[Trofosides A and B and other cytostatic steroid-derived compounds from the Far East starfish Trofodiscus über].

PubMed

Levina, E V; Kalinovskiĭ, A I; Andriiashchenko, P V; Menzorova, N I; Dmitrenok, P S

2007-01-01

Three new polar steroids identified as trofoside A, (20R,24S)-24-O-(3-O-methyl-beta-D-xylopyranosyl)-3beta,6alpha,8,15beta,24-pentahydroxy-5alpha-cholestane, its 22(23)-dehydro derivative (trofoside B), and 15-sulfoxy-(20R,24S)-5alpha-cholestane-3beta,6beta,8,15alpha,24-pentaol sodium salt, were isolated from Trofodiscus uber starfish extracts collected in the Sea of Okhotsk. Two known compounds, trofoside A aglycone, (20R,24S)-3beta,6alpha,8,15beta,24-pentahydroxy-5alpha-cholestane, and triseramide, (20R,24R,25S,22E)-24-methyl-3beta,6alpha,8,15beta-tetrahydroxy-5alpha-cholest-22-en-27-oic acid (2-sulfoethyl)amide sodium salt, were also found. The structures of the isolated polyoxysteroids were established from their spectra. Minimal concentrations causing degradation of unfertilized egg-cells of the sea-urchin Strongylocentrotus intermedius (C(min)) and terminating the cell division at the stage of the first division (C(min) embr.), as well as the concentrations causing 50% immobilization of sperm cells (ImC50) and inhibiting their ability to fertilize egg-cells by 50% (IC50) were determined for the isolated compounds. Of three compounds highly toxic in embryos and sea-urchin sperm cells, the polyol with a sulfo group in the steroid core was the most active; two glycosides with monosaccharide chains located at C3 and C24 atoms were less toxic. Note that all the compounds with the spermiotoxic activities differently affected the embryo development. The positions of monosaccharide residues in the core considerably influence the compound activity. For example, both mono- and double chained glycosides with the monosaccharide fragment at C3 and C24 atoms are active against sea-urchin sperm cells and embryos, whereas the C24 glycosylated trofoside A does not affect embryos and displays a poor spermiotoxicity.

Neomycin is a platelet-derived growth factor (PDGF) antagonist that allows discrimination of PDGF alpha- and beta-receptor signals in cells expressing both receptor types.

PubMed

Vassbotn, F S; Ostman, A; Siegbahn, A; Holmsen, H; Heldin, C H

1992-08-05

The aminoglycoside neomycin has recently been found to affect certain platelet-derived growth factor (PDGF) responses in C3H/10T1/2 C18 fibroblasts. Using porcine aortic endothelial cells transfected with PDGF alpha- or beta-receptors, we explored the possibility that neomycin interferes with the interaction between the different PDGF isoforms and their receptors. We found that neomycin (5 mM) inhibited the binding of 125I-PDGF-BB to the alpha-receptor with only partial effect on the binding of 125I-PDGF-AA; in contrast, the binding of 125I-PDGF-BB to the beta-receptor was not affected by the aminoglycoside. Scatchard analyses showed that neomycin (5 mM) decreased the number of binding sites for PDGF-BB on alpha-receptor-expressing cells by 87%. Together with cross-competition studies with 125I-labeled PDGF homodimers, the effect of neomycin indicates that PDGF-AA and PDGF-BB bind to both common and unique structures on the PDGF alpha-receptor. Neomycin specifically inhibited the autophosphorylation of the alpha-receptor by PDGF-BB, with less effect on the phosphorylation induced by PDGF-AA and no effect on the phosphorylation of the beta-receptor by PDGF-BB. Thus, neomycin is a PDGF isoform- and receptor-specific antagonist that provides a possibility to compare the signal transduction pathways of alpha- and beta-receptors in cells expressing both receptor types. This approach was used to show that activation of PDGF beta-receptors by PDGF-BB mediated a chemotactic response in human fibroblasts, whereas activation of alpha-receptors by the same ligand inhibited chemotaxis.

Antiapoptotic property of human alpha-synuclein in neuronal cell lines is associated with the inhibition of caspase-3 but not caspase-9 activity.

PubMed

Li, Wenxue; Lee, Michael K

2005-06-01

Abnormalities of alpha-synuclein (alpha-Syn) are mechanistically linked to Parkinson's disease (PD) and other alpha-synucleinopathies. To gain additional insights into the relationships between alpha-Syn expression and cell death, we examined the effects of expressing human alpha-Syn (Hualpha-Syn) variants on the cellular vulnerability to apoptotic stimuli. We show that the expression of wild-type (WT) and A30P mutant, but not A53T mutant, Hualpha-Syn leads to the protection of neuronal cell lines from apoptosis but not necrosis. Significantly, Hualpha-Syn did not protect non-neuronal cell lines from apoptosis. We also show that A53T mutant is a loss of function in regards to the antiapoptotic property since the expression of WT Hualpha-Syn with an excess of A53T mutant Hualpha-Syn leads to protection of the cells from apoptosis. The antiapoptotic property is specific to human alpha-Syn as neither beta-Syn nor mouse alpha-Syn protected cells from apoptosis, and the carboxy-terminal 20 amino acids are required for the antiapoptotic property. Analyses of capase-3 and caspase-9 activation reveal that the antiapoptotic property of Hualpha-Syn in neuronal cell lines is associated with the attenuation of caspase-3 activity without affecting the caspase-9 activity or the levels of cleaved, active caspase-3. We conclude that Hualpha-Syn modulates the activity of cleaved caspase-3 product in neuronal cell lines.

Studies on the synergistic effect of androgen on the stimulation of progestin secretion by FSH in cultured rat granulosa cells: progesterone metabolism and the effect of androgens.

PubMed

Nimrod, A

1977-09-01

Metabolic transformations of progesterone in cultures of granulosa cells from immature hypophysectomized rats treated with diethylstilbestrol were studied in relation to the synergistic action of exogenous androgen and FSH on progestin (progesterone and 20alpha-dihydroprogesterone) accumulation. Androstenedione (Ad; 10 ng/ml) enhanced the sensitivity of rat granulosa cells to this steroidogenic action of FSH, lowering the threshold of the response from greater than 4 ng/ml (FSH alone) to 0.8 ng/ml in the presence of Ad. A synergistic effect with FSH was also shown by various 5alpha-androstane derivatives. They were, however, less effective than the parent delta4-3 keto androstenes. Progesterone underwent extensive 5alpha-reduction during culture, leading to accumulation of endogenous 5alpha-pregnane compounds, and to transformation of labelled progesterone into 5 alpha-reduced radiometabolites. These compounds corresponded in gas-liquid and thin-layer chromatographic behaviour to 3alpha-hydroxy-5alpha-pregnan-20-one, 20alpha-hydroxy-5alpha-pregnan-3-one and 5alpha-pregnane-3alpha,20alpha-diol. The rate of 5alpha-reduction of progestins was not affected by the presence of exogenous Ad (1 microgram/ml), ruling out the possibility that the effect of androgen on progestin accumulation depends on competitive inhibition of 5alpha-reductase. An involvement of androgen of thecal origin in the enhancement of the sensitivity of the FSH-responsive mechanism in granulosa cells is suggested.

Genetic ablation of the alpha 6-integrin subunit in Tie1Cre mice enhances tumour angiogenesis.

PubMed

Germain, Mitchel; De Arcangelis, Adèle; Robinson, Stephen D; Baker, Marianne; Tavora, Bernardo; D'Amico, Gabriela; Silva, Rita; Kostourou, Vassiliki; Reynolds, Louise E; Watson, Alan; Jones, J Louise; Georges-Labouesse, Elisabeth; Hodivala-Dilke, Kairbaan

2010-02-01

Laminins are expressed highly in blood vessel basement membranes and have been implicated in angiogenesis. alpha6beta1- and alpha6beta4-integrins are major receptors for laminins in endothelial cells, but the precise role of endothelial alpha6-integrin in tumour angiogenesis is not clear. We show that blood vessels in human invasive ductal carcinoma of the breast have decreased expression of the alpha6-integrin-subunit when compared with normal breast tissue. These data suggest that a decrease in alpha6-integrin-subunit expression in endothelial cells is associated with tumour angiogenesis. To test whether the loss of the endothelial alpha6-integrin subunit affects tumour growth and angiogenesis, we generated alpha6fl/fl-Tie1Cre+ mice and showed that endothelial deletion of alpha6-integrin is sufficient to enhance tumour size and tumour angiogenesis in both murine B16F0 melanoma and Lewis cell lung carcinoma. Mechanistically, endothelial alpha6-integrin deficiency elevated significantly VEGF-mediated angiogenesis both in vivo and ex vivo. In particular, alpha6-integrin-deficient endothelial cells displayed increased levels of VEGF-receptor 2 (VEGFR2) and VEGF-mediated downstream ERK1/2 activation. By developing the first endothelial-specific alpha6-knockout mice, we show that the expression of the alpha6-integrin subunit in endothelial cells acts as a negative regulator of angiogenesis both in vivo and ex vivo. Copyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Lentiviral gene therapy of murine hematopoietic stem cells ameliorates the Pompe disease phenotype.

PubMed

van Til, Niek P; Stok, Merel; Aerts Kaya, Fatima S F; de Waard, Monique C; Farahbakhshian, Elnaz; Visser, Trudi P; Kroos, Marian A; Jacobs, Edwin H; Willart, Monique A; van der Wegen, Pascal; Scholte, Bob J; Lambrecht, Bart N; Duncker, Dirk J; van der Ploeg, Ans T; Reuser, Arnold J J; Verstegen, Monique M; Wagemaker, Gerard

2010-07-01

Pompe disease (acid alpha-glucosidase deficiency) is a lysosomal glycogen storage disorder characterized in its most severe early-onset form by rapidly progressive muscle weakness and mortality within the first year of life due to cardiac and respiratory failure. Enzyme replacement therapy prolongs the life of affected infants and supports the condition of older children and adults but entails lifelong treatment and can be counteracted by immune responses to the recombinant enzyme. We have explored the potential of lentiviral vector-mediated expression of human acid alpha-glucosidase in hematopoietic stem cells (HSCs) in a Pompe mouse model. After mild conditioning, transplantation of genetically engineered HSCs resulted in stable chimerism of approximately 35% hematopoietic cells that overexpress acid alpha-glucosidase and in major clearance of glycogen in heart, diaphragm, spleen, and liver. Cardiac remodeling was reversed, and respiratory function, skeletal muscle strength, and motor performance improved. Overexpression of acid alpha-glucosidase did not affect overall hematopoietic cell function and led to immune tolerance as shown by challenge with the human recombinant protein. On the basis of the prominent and sustained therapeutic efficacy without adverse events in mice we conclude that ex vivo HSC gene therapy is a treatment option worthwhile to pursue.

Wnt5 is required for notochord cell intercalation in the ascidian Halocynthia roretzi.

PubMed

Niwano, Tomoko; Takatori, Naohito; Kumano, Gaku; Nishida, Hiroki

2009-08-25

In the embryos of various animals, the body elongates after gastrulation by morphogenetic movements involving convergent extension. The Wnt/PCP (planar cell polarity) pathway plays roles in this process, particularly mediolateral polarization and intercalation of the embryonic cells. In ascidians, several factors in this pathway, including Wnt5, have been identified and found to be involved in the intercalation process of notochord cells. In the present study, the role of the Wnt5 genes, Hr-Wnt5alpha (Halocynthia roretzi Wnt5alpha) and Hr-Wnt5beta, in convergent extension was investigated in the ascidian H. roretzi by injecting antisense oligonucleotides and mRNAs into single precursor blastomeres of various tissues, including notochord, at the 64-cell stage. Hr-Wnt5alpha is expressed in developing notochord and was essential for notochord morphogenesis. Precise quantitative control of its expression level was crucial for proper cell intercalation. Overexpression of Wnt5 proteins in notochord and other tissues that surround the notochord indicated that Wnt5alpha plays a role within the notochord, and is unlikely to be the source of polarizing cues arising outside the notochord. Detailed mosaic analysis of the behaviour of individual notochord cells overexpressing Wnt5alpha indicated that a Wnt5alpha-manipulated cell does not affect the behaviour of neighbouring notochord cells, suggesting that Wnt5alpha works in a cell-autonomous manner. This is further supported by comparison of the results of Wnt5alpha and Dsh (Dishevelled) knockdown experiments. In addition, our results suggest that the Wnt/PCP pathway is also involved in mediolateral intercalation of cells of the ventral row of the nerve cord (floor plate) and the endodermal strand. The present study highlights the role of the Wnt5alpha signal in notochord convergent extension movements in ascidian embryos. Our results raise the novel possibility that Wnt5alpha functions in a cell-autonomous manner in activation of the Wnt/PCP pathway to polarize the protrusive activity that drives convergent extension.

E-cadherin interactions regulate beta-cell proliferation in islet-like structures.

PubMed

Carvell, Melanie J; Marsh, Phil J; Persaud, Shanta J; Jones, Peter M

2007-01-01

Islet function is dependent on cells within the islet interacting with each other. E-cadherin (ECAD) mediates Ca(2+)-dependent homophilic cell adhesion between b-cells within islets and has been identified as a tumour suppressor. We generated clones of the MIN6 beta-cell line that stably over- (S) and under-express (alphaS) ECAD. Modified expression of ECAD was confirmed by quantitative RT-PCR, immunoblotting and immunocytochemistry. Preproinsulin mRNA, insulin content and basal rates of insulin secretion were higher in S cells compared to aS and control (V) cells. However, stimulated insulin secretory responses were unaffected by ECAD expression levels. ECAD expression did affect proliferation, with enhanced ECAD expression being associated with reduced proliferation and vice versa. Formation of islet-like structures was associated with a significant reduction in proliferation of V and S cells but not alphaS cells. These data suggest that ECAD expression levels do not modulate insulin secretory function but are consistent with a role for ECAD in the regulation of beta-cell proliferation. Copyright (c) 2007 S. Karger AG, Basel.

The effects of thalidomide on the stimulation of NF-kappaB activity and TNF-alpha production by lipopolysaccharide in a human colonic epithelial cell line.

PubMed

Kim, You Sun; Kim, Joo Sung; Jung, Hyun Chae; Song, In Sung

2004-04-30

The immunomodulatory and anti-inflammatory effects of thalidomide are associated with inhibition of TNF-alpha levels. However, the mechanism by which thalidomide reduces TNF-alpha production remains elusive. NF-kappaB is known to play a central role in regulating inflammatory responses in patients with inflammatory bowel disease (IBD). We tested whether thalidomide acts through inhibiting NF-kappaB activity. HT-29 cells were stimulated with LPS (1 microg/ml) alone, or after pretreatment with thalidomide (100 microg/ml), and NF-kappaB activity was determined by gel mobility shift assays. RT-PCR was used to measure expression of the proinflammatory cytokine genes TNF-alpha, IL-1beta and IL-8. The level of TNF-alpha mRNA was also analyzed by real-time quantitative RT-PCR, and TNF-alpha protein was measured by ELISA. Thalidomide pretreatment did not affect NF-kappaB activity in HT-29 cells stimulated with LPS but production of TNF-alpha was depressed. Thalidomide was found to accelerate the degradation of TNF-alpha mRNA, but had little effect on IL-1beta or IL-8. These observations suggest that the immunomodulatory effect of thalidomide in colonic epithelial cells is associated with inhibition of TNF-alpha. However, it does not act by inhibiting NF-kappaB but rather by inducing degradation of TNF-alpha mRNA.

Rab-coupling protein coordinates recycling of alpha5beta1 integrin and EGFR1 to promote cell migration in 3D microenvironments.

PubMed

Caswell, Patrick T; Chan, May; Lindsay, Andrew J; McCaffrey, Mary W; Boettiger, David; Norman, Jim C

2008-10-06

Here we show that blocking the adhesive function of alphavbeta3 integrin with soluble RGD ligands, such as osteopontin or cilengitide, promoted association of Rab-coupling protein (RCP) with alpha5beta1 integrin and drove RCP-dependent recycling of alpha5beta1 to the plasma membrane and its mobilization to dynamic ruffling protrusions at the cell front. These RCP-driven changes in alpha5beta1 trafficking led to acquisition of rapid/random movement on two-dimensional substrates and to a marked increase in fibronectin-dependent migration of tumor cells into three-dimensional matrices. Recycling of alpha5beta1 integrin did not affect its regulation or ability to form adhesive bonds with substrate fibronectin. Instead, alpha5beta1 controlled the association of EGFR1 with RCP to promote the coordinate recycling of these two receptors. This modified signaling downstream of EGFR1 to increase its autophosphorylation and activation of the proinvasive kinase PKB/Akt. We conclude that RCP provides a scaffold that promotes the physical association and coordinate trafficking of alpha5beta1 and EGFR1 and that this drives migration of tumor cells into three-dimensional matrices.

Inhibitory function of adapter-related protein complex 2 alpha 1 subunit in the process of nuclear translocation of human immunodeficiency virus type 1 genome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kitagawa, Yukiko; Department of Oral and Maxillofacial Surgery II, Osaka University, Osaka 565-0871; Kameoka, Masanori

2008-03-30

The transfection of human cells with siRNA against adapter-related protein complex 2 alpha 1 subunit (AP2{alpha}) was revealed to significantly up-regulate the replication of human immunodeficiency virus type 1 (HIV-1). This effect was confirmed by cell infection with vesicular stomatitis virus G protein-pseudotyped HIV-1 as well as CXCR4-tropic and CCR5-tropic HIV-1. Viral adsorption, viral entry and reverse transcription processes were not affected by cell transfection with siRNA against AP2{alpha}. In contrast, viral nuclear translocation as well as the integration process was significantly up-regulated in cells transfected with siRNA against AP2{alpha}. Confocal fluorescence microscopy revealed that a subpopulation of AP2{alpha} wasmore » not only localized in the cytoplasm but was also partly co-localized with lamin B, importin {beta} and Nup153, implying that AP2{alpha} negatively regulates HIV-1 replication in the process of nuclear translocation of viral DNA in the cytoplasm or the perinuclear region. We propose that AP2{alpha} may be a novel target for disrupting HIV-1 replication in the early stage of the viral life cycle.« less

Effect of pro-inflammatory mediators on membrane-associated mucins expressed by human ocular surface epithelial cells.

PubMed

Albertsmeyer, Ann-Christin; Kakkassery, Vinodh; Spurr-Michaud, Sandra; Beeks, Olivia; Gipson, Ilene K

2010-03-01

Membrane-associated mucins are altered on the ocular surface in non-Sjögren's dry eye. This study sought to determine if inflammatory mediators, present in tears of dry eye patients, regulate membrane-associated mucins MUC1 and -16 at the level of gene expression, protein biosynthesis and/or ectodomain release. A human corneal limbal epithelial cell line (HCLE), which produces membrane-associated mucins, was used. Cells were treated with interleukin (IL)-6, -8, or -17, tumor necrosis factor-alpha (TNF-alpha), and Interferon-gamma (IFN-gamma), or a combination of TNF-alpha and IFN-gamma, or IFN-gamma and IL-17, for 1, 6, 24, or 48 h. Presence of receptors for these mediators was verified by RT-PCR. Effects of the cytokines on expression levels of MUC1 and -16 were determined by real-time PCR, and on mucin protein biosynthesis and ectodomain release in cell lysates and culture media, respectively, by immunoblot analysis. TNF-alpha and IFN-gamma each significantly induced MUC1 expression, cellular protein content and ectodomain release over time. Combined treatment with the two cytokines was not additive. By comparison, one of the inflammatory mediators, IFN-gamma, affected all three parameters-gene expression, cellular protein, and ectodomain release-for MUC16. Combined treatment with TNF-alpha and IFN-gamma showed effects similar to IFN-gamma alone, except that ectodomain release followed that of TNF-alpha, which induced MUC16 ectodomain release. In conclusion, inflammatory mediators present in tears of dry eye patients can affect MUC1 and -16 on corneal epithelial cells and may be responsible for alterations of surface mucins in dry eye.

Depletion of the cellular levels of Bag-1 proteins attenuates phorbol ester-induced downregulation of I{kappa}B{alpha} and nuclear accumulation of NF-{kappa}B

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maier, Jana V., E-mail: Jana.maier@kit.edu; Volz, Yvonne; Berger, Caroline

2010-10-22

Research highlights: {yields}Bag-1 depletion only marginally affects the action of the glucocorticoid receptor but strongly regulates the activity of NF-{kappa}B. {yields}Bag-1 depletion attenuates phosphorylation and degradation of I{kappa}B{alpha} and nuclear accumulation of NF-{kappa}B p65 and p50. {yields}Bag-1 interacts with I{kappa}B{alpha} and partially restores I{kappa}B{alpha} and NF-{kappa}B activation in Bag-1 depleted cells. -- Abstract: Bag-1 consists in humans of four isoforms generated from the same RNA by alternative translation. Overexpression of single Bag-1 isoforms has identified Bag-1 as a negative regulator of action of many proteins including the glucocorticoid receptor (GR). Here we have analysed the ability of Bag-1 to regulatemore » the transrepression function of the GR. Silencing Bag-1 expression only marginally affects the transrepression action of the GR but decreased the action of the transcription factor NF-{kappa}B. Furthermore phosphorylation and degradation of the inhibitor protein I{kappa}B{alpha} and nuclear accumulation of p65 and p50 NF-{kappa}B proteins in response to phorbol ester was attenuated following Bag-1 depletion in HeLa cells. Reconstitution of Bag-1 in depleted cells partially restored I{kappa}B{alpha} and NF-{kappa}B activation. Knock-down of Bag-1 expression also did not significantly alter GR-mediated transactivation but affected the basal transcription of some of the target genes. Thus Bag-1 proteins function as regulators of the action of selective transcription factors.« less

In vitro evaluation of the immunotoxic potential of perfluorinated compounds (PFCs)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Corsini, Emanuela, E-mail: emanuela.corsini@unimi.it; Avogadro, Anna; Galbiati, Valentina

2011-01-15

There is evidence from both epidemiology and laboratory studies that perfluorinated compounds may be immunotoxic, affecting both cell-mediated and humoral immunity. The overall goal of this study was to investigate the mechanisms underlying the immunotoxic effects of perfluorooctane sulfonate (PFOS) and perfluorooctane acid (PFOA), using in vitro assays. The release of the pro-inflammatory cytokines IL-6, IL-8, and TNF-{alpha} was evaluated in lipolysaccharide (LPS)-stimulated human peripheral blood leukocytes and in the human promyelocytic cell line THP-1, while the release of IL-4, IL-10 and IFN-{gamma} was evaluated in phytohaemagglutinin (PHA)-stimulated peripheral blood leukocytes. PFOA and PFOS suppressed LPS-induced TNF-{alpha} production in primarymore » human cultures and THP-1 cells, while IL-8 was suppressed only in THP-1 cells. IL-6 release was decreased only by PFOS. Both PFOA and PFOS decreased T-cell derived, PHA-induced IL-4 and IL-10 release, while IFN-{gamma} release was affected only by PFOS. In all instances, PFOS was more potent than PFOA. Mechanistic investigations carried out in THP-1 cells demonstrated that the effect on cytokine release was pre-transcriptional, as assessed by a reduction in LPS-induced TNF-{alpha} mRNA expression. Using siRNA, a role for PPAR-{alpha} could be demonstrated for PFOA-induced immunotoxicity, while an inhibitory effect on LPS-induced I-{kappa}B degradation could explain the immunomodulatory effect of PFOS. The dissimilar role of PPAR-{alpha} in PFOA and PFOS-induced immunotoxicity was consistent with the differing effects observed on LPS-induced MMP-9 release: PFOA, as the PPAR-{alpha} agonist fenofibrate, modulated the release, while PFOS had no effect. Overall, these studies suggest that PFCs directly suppress cytokine secretion by immune cells, and that PFOA and PFOS have different mechanisms of action.« less

Bovine alpha-lactalbumin stimulates mucus metabolism in gastric mucosa.

PubMed

Ushida, Y; Shimokawa, Y; Toida, T; Matsui, H; Takase, M

2007-02-01

Bovine alpha-lactalbumin (alpha-LA), a major milk protein, exerts strong gastroprotective activity against rat experimental gastric ulcers induced by ethanol or stress. To elucidate the mechanisms underlying this activity, the influence of alpha-LA on gastric mucus metabolism was investigated in vitro and in vivo. For the in vitro study, RGM1 cells (a rat gastric epithelial cell line) were selected for observation of the direct activity of alpha-LA on gastric mucosal cells and cultured in the presence of either alpha-LA or ovalbumin (OVA), a reference protein showing no gastroprotective activity. Amounts of synthesized and secreted mucin, a major component of mucus, were determined using [3H]glucosamine as a tracer, and prostaglandin E2 (PGE2) levels in the culture medium were determined by RIA. For the in vivo study, the thickness of the mucus gel layer, a protective barrier for gastric mucosa, was evaluated histochemically in rat gastric mucosa. alpha-Lactalbumin (3 mg/mL) significantly stimulated mucin synthesis and secretion in RGM1 cells and also increased PGE2 levels in the culture medium. In contrast, OVA showed no enhancing effects under identical conditions. Neither indomethacin, a cyclo-oxygenase inhibitor, nor AH23848, a prostaglandin EP4 receptor antagonist, affected alpha-LA-induced enhancement of mucin synthesis and secretion. In vivo, oral administration of alpha-LA (300 mg/kg x 3 times/d x 7 d) increased the thickness of the mucus gel layer in rats. These results indicate that alpha-LA fortifies the mucus gel layer by stimulating mucin production and secretion in gastric mucus-producing cells, and that this enhancing effect is independent of endogenous PGE2. Comparison of the efficacy of alpha-LA with OVA suggests that the activities observed in RGM1 cells are closely related to the gastroprotective effects in rat gastric ulcer models. In conclusion, alpha-LA stimulates mucus metabolism, and this action may be responsible for its gastroprotective activity.

Mode of alpha-amylase production by the shochu koji mold Aspergillus kawachii.

PubMed

Nagamine, Kazuki; Murashima, Kenji; Kato, Taku; Shimoi, Hitoshi; Ito, Kiyoshi

2003-10-01

Aspergillus kawachii produces two kinds of alpha-amylase, one is an acid-unstable alpha-amylase and the other is an acid-stable alpha-amylase. Because the quality of the shochu depends strongly on the activities of the alpha-amylases, the culture conditions under which these alpha-amylases are produced were examined. In liquid culture, acid-unstable alpha-amylase was produced abundantly, but, acid-stable alpha-amylase was not produced. The acid-unstable alpha-amylase was produced significantly when glycerol or glucose was used as a carbon source, similarly to the use of inducers such as starch or maltose. In liquid culture, A. kawachii assimilated starch at pH 3.0, but no alpha-amylase activity was recognized in the medium. Instead, the alpha-amylase was found to be trapped in the cell wall. The trapped form was identified as acid-unstable alpha-amylase. Usually, acid-unstable alpha-amylase is unstable at pH 3.0, so its stability appeared to be due to its immobilization in the cell wall. In solid-state culture, both kinds of alpha-amylase were produced. The production of acid-stable alpha-amylase seems to be solid-state culture-specific and was affected by the moisture content in the solid medium.

Selective suppression of cytokine secretion in whole blood cell cultures of patients with colorectal cancer.

PubMed Central

Lahm, H.; Schindel, M.; Frikart, L.; Cerottini, J. P.; Yilmaz, A.; Givel, J. C.; Fischer, J. R.

1998-01-01

We have investigated the secretion of interferon alpha (IFN-alpha), IFN-gamma, interleukin-1alpha (IL-1alpha), IL-1beta, IL-2 and tumour necrosis factor alpha (TNF-alpha) in whole blood cell cultures (WBCCs) of colorectal cancer patients upon mitogen stimulation. Whereas the values for IL-1beta and TNF-alpha remained virtually unchanged in comparison with healthy control subjects, WBCCs of colorectal cancer patients secreted significantly lower amounts of IFN-alpha (P < 0.005), IFN-gamma (P < 0.0001), IL-1alpha (P < 0.0001) and IL-2 (P < 0.05). This reduction correlated with the progression of the disease. The total leucocyte and monocyte population were almost identical in both groups. In contrast, a dramatic depletion of lymphocytes was observed in colorectal cancer patients, which affected both lymphocyte counts (P < 0.0005) and their distribution (P < 0.0001). Our results suggest a selective suppression of cytokines in colorectal cancer patients that is related to tumour burden. Several mechanisms might account for this phenomenon, one of which might be lymphocyte depletion. PMID:9792144

Activation of the alpha-globin gene expression correlates with dramatic upregulation of nearby non-globin genes and changes in local and large-scale chromatin spatial structure.

PubMed

Ulianov, Sergey V; Galitsyna, Aleksandra A; Flyamer, Ilya M; Golov, Arkadiy K; Khrameeva, Ekaterina E; Imakaev, Maxim V; Abdennur, Nezar A; Gelfand, Mikhail S; Gavrilov, Alexey A; Razin, Sergey V

2017-07-11

In homeotherms, the alpha-globin gene clusters are located within permanently open genome regions enriched in housekeeping genes. Terminal erythroid differentiation results in dramatic upregulation of alpha-globin genes making their expression comparable to the rRNA transcriptional output. Little is known about the influence of the erythroid-specific alpha-globin gene transcription outburst on adjacent, widely expressed genes and large-scale chromatin organization. Here, we have analyzed the total transcription output, the overall chromatin contact profile, and CTCF binding within the 2.7 Mb segment of chicken chromosome 14 harboring the alpha-globin gene cluster in cultured lymphoid cells and cultured erythroid cells before and after induction of terminal erythroid differentiation. We found that, similarly to mammalian genome, the chicken genomes is organized in TADs and compartments. Full activation of the alpha-globin gene transcription in differentiated erythroid cells is correlated with upregulation of several adjacent housekeeping genes and the emergence of abundant intergenic transcription. An extended chromosome region encompassing the alpha-globin cluster becomes significantly decompacted in differentiated erythroid cells, and depleted in CTCF binding and CTCF-anchored chromatin loops, while the sub-TAD harboring alpha-globin gene cluster and the upstream major regulatory element (MRE) becomes highly enriched with chromatin interactions as compared to lymphoid and proliferating erythroid cells. The alpha-globin gene domain and the neighboring loci reside within the A-like chromatin compartment in both lymphoid and erythroid cells and become further segregated from the upstream gene desert upon terminal erythroid differentiation. Our findings demonstrate that the effects of tissue-specific transcription activation are not restricted to the host genomic locus but affect the overall chromatin structure and transcriptional output of the encompassing topologically associating domain.

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

PubMed

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

2014-01-21

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). 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. 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 (1)H-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. 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 conventional drugs.

p38 Mitogen-Activated Protein Kinase in Metastasis Associated with Transforming Growth Factor Beta

DTIC Science & Technology

2006-06-01

Functional Analysis of cells • Expression of HA-tagged TβRI • Signaling (p-Smad2, p- HSP27 , p-p38) • Transcription (SBE-Lux, 3TP-Lux) • Proliferation...when they also affected lls expressing DN-p38alpha phosphorylation of HSP27 was effectively blocked (data not shown). However, cells e studies show...231-ALK5-T204D cells treated with kinase inhibitors U0126, SB202190, SP600125 (JNK). (B) Immunoblot of phospho-Smad2/3, phospho- HSP27 , and alpha

Alpha-Tocopherol modulates transcriptional activities that affect essential biological processes in Bovine Cells

USDA-ARS?s Scientific Manuscript database

Alpha-tocopherol is the major isoform of vitamin E. after nearly 100 years of research and countless publications, the physiological functions of vitamin E remain mysterious to a certain degree. Nevertheless, vitamin E is one of the most commonly used single nutrient supplements. Recent data has su...

Kruppel-like factor 2 inhibits hypoxia-inducible factor 1alpha expression and function in the endothelium.

PubMed

Kawanami, Daiji; Mahabeleshwar, Ganapati H; Lin, Zhiyong; Atkins, G Brandon; Hamik, Anne; Haldar, Saptarsi M; Maemura, Koji; Lamanna, Joseph C; Jain, Mukesh K

2009-07-31

Hypoxia-inducible factor 1 (HIF-1) is a central regulator of the hypoxic response in many cell types. In endothelial cells, HIF-1 induces the expression of key proangiogenic factors to promote angiogenesis. Recent studies have identified Kruppel-like factor 2 (KLF2) as a potent inhibitor of angiogenesis. However, the role of KLF2 in regulating HIF-1 expression and function has not been evaluated. KLF2 expression was induced acutely by hypoxia in endothelial cells. Adenoviral overexpression of KLF2 inhibited hypoxia-induced expression of HIF-1alpha and its target genes such as interleukin 8, angiopoietin-2, and vascular endothelial growth factor in endothelial cells. Conversely, knockdown of KLF2 increased expression of HIF-1alpha and its targets. Furthermore, KLF2 inhibited hypoxia-induced endothelial tube formation, whereas endothelial cells from mice with haploinsufficiency of KLF2 showed increased tube formation in response to hypoxia. Consistent with this ex vivo observation, KLF2 heterozygous mice showed increased microvessel density in the brain. Mechanistically, KLF2 promoted HIF-1alpha degradation in a von Hippel-Lindau protein-independent but proteasome-dependent manner. Finally, KLF2 disrupted the interaction between HIF-1alpha and its chaperone Hsp90, suggesting that KLF2 promotes degradation of HIF-1alpha by affecting its folding and maturation. These observations identify KLF2 as a novel inhibitor of HIF-1alpha expression and function. Therefore, KLF2 may be a target for modulating the angiogenic response in disease states.

Increased proliferation of endothelial cells with overexpression of soluble TNF-alpha receptor I gene.

PubMed

Sugano, Masahiro; Tsuchida, Keiko; Tomita, Hideharu; Makino, Naoki

2002-05-01

Vascular endothelial growth factor (VEGF) can overcome a potential anti-angiogenic effect of TNF-alpha by inhibiting endothelial apoptosis induced by this cytokine. Soluble TNF-alpha receptor I (sTNFRI) is an extracellular domain of TNFRI and antagonizes the activity of TNF-alpha. Here we report that sTNFRI is able to stimulate the growth of endothelial cells not by antagonizing TNF-alpha. Exogenously added recombinant human sTNFRI stimulated significantly more cell growth of human umbilical venous endothelial cells (HUVEC) with a low dose (50-200 pg/ml) compared with smooth muscle cells. In contrast, monoclonal antibody against TNF-alpha did not stimulate growth of human HUVEC. The sTNFRI expression plasmid (pcDNA3.1 plasmid) was introduced into the cell culture using OPTI-MEM, lipofectin and transferrin. Growth of HUVEC transfected with sTNFRI vector also increased significantly compared with those transfected with control vector. HUVEC transfected with sTNFRI vector increased the extracellular domain of TNFRI mRNA levels, but did not affect the intracellular domain of TNFRI mRNA levels. Accumulation of sTNFRI significantly increased in conditioned medium from HUVEC transfected with sTNFRI vector compared with those transfected with control vector. HUVEC transfected with sTNFRI vector not only increased sTNFRI but also prevented shedding of sTNFRI from TNFRI. The TNF-alpha -induced internucleosomic fragmentation was also significantly prevented in HUVEC transfected with sTNFRI vector compared with those transfected with control vector. These results suggest that instead of growth factors such as VEGF, local transfection of the sTNFRI gene may have potential therapeutic value in vascular diseases in which TNF-alpha is also usually highly expressed.

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

PubMed

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

2003-04-01

Localization of plasmin activity on leukocyte surfaces plays a critical role in fibrinolysis as well as in pathological and physiological processes in which cells must degrade the extracellular matrix in order to migrate. The binding of plasminogen to leukocytic cell lines induces a 30- to 80-fold increase in the rate of plasminogen activation by tissue-type (tPA) and urokinase-type (uPA) plasminogen activators. In the present study we have examined the role of alpha-enolase in plasminogen activation on the cell surface. We produced and characterized a monoclonal antibody (MAb) 11G1 against purified alpha-enolase, which abrogated about 90% of cell-dependent plasminogen activation by either uPA or tPA on leukocytoid cell lines of different lineages: B-lymphocytic, T-lymphocytic, granulocytic, and monocytic cells. In addition, MAb 11G1 also blocked enhancement of plasmin formation by peripheral blood neutrophils and monocytes. In contrast, MAb 11G1 did not affect plasmin generation in the presence of fibrin, indicating that this antibody did not interact with fibrinolytic components in the absence of cells. These data suggest that, although leukocytic cells display several molecules that bind plasminogen, alpha-enolase is responsible for the majority of the promotion of plasminogen activation on the surfaces of leukocytic cells. Copyright 2003 Wiley-Liss, Inc.

Transcriptional regulation of human retinoic acid receptor-alpha (RAR-{alpha}) by Wilms` tumour gene product

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goodyer, P.R.; Torban, E.; Dehbi, M.

1994-09-01

The Wilms` tumor gene encodes a 47-49 kDa transcription factor expressed in kidney, gonads and mesothelium during embryogenesis. Inherited mutations of WT1 lead to aberrant urogenital development and Wilms` tumor, but the role of WT1 in development is not fully understood. Since the human RAR-{alpha} gene contains a potential WT1 binding site at its 5{prime} end, we studied the effect of WT1 co-transfection on expression of an RAR-{alpha} promoter/CAT reporter construct in COS cells. COS cells were plated at 5X10{sup 5} cells/dish in DMEM with 10% FBS and transfected by the Ca/PO4 method with an expression plasmid containing the full-lengthmore » WT1 (-/-) cDNA under the control of the CMV promoter, plasmid containing the RAR-{alpha} promoter (-519 to +36)/CAT reporter and TK/growth hormone plasmid to control for efficiency of transfection. CAT/GH activity at 48 hours was inhibited by co-transfection with increasing amounts of WT1 (-/-); maximum inhibition = 5% of control. WT1 co-transfection did not affect expression of TKGH, nor of a CMV-CAT vector. Expression of WT1 protein in tranfected COS cells was demonstrated by Western blotting. Minimal inhibiton of RAR-{alpha}/CAT activity was seen when cells were co-transfected with vectors containing WT1 deletion mutants, alternate WT1 splicing variants, or WT1 (-/-) cDNA bearing a mutation identified in a patient with Drash syndrome. Gel shift assays indicated binding of WT1 to RAR-{alpha} cDNA but not to an RAR-{alpha} deletion mutant lacking the GCGGGGGGCG site. These observations suggest that WT1 may function to regulate RAR-{alpha} expression during normal development.« less

Selective inhibition of alpha1B-adrenergic receptor expression and function using a phosphorothioate antisense oligodeoxynucleotide.

PubMed

Gonzalez-Cabrera, P J; Iversen, P L; Liu, M F; Scofield, M A; Jeffries, W B

1998-06-01

To investigate alpha1B-adrenoceptor function, we developed a phosphorothioate antisense oligodeoxynucleotide (AO) to inhibit the expression of the alpha1B-adrenoceptor subtype in DDT1 MF2 cells. We measured the cellular uptake of the AO and its effect on alpha1B-adrenoceptor mRNA expression, protein density, and coupling to phospholipase C. Cells treated with either a control oligodeoxynucleotide (CO) or medium alone served as control groups. Confocal microscopy demonstrated that DDT1 MF2 cells internalized carboxyfluorescein-labeled (FAM) AO within 30 min. Analysis of cellular lysates showed that approximately 50% of the intracellular FAM-AO was present as an intact 18-mer for up to 48 hr. Incubation of cells with AO for 48 hr decreased alpha1B-adrenoceptor density ([3H]prazosin Bmax) versus control groups by 12% (1 microM AO) and 72% (10 microM AO). In time course experiments, AO (10 microM) reduced alpha1B-adrenoceptor density by 28, 64, and 68% versus controls after 24, 48, and 72 hr of exposure, respectively. alpha1B-Adrenoceptor mRNA concentration (measured by RT-PCR) was reduced by 25% in cells treated for 48 hr with 10 microM AO versus controls. AO pretreatment (10 microM, 48 hr) reduced the maximum response to agonist-stimulated [3H]inositol phosphate accumulation. The maximal response of the full agonist norepinephrine was reduced by 30% after AO treatment, and by 73% for the partial agonist naphazoline. In contrast, AO did not affect histamine-stimulated total [3H]inositol phosphate accumulation. Thus, AO effectively reduced alpha1B-adrenoceptor subtype expression and function in vitro, suggesting a potential to selectively inhibit alpha1B-adrenoceptor function in vivo.

Phthalocyanine tetrasulfonates affect the amyloid formation and cytotoxicity of alpha-synuclein.

PubMed

Lee, Eui-Nam; Cho, Hyun-Ju; Lee, Choong-Hwan; Lee, Daekyun; Chung, Kwang Chul; Paik, Seung R

2004-03-30

Alpha-synuclein is a pathological component of Parkinson's disease by constituting the filamentous component of Lewy bodies. Phthalocyanine (Pc) effects on the amyloidosis of alpha-synuclein have been examined. The copper complex of phthalocyanine tetrasulfonate (PcTS-Cu(2+)) caused the self-oligomerization of alpha-synuclein while Pc-Cu(2+) did not affect the protein, indicating that introduction of the sulfonate groups was critical for the selective protein interaction. The PcTS-Cu(2+) interaction with alpha-synuclein has occurred predominantly at the N-terminal region of the protein with a K(d) of 0.83 microM apart from the hydrophobic NAC (non-Abeta component of Alzheimer's disease amyloid) segment. Phthalocyanine tetrasulfonate (PcTS) lacking the intercalated copper ion also showed a considerable affinity toward alpha-synuclein with a K(d) of 3.12 microM, and its binding site, on the other hand, was located at the acidic C-terminus. These mutually exclusive interactions between PcTS and PcTS-Cu(2+) toward alpha-synuclein resulted in distinctive features on the kinetics of protein aggregation, morphologies of the final aggregates, and their in vitro cytotoxicities. The PcTS actually suppressed the fibrous amyloid formation of alpha-synuclein, but it produced the chopped-wood-looking protein aggregates. The aggregates showed rather low toxicity (9.5%) on human neuroblastoma cells (SH-SY5Y). In fact, the PcTS was shown to effectively rescue the cell death of alpha-synuclein overexpressing cells caused by the lactacystin treatment as a proteasome inhibitor. The anti-aggregative and anti-amyloidogenic properties of PcTS were also demonstrated with alcohol dehydrogenase, glutathione S-transferase, and amyloid beta/A4 protein under their aggregative conditions. The PcTS-Cu(2+), on the other hand, promoted the protein aggregation of alpha-synuclein, which gave rise to the fibrillar protein aggregates whose cytotoxicity became significant to 35.8%. Taken together, the data provided in this study indicate that PcTS/PcTS-Cu(2+) could be considered as possible candidates for the development of therapeutic or prophylactic strategies against the alpha-synuclein-related neurodegenerative disorders.

