesion sites are enriched in occupied beta(1) integrins and precede the formation of focal adhesions devoid of ICAP-1alpha and nm23-H2, indicating the dynamic segregation of components of matrix adhesions. This peripheral staining of ICAP-1alpha and nm23-H2 is only observed in cells spreading on fibronectin and collagen and is absent in cells spreading on poly-l-lysine, vitronectin, or laminin. This is consistent with the fact that targeting of both ICAP-1alpha and nm23-H2 to the cell periphery is dependent on beta(1) integrin engagement rather than being a consequence of cell adhesion. This finding represents the first evidence that the tumor suppressor nm23-H2 could act on beta(1) integrin-mediated cell adhesion by interacting with one of the integrin partners, ICAP-1alpha. PMID:11919189

  14. Tumor necrosis factor-alpha induces nuclear factor-kappaB-dependent TRPC1 expression in endothelial cells.

    PubMed

    Paria, Biman C; Malik, Asrar B; Kwiatek, Angela M; Rahman, Arshad; May, Michael J; Ghosh, Sankar; Tiruppathi, Chinnaswamy

    2003-09-26

    We investigated the role of tumor necrosis factor-alpha (TNF-alpha) in activating the store-operated Ca2+ channels in endothelial cells via the expression of transient receptor potential channel (TRPC) isoforms. We observed that TNF-alpha exposure of human umbilical vein endothelial cells resulted in TRPC1 mRNA and protein expression, whereas it had no effect on TRPC3, TRPC4, or TRPC5 expression. The TRPC1 expression was associated with increased Ca2+ influx after intracellular Ca2+ store depletion with either thrombin or thapsigargin. We cloned the 5'-regulatory region of the human TRPC1 (hTRPC1) gene which contained a TATA box and CCAAT sequence close to the transcription initiation site. We also identified four nuclear factor-kappaB (NF-kappaB)-binding sites in the 5'-regulatory region. To address the contribution of NF-kappaB in the mechanism of TRPC1 expression, we determined the effects of TNF-alpha on expression of the reporter luciferase after transfection of hTRPC1 promoter-luciferase (hTRPC1-Pro-Luc) construct in the human dermal microvascular endothelial cell line. Reporter activity increased >4-fold at 4 h after TNF-alpha challenge. TNF-alpha-induced increase in reporter activity was markedly reduced by co-expression of either kinase-defective IKKbeta kinase mutant or non-phosphorylatable IkappaB mutant. Treatment with NEMO-binding domain peptide, which prevents NF-kappaB activation by selectively inhibiting IKKgamma interaction with IKK complex, also blocked the TNF-alpha-induced TRPC1 expression. Thus, TNF-alpha induces TRPC1 expression through an NF-kappaB-dependent pathway in endothelial cells, which can trigger augmented Ca2+ entry following Ca2+ store depletion. The augmented Ca2+ entry secondary to TRPC1 expression may be an important mechanism of endothelial injury induced by TNF-alpha. PMID:12855710

  15. Familial giant cell hepatitis associated with synthesis of 3 beta, 7 alpha-dihydroxy-and 3 beta,7 alpha, 12 alpha-trihydroxy-5-cholenoic acids.

    PubMed Central

    Clayton, P T; Leonard, J V; Lawson, A M; Setchell, K D; Andersson, S; Egestad, B; Sjövall, J

    1987-01-01

    Urinary bile acids from a 3-mo-old boy with cholestatic jaundice were analyzed by ion exchange chromatography and gas chromatography-mass spectrometry (GC-MS). This suggested the presence of labile sulfated cholenoic acids with an allylic hydroxyl group, a conclusion supported by analysis using fast atom bombardment mass spectrometry (FAB-MS). The compounds detected by FAB-MS were separated by thin layer chromatography and high performance liquid chromatography. The sulfated bile acids could be solvolyzed in acidified tetrahydrofuran, and glycine conjugates were partially hydrolyzed by cholylglycine hydrolase. Following solvolysis, deconjugation, and methylation with diazomethane, the bile acids were identified by GC-MS of trimethylsilyl derivatives. The major bile acids in the urine were 3 beta,7 alpha-dihydroxy-5-cholenoic acid 3-sulfate, 3 beta,7 alpha,12 alpha-trihydroxy-5-cholenoic acid monosulfate, and their glycine conjugates. Chenodeoxycholic acid and cholic acid were undetectable in urine and plasma. The family pedigree suggested that abnormal bile acid synthesis was an autosomal recessive condition leading to cirrhosis in early childhood. PMID:3470305

  16. 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; The Center for Advanced Medical Engineering and Informatics, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871 ; Ishimoto, Kenji; Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871 ; Tanaka, Toshiya; Hamakubo, Takao; Sakai, Juro; Kodama, Tatsuhiko; Doi, Takefumi; The Center for Advanced Medical Engineering and Informatics, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871; Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871

    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.

  17. Interleukin-2 and interferon-alpha-2a outpatient therapy for metastatic renal cell carcinoma.

    PubMed

    Lipton, A; Harvey, H; Givant, E; Hopper, K; Lawler, J; Matthews, Y; Hirsh, M; Zeffren, J

    1993-02-01

    The combination of interleukin-2 (IL-2) and interferon-alpha-2a (IFN-alpha-2a) has synergistic bioactivity in numerous preclinical model systems. Thirty-nine patients with metastatic renal cell cancer were treated with continuous intravenous infusion IL-2 for 4-5 days plus intramuscular IFN-alpha-2a 2-3 days a week for 4 consecutive weeks. A 2- to 4-week rest period was permitted after each 4 weeks of treatment. Thirty-one of the 39 patients were assessable for response determination. Response rate (six complete+seven partial remissions) was 33.3% for all patients, or 41.9% when the analysis was restricted to the 31 evaluable patients. Three patients were unable to tolerate treatment due to anorexia, weight loss, and severe fatigue. This therapy was relatively well tolerated in the outpatient setting in the other patients despite fever, chills, fatigue, anorexia, and weight loss. There was no correlation of response with site of metastases or bulk of disease. PMID:8318497

  18. Heat shock proteins in the retina: Focus on HSP70 and alpha crystallins in ganglion cell survival.

    PubMed

    Piri, Natik; Kwong, Jacky M K; Gu, Lei; Caprioli, Joseph

    2016-05-01

    Heat shock proteins (HSPs) belong to a superfamily of stress proteins that are critical constituents of a complex defense mechanism that enhances cell survival under adverse environmental conditions. Cell protective roles of HSPs are related to their chaperone functions, antiapoptotic and antinecrotic effects. HSPs' anti-apoptotic and cytoprotective characteristics, their ability to protect cells from a variety of stressful stimuli, and the possibility of their pharmacological induction in cells under pathological stress make these proteins an attractive therapeutic target for various neurodegenerative diseases; these include Alzheimer's, Parkinson's, Huntington's, prion disease, and others. This review discusses the possible roles of HSPs, particularly HSP70 and small HSPs (alpha A and alpha B crystallins) in enhancing the survival of retinal ganglion cells (RGCs) in optic neuropathies such as glaucoma, which is characterized by progressive loss of vision caused by degeneration of RGCs and their axons in the optic nerve. Studies in animal models of RGC degeneration induced by ocular hypertension, optic nerve crush and axotomy show that upregulation of HSP70 expression by hyperthermia, zinc, geranyl-geranyl acetone, 17-AAG (a HSP90 inhibitor), or through transfection of retinal cells with AAV2-HSP70 effectively supports the survival of injured RGCs. RGCs survival was also stimulated by overexpression of alpha A and alpha B crystallins. These findings provide support for translating the HSP70- and alpha crystallin-based cell survival strategy into therapy to protect and rescue injured RGCs from degeneration associated with glaucomatous and other optic neuropathies. PMID:27017896

  19. A single base mutation in an I-A alpha-chain gene alters T-cell recognition.

    PubMed

    Griffith, I J; Choi, E M; Glimcher, L H

    1987-02-01

    The interaction between the clonally selected T-cell antigen receptor, antigen, and Ia molecule is poorly understood at the molecular level. A cell line bearing an altered I-Ak alpha-chain (Ak alpha) molecule has been examined in order to provide more information about the relationship between Ia structure and function. The cell line, 3J9, was derived from the TA3 B-cell hybridoma through a series of negative and positive immunoselection steps. The 3J9 mutant lacked the binding site recognized by the Ak alpha-specific monoclonal antibody 39J and failed to present antigen to two T-cell hybridomas out of a large panel of I-Ak-restricted T-cell hybridomas examined. Sequence analysis of the mutant Ak alpha gene showed a single base transition (G----A) that resulted in a glutamic acid to lysine substitution at amino acid 75 of the alpha 1 domain. This mutation confirms the importance of amino acid 75 in the expression of the Ia.19 epitope, demonstrates the involvement of this region in the presentation of antigen to specific T cells, and provides a further example of the multiple functional domains on the Ia molecule that are involved in antigen presentation. PMID:3493486

  20. A single base mutation in an I-A alpha-chain gene alters T-cell recognition.

    PubMed Central

    Griffith, I J; Choi, E M; Glimcher, L H

    1987-01-01

    The interaction between the clonally selected T-cell antigen receptor, antigen, and Ia molecule is poorly understood at the molecular level. A cell line bearing an altered I-Ak alpha-chain (Ak alpha) molecule has been examined in order to provide more information about the relationship between Ia structure and function. The cell line, 3J9, was derived from the TA3 B-cell hybridoma through a series of negative and positive immunoselection steps. The 3J9 mutant lacked the binding site recognized by the Ak alpha-specific monoclonal antibody 39J and failed to present antigen to two T-cell hybridomas out of a large panel of I-Ak-restricted T-cell hybridomas examined. Sequence analysis of the mutant Ak alpha gene showed a single base transition (G----A) that resulted in a glutamic acid to lysine substitution at amino acid 75 of the alpha 1 domain. This mutation confirms the importance of amino acid 75 in the expression of the Ia.19 epitope, demonstrates the involvement of this region in the presentation of antigen to specific T cells, and provides a further example of the multiple functional domains on the Ia molecule that are involved in antigen presentation. PMID:3493486

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

    SciTech Connect

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

    2011-06-01

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

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

    PubMed Central

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

    1998-01-01

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

  3. Targeting of surface alpha-enolase inhibits the invasiveness of pancreatic cancer cells

    PubMed Central

    Chattaragada, Michelle S.; Rolla, Simona; Conti, Laura; Bestagno, Marco; Zentilin, Lorena; Yang, Sheng-Hui; Migliorini, Paola; Cappello, Paola; Burrone, Oscar; Novelli, Francesco

    2015-01-01

    Pancreatic Ductal Adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by rapid progression, invasiveness and resistance to treatment. We have previously demonstrated that most PDAC patients have circulating antibodies against the glycolytic enzyme alpha-enolase (ENO1), which correlates with a better response to therapy and survival. ENO1 is a metabolic enzyme, also expressed on the cell surface where it acts as a plasminogen receptor. ENO1 play a crucial role in cell invasion and metastasis by promoting plasminogen activation into plasmin, a serine-protease involved in extracellular matrix degradation. The aim of this study was to investigate the role of ENO1 in PDAC cell invasion. We observed that ENO1 was expressed on the cell surface of most PDAC cell lines. Mouse anti-human ENO1 monoclonal antibodies inhibited plasminogen-dependent invasion of human PDAC cells, and their metastatic spreading in immunosuppressed mice was inhibited. Notably, a single administration of Adeno-Associated Virus (AAV)-expressing cDNA coding for 72/1 anti-ENO1 mAb reduced the number of lung metastases in immunosuppressed mice injected with PDAC cells. Overall, these data indicate that ENO1 is involved in PDAC cell invasion, and that administration of an anti-ENO1 mAb can be exploited as a novel therapeutic option to increase the survival of metastatic PDAC patients. PMID:25860938

  4. Osteogenic potential of alpha smooth muscle actin expressing muscle resident progenitor cells.

    PubMed

    Matthews, Brya G; Torreggiani, Elena; Roeder, Emilie; Matic, Igor; Grcevic, Danka; Kalajzic, Ivo

    2016-03-01

    Heterotopic ossification (HO) is a pathological process where bone forms in connective tissues such as skeletal muscle. Previous studies have suggested that muscle-resident non-myogenic mesenchymal progenitors are the likely source of osteoblasts and chondrocytes in HO. However, the previously identified markers of muscle-resident osteoprogenitors label up to half the osteoblasts within heterotopic lesions, suggesting other cell populations are involved. We have identified alpha smooth muscle actin (αSMA) as a marker of osteoprogenitor cells in bone and periodontium, and of osteo-chondro progenitors in the periosteum during fracture healing. We therefore utilized a lineage tracing approach to evaluate whether αSMACreERT2 identifies osteoprogenitors in the muscle. We show that in the muscle, αSMACreERT2 labels both perivascular cells, and satellite cells. αSMACre-labeled cells undergo osteogenic differentiation in vitro and form osteoblasts and chondrocytes in BMP2-induced HO in vivo. In contrast, Pax7CreERT2-labeled muscle satellite cells were restricted to myogenic differentiation in vitro, and rarely contributed to HO in vivo. Our data indicate that αSMACreERT2 labels a large proportion of osteoprogenitors in skeletal muscle, and therefore represents another marker of muscle-resident cells with osteogenic potential under HO-inducing stimulus. In contrast, muscle satellite cells make minimal contribution to bone formation in vivo. PMID:26721734

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1999-04-01

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

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

    SciTech Connect

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

    2013-11-01

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

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

    SciTech Connect

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

    2005-04-15

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

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

    SciTech Connect

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

    2008-04-04

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

  11. Distributions of cell populations within. cap alpha. -particle range of plutonium deposits in the rat and beagle testis

    SciTech Connect

    Miller, S.C.; Rowland, H.G.; Bowman, B.M.

    1985-01-01

    Plutonium is not uniformly distributed in testicular tissues; thus some cell populations may receive larger or smaller radiation exposures than would be expected if the nuclide were uniformly distributed. The distributions of cell populations within ..cap alpha..-particle range of Pu deposits in rat and beagle testes were determined. The data were collected from autoradiographs of testicular tissues containing /sup 241/Pu. A cell distribution factor (CDF) was determined for each cell population and is defined as the average number of each cell type within ..cap alpha..-particle range of each observed Pu deposit relative to the number of each cell type that would be expected within ..cap alpha..-particle range of each Pu deposit, if the deposits were distributed uniformly. In addition, the percentage of the spermatogonial stem cell population within ..cap alpha..-particle range of Pu deposits was determined. The largest CDFs seen in both species were in the interstitial tissues, particulary for Leydig cells. Because the organization of testicular tissues in the beagle is quite different from rodents but more similar to human, the results from this study suggest that extrapolations from rodents to humans may tend to overestimate the potential for radiation exposure to spermatogonial stem cells as well as the fraction of the spermatogonial stem cell population at risk to exposure from internally deposited /sup 239/Pu.

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

    PubMed

    Kyprianou, N; Benning, C M

    2000-08-15

    Recent evidence from our laboratory has demonstrated that alpha1-adrenoceptor antagonists doxazosin and terazosin induced apoptosis in prostate epithelial and smooth muscle cells in patients with benign prostatic hypertrophy (BPH; J. Urol., 159: 1810-1815, 1998; J. Urol., 161: 2002-2007, 1999). In this study, we investigated the biological action of three alpha1-adrenoceptor antagonists, doxazosin, terazosin, and tamsulosin, against prostate cancer cell growth. The antigrowth effect of the three alpha1-adrenoceptor antagonists was examined in two human prostate cancer cell lines, PC-3 and DU-145, and a prostate smooth muscle cell primary culture, SMC-1, on the basis of: (a) cell viability assay; (b) rate of DNA synthesis; and (c) induction of apoptosis. Our results indicate that treatment of prostate cancer cells with doxazosin or terazosin results in a significant loss of cell viability, via induction of apoptosis in a dose-dependent manner, whereas tamsulosin had no effect on prostate cell growth. Neither doxazosin nor terazosin exerted a significant effect on the rate of cell proliferation in prostate cancer cells. Exposure to phenoxybenzamine, an irreversible inhibitor of alpha1-adrenoceptors, does not abrogate the apoptotic effect of doxazosin or terazosin against human prostate cancer or smooth muscle cells. This suggests that the apoptotic activity of doxazosin and terazosin against prostate cells is independent of their capacity to antagonize alpha1-adrenoceptors. Furthermore, an in vivo efficacy trial demonstrated that doxazosin administration (at tolerated pharmacologically relevant doses) in SCID mice bearing PC-3 prostate cancer xenografts resulted in a significant inhibition of tumor growth. These findings demonstrate the ability of doxazosin and terazosin (but not tamsulosin) to suppress prostate cancer cell growth in vitro and in vivo by inducing apoptosis without affecting cell proliferation. This evidence provides the rationale for targeting both

  13. Hepatosplenic alpha/beta T-cell lymphoma masquerading as cirrhosis

    PubMed Central

    Hariton, Eduardo; Kothari, Darshan; Pihan, German A.; Robson, Simon C.

    2013-01-01

    A 59-year-old man with diabetes mellitus, prior hepatitis B infection and recently diagnosed cirrhosis with prior Babesiosis presented to our institution from an outside hospital with six months of worsening abdominal pain, myalgias and fevers. On admission, physical examination revealed jaundice, hepatosplenomegaly and diffuse lymphadenopathy. Laboratory investigations demonstrated mild anemia, thrombocytopenia, hyperbilirubinemia and elevated lactate dehydrogenase. Tests for human immunodeficiency virus, and active Babesia microti infection were negative, however Epstein-Barr virus DNA by quantitative PCR was markedly elevated. CT scan revealed features suggestive of a cirrhotic liver without focal mass lesions as well as massive splenomegaly with axillary, retroperitoneal and inguinal lymphadenopathy. Bone marrow and lymph node biopsies were obtained which ultimately revealed hepatosplenic T-cell lymphoma. The patient’s initial liver biopsy from five months prior to presentation was re-evaluated by our institution’s pathologists. Histologic analysis showed hepatic sinusoidal and portal infiltration of atypical lymphocytes morphologically identical to those present on the more recently excised lymph node tissue. The hepatic sinusoidal lymphoid cells were strongly positive for CD2, CD3 and CD5 whereas CD4, CD8 stained only minor subsets of the T cells. Subsequent flow cytometric immunophenotypying of peripheral blood identified T-cell receptor alpha/beta positive cells that lacked CD4 and CD8 (double negative alpha/beta T cells). Given the established bone marrow involvement, he was diagnosed with stage IV disease and treated with chemotherapy. His clinical course involved multiple hospitalizations complicated by hyponatremia, neutropenic fevers and pulmonary emboli. Following his fourth cycle of chemotherapy, he developed worsening liver failure and expired approximately three months after initial diagnosis of lymphoma. Hepatosplenic lymphoma of alpha/beta T

  14. The Role of Plasmacytoid Dendritic Cells in Innate and Adaptive Immune Responses against Alpha Herpes Virus Infections

    PubMed Central

    Schuster, Philipp; Boscheinen, Jan Bernardin; Tennert, Karin; Schmidt, Barbara

    2011-01-01

    In 1999, two independent groups identified plasmacytoid dendritic cells (PDC) as major type I interferon- (IFN-) producing cells in the blood. Since then, evidence is accumulating that PDC are a multifunctional cell population effectively coordinating innate and adaptive immune responses. This paper focuses on the role of different immune cells and their interactions in the surveillance of alpha herpes virus infections, summarizes current knowledge on PDC surface receptors and their role in direct cell-cell contacts, and develops a risk factor model for the clinical implications of herpes simplex and varicella zoster virus reactivation. Data from studies involving knockout mice and cell-depletion experiments as well as human studies converge into a “spider web”, in which the direct and indirect crosstalk between many cell populations tightly controls acute, latent, and recurrent alpha herpes virus infections. Notably, cells involved in innate immune regulations appear to shape adaptive immune responses more extensively than previously thought. PMID:22312349

  15. Alpha-quartz-induced chemokine expression by rat lung epithelial cells: effects of in vivo and in vitro particle exposure.

    PubMed

    Driscoll, K E; Howard, B W; Carter, J M; Asquith, T; Johnston, C; Detilleux, P; Kunkel, S L; Isfort, R J

    1996-11-01

    Chemokines are chemotactic cytokines that can play a key role in leukocyte recruitment to sites of tissue injury or infection. Previous studies have demonstrated that exposure to alpha-quartz as well as other noxious particles increases chemokine gene expression in rat lung, although the cells responsible for chemokine expression and the mechanisms underlying this response have remained unclear. The present studies demonstrate that exposure of rats to alpha-quartz induced expression of mRNA for the chemokine macrophage-inflammatory protein (MIP)-2 in epithelial cells lining the terminal bronchioles and alveolar ducts as well as macrophages and alveolar type II cells in the more distal lung. Treatment of rats with an anti-MIP-2 antiserum before alpha-quartz exposure markedly attenuated neutrophilic infiltration of the lungs demonstrating an important role for MIP-2 in alpha-quartz-induced pulmonary inflammation. In vitro exposure of primary cultures of rat alveolar type II cells or the rat alveolar type II cell line RLE-6TN to tumor necrosis factor-alpha, endotoxin, or alpha-quartz increased mRNA for MIP-2 as well as the structurally and functionally similar chemokine cytokine-induced neutrophil chemoattractant but not the chemokine MIP-1 alpha. The alpha-quartz-induced increase in epithelial MIP-2 mRNA resulted, at least in part, from increased gene transcription and was associated with the release of active MIP-2 protein. Induction of RLE-6TN MIP-2 and cytokine-induced neutrophil chemoattractant mRNA expression was not unique to alpha-quartz, being also increased by crocidolite asbestus fibers but not by titanium dioxide or MMVF-10 glass fibers. These findings indicate that epithelial cells contribute to chemokine expression in rat lung after exposure to alpha-quartz and potentially other noxious particles and suggest that alpha-quartz-activated MIP-2 expression in vivo results, at least in part, from a direct action of the particles on the lung epithelium. PMID

  16. Effect of salivary gland adenocarcinoma cell-derived alpha-N-acetylgalactosaminidase on the bioactivity of macrophage activating factor.

    PubMed

    Matsuura, Takashi; Uematsu, Takashi; Yamaoka, Minoru; Furusawa, Kiyofumi

    2004-03-01

    The aim of this study was to clarify the effects of alpha-N-acetylgalactosaminidase (alpha-NaGalase) produced by human salivary gland adenocarcinoma (SGA) cells on the bioactivity of macrophage-activating factor (GcMAF). High exo-alpha-NaGalase activity was detected in the SGA cell line HSG. HSG alpha-NaGalase had both exo- and endo-enzyme activities, cleaving the Gal-GalNAc and GalNAc residues linked to Thr/Ser but not releasing the [NeuAc2-6]GalNac residue. Furthermore, GcMAF enzymatically prepared from the Gc protein enhanced the superoxide-generation capacity and phagocytic activity of monocytes/macrophages. However, GcMAF treated with purified alpha-NaGalase did not exhibit these effects. Thus, HSG possesses the capacity to produce larger quantities of alpha-NaGalase, which inactivates GcMAF produced from Gc protein, resulting in reduced phagocytic activity and superoxide-generation capacity of monocytes/macrophages. The present data strongly suggest that HSG alpha-NaGalase acts as an immunodeficiency factor in cancer patients. PMID:14767536

  17. Mountain grown ginseng induces apoptosis in HL-60 cells and its mechanism have little relation with TNF-alpha production.

    PubMed

    Koo, Hyun-Na; Jeong, Hyun-Ja; Choi, In-Young; An, Hyo-Jin; Moon, Phil-Dong; Kim, Seong-Jin; Jee, Seon-Young; Um, Jae-Young; Hong, Seung-Heon; Shin, Soon-Shik; Yang, Deok-Chun; Seo, Yong-Suk; Kim, Hyung-Min

    2007-01-01

    The root of ginseng is one of the most popular natural tonics in Oriental countries. Ginseng grown in the wild, deep in the mountains, is known as Sansam (mountain grown ginseng, MGG). MGG belongs to Araliaceae and Panax. In this study, we investigated the effects of MGG on the cytotoxicity, induction of apoptosis and the putative pathways of its actions in human promyelocytic leukemia cells, HL-60. Using apoptosis analysis, we found that MGG is a potent inducer of apoptosis, but it has less effect on human peripheral blood mononuclear cells. Caspase-3 activation and subsequent apoptotic cell death in MGG-treated cells were partially blocked by the caspase-3 inhibitor, Z-DEVD-FMK. MGG also inhibited the caspase-8 activity. To determine whether MGG-induced apoptosis is involved in tumor necrosis factor-alpha (TNF-alpha) secretion, TNF-alpha secretion was quantified by enzyme-linked immunosorbent assay (ELISA) method. Unexpectedly, MGG significantly decreased the TNF-alpha secretion compared to the control. These results suggest that MGG-induced cytotoxicity have little relation with the secretion of TNF-alpha in HL-60 cells. Furthermore, MGG with rIFN-gamma synergistically increased nitric oxide (NO) production in mouse peritoneal macrophages. Taken together, our data indicate that MGG is a potent inducer of apoptosis on HL-60 cells and these abilities could be used clinically for the treatment of cancer. PMID:17265560

  18. Characterization of human DNGR-1+ BDCA3+ leukocytes as putative equivalents of mouse CD8alpha+ dendritic cells.

    PubMed

    Poulin, Lionel Franz; Salio, Mariolina; Griessinger, Emmanuel; Anjos-Afonso, Fernando; Craciun, Ligia; Chen, Ji-Li; Keller, Anna M; Joffre, Olivier; Zelenay, Santiago; Nye, Emma; Le Moine, Alain; Faure, Florence; Donckier, Vincent; Sancho, David; Cerundolo, Vincenzo; Bonnet, Dominique; Reis e Sousa, Caetano

    2010-06-01

    In mouse, a subset of dendritic cells (DCs) known as CD8alpha+ DCs has emerged as an important player in the regulation of T cell responses and a promising target in vaccination strategies. However, translation into clinical protocols has been hampered by the failure to identify CD8alpha+ DCs in humans. Here, we characterize a population of human DCs that expresses DNGR-1 (CLEC9A) and high levels of BDCA3 and resembles mouse CD8alpha+ DCs in phenotype and function. We describe the presence of such cells in the spleens of humans and humanized mice and report on a protocol to generate them in vitro. Like mouse CD8alpha+ DCs, human DNGR-1+ BDCA3hi DCs express Necl2, CD207, BATF3, IRF8, and TLR3, but not CD11b, IRF4, TLR7, or (unlike CD8alpha+ DCs) TLR9. DNGR-1+ BDCA3hi DCs respond to poly I:C and agonists of TLR8, but not of TLR7, and produce interleukin (IL)-12 when given innate and T cell-derived signals. Notably, DNGR-1+ BDCA3+ DCs from in vitro cultures efficiently internalize material from dead cells and can cross-present exogenous antigens to CD8+ T cells upon treatment with poly I:C. The characterization of human DNGR-1+ BDCA3hi DCs and the ability to grow them in vitro opens the door for exploiting this subset in immunotherapy. PMID:20479117

  19. Low doses of alpha particles do not induce sister chromatid exchanges in bystander Chinese hamster cells defective in homologous recombination

    SciTech Connect

    Nagasawa, H; Wilson, P F; Chen, D J; Thompson, L H; Bedford, J S; Little, J B

    2007-10-26

    We reported previously that the homologous recombinational repair (HRR)-deficient Chinese hamster mutant cell line irs3 (deficient in the Rad51 paralog Rad51C) showed only a 50% spontaneous frequency of sister chromatid exchange (SCE) as compared to parental wild-type V79 cells. Furthermore, when irradiated with very low doses of alpha particles, SCEs were not induced in irs3 cells, as compared to a prominent bystander effect observed in V79 cells (Nagasawa et al., Radiat. Res. 164, 141-147, 2005). In the present study, we examined additional Chinese hamster cell lines deficient in the Rad51 paralogs Rad51C, Rad51D, Xrcc2, and Xrcc3 as well as another essential HRR protein, Brca2. Spontaneous SCE frequencies in non-irradiated wild-type cell lines CHO, AA8 and V79 were 0.33 SCE/chromosome, whereas two Rad51C-deficient cell lines showed only 0.16 SCE/chromosome. Spontaneous SCE frequencies in cell lines defective in Rad51D, Xrcc2, Xrcc3, and Brca2 ranged from 0.23-0.33 SCE/chromosome, 0-30% lower than wild-type cells. SCEs were induced significantly 20-50% above spontaneous levels in wild-type cells exposed to a mean dose of 1.3 mGy of alpha particles (<1% of nuclei traversed by an alpha particle). However, induction of SCEs above spontaneous levels was minimal or absent after {alpha}-particle irradiation in all of the HRR-deficient cell lines. These data suggest that Brca2 and the Rad51 paralogs contribute to DNA damage repair processes induced in bystander cells (presumably oxidative damage repair in S-phase cells) following irradiation with very low doses of alpha particles.

  20. A special repertoire of alpha:beta T cells in neonatal mice.

    PubMed Central

    Bogue, M; Candéias, S; Benoist, C; Mathis, D

    1991-01-01

    According to several functional criteria, the mature thymocytes of neonatal and adult mice are distinctly different. We wondered whether these differences in function might have a structural correlate: do neonates have a distinct repertoire of alpha:beta T cells? In this study, we have exploited the power of polymerase chain reaction technology to generate large numbers of T cell receptor sequences from sorted thymocyte populations from newborn and adult mice. The newborn-derived sequences show very few N nucleotide additions, usually the major source of diversity in T cell receptors. Most interestingly, the paucity of N insertions appears to be exaggerated by selection events that operate during T cell differentiation in the thymus. The significance of these results is largely: (i) that they parallel recent findings on the B cell repertoire in neonates, raising questions about the reactivities specified by such a special repertoire; and (ii) that they suggest a means to 'tag' T cells exported perinatally, allowing one to test the premise that autoreactive T cells derive preferentially from the newborn repertoire. Images PMID:1834457

  1. Clostridium perfringens Alpha-Toxin Induces Gm1a Clustering and Trka Phosphorylation in the Host Cell Membrane.

    PubMed

    Takagishi, Teruhisa; Oda, Masataka; Kabura, Michiko; Kurosawa, Mie; Tominaga, Kaori; Urano, Shiori; Ueda, Yoshibumi; Kobayashi, Keiko; Kobayashi, Toshihide; Sakurai, Jun; Terao, Yutaka; Nagahama, Masahiro

    2015-01-01

    Clostridium perfringens alpha-toxin elicits various immune responses such as the release of cytokines, chemokines, and superoxide via the GM1a/TrkA complex. Alpha-toxin possesses phospholipase C (PLC) hydrolytic activity that contributes to signal transduction in the pathogenesis of gas gangrene. Little is known about the relationship between lipid metabolism and TrkA activation by alpha-toxin. Using live-cell fluorescence microscopy, we monitored transbilayer movement of diacylglycerol (DAG) with the yellow fluorescent protein-tagged C1AB domain of protein kinase C-γ (EYFP-C1AB). DAG accumulated at the marginal region of the plasma membrane in alpha toxin-treated A549 cells, which also exhibited GM1a clustering and TrkA phosphorylation. Annexin V binding assays showed that alpha-toxin induced the exposure of phosphatidylserine on the outer leaflet of the plasma membrane. However, H148G, a variant toxin which binds cell membrane and has no enzymatic activity, did not induce DAG translocation, GM1a clustering, or TrkA phosphorylation. Alpha-toxin also specifically activated endogenous phospholipase Cγ-1 (PLCγ-1), a TrkA adaptor protein, via phosphorylation. U73122, an endogenous PLC inhibitor, and siRNA for PLCγ-1 inhibited the formation of DAG and release of IL-8. GM1a accumulation and TrkA phosphorylation in A549 cells treated with alpha-toxin were also inhibited by U73122. These results suggest that the flip-flop motion of hydrophobic lipids such as DAG leads to the accumulation of GM1a and TrkA. We conclude that the formation of DAG by alpha-toxin itself (first step) and activation of endogenous PLCγ-1 (second step) leads to alterations in membrane dynamics, followed by strong phosphorylation of TrkA. PMID:25910247

  2. Clostridium perfringens Alpha-Toxin Induces Gm1a Clustering and Trka Phosphorylation in the Host Cell Membrane

    PubMed Central

    Takagishi, Teruhisa; Oda, Masataka; Kabura, Michiko; Kurosawa, Mie; Tominaga, Kaori; Urano, Shiori; Ueda, Yoshibumi; Kobayashi, Keiko; Kobayashi, Toshihide; Sakurai, Jun; Terao, Yutaka; Nagahama, Masahiro

    2015-01-01

    Clostridium perfringens alpha-toxin elicits various immune responses such as the release of cytokines, chemokines, and superoxide via the GM1a/TrkA complex. Alpha-toxin possesses phospholipase C (PLC) hydrolytic activity that contributes to signal transduction in the pathogenesis of gas gangrene. Little is known about the relationship between lipid metabolism and TrkA activation by alpha-toxin. Using live-cell fluorescence microscopy, we monitored transbilayer movement of diacylglycerol (DAG) with the yellow fluorescent protein-tagged C1AB domain of protein kinase C-γ (EYFP-C1AB). DAG accumulated at the marginal region of the plasma membrane in alpha toxin-treated A549 cells, which also exhibited GM1a clustering and TrkA phosphorylation. Annexin V binding assays showed that alpha-toxin induced the exposure of phosphatidylserine on the outer leaflet of the plasma membrane. However, H148G, a variant toxin which binds cell membrane and has no enzymatic activity, did not induce DAG translocation, GM1a clustering, or TrkA phosphorylation. Alpha-toxin also specifically activated endogenous phospholipase Cγ-1 (PLCγ-1), a TrkA adaptor protein, via phosphorylation. U73122, an endogenous PLC inhibitor, and siRNA for PLCγ-1 inhibited the formation of DAG and release of IL-8. GM1a accumulation and TrkA phosphorylation in A549 cells treated with alpha-toxin were also inhibited by U73122. These results suggest that the flip-flop motion of hydrophobic lipids such as DAG leads to the accumulation of GM1a and TrkA. We conclude that the formation of DAG by alpha-toxin itself (first step) and activation of endogenous PLCγ-1 (second step) leads to alterations in membrane dynamics, followed by strong phosphorylation of TrkA. PMID:25910247

  3. Interleukin-6 enhances insulin secretion by increasing glucagon-like peptide-1 secretion from L cells and alpha cells

    PubMed Central

    Ellingsgaard, Helga; Hauselmann, Irina; Schuler, Beat; Habib, Abdella M; Baggio, Laurie L; Meier, Daniel T; Eppler, Elisabeth; Bouzakri, Karim; Wueest, Stephan; Muller, Yannick D; Hansen, Ann Maria Kruse; Reinecke, Manfred; Konrad, Daniel; Gassmann, Max; Reimann, Frank; Halban, Philippe A; Gromada, Jesper; Drucker, Daniel J; Gribble, Fiona M; Ehses, Jan A; Donath, Marc Y

    2014-01-01

    Exercise, obesity and type 2 diabetes are associated with elevated plasma concentrations of interleukin-6 (IL-6). Glucagon-like peptide-1 (GLP-1) is a hormone that induces insulin secretion. Here we show that administration of IL-6 or elevated IL-6 concentrations in response to exercise stimulate GLP-1 secretion from intestinal L cells and pancreatic alpha cells, improving insulin secretion and glycemia. IL-6 increased GLP-1 production from alpha cells through increased proglucagon (which is encoded by GCG) and prohormone convertase 1/3 expression. In models of type 2 diabetes, the beneficial effects of IL-6 were maintained, and IL-6 neutralization resulted in further elevation of glycemia and reduced pancreatic GLP-1. Hence, IL-6 mediates crosstalk between insulin-sensitive tissues, intestinal L cells and pancreatic islets to adapt to changes in insulin demand. This previously unidentified endocrine loop implicates IL-6 in the regulation of insulin secretion and suggests that drugs modulating this loop may be useful in type 2 diabetes. PMID:22037645

  4. Interleukin-6 enhances insulin secretion by increasing glucagon-like peptide-1 secretion from L cells and alpha cells.

    PubMed

    Ellingsgaard, Helga; Hauselmann, Irina; Schuler, Beat; Habib, Abdella M; Baggio, Laurie L; Meier, Daniel T; Eppler, Elisabeth; Bouzakri, Karim; Wueest, Stephan; Muller, Yannick D; Hansen, Ann Maria Kruse; Reinecke, Manfred; Konrad, Daniel; Gassmann, Max; Reimann, Frank; Halban, Philippe A; Gromada, Jesper; Drucker, Daniel J; Gribble, Fiona M; Ehses, Jan A; Donath, Marc Y

    2011-01-01

    Exercise, obesity and type 2 diabetes are associated with elevated plasma concentrations of interleukin-6 (IL-6). Glucagon-like peptide-1 (GLP-1) is a hormone that induces insulin secretion. Here we show that administration of IL-6 or elevated IL-6 concentrations in response to exercise stimulate GLP-1 secretion from intestinal L cells and pancreatic alpha cells, improving insulin secretion and glycemia. IL-6 increased GLP-1 production from alpha cells through increased proglucagon (which is encoded by GCG) and prohormone convertase 1/3 expression. In models of type 2 diabetes, the beneficial effects of IL-6 were maintained, and IL-6 neutralization resulted in further elevation of glycemia and reduced pancreatic GLP-1. Hence, IL-6 mediates crosstalk between insulin-sensitive tissues, intestinal L cells and pancreatic islets to adapt to changes in insulin demand. This previously unidentified endocrine loop implicates IL-6 in the regulation of insulin secretion and suggests that drugs modulating this loop may be useful in type 2 diabetes. PMID:22037645

  5. Transforming growth factor alpha treatment alters intracellular calcium levels in hair cells and protects them from ototoxic damage in vitro.

    PubMed

    Staecker, H; Dazert, S; Malgrange, B; Lefebvre, P P; Ryan, A F; Van de Water, T R

    1997-07-01

    To determine if transforming growth factor alpha (TGF alpha) pretreatment protects hair cells from aminoglycoside induced injury by modifying their intracellular calcium concentration, we assayed hair cell calcium levels in organ of Corti explants both before and after aminoglycoside (i.e. neomycin, 10(-3) M) exposure either with or without growth factor pretreatment. After TGF alpha (500 ng/ml) treatment, the intracellular calcium level of hair cells showed a five-fold increase as compared to the levels observed in the hair cells of control cultures. After ototoxin exposure, calcium levels in hair cells of control explants showed an increase relative to their baseline levels, while in the presence of growth factors pretreatment, hair cells showed a relative reduction in calcium levels. Pretreatment of organ of Corti explants afforded significant protection of hair cell stereocilia bundle morphology from ototoxic damage when compared to explants exposed to ototoxin alone. This study correlates a rise in hair cell calcium levels with the otoprotection of hair cells by TGF alpha in organ of Corti explants. PMID:9263032

  6. Structure and diversity of the T-cell receptor alpha chain in the Mexican axolotl.

    PubMed

    Fellah, J S; Kerfourn, F; Dumay, A M; Aubet, G; Charlemagne, J

    1997-01-01

    Polymerase chain reaction was used to isolate cDNA clones encoding putative T-cell receptor (TCR) alpha chains in an amphibian, the Mexican axolotl (Ambystoma mexicanum). Five TCRalpha-V chain-encoding segments were identified, each belonging to a separate family. The best identity scores for these axolotl TCRalpha-V segments were all provided by sequences belonging to the human TCRalpha-V1 family and the mouse TCRalpha-V3 and TCRalpha-V8 families. A total of 14 different TCRA-J segments were identified from 44 TCRA-V/TCRA-J regions sequenced, suggesting that a large repertoire of TCRA-J segments is a characteristic of most vertebrates. The structure of the axolotl CDR3 alpha chain loop is in good agreement with that of mammals, including a majority of small hydrophobic residues at position 92 and of charged, hydrophilic, or polar residues at positions 93 and 94, which are highly variable and correspond to the TCRA-V/J junction. This suggests that some positions of the axolotl CDR3 alpha chain loop are positively selected during T-cell differentiation, particularly around residue 93 that could be selected for its ability to makes contacts with major histocompatibility complex-associated antigenic peptides, as in mammals. The axolotl Calpha domain had the typical structure of mammalian and avian Calpha domains, including the charged residues in the TM segment that are thought to interact with other proteins in the membrane, as well as most of the residues forming the conserved antigen receptor transmembrane motif. PMID:9002443

  7. Association of solubilized angiotensin II receptors with phospholipase C-alpha in murine neuroblastoma NIE-115 cells.

    PubMed

    Mah, S J; Ades, A M; Mir, R; Siemens, I R; Williamson, J R; Fluharty, S J

    1992-08-01

    The peptide angiotensin II (AngII) has been reported to stimulate phosphoinositide-specific phospholipase C (PLC) activity in the murine neuroblastoma cell line N1E-115. In the present study, polyclonal antibodies raised against a PLC isoenzyme, PLC-alpha, reacted with a 60-kDa protein present in both membrane and cytosolic fractions of differentiated N1E-115 cells. In order to examine the possible association of PLC-alpha with cell surface AngII receptors (AngII-Rs), membranes from differentiated N1E-115 cells were solubilized, using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). CHAPS (1%) solubilized AngII-Rs, from N1E-115 cells, that maintained their high affinity for agonists. Gel filtration analysis of the solubilized membranes revealed that the majority of the specific binding of 125I-AngII eluted as a large protein complex with a molecular mass of 380 kDa and that agonist binding was partially reduced by guanosine-5'-O-(3-thio)triphosphate (GTP gamma S), within this complex. CHAPS also effectively solubilized immunoreactive PLC-alpha, from N1E-115 cell membranes, that was similarly present within the 380-kDa AngII-binding complex. Anti-PLC-alpha antisera immunoprecipitated approximately 16% of the total phosphatidylinositol-4,5-bisphosphate-specific PLC activity in the 1% CHAPS extract and 40% of cytosolic PLC activity. Moreover, a 60-kDa 35S-Trans S-labeled protein, comigrating with immunoreactive PLC-alpha, was immunoprecipitated from the 1% CHAPS extract by the antisera. In addition, anti-PLC-alpha antisera immunoprecipitated approximately 20% of solubilized AngII-Rs prebound with 125I-AngII but failed to precipitate receptors prebound with the antagonist 125I-Sarc1,Ile8-AngII. The anti-PLC-alpha antisera also immunoprecipitated AngII-Rs when intact membranes were labeled with 125I-AngII before solubilization in 1% CHAPS, suggesting that the AngII-R interaction with PLC-alpha was not the result of detergent

  8. The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor regulates cell surface plasminogen activator activity on human trophoblast cells.

    PubMed

    Zhang, J C; Sakthivel, R; Kniss, D; Graham, C H; Strickland, D K; McCrae, K R

    1998-11-27

    The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor (LRP/alpha2MR) mediates the internalization of numerous ligands, including prourokinase (pro-UK) and complexes between two-chain urokinase (tc-u-PA) and plasminogen activator inhibitor type-1 (PAI-1). It has been suggested that through its ability to internalize these ligands, LRP/alpha2MR may regulate the expression of plasminogen activator activity on cell surfaces; this hypothesis, however, has not been experimentally confirmed. To address this issue, we assessed the ability of LRP/alpha2MR to regulate plasminogen activator activity on human trophoblast cells, which express both LRP/alpha2MR and the urokinase receptor (uPAR). Trophoblasts internalized and degraded exogenous 125I-pro-UK (primarily following its conversion to tc-u-PA and incorporation into tc-u-PA.PAI complexes) in an LRP/alpha2MR-dependent manner, which was inhibited by the LRP/alpha2MR receptor-associated protein. Receptor-associated protein also caused a approximately 50% reduction in cell surface plasminogen activator activity and delayed the regeneration of unoccupied uPAR by cells on which uPAR were initially saturated with pro-UK. Identical effects were caused by anti-LRP/alpha2MR antibodies. These results demonstrate that LRP/alpha2MR promotes the expression of cell surface plasminogen activator activity on trophoblasts by facilitating the clearance of tc-u-PA.PAI complexes and regeneration of unoccupied cell surface uPAR. PMID:9822706

  9. A case of an alpha-fetoprotein-producing intrahepatic cholangiocarcinoma suggests probable cancer stem cell origin.

    PubMed

    Ishikawa, Kenji; Sasaki, Atsushi; Haraguchi, Naotsugu; Yoshikawa, Yasuji; Mori, Masaki

    2007-03-01

    Recent evidence suggests that some cancers may originate from cancer stem cells, which may derive from carcinogenesis of normal stem cells. A hepatic progenitor cell population, which gives rise to hepatocytes and cholangiocytes, has been suggested in humans, though whether these cells can give rise to malignant tumors has not been confirmed. We report here a case of an alpha-fetoprotein (AFP)-producing intrahepatic cholangiocarcinoma (ICC) in an 81-year-old woman with chronic hepatitis C viral infection, suggesting malignant transformation of hepatic stem cells as a mechanism for hepatic neoplasia. Abdominal computed tomography revealed a low-density mass with surrounding enhancement measuring 5 cm x 5 cm in segments IV and VIII of the liver. The preoperative serum levels of tumor markers were 1.7 ng/ml of carcinoembryonic antigen, 22 mAU/ml of protein induced by vitamin K absence or antagonist II, 43.4 U/ml of carbohydrate antigen 19-9, and 1,560 ng/ml of AFP. Following central bisegmentectomy of the liver, serum AFP levels decreased dramatically. Histologically, the tumor cells showed indistinct glandular structures with abundant fibrous stroma. Immunohistochemical analysis demonstrated that the neoplastic cells reacted strongly to antibodies against AFP and cytokeratin (CK) 7. In addition, cancer cells showed partially positive reaction to anti-CK14, a liver stem cell marker, and to anticluster designation (CD) 133, a hematopoietic stem cell marker, and negative reaction to antihepatocyte paraffin (HepPar) 1. These data may indicate that the tumor was derived from a normal liver stem cell that underwent oncogenic transformation. PMID:17405896

  10. RhoA-dependent switch between alpha2beta1 and alpha3beta1 integrins is induced by laminin-5 during early stage of HT-29 cell differentiation.

    PubMed

    Gout, S P; Jacquier-Sarlin, M R; Rouard-Talbot, L; Rousselle, P; Block, M R

    2001-10-01

    Integrin-mediated interactions between the basement membrane and epithelial cells control the differentiation of epithelia. We characterized the modulation of adhesive behaviors to basement membrane proteins and of integrin function in the human colon adenocarcinoma HT-29 cell line, which differentiates into enterocytes after the substitution of galactose for glucose in the medium. We demonstrate an increased capability of these cells to adhere to collagen type IV during the early stage of differentiation. This effect occurs without any changes in integrin cell surface expression but rather results from an alpha2beta1/alpha3beta1 integrin switch, alpha3beta1 integrin becoming the major collagen receptor. The increase in laminin-5 secretion and deposit on the matrix is a key factor in the mechanism regulating cell adhesion, because it is responsible for the activation of alpha3beta1 integrin. Furthermore, down-regulation of RhoA GTPase activity occurs during HT-29 cell differentiation and correlates with the activation of the integrin alpha3beta1. Indeed, C3 transferase, a RhoA GTPase inhibitor, induces a similar alpha2beta1/alpha3beta1 switch in undifferentiated HT-29 cells. These results indicate that the decrease in RhoA activation is the biochemical mechanism underlying this integrin switch observed during cell differentiation. The physiological relevance of such modulation of integrin activity in the functioning of the crypt-villus axis is discussed. PMID:11598208

  11. The synergistic necrohemorrhagic action of Clostridium perfringens perfringolysin and alpha toxin in the bovine intestine and against bovine endothelial cells

    PubMed Central

    2013-01-01

    Bovine necrohemorrhagic enteritis is a major cause of mortality in veal calves. Clostridium perfringens is considered as the causative agent, but there has been controversy on the toxins responsible for the disease. Recently, it has been demonstrated that a variety of C. perfringens type A strains can induce necrohemorrhagic lesions in a calf intestinal loop assay. These results put forward alpha toxin and perfringolysin as potential causative toxins, since both are produced by all C. perfringens type A strains. The importance of perfringolysin in the pathogenesis of bovine necrohemorrhagic enteritis has not been studied before. Therefore, the objective of the current study was to evaluate the role of perfringolysin in the development of necrohemorrhagic enteritis lesions in calves and its synergism with alpha toxin. A perfringolysin-deficient mutant, an alpha toxin-deficient mutant and a perfringolysin alpha toxin double mutant were less able to induce necrosis in a calf intestinal loop assay as compared to the wild-type strain. Only complementation with both toxins could restore the activity to that of the wild-type. In addition, perfringolysin and alpha toxin had a synergistic cytotoxic effect on bovine endothelial cells. This endothelial cell damage potentially explains why capillary hemorrhages are an initial step in the development of bovine necrohemorrhagic enteritis. Taken together, our results show that perfringolysin acts synergistically with alpha toxin in the development of necrohemorrhagic enteritis in a calf intestinal loop model and we hypothesize that both toxins act by targeting the endothelial cells. PMID:23782465

  12. Purification and characterization of the glycoprotein hormone. cap alpha. -subunit-like material secreted by HeLa cells

    SciTech Connect

    Cox, G.S.; Rimerman, R.A.

    1988-08-23

    The protein secreted by HeLa cells that cross-reacts with antiserum developed against the ..cap alpha..-subunit of human chorionic gonadotropin (hCG) has been purified approximately 30,000-fold from concentrated culture medium by organic solvent fractionation followed by ion exchange, gel filtration, and lectin affinity chromatography. The final preparation had a specific activity (by RIA) of 6.8 x 10/sup 5/ ng of ..cap alpha../mg of protein and appeared homogeneous by electrophoresis on reducing/denaturing polyacrylamide gels (SDS-PAGE). Amino acid analysis indicated that HeLa-..cap alpha.. had a composition very similar to that of the urinary hCG ..cap alpha..-subunit. However, comparison of hCG-..cap alpha.. and HeLa-..cap alpha.. demonstrated that the tumor-associated subunit was not identical with its normal counterpart. The purified tumor protein had an apparent molecular weight greater than that of the urinary ..cap alpha..-subunit when analyzed by SDS-PAGE, and this difference was even greater when a partially purified preparation was examined by an immunoblot technique (Western). Isoelectric focusing of the HeLa and hCG subunits demonstrated that the tumor protein had a lower pI. Immunoprecipitation and electrophoresis of ..cap alpha..-subunit from HeLa cultures labeled with (/sup 3/H)fucose indicated that the tumor subunit was fucosylated, whereas analysis of hCG-..cap alpha.. hydrosylates by HPLC confirmed previous reports that the placental subunit does not contain fucose. The results indicate that, regardless of whether or not a single ..cap alpha..-subunit gene is being expressed in both normal and neoplastic tissues, posttranslational modifications lead to a highly altered subunit in the tumor. The differences observed may be useful in diagnosing neoplastic vs hyperplastic conditions and may lend insight into the mechanism of ectopic hormone production by tumors.

  13. PGC-1alpha induces dynamic protein interactions on the ERRalpha gene multi-hormone response element nucleosome in kidney cells.

    PubMed

    Wang, Liangli; Li, Yin; Hu, Peng; Teng, Christina T

    2008-12-15

    ERR (oestrogen-related receptor)-alpha modulates the oestrogen signalling pathway and regulates genes participating in the physiological energy balance programme. Oestrogen and PGC-1alpha (peroxisome proliferator-activated receptor-gamma coactivator-1alpha), the master regulator of the energy homoeostasis programme, both regulate the expression of ERRalpha through the MHRE (multi-hormone response element) of the ERRalpha gene. Although the molecular mechanism of oestrogen action on ERRalpha regulation is well characterized, the mechanism of PGC-1alpha induction is unclear. In this study, we examine chromatin structural changes and protein interactions at the MHRE nucleosome in response to PGC-1alpha expression in HK2 human kidney cells. We mapped the nucleosome positions of the ERRalpha gene promoter and examined the changes of histone acetylation in response to PGC-1alpha expression. The interactions of DNA-binding proteins, ERRalpha and ERRgamma, co-activators {CBP [CREB (cAMP-response-element-binding protein)-binding protein], p300, PCAF (p300/CBP-associated factor)}, co-repressor [RIP140 (receptor-interacting protein of 140 kDa)] and RNA polymerase II at the MHRE nucleosome region were investigated over time before and after PGC-1alpha expression in the HK2 cells. We found a dynamic cyclic interaction of these proteins shortly after PGC-1alpha expression and a slower cycling interaction, with fewer proteins involved, 20 h later. By using the siRNA (small interfering RNA) knockdown approach, we discovered that ERRgamma was involved in the initial phase, but not in the later phase, of PGC-1alpha-induced ERRalpha expression. PMID:18673300

  14. PTH-related protein upregulates integrin {alpha}6{beta}4 expression and activates Akt in breast cancer cells

    SciTech Connect

    Shen Xiaoli; Falzon, Miriam . E-mail: mfalzon@utmb.edu

    2006-11-15

    Breast cancer is the most common carcinoma that metastasizes to bone. Tumor-produced parathyroid hormone-related protein (PTHrP), a known stimulator of osteoclastic bone resorption, is a major mediator of the osteolytic process in breast cancer. We have previously shown that PTHrP increases breast cancer cell proliferation, survival, migration, and pro-invasive integrin {alpha}6{beta}4 expression. To determine the role of integrin {alpha}6{beta}4 in these PTHrP-mediated effects, we utilized two strategies to modulate expression of the {alpha}6 and {beta}4 subunits in parental and PTHrP-overexpressing MDA-MB-231 and MCF-7 cells: overexpression of {alpha}6{beta}4 by transfection with constructs encoding the {alpha}6 and {beta}4 subunits, and suppression of endogenous {alpha}6{beta}4 expression by transfection with siRNAs targeting these subunits. We now show that the effects of PTHrP are mediated via upregulation of integrin {alpha}6{beta}4 expression. We also show that integrin {alpha}6{beta}4 expression is modulated at the mRNA level, indicating a transcriptional and/or post-transcriptional mechanism of action for PTHrP. PTHrP expression also increased the levels of phosphorylated Akt, with a consequent increase in the levels of phosphorylated (inactive) glycogen synthase kinase-3 (GSK-3). The role of PTHrP in breast cancer growth and metastasis may thus be mediated via upregulation of integrin {alpha}6{beta}4 expression and Akt activation, with consequent inactivation of GSK-3.

  15. Regression of hepatocarcinoma cells using RNA aptamer specific to alpha-fetoprotein

    SciTech Connect

    Lee, Young Ju; Lee, Seong-Wook

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Identification of RNA aptamer specific to AFP with high affinity. Black-Right-Pointing-Pointer Specific induction of HCC proliferation by AFP. Black-Right-Pointing-Pointer Efficient increase in oncogene expression by AFP. Black-Right-Pointing-Pointer Efficient inhibition of AFP-mediated HCC proliferation by the aptamer. Black-Right-Pointing-Pointer Efficient suppression of AFP-induced oncogene expression of by the aptamer. -- Abstract: Alpha-fetoprotein (AFP) is a cancer-associated fetal protein and has long been utilized as a serum fetal defect/tumor marker to monitor distress/disease progression. In addition, AFP is closely associated with the proliferation of hepatocellular carcinoma. Thus, direct targeting of AFP has been recommended for a therapeutic strategy against hepatocellular carcinoma. In this study, we developed and characterized an RNA aptamer that specifically bound to the alpha-fetoprotein using SELEX technology. The aptamer interacted with the AFP with a K{sub D} of {approx}33 nM. Importantly, the identified aptamer specifically and efficiently inhibited the AFP-mediated proliferation of hepatocarcinoma cells in a dose dependent manner. Moreover, the aptamer efficiently down-regulated AFP-induced expression of oncogenes in the cells. These results indicate that an AFP-specific RNA aptamer could be a useful therapeutic and diagnostic agent against AFP-related hepatocellular carcinoma.

  16. Differences between normal and alpha-synuclein overexpressing SH-SY5Y neuroblastoma cells after Abeta(1-42) and NAC treatment.

    PubMed

    Hunya, Akos; Földi, István; Szegedi, Viktor; Soós, Katalin; Zarándi, Márta; Szabó, Antal; Zádori, Dénes; Penke, Botond; Datki, Zsolt L

    2008-03-28

    Alpha-synuclein (alphaSN) plays a major role in numerous neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease. Intracellular inclusions containing aggregated alphaSN have been reported in Alzheimer's and Parkinson's affected brains. Moreover, a proteolytic fragment of alphaSN, the so-called non-amyloid component of Alzheimer's disease amyloid (NAC) was found to be an integral part of Alzheimer's dementia related plaques. Despite the extensive research on this topic, the exact toxic mechanism of alphaSN remains elusive. We have taken the advantage of an alphaSN overexpressing SH-SY5Y cell line and investigated the effects of classical apoptotic factors (e.g. H(2)O(2), amphotericin B and ruthenium red) and aggregated disease-related peptides on cell viability compared to wild type neuroblastoma cells. It was found that alphaSN overexpressing cells are more sensitive to aggregated peptides treatment than normal expressing counterparts. In contrast, cells containing elevated amount of alphaSN were less vulnerable to classical apoptotic stressors than wild type cells. In addition, alphaSN overexpression is accompanied by altered phenotype, attenuated proliferation kinetics, increased neurite arborisation and decreased cell motility. Based on these results, the alphaSN overexpressing cell lines may represent a good and effective in vitro model for Alzheimer's and Parkinson's disease. PMID:18355641

  17. Choline kinase-alpha by regulating cell aggressiveness and drug sensitivity is a potential druggable target for ovarian cancer

    PubMed Central

    Granata, A; Nicoletti, R; Tinaglia, V; De Cecco, L; Pisanu, M E; Ricci, A; Podo, F; Canevari, S; Iorio, E; Bagnoli, M; Mezzanzanica, D

    2014-01-01

    Background: Aberrant choline metabolism has been proposed as a novel cancer hallmark. We recently showed that epithelial ovarian cancer (EOC) possesses an altered MRS-choline profile, characterised by increased phosphocholine (PCho) content to which mainly contribute over-expression and activation of choline kinase-alpha (ChoK-alpha). Methods: To assess its biological relevance, ChoK-alpha expression was downmodulated by transient RNA interference in EOC in vitro models. Gene expression profiling by microarray analysis and functional analysis was performed to identify the pathway/functions perturbed in ChoK-alpha-silenced cells, then validated by in vitro experiments. Results: In silenced cells, compared with control, we observed: (I) a significant reduction of both CHKA transcript and ChoK-alpha protein expression; (II) a dramatic, proportional drop in PCho content ranging from 60 to 71%, as revealed by 1H-magnetic spectroscopy analysis; (III) a 35–36% of cell growth inhibition, with no evidences of apoptosis or modification of the main cellular survival signalling pathways; (IV) 476 differentially expressed genes, including genes related to lipid metabolism. Ingenuity pathway analysis identified cellular functions related to cell death and cellular proliferation and movement as the most perturbed. Accordingly, CHKA-silenced cells displayed a significant delay in wound repair, a reduced migration and invasion capability were also observed. Furthermore, although CHKA silencing did not directly induce cell death, a significant increase of sensitivity to platinum, paclitaxel and doxorubicin was observed even in a drug-resistant context. Conclusion: We showed for the first time in EOC that CHKA downregulation significantly decreased the aggressive EOC cell behaviour also affecting cells' sensitivity to drug treatment. These observations open the way to further analysis for ChoK-alpha validation as a new EOC therapeutic target to be used alone or in combination with

  18. Estrogen receptor alpha is cell cycle-regulated and regulates the cell cycle in a ligand-dependent fashion.

    PubMed

    JavanMoghadam, Sonia; Weihua, Zhang; Hunt, Kelly K; Keyomarsi, Khandan

    2016-06-17

    Estrogen receptor alpha (ERα) has been implicated in several cell cycle regulatory events and is an important predictive marker of disease outcome in breast cancer patients. Here, we aimed to elucidate the mechanism through which ERα influences proliferation in breast cancer cells. Our results show that ERα protein is cell cycle-regulated in human breast cancer cells and that the presence of 17-β-estradiol (E2) in the culture medium shortened the cell cycle significantly (by 4.5 hours, P < 0.05) compared with unliganded conditions. The alterations in cell cycle duration were observed in the S and G2/M phases, whereas the G1 phase was indistinguishable under liganded and unliganded conditions. In addition, ERα knockdown in MCF-7 cells accelerated mitotic exit, whereas transfection of ERα-negative MDA-MB-231 cells with exogenous ERα significantly shortened the S and G2/M phases (by 9.1 hours, P < 0.05) compared with parental cells. Finally, treatment of MCF-7 cells with antiestrogens revealed that tamoxifen yields a slower cell cycle progression through the S and G2/M phases than fulvestrant does, presumably because of the destabilizing effect of fulvestrant on ERα protein. Together, these results show that ERα modulates breast cancer cell proliferation by regulating events during the S and G2/M phases of the cell cycle in a ligand-dependent fashion. These results provide the rationale for an effective treatment strategy that includes a cell cycle inhibitor in combination with a drug that lowers estrogen levels, such as an aromatase inhibitor, and an antiestrogen that does not result in the degradation of ERα, such as tamoxifen. PMID:27049344

  19. Integrin-Alpha IIb Identifies Murine Lymph Node Lymphatic Endothelial Cells Responsive to RANKL

    PubMed Central

    Cordeiro, Olga G.; Chypre, Mélanie; Brouard, Nathalie; Rauber, Simon; Alloush, Farouk; Romera-Hernandez, Monica; Bénézech, Cécile; Li, Zhi; Eckly, Anita; Coles, Mark C.; Rot, Antal; Yagita, Hideo; Léon, Catherine; Ludewig, Burkhard; Cupedo, Tom; Lanza, François; Mueller, Christopher G.

    2016-01-01

    Microenvironment and activation signals likely imprint heterogeneity in the lymphatic endothelial cell (LEC) population. Particularly LECs of secondary lymphoid organs are exposed to different cell types and immune stimuli. However, our understanding of the nature of LEC activation signals and their cell source within the secondary lymphoid organ in the steady state remains incomplete. Here we show that integrin alpha 2b (ITGA2b), known to be carried by platelets, megakaryocytes and hematopoietic progenitors, is expressed by a lymph node subset of LECs, residing in medullary, cortical and subcapsular sinuses. In the subcapsular sinus, the floor but not the ceiling layer expresses the integrin, being excluded from ACKR4+ LECs but overlapping with MAdCAM-1 expression. ITGA2b expression increases in response to immunization, raising the possibility that heterogeneous ITGA2b levels reflect variation in exposure to activation signals. We show that alterations of the level of receptor activator of NF-κB ligand (RANKL), by overexpression, neutralization or deletion from stromal marginal reticular cells, affected the proportion of ITGA2b+ LECs. Lymph node LECs but not peripheral LECs express RANK. In addition, we found that lymphotoxin-β receptor signaling likewise regulated the proportion of ITGA2b+ LECs. These findings demonstrate that stromal reticular cells activate LECs via RANKL and support the action of hematopoietic cell-derived lymphotoxin. PMID:27010197

  20. Behaviour of oleic acid-depleted bovine alpha-lactalbumin made LEthal to tumor cells (BAMLET).

    PubMed

    Hoque, Mehboob; Gupta, Jyoti; Rabbani, Gulam; Khan, Rizwan Hasan; Saleemuddin, M

    2016-05-24

    Oleic acid (OA) complexes of human alpha-lactalbumin (α-LA) and several other proteins are effective in the killing of a variety of tumor cells. While debate on whether the key component of the complexes is the OA or protein continues, studies probing the mechanism of action of the complexes at the tumor cell surface or in the cell interior assume the action of a molecule in the form of an undissociated complex. Recent evidence however suggests that OA complexes of protein are stripped of bound OA on interaction with artificial or natural membranes before entering the cell. Properties of BAMLET stripped of its OA by exposure to erythrocytes (ET-BAMLET) were investigated in the study. ET-BAMLET resembled α-LA in its inability to induce hemolysis of erythrocytes and behaviour in a gel filtration column. Spectroscopy techniques-fluorescence, far- and near UV CD as well as calorimetry and proteolysis however suggest the molecule to be different both from native α-LA and the apo form. Remarkably, unlike native α-LA and apo-α-LA, ET-BAMLET binds OA and turns hemolytic by simple mixing with the fatty acid around neutral pH. Since BAMLET/HAMLET incubated cells take up large amounts of OA, the study suggests the possibility of ET-BAMLET combining with OA and reforming the complex inside the cells. PMID:27109252

  1. 17{alpha}-Estradiol arrests cell cycle progression at G{sub 2}/M and induces apoptotic cell death in human acute leukemia Jurkat T cells

    SciTech Connect

    Jun, Do Youn; Park, Hae Sun; Kim, Jun Seok; Kim, Jong Sik; Park, Wan; Song, Bang Ho; Kim, Hee-Sook; Taub, Dennis; Kim, Young Ho

    2008-09-15

    A pharmacological dose (2.5-10 {mu}M) of 17{alpha}-estradiol (17{alpha}-E{sub 2}) exerted a cytotoxic effect on human leukemias Jurkat T and U937 cells, which was not suppressed by the estrogen receptor (ER) antagonist ICI 182,780. Along with cytotoxicity in Jurkat T cells, several apoptotic events including mitochondrial cytochrome c release, activation of caspase-9, -3, and -8, PARP degradation, and DNA fragmentation were induced. The cytotoxicity of 17{alpha}-E{sub 2} was not blocked by the anti-Fas neutralizing antibody ZB-4. While undergoing apoptosis, there was a remarkable accumulation of G{sub 2}/M cells with the upregulatoin of cdc2 kinase activity, which was reflected in the Thr56 phosphorylation of Bcl-2. Dephosphorylation at Tyr15 and phosphorylation at Thr161 of cdc2, and significant increase in the cyclin B1 level were underlying factors for the cdc2 kinase activation. Whereas the 17{alpha}-E{sub 2}-induced apoptosis was completely abrogated by overexpression of Bcl-2 or by pretreatment with the pan-caspase inhibitor z-VAD-fmk, the accumulation of G{sub 2}/M cells significantly increased. The caspase-8 inhibitor z-IETD-fmk failed to influence 17{alpha}-E{sub 2}-mediated caspase-9 activation, but it markedly reduced caspase-3 activation and PARP degradation with the suppression of apoptosis, indicating the contribution of caspase-8; not as an upstream event of the mitochondrial cytochrome c release, but to caspase-3 activation. In the presence of hydroxyurea, which blocked the cell cycle progression at the G{sub 1}/S boundary, 17{alpha}-E{sub 2} failed to induce the G{sub 2}/M arrest as well as apoptosis. These results demonstrate that the cytotoxicity of 17{alpha}-E{sub 2} toward Jurkat T cells is attributable to apoptosis mainly induced in G{sub 2}/M-arrested cells, in an ER-independent manner, via a mitochondria-dependent caspase pathway regulated by Bcl-2.

  2. Andrographolide down-regulates hypoxia-inducible factor-1{alpha} in human non-small cell lung cancer A549 cells

    SciTech Connect

    Lin, Hui-Hsuan; Tsai, Chia-Wen; Chou, Fen-Pi; Wang, Chau-Jong; Hsuan, Shu-Wen; Wang, Cheng-Kun; Chen, Jing-Hsien

    2011-02-01

    Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess multiple pharmacological activities. In our previous study, Andro had been shown to inhibit non-small cell lung cancer (NSCLC) A549 cell migration and invasion via down-regulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Here we demonstrated that Andro inhibited the expression of hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) in A549 cells. HIF-1{alpha} plays an important role in tumor growth, angiogenesis and lymph node metastasis of NSCLC. The Andro-induced decrease of cellular protein level of HIF-1{alpha} was correlated with a rapid ubiquitin-dependent degradation of HIF-1{alpha}, and was accompanied by increased expressions of hydroxyl-HIF-1{alpha} and prolyl hydroxylase (PHD2), and a later decrease of vascular endothelial growth factor (VEGF) upon the treatment of Andro. The Andro-inhibited VEGF expression appeared to be a consequence of HIF-1{alpha} inactivation, because its DNA binding activity was suppressed by Andro. Molecular data showed that all these effects of Andro might be mediated via TGF{beta}1/PHD2/HIF-1{alpha} pathway, as demonstrated by the transfection of TGF{beta}1 overexpression vector and PHD2 siRNA, and the usage of a pharmacological MG132 inhibitor. Furthermore, we elucidated the involvement of Andro in HIF-1{alpha} transduced VEGF expression in A549 cells and other NSCLC cell lines. In conclusion, these results highlighted the potential effects of Andro, which may be developed as a chemotherapeutic or an anti-angiogenesis agent for NSCLC in the future.

  3. Cell-associated alpha-amylases of butyrate-producing Firmicute bacteria from the human colon.

    PubMed

    Ramsay, Alan G; Scott, Karen P; Martin, Jenny C; Rincon, Marco T; Flint, Harry J

    2006-11-01

    Selected butyrate-producing bacteria from the human colon that are related to Roseburia spp. and Butyrivibrio fibrisolvens showed a good ability to utilize a variety of starches for growth when compared with the Gram-negative amylolytic anaerobe Bacteroides thetaiotaomicron. A major cell-associated amylase of high molecular mass (140-210 kDa) was detected in each strain by SDS-PAGE zymogram analysis, and genes corresponding to these enzymes were analysed for two representative strains. Amy13B from But. fibrisolvens 16/4 is a multi-domain enzyme of 144.6 kDa that includes a family 13 glycoside hydrolase domain, and duplicated family 26 carbohydrate-binding modules. Amy13A (182.4 kDa), from Roseburia inulinivorans A2-194, also includes a family 13 domain, which is preceded by two repeat units of approximately 116 aa rich in aromatic residues, an isoamylase N-terminal domain, a pullulanase-associated domain, and an additional unidentified domain. Both Amy13A and Amy13B have N-terminal signal peptides and C-terminal cell-wall sorting signals, including a modified LPXTG motif similar to that involved in interactions with the cell surface in other Gram-positive bacteria, a hydrophobic transmembrane segment, and a basic C terminus. The overexpressed family 13 domains showed an absolute requirement for Mg2+ or Ca2+ for activity, and functioned as 1,4-alpha-glucanohydrolases (alpha-amylases; EC 3.2.1.1). These major starch-degrading enzymes thus appear to be anchored to the cell wall in this important group of human gut bacteria. PMID:17074899

  4. Canarypox Virus-Induced Maturation of Dendritic Cells Is Mediated by Apoptotic Cell Death and Tumor Necrosis Factor Alpha Secretion

    PubMed Central

    Ignatius, Ralf; Marovich, Mary; Mehlhop, Erin; Villamide, Loreley; Mahnke, Karsten; Cox, William I.; Isdell, Frank; Frankel, Sarah S.; Mascola, John R.; Steinman, Ralph M.; Pope, Melissa

    2000-01-01

    Recombinant avipox viruses are being widely evaluated as vaccines. To address how these viruses, which replicate poorly in mammalian cells, might be immunogenic, we studied how canarypox virus (ALVAC) interacts with primate antigen-presenting dendritic cells (DCs). When human and rhesus macaque monocyte-derived DCs were exposed to recombinant ALVAC, immature DCs were most susceptible to infection. However, many of the infected cells underwent apoptotic cell death, and dying infected cells were engulfed by uninfected DCs. Furthermore, a subset of DCs matured in the ALVAC-exposed DC cultures. DC maturation coincided with tumor necrosis factor alpha (TNF-α) secretion and was significantly blocked in the presence of anti-TNF-α antibodies. Interestingly, inhibition of apoptosis with a caspase 3 inhibitor also reduced some of the maturation induced by exposure to ALVAC. This indicates that both TNF-α and the presence of primarily apoptotic cells contributed to DC maturation. Therefore, infection of immature primate DCs with ALVAC results in apoptotic death of infected cells, which can be internalized by noninfected DCs driving DC maturation in the presence of the TNF-α secreted concomitantly by exposed cells. This suggests an important mechanism that may influence the immunogenicity of avipox virus vectors. PMID:11070033

  5. Disturbance of Tumor Necrosis Factor Alpha-Mediated Beta Interferon Signaling in Cervical Carcinoma Cells

    PubMed Central

    Bachmann, Anastasia; Hanke, Brigitte; Zawatzky, Rainer; Soto, Ubaldo; van Riggelen, Jan; zur Hausen, Harald; Rösl, Frank

    2002-01-01

    In the present study we show that malignant human papillomavirus (HPV)-positive cells lost their ability to synthesize endogenous beta interferon (IFN-β) upon tumor necrosis factor alpha (TNF-α) treatment. IFN-β transcription, however, was reinducible in nonmalignant HPV-positive cells, which was confirmed in functional protection assays against encephalomyocarditis virus or vesicular stomatitis virus infections. Addition of neutralizing antibodies against IFN-β blocked the antiviral effect, excluding the possibility that other IFN types were involved. Conversely, both malignant and immortalized cells could be protected against viral cytolysis when either IFN-β, IFN-α, or IFN-γ was added exogenously. This indicates that only the cross talk between TNF-α and the IFN-β pathways, and not IFN-α/β and IFN-γ signaling in general, is perturbed in cervical carcinoma cells. Notably, full virus protection was restricted exclusively to nonmalignant cells, indicating that the antiviral effect correlates with the growth-inhibitory and virus-suppressive properties of TNF-α. The IFN-regulatory factors IRF-1 and p48 (ISGF3γ) emerged as key regulatory molecules in the differential IFN-β response, since their transcription was either absent or only inefficiently enhanced in tumorigenic cells upon treatment with TNF-α. Inducibility of both genes, however, became reestablished in cervical carcinoma cells, which were complemented to nontumorigenicity after somatic cell hybridization. Complementation was paralleled by the entire reconstitution of cytokine-mediated IFN-β expression and the ability of TNF-α to exert an antiviral state. In contrast, under conditions where tumor suppression was not accomplished upon somatic cell hybridization, neither expression of IRF-1, p48, and IFN-β nor antiviral activity could be restored. PMID:11739693

  6. Hypoxia Inducible Factor 1 Alpha Is Expressed in Germ Cells throughout the Murine Life Cycle

    PubMed Central

    Gardner, Lauren H.; Mathews, Juanita; Yamazaki, Yuki; Allsopp, Richard C.