Histaminergic regulation of natural killer cell-mediated clearance of tumour cells in mice.

PubMed

Asea, A; Hermodsson, S; Hellstrand, K

1996-01-01

Treatment of Swiss albino mice with histamine enhanced the clearance of natural killer (NK)-cell sensitive YAC-1 lymphoma and B16/F10 melanoma cells from lung tissue in vivo, but did not affect the elimination of NK-cell-insensitive P815 mastocytoma cells. The effect of histamine was apparently mediated by H2-type histamine receptors (H2R) since it was blocked by ranitidine, and H2R antagonist. Histamine did not affect clearance of tumour cells in animals depleted of NK cells in vivo by treatment with antibodies to asialo-GM1 or NK1.1. The effect of histamine was time-dependent: pretreatment with histamine for 3 h significantly augmented the clearance of YAC-1 cells, whereas, pretreatment with histamine for 5 min was ineffective. Histamine potentiated the anti-tumour properties of NK-cell activators such as interleukin-2 (IL-2) or interferon-alpha (IFN-alpha) in vivo. None of these lymphokines significantly affected the clearance of YAC-1 cells unless animals were concomitantly treated with histamine. Treatment with ranitidine alone reduced the in vivo clearance of YAC-1 cells from lungs but did not affect the clearance of NK-cell-insensitive P815 cells. Effects of ranitidine on NK-cell function in vivo were not shared by a chemical control to ranitidine, AH20239AA, thus indicating that the inhibition of NK-cells results from H2R antagonism rather than non-specific toxicity. It is concluded that histaminergic mechanisms may be involved in the regulation of NK cell function in vivo.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Papaconstantinou, J.; Stewart, J.A.; Rabek, J.P.

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 thatmore » 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.« less

An activating G{sub s}{alpha} mutation is present in fibrous dysplasia of bone in the McCune-Albright syndrome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shenker, A.; Weinstein, L.S.; Spiegel, A.M.

1994-09-01

McCune-Albright syndrome (MAS) is a sporadic disease characterized by polyostotic fibrous dysplasia, cafe-au-lait spots, and multiple endocrinopathies. The etiology of fibrous dysplasia is unknown. Activating mutations of codon 201 in the gene encoding the {alpha}-subunit of G{sub s}, the G-protein that stimulates adenylyl cyclase, have been found in all affected MAS tissues that have been studied. Initial attempts to amplify DNA from decalcified paraffin-embedded bone specimens from frozen surgical bone specimens from five MAS patients using polymerase chain reaction and allele-specific oligonucleotide hybridization. Most of the cells in four specimens of dysplastic bone contained a heterozygous mutation encoding substitution ofmore » Arg{sup 201} of G{sub s}{alpha} with His, but the mutation was barely detectable in peripheral blood specimens from the patients. Only a small amount of mutant allele was detected in a specimen of normal cortical bone from the fifth patient, although this patients had a high proportion of mutation in other, affected tissues. The mosaic distribution of mutant alleles is consistent with an embryological somatic cell mutation of the G{sub s}{alpha} gene in MAS. The presence of an activating mutation of G{sub s}{alpha} in osteoblastic progenitor cells may cause them to exhibit increased proliferation and abnormal differentiation, thereby producing the lesions of fibrous dysplasia. 43 refs., 2 figs.« less

Role of a guanine nucleotide-binding protein in. cap alpha. /sub 1/-adrenergic receptor-mediated Ca/sup 2 +/ mobilization in DDT/sub 1/ MF-2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cornett, L.E.; Norris, J.S.

1987-11-01

In this study the mechanisms involved in ..cap alpha../sub 1/-adrenergic receptor-mediated Ca/sup 2 +/ mobilization at the level of the plasma membrane were investigated. Stimulation of /sup 45/Ca/sup 2 +/ efflux from saponin-permeabilized DDT/sub 1/ MF-2 cells was observed with the addition of either the ..cap alpha../sub 1/-adrenergic agonist phenylephrine and guanosine-5'-triphosphate or the nonhydrolyzable guanine nucleotide guanylyl-imidodiphosphate. In the presence of (/sup 32/P) NAD, pertussis toxin was found to catalyze ADP-ribosylation of a M/sub r/ = 40,500 (n = 8) peptide in membranes prepared from DDT/sub 1/, MF-2 cells, possibly the ..cap alpha..-subunit of N/sub i/. However, stimulation ofmore » unidirectional /sup 45/Ca/sup 2 +/ efflux by phenylephrine was not affected by previous treatment of cells with 100 ng/ml pertussis toxin. These data suggest that the putative guanine nucleotide-binding protein which couples the ..cap alpha../sub 1/-adrenergic receptor to Ca/sup 2 +/ mobilization in DDT/sub 1/ MF-2 cells is not a pertussis toxin substrate and may possibly be an additional member of guanine nucleotide binding protein family.« less

Interactions between integrin receptors and fibronectin are required for calvarial osteoblast differentiation in vitro

NASA Technical Reports Server (NTRS)

Moursi, A. M.; Globus, R. K.; Damsky, C. H.

1997-01-01

We previously showed that anti-fibronectin antibodies or soluble fibronectin fragments containing the central cell-binding domain inhibit formation of mineralized nodules by fetal calvarial osteoblasts in vitro. These findings suggest a critical role for fibronectin in osteoblast differentiation and morphogenesis. In this study we tested the hypothesis that fibronectin's effects on osteogenesis are mediated via direct interactions with integrin receptors for fibronectin on osteoblasts. Immunocytochemical analysis identified the integrin fibronectin receptor alpha5ss1 in fetal rat calvarial tissue and in cultured osteoblasts at all stages of differentiation. Three other integrins, alpha3ss1, alpha8ss1 and alphavss3, which can bind fibronectin, as well as other matrix components, were also identified in tissue and at all stages of cell culture. Immunoprecipitation data showed that alpha5ss1 levels are constant throughout osteoblast differentiation whereas levels of alpha3ss1 and alpha8ss1 decline in mature mineralized cultures. To determine whether integrin fibronectin receptors are required for osteoblast formation of mineralized nodules, we examined the extent of nodule formation in the presence and absence of function-perturbing anti-integrin antibodies. The antibodies were present continuously in cultures beginning at confluence (day 3), and nodule formation was measured at days 10 and 20. An anti-alpha5 integrin subunit antibody reduced nodule formation to less than 5% of control values at both time points. Inhibition of nodule formation was reversible and did not affect cell attachment and viability. Function-perturbing antibodies against alpha3ss1 and alpha8ss1 also reduced nodule formation, to less than 20% of control values. In contrast, function-perturbing antibodies to alphavss3 and alphavss5 did not affect nodule formation, indicating that the inhibitions noted were indeed specific. To determine the effect of antibody treatment on gene expression, steady-state mRNA expression was examined and found to be suppressed for osteoblast markers alkaline phosphatase and osteocalcin. Together, these results indicate that direct osteoblast interactions with the extracellular matrix are mediated by a select group of integrin receptors that includes alpha5ss1, alpha3ss1 and alpha8ss1. We further conclude that the specific alpha5ss1 fibronectin receptor mediates critical interactions between osteoblasts and fibronectin required for both bone morphogenesis and osteoblast differentiation.

Dissecting the role of matrix metalloproteinases (MMP) and integrin alpha(v)beta3 in angiogenesis in vitro: absence of hemopexin C domain bioactivity, but membrane-Type 1-MMP and alpha(v)beta3 are critical.

PubMed

Nisato, Riccardo E; Hosseini, Ghamartaj; Sirrenberg, Christian; Butler, Georgina S; Crabbe, Thomas; Docherty, Andrew J P; Wiesner, Matthias; Murphy, Gillian; Overall, Christopher M; Goodman, Simon L; Pepper, Michael S

2005-10-15

Matrix metalloproteinase (MMP)-2 and its hemopexin C domain autolytic fragment (also called PEX) have been proposed to be crucial for angiogenesis. Here, we have investigated the dependency of in vitro angiogenesis on MMP-mediated extracellular proteolysis and integrin alpha(v)beta3-mediated cell adhesion in a three-dimensional collagen I model. The hydroxamate-based synthetic inhibitors BB94, CT1399, and CT1847 inhibited endothelial cell invasion, as did neutralizing anti-membrane-type 1-MMP (MT1-MMP) antibodies and tissue inhibitor of MMP (TIMP)-2 and TIMP-3 but not TIMP-1. This confirmed the pivotal importance of MT1-MMP over other MMPs in this model. Invasion was also inhibited by a nonpeptidic antagonist of integrin alpha(v)beta3, EMD 361276. Although PEX strongly inhibited pro-MMP-2 activation, when contaminating lipopolysaccharide was neutralized, PEX neither affected angiogenesis nor bound integrin alpha(v)beta(3). Moreover, no specific binding of pro-MMP-2 to integrin alpha(v)beta3 was found, whereas only one out of four independently prepared enzymatically active MMP-2 preparations could bind integrin alpha(v)beta3 , and this in a PEX-independent manner. Likewise, integrin alpha(v)beta3 -expressing cells did not bind MMP-2-coated surfaces. Hence, these findings show that endothelial cell invasion of collagen I gels is MT1-MMP and alpha(v)beta3 - dependent but MMP-2 independent and does not support a role for PEX in alpha(v)beta3 integrin binding or in modulating angiogenesis in this system.

The Val192Leu mutation in the alpha-subunit of beta-hexosaminidase A is not associated with the B1-variant form of Tay-Sachs disease.

PubMed Central

Hou, Y.; Vavougios, G.; Hinek, A.; Wu, K. K.; Hechtman, P.; Kaplan, F.; Mahuran, D. J.

1996-01-01

Substitution mutations adversely affecting the alpha-subunit of beta-hexosaminidase A (alphabeta) (EC 3.2.1.52) result in Tay-Sachs disease. The majority affect the initial folding of the pro-alpha chain in the endoplasmic reticulum, resulting in its retention and degradation. A much less common occurrence is a mutation that specifically affects an "active-site" residue necessary for substrate binding and/or catalysis. In this case, hexosaminidase A is present in the lysosome, but it lacks all alpha-specific activity. This biochemical phenotype is referred to as the "B1-variant form" of Tay-Sachs disease. Kinetic analysis of suspected B1-variant mutations is complex because hexosaminidase A is heterodimeric and both subunits possess similar active sites. In this report, we examine a previously identified B1-variant mutation, alpha-Val192Leu. Chinese hamster ovary cells were permanently cotransfected with an alpha-cDNA-construct encoding the substitution and a mutant beta-cDNA (beta-Arg211Lys), encoding a beta-subunit that is inactive but normal in all other respects. We were surprised to find that the Val192Leu substitution, produced a pro-alpha chain that did not form alpha-beta dimers and was not transported to the lysosome. Finally, we reexamined the hexosaminidase activity and protein levels in the fibroblasts from the original patient. These data were also not consistent with the biochemical phenotype of the B1 variant of Tay-Sachs disease previously reported to be present. Thus, we conclude that the Val192Leu substitution does not specifically affect the alpha-active site. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8659543

The Val192Leu mutation in the alpha-subunit of beta-hexosaminidase A is not associated with the B1-variant form of Tay-Sachs disease.

PubMed

Hou, Y; Vavougios, G; Hinek, A; Wu, K K; Hechtman, P; Kaplan, F; Mahuran, D J

1996-07-01

Substitution mutations adversely affecting the alpha-subunit of beta-hexosaminidase A (alphabeta) (EC 3.2.1.52) result in Tay-Sachs disease. The majority affect the initial folding of the pro-alpha chain in the endoplasmic reticulum, resulting in its retention and degradation. A much less common occurrence is a mutation that specifically affects an "active-site" residue necessary for substrate binding and/or catalysis. In this case, hexosaminidase A is present in the lysosome, but it lacks all alpha-specific activity. This biochemical phenotype is referred to as the "B1-variant form" of Tay-Sachs disease. Kinetic analysis of suspected B1-variant mutations is complex because hexosaminidase A is heterodimeric and both subunits possess similar active sites. In this report, we examine a previously identified B1-variant mutation, alpha-Val192Leu. Chinese hamster ovary cells were permanently cotransfected with an alpha-cDNA-construct encoding the substitution and a mutant beta-cDNA (beta-Arg211Lys), encoding a beta-subunit that is inactive but normal in all other respects. We were surprised to find that the Val192Leu substitution, produced a pro-alpha chain that did not form alpha-beta dimers and was not transported to the lysosome. Finally, we reexamined the hexosaminidase activity and protein levels in the fibroblasts from the original patient. These data were also not consistent with the biochemical phenotype of the B1 variant of Tay-Sachs disease previously reported to be present. Thus, we conclude that the Val192Leu substitution does not specifically affect the alpha-active site.

[Gastroduodenal mucosa sensitivity to estrogen in ulcers complicated by hemorrhage].

PubMed

Duzhiy, I D; Romanyuk, A M; Kharchenko, S V; Moskalenko, R A; Pyatykop, G I; Lyndin, M S

2015-02-01

Expression of alpha-receptors of estrogen (RE) in accordance to immunohistochemical (IHC) labeling in gastroduodenal mucosa cells was studied up in patients, suffering the ulcer disease and without it. In 4 patients (group I) a gastroduodenal mucosa affection was revealed, they were operated on for hemorrhage from gastroduodenal ulcers; in 3 patients (group II) gastroduodenal mucosa affection was not observed; in 4 patients (group III, control), a mammary gland cancer was diagnosed, a positive reaction on alpha-RE was noted. In groups I and II the biopsies were studied, obtained from pylorus and gastric fundus, as well as from duodenal ampula, and in a group III--obtained from the tumor. In a control group a positive labeling of nuclei was revealed in biopsies. In patients of groups I and II the alpha-RE expression by cellular nuclei was not revealed, but, the lots of positive IHC labeling of cytoplasm in glandular and stromal mucosal cells of the investigated gut were noted. Positive IHC labeling of cytoplasm for alpha-RE witnesses about sensitivity to them in norma and pathological processes. But, a trustworthy difference of alpha-RE expression by cellular nuclei was not noted. For confirmation or denial of this hypothesis further clinical and IHC investigations are needed.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dehne, Nathalie, E-mail: dehne@biochem.uni-frankfurt.de; Hintereder, Gudrun, E-mail: Gudrun.Hintereder@kgu.de; Bruene, Bernhard, E-mail: bruene@pathobiochemie1.de

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.more » 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.« less

A synthetic peptide derived from alpha-fetoprotein inhibits the estradiol-induced proliferation of mammary tumor cells in culture through the modulation of p21.

PubMed

Sierralta, Walter D; Epuñan, María J; Reyes, José M; Valladares, Luis E; Pino, Ana M

2008-01-01

A stable cyclized 9-mer peptide (cP) containing the active site of alpha-alpha fetoprotein (alphaFP) has been shown to be effective for prevention of estrogen-stimulated tumor cell proliferation in culture or of xenographt growth in immunodeficient mice. cP does not block 17beta-estradiol (E2) binding to its receptors, but rather appears to interfere with intracellular processing of the signal that supports growth. To obtain insight on that mechanism we studied the effect of cP on the proliferation of MCF-7 cells in culture. Proliferation in the presence of 2 microM E2 is decreased up to 40% upon addition of 2 microg ml(-1) cP to the medium; the presence of cP did not increase cell death, cP reduced also the proliferation of estrogen-dependent ZR75-1 cells but had no effect on autonomous MDA-MB-231 cells, cP did not modify the number of binding sites for labeled E2 or affected cell death. We detected increased nuclear p21Cip1 immunoreactivity after cP treatment. Our results suggest that cP acts via p21Cip1 to slow the process of MCF-7 cells through the cycle.

Gene expression profiles in chondrosarcoma cells subjected to cyclic stretching and hydrostatic pressure. A cDNA array study.

PubMed

Karjalainen, Hannu M; Sironen, Reijo K; Elo, Mika A; Kaarniranta, Kai; Takigawa, Masaharu; Helminen, Heikki J; Lammi, Mikko J

2003-01-01

Mechanical forces have a profound effect on cartilage tissue and chondrocyte metabolism. Strenuous loading inhibits the cellular metabolism, while optimal level of loading at correct frequency raises an anabolic response in chondrocytes. In this study, we used Atlas Human Cancer cDNA array to investigate mRNA expression profiles in human chondrosarcoma cells stretched 8% for 6 hours at a frequency of 0.5 Hz. In addition, cultures were exposed to continuous and cyclic (0.5 Hz) 5 MPa hydrostatic pressure. Cyclic stretch had a more profound effect on the gene expression profiles than 5 MPa hydrostatic pressure. Several genes involved with the regulation of cell cycle were increased in stretched cells, as well as mRNAs for PDGF-B, glucose-1-phosphate uridylyltransferase, Tiam1, cdc37 homolog, Gem, integrin alpha6, and matrix metalloproteinase-3. Among down-regulated genes were plakoglobin, TGF-alpha, retinoic acid receptor-alpha and Wnt8b. A smaller number of changes was detected after pressure treatments. Plakoglobin was increased under cyclic and continuous 5 MPa hydrostatic pressure, while mitogen-activated protein kinase-9, proliferating cell nuclear antigen, Rad6, CD9 antigen, integrins alphaE and beta8, and vimentin were decreased. Cyclic and continuous pressurization induces a number of specific changes. In conclusion, a different set of genes were affected by three different types of mechanical stimuli applied on chondrosarcoma cells.

{beta}-Catenin regulates airway smooth muscle contraction.

PubMed

Jansen, Sepp R; Van Ziel, Anna M; Baarsma, Hoeke A; Gosens, Reinoud

2010-08-01

beta-Catenin is an 88-kDa member of the armadillo family of proteins that is associated with the cadherin-catenin complex in the plasma membrane. This complex interacts dynamically with the actin cytoskeleton to stabilize adherens junctions, which play a central role in force transmission by smooth muscle cells. Therefore, in the present study, we hypothesized a role for beta-catenin in the regulation of smooth muscle force production. beta-Catenin colocalized with smooth muscle alpha-actin (sm-alpha-actin) and N-cadherin in plasma membrane fractions and coimmunoprecipitated with sm-alpha-actin and N-cadherin in lysates of bovine tracheal smooth muscle (BTSM) strips. Moreover, immunocytochemistry of cultured BTSM cells revealed clear and specific colocalization of sm-alpha-actin and beta-catenin at the sites of cell-cell contact. Treatment of BTSM strips with the pharmacological beta-catenin/T cell factor-4 (TCF4) inhibitor PKF115-584 (100 nM) reduced beta-catenin expression in BTSM whole tissue lysates and in plasma membrane fractions and reduced maximal KCl- and methacholine-induced force production. These changes in force production were not accompanied by changes in the expression of sm-alpha-actin or sm-myosin heavy chain (MHC). Likewise, small interfering RNA (siRNA) knockdown of beta-catenin in BTSM strips reduced beta-catenin expression and attenuated maximal KCl- and methacholine-induced contractions without affecting sm-alpha-actin or sm-MHC expression. Conversely, pharmacological (SB-216763, LiCl) or insulin-induced inhibition of glycogen synthase kinase-3 (GSK-3) enhanced the expression of beta-catenin and augmented maximal KCl- and methacholine-induced contractions. We conclude that beta-catenin is a plasma membrane-associated protein in airway smooth muscle that regulates active tension development, presumably by stabilizing cell-cell contacts and thereby supporting force transmission between neighboring cells.

Thalidomide inhibits inflammatory and angiogenic activation of human intestinal microvascular endothelial cells (HIMEC).

PubMed

Rafiee, Parvaneh; Stein, Daniel J; Nelson, Victoria M; Otterson, Mary F; Shaker, Reza; Binion, David G

2010-02-01

The glutamic acid derivative thalidomide is a transcriptional inhibitor of TNF-alpha but is also known to affect human blood vessels, which may underlie its teratogenicity. Thalidomide has been used in the treatment of refractory Crohn's disease (CD), but the therapeutic mechanism is not defined. We examined the effect of thalidomide on primary cultures of human intestinal microvascular endothelial cells (HIMEC), the relevant endothelial cell population in inflammatory bowel disease (IBD), to determine its effect on endothelial activation, leukocyte interaction, and VEGF-induced angiogenesis. HIMEC cultures were pretreated with thalidomide before activation with either TNF-alpha/LPS or VEGF. A low-shear-stress flow adhesion assay with either U-937 or whole blood was used to assess HIMEC activation following TNF-alpha/LPS, and a Wright's stain identified adherent leukocytes. Expression of cell adhesion molecules (E-selectin, intercellular adhesion molecule-1, vascular cell adhesion molecule-1) was assessed using radioimmunoassay. Effects of thalidomide on NF-kappaB activation, cyclooxygenase (COX)-2, and inducible nitric oxide synthase (iNOS) expression in TNF-alpha/LPS-activated HIMEC were determined by RT-PCR and Western blotting. Thalidomide blocked adhesion of both U-937 and whole blood leukocytes by 50% in HIMEC, inhibiting binding of all classes of leukocytes. Thalidomide also blocked NF-kappaB and cell adhesion molecule expression in HIMEC. In marked contrast, thalidomide did not affect either iNOS or COX-2 expression, two key molecules that play a role in the downregulation of HIMEC activation. VEGF-induced HIMEC transmigration, growth, proliferation, tube formation, and Akt phosphorylation were significantly inhibited by thalidomide. In summary, thalidomide exerted a potent effect on HIMEC growth and activation, suggesting that it may also function via an endothelial mechanism in the treatment of CD.

Thalidomide inhibits lipopolysaccharide-induced tumor necrosis factor-alpha production via down-regulation of MyD88 expression.

PubMed

Noman, Abu Shadat M; Koide, Naoki; Hassan, Ferdaus; I-E-Khuda, Imtiaz; Dagvadorj, Jargalsaikhan; Tumurkhuu, Gantsetseg; Islam, Shamima; Naiki, Yoshikazu; Yoshida, Tomoaki; Yokochi, Takashi

2009-02-01

The effect of thalidomide on lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-alpha production was studied by using RAW 264.7 murine macrophage-like cells. Thalidomide significantly inhibited LPS-induced TNF-alpha production. Thalidomide prevented the activation of nuclear factor (NF)-KB by down-regulating phosphorylation of inhibitory KB factor (IKB), and IKB kinase (IKK)-alpha and IKK-beta Moreover, thalidomide inhibited LPS-induced phosphorylation of AKT, p38 and stress-activated protein kinase (SAPK)/JNK. The expression of myeloid differentiation factor 88 (MyD88) protein and mRNA was markedly reduced in thalidomide-treated RAW 264.7 cells but there was no significant alteration in the expression of interleukin-1 receptor-associated kinase (IRAK) 1 and TNF receptor-associated factor (TRAF) 6 in the cells. Thalidomide did not affect the cell surface expression of Toll-like receptor (TLR) 4 and CD14, suggesting the impairment of intracellular LPS signalling in thalidomide-treated RAW 264.7 cells. Thalidomide significantly inhibited the TNF-alpha production in response to palmitoyl-Cys(RS)-2,3-di(palmitoyloxy) propyl)-Ala-Gly-OH (Pam(3)Cys) as a MyD88-dependent TLR2 ligand. Therefore, it is suggested that thalidomide might impair LPS signalling via down-regulation of MyD88 protein and mRNA and inhibit LPS-induced TNF-alpha production. The putative mechanism of thalidomide-induced MyD88 down-regulation is discussed.

Proteomic profiling of bone marrow mesenchymal stem cells upon TGF-beta stimulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Daojing; Park, Jennifer S.; Chu, Julia S.F.

Bone marrow mesenchymal stem cells (MSCs) can differentiate into different types of cells, and have tremendous potential for cell therapy and tissue engineering. Transforming growth factor {beta}1 (TGF-{beta}) plays an important role in cell differentiation and vascular remodeling. We showed that TGF-{beta} induced cell morphology change and an increase in actin fibers in MSCs. To determine the global effects of TGF-{beta} on MSCs, we employed a proteomic strategy to analyze the effect of TGF-{beta} on the human MSC proteome. By using two-dimensional gel electrophoresis and electrospray ionization coupled to Quadrupole/time-of-flight tandem mass spectrometers, we have generated a proteome reference mapmore » of MSCs, and identified {approx}30 proteins with an increase or decrease in expression or phosphorylation in response to TGF-{beta}. The proteins regulated by TGF-{beta} included cytoskeletal proteins, matrix synthesis proteins, membrane proteins, metabolic enzymes, etc. TGF-{beta} increased the expression of smooth muscle (SM) {alpha}-actin and decreased the expression of gelsolin. Over-expression of gelsolin inhibited TGF-{beta}-induced assembly of SM {alpha}-actin; on the other hand, knocking down gelsolin expression enhanced the assembly of {alpha}-actin and actin filaments without significantly affecting {alpha}-actin expression. These results suggest that TGF-{beta} coordinates the increase of {alpha}-actin and the decrease of gelsolin to promote MSC differentiation. This study demonstrates that proteomic tools are valuable in studying stem cell differentiation and elucidating the underlying molecular mechanisms.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Meiqing; Ye, Zhenmin; Umeshappa, Keshav Sokke

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 recipientsmore » 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.« less

The Val{sup 192}Leu mutation in the {alpha}-subunit of {beta}-hexosaminidase A is not associated with the B1-variant form of Tay-Sachs disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hou, Y.; Vavougios, G.; Hinek, A.

1996-07-01

Substitution mutations adversely affecting the {alpha}-subunit of {beta}-hexosaminidase A ({alpha}{beta}) (EC 3.2.1.52) result in Tay-Sachs disease. The majority affect the initial folding of the pro-{alpha} chain in the endoplasmic reticulum, resulting in its retention and degradation. A much less common occurrence is a mutation that specifically affects an {open_quotes}active-site{close_quotes} residue necessary for substrate binding and/or catalysis. In this case, hexosaminidase A is present in the lysosome, but it lacks all {alpha}-specific activity. This biochemical phenotype is referred to as the {open_quotes}B1-variant form{close_quotes} of Tay-Sachs disease. Kinetic analysis of suspected B1-variant mutations is complex because hexosaminidase A is heterodimeric and bothmore » subunits possess similar active sites. In this report, we examine a previously identified B1-variant mutation, {alpha}-Val{sup 192}Leu. Chinese hamster ovary cells were permanently cotransfected with an {alpha}-cDNA-construct encoding the substitution and a mutant {beta}-cDNA ({beta}-Arg{sup 211}Lys), encoding a {beta}-subunit that is inactive but normal in all other respects. We were surprised to find that the Val{sup 192}Leu substitution produced a pro-{alpha} chain that did not form {alpha}-{beta} dimers and was not transported to the lysosome. Finally, we reexamined the hexosaminidase activity and protein levels in the fibroblasts from the original patient. These data were also not consistent with the biochemical phenotype of the B1 variant of Tay-Sachs disease previously reported to be present. Thus, we conclude that the Val{sup 192}Leu substitution does not specifically affect the {alpha}-active site. 23 refs., 4 figs., 2 tabs.« less

Inflammation activates the interferon signaling pathways in taste bud cells.

PubMed

Wang, Hong; Zhou, Minliang; Brand, Joseph; Huang, Liquan

2007-10-03

Patients with viral and bacterial infections or other inflammatory illnesses often experience taste dysfunctions. The agents responsible for these taste disorders are thought to be related to infection-induced inflammation, but the mechanisms are not known. As a first step in characterizing the possible role of inflammation in taste disorders, we report here evidence for the presence of interferon (IFN)-mediated signaling pathways in taste bud cells. IFN receptors, particularly the IFN-gamma receptor IFNGR1, are coexpressed with the taste cell-type markers neuronal cell adhesion molecule and alpha-gustducin, suggesting that both the taste receptor cells and synapse-forming cells in the taste bud can be stimulated by IFN. Incubation of taste bud-containing lingual epithelia with recombinant IFN-alpha and IFN-gamma triggered the IFN-mediated signaling cascades, resulting in the phosphorylation of the downstream STAT1 (signal transducer and activator of transcription protein 1) transcription factor. Intraperitoneal injection of lipopolysaccharide or polyinosinic:polycytidylic acid into mice, mimicking bacterial and viral infections, respectively, altered gene expression patterns in taste bud cells. Furthermore, the systemic administration of either IFN-alpha or IFN-gamma significantly increased the number of taste bud cells undergoing programmed cell death. These findings suggest that bacterial and viral infection-induced IFNs can act directly on taste bud cells, affecting their cellular function in taste transduction, and that IFN-induced apoptosis in taste buds may cause abnormal cell turnover and skew the representation of different taste bud cell types, leading to the development of taste disorders. To our knowledge, this is the first study providing direct evidence that inflammation can affect taste buds through cytokine signaling pathways.

Thalassemia

MedlinePlus

... disorder that affects the way the body makes hemoglobin. Hemoglobin is the part of red blood cells that ... alpha globin and beta globin; these proteins make hemoglobin. In thalassemia, a gene that makes these proteins ...

Anticancer effects of phenoxazine derivatives revealed by inhibition of cell growth and viability, disregulation of cell cycle, and apoptosis induction in HTLV-1-positive leukemia cells.

PubMed

Miyano-Kurosaki, Naoko; Ikegami, Kou; Kurosaki, Kunihiko; Endo, Takahiko; Aoyagi, Hoshimi; Hanami, Mari; Yasumoto, Jun; Tomoda, Akio

2009-05-01

Adult T-cell leukemia (ATL) is a malignant tumor of human CD4(+) T cells infected with a human retrovirus, T lymphotropic virus type-1 (HTLV-1). The aim of the present study was to investigate the apoptotic effects of phenoxazines, 2-amino-4,4alpha-dihydro-4alpha,7-dimethyl-3H-phenoxazine-3-one (Phx-1), 3-amino-1,4alpha-dihydro-4alpha,8-dimethyl-2H-phenoxazine-2-one (Phx-2), and 2-aminophenoxazine-3-one (Phx-3) on a T cell leukemia cell line from ATL patients, MT-1 cells; HTLV-1 transformed T-cell lines, HUT-102 cells and MT-2 cells; and an HTLV-1-negative rat sarcoma cell line, XC cells. Among these phenoxazines, Phx-3 at concentrations of less than 10 microg/ml extensively inhibited growth and cell viability; arrested cell cycles at sub G(0)/G(1) phase; and augmented apoptosis of MT-1, HUT-102, and MT-2 cells. However, these phenoxazines did not affect the cell viability of an HTLV-1-negative rat sarcoma cell line, XC cells, and phytohemaggutinin-activated human peripheral blood mononuclear cells, although they markedly inhibited the growth of these cells. The transmission of HTLV-1 from HTLV-1-positive cells (MT-2 cells) to HTLV-1-negative cells (XC cells) was considered to be prevented by Phx-1, Phx-2, or Phx-3 because the syncytium formation between these cells was inhibited markedly in the presence of these phenoxazines. The present results suggest that Phx-1, Phx-2, and, in particular, Phx-3 may be useful as therapeutic agents against ATL, which is extremely refractory to current therapies.

Advancing Stem Cell Models of Alpha-Synuclein Gene Regulation in Neurodegenerative Disease.

PubMed

Piper, Desiree A; Sastre, Danuta; Schüle, Birgitt

2018-01-01

Alpha-synuclein ( non A4 component of amyloid precursor, SNCA, NM_000345.3 ) plays a central role in the pathogenesis of Parkinson's disease (PD) and related Lewy body disorders such as Parkinson's disease dementia, Lewy body dementia, and multiple system atrophy. Since its discovery as a disease-causing gene in 1997, alpha-synuclein has been a central point of scientific interest both at the protein and gene level. Mutations, including copy number variants, missense mutations, short structural variants, and single nucleotide polymorphisms, can be causative for PD and affect conformational changes of the protein, can contribute to changes in expression of alpha-synuclein and its isoforms, and can influence regulation of temporal as well as spatial levels of alpha-synuclein in different tissues and cell types. A lot of progress has been made to understand both the physiological transcriptional and epigenetic regulation of the alpha-synuclein gene and whether changes in transcriptional regulation could lead to disease and neurodegeneration in PD and related alpha-synucleinopathies. Although the histopathological changes in these neurodegenerative disorders are similar, the temporal and spatial presentation and progression distinguishes them which could be in part due to changes or disruption of transcriptional regulation of alpha-synuclein. In this review, we describe different genetic alterations that contribute to PD and neurodegenerative conditions and review aspects of transcriptional regulation of the alpha-synuclein gene in the context of the development of PD. New technologies, advanced gene engineering and stem cell modeling, are on the horizon to shed further light on a better understanding of gene regulatory processes and exploit them for therapeutic developments.

Stress and substance P but not the substance P-metabolite SP5-11 trigger murine abortion by augmenting TNF-alpha levels at the feto-maternal interface.

PubMed

Fest, S; Zenclussen, A C; Joachim, R; Hagen, E; Demuth, H-U; Hoffmann, T

2006-01-01

In a well-established murine abortion model, stress is thought to trigger fetal rejection by inducing a proinflammatory immune response via substance P (SP), being tumour necrosis factor (TNF)-alpha-producing CD8+ T cells involved. Interestingly, the SP metabolite SP5-11 also binds to SP receptors and mediates SP-like effects on immune cells at sites of inflammation. No data were available regarding the effects of SP5-11 on pregnancy outcome in the CBA/J x DBA/2J abortion-prone combination. We investigated the influence of SP5-11 in contrast to stress or SP on the abortion rate and the cytokine production by lymphocytes as well as on the levels of CD8+ T cells. Stress and SP boosted the abortion rate and increased the percentage of type 1 [TNF-alpha, interferon-gamma, interleukin (IL)-12] and type 2 (IL-4 and IL-10) cytokine-producing lymphocytes in blood and decidua, predominantly CD8+ T cells. Interestingly, SP5-11 did not significantly affect the abortion rate or cytokine production in the decidua, while increasing the Th1 and Th2 cytokine production systemically. Our data suggest that stress and SP induce abortion by augmenting the local levels of TNF-alpha, which seems therefore to be a potent trigger of miscarriage. On the contrary, the SP metabolite SP5-11 only affects the systemic cytokine production without boosting the abortion rate in this experimental model.

Expression of the alpha and beta subunits of Ca2+/calmodulin kinase II in the cerebellum of jaundiced Gunn rats during development: a quantitative light microscopic analysis.

PubMed

Conlee, J W; Shapiro, S M; Churn, S B

2000-04-01

The homozygous (jj) jaundiced Gunn rat model for hyperbilirubinemia displays pronounced cerebellar hypoplasia. To examine the cellular mechanisms involved in bilirubin toxicity, this study focused on the effect of hyperbilirubinemia on calcium/calmodulin-dependent kinase II (CaM kinase II). CaM kinase II is a neuronally enriched enzyme which performs several important functions. Immunohistochemical analysis of alternating serial sections were performed using monoclonal antibodies for the alpha and beta subunits of CaM kinase II. Measurements were made of the total numbers of stained cells in each of the deep cerebellar nuclei and of Purkinje and granule cell densities in cerebellar lobules II, VI, and IX. The beta subunit was present in Purkinje cells and deep cerebellar nuclei of both groups at all ages, but only granule cells which had migrated through the Purkinje cell layer showed staining for beta subunit; external granule cells were completely negative. Many Purkinje cells had degenerated in the older animals, and the percent of granule cells stained for beta subunit was significantly reduced. The alpha subunit was found exclusively in Purkinje cells, although its appearance was delayed in the jaundiced animals. Sulfadimethoxine was administered to some jj rats 24 h or 15 days prior to sacrifice to increase brain bilirubin concentration. Results showed that bilirubin exposure modulated both alpha and beta CaM kinase II subunit expression in selective neuronal populations, but sulfadimethoxine had no acute effect on enzyme immunoreactivity. Thus, developmental expression of the alpha and beta subunits of CaM kinase II was affected by chronic bilirubin exposure during early postnatal development of jaundiced Gunn rats.

Predominant Improvement of Alpha Cell Function after Steroid Therapy in a Patient with Autoimmune Pancreatitis: Case Report.

PubMed

Takeshima, Ken; Ariyasu, Hiroyuki; Iwakura, Hiroshi; Kawai, Shintaro; Uraki, Shinsuke; Inaba, Hidefumi; Furuta, Machi; Warigaya, Kenji; Murata, Shin-Ichi; Akamizu, Takashi

2018-06-01

Autoimmune pancreatitis (AIP) is a subset of inflammatory pancreatic disease, responsive to corticosteroid therapy. It is prone to being affected by diabetes mellitus, but the effectiveness of steroid therapy on pancreatic endocrine function is still controversial. We present a case of AIP, focusing on pancreatic endocrine function after steroid therapy. The patient was referred to our hospital with exacerbation of diabetic control and pancreatic swelling. By admission, the insulin secretory capacity was severely impaired. The patient was diagnosed with AIP and treated with prednisolone, resulting in marked improvement of the pancreatic swelling. Glycemic control worsened transiently after initiation of steroid therapy, but insulin requirements decreased along with tapering prednisolone dosage. Pancreatic cytology showed that the acinar structure had been destroyed, and the islets had disappeared. Insulin and glucagon immunostaining revealed slightly scattered alpha and beta cells within the fibrotic stroma. The patient notably showed improved pancreatic alpha cell function predominantly after steroid therapy, despite partial improvement of beta cell function. An imbalance between alpha and beta cell function may contribute to insufficient diabetic control in some patients with AIP. The pancreatic endocrine function test in combination with pancreatic cytology could be helpful when considering the treatment strategy for diabetic control in patients with AIP.

Inhibition of hypoxia inducible factor-1alpha by dihydroxyphenylethanol, a product from olive oil, blocks microsomal prostaglandin-E synthase-1/vascular endothelial growth factor expression and reduces tumor angiogenesis.

PubMed

Terzuoli, Erika; Donnini, Sandra; Giachetti, Antonio; Iñiguez, Miguel A; Fresno, Manuel; Melillo, Giovanni; Ziche, Marina

2010-08-15

2-(3,4-dihydroxyphenil)-ethanol (DPE), a polyphenol present in olive oil, has been found to attenuate the growth of colon cancer cells, an effect presumably related to its anti-inflammatory activity. To further explore the effects of DPE on angiogenesis and tumor growth we investigated the in vivo efficacy of DPE in a HT-29 xenograft model and in vitro activities in colon cancer cells exposed to interleukin-1beta (IL-1beta) and prostaglandin E-2 (PGE-2). DPE (10 mg/kg/day for 14 days) inhibited tumor growth, reducing vessel lumina and blood perfusion to tumor, and diminished expression of hypoxia inducible factor-1alpha (HIF-1alpha), vascular endothelial growth factor (VEGF), and microsomal prostaglandin-E synthase-1 (mPGEs-1). In vitro, DPE (100 mumol/L) neither affected cell proliferation nor induced apoptosis in HT-29 and WiDr cells. DPE prevented the IL-1beta-mediated increase of mPGEs-1 expression and PGE-2 generation, as it did the silencing of HIF-1alpha. Moreover, DPE blocked mPGEs-1-dependent expression of VEGF and inhibited endothelial sprouting induced by tumor cells in a coculture system. PGE-2 triggers a feed-forward loop involving HIF-1alpha, which impinges on mPGEs-1 and VEGF expression, events prevented by DPE via extracellular signal-related kinase 1/2. The reduction of PGE-2 and VEGF levels, caused by DPE, was invariably associated with a marked decrease in HIF-1alpha expression and activity, independent of proteasome activity, indicating that the DPE effects on tumor growth and angiogenesis are dependent on the inhibition of HIF-1alpha translation. We show that the in vivo DPE antitumor effect is associated with anti-inflammatory and antiangiogenic activities resulting from the downregulation of the HIF-1alpha/mPGEs-1/VEGF axis.

15-Deoxy-{delta}{sup 12,14}-prostaglandin J2 (15d-PGJ2) mediates repression of TNF-{alpha} by decreasing levels of acetylated histone H3 and H4 at its promoter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Engdahl, Ryan; Monroy, M. Alexandra; Temple University School of Medicine, Department of Anatomy and Cell Biology, 3400 North Broad Street, Philadelphia, PA 19140

2007-07-20

Prostaglandin metabolite 15-Deoxy-{delta}{sup 12,14}-prostaglandin J2 (15d-PGJ2) is known to inhibit a number of pro-inflammatory cytokines as well as being a ligand for nuclear receptor PPAR{gamma}. We investigated the ability of 15d-PGJ2 to inhibit TNF-{alpha} gene expression through mechanisms that involve histone modification. Pretreatment with 15d-PGJ2 (10 {mu}M) inhibited LPS-stimulated TNF-{alpha} mRNA in THP-1 monocytes or PMA-differentiated cells to nearly basal levels. A specific PPAR{gamma} ligand, GW1929, failed to inhibit LPS-induced TNF-{alpha} mRNA expression nor did a PPAR{gamma} antagonist, GW9662, alter the repression of TNF-{alpha} mRNA in LPS-stimulated cells pretreated with 15d-PGJ2 suggesting a PPAR{gamma}-independent inhibition of TNF-{alpha} mRNA in THP-1more » cells. Transfection studies with a reporter construct and subsequent treatment with 15d-PGJ2 demonstrated a dose-dependent inhibition of the TNF-{alpha} promoter. Additional studies demonstrated that inhibition of histone deacetylases with trichostatin A (TSA) or overexpression of histone acetyltransferase CBP could overcome 15d-PGJ2-mediated repression of the TNF-{alpha} promoter, suggesting that an important mechanism whereby 15d-PGJ2 suppresses a cytokine is through factors that regulate histone modifications. To examine the endogenous TNF-{alpha} promoter, chromatin immunoprecipitations (ChIP) were performed. ChIP assays demonstrated that LPS stimulation induced an increase in histone H3 and H4 acetylation at the TNF-{alpha} promoter, which was reduced in cells pretreated with 15d-PGJ2. These results highlight the ability of acetylation and deacetylation factors to affect the TNF-{alpha} promoter and demonstrate that an additional important mechanism whereby 15d-PGJ2 mediates TNF-{alpha} transcriptional repression by altering levels of acetylated histone H3 and H4 at its promoter.« less

Subunit assembly of hemoglobin: an important determinant of hematologic phenotype.