    2016-01-01

    Pluripotent stem cells of the early embryo, and germ line cells, are essential to ensure uncompromised development to adulthood as well as species propagation, respectively. Recently, the transcription factor hypoxia inducible factor 1 alpha (Hif1α) has been shown to have important roles in embryonic stem cells; in particular, regulation of conversion to glycolytic metabolism and, as we have shown, maintenance of functional levels of telomerase. In the present study, we sought to assess whether Hif1α was also expressed in the primitive cells of the murine embryo. We observed expression of Hif1α in pre-implantation embryos, specifically the 2-cell stage, morula, and blastocyst. Robust Hif1α expression was also observed in male and female primordial germ cells. We subsequently assessed whether Hif1α was expressed in adult male and female germ cells. In the testis, Hif1α was robustly expressed in spermatogonial cells, in both juvenile (6-week old) and adult (3-month old) males. In the ovaries, Hif1α was expressed in mature oocytes from adult females, as assessed both in situ and in individual oocytes flushed from super-ovulated females. Analysis of Hif1α transcript levels indicates a mechanism of regulation during early development that involves stockpiling of Hif1α protein in mature oocytes, presumably to provide protection from hypoxic stress until the gene is re-activated at the blastocyst stage. Together, these observations show that Hif1α is expressed throughout the life-cycle, including both the male and female germ line, and point to an important role for Hif1α in early progenitor cells. PMID:27148974

  7. Positive selection of invariant V alpha 14+ T cells by non-major histocompatibility complex-encoded class I-like molecules expressed on bone marrow-derived cells.

    PubMed Central

    Adachi, Y; Koseki, H; Zijlstra, M; Taniguchi, M

    1995-01-01

    V alpha 14+ T cells are a unique subset expressing an invariant T-cell antigen receptor alpha chain encoded by V alpha 14 and J alpha 281 gene fragments with a 1-nt N region. Most invariant V alpha 14+ T cells develop in extrathymic organs, independent of thymus, and expand at a high frequency in various mouse strains regardless of major histocompatibility complex (MHC) haplotype. In this paper, we show that the positive selection of invariant V alpha 14+ T cells requires a beta 2-microglobulin-associated MHC class I-like molecule not linked to the MHC on chromosome 17. This was determined by linkage analysis on DNA from recombinant mice generated by crossing a C57BL/6 mouse with a wild mouse, Mus musculus molossinus, that is negative for invariant V alpha 14 TCR expression. However, the peptide transporter TAP1 is not necessary for positive selection of invariant V alpha 14+ T cells, indicating the direct recognition of the MHC class I-like molecule without peptide by the invariant V alpha 14 TCR. Further, experiments with bone marrow-chimeric mice show that invariant V alpha 14+ T cells in the periphery are selected by bone marrow cells, suggesting a unique lineage of V alpha 14+ T cells differentiated through a selection process distinct from that of conventional alpha beta TCR+ T cells. Images Fig. 1 Fig. 2 Fig. 3 PMID:7862661

  8. A study of membrane protein defects and alpha hemoglobin chains of red blood cells in human beta thalassemia

    SciTech Connect

    Rouyer-Fessard, P.; Garel, M.C.; Domenget, C.; Guetarni, D.; Bachir, D.; Colonna, P.; Beuzard, Y. )

    1989-11-15

    The soluble pool of alpha hemoglobin chains present in blood or bone marrow cells was measured with a new affinity method using a specific probe, beta A hemoglobin chain labeled with ({sup 3}H)N-ethylmaleimide. This pool of soluble alpha chains was 0.067 {plus minus} 0.017% of hemoglobin in blood of normal adult, 0.11 {plus minus} 0.03% in heterozygous beta thalassemia and ranged from 0.26 to 1.30% in homozygous beta thalassemia intermedia. This elevated pool of soluble alpha chains observed in human beta thalassemia intermedia decreased 33-fold from a value of 10% of total hemoglobin in bone marrow cells to 0.3% in the most dense red blood cells. The amount of insoluble alpha chains was measured by using the polyacrylamide gel electrophoresis in urea and Triton X-100. In beta thalassemia intermedia the amount of insoluble alpha chains was correlated with the decreased spectrin content of red cell membrane and was associated with a decrease in ankyrin and with other abnormalities of the electrophoretic pattern of membrane proteins. The loss and topology of the reactive thiol groups of membrane proteins was determined by using ({sup 3}H)N-ethylmaleimide added to membrane ghosts prior to urea and Triton X-100 electrophoresis. Spectrin and ankyrin were the major proteins with the most important decrease of thiol groups.

  9. Tumour necrosis factor-alpha and interferon-gamma production measured at the single cell level in normal and inflamed human intestine.

    PubMed Central

    MacDonald, T T; Hutchings, P; Choy, M Y; Murch, S; Cooke, A

    1990-01-01

    The spot-ELISA technique has been used to enumerate the frequency of cells secreting tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), isolated from biopsies of normal intestine and from biopsies of children with inflammatory bowel disease. TNF-alpha production was undetectable in six out of 12 biopsies from normal intestine and in the other six biopsies it ranged from 60 to 580 TNF-alpha-secreting cells/10(6) isolated intestinal cells. In contrast, cells isolated from biopsies of children with Crohn's disease (n = 9) all showed elevated frequencies of TNF-alpha-secreting cells (500-12,000 secreting cells/10(6) cells). In ulcerative colitis, four out of eight children had increased production of TNF-alpha and in children with indeterminate colitis two out of three had elevated levels. There was no correlation between plasma TNF-alpha levels and the number of intestinal cells secreting TNF-alpha. In controls and all groups of patients IFN-gamma-secreting cells were uncommon. These results suggest that TNF-alpha is an important mediator of inflammation in the human gut, and, furthermore, may play a role in the growth failure frequently seen in children with inflammatory bowel disease. PMID:2117510

  10. Interferon-alpha suppresses proliferation of chronic myelogenous leukemia cells K562 by extending cell cycle S-phase without inducing apoptosis.

    PubMed

    Grebenová, Dana; Kuzelová, Katerina; Fuchs, Ota; Halada, Petr; Havlícek, Vladimír; Marinov, Iuri; Hrkal, Zbynĕk

    2004-01-01

    We examined the effects of interferon-alpha (IFN-alpha) treatment on the growth, cell cycle, proliferation, and apoptotic parameters as well as adhesive properties and proteome of chronic myelogenous leukemia (CML)-derived K562 cells. IFN-alpha treatment (200 to 600 U/ml, 24 to 72 h) suppressed growth and caused accumulation of K562 cells in the S-phase of cell cycle (increase in S-phase cells by up to 52% in comparison with the untreated controls) at the expenses of cells in G1-phase. No transition of cells to G0-phase occurred as followed from Ki-67 protein determination. Although the level of chimeric gene product, BCR-ABL mRNA coding for BCR-ABL protein with anti-apoptotic properties, decreased by 30%, apoptosis was not triggered as judged from Annexin-V, APO2.7, and TUNEL assays. Adhesion of K562 cells to fibronectin-coated surfaces increased by up to 52% as determined by calcein assay. The proteomic analysis (2-D electrophoresis in combination with mass spectrometry, MALDI-MS) revealed a single protein, ubiquitine cross-reactive protein (UBCR), whose level markedly increased due to IFN-alpha treatment. The ubiquitination-like directed degradation processes may thus play a role in the mechanism of IFN-alpha antiproliferative effects. PMID:14757443

  11. SIP1/NHERF2 enhances estrogen receptor alpha transactivation in breast cancer cells

    PubMed Central

    Meneses-Morales, Ivan; Tecalco-Cruz, Angeles C.; Barrios-García, Tonatiuh; Gómez-Romero, Vania; Trujillo-González, Isis; Reyes-Carmona, Sandra; García-Zepeda, Eduardo; Méndez-Enríquez, Erika; Cervantes-Roldán, Rafael; Pérez-Sánchez, Víctor; Recillas-Targa, Félix; Mohar-Betancourt, Alejandro; León-Del-Río, Alfonso

    2014-01-01

    The estrogen receptor alpha (ERα) is a ligand-activated transcription factor that possesses two activating domains designated AF-1 and AF-2 that mediate its transcriptional activity. The role of AF-2 is to recruit coregulator protein complexes capable of modifying chromatin condensation status. In contrast, the mechanism responsible for the ligand-independent AF-1 activity and for its synergistic functional interaction with AF-2 is unclear. In this study, we have identified the protein Na+/H+ Exchanger RegulatoryFactor 2 (NHERF2) as an ERα-associated coactivator that interacts predominantly with the AF-1 domain of the nuclear receptor. Overexpression of NHERF2 in breast cancer MCF7 cells produced an increase in ERα transactivation. Interestingly, the presence of SRC-1 in NHERF2 stably overexpressing MCF7 cells produced a synergistic increase in ERα activity. We show further that NHERF2 interacts with ERα and SRC-1 in the promoter region of ERα target genes. The binding of NHERF2 to ERα in MCF7 cells increased cell proliferation and the ability of MCF7 cells to form tumors in a mouse model. We analyzed the expression of NHERF2 in breast cancer tumors finding a 2- to 17-fold increase in its mRNA levels in 50% of the tumor samples compared to normal breast tissue. These results indicate that NHERF2 is a coactivator of ERα that may participate in the development of estrogen-dependent breast cancer tumors. PMID:24771346

  12. Identification of gene-based responses in human blood cells exposed to alpha particle radiation

    PubMed Central

    2014-01-01

    Background The threat of a terrorist-precipitated nuclear event places humans at danger for radiological exposures. Isotopes which emit alpha (α)-particle radiation pose the highest risk. Currently, gene expression signatures are being developed for radiation biodosimetry and triage with respect to ionizing photon radiation. This study was designed to determine if similar gene expression profiles are obtained after exposures involving α-particles. Methods Peripheral blood mononuclear cells (PBMCs) were used to identify sensitive and robust gene-based biomarkers of α-particle radiation exposure. Cells were isolated from healthy individuals and were irradiated at doses ranging from 0-1.5 Gy. Microarray technology was employed to identify transcripts that were differentially expressed relative to unirradiated cells 24 hours post-exposure. Statistical analysis identified modulated genes at each of the individual doses. Results Twenty-nine genes were common to all doses with expression levels ranging from 2-10 fold relative to control treatment group. This subset of genes was further assessed in independent complete white blood cell (WBC) populations exposed to either α-particles or X-rays using quantitative real-time PCR. This 29 gene panel was responsive in the α-particle exposed WBCs and was shown to exhibit differential fold-changes compared to X-irradiated cells, though no α-particle specific transcripts were identified. Conclusion Current gene panels for photon radiation may also be applicable for use in α-particle radiation biodosimetry. PMID:25017500

  13. Alpha-lipoic acid enhances DMSO-induced cardiomyogenic differentiation of P19 cells.

    PubMed

    Shen, Xinghua; Yang, Qinghui; Jin, Peng; Li, Xueqi

    2014-09-01

    Alpha-lipoic acid (α-LA) is a potent antioxidant that acts as an essential cofactor in mitochondrial dehydrogenase reactions. α-LA has been shown to possess anti-inflammatory and cytoprotective properties, and is used to improve symptoms of diabetic neuropathy. However, the role of α-LA in stem cell differentiation and the underlying molecular mechanisms remain unknown. In the present study, we showed that α-LA significantly promoted dimethyl sulfoxide (DMSO)-induced cardiomyogenic differentiation of mouse embryonic carcinoma P19 cells. α-LA dose dependently increased beating embryonic body (EB) percentages of DMSO-differentiated P19 cells. The expressions of cardiac specific genes TNNT2, Nkx2.5, GATA4, MEF2C, and MLC2V and cardiac isoform of troponin T (cTnT)-positively stained cell population were significantly up-regulated by the addition of α-LA. We also demonstrated that the differentiation time after EB formation was critical for α-LA to take effect. Interestingly, without DMSO treatment, α-LA did not stimulate the cardiomyogenic differentiation of P19 cells. Further investigation indicated that collagen synthesis-enhancing activity, instead of the antioxidative property, plays a significant role in the cardiomyogenic differentiation-promoting function of α-LA. These findings highlight the potential use of α-LA for regenerative therapies in heart diseases. PMID:25112287

  14. Basal cell proliferation in female SKH-1 mice treated with alpha- and beta-hydroxy acids.

    PubMed

    Sams, R L; Couch, L H; Miller, B J; Okerberg, C V; Warbritton, A; Wamer, W G; Beer, J Z; Howard, P C

    2001-08-15

    Alpha- and beta-hydroxy acids are compounds that have been used extensively in cosmetic and dermatological formulations. Clinical and qualitative effects of alpha- and beta-hydroxy acids have been well characterized, but little is known about their mechanism of action or acute and chronic biochemical effects. In the present study, we examined the acute proliferative effects of glycolic and salicylic acids on cell proliferation in the epidermis of SKH-1 female mice, using BrdU incorporation as a marker of epidermal proliferation. In preliminary experiments, we observed an increase in the rate of proliferation after 3 days of treatment with 10% glycolic acid-containing cream and this was sustained throughout a 6.5-week (treatment 5 days/week) time course compared with untreated control animals. After each treatment with cream containing glycolic acid there was a wave of proliferation that was maximal 12 to 16 h (significant at p < 0.05) after treatment, followed by a subsequent increase in epidermal thickness at 18 to 20 h (significant at p < 0.05). The effects of the concentration and pH level of glycolic acid- and salicylic acid-containing creams on the rate of proliferation and increases in skin thickness in SKH-1 epidermis were also investigated. We observed a dose-dependent increase in epidermal proliferation of animals treated with either glycolic or salicylic acid. A similar time-dependent response was observed in the epidermal thickness in animals treated with salicylic acid, but not with glycolic acid. Differences in pH (3.5 or 4.0) had no significant effect on either epidermal proliferation or skin thickness. The data that we present here should be useful in characterizing not only the beneficial but also the adverse effects that occur following acute or chronic usage of alpha-hydroxy acids. PMID:11509029

  15. DR alpha beta dimers released from complexes with invariant chain fail to stimulate alloreactive T cell clones.

    PubMed

    Demotz, S

    1993-09-01

    To demonstrate that DR alpha beta dimers still complexed to invariant chain (Ii) have not yet acquired peptides recognized by alloreactive T cells, complexes between DR molecules and Ii isolated from Epstein-Barr-virus (EBV)-transformed B cells were analyzed by affinity chromatography and gel filtration. First, it was shown that DR/Ii complexes inserted into artificial planar membranes (PM) failed to stimulate proliferative response of five alloreactive T cell clones and a polyclonal alloreactive T cell line, while PM bearing mature DR alpha beta dimers from the same EBV-B cells were stimulatory for the T cell clones and the T cell line. These findings indicate that either Ii inhibits binding of peptides to DR molecules or Ii hinders T cells recognition of peptide/DR complexes. Second, to discriminate between these two possibilities, DR alpha beta dimers, which were artificially released from complexes between DR molecules and Ii, were inserted into PM. These DR alpha beta dimers were devoid of alloreactive stimulatory capacity while fully capable of binding and presenting a tetanus toxin synthetic peptide to a specific T cell clone, indicating that DR molecules released from complexes with Ii are empty. This study, by showing that DR molecules bound to Ii do not bear peptides recognized by alloreactive T cells, supports the notion that association of Ii with class II major histocompatibility complex (MHC) molecules prevents premature peptide loading and hence favors encounter with peptides derived from proteins of the extracellular compartment. Since allogeneic class II MHC molecules released from complexes with Ii were not stimulatory for five out of five alloreactive T cell clones and a polyclonal alloreactive T cell line, these data also indicate that, in most cases, alloreactive T cells recognize ligands constituted by complexes between allogeneic class II MHC molecules and specific peptides which derive from the antigen-presenting cells themselves or serum

  16. The p38alpha/beta MAPK functions as a molecular switch to activate the quiescent satellite cell.

    PubMed

    Jones, Nathan C; Tyner, Kristina J; Nibarger, Lisa; Stanley, Heather M; Cornelison, Dawn D W; Fedorov, Yuri V; Olwin, Bradley B

    2005-04-11

    Somatic stem cells cycle slowly or remain quiescent until required for tissue repair and maintenance. Upon muscle injury, stem cells that lie between the muscle fiber and basal lamina (satellite cells) are activated, proliferate, and eventually differentiate to repair the damaged muscle. Satellite cells in healthy muscle are quiescent, do not express MyoD family transcription factors or cell cycle regulatory genes and are insulated from the surrounding environment. Here, we report that the p38alpha/beta family of mitogen-activated protein kinases (MAPKs) reversibly regulates the quiescent state of the skeletal muscle satellite cell. Inhibition of p38alpha/beta MAPKs (a) promotes exit from the cell cycle, (b) prevents differentiation, and (c) insulates the cell from most external stimuli allowing the satellite cell to maintain a quiescent state. Activation of satellite cells and p38alpha/beta MAPKs occurs concomitantly, providing further support that these MAPKs function as a molecular switch for satellite cell activation. PMID:15824134

  17. Tumor necrosis factor alpha promotes the expression of immunosuppressive proteins and enhances the cell growth in a human bone marrow-derived stem cell culture

    SciTech Connect

    Miettinen, Johanna A.; Pietilae, Mika; Salonen, Riikka J.; Ohlmeier, Steffen; Ylitalo, Kari; Huikuri, Heikki V.; Lehenkari, Petri

    2011-04-01

    Mesenchymal stem cells (MSCs) are widely used in experimental treatments for various conditions that involve normal tissue regeneration via inflammatory repair. It is known that MSCs can secrete multiple soluble factors and suppress inflammation. Even though the effect of MSCs on inflammation has been extensively studied, the effect of inflammation on MSCs is poorly understood. One of the major cytokines released at the site of inflammation is tumor necrosis factor alpha (TNF-{alpha}) which is known to induce MSC invasion and proliferation. Therefore, we wanted to test the effects of TNF-{alpha} exposure on MSCs derived from human bone marrow. We found, as expected, that cell proliferation was significantly enhanced during TNF-{alpha} exposure. However, according to the cell surface marker analysis, the intensity of several antigens in the minimum criteria panel for MSCs proposed by International Society of Cellular Therapy (ISCT) was decreased dramatically, and in certain cases, the criteria for MSCs were not fulfilled. In addition, TNF-{alpha} exposure resulted in a significant but transient increase in human leukocyte antigen and CD54 expression. Additional proteomic analysis by two-dimensional difference gel electrophoresis and mass spectrometry revealed three proteins whose expression levels decreased and 8 proteins whose expression levels increased significantly during TNF-{alpha} exposure. The majority of these proteins could be linked to immunosuppressive and signalling pathways. These results strongly support reactive and immunosuppressive activation of MSCs during TNF-{alpha} exposure, which might influence MSC differentiation stage and capacity.

  18. CHO cell repair of single-strand and double-strand DNA breaks induced by gamma- and alpha-radiations.

    PubMed

    Cole, A; Shonka, F; Corry, P; Cooper, W G

    1975-01-01

    Neutral and alkaline sucrose gradient sedimentation analysis was used to measure double- and single-strand breaks in the DNA of Chinese hamster ovary (CHO) cells exposed to either gamma- or alpha-radiation. After irradiation, cells were incubated for 15-180 min to test the ability of the cell to rejoin the DNA breaks. Essentially complete rejoining was observed for single-strand breaks induced by gamma- or alpha-doses below 20 krad and for double-strand breaks induced by gamma doses below 60 krad. Approximately 80% rejoining was observed for double-strand breaks induced by alpha doses below 40 krad. At higher doses, the repair system appeared to saturate in such a way that essentially no additional breaks were rejoined. PMID:1191188

  19. Pro-gliogenic effect of IL-1alpha in the differentiation of embryonic neural precursor cells in vitro.

    PubMed

    Ajmone-Cat, Maria Antonietta; Cacci, Emanuele; Ragazzoni, Ylenia; Minghetti, Luisa; Biagioni, Stefano

    2010-05-01

    Inflammation is regarded as a main obstacle to brain regeneration. Major detrimental effects are attributed to microglial/macrophagic products, such as TNF-alpha and interleukin (IL)-6. The role of cytokines of the IL-1 family, particularly of IL-1alpha, in the modulation of neural precursor cell (NPC) properties is less characterized. IL-1alpha is one of the most abundant cytokines released upon acute stimulation of microglia with lipopolysaccharide and is down-regulated upon chronic stimulation. As we recently demonstrated, acutely activated microglia reduces NPC survival, prevent neuronal differentiation and promote glial differentiation. Chronically activated microglia are instead permissive to NPC survival and neuronal differentiation, and less effective in promoting astrocytic differentiation. We thus investigated whether IL-1alpha could contribute to the effects of acutely activated microglia on NPC. We found that NPC express functional IL-1 receptors and that exposure to recombinant IL-1alpha strongly enhances NPC differentiation into astrocytes, without affecting cell viability and neuronal differentiation. In the same conditions, recombinant IL-1beta has pro-gliogenic effects at concentrations 10-fold higher than those found in activated microglial conditioned media. Interestingly, immunodepletion of IL-1alpha in activated microglial conditioned media fails to revert microglial pro-gliogenic action and slightly enhances neuronal differentiation, revealing that other microglial-derived factors contribute to the modulation of NPC properties. PMID:20236219

  20. Interaction of the human cytomegalovirus particle with the host cell induces hypoxia-inducible factor 1 alpha

    SciTech Connect

    McFarlane, Steven; Nicholl, Mary Jane; Sutherland, Jane S.; Preston, Chris M.

    2011-05-25

    The cellular protein hypoxia-inducible factor 1 alpha (HIF-1{alpha}) was induced after infection of human fibroblasts with human cytomegalovirus (HCMV). HCMV irradiated with ultraviolet light (uv-HCMV) also elicited the effect, demonstrating that the response was provoked by interaction of the infecting virion with the cell and that viral gene expression was not required. Although induction of HIF-1{alpha} was initiated by an early event, accumulation of the protein was not detected until 9 hours post infection, with levels increasing thereafter. Infection with uv-HCMV resulted in increased abundance of HIF-1{alpha}-specific RNA, indicating stimulation of transcription. In addition, greater phosphorylation of the protein kinase Akt was observed, and the activity of this enzyme was required for induction of HIF-1{alpha} to occur. HIF-1{alpha} controls the expression of many cellular gene products; therefore the findings reveal new ways in which interaction of the HCMV particle with the host cell may cause significant alterations to cellular physiology.

  1. A role for protein kinase CK2 in cell proliferation: evidence using a kinase-inactive mutant of CK2 catalytic subunit alpha.

    PubMed

    Lebrin, F; Chambaz, E M; Bianchini, L

    2001-04-12

    Protein kinase CK2 is an ubiquitous and pleiotropic Ser/Thr protein kinase composed of two catalytic (alpha and/or alpha') and two regulatory (beta) subunits generally combined to form alpha(2)beta(2), alphaalpha'beta(2), or alpha'(2)beta(2) heterotetramers. To gain more insight into the role of CK2 in the control of proliferation in mammalian cells, overexpression of isolated CK2 subunits alpha, alpha', or beta was carried out in two fibroblast cell lines: NIH3T3 and CCL39. To interfere with CK2 cellular functions, cells were also transfected with a kinase-inactive mutant of CK2alpha catalytic subunit: CK2alpha-K68A. In NIH3T3 cells, overexpression of either wild-type subunit (alpha, alpha' or beta) had no effect on cell proliferation. In contrast, overexpression of the CK2alpha kinase-deficient mutant induced a marked inhibition of cell proliferation. This resulted from a defect in G1/S progression as demonstrated in transient transfection experiments in both NIH3T3 and CCL39 cells using BrdU incorporation measurements and in CCL39 clones stably overexpressing the CK2alpha-K68A mutant by growth curve analysis. We demonstrated that the kinase-negative mutant has the capacity to integrate the endogenous CK2 subunit pool both as an isolated kinase-inactive alpha subunit and as associated to the beta subunit in a kinase-inactive tetramer. Finally we showed that expression of the kinase-inactive mutant interferes with phosphorylation of an endogenous CK2 substrate; we speculate that optimal phosphorylation of target proteins by CK2 is required to achieve optimal cell cycle progression. PMID:11360185

  2. The IL-6/sIL-6R treatment of a malignant melanoma cell line enhances susceptibility to TNF-{alpha}-induced apoptosis

    SciTech Connect

    Wagley, Yadav; Yoo, Yung-Choon; Seo, Han Geuk; Rhee, Man Hee; Kim, Tae-Hyoung; Kang, Keon Wook; Nah, Seung-Yeol; Oh, Jae-Wook . E-mail: ohjw@mail.chosun.ac.kr

    2007-03-23

    Melanoma is an intractable tumor that has shown very impressive and promising response to local administration of high dose recombinant TNF-{alpha} in combination with IFN-{gamma} in clinical studies. In this study, we investigated the effect of IL-6/sIL-6R on TNF-{alpha}-resistant B16/F10.9 melanoma cells. A low dose of TNF-{alpha} or IL-6/sIL-6R had minimal affect on the cell growth. However, the highly active fusion protein of sIL-6R and IL-6 (IL6RIL6), covalently linked by a flexible peptide, sensitized TNF-{alpha}-resistant F10.9 melanoma cells to TNF-{alpha}-induced apoptosis. Stimulation of the cells with IL6RIL6 plus TNF-{alpha} resulted in both the activation of caspase-3 and the reduction of bcl-2 expression. Flow cytometry analysis showed that IL6RIL6-upregulated TNF-R55 and TNF-R75 expression, suggesting an increase in TNF-{alpha} responsiveness by IL6RIL6 resulting from the induction of TNF receptors. Moreover, exposure of F10.9 cells to neutralizing antibody to TNF-R55 significantly inhibited IL6RIL6/TNF-{alpha}-induced cytotoxicity. These results suggest that the IL6/sIL6R/gp130 system, which sensitizes TNF-{alpha}-resistant melanoma cells to TNF-{alpha}-induced apoptosis, may provide a new target for immunotherapy.

  3. Prostaglandin F(2alpha) suppresses early phase of adipogenesis, but is not associated with osteoblastogenesis in mouse mesenchymal stem cells.

    PubMed

    Fujimori, Ko; Ueno, Toshiyuki; Amano, Fumio

    2010-09-01

    In this study, we investigated the roles of prostaglandin (PG) F(2alpha) in the differentiation of mouse ST2 mesenchymal stem cells (MSC) into adipocytes and osteoblasts. PGF(2alpha) was not produced in the undifferentiated ST2 MSC, but its highest level of production was detected at 3h after the initiation of adipogenesis and then quickly decreased. On the contrary, apparent PGF(2alpha) production was not detected during the osteoblastogenesis of ST2 MSC. The PGF(2alpha) production pattern during adipogenesis well resembled the expression profiles of aldo-keto reductase (AKR) 1B3, which acted as the PGF(2alpha) synthase, and cyclooxygenase-2 genes; but the pattern showed a slight delay compared with these profiles. The siRNA for AKR1B3, but not that for AKR1B8 or 1B10, decreased PGF(2alpha) production and enhanced the expression of adipogenic genes, but did not affect the mRNA levels of osteoblastogenic genes, during the adipogenesis of ST2 MSC. The FP receptor was expressed during adipogenesis of ST2 MSC, and its agonist or antagonist suppressed or enhanced, respectively, the lipid accumulation and the adipogenic gene expression; but this receptor was not associated with the osteoblastogenesis. These results indicate that AKR1B3-mediated PGF(2alpha) suppressed the early phase of adipogenesis through FP receptors, but did not affect osteoblastogenesis in ST2 MSC. Therefore, PGF(2alpha) suppressed the progression of early phase adipogenesis after determination of the cell fate that causes MSC to differentiate into adipocytes. PMID:20601070

  4. In vitro protective effects of two extracts from bergamot peels on human endothelial cells exposed to tumor necrosis factor-alpha (TNF-alpha).

    PubMed

    Trombetta, Domenico; Cimino, Francesco; Cristani, Mariateresa; Mandalari, Giuseppina; Saija, Antonella; Ginestra, Giovanna; Speciale, Antonio; Chirafisi, Joselita; Bisignano, Giuseppe; Waldron, Keith; Narbad, Arjan; Faulds, Craig B

    2010-07-28

    Bergamot ( Citrus bergamia Risso) is a less commercialized Citrus fruit, mainly used for its essential oil extracted from the peel. Bergamot peel (BP) represents about 60% of the processed fruits and is regarded as primary waste. However, it contains good amounts of useful compounds, such as pectins and flavonoids. Many of the bioactivities of Citrus flavonoids appear to impact vascular endothelial cells. Herein, we report the protective effect of two flavonoid-rich extracts from BP (endowed with radical-scavenging properties and lacking genotoxic activity) against alterations in cell modifications induced by the pleiotropic inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) on human umbilical vein endothelial cells (HUVECs), as demonstrated by monitoring intracellular levels of malondialdehyde/4-hydroxynonenal, reduced and oxidized glutathione and superoxide dismutase activity, and the activation status of nuclear factor-kappaB (NF-kappaB). Thus, BP appears to be a potential source of natural antioxidant/anti-inflammatory phytocomplexes to be employed as ingredients of nutraceutical products or functional foods. PMID:20578719

  5. TNF-alpha-induced apoptosis is prevented by erythropoietin treatment on SH-SY5Y cells

    SciTech Connect

    Pregi, Nicolas Wenker, Shirley; Vittori, Daniela; Leiros, Claudia Perez; Nesse, Alcira

    2009-02-01

    The growth factor erythropoietin (Epo) has shown neuronal protective action in addition to its well known proerythroid activity. Furthermore, Epo has dealt with cellular inflammation by inhibiting the expression of several proinflammatory cytokines, such as IL-1 and TNF-{alpha}. The action of TNF can have both apoptotic and antiapoptotic consequences due to altered balance between different cell signalling pathways. This work has focused on the apoptotic effects of this cytokine and the potential protective action of Epo. The model we used was neuroblastoma SH-SY5Y cells cultured in the presence of 25 ng/ml TNF-{alpha} or pretreated with 25 U/ml Epo for 12 h before the addition of TNF-{alpha}. Apoptosis was evaluated by differential cell count after Hoechst staining, analysis of DNA ladder pattern, and measurement of caspase activity. Despite its ability to induce NF-{kappa}B nuclear translocation, TNF-{alpha} induced cell death, which was found to be associated to upregulation of TNF Receptor 1 expression. On the other hand, cells activated by Epo became resistant to cell death. Prevention of death receptor upregulation and caspase activation may explain this antiapoptotic effect of Epo, which may be also favoured by the induction of a higher expression of protective factors, such as Bcl-2 and NF-{kappa}B, through mechanisms involving Jak/STAT and PI3K signalling pathways.

  6. The integrin alpha 6 beta 1 promotes the survival of metastatic human breast carcinoma cells in mice.

    PubMed Central

    Wewer, U. M.; Shaw, L. M.; Albrechtsen, R.; Mercurio, A. M.

    1997-01-01

    The role of the integrin alpha 6 beta 1 in breast carcinoma progression was studied by targeted elimination of this integrin in MDA-MB-435 cells, a human breast carcinoma cell line that is highly metastatic in athymic mice. The strategy used is based on the finding that expression of a cytoplasmic domain deletion mutant of the beta 4-integrin subunit (beta 4-delta CYT) in MDA-MB-435 cells eliminates formation of the alpha 6 beta 1 heterodimer. MDA-MB-435 cells that lacked alpha 6 beta 1 expression (beta 4-delta CYT transfectants) formed tumors in athymic mice that were suppressed in their growth and that exhibited a significant increase in apoptosis in comparison to the control tumors. Unlike the control MDA-MB-435 cells, the beta 4-delta CYT transfectants were unable to establish metastatic foci in the lungs. Also, the control transfectants grew substantially better than the beta 4-delta CYT transfectants in the liver after intrahepatic injection because of extensive apoptosis in the beta 4-delta CYT transfectants. These data suggest that a major function of the alpha 6 beta 1 integrin in breast carcinoma is to facilitate tumorigenesis and promote tumor cell survival in distant organs. Images Figure 1 Figure 2 Figure 3 PMID:9358743

  7. 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. PMID:2175958

  8. T-cell alpha beta + and gamma delta + deficient mice display abnormal but distinct phenotypes toward a natural, widespread infection of the intestinal epithelium.

    PubMed Central

    Roberts, S J; Smith, A L; West, A B; Wen, L; Findly, R C; Owen, M J; Hayday, A C

    1996-01-01

    Vertebrate immune systems contain T cells bearing either alpha beta or gamma delta T-cell antigen receptors (TCRs). alpha beta T cells perform all well-characterized T-cell effector functions, while the biological functions of gamma delta + cells remain unclear. Of particular interest is the role of gamma delta + cells during epithelial infections, since gamma delta + cells are commonly abundant within epithelia. Eimeria spp. are intracellular protozoa that infect epithelia of most vertebrates, causing coccidiosis. This study shows that in response to Eimeria vermiformis, mice lacking alpha beta T cells display defects in protective immunity, while mice lacking gamma delta + cells display exaggerated intestinal damage, apparently due to a failure to regulate the consequences of the alpha beta T cell response. An immuno-downregulatory role during infection, and during autoimmune disease, may be a general one for gamma delta + cells. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8876213

  9. Combination of interferon-alpha and 5-fluorouracil inhibits endothelial cell growth directly and by regulation of angiogenic factors released by tumor cells

    PubMed Central

    2009-01-01

    Background The combination therapy of interferon (IFN)-alpha and 5-fluorouracil (5-FU) improved the prognosis of the patients with hepatocellular carcinoma (HCC). To determine the molecular mechanisms of the anti-tumor and anti-angiogenic effects, we examined the direct anti-proliferative effects on human umbilical vein endothelial cells (HUVEC) and indirect effects by regulating secretion of angiogenic factors from HCC cells. Methods The direct effects on HUVEC were examined by TUNEL, Annexin-V assays and cell cycles analysis. For analysis of the indirect effects, the apoptosis induced by the conditioned medium from HCC cell treated by IFN-alpha/5-FU and expression of angiogenic factors was examined. Results IFN-alpha and 5-FU alone had anti-proliferative properties on HUVEC and their combination significantly inhibited the growth (compared with control, 5-FU or IFN alone). TUNEL and Annexin-V assays showed no apoptosis. Cell cycle analysis revealed that IFN-alpha and 5-FU delayed cell cycle progression in HUVEC with S-phase accumulation. The conditioned medium from HuH-7 cells after treatment with IFN/5-FU significantly inhibited HUVEC growth and induced apoptosis, and contained high levels of angiopoietin (Ang)-1 and low levels of vascular endothelial growth factor (VEGF) and Ang-2. Knockdown of Ang-1 in HuH-7 cells abrogated the anti-proliferative effects on HUVEC while knockdown of Ang-2 partially rescue the cells. Conclusion These results suggested that IFN-alpha and 5-FU had direct growth inhibitory effects on endothelial cells, as well as anti-angiogenic effects through regulation of angiogenic factors released from HCC cells. Modulation of VEGF and Angs secretion by IFN-alpha and 5-FU may contribute to their anti-angiogenic and anti-tumor effects on HCC. PMID:19821965

  10. IDENTIFICATION OF PROTEASOME ALPHA6 SUBUNIT ASSOCIATED WITH DELTAMETHRIN RESISTANCE IN Drosophila melanogaster Kc CELLS.

    PubMed

    Hu, Junli; Xu, Qin; Chi, Qingping; Liu, Wei; Li, Fengliang; Cheng, Luogen

    2016-02-01

    Differential expression of the proteasome alpha6 (prosalpha6) was previously reported between Plutella xylostella strains that are resistant or susceptible to the pesticide deltamethrin (DM). This finding indicated that the prosalpha6 may be involved in DM resistance. In this article, qPCR analysis revealed that the prosalpha6 was also significantly upregulated in Drosophila Kc cells treated with DM. To better understand the contribution of prosalpha6 in DM resistance, RNA interference, heterologous expression, and a proteasome inhibitor (MG-132) were used. MG-132 was used to suppress proteasomal activity, and the dsRNA was designed to block the function of prosalpha6. The results indicated that both MG-132 and prosalpha6 knockdown decreased the cellular viability following DM treatment. Prosalpha6 was cloned and transfected into Drosophila Kc cells. The result showed that overexpression of prosalpha6 in Drosophila Kc cells conferred some protection against DM. Taken together, our results indicate that prosalpha6 is involved in Drosophila cells DM resistance. PMID:26764169

  11. [Alpha-1 Antitrypsin Affects U0126-Induced Cytotoxicity in Colon Cancer Cell Line (HCT116)].