PubMed

Bunn, H F

1987-01-01

Hemoglobin's physiologic properties depend on the orderly assembly of its subunits in erythropoietic cells. The biosynthesis of alpha- and beta-globin polypeptide chains is normally balanced. Heme rapidly binds to the globin subunit, either during translation or shortly thereafter. The formation of the alpha beta-dimer is facilitated by electrostatic attraction of a positively charged alpha-subunit to a negatively charged beta-subunit. The alpha beta-dimer dissociates extremely slowly. The difference between the rate of dissociation of alpha beta- and alpha gamma-dimers with increasing pH explains the well-known alkaline resistance of Hb F. Two dimers combine to form the functioning alpha 2 beta 2-tetramer. This model of hemoglobin assembly explains the different levels of positively charged and negatively charged mutant hemoglobins that are encountered in heterozygotes and the effect of alpha-thalassemia and heme deficiency states in modifying the level of the variant hemoglobin as well as Hb A2. Electrostatic interactions also affect the binding of hemoglobin to the cytoplasmic surface of the red cell membrane and may underlie the formation of target cells. Enhanced binding of positively charged variants such as S and C trigger a normally dormant pathway for potassium and water loss. Thus, the positive charge on beta c is responsible for the two major contributors to the pathogenesis of Hb SC disease: increased proportion of Hb S and increased intracellular hemoglobin concentration. It is likely that electrostatic interactions play an important role in the assembly of a number of other multisubunit macromolecules, including membrane receptors, cytoskeletal proteins, and DNA binding proteins.

Effect of heat treatment and enzymatic digestion on the B cell epitopes of cow's milk proteins.

PubMed

Morisawa, Y; Kitamura, A; Ujihara, T; Zushi, N; Kuzume, K; Shimanouchi, Y; Tamura, S; Wakiguchi, H; Saito, H; Matsumoto, K

2009-06-01

Processing milk leads to changes in clinical allergenicity. However, the mechanism by which heat treatment affects the allergenicity of milk proteins is not fully understood. We investigated the effect of heat treatment and enzymatic digestion on the allergenicity of B cell epitopes of milk proteins using a histamine release assay. Human basophils were passively sensitized using sera from 10 patients with allergies to cow's milk. All the patients experienced symptoms immediately after ingesting milk. The human basophils were obtained from umbilical cord blood mononuclear cells after culturing the mononuclear cells for 3-4 weeks in the presence of IL-3. After sensitization with 10% patient sera for 48 h, the cells were stimulated with untreated, heat-treated, or heat-treated and pepsin-and-trypsin-digested beta-lactoglobulin or alpha-casein for 1 h. The histamine concentrations in the supernatants were then measured by radioimmunoassay. Heat treatment alone did not alter the molecular weight of beta-lactoglobulin or alpha-casein. Heat treatment of beta-lactoglobulin significantly increased its susceptibility to enzymatic digestion in a time- and temperature-dependent manner and reduced its ability to induce histamine release from sensitized basophils. Similar findings were not observed for alpha-casein. The combination of heat treatment and enzymatic digestion reduced the abilities of both beta-lactoglobulin and alpha-casein to induce histamine release from passively sensitized basophils. Heat treatment reduced the allergenicity of beta-lactoglobulin by inducing conformational changes and by increasing its susceptibility to enzymatic digestion, both of which disrupted B cell epitopes, whereas heat treatment alone did not alter the allergenicity of alpha-casein.

Integrin {alpha}{beta}1, {alpha}{sub v}{beta}, {alpha}{sub 6}{beta} effectors p130Cas, Src and talin regulate carcinoma invasion and chemoresistance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sansing, Hope A.; Sarkeshik, Ali; Yates, John R.

2011-03-11

Research highlights: {yields} Proteomics of clustered integrin {alpha}{beta}1, {alpha}{sub v}{beta}, {alpha}{sub 6}{beta} receptors in oral carcinoma. {yields} p130Cas, Dek, Src and talin regulate oral carcinoma invasion. {yields} p130Cas, talin, Src and zyxin regulate oral carcinoma resistance to cisplatin. -- Abstract: Ligand engagement by integrins induces receptor clustering and formation of complexes at the integrin cytoplasmic face that controls cell signaling and cytoskeletal dynamics critical for adhesion-dependent processes. This study searches for a subset of integrin effectors that coordinates both tumor cell invasion and resistance to the chemotherapeutic drug cisplatin in oral carcinomas. Candidate integrin effectors were identified in a proteomicsmore » screen of proteins recruited to clustered integrin {alpha}{beta}1, {alpha}{sub v}{beta} or {alpha}{sub 6}{beta} receptors in oral carcinomas. Proteins with diverse functions including microtubule and actin binding proteins, and factors involved in trafficking, transcription and translation were identified in oral carcinoma integrin complexes. Knockdown of effectors in the oral carcinoma HN12 cells revealed that p130Cas, Dek, Src and talin were required for invasion through Matrigel. Disruption of talin or p130Cas by RNA interference increased resistance to cisplatin, whereas targeting Dek, Src or zyxin reduced HN12 resistance to cisplatin. Analysis of the spreading of HN12 cells on collagen I and laminin I revealed that a decrease in p130Cas or talin expression inhibited spreading on both matrices. Interestingly, a reduction in zyxin expression enhanced spreading on laminin I and inhibited spreading on collagen I. Reduction of Dek, Src, talin or zyxin expression reduced HN12 proliferation by 30%. Proliferation was not affected by a reduction in p130Cas expression. We conclude that p130Cas, Src and talin function in both oral carcinoma invasion and resistance to cisplatin.« less

Partial Oxygen Pressure Affects the Expression of Prognostic Biomarkers HIF-1 Alpha, Ki67, and CK20 in the Microenvironment of Colorectal Cancer Tissue.

PubMed

Zhang, Lirong; Hu, Yu; Xi, Ning; Song, Jie; Huang, Wenjing; Song, Shanshan; Liu, Yiting; Liu, Xianying; Xie, Yingjun

2016-01-01

Hypoxia is prognostically important in colorectal cancer (CRC) therapy. Partial oxygen pressure (pO 2 ) is an important parameter of hypoxia. The correlation between pO 2 levels and expression levels of prognostic biomarkers was measured in CRC tissues. Human CRC tissues were collected and pO 2 levels were measured by OxyLite. Three methods for tissue fixation were compared, including formalin, Finefix, and Finefix-plus-microwave. Immunohistochemistry (IHC) staining was conducted by using the avidin-biotin complex technique for detecting the antibodies to hypoxia inducible factor-1 (HIF-1) alpha, cytokeratin 20 (CK20), and cell proliferation factor Ki67. The levels of pO 2 were negatively associated with the size of CRC tissues. Finefix-plus-microwave fixation has the potential to replace formalin. Additionally, microwave treatment improved Finefix performance in tissue fixation and protein preservation. The percentage of positive cells and gray values of HIF-1 alpha, CK20, and Ki67 were associated with CRC development ( P < 0.05). The levels of pO 2 were positively related with the gray values of Ki67 and negatively related with the values of HIF-1 alpha and CK20 ( P < 0.05). Thus, the levels of microenvironmental pO 2 affect the expression of predictive biomarkers HIF-1 alpha, CK20, and Ki67 in the development of CRC tissues.

Integrin alphaIIb-subunit cytoplasmic domain mutations demonstrate a requirement for tyrosine phosphorylation of beta3-subunits in actin cytoskeletal organization.

PubMed

Yamodo, Innocent H; Blystone, Scott D

2004-01-01

Using truncated or mutated alphaIIb integrin cytoplasmic domains fused to the alphaV extracellular domain and expressed with the beta3 integrin subunit, we demonstrate that the double mutation of proline residues 998 and 999 to alanine (PP998/999AA), previously shown to disturb the C-terminal conformation of the alphaIIb integrin cytoplasmic domain, prevents tyrosine phosphorylation of beta3 integrin induced by Arg-Gly-Asp peptide ligation. This mutation also inhibits integrin mediated actin assembly and cell adhesion to vitronectin. In contrast, progressive truncation of the alphaIIb-subunit cytoplasmic domain did not reproduce these effects. Interestingly, the PP998/999AA mutations of alphaIIb did not affect beta3 tyrosine phosphorylation, cell adhesion, or actin polymerization induced by manganese. Exogenous addition of manganese was sufficient to rescue beta3 phosphorylation, cell adhesion, and actin assembly in cells expressing the PP998/999AA mutation when presented with a vitronectin substrate. Further, induction of the high affinity conformation of this mutant beta3 integrin by incubation with either Arg-Gly-Asp peptide or exogenous manganese was equivalent. These results suggest that the extracellular structure of beta3 integrins in the high affinity conformation is not directly related to the structure of the cytoplasmic face of the integrin. Moreover, the requirement for beta3 phosphorylation is demonstrated without mutation of the beta3 subunit. In support of our previous hypothesis of a role for beta3 phosphorylation in adhesion, these studies demonstrate a strong correlation between beta3 tyrosine phosphorylation and assembly of a cytoskeleton competent to support firm cell adhesion.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Hyeon Ho; Lee, Youngae; Laboratory of Cutaneous Aging Research, Department of Dermatology, Clinical Research Institutes, Seoul National University Hospital, 28 Yongon-dong, Jongno-gu, Seoul 110-744

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.more » 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.« less

Guinea pig hepatocyte alpha 1A-adrenoceptors: characterization, signal transduction and regulation.

PubMed

García-Sáinz, J A; Romero-Avila, T; Olivares-Reyes, J A; Macías-Silva, M

1992-11-02

Activation of guinea pig hepatocyte alpha 1-adrenoceptors increases phosphatidylinositol (PI) labeling, [3H]inositol phosphate production and phosphorylase activity. These adrenergic actions were not altered by pretreatment with chlorethylclonidine but were blocked by 5-methyl urapidil and prazosin (the former being 3- to 10-fold more potent than the latter), indicating that alpha 1A-adrenoceptors were involved. When the cells were incubated in buffer without calcium and containing EGTA, the alpha 1A-adrenergic stimulation of PI labeling was diminished but not abolished and that of phosphorylase was not affected. The alpha 1A-adrenergic effects were insensitive to pertussis toxin treatment. Phorbol myristate acetate inhibited the alpha 1A-adrenergic actions, although at relatively large concentrations, and also those of other agents such as angiotensin II and NaF. Our data clearly indicate that guinea pig hepatocytes express alpha 1A-adrenoceptors whose activation stimulates phosphoinositide turnover, via a pertussis toxin-insensitive process; the alpha 1A-adrenergic effects were at least partially independent of extracellular calcium.

Intraarticular glucocorticoid treatment reduces inflammation in synovial cell infiltrations more efficiently than in synovial blood vessels.

PubMed

af Klint, Erik; Grundtman, Cecilia; Engström, Marianne; Catrina, Anca Irinel; Makrygiannakis, Dimitrios; Klareskog, Lars; Andersson, Ulf; Ulfgren, Ann-Kristin

2005-12-01

To investigate whether intraarticular (IA) glucocorticoid (GC) therapy diminishes synovial cell infiltration, vascularity, expression of proinflammatory cytokines, and adhesion molecule levels in patients with chronic arthritides. Thirty-one patients with chronic arthritides received a single IA injection of triamcinolone hexacetonide to treat active large-joint inflammation. Synovial biopsy specimens were obtained with arthroscopic guidance before and 9-15 days after injection. The presence of T lymphocytes, macrophages, intercellular adhesion molecule 1 (ICAM-1), vascular endothelial growth factor (VEGF), the pan-endothelial marker CD31, and the proinflammatory cytokines interleukin-1alpha (IL-1alpha), IL-1beta, tumor necrosis factor (TNF), and high mobility group box chromosomal protein 1 (HMGB-1) was studied by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction. IA GC treatment resulted in good clinical response in 29 of 31 joints. After therapeutic intervention, the number of synovial T lymphocytes declined, whereas the number of macrophages remained unchanged. Overall synovial protein expression of TNF, IL-1beta, extranuclear HMGB-1, VEGF, and ICAM-1 was reduced at followup tissue sampling, while no significant effects were observed regarding vascularity. In contrast, expression of IL-1alpha, VEGF, and cytoplasmic HMGB-1 protein in vascular endothelial cells was not affected. GC therapy down-regulated levels of messenger RNA encoding IL-1alpha and IL-1beta, but not TNF or HMGB-1. Synovial cell infiltration and proinflammatory cytokine expression were affected in a multifaceted manner by IA GC treatment. Marked reduction of synovial T lymphocytes, TNF, IL-1beta, extranuclear HMGB-1, ICAM-1, and VEGF occurred in association with beneficial clinical effects. Unexpectedly, macrophage infiltration and proinflammatory endothelial cytokine expression remained unchanged. These findings may reflect mechanisms controlling the transiency of clinical improvement frequently observed after IA GC injection.

The molecular chaperone alphaA-crystallin enhances lens epithelial cell growth and resistance to UVA stress.

PubMed

Andley, U P; Song, Z; Wawrousek, E F; Bassnett, S

1998-11-20

alphaA-Crystallin (alphaA) is a member of the small heat shock protein (sHSP) family and has the ability to prevent denatured proteins from aggregating in vitro. Lens epithelial cells express relatively low levels of alphaA, but in differentiated fiber cells, alphaA is the most abundant soluble protein. The lenses of alphaA-knock-out mice develop opacities at an early age, implying a critical role for alphaA in the maintenance of fiber cell transparency. However, the function of alpha-crystallin in the lens epithelium is unknown. To investigate the physiological function of alphaA in lens epithelial cells, we used the following two systems: alphaA knock-out (alphaA(-/-)) mouse lens epithelial cells and human lens epithelial cells that overexpress alphaA. The growth rate of alphaA(-/-) mouse lens epithelial cells was reduced by 50% compared with wild type cells. Cell cycle kinetics, measured by fluorescence-activated cell sorter analysis of propidium iodide-stained cells, indicated a relative deficiency of alphaA(-/-) cells in the G2/M phases. Exposure of mouse lens epithelial cells to physiological levels of UVA resulted in an increase in the number of apoptotic cells in the cultures. Four hours after irradiation the fraction of apoptotic cells in the alphaA(-/-) cultures was increased 40-fold over wild type. In cells lacking alphaA, UVA exposure modified F-actin, but actin was protected in cells expressing alphaA. Stably transfected cell lines overexpressing human alphaA were generated by transfecting extended life span human lens epithelial cells with the mammalian expression vector construct pCI-neoalphaA. Cells overexpressing alphaA were resistant to UVA stress, as determined by clonogenic survival. alphaA remained cytoplasmic after exposure to either UVA or thermal stress indicating that, unlike other sHSPs, the protective effect of alphaA was not associated with its relocalization to the nucleus. These results indicate that alphaA has important cellular functions in the lens over and above its well characterized role in refraction.

Distribution of sialic acid receptors and influenza A virus of avian and swine origin in experimentally infected pigs.

PubMed

Trebbien, Ramona; Larsen, Lars E; Viuff, Birgitte M

2011-09-08

Pigs are considered susceptible to influenza A virus infections from different host origins because earlier studies have shown that they have receptors for both avian (sialic acid-alpha-2,3-terminal saccharides (SA-alpha-2,3)) and swine/human (SA-alpha-2,6) influenza viruses in the upper respiratory tract. Furthermore, experimental and natural infections in pigs have been reported with influenza A virus from avian and human sources. This study investigated the receptor distribution in the entire respiratory tract of pigs using specific lectins Maackia Amurensis (MAA) I, and II, and Sambucus Nigra (SNA). Furthermore, the predilection sites of swine influenza virus (SIV) subtypes H1N1 and H1N2 as well as avian influenza virus (AIV) subtype H4N6 were investigated in the respiratory tract of experimentally infected pigs using immunohistochemical methods. SIV antigen was widely distributed in bronchi, but was also present in epithelial cells of the nose, trachea, bronchioles, and alveolar type I and II epithelial cells in severely affected animals. AIV was found in the lower respiratory tract, especially in alveolar type II epithelial cells and occasionally in bronchiolar epithelial cells. SA-alpha-2,6 was the predominant receptor in all areas of the respiratory tract with an average of 80-100% lining at the epithelial cells. On the contrary, the SA-alpha-2,3 was not present (0%) at epithelial cells of nose, trachea, and most bronchi, but was found in small amounts in bronchioles, and in alveoli reaching an average of 20-40% at the epithelial cells. Interestingly, the receptor expression of both SA-alpha-2,3 and 2,6 was markedly diminished in influenza infected areas compared to non-infected areas. A difference in predilection sites between SIV and AIV virus was found, and this difference was in accordance with the distribution of the SA-alpha-2,6 and SA-alpha-2,3 receptor, respectively. The results indicated that the distribution of influenza A virus receptors in pigs are similar to that of humans and therefore challenge the theory that the pig acts as a mixing vessel between human and avian influenza viruses. Furthermore, it was shown that AIV prefers to infect alveolar type II epithelial cells in pigs. This corresponds with findings in humans emphasising the resemblance between the two species.

Genetic manipulation of murine embryonic stem cells with enhanced green fluorescence protein and sulfatase-modifying factor I genes.

PubMed

Zhao, Guoying; Karageorgos, Litsa; Hutchinson, Rhonda G; Hopwood, John J; Hemsley, Kim

2010-05-01

Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder (LSD) in which an absence of sulfamidase results in incomplete degradation and subsequent accumulation of its substrate, heparan sulfate. Most neurodegenerative LSD remain untreatable. However, therapy options, such as gene, enzyme end cell therapy, are under investigation. Previously, we have constructed an embryonic stem (ES) cell line (NS21) that over-expresses human sulphamidase as a potential treatment for murine MPS IIIA. In the present study the sulfatase-modifying factor I (SUMF1) and enhanced green fluorescence protein (eGFP) genes were co-introduced under a cytomegalovirus (CMV) promoter into NS21 cells, to enhance further sulfamidase activity and provide a marker for in vivo cell tracking, respectively. eGFP was also introduced under the control of the human elongation factor-1alpha (hEF-1alpha) promoter to compare the stability of transgene expression. During differentiation of ES cells into glial precursors, SUMF1 was down-regulated and was hardly detectable by day 18 of differentiation. Likewise, eGFP expression was heterogeneous and highly unstable. Use of a human EF-1alpha promoter resulted in more homogeneous eGFP expression, with approximately 50% of cells eGFP positive following differentiation into glial precursors. Compared with NS21 cells, the outgrowth of eGFP-expressing cells was not as confluent when differentiated into glial precursors. Our data suggest that SUMF1 enhances sulfamidase activity in ES cells, hEF-1alpha is a stronger promoter than CMV for ES cells and over-expression of eGFP may affect cell growth and contribute to unstable gene expression.

alpha-Adrenergic-mediated activation of human reconstituted fibrinogen receptor (integrin alphaIIbbeta3) in Chinese hamster ovary cells.

PubMed

Butta, Nora; Larrucea, Susana; Gonzalez-Manchon, Consuelo; Alonso, Sonia; Parrilla, Roberto

2004-12-01

This work reports the functional studies of CHO cells coexpressing alpha-adrenergic (alphaAR) and human fibrinogen (Fg) receptors (integrin alphaIIbbeta3). Stimulation of these cells with alpha-agonists produced a transient rise in the free cytosolic calcium (Ca(++)) accompanied by enhanced binding to soluble Fg, and these effects were prevented by specific alphaAR antagonists. The alpha-adrenergic-induced activation of alphaIIbbeta3 in CHO-alphaIIbbeta3-alphaAR increased the rate of adhesion and extension of cells onto Fg coated plates, and also induced a soluble Fg- and alphaIIbbeta3-dependent formation of cell aggregates, whereas no effects were observed by the stimulation of CHO-alphaIIbbeta3 cells. alpha-Adrenergic antagonists, the ligand mimetic peptide RGDS, pertussis toxin (PTX), or EDTA, they all prevented the alpha-adrenergic stimulation of adhesion and aggregation. However, inhibition of PKC prevented the alpha-adrenergic stimulation of cell adherence, whereas blocking the intracellular Ca(++) mobilization impeded the stimulation of cell aggregation. The alpha-adrenergic activation was associated with phosphorylation of a protein of approximately 100 kDa and proteins of the MAPK family. The former was selectively phosphorylated by alpha-adrenergic stimulation whereas the latter were phosphorylated by the binding of cells to Fg and markedly intensified by alpha-adrenergic stimulation.

Methylation of an alpha-foetoprotein gene intragenic site modulates gene activity.

PubMed Central

Opdecamp, K; Rivière, M; Molné, M; Szpirer, J; Szpirer, C

1992-01-01

By comparing the methylation pattern of Mspl/Hpall sites in the 5' region of the mouse alpha-foetoprotein (AFP) gene of different cells (hepatoma cells, foetal and adult liver, fibroblasts), we found a correlation between gene expression and unmethylation of a site located in the first intron of the gene. Other sites did not show this correlation. In transfection experiments of unmethylated and methylated AFP-CAT chimeric constructions, we then showed that methylation of the intronic site negatively modulates expression of CAT activity. We also found that a DNA segment centered on this site binds nuclear proteins; however methylation did not affect protein binding. Images PMID:1371343

Physiologic oxygen concentration enhances the stem-like properties of CD133+ human glioblastoma cells in vitro.

PubMed

McCord, Amy M; Jamal, Muhammad; Shankavaram, Uma T; Shankavarum, Uma T; Lang, Frederick F; Camphausen, Kevin; Tofilon, Philip J

2009-04-01

In vitro investigations of tumor stem-like cells (TSC) isolated from human glioblastoma (GB) surgical specimens have been done primarily at an atmospheric oxygen level of 20%. To determine whether an oxygen level more consistent with in situ conditions affects their stem cell-like characteristics, we compared GB TSCs grown under conditions of 20% and 7% oxygen. Growing CD133(+) cells sorted from three GB neurosphere cultures at 7% O(2) reduced their doubling time and increased the self-renewal potential as reflected by clonogenicity. Furthermore, at 7% oxygen, the cultures exhibited an enhanced capacity to differentiate along both the glial and neuronal pathways. As compared with 20%, growth at 7% oxygen resulted in an increase in the expression levels of the neural stem cell markers CD133 and nestin as well as the stem cell markers Oct4 and Sox2. In addition, whereas hypoxia inducible factor 1alpha was not affected in CD133(+) TSCs grown at 7% O(2), hypoxia-inducible factor 2alpha was expressed at higher levels as compared with 20% oxygen. Gene expression profiles generated by microarray analysis revealed that reducing oxygen level to 7% resulted in the up-regulation and down-regulation of a significant number of genes, with more than 140 being commonly affected among the three CD133(+) cultures. Furthermore, Gene Ontology categories up-regulated at 7% oxygen included those associated with stem cells or GB TSCs. Thus, the data presented indicate that growth at the more physiologically relevant oxygen level of 7% enhances the stem cell-like phenotype of CD133(+) GB cells.

Gonadotropin-releasing hormone (GnRH) agonist triptorelin inhibits estradiol-induced serum response element (SRE) activation and c-fos expression in human endometrial, ovarian and breast cancer cells.

PubMed

Gründker, Carsten; Günthert, Andreas R; Hellriegel, Martin; Emons, Günter

2004-11-01

The majority of human endometrial (>80%), ovarian (>80%) and breast (>50%) cancers express GnRH receptors. Their spontaneous and epidermal growth-factor-induced proliferation is dose- and time-dependently reduced by treatment with GnRH and its agonists. In this study, we demonstrate that the GnRH agonist triptorelin inhibits estradiol (E2)-induced cancer cell proliferation. The proliferation of quiescent estrogen receptor alpha (ER alpha)-/ER beta-positive, but not of ER alpha-negative/ER beta-positive endometrial, ovarian and breast cancer cell lines, was significantly stimulated (P<0.001) (ANOVA) after treatment with E2 (10(-8) M). This effect was time- and dose-dependently antagonized by simultaneous treatment with triptorelin. The inhibitory effect was maximal at 10(-5) M concentration of triptorelin (P<0.001). In addition, we could show that, in ER alpha-/ER beta-positive cell lines, E2 induces activation of serum response element (SRE) and expression of the immediate early-response gene c-fos. These effects were blocked by triptorelin (P<0.001). E2-induced activation of estrogen-response element (ERE) was not affected by triptorelin. The transcriptional activation of SRE by E2 is due to ER alpha activation of the mitogen-activated protein kinase (MAPK) pathway. This pathway is impeded by GnRH, resulting in a reduction of E2-induced SRE activation and, in consequence, a reduction of E2-induced c-fos expression. This causes downregulation of E2-induced cancer cell proliferation.

A pro-inflammatory role of deubiquitinating enzyme cylindromatosis (CYLD) in vascular smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Shuai; Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC 29208; Lv, Jiaju

2012-03-30

Highlights: Black-Right-Pointing-Pointer Cyld deficiency suppresses pro-inflammatory phenotypic switch of VSMCs. Black-Right-Pointing-Pointer Cyld deficiency inhibits MAPK rather than NF-kB activity in inflamed VSMCs. Black-Right-Pointing-Pointer CYLD is up-regulated in the coronary artery with neointimal hyperplasia. -- Abstract: CYLD, a deubiquitinating enzyme (DUB), is a critical regulator of diverse cellular processes, ranging from proliferation and differentiation to inflammatory responses, via regulating multiple key signaling cascades such as nuclear factor kappa B (NF-{kappa}B) pathway. CYLD has been shown to inhibit vascular lesion formation presumably through suppressing NF-{kappa}B activity in vascular cells. However, herein we report a novel role of CYLD in mediating pro-inflammatory responsesmore » in vascular smooth muscle cells (VSMCs) via a mechanism independent of NF-{kappa}B activity. Adenoviral knockdown of Cyld inhibited basal and the tumor necrosis factor alpha (TNF{alpha})-induced mRNA expression of pro-inflammatory cytokines including monocyte chemotactic protein-1 (Mcp-1), intercellular adhesion molecule (Icam-1) and interleukin-6 (Il-6) in rat adult aortic SMCs (RASMCs). The CYLD deficiency led to increases in the basal NF-{kappa}B transcriptional activity in RASMCs; however, did not affect the TNF{alpha}-induced NF-{kappa}B activity. Intriguingly, the TNF{alpha}-induced I{kappa}B phosphorylation was enhanced in the CYLD deficient RASMCs. While knocking down of Cyld decreased slightly the basal expression levels of I{kappa}B{alpha} and I{kappa}B{beta} proteins, it did not alter the kinetics of TNF{alpha}-induced I{kappa}B protein degradation in RASMCs. These results indicate that CYLD suppresses the basal NF-{kappa}B activity and TNF{alpha}-induced I{kappa}B kinase activation without affecting TNF{alpha}-induced NF-{kappa}B activity in VSMCs. In addition, knocking down of Cyld suppressed TNF{alpha}-induced activation of mitogen activated protein kinases (MAPKs) including extracellular signal-activated kinases (ERK), c-Jun N-terminal kinase (JNK), and p38 in RASMCs. TNF{alpha}-induced RASMC migration and monocyte adhesion to RASMCs were inhibited by the Cyld knockdown. Finally, immunochemical staining revealed a dramatic augment of CYLD expression in the injured coronary artery with neointimal hyperplasia. Taken together, our results uncover an unexpected role of CYLD in promoting inflammatory responses in VSMCs via a mechanism involving MAPK activation but independent of NF-{kappa}B activity, contributing to the pathogenesis of vascular disease.« less

Neutrophil-derived alpha defensins control inflammation by inhibiting macrophage mRNA translation

PubMed Central

Tomlinson, Gareth H.; Miles, Katherine; Smith, Richard W. P.; Rossi, Adriano G.; Hiemstra, Pieter S.; van ’t Wout, Emily F. A.; Dean, Jonathan L. E.; Gray, Nicola K.; Lu, Wuyuan; Gray, Mohini

2016-01-01

Neutrophils are the first and most numerous cells to arrive at the site of an inflammatory insult and are among the first to die. We previously reported that alpha defensins, released from apoptotic human neutrophils, augmented the antimicrobial capacity of macrophages while also inhibiting the biosynthesis of proinflammatory cytokines. In vivo, alpha defensin administration protected mice from inflammation, induced by thioglychollate-induced peritonitis or following infection with Salmonella enterica serovar Typhimurium. We have now dissected the antiinflammatory mechanism of action of the most abundant neutrophil alpha defensin, Human Neutrophil Peptide 1 (HNP1). Herein we show that HNP1 enters macrophages and inhibits protein translation without inducing the unfolded-protein response or affecting mRNA stability. In a cell-free in vitro translation system, HNP1 powerfully inhibited both cap-dependent and cap-independent mRNA translation while maintaining mRNA polysomal association. This is, to our knowledge, the first demonstration of a peptide released from one cell type (neutrophils) directly regulating mRNA translation in another (macrophages). By preventing protein translation, HNP1 functions as a “molecular brake” on macrophage-driven inflammation, ensuring both pathogen clearance and the resolution of inflammation with minimal bystander tissue damage. PMID:27044108

Functions of fukutin, a gene responsible for Fukuyama type congenital muscular dystrophy, in neuromuscular system and other somatic organs.

PubMed

Yamamoto, Tomoko; Shibata, Noriyuki; Saito, Yoshiaki; Osawa, Makiko; Kobayashi, Makio

2010-06-01

Fukuyama type congenital muscular dystrophy (FCMD) is an autosomal recessive disease, exhibiting muscular dystrophy, and central nervous system (CNS) and ocular malformations. It is included in alpha-dystroglycanopathy, a group of muscular dystrophy showing reduced glycosylation of alpha-dystroglycan. alpha-Dystroglycan is one of the components of dystrophin-glycoprotein complex linking extracellular and intracellular proteins. The sugar chains of alpha-dystroglycan are receptors for extracellular matrix proteins such as laminin. Fukutin, a gene responsible for FCMD, is presumably related to the glycosylation of alpha-dystroglycan like other causative genes of alpha-dystroglycanopathy. The CNS lesion of FCMD is characterized by cobblestone lissencephaly, associated with decreased glycosylation of alpha-dystroglycan in the glia limitans where the basement membrane is formed. Astrocytes whose endfeet form the glia limitans seem to be greatly involved in the genesis of the CNS lesion. Fukutin is probably necessary for astrocytic function. Other components of the CNS may also need fukutin, such as migration and synaptic function in neurons. However, roles of fukutin in oligodendroglia, microglia, leptomeninges and capillaries are unknown at present. Fukutin is expressed in various somatic organs as well, and appears to work differently between epithelial cells and astrocytes. In the molecular level, since the dystrophin-glycoprotein complex is linked to cell signaling pathways involving c-src and c-jun, fukutin may be able to affect cell proliferation/survival. Fukutin was localized in the nucleus on cancer cell lines. With the consideration that mutations of fukutin give rise to wide spectrum of the clinical phenotype, more unknown functions of fukutin besides the glycosylation of alpha-dystroglycan can be suggested. Trials for novel treatments including gene therapy are in progress in muscular dystrophies. Toward effective therapies with minimal side effects, precise evaluation of the pathomechanism of FCMD and the function of fukutin would be required.

Differential regulation of the cell cycle by alpha1-adrenergic receptor subtypes.

PubMed

Gonzalez-Cabrera, Pedro J; Shi, Ting; Yun, June; McCune, Dan F; Rorabaugh, Boyd R; Perez, Dianne M

2004-11-01

Alpha(1)-Adrenergic receptors have been implicated in growth-promoting pathways. A microarray study of individual alpha(1)-adrenergic receptor subtypes (alpha(1A), alpha(1B), and alpha(1D)) expressed in Rat-1 fibroblasts revealed that epinephrine altered the transcription of several cell cycle regulatory genes in a direction consistent with the alpha(1A)- and alpha(1D)-adrenergic receptors mediating G(1)-S cell cycle arrest and the alpha(1B-)mediating cell-cycle progression. A time course indicated that in alpha(1A) cells, epinephrine stimulated a G(1)-S arrest, which began after 8 h of stimulation and maximized at 16 h, at which point was completely blocked with cycloheximide. The alpha(1B)-adrenergic receptor profile also showed unchecked cell cycle progression, even under low serum conditions and induced foci formation. The G(1)-S arrest induced by alpha(1A)- and alpha(1D)-adrenergic receptors was associated with decreased cyclin-dependent kinase-6 and cyclin E-associated kinase activities and increased expression of the cyclin-dependent kinase inhibitor p27(Kip1), all of which were blocked by prazosin. There were no differences in kinase activities and/or expression of p27(Kip1) in epinephrine alpha(1B)-AR fibroblasts, although the microarray did indicate differences in p27(Kip1) RNA levels. Cell counts proved the antimitotic effect of epinephrine in alpha(1A) and alpha(1D) cells and indicated that alpha(1B)-adrenergic receptor subtype expression was sufficient to cause proliferation of Rat-1 fibroblasts independent of agonist stimulation. Analysis in transfected PC12 cells also confirmed the alpha(1A)- and alpha(1B)-adrenergic receptor effect. The alpha(1B)-subtype native to DDT1-MF2 cells, a smooth muscle cell line, caused progression of the cell cycle. These results indicate that the alpha(1A)- and alpha(1D)-adrenergic receptors mediate G(1)-S cell-cycle arrest, whereas alpha(1B)-adrenergic receptor expression causes a cell cycle progression and may induce transformation in sensitive cell lines.

Increased virulence and competitive advantage of a/alpha over a/a or alpha/alpha offspring conserves the mating system of Candida albicans.

PubMed

Lockhart, Shawn R; Wu, Wei; Radke, Joshua B; Zhao, Rui; Soll, David R

2005-04-01

The majority of Candida albicans strains in nature are a/alpha and must undergo homozygosis to a/a or alpha/alpha to mate. Here we have used a mouse model for systemic infection to test the hypothesis that a/alpha strains predominate in nature because they have a competitive advantage over a/a and alpha/alpha offspring in colonizing hosts. Single-strain injection experiments revealed that a/alpha strains were far more virulent than either their a/a or alpha/alpha offspring. When equal numbers of parent a/alpha and offspring a/a or alpha/alpha cells were co-injected, a/alpha always exhibited a competitive advantage at the time of extreme host morbidity or death. When equal numbers of an engineered a/a/alpha2 strain and its isogenic a/a parent strain were co-injected, the a/a/alpha2 strain exhibited a competitive advantage at the time of host morbidity or death, suggesting that the genotype of the mating-type (MTL) locus, not associated genes on chromosome 5, provides a competitive advantage. We therefore propose that heterozygosity at the MTL locus not only represses white-opaque switching and genes involved in the mating process, but also affects virulence, providing a competitive advantage to the a/alpha genotype that conserves the mating system of C. albicans in nature.

Alpha-Mannosidosis: Therapeutic Strategies.

PubMed

Ceccarini, Maria Rachele; Codini, Michela; Conte, Carmela; Patria, Federica; Cataldi, Samuela; Bertelli, Matteo; Albi, Elisabetta; Beccari, Tommaso

2018-05-17

Alpha-mannosidosis (α-mannosidosis) is a rare lysosomal storage disorder with an autosomal recessive inheritance caused by mutations in the gene encoding for the lysosomal α-d-mannosidase. So far, 155 variants from 191 patients have been identified and in part characterized at the biochemical level. Similarly to other lysosomal storage diseases, there is no relationship between genotype and phenotype in alpha-mannosidosis. Enzyme replacement therapy is at the moment the most effective therapy for lysosomal storage disease, including alpha-mannosidosis. In this review, the genetic of alpha-mannosidosis has been described together with the results so far obtained by two different therapeutic strategies: bone marrow transplantation and enzyme replacement therapy. The primary indication to offer hematopoietic stem cell transplantation in patients affected by alpha-mannosidosis is preservation of neurocognitive function and prevention of early death. The results obtained from a Phase I⁻II study and a Phase III study provide evidence of the positive clinical effect of the recombinant enzyme on patients with alpha-mannosidosis.

Human T-cell lymphotropic virus type 1-infected T lymphocytes impair catabolism and uptake of glutamate by astrocytes via Tax-1 and tumor necrosis factor alpha.

PubMed

Szymocha, R; Akaoka, H; Dutuit, M; Malcus, C; Didier-Bazes, M; Belin, M F; Giraudon, P

2000-07-01

Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of a chronic progressive myelopathy called tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). In this disease, lesions of the central nervous system (CNS) are associated with perivascular infiltration by lymphocytes. We and others have hypothesized that these T lymphocytes infiltrating the CNS may play a prominent role in TSP/HAM. Here, we show that transient contact of human or rat astrocytes with T lymphocytes chronically infected by HTLV-1 impairs some of the major functions of brain astrocytes. Uptake of extracellular glutamate by astrocytes was significantly decreased after transient contact with infected T cells, while the expression of the glial transporters GLAST and GLT-1 was decreased. In two-compartment cultures avoiding direct cell-to-cell contact, similar results were obtained, suggesting possible involvement of soluble factors, such as cytokines and the viral protein Tax-1. Recombinant Tax-1 and tumor necrosis factor alpha (TNF-alpha) decreased glutamate uptake by astrocytes. Tax-1 probably acts by inducing TNF-alpha, as the effect of Tax-1 was abolished by anti-TNF-alpha antibody. The expression of glutamate-catabolizing enzymes in astrocytes was increased for glutamine synthetase and decreased for glutamate dehydrogenase, the magnitudes of these effects being correlated with the level of Tax-1 transcripts. In conclusion, Tax-1 and cytokines produced by HTLV-1-infected T cells impair the ability of astrocytes to manage the steady-state level of glutamate, which in turn may affect neuronal and oligodendrocytic functions and survival.

Gastroschisis in the rat model is associated with a delayed maturation of intestinal pacemaker cells and smooth muscle cells.

PubMed

Midrio, P; Faussone-Pellegrini, M S; Vannucchi, M G; Flake, A W

2004-10-01

A pacemaker system is required for peristalsis generation. The interstitial cells of Cajal (ICC) are considered the intestinal pacemaker, and are identified by expression of the c-kit gene--encoded protein. Gastroschisis is characterized by a severe gastrointestinal dysmotility in newborns. In spite of this clinical picture, few studies have focused on smooth muscle cells (SMC) morphology and none on ICC. Therefore, their morphology has been studied in fetuses at term in the rat model of gastroschisis. At 18.5 day's gestation (E18.5), 10 rat fetuses were killed, 10 underwent surgical creation of gastroschisis, and 10 underwent manipulation only. The small intestine of the latter 2 groups was harvested at E21.5. Specimens were processed for H&E, c-kit and actin (alpha smooth muscle antibody [alpha-SMA]) immunohistochemistry, and transmission electron microscopy (TEM). In the controls, SMC were c-kit+ and alpha-SMA+, with labeling intensity increasing by age. At E21.5, some cells around the Auerbach's plexus were more intensely c-kit+, and differentiating ICC were seen under TEM at this level. Gastroschisis fetuses had no c-kit+ cells referable to ICC. In the more damaged loops, SMC were very faintly c-kit+ and alpha-SMA+. Under TEM, there were few differentiated SMC and no presumptive ICC. In the less-damaged loops, SMC were faintly c-kit+ and alpha-SMA+ and had ultrastructural features intermediate between those of E18.5 and E21.5 controls; ICC were very immature. ICC and SMC differentiation is delayed in gastroschisis with the most damaged loops showing the most incomplete picture. These findings might help in understanding the delayed onset of peristalsis and the variable time-course of the recover seen in babies affected by gastroschisis.

Effect of alpha-tocopherol on bovine in vitro fertilization.

PubMed

Marques, A; Santos, P; Antunes, G; Chaveiro, A; Moreira da Silva, F

2010-02-01

The objectives of this work are to determine if exogenous supplementation with alpha-tocopherol increases the in vitro fertilization (IVF) rate of bovine oocytes and improves viability of selected spermatozoa after 'swim-up'. The percentage of fertilized oocytes was significantly but negatively correlated (r = -0.941, p < 0.01) with the concentration of alpha-tocopherol. The control resulted in 95% of fertilized oocytes, which decreased as follows: 25 microM alpha-tocopherol (alpha25) 86%, 50 microM alpha-tocopherol (alpha50) 74%, 100 microM alpha-tocopherol (alpha100) 66% and 200 microM alpha-tocopherol (alpha200) 56%. Relatively to sperm viability after 'swim-up' with alpha-tocopherol supplementation, this antioxidant proved to have a beneficial effect as its concentration increased up to alpha50, decreasing for the concentrations of alpha100 and alpha200. Control resulted in 83% of live cells and 16% of dead cells; alpha25 resulted in 88% of live cells and 12% of dead cells; alpha50 resulted in 91% of live cells and 9% of dead cells; alpha100 resulted in 67% of live cells and 33% of dead cells; and finally alpha200 resulted in 57% of live cells and 42% of dead cells. In summary, the present study allows to conclude that, in our conditions, supplementation with the antioxidant alpha-tocopherol in IVF of bovine oocytes has a detrimental effect on fertilization rates. Nevertheless, exogenous supplementation with alpha-tocopherol at a concentration of 50 mM in the sperm-TALP media during the 'swim-up' technique has a significant beneficial effect on the selected spermatozoa viability.

Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Soeda, Junpei; Morgan, Maelle; McKee, Chad

Highlights: Black-Right-Pointing-Pointer Cigarette smoke may induce liver fibrosis via nicotine receptors. Black-Right-Pointing-Pointer Nicotine induces proliferation of hepatic stellate cells (HSCs). Black-Right-Pointing-Pointer Nicotine activates hepatic fibrogenic pathways. Black-Right-Pointing-Pointer Nicotine receptor antagonists attenuate HSC proliferation. Black-Right-Pointing-Pointer Nicotinic receptor antagonists may have utility as novel anti-fibrotic agents. -- Abstract: Background and aims: Cigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine - which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells inmore » the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH). Methods: hHSC were isolated from healthy human livers and nAChR expression analyzed - RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-{alpha}2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-{beta}1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH). Results: hHSCs express muscle type ({alpha}1, {beta}1, delta and epsilon) and neuronal type ({alpha}3, {alpha}6, {alpha}7, {beta}2 and {beta}4) nAChR subunits at the mRNA level. Among these subunits, {alpha}3, {alpha}7, {beta}1 and {epsilon} were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-{alpha}2 and TGF-{beta}1 mRNA expression were significantly upregulated by nicotine and inhibited by mecamylamine. {alpha}1 and {alpha}3-nAChR mRNA expression was significantly upregulated in NASH fibrosis compared to normal livers. Conclusion: Nicotine at levels in smokers' blood is pro-fibrogenic, through actions on hHSCs expressed nAChRs. Therefore, CS, via its nicotine content, may worsen liver fibrosis. Moreover, nicotinic receptor antagonists may have utility as novel anti-fibrotic agents.« less

Maternal Obesity Accelerates Fetal Pancreatic Beta Cell but not Alpha Cell Development in the Sheep: Prenatal and Postnatal Consequences

USDA-ARS?s Scientific Manuscript database

Maternal obesity affects offspring weight, body composition and organ function, increasing diabetes and metabolic syndrome risk. We determined effects of maternal obesity and a high energy diet on fetal pancreatic development. Sixty days prior to breeding. ewes were assigned to control (C, 100% of N...

Down-regulation of Jab1, HIF-1alpha, and VEGF by Moloney murine leukemia virus-ts1 infection: a possible cause of neurodegeneration.

PubMed

Lungu, Gina F; Stoica, George; Wong, Paul K Y

2008-05-01

Moloney murine leukemia virus-temperature sensitive (MoMuLV-ts1)-mediated neuronal death is a result of both loss of glial support and release of cytokines and neurotoxins from ts1-infected glial cells. Here the authors propose vascular endothelial growth factor (VEGF) down-regulation as another contributory factor in neuronal degeneration induced by ts1 infection. To determine how ts1 affects VEGF expression in ts1-infected brain, the authors examined the expression of several proteins that are important in regulating the expression of VEGF. The authors found significant decreases in Jun-activating domain-binding protein 1 (Jab1), hypoxia-inducible factor (HIF)-1alpha, and VEGF levels and increases in p53 protein levels in ts1-infected brains compared to noninfected control brains. The authors suggest that a decrease Jab1 expression in ts1 infection leads to accumulation of p53, which binds to HIF-1alpha to accelerate its degradation. A rapid degradation of HIF-1alpha leads to decreased VEGF production and secretion. Considering that endothelial cells are the most conspicuous in virus replication and production in ts1 infection, but are not killed by the infection, the authors examined the expression of these proteins using infected and noninfected mouse cerebrovascular endothelial (CVE) cells. The ts1- infected CVE cells showed decreased Jab1, HIF-1alpha, and VEGF mRNA and protein levels and increased p53 protein levels compared with noninfected cells, consistent with the results found in vivo. These results confirm that ts1 infection results in insufficient secretion of VEGF from endothelial cells and may result in decreased neuroprotection. This study suggested that ts1-mediated neuropathology in mice may result from changes in expression and activity of Jab1, p53, and HIF-1alpha, with a final target on VEGF expression and neuronal degeneration.

Characterization of alpha(4)beta(1) (CD49d/CD29) on equine leukocytes: potential utility of a potent alpha(4)beta(1) (CD49d/CD29) receptor antagonist in the treatment of equine heaves (recurrent airway obstruction).

PubMed

Treonze, Kelly M; Alves, Kenneth; Fischer, Paul; Hagmann, William K; Hora, Donald; Kulick, Alison; Vakerich, Ken; Smith, Nicholas D; Lingham, Russell B; Maniar, Salony; Reger, Thomas S; Zunic, Jasmine; Munoz, Benito; Prasit, Peppi; Nicholson, Donald; Si, Qian; Judd, Keith; Nicolich, Susan; Kellerhouse, Patricia; Thompson, Donald; Mumford, Richard A

2009-07-15

The purpose of this study was to characterize the alpha(4)beta(1) receptor (CD49d/CD29, very late antigen-4, VLA-4) on circulating equine leukocytes and to evaluate the intrinsic potency of an alpha(4)beta(1) receptor antagonist (Compound B) in the horse. Ultimately, these studies would allow us to determine the suitability of treating recurrent airway obstruction (RAO; heaves) affected horses by blocking the cellular recruitment of lymphocytes and neutrophils into the lung. The data demonstrates the alpha(4)beta(1) integrin is present on horse lymphocytes and neutrophils (fluorescence-assisted cell sorter, FACS) and can bind low molecular weight alpha(4)beta(1) antagonists (Compounds A and B) with high affinity. K(D) values for the binding of Compound A to non-activated alpha(4)beta(1) on isolated horse PBMCs (peripheral blood mononuclear cells) and activated neutrophils were 17 pM and 27 pM, respectively. Compound B was identified as a suitable antagonist for performing a series of in vivo experiments. Compound B was found to possess excellent potency in horse whole blood, possessing IC(50) and IC(90) values of 39 pM and 172 pM, respectively. This represents a 3.9-fold molar excess of drug over the alpha(4)beta(1) concentration in blood. Following oral administration of Compound B (5 mg/kg) to beagle dogs and rhesus monkeys, rapid and sustained alpha(4)beta(1) receptor occupancy (>80%) was achieved and maintained for a period of 24 h. When Compound B was administered intravenously to the horse, by either a slow or rapid infusion at a dose of 0.3 mg/kg, receptor blockade of >80% was observed out to 24 h with a concomitant leukocytosis. We believe that Compound B possesses suitable intrinsic and pharmacological properties to be evaluated clinically in horses affected by RAO.

The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by alpha-Cyano-4-hydroxycinnamate and related compounds.

PubMed

Halestrap, A P; Denton, R M

1975-04-01

1. Effects of alpha-cyano-4-hydroxycinnamate and alpha-cyanocinnamate on a number of enzymes involved in pyruvate metabolism have been investigated. Little or no inhibition was observed of any enzyme at concentrations that inhibit completely mitochondrial pyruvate transport. At much higher concentrations (1 mM) some inhibition of pyruvate carboxylase was apparent. 2. Alpha-Cyano-4-hydroxycinnamate (1-100 muM) specifically inhibited pyruvate oxidation by mitochondria isolated from rat heart, brain, kidney and from blowfly flight muscle; oxidation of other substrates in the presence or absence of ADP was not affected. Similar concentrations of the compound also inhibited the carboxylation of pyruvate by rat liver mitochondria and the activation by pyruvate of pyruvate dehydrogenase in fat-cell mitochondria. These findings imply that pyruvate dehydrogenase, pyruvate dehydrogenase kinase and pyruvate carboxylase are exposed to mitochondrial matrix concentrations of pyruvate rather than to cytoplasmic concentrations. 3. Studies with whole-cell preparations incubated in vitro indicate that alpha-cyano-4-hydroxycinnamate or alpha-cyanocinnamate (at concentrations below 200 muM) can be used to specifically inhibit mitochondrial pyruvate transport within cells and thus alter the metabolic emphasis of the preparation. In epididymal fat-pads, fatty acid synthesis from glucose and fructose, but not from acetate, was markedly inhibited. No changes in tissue ATP concentrations were observed. The effects on fatty acid synthesis were reversible. In kidney-cortex slices, gluconeogenesis from pyruvate and lactate but not from succinate was inhibited. In the rat heart perfused with medium containing glucose and insulin, addition of alpha-cyanocinnamate (200 muM) greatly increased the output and tissue concentrations of lactate plus pyruvate but decreased the lactate/pyruvate ratio. 4. The inhibition by cyanocinnamate derivatives of pyruvate transport across the cell membrane of human erythrocytes requires much higher concentrations of the derivatives than the inhibition of transport across the mitochondrial membrane. Alpha-Cyano-4-hydroxycinnamate appears to enter erythrocytes on the cell-membrane pyruvate carrier. Entry is not observed in the presence of albumin, which may explain the small effects when these compounds are injected into whole animals.

Induction of experimental bone metastasis in mice by transfection of integrin alpha 4 beta 1 into tumor cells.

PubMed Central

Matsuura, N.; Puzon-McLaughlin, W.; Irie, A.; Morikawa, Y.; Kakudo, K.; Takada, Y.

1996-01-01

Cell adhesion receptors (eg, integrins and CD44) play an important role in invasion and metastasis during tumor progression. The increase in integrin alpha 4 beta 1 expression on primary melanomas has been reported to significantly correlate with the development of metastases. alpha 4 beta 1 is a cell surface heterodimer that mediates cell-cell and cell-extracellular matrix interactions through adhesion to vascular cell adhesion molecule (VCAM)-1 and to the IIICS region of fibronectin. To test the effects of alpha 4 beta 1 expression on tumor cell metastasis, Chinese hamster ovary cells were transfected with human alpha 4 cDNA. Whereas alpha 4-negative Chinese hamster ovary cells developed only pulmonary metastasis, alpha 4-positive Chinese hamster ovary cells developed bone and pulmonary metastasis in 3 to 4 weeks when injected intravenously into nude mice. Bone metastasis was inhibited by antibody against alpha 4 or VCAM-1. Expression of alpha 3 beta 1, alpha 6 beta 1, or alpha V beta 1 did not induce bone metastasis. Expression of alpha 4 beta 1 also induced bone metastasis in K562 human erythroleukemia cells injected into SCID mice. These results demonstrate that alpha 4 beta 1 can induce tumor cell trafficking to bone, probably via interaction with VCAM-1 that is constitutively expressed on bone marrow stromal cells. Images Figure 1 Figure 3 PMID:8546226

Administration of progesterone after trauma and hemorrhagic shock prevents hepatocellular injury.

PubMed

Kuebler, Joachim F; Yokoyama, Yukihiro; Jarrar, Doraid; Toth, Balazs; Rue, Loring W; Bland, Kirby I; Wang, Ping; Chaudry, Irshad H

2003-07-01

Administration of a single dose of progesterone following trauma and hemorrhage in progesterone-deficient rats would ameliorate the inflammatory response and hepatocellular damage. A university laboratory. Ovariectomized female Sprague-Dawley rats (250-350 g; Charles River Laboratories, Wilmington, Mass) underwent a 5-cm midline laparotomy (ie, induction of soft tissue trauma), were bled to a mean arterial blood pressure of 35 mm Hg for about 90 minutes, and then were resuscitated using Ringer lactate solution. Progesterone (25 mg/kg of body weight) or vehicle was administered subcutaneously at the end of resuscitation. In additional animals, Kupffer cells were isolated following trauma, hemorrhage, and resuscitation and treated in vitro with progesterone, lipopolysaccharide, or both. Six hours following resuscitation, plasma tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) levels and liver myeloperoxidase activity were determined. Hepatocellular function (maximum velocity of indocyanine green clearance [Vmax] and the efficiency of the active transport or Michaelis-Menten constant [Km]) and plasma levels of transaminases were measured 20 hours after resuscitation. Kupffer cell IL-6 and TNF-alpha production were assessed. Plasma levels of TNF-alpha, IL-6, aspartate aminotransferase, and alanine aminotransferase, as well as hepatic myeloperoxidase activity were increased, whereas indocyanine green clearance was depressed in vehicle-treated rats following trauma-hemorrhage. Animals treated with progesterone showed significantly reduced levels of the TNF-alpha, IL-6, and transaminases as well as reduced myeloperoxidase activity in the liver. Progesterone-treated animals showed increased Vmax and Kmax values for indocyanine green. In vitro treatment of Kupffer cells with progesterone decreased TNF-alpha production but did not affect the production of IL-6. Progesterone administration following trauma-hemorrhage ameliorates the proinflammatory response and, subsequently, the hepatocellular injury via direct action on immunocompetent cells.

Alterations in dendritic cell function in aged mice: potential implications for immunotherapy design.

PubMed

Paula, Carine; Motta, Adriana; Schmitz, Carla; Nunes, Claudia P; Souza, Ana Paula; Bonorino, Cristina

2009-02-01

It is known that immune system functions decrease with age, and that adaptive immune responses, especially CD4+ T cell function, seem to be the main affected point in immunity with aging. Dendritic cells (DC) are the major antigen presenting cell (APC), and at least part of the defects observed in adaptive immunity of aged individuals could be due to diminished potential of bone marrow to generate new DC, or defects in DC function. In this study, we investigated if the ability of aged bone marrow (BM) to generate new DC in vitro, as well as aged BM-derived DC responses to lypopolysaccharide (LPS). Because DC are important tools in newly developing anti-tumor therapies, we also studied the ability of aged DC to phagocytose and present antigen from necrotic tumor cells. We found that aged BM generated fewer DC in vitro compared to young BM. While LPS-induced DC maturation is reduced in DC of aged mice, a high TNF-alpha production is observed in aged DC even without LPS stimulation. While phagocytosis of tumor cells is not affected by age, and DC derived from aged BM show a higher TNF-alpha production in response to phagocytosis, presentation of tumor antigens was decreased in aged DC. Because class II upregulation in response to phagocytosis was similar between aged and young DC, this could indicate an age associated processing defect in the exogenous pathway. These findings suggest that age of BM used to generate DC does not impair their phagocytic ability or TNF-alpha production, however leads to a decreased yield in mature DC, reduced response to LPS, and diminished antigen processing/presentation potential. Our results are relevant to optimization DC-based vaccine design for aged populations.

Ubiquitin carboxyl terminal hydrolase L1 negatively regulates TNF{alpha}-mediated vascular smooth muscle cell proliferation via suppressing ERK activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ichikawa, Tomonaga; Li, Jinqing; Dong, Xiaoyu

2010-01-01

Deubiquitinating enzymes (DUBs) appear to be critical regulators of a multitude of processes such as proliferation, apoptosis, differentiation, and inflammation. We have recently demonstrated that a DUB of ubiquitin carboxyl terminal hydrolase L1 (UCH-L1) inhibits vascular lesion formation via suppressing inflammatory responses in vasculature. However, the precise underlying mechanism remains to be defined. Herein, we report that a posttranscriptional up-regulation of UCH-L1 provides a negative feedback to tumor necrosis factor alpha (TNF{alpha})-mediated activation of extracellular signal-regulated kinases (ERK) and proliferation in vascular smooth muscle cells (VSMCs). In rat adult VSMCs, adenoviral over-expression of UCH-L1 inhibited TNF{alpha}-induced activation of ERK andmore » DNA synthesis. In contrast, over-expression of UCH-L1 did not affect platelet derived growth factor (PDGF)-induced VSMC proliferation and activation of growth stimulating cascades including ERK. TNF{alpha} hardly altered UCH-L1 mRNA expression and stability; however, up-regulated UCH-L1 protein expression via increasing UCH-L1 translation. These results uncover a novel mechanism by which UCH-L1 suppresses vascular inflammation.« less

Marrow stromal cells from patients affected by MPS I differentially support haematopoietic progenitor cell development.

PubMed

Baxter, M A; Wynn, R F; Schyma, L; Holmes, D K; Wraith, J E; Fairbairn, L J; Bellantuono, I

2005-01-01

Bone marrow transplantation is the therapy of choice in patients affected by MPS I (Hurler syndrome), but a high incidence of rejection limits the success of this treatment. The deficiency of alpha-L-iduronidase (EC 1.2.3.76), one of the enzymes responsible for the degradation of glycosaminoglycans, results in accumulation of heparan and dermatan sulphate in these patients. Heparan sulphate and dermatan sulphate are known to be important components of the bone marrow microenvironment and critical for haematopoietic cell development. In this study we compared the ability of marrow stromal cells from MPS I patients and healthy donors to support normal haematopoiesis in Dexter-type long term culture. We found an inverse stroma/supernatant ratio in the number of clonogenic progenitors, particularly the colony-forming unit granulocyte-machrophage in MPS I cultures when compared to normal controls. No alteration in the adhesion of haematopoietic cells to the stroma of MPS I patients was found, suggesting that the altered distribution in the number of clonogenic progenitors is probably the result of an accelerated process of differentiation and maturation. The use of alpha-L-iduronidase gene-corrected marrow stromal cells re-established normal haematopoiesis in culture, suggesting that correction of the bone marrow microenvironment with competent enzyme prior to transplantation might help establishment of donor haematopoiesis.

Integrin-mediated transforming growth factor-beta activation regulates homeostasis of the pulmonary epithelial-mesenchymal trophic unit.

PubMed

Araya, Jun; Cambier, Stephanie; Morris, Alanna; Finkbeiner, Walter; Nishimura, Stephen L

2006-08-01

Trophic interactions between pulmonary epithelial and mesenchymal cell types, known as the epithelial-mesenchymal trophic unit (EMTU), are crucial in lung development and lung disease. Transforming growth factor (TGF)-beta is a key factor in mediating these interactions, but it is expressed in a latent form that requires activation to be functional. Using intact fetal tracheal tissue and primary cultures of fetal tracheal epithelial cells and fibroblasts, we demonstrate that a subset of integrins, alpha(v)beta(6) and alpha(v)beta(8), are responsible for almost all of the TGF-beta activation in the EMTU. Both alpha(v)beta(8) and alpha(v)beta(6) contribute to fetal tracheal epithelial activation of TGF-beta, whereas only alpha(v)beta(8) contributes to fetal tracheal fibroblast activation of TGF-beta. Interestingly, fetal tracheal epithelial alpha(v)beta(8)-mediated TGF-beta activation can be enhanced by phorbol esters, likely because of the increased activity of MT1-MMP, an essential co-factor in alpha(v)beta(8)-mediated activation of TGF-beta. Autocrine alpha(v)beta(8)-mediated TGF-beta activation by fetal tracheal fibroblasts results in suppression of both transcription and secretion of hepatocyte growth factor, which is sufficient to affect phosphorylation of the airway epithelial hepatocyte growth factor receptor, c-Met, as well as airway epithelial proliferation in a co-culture model of the EMTU. These findings elucidate the function and complex regulation of integrin-mediated activation of TGF-beta within the EMTU.

Involvement of the Clock Gene Rev-erb alpha in the Regulation of Glucagon Secretion in Pancreatic Alpha-Cells

PubMed Central

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

2013-01-01

Disruption of pancreatic clock genes impairs pancreatic beta-cell function, leading to the onset of diabetes. Despite the importance of pancreatic alpha-cells in the regulation of glucose homeostasis and in diabetes pathophysiology, nothing is known about the role of clock genes in these cells. Here, we identify the clock gene Rev-erb alpha as a new intracellular regulator of glucagon secretion. Rev-erb alpha down-regulation by siRNA (60–70% inhibition) in alphaTC1-9 cells inhibited low-glucose induced glucagon secretion (p<0.05) and led to a decrease in key genes of the exocytotic machinery. The Rev-erb alpha agonist GSK4112 increased glucagon secretion (1.6 fold) and intracellular calcium signals in alphaTC1-9 cells and mouse primary alpha-cells, whereas the Rev-erb alpha antagonist SR8278 produced the opposite effect. At 0.5 mM glucose, alphaTC1-9 cells exhibited intrinsic circadian Rev-erb alpha expression oscillations that were inhibited by 11 mM glucose. In mouse primary alpha-cells, glucose induced similar effects (p<0.001). High glucose inhibited key genes controlled by AMPK such as Nampt, Sirt1 and PGC-1 alpha in alphaTC1-9 cells (p<0.05). AMPK activation by metformin completely reversed the inhibitory effect of glucose on Nampt-Sirt1-PGC-1 alpha and Rev-erb alpha. Nampt inhibition decreased Sirt1, PGC-1 alpha and Rev-erb alpha mRNA expression (p<0.01) and glucagon release (p<0.05). These findings identify Rev-erb alpha as a new intracellular regulator of glucagon secretion via AMPK/Nampt/Sirt1 pathway. PMID:23936124

Receptor-selective retinoids implicate retinoic acid receptor alpha and gamma in the regulation of bmp-2 and bmp-4 in F9 embryonal carcinoma cells.

PubMed

Rogers, M B

1996-01-01

The effect of retinoids on malignant cells and embryos indicates that retinoids influence the expression of growth factors or alter the response of cells to growth factors. The bone morphogenetic proteins, Bmp-2 and Bmp-4, are candidates for such growth factors because retinoic acid (RA) treatment of F9 embryonal carcinoma cells induced Bmp-2 mRNA, while simultaneously repressing Bmp-4 levels. Also, recombinant Bmp-2 affected the growth and differentiation of these cells. Regulation of each gene was concentration dependent and required continuous RA treatment. The short half-lives of the Bmp-2 (75 +/- 11 min) and Bmp-4 (70 +/- 4 min) mRNAs suggest that their abundance is primarily controlled at the transcriptional level. To determine which RA receptor (RAR) controls bmp-2 and bmp-4 expression, F9 cells were exposed to various receptor-selective retinoids. RAR alpha- and gamma-selective retinoids induced Bmp-2 and repressed Bmp-4 equally as well as all-trans RA. In contrast, a RAR beta-selective retinoid had little effect on Bmp-2 induction but repressed Bmp-4. A RAR alpha-selective antagonist inhibited all-trans RA stimulation of Bmp-2, although not as dramatically as a RAR beta gamma-selective antagonist. No differences were observed between Bmp levels in all-trans RA and 9-cis RA-treated cells, indicating that the RXRs play little part in controlling these genes. The results are consistent with RAR alpha and gamma-controlled Bmp-2 and Bmp-4 regulation.

Distributional map of the terminal and sub-terminal sugar residues of the glycoconjugates in the prepubertal and postpubertal testis of a subject affected by complete androgen insensitivity syndrome (Morris's syndrome): lectin histochemical study.

PubMed

Gheri, G; Vannelli, G B; Marini, M; Zappoli Thyrion, G D; Gheri, R G; Sgambati, E

2004-01-01

In the present research we have investigated the distribution of the sugar residues of the glycoconjugates in the prepubertal and postpubertal testes of a subject with Morris's syndrome (CAIS, Complete Androgen Insensitivity Syndrome). For this purpose a battery of six horseradish peroxidase-conjugated lectins was used (SBA, PNA, WGA, ConA, LTA and UEAI). We have obtained a complete distributional map of the terminal and sub-terminal oligosaccharides in the tunica albuginea, interstitial tissue, lamina propria of the seminiferous tubules, Leydig cells, Sertoli cells, spermatogonia, mastocytes and endothelial cells. Furthermore the present study has shown that a large amount of sugar residues were detectable in the prepubertal and postpubertal testes but that some differences exist with particular regard to the Sertoli cells. The Sertoli cells and the Leydig cells of the retained prepubertal testis of the patient affected by Morris's syndrome were characterized by the presence of alpha-L-fucose, which was absent in the retained prepubertal testis of the normal subjects. Comparing the results on the postpubertal testis with those obtained on the same aged testis of healthy subjects we have demonstrated that alpha-L-fucose in the Sertoli and Leydig cells and D-galactose-N-acetyl-D-galactosamine in the Leydig cells are a unique feature of the subject affected by Morris's syndrome. D-galactose (ss1,3)-N-acetyl-D-galactosamine and sialic acid, which are present in the Leydig cells of the normal testis were never observed in the same cells of the postpubertal testis of the CAIS patient.

Molecular pathways mediating differential responses to lipopolysaccharide between human and baboon arterial endothelial cells.

PubMed

Shi, Qiang; Cox, Laura A; Glenn, Jeremy; Tejero, Maria E; Hondara, Vida; Vandeberg, John L; Wang, Xing Li

2010-02-01

1. Vascular inflammation plays a critical role in atherogenesis. Previously, we showed that baboon arterial endothelial cells (BAEC) were hyporesponsive to lipopolysaccharide (LPS) compared with human arterial endothelial cells (HAEC). 2. In the present study, we investigated mechanisms underlying differential responses between HAEC and BAEC to tumour necrosis factor (TNF)-alpha and LPS. 3. Both HAEC and BAEC responded similarly to TNF-alpha. However, BAEC showed retarded responses to LPS in expression of E-selectin, intercellular adhesion molecule-1, monocyte chemotactic protein-1 and interleukin-8 (P < 0.05). These changes were confirmed at the mRNA level. Tumour necrosis factor-alpha activated nuclear factor-kappaB members such as p50, p52, p65, c-rel and RelB in both HAEC and BAEC. In contrast, LPS activated p50 and p65 only in HAEC. Using microarray assays, we found that TNF receptor-associated factor 2 (TRAF-2), TNF receptor superfamily, member 1A-associated via death domain (TRADD) and nuclear factors such as nuclear factor of kappa in B-cells inhibitor, alpha (NFKBIA) and nuclear factor of kappa in B-cells inhibitor, beta (NFKBIB) were upregulated by LPS only in HAEC. Although the baseline expression of Toll-like receptor (TLR) 4 was low in both HAEC and BAEC, TNF-alpha activated TLR4 expression in both cell types. Although LPS increased TLR4 expression only in HAEC, human and baboon peripheral blood mononuclear cells exhibited similar TLR4 expression and response to LPS. Transfecting BAEC with TLR4/myeloid differentiation protein-2 overexpression vector conferred BAEC responsiveness to LPS. 4. The findings of the present study indicate that an altered TLR4 system may be responsible for the resistance of baboon endothelial cells to LPS. Given the importance of TLR4 in human immune responses and vascular diseases, the natural resistance of baboons to LPS/TLR4-initiated inflammation could make the baboon a valuable animal model in which to study how inflammation affects atherogenesis.

Immunological alterations during the clinical and recovery phases of experimental swine dysentery.

PubMed

Jonasson, Robert; Andersson, Märit; Råsbäck, Therese; Johannisson, Anders; Jensen-Waern, Marianne

2006-07-01

The aim of this study was to examine changes in the systemic immune response during the incubation period and following the onset of clinical swine dysentery, including the recovery period. Ten healthy conventional pigs were inoculated with Brachyspira hyodysenteriae. Blood was sampled at pre-inoculation, at days 4 and 14 post-inoculation, during the first 4 days with clinical signs of dysentery and at days 1, 3, 7, 11 and 15 of the recovery period. Eight pigs developed haemorrhagic diarrhoea. Flow-cytometric analyses of lymphocyte subpopulations showed that all animals, including the two that remained healthy, had an increase in CD8alpha+ CD4- cells and gammadelta T cells at days 4 and 14 post-inoculation. In addition, an increase in CD4+ CD8alpha+ cells and CD8alpha+ CD8beta+ cells was observed at days 4 and 14 post-inoculation in animals that developed dysentery. During clinical signs of dysentery, the acute-phase protein serum amyloid A was increased. There was a two- to threefold increase in both neutrophils and monocytes during signs of dysentery and at the beginning of the recovery period. The numbers of CD8alpha+ CD8beta- CD4-, CD45RA- lymphocytes also increased during the dysentery period. Circulating CD21+ cells and CD21+ CD45RA- cells decreased at the end of the incubation period, during signs of dysentery and at the beginning of the recovery period. The dysentery-affected animals developed antibodies to B. hyodysenteriae-specific antigens (approximately 16 kDa and approximately 30 kDa) from the first day of recovery, and gammadelta T cells showed an increase during the recovery period. In comparison with pre-inoculation, increased numbers of monocytes, neutrophils, CD8alpha+ CD8beta- CD4- lymphocytes and CD45RA- lymphocytes were observed during clinical dysentery. Increased numbers of neutrophils, gammadelta T cells and specific antibodies were seen during the recovery period.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saji, Chiaki; Higashi, Chizuka; Niinaka, Yasufumi

Highlights: Black-Right-Pointing-Pointer Constitutive NF-{kappa}B signaling is essential for the survival and growth of PEL cells. Black-Right-Pointing-Pointer NF-{kappa}B signaling is upregulated by the proteasome-dependent degradation of I{kappa}B{alpha}. Black-Right-Pointing-Pointer Proteasome inhibitors suppress NF-{kappa}B signaling and induce apoptosis in PEL cells through stabilization of I{kappa}B{alpha}. Black-Right-Pointing-Pointer Proteasome inhibitors suppress viral replication in PEL cells during lytic KSHV infection. -- Abstract: Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV). This study provides evidence that proteasomal activity is required for both survival of PEL cells stably harboring the KSHV genome and viral replication of KSHV. We evaluated the cytotoxicmore » effects of proteasome inhibitors on PEL cells. The proteasome inhibitors MG132, lactacystin, and proteasome inhibitor I dramatically inhibited cell proliferation and induced apoptosis of PEL cells through the accumulation of p21 and p27. Furthermore, proteasome inhibitors induced the stabilization of NF-{kappa}B inhibitory molecule (I{kappa}B{alpha}) and suppressed the transcriptional activity of NF-{kappa}B in PEL cells. The NF-{kappa}B specific inhibitor BAY11-7082 also induced apoptosis in PEL cells. The constitutive activation of NF-{kappa}B signaling is essential for the survival and growth of B cell lymphoma cells, including PEL cells. NF-{kappa}B signaling is upregulated by proteasome-dependent degradation of I{kappa}B{alpha}. The suppression of NF-{kappa}B signaling by proteasome inhibitors may contribute to the induction of apoptosis in PEL cells. In addition, proteasome activity is required for KSHV replication in KSHV latently infected PEL cells. MG132 reduced the production of progeny virus from PEL cells at low concentrations, which do not affect PEL cell growth. These findings suggest that proteasome inhibitors may represent a novel strategy for the treatment of KSHV infection and KSHV-associated lymphomas.« less

Staurosporine, but not Ro 31-8220, induces interleukin 2 production and synergizes with interleukin 1alpha in EL4 thymoma cells.

PubMed

Mahon, T M; Matthews, J S; O'Neill, L A

1997-07-01

Protein kinase C (PKC) has been implicated in interleukin 1 (IL1) signal transduction in a number of cellular systems, either as a key event in IL1 action or as a negative regulator. Here we have examined the effects of two PKC inhibitors, staurosporine and the more selective agent Ro 31-8220, on IL1 responses in the murine thymoma line EL4.NOB-1. A 1 h pulse of staurosporine was found to strongly potentiate the induction of IL2 by IL1alpha in these cells. In contrast, neither a pulse nor prolonged incubation with Ro 31-8220 affected the response to IL1alpha. Both agents blocked the response to PMA, however. A 1 h pulse of staurosporine was also found to induce IL2 production on its own, activate the transcription factor nuclear factor kappaB (NFkappaB) and increase the expression of a NFkappaB-linked reporter gene. It synergized with IL1alpha in all of these responses. Ro 31-8220 was again without effect, although both staurosporine and Ro 31-8220 blocked the activation of NFkappaB by PMA. Finally, staurosporine caused the translocation of PKC-alpha and -epsilon, and to a lesser extent PKC-beta, but not PKC-θ or -zeta, from the cytosol to the membrane, although a similar effect was observed with Ro 31-8220. The results suggest that PKC is not involved in IL1alpha signalling in EL4 cells. Furthermore, the potentiating effect of staurosporine on IL1alpha action does not involve PKC inhibition, and is likely to be at the level of NFkappaB activation.

G protein abnormalities in pituitary adenomas.

PubMed

Spada, A; Lania, A; Ballarè, E

1998-07-25

It has been demonstrated that the majority of secreting and nonsecreting adenomas is monoclonal in origin suggesting that these neoplasia arise from the replication of a single mutated cell, in which growth advantage results from either activation of protooncogenes or inactivation of antioncogenes. Although a large number of genes has been screened for mutations, only few genetic abnormalities have been found in pituitary tumors such as allelic deletion of chromosome 11q13 where the MEN-1 gene has been localised, and mutations in the gene encoding the alpha subunit of the stimulatory Gs and Gi2 protein. These mutations constitutively activate the alpha subunit of the Gs and Gi2 protein by inhibiting their intrinsic GTPase activity. Both Gs alpha and Gi2alpha can be considered products of protooncogenes (gsp and gip2, respectively) since gain of function mutations that activate mitogenic signals have been recognized in human tumors. Gsp oncogene is found in 30-40% of GH-secreting adenomas, in a low percentage of nonfunctioning and ACTH-secreting pituitary adenomas, in toxic thyroid adenomas and differentiated thyroid carcinomas. The same mutations, occurred early in embriogenesis, have been also identified in tissues from patients affected with the McCune Albright syndrome. These mutations result in an increased cAMP production and in the subsequent overactivation of specific pathways involved in both cell growth and specific programmes of cell differentiation. By consequence, the endocrine tumors expressing gsp oncogene retain differentiated functions. The gip2 oncogene has been identified in about 10% of nonfunctioning pituitary adenomas, in tumors of the ovary and the adrenal cortex. However, it remains to be established whether Gi proteins activate mitogenic signals in pituitary cells. Since Gi proteins are involved in mediating the effect of inhibitory neurohormones on intracellular effectors, it has been proposed that in pituitary tumors the low expression of these proteins, particularly Gi1-3alpha, may contribute to uncontrolled pituitary cells growth by preventing the transduction of inhibitory signals. While by in vitro mutagenesis it has been demonstrated that activated mutant of Gq alpha, G12alpha, G13alpha and Gz alpha are fully oncogenic, it remains to be proved whether or not these abnormalities might naturally occur in human tumors and, in particular, in pituitary adenomas.

Chromosomal aberrations and delays in cell progression induced by x-rays in Tradescantia clone 02 meristems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Geard, C.R.

1983-01-01

In root meristems of Tradescantia clone 02 (developed by Sparrow and his colleagues for mutation studies), X-rays interfere with the progression of cells through the cell cycle and induce chromosomal aberrations in a dose-dependent manner consistent with linear-quadratic kinetics. Sequential mitotic cell accumulations after irradiation indicate that sensitivity to aberration induction is probably greatest in cells from late S to early G2, with chromatid interchanges the most frequent aberration type and all aberrations consistent with initiation from the interaction between two lesions. The ratio of the coefficients in the linear (..cap alpha..) and the quadratic (..beta..) terms (..cap alpha../..beta..) ismore » equal to the dose average of specific energy produced by individual particles in the site where interaction takes place. The ratio ..cap alpha../..beta.. for chromosomal aberrations is similar to that previously found for X-ray-induced mutation in Tradescantia stamen hairs, supporting the proposal that radiation-induced mutational events are due to chromosomal aberrations with interaction distances of about 1..mu..m. Abrahamson and co-workers have noted that both ..cap alpha../..beta.. ratios appear to be related to nuclear target size and are similar for chromosomal and mutational endpoints in the same organism. These findings support this concept; however, it is apparent that any situation which diminishes yield at high doses (e.g., mitotic delay) will probably affect the ..beta.. component. 23 references, 5 figures, 2 tables.« less

Chromosomal aberrations and delays in cell progression induced by x-rays in Tradescantia clone 02 meristems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Geard, C.R.

1983-01-01

In root meristems of Tradescantia clone 02 (developed by Sparrow and his colleagues for mutation studies), X-rays interfere with the progression of cells through the cell cycle and induce chromosomal aberrations in a dose-dependent manner consistent with linear-quadratic kinetics. Sequential mitotic cell accumulations after irradiation indicate that sensitivity to aberrration induction is probably greatest in cells from late S to early G2, with chromatid interchanges the most frequent aberration type and all aberrations consistent with intiation from the interaction between two lesions. The ratio of the coefficients in the linear (..cap alpha..) and the quadratic (..beta..) terms (..cap alpha../..beta..) ismore » equal to the dose average of specific energy produced by individual particles in the site where interaction takes place. The ratio ..cap alpha../..beta.. for chromosomal aberrations is similar to that previously found for X-ray-induced mutation in Tradescantia stamen hairs, supporting the proposal that radiation-induced mutational events are due to chromosomal aberrations with interaction distances of about 1 ..mu..m. Abrahmson and co-workers have noted that both ..cap alpha../..beta.. ratios appear to be related to nuclear target size and are similar for chromosomal and mutational endpoints in the same organism. These findings support this concept; however, it is apparent that any situation which diminishes yield at high doses (e.g., mitotic delay) will primarily affect the ..beta.. component, resulting in low assessments of interaction site diameters.« less

Different susceptibility of rat pancreatic alpha and beta cells to hypoxia.

PubMed

Bloch, Konstantin; Vennäng, Julia; Lazard, Daniel; Vardi, Pnina

2012-06-01

Insulin-producing beta cells are known to be highly susceptible to hypoxia, which is a major factor in their destruction after pancreatic islet transplantation. However, whether the glucagon-producing pancreatic islet alpha cells are sensitive to hypoxia is not known. Our objective was to compare the sensitivity of alpha and beta cells to hypoxia. Isolated rat pancreatic islets were exposed to hypoxia (1% oxygen, 94% N(2), 5% CO(2)) for 3 days. The viability of the alpha and beta cells, as well as the stimulus-specific secretion of glucagon and insulin, was evaluated. A quantitative analysis of the proportion of beta to alpha cells indicated that, under normoxic conditions, islet cells were composed mainly of beta cells (87 ± 3%) with only 13 ± 3% alpha cells. Instead, hypoxia treatment significantly increased the proportion of alpha cells (40 ± 13%) and decreased the proportion of beta cells to 60 ± 13%. Using the fluorescent TUNEL assay we found that only a few percent of beta cells and alpha cells were apoptotic in normoxia. In contrast, hypoxia induced an abundance of apoptotic beta cells (61 ± 22%) and had no effect on the level of apoptosis in alpha cells. In conclusion, this study demonstrates that hypoxia results in severe functional abnormality in both beta and alpha cells while alpha cells display significantly decreased rate of apoptosis compared to intensive apoptotic injury of beta cells. These findings have implications for the understanding of the possible role of hypoxia in the pathophysiology of diabetes.

Characterization of CD4 and CD8 T Cell Responses in MuSK Myasthenia Gravis

PubMed Central

Yi, JS; Guidon, A; Sparks, S; Osborne, R; Juel, VC; Massey, JM; Sanders, DB; Weinhold, KJ; Guptill, JT

2014-01-01

Muscle specific tyrosine kinase myasthenia gravis (MuSK MG) is a form of autoimmune MG that predominantly affects women and has unique clinical features, including prominent bulbar weakness, muscle atrophy, and excellent response to therapeutic plasma exchange. Patients with MuSK MG have predominantly IgG4 autoantibodies directed against MuSK on the postsynaptic muscle membrane. Lymphocyte functionality has not been reported in this condition. The goal of this study was to characterize T-cell responses in patients with MuSK MG. Intracellular production of IFN-gamma, TNF-alpha, IL-2, IL-17, and IL-21 by CD4+ and CD8+ T-cells was measured by polychromatic flow cytometry in peripheral blood samples from 11 Musk MG patients and 10 healthy controls. Only one MuSK MG patient was not receiving immunosuppressive therapy. Regulatory T-cells (Treg) were also included in our analysis to determine if changes in T cell function were due to altered Treg frequencies. CD8+ T-cells from MuSK MG patients had higher frequencies of polyfunctional responses than controls, and CD4+ T-cells had higher IL-2, TNF-alpha, and IL-17. MuSK MG patients had a higher percentage of CD4+ T-cells producing combinations of IFN-gamma/IL-2/TNF-gamma, TNF-alpha/IL-2, and IFN-gamma/TNF-alpha. Interestingly, Treg numbers and CD39 expression were not different from control values. MuSK MG patients had increased frequencies of Th1 and Th17 cytokines and were primed for polyfunctional proinflammatory responses that cannot be explained by a defect in Treg function or number. PMID:24378287

Alpha Interferon Restricts Human T-Lymphotropic Virus Type 1 and 2 De Novo Infection through PKR Activation

PubMed Central

Cachat, Anne; Chevalier, Sébastien Alain; Alais, Sandrine; Ko, Nga Ling; Ratner, Lee; Journo, Chloé; Dutartre, Hélène

2013-01-01

Type I interferon (IFN-I) inhibits the replication of different viruses. However, the effect of IFN-I on the human T-lymphotropic virus type 1 (HTLV-1) viral cycle is controversial. Here, we investigated the consequences of IFN-α addition for different steps of HTLV-1 and HTLV-2 infection. We first show that alpha interferon (IFN-α) efficiently impairs HTLV-1 and HTLV-2 de novo infection in a T cell line and in primary lymphocytes. Using pseudotyped viruses expressing HTLV-1 envelope, we then show that cell-free infection is insensitive to IFN-α, demonstrating that the cytokine does not affect the early stages of the viral cycle. In contrast, intracellular levels of Gag, Env, or Tax protein are affected by IFN-α treatment in T cells, primary lymphocytes, or 293T cells transfected with HTLV-1 or HTLV-2 molecular clones, demonstrating that IFN-α acts during the late stages of infection. We show that IFN-α does not affect Tax-mediated transcription and acts at a posttranscriptional level. Using either small interfering RNA (siRNA) directed against PKR or a PKR inhibitor, we demonstrate that PKR, whose expression is induced by interferon, plays a major role in IFN-α-induced HTLV-1/2 inhibition. These results indicate that IFN-α has a strong repressive effect on the HTLV-1 and HTLV-2 viral cycle during de novo infection of cells that are natural targets of the viruses. PMID:24089560

Multiple alpha subunits of integrin are involved in cell-mediated responses of the Manduca immune system.

PubMed

Zhuang, Shufei; Kelo, Lisha; Nardi, James B; Kanost, Michael R

2008-01-01

The cell-mediated responses of the insect innate immune system-phagocytosis, nodulation, encapsulation-involve multiple cell adhesion molecules of hemocyte surfaces. A hemocyte-specific (HS) integrin and a member of the immunoglobulin (Ig) superfamily (neuroglian) are involved in the encapsulation response of hemocytes in Manduca sexta. In addition, two new integrin alpha (alpha) subunits have been found on these hemocytes. The alpha2 subunit is mainly expressed in epidermis and Malphigian tubules, whereas the alpha3 subunit is primarily expressed on hemocytes and fat body cells. Of the three known alpha subunits, the alpha1 subunit found in HS integrin is the predominant subunit of hemocytes. Cell adhesion assays indicate that alpha2 belongs to the integrin family with RGD-binding motifs, confirming the phylogenetic analysis of alpha subunits based on the amino-acid sequence alignment of different alpha subunits. Double-stranded RNAs (dsRNAs) targeting each of these three integrin alpha subunits not only specifically decreased transcript expression of each alpha subunit in hemocytes, but also abolished the cell-mediated encapsulation response of hemocytes to foreign surfaces. The individual alpha subunits of M. sexta integrins, like their integrin counterparts in mammalian immune systems, have critical, individual roles in cell-substrate and cell-cell interactions during immune responses.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jiwon; Lee, Suk Hyung; Korea University of Science and Technology, Yusong, Daejeon 305-333

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-kappamore » 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.« less

Rotenone Upregulates Alpha-Synuclein and Myocyte Enhancer Factor 2D Independently from Lysosomal Degradation Inhibition

PubMed Central

Stefanoni, Giovanni; Melchionda, Laura; Riva, Chiara; Brighina, Laura

2013-01-01

Dysfunctions of chaperone-mediated autophagy (CMA), the main catabolic pathway for alpha-synuclein, have been linked to the pathogenesis of Parkinson's disease (PD). Since till now there is limited information on how PD-related toxins may affect CMA, in this study we explored the effect of mitochondrial complex I inhibitor rotenone on CMA substrates, alpha-synuclein and MEF2D, and effectors, lamp2A and hsc70, in a human dopaminergic neuroblastoma SH-SY5Y cell line. Rotenone induced an upregulation of alpha-synuclein and MEF2D protein levels through the stimulation of their de novo synthesis rather than through a reduction of their CMA-mediated degradation. Moreover, increased MEF2D transcription resulted in higher nuclear protein levels that exert a protective role against mitochondrial dysfunction and oxidative stress. These results were compared with those obtained after lysosome inhibition with ammonium chloride. As expected, this toxin induced the cytosolic accumulation of both alpha-synuclein and MEF2D proteins, as the result of the inhibition of their lysosome-mediated degradation, while, differently from rotenone, ammonium chloride decreased MEF2D nuclear levels through the downregulation of its transcription, thus reducing its protective function. These results highlight that rotenone affects alpha-synuclein and MEF2D protein levels through a mechanism independent from lysosomal degradation inhibition. PMID:23984410

TNF-alpha sensitizes HT-29 colonic epithelial cells to intestinal lactobacilli.