    PubMed

    Ljujic, M; Mijatovic, S; Bulatovic, M Z; Mojic, M; Maksimovic-Ivanic, D; Radojkovic, D; Topic, A

    2016-01-01

    Alpha-1-antitrypsin (AAT), an acute phase protein, is the principal circulatory anti-protease. This multifunctional protein is encoded by the SERPINA1 gene. Although AAT was recognised as a potential tumour marker, its role in cancer biology remains unknown. Given that it has been demonstrated that AAT has an anti-apoptotic property against non-malignant cells, we aimed to investigate whether AAT affects apoptosis in a colon cancer cell line (HCT116). The presence of AAT in the HCT116 cell culture antagonized cytotoxicity of blockers of MEK1/2, PI3K/Akt pathways as well as NF-κB. The dominantly recovered cell viability was observed in the co-treatment with MEK1/2 inhibitor U0126. In addition, it was revealed that AAT almost completely abolished U0126-induced apoptosis through maintenance of the autophagy process. Our study revealed for the first time that the observed cyto-protection triggered by AAT was accompanied by sustained autophagy which opposed apoptosis. These results may contribute to understanding of the role of AAT in cancer development and evaluation of efficacy of cancer therapy. PMID:27028823

  12. Production of inositol trisphosphates upon. cap alpha. -adrenergic stimulation in BC3H-1 muscle cells

    SciTech Connect

    Ambler, S.K.; Thompson, B.; Brown, J.H.; Taylor, P.

    1986-05-01

    Activation of ..cap alpha../sub 1/-adrenergic receptors in BC3H-1 muscle cells rapidly mobilizes intracellular and results in a paradoxically slower accumulation of inositol trisphosphate. A possible explanation for this discrepancy may be provided by the recent findings of Irvine et al. of additional Ins P3 isomers besides the Ca/sup + +/-mobilizing isomer, Ins 1,4,5-P3. They have eluted and separated the inositol phosphates of BC3H-1 cells with an NH/sub 4//sup +/ x HCO/sub 2//sup -//H/sub 3/PO/sub 4/ gradient on a Whatman Partisil 10SAX column using Hewlett-Packard HPLC. Commercial (/sup 3/H)Ins 1,4,5-P3 and (/sup 3/H)inositol phosphates from carbachol-stimulated parotid glands were used as standards. Little or no Ins 1,3,4-P3 could be detected in control or phenylephrine-treated BC3H-1 cells. Ins 1,4,5-P3 followed the pattern of agonist stimulation observed previously. As a positive control, Ins P3 isomers were also measured in 1321N1 astrocytoma cells. Muscarinic stimulation of 1321N1 cells results in both the rapid accumulation of Ins P3 and Ca/sup + +/ mobilization. There is no detectable basal Ins 1,3,4-P3, but carbachol stimulates a rapid production of this compound in 1321N1 cells. Agonist activation also results in a rapid increase in Ins 1,4,5-P3 above basal values. These studies indicate that Ins 1,3,4-P3 does not contribute to the InsP3 signal in BC3H-1 cells and multiple mechanisms may exist for the coupling of receptors to PI turnover.

  13. Wortmannin influences hypoxia-inducible factor-1 alpha expression and glycolysis in esophageal carcinoma cells

    PubMed Central

    Zeng, Ling; Zhou, Hai-Yun; Tang, Na-Na; Zhang, Wei-Feng; He, Gui-Jun; Hao, Bo; Feng, Ya-Dong; Zhu, Hong

    2016-01-01

    AIM: To investigate the influence of phosphatidylinositol-3-kinase protein kinase B (PI3K/AKT)-HIF-1α signaling pathway on glycolysis in esophageal carcinoma cells under hypoxia. METHODS: Esophageal carcinoma cell lines Eca109 and TE13 were cultured under hypoxia environment, and the protein, mRNA and activity levels of hypoxia inducible factor-1 alpha (HIF-1α), glucose transporter 1, hexokinase-II, phosphofructokinase 2 and lactate dehydrogenase-A were determined. Supernatant lactic acid concentrations were also detected. The PI3K/AKT signaling pathway was then inhibited with wortmannin, and the effects of hypoxia on the expression or activities of HIF-1α, associated glycolytic enzymes and lactic acid concentrations were observed. Esophageal carcinoma cells were then transfected with interference plasmid with HIF-1α-targeting siRNA to assess impact of the high expression of HIF-1α on glycolysis. RESULTS: HIF-1α is highly expressed in the esophageal carcinoma cell lines tested, and with decreasing levels of oxygen, the expression of HIF-1α and the associated glycolytic enzymes and the extracellular lactic acid concentration were enhanced in the esophageal carcinoma cell lines Eca109 and TE13. In both normoxia and hypoxic conditions, the level of glycolytic enzymes and the secretion of lactic acid were both reduced by wortmannin. The expression and activities of glycolytic enzymes and the lactic acid concentration in cells were reduced by inhibiting HIF-1α, especially the decreasing level of glycolysis was significant under hypoxic conditions. CONCLUSION: The PI3K/AKT pathway and HIF-1α are both involved in the process of glycolysis in esophageal cancer cells. PMID:27239113

  14. High glucose concentrations attenuate hypoxia-inducible factor-1{alpha} expression and signaling in non-tumor cells

    SciTech Connect

    Dehne, Nathalie; Bruene, Bernhard

    2010-04-15

    Hypoxia-inducible factor (HIF) is the major transcription factor mediating adaption to hypoxia e.g. by enhancing glycolysis. In tumor cells, high glucose concentrations are known to increase HIF-1{alpha} expression even under normoxia, presumably by enhancing the concentration of tricarboxylic acid cycle intermediates, while reactions of non-tumor cells are not well defined. Therefore, we analyzed cellular responses to different glucose concentrations in respect to HIF activation comparing tumor to non-tumor cells. Using cells derived from non-tumor origin, we show that HIF-1{alpha} accumulation was higher under low compared to high glucose concentrations. Low glucose allowed mRNA expression of HIF-1 target genes like adrenomedullin. Transfection of C{sub 2}C{sub 12} cells with a HIF-1{alpha} oxygen-dependent degradation domaine-GFP fusion protein revealed that prolyl hydroxylase (PHD) activity is impaired at low glucose concentrations, thus stabilizing the fusion protein. Mechanistic considerations suggested that neither O{sub 2} redistribution nor an altered redox state explains impaired PHD activity in the absence of glucose. In order to affect PHD activity, glucose needs to be metabolized. Amino acids present in the medium also diminished HIF-1{alpha} expression, while the addition of fatty acids did not. This suggests that glucose or amino acid metabolism increases oxoglutarate concentrations, which enhances PHD activity in non-tumor cells. Tumor cells deprived of glutamine showed HIF-1{alpha} accumulation in the absence of glucose, proposing that enhanced glutaminolysis observed in many tumors enables these cells to compensate reduced oxoglutarate production in the absence of glucose.

  15. Organization of the V gene segments in mouse T-cell antigen receptor [alpha]/[delta] locus

    SciTech Connect

    Wang, K.; Klotz, J.L.; Kiser, G.; Bristol, G.; Hays, E.; Lai, E.; Gese, E.; Kronenberg, M.; Hood, L. )

    1994-04-01

    The mouse T-cell receptor (TCR) [alpha]/[delta] was mapped using 17 V[alpha] and 4 V[delta] subfamily-specific probes. Four complementary methods were used: (1) an estimate of the V gene repertoire by Southern blot analysis of genomic DNA with subfamily-specific probes; (2) an analysis of V gene segments deleted by TCR gene rearrangements from a panel of T-cell tumors and hybridomas; (3) an analysis of overlapping clusters of cosmid clones; and (4) an analysis of large DNA fragments separated by field-inversion gel electrophoresis. The [alpha]/[delta] locus spans about 1 Mb. The distance between the 3[prime]-most V gene segments (V[delta]1) and the [delta] constant gene (C[delta]) is no more than 150 kb. Sixty-six V gene segments have been mapped physically on cosmids. The members of individual V[alpha] gene segments subfamilies are dispersed throughout the locus. In contrast, the V[delta] gene segments V[delta]1 to 5 are clustered at the 3[prime] end of the V gene segments cluster. At least two DNA segment duplications, 45 to 80 kb in length, are present in the locus. These data provide information on the evolution of the [alpha]/[delta] locus and on organizational features that might influence the expression of specific V gene segments in [gamma][delta] cells. 35 refs., 5 figs., 2 tabs.

  16. Transforming growth factor-alpha promotes mammary tumorigenesis through selective survival and growth of secretory epithelial cells.

    PubMed Central

    Smith, G. H.; Sharp, R.; Kordon, E. C.; Jhappan, C.; Merlino, G.

    1995-01-01

    Transforming growth factor (TGF)-alpha stimulates the growth and development of mammary epithelial cells and is implicated in the pathogenesis of human breast cancer. In this report we evaluate the consequences of overexpressing TGF-alpha in the mammary gland of transgenic mice and examine associated cellular mechanisms. When operating on a FVB/N genetic background (line MT100), TGF-alpha induced the stochastic development of mammary adenomas and adenocarcinomas f secretory epithelial origin in 64% of multiparous females. In contrast, tumors were exceedingly rare in virgin MT100 females, MT100 males, and multiparous FVB/N females. In MT100 females multiple foci of hyperplastic secretory lesions preceded the development of frank tumors; these initial lesions appeared during the involution period after the first lactation. Serial transplantation of these hyperplasias indicated an absence of proliferative immortality. Nevertheless, they gave rise to tumors at a low frequency and after a prolonged latency in virgin hosts; in multiparous hosts, tumors developed earlier and at a high incidence. The TGF-alpha transgene was highly expressed in hyperplasias and tumors but not in virgin and nonlesion-bearing tissue, suggesting that TGF-alpha overexpression provides a selective growth advantage. TGF-alpha also induced at lactation a 6.4-fold increase in DNA synthesis in MT100 epithelial cells, many of which were binucleated. MT100 mammary tissue experienced an obvious delay in involution, resulting in the postlactational survival of a significant population of unregressed secretory epithelial cells. In contrast, another line of transgenic mice on a CD-1 genetic background (MT42), in which TGF-alpha overexpression induced liver but not mammary tumors, failed to demonstrate postlactational epithelial cell survival. These data show that TGF-alpha promotes mammary tumorigenesis in multiparous MT100 mice by stimulating secretory epithelial cell proliferation during lactation and

  17. Steroid signalling in human ovarian surface epithelial cells: the response to interleukin-1alpha determined by microarray analysis.

    PubMed

    Rae, M T; Niven, D; Ross, A; Forster, T; Lathe, R; Critchley, H O D; Ghazal, P; Hillier, S G

    2004-10-01

    The human ovarian surface epithelium (HOSE) is a common site of gynaecological disease including endometriosis and ovarian cancer, probably due to serial injury-repair events associated with successive ovulations. To comprehend the importance of steroid signalling in the regulation of the HOSE, we used a custom microarray to catalogue the expression of over 250 genes involved in the synthesis and reception of steroid hormones, sterols and retinoids. The array included a subset of non-steroidogenic genes commonly involved in pro-/anti-inflammatory signalling. HOSE cells donated by five patients undergoing surgery for non-malignant gynaecological conditions were cultured for 48 h in the presence and absence of 500 pg/ml interleukin-1alpha (IL-1alpha). Total RNA was reverse-transcribed into biotin-labelled cDNA, which was hybridised to the array and visualised by gold-particle resonance light scattering and charge-coupled device (CCD) camera detection. Results for selected genes were verified by quantitative reverse-transcription PCR. In five out of five cases, untreated HOSE cells expressed genes encoding enzymes required for de novo biosynthesis of cholesterol from acetate and subsequent formation of C21-pregnane and C19-androstane steroids. Consistent with the inability of HOSE cells to synthesise glucocorticoids, oestrogens or 5alpha-reduced androgens de novo, CYP21, CYP19 and 5alpha-reductase were not detected. The only steroidogenic gene significantly up-regulated by IL-1alpha was 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1). Other cytokine-induced genes were IL-6, IL-8, nuclear factor kappaB (NFkappaB) inhibitor alpha, metallothionein-IIA and lysyl oxidase: inflammation-associated genes that respond to glucocorticoids. The only steroidogenic gene significantly suppressed by IL-1alpha was 3betaHSD1. Other genes suppressed by IL-1alpha were aldehyde dehydrogenase (ALDH) 1, ALDH 10, gonadotrophin hormone-releasing hormone receptor, peroxisome

  18. [beta]-hexosaminidase isozymes from cells cotransfected with [alpha] and [beta] cDNA constructs: Analysis of the [alpha]-subunit missense mutation associated with the adult form of Tay-Sachs disease

    SciTech Connect

    Brown, C.A.; Mahuran, D.J. )

    1993-08-01

    In vitro mutagenesis and transient expression in COS cells has been used to associate a missense mutation with a clinical or biochemical phenotype. Mutations affecting the [alpha]-subunit of [beta]-hexosaminidase A ([alpha][beta]) (E.C.3.2.1.52) result in Tay-Sachs disease. Because hexosaminidase A is heterodimeric, analysis of [alpha]-chain mutations is not straightforward. The authors examine three approaches utilizing previously identified mutations affecting [alpha]-chain folding. These involve transfection of (1) the [alpha] cDNA alone; (2) a [beta] cDNA construct encoding a [beta]-subunit substituted at a position homologous to that of the [alpha]-subunit, and (3) both [alpha] and [beta] cDNAs. The latter two procedures amplified residual activity levels over that of patient samples, an effect not previously found with mutations affecting an [open quotes]active[close quotes] [alpha]Arg residue. This effect may help to discriminate between protein-folding and active-site mutations. The authors conclude that, with proper controls, the latter method of cotransfection can be used to evaluate the effects and perhaps to predict the clinical course of some [alpha]-chain mutations. Using this technique, they demonstrate that the adult-onset Tay-Sachs mutation, [alpha]Gly[yields]Ser[sup 269], does not directly affect [alpha][beta] dimerization but exerts an indirect effect on the dimer through destabilizing the folded [alpha]-subunit at physiological temperatures. Two other [alpha] mutations linked to more severe phenotypes appear to inhibit the initial folding of the subunit. 36 refs., 2 figs., 5 tabs.

  19. Direct and remote modulation of L-channels in chromaffin cells: distinct actions on alpha1C and alpha1D subunits?

    PubMed

    Baldelli, Pietro; Hernández-Guijo, Jesus Miguel; Carabelli, Valentina; Novara, Monica; Cesetti, Tiziana; Andrés-Mateos, Eva; Montiel, Carmen; Carbone, Emilio

    2004-02-01

    Understanding precisely the functioning of voltage-gated Ca2+ channels and their modulation by signaling molecules will help clarifying the Ca(2+)-dependent mechanisms controlling exocytosis in chromaffin cells. In recent years, we have learned more about the various pathways through which Ca2+ channels can be up- or down-modulated by hormones and neurotransmitters and how these changes may condition chromaffin cell activity and catecolamine release. Recently, the attention has been focused on the modulation of L-channels (CaV 1), which represent the major Ca2+ current component in rat and human chromaffin cells. L-channels are effectively inhibited by the released content of secretory granules or by applying mixtures of exogenous ATP, opioids, and adrenaline through the activation of receptor-coupled G proteins. This unusual inhibition persists in a wide range of potentials and results from a direct (membrane-delimited) interaction of G protein subunits with the L-channels co-localized in membrane microareas. Inhibition of L-channels can be reversed when the cAMP/PKA pathway is activated by membrane permeable cAMP analog or when cells are exposed to isoprenaline (remote action), suggesting the existence of parallel and opposite effects on L-channel gating by distinctly activated membrane autoreceptors. Here, the authors review the molecular components underlying these two opposing signaling pathways and present new evidence supporting the presence of two L-channel types in rat chromaffin cells (alpha1C and alpha1D), which open new interesting issues concerning Ca(2+)-channel modulation. In light of recent findings on the regulation of exocytosis by Ca(2+)-channel modulation, the authors explore the possible role of L-channels in the autocontrol of catecholamine release. PMID:15034224

  20. Nicotine promotes cell proliferation via {alpha}7-nicotinic acetylcholine receptor and catecholamine-synthesizing enzymes-mediated pathway in human colon adenocarcinoma HT-29 cells

    SciTech Connect

    Wong, Helen Pui Shan; Yu Le; Lam, Emily Kai Yee; Tai, Emily Kin Ki; Wu, William Ka Kei; Cho, Chi Hin . E-mail: chcho@cuhk.edu.hk

    2007-06-15

    Cigarette smoking has been implicated in colon cancer. Nicotine is a major alkaloid in cigarette smoke. In the present study, we showed that nicotine stimulated HT-29 cell proliferation and adrenaline production in a dose-dependent manner. The stimulatory action of nicotine was reversed by atenolol and ICI 118,551, a {beta}{sub 1}- and {beta}{sub 2}-selective antagonist, respectively, suggesting the role of {beta}-adrenoceptors in mediating the action. Nicotine also significantly upregulated the expression of the catecholamine-synthesizing enzymes [tyrosine hydroxylase (TH), dopamine-{beta}-hydroxylase (D{beta}H) and phenylethanolamine N-methyltransferase]. Inhibitor of TH, a rate-limiting enzyme in the catecholamine-biosynthesis pathway, reduced the actions of nicotine on cell proliferation and adrenaline production. Expression of {alpha}7-nicotinic acetylcholine receptor ({alpha}7-nAChR) was demonstrated in HT-29 cells. Methyllycaconitine, an {alpha}7-nAChR antagonist, reversed the stimulatory actions of nicotine on cell proliferation, TH and D{beta}H expression as well as adrenaline production. Taken together, through the action on {alpha}7-nAChR nicotine stimulates HT-29 cell proliferation via the upregulation of the catecholamine-synthesis pathway and ultimately adrenaline production and {beta}-adrenergic activation. These data reveal the contributory role {alpha}7-nAChR and {beta}-adrenoceptors in the tumorigenesis of colon cancer cells and partly elucidate the carcinogenic action of cigarette smoke on colon cancer.

  1. A Stress-Induced Small RNA Modulates Alpha-Rhizobial Cell Cycle Progression

    PubMed Central

    Robledo, Marta; Frage, Benjamin; Wright, Patrick R.; Becker, Anke

    2015-01-01

    Mechanisms adjusting replication initiation and cell cycle progression in response to environmental conditions are crucial for microbial survival. Functional characterization of the trans-encoded small non-coding RNA (trans-sRNA) EcpR1 in the plant-symbiotic alpha-proteobacterium Sinorhizobium meliloti revealed a role of this class of riboregulators in modulation of cell cycle regulation. EcpR1 is broadly conserved in at least five families of the Rhizobiales and is predicted to form a stable structure with two defined stem-loop domains. In S. meliloti, this trans-sRNA is encoded downstream of the divK-pleD operon. ecpR1 belongs to the stringent response regulon, and its expression was induced by various stress factors and in stationary phase. Induced EcpR1 overproduction led to cell elongation and increased DNA content, while deletion of ecpR1 resulted in reduced competitiveness. Computationally predicted EcpR1 targets were enriched with cell cycle-related mRNAs. Post-transcriptional repression of the cell cycle key regulatory genes gcrA and dnaA mediated by mRNA base-pairing with the strongly conserved loop 1 of EcpR1 was experimentally confirmed by two-plasmid differential gene expression assays and compensatory changes in sRNA and mRNA. Evidence is presented for EcpR1 promoting RNase E-dependent degradation of the dnaA mRNA. We propose that EcpR1 contributes to modulation of cell cycle regulation under detrimental conditions. PMID:25923724

  2. Inhibition of MAP kinase promotes the recruitment of corepressor SMRT by tamoxifen-bound estrogen receptor alpha and potentiates tamoxifen action in MCF-7 cells

    SciTech Connect

    Hong, Wei; Chen, Linfeng; Li, Juan; Yao, Zhi

    2010-05-28

    Estrogen receptor alpha (ER{alpha}), a ligand controlled transcription factor, plays an important role in breast cancer growth and endocrine therapy. Tamoxifen (TAM) antagonizes ER{alpha} activity and has been applied in breast cancer treatment. TAM-bound ER{alpha} associates with nuclear receptor-corepressors. Mitogen-activated protein kinase (MAPK) has been elucidated to result in cross-talk between growth factor and ER{alpha} mediated signaling. We show that activated MAPK represses interaction of TAM-bound ER{alpha} with silencing mediator for retinoid and thyroid hormone receptors (SMRT) and inhibits the recruitment of SMRT by ER{alpha} to certain estrogen target genes. Blockade of MAPK signaling cascade with MEK inhibitor U0126 promotes the interaction and subsequently inhibits ER{alpha} activity via enhanced recruitment of SMRT, leading to reduced expression of ER{alpha} target genes. The growth rate of MCF-7 cells was decelerated when treated with both TAM and U0126. Moreover, the growth of MCF-7 cells stably expressing SMRT showed a robust repression in the presence of TAM and U0126. These results suggest that activated MAPK signaling cascade attenuates antagonist-induced recruitment of SMRT to ER{alpha}, suggesting corepressor mediates inhibition of ER{alpha} transactivation and breast cancer cell growth by antagonist. Taken together, our finding indicates combination of antagonist and MAPK inhibitor could be a helpful approach for breast cancer therapy.

  3. Hepatitis B Virus X Protein Driven Alpha Fetoprotein Expression to Promote Malignant Behaviors of Normal Liver Cells and Hepatoma Cells

    PubMed Central

    Zhu, Mingyue; Lu, Yan; Li, Wei; Guo, Junli; Dong, Xu; Lin, Bo; Chen, Yi; Xie, Xieju; Li, Mengsen

    2016-01-01

    Background: The infection of Hepatitis B virus (HBV) is closely associated with the development of hepatocellular carcinoma(HCC), HBV-X protein(HBx) is able to induce expression of alpha-fetoprotein(AFP) in normal liver cells, and AFP harbors a function to promote malignant transformation of normal liver cells, but the role AFP playing in malignant behaviors of HCC cells is still unclear. Methods: Fifty-six liver tissue samples were collected from the clinical patients through hepatectomy(include normal liver tissues, HBV-related hepatitis liver tissues and HBV-related HCC tissues), and diagnosis of these tissues by pathology section, expression of AFP, Ras and CXCR4 were evidenced by immunohisochemical staining and Western blotting; The proliferation of human normal liver cells line L-02 cells and human hepatoma cells line, HLE cells(non AFP-producing) were performed by MTT method; Repaired capacity of L-02 and HLE cells were compared by wound healing assay; Migration and invasion of these cells were analyzed by Transwell chamber assay; HBx expressed vectors(pcDNA3.1-HBx) were constructed and transfected into L-02 and HLE cells, effects of pcDNA3.1-HBx on the malignant behaviors were also detected by MTT, Transwell chamber assay and the expression of AFP, Ras and CXCR4 were evidenced by Western blotting. Results: we found that expression of AFP, Ras and CXCR4 in HBV-related HCC and lymph nodes metastasis tissues were significantly elevated compared with HBV-related HCC, non metastasis tissues and HBV-related hepatitis tissues; Expression of AFP, Ras and CXCR4 in HBV-related hepatitis tissues were significantly enhanced compared with normal liver tissues; The growth ratio, migratory and invasive ability, expression of AFP, Ras and CXCR4 of the cells were outstanding promoted while L-02 and HLE cells were transfected with pcDNA3.1-HBx vectors. The proliferation ratio, migration and invasion ability, and expression of Ras and CXCR4 were significantly inhibited while

  4. Galectin-1 suppresses alpha2(I) collagen through Smad3 in renal epithelial cells.

    PubMed

    Okano, K; Uchida, K; Nitta, K; Hayashida, T

    2008-10-01

    Transforming growth factor (TGF-beta1) promotes renal fibrogenesis through activation of Smads. Galectin-1 is reported to prevent experimental glomerulonephritis. Here we investigated the fact that transfected galectin-1 significantly suppressed the transcription of alpha2(I) collagen (COL1A2) in TGF-beta1- activated human renal epithelial cells. Conversely, galectin-1 silencing RNA reduced secretion of type I collagen by HKC cells. Galectin-1 significantly decreased activation of a TGF-beta1-responsive reporter construct and of a minimal reporter construct that contains four repeats of the Smad binding element (SBE). Galectin-1 had no effect on phosphorylation of Smad3 at the linker region and C-terminus, whereas it decreased affinity of Smad3 to the SBE. Additionally, the inhibitory effect of galectin-1 disappeared using a mutated reporter construct, 376 m-LUC, in which a potential Smad recognition site within the promoter is mutated. Taken together, the results suggest that galectin-1 decreases Smad3-complex from binding to the SBE, down-regulating transcription of COL1A2 in TGF-beta1-stimulated renal epithelial cells. PMID:18791848

  5. Induction of manganese superoxide dismutase by tumour necrosis factor-alpha in human endometrial stromal cells.

    PubMed

    Karube-Harada, A; Sugino, N; Kashida, S; Takiguchi, S; Takayama, H; Yamagata, Y; Nakamura, Y; Kato, H

    2001-11-01

    The present study was undertaken to investigate the effect of tumour necrosis factor-alpha (TNFalpha) on superoxide dismutase (SOD) expression in human endometrial stromal cells (ESC) and to determine whether there is a difference in responsiveness to TNFalpha between ESC and decidualized ESC. TNFalpha increased manganese-SOD (Mn-SOD) mRNA level and Mn-SOD activity in a dose-dependent manner in ESC. The concentration of TNFalpha required for an effect was lower for decidualized ESC than for non-decidualized ESC. TNFalpha had no effect on copper-zinc-SOD (Cu,Zn-SOD) expression in either type of cell. Incubation of ESC with actinomycin D, an RNA synthesis inhibitor, blocked TNFalpha-induced Mn-SOD mRNA expression, but cycloheximide, a protein synthesis inhibitor, had no effect. H7, an inhibitor of protein kinase C (PKC), also inhibited TNFalpha-stimulated Mn-SOD mRNA expression in both types of cells. These findings suggest that TNFalpha-induced Mn-SOD expression is regulated at the transcription level and mediated by PKC-dependent phosphorylation and that de-novo protein synthesis is not required for the TNFalpha effect. In summary, TNFalpha induces Mn-SOD expression in human ESC. This phenomenon may be important for protection of ESC from cytokine-mediated oxidative stress. PMID:11675473

  6. The severity of alpha-particle-induced DNA damage is revealed by exposure to cell-free extracts

    SciTech Connect

    Hodgkins, P.S.; O`Neill, P.; Stevens, D.; Fairman, M.P.

    1996-12-01

    The rejoining of single-strand breaks induced by {alpha}-particle and {gamma} irradiation in plasmid DNA under two scavenging conditions has been compared. At the two scavenger conditions has been compared. At the two scavenger capacities used of 1.5 {times} 10{sup 7} and 3 {times} 10{sup 8}s{sup {minus}1} using Tris-HCl as the scavenger, the ratio of single- to double-strand breaks for {alpha} particles is fivefold less than the corresponding ratios for {gamma} irradiation. The repair of such radiation-induced single-strand breaks has been examined using a cell-free system derived from human whole-cell extracts. We show that the rejoining of single-strand breaks for both {alpha}-particle- and {gamma}-irradiated plasmid is dependent upon the scavenging capacity and that the efficiency of rejoining of {alpha}-particle-induced single-strand breaks is significantly less than that observed for {gamma}-ray-induced breaks. In addition, for DNA that had been irradiated under conditions that mimic the cellular environment with respect to the radical scavenging capacity, 50 of {alpha}-particle-induced single-strand breaks are converted to double-strand breaks, in contrast with only {approximately}12% conversion of {gamma}-ray-induced single-strand breaks, indicating that the initial damage caused by {alpha} particles is more severe. These studies provide experimental evidence for increased clustering of damage which may have important implications for the induction of cancer by low-level {alpha}-particle sources such as domestic radon. 37 refs., 5 figs., 1 tab.

  7. Structural and Functional Studies of Ig[alpha beta] and Its Assembly with the B Cell Antigen Receptor

    SciTech Connect

    Radaev, Sergei; Zou, Zhongcheng; Tolar, Pavel; Nguyen, Khanh; Nguyen, AnhThao; Krueger, Peter D.; Stutzman, Nicole; Pierce, Susan; Sun, Peter D.

    2010-11-09

    The B cell antigen receptor (BCR) plays an essential role in all phases of B cell development. Here we show that the extracellular domains of murine and human Ig{beta} form an I-set immunoglobulin-like structure with an interchain disulfide between cysteines on their G strands. Structural and sequence analysis suggests that Ig{alpha} displays a similar fold as Ig{beta}. An Ig{alpha}{beta} heterodimer model was generated based on the unique disulfide-bonded Ig{beta} dimer. Solution binding studies showed that the extracellular domains of Ig{alpha}{beta} preferentially recognize the constant region of BCR with chain specificity, suggesting a role for Ig{alpha}{beta} to enhance BCR chain signaling. Cluster mutations on Ig{alpha}, Ig{beta}, and a membrane-bound form of immunoglobulin (mIgM) based on the structural model identified distinct areas of potential contacts involving charged residues on both subunits of the coreceptor and the C4 domain of mIgM. These studies provide the first structural model for understanding BCR function.

  8. Differential effects of n-3 and n-6 fatty acids on prostaglandin F2alpha production by bovine endometrial cells.

    PubMed

    Caldari-Torres, C; Rodriguez-Sallaberry, C; Greene, E S; Badinga, L

    2006-03-01

    Recent studies have implicated n-3 polyunsaturated fatty acids in the reduction of eicosanoid production in the bovine uterus. The objective of this study was to determine whether the effect of eicosapentaenoic acid (EPA; C(20:5), n-3) on PGF2alpha production by bovine endometrial (BEND) cells is influenced by the quantity of linoleic acid (C(18:2), n-6) in the incubation medium. Confluent BEND cells were incubated in the absence (control) or presence of 100 microM of EPA for 24 h. After incubation, cells were rinsed and then stimulated with phorbol 12,13-dibutyrate (PDBu; 100 ng/mL) for 6 h. Additional sets of culture dishes were treated with a combination of EPA and increasing n-6/n-3 fatty acid ratios for 24 h and then challenged with PDBu for 6 h. The PDBu stimulated PGF2alpha secretion and upregulated steady-state concentrations of prostaglandin endoperoxide synthase-2 and peroxisome proliferator-activated receptor delta mRNA within 6 h. Preincubation of BEND cells with EPA for 24 h decreased PGF2alpha response to phorbol ester, but had no detectable effects on prostaglandin endoperoxide synthase-2 or peroxisome proliferator-activated receptor delta mRNA abundance in PDBu-stimulated BEND cells. The inhibitory effect of EPA on PGF2alpha production was reverted in BEND cells treated with an increasing n-6-to-n-3 fatty acid ratio. Findings indicate that the net inhibition of endometrial PGF2alpha bioynthesis by n-3 fatty acids may vary depending on the ratio of n-6 to n-3 fatty acids in the uterus. PMID:16507691

  9. Down-regulation of class II phosphoinositide 3-kinase alpha expression below a critical threshold induces apoptotic cell death.

    PubMed

    Elis, Winfried; Triantafellow, Ellen; Wolters, Natalie M; Sian, Katie R; Caponigro, Giordano; Borawski, Jason; Gaither, L Alex; Murphy, Leon O; Finan, Peter M; Mackeigan, Jeffrey P

    2008-04-01

    Members of the phosphoinositide 3-kinase (PI3K) family collectively control multiple cellular responses, including proliferation, growth, chemotaxis, and survival. These diverse effects can partly be attributed to the broad range of downstream effectors being regulated by the products of these lipid kinases, the 3'-phosphoinositides. However, an additional layer of complexity is introduced by the existence of multiple PI3K enzyme isoforms. Much has been learned over the last years on the roles of the classes I and III PI3K members in cellular signaling, but little is known about the isoform-specific tasks done by the class II PI3Ks (C2alpha, beta, and gamma). In this study, we used quantitative reverse transcription-PCR and RNA interference in mammalian cells to gain further insight into the function of these lesser studied PI3K enzymes. We find that PI3K-C2alpha, but not PI3K-C2beta, has an important role in controlling cell survival and by using a panel of RNA interference reagents, we were able to determine a critical threshold of PI3K-C2alpha mRNA levels, below which the apoptotic program is switched on, via the intrinsic cell death pathway. In addition, knockdown of PI3K-C2alpha to levels that by themselves do not induce apoptosis sensitize cells to the anticancer agent Taxol (paclitaxel). Lastly, we report that lowering the levels of PI3K-C2alpha in a number of cancer cell lines reduces their proliferation and cell viability, arguing that PI3K inhibitors targeting not only the class Ialpha isoform but also class IIalpha may contribute to an effective anticancer strategy. PMID:18403640

  10. RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND THE HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY

    EPA Science Inventory

    Rainbow Trout Androgen Receptor Alpha And Human Androgen Receptor: Comparisons in the COS Whole Cell Binding Assay
    Mary C. Cardon, L. Earl Gray, Jr. and Vickie S. Wilson
    U.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology Division, Research Triangle...

  11. BINDING OF STEROIDS AND ENVIRONMENTAL CHEMICALS TO THE RAINBOW TROUT ANDROGEN RECEPTOR ALPHA EXPRESSED IN COS CELLS

    EPA Science Inventory

    Binding of Steroids and Environmental Chemicals to the Rainbow Trout Androgen Receptor Alpha Expressed in COS Cells.

    Mary C. Cardon, L. Earl Gray. Jr., Phillip C. Hartig and Vickie S. Wilson
    U.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology...

  12. RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND THE HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY

    EPA Science Inventory

    RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY.
    MC Cardon, PC Hartig,LE Gray, Jr. and VS Wilson.
    U.S. EPA, ORD, NHEERL, RTD, Research Triangle Park, NC, USA.
    Typically, in vitro hazard assessments for ...

  13. Rapid response to 2'-deoxycoformycin in advanced hairy cell leukemia after failure of interferons alpha and gamma.

    PubMed

    Lembersky, B C; Ratain, M J; Westbrook, C; Golomb, H M

    1988-01-01

    A patient with advanced hairy cell leukemia initially had a short-lived minor response to interferon alpha therapy and failed to respond to interferon gamma. Subsequent treatment with 2'-deoxycoformycin (dCF) administered biweekly for 12 wk resulted in a complete hematological remission which has continued for 16 months without additional therapy. PMID:3128105

  14. Epstein-Barr Virus BARF1 Protein Is Dispensable for B-Cell Transformation and Inhibits Alpha Interferon Secretion from Mononuclear Cells

    PubMed Central

    Cohen, Jeffrey I.; Lekstrom, Kristen

    1999-01-01

    The Epstein-Barr virus (EBV) BARF1 gene encodes a soluble colony-stimulating factor 1 (CSF-1) receptor that neutralizes the effects of CSF-1 in vitro. To study the effect of BARF1 on EBV-induced transformation, we added recombinant BARF1 to B cells in the presence of EBV. BARF1 did not enhance transformation of B cells by EBV in vitro. To study the role of BARF1 in the context of EBV infection, we constructed a recombinant EBV mutant with a large deletion followed by stop codons in the BARF1 gene as well as a recombinant virus with a wild-type BARF1 gene. While BARF1 has previously been shown to act as an oncogene in several cell lines, the EBV BARF1 deletion mutant transformed B cells and initiated latent infection, and the B cells transformed with the BARF1 mutant virus induced tumors in SCID mice with an efficiency similar to that of the wild-type recombinant virus. Since human CSF-1 stimulates secretion of alpha interferon from mononuclear cells and BARF1 encodes a soluble CSF-1 receptor, we examined whether recombinant BARF1 or BARF1 derived from EBV-infected B cells could inhibit alpha interferon secretion. Recombinant BARF1 inhibited alpha interferon secretion by mononuclear cells in a dose-dependent fashion. The B cells transformed with mutant BARF1 EBV showed reduced inhibition of alpha interferon secretion by human mononuclear cells when compared with the B cells transformed with wild-type recombinant virus. These experiments indicate that BARF1 expressed from the EBV genome directly inhibits alpha interferon secretion, which may modulate the innate host response to the virus. PMID:10438853

  15. Lycopene is a more potent inhibitor of human cancer cell proliferation than either alpha-carotene or beta-carotene.

    PubMed

    Levy, J; Bosin, E; Feldman, B; Giat, Y; Miinster, A; Danilenko, M; Sharoni, Y

    1995-01-01

    The antiproliferative properties of lycopene, the major tomato carotenoid, were compared with those of alpha- and beta-carotene. Lycopene, delivered in cell culture medium from stock solutions in tetrahydrofuran, strongly inhibited proliferation of endometrial (Ishikawa), mammary (MCF-7), and lung (NCI-H226) human cancer cells with half-maximal inhibitory concentration of 1-2 microM; alpha- and beta-carotene were far less effective inhibitors. For example, in Ishikawa cells, a 4-fold higher concentration of alpha-carotene or a 10-fold higher concentration of beta-carotene was needed for the same order of growth suppression. The inhibitory effect of lycopene was detected after 24 hours of incubation, and it was maintained for at least three days. In contrast to cancer cells, human fibroblasts were less sensitive to lycopene, and the cells gradually escaped growth inhibition over time. In addition to its inhibitory effect on basal endometrial cancer cell proliferation, lycopene also suppressed insulin-like growth factor-I-stimulated growth. Insulin-like growth factors are major autocrine/paracrine regulators of mammary and endometrial cancer cell growth. Therefore, lycopene interference in this major autocrine/paracrine system may open new avenues for research on the role of lycopene in the regulation of endometrial cancer and other tumors. PMID:8610045

  16. Telmisartan increases lipoprotein lipase expression via peroxisome proliferator-activated receptor-alpha in HepG2 cells.