PubMed

McCracken, Vance J; Chun, Taehoon; Baldeón, Manuel E; Ahrné, Siv; Molin, Göran; Mackie, Roderick I; Gaskins, H Rex

2002-09-01

The ability of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) to influence epithelial interleukin (IL)-8 responses to the intestinal bacterium Lactobacillus plantarum 299v was analyzed in the human HT-29 colonic epithelial cell line. In the absence of TNF-alpha, IL-8 mRNA expression was not detectable by Northern blot analysis in HT-29 cells alone or in HT-29 cells co-cultured with L. plantarum 299v. However, TNF-alpha induced IL-8 mRNA expression, and co-culture of TNF-alpha-treated HT-29 cells with L. plantarum 299v significantly increased IL-8 mRNA expression above levels induced by TNF-alpha alone in an adhesion-dependent manner. The increase in IL-8 mRNA expression was not observed in TNF-alpha-treated HT-29/L. plantarum 299v co-cultures using heat-killed lactobacilli or when L. plantarum adhesion was prevented using mannoside or a trans-well membrane. Paradoxically, IL-8 secretion was decreased in TNF-alpha-treated HT-29 cells with L. plantarum 299v relative to cells treated with TNF-alpha alone. TNF-alpha-mediated responsiveness to L. plantarum 299v was further investigated by analyzing expression of a coreceptor for bacterial cell wall products CD14. HT-29 cells expressed CD14 mRNA and cell-surface CD14; however, TNF-alpha did not alter CD14 mRNA or cell-surface expression, and blockade of CD14 with monoclonal antibody MY4 did not alter the IL-8 response to L. plantarum 299v in TNF-alpha-treated HT-29 cells. These results indicate that although TNF-alpha sensitizes HT-29 epithelial cells to intestinal lactobacilli, the bacteria exert a protective effect by downregulating IL-8 secretion.

Noradrenaline inhibits lipopolysaccharide-induced tumor necrosis factor and interleukin 6 production in human whole blood.

PubMed Central

van der Poll, T; Jansen, J; Endert, E; Sauerwein, H P; van Deventer, S J

1994-01-01

Sepsis and lipopolysaccharide (LPS) trigger the systemic release of both cytokines and catecholamines. Cytokines are known to be capable of eliciting a stress hormone response in vivo. The present study sought insight into the effect of noradrenaline on LPS-induced release of tumor necrosis factor alpha (TNF) and interleukin 6 (IL-6) in human whole blood. Whole blood was incubated with LPS for 4 h at 37 degrees C in the presence and absence of noradrenaline and/or specific alpha and beta antagonists and agonists. Noradrenaline caused a dose-dependent inhibition of LPS-induced TNF and IL-6 production. This effect could be completely prevented by addition of the specific beta 1, antagonist metoprolol, while it was not affected by the alpha antagonist phentolamine. Specific beta-adrenergic stimulation by isoprenaline mimicked the inhibiting effect of noradrenaline on LPS-evoked cytokine production, whereas alpha-adrenergic stimulation by phenylephrine had no effect. Fluorescence-activated cell sorter analysis demonstrated that beta-adrenergic stimulation had no effect on LPS binding to and internalization into mononuclear cells or on the expression of CD14, the major receptor for LPS on mononuclear cells. In acute sepsis, enhanced release of noradrenaline may be part of a negative feedback mechanism meant to inhibit ongoing TNF and IL-6 production. PMID:8168970

Vitamin E and mast cells.

PubMed

Zingg, Jean-Marc

2007-01-01

Mast cells play an important role in the immune system by interacting with B and T cells and by releasing several mediators involved in activating other cells. Hyperreactivity of mast cells and their uncontrolled accumulation in tissues lead to increased release of inflammatory mediators contributing to the pathogenesis of several diseases such as rheumatoid arthritis, atherosclerosis, multiple sclerosis, and allergic disorders such as asthma and allergic rhinitis. Interference with mast cell proliferation, survival, degranulation, and migration by synthetic or natural compounds may represent a preventive strategy for the management of these diseases. Natural vitamin E covers a group of eight analogues-the alpha-, beta-, gamma-, and delta-tocopherols and the alpha-, beta-, gamma-, and delta-tocotrienols, but only alpha-tocopherol is efficiently retained by the liver and distributed to peripheral tissues. Mast cells preferentially locate in the proximity of tissues that interface with the external environment (the epithelial surface of the skin, the gastrointestinal mucosa, and the respiratory system), what may render them accessible to treatments with inefficiently retained natural vitamin E analogues and synthetic derivatives. In addition to scavenging free radicals, the natural vitamin E analogues differently modulate signal transduction and gene expression in several cell lines; in mast cells, protein kinase C, protein phosphatase 2A, and protein kinase B are affected by vitamin E, leading to the modulation of proliferation, apoptosis, secretion, and migration. In this chapter, the possibility that vitamin E can prevent diseases with mast cells involvement by modulating signal transduction and gene expression is evaluated.

Decursin and PDBu: two PKC activators distinctively acting in the megakaryocytic differentiation of K562 human erythroleukemia cells.

PubMed

Kim, Hyeon Ho; Ahn, Kyung Seop; Han, Hogyu; Choung, Se Young; Choi, Sang-Yun; Kim, Ik-Hwan

2005-12-01

Protein kinase C (PKC) plays an important role in the proliferation and differentiation of various cell types including normal and leukemic hematopoietic cells. Phorbol 12,13-dibutyrate (PDBu) induces the megakaryocytic differentiation of K562 human erythroleukemia cells through PKC activation. Decursin, a pyranocoumarin from Angelica gigas, exhibits the cytotoxic effects on various human cancer cell lines and in vitro PKC activation. We report here the differences between two PKC activators, tumor-suppressing decursin and tumor-promoting PDBu, in their actions on the megakaryocytic differentiation of K562 cells. First of all, decursin inhibited PDBu-induced bleb formation in K562 cells. Decursin also inhibited the PDBu-induced megakaryocytic differentiation of K562 cells that is characterized by an increase in substrate adhesion, the secretion of granulocyte/macrophage colony stimulating factor (GM-CSF) and interleukin-6 (IL-6), and the surface expression of integrin beta3. The binding of PDBu to PKC was competitively inhibited by decursin. Decursin induced the more rapid down-regulation of PKC alpha and betaII isozymes than that induced by PDBu in K562 cells. Unlike PDBu, decursin promoted the translocation of PKC alpha and betaII to the nuclear membrane. Decursin-induced faster down-regulation and nuclear translocation of PKC alpha and betaII were not affected by the presence of PDBu. All these results indicate that decursin and phorbol ester are PKC activators distinctively acting in megakaryocytic differentiation and PKC modulation in K562 leukemia cells.

CD72 ligation regulates defective naive newborn B cell responses.

PubMed

Howard, L M; Reen, D J

1997-02-01

The biological basis for reduced Ig production by naive newborn B cells compared to adult peripheral blood B cells is not fully understood. In a Con A + IL-2 T cell-dependent system using "competent" adult T cells, adult B cells produced large amounts of IgM, IgG, and IgA, while cord B cells were restricted to low levels of only IgM production. Cord B cell activation was also diminished. The contribution of specific B-T cell contact-mediated events to the diminished cord B cell response in this system, using mAbs to CD40, CD28, CD80, and CD72, were investigated, as well as regulation of B cell Ig production by cytokines. alphaCD72 ligation increased cord B cell activation and IgM production, but did not affect adult B cells. Blocking alphaCD40 mAb inhibited cord B cell Ig production completely, but only partly inhibited adult B cell Ig production even at high concentration, suggesting a greater sensitivity of cord B cells to disruption of the CD40-CD40L interaction. Addition of IL-10 did not increase cord B cell Ig production, while adult B cell Ig production was increased. However, combined addition of IL-10 and alphaCD72 significantly increased cord B cell Ig production over that in the presence of either alphaCD72 or IL-10 alone, but had no effect on adult B cells over that of IL-10 alone. These data suggest that the diminished T cell-dependent response of cord B cells is due to reduced or absent CD72 ligation. CD72 ligation plays an important role in the induction of primary responses by naive B cells. CD72 modulation of naive B cell sensitivity to IL-10 stimulation may have implications in the induction of class switch, which is deficient in newborn B cells. Since all T cells express CD5 constitutively, these data also suggest the existence of another ligand for CD72.

A cyclized peptide derived from alpha fetoprotein inhibits the proliferation of ER-positive canine mammary cancer cells.

PubMed

Torres, Cristian Gabriel; Pino, Ana María; Sierralta, Walter Daniel

2009-06-01

The effects of estradiol (E2) and of an AFP-derived cyclized peptide (cP) on the proliferation of primary cultures of cancer cells isolated from spontaneous canine mammary tumors were studied. The cellular response to E2 and cP was related to the expression of estradiol receptor (isoforms alpha and beta). In ER-positive cells, 2 nM estradiol increased cell proliferation and the phosphorylation of ERK1/2; 2 microg/ml cP inhibited all these effects. Estradiol also increased HER2 immunoreactivity in ER-positive cells, an effect that was reverted to its basal values by cP. Estradiol stimulated in these cells the release of MMP2 and MMP9 and the shedding of HB-EGF, effects that the cP did not affect. ER-negative cells were refractory to estradiol or cP. All canine mammary tumor cells in culture responded to treatments analogously to human mammary cancer cells. Our results support the proposal of cP as a new, potentially effective therapeutic agent for the management of mammary cancer.

Prolonged tomato juice consumption has no effect on cell-mediated immunity of well-nourished elderly men and women.

PubMed

Watzl, B; Bub, A; Blockhaus, M; Herbert, B M; Lührmann, P M; Neuhäuser-Berthold, M; Rechkemmer, G

2000-07-01

The immunomodulatory potential of carotenoids has been investigated thoroughly only for beta-carotene. Data on the immunomodulatory activity of other carotenoids such as lycopene are scarce. The objective of this study was to investigate the effects of prolonged tomato juice consumption on cell-mediated immunity of well-nourished healthy elderly persons. In an intervention study, 33 female and 20 male subjects (aged 63-86 y) consumed 330 mL/d tomato juice (47.1 mg/d lycopene) or mineral water for 8 wk. Immune status was assessed by measuring number and lytic activity of natural killer (NK) cells, secretion of cytokines [interleukin (IL)-2, IL-4, tumor necrosis factor-alpha (TNF-alpha)] by activated peripheral blood mononuclear cells (PBMC), lymphocyte proliferation, and delayed-type hypersensitivity (DTH) skin responses. Tomato juice consumption resulted in significantly increased plasma lycopene and beta-carotene concentrations over time. In both treatment groups, TNF-alpha and IL-4 secretion were increased at the end of the intervention period, whereas IL-2 secretion was decreased. Tomato juice consumption had no effect on lymphocyte proliferation, DTH or the number of NK cells. Lytic activity of NK cells was increased in both groups at the end of the intervention period. In conclusion, these results show that prolonged tomato juice consumption increased plasma lycopene concentrations without significantly affecting cell-mediated immunity in well-nourished elderly subjects.

Islets of Langerhans in the parakeet, Psittacula krameri.

PubMed

Gupta, Y K; Kumar, S

1980-01-01

The pancreatic gland of Psittacula krameri is divisible into 4 lobes i.e. dorsal, ventral, third and splenic. The endocrine part is composed of alpha 1-, alpha 2- and beta-cells. The islets are of 4 kinds viz., alpha islets (having alpha 1- and alpha 2-cells), beta islets (having beta- and alpha 1-cells), pure beta islets (consisting of beta-cells exclusively) and mixed islets (with beta-, alpha 1- and alpha 2-cells). The distribution of alpha islets is mostly restricted to the splenic and third lobes whereas the beta islets are found in all 4 lobes. Though the alpha islets are only few in the dorsal lobe, their size is best developed in the third and dorsal lobes. Sometimes beta and alpha islets are present in very close proximity but their cells never mingle. An interesting feature was the complete absence of alpha islets from the ventral lobe.A relative abundance of alpha 2- cells in this bird seems to be associated with its comparatively higher blood glucose level and frugivorous habit. Tinctorial reactions suggest that the insulin content of the endocrine pancreas is low. There were no seasonal changes in the islet tissue of P. krameri.

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 any of the known prostaglandin receptor types.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yao, Congjun; Evans, Chheng-Orn; Stevens, Victoria L.

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 PCNAmore » 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.« less

Particle-in-cell simulation of an electronegative plasma under direct current bias studied in a large range of electronegativity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oudini, N.; Laboratoire des plasmas de Decharges, Centre de Developement des Technologies Avancees, Cite du 20 Aout BP 17 Baba Hassen, 16081 Algiers; Raimbault, J.-L.

2013-04-15

A one-dimensional electronegative plasma situated between two symmetrical parallel electrodes under DC bias is studied by Particle-In-Cell simulation with Monte Carlo Collisions. By varying the electronegativity {alpha}{identical_to}n{sub -}/n{sub e} from the limit of electron-ion plasmas (negative ion free) to ion-ion plasmas (electron free), the sheaths formation, the negative ion flux flowing towards the electrodes, and the particle velocities at the sheath edges are investigated. Depending on {alpha}, it is shown that the electronegative plasma behavior can be described by four regimes. In the lowest regime of {alpha}, i.e., {alpha} < 50, negative ions are confined by two positive sheaths withinmore » the plasma, while in the higher regimes of {alpha}, a negative sheath is formed and the negative ion flux can be extracted from the bulk plasma. In the two intermediate regimes of {alpha}, i.e., 50 < {alpha} < 10{sup 5}, both the electron and the negative ion fluxes are involved in the neutralization of the positive ions flux that leaves the plasma. In particular, we show that the velocity of the negative ions entering the negative sheath is affected by the presence of the electrons, and is not given by the modified Bohm velocity generally accepted for electronegative plasmas. For extremely high electronegativity, i.e., {alpha} > 10{sup 5}, the presence of electrons in the plasma is marginal and the electronegative plasma can be considered as an ion-ion plasma (electron free).« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Dohee; Yang, Jae-Yeon; Shin, Chan Soo, E-mail: csshin@snu.ac.kr

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 Runx2more » 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.« less

Modulation of Mycoplasma arthritidis-derived superantigen-induced cytokine gene expression by dexamethasone and interleukin-4.

PubMed

Mehindate, K; al-Daccak, R; Rink, L; Mecheri, S; Hébert, J; Mourad, W

1994-11-01

Activation of human monocytes or monocytic cell lines with all known stimuli coordinately induces the gene expression of various cytokines, including tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and the IL-1 receptor antagonist (IL-1Ra). In contrast, superantigens induce TNF-alpha and IL-1 beta but fail to affect IL-1Ra gene expression, suggesting that activation of monocytes via major histocompatibility complex class II is distinct from other signal transduction pathways. In the present study, we analyzed the regulation of the Mycoplasma arthritidis-derived superantigen (MAM)-induced IL-1 beta and TNF-alpha gene expression by studying the effects of two different anti-inflammatory agents: dexamethasone (DEX) and the T-cell-derived cytokine IL-4. Both agents contributed to the downregulation of MAM-induced IL-1 beta and TNF-alpha gene expression. They accelerated the normal decline of the gene expression of both MAM-induced cytokines by decreasing the stability of mRNAs via the induction or enhanced synthesis of one or more regulatory proteins. In addition, IL-4, but not DEX, induced a strong and rapid expression of IL-1Ra mRNA in MAM-stimulated and unstimulated THP-1 cells in a de novo protein synthesis-independent manner. The capacity of IL-4 to induce IL-1Ra gene expression reinforces its anti-inflammatory activity. This study illustrates some of the mechanisms by which MAM-induced proinflammatory monokine gene expression can be downregulated by IL-4 and DEX.

Combinations of ERK and p38 MAPK inhibitors ablate tumor necrosis factor-alpha (TNF-alpha ) mRNA induction. Evidence for selective destabilization of TNF-alpha transcripts.

PubMed

Rutault, K; Hazzalin, C A; Mahadevan, L C

2001-03-02

Tumor necrosis factor-alpha (TNF-alpha) is a potent proinflammatory cytokine whose synthesis and secretion are implicated in diverse pathologies. Hence, inhibition of TNF-alpha transcription or translation and neutralization of its protein product represent major pharmaceutical strategies to control inflammation. We have studied the role of ERK and p38 mitogen-activated protein (MAP) kinase in controlling TNF-alpha mRNA levels in differentiated THP-1 cells and in freshly purified human monocytes. We show here that it is possible to produce virtually complete inhibition of lipopolysaccharide-stimulated TNF-alpha mRNA accumulation by using a combination of ERK and p38 MAP kinase inhibitors. Furthermore, substantial inhibition is achievable using combinations of 1 microm of each inhibitor, whereas inhibitors used individually are incapable of producing complete inhibition even at high concentrations. Finally, addressing mechanisms involved, we show that inhibition of p38 MAP kinase selectively destabilizes TNF-alpha transcripts but does not affect degradation of c-jun transcripts. These results impinge on the controversy in the literature surrounding the mode of action of MAP kinase inhibitors on TNF-alpha mRNA and suggest the use of combinations of MAP kinase inhibitors as an effective anti-inflammatory strategy.

Alpha-tocopherol and alpha-tocopheryl quinone levels in cervical intraepithelial neoplasia and cervical cancer.

PubMed

Palan, Prabhudas R; Woodall, Angela L; Anderson, Patrick S; Mikhail, Magdy S

2004-05-01

alpha-Tocopherol is a potent antioxidant that protects cell membranes against oxidative damage. Red blood cell alpha-tocopherol levels reflect membrane alpha-tocopherol concentrations, and altered levels may suggest membrane damage. The objective of this study was to determine the levels of alpha-tocopherol and alpha-tocopheryl quinone, the oxidized product of alpha-tocopherol, in plasma and red blood cells that were obtained from control subjects and patients with cervical intraepithelial neoplasia and cervical cancer. In this cross-sectional study, 72 women, (32 African American and 40 Hispanic) were recruited. Among these subjects, 37 women had cervical intraepithelial neoplasia; 14 women had cervical cancer, and 21 women were considered control subjects, who had normal Papanicolaou test results. alpha-Tocopherol and alpha-tocopheryl quinone levels were determined in red blood cell and plasma by high-pressure liquid chromatography. Plasma levels of alpha-tocopherol and alpha-tocopheryl quinone were decreased significantly (P=.012 and=.005, respectively, by Kruskal-Wallis test) in study groups compared with the control group; red blood cell levels of alpha-tocopherol and alpha-tocopheryl quinone were not altered significantly. The lower alpha-tocopherol level that was observed in this study is consistent with our previous reports of decreased antioxidant concentrations and increased oxidative stress in women with cervical intraepithelial neoplasia. Unaltered red blood cell alpha-tocopherol and alpha-tocopheryl quinone levels suggest undamaged cell membrane. Further studies are needed to investigate the potential role of oxidative stress in cervical intraepithelial neoplasia.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Ling; Reinach, Peter; Lu, Luo

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 inmore » 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.« less

Alstonine as a potential fluorescent marker for tiny tumor detection and imaging

NASA Astrophysics Data System (ADS)

Viallet, Pierre M.; Vo-Dinh, Tuan; Salmon, Jean-Marie; Watts, Wendi; Rocchi, Emmanuelle; Isola, Narayana R.; Rebillard, Xavier

1997-06-01

3,4,5,6,16,17-Hexadehydro-16-(methoxycarbolyl)-19(alpha) - methyl-20(alpha) -oxyohimbanium (alstonine) is a fluorescent alcaloid which is known to stain tumor cells more efficiently than normal. The interactions between alstonine and biological macromolecules were first investigated to provide the rationale for preferential labelling. Molecular filtration and spectrosfluorometric techniques with different macromolecules and isopolynucleotides have demonstrated that binding occurs only in the presence of uridyl rings. For the binding affect only the fluorescence intensity of alstonine it can be assumed that it involves only the side chain of the fluorescent compound. The capability for preferential staining was verified in culture using SK-OV-3 cells and rat hepatocarcinoma cells as tumor cells and Mouse fibroblasts or rat liver cells as controls. Techniques of image analysis have demonstrated the efficiency of cellular labelling even in aggregates of rat hepatocarcinoma. These experiments lead the way to the detection of tiny tumors developed on thin visceral walls, using a fiber optic device.

Raman and nuclear magnetic resonance investigation of alkali metal vapor interaction with alkene-based anti-relaxation coating

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tretiak, O. Yu., E-mail: otretiak@genphys.ru; Balabas, M. V.; Blanchard, J. W.

2016-03-07

The use of anti-relaxation coatings in alkali vapor cells yields substantial performance improvements compared to a bare glass surface by reducing the probability of spin relaxation in wall collisions by several orders of magnitude. Some of the most effective anti-relaxation coating materials are alpha-olefins, which (as in the case of more traditional paraffin coatings) must undergo a curing period after cell manufacturing in order to achieve the desired behavior. Until now, however, it has been unclear what physicochemical processes occur during cell curing, and how they may affect relevant cell properties. We present the results of nondestructive Raman-spectroscopy and magnetic-resonancemore » investigations of the influence of alkali metal vapor (Cs or K) on an alpha-olefin, 1-nonadecene coating the inner surface of a glass cell. It was found that during the curing process, the alkali metal catalyzes migration of the carbon-carbon double bond, yielding a mixture of cis- and trans-2-nonadecene.« less

The genomic response of a human uterine endometrial adenocarcinoma cell line to 17alpha-ethynyl estradiol.

PubMed

Naciff, Jorge M; Khambatta, Zubin S; Thomason, Ryan G; Carr, Gregory J; Tiesman, Jay P; Singleton, David W; Khan, Sohaib A; Daston, George P

2009-01-01

We have determined the gene expression profile induced by 17 alpha-ethynyl estradiol (EE) in Ishikawa cells, a human uterine-derived estrogen-sensitive cell line, at various doses (1 pM, 100 pM, 10 nM, and 1 microM) and time points (8, 24, and 48 h). The transcript profiles were compared between treatment groups and controls (vehicle-treated) using high-density oligonucleotide arrays to determine the expression level of approximately 38,500 human genes. By trend analysis, we determined that the expression of 2560 genes was modified by exposure to EE in a dose- and time-dependent manner (p

Coordination of the Ser2056 and Thr2609 Clusters of DNA-PKcs in Regulating Gamma Rays and Extremely Low Fluencies of Alpha-Particle Irradiation to G0/G1 Phase Cells.

PubMed

Nagasawa, Hatsumi; Lin, Yu-Fen; Kato, Takamitsu A; Brogan, John R; Shih, Hung-Ying; Kurimasa, Akihiro; Bedford, Joel S; Chen, Benjamin P C; Little, John B

2017-02-01

The catalytic subunit of DNA dependent protein kinase (DNA-PKcs) and its kinase activity are critical for mediation of non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSB) in mammalian cells after gamma-ray irradiation. Additionally, DNA-PKcs phosphorylations at the T2609 cluster and the S2056 cluster also affect DSB repair and cellular sensitivity to gamma radiation. Previously we reported that phosphorylations within these two regions affect not only NHEJ but also homologous recombination repair (HRR) dependent DSB repair. In this study, we further examine phenotypic effects on cells bearing various combinations of mutations within either or both regions. Effects studied included cell killing as well as chromosomal aberration induction after 0.5-8 Gy gamma-ray irradiation delivered to synchronized cells during the G 0 /G 1 phase of the cell cycle. Blocking phosphorylation within the T2609 cluster was most critical regarding sensitization and depended on the number of available phosphorylation sites. It was also especially interesting that only one substitution of alanine in each of the two clusters separately abolished the restoration of wild-type sensitivity by DNA-PKcs. Similar patterns were seen for induction of chromosomal aberrations, reflecting their connection to cell killing. To study possible change in coordination between HRR and NHEJ directed repair in these DNA-PKcs mutant cell lines, we compared the induction of sister chromatid exchanges (SCEs) by very low fluencies of alpha particles with mutant cells defective in the HRR pathway that is required for induction of SCEs. Levels of true SCEs induced by very low fluence of alpha-particle irradiation normally seen in wild-type cells were only slightly decreased in the S2056 cluster mutants, but were completely abolished in the T2609 cluster mutants and were indistinguishable from levels seen in HRR deficient cells. Again, a single substitution in the S2056 together with a single substitution in the T2609 cluster abolished SCE formation and thus also effectively interferes with HRR.

Frontal alpha asymmetry neurofeedback for the reduction of negative affect and anxiety.

PubMed

Mennella, Rocco; Patron, Elisabetta; Palomba, Daniela

2017-05-01

Frontal alpha asymmetry has been proposed to underlie the balance between approach and withdrawal motivation associated to each individual's affective style. Neurofeedback of EEG frontal alpha asymmetry represents a promising tool to reduce negative affect, although its specific effects on left/right frontal activity and approach/withdrawal motivation are still unclear. The present study employed a neurofeedback training to increase frontal alpha asymmetry (right - left), in order to evaluate discrete changes in alpha power at left and right sites, as well as in positive and negative affect, anxiety and depression. Thirty-two right-handed females were randomly assigned to receive either the neurofeedback on frontal alpha asymmetry, or an active control training (N = 16 in each group). The asymmetry group showed an increase in alpha asymmetry driven by higher alpha at the right site (p < 0.001), as well as a coherent reduction in both negative affect and anxiety symptoms (ps < 0.05), from pre-to post-training. No training-specific modulation emerged for positive affect and depressive symptoms. These findings provide a strong rationale for the use of frontal alpha asymmetry neurofeedback for the reduction of negative affect and anxiety in clinical settings. Copyright © 2017 Elsevier Ltd. All rights reserved.

RACK1 binds to Smad3 to modulate transforming growth factor-beta1-stimulated alpha2(I) collagen transcription in renal tubular epithelial cells.

PubMed

Okano, Kazuhiro; Schnaper, H William; Bomsztyk, Karol; Hayashida, Tomoko

2006-09-08

Although it is clear that transforming growth factor-beta1 (TGF-beta1) is critical for renal fibrogenesis, the complexity of the involved mechanisms is increasingly apparent. TGF-beta1 stimulates phosphorylation of Smad2/3 and activates other signaling molecules as well. The molecular link between these other kinases and Smads is not known. We sought new binding partners for Smad3 in renal cells and identified receptor for activated protein kinase C 1 (RACK1) as a novel binding partner of Smad3. The linker region of Smad3 and the tryptophan-aspartic acid repeat 6 and 7 of RACK1 are sufficient for the association. RACK1 also interacts with Smad3 in the human kidney epithelial cell line, HKC. Silencing RACK1 increases transcriptional activity of TGF-beta1-responsive promoter sequences of the Smad binding element (SBE), p3TP-Lux, and alpha2(I) collagen. Conversely, overexpressed RACK1 negatively modulates alpha2(I) collagen transcriptional activity in TGF-beta1-stimulated cells. RACK1 did not affect phosphorylation of Smad3 at the C terminus or in the linker region. However, RACK1 reduced direct binding of Smad3 to the SBE motif. Mutating a RACK1 tyrosine at residue 246, but not at 228, decreased the inhibitory effect of RACK1 on both alpha2(I) collagen promoter activity and Smad binding to SBE induced by TGF-beta1. These results suggest that RACK1 modulates transcription of alpha2(I) collagen by TGF-beta1 through interference with Smad3 binding to the gene promoter.

Expression of transforming growth factor-beta1, -beta2 and -beta3 in normal and diseased canine mitral valves.

PubMed

Aupperle, H; März, I; Thielebein, J; Schoon, H-A

2008-01-01

The pathogenesis of chronic valvular disease (CVD) in dogs remains unclear, but activation and proliferation of valvular stromal cells (VSC) and their transdifferentiation into myofibroblast-like cells has been described. These alterations may be influenced by transforming growth factor-beta (TGF-beta), a cytokine involved in extracellular matrix (ECM) regulation and mesenchymal cell differentiation. The present study investigates immunohistochemically the expression of TGF-beta1, -beta2, -beta3 and smooth muscle alpha actin (alpha-SMA) in normal canine mitral valves (MVs) (n=10) and in the valves of dogs with mild (n=7), moderate (n=14) and severe (n=9) CVD. In normal mitral valves there was no expression of alpha-SMA but VSC displayed variable expression of TGF-beta1 (10% of VSC labelled), TGF-beta2 (1-5% labelled) and TGF-beta3 (50% labelled). In mild CVD the affected atrialis contain activated and proliferating alpha-SMA-positive VSC, which strongly expressed TGF-beta1 and -beta3, but only 10% of these cells expressed TGF-beta2. In unaffected areas of the leaflet there was selective increase in expression of TGF-beta1 and -beta3. In advanced CVD the activated subendothelial VSC strongly expressed alpha-SMA, TGF-beta1 and -beta3. Inactive VSC within the centre of the nodules had much less labelling for TGF-beta1 and -beta3. TGF-beta1 labelling was strong within the ECM. These data suggest that TGF-beta plays a role in the pathogenesis of CVD by inducing myofibroblast-like differentiation of VSC and ECM secretion. Changed haemodynamic forces and expression of matrix metalloproteinases (MMPs) may in turn regulate TGF-beta expression.

The role of DNA repair in herpesvirus pathogenesis.

PubMed

Brown, Jay C

2014-10-01

In cells latently infected with a herpesvirus, the viral DNA is present in the cell nucleus, but it is not extensively replicated or transcribed. In this suppressed state the virus DNA is vulnerable to mutagenic events that affect the host cell and have the potential to destroy the virus' genetic integrity. Despite the potential for genetic damage, however, herpesvirus sequences are well conserved after reactivation from latency. To account for this apparent paradox, I have tested the idea that host cell-encoded mechanisms of DNA repair are able to control genetic damage to latent herpesviruses. Studies were focused on homologous recombination-dependent DNA repair (HR). Methods of DNA sequence analysis were employed to scan herpesvirus genomes for DNA features able to activate HR. Analyses were carried out with a total of 39 herpesvirus DNA sequences, a group that included viruses from the alpha-, beta- and gamma-subfamilies. The results showed that all 39 genome sequences were enriched in two or more of the eight recombination-initiating features examined. The results were interpreted to indicate that HR can stabilize latent herpesvirus genomes. The results also showed, unexpectedly, that repair-initiating DNA features differed in alpha- compared to gamma-herpesviruses. Whereas inverted and tandem repeats predominated in alpha-herpesviruses, gamma-herpesviruses were enriched in short, GC-rich initiation sequences such as CCCAG and depleted in repeats. In alpha-herpesviruses, repair-initiating repeat sequences were found to be concentrated in a specific region (the S segment) of the genome while repair-initiating short sequences were distributed more uniformly in gamma-herpesviruses. The results suggest that repair pathways are activated differently in alpha- compared to gamma-herpesviruses. Copyright © 2014. Published by Elsevier Inc.

Mouse macrophages primed with alendronate down-regulate monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) production in response to Toll-like receptor (TLR) 2 and TLR4 agonist via Smad3 activation.

PubMed

Masuda, Takahiro; Deng, Xue; Tamai, Riyoko

2009-08-01

Alendronate is one of the nitrogen-containing bisphosphonates (NBPs) used as anti-bone resorptive drugs. However, NBPs have inflammatory side effects including osteomyelitis and osteonecrosis of the jaw. In the present study, we examined the effects of alendronate on chemokine production by the macrophage-like cell line, J774.1, when incubated with Pam(3)CSK(4) (a Toll-like receptor (TLR) 2 agonist) and Lipid A (a TLR4 agonist). Pretreatment of J774.1 cells with alendronate decreased the production of TLR ligand-induced monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) but did not influence nuclear factor-kappaB (NF-kappaB) activation. While this agent induced caspase-8 activation, a caspase-8 inhibitor did not affect the decrease in MCP-1 production by alendronate and TLR ligands. Thus, the alendronate-mediated decrease in chemokine production was independent of NF-kappaB and caspase-8 activation. Although transforming growth factor-beta1 (TGF-beta1) is known to inhibit chemokine production by various cell types via Smad3 activation, pretreatment with alendronate did not increase TGF-beta1 production by J774.1 cells incubated in the presence or absence of TLR ligands. However, alendronate directly activated Smad3. These results suggest that by down-regulating MCP-1 and MIP-1alpha production via Smad3, long-term use of alendronate might inhibit normal activation and migration of osteoclasts and cause osteonecrosis.

Endothelial NOS is required for SDF-1alpha/CXCR4-mediated peripheral endothelial adhesion of c-kit+ bone marrow stem cells.

PubMed

Kaminski, Alexander; Ma, Nan; Donndorf, Peter; Lindenblatt, Nicole; Feldmeier, Gregor; Ong, Lee-Lee; Furlani, Dario; Skrabal, Christian A; Liebold, Andreas; Vollmar, Brigitte; Steinhoff, Gustav

2008-01-01

In the era of intravascular approaches for regenerative cell therapy, the underlying mechanisms of stem cell migration to non-marrow tissue have not been clarified. We hypothesized that next to a local inflammatory response implying adhesion molecule expression, endothelial nitric oxide synthase (eNOS)-dependent signaling is required for stromal- cell-derived factor-1 alpha (SDF-1alpha)-induced adhesion of c-kit+ cells to the vascular endothelium. SDF-1alpha/tumor necrosis factor-alpha (TNF-alpha)-induced c-kit+-cell shape change and migration capacity was studied in vitro using immunohistochemistry and Boyden chamber assays. In vivo interaction of c-kit+ cells from bone marrow with the endothelium in response to SDF-1alpha/TNF-alpha stimulation was visualized in the cremaster muscle microcirculation of wild-type (WT) and eNOS (-/-) mice using intravital fluorescence microscopy. In addition, NOS activity was inhibited with N-nitro-L-arginine-methylester-hydrochloride in WT mice. To reveal c-kit+-specific adhesion behavior, endogenous leukocytes (EL) and c-kit+ cells from peripheral blood served as control. Moreover, intercellular adhesion molecule-1 (ICAM-1) and CXCR4 were blocked systemically to determine their role in inflammation-related c-kit+-cell adhesion. In vitro, SDF-1alpha enhanced c-kit+-cell migration. In vivo, SDF-1alpha alone triggered endothelial rolling-not firm adherence-of c-kit+ cells in WT mice. While TNF-alpha alone had little effect on adhesion of c-kit+ cells, it induced maximum endothelial EL adherence. However, after combined treatment with SDF-1alpha+TNF-alpha, endothelial adhesion of c-kit+ cells increased independent of their origin, while EL adhesion was not further incremented. Systemic treatment with anti-ICAM-1 and anti-CXCR4-monoclonal antibody completely abolished endothelial c-kit+-cell adhesion. In N-nitro-L-arginine-methylester-hydrochloride-treated WT mice as well as in eNOS (-/-) mice, firm endothelial adhesion of c-kit+ cells was entirely abrogated, while EL adhesion was significantly increased. The chemokine SDF-1alpha mediates firm adhesion c-kit+ cells only in the presence of TNF-alpha stimulation via an ICAM-1- and CXCR4-dependent mechanism. The presence of eNOS appears to be a crucial and specific factor for firm c-kit+-cell adhesion to the vascular endothelium.

Expression and in vitro regulation of integrins by normal human urothelial cells.

PubMed

Southgate, J; Kennedy, W; Hutton, K A; Trejdosiewicz, L K

1995-08-01

Integrins are thought to be essential adhesion receptors for the maintenance of tissue histioarchitecture. The purpose of this study was to determine integrin expression patterns in the human stratified transitional epithelium of the urinary tract (urothelium). In situ expression patterns were compared with in vitro expression, using a normal cell culture model system in which the effects of cell stratification can be studied independently of differentiation. By immunohistological criteria, the urothelia of bladder, ureter and renal pelvis expressed alpha 2 beta 1 and alpha 3 beta 1 integrins in all layers at intercellular junctions, and cytoplasmically in the lower strata. By contrast, alpha 6 beta 4 and occasionally alpha v beta 4 were expressed only by basal cells and localised to the basal lamina. These expression patterns were unaltered in specimens where an inflammatory cell infiltrate was present. In long-term cultures of normal urothelial cells maintained in a low-Ca++ serum-free medium, the monolayer cultures expressed alpha 2 beta 1, alpha 3 beta 1 and alpha 5 beta 1 integrins at intercellular junctions and in cytoplasmic inclusions, whereas alpha 6 beta 4 was distributed in a random pattern over the substratum. Increasing exogenous Ca++ concentrations induced cell stratification and desmosome formation, but not cytodifferentiation. Under these conditions, alpha 6 beta 4 became cell-, rather than substratum-associated, localising particularly to filopodia and lamellipodia. Quantitation of integrin expression by flow cytometry confirmed increased surface expression of alpha 6 beta 4 in high Ca++ media, and also of alpha 3 and alpha 5, but not alpha 2, subunits. These results suggest that alpha 2 beta 1 and alpha 3 beta 1 integrins, although differentially regulated, are mainly involved in homotypic cell-cell interactions and the maintenance of a stratified morphology, whereas alpha 6 beta 4 is the principal integrin involved in substratum adhesion.

Short-term, serum-free, static culture of cord blood-derived CD34+ cells: effects of FLT3-L and MIP-1alpha on in vitro expansion of hematopoietic progenitor cells.

PubMed

Capmany, G; Querol, S; Cancelas, J A; García, J

1999-08-01

The use of ex vivo expanded cells has been suggested as a possible means to accelerate the speed of engraftment in cord blood (CB) transplantation. The aim of this study was to fix the optimal condition for the generation of committed progenitors without affecting the stem cell compartment. Analysis of the effects of FLT3-L and MIP-1alpha when combined with SCF, IL-3 and IL-6, in short-term (6 days), serum-free expansion cultures of CB-selected CD34+ cells. An important expansion was obtained that ranged between 8-15 times for CFU-GM, 21-51 times for the BFU-E/CFU-Mix population and 11 to 30 times for CD34+ cells assessed by flow cytometry. From the combinations tested, those in which FLT3-L was present had a significant increase in the expansion of committed progenitors, while the presence of MIP-1alpha had a detrimental effect on the generation of more differentiated cells. However, stem cell candidates assessed by week 5 CAFC assay could be maintained in culture when both MIP-1a and FLT3-L were present (up to 91% recovery). This culture system was also able to expand megakaryocytic precursors as determined by the co-expression of CD34 and CD61 antigens (45-70 times), in spite of the use of cytokines non-specific for the megakaryocytic lineage. The results obtained point to the combination of SCF, IL-3, IL-6, FLT3-L and MIP-1alpha as the best suited for a pre-clinical short-term serum-free static ex vivo expansion protocol of CB CD34+ cells, since it can generate large numbers of committed progenitor cells as well as maintaining week 5 CAFC.

Inhibition of lymphocyte proliferation and antibody production in vitro by silica, talc, bentonite or Corynebacterium parvum: involvement of peroxidative processes.

PubMed

Hoffeld, J T

1983-05-01

This study was undertaken to determine whether and by what means particles which induce granulomata in vivo can affect murine spleen lymphoproliferative and antibody responses in vitro. Particles of silica, talc, Bentonite or C. parvum cells inhibited lipopolysaccharide- or concanavalin A-stimulated proliferation and sheep red blood cell-induced antibody response in vitro. The inhibition required at least 48 hours exposure of the cells to the particles. The late onset of inhibition and its reproducibility at different cell or mitogen concentrations implicated particle-induced injury to both phagocytes and lymphocytes. Either alpha-tocopherol or 2-mercaptoethanol prevented the particle-induced inhibition of spleen cell responses. alpha-Tocopherol and 2-mercaptoethanol have in common the capacity to protect cells against membrane lipid peroxidation. The inhibitory peroxidative process(es) implicated by these studies are most likely attributable to: (a) stimulation of oxidative metabolism of phagocytic cells by particles; and (b) iron-catalyzed peroxidation directly by the particles. These data may be relevant in understanding the pathogenesis of and devising therapeutic approaches toward various granulomatous conditions.

Mycophenolate mofetil modulates adhesion receptors of the beta1 integrin family on tumor cells: impact on tumor recurrence and malignancy

PubMed Central

Engl, Tobias; Makarević, Jasmina; Relja, Borna; Natsheh, Iyad; Müller, Iris; Beecken, Wolf-Dietrich; Jonas, Dietger; Blaheta, Roman A

2005-01-01

Background Tumor development remains one of the major obstacles following organ transplantation. Immunosuppressive drugs such as cyclosporine and tacrolimus directly contribute to enhanced malignancy, whereas the influence of the novel compound mycophenolate mofetil (MMF) on tumor cell dissemination has not been explored. We therefore investigated the adhesion capacity of colon, pancreas, prostate and kidney carcinoma cell lines to endothelium, as well as their beta1 integrin expression profile before and after MMF treatment. Methods Tumor cell adhesion to endothelial cell monolayers was evaluated in the presence of 0.1 and 1 μM MMF and compared to unstimulated controls. beta1 integrin analysis included alpha1beta1 (CD49a), alpha2beta1 (CD49b), alpha3beta1 (CD49c), alpha4beta1 (CD49d), alpha5beta1 (CD49e), and alpha6beta1 (CD49f) receptors, and was carried out by reverse transcriptase-polymerase chain reaction, confocal microscopy and flow cytometry. Results Adhesion of the colon carcinoma cell line HT-29 was strongly reduced in the presence of 0.1 μM MMF. This effect was accompanied by down-regulation of alpha3beta1 and alpha6beta1 surface expression and of alpha3beta1 and alpha6beta1 coding mRNA. Adhesion of the prostate tumor cell line DU-145 was blocked dose-dependently by MMF. In contrast to MMF's effects on HT-29 cells, MMF dose-dependently up-regulated alpha1beta1, alpha2beta1, alpha3beta1, and alpha5beta1 on DU-145 tumor cell membranes. Conclusion We conclude that MMF possesses distinct anti-tumoral properties, particularly in colon and prostate carcinoma cells. Adhesion blockage of HT-29 cells was due to the loss of alpha3beta1 and alpha6beta1 surface expression, which might contribute to a reduced invasive behaviour of this tumor entity. The enhancement of integrin beta1 subtypes observed in DU-145 cells possibly causes re-differentiation towards a low-invasive phenotype. PMID:15644133

Ca2+ permeability through rat cloned alpha9-containing nicotinic acetylcholine receptors.

PubMed

Fucile, Sergio; Sucapane, Antonietta; Eusebi, Fabrizio

2006-04-01

We investigated the functional properties of rat alpha9 and alpha9alpha10 nicotinic acetylcholine receptors (nAChRs) expressed by transient transfection in the rat GH4C1 cell line, using both Ca(2+) imaging and whole-cell recording. Acute applications of ACh generated short-delay fast-rising and quick-decaying Ca(2+) transients, suppressed in Ca(2+)-free medium and invariably accompanied by the activation of whole-cell inward currents. The mean amplitude of ACh-induced currents was as small as -16 pA in alpha9 subunit cDNA-transfected GH4C1 cells (alpha9-GH4C1), while they were much larger (range: -150 to -300 pA) in alpha9alpha10 subunit cDNAs-transfected GH4C1 cells (alpha9alpha10-GH4C1). Currents were not activated by nicotine, were blocked by methyllycaconitine and were ACh concentration-dependent. Because the Ca(2+) permeability of alpha9-containing nAChRs has been estimated in immortalized cochlear UB/OC-2 mouse cells, we also characterized the ACh-induced responses in these cells. Unlike alpha9- and alpha9alpha10-GH4C1 cells, UB/OC-2 cells responded to ACh with both long-delay methyllycaconitine-insensitive whole-cell currents and long-lasting Ca(2+) transients, the latter being detected in the absence of Ca(2+) in the extracellular medium and being suppressed by the Ca(2+)-ATPase inhibitor thapsigargin, known to deplete IP(3)-sensitive stores. These results indicated the involvement of muscarinic nAChRs and the lack of functional ACh-gated receptor channels in UB/OC-2 cells. Thus, we measured the fractional Ca(2+) current (P(f), i.e. the percentage of total current carried by Ca(2+) ions) in alpha9alpha10-GH4C1, obtaining a P(f) value of 22 +/- 4%; this is the largest value estimated to date for a ligand-gated receptor channel. The physiological role played by Ca(2+) entry through alpha9-containing nAChRs gated by ACh is discussed.

Construction and properties of a cell line constitutively expressing the herpes simplex virus glycoprotein B dependent on functional alpha 4 protein synthesis.

PubMed Central

Arsenakis, M; Hubenthal-Voss, J; Campadelli-Fiume, G; Pereira, L; Roizman, B

1986-01-01

We report the construction of a cell line constitutively expressing the glycoprotein B (gB) of herpes simplex virus (HSV) 1. The cell line was constructed in two steps. In the first, a baby hamster kidney cell line was transfected with the DNA of a plasmid containing the neomycin phosphotransferase gene that confers resistance to the antibiotic G418 and the gene specifying a temperature-sensitive (ts-) alpha 4 protein of HSV-1, the major viral regulatory protein. A clonal cell line, alpha 4/c113, selected for resistance to the antibiotic G418, expressed high levels of alpha 4 protein constitutively. Superinfection of these cells with HSV-2 resulted in twofold induction of the resident HSV-1 alpha 4 gene. In the second step, alpha 4/c113 cells were transfected with the DNA of a plasmid carrying the gB gene and the mouse methotrexate resistance dihydrofolate reductase gene. A clonal cell line, alpha 4/c113/gB, selected for methotrexate resistance expressed gB constitutively. Expression of both gB and alpha 4 continued unabated for at least 32 serial passages. Cells passaged serially in medium containing both methotrexate and G418 after passage 10 contained a higher copy number of the alpha 4 gene and produced larger amounts of both gB and alpha 4 proteins than did cells maintained in medium containing methotrexate alone. Expression of gB was dependent on the presence of functional alpha 4 protein inasmuch as expression of gB ceased on shift up to nonpermissive temperatures, when shifted to permissive temperatures, the cell line reinitiated expression of gB after a delay commensurate with the length of incubation at the nonpermissive temperature, and the cell-resident HSV-1 gB gene was expressed at the nonpermissive temperature in cells infected with a recombinant expressing a ts+ alpha 4 protein and an HSV-2 gB. The properties of the alpha 4/c113 cell line suggest that it may express other viral genes induced by alpha 4 protein constitutively, provided that the product is not toxic to the cells. Images PMID:3022001

GnRH signalling pathways and GnRH-induced homologous desensitization in a gonadotrope cell line (alphaT3-1).

PubMed

Poulin, B; Rich, N; Mas, J L; Kordon, C; Enjalbert, A; Drouva, S V

1998-07-25

Exposure of the gonadotrope cells to gonadotropin-releasing hormone (GnRH) reduces their responsiveness to a new GnRH stimulation (homologous desensitization). The time frame as well as the mechanisms underlying this phenomenon are yet unclear. We studied in a gonadotrope cell line (alphaT3-1) the effects of short as well as long term GnRH pretreatments on the GnRH-induced phospholipases-C (PLC), -A2 (PLA2) and -D (PLD) activities, by measuring the production of IP3, total inositol phosphates (IPs), arachidonic acid (AA) and phosphatidylethanol (PEt) respectively. We demonstrated that although rapid desensitization of GnRH-induced IP3 formation did not occur in these cells, persistent stimulation of cells with GnRH or its analogue resulted in a time-dependent attenuation of GnRH-elicited IPs formation. GnRH-induced IPs desensitization was potentiated after direct activation of PKC by the phorbol ester TPA, suggesting the involvement of distinct mechanisms in the uncoupling exerted by either GnRH or TPA on GnRH-stimulated PI hydrolysis. The levels of individual phosphoinositides remained unchanged under any desensitization condition applied. Interestingly, while the GnRH-induced PLA2 activity was rapidly desensitized (2.5 min) after GnRH pretreatments, the neuropeptide-evoked PLD activation was affected at later times, indicating an important time-dependent contribution of these enzymatic activities in the sequential events underlying the GnRH-induced homologous desensitization processes in the gonadotropes. Under GnRH desensitization conditions, TPA was still able to induce PLD activation and to further potentiate the GnRH-evoked PLD activity. AlphaT3-1 cells possess several PKC isoforms which, except PKCzeta, were differentially down-regulated by TPA (PKCalpha, betaII, delta, epsilon, eta) or GnRH (PKCbetaII, delta, epsilon, eta). In spite of the presence of PKC inhibitors or down-regulation of PKC isoforms by TPA, the desensitizing effect of the neuropeptide on GnRH-induced IPs, AA and PEt formation remained unchanged. In conclusion, in alphaT3-1 cells the GnRH-induced homologous desensitization affects the GnRH coupling with PLC, PLA2 and PLD by mechanism(s) which do not implicate TPA-sensitive PKC isoforms, but likely reflect time-dependent modification(s) on the activation processes of the enzymes.

Lens epithelial cells derived from alphaB-crystallin knockout mice demonstrate hyperproliferation and genomic instability.

PubMed

Andley, U P; Song, Z; Wawrousek, E F; Brady, J P; Bassnett, S; Fleming, T P

2001-01-01

alphaB-crystallin is a member of the small heat shock protein family and can act as a molecular chaperone preventing the in vitro aggregation of other proteins denatured by heat or other stress conditions. Expression of alphaB-crystallin increases in cells exposed to stress and enhanced in tumors of neuroectodermal origin and in many neurodegenerative diseases. In the present study, we examined the properties of lens epithelial cells derived from mice in which the alphaB-crystallin gene had been knocked out. Primary rodent cells immortalize spontaneously in tissue culture with a frequency of 10(-5) to 10(-6). Primary lens epithelial cells derived from alphaB-crystallin-/- mice produced hyperproliferative clones at a frequency of 7.6 x 10(-2), four orders of magnitude greater than predicted by spontaneous immortalization (1). Hyperproliferative alphaB-crystallin-/- cells were shown to be truly immortal since they have been passaged for more than 100 population doublings without any diminution in growth potential. In striking contrast to the wild-type cells, which were diploid, the alphaB-crystallin-/- cultures had a high proportion of tetraploid and higher ploidy cells, indicating that the loss of alphaB-crystallin is associated with an increase in genomic instability. Further evidence of genomic instability of alphaB-crystallin-/- cells was observed when primary cultures were infected with Ad12-SV40 hybrid virus. In striking contrast to wild-type cells, alphaB-crystallin-/- cells expressing SV40 T antigen exhibited a widespread cytocidal response 2 to 3 days after attaining confluence, indicating that SV40 T antigen enhanced the intrinsic genomic instability of alphaB-crystallin-/- lens epithelial cells. These observations suggest that the widely distributed molecular chaperone alphaB-crystallin may play an important nuclear role in maintaining genomic integrity.

Dependence and independence of survival parameters on linear energy transfer in cells and tissues

PubMed Central

Ando, Koichi; Goodhead, Dudley T.

2016-01-01

Carbon-ion radiotherapy has been used to treat more than 9000 cancer patients in the world since 1994. Spreading of the Bragg peak is necessary for carbon-ion radiotherapy, and is designed based on the linear–quadratic model that is commonly used for photon therapy. Our recent analysis using in vitro cell kills and in vivo mouse tissue reaction indicates that radiation quality affects mainly the alpha terms, but much less the beta terms, which raises the question of whether this is true in other biological systems. Survival parameters alpha and beta for 45 in vitro mammalian cell lines were obtained by colony formation after irradiation with carbon ions, fast neutrons and X-rays. Relationships between survival parameters and linear energy transfer (LET) below 100 keV/μm were obtained for 4 mammalian cell lines. Mouse skin reaction and tumor growth delay were measured after fractionated irradiation. The Fe-plot provided survival parameters of the tissue reactions. A clear separation between X-rays and high-LET radiation was observed for alpha values, but not for beta values. Alpha values/terms increased with increasing LET in any cells and tissues studied, while beta did not show a systematic change. We have found a puzzle or contradiction in common interpretations of the linear-quadratic model that causes us to question whether the model is appropriate for interpreting biological effectiveness of high-LET radiation up to 500 keV/μm, probably because of inconsistency in the concept of damage interaction. A repair saturation model proposed here was good enough to fit cell kill efficiency by radiation of wide-ranged LET. A model incorporating damage complexity and repair saturation would be suitable for heavy-ion radiotherapy. PMID:27380803

Nuclear p21-activated kinase 1 in breast cancer packs off tamoxifen sensitivity.

PubMed

Rayala, Suresh K; Molli, Poonam R; Kumar, Rakesh

2006-06-15

There is significant clinical interest in the factors that influence the development of tamoxifen resistance in estrogen receptor-alpha (ER-alpha)-positive breast cancers. Recent studies suggest that in ER-positive breast tumor cells, elevated protein levels, and in particular, nuclear localization of p21-activated kinase 1 (PAK1), is associated with the progressive limitation of tamoxifen sensitivity. These phenotypic effects of PAK1 in model systems are mechanistically linked with the ability of PAK1 to phosphorylate ER-alpha on serine 305 and subsequent secondary activation of serine 118. These findings prompt further investigation of how nuclear signaling by PAK1 may affect estrogen's action and whether tamoxifen resistance might be prevented or reversed by PAK1 inhibition.

Localized defects in radiation-damaged zircon

PubMed

Rios; Malcherek; Salje; Domeneghetti

2000-12-01

The crystal structure of a radiation-damaged natural zircon, ZrSiO(4) (alpha-decay radiation dose is ca 1.8 x 10(18) alpha-decay events g(-1)), has been determined. The anisotropic unit-cell swelling observed in the early stages of the amorphization process (0.17% along the a axis and 0.62% along the c axis compared with the undamaged material) is a consequence of the anisotropy of the expansion of ZrO(8) polyhedra. Larger anisotropic displacement parameters were found for Zr and O atoms, indicating that the distortion produced by alpha particle-induced localized defects mainly affects the ZrO(8) unit. The overall shape of SiO(4) tetrahedra remains essentially undistorted, while Si-O bonds are found to lengthen by 0.43%.

Immunoregulatory and antioxidant performance of alpha-tocopherol and selenium on human lymphocytes.

PubMed

Lee, Chung-Yung Jetty; Wan, Jennifer Man-Fan

2002-05-01

The role of alpha-tocopherol (alpha-toco) and selenium (Se) on human lymphocyte oxidative stress and T-cells proliferation were studied by flow cytometry. We measured the hydrogen peroxide and glutathione levels in cultured human T-lymphocytes and the proliferation of their subsets: T-helper/inducer, T-suppressor/cytotoxic, and natural killer and interleukin-2 receptors upon stimulation by the mitogens phytohemaglutinin (PHA) and lipopolysaccharide (LPS). The results indicate that early stimulation by mitogens is affected by the glutathione and hydrogen peroxide status of the T-lymphocytes. The addition of 100 microM or 500 microM alpha-toco or 0.5 microM Se alone shows weak antioxidant and immunostimulant properties. When combined, an enhanced antioxidant and immunoregulatory effect was observed. The present findings indicate that alpha-toco and Se have interactive effects as oxygen radical scavengers, thus promoting human lymphocyte response to antigens. This suggests that micronutrient status is an important factor in considering when interpreting the results of in vitro assays of lymphocyte function.

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.

The compounds from the hollyhock extract (Althaea rosea Cav. var. nigra) affect the aromatization in rat testicular cells in vivo and in vitro.

PubMed

Papiez, Monika; Gancarczyk, Monika; Bilińska, Barbara

2002-01-01

Among medicinal plants, extract from the hollyhock flowers is a source of antocyanides and flavonoids. The latter compounds belong, among others, to phytoestrogens (plant-derived dietary estrogens). The important role of estrogens in the testis is now well documented, and phytoestrogens, which may act as estrogen agonists or estrogen antagonists can also alter the reproductive function of the male. The aim of this study was to show whether the exposure of male rats to the aqueous hollyhock extract could affect the process of aromatization in their testes and in cultured Leydig cells. This was investigated by immunocytochemistry and radioimmunological assays. Immunoreactivities for aromatase and estrogen receptor beta were weaker both in testicular sections and cultured Leydig cells after hollyhock extract administration when compared to the controls, while the intensity of immunoreaction for estrogen receptor alpha remained unchanged. A lower level of estradiol secreted by cultured Leydig cells from the experimental group positively correlated with a direct inhibition of aromatase activity. Additionally, a quantitative analysis of flavonoid fraction from the hollyhock extract revealed the presence of quercetin and kaempferol. It seems that a weak antiestrogenic activity of flavonoid compounds present in the hollyhock extract is mediated through aromatase and estrogen receptor beta rather than by estrogen receptor alpha.

Lead acetate induces EGFR activation upstream of SFK and PKC{alpha} linkage to the Ras/Raf-1/ERK signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, C.-Y.; Wang, Y.-T.; Tzeng, D.-W.

2009-03-01

Lead acetate (Pb), a probable human carcinogen, can activate protein kinase C (PKC) upstream of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Yet, it remains unclear whether Pb activation of PKC {yields} ERK1/2 involves receptor/non-receptor tyrosine kinases and the Ras signaling transducer. Here we demonstrate a novel mechanism elicited by Pb for transmitting ERK1/2 signaling in CL3 human non-small-cell lung adenocarcinoma cells. Pb induction of higher steady-state levels of Ras-GTP was essential for increasing phospho-Raf-1{sup S338} and phospho-ERK1/2. Pre-treatment of the cells with a conventional PKC inhibitor Goe6976 or depleting PKC{alpha} using specific small interfering RNA blocked Pb induction ofmore » Ras-GTP. Pb also activated cellular tyrosine kinases. Specific pharmacological inhibitors, PD153035 for epidermal growth factor receptor (EGFR) and SU6656 for Src family tyrosine kinases (SFK), but not AG1296 for platelet-derived growth factor receptor, could suppress the Pb-induced tyrosine kinases, PKC{alpha}, Ras-GTP, phospho-Raf-1{sup S338} and phospho-ERK1/2. Furthermore, phosphorylation of tyrosines on the EGFR multiple autophosphorylation sites and the conserved SFK autophosphorylation site occurred during exposure of cells to Pb for 1-5 min and 5-30 min, respectively. Intriguingly, Pb activation of EGFR required the intrinsic kinase activity but not dimerization of the receptor. Inhibition of SFK or PKC{alpha} activities did not affect EGFR phosphorylation, while knockdown of EGFR blocked SFK phosphorylation and PKC{alpha} activation following Pb. Together, these results indicate that immediate activation of EGFR in response to Pb is obligatory for activation of SFK and PKC{alpha} and subsequent the Ras-Raf-1-MKK1/2-ERK1/2 signaling cascade.« less

[Experimental study of IFN-alpha and IFN-gamma on reversing ATRA-resistance in MR2 cell line].

PubMed

He, Peng-Cheng; Zhang, Mei; Li, Jing; Cao, Yun-Xin; Cai, Rui-Bo; Liu, Ya-Lin

2007-03-01

To explore the possibility and the possible mechanism of reversing ATRA-resistance in MR2 cells by using IFN-alpha and IFN-gamma in combination with all-trans retinoic acid (ATRA). After MR2 cells(ATRA-resistance cell line) were treated with IFN-alpha, IFN-gamma and ATRA alone or IFN-alpha and IFN-gamma in combination with ATRA respectively, the cell proliferation was tested by MTT colorimetry, the cell differentiation was tested through light microscope, by NBT test and flow cytometry (FCM). The expression of promyelocytic leukemia (PML) protein was observed by indirect immunofluorescence staining. Both IFN-alpha and IFN-gamma could inhibit the proliferation of MR2 cells. The effects were more obviously in both IFN-alpha+ATRA group and IFN-gamma+ATRA group. But there were no significant difference between either IFN-alpha group and IFN-gamma group or IFN-alpha+ATRA group and IFN-gamma+ATRA group (P>0.05). Both IFN could also induce the differentiation of MR2 cells. The effects of IFN-alpha+ATRA group and IFN-gamma+ATRA group were more obvious. However, the differentiation of MR2 cells induced by IFN-gamma+ATRA group was more higher than that by IFN-alpha+ATRA group (P<0.05). Both IFN could induce the expression of PML protein. The reversing effcet of IFN-gamma+ATRA group on ATRA-resistence in MR2 cells are more powerful than that of IFN-alpha+ATRA group, which may be related to the different signal transduction pathway of IFN-alpha and IFN-gamma.

Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, Meisheng; Tran, V.T.; Fong, H.K.W.

1991-05-01

The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein {alpha} subunits (G{alpha}) including G{sub s}{alpha}, G{sub i-1}{alpha}, G{sub i-2}{alpha}, G{sub i-3}{alpha}, and G{sub z}{alpha} (or G{sub x}{alpha}), where G{sub s} and G{sub i} are proteins that stimulate or inhibit adenylyl cyclase, respectively, and G{sub z} is a protein that may mediate pertussis toxin-insensitive events. Other G{alpha}-related mRNA transcripts were detected in fetal RPE cells by low-stringency hybridization to G{sub i-2}{alpha} and G{sub s}{alpha}more » protein-coding cDNA probes. The diversity of G proteins in RPE cells was further studied by cDNA amplification with reverse transcriptase and the polymerase chain reaction. This approach revealed that, besides the above mentioned members of the G{alpha} gene family, at least two other G{alpha} subunits are expressed in RPE cells. Human retinal cDNA clones that encode one of the additional G{alpha} subunits were isolated and characterized. The results indicate that this G{alpha} subunit belongs to a separate subfamily of G proteins that may be insensitive to inhibition by pertussis toxin.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jeeyun; Im, Young-Hyuck; Jung, Hae Hyun

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 A549more » cells.« less

New study on the correlation between carbon dioxide concentration in the environment and radon monitor devices.

PubMed

Shahrokhi, A; Burghele, B D; Fábián, F; Kovács, T

2015-12-01

The influence of high geogenic carbon dioxide concentrations on monitoring devices might present a significant challenge to the measurement of radon concentrations in environments with a high level of carbon dioxide concentration such as volcano sites, mofettes, caves, etc. In this study, the influence of carbon dioxide concentration on several different types of radon monitor devices - including Alpha Spectrometry (Sarad RTM 2200, EQF 3220, RAD7), Ionizing Chamber (AlphaGUARD PQ2000 PRO) and Active Cell (Active scintillation cell, Pylon 300A) - was examined to represent new aspects of radon measuring in environments with carbon dioxide. In light of the results, all measuring devices were exposed to variable conditions affected by carbon dioxide concentration, except for the AlphaGUARD, which was kept in a steady state throughout the experiment. It was observed that alpha spectroscopy devices were affected by carbon dioxide, since measured radon concentrations decreased in the presence of 70% and 90% carbon dioxide concentrations by 26.5 ± 2% and 14.5 ± 2.5% for EQF 3220, and 32 ± 2% and 35.5 ± 2% for RTM 2200. However, the ionizing chamber instrument was unaffected by changes in carbon dioxide concentration. It was determined that the RAD7 performed relatively inefficiently in the presence of carbon dioxide concentrations higher than 67% by an overall efficiency factor of approximately 0.52, confirming that it is not an admissible radon monitor instrument in environments with high carbon dioxide concentrations. Copyright © 2015 Elsevier Ltd. All rights reserved.

Distinct expression pattern of IFN-alpha and TNF-alpha in juvenile idiopathic arthritis synovial tissue.

PubMed

Gattorno, M; Chicha, L; Gregorio, A; Ferlito, F; Rossi, F; Jarrossay, D; Lanzavecchia, A; Martini, A; Manz, M G

2007-04-01

Recent laboratory and clinical data suggest that two prototype autoimmune diseases, systemic lupus erythematosus and rheumatoid arthritis are mainly driven by distinct cytokines, interferon (IFN)-alpha and tumour necrosis factor (TNF)-alpha, respectively. We here investigated the presence and characteristics of natural type I IFN-producing cells (IPCs), as well as IFN-alpha and TNF-alpha expression at sites of inflammation in juvenile idiopathic arthritis (JIA). Peripheral blood (PB) and synovial fluid (SF) mononuclear cells (MNCs) (n = 25 each) from JIA patients with active disease were studied. IPCs were identified as BCDA-2(+)CD123(+)HLA-DR(+)CD45RA(+) cells, and dendritic cells (DCs) as CD11c(+)CD14(-/low)lin(-) cells by flow cytometry. IPCs and DCs were analysed for Toll-like receptor-7 and -9 mRNA expression by real-time polymerase chain reaction. IFN-alpha was measured by enzyme-linked immunosorbent assay in serum, SF and in supernatants of influenza virus-infected, cultured IPCs. Synovial tissues of n = 6 additional JIA patients were analysed by immunohistochemistry using mAbs against CD123, IFN-alpha, TNF-alpha, CD3, CD19 and CD138. IPCs were enriched in SF MNCs compared with PB MNCs in all JIA patients. Influenza-induced, but no spontaneous IFN-alpha release was detected from SF IPCs, and serum and SF IFN-alpha levels were not elevated. Nonetheless, in synovial tissue IFN-alpha producing cells accumulated at inflammatory lymph-follicular-like structures, while TNF-alpha producing cells were mostly found at the lining and sublining layers. These data suggest that besides TNF-alpha-expressing cells, IFN-alpha-producing IPCs are involved in initiation, maintenance or regulation of the inflammatory response in JIA.

Altered expression of extracellular matrix molecules and their receptors in chronic pancreatitis and pancreatic adenocarcinoma in comparison with normal pancreas.

PubMed

Shimoyama, S; Gansauge, F; Gansauge, S; Oohara, T; Beger, H G

1995-12-01

The aim of this study was to elucidate the expression and distribution patterns of both integrins and extracellular matrix (ECM) molecules in chronic pancreatitis (CP) and pancreatic adenocarcinoma (PC) compared with normal pancreas (NP). Expression of nine alpha-subunits (alpha 2-alpha 6, alpha V, alpha L, alpha M, and alpha X), four beta-subunits (beta 1, beta 3-beta 5), and four ECM molecules (type IV collagen, laminin, fibronectin, and vitronectin) was investigated immunohistochemically. In CP, all integrins except alpha V showed nearly the same staining patterns compared with NP. Some acinar cells in CP expressed alpha V. Whereas alpha 2, alpha 3, and alpha 6 expression was stronger and diffuse, no alpha 5 expression was seen in PC. Basement membrane (BM) showed continuous staining in CP, whereas it showed discontinuous/absent staining in PC with antitype IV collagen, laminin, and vitronectin antibodies. Some carcinoma cells showed reverse correlation between alpha 2, alpha 3, and alpha 6 expression and type IV collagen and laminin expression. Fibronectin showed diffuse stromal expression in CP and PC. Some acinar cells or duct cells in CP carcinoma cells in PC showed intracellular VN expression. These results suggest that these integrins and ECM molecules are involved in inflammatory and malignant processes in pancreas.

Suppressive effects of ketamine on macrophage functions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang Yi; Department of Anesthesiology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan; Chen, T.-L.

2005-04-01

Ketamine is an intravenous anesthetic agent. Clinically, induction of anesthesia with ketamine can cause immunosuppression. Macrophages play important roles in host defense. In this study, we attempted to evaluate the effects of ketamine on macrophage functions and its possible mechanism using mouse macrophage-like Raw 264.7 cells as the experimental model. Exposure of macrophages to 10 and 100 {mu}M ketamine, which correspond to 0.1 and 1 times the clinically relevant concentration, for 1, 6, and 24 h had no effect on cell viability or lactate dehydrogenase release. When the administered concentration reached 1000 {mu}M, ketamine caused a release of lactate dehydrogenasemore » and cell death. Ketamine, at 10 and 100 {mu}M, did not affect the chemotactic activity of macrophages. Administration of 1000 {mu}M ketamine in macrophages resulted in a decrease in cell migration. Treatment of macrophages with ketamine reduced phagocytic activities. The oxidative ability of macrophages was suppressed by ketamine. Treatment with lipopolysaccharide induced TNF-{alpha}, IL-1{beta}, and IL-6 mRNA in macrophages. Administration of ketamine alone did not influence TNF-{alpha}, IL-1{beta}, or IL-6 mRNA production. Meanwhile, cotreatment with ketamine and lipopolysaccharide significantly inhibited lipopolysaccharide-induced TNF-{alpha}, IL-1{beta}, and IL-6 mRNA levels. Exposure to ketamine led to a decrease in the mitochondrial membrane potential. However, the activity of mitochondrial complex I NADH dehydrogenase was not affected by ketamine. This study shows that a clinically relevant concentration of ketamine (100 {mu}M) can suppress macrophage function of phagocytosis, its oxidative ability, and inflammatory cytokine production possibly via reduction of the mitochondrial membrane potential instead of direct cellular toxicity.« less

Comparison of alpha 1A- and alpha 1B-adrenoceptor coupling to inositol phosphate formation in rat kidney.

PubMed

Büscher, R; Erdbrügger, W; Philipp, T; Brodde, O E; Michel, M C

1994-12-01

We have compared the coupling mechanisms of rat renal alpha 1A- and alpha 1B-like adrenoceptors to inositol phosphate formation. The experiments were performed in parallel in native renal tissue preparations and in those where alpha 1B-adrenoceptors had been inactivated by treatment with 10 mumol/l chloroethylclonidine for 30 min at 37 degrees C; renal slices were used in most experiments but isolated renal cells were also used in some cases. The Ca2+ chelating agent, EGTA (5 mmol/l), reduced noradrenaline-stimulated inositol phosphate formation in native but enhanced it in chloroethylclonidine-treated renal slices. The inhibitory effect of EGTA was not mimicked by 100 nmol/l nifedipine. Inactivation of 87% of cellular Gi by 16-20 h treatment with 500 ng/ml pertussis toxin did not significantly affect noradrenaline-stimulated inositol phosphate formation in isolated renal cells but abolished the inhibitory effect of chloroethylclonidine. The adenylate cyclase activator, forskolin (20 mumol/l), inhibited noradrenaline-stimulated inositol phosphate formation in native and chloroethylclonidine-treated slices, and the inhibitory effects of chloroethylclonidine treatment and forskolin were additive. We conclude that in rat kidney inositol phosphate formation via alpha 1B-like adrenoceptors may involve the influx of extracellular Ca2+ and a pertussis toxin-sensitive G-protein but is insensitive to inhibition by forskolin. In contrast alpha 1A-like adrenoceptor-mediated inositol phosphate formation does not require the presence of extracellular Ca2+ or of Gi and is sensitive to inhibition by forskolin. In comparison to published data from other model systems we further conclude that the signaling mechanisms of alpha 1-adrenoceptor subtypes may depend on their cellular environment.

A pseudogene long noncoding RNA network regulates PTEN transcription and translation in human cells

PubMed Central

Johnsson, Per; Ackley, Amanda; Vidarsdottir, Linda; Lui, Weng-Onn; Corcoran, Martin; Grandér, Dan; Morris, Kevin V.

2013-01-01

PTEN is a tumor suppressor gene that has been shown to be under the regulatory control of a PTEN pseudogene expressed noncoding RNA, PTENpg1. Here, we characterize a previously unidentified PTENpg1 encoded antisense RNA (asRNA), which regulates PTEN transcription and PTEN mRNA stability. We find two PTENpg1 asRNA isoforms, alpha and beta. The alpha isoform functions in trans, localizes to the PTEN promoter, and epigenetically modulates PTEN transcription by the recruitment of DNMT3a and EZH2. In contrast, the beta isoform interacts with PTENpg1 through an RNA:RNA pairing interaction, which affects PTEN protein output via changes of PTENpg1 stability and microRNA sponge activity. Disruption of this asRNA-regulated network induces cell cycle arrest and sensitizes cells to doxorubicin, suggesting a biological function for the respective PTENpg1 expressed asRNAs. PMID:23435381

Paracrine GABA and insulin regulate pancreatic alpha cell proliferation in a mouse model of type 1 diabetes.

PubMed

Feng, Allen L; Xiang, Yun-Yan; Gui, Le; Kaltsidis, Gesthika; Feng, Qingping; Lu, Wei-Yang

2017-06-01

This study aimed to elucidate the mechanism of increased proliferation of alpha cells in recent-onset type 1 diabetes. Pancreatic beta cells express GAD and produce γ-aminobutyric acid (GABA), which inhibits alpha cell secretion of glucagon. We explored the roles of GABA in alpha cell proliferation in conditions corresponding to type 1 diabetes in a mouse model and in vitro. Type 1 diabetes was induced by injecting the mice with streptozotocin (STZ). Some of the STZ-injected mice were treated with GABA (10 mg/kg daily) for 12 days. Isolated pancreatic islets were treated with STZ or STZ together with GABA for 2 days. The effects of GABA treatment on STZ-induced alpha cell proliferation in vivo and in vitro were assessed. The effect of muscimol, a GABA receptor agonist, on αTC1-6 cell proliferation was also examined. STZ injection substantially decreased levels of GAD, GABA and insulin in pancreatic beta cells 12 h after injection; this was followed by an upsurge of phosphorylated mechanistic target of rapamycin (p-mTOR) in the alpha cells at day 1, and a significant increase in alpha cell mass at day 3. Treating STZ-injected mice with GABA largely restored the immunodetectable levels of insulin and GAD in the beta cells and significantly decreased the number of aldehyde dehydrogenase 1 family, member A3 (ALDH1a3)-positive cells, alpha cell mass and hyperglucagonaemia. STZ treatment also increased alpha cell proliferation in isolated islets, which was reversed by co-treatment with GABA. Muscimol, together with insulin, significantly lowered the level of cytosolic Ca 2+ and p-mTOR, and decreased the proliferation rate of αTC1-6 cells. GABA signalling critically controls the alpha cell population in pancreatic islets. Low intraislet GABA may contribute to alpha cell hyperplasia in early type 1 diabetes.

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

2016-08-08

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.

Coenzyme Q{sub 10} and alpha-tocopherol protect against amitriptyline toxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cordero, Mario D.; Dpto. Citologia e Histologia Normal y Patologica, Facultad de Medicina. Universidad de Sevilla. 41009 Sevilla; Moreno-Fernandez, Ana Maria

Since amitriptyline is a very frequently prescribed antidepressant drug, it is not surprising that amitriptyline toxicity is relatively common. Amitriptyline toxic systemic effects include cardiovascular, autonomous nervous, and central nervous systems. To understand the mechanisms of amitriptyline toxicity we studied the cytotoxic effects of amitriptyline treatment on cultured primary human fibroblasts and zebrafish embryos, and the protective role of coenzyme Q{sub 10} and alpha-tocopherol, two membrane antioxidants. We found that amitriptyline treatment induced oxidative stress and mitochondrial dysfunction in primary human fibroblasts. Mitochondrial dysfunction in amitriptyline treatment was characterized by reduced expression levels of mitochondrial proteins and coenzyme Q{sub 10},more » decreased NADH:cytochrome c reductase activity, and a drop in mitochondrial membrane potential. Moreover, and as a consequence of these toxic effects, amitriptyline treatment induced a significant increase in apoptotic cell death activating mitochondrial permeability transition. Coenzyme Q{sub 10} and alpha-tocopherol supplementation attenuated ROS production, lipid peroxidation, mitochondrial dysfunction, and cell death, suggesting that oxidative stress affecting cell membrane components is involved in amitriptyline cytotoxicity. Furthermore, amitriptyline-dependent toxicity and antioxidant protection were also evaluated in zebrafish embryos, a well established vertebrate model to study developmental toxicity. Amitriptyline significantly increased embryonic cell death and apoptosis rate, and both antioxidants provided a significant protection against amitriptyline embryotoxicity.« less

Boron modulates extracellular matrix and TNF alpha synthesis in human fibroblasts.

PubMed

Benderdour, M; Hess, K; Dzondo-Gadet, M; Nabet, P; Belleville, F; Dousset, B

1998-05-29

Boric acid was not mitogenic for human fibroblasts and it did not change cell viability until 0.5% (w/v). Boric acid treatment affected the metabolism of human dermal fibroblasts in culture, decreasing the synthesis of extracellular matrix macromolecules such as proteoglycans, collagen, and total proteins. It also increased the release of these molecules into the culture medium. The principal proteins secreted into the medium after boric acid treatment had molecular masses of 90, 70, 58, 49, and 43 kDa and faint bands were detected by electrophoresis between 14 and 30 kDa. hsp 70 and TNF alpha were detected among the secreted proteins by immunoblotting, and the amount of TNF alpha released was quantified by radioimmunoassay. Total mRNA levels were higher after boric acid treatment and peaked after 6 h of treatment. TNF alpha mRNA was undetectable in unstimulated fibroblasts and two TNF alpha mRNA bands were detected after stimulation: immature mRNA (4.8 kb) and mature TNF alpha mRNA (1.9 kb). Thus, the effects of boric acid observed in wound repair in vivo may be due to TNF alpha synthesis and secretion.

Tumour necrosis factor-alpha impairs neuronal differentiation but not proliferation of hippocampal neural precursor cells: Role of Hes1.

PubMed

Keohane, Aoife; Ryan, Sinead; Maloney, Eimer; Sullivan, Aideen M; Nolan, Yvonne M

2010-01-01

Tumour necrosis factor-alpha (TNFalpha) is a pro-inflammatory cytokine, which influences neuronal survival and function yet there is limited information available on its effects on hippocampal neural precursor cells (NPCs). We show that TNFalpha treatment during proliferation had no effect on the percentage of proliferating cells prepared from embryonic rat hippocampal neurosphere cultures, nor did it affect cell fate towards either an astrocytic or neuronal lineage when cells were then allowed to differentiate. However, when cells were differentiated in the presence of TNFalpha, significantly reduced percentages of newly born and post-mitotic neurons, significantly increased percentages of astrocytes and increased expression of TNFalpha receptors, TNF-R1 and TNF-R2, as well as expression of the anti-neurogenic Hes1 gene, were observed. These data indicate that exposure of hippocampal NPCs to TNFalpha when they are undergoing differentiation but not proliferation has a detrimental effect on their neuronal lineage fate, which may be mediated through increased expression of Hes1. Copyright 2009 Elsevier Inc. All rights reserved.

Modulation of Mycoplasma arthritidis-derived superantigen-induced cytokine gene expression by dexamethasone and interleukin-4.

PubMed Central

Mehindate, K; al-Daccak, R; Rink, L; Mecheri, S; Hébert, J; Mourad, W

1994-01-01

Activation of human monocytes or monocytic cell lines with all known stimuli coordinately induces the gene expression of various cytokines, including tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and the IL-1 receptor antagonist (IL-1Ra). In contrast, superantigens induce TNF-alpha and IL-1 beta but fail to affect IL-1Ra gene expression, suggesting that activation of monocytes via major histocompatibility complex class II is distinct from other signal transduction pathways. In the present study, we analyzed the regulation of the Mycoplasma arthritidis-derived superantigen (MAM)-induced IL-1 beta and TNF-alpha gene expression by studying the effects of two different anti-inflammatory agents: dexamethasone (DEX) and the T-cell-derived cytokine IL-4. Both agents contributed to the downregulation of MAM-induced IL-1 beta and TNF-alpha gene expression. They accelerated the normal decline of the gene expression of both MAM-induced cytokines by decreasing the stability of mRNAs via the induction or enhanced synthesis of one or more regulatory proteins. In addition, IL-4, but not DEX, induced a strong and rapid expression of IL-1Ra mRNA in MAM-stimulated and unstimulated THP-1 cells in a de novo protein synthesis-independent manner. The capacity of IL-4 to induce IL-1Ra gene expression reinforces its anti-inflammatory activity. This study illustrates some of the mechanisms by which MAM-induced proinflammatory monokine gene expression can be downregulated by IL-4 and DEX. Images PMID:7927746

N-acetylcysteine attenuates TNF-alpha-induced human vascular endothelial cell apoptosis and restores eNOS expression.

PubMed

Xia, Zhengyuan; Liu, Min; Wu, Yong; Sharma, Vijay; Luo, Tao; Ouyang, Jingping; McNeill, John H

2006-11-21

The circulatory inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is increased in pathological conditions, such as diabetes, which initiate or exacerbate vascular endothelial injury. Both nitric oxide (NO) and reactive oxygen species may play a dual role (i.e., inhibiting or promoting) in TNF-alpha-induced endothelial cell apoptosis. We investigated the effects of the antioxidant N-acetylcysteine on TNF-alpha-induced apoptosis in human vascular endothelial cell (cell line ECV304) apoptosis, NO production and lipid peroxidation. Cultured vascular endothelial cell (ECV304) were either not treated (control), or treated with TNF-alpha (40 ng/ml) alone or TNF-alpha in the presence of N-acetylcysteine at 30 mmol/l or 1 mmol/l, respectively, for 24 h. Cell viability was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cell apoptosis was assessed by flow cytometry. TNF-alpha-induced endothelial cell apoptosis was associated with increased inducible NO synthase but reduced endothelial NO synthase (eNOS) protein expression. NO production and the levels of the lipid peroxidation product malondialdehyde were concomitantly increased. Treatment with NAC at 30 mmol/l restored eNOS expression and further increased NO production as compared to TNF-alpha alone, resulting in improved cell viability and reduced apoptosis. This was accompanied by increased superoxide dismutase activity, increased glutathione peroxidase production and reduced malondialdehyde levels. N-acetylcysteine at 1 mmol/l, however, did not have significant effects on TNF-alpha-induced endothelial cell apoptosis and cell viability despite it slightly enhanced glutathione peroxidase production. N-acetylcysteine attenuation of TNF-alpha-induced human vascular endothelial cell apoptosis is associated with the restoration of eNOS expression.

Functional coupling of rat myometrial alpha 1-adrenergic receptors to Gh alpha/tissue transglutaminase 2 during pregnancy.

PubMed

Dupuis, Morgan; Lévy, Arlette; Mhaouty-Kodja, Sakina

2004-04-30

Gh alpha protein, which exhibits both transglutaminase and GTPase activities, represents a new class of GTP-binding proteins. In the present study, we characterized Gh alpha in rat uterine smooth muscle (myometrium) and followed its expression during pregnancy by reverse transcription-PCR and Western blot. We also measured transglutaminase and GTP binding functions and used a smooth muscle cell line to evaluate the role of Gh alpha in cell proliferation. The results show that pregnancy is associated with an up-regulation of Gh alpha expression at both the mRNA and protein level. Gh alpha induced during pregnancy is preferentially localized to the plasma membrane. This was found associated with an increased ability of plasma membrane preparations to catalyze Ca(2+)-dependent incorporation of [(3)H]putrescine into casein in vitro. In the cytosol, significant changes in the level of immunodetected Gh alpha and transglutaminase activity were seen only at term. Activation of alpha1-adrenergic receptors (alpha1-AR) enhanced photoaffinity labeling of plasma membrane Gh alpha. Moreover, the level of alpha1-AR-coupled Gh alpha increased progressively with pregnancy, which parallels the active period of myometrial cell proliferation. Overexpression of wild type Gh alpha in smooth muscle cell line DDT1-MF2 increased alpha1-AR-induced [(3)H]thymidine incorporation. A similar response was obtained in cells expressing the transglutaminase inactive mutant (C277S) of Gh alpha. Together, these findings underscore the role of Gh alpha as signal transducer of alpha1-AR-induced smooth muscle cell proliferation. In this context, pregnant rat myometrium provides an interesting physiological model to study the mechanisms underlying the regulation of the GTPase function of Gh alpha

FcepsilonRI-alpha siRNA inhibits the antigen-induced activation of mast cells.

PubMed

Safaralizadeh, Reza; Soheili, Zahra-Soheila; Deezagi, Abdolkhaleg; Pourpak, Zahra; Samiei, Shahram; Moin, Mostafa

2009-12-01

FcepsilonRI, The high affinity receptor for IgE plays a critical role in triggering the allergic reactions. It is responsible for inducing mast cell degranulation and deliberation of allergy mediators when it is aggregated by allergen and IgE complexes. FcepsilonRI on the mast cells consists of three subunits; alpha chain directly binds IgE, beta chain and dimmer of gamma chains together mediate intracellular signaling. Cross-linking of IgE-bound FcepsilonRI on the surface of mast cells and basophils by the multivalent antigen induces release of chemical mediators. The present in vitro study was designed to investigate the effect of synthetic FcepsilonRI-alpha siRNA on the antigen-induced activation of MC/9 cells. MC/9 cells which are murine mast cells were transfected by FcepsilonRI-alpha siRNA and negative control siRNA. After 6 h, anti-DNP (Dinitrophenyl) IgE was used for the cells sensitization. Then the cells were challenged with Dinitrophenyl-Human Serum Albumin (DNP-HSA) for mast cell degranulation induction before collection of supernatants. The amount of mRNA and protein expression was measured by Real Time PCR and western blot analysis, respectively. Determination of the expression rate of FcepsilonRI-alpha on cell surface was achieved by flow cytometry. ELISA and spectrophotometry methods were used subsequently for measuring the effects of FcepsilonRI-alpha siRNA on antigen-induced histamine and beta-hexosaminidase release. FcepsilonRI-alpha siRNA treated cells showed significant decrease in FcepsilonRI-alpha mRNA and protein expression in comparison to control cells. FcepsilonRI-mediated mast cell release of beta-hexosaminidase and histamine were also inhibited. In this study it was shown that FcepsilonRI-alpha siRNA could suppress FcepsilonRI-alpha expression and inhibited degranulation and histamine release in antigen-stimulated MC/9 cells. In conclusion, knock-down of FcepsilonRI-alpha by siRNA could be a promising method for inhibition of the mast cell-mediated allergic reactions.