    PubMed

    Yin, Shi Nan; Liu, Min; Jing, Dan Qing; Mu, Yi Ming; Lu, Ju Ming; Pan, Chang Yu

    2014-01-01

    In addition to their hypotensive properties, angiotensin receptor blockers (ARBs) have been shown to exert clinical antidyslipidemic effects. The mechanism underlying these ARB lipid metabolic effects remains unclear. Some ARBs, for example, telmisartan, activate peroxisome proliferator-activated activated receptor-gamma (PPAR-gamma). We hypothesized that PPAR-gamma-activating ARBs might exert antidyslipidemic effects via PPAR-alpha. In this study, we assessed the effect of telmisartan on the expression of PPAR-alpha and lipoprotein lipase (LPL). PPAR-alpha expression was detected by reverse-transcription polymerase chain reaction and Western blot in HepG2 hepatocytes as well as differentiated C2C12 myocytes treated with increasing concentrations of telmisartan (0.1-10 μmol/L) for 48 h. Results showed that 1 μmol/L and 10 μmol/L telmisartan significantly increased the expression of PPAR-alpha mRNA and protein in HepG2 cells (p < 0.01). No effect was shown in differentiated C2C12 cells. Similarly, 1 µmol/L and 10 μmol/L telmisartan significantly increased the expression of LPL mRNA and protein in HepG2 cells (p < 0.01), and this increase was significantly (p < 0.01) inhibited by the PPAR-alpha-specific antagonist MK886. These results indicate that certain of the antidyslipidemic effects of telmisartan might be mediated via increased PPAR-alpha-dependent induction of LPL expression. PMID:24067162

  17. Novel ring A stereoisomers of 2-methyl-1alpha,25-dihydroxyvitamin D(3) and 2-methyl-20-epi-1alpha,25-dihydroxyvitamin D(3): transactivation of target genes and modulation of differentiation in human promyelocytic leukemia (HL-60) cells.

    PubMed

    Nakagawa, K; Kurobe, M; Ozono, K; Konno, K; Fujishima, T; Takayama, H; Okano, T

    2000-03-15

    We evaluated the biological activity of two sets of ring A stereoisomers of 2-methyl-1alpha,25-dihydroxyvitamin D(3) (2-methyl-1alpha,25(OH)(2)D(3)) and 2-methyl-20-epi-1alpha, 25-dihydroxyvitamin D(3) (2-methyl-20-epi-1alpha,25(OH)(2)D(3)) in terms of the following: transactivation of a rat 25-hydroxyvitamin D(3)-24-hydroxylase gene promoter including two vitamin D response elements (VDREs) and a human osteocalcin gene promoter including a VDRE in transfected human osteosarcoma (MG-63) cells; a vitamin D receptor (VDR)-mediated response using a VDR-GAL4 one-hybrid luciferase reporter system and a retinoid X receptor alpha (RXRalpha)-mediated response using an expressed VDR/RXRalpha-GAL4 modified two-hybrid luciferase reporter system in transfected human epitheloid carcinoma, cervix (HeLa) cells; and modulation of cell surface CD11b antigen expression in human leukemia (HL-60) cells. All the diastereomers of both analogues exhibited unique biological activity profiles depending upon the configurations of the C-1 and C-3 hydroxyl groups, the C-2 methyl group in ring A, and the C-20 methyl group in the side chain. Of the eight possible diastereomers of the 2-methyl analogues, 2alpha-methyl-1alpha,25(OH)(2)D(3) was the most potent and exhibited comparable or even greater biological potency than 1alpha,25(OH)(2)D(3). Of the eight possible diastereomers of the 2-methyl-20-epi analogues, 2alpha-methyl-20-epi-1alpha,25(OH)(2)D(3) was the most potent and exhibited 100- to 200-fold higher transcriptional potencies than 1alpha,25(OH)(2)D(3) and exceptionally high cell regulatory activities. 2beta-methyl-20-epi-1alpha,25(OH)(2)D(3) was nearly as potent as its 2-epimer, 2alpha-methyl-20-epi-1alpha,25(OH)(2)D(3), whereas its 20-epimer, 2beta-methyl-1alpha,25(OH)(2)D(3), was almost completely biologically inactive. In these respects, it can be postulated that the double modification of 2-methyl substitution and 20-epimerization to 1alpha,25(OH)(2)D(3) induces remarkable changes

  18. Chemokine gene expression in the murine renal cell carcinoma, RENCA, following treatment in vivo with interferon-alpha and interleukin-2.

    PubMed Central

    Sonouchi, K.; Hamilton, T. A.; Tannenbaum, C. S.; Tubbs, R. R.; Bukowski, R.; Finke, J. H.

    1994-01-01

    The expression of three chemoattractant cytokine (chemokine) messenger (m)RNAs in the murine renal cell carcinoma (RENCA) from mice treated with a combination of interferon-alpha (IFN-alpha) and interleukin-2 was examined and related to tumor infiltration by inflammatory leukocytes. Using a semi-quantitative reverse transcriptase polymerase chain reaction assay, mRNAs encoding the KC, JE, and IP-10 genes were all elevated in tumor tissue from mice treated systemically with IFN-alpha/interleukin-2 for 4 days. Similarly, the mRNA for tumor necrosis factor-alpha (TNF-alpha) was also increased in tumors from treated as compared to control animals. The same tumors showed a significant increase in Mac-1+ leukocytes, which correlated well with the increase in chemokine and TNF-alpha gene expression. The renal cell carcinoma tumor itself may be responsible for the expression of chemokine genes in the tumor bed following cytokine therapy. Cultures of freshly explanted RENCA cells expressed significant levels of chemokine mRNAs when stimulated in vitro with IFN alpha, IFN gamma, and/or interleukin-2, demonstrating that this tumor cell has potential for expression of these genes in vivo. In contrast, TNF-alpha expression was not detected in cultured tumor cells. Thus TNF-alpha may be expressed by infiltrating monocytes following exposure to recombinant cytokine therapy. Images Figure 1 Figure 2 Figure 4 PMID:8160774

  19. Alpha-bungarotoxin binding to target cell in a developing visual system by carboxylated nanodiamond

    NASA Astrophysics Data System (ADS)

    Liu, Kuang-Kai; Chen, Mei-Fang; Chen, Po-Yi; Lee, Tony J. F.; Cheng, Chia-Liang; Chang, Chia-Ching; Ho, Yen-Peng; Chao, Jui-I.

    2008-05-01

    Biological molecules conjugating with nanoparticles are valuable for applications including bio-imaging, bio-detection, and bio-sensing. Nanometer-sized diamond particles have excellent electronic and chemical properties for bio-conjugation. In this study, we manipulated the carboxyl group produced on the surface of nanodiamond (carboxylated nanodiamond, cND) for conjugating with alpha-bungarotoxin (α-BTX), a neurotoxin derived from Bungarus multicinctus with specific blockade of alpha7-nicotinic acetylcholine receptor (α7-nAChR). The electrostatic binding of cND-α-BTX was mediated by the negative charge of the cND and the positive charge of the α-BTX in physiological pH conditions. Sodium dodecyl sulfate-polyacrylamide gel analysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS) spectra displayed that α-BTX proteins were conjugated with cND particles via non-covalent bindings. The green fluorescence of the cND particles combining with the red fluorescence of tetramethylrhodamine-labeled α-BTX presented a yellow color at the same location, which indicated that α-BTX proteins were conjugated with cND particles. Xenopus laevis's oocytes expressed the human α7-nAChR proteins by microinjection with α7-nAChR mRNA. The cND-α-BTX complexes were bound to α7-nAChR locating on the cell membrane of oocytes and human lung A549 cancer cells analyzed by laser scanning confocal microscopy. The choline-evoked α7-nAChR-mediated inward currents of the oocytes were blocked by cND-α-BTX complexes in a concentration-dependent manner using two-electrode voltage-clamp recording. Furthermore, the fluorescence intensity of cND-α-BTX binding on A549 cells could be quantified by flow cytometry. These results indicate that cND-conjugated α-BTX still preserves its biological activity in blocking the function of α7-nAChR, and provide a visual system showing the binding of α-BTX to α7-nAChR.

  20. EGCG decreases binding of calcium oxalate monohydrate crystals onto renal tubular cells via decreased surface expression of alpha-enolase.

    PubMed

    Kanlaya, Rattiyaporn; Singhto, Nilubon; Thongboonkerd, Visith

    2016-06-01

    Crystal retention on tubular cell surface inside renal tubules is considered as the earliest and crucial step for kidney stone formation. Therapeutics targeting this step would cease the development of kidney stone. This study thus aimed to investigate the potential role of epigallocatechin-3-gallate (EGCG), a major antioxidant found in green tea leaves, in the reduction of calcium oxalate monohydrate (COM) crystal binding onto renal tubular cells. Pretreatment of the cells with EGCG for up to 6 h significantly diminished crystal-binding capability in a dose-dependent manner. Indirect immunofluorescence assay without and with cell permeabilization followed by laser-scanning confocal microscopy revealed that EGCG significantly reduced surface expression of alpha-enolase, whereas its intracellular level was increased. Western blot analysis confirmed such contradictory changes in membrane and cytosolic fractions of EGCG-treated cells, whereas the total level in whole cell lysate remained unchanged. Moreover, overexpression of surface alpha-enolase and enhancement of cell-crystal adhesion induced by 10 mM sodium oxalate were completely abolished by EGCG. Taken together, these data indicate that EGCG decreases binding of COM crystals onto renal tubular cells by decreasing the surface expression of alpha-enolase via re-localization or inhibition of alpha-enolase shuttling from the cytoplasm to the plasma membrane. These findings may also explain the effects of EGCG in reducing COM crystal deposition in previous animal models of kidney stone disease. Thus, EGCG may be useful for the prevention of new or recurrent stone formation. PMID:26898643

  1. The effects of HIF-1alpha on gene expression profiles of NCI-H446 human small cell lung cancer cells

    PubMed Central

    2009-01-01

    Background Gene targeted therapy refers to any therapy focused on one of the many biological features of the tumor. Such features are mediated by specific genes that are involved in tumor metastasis, recurrence, poor response to chemotherapy and others. Hypoxia is an important pathognomonic feature of many malignant tumors including SCLC (small cell lung cancer). HIF-1alpha, which is induced by hypoxia, is the most important regulatory factor of many specific genes that can influence the biological features of tumors. Methods In this study, we tried to elucidate the changes in gene expression profiles of SCLC NCI-H446 cells mediated by HIF-1alpha. According to different treatments of cells, three experimental pairwise comparisons were designed: hypoxia group vs. control group, Ad5-HIF-1alpha group vs. Ad5 group, and Ad5-siHIF-1 alpha group Vs Ad5 group. Results Results from the analysis of gene expression profiles indicated that there were 65 genes upregulated and 28 genes downregulated more than two-fold in all three experimental pairwise comparisons. These genes were involved in transport, signal-transduction, cell adhesion/motility, growth factor/cytokines, transcription, inflammatory response, metabolic process, in addition to others. SOCS1, IGFBP5, IL-6 and STAT3 were also upregulated at protein level. SOCS1 could significantly induce apoptosis and suppress growth of NCI-H446 cells but HIF-1alpha could induce growth and suppress apoptosis. Conclusions Through this research, we are trying to find novel functional genes that are mediated by HIF-1alpha and provide the theoretical basis for new therapeutic targets. HIF-1 alpha maybe upregulate the expression of SOCS1 through mediation of STAT3 and IL-6. In addition, SOCS1 could significantly induce apoptosis and suppress growth of NCI-H446 cells. This was contrary to HIF-1alpha and it indicated that there might be an antagonism effect between HIF-1alpha and SOCS1 on regulating growth and apoptosis of NCI-H446

  2. ER Alpha Rapid Signaling Is Required for Estrogen Induced Proliferation and Migration of Vascular Endothelial Cells

    PubMed Central

    Lu, Qing; Schnitzler, Gavin R.; Ueda, Kazutaka; Iyer, Lakshmanan K.; Diomede, Olga I.; Andrade, Tiffany; Karas, Richard H.

    2016-01-01

    Estrogen promotes the proliferation and migration of vascular endothelial cells (ECs), which likely underlies its ability to accelerate re-endothelialization and reduce adverse remodeling after vascular injury. In previous studies, we have shown that the protective effects of E2 (the active endogenous form of estrogen) in vascular injury require the estrogen receptor alpha (ERα). ERα transduces the effects of estrogen via a classical DNA binding, “genomic” signaling pathway and via a more recently-described “rapid” signaling pathway that is mediated by a subset of ERα localized to the cell membrane. However, which of these pathways mediates the effects of estrogen on endothelial cells is poorly understood. Here we identify a triple point mutant version of ERα (KRR ERα) that is specifically defective in rapid signaling, but is competent to regulate transcription through the “genomic” pathway. We find that in ECs expressing wild type ERα, E2 regulates many genes involved in cell migration and proliferation, promotes EC migration and proliferation, and also blocks the adhesion of monocytes to ECs. ECs expressing KRR mutant ERα, however, lack all of these responses. These observations establish KRR ERα as a novel tool that could greatly facilitate future studies into the vascular and non-vascular functions of ERα rapid signaling. Further, they support that rapid signaling through ERα is essential for many of the transcriptional and physiological responses of ECs to E2, and that ERα rapid signaling in ECs, in vivo, may be critical for the vasculoprotective and anti-inflammatory effects of estrogen. PMID:27035664

  3. Stimulation of prostaglandin E2-synthesis by noradrenaline in primary cell cultures from rabbit splenic pulpa is mediated by atypical alpha-adrenoceptors.

    PubMed

    Brückner-Schmidt, R; Jackisch, R; Hertting, G

    1981-02-01

    In primary cell cultures originating from rabbit splenic pulpa the effects of various adrenoceptor agonists on prostaglandin (PG)-synthesis were studied. The cells - microscopically identified as fibroblasts - released PGs into the medium: especially PGE2 besides small amounts of PGF2alpha and PGD2. Noradrenaline increased dose-dependently the amount of PGs released into the medium. Besides noradrenaline, only the catecholamines adrenaline and alpha-methylnoradrenaline strongly activated PG-synthesis. Other alpha-adrenoceptor agonists like the phenylethylamine and imidazoline derivatives were only weak agonists or completely ineffective. All adrenoceptor agonists without intrinsic activity in these cells antagonized the noradrenaline effect on PG-synthesis, the imidazolines being more potent antagonists than the phenylethylamines. The beta-adrenoceptor agonist isoprenaline stimulated PG-synthesis at high concentration only. The effects of both noradrenaline and isoprenaline were inhibited by low concentrations of phentolamine phenoxybenzamine, but not by propranolol. The preferential alpha2-adrenoceptor antagonists yohimbine and rauwolscine were about 50 times more potent in blocking the noradrenaline effect on PG-synthesis than the more alpha1-specific antagonist corynanthine. However, prazosin, another alpha1-adrenoceptor antagonist, was about equipotent with yohimbine. It is concluded that noradrenaline elicits PG-synthesis in rabbit splenic fibroblasts via alpha-adrenoceptor stimulation. The alpha-adrenoceptor involved has properties which are different from those reported so far for alpha1- or alpha2-adrenoceptors. PMID:6268994

  4. Affinity maturation of anti-TNF-alpha scFv with somatic hypermutation in non-B cells.

    PubMed

    Chen, Shaopeng; Qiu, Junkang; Chen, Chuan; Liu, Chunchun; Liu, Yuheng; An, Lili; Jia, Junying; Tang, Jie; Wu, Lijun; Hang, Haiying

    2012-06-01

    Activation-induced cytidine deaminase (AID) is required for the generation of antibody diversity through initiating both somatic hypermutation (SHM) and class switch recombination. A few research groups have successfully used the feature of AID for generating mutant libraries in directed evolution of target proteins in B cells in vitro. B cells, cultured in suspension, are not convenient for transfection and cloning. In this study, we established an AID-based mutant accumulation and sorting system in adherent human cells. Mouse AID gene was first transfected into the human non-small cell lung carcinoma H1299 cells, and a stable cell clone (H1299-AID) was selected. Afterwards, anti-hTNF-α scFv (ATscFv) was transfected into H1299-AID cells and ATscFv was displayed on the surface of H1299-AID cells. By 4-round amplification/flow cytometric sorting for cells with the highest affinities to hTNF-alpha, two ATscFv mutant gene clones were isolated. Compared with the wild type ATscFv, the two mutants were much more efficient in neutralizing cytotoxicity of hTNF-alpha. The results indicate that directed evolution by somatic hypermutation can be carried out in adherent non-B cells, which makes directed evolution in mammalian cells easier and more efficient. PMID:22467272

  5. Glucocorticoids inhibit coordinated translation of. cap alpha. - and. beta. -globin mRNAs in Friend erythroleukemia cells

    SciTech Connect

    Papaconstantinou, J.; Stewart, J.A.; Rabek, J.P.; McClintock, P.R.; Wong, E.Y.

    1983-12-01

    The dimethylsulfoxide (Me/sub 2/SO)-mediated induction of hemoglobin synthesis in Friend erythroleukemia cells is inhibited by the glucocorticoids hydrocortisone, dexamethasone, and fluocinolone acetonide; hydrocortisone, at concentrations of 10/sup -5/ to 10/sup -8/ M inhibits by 90-30% and fluocinolone acetonide at concentrations of 10/sup -8/ to 10/sup -11/ M shows a greater than 90% inhibition. At these concentrations the hormones have no effect on cell growth or viability. In this study it has been shown that there is a group of proteins, including the ..cap alpha..- and ..beta..-globins, whose regulation is associated with the induction of Friend erythroleukemia cell differentiation, and that the expression of these, in addition to ..cap alpha..- and ..beta..-globin, is affected by glucocorticoids. It is concluded that, although the translation of ..cap alpha..- and ..beta..-globin mRNA is a major site of inhibition by glucocorticoids, there is a detectable amount of ..cap alpha..- and ..beta..-globin mRNA translation which results in unequal amounts of globin synthesis and an overall more potent inhibition of hemoglobin formation.

  6. Isoforms of alpha1E voltage-gated calcium channels in rat cerebellar granule cells--detection of major calcium channel alpha1-transcripts by reverse transcription-polymerase chain reaction.

    PubMed

    Schramm, M; Vajna, R; Pereverzev, A; Tottene, A; Klöckner, U; Pietrobon, D; Hescheler, J; Schneider, T

    1999-01-01

    In primary cultures of rat cerebellar granule cells, transcripts of voltage-gated Ca2+ channels have been amplified by reverse transcription-polymerase chain reaction and identified by sequencing of subcloned polymerase chain reaction products. In these neurons cultured for six to eight days in vitro, fragments of the three major transcripts alpha1C, alpha1A, and alpha1E are detected using degenerated oligonucleotide primer pairs under highly stringent conditions. Whole-cell Ca2+ current recordings from six to eight days in vitro granule cells show that most of the current is due to L-type (25%), P-type (33%) and R-type (30%) Ca2+ channels. These data support the correlation between alpha1A and P-type Ca2+ channels (G1) and between alpha1E and R-type channels (G2 and G3). By including specific primer pairs for alpha1E the complimentary DNA fragments of indicative regions of alpha1E isoforms are amplified corresponding to the three most variable regions of alpha1E, the 5'-end, the II/III-loop, and the central part of the 3'-end. Although the complementary DNA fragments of the 5'-end of rat alpha1E yield a uniform reverse transcription-polymerase chain reaction product, its structure is unusual in the sense that it is longer than in the cloned rat alpha1E complementary DNA. It corresponds to the alpha1E isoform reported for mouse and human brain and is also expressed in cerebellum and cerebrum of rat brain as the major or maybe even the only variant of alpha1E. While fragments of a new rat alpha1E isoform are amplified from the 5'-end, three known fragments of the II/III-loop and two known isoforms homologue to the 3'-coding region are detected, which in the last case are discriminated by a 129 base pair insertion. The shift of the alpha1E expression from a pattern seen in cerebellum (alpha1Ee) to a pattern identified in other regions of the brain (alpha1E-3) is discussed. These data show that: (i) alpha1E is expressed in rat brain as a structural homologue to the

  7. VEGF Secretion is Inhibited by Interferon-Alpha in Several Melanoma Cell Lines

    PubMed Central

    Raig, Ene T.; Jones, Natalie B.; Varker, Kimberly A.; Benniger, Kristen; Go, Michael R.; Biber, Jennifer L.; Lesinski, Gregory B.

    2008-01-01

    Interferon-alpha (IFN-α) is employed in the treatment of malignant melanoma; however, it mediates regression of disease in only 10–15% of patients. Currently, its mechanism of action is uncharacterized. Low-dose IFN-α exerts anti-angiogenic effects when used in the treatment of life-threatening hemangiomas of infancy, suggesting anti-angiogenesis as a mechanism of action. IFN-α may exert its anti-tumor effect in the setting of advanced malignancy by inhibiting the secretion of vascular endothelial growth factor (VEGF), a pro-angiogenic substance. We hypothesized that IFN-α would decrease the release of VEGF by melanoma tumors. We studied the effect of IFN-α on VEGF production in nine human melanoma cell lines. We also examined VEGF levels in 49 patients with advanced malignancies who received low-dose IFN-α and interleukin-12 (IL-12) on an NCI-sponsored phase I trial. Human melanoma cell lines produced varying amounts of VEGF in vitro (60–1500 pg/mL at 48 h). Certain melanoma cell lines such as 18105 MEL secreted low levels of VEGF (152 pg/mL) after 48 h of culture, whereas other lines secreted very high levels (FO-1 3,802 pg/mL). Treatment of melanoma cells with IFN-α (2000 U/mL) decreased VEGF secretion by 40–60% in VEGF-high cell lines; however, this effect was not demonstrated in VEGF-low cell lines. In cancer patients, pretreatment VEGF plasma levels varied from 471 to 4200 pg/mL. A decrease in VEGF plasma levels after treatment directly correlated with the number of treatment cycles administered (Pearson correlation, p = 0.04). In summary, IFN-α inhibits VEGF secretion by melanoma cell lines in vitro and may have similar actions in malignancies that respond to IFN-α treatment. PMID:18771339

  8. A nuclear pathway for alpha 1-adrenergic receptor signaling in cardiac cells.

    PubMed Central

    Ardati, A; Nemer, M

    1993-01-01

    alpha 1-Adrenergic agonists and antagonists constitute an important class of therapeutic agents commonly used for the treatment of various cardiovascular diseases like hypertension, congestive heart failure and supraventricular tachycardia. At the heart level, activation of alpha 1-adrenergic receptors is associated with marked morphological and genetic changes. These include enhancement of contractility, myocardial growth (hypertrophy) and release of the heart major secretory product, atrial natriuretic factor (ANF). However, the signal transduction pathways which link extracellular activation of the receptors to cellular and genetic changes are not well understood. Using primary cardiocyte cultures from neonate rat hearts, an alpha 1-adrenergic regulatory sequence has been identified in the 5' flanking region of the ANF gene. This sequence, which is necessary and sufficient for transcriptional activation in response to the alpha 1-specific agonist phenylephrine, interacts with novel zinc-dependent proteins which are induced by alpha 1-adrenergic stimulation. Consistent with a conserved regulatory mechanism, the alpha 1 response element is highly conserved between rodent, bovine and human ANF genes, and is also present in the promoter region of other alpha 1-responsive cardiac genes. The identification of a nuclear pathway for alpha 1-receptor signaling will be useful for elucidating the intracellular effectors of alpha 1-adrenergic receptors. Images PMID:8262057

  9. Bench-level characterization of a CMOS standard-cell D-latch using alpha-particle sensitive test circuits

    SciTech Connect

    Blaes, B.R.; Soli, G.A.; Buehler, M.G. )

    1991-12-01

    This paper describes a methodology for predicting the SEU susceptibility of a standard-cell D-latch using an alpha-particle sensitive SRAM, SPICE critical charge simulation results, and alpha-particle interaction physics. Measurements were made on a 1.6-{mu}m n-well CMOS 4k-bit test SRAM irradiated with an Am-241 alpha-particle source. A collection depth of 6.09 {mu}m was determined using these results and TRIM computer code. Using this collection depth and SPICE derived critical charge results on the latch design, an LET threshold of 34 Mev cm{sup 2}/mg was predicted. Heavy ion tests were then performed on the latch and an LET threshold of 41 MeV cm{sup 2}/mg was determined.

  10. Bench-level characterization of a CMOS standard-cell D-latch using alpha-particle sensitive test circuits

    NASA Technical Reports Server (NTRS)

    Blaes, B. R.; Soli, G. A.; Buehler, M. G.

    1991-01-01

    A methodology is described for predicting the SEU susceptibility of a standard-cell D-latch using an alpha-particle sensitive SRAM, SPICE critical charge simulation results, and alpha-particle interaction physics. Measurements were made on a 1.6-micron n-well CMOS 4-kb test SRAM irradiated with an Am-241 alpha-particle source. A collection depth of 6.09 micron was determined using these results and TRIM computer code. Using this collection depth and SPICE derived critical charge results on the latch design, an LET threshold of 34 MeV sq cm/mg was predicted. Heavy ion tests were then performed on the latch and an LET threshold of 41 MeV sq cm/mg was determined.

  11. Etomidate, propofol and the neurosteroid THDOC increase the GABA efficacy of recombinant alpha4beta3delta and alpha4beta3 GABA A receptors expressed in HEK cells.

    PubMed

    Meera, Pratap; Olsen, Richard W; Otis, Thomas S; Wallner, Martin

    2009-01-01

    General anesthetics, once thought to exert their effects through non-specific membrane effects, have highly specific ion channel targets that can silence neuronal populations in the nervous system, thereby causing unconsciousness and immobility, characteristic of general anesthesia. Inhibitory GABA(A) receptors (GABA(A)Rs), particularly highly GABA-sensitive extrasynaptic receptor subtypes that give rise to sustained inhibitory currents, are uniquely sensitive to GABA(A)R-active anesthetics. A prominent population of extrasynaptic GABA(A)Rs is made up of alpha4, beta2 or beta3, and delta subunits. Considering the demonstrated importance of GABA receptor beta3 subunits for in vivo anesthetic effects of etomidate and propofol, we decided to investigate the effects of GABA anesthetics on "extrasynaptic" alpha4beta3delta and also binary alpha4beta3 receptors expressed in human embryonic kidney (HEK) cells. Consistent with previous work on similar receptor subtypes we show that maximal GABA currents through "extrasynaptic" alpha4beta3delta receptors, receptors defined by sensitivity to EtOH (30mM) and the beta-carboline beta-CCE (1microM), are enhanced by the GABA(A)R-active anesthetics etomidate, propofol, and the neurosteroid anesthetic THDOC. Furthermore, we show that receptors formed by alpha4beta3 subunits alone also show high GABA sensitivity and that saturating GABA responses of alpha4beta3 receptors are increased to the same extent by etomidate, propofol, and THDOC as are alpha4beta3delta receptors. Therefore, both alpha4beta3 and alpha4beta3delta receptors show low GABA efficacy, and GABA is also a partial agonist on certain binary alphabeta receptor subtypes. Increasing GABA efficacy on alpha4/6beta3delta and alpha4beta3 receptors is likely to make an important contribution to the anesthetic effects of etomidate, propofol and the neurosteroid THDOC. PMID:18778723

  12. Toward selective CK2alpha and CK2alpha' inhibitors: Development of a novel whole-cell kinase assay by Autodisplay of catalytic CK2alpha'.

    PubMed

    Bollacke, Andre; Nienberg, Christian; Le Borgne, Marc; Jose, Joachim

    2016-03-20

    Human protein kinase CK2 is an emerging target for the development of novel anti-cancer therapeutics. CK2 is a tetramer composed of two catalytically active α- and/or α'-subunits, bound to a dimer of the regulatory β-subunit. Inhibitors targeting one of the two isoforms of the catalytically active CK2-subunit (α- and α') are important to study the distinct functions of these isoforms toward different CK2 associated pathologies. The present study for the first time describes the successful Autodisplay of the CK2α'-subunit, the paralogous isoform of CK2α. Expression on the cell surface of E. coli of CK2α' alone and in combination with the regulatory CK2β-subunit was confirmed by outer membrane isolation and protease accessibility test. Kinase activity of surface displayed CK2 could be detected with a CE-based assay and was found to be 3.06×10(-6) μmol/min for CK2α' alone and 1.02×10(-5) μmol/min when expressed in combination with CK2β. The comparison of the influence of NaCl on activity of the α'-subunit alone and in combination with the non-catalytically active β-subunit indicated interaction of both subunits on the cell surface. TMCB (4,5,6,7-tetrabromo-2-(dimethylamino)-1H-benzo[d]imidazol-1-yl)acetic acid), a known CK2 inhibitor described with distinct Ki values of 83 nM and 21 nM for the two different catalytic CK2 subunits α and α' was used for testing. First, inhibition of TMCB toward the purified CK2 holoenzyme CK2α2β2 was determined and resulted in a Ki value of 10.1 nM. Second, Ki values were determined with the surface displayed isoform CK2 holoenzymes and turned out to be of 31.1 nM for CK2α2β2 and 19.6 nM for CK2α'2β2. The inhibition data as obtained represented the distinct affinities of TMCB toward the two isoform holoenzymes. This indicated, that the surface display of CKα and CK2α', in the context of the corresponding holoenzymes, can be used to identify selective compounds. A set of twelve ATP competitive CK2 inhibitors

  13. Hypoxia reduces constitutive and TNF-{alpha}-induced expression of monocyte chemoattractant protein-1 in human proximal renal tubular cells

    SciTech Connect

    Li Xuan; Kimura, Hideki . E-mail: hkimura@fmsrsa.fukui-med.ac.jp; Hirota, Kiichi; Sugimoto, Hidehiro; Yoshida, Haruyoshi

    2005-10-07

    Chronic hypoxia has been reported to be associated with macrophage infiltration in progressive forms of kidney disease. Here, we investigated the regulatory effects of hypoxia on constitutive and TNF-{alpha}-stimulated expression of monocyte chemoattractant protein-1 (MCP-1) in cultured human proximal renal tubular cells (HPTECs). Hypoxia reduced constitutive MCP-1 expression at the mRNA and protein levels in a time-dependent fashion for up to 48 h. Hypoxia also inhibited MCP-1 up-regulation by TNF-{alpha}. Treatment with actinomycin D showed that hypoxic down-regulation of MCP-1 expression resulted mainly from a decrease in the transcription but not the mRNA stability. Immunoblot and immunofluorescence analyses revealed that treatment with hypoxia or an iron chelator, desferrioxamine, induced nuclear accumulation of hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) in HPTECs. Desferrioxamine mimicked hypoxia in the reduction of MCP-1 expression. However, overexpression of a dominant negative form of HIF-1{alpha} did not abolish the hypoxia-induced reduction of MCP-1 expression in HPTECs. These results suggest that hypoxia is an important negative regulator of monocyte chemotaxis to the renal inflamed interstitium, by reducing MCP-1 expression partly via hypoxia-activated signals other than the HIF-1 pathway.

  14. Laminin alpha 5, a major transcript of normal and malignant rat liver epithelial cells, is differentially expressed in developing and adult liver.

    PubMed

    Seebacher, T; Medina, J L; Bade, E G

    1997-11-25

    The laminin family of extracellular matrix glycoproteins plays a major role in cell migration and differentiation and in tumor cell invasion. As previously shown, the laminin deposited by normal and malignant rat liver epithelial cells in their extracellular matrix (ECM) and into their ECM migration tracks does not contain a typical (EHS-like) alpha 1 heavy chain. By RT-PCR screening we have now identified two alpha chains among a total of five additional laminin chains produced by these cells. Three of the newly identified chains were not previously known for the rat. Their sequences have been deposited in the EMBL nucleotide sequence data bank. The alpha 5 chain now identified is expressed at comparably high levels by both the normal and the malignant liver epithelial cells. The chain is also expressed in fetal liver together with the alpha 2 and beta 2 chains, but it is only vestigially expressed in the mature organ as shown by RT-PCR. These results suggest for alpha 5 a role in development and production of the chain by only a small subset of cells in adult liver. At the level of detection used, no changes were observed in regenerating liver after partial hepatectomy. In addition to the alpha 5 chain, the cultured cells express the beta 1 and beta 2 light chains, indicating the expression of more than one laminin isoform by the same cell line. The expression of the alpha 5 chain and of the other new non-EHS isoform chains was also analyzed in various tissues. The malignant liver epithelial cells, but not their nontumorigenic parental cells, also express, in addition to the alpha 5 chain the alpha 2 chain, which is expressed at high level by the NBT II bladder carcinoma cell line, suggesting a relationship with malignancy. PMID:9417868

  15. Knockdown of {alpha}II spectrin in normal human cells by siRNA leads to chromosomal instability and decreased DNA interstrand cross-link repair

    SciTech Connect

    McMahon, Laura W.; Zhang Pan; Sridharan, Deepa M.; Lefferts, Joel A.; Lambert, Muriel W.

    2009-04-03

    Nonerythroid {alpha}-spectrin ({alpha}IISp) is a structural protein involved in repair of DNA interstrand cross-links and is deficient in cells from patients with Fanconi anemia (FA), which are defective in ability to repair cross-links. In order to further demonstrate the importance of the role that {alpha}IISp plays in normal human cells and in the repair defect in FA, {alpha}IISp was knocked down in normal cells using siRNA. Depletion of {alpha}IISp in normal cells by siRNA resulted in chromosomal instability and cellular hypersensitivity to DNA interstrand cross-linking agents. An increased number of chromosomal aberrations were observed and, following treatment with a DNA interstrand cross-linking agent, mitomycin C, cells showed decreased cell growth and survival and decreased formation of damage-induced {alpha}IISp and XPF nuclear foci. Thus depletion of {alpha}IISp in normal cells leads to a number of defects observed in FA cells, such as chromosome instability and a deficiency in cross-link repair.

  16. Enhanced homologous recombination is induced by alpha-particle radiation in somatic cells of Arabidopsis thaliana

    NASA Astrophysics Data System (ADS)

    Bian, Po; Liu, Ping; Wu, Yuejin

    Almost 9 percent of cosmic rays which strike the earth's atmosphere are alpha particles. As one of the ionizing radiations (IR), its biological effects have been widely studied. However, the plant genomic instability induced by alpha-particle radiation was not largely known. In this research, the Arabidopsis thaliana transgenic for GUS recombination substrate was used to evaluate the genomic instability induced by alpha-particle radiation (3.3MeV). The pronounced effects of systemic exposure to alpha-particle radiation on the somatic homologous recombination frequency (HRF) were found at different doses. The 10Gy dose of radiation induced the maximal HRF which was 1.9-fold higher than the control. The local radiation of alpha-particle (10Gy) on root also resulted in a 2.5-fold increase of somatic HRF in non-radiated aerial plant, indicating that the signal(s) of genomic instability was transferred to non-radiated parts and initiated their genomic instability. Concurrent treatment of seedlings of Arabidopsis thaliana with alpha-particle and DMSO(ROS scavenger) both in systemic and local radiation signifi- cantly suppressed the somatic HR, indicating that the free radicals produced by alpha-particle radiation took part in the production of signal of genomic instability rather than the signal transfer. Key words: alpha-particle radiation, somatic homologous recombination, genomic instability

  17. Pur-Alpha Induces JCV Gene Expression and Viral Replication by Suppressing SRSF1 in Glial Cells

    PubMed Central

    Sariyer, Ilker Kudret; Sariyer, Rahsan; Otte, Jessica; Gordon, Jennifer

    2016-01-01

    Objective PML is a rare and fatal demyelinating disease of the CNS caused by the human polyomavirus, JC virus (JCV), which occurs in AIDS patients and those on immunosuppressive monoclonal antibody therapies (mAbs). We sought to identify mechanisms that could stimulate reactivation of JCV in a cell culture model system and targeted pathways which could affect early gene transcription and JCV T-antigen production, which are key steps of the viral life cycle for blocking reactivation of JCV. Two important regulatory partners we have previously identified for T-antigen include Pur-alpha and SRSF1 (SF2/ASF). SRSF1, an alternative splicing factor, is a potential regulator of JCV whose overexpression in glial cells strongly suppresses viral gene expression and replication. Pur-alpha has been most extensively characterized as a sequence-specific DNA- and RNA-binding protein which directs both viral gene transcription and mRNA translation, and is a potent inducer of the JCV early promoter through binding to T-antigen. Methods and Results Pur-alpha and SRSF1 both act directly as transcriptional regulators of the JCV promoter and here we have observed that Pur-alpha is capable of ameliorating SRSF1-mediated suppression of JCV gene expression and viral replication. Interestingly, Pur-alpha exerted its effect by suppressing SRSF1 at both the protein and mRNA levels in glial cells suggesting this effect can occur independent of T-antigen. Pur-alpha and SRSF1 were both localized to oligodendrocyte inclusion bodies by immunohistochemistry in brain sections from patients with HIV-1 associated PML. Interestingly, inclusion bodies were typically positive for either Pur-alpha or SRSF1, though some cells appeared to be positive for both proteins. Conclusions Taken together, these results indicate the presence of an antagonistic interaction between these two proteins in regulating of JCV gene expression and viral replication and suggests that they play an important role during viral

  18. High-affinity ouabain binding by yeast cells expressing Na+, K(+)-ATPase alpha subunits and the gastric H+, K(+)-ATPase beta subunit.

    PubMed Central

    Eakle, K A; Kim, K S; Kabalin, M A; Farley, R A

    1992-01-01

    Recently, a beta subunit for the rat gastric H+,K(+)-ATPase (HK beta), which is structurally similar to the beta subunit of Na+, K(+)-ATPase, has been cloned and characterized. Using heterologous expression in yeast, we have tested the specificity of beta subunit assembly with different isoforms of the alpha subunit of Na+, K(+)-ATPase. Coexpression in yeast cells of the HK beta with both the sheep alpha 1 subunit and the rat alpha 3 subunit isoforms of Na+, K(+)-ATPase (alpha 1 and alpha 3, respectively) leads to the appearance of high-affinity ouabain-binding sites in yeast membranes. These ouabain-binding sites (alpha 1 plus HK beta, alpha 3 plus HK beta) have a high affinity for ouabain (Kd, 5-10 nM) and are expressed at levels similar to those formed with the rat beta 1 subunit of Na+, K(+)-ATPase (beta 1) (alpha 1 plus beta 1 or alpha 3 plus beta 1). Potassium acts as a specific antagonist of ouabain binding by alpha 1 plus HK beta and alpha 3 plus HK beta just like sodium pumps formed with beta 1. Sodium pumps formed with the HK beta, however, show quantitative differences in their affinity for ouabain and in the antagonism of K+ for ouabain binding. These data suggest that the structure of the beta subunit may play a role in sodium pump function. Images PMID:1313569

  19. High-affinity ouabain binding by yeast cells expressing Na+, K(+)-ATPase alpha subunits and the gastric H+, K(+)-ATPase beta subunit.

    PubMed

    Eakle, K A; Kim, K S; Kabalin, M A; Farley, R A

    1992-04-01

    Recently, a beta subunit for the rat gastric H+,K(+)-ATPase (HK beta), which is structurally similar to the beta subunit of Na+, K(+)-ATPase, has been cloned and characterized. Using heterologous expression in yeast, we have tested the specificity of beta subunit assembly with different isoforms of the alpha subunit of Na+, K(+)-ATPase. Coexpression in yeast cells of the HK beta with both the sheep alpha 1 subunit and the rat alpha 3 subunit isoforms of Na+, K(+)-ATPase (alpha 1 and alpha 3, respectively) leads to the appearance of high-affinity ouabain-binding sites in yeast membranes. These ouabain-binding sites (alpha 1 plus HK beta, alpha 3 plus HK beta) have a high affinity for ouabain (Kd, 5-10 nM) and are expressed at levels similar to those formed with the rat beta 1 subunit of Na+, K(+)-ATPase (beta 1) (alpha 1 plus beta 1 or alpha 3 plus beta 1). Potassium acts as a specific antagonist of ouabain binding by alpha 1 plus HK beta and alpha 3 plus HK beta just like sodium pumps formed with beta 1. Sodium pumps formed with the HK beta, however, show quantitative differences in their affinity for ouabain and in the antagonism of K+ for ouabain binding. These data suggest that the structure of the beta subunit may play a role in sodium pump function. PMID:1313569

  20. Comparison of alpha-Type-1 polarizing and standard dendritic cell cytokine cocktail for maturation of therapeutic monocyte-derived dendritic cell preparations from cancer patients.

    PubMed

    Trepiakas, Redas; Pedersen, Anders Elm; Met, Ozcan; Hansen, Morten H; Berntsen, Annika; Svane, Inge Marie

    2008-06-01

    The current "gold standard" for generation of dendritic cell (DC) used in DC-based cancer vaccine studies is maturation of monocyte-derived DCs with tumor necrosis factor-alpha (TNF-alpha)/IL-1beta/IL-6 and prostaglandin E(2) (PGE(2)). Recently, a protocol for producing so-called alpha-Type-1 polarized dendritic cells (alphaDC1) in serum-free medium was published based on maturation of monocyte-derived DCs with TNF-alpha/IL-1-beta/polyinosinic:polycytidylic acid (poly-I:C)/interferon (IFN)-alpha and IFN-gamma. This DC maturation cocktail was described to fulfill the criteria for optimal DC generation and to be superior to the standard DC (sDC) cocktail as it induced fully mature DCs with potent IL-12p70 secretion together with CCR7 expression which is necessary for priming of a TH1 response and for migration to the draining lymph node, respectively. In this study, we tested the adaptation of alphaDC1 maturation cocktail to a protocol for clinical grade DC generation from cancer patients performed in X-VIVO 15 medium. We showed that alphaDC1 in this protocol induce lower up-regulation of CD83 and several other maturation markers, co-stimulatory molecules and CCR7 together with higher up-regulation of inhibitory molecules such as PD-L1, ILT2, ILT3 as compared to sDC. Although alphaDC1 matured DCs secreted more IL-12p70 and IL-23 these DCs had lower or similar stimulatory capacity compared to sDCs when used as stimulating cells in mixed lymphocyte reaction (MLR) or for induction of autologous influenza antigen specific T lymphocytes. Thus, our observations underline that alphaDC1 maturation cannot be directly adapted to alternative protocols for DC generation. Also, this study indicates the necessity for further investigation of correlation between in vitro DC parameters and their in vivo efficacy in clinical vaccination trials. PMID:18450338

  1. alpha-Amylase production in high cell density submerged cultivation of Aspergillus oryzae and A. nidulans.

    PubMed

    Agger, T; Spohr, A B; Nielsen, J

    2001-01-01

    The effect of biomass concentration on the formation of Aspergillus oryzae alpha-amylase during submerged cultivation with A. oryzae and recombinant A. nidulans strains has been investigated. It was found that the specific rate of alpha-amylase formation in chemostats decreased significantly with increasing biomass concentration in the range of approx. 2-12 g dry weight kg(-1). When using a recombinant A. nidulans strain in which the gene responsible for carbon catabolite repression of the A. oryzae alpha-amylase gene (creA) was deleted, no significant decrease in the specific rate of alpha-amylase formation was observed. On the basis of the experimental results, it is suggested that the low value of the specific alpha-amylase productivity observed at high biomass concentration is caused by slow mixing of the concentrated feed solution in the viscous fermentation medium. PMID:11234963

  2. S1 nuclease analysis of alpha-globin gene expression in preleukemic patients with acquired hemoglobin H disease after transfer to mouse erythroleukemia cells.

    PubMed

    Helder, J; Deisseroth, A

    1987-04-01

    The loss of alpha-globin gene transcriptional activity rarely occurs as an acquired abnormality during the evolution of myeloproliferative disease or preleukemia. To test whether the mutation responsible for the loss of alpha-globin gene expression (hemoglobin H disease) in these patients is linked with the alpha-globin genes on chromosome 16, we transferred chromosome 16 from preleukemic patients with acquired hemoglobin H disease to mouse erythroleukemia cells and measured the transcriptional activity of the human alpha-globin genes. After transfer to mouse erythroleukemia cells, the expression of human alpha-globin genes from the peripheral blood or marrow cells of preleukemic patients with acquired hemoglobin H disease was similar to that of human alpha-globin genes transferred to mouse erythroleukemia cells from normal donors. These data showed that factor(s) in the mouse erythroleukemia cell can genetically complement the alpha-globin gene defect in these preleukemia patients with acquired hemoglobin H disease and suggest that altered expression of a gene in trans to the alpha-globin gene may be responsible for the acquisition of hemoglobin H disease in these patients. PMID:3031681

  3. Rescue of type I collagen-deficient phenotype by retroviral-vector-mediated transfer of human pro alpha 1(I) collagen gene into Mov-13 cells.

    PubMed Central

    Stacey, A; Mulligan, R; Jaenisch, R

    1987-01-01

    A full-length cDNA clone corresponding to the human pro alpha 1(I) collagen gene was isolated and inserted into a retrovirus vector. Cell lines were obtained which produced recombinant viruses transducing the collagen cDNA (HUC virus). To test whether the transduced cDNA was functional, Mov-13 mouse cells were infected with the virus. These cells do not produce any type I collagen due to an insertional mutation of the pro alpha 1(I) gene which blocks transcription. While normal amounts of pro alpha 2(I) RNA were synthesized, no alpha 2(I) collagen chains were detectable in the mutant Mov-13 cells. Infection with HUC virus, however, resulted in the production of stable type I collagen, which was secreted into the medium. Analysis of pepsin-resistant proteins indicated that interspecies heterotrimers consisting of human alpha 1(I) and mouse alpha 2(I) collagen chains were secreted by the infected Mov-13 cells. Our results show that pro alpha (I) collagen chains from species as distant as human and mouse can associate to form stable type I collagen. The availability of a retrovirus vector transducing a functional pro alpha 1(I) collagen gene combined with the Mov-13 mutant system should enable us to study the effect of specific mutations on the synthesis, assembly, and function of type I collagen, not only in tissue culture but also in the animal. Images PMID:3599181

  4. Alpha 1 Antitrypsin Inhibits Dendritic Cell Activation and Attenuates Nephritis in a Mouse Model of Lupus

    PubMed Central

    Elshikha, Ahmed S.; Lu, Yuanqing; Chen, Mong-Jen; Akbar, Mohammad; Zeumer, Leilani; Ritter, Andrea; Elghamry, Hanaa; Mahdi, Mahmoud A.; Morel, Laurence; Song, Sihong

    2016-01-01

    Systemic lupus erythematosus (SLE) is an autoimmune disorder with a worldwide distribution and considerable mortality and morbidity. Although the pathogenesis of this disease remains elusive, over-reactive dendritic cells (DCs) play a critical role in the disease development. It has been shown that human alpha-1 antitrypsin (hAAT) has protective effects in type 1 diabetes and rheumatoid arthritis mouse models. In the present study, we tested the effect of AAT on DC differentiation and functions, as well as its protective effect in a lupus-prone mouse model. We showed that hAAT treatment significantly inhibited LPS (TLR4 agonist) and CpG (TLR9 agonist) -induced bone-marrow (BM)-derived conventional and plasmacytoid DC (cDC and pDC) activation and reduced the production of inflammatory cytokines including IFN-I, TNF-α and IL-1β. In MRL/lpr mice, hAAT treatment significantly reduced BM-derived DC differentiation, serum autoantibody levels, and importantly attenuated renal pathology. Our results for the first time demonstrate that hAAT inhibits DC activation and function, and it also attenuates autoimmunity and renal damage in the MRL/lpr lupus model. These results imply that hAAT has a therapeutic potential for the treatment of SLE in humans. PMID:27232337

  5. Pseudomonas aeruginosa Activates PKC-Alpha to Invade Middle Ear Epithelial Cells

    PubMed Central

    Mittal, Rahul; Grati, M’hamed; Yan, Denise; Liu, Xue Z.

    2016-01-01

    Otitis media (OM) is a group of complex inflammatory disorders affecting the middle ear which can be acute or chronic. Chronic suppurative otitis media (CSOM) is a form of chronic OM characterized by tympanic membrane perforation and discharge. Despite the significant impact of CSOM on human population, it is still an understudied and unexplored research area. CSOM is a leading cause of hearing loss and life-threatening central nervous system complications. Bacterial exposure especially Pseudomonas aeruginosa is the most common cause of CSOM. Our previous studies have demonstrated that P. aeruginosa invades human middle ear epithelial cells (HMEECs). However, molecular mechanisms leading to bacterial invasion of HMEECs are not known. The aim of this study is to characterize the role of PKC pathway in the ability of P. aeruginosa to colonize HMEECs. We observed that otopathogenic P. aeruginosa activates the PKC pathway, specifically phosphorylation of PKC-alpha (PKC-α) in HMEECs. The ability of otopathogenic P. aeruginosa to phosphorylate PKC-α depends on bacterial OprF expression. The activation of PKC-α was associated with actin condensation. Blocking the PKC pathway attenuated the ability of bacteria to invade HMEECs and subsequent actin condensation. This study, for the first time, demonstrates that the host PKC-α pathway is involved in invasion of HMEECs by P. aeruginosa and subsequently to cause OM. Characterizing the role of the host signaling pathway in the pathogenesis of CSOM will provide novel avenues to design effective treatment modalities against the disease. PMID:26973629

  6. Alpha 1 Antitrypsin Inhibits Dendritic Cell Activation and Attenuates Nephritis in a Mouse Model of Lupus.

    PubMed

    Elshikha, Ahmed S; Lu, Yuanqing; Chen, Mong-Jen; Akbar, Mohammad; Zeumer, Leilani; Ritter, Andrea; Elghamry, Hanaa; Mahdi, Mahmoud A; Morel, Laurence; Song, Sihong

    2016-01-01

    Systemic lupus erythematosus (SLE) is an autoimmune disorder with a worldwide distribution and considerable mortality and morbidity. Although the pathogenesis of this disease remains elusive, over-reactive dendritic cells (DCs) play a critical role in the disease development. It has been shown that human alpha-1 antitrypsin (hAAT) has protective effects in type 1 diabetes and rheumatoid arthritis mouse models. In the present study, we tested the effect of AAT on DC differentiation and functions, as well as its protective effect in a lupus-prone mouse model. We showed that hAAT treatment significantly inhibited LPS (TLR4 agonist) and CpG (TLR9 agonist) -induced bone-marrow (BM)-derived conventional and plasmacytoid DC (cDC and pDC) activation and reduced the production of inflammatory cytokines including IFN-I, TNF-α and IL-1β. In MRL/lpr mice, hAAT treatment significantly reduced BM-derived DC differentiation, serum autoantibody levels, and importantly attenuated renal pathology. Our results for the first time demonstrate that hAAT inhibits DC activation and function, and it also attenuates autoimmunity and renal damage in the MRL/lpr lupus model. These results imply that hAAT has a therapeutic potential for the treatment of SLE in humans. PMID:27232337

  7. [Treatment of advanced renal cell carcinoma with interferon alpha and OK-432 (streptococcal preparation)].

    PubMed

    Shinoda, M; Naide, Y

    1992-11-01

    A total of 12 patients with advanced renal cell carcinoma received interferon alpha (3 million units intramuscularly 6 times weekly) and OK-432 (5 KE (Klinische Einheit) intramuscularly twice weekly). Metastatic lesions appeared before operation in six patients and after operation in six patients. Among them 5 patients had received interferon therapy and this combination therapy was started after the judgment of progressive disease for interferon therapy. Eleven pulmonary and 5 bone metastases were evaluable. The median duration of the combination therapy was 89.3 weeks. There were 4 partial responses and no complete responses among the 12 patients, giving a response rate of 33.3%. The median duration of response was 25 months, with a range of 6 to 54 months. Responses were seen predominantly in patients in whom metastases appeared after operation (3 of 4 responders). However, regarding the individual organs, two complete and 2 partial responses were observed among 11 pulmonary metastases and 2 partial responses among 5 bone metastases. The survival period after discovery of the metastasis was 10 to 67 months and the 5-year survival rate was 70.5%. Almost all patients had fever and induration at the injection site. Other side effects included leukopenia, anorexia, and depression. This combination therapy is thought to be effective against bone or other organs metastasis resistant to interferon alone. PMID:1485585

  8. TNF-{alpha} promotes human retinal pigment epithelial (RPE) cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression through activation of Akt/mTORC1 signaling

    SciTech Connect

    Wang, Cheng-hu; Cao, Guo-Fan; Jiang, Qin; Yao, Jin

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer TNF-{alpha} induces MMP-9 expression and secretion to promote RPE cell migration. Black-Right-Pointing-Pointer MAPK activation is not critical for TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer Akt and mTORC1 signaling mediate TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer SIN1 knockdown showed no significant effect on MMP-9 expression by TNF-{alpha}. -- Abstract: Tumor necrosis factor-alpha (TNF-{alpha}) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-{alpha} promotes human RPE cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression. Inhibition of MMP-9 by its inhibitor or its neutralizing antibody inhibited TNF-{alpha}-induced in vitro RPE cell migration. Reversely, exogenously-added active MMP-9 promoted RPE cell migration. Suppression Akt/mTOR complex 1(mTORC1) activation by LY 294002 and rapamycin inhibited TNF-{alpha}-mediated MMP-9 expression. To introduce a constitutively active Akt (CA-Akt) in cultured RPE cells increased MMP-9 expression, and to block mTORC1 activation by rapamycin inhibited its effect. RNA interference (RNAi)-mediated silencing of SIN1, a key component of mTOR complex 2 (mTORC2), had no effect on MMP-9 expression or secretion. In conclusion, this study suggest that TNF-{alpha} promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.

  9. Transcriptional and Secretomic Profiling of Epidermal Cells Exposed to Alpha Particle Radiation

    PubMed Central

    Chauhan, Vinita; Howland, Matthew; Greene, Hillary Boulay; Wilkins, Ruth C

    2012-01-01

    Alpha (α)-particle emitters are probable isotopes to be used in a terrorist attack. The development of biological assessment tools to identify those who have handled these difficult to detect materials would be an asset to our current forensic capacity. In this study, for the purposes of biomarker discovery, human keratinocytes were exposed to α-particle and X-radiation (0.98 Gy/h at 0, 0.5, 1.0, 1.5 Gy) and assessed for differential gene and protein expression using microarray and Bio-Plex technology, respectively. Secretomic analysis of supernatants showed expression of two pro-inflammatory cytokines (IL-13 and PDGF-bb) to be exclusively affected in α-particle exposed cells. The highest dose of α-particle radiation modulated a total of 67 transcripts (fold change>|1.5|, (False discovery rate) FDR<0.05) in exposed cells. Several genes which responded with high expression levels (>2 fold) included KIF20A, NEFM, C7orf10, HIST1H2BD, BMP6, and HIST1H2AC. Among the high expressing genes, five (CCNB2, BUB1, NEK2, CDC20, AURKA) were also differentially expressed at the medium (1.0 Gy) dose however, these genes were unmodulated following exposure to X-irradiation. Networks of these genes clustered around tumor protein-53 and transforming growth factor-beta signaling. This study has identified some potential gene /protein responses and networks that may be validated further to confirm their specificity and potential to be signature biomarkers of α-particle exposure. PMID:23002402

  10. Gene amplification and microsatellite instability induced in tumorigenic human bronchial epithelial cells by alpha particles and heavy ions

    NASA Technical Reports Server (NTRS)

    Piao, C. Q.; Hei, T. K.; Hall, E. J. (Principal Investigator)

    2001-01-01

    Gene amplification and microsatellite alteration are useful markers of genomic instability in tumor and transformed cell lines. It has been suggested that genomic instability contributes to the progression of tumorigenesis by accumulating genetic changes. In this study, amplification of the carbamyl-P-synthetase, aspartate transcarbamylase, dihydro-orotase (CAD) gene in transformed and tumorigenic human bronchial epithelial (BEP2D) cells induced by either alpha particles or (56)Fe ions was assessed by measuring resistance to N-(phosphonacetyl)-l-aspartate (PALA). In addition, alterations of microsatellite loci located on chromosomes 3p and 18q were analyzed in a series of primary and secondary tumor cell lines generated in nude mice. The frequency of PALA-resistant colonies was 1-3 x 10(-3) in tumor cell lines, 5-8 x 10(-5) in transformed cells prior to inoculation into nude mice, and less than 10(-7) in control BEP2D cells. Microsatellite alterations were detected in all 11 tumor cell lines examined at the following loci: D18S34, D18S363, D18S877, D3S1038 and D3S1607. No significant difference in either PALA resistance or microsatellite instability was found in tumor cell lines that were induced by alpha particles compared to those induced by (56)Fe ions.

  11. Effects of meal size and composition on incretin, alpha-cell, and beta-cell responses.

    PubMed

    Rijkelijkhuizen, Josina M; McQuarrie, Kelly; Girman, Cynthia J; Stein, Peter P; Mari, Andrea; Holst, Jens J; Nijpels, Giel; Dekker, Jacqueline M

    2010-04-01

    The incretins glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) regulate postprandial insulin release from the beta-cells. We investigated the effects of 3 standardized meals with different caloric and nutritional content in terms of postprandial glucose, insulin, glucagon, and incretin responses. In a randomized crossover study, 18 subjects with type 2 diabetes mellitus and 6 healthy volunteers underwent three 4-hour meal tolerance tests (small carbohydrate [CH]-rich meal, large CH-rich meal, and fat-rich meal). Non-model-based and model-based estimates of beta-cell function and incremental areas under the curve of glucose, insulin, C-peptide, glucagon, GLP-1, and GIP were calculated. Mixed models and Friedman tests were used to test for differences in meal responses. The large CH-rich meal and fat-rich meal resulted in a slightly larger insulin response as compared with the small CH-rich meal and led to a slightly shorter period of hyperglycemia, but only in healthy subjects. Model-based insulin secretion estimates did not show pronounced differences between meals. Both in healthy individuals and in those with diabetes, more CH resulted in higher GLP-1 release. In contrast with the other meals, GIP release was still rising 2 hours after the fat-rich meal. The initial glucagon response was stimulated by the large CH-rich meal, whereas the fat-rich meal induced a late glucagon response. Fat preferentially stimulates GIP secretion, whereas CH stimulates GLP-1 secretion. Differences in meal size and composition led to differences in insulin and incretin responses but not to differences in postprandial glucose levels of the well-controlled patients with diabetes. PMID:19846181

  12. Integrin alpha 11 regulates IGF2 expression in fibroblasts to enhance tumorigenicity of human non-small-cell lung cancer cells.

    PubMed

    Zhu, Chang-Qi; Popova, Svetlana N; Brown, Ewan R S; Barsyte-Lovejoy, Dalia; Navab, Roya; Shih, Warren; Li, Ming; Lu, Ming; Jurisica, Igor; Penn, Linda Z; Gullberg, Donald; Tsao, Ming-Sound

    2007-07-10

    Integrin alpha11 (ITGA11/alpha11) is localized to stromal fibroblasts and commonly overexpressed in non-small-cell lung carcinoma (NSCLC). We hypothesized that stromal alpha11 could be important for the tumorigenicity of NSCLC cells. SV40 immortalized mouse embryonic fibroblasts established from wild-type (WT) and Itga11-deficient [knockout (KO)] mice were tested for their tumorigenicity in immune-deficient mice when implanted alone or coimplanted with the A549 human lung adenocarcinoma cells. A549 coimplanted with the fibroblasts showed a markedly enhanced tumor growth rate compared with A549, WT, or KO, which alone formed only small tumors. Importantly, the growth was significantly greater for A549+WT compared with A549+KO tumors. Reexpression of human alpha11 cDNA in KO cells rescued a tumor growth rate to that comparable with the A549+WT tumors. These findings were validated in two other NSCLC cell lines, NCI-H460 and NCI-H520. Gene expression profiling indicated that IGF2 mRNA expression level was >200 times lower in A549+KO compared with A549+WT tumors. Stable short-hairpin RNA (shRNA) down-regulation of IGF2 in WT (WT(shIGF2)) fibroblasts resulted in a decreased growth rate of A549+WT(shIGF2), compared with A549+WT tumors. The results indicate that alpha11 is an important stromal factor in NSCLC and propose a paradigm for carcinoma-stromal interaction indirectly through interaction between the matrix collagen and stromal fibroblasts to stimulate cancer cell growth. PMID:17600088

  13. Immunodominant regions for T helper-cell sensitization on the human nicotinic receptor alpha subunit in myasthenia gravis.

    PubMed Central

    Protti, M P; Manfredi, A A; Straub, C; Howard, J F; Conti-Tronconi, B M

    1990-01-01

    In myasthenia gravis an autoimmune response against the nicotinic acetylcholine receptor (AChR) occurs. The alpha subunit of the AChR contains both the epitope(s) that dominates the antibody response (main immunogenic region) and epitopes involved in T helper cell sensitization. In this study, overlapping synthetic peptides corresponding to the complete AChR alpha-subunit sequence were used to propagate polyclonal AChR-specific T helper cell lines from four myasthenic patients of different HLA types. Response of the T helper lines to the individual peptides was studied. Four immunodominant sequence segments were identified--i.e., residues 48-67, 101-120, 304-322, and 419-437. These regions did not include residues known to form the main immunogenic region or the cholinergic binding site, and they frequently contained sequence motifs that have been proposed to be related to T-epitope formation. Images PMID:2145582

  14. alpha-Factor-mediatd modification of a 32P-labeled protein by MATa cells of Saccharomyces cerevisiae.

    PubMed

    Finkelstein, D B; McAlister, L

    1981-03-10

    Addition of the polypeptide mating pheromone alpha-factor to haploid MATa cells of Saccharomyces cerevisiae results in the modification of a 32P-labeled protein (P17) with an apparent Mr of 17,000 to a form having an apparent Mr of 17,500 (P17). 32P associated with both P17 and P17 exhibits an unusually rapid rate of turnover. The conversion of P17 to P17 precedes the appearance of morphologically abnormal cells and, in contrast to other responses elicited by this pheromone, this change in apparent molecular weight does not require protein synthesis. Upon removal of alpha-factor, the P17/P17 ratio returns to pretreatment levels. PMID:7007388

  15. Magnetism and mineralogy of Almahata Sitta polymict ureilite (= asteroid 2008 TC3): Implications for the ureilite parent body magnetic field

    NASA Astrophysics Data System (ADS)

    Hoffmann, Viktor H.; Hochleitner, Rupert; Torii, Masayuki; Funaki, Minoru; Mikouchi, Takashi; Kaliwoda, Melanie; Jenniskens, Peter; Shaddad, Muawia H.

    2011-10-01

    The Almahata Sitta meteorite is the first case of recovered extraterrestrial material originating from an asteroid that was detected in near Earth space shortly before entering and exploding in the high atmosphere. The aims of our project within the 2008 TC3 consortium were investigating Almahata Sitta's (AS) magnetic signature, phase composition and mineralogy, focussing on the opaque minerals, and gaining new insights into the magnetism of the ureilite parent body (UPB). We report on the general magnetic properties and behavior of Almahata Sitta and try to place the results in context with the existing data set on ureilites and ureilite parent body models. The magnetic signature of AS is dominated by a set of low-Ni kamacites with large grain sizes. Additional contributions come from micron-sized kamacites, suessite, (Cr) troilite, and daubreelite, mainly found in the olivine grains adjacent to carbon-rich veins. Our results show that the paleomagnetic signal is of extraterrestrial origin as can be seen by comparing with laboratory produced magnetic records (IRM). Four types of kamacite (I-IV) have been recognized in the sample. The elemental composition of the ureilite vein metal Kamacite I (particularly Co) clearly differs from the other kamacites (II-IV), which are considered to be indigenous. Element ratios of kamacite I indicate that it was introduced into the UPB by an impactor, supporting the conclusions of Gabriel and Pack (2009).

  16. Brief report: a new profile of terminal N-acetyllactosamines glycans on pig red blood cells and different expression of alpha-galactose on Sika deer red blood cells and nucleated cells.

    PubMed

    Tan, Yingxia; Gong, Feng; Li, Subo; Ji, Shouping; Lu, Yanping; Gao, Hongwei; Xu, Hua; Zhang, Yangpei

    2010-05-01

    It has been reported that: (1) large variations were found in the number of sialic acid (SA) capped with N-acetyllactosamines (SA-Galbeta1-4GlcNAc-R) and alpha-Gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R) or uncapped N-acetyllactosamines (Galbeta1-4GlcNAc-R) on different mammalian red blood cells, and on nucleated cells originating from a given tissue in various species; (2) goat, sheep, horse and mouse red blood cells lack alpha-Gal epitopes, despite the expression of this epitope on a variety of nucleated cells in these species, including lymphocytes differentiated from the same hematopoietic origin. In this study, flow cytometry and Western blot analyses of pig red blood cells showed that alpha-Gal epitopes on pig red cells developed concomitantly after treatment with neuraminidase, suggesting that the terminal N-acetyllactosaminide glycans were capped with SA-alpha-Gal epitopes. Whereas, the expression of the alpha-Gal epitopes on red blood cells from Sika deer (Cevus nippon hortulorum) were found to be absent even though the epitopes were present on their white blood cells. Thus, these results add new data not only for the terminal carbohydrate structures on cell surface glycans of various mammalian cells, but also for wide variety of epitope expression on the cells from different tissues, which might be useful for understanding their unique states resulting from differentiation and evolution. PMID:20422448

  17. A DC-81-indole conjugate agent suppresses melanoma A375 cell migration partially via interrupting VEGF production and stromal cell-derived factor-1{alpha}-mediated signaling

    SciTech Connect

    Hsieh, Ming-Chu; Hu, Wan-Ping; Yu, Hsin-Su; Wu, Wen-Chuan; Chang, Long-Sen; Kao, Ying-Hsien; Wang, Jeh-Jeng

    2011-09-01

    Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) chemicals are antitumor antibiotics inhibiting nucleic acid synthesis. An indole carboxylate-PBD hybrid with six-carbon spacer structure (IN6CPBD) has been previously demonstrated to induce melanoma cell apoptosis and reduce metastasis in mouse lungs. This study aimed at investigating the efficacy of the other hybrid compound with four-carbon spacer (IN4CPBD) and elucidating its anti-metastatic mechanism. Human melanoma A375 cells with IN4CPBD treatment underwent cytotoxicity and apoptosis-associated assays. Transwell migration assay, Western blotting, and ELISA were used for mechanistic study. IN4CPBD exhibited potent melanoma cytotoxicity through interrupting G1/S cell cycle progression, increasing DNA fragmentation and hypodipoidic DNA contents, and reducing mitochondrial membrane potential. Caspase activity elevation suggested that both intrinsic and extrinsic pathways were involved in IN4CPBD-induced melanoma apoptosis. IN4CPBD up-regulated p53 and p21, thereby concomitantly derailing the equilibrium between Bcl-2 and Bax levels. Transwell migration assay demonstrated that stromal cell-derived factor-1{alpha} (SDF-1{alpha}) stimulated A375 cell motility, while kinase inhibitors treatment confirmed that Rho/ROCK, Akt, ERK1/2, and p38 MAPK pathways were involved in SDF-1{alpha}-enhanced melanoma migration. IN4CPBD not only abolished the SDF-1{alpha}-enhanced chemotactic motility but also suppressed constitutive MMP-9 and VEGF expression. Mechanistically, IN4CPBD down-regulated Akt, ERK1/2, and p38 MAPK total proteins and MYPT1 phosphorylation. In conclusion, beyond the fact that IN4CPBD induces melanoma cell apoptosis at cytotoxic dose, the interruption in the VEGF expression and the SDF-1{alpha}-related signaling at cytostatic dose may partially constitute the rationale for its in vivo anti-metastatic potency. - Research Highlights: > A novel carboxylate-PBD hybrid as anti-melanoma drug. > IN4CPBD interrupts melanoma cell

  18. Anoikis-resistant MDCK cells carrying susceptibilities to TNF-alpha and verotoxin that are suitable for influenza virus cultivation.

    PubMed

    Tsutsumi, Reiko; Fujisaki, Shigemi; Shozushima, Masanori; Saito, Koichi; Sato, Shigehiro

    2006-10-01

    Madin-Darby canine kidney (MDCK) cells were originally anchorage-dependent epithelial cells. Here, we have isolated a novel MDCK-derived cell population, termed 6 M-4, by means of culturing MDCK cells in suspension for nearly 6 months in the presence of Streptomyces griseus metalloendopeptidase (MEP). The isolated cells showed unique proliferation characteristics, which differed from parental MDCK cells. They proliferated adherently on a polystyrene matrix, but proliferated non-adherently both in the presence of MEP and on a non-adhesive matrix coated with poly 2-methacryloyloxyethyl phosphorylcholine (MPC). The 6 M-4 cells consisted of at least two cell types. One type, termed 6 M-4-TR7, would not grow in soft agar and showed a novel phenotype in that the cells were susceptible to both TNF-alpha and verotoxin 1 (VT1). In addition, the isolated adhesion-independent cells sustained epithelial traits of parental MDCK cells. We further show that these MDCK-derivative cells are suitable for influenza virus cultivation. Hemagglutination (HA) titers of influenzaviruses A and B were increased in the suspension culture of 6 M-4-TR7 cells supplemented with the MEP in comparison to adherently growing cells in the presence of trypsin. PMID:19002866

  19. T cell receptor alpha-chain gene rearrangements in B-precursor leukemia are in contrast to the findings in T cell acute lymphoblastic leukemia. Comparative study of T cell receptor gene rearrangement in childhood leukemia.

    PubMed Central

    Hara, J; Benedict, S H; Mak, T W; Gelfand, E W

    1987-01-01

    We have analyzed T cell receptor alpha-chain gene configuration using three genomic joining (J) region probes in 64 children with acute lymphoblastic leukemia (ALL). 11 out of 18 T-ALLs were T3 positive; alpha-chain gene rearrangements were demonstrated in only two of 18, indicating that the majority of T-ALLs would have rearrangements involving J alpha segments located upstream of these probes. In contrast, 15 out of 46 B-precursor ALLs showed rearrangements of the alpha-chain gene and J alpha segments located approximately 20-30 kb upstream of the constant region were involved in 13 of these patients. Nine of 15 B-precursor ALLs with rearranged alpha-chain genes had rearrangements of both gamma- and beta-chain genes, whereas the remaining six had no rearrangements of gamma- and beta-chain genes. These findings indicated that alpha-chain gene rearrangement is not specific for T lineage cells and gamma- and/or beta-chain gene rearrangement does not appear essential for alpha-chain gene rearrangement, at least in B-precursor leukemic cells. Images PMID:3500187

  20. Divergent effects of 17-{beta}-estradiol on human vascular smooth muscle and endothelial cell function diminishes TNF-{alpha}-induced neointima formation

    SciTech Connect

    Nintasen, Rungrat; Riches, Kirsten; Mughal, Romana S.; Viriyavejakul, Parnpen; Chaisri, Urai; Maneerat, Yaowapa; Turner, Neil A.; Porter, Karen E.

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer TNF-{alpha} augments neointimal hyperplasia in human saphenous vein. Black-Right-Pointing-Pointer TNF-{alpha} induces detrimental effects on endothelial and smooth muscle cell function. Black-Right-Pointing-Pointer Estradiol exerts modulatory effects on TNF-induced vascular cell functions. Black-Right-Pointing-Pointer The modulatory effects of estradiol are discriminatory and cell-type specific. -- Abstract: Coronary heart disease (CHD) is a condition characterized by increased levels of proinflammatory cytokines, including tumor necrosis factor-{alpha} (TNF-{alpha}). TNF-{alpha} can induce vascular endothelial cell (EC) and smooth muscle cell (SMC) dysfunction, central events in development of neointimal lesions. The reduced incidence of CHD in young women is believed to be due to the protective effects of estradiol (E2). We therefore investigated the effects of TNF-{alpha} on human neointima formation and SMC/EC functions and any modulatory effects of E2. Saphenous vein (SV) segments were cultured in the presence of TNF-{alpha} (10 ng/ml), E2 (2.5 nM) or both in combination. Neointimal thickening was augmented by incubation with TNF-{alpha}, an effect that was abolished by co-culture with E2. TNF-{alpha} increased SV-SMC proliferation in a concentration-dependent manner that was optimal at 10 ng/ml (1.5-fold increase), and abolished by E2 at all concentrations studied (1-50 nM). Surprisingly, E2 itself at low concentrations (1 and 5 nM) stimulated SV-SMC proliferation to a level comparable to that of TNF-{alpha} alone. SV-EC migration was significantly impaired by TNF-{alpha} (42% of control), and co-culture with E2 partially restored the ability of SV-EC to migrate and repair the wound. In contrast, TNF-{alpha} increased SV-SMC migration by 1.7-fold, an effect that was completely reversed by co-incubation with E2. Finally, TNF-{alpha} potently induced ICAM-1 and VCAM-1 expression in both SV-EC and SV-SMC. However there

  1. Effects of pentoxifylline on TNF-alpha production by peripheral blood mononuclear cells in patients with nonalcoholic steatohepatitis.