Comparison of cell wall proteins of Saccharomyces cerevisiae as anchors for cell surface expression of heterologous proteins.

PubMed Central

Van der Vaart, J M; te Biesebeke, R; Chapman, J W; Toschka, H Y; Klis, F M; Verrips, C T

1997-01-01

The carboxyl-terminal regions of five cell wall proteins (Cwp1p, Cwp2p, Ag alpha 1p, Tip1p, and Flo1p) and three potential cell wall proteins (Sed1p, YCR89w, and Tir1p) all proved capable of immobilizing alpha-galactosidase in the cell wall of Saccharomyces cerevisiae. The fraction of the total amount of fusion protein that was localized to the cell wall varied depending on the anchor domain used. The highest proportion of cell wall incorporation was achieved with Cwp2p, Ag alpha 1p, or Sed1p as an anchor. Although 80% of these fusion proteins were incorporated in the cell wall, the total production of alpha-galactosidase-Ag alpha 1p was sixfold lower than that of alpha-galactosidase-Cwp2p and eightfold lower than that of alpha-galactosidase-Sed1p. Differences in mRNA levels were not responsible for this discrepancy, nor was an intracellular accumulation of alpha-galactosidase-Ag alpha 1p detectable. A lower translation efficiency of the alpha-galactosidase-AG alpha 1 fusion construct is most likely to be responsible for the low level of protein production. alpha-Galactosidase immobilized by the carboxyl-terminal 67 amino acids of Cwp2p was most effective in the hydrolysis of the high-molecular-weight substrate guar gum from Cyamopsis tetragonoloba. This indicates that the use of a large anchoring domain does not necessarily result in a better exposure of the immobilized enzyme to the exterior of the yeast cell. PMID:9023939

Bioactive interleukin-1alpha is cytolytically released from Candida albicans-infected oral epithelial cells.

PubMed

Dongari-Bagtzoglou, A; Kashleva, H; Villar, C Cunha

2004-12-01

Oral epithelial cells are primary targets of Candida albicans in the oropharynx and may regulate the inflammatory host response to this pathogen. This investigation studied the mechanisms underlying interleukin-1alpha (IL-1alpha) release by oral epithelial cells and the role of IL-1alpha in regulating the mucosal inflammatory response to C. albicans. Infected oral epithelial cells released processed IL-1alpha protein in culture supernatants. The IL-1alpha generated was stored intracellularly and was released upon cell lysis. This was further supported by the fact that different C. albicans strains induced variable IL-1alpha release, depending on their cytolytic activity. IL-1alpha from C. albicans-infected oral epithelial cells upregulated proinflammatory cytokine secretion (IL-8 and GM-CSF) in uninfected oral epithelial or stromal cells. Our studies suggest that production of IL-1alpha, IL-8 and GM-CSF may take place in the oral mucosa in response to lytic infection of epithelial cells with C. albicans. This process can act as an early innate immune surveillance system and may contribute to the clinicopathologic signs of infection in the oral mucosa.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Xiaomin; Beijing Key Laboratory for Protein Therapeutics, Tsinghua University, Beijing 100084; Cancer Biology Laboratory, School of Life Sciences, Tsinghua University, Beijing 100084

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 andmore » 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.« less

Self and viral peptides can initiate lysis by autologous natural killer cells.

PubMed

Mandelboim, O; Wilson, S B; Valés-Gómez, M; Reyburn, H T; Strominger, J L

1997-04-29

Natural killer (NK) cells are inhibited by specific allotypes of class I major histocompatibility complex ligands recognized by polymorphic inhibitory receptors (e.g., NKIR1 and NKIR2). NK1- and NK2-specific clones recognize two groups of HLA-C allotypes that are distinguished by a dimorphism at residue 80 in the alpha1 helix (alphaLys-80 and alphaAsn-80, respectively). "Empty" HLA-Cw7 expressed in peptide transporter-deficient cells and HLA-Cw7 loaded with several peptides each functioned as inhibitory ligands for NK2 lines and clones. However, loading of HLA-Cw7 with two other peptides derived from glutamic acid decarboxylase or coxsackie virus (each of which has been associated with autoimmune diabetes mellitus) abrogated this inhibitory recognition. Both peptides contained Lys at P8 of the epitope. Substitution of P8 with Ala or two other basic amino acids, His and Arg, resulted in peptides that were inhibitory, as were peptides with P8 Val, Glu, or Asn. The manner in which a Lys at P8 might affect recognition is discussed, together with a hypothesis for a novel mechanism by which an autoimmune disease might be initiated.

Anti-inflammatory effect of linear polarized infrared irradiation on interleukin-1beta-induced chemokine production in MH7A rheumatoid synovial cells.

PubMed

Shibata, Yasuko; Ogura, Naomi; Yamashiro, Keisuke; Takashiba, Shogo; Kondoh, Toshirou; Miyazawa, Keiji; Matsui, Masaru; Abiko, Yoshimitsu

2005-12-01

We examined the anti-inflammatory effect of infrared linear polarized light irradiation on the MH7A rheumatoid fibroblast-like synoviocytes (FLS) stimulated with the proinflammatory cytokine interleukin (IL)-1beta. Expression of messenger ribonucleic acids (mRNAs) encoding IL-8, RANTES (regulated upon activation, normal T cell expressed and secreted), growth-related gene alpha (GROalpha), and macrophage inflammatory protein-1alpha (MIP1alpha) was measured using real-time reverse transcription polymerase chain reaction, and the secreted proteins were measured in the conditioned media using enzyme-linked immunosorbent assays. We found that irradiation with linear polarized infrared light suppressed IL-1beta-induced expression of IL-8 mRNA and, correspondingly, the synthesis and release of IL-8 protein in MH7A cells. This anti-inflammatory effect was equivalent to that obtained with the glucocorticoid dexamethasone. Likewise, irradiation suppressed the IL-1beta-induced expression of RANTES and GROalpha mRNA. These results suggest that the irradiation of the areas around the articular surfaces of joints affected by rheumatoid arthritis (RA) using linear polarized light may represent a useful new approach to treatment.

Integrin expression and integrin-mediated adhesion in vitro of human multipotent stromal cells (MSCs) to endothelial cells from various blood vessels.

PubMed

Semon, Julie A; Nagy, Lauren H; Llamas, Claire B; Tucker, H Alan; Lee, Ryang Hwa; Prockop, Darwin J

2010-07-01

Multipotent mesenchymal stromal cells (MSCs) home to damaged tissue by processes partly regulated by integrins. Integrin subunits expressed by MSCs were identified by flow cytometry (FC), immunocytochemistry (IC), and a panel of integrin-binding antibodies. In subconfluent cultures, over 80% of MSCs expressed integrin subunits beta1, beta2, and alpha3, 20%-55% expressed alpha1, alpha2, alpha4, alpha5, alpha6, and alphaV, and about 10% expressed beta3 when assayed by FC. None of the cells expressed significant levels of 13 other integrins as assayed by FC, but seven of the 13 integrins were detected by IC: beta5, alpha7, alpha8, alpha9, alpha11, alphaX, and alphaD. Expression of some integrins changed with MSC confluency: integrins beta3, alpha1, alpha3, alpha5, and alphaV increased, and alpha6 decreased. Furthermore, alpha4 was the only integrin to vary among preparations of MSCs from different donors. The results resolved some discrepancies in the literature concerning integrin expression by MSCs. We also investigated the role of specific integrins in MSC adhesion to endothelial cells (ECs) from the pulmonary artery (HPAEC), cardiac-derived microvasculature (HMVEC-C), and umbilical veins (HUVEC). In experiments with blocking antibodies to beta integrins, anti-beta5 reduced MSC adhesion to all types of ECs, anti-beta1 to both HUVEC and HPAEC, anti-beta3 to HUVEC, and anti-beta2 to HMVEC-C. With blocking antibodies to alpha integrins, anti-alphaX reduced adhesion to HPAEC and HMVEC-C, anti-alphaV to HPAEC, and both anti-alpha7 and anti-alphaD to HMVEC-C. Thus, MSCs use diverse integrins to adhere to EC from various blood vessels in vitro.

Estrogen receptor alpha modulates toll-like receptor signaling in murine lupus

PubMed Central

Cunningham, Melissa A.; Naga, Osama S.; Eudaly, Jackie G.; Scott, Jennifer L.; Gilkeson, Gary S.

2013-01-01

Systemic lupus erythematosus (SLE) is a disease that disproportionately affects females. Despite significant research effort, the mechanisms underlying the female predominance in this disease are largely unknown. Previously, we showed that estrogen receptor alpha knockout (ERαKO) lupus prone female mice had significantly less pathologic renal disease and proteinuria, and significantly prolonged survival. Since autoantibody levels and number and percentage of B/T cells were not significantly impacted by ERα genotype, we hypothesized that the primary benefit of ERα deficiency in lupus nephritis was via modulation of the innate immune response. Using BMDCs and spleen cells/B cells from female wild-type or ERαKO mice, we found that ERαKO-derived cells have a significantly reduced inflammatory response after stimulation with TLR agonists. Our results indicate that the inflammatory response to TLR ligands is significantly impacted by the presence of ERα despite the absence of estradiol, and may partially explain the protective effect of ERα deficiency in lupus-prone animals. PMID:22659029

Zinc oxide nanoparticles and SH-SY5Y cell line

NASA Astrophysics Data System (ADS)

Zheng, Jinghui

The Arctic and sub-arctic regions are impacted by the growth of the global nanotechnology industry. Nanomaterials have unique chemical and physical properties that may lead to toxicological effects that interfere with normal cellular metabolism. Zinc oxide nanoparticles (ZnO NPs) are now very common and widely used in daily life. In industry, ZnO NPs are used to protect different materials from damage caused by UV exposure. The scientific literature suggests that ZnO NPs can have negative impacts on both living organisms and plants. However, there is a paucity of research on the mechanisms by which ZnO NPs may affect the neuronal cells. This study investigates how ZnO NPs interact with the neuroblastoma cell line SH-SY5Y. Using transmission electron microscopy, we observed that the ZnO NPs form 36 nm particles on average, and increase the level of vascular endothelial growth factor (VEGF) in extracellular fluid, as measured by an enzyme-linked immunosorbent assay (ELISA). Moreover, ZnO NPs, in presence of tumor necrosis factor-alpha (TNF-alpha), can also decrease the level of extracellular VEGF compared with TNF-alpha treatment alone. These findings suggest the basis for more studies on understanding the mechanism by which ZnO NPs impact cytokine signaling. Another direction is using ELISA technology to observe the interactions of NPs with different cell types such as neuronal stem cells.

Metabolism of two Go alpha isoforms in neuronal cells during differentiation.

PubMed

Brabet, P; Pantaloni, C; Bockaert, J; Homburger, V

1991-07-15

We have previously shown that undifferentiated N1E-115 neuroblastoma cells express only one isoform of Go alpha (pI = 5.8), whereas differentiated neuroblastoma cells expressed, in addition to this isoform, another Go alpha with a more acidic pI (5.55). Moreover, primary cultures of cerebellar granule cells, which are extremely well differentiated cells yielding a high density of synapses, expressed only a single Go alpha isoform with a pI of 5.55 (Brabet, P., Pantaloni, C., Rodriguez Martinez, J., Bockaert, J., and Homburger, V. (1990) J. Neurochem. 54, 1310-1320). In this report, using biosynthetic labeling with [35S]methionine and specific quantitative immunoprecipitation with a polyclonal antibody raised against the purified Go alpha protein, we have determined 1) the degradation rate of total Go alpha (sum of the two isoforms) in differentiated as well as in undifferentiated neuroblastoma cells and in cerebellar granule cells, 2) the degradation rates of each isoform in differentiated neuroblastoma cells. The t 1/2 for total Go alpha protein degradation was very different in the three neuronal cell populations and was 28 +/- 5 h (n = 5), 58 +/- 9 h (n = 5), and 154 +/- 22 h (n = 6) in undifferentiated, differentiated neuroblastoma, and granule cells, respectively. Using two-dimensional gel analysis of immunoprecipitates, we have also determined the individual t 1/2 for degradation of each Go alpha isoform in differentiated neuroblastoma cells, in which the two Go alpha isoforms were expressed. Results indicated that the two Go alpha isoforms exhibit similar t1/2 for degradation (49 +/- 5 h, n = 3). Thus, the t1/2 for degradation of the more basic Go alpha isoform is higher in differentiated neuroblastoma cells (49 +/- 5 h, n = 3) than in undifferentiated neuroblastoma cells (28 +/- 5 h, n = 5) which expressed only the more basic Go alpha isoform. It can be concluded that the degradation rate of the more basic Go alpha isoform is not a characteristic of the protein itself but depends on the state of the cell differentiation. The comparison between the t1/2 for degradation of the more acidic Go alpha isoform is differentiated neuroblastoma cells (51 +/- 6 h, n = 3) with that of cerebellar granule cells (154 +/- 22 h, n = 6) suggests that there is also a decrease in the degradation rate of the more acidic Go alpha isoform during differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Requirement for IFN-gamma in IL-12 production induced by collaboration between v(alpha)14(+) NKT cells and antigen-presenting cells.

PubMed

Yang, Y F; Tomura, M; Ono, S; Hamaoka, T; Fujiwara, H

2000-12-01

Two cytokines IL-4 and IL-12 are known to determine the balance between T(h)1 and T(h)2 development. In addition to IL-4 production of V(alpha)14(+) NKT cells, they have recently been demonstrated to have the capacity to stimulate IL-12 production by antigen-presenting cells (APC). This study demonstrates that IFN-gamma is absolutely required for the NKT cell-stimulated IL-12 production. Culture of B cell-depleted spleen cells from C57BL/6 mice with alpha-galactosylceramide (alpha-GalCer) capable of selectively stimulating V(alpha)14/J(alpha)281(+) NKT cells resulted in the production of IL-12 together with IL-4. Whereas IL-4 production occurred in culture of IFN-gamma(-/-) C57BL/6 splenocytes, the same culture failed to generate IL-12 production. While IL-12 production induced during culture of V(alpha)14(+) NKT cells and APC depended on the interaction between CD40 ligand on NKT cells and CD40 on APC, the expression levels of these key molecules were comparable in cells from wild-type and IFN-gamma(-/-) mice. Addition of rIFN-gamma to alpha-GalCer stimulated IFN-gamma(-/-) splenocyte culture, and administration of rIFN-gamma to alpha-GalCer-injected IFN-gamma(-/-) mice resulted in the restoration of IL-12 production in vitro and in vivo. These results illustrate a mandatory role for IFN-gamma in V(alpha)14(+) NKT cell-stimulated IL-12 production by APC.

alpha(4)beta(7) independent pathway for CD8(+) T cell-mediated intestinal immunity to rotavirus.

PubMed

Kuklin, N A; Rott, L; Darling, J; Campbell, J J; Franco, M; Feng, N; Müller, W; Wagner, N; Altman, J; Butcher, E C; Greenberg, H B

2000-12-01

Rotavirus (RV), which replicates exclusively in cells of the small intestine, is the most important cause of severe diarrhea in young children worldwide. Using a mouse model, we show that expression of the intestinal homing integrin alpha(4)ss(7) is not essential for CD8(+) T cells to migrate to the intestine or provide immunity to RV. Mice deficient in ss7 expression (ss7(-/-)) and unable to express alpha(4)ss(7) integrin were found to clear RV as quickly as wild-type (wt) animals. Depletion of CD8(+) T cells in ss7(-/-) animals prolonged viral shedding, and transfer of immune ss7(-/-) CD8(+) T cells into chronically infected Rag-2-deficient mice resolved RV infection as efficiently as wt CD8(+) T cells. Paradoxically, alpha(4)ss(7)(hi) memory CD8(+) T cells purified from wt mice that had been orally immunized cleared RV more efficiently than alpha(4)ss(7)(low) CD8(+) T cells. We explained this apparent contradiction by demonstrating that expression of alpha(4)ss(7) on effector CD8(+) T cells depends upon the site of initial antigen exposure: oral immunization generates RV-specific CD8(+) T cells primarily of an alpha(4)ss(7)(hi) phenotype, but subcutaneous immunization yields both alpha(4)ss(7)(hi) and alpha(4)ss(7)(low) immune CD8(+) T cells with anti-RV effector capabilities. Thus, alpha(4)ss(7) facilitates normal intestinal immune trafficking to the gut, but it is not required for effective CD8(+) T cell immunity.

Uroepithelial cells are part of a mucosal cytokine network.

PubMed Central

Hedges, S; Agace, W; Svensson, M; Sjögren, A C; Ceska, M; Svanborg, C

1994-01-01

This study compared the cytokine production of uroepithelial cell lines in response to gram-negative bacteria and inflammatory cytokines. Human kidney (A498) and bladder (J82) epithelial cell lines were stimulated with either Escherichia coli Hu734, interleukin 1 alpha (IL-1 alpha), or tumor necrosis factor alpha (TNF-alpha). Supernatant samples were removed, and the RNA was extracted from cells at 0, 2, 6, and 24 h. The secreted cytokine levels were determined by bioassay or immunoassay; mRNA was examined by reverse transcription-PCR. The two cell lines secreted IL-6 and IL-8 constitutively. IL-6 and IL-8 mRNA were constitutively produced in both cell lines; IL-1 beta mRNA was detected in J82 cells. IL-1 alpha induced significantly higher levels of IL-6 secretion than did E. coli Hu734 or TNF-alpha. IL-1 alpha and TNF-alpha induced significantly higher levels of IL-8 secretion than did E. coli Hu734. Secreted IL-1 beta was not detected; IL-1 alpha and TNF-alpha were not detected above the levels used for stimulation. IL-1 alpha, IL-1 beta, IL-6, and IL-8 mRNAs were detected in both cell lines after exposure to the stimulants. TNF-alpha mRNA was occasionally detected in the J82 cell line after TNF-alpha stimulation. Cytokine (IL-6 and IL-8) and control (glyceraldehyde 3-phosphate dehydrogenase [G3PDH] and beta-actin) mRNA concentrations were quantitated with internal PCR standards. Cytokine mRNA levels relative to beta-actin mRNA levels were the highest in E. coli-stimulated cells. In comparison, the cytokine mRNA levels relative to G3PDH mRNA levels were the highest in IL-1 alpha-stimulated cells. beta-Actin mRNA levels decreased after bacterial stimulation but not after cytokine stimulation, while G3PDH mRNA levels increased in response to all of the stimulants tested. These results suggested that E. coli Hu734 lowered the beta-actin mRNA levels in uroepithelial cells, thus distorting the IL-6 and IL-8 mRNA levels relative to this control. In summary, E. coli IL-1 alpha and TNF-alpha were found to activate the de novo synthesis and secretion of IL-6 and IL-8 in uroepithelial cells. These results emphasize the role of epithelial cells in cytokine-mediated responses during the early stages of infection. Images PMID:8188354

Dose-response effects of estrogenic mycotoxins (zearalenone, alpha- and beta-zearalenol) on motility, hyperactivation and the acrosome reaction of stallion sperm

PubMed Central

2011-01-01

Background The aim of this study was to investigate the in vitro effects of the Fusarium fungus-derived mycotoxin, zearalenone and its derivatives alpha-zearalenol and beta-zearalenol on motility parameters and the acrosome reaction of stallion sperm. Since the toxic effects of zearalenone and its derivatives are thought to result from their structural similarity to 17beta-estradiol, 17beta-estradiol was used as a positive control for 'estrogen-like' effects. Methods Stallion spermatozoa were exposed in vitro to zearalenone, alpha-zearalenol, beta-zearalenol or 17beta-estradiol at concentrations ranging from 1 pM - 0.1 mM. After 2 hours exposure, motility parameters were evaluated by computer-assisted analysis, and acrosome integrity was examined by flow cytometry after staining with fluoroscein-conjugated peanut agglutinin. Results Mycotoxins affected sperm parameters only at the highest concentration tested (0.1 mM) after 2 hours exposure. In this respect, all of the compounds reduced the average path velocity, but only alpha-zearalenol reduced percentages of motile and progressively motile sperm. Induction of motility patterns consistent with hyperactivation was stimulated according to the following rank of potency: alpha-zearalenol >17beta-estradiol > zearalenone = beta-zearalenol. The hyperactivity-associated changes observed included reductions in straight-line velocity and linearity of movement, and an increase in the amplitude of lateral head displacement, while curvilinear velocity was unchanged. In addition, whereas alpha- and beta- zearalenol increased the percentages of live acrosome-reacted sperm, zearalenone and 17beta-estradiol had no apparent effect on acrosome status. In short, alpha-zearalenol inhibited normal sperm motility, but stimulated hyperactive motility in the remaining motile cells and simultaneously induced the acrosome reaction. Beta-zearalenol induced the acrosome reaction without altering motility. Conversely, zearalenone and 17beta-estradiol did not induce the acrosome reaction but induced hyperactive motility albeit to a different extent. Conclusions Apparently, the mycotoxin zearalenone has 17beta-estradiol-like estrogenic activity that enables it to induce hyperactivated motility of equine sperm cells, whereas the zearalenol derivatives induce premature completion of the acrosome reaction and thereby adversely affect stallion sperm physiology. The alpha form of zearalenol still possessed the estrogenic ability to induce hyperactivated motility, whereas its beta stereo-isomere had lost this property. PMID:21970729

Neuroblastoma differentiation involves the expression of two isoforms of the alpha-subunit of Go.

PubMed

Brabet, P; Pantaloni, C; Rodriguez, M; Martinez, J; Bockaert, J; Homburger, V

1990-04-01

The regulation of GTP-binding proteins (G proteins) was examined during the course of differentiation of neuroblastoma N1E-115 cells. N1E-115 cell membranes possess three Bordetella pertussis toxin (PTX) substrates assigned to alpha-subunits (G alpha) of Go (a G protein of unknown function) and "Gi (a G protein inhibitory to adenylate cyclase)-like" proteins and one substrate of Vibrio cholerae toxin corresponding to an alpha-subunit of Gs (a G protein stimulatory to adenylate cyclase). In undifferentiated cells, only one form of Go alpha was found, having a pI of 5.8 Go alpha content increased by approximately twofold from the undifferentiated state to 96 h of cell differentiation. This is mainly due to the appearance of another Go alpha form having a pI of 5.55. Both Go alpha isoforms have similar sizes on sodium dodecyl sulfate-polyacrylamide gels, are recognized by polyclonal antibodies to bovine brain Go alpha, are ADP-ribosylated by PTX, and are covalently myristylated in whole N1E-115 cells. In addition, immunofluorescent staining of N1E-115 cells with Go alpha antibodies revealed that association of Go alpha with the plasma membrane appears to coincide with the expression of the most acidic isoform and morphological cell differentiation. In contrast, the levels of both Gi alpha and Gs alpha did not significantly change, whereas that of the common beta-subunit increased by approximately 30% over the same period. These results demonstrate specific regulation of the expression of Go alpha during neuronal differentiation.

LPS receptor CD14 participates in release of TNF-alpha in RAW 264.7 and peritoneal cells but not in kupffer cells.

PubMed

Lichtman, S N; Wang, J; Lemasters, J J

1998-07-01

Lipopolysaccharide (LPS) is a bacterial polymer that stimulates macrophages to release tumor necrosis factor-alpha (TNF-alpha). In macrophages (RAW 264.7 and peritoneal cells), LPS binds to the CD14 surface receptor as the first step toward signaling. Liver macrophages, Kupffer cells, are the most numerous fixed-tissue macrophage in the body. The presence of CD14 on Kupffer cells and its role in LPS stimulation of TNF-alpha were examined. TNF-alpha release by Kupffer cells after LPS stimulation was the same in the presence and absence of serum. RAW 264.7 and peritoneal cells, which utilize the CD14 receptor, released significantly less TNF-alpha after LPS stimulation in the absence of serum because of the absence of LPS-binding protein. Phosphatidylinositol-phospholipase C treatment, which cleaves the CD14 receptor, decreased LPS-stimulated TNF-alpha release by RAW 264.7 cells but not by Kupffer cells. Deacylated LPS (dLPS) competes with LPS at the CD14 receptor when incubated in a ratio of 100:1 (dLPS/LPS). Such competition blocked LPS-stimulated TNF-alpha release from RAW 264.7 cells but not from Kupffer cells. Western and fluorescence-activated cell sorter analysis directly demonstrated the presence of CD14 on RAW 264.7 cells and murine peritoneal cells but showed only minimal amounts of CD14 in murine Kupffer cells. LPS stimulation did not increase the amount of CD14 detectable on mouse Kupffer cells. CD14 expression is very low in Kupffer cells, and LPS-stimulated TNF-alpha release is independent of CD14 in these cells.

Distinct freshwater and seawater isoforms of Na+/K+-ATPase in gill chloride cells of Atlantic salmon

USGS Publications Warehouse

McCormick, Stephen D.; Regish, A.M.; Christensen, A.K.

2009-01-01

Gill Na(+)/K(+)-ATPase (NKA) in teleost fishes is involved in ion regulation in both freshwater and seawater. We have developed and validated rabbit polyclonal antibodies specific to the NKA alpha1a and alpha1b protein isoforms of Atlantic salmon (Salmo salar Linnaeus), and used western blots and immunohistochemistry to characterize their size, abundance and localization. The relative molecular mass of NKA alpha1a is slightly less than that for NKA beta1b. The abundance of gill NKA alpha1a was high in freshwater and became nearly undetectable after seawater acclimation. NKA alpha1b was present in small amounts in freshwater and increased 13-fold after seawater acclimation. Both NKA isoforms were detected only in chloride cells. NKA alpha1a was located in both filamental and lamellar chloride cells in freshwater, whereas in seawater it was present only as a faint background in filamental chloride cells. In freshwater, NKA alpha1b was found in a small number of filamental chloride cells, and after seawater acclimation it was found in all chloride cells on the filament and lamellae. Double simultaneous immunofluorescence indicated that NKA alpha1a and alpha1b are located in different chloride cells in freshwater. In many chloride cells in seawater, NKA alpha1b was present in greater amounts in the subapical region than elsewhere in the cell. The combined patterns in abundance and immunolocalization of these two isoforms can explain the salinity-related changes in total NKA and chloride cell abundance. The results indicate that there is a freshwater and a seawater isoform of NKA alpha-subunit in the gills of Atlantic salmon and that they are present in distinct chloride cells.

1alpha,25-dihydroxy-vitamin-D3 as new immunotherapy in treatment of recurrent spontaneous abortion.

PubMed

Bubanovic, I

2004-01-01

Recurrent spontaneous abortion (RSA) is serious health problem affecting 2-5% of reproducing couples worldwide. It has long been suspected that nearly 80% of the unexplained RSAs are due to immunologic causes. Although the major tissue confronting the mother's immune system is the placental villous trophoblast, the immunological risk to the developing embryo is not great until the time of implantation. In addition, trophoblast is not sensible to lysis by NK cells, TNF-alpha or macrophages, but may be killed by lymphokine activated NK cells (LAK) and may undergo apoptosis in response to TNF-alpha and/or IFN-gamma in vitro. The two most commonly used treatments for RSA are intravenous immunoglobulin (IVIg) and alloimmunization with partner's leukocytes (LIT). We promote vitamin D3 as new immunomodulatory agent in treatment of RSA. Different mechanisms have been proposed to account for the immunosuppressive effect of 1alpha, 25-dihydroxy-vitamin-D3 (VD3). Portion of the VD3 activity involves the downregulation of IL-2, IFN-gamma and TNF-alpha genes transcription. Because immunomodulatory effects of VD3 are very similar to IL-10 effects, acting of VD3 in immunotherapy of RSA syndrome, preeclamptic and eclamptic pregnancy, as well as PIH syndrome, is very reasonable. We propose using of VD3 as immunotherapy or adjuvant therapy in combination with classic immunotherapies of endangered pregnancies.

Involvement of estrogen receptor variant ER-alpha36, not GPR30, in nongenomic estrogen signaling.

PubMed

Kang, Lianguo; Zhang, Xintian; Xie, Yan; Tu, Yaping; Wang, Dong; Liu, Zhenming; Wang, Zhao-Yi

2010-04-01

Accumulating evidence suggested that an orphan G protein-coupled receptor (GPR)30, mediates nongenomic responses to estrogen. The present study was performed to investigate the molecular mechanisms underlying GPR30 function. We found that knockdown of GPR30 expression in breast cancer SK-BR-3 cells down-regulated the expression levels of estrogen receptor (ER)-alpha36, a variant of ER-alpha. Introduction of a GPR30 expression vector into GPR30 nonexpressing cells induced endogenous ER-alpha36 expression, and cotransfection assay demonstrated that GPR30 activated the promoter activity of ER-alpha36 via an activator protein 1 binding site. Both 17beta-estradiol (E2) and G1, a compound reported to be a selective GPR30 agonist, increased the phosphorylation levels of the MAPK/ERK1/2 in SK-BR-3 cells, which could be blocked by an anti-ER-alpha36-specific antibody against its ligand-binding domain. G1 induced activities mediated by ER-alpha36, such as transcription activation activity of a VP16-ER-alpha36 fusion protein and activation of the MAPK/ERK1/2 in ER-alpha36-expressing cells. ER-alpha36-expressing cells, but not the nonexpressing cells, displayed high-affinity, specific E2 and G1 binding, and E2- and G1-induced intracellular Ca(2+) mobilization only in ER-alpha36 expressing cells. Taken together, our results demonstrated that previously reported activities of GPR30 in response to estrogen were through its ability to induce ER-alpha36 expression. The selective G protein-coupled receptor (GPR)30 agonist G1 actually interacts with ER-alpha36. Thus, the ER-alpha variant ER-alpha36, not GPR30, is involved in nongenomic estrogen signaling.

Immunological characterization of eristostatin and echistatin binding sites on alpha IIb beta 3 and alpha V beta 3 integrins.

PubMed Central

Marcinkiewicz, C; Rosenthal, L A; Mosser, D M; Kunicki, T J; Niewiarowski, S

1996-01-01

Two disintegrins with a high degree of amino acid sequence similarity, echistatin and eristostatin, showed a low level of interaction with Chinese hamster ovary (CHO) cells, but they bound to CHO cells transfected with alpha IIb beta 3 genes (A5 cells) and to CHO cells transfected with alpha v beta 3 genes (VNRC3 cells) in a reversible and saturable manner. Scatchard analysis revealed that eristostatin bound to 816000 sites per A5 cell (Kd 28 nM) and to 200000 sites (Kd 14 nM) per VNRC3 cell respectively. However, VNRC3 cells did not bind to immobilized eristostatin. Echistatin bound to 495000 sites (Kd 53 nM) per A5 cell and to 443000 sites (Kd 20 nM) per VNRC3 cell. As determined by flow cytometry, radiobinding assay and adhesion studies, binding of both disintegrins to A5 cells and resting platelets and binding of echistatin to VNRC3 cells resulted in the expression of ligand-induced binding sites (LIBS) on the beta 3 subunit. Eristostatin inhibited, more strongly than echistatin, the binding of three monoclonal antibodies: OPG2 (RGD motif dependent), A2A9 (alpha IIb beta 3 complex dependent) and 7E3 (alpha IIb beta 3 and alpha v beta 3 complex dependent) to A5 cells, to resting and to activated platelets and to purified alpha IIb beta 3. Experiments in which echistatin and eristostatin were used alone or in combination to inhibit the binding of 7E3 and OPG2 antibodies to resting platelets suggested that these two disintegrins bind to different but overlapping sites on alpha IIb beta 3 integrin. Monoclonal antibody LM 609 and echistatin seemed to bind to different sites on alpha v beta 3 integrin. However, echistatin inhibited binding of 7E3 antibody to VNRC3 cells and to purified alpha v beta 3 suggesting that alpha v beta 3 and alpha IIb beta 3 might share the same epitope to which both echistatin and 7E3 bind. Eristostatin had no effect in these systems, providing further evidence that it binds to a different epitope on alpha v beta 3. PMID:8760368

Effects of TNF-alpha on Endothelial Cell Collective Migration

NASA Astrophysics Data System (ADS)

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

2013-03-01

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

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

PubMed Central

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

2016-01-01

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fukada, So-ichiro; Yamamoto, Yukiko; Segawa, Masashi

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 dramaticallymore » 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.« less

[Immune diagnostics of disorders in the IFN-alpha/NK-cell system in patients with frequently recurrent herpes simplex].

PubMed

Karsonova, A V; Shulzhenko, A E; Karaulov, A V

2014-01-01

Study of features of NK-cell response to the effect of recombinant IFN-alpha in complex with evaluation of the ability to synthesize inherent IFN-alpha in patients with frequently recurrent herpes simplex (FRHS). 48 patients with genital (n = 31), labial (n = 10) and mixed localization (n = 7) FRHS diagnosis were observed. 31 healthy donors composed the control group. MC were cultivated in the presence of a recombinant human IFN-alpha2b at the concentration of 10, 100 and 1000 U/ml for 24 hours. NK-cell response to the effect of IFN-alpha was evaluated after 24 hours using flow cytometry by degranulation reaction and in the NK-activity test. IFN-alpha synthesis was evaluated in HSV-1, HSV-2 and Newcastle disease virus stimulated cell supernatants by EIA method. Patients with FRHS were established to be a heterogeneous group by parameters in the IFN-alpha/NK-cell cytotoxicity system. 2 types of NK-cell response to the stimulation by recombinant IFN-alpha were identified. Type A is characterized by a decrease of NK-cell response to IFN-alpha in the remission phase and does not have this defect in the exacerbation phase. Synthesis of inherent IFN-alpha in response to viral inductors for type A was comparable with the response in healthy donors in both phases. On the contrary type B having normal sensitivity of NK-cells to IFN-alpha in the remission phase is characterized by a decrease of this parameter in the exacerbation phase for more than 3 times. Synthesis of inherent IFN-alpha in response to viral inductors during type B is increased in the remission phase and decreased in the exacerbation phase. During immune-correcting therapy of FRHS a personalized approach taking into account features of NK-cell response to IFN-alpha is necessary, because types A and B have principal differences by cytotoxicity parameters of NK-cells and their change under the effect of IFN-alpha, as well as by parameters of IFN-alpha synthesis in response to viral inductors at various phases of the clinical process.

Alpha1B-adrenoceptor signaling and cell motility: GTPase function of Gh/transglutaminase 2 inhibits cell migration through interaction with cytoplasmic tail of integrin alpha subunits.

PubMed

Kang, Sung Koo; Yi, Kye Sook; Kwon, Nyoun Soo; Park, Kwang-Hyun; Kim, Uh-Hyun; Baek, Kwang Jin; Im, Mie-Jae

2004-08-27

A multifunctional enzyme, G(h), is a GTP-binding protein that couples to the alpha(1B)-adrenoreceptor and stimulates phospholipase C-delta1 but also displays transglutaminase 2 (TG2) activity. G(h)/TG2 has been implicated to play a role in cell motility. In this study we have examined which function of G(h)/TG2 is involved in this cellular response and the molecular basis. Treatment of human aortic smooth muscle cell with epinephrine inhibits migration to fibronectin and vitronectin, and the inhibition is blocked by the alpha(1)-adrenoreceptor antagonist prazosin or chloroethylclonidine. Up-regulation or overexpression of G(h)/TG2 in human aortic smooth muscle cells, DDT1-MF2, or human embryonic kidney cells, HEK 293 cells, results in inhibition of the migratory activity, and stimulation of the alpha(1B)-adrenoreceptor with the alpha(1) agonist further augments the inhibition of migration of human aortic smooth muscle cells and DDT1-MF2. G(h)/TG2 is coimmunoprecipitated by an integrin alpha(5) antibody and binds to the cytoplasmic tail peptide of integrins alpha(5), alpha(v), and alpha(IIb) subunits in the presence of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS). Mutation of Lys-Arg residues in the GFFKR motif, present in the alpha(5)-tail, significantly reduces the binding of GTPgammaS-G(h)/TG2. Moreover, the motif-containing integrin alpha(5)-tail peptides block G(h)/TG2 coimmunoprecipitation and reverse the inhibition of the migratory activity of HEK 293 cells caused by overexpression G(h)/TG2. These results provide evidence that G(h) function initiates the modulation of cell motility via association of GTP-bound G(h)/TG2 with the GFFKR motif located in integrin alpha subunits.

Trimethyltin-activated cyclooxygenase stimulates tumor necrosis factor-alpha release from glial cells through reactive oxygen species.

PubMed

Viviani, B; Corsini, E; Pesenti, M; Galli, C L; Marinovich, M

2001-04-15

Exposure of a primary culture of glial cells to the classical neurotoxicant trimethyltin (TMT) results in the release of prostaglandin (PG)E(2) and tumor necrosis factor (TNF)-alpha. Prior treatment of glial cells with either the nonspecific inhibitor of cyclooxygenase and lypoxygenase eicosatetraynoic acid (ETYA) or the cyclooxygenase inhibitor indomethacin completely prevented TMT-induced PGE(2) production and TNF-alpha release, suggesting a role for cyclooxygenase metabolites in TMT-induced TNF-alpha release. Exposure of glial cells to increasing concentrations of PGE(2) or other prostanoids did not increase TNF-alpha synthesis, while the presence of exogenous PGE(2) during treatment of glial cells with TMT actually suppressed TNF-alpha release. The activation of arachidonic acid metabolism produces reactive oxygen species (ROS). Scavenging of ROS by means of the antioxidant trolox prevented the TMT-induced release of TNF-alpha from glial cells, while indomethacin was found to suppress ROS formation induced by 1 microM TMT in glial cells. These results suggest that activation of arachidonic acid metabolism causes TNF-alpha release through the production of ROS rather than PGE(2). Indeed, PGE(2) may exert negative feedback on the release of TNF-alpha. Copyright 2001 Academic Press.

Impaired coactivator activity of the Gly{sub 482} variant of peroxisome proliferator-activated receptor {gamma} coactivator-1{alpha} (PGC-1{alpha}) on mitochondrial transcription factor A (Tfam) promoter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Yon-Sik; Hong, Jung-Man; Lim, Sunny

2006-06-09

Mitochondrial dysfunction may cause diabetes or insulin resistance. Peroxisome proliferation-activated receptor-{gamma} (PPAR-{gamma}) coactivator-1 {alpha} (PGC-1{alpha}) increases mitochondrial transcription factor A (Tfam) resulting in mitochondrial DNA content increase. An association between a single nucleotide polymorphism (SNP), G1444A(Gly482Ser), of PGC-1{alpha} coding region and insulin resistance has been reported in some ethnic groups. In this study, we investigated whether a change of glycine to serine at codon 482 of PGC-1{alpha} affected the Tfam promoter activity. The cDNA of PGC-1{alpha} variant bearing either glycine or serine at 482 codon was transfected into Chang human hepatocyte cells. The PGC-1{alpha} protein bearing glycine had impaired coactivatormore » activity on Tfam promoter-mediated luciferase. We analyzed the PGC-1{alpha} genotype G1444A and mitochondrial DNA (mtDNA) copy number from 229 Korean leukocyte genomic DNAs. Subjects with Gly/Gly had a 20% lower amount of peripheral blood mtDNA than did subjects with Gly/Ser and Ser/Ser (p < 0.05). No correlation was observed between diabetic parameters and PGC-1{alpha} genotypes in Koreans. These results suggest that PGC-1{alpha} variants with Gly/Gly at 482nd amino acid may impair the Tfam transcription, a regulatory function of mitochondrial biogenesis, resulting in dysfunctional mtDNA replication.« less

A defective retroviral vector encoding human interferon-alpha2 can transduce human leukemic cell lines.

PubMed

Austruy, E; Bagnis, C; Carbuccia, N; Maroc, C; Birg, F; Dubreuil, P; Mannoni, P; Chabannon, C

1998-01-01

Using the LXSN backbone, a defective retroviral vector (LISN) was constructed that encodes the human interferon (IFN)-alpha2 (hIFN-alpha2) gene and the neomycin resistance gene; the hIFN-alpha2 gene was cloned from human placental genomic DNA. High titers of the LISN retrovirus were produced by the amphotropic packaging cell line GP+envAM12. LISN is able to infect three human hematopoietic and leukemic cell lines: K562, LAMA-84, and TF-1. G418-resistant cells were detected in a similar proportion after infection with either the LISN retroviral vector or the LnLSN retroviral vector (encoding the nlsLacZ gene instead of hIFN-alpha2), suggesting that hIFN-alpha2 does not inhibit (or only partially inhibits) the production of retroviral particles by the packaging cell line and the infection of human cells. LISN-infected cells express and secrete hIFN-alpha2 as demonstrated by Northern blot analysis of poly(A)+ RNA, detection of the intracellular protein by fluorescence-activated cell sorter analysis, and detection of secreted hIFN-alpha in cell supernatants using an enzyme-linked immunosorbent assay. Retrovirally produced hIFN-alpha2 is biologically active, as demonstrated by the partial inhibition of the growth of K562 and TF-1, the modulation of the expression of cell surface antigens, the induction of the (2'-5') oligoadenylate synthetase, and, for LAMA-84, the down-modulation of the BCR-ABL protein. We conclude that the infection of human leukemic cell lines with a retroviral vector encoding hIFN-alpha2 is feasible and induces the expected biological effects. This experimental model will be useful in investigating the possibility of transducing normal and leukemic cells and hematopoietic progenitors and in determining the consequences of the autocrine production of hIFN-alpha2 on the behavior of these cells.

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

PubMed

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

2006-02-21

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

Allelic exchange of pheromones and their receptors reprograms sexual identity in Cryptococcus neoformans.

PubMed

Stanton, Brynne C; Giles, Steven S; Staudt, Mark W; Kruzel, Emilia K; Hull, Christina M

2010-02-26

Cell type specification is a fundamental process that all cells must carry out to ensure appropriate behaviors in response to environmental stimuli. In fungi, cell identity is critical for defining "sexes" known as mating types and is controlled by components of mating type (MAT) loci. MAT-encoded genes function to define sexes via two distinct paradigms: 1) by controlling transcription of components common to both sexes, or 2) by expressing specially encoded factors (pheromones and their receptors) that differ between mating types. The human fungal pathogen Cryptococcus neoformans has two mating types (a and alpha) that are specified by an extremely unusual MAT locus. The complex architecture of this locus makes it impossible to predict which paradigm governs mating type. To identify the mechanism by which the C. neoformans sexes are determined, we created strains in which the pheromone and pheromone receptor from one mating type (a) replaced the pheromone and pheromone receptor of the other (alpha). We discovered that these "alpha(a)" cells effectively adopt a new mating type (that of a cells); they sense and respond to alpha factor, they elicit a mating response from alpha cells, and they fuse with alpha cells. In addition, alpha(a) cells lose the alpha cell type-specific response to pheromone and do not form germ tubes, instead remaining spherical like a cells. Finally, we discovered that exogenous expression of the diploid/dikaryon-specific transcription factor Sxi2a could then promote complete sexual development in crosses between alpha and alpha(a) strains. These data reveal that cell identity in C. neoformans is controlled fully by three kinds of MAT-encoded proteins: pheromones, pheromone receptors, and homeodomain proteins. Our findings establish the mechanisms for maintenance of distinct cell types and subsequent developmental behaviors in this unusual human fungal pathogen.