    PubMed

    Duman, Deniz G; Ozdemir, Filiz; Birben, Esra; Keskin, Ozlem; Ekşioğlu-Demiralp, Emel; Celikel, Cigdem; Kalayci, Omer; Kalayci, Cem

    2007-10-01

    Pentoxifylline (POF) is a new candidate for the treatment of nonalcoholic steatohepatitis (NASH). Its effects on the cytokine production in patients with NASH are not completely understood. This study was designed to investigate the effect of POF on TNF-alpha production by peripheral blood mononuclear cells (PBMC) in patients with NASH. After preliminary experiments in healthy control subjects to determine the range of POF concentration to be used in NASH patients, PBMCs from patients with NASH (n = 13) were cultured in the presence of lipopolysaccharide (LPS, 100 ng/ml) and various concentrations of POF for 24 hr. Concentrations of TNF-alpha in culture supernatants were measured by ELISA and the transcriptional activity was determined by RT-PCR. As dictated by the results of our preliminary study in PBMC from healthy control subjects, we treated LPS stimulated PBMCs from NASH patients with 10, 100, and 500 microg/ml of POF. Stimulation of PBMCs from NASH patients with LPS resulted in a strong up-regulation of TNF-alpha production from median 355.9 (interquartile range, 206.7-463.5) pg/ml to 1,670 pg/ml (interquartile range, 1,121-2,414) pg/ml. In this LPS-stimulated culture system, POF caused a dose-dependent suppression of TNF-alpha levels (P < 0.001, ANOVA on ranks for repeated measures). TNF-alpha levels in culture supernatants decreased to 870.3 (range, 598.3-2,077) pg/ml with 10 microg/ml of POF treatment, and to levels similar to those obtained in baseline unstimulated cultures (133.4 (range, 95.8-1518.5) pg/ml) at 100 microg/ml. At 500 microg/ml, POF suppressed TNF-alpha production to levels significantly lower than that obtained in unstimulated (baseline) culture supernatants (76.3 (range, 33-94.5) pg/ml; P = 0.001). The mRNA expression was consistent with the effects on protein concentration. Demographic characteristics of the patients, laboratory results, such as AST, ALT, alkaline phosphatase, GGT, and triglyceride levels, and the liver histology did

  2. Cytokine-inducible expression in endothelial cells of an I kappa B alpha-like gene is regulated by NF kappa B.

    PubMed Central

    de Martin, R; Vanhove, B; Cheng, Q; Hofer, E; Csizmadia, V; Winkler, H; Bach, F H

    1993-01-01

    The transient expression of many different genes is mediated by the inducible transcription factor p50-p65 NF kappa B, which in turn is regulated by complex formation with its inhibitor I kappa B alpha. We describe here that in porcine aortic endothelial cells, either IL-1 alpha, TNF alpha or LPS upregulates an inhibitor of NF kappa B which we refer to as ECI-6. ECI-6 is by structural and functional criteria an I kappa B alpha protein, the porcine homologue of MAD-3, pp40 and RL/IF-1. We have studied the promoter of the ECI-6/I kappa B alpha gene and provide three lines of evidence that its expression is directly regulated by NF kappa B. First, the 5' regulatory region of ECI-6/I kappa B alpha contains two sites that bind NF kappa B in electrophoretic mobility shift assays. Second, expression following transfection of an ECI-6/I kappa B alpha promoter-luciferase reporter construct is dependent on a co-transfected NF kappa B-p65 subunit. Third, pretreatment of endothelial cells with antioxidants, agents that inhibit activation of NF kappa B, inhibit the expression of ECI-6/I kappa B alpha. We conclude that the regulated expression of ECI-6/I kappa B alpha could represent a novel feedback mechanism by which NF kappa B downregulates its own activity after transient activation of target genes has been achieved. Images PMID:8334993

  3. Prevention by alpha-tocopherol and rutin of glutathione and ATP depletion induced by oxidized LDL in cultured endothelial cells.

    PubMed Central

    Schmitt, A.; Salvayre, R.; Delchambre, J.; Nègre-Salvayre, A.

    1995-01-01

    1. Oxidized low density lipoproteins (LDL) are thought to play an important role in atherogenesis. Mildly oxidized LDL are cytotoxic to cultured endothelial cells. Toxic doses of oxidized LDL promote the peroxidation of cellular lipids (beginning at 6 h and being maximal after 12 h of pulse with oxidized LDL) and glutathione and ATP depletion (beginning after 15 h of pulse and evolving concurrently with the cytotoxicity). 2. Antioxidants from 3 different classes (rutin, ascorbic acid and alpha-tocopherol) were compared as to their ability to inhibit the cytotoxic effect of oxidized LDL to endothelial cells. 3. Effective concentrations of alpha-tocopherol inhibited cellular lipid peroxidation, glutathione and ATP depletion and the cytotoxic effect. 4. Ascorbic acid was less effective than alpha-tocopherol and rutin, and exhibited a dose-dependent biphasic effect in the presence of oxidized LDL. 5. Effective concentrations of rutin inhibited glutathione and ATP depletion as well as cytotoxicity, but did not block cellular lipid peroxidation. This suggests that the glutathione and ATP depletion is directly correlated to the cytotoxicity of oxidized LDL, whereas cellular lipid peroxidation is probably not directly the cause of cellular damage leading to cell death. 6. The association of antioxidants of 3 different classes allowed the suppression of the biphasic effect of ascorbic acid and increased the efficacy of the protective effect. The potential consequences for prevention of the pathogenic role of oxidized LDL in endothelial injury are discussed. PMID:8640336

  4. Alpha-beta T cells provide protection against lethal encephalitis in the murine model of VEEV infection

    SciTech Connect

    Paessler, Slobodan Yun, Nadezhda E.; Judy, Barbara M.; Dziuba, Natallia; Zacks, Michele A.; Grund, Anna H.; Frolov, Ilya; Campbell, Gerald A.; Weaver, Scott C.; Estes, D. Mark

    2007-10-25

    We evaluated the safety and immunogenicity of a chimeric alphavirus vaccine candidate in mice with selective immunodeficiencies. This vaccine candidate was highly attenuated in mice with deficiencies in the B and T cell compartments, as well as in mice with deficient gamma-interferon responsiveness. However, the level of protection varied among the strains tested. Wild type mice were protected against lethal VEEV challenge. In contrast, alpha/beta ({alpha}{beta}) TCR-deficient mice developed lethal encephalitis following VEEV challenge, while mice deficient in gamma/delta ({gamma}{delta}) T cells were protected. Surprisingly, the vaccine potency was diminished by 50% in animals lacking interferon-gamma receptor alpha chain (R1)-chain and a minority of vaccinated immunoglobulin heavy chain-deficient ({mu}MT) mice survived challenge, which suggests that neutralizing antibody may not be absolutely required for protection. Prolonged replication of encephalitic VEEV in the brain of pre-immunized mice is not lethal and adoptive transfer experiments indicate that CD3{sup +} T cells are required for protection.

  5. Neutrophil killing of human umbilical vein endothelial cells is oxygen radical-mediated and enhanced by TNF-. alpha

    SciTech Connect

    Dame, M.K.; Varani, J.; Weinberg, J.M.; Ward, P.A. )

    1991-03-11

    Human umbilical vein endothelial cells are sensitive to killing by activated human neutrophils. Killing is inhibited in the presence of catalase and deferoxamine mesylate but not soybean trypsin inhibitor. Reagent hydrogen peroxide can substitute for activated neutrophils in producing endothelial cell injury. These data suggest that lethal injury is due to the production of oxygen radicals by activated neutrophils. In these respects, the human umbilical vein endothelial cells are similar to rat pulmonary artery endothelial cells in that pretreatment with TNF-{alpha} increases sensitivity to injury by activated neutrophils. In part, the increased endothelial cell sensitivity to killing by neutrophils may be due to up-regulation of surface adhesion molecules. However, it was observed that cells passaged more than two times in culture did not demonstrate increased killing after treatment with TNF-{alpha} while up-regulation of neutrophil adhesion could be detected through several additional passages. Although the human umbilical vein endothelial cells are qualitatively similar to rat pulmonary artery endothelial cells in their sensitivity to killing, they are quantitatively much more resistant. What accounts for the relative resistance of the human umbilical vein endothelial cells is not fully understood. In the rat pulmonary artery endothelial cells, killing is known to be dependent on an intraendothelial source of iron. Pre-treatment of the human umbilical vein endothelial cells with 8-hydroxyquinoline-bound iron increased their sensitivity to oxidant injury. These data suggest that the availability of iron within the human umbilical vein endothelial cells may be a limiting factor in sensitivity to oxygen radical-mediated injury.

  6. Production of interferon-gamma and tumour necrosis factor-alpha by human T-cell clones expressing different forms of the gamma delta receptor.

    PubMed Central

    Christmas, S E; Meager, A

    1990-01-01

    Panels of human T-cell clones bearing the gamma delta T-cell receptor (TcR) were obtained from peripheral blood and decidual tissue and maintained in the presence of interleukin-2 (IL-2). TcR V gamma and V delta gene expression was determined in 40 TcR delta 1+ clones using the gamma delta T-cell subset markers Ti gamma A and delta TCS1, in conjunction with Southern blot analysis using TcR J gamma and J delta probes. gamma delta T-cell clones, together with control alpha beta T-cell clones derived from the same lymphocyte populations, were stimulated with phytohaemagglutinin (PHA) and their ability to produce interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) tested using specific ELISA. Many clones representative of the major peripheral V gamma 9/V delta 2J1 subset produced high amounts of both cytokines and mean levels were not significantly different from those produced by alpha beta T-cell clones. Panels of clones expressing V gamma 9 and V delta 2J1 produced significantly higher levels of TNF-alpha than clones not expressing V delta 2J1 and those expressing V delta 1J1. There was no relationship between levels of IFN-gamma and TNF-alpha produced by individual gamma delta T-cell clones and also no relationship between their non-major histocompatibility complex (MHC)-restricted cytotoxic activity and levels of either cytokine. There was a significant tendency for gamma delta T-cell clones to produce more TNF-alpha than IFN-gamma in comparison to alpha beta T-cell clones. The significance of these findings is discussed in the light of the reported differences in distribution in vivo of V delta 1J1+ and V delta 2J1+ cells. Images Figure 1 PMID:2126252

  7. Subcutaneous administration of interleukin 2 and interferon-alpha-2b in advanced renal cell carcinoma: a confirmatory study.

    PubMed Central

    Facendola, G.; Locatelli, M. C.; Pizzocaro, G.; Piva, L.; Pegoraro, C.; Pallavicini, E. B.; Signaroldi, A.; Meregalli, M.; Lombardi, F.; Beretta, G. D.

    1995-01-01

    Recent clinical studies have suggested that the combination of subcutaneous recombinant human interleukin 2 (rIL-2) and interferon alpha (rIFN-alpha) is especially promising in advanced renal cell carcinoma. We assessed the safety, activity and toxicity of home therapy with these two agents in 50 patients. Each treatment cycle consisted of a 2 day pulse phase, with 9 x 10(6) IU m-2 of rIL-2 being given subcutaneously every 12 h, followed by a 6 week maintenance phase during which rIL-2 1.8 x 10(6) IU m-2 was administered subcutaneously every 12 h on days 1-5 and rIFN-alpha 2b 5 x 10(6) IU m-2 once a day on days 1, 3 and 5. Objective responses (CR+PR) occurred in 9/50 (18%) patients, six of whom (12%) achieved a complete response. Disease stabilisation was observed in 17 cases (34%) and 18 patients progressed during therapy. In the other six cases, treatment was interrupted early for toxicity or patient refusal. One patient died of myocardial infarction during the second cycle. The overall median survival was 12 months. Home therapy with subcutaneous rIL-2 + rIFN-alpha 2b proved to be active, feasible and moderately toxic, but serious adverse events can sometimes occur. PMID:8519672

  8. Ceramides and cell signaling molecules in psoriatic epidermis: reduced levels of ceramides, PKC-alpha, and JNK.

    PubMed

    Lew, Bark-Lynn; Cho, Yunhi; Kim, Jungmin; Sim, Woo-Young; Kim, Nack-In

    2006-02-01

    Ceramides are the main lipids in the stratum corneum and are generated during cellular stress and apoptosis by de novo synthesis or by the action of sphingomyelinase. In addition, they are lipid second messengers produced by sphingolipid metabolism and trigger important cell responses, including protein kinase C-alpha (PKC-alpha) activation and the stimulation of signal transduction pathways with apoptosis and stress-activated protein kinases (SAPK), such as c-jun N-terminal kinase (JNK). Thus, ceramides have anti-proliferative and apoptotic effects. This study measured the changes in the levels of epidermal ceramides and ceramide-related apoptotic signaling molecules in psoriasis patients. Samples from lesional and non-lesional epidermis were obtained from psoriasis patients. Total ceramides were fractionated using thin-layer chromatography, and the levels of PKC-alpha and JNK expression were measured using Western blot analysis with specific antibodies. The ceramide level was reduced significantly, and this was associated with the downregulation of apoptotic signaling molecules, such as PKC-alpha and JNK, in the lesional epidermis of psoriasis patients. These results suggest that the decreased level of ceramides downregulates the apoptotic pathway, leading to epidermal proliferation in psoriasis. PMID:16479073

  9. 12S-lipoxygenase protein associates with {alpha}-actin fibers in human umbilical artery vascular smooth muscle cells

    SciTech Connect

    Weisinger, Gary . E-mail: gary_w@tasmc.health.gov.il; Limor, Rona; Marcus-Perlman, Yonit; Knoll, Esther; Kohen, Fortune; Schinder, Vera; Firer, Michael; Stern, Naftali

    2007-05-11

    The current study sets out to characterize the intracellular localization of the platelet-type 12S-lipoxygenase (12-LO), an enzyme involved in angiotensin-II induced signaling in vascular smooth muscle cells (VSMC). Immunohistochemical analysis of VSMC in vitro or human umbilical arteries in vivo showed a clear cytoplasmic localization. On immunogold electron microscopy, 12-LO was found primarily associated with cytoplasmic VSMC muscle fibrils. Upon angiotensin-II treatment of cultured VSMC, immunoprecipitated 12-LO was found bound to {alpha}-actin, a component of the cytoplasmic myofilaments. 12-LO/{alpha}-actin binding was blocked by VSMC pretreatment with the 12-LO inhibitors, baicalien or esculetine and the protein synthesis inhibitor, cycloheximide. Moreover, the binding of 12-LO to {alpha}-actin was not associated with 12-LO serine or tyrosine phosphorylation. These observations suggest a previously unrecognized angiotensin-II dependent protein interaction in VSMC through which 12-LO protein may be trafficked, for yet undiscovered purposes towards the much more abundantly expressed cytoskeletal protein {alpha}-actin.

  10. Sphingosine kinase 1: a new modulator of hypoxia inducible factor 1alpha during hypoxia in human cancer cells.

    PubMed

    Ader, Isabelle; Brizuela, Leyre; Bouquerel, Pierre; Malavaud, Bernard; Cuvillier, Olivier

    2008-10-15

    Here, we provide the first evidence that sphingosine kinase 1 (SphK1), an oncogenic lipid kinase balancing the intracellular level of key signaling sphingolipids, modulates the transcription factor hypoxia inducible factor 1alpha (HIF-1alpha), master regulator of hypoxia. SphK1 activity is stimulated under low oxygen conditions and regulated by reactive oxygen species. The SphK1-dependent stabilization of HIF-1alpha levels is mediated by the Akt/glycogen synthase kinase-3beta signaling pathway that prevents its von Hippel-Lindau protein-mediated degradation by the proteasome. The pharmacologic and RNA silencing inhibition of SphK1 activity prevents the accumulation of HIF-1alpha and its transcriptional activity in several human cancer cell lineages (prostate, brain, breast, kidney, and lung), suggesting a canonical pathway. Therefore, we propose that SphK1 can act as a master regulator for hypoxia, giving support to its inhibition as a valid strategy to control tumor hypoxia and its molecular consequences. PMID:18922940

  11. Effects of tumor necrosis factor-alpha and interferon-gamma on expressions of matrix metalloproteinase-2 and -9 in human bladder cancer cells.

    PubMed

    Shin, K Y; Moon, H S; Park, H Y; Lee, T Y; Woo, Y N; Kim, H J; Lee, S J; Kong, G

    2000-10-31

    We have investigated the effects of tumor necrosis factor-alpha (TNF-alpha) and interferon (INF-gamma), the potent Bacillus Calmette-Guerin (BCG)-induced cytokines on the production of MMP-2, MMP-9, TIMP-1, TIMP-2 and MT1-MMP in high grade human bladder cancer cell lines, T-24, J-82 and HT-1376 cell lines. MMP-2 expression and activity were decreased in T-24 cells treated with both cytokines in a dose dependent manner. However, J-82 cells treated with TNF-alpha and INF-gamma revealed dose dependent increases of MMP-9 expression and activity with similar baseline expression and activity of MMP-2. HT-1376 cells after exposure to TNF-alpha only enhanced the expression and activity of MMP-9. These results indicate that TNF-alpha and INF-gamma could regulate the production of MMP-2 or MMP-9 on bladder cancer cells and their patterns of regulation are cell specific. Furthermore, this diverse response of bladder cancer cells to TNF-alpha and INF-gamma suggests that BCG immunotherapy may enhance the invasiveness of bladder cancer in certain conditions with induction of MMPs. PMID:10996723

  12. TNF-alpha-dependent activation of NF-kappa B in human osteoblastic HOS-TE85 cells is repressed in vector-averaged gravity using clinostat rotation.

    PubMed

    Kobayashi, K; Kambe, F; Kurokouchi, K; Sakai, T; Ishiguro, N; Iwata, H; Koga, K; Gruener, R; Seo, H

    2000-12-01

    Effects of vector-averaged gravity on tumor necrosis factor (TNF)-alpha-dependent activation of nuclear factor kappa B (NF-kappa B) in human osteoblastic HOS-TE85 cells were investigated by culturing the cells using clinostat rotation (clinorotation). Cell cultures were rotated for 72 h at 40 rpm in a clinostat. At the end of clinorotation, the cells were treated with TNF-alpha for 30 min under stationary conditions. Electrophoretic mobility shift assays revealed that TNF-alpha-dependent activation of NF-kappa B was markedly reduced in the clinorotated cells when compared with the cells in control stationary cultures or after horizontal rotation (motional controls). The NF-kappa B-dependent transactivation was also impaired in the clinorotated cells, as evidenced by a transient transfection assay with a reporter plasmid containing multimerized NF-kappa B sites. Consistent with these findings, the TNF-alpha-dependent induction of endogenous NF-kappa B-responsive genes p105, I kappa B-alpha, and IL-8, was significantly attenuate in clinorotated cells. These results demonstrate that vector-averaged gravity inhibits the responsiveness of osteoblasts to TNF-alpha by repressing NF-kappa B activation. PMID:11112449

  13. Propanil inhibits tumor necrosis factor-alpha production by reducing nuclear levels of the transcription factor nuclear factor-kappab in the macrophage cell line ic-21.

    PubMed

    Frost, L L; Neeley, Y X; Schafer, R; Gibson, L F; Barnett, J B

    2001-05-01

    Tumor necrosis factor-alpha (TNF-alpha) is an essential proinflammatory cytokine whose production is normally stimulated by bacterial cell wall components, such as lipopolysaccharide (LPS), during an infection. Macrophages stimulated with LPS in vitro produce several cytokines, including TNF-alpha. LPS-stimulated primary mouse macrophages produced less TNF-alpha protein and message after treatment with the herbicide propanil (Xie et al., Toxicol. Appl. Pharmacol. 145, 184-191, 1997). Nuclear factor-kappaB (NF-kappaB) tightly regulates TNF-alpha transcription. Therefore, as a step toward understanding the mechanism of the effect of propanil on TNF-alpha transcription, IC-21 cells were transfected with a TNF-alpha promoter-luciferase construct, and the effect of propanil on luciferase activity was measured. Cells transfected with promoter constructs containing a kappaB site showed decreased luciferase activity relative to controls after propanil treatment. These observations implicated NF-kappaB binding as an intracellular target of propanil. Further studies demonstrated a marked reduction in the nuclear levels of the stimulatory p65 subunit of NF-kappaB after propanil treatment, as measured by fluorescence confocal microscopy and Western blot analysis. The p50 subunit of NF-kappaB was not found to be reduced after propanil exposure by Western blot. Electrophoretic mobility gel shift assays showed decreased DNA binding of both p65/p50 heterodimers and p50/p50 homodimers to the kappaB3 site of the TNF-alpha promoter of propanil-treated cells. The marked reduction in nuclear p65/p50 NF-kappaB levels and diminished binding to the TNF-alpha promoter in propanil-treated cells are consistent with reduced TNF-alpha levels induced by LPS. PMID:11312646

  14. Mutant HNF-1{alpha} and mutant HNF-1{beta} identified in MODY3 and MODY5 downregulate DPP-IV gene expression in Caco-2 cells

    SciTech Connect

    Gu Ning; Adachi, Tetsuya; Matsunaga, Tetsuro; Takeda, Jun; Tsujimoto, Gozoh; Ishihara, Akihiko; Yasuda, Koichiro; Tsuda, Kinsuke . E-mail: jinkan@tom.life.h.kyoto-u.ac.jp

    2006-08-04

    Dipeptidylpeptidase IV (DPP-IV) is a well-documented drug target for the treatment of type 2 diabetes. Hepatocyte nuclear factors (HNF)-1{alpha} and HNF-1{beta}, known as the causal genes of MODY3 and MODY5, respectively, have been reported to be involved in regulation of DPP-IV gene expression. But, it is not completely clear (i) that they play roles in regulation of DPP-IV gene expression, and (ii) whether DPP-IV gene activity is changed by mutant HNF-1{alpha} and mutant HNF-1{beta} in MODY3 and MODY5. To explore these questions, we investigated transactivation effects of wild HNF-1{alpha} and 13 mutant HNF-1{alpha}, as well as wild HNF-1{beta} and 2 mutant HNF-1{beta}, on DPP-IV promoter luciferase gene in Caco-2 cells by means of a transient experiment. Both wild HNF-1{alpha} and wild HNF-1{beta} significantly transactivated DPP-IV promoter, but mutant HNF-1{alpha} and mutant HNF-1{beta} exhibited low transactivation activity. Moreover, to study whether mutant HNF-1{alpha} and mutant HNF-1{beta} change endogenous DPP-IV enzyme activity, we produced four stable cell lines from Caco-2 cells, in which wild HNF-1{alpha} or wild HNF-1{beta}, or else respective dominant-negative mutant HNF-1{alpha}T539fsdelC or dominant-negative mutant HNF-1{beta}R177X, was stably expressed. We found that DPP-IV gene expression and enzyme activity were significantly increased in wild HNF-1{alpha} cells and wild HNF-1{beta} cells, whereas they decreased in HNF-1{alpha}T539fsdelC cells and HNF-1{beta}R177X cells, compared with DPP-IV gene expression and enzyme activity in Caco-2 cells. These results suggest that both wild HNF-1{alpha} and wild HNF-1{beta} have a stimulatory effect on DPP-IV gene expression, but that mutant HNF-1{alpha} and mutant HNF-1{beta} attenuate the stimulatory effect.

  15. Adhesive hierarchy involving the cell adhesion molecules L1, CD24, and alpha 6 integrin in murine neuroblastoma N2A cells.

    PubMed

    Kadmon, G; Imhof, B A; Altevogt, P; Schachner, M

    1995-09-01

    The aggregation rate of resuspended neuroblastoma N2A cells depends on the density of the cells in culture prior to their resuspension: isolated, fast growing cells have a weak tendency to aggregate whereas confluent, slowly growing cells reaggregate very strongly. L1 antibody 557 strongly inhibited the slow aggregation of isolated, fast growing cells but not the reaggregation of confluent cells. CD24 (nectadrin) antibodies did not affect the aggregation of isolated or confluent cells but stimulated the aggregation of subconfluent cells. In all stages aggregation was not inhibited when antibody 557 was used together with CD24 antibodies at 37 degrees C in the presence of divalent cations. EA-1 antibody to alpha 6 integrin chain stimulated the aggregation of subconfluent cells but inhibited the reaggregation of confluent cells. Therefore, L1 appears to be an early recognition molecule mediating weak primary adhesion. CD24 appears to participate in activating secondary adhesion mechanisms during primary adhesion, possibly in cooperation with L1, and alpha 6 integrin seems to serve as a secondary, strong adhesion molecule that in early adhesion phases also mediates the activation of itself or of other adhesion mechanisms. These results indicate that neural cells might employ a strategy of adhesion cascade in establishing stable contacts. PMID:7669058

  16. cap alpha. -transforming growth factor secreted by untransformed bovine anterior pituitary cells in culture. II. Identification using a sequence-specific monoclonal antibody

    SciTech Connect

    Kobrin, M.S.; Samsoondar, J.; Kudlow, J.E.

    1986-11-05

    Untransformed bovine anterior pituitary cells cultured in serum-free defined medium secrete an epidermal growth factor (EGF)-like peptide with an amino acid composition similar to rat or human ..cap alpha..-transforming growth factor (..cap alpha..TGF). To further characterize the bovine pituitary ..cap alpha..TGF, it was compared to a human ..cap alpha..TGF partially purified from the conditioned medium of a human melanoma cell line. An anti-..cap alpha..TGF monoclonal antibody, MF9, was produced from hybridomas derived from mice immunized with a 17-residue synthetic peptide corresponding to the carboxyl-terminal sequence of rat ..cap alpha..TGF. The hybridoma supernatants were initially screened for the ability to immunoprecipitate /sup 125/I-peptide and then tested for recognition of human ..cap alpha..TGF. Only 2 of 36 antipeptide antibodies recognized the native ..cap alpha..TGF. The binding of /sup 125/I-peptide to MF9 was displaced by human ..cap alpha..TGF but not by EGF. Bovine pituitary ..cap alpha..TGF also displaced the binding of /sup 125/I-peptide to MF9 in a similar manner to human ..cap alpha..TGF. Both iodinated human and bovine pituitary ..cap alpha..TGF were immunoprecipitated by MF9 whereas /sup 125/I-EGF was not. Tryptic digests of both /sup 125/I-..cap alpha..TGFs chromatographed to give a single, indistinguishable peak of iodinated material on a reverse-phase C/sub 18/ high performance liquid chromatography column when eluted with two different solvent systems, suggesting the generation of a single and identical tyrosine-containing tryptic peptide from both ..cap alpha..TGFs. The comparisons of the bovine pituitary and human melanoma ..cap alpha..TGF using a sequence-specific monoclonal antibody and peptide mapping suggest that these ..cap alpha..TGFs are related and that ..cap alpha..TGF production is not limited to transformed or fetal sources.

  17. 1Alpha,25-dihydroxyvitamin D3 inhibits programmed cell death in HL-60 cells by activation of sphingosine kinase.

    PubMed

    Kleuser, B; Cuvillier, O; Spiegel, S

    1998-05-01

    Sphingolipid breakdown products [ceramide, sphingosine, and sphingosine-1-phosphate (SPP)] are emerging as a new class of bioactive molecules. In agreement with previous studies, treatment of human promyelocytic leukemia HL-60 cells with 1-alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] induced a transient increase of ceramide levels within 2 h, which then returned to basal levels within 8 h. In contrast, sphingosine kinase activity increased more slowly and reached maximal levels only after 20 h of exposure, leading to a concomitant increase in SPP level. Unlike treatments with cell-permeable ceramide analogues or sphingomyelinase, which induce apoptosis, 1,25-(OH)2D3 did not induce apoptosis, despite the early formation of ceramide. Moreover, prolonged treatment of HL-60 cells with 1,25-(OH)2D3 suppressed ceramide-induced apoptosis. There was a correlation between the time course and dose response of the activation of sphingosine kinase by 1,25-(OH)2D3 and the protection against apoptosis. In contrast, treatment with all-trans-retinoic acid neither stimulated sphingosine kinase activity nor protected cells from ceramide-induced apoptosis. Treatment with SPP protected HL-60 cells from ceramide-induced apoptosis, and N,N-dimethylsphingosine (DMS), a competitive inhibitor of sphingosine kinase, prevented the survival effect of 1,25-(OH)2D3. The effect of DMS was counteracted by SPP, suggesting that SPP is a critical component of the cytoprotective effect of 1,25-(OH)2D3. Chelerythrine chloride, an inhibitor of protein kinase C, markedly reduced sphingosine kinase activity and the apoptosis-sparing effect of 1,25-(OH)2D3, and conversely, the tumor promoter 12-O-tetradecanoylphorhol-13-acetate not only suppressed ceramide-induced apoptosis but also stimulated sphingosine kinase activity. Moreover, the protective effect of 12-O-tetradecanoylphorbol-13-acetate was blocked by DMS. Collectively, our observations indicate that the cytoprotective effect of 1,25-(OH)2D3 is

  18. Effect of Tumour Necrosis Factor-Alpha on Estrogen Metabolic Pathways in Breast Cancer Cells

    PubMed Central

    Kamel, Marwa; Shouman, Samia; El-Merzebany, Mahmoud; Kilic, Gokhan; Veenstra, Timothy; Saeed, Muhammad; Wagih, Mohamed; Diaz-Arrastia, Concepcion; Patel, Deepa; Salama, Salama

    2012-01-01

    Tumor necrosis factor-alpha (TNF-α) is a proinflammatory cytokine that has been linked to breast cancer development. Estrogen metabolic pathway is also involved in breast carcinogenesis and DNA adducts formation. In this study we investigated the effect of TNF-α on the estrogen metabolic pathway in MCF-7, a breast cancer cell line. Capillary liquid chromatography/mass spectrometry (LC/MS) and High performance liquid chromatography (HPLC) were used for analysis of estrogen metabolites and estrogen-DNA adducts levels respectively. Reporter gene assay, Real time reverse transcription polymerase chain reaction (real time RT-PCR) and Western blot were used to assess the expression of estrogen metabolizing genes and enzymes. TNF-α significantly increased the total EM and decreased the estrone (E1) / 17-β estradiol (E2) ratio. Moreover, it altered the expression of genes and enzymes involved in E2 activation and deactivation pathways e.g. Cytochrome P-450 1A1 (CYP1A1), Cytochrome P-450 1B1 (CYP1B1), Catechol-O-methyl transferase (COMT) and Nicotinamide adenine dinucleotide phosphate-quinone oxidoreductase 1 (NQO1). In addition, there were increased levels of some catechol estrogens e.g. 4-hydroxy-estrone (4-OHE1) and 2-hydroxyestradiol (2-OHE2) with decreased levels of methylated catechols e.g. 2-methoxy estradiol (2-MeOE2). DNA adducts especially 4-OHE1-[2]-1-N3 Adenine was significantly increased. TNF-α directs the estrogen metabolism into more hormonally active and carcinogenic products in MCF-7. This may implicate a new possible explanation for inflammation associated breast cancer. PMID:22866165

  19. Alpha-1 Antitrypsin Mitigates the Inhibition of Airway Epithelial Cell Repair by Neutrophil Elastase.

    PubMed

    Garratt, Luke W; Sutanto, Erika N; Ling, Kak-Ming; Looi, Kevin; Iosifidis, Thomas; Martinovich, Kelly M; Shaw, Nicole C; Buckley, Alysia G; Kicic-Starcevich, Elizabeth; Lannigan, Francis J; Knight, Darryl A; Stick, Stephen M; Kicic, Anthony

    2016-03-01

    Neutrophil elastase (NE) activity is associated with many destructive lung diseases and is a predictor for structural lung damage in early cystic fibrosis (CF), which suggests normal maintenance of airway epithelium is prevented by uninhibited NE. However, limited data exist on how the NE activity in airways of very young children with CF affects function of the epithelia. The aim of this study was to determine if NE activity could inhibit epithelial homeostasis and repair and whether any functional effect was reversible by antiprotease alpha-1 antitrypsin (α1AT) treatment. Viability, inflammation, apoptosis, and proliferation were assessed in healthy non-CF and CF pediatric primary airway epithelial cells (pAECnon-CF and pAECCF, respectively) during exposure to physiologically relevant NE. The effect of NE activity on pAECCF wound repair was also assessed. We report that viability after 48 hours was significantly decreased by 100 nM NE in pAECnon-CF and pAECCF owing to rapid cellular detachment that was accompanied by inflammatory cytokine release. Furthermore, both phenotypes initiated an apoptotic response to 100 nM NE, whereas ≥ 50 nM NE activity significantly inhibited the proliferative capacity of cultures. Similar concentrations of NE also significantly inhibited wound repair of pAECCF, but this effect was reversed by the addition of α1AT. Collectively, our results demonstrate free NE activity is deleterious for epithelial homeostasis and support the hypothesis that proteases in the airway contribute directly to CF structural lung disease. Our results also highlight the need to investigate antiprotease therapies in early CF disease in more detail. PMID:26221769

  20. Mechanisms of Alpha-Synuclein Action on Neurotransmission: Cell-Autonomous and Non-Cell Autonomous Role

    PubMed Central

    Emanuele, Marco; Chieregatti, Evelina

    2015-01-01

    Mutations and duplication/triplication of the alpha-synuclein (αSyn)-coding gene have been found to cause familial Parkinson’s disease (PD), while genetic polymorphisms in the region controlling the expression level and stability of αSyn have been identified as risk factors for idiopathic PD, pointing to the importance of wild-type (wt) αSyn dosage in the disease. Evidence that αSyn is present in the cerebrospinal fluid and interstitial brain tissue and that healthy neuronal grafts transplanted into PD patients often degenerate suggests that extracellularly-released αSyn plays a role in triggering the neurodegenerative process. αSyn’s role in neurotransmission has been shown in various cell culture models in which the protein was upregulated or deleted and in knock out and transgenic animal, with different results on αSyn’s effect on synaptic vesicle pool size and mobilization, αSyn being proposed as a negative or positive regulator of neurotransmitter release. In this review, we discuss the effect of αSyn on pre- and post-synaptic compartments in terms of synaptic vesicle trafficking, calcium entry and channel activity, and we focus on the process of exocytosis and internalization of αSyn and on the spreading of αSyn-driven effects due to the presence of the protein in the extracellular milieu. PMID:25985082

  1. The inhibitory effect of dexamethasone on platelet-derived growth factor-induced vascular smooth muscle cell migration through up-regulating PGC-1{alpha} expression

    SciTech Connect

    Xu, Wei; Guo, Ting; Zhang, Yan; Jiang, Xiaohong; Zhang, Yongxian; Zen, Ke; Yu, Bo; Zhang, Chen-Yu

    2011-05-01

    Dexamethasone has been shown to inhibit vascular smooth muscle cell (VSMC) migration, which is required for preventing restenosis. However, the mechanism underlying effect of dexamethasone remains unknown. We have previously demonstrated that peroxisome proliferator-activated receptor gamma (PPAR{gamma}) coactivator-1 alpha (PGC-1{alpha}) can inhibit VSMC migration and proliferation. Here, we investigated the role of PGC-1{alpha} in dexamethasone-reduced VSMC migration and explored the possible mechanism. We first examined PGC-1{alpha} expression in cultured rat aortic VSMCs. The results revealed that incubation of VSMCs with dexamethasone could significantly elevate PGC-1{alpha} mRNA expression. In contrast, platelet-derived growth factor (PDGF) decreased PGC-1{alpha} expression while stimulating VSMC migration. Mechanistic study showed that suppression of PGC-1{alpha} by small interfering RNA strongly abrogated the inhibitory effect of dexamethasone on VSMC migration, whereas overexpression of PGC-1{alpha} had the opposite effect. Furthermore, an analysis of MAPK signal pathways showed that dexamethasone inhibited ERK and p38 MAPK phosphorylation in VSMCs. Overexpression of PGC-1{alpha} decreased both basal and PDGF-induced p38 MAPK phosphorylation, but it had no effect on ERK phosphorylation. Finally, inhibition of PPAR{gamma} activation by a PPAR{gamma} antagonist GW9662 abolished the suppressive effects of PGC-1{alpha} on p38 MAPK phosphorylation and VSMC migration. These effects of PGC-1{alpha} were enhanced by a PPAR{gamma} agonist troglitazone. Collectively, our data indicated for the first time that one of the anti-migrated mechanisms of dexamethasone is due to the induction of PGC-1{alpha} expression. PGC-1{alpha} suppresses PDGF-induced VSMC migration through PPAR{gamma} coactivation and, consequently, p38 MAPK inhibition.

  2. Glioblastoma expression of vitronectin and the alpha v beta 3 integrin. Adhesion mechanism for transformed glial cells.

    PubMed Central

    Gladson, C L; Cheresh, D A

    1991-01-01

    Glioblastoma multiforme, the most malignant astroglial-derived tumor, grows as an adherent mass and locally invades normal brain. An examination of adult cerebral glioblastoma biopsy material for the expression of adhesive proteins that might potentiate adhesion and invasion demonstrated tumor cell-associated vitronectin (5/5). In contrast, vitronectin was not detected associated with glial cells in low grade astroglial tumors (0/4), reactive astrogliosis (0/4), or in normal adult cortex and cerebral white matter (0/5). Also, a wide variety of other adhesive ligands were absent from the glioblastoma tumor parenchyma. The alpha v beta 3 integrin was the only vitronectin receptor identified in glioblastoma tumors in situ, and was also not expressed on low grade astroglial-derived tumors, reactive astrogliosis, or on glia or neurons in normal adult cortex and cerebral white matter. In a cell attachment assay, cultured glioblastoma cells attached to the parenchyma of glioblastoma tumor cryostat sections at the sites of vitronectin expression, but failed to attach to normal brain. This adhesion was inhibited by antibodies directed against vitronectin, the alpha v beta 3 integrin, and with an Arg-Gly-Asp-containing peptide. These data provide evidence for a cell adhesion mechanism in glioblastoma tumors that might potentiate glioblastoma cell invasion of normal brain. Images PMID:1721625

  3. Expressions of endothelin-1, fibronectin, and interleukin-1alpha of human umbilical vein endothelial cells under prolonged culture.

    PubMed

    Kiyonaga, H; Doi, Y; Karasaki, Y; Arashidani, K; Itoh, H; Fujimoto, S

    2001-03-01

    We examined human umbilical vein endothelial cells (HUVECs) under prolonged culture by electron microscopy and by light and electron immunocytochemistry including double immunolabeling. Based on the cell area of HUVECs through multiple passages, we divided the cells into first, second, and third stages, which exhibited distinct morphological and immunocytochemical characteristics. During the first stage, HUVECs were polygonal in shape and had already formed the monolayer confluence. During the second stage, they were characterized by an increased number of Weibel-Palade (WP) bodies, which were actively segregated from Golgi cisterns. Endothelin (ET)-1 and von Willebrand factor, an endothelial cell marker, were occasionally colocalized in WP bodies. The increase in WP bodies correlated with high ET-1 concentration in the cultured medium, suggesting that these inclusions are involved in storage and release of ET-1 in a manner indicating a regulatory pathway. During the third stage, fibronectin and interleukin (IL)-1alpha were expressed in HUVECs as well as in multinucleated giant cells, which originated from HUVECs, but WP bodies decreased in number in association with a decrease in ET-1 immunoreactivity and concentration. The foregoing changes in immunoreactivities for ET-1, fibronectin, and IL-1alpha affecting HUVECs under prolonged culture may reflect a senescent process of these cells. PMID:11479772

  4. The role of 14-3-3{beta} in transcriptional activation of estrogen receptor {alpha} and its involvement in proliferation of breast cancer cells

    SciTech Connect

    Kim, Yoonseo; Kim, Hyungjin; Jang, Sung-Wuk; Ko, Jesang

    2011-10-14

    Highlights: {yields} 14-3-3{beta} interacts with ER{alpha} and the interaction is Akt-dependent. {yields} 14-3-3{beta} regulates the transcriptional activity of ER{alpha} in a ligand-dependent manner. {yields} 14-3-3{beta} increases expressions of ER{alpha} target genes. {yields} 14-3-3{beta} increases breast cancer cell proliferation. -- Abstract: The estrogen receptor (ER) functions as a transcription factor that mediates the effects of estrogen. ER{alpha}, which plays a crucial role in the development and progression of breast cancer, is activated by estrogen binding, leading to receptor phosphorylation, dimerization, and recruitment of co-activators and chaperons to the estrogen-bound receptor complex. The 14-3-3 proteins bind to target proteins via phosphorylation and influence many cellular events by altering their subcellular localization or acting as a chaperone. However, regulation of ER{alpha} expression and transactivation by the 14-3-3 proteins has not been reported. We demonstrate that 14-3-3{beta} functions as a positive regulator of ER{alpha} through a direct protein-protein interaction in an estrogen-dependent manner. Ectopic expression of 14-3-3{beta} stimulated ER{alpha}-mediated transcriptional activity in MCF-7 breast cancer cells. Enhanced ER{alpha} transcriptional activity due to 14-3-3{beta} increased the expressions of the endogenous ER{alpha} target genes, leading to proliferation of breast cancer cells. We suggest that 14-3-3{beta} has oncogenic potential in breast cancer via binding to ER{alpha} and activation of the transcriptional activity of ER{alpha}.

  5. Characteristics and mechanisms of the bystander response in monolayer cell cultures exposed to very low fluences of alpha particles

    NASA Astrophysics Data System (ADS)

    Little, John B.; Azzam, Edouard I.; de Toledo, Sonia M.; Nagasawa, Hatsumi

    2005-02-01

    When confluent cultures of mammalian cells are irradiated with very low fluences of alpha particles whereby only occasional cells receive any radiation exposure, genetic changes are observed in the non-irradiated ("bystander") cells. Upregulation of the p53 damage-response pathway as well as activation of proteins in the MAPK family occurred in bystander cells; p53 was phosphorylated on the serine 15 residue suggesting that the upregulation of p53 was a consequence of DNA damage. Damage signals were transmitted to bystander cells through gap junctions, as confirmed by the use of genetically manipulated cells including connexin43 knockouts. Expression of connexin43 was markedly enhanced by irradiation. A moderate bystander effect was observed for specific gene mutations and chromosomal aberrations. This effect was markedly enhanced in cells defective in the non-homologous end joining DNA repair pathway. Finally, an upregulation of oxidative metabolism occurred in bystander cells; the increased levels of reactive oxygen species appeared to be derived from flavine-containing oxidase enzymes. We hypothesize that genetic effects observed in non-irradiated bystander cells are a consequence of oxidative base damage; >90% of mutations in bystander cells were point mutations. When bystander cells cannot repair DNA double strand breaks, they become much more sensitive to the induction of chromosomal aberrations and mutations, the latter consisting primarily of deletion mutants. While we propose that the genetic effects occurring in bystander cells are a consequence of oxidative stress, the nature of the signal that initiates this process remains to be determined.

  6. Alpha-ketoglutarate enhances milk protein synthesis by porcine mammary epithelial cells.

    PubMed

    Jiang, Qian; He, Liuqin; Hou, Yongqing; Chen, Jiashun; Duan, Yehui; Deng, Dun; Wu, Guoyao; Yin, Yulong; Yao, Kang

    2016-09-01

    Alpha-ketoglutarate (AKG), a key intermediate in the Krebs cycle, has been reported to promote protein synthesis through activating mechanistic targeting of rapamycin (mTOR) in enterocytes. The study tested the hypothesis that AKG may enhance growth and milk protein synthesis in porcine mammary epithelial cells (PMECs). PMECs were cultured for 96 h in Dulbecco's modified Eagle's-F12 Ham medium (DMEM-F12) containing prolactin (2 µg/ml) and AKG (0 or 1.5 mM). At the end of 96-h culture, the abundance of apoptosis-related proteins (caspase-3, caspase-9), milk-specific proteins (α-lactalbumin and β-casein), mTOR signaling proteins (mTOR, p-mTOR, PERK, p-PERK, eIF2a, P70S6K and p-P70S6K), and endoplasmic reticulum stress (ERS)-associated proteins (BiP and CHOP) in PMEC were determined. Addition of AKG dose-dependently enhanced cell viability in the absence or presence of prolactin, with optimal concentrations of AKG being at 1.0 and 1.5 mM, respectively. In the presence of prolactin, addition of 1.5 mM AKG: (1) decreased (P < 0.05) the abundance of caspase-3 and caspase-9 by 21 and 39 %; (2) enhanced (P < 0.05) the phosphorylation of p-mTOR and p-P70S6K by 39 and 89 %, respectively; (3) increased (P < 0.05) the production of β-casein and α-lactalbumin by 16 and 20 %, respectively; (4) attenuated (P < 0.05) the expression of CHOP by 34 % but promoted (P < 0.05) the expression of BiP by 46 %; (5) increased (P < 0.05) the secretion of lactose by 15 %, when compared to the 0 mM AKG group. Rapamycin (50 nM; an inhibitor of mTOR) attenuated (P < 0.05) the stimulatory effect of AKG on mTOR signaling and syntheses of milk protein and lactose, while relieving (P < 0.05) an inhibitory effect of AKG on expression of proteins related to ERS. Collectively, our results indicate that AKG enhances milk protein production by modulating mTOR and ERS signaling pathways in PMECs. PMID:27188418

  7. Major vault protein forms complexes with hypoxia-inducible factor (HIF)-1alpha and reduces HIF-1alpha level in ACHN human renal adenocarcinoma cells.

    PubMed

    Iwashita, Ken-ichi; Ikeda, Ryuji; Takeda, Yasuo; Sumizawa, Tomoyuki; Furukawa, Tatsuhiko; Yamaguchi, Tatsuya; Akiyama, Shin-ichi; Yamada, Katsushi

    2010-04-01

    Vaults are evolutionarily highly conserved ribonucleoprotein (RNP) particles with a hollow barrel-like structure. Although roles in multidrug resistance and innate immunity have been suggested, the physiological function of vaults remains unclear. Major vault protein (MVP), the main component of the vault particle, has been reported to be induced by hypoxia. However, there are no reports about the effect of vaults on cellular responses to hypoxia. We thus examined whether vaults are implicated in cellular responses to hypoxia. In this study, we focused on hypoxia-inducible factor-1alpha (HIF-1alpha), which is a master regulator of hypoxic responses, and found that: (i) MVP knockdown by RNA interference increases HIF-1alpha protein levels induced by hypoxia and hypoxia mimetics; (ii) MVP knockdown does not affect HIF-1alpha mRNA levels, but decreases the ubiquitination and degradation of HIF-1alpha protein; and (iii) vaults form complexes with HIF-1alpha, PHD2, and pVHL. Taken together, these results suggest that vaults function as scaffolds in HIF-1alpha degradation pathway and promote the ubiquitination and degradation of HIF-1alpha. PMID:20175781

  8. Proinflammatory response of alveolar epithelial cells is enhanced by alveolar macrophage-produced TNF-alpha during pulmonary ischemia-reperfusion injury.

    PubMed

    Sharma, Ashish K; Fernandez, Lucas G; Awad, Alaa S; Kron, Irving L; Laubach, Victor E

    2007-07-01

    Pulmonary ischemia-reperfusion (IR) injury entails acute activation of alveolar macrophages followed by neutrophil sequestration. Although proinflammatory cytokines and chemokines such as TNF-alpha and monocyte chemoattractant protein-1 (MCP-1) from macrophages are known to modulate acute IR injury, the contribution of alveolar epithelial cells to IR injury and their intercellular interactions with other cell types such as alveolar macrophages and neutrophils remain unclear. In this study, we tested the hypothesis that following IR, alveolar macrophage-produced TNF-alpha further induces alveolar epithelial cells to produce key chemokines that could then contribute to subsequent lung injury through the recruitment of neutrophils. Cultured RAW264.7 macrophages and MLE-12 alveolar epithelial cells were subjected to acute hypoxia-reoxygenation (H/R) as an in vitro model of pulmonary IR. H/R (3 h/1 h) significantly induced KC, MCP-1, macrophage inflammatory protein-2 (MIP-2), RANTES, and IL-6 (but not TNF-alpha) by MLE-12 cells, whereas H/R induced TNF-alpha, MCP-1, RANTES, MIP-1alpha, and MIP-2 (but not KC) by RAW264.7 cells. These results were confirmed using primary murine alveolar macrophages and primary alveolar type II cells. Importantly, using macrophage and epithelial coculture methods, the specific production of TNF-alpha by H/R-exposed RAW264.7 cells significantly induced proinflammatory cytokine/chemokine expression (KC, MCP-1, MIP-2, RANTES, and IL-6) by MLE-12 cells. Collectively, these results demonstrate that alveolar type II cells, in conjunction with alveolar macrophage-produced TNF-alpha, contribute to the initiation of acute pulmonary IR injury via a proinflammatory cascade. The release of key chemokines, such as KC and MIP-2, by activated type II cells may thus significantly contribute to neutrophil sequestration during IR injury. PMID:17416740

  9. Heterogeneous distributions of amino acids provide evidence of multiple sources within the Almahata Sitta parent body, asteroid 2008 TC3

    NASA Astrophysics Data System (ADS)

    Burton, Aaron S.; Glavin, Daniel P.; Callahan, Michael P.; Dworkin, Jason P.; Jenniskens, Peter; Shaddad, Muawia H.

    2011-11-01

    Two new fragments of the Almahata Sitta meteorite and a sample of sand from the related strewn field in the Nubian Desert, Sudan, were analyzed for two to six carbon aliphatic primary amino acids by ultrahigh performance liquid chromatography with UV-fluorescence detection and time-of-flight mass spectrometry (LC-FT/ToF-MS). The distribution of amino acids in fragment #25, an H5 ordinary chondrite, and fragment #27, a polymict ureilite, were compared with results from the previously analyzed fragment #4, also a polymict ureilite. All three meteorite fragments contain 180-270 parts-per-billion (ppb) of amino acids, roughly 1000-fold lower than the total amino acid abundance of the Murchison carbonaceous chondrite. All of the Almahata Sitta fragments analyzed have amino acid distributions that differ from the Nubian Desert sand, which primarily contains L-α-amino acids. In addition, the meteorites contain several amino acids that were not detected in the sand, indicating that many of the amino acids are extraterrestrial in origin. Despite their petrological differences, meteorite fragments #25 and #27 contain similar amino acid compositions; however, the distribution of amino acids in fragment #27 was distinct from those in fragment #4, even though both are polymict ureilites from the same parent body. Unlike in CM2 and CR2/3 meteorites, there are low relative abundances of α-amino acids in the Almahata Sitta meteorite fragments, which suggest that Strecker-type chemistry was not a significant amino acid formation mechanism. Given the high temperatures that asteroid 2008 TC3 appears to have experienced and lack of evidence for aqueous alteration on the asteroid, it is possible that the extraterrestrial amino acids detected in Almahata Sitta were formed by Fischer-Tropsch/Haber-Bosch type gas-grain reactions at elevated temperatures.

  10. Transcriptional Response of Human Cells to Microbeam Irradiation with 2.1 MeV Alpha Particles

    NASA Astrophysics Data System (ADS)

    Hellweg, C. E.; Bogner, S.; Spitta, L.; Arenz, A.; Baumstark-Khan, C.; Greif, K. D.; Giesen, U.

    Within the next decades an increasing number of human beings in space will be simultaneously exposed to different stimuli especially microgravity and radiation To assess the risks for humans during long-duration space missions the complex interplay of these parameters at the cellular level must be understood Cellular stress protection responses lead to increased transcription of several genes via modulation of transcription factors Activation of the Nuclear Factor kappa B NF- kappa B pathway as a possible anti-apoptotic route represents such an important cellular stress response A screening assay for detection of NF- kappa B-dependent gene activation using the destabilized variant of Enhanced Green Fluorescent Protein d2EGFP as reporter protein had been developed It consists of Human Embryonic Kidney HEK 293 Cells stably transfected with a receptor-reporter-construct carrying d2EGFP under the control of a NF- kappa B response element Clones positive for Tumor Necrosis Factor alpha TNF- alpha inducible d2EGFP expression were selected as cellular reporters Irradiation was performed either with X-rays 150 kV 19 mA at DLR Cologne or with 2 1 MeV alpha particles LET sim 160 keV mu m at PTB Braunschweig After irradiation the following biological endpoints were determined i cell survival via the colony forming ability test ii time-dependent activation of NF- kappa B dependent d2EGFP gene expression using flow cytometry iii quantitative RT-PCR

  11. Nonlinear relationship between alpha 1-adrenergic receptor occupancy and norepinephrine-stimulated calcium flux in cultured vascular smooth muscle cells

    SciTech Connect

    Colucci, W.S.; Brock, T.A.; Gimbrone, M.A. Jr.; Alexander, R.W.

    1985-05-01

    To determine the relationship between vascular alpha 1-adrenergic receptor occupancy and receptor-coupled calcium flux, the authors have studied (/sup 3/H)prazosin binding and l-norepinephrine-induced /sup 45/Ca efflux in cultured vascular smooth muscle cells isolated from the rabbit aorta. In a crude cellular homogenate, (/sup 3/H)prazosin bound to a single high affinity site, whereas l-norepinephrine (NE) binding was best described by a two-site model. NE-stimulated /sup 45/Ca efflux was concentration-dependent (EC/sup 50/ = 108 nM) and potently inhibited by prazosin (IC/sup 50/ = 0.15 nM). For the total receptor pool identified by (/sup 3/H)prazosin binding, the relationship between receptor occupancy by NE and NE-stimulated /sup 45/Ca efflux was markedly nonlinear, such that 50% of maximum NE-stimulated efflux occurred with occupancy of only approximately 7% of receptors. These two experimental approaches provide direct evidence for the presence in cultured rabbit aortic smooth muscle cells of a sizable pool of alpha 1-adrenergic receptors in excess of those needed for maximum NE-stimulated /sup 45/Ca efflux. This evidence of ''spare'' receptors, together with the finding of two affinity states of agonist binding, raises the possibility of functional heterogeneity of alpha 1-adrenergic receptors in this system.

  12. Sphingosine kinase-1 mediates TNF-alpha-induced MCP-1 gene expression in endothelial cells: upregulation by oscillatory flow.

    PubMed

    Chen, Xi-Lin; Grey, Janice Y; Thomas, Suzanne; Qiu, Fei-Hua; Medford, Russell M; Wasserman, Martin A; Kunsch, Charles

    2004-10-01

    Atherosclerosis is a focal inflammatory disease and preferentially occurs in areas of low fluid shear stress and oscillatory flow, whereas the risk of atherosclerosis is decreased in regions of high fluid shear stress and steady laminar flow. Sphingosine kinase-1 (SphK1) catalyzes the conversion of sphingosine to sphingosine-1 phosphate (S1P), a sphingolipid metabolite that plays important roles in angiogenesis, inflammation, and cell growth. In the present study, we demonstrated that exposure of human aortic endothelial cells to oscillatory flow (shear stress, +/-5 dyn/cm(2) for 48 h) resulted in a marked increase in SphK1 mRNA levels compared with endothelial cells kept in static culture. In contrast, laminar flow (shear stress, 20 dyn/cm(2) for 48 h) decreased SphK1 mRNA levels. We further investigated the role of SphK1 in TNF-alpha-induced expression of inflammatory genes, such as monocyte chemoattractant protein-1 (MCP-1) and VCAM-1 by using small interfering RNA (siRNA) specifically for SphK1. Treatment of endothelial cells with SphK1 siRNA suppressed TNF-alpha-induced increase in MCP-1 mRNA levels, MCP-1 protein secretion, and activation of p38 MAPK. SphK1 siRNA also inhibited TNF-alpha-induced cell surface expression of VCAM-1, but not ICAM-1, protein. Exposure of endothelial cells to S1P led to an increase in MCP-1 protein secretion and MCP-1 mRNA levels and activation of NF-kappaB-mediated transcriptional activity. Treatment of endothelial cells with the p38 MAPK inhibitor SB-203580 suppressed S1P-induced MCP-1 protein secretion. These data suggest that SphK1 mediates TNF-alpha-induced MCP-1 gene expression through a p38 MAPK-dependent pathway and may participate in oscillatory flow-mediated proinflammatory signaling pathway in the vasculature. PMID:15191888

  13. An enhanced and sensitive autocrine stimulation by transforming growth factor-alpha is acquired in the brain metastatic variant of a human non-small-cell lung cancer cell line.

    PubMed

    Fang, K

    1996-12-01

    Transforming growth factor-alpha (TGF-alpha)-mediated autocrine regulation in human non-small-cell lung cancer (NSCLC) cells NCI-H226 and its brain metastatic variant H226Br were compared. An enhanced TGF-alpha-induced dose-dependent mitogenic responsiveness in H226Br cells was observed. Neutralising antibody that binds TGF-alpha inhibits H226Br cell growth more effectively than NCI-H226 cell growth. Binding assay with 125I-labelled epidermal growth factor (EGF) revealed that H226Br has two types of EGF receptors (EGFRs), whereas the parental cell line, NCI-H226, has only one. H226Br cells contain twice as many EGFRs as H226 cells, as proved by Scatchard analysis and immune kinase assay. Northern analysis indicated that there is more EGFR transcript in H226Br than in NCI-H226, indicating a transcriptional EGFR gene elevation during metastasis progression. The level of accumulated immunoactive TGF-alpha is lower in the conditioned medium of H226Br than in that of NCI-H226. demonstrating down-regulation of TGF-alpha transcript. The accumulated data suggest an elevated and sensitive autocrine modulation by TGF-alpha and EGFR in immortalising the brain metastatic variant cells that were derived from a human NSCLC squamous cell line. PMID:8956792

  14. Human and rat mast cell high-affinity immunoglobulin E receptors: Characterization of putative. alpha. -chain gene products

    SciTech Connect

    Shimizu, Akira; Benfey, P.N.; Leder, P. ); Tepler, I. Brigham and Women's Hospital, Boston, MA ); Berenstein, E.H.; Siraganian, R.P. )

    1988-03-01

    The authors have cloned and determined the entire nucleotide sequence of cDNAs corresponding to the putative {alpha} subunits of the human and rat mast cell high-affinity IgE receptors. Both human and rat cDNAs encode an NH{sub 2}-terminal signal peptide, two immunoglobulin-like extracellular domains (encoded by discrete exons), a hydrophobic transmembrane region, and a positively charged cytoplasmic tail. The human and rat {alpha} subunits share an overall homology with one another and the immunoglobulin gene family, suggesting that they arose from a common ancestral gene and continue to share structural homology with their ligands. In addition, the rat gene is transcribed into at least three distinct forms, each of which yields a somewhat different coding sequence.

  15. Encapsulation of eukaryotic cells in alginate microparticles: cell signaling by TNF-alpha through capsular structure of cystic fibrosis cells.

    PubMed

    Mazzitelli, Stefania; Borgatti, Monica; Breveglieri, Giulia; Gambari, Roberto; Nastruzzi, Claudio

    2011-06-01

    Entrapment of mammalian cells in natural or synthetic biomaterials represents an important tool for both basic and applied research in tissue engineering. For instance, the encapsulation procedures allow to physically isolate cells from the surrounding environment, after their transplantation maintaining the normal cellular physiology. The first part of the current paper describes different microencapsulation techniques including bulk emulsion technique, vibrating-nozzle procedure, gas driven mono-jet device protocol and microfluidic based approach. In the second part, the application of a microencapsulation procedure to the embedding of IB3-1 cells is also described. IB3-1 is a bronchial epithelial cell line, derived from a cystic fibrosis (CF) patient. Different experimental parameters of the encapsulation process were analyzed, including frequency and amplitude of vibration, polymer pumping rate and distance between the nozzle and the gelling bath. We have found that the microencapsulation procedure does not alter the viability of the encapsulated IB3-1 cells. The encapsulated IB3-1 cells were characterized in term of protein secretion, analysing the culture medium by Bio-Plex strategy. The analyzed factors include members of the interleukin family (IL-6), chemokines (IL-8 and MCP-1) and growth factors (G-CSF). The experiments demonstrated that most of the analyzed proteins, were secreted both by the free and encapsulated cells, even if in a different extent. PMID:21484183

  16. Alpha-tubulin enhanced renal tubular cell proliferation and tissue repair but reduced cell death and cell-crystal adhesion.

    PubMed

    Manissorn, Juthatip; Khamchun, Supaporn; Vinaiphat, Arada; Thongboonkerd, Visith

    2016-01-01

    Adhesion of calcium oxalate (CaOx) crystals on renal tubular epithelial cells is a critical event for kidney stone disease that triggers many cascades of cellular response. Our previous expression proteomics study identified several altered proteins in MDCK renal tubular cells induced by CaOx crystals. However, functional significance of those changes had not been investigated. The present study thus aimed to define functional roles of such proteome data. Global protein network analysis using STRING software revealed α-tubulin, which was decreased, as one of central nodes of protein-protein interactions. Overexpression of α-tubulin (pcDNA6.2-TUBA1A) was then performed and its efficacy was confirmed. pcDNA6.2-TUBA1A could maintain levels of α-tubulin and its direct interacting partner, vimentin, after crystal exposure. Also, pcDNA6.2-TUBA1A successfully reduced cell death to almost the basal level and increased cell proliferation after crystal exposure. Additionally, tissue repair capacity was improved in pcDNA6.2-TUBA1A cells. Moreover, cell-crystal adhesion was reduced by pcDNA6.2-TUBA1A. Finally, levels of potential crystal receptors (HSP90, HSP70, and α-enolase) on apical membrane were dramatically reduced to basal levels by pcDNA6.2-TUBA1A. These findings implicate that α-tubulin has protective roles in kidney stone disease by preventing cell death and cell-crystal adhesion, but on the other hand, enhancing cell proliferation and tissue repair function. PMID:27363348

  17. Alpha-tubulin enhanced renal tubular cell proliferation and tissue repair but reduced cell death and cell-crystal adhesion

    PubMed Central

    Manissorn, Juthatip; Khamchun, Supaporn; Vinaiphat, Arada; Thongboonkerd, Visith

    2016-01-01

    Adhesion of calcium oxalate (CaOx) crystals on renal tubular epithelial cells is a critical event for kidney stone disease that triggers many cascades of cellular response. Our previous expression proteomics study identified several altered proteins in MDCK renal tubular cells induced by CaOx crystals. However, functional significance of those changes had not been investigated. The present study thus aimed to define functional roles of such proteome data. Global protein network analysis using STRING software revealed α-tubulin, which was decreased, as one of central nodes of protein-protein interactions. Overexpression of α-tubulin (pcDNA6.2-TUBA1A) was then performed and its efficacy was confirmed. pcDNA6.2-TUBA1A could maintain levels of α-tubulin and its direct interacting partner, vimentin, after crystal exposure. Also, pcDNA6.2-TUBA1A successfully reduced cell death to almost the basal level and increased cell proliferation after crystal exposure. Additionally, tissue repair capacity was improved in pcDNA6.2-TUBA1A cells. Moreover, cell-crystal adhesion was reduced by pcDNA6.2-TUBA1A. Finally, levels of potential crystal receptors (HSP90, HSP70, and α-enolase) on apical membrane were dramatically reduced to basal levels by pcDNA6.2-TUBA1A. These findings implicate that α-tubulin has protective roles in kidney stone disease by preventing cell death and cell-crystal adhesion, but on the other hand, enhancing cell proliferation and tissue repair function. PMID:27363348

  18. Hit rates and radiation doses to nuclei of bone lining cells from alpha-particle-emitting radionuclides

    NASA Technical Reports Server (NTRS)

    Polig, E.; Jee, W. S.; Kruglikov, I. L.

    1992-01-01

    Factors relating the local concentration of a bone-seeking alpha-particle emitter to the mean hit rate have been determined for nuclei of bone lining cells using a Monte Carlo procedure. Cell nuclei were approximated by oblate spheroids with dimensions and location taken from a previous histomorphometric study. The Monte Carlo simulation is applicable for planar and diffuse labels at plane or cylindrical bone surfaces. Additionally, the mean nuclear dose per hit, the dose mean per hit, the mean track segment length and its second moment, the percentage of stoppers, and the frequency distribution of the dose have been determined. Some basic features of the hit statistics for bone lining cells have been outlined, and the consequences of existing standards of radiation protection with regard to the hit frequency to cell nuclei are discussed.

  19. T-cell abnormalities after mediastinal irradiation for lung cancer. The in vitro influence of synthetic thymosin alpha-1

    SciTech Connect

    Schulof, R.S.; Chorba, T.L.; Cleary, P.A.; Palaszynski, S.R.; Alabaster, O.; Goldstein, A.L.

    1985-03-01

    The effects of mediastinal irradiation (RT) on the numbers and functions of purified peripheral blood T-lymphocytes from patients with locally advanced non-small cell lung cancer were evaluated. The patients were candidates for a randomized trial to evaluate the immunorestorative properties of synthetic thymosin alpha-1. Twenty-one patients studied before RT did not exhibit any significant difference in T-cell numbers or function compared to age-matched healthy subjects. However, 41 patients studied within 1 week after completing RT exhibited significant depressions of E-rosette-forming cells at 4 degrees C (E4 degrees-RFC)/mm3, E-rosette-forming cells at 29 degrees C (E29 degrees-RFC)/mm3, OKT3/mm3, OKT4/mm3, and OKT8/mm3 (P . 0.0001); total T-cell percentages (%OKT3, P . 0.01); and T-cell proliferative responses in mixed lymphocyte cultures (MLR) (P . 0.01) and to the mitogen phytohemagglutinin under suboptimal conditions (P less than or equal to 0.03). Nine patients studied before and after RT showed a significant increase in OKT4/OKT8 (P . 0.01) following RT. A short-term in vitro incubation with thymosin alpha-1 could enhance MLR of T-cells in 12 of 27 patients with post-RT abnormalities. In 13 patients who were treated with placebo, the RT-induced depression of T-cell numbers and function persisted for at least 3 to 4 months. In addition, in 12 patients progressive decreases developed in %E4 degrees-RFC, %OKT3, %OKT4, and OKT4/OKT8, which always preceded clinical relapse.

  20. An alpha-fetoprotein-producing human gallbladder carcinoma cell line (KMG-C) shows more aggressive biologic behavior than an alpha-fetoprotein-non-producing cell line.

    PubMed

    Maruiwa, M; Yano, H; Fukuda, K; Ogasawara, S; Hisaka, T; Yamaguchi, R; Kojiro, M

    1996-06-01

    We have established a new human alphafetoprotein (AFP)-producing gallbladder carcinoma (GBC) cell line, termed KMG-C, from a 67-year-old Japanese male. KMG-C and its reconstituted tumors in nude mice showed the morphological features of an adenocarcinoma. Functionally, KMG-C secreted AFP, carcinoembryonic antigen, and carbohydrate antigen 19-9, as well as 7 serum proteins, including albumin and C-reactive protein. KMG-C showed more malignant biological behavior than an AFP-nonproducing GBC cell line, KMG-A, established originally from the tumor of the same patient; KMG-C had a shorter doubling time, higher tumorigenicity, and an aneuploid DNA index. Our results suggest that AFP-producing GBC cells may have more malignant biological characteristics than AFP-non-producing GBC cells in GBCs having both of these components. PMID:21544480

  1. Dcas is required for importin-alpha3 nuclear export and mechano-sensory organ cell fate specification in Drosophila.

    PubMed

    Tekotte, Hildegard; Berdnik, Daniela; Török, Tibor; Buszczak, Michael; Jones, Lynn M; Cooley, Lynn; Knoblich, Jürgen A; Davis, Ilan

    2002-04-15

    We have studied the in vivo function and tissue specificity of Dcas, the Drosophila ortholog of CAS, the importin beta-like export receptor for importin alpha. While dcas mRNA is specifically expressed in the embryonic central nervous system, Dcas protein is maternally supplied to all embryonic cells and its nuclear/cytoplasmic distribution varies in different tissues and times in development. Unexpectedly, hypomorphic alleles of dcas show specific transformations in mechano-sensory organ cell identity, characteristic of mutations that increase Notch signaling. Dcas is essential for efficient importin-alpha3 nuclear export in mechano-sensory cells and the surrounding epidermal cells and is indirectly required for the import of one component of the Notch pathway, but not others tested. We interpret the specificity of the dcas phenotype as indicating that one or more Notch signaling components are particularly sensitive to a disruption in nuclear protein import. We propose that mutations in house keeping genes often cause specific developmental phenotypes, such as those observed in many human genetic disorders. PMID:11944946

  2. 7-ketocholesterol incorporation into sphingolipid/cholesterol-enriched (lipid raft) domains is impaired by vitamin E: a specific role for alpha-tocopherol with consequences on cell death.

    PubMed

    Royer, Marie-Charlotte; Lemaire-Ewing, Stéphanie; Desrumaux, Catherine; Monier, Serge; Pais de Barros, Jean-Paul; Athias, Anne; Néel, Dominique; Lagrost, Laurent

    2009-06-01

    Cholesterol oxides, in particular 7-ketocholesterol, are proatherogenic compounds that induce cell death in the vascular wall when localized in lipid raft domains of the cell membrane. Deleterious effects of 7-ketocholesterol can be prevented by vitamin E, but the molecular mechanism involved is unclear. In this study, unlike gamma-tocopherol, the alpha-tocopherol vitamin E form was found to prevent 7-ketocholesterol-mediated apoptosis of A7R5 smooth muscle cells. To be operative, alpha-tocopherol needed to be added to the cells before 7-ketocholesterol, and its anti-apoptotic effect was reduced and even suppressed when added together or after 7-ketocholesterol, respectively. Both pre- and co-treatment of the cells with alpha-tocopherol resulted in the redistribution of 7-ketocholesterol out of the sphingolipid/cholesterol-enriched (lipid raft) domains. In turn, fewer amounts of alpha-tocopherol associated with lipid rafts on 7-ketocholesterol-pretreated cells compared with untreated cells, with no prevention of cell death in this case. In further support of the implication of lipid raft domains, the dephosphorylation/inactivation of Akt-PKB was involved in the 7-ketocholesterol-induced apoptosis. Akt-PKB dephosphorylation was prevented by alpha-tocopherol, but not gamma-tocopherol pretreatment. PMID:19351882

  3. Endothelial monocyte activating polypeptide-II modulates endothelial cell responses by degrading hypoxia-inducible factor-1alpha through interaction with PSMA7, a component of the proteasome

    SciTech Connect

    Tandle, Anita T.; Calvani, Maura; Uranchimeg, Badarch; Zahavi, David; Melillo, Giovanni; Libutti, Steven K.

    2009-07-01

    The majority of human tumors are angiogenesis dependent. Understanding the specific mechanisms that contribute to angiogenesis may offer the best approach to develop therapies to inhibit angiogenesis in cancer. Endothelial monocyte activating polypeptide-II (EMAP-II) is an anti-angiogenic cytokine with potent effects on endothelial cells (ECs). It inhibits EC proliferation and cord formation, and it suppresses primary and metastatic tumor growth in-vivo. However, very little is known about the molecular mechanisms behind the anti-angiogenic activity of EMAP-II. In the present study, we explored the molecular mechanism behind the anti-angiogenic activity exerted by this protein on ECs. Our results demonstrate that EMAP-II binds to the cell surface {alpha}5{beta}1 integrin receptor. The cell surface binding of EMAP-II results in its internalization into the cytoplasmic compartment where it interacts with its cytoplasmic partner PSMA7, a component of the proteasome degradation pathway. This interaction increases hypoxia-inducible factor 1-alpha (HIF-1{alpha}) degradation under hypoxic conditions. The degradation results in the inhibition of HIF-1{alpha} mediated transcriptional activity as well as HIF-1{alpha} mediated angiogenic sprouting of ECs. HIF-1{alpha} plays a critical role in angiogenesis by activating a variety of angiogenic growth factors. Our results suggest that one of the major anti-angiogenic functions of EMAP-II is exerted through its inhibition of the HIF-1{alpha} activities.

  4. New tools for human fat cell alpha-2A adrenoceptor characterization. Identification on membranes and on intact cells using the new antagonist (3H)RX821002

    SciTech Connect

    Galitzky, J.; Larrouy, D.; Berlan, M.; Lafontan, M. )

    1990-01-01

    The pharmacology of the alpha-2 adrenoceptor of the human adipocyte was improved by using some new alpha-2 antagonists from different chemical families (imidazolines, benzazepines and benzofuroquinolizines) in biological and binding assays. Moreover, investigations were also carried out to define the binding properties of a new imidazolinic antagonist, RX821002 (2-(2-methoxy-1,4-benzodioxan-2yl)-2-imidazoline), which could be a potential radioligand. (3H)RX821002 binding was very rapid and reversible. Saturation isotherms indicated that (3H)RX821002 labeled, with high affinity, a homogeneous population of noninteracting binding sites with a mean Kd of 0.98 +/- 0.05 nM (n = 6). The binding of (3H)RX821002 on the human fat cell alpha-2 adrenoceptor displayed a specificity which is strictly similar to that obtained with (3H)rauwolscine and which is classical for an alpha-2 A adrenoceptor. The binding parameters of (3H)RX821002 were compared with those obtained with the classical alpha-2 antagonist (3H)yohimbine. Analysis of the data indicate: (1) that (3H)RX821002 exhibited higher affinity; (2) that the nonspecific binding of (3H)RX821002 was very low; (3) that the total number of sites (maximum binding values) defined with (3H)RX821002 was significantly higher than that defined with (3H)yohimbine. This difference was not due to a specific preferential labeling of one of the two affinity states of the receptor, but suggested that (3H)yohimbine does not label the whole receptor population; (4) that (3H)RX821002 specific binding wa