Oleic acid and peanut oil high in oleic acid reverse the inhibitory effect of insulin production of the inflammatory cytokine TNF-alpha both in vitro and in vivo systems.

PubMed

Vassiliou, Evros K; Gonzalez, Andres; Garcia, Carlos; Tadros, James H; Chakraborty, Goutam; Toney, Jeffrey H

2009-06-26

Chronic inflammation is a key player in pathogenesis. The inflammatory cytokine, tumor necrosis factor-alpha is a well known inflammatory protein, and has been a therapeutic target for the treatment of diseases such as Rheumatoid Arthritis and Crohn's Disease. Obesity is a well known risk factor for developing non-insulin dependent diabetes melitus. Adipose tissue has been shown to produce tumor necrosis factor-alpha, which has the ability to reduce insulin secretion and induce insulin resistance. Based on these observations, we sought to investigate the impact of unsaturated fatty acids such as oleic acid in the presence of TNF-alpha in terms of insulin production, the molecular mechanisms involved and the in vivo effect of a diet high in oleic acid on a mouse model of type II diabetes, KKAy. The rat pancreatic beta cell line INS-1 was used as a cell biological model since it exhibits glucose dependent insulin secretion. Insulin production assessment was carried out using enzyme linked immunosorbent assay and cAMP quantification with competitive ELISA. Viability of TNF-alpha and oleic acid treated cells was evaluated using flow cytometry. PPAR-gamma translocation was assessed using a PPRE based ELISA system. In vivo studies were carried out on adult male KKAy mice and glucose levels were measured with a glucometer. Oleic acid and peanut oil high in oleic acid were able to enhance insulin production in INS-1. TNF-alpha inhibited insulin production but pre-treatment with oleic acid reversed this inhibitory effect. The viability status of INS-1 cells treated with TNF-alpha and oleic acid was not affected. Translocation of the peroxisome proliferator- activated receptor transcription factor to the nucleus was elevated in oleic acid treated cells. Finally, type II diabetic mice that were administered a high oleic acid diet derived from peanut oil, had decreased glucose levels compared to animals administered a high fat diet with no oleic acid. Oleic acid was found to be effective in reversing the inhibitory effect in insulin production of the inflammatory cytokine TNF-alpha. This finding is consistent with the reported therapeutic characteristics of other monounsaturated and polyunsaturated fatty acids. Furthermore, a diet high in oleic acid, which can be easily achieved through consumption of peanuts and olive oil, can have a beneficial effect in type II diabetes and ultimately reverse the negative effects of inflammatory cytokines observed in obesity and non insulin dependent diabetes mellitus.

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

PubMed

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

1997-03-01

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

Downregulation of the Petunia hybrida alpha-expansin gene PhEXP1 reduces the amount of crystalline cellulose in cell walls and leads to phenotypic changes in petal limbs.

PubMed

Zenoni, Sara; Reale, Lara; Tornielli, Giovanni Battista; Lanfaloni, Luisa; Porceddu, Andrea; Ferrarini, Alberto; Moretti, Chiaraluce; Zamboni, Anita; Speghini, Adolfo; Ferranti, Francesco; Pezzotti, Mario

2004-02-01

The expansins comprise a family of proteins that appear to be involved in the disruption of the noncovalent bonds between cellulose microfibrils and cross-linking glycans, thereby promoting wall creep. To understand better the expansion process in Petunia hybrida (petunia) flowers, we isolated a cDNA corresponding to the PhEXP1 alpha-expansin gene of P. hybrida. Evaluation of the tissue specificity and temporal expression pattern demonstrated that PhEXP1 is preferentially expressed in petal limbs during development. To determine the function of PhEXP1, we used a transgenic antisense approach, which was found to cause a decrease in petal limb size, a reduction in the epidermal cell area, and alterations in cell wall morphology and composition. The diminished cell wall thickness accompanied by a reduction in crystalline cellulose indicates that the activity of PhEXP1 is associated with cellulose metabolism. Our results suggest that expansins play a role in the assembly of the cell wall by affecting either cellulose synthesis or deposition.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cowell, Rita M.; Department of Neurology, University of Michigan, Ann Arbor, MI 48109; Talati, Pratik

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 3more » 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.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jambor de Sousa, Ulrike L.; Koss, Michael D.; Fillies, Marion

2005-12-16

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

Loss of tumorigenic potential by human lung tumor cells in the presence of antisense RNA specific to the ectopically synthesized alpha subunit of human chorionic gonadotropin.

PubMed

Rivera, R T; Pasion, S G; Wong, D T; Fei, Y B; Biswas, D K

1989-06-01

A clonal strain of human lung tumor cells in culture (ChaGo), derived from a bronchogenic carcinoma, synthesizes and secretes large amounts of alpha (alpha) and a comparatively lower level of beta (beta) subunit of the glycoprotein hormone, human chorionic gonadotropin (HCG). ChaGo cells lost their characteristic anchorage-independent growth phenotype in the presence of anti-alpha-HCG antibody. The effect of the antibody was partially reversed by addition of alpha-HCG to the culture medium. ChaGo cells were transfected with an expression vector (pRSV-anti-alpha-HCG), that directs synthesis of RNA complementary to alpha-HCG mRNA. The transfectants produced alpha-HCG antisense RNA which was associated with the reduced level of alpha-HCG. Transfectants also displayed several altered phenotypic properties, including altered morphology, less mitosis, reduced growth rate, loss of anchorage-independent growth, and loss of tumorigenicity in nude mice. Treatment of transfectants with 8,bromo-cAMP resulted in increased accumulation of alpha-HCG mRNA, no change in the level of alpha-HCG antisense RNA, release of the inhibition of [3H]thymidine incorporation, and restoration of anchorage-independent growth phenotype. The overexpression of c-myc, observed in ChaGo cells, was unaffected by the reduced level of alpha-HCG. These results suggest that ectopic synthesis of the alpha subunit of HCG plays a functional role in the transformation of these human lung cells.

Targeted drug delivery and enhanced intracellular release using functionalized liposomes

NASA Astrophysics Data System (ADS)

Garg, Ashish

The ability to target cancer cells using an appropriate drug delivery system can significantly reduce the associated side effects from cancer therapies and can help in improving the overall quality of life, post cancer survival. Integrin alpha5beta1 is expressed on several types of cancer cells, including colon cancer and plays an important role in tumor growth and metastasis. Thus, the ability to target the integrin alpha 5beta1 using an appropriate drug delivery nano-vector can significantly help in inhibiting tumor growth and reducing tumor metastasis. The work in this thesis focuses on designing and optimizing, functionalized stealth liposomes (liposomes covered with polyethylene glycol (PEG)) that specifically target the integrin alpha5beta1. The PEG provides a steric barrier allowing the liposomes to circulate in the blood for longer duration and the functionalizing moiety, PR_b peptide specifically recognizes and binds to integrin alpha5beta1 expressing cells. The work demonstrates that by optimizing the amount of PEG and PR_b on the liposomal interface, nano-vectors can be engineered that bind to CT26.WT colon cancer cells in a specific manner and internalize through alpha 5beta1-mediated endocytosis. To further improve the efficacy of the system, PR_b functionalized pH-sensitive stealth liposomes that exhibit triggered release under mild acidic conditions present in endocytotic vesicles were designed. The study showed that PR_b functionalized pH-sensitive stealth liposomes, undergo destabilization under mildly acidic conditions and incorporation of the PR_b peptide does not significantly affect the pH-sensitivity of the liposomes. PR_b functionalized pH-sensitive stealth liposomes bind to CT26.WT colon carcinoma cells that express integrin alpha5beta 1, undergo cellular internalization, and release their load intracellularly in a short period of time as compared to other formulations. PR_b-targeted pH-sensitive stealth liposomes encapsulating 5-fluorouracil (5-FU) show significantly higher cytotoxicity than the PR_b-targeted inert stealth liposomes and the non-targeted stealth liposomes (both pH-sensitive and inert). The studies demonstrated that optimized PR_b functionalized pH sensitive liposomes have the potential to deliver a payload, such as chemotherapeutic agents, directly to colon cancer cells in an efficient and specific manner.

Human cancers converge at the HIF-2alpha oncogenic axis.

PubMed

Franovic, Aleksandra; Holterman, Chet E; Payette, Josianne; Lee, Stephen

2009-12-15

Cancer development is a multistep process, driven by a series of genetic and environmental alterations, that endows cells with a set of hallmark traits required for tumorigenesis. It is broadly accepted that growth signal autonomy, the first hallmark of malignancies, can be acquired through multiple genetic mutations that activate an array of complex, cancer-specific growth circuits [Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57-70; Vogelstein B, Kinzler KW (2004) Cancer genes and the pathways they control. Nat Med 10:789-799]. The superfluous nature of these pathways is thought to severely limit therapeutic approaches targeting tumor proliferation, and it has been suggested that this strategy be abandoned in favor of inhibiting more systemic hallmarks, including angiogenesis (Ellis LM, Hicklin DJ (2008) VEGF-targeted therapy: Mechanisms of anti-tumor activity. Nat Rev Cancer 8:579-591; Stommel JM, et al. (2007) Coactivation of receptor tyrosine kinases affects the response of tumor cells to targeted therapies. Science 318:287-290; Kerbel R, Folkman J (2002) Clinical translation of angiogenesis inhibitors. Nat Rev Cancer 2:727-739; Kaiser J (2008) Cancer genetics: A detailed genetic portrait of the deadliest human cancers. Science 321:1280-1281]. Here, we report the unexpected observation that genetically diverse cancers converge at a common and obligatory growth axis instigated by HIF-2alpha, an element of the oxygen-sensing machinery. Inhibition of HIF-2alpha prevents the in vivo growth and tumorigenesis of highly aggressive glioblastoma, colorectal, and non-small-cell lung carcinomas and the in vitro autonomous proliferation of several others, regardless of their mutational status and tissue of origin. The concomitant deactivation of select receptor tyrosine kinases, including the EGFR and IGF1R, as well as downstream ERK/Akt signaling, suggests that HIF-2alpha exerts its proliferative effects by endorsing these major pathways. Consistently, silencing these receptors phenocopies the loss of HIF-2alpha oncogenic activity, abrogating the serum-independent growth of human cancer cells in culture. Based on these data, we propose an alternative to the predominant view that cancers exploit independent autonomous growth pathways and reveal HIF-2alpha as a potentially universal culprit in promoting the persistent proliferation of neoplastic cells.

Natural protection from zoonosis by alpha-gal epitopes on virus particles in xenotransmission.

PubMed

Kim, Na Young; Jung, Woon-Won; Oh, Yu-Kyung; Chun, Taehoon; Park, Hong-Yang; Lee, Hoon-Taek; Han, In-Kwon; Yang, Jai Myung; Kim, Young Bong

2007-03-01

Clinical transplantation has become one of the preferred treatments for end-stage organ failure, and one of the novel approaches being pursued to overcome the limited supply of human organs involves the use of organs from other species. The pig appears to be a near ideal animal due to proximity to humans, domestication, and ability to procreate. The presence of Gal-alpha1,3-Gal residues on the surfaces of pig cells is a major immunological obstacle to xenotransplantation. Alpha1,3galactosyltransferase (alpha1,3GT) catalyzes the synthesis of Gal alpha 1-3Gal beta 1-4GlcNAc-R (alpha-gal epitope) on the glycoproteins and glycolipids of non-primate mammals, but this does not occur in humans. Moreover, the alpha-gal epitope causes hyperacute rejection of pig organs in humans, and thus, the elimination of this antigen from pig tissues is highly desirable. Recently, concerns have been raised that the risk of virus transmission from such pigs may be increased due to the absence of alpha-gal on their viral particles. In this study, transgenic cells expressing alpha1,3GT were selected using 1.25 mg/ml neomycin. The development of HeLa cells expressing alpha1,3GT now allows accurate studies to be conducted on the function of the alpha-gal epitope in xenotransmission. The expressions of alpha-gal epitopes on HeLa/alpha-gal cells were demonstrated by flow cytometry and confocal microscopy using cells stained with IB4-fluorescein isothiocyanate lectin. Vaccinia viruses propagated in HeLa/alpha-gal cells also expressed alpha-gal on their viral envelopes and were more sensitive to inactivation by human sera than vaccinia virus propagated in HeLa cells. Moreover, neutralization of vaccinia virus was inhibited in human serum by 10 mm ethylene glycol bis(beta-aminoethylether)tetraacetic acid (EDTA) treatment. Our data indicated that alpha-gal epitopes are one of the major barriers to zoonosis via xenotransmission.

Photodynamic therapy (PDT) as a biological modifier

NASA Astrophysics Data System (ADS)

Obochi, Modestus; Tao, Jing-Song; Hunt, David W. C.; Levy, Julia G.

1996-04-01

The capacity of photosensitizers and light to ablate cancerous tissues and unwanted neovasculature constitutes the classical application of photodynamic therapy (PDT). Cell death results from either necrotic or apoptotic processes. The use of photosensitizers and light at doses which do not cause death has been found to affect changes in certain cell populations which profoundly effect their expression of cell surface molecules and secretion of cytokines, thereby altering the functional attributes of the treated cells. Cells of the immune system and the skin may be sensitive to modulation by 'sub-lethal PDT.' Ongoing studies have been conducted to assess, at the molecular level, changes in both lymphocytes and epidermal cells (EC) caused by treatment with low levels of benzoporphyrin derivative monoacid ring A (BPD) (a photosensitizer currently in clinical trials for cancer, psoriasis, endometriosis and age-related macular degeneration) and light. Treatment of skin with BPD and light, at levels which significantly enhanced the length of murine skin allograft acceptance, have been found to down-regulate the expression of Langerhans cell (LC) surface antigen molecules [major histocompatibility complex (MHC) class II and intracellular adhesion molecule (ICAM)-1] and the formation of some cytokines (tumor necrosis factor-alpha (TNF- (alpha) ).

A single mutation results in diploid gamete formation and parthenogenesis in a Drosophila yemanuclein-alpha meiosis I defective mutant.

PubMed

Meyer, Régis E; Delaage, Michèle; Rosset, Roland; Capri, Michèle; Aït-Ahmed, Ounissa

2010-11-16

Sexual reproduction relies on two key events: formation of cells with a haploid genome (the gametes) and restoration of diploidy after fertilization. Therefore the underlying mechanisms must have been evolutionary linked and there is a need for evidence that could support such a model. We describe the identification and the characterization of yem1, the first yem-alpha mutant allele (V478E), which to some extent affects diploidy reduction and its restoration. Yem-alpha is a member of the Ubinuclein/HPC2 family of proteins that have recently been implicated in playing roles in chromatin remodeling in concert with HIRA histone chaperone. The yem1 mutant females exhibited disrupted chromosome behavior in the first meiotic division and produced very low numbers of viable progeny. Unexpectedly these progeny did not display paternal chromosome markers, suggesting that they developed from diploid gametes that underwent gynogenesis, a form of parthenogenesis that requires fertilization. We focus here on the analysis of the meiotic defects exhibited by yem1 oocytes that could account for the formation of diploid gametes. Our results suggest that yem1 affects chromosome segregation presumably by affecting kinetochores function in the first meiotic division. This work paves the way to further investigations on the evolution of the mechanisms that support sexual reproduction.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rajalin, Ann-Marie; Pollock, Hanna; Aarnisalo, Piia, E-mail: piia.aarnisalo@helsinki.fi

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 sialoproteinmore » (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.« less

Ceftiofur impairs pro-inflammatory cytokine secretion through the inhibition of the activation of NF-{kappa}B and MAPK

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ci Xinxin; Song Yu; Zeng Fanqin

2008-07-18

Ceftiofur is a new broad-spectrum, third-generation cephalosporin antibiotic for veterinary use. Immunopharmacological studies can provide new information on the immunomodulatory activities of some drugs, including their effect on cytokine productions. For this reason, we investigated the effect of ceftiofur on cytokine productions in vitro. We found that ceftiofur can downregulate tumor necrosis factor-{alpha} (TNF-{alpha}), interleukin-1{beta} (IL-1{beta}), and interleukin-6 (IL-6), but did not affect interleukin-10 (IL-10) production. We further investigated signal transduction mechanisms to determine how ceftiofur affects. RAW 264.7 cells were pretreated with 1, 5, or 10 mg/L of ceftiofur 1 h prior to treatment with 1 mg/L of LPS.more » Thirty minutes later, cells were harvested and mitogen activated protein kinases (MAPKs) activation was measured by Western blot. Alternatively, cells were fixed and nuclear factor-{kappa}B (NF-{kappa}B) activation was measured using immunocytochemical analysis. Signal transduction studies showed that ceftiofur significantly inhibited extracellular signal-regulated kinase (ERK), p38, and c-jun NH{sub 2}-terminal kinase (JNK) phosphorylation protein expression. Ceftiofur also inhibited p65-NF-{kappa}B translocation into the nucleus. Therefore, ceftiofur may inhibit LPS-induced production of inflammatory cytokines by blocking NF-{kappa}B and MAPKs signaling in RAW264.7 cells.« less

Preliminary in vivo efficacy studies of a recombinant rhesus anti-alpha(4)beta(7) monoclonal antibody.

PubMed

Pereira, L E; Onlamoon, N; Wang, X; Wang, R; Li, J; Reimann, K A; Villinger, F; Pattanapanyasat, K; Mori, K; Ansari, A A

2009-01-01

Recent findings established that primary targets of HIV/SIV are lymphoid cells within the gastrointestinal (GI) tract. Focus has therefore shifted to T-cells expressing alpha(4)beta(7) integrin which facilitates trafficking to the GI tract via binding to MAdCAM-1. Approaches to better understand the role of alpha(4)beta(7)+ T-cells in HIV/SIV pathogenesis include their depletion or blockade of their synthesis, binding and/or homing capabilities in vivo. Such studies can ideally be conducted in rhesus macaques (RM), the non-human primate model of AIDS. Characterization of alpha(4)beta(7) expression on cell lineages in RM blood and GI tissues reveal low densities of expression by NK cells, B-cells, naïve and TEM (effector memory) T-cells. High densities were observed on TCM (central memory) T-cells. Intravenous administration of a single 50mg/kg dose of recombinant rhesus alpha(4)beta(7) antibody resulted in significant initial decline of alpha(4)beta(7)+ lymphocytes and sustained coating of the alpha(4)beta(7) receptor in both the periphery and GI tissues.

Hypoxia preconditioning protection of corneal stromal cells requires HIF1alpha but not VEGF.

PubMed

Xing, Dongmei; Bonanno, Joseph A

2009-05-18

Hypoxia preconditioning protects corneal stromal cells from stress-induced death. This study determined whether the transcription factor HIF-1alpha (Hypoxia Inducible Factor) is responsible and whether this is promulgated by VEGF (Vascular Endothelial Growth Factor). Cultured bovine stromal cells were preconditioned with hypoxia in the presence of cadmium chloride, a chemical inhibitor of HIF-1alpha, and HIF-1alpha siRNA to test if HIF-1alpha activity is needed for hypoxia preconditioning protection from UV-irradiation induced cell death. TUNEL assay was used to detect cell apoptosis after UV-irradiation. RT-PCR and western blot were used to detect the presence of HIF-1alpha and VEGF in transcriptional and translational levels. During hypoxia (0.5% O2), 5 muM cadmium chloride completely inhibited HIF-1alpha expression and reversed the protection by hypoxia preconditioning. HIF-1alpha siRNA (15 nM) reduced HIF-1alpha expression by 90% and produced a complete loss of protection provided by hypoxia preconditioning. Since VEGF is induced by hypoxia, can be HIF-1alpha dependent, and is often protective, we examined the changes in transcription of VEGF and its receptors after 4 h of hypoxia preconditioning. VEGF and its receptors Flt-1 and Flk-1 are up-regulated after hypoxia preconditioning. However, the transcription and translation of VEGF were paradoxically increased by siHIF-1alpha, suggesting that VEGF expression in stromal cells is not down-stream of HIF-1alpha. These findings demonstrate that hypoxia preconditioning protection in corneal stromal cells requires HIF-1alpha, but that VEGF is not a component of the protection.

Analysis of PGC-1{alpha} variants Gly482Ser and Thr612Met concerning their PPAR{gamma}2-coactivation function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nitz, Inke; Ewert, Agnes; Klapper, Maja

2007-02-09

Peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha} (PGC-1{alpha}) is a cofactor involved in adaptive thermogenesis, fatty acid oxidation, and gluconeogenesis. Dysfunctions of this protein are likely to contribute to the development of obesity and the metabolic syndrome. This is in part but not definitely confirmed by results of population studies. The aim of this study was to investigate if common genetic variants rs8192678 (Gly482Ser) and rs3736265 (Thr612Met) in the PGC-1{alpha} gene lead to a functional consequence in cofactor activity using peroxisome proliferator-activated receptor-{gamma} 2 (PPAR{gamma}2) as interacting transcription factor. Reporter gene assays in HepG2 cells with wildtype and mutant proteins of both PGC1{alpha}more » and PPAR{gamma}2 (Pro12Ala, rs1801282) using the acyl-CoA-binding protein (ACBP) promoter showed no difference in coactivator activity. This is First study implicating that the Gly482Ser and Thr612Met polymorphisms in PGC-1{alpha} and Pro12Ala polymorphism in PPAR{gamma}2 do not affect the functional integrity of these proteins.« less

TNF-{alpha} upregulates the A{sub 2B} adenosine receptor gene: The role of NAD(P)H oxidase 4

DOE Office of Scientific and Technical Information (OSTI.GOV)

St Hilaire, Cynthia; Koupenova, Milka; Carroll, Shannon H.

2008-10-24

Proliferation of vascular smooth muscle cells (VSMC), oxidative stress, and elevated inflammatory cytokines are some of the components that contribute to plaque formation in the vasculature. The cytokine tumor necrosis factor-alpha (TNF-{alpha}) is released during vascular injury, and contributes to lesion formation also by affecting VSMC proliferation. Recently, an A{sub 2B} adenosine receptor (A{sub 2B}AR) knockout mouse illustrated that this receptor is a tissue protector, in that it inhibits VSMC proliferation and attenuates the inflammatory response following injury, including the release of TNF-{alpha}. Here, we show a regulatory loop by which TNF-{alpha} upregulates the A{sub 2B}AR in VSMC in vitromore » and in vivo. The effect of this cytokine is mimicked by its known downstream target, NAD(P)H oxidase 4 (Nox4). Nox4 upregulates the A{sub 2B}AR, and Nox inhibitors dampen the effect of TNF-{alpha}. Hence, our study is the first to show that signaling associated with Nox4 is also able to upregulate the tissue protecting A{sub 2B}AR.« less

[Observation on alpha-SMA during Erigeron Breviscapus (Vant) Hand-Mazz obstructs the evolution of carcinogenesis of golden hamster cheek pouch].

PubMed

Zhou, C T; Zhang, S L; Ding, R Y; Hua, L; Zhong, W J

2000-06-01

To observe dynamically that Erigeron Breviscapus (Vant) Hand-Mazz (HEr) affects the expression of alpha-smooth muscle actin (alpha-SMA). To discuss the probable mechanism of obstructing leukoplakia carcinogenesis of this medicine. 120 golden hamsters were randomly divided into model group (48), HEr group (48) and control group (6). HEr was applied to obstruct the evolution of carcinogenesis of golden hamster cheek pouch. Immunohistochemistry was used to detect the expression level of alpha-SMA with cheek pouch specimen that besmears DMBA in 4-9 weeks. Results were compared with model group. Vessel density dyed with alpha-SMA continuously of HEr group was 65.76 significantly higher than that of model group 42.12 (P<0.001). High classification cases in HEr group were much more than model group when cases were divided into five groups as follow: 100%, 50%, 20%, 10%, 3% (P<0.01). HEr can raise the expression level of alpha-SMA exactly during the evolution of leukoplakia carcinogenesis of golden hamster, which shows that this medicine obstructs carcinogenesis by keeping the normal physiological function of vascular myoepithelial cell and integrity of vascular basement membrane.

The small-molecule TNF-alpha modulator, UTL-5g, reduces side effects induced by cisplatin and enhances the therapeutic effect of cisplatin in vivo.

PubMed

Shaw, JiaJiu; Chen, Ben; Huang, Wen-Hsin; Lee, An-Rong; Media, Joseph; Valeriote, Frederick A

2011-01-01

We investigated a small-molecule modulator of tumor necrosis factor alpha (TNF-alpha), UTL-5g (also referred to as GBL-5g), as a potential chemoprotective agent against cisplatin-induced side effects including nephrotoxicity, hepatotoxicity and hematotoxicity. Pretreatment of UTL-5g i.p. in BDF1 mice reduced the levels of blood urea nitrogen (BUN) and creatinine induced by cisplatin treatment. The levels of both aspartate transaminase (AST) and alanine transaminase (ALT) in these animals were also reduced by UTL-5g. Pretreatment of UTL-5g did not significantly affect the number of white blood cells (WBC) under current experimental conditions, yet it markedly increased blood platelet counts by more than threefold. Therapeutic assessment in SCID mice inoculated with human HCT-15 tumor cells showed that UTL-5g did not attenuate the anti-tumor effect of cisplatin but increased the therapeutic efficacy of cisplatin. The LD50 of UTL-5g was determined to be > 2,000 mg/kg by an acute toxicity study. In summary, our studies showed that 1) UTL-5g significantly reduces nephrotoxicity and hepatotoxicity induced by cisplatin in mice, presumably by lowering the levels of TNF-alpha, 2) UTL-5g markedly increased blood platelet counts in mice and 3) UTL-5g treatment increased the therapeutic efficacy of cisplatin against HCT-15 cells inoculated in SCID mice.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abdelalim, Essam Mohamed, E-mail: essam_abdelalim@yahoo.com; Department of Cytology and Histology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia 41522; 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 ofmore » 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.« less

TNF-alpha induction of GM2 expression on renal cell carcinomas promotes T cell dysfunction.

PubMed

Raval, Gira; Biswas, Soumika; Rayman, Patricia; Biswas, Kaushik; Sa, Gaurisankar; Ghosh, Sankar; Thornton, Mark; Hilston, Cynthia; Das, Tanya; Bukowski, Ronald; Finke, James; Tannenbaum, Charles S

2007-05-15

Previous studies from our laboratory demonstrated the role of tumor-derived gangliosides as important mediators of T cell apoptosis, and hence, as one mechanism by which tumors evade immune destruction. In this study, we report that TNF-alpha secreted by infiltrating inflammatory cells and/or genetically modified tumors augments tumor-associated GM2 levels, which leads to T cell death and immune dysfunction. The conversion of weakly apoptogenic renal cell carcinoma (RCC) clones to lines that can induce T cell death requires 3-5 days of TNF-alpha pretreatment, a time frame paralleling that needed for TNF-alpha to stimulate GM2 accumulation by SK-RC-45, SK-RC-54, and SK-RC-13. RCC tumor cell lines permanently transfected with the TNF-alpha transgene are similarly toxic for T lymphocytes, which correlates with their constitutively elevated levels of GM2. TNF-alpha increases GM2 ganglioside expression by enhancing the mRNA levels encoding its synthetic enzyme, GM2 synthase, as demonstrated by both RT-PCR and Southern analysis. The contribution of GM2 gangliosides to tumor-induced T cell death was supported by the finding that anti-GM2 Abs significantly blocked T cell apoptosis mediated by TNF-alpha-treated tumor cells, and by the observation that small interfering RNA directed against TNF-alpha abrogated GM2 synthase expression by TNF-transfected SK-RC-45, diminished its GM2 accumulation, and inhibited its apoptogenicity for T lymphocytes. Our results indicate that TNF-alpha signaling promotes RCC-induced killing of T cells by stimulating the acquisition of a distinct ganglioside assembly in RCC tumor cells.

Activated release of membrane-anchored TGF-alpha in the absence of cytosol

PubMed Central

1993-01-01

The ectodomain of proTGF-alpha, a membrane-anchored growth factor, is converted into soluble TGF-alpha by a regulated cellular proteolytic system that recognizes proTGF-alpha via the C-terminal valine of its cytoplasmic tail. In order to define the biochemical components involved in proTGF-alpha cleavage, we have used cells permeabilized with streptolysin O (SLO) that have been extensively washed to remove cytosol. PMA, acting through a Ca(2+)-independent protein kinase C, activates cleavage as efficiently in permeabilized cells as it does in intact cells. ProTGF-alpha cleavage is also stimulated by GTP gamma S through a mechanism whose pharmacological properties suggest the involvement of a heterotrimeric G protein acting upstream of the PMA- sensitive Ca(2+)-independent protein kinase C. Activated proTGF-alpha cleavage is dependent on ATP hydrolysis, appears not to require vesicular traffic, and acts specifically on proTGF-alpha that has reached the cell surface. These results indicate that proTGF-alpha is cleaved from the cell surface by a regulated system whose signaling, recognition, and proteolytic components are retained in cells devoid of cytosol. PMID:8314849

Transforming growth factor-{alpha} enhances corneal epithelial cell migration by promoting EGFR recycling.

PubMed

McClintock, Jennifer L; Ceresa, Brian P

2010-07-01

PURPOSE. The goal of this study was to determine the molecular mechanism by which transforming growth factor-alpha (TGF-alpha) is a more potent activator of epidermal growth factor receptor (EGFR)-mediated corneal wound healing than epidermal growth factor (EGF). METHODS. Telomerase immortalized human corneal epithelial (hTCEpi) cells and primary human corneal epithelial cells were tested for their ability to migrate in response to EGF and TGF-alpha. In parallel, the endocytic trafficking of the EGFR in response to these same ligands was examined using indirect immunofluorescence, immunoblots, and radioligand binding. RESULTS. TGF-alpha, compared with EGF, is a more potent activator of corneal epithelial cell migration. Although both TGF-alpha and EGF were able to induce EGFR internalization and phosphorylation, only those receptors that were stimulated with EGF progressed to lysosomal degradation. EGFRs stimulated with TGF-alpha recycled back to the plasma membrane, where they could be reactivated with ligand. CONCLUSIONS. This study reveals that EGFR-mediated cell migration is limited by ligand-stimulated downregulation of the EGFR. This limitation can be overcome by treating cells with TGF-alpha because TGF-alpha stimulates EGFR endocytosis, but not degradation. After internalization of the TGF-alpha/EGFR complex, EGFR recycles back to the plasma membrane, where it can be restimulated. This sequence of events provides the receptor multiple opportunities for stimulation. Thus, stimulation with TGF-alpha prolongs EGFR signaling compared with EGF.

The role of alpha3beta1 integrin in determining the supramolecular organization of laminin-5 in the extracellular matrix of keratinocytes.

PubMed

deHart, Gregory W; Healy, Kevin E; Jones, Jonathan C R

2003-02-01

Analyses of mice with targeted deletions in the genes for alpha3 and beta1 integrin suggest that the alpha3beta1 integrin heterodimer likely determines the organization of the extracellular matrix within the basement membrane of skin. Here we tested this hypothesis using keratinocytes derived from alpha3 integrin-null mice. We have compared the organizational state of laminin-5, a ligand of alpha3beta1 integrin, in the matrix of wild-type keratinocytes with that of laminin-5 in the matrix of alpha3 integrin-null cells. Laminin-5 distributes diffusely in arc structures in the matrix of wild-type mouse keratinocytes, whereas laminin-5 is organized into linear, spike-like arrays by the alpha3 integrin-null cells. The fact that alpha3 integrin-null cells are deficient in their ability to assemble a proper laminin-5 matrix is also shown by their failure to remodel laminin-5 when plated onto surfaces coated with purified laminin-5 protein. In sharp contrast, wild-type keratinocytes organize exogenously added laminin-5 into discrete ring-like organizations. These findings led us next to assess whether differences in laminin-5 organization in the matrix of the wild-type and alpha3 integrin-null cells impact cell behavior. Our results indicate that alpha3 integrin-null cells are more motile than their wild-type counterparts and leave extensive trails of laminin-5 over the surface on which they move. Moreover, HEK 293 cells migrate significantly more on the laminin-5-rich matrix derived from the alpha3 integrin-null cells than on the wild-type keratinocyte laminin-5 matrix. In addition, alpha3 integrin-null cells show low strength of adhesion to surfaces coated with purified laminin-5 compared to wild-type cells although both the wild type and the alpha3 integrin-null keratinocytes adhere equally strongly to laminin-5 that has been organized into arrays by other epithelial cells. These data suggest: (1) that alpha3beta1 integrin plays an important role in determining the incorporation of laminin-5 into its proper higher-order structure within the extracellular matrix of keratinocytes and (2) that the organizational state of laminin-5 has an influence on laminin-5 matrix function. Copyright 2003 Elsevier Science (USA)

Reactive oxygen species-generating mitochondrial DNA mutation up-regulates hypoxia-inducible factor-1alpha gene transcription via phosphatidylinositol 3-kinase-Akt/protein kinase C/histone deacetylase pathway.

PubMed

Koshikawa, Nobuko; Hayashi, Jun-Ichi; Nakagawara, Akira; Takenaga, Keizo

2009-11-27

Lewis lung carcinoma-derived high metastatic A11 cells constitutively overexpress hypoxia-inducible factor (HIF)-1alpha mRNA compared with low metastatic P29 cells. Because A11 cells exclusively possess a G13997A mutation in the mitochondrial NADH dehydrogenase subunit 6 (ND6) gene, we addressed here a causal relationship between the ND6 mutation and the activation of HIF-1alpha transcription, and we investigated the potential mechanism. Using trans-mitochondrial cybrids between A11 and P29 cells, we found that the ND6 mutation was directly involved in HIF-1alpha mRNA overexpression. Stimulation of HIF-1alpha transcription by the ND6 mutation was mediated by overproduction of reactive oxygen species (ROS) and subsequent activation of phosphatidylinositol 3-kinase (PI3K)-Akt and protein kinase C (PKC) signaling pathways. The up-regulation of HIF-1alpha transcription was abolished by mithramycin A, an Sp1 inhibitor, but luciferase reporter and chromatin immunoprecipitation assays indicated that Sp1 was necessary but not sufficient for HIF-1alpha mRNA overexpression in A11 cells. On the other hand, trichostatin A, a histone deacetylase (HDAC) inhibitor, markedly suppressed HIF-1alpha transcription in A11 cells. In accordance with this, HDAC activity was high in A11 cells but low in P29 cells and in A11 cells treated with the ROS scavenger ebselene, the PI3K inhibitor LY294002, and the PKC inhibitor Ro31-8220. These results suggest that the ROS-generating ND6 mutation increases HIF-1alpha transcription via the PI3K-Akt/PKC/HDAC pathway, leading to HIF-1alpha protein accumulation in hypoxic tumor cells.

Signal-transducing mechanisms of ketamine-caused inhibition of interleukin-1{beta} gene expression in lipopolysaccharide-stimulated murine macrophage-like Raw 264.7 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, T.-L.; Chang, C.-C.; Lin, Y.-L.

2009-10-01

Ketamine may affect the host immunity. Interleukin-1{beta} (IL-1{beta}), IL-6, and tumor necrosis factor-{alpha} (TNF-{alpha}) are pivotal cytokines produced by macrophages. This study aimed to evaluate the effects of ketamine on the regulation of inflammatory cytokine gene expression, especially IL-1{beta}, in lipopolysaccharide (LPS)-activated murine macrophage-like Raw 264.7 cells and its possible signal-transducing mechanisms. Administration of Raw 264.7 cells with a therapeutic concentration of ketamine (100 {mu}M), LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. Exposure to 100 {mu}M ketamine decreased the binding affinity of LPS and LPS-binding protein but didmore » not affect LPS-induced RNA and protein synthesis of TLR4. Treatment with LPS significantly increased IL-1{beta}, IL-6, and TNF-{alpha} gene expressions in Raw 264.7 cells. Ketamine at a clinically relevant concentration did not affect the synthesis of these inflammatory cytokines, but significantly decreased LPS-caused increases in these cytokines. Immunoblot analyses, an electrophoretic mobility shift assay, and a reporter luciferase activity assay revealed that ketamine significantly decreased LPS-induced translocation and DNA binding activity of nuclear factor-kappa B (NF{kappa}B). Administration of LPS sequentially increased the phosphorylations of Ras, Raf, MEK1/2, ERK1/2, and IKK. However, a therapeutic concentration of ketamine alleviated such augmentations. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA reduced cellular TLR4 amounts and ameliorated LPS-induced RAS activation and IL-1{beta} synthesis. Co-treatment with ketamine and TLR4 siRNA synergistically ameliorated LPS-caused enhancement of IL-1{beta} production. Results of this study show that a therapeutic concentration of ketamine can inhibit gene expression of IL-1{beta} possibly through suppressing TLR4-mediated signal-transducing phosphorylations of Ras, Raf, MEK1/2, ERK1/2, and IKK and subsequent translocation and transactivation of NF{kappa}B.« less

Mutant HNF-1{alpha} and mutant HNF-1{beta} identified in MODY3 and MODY5 downregulate DPP-IV gene expression in Caco-2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gu Ning; Laboratory of Neurochemistry, Graduate School of Human and Environmental Studies, Kyoto University, Kyoto; Adachi, Tetsuya

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 2more » 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.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamauchi, Akiyo; Laboratory of Molecular Genetics, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai 980-8578; Department of Biochemistry, Nara Medical University, Kashihara 634-8521

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-expressingmore » 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}.« less

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.

Bullous pemphigoid and pemphigus vulgaris: correlated behaviour of serum VEGF, sE-selectin and TNF-alpha levels.

PubMed

Ameglio, F; D'Auria, L; Cordiali-Fei, P; Mussi, A; Valenzano, L; D'Agosto, G; Ferraro, C; Bonifati, C; Giacalone, B

1997-01-01

Recently, we reported that soluble E-selectin (sE-selectin), an isoform of the cell membrane E-selectin, an adhesion molecule synthesized only by endothelial cells, is significantly increased in sera of the patients with bullous pemphigoid (PB) or pemphigus vulgaris. A significant correlation was also found between the serum sE-selectin levels and the number of skin lesions, suggesting the possible use of this molecule to gauge disease intensity before therapy. One of the sE-selectin inducers is tumor nerosis factor-alpha (TNF-alpha), that is also able to enhance vascular endothelial growth factor (VEGF), a strong endothelium activator. On the basis of these observations, the present study was conducted to analyze the serum levels of VEGF, sE-selectin, and TNF-alpha in 8 patients with BP (age: 82, range 54-87, 7 males, 1 female) and in 6 patients affected affected with PV (age: 55, range 44-65; 5 males, 1 female) and to verify possible correlations between these variables and the disease activity, In addition, serum sE-selectin levels were measured over time and compared with the serum anti-epithelium antibodies titers. The sE-selectin, VEGF and TNF-alpha levels were measured in the samples by means of commercially available ELISA kit. The same samples were also employed to measure the anti-epithelium antibody titers. Serum VEGF, sE-selectin and TNF-alpha levels were significantly correlated each other (p at least < 0.01). All three variables were also significantly correlated with the number of lesions (p at least < 0.01). Serum VEGF levels were found increased (median = 178 pg/ml, range 37-595) as compared to 28 healthy controls (median = 135 pg/ml, range 18/269, p < 0.05). Also serum TNF-alpha levels were found increased (median = 5.5 pg/ml, range < 0.1-41.0) as compared to 28 healthy controls (median < 0.1 pg/ml, range < 0.1-5.3), p < 0.01). When the patients were observed over time, serum sE-selectin levels highly correlated with the disease intensity in both dermatoses, although with different regression curves. These data further underline the endothelium involvement in these bullous dermatoses and stress the possibility of employing sE-selectin as a non-specific follow-up marker of both BP and PV.

Upregulation of estrogen receptor expression in the uterus of ovariectomized B6C3F1 mice and Ishikawa cells treated with bromoethane

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aoyama, Hiroaki; Couse, John F.; Hewitt, Sylvia C.

2005-12-15

In a 2-year NTP bioassay, Bromoethane (BE) was found to induce endometrial neoplasms in the uterus of B6C3F1 mice [; ]. In women, hormonal influences, such as 'unopposed' estrogenic stimulus, have been implicated as important etiologic factors in uterine cancer. BE, however, does not affect the serum concentrations of sex hormones in female B6C3F1 mice [] and the mechanism of BE-induced uterine carcinogenesis still remains unclear. In the present study, we examined the estrogenic effects of BE on the uterus of ovariectomized B6C3F1 mice and on Ishikawa cells. Groups of 6 mice were given daily s.c. injections of 0, 100,more » 500 or 1000 mg BE/kg for 3 consecutive days. Mice treated with 17{beta}-estradiol served as positive controls. Mice were necropsied 24 h after the final injection, and uteri were weighed and examined histologically and immunohistochemically along with the vagina. Changes observed in the estrogen-treated mice included increased uterine weights, edema and inflammation of the endometrium, increased epithelial layers of the uterine and vaginal lumens and keratinization of the vaginal epithelium. In the BE-treated mice, no such changes occurred; however, immunohistochemical staining of the uterus revealed a significant increase in immunoexpression of the estrogen receptor alpha (ER{alpha}) in the two higher dose groups. Analysis of mRNA also showed slightly increased uterine ER{alpha} expression in these groups. Upregulated expression of ER{alpha} was confirmed in BE-treated Ishikawa cells, in which Western blotting analyses identified an intense signal at approximately 66 kDa, which is consistent with ER{alpha}. These data suggest that upregulated expression of ER{alpha} may be important in the induction of endometrial neoplasms in BE-treated mice.« less

Specific antibodies against Go isoforms reveal the early expression of the Go2 alpha subunit and appearance of Go1 alpha during neuronal differentiation.

PubMed

Rouot, B; Charpentier, N; Chabbert, C; Carrette, J; Zumbihl, R; Bockaert, J; Homburger, V

1992-02-01

We have previously identified two isoforms of Go alpha in membranes of N1E-115 neuroblastoma cells, using an antibody raised against the purified Go alpha subunit; one isoform of the Go alpha subunit (pI 5.80) is present in undifferentiated cells, whereas a more acidic isoform (pI 5.55) appears during differentiation [J. Neurochem. 54:1310-1320 (1990)]. Recently, the Go alpha gene has been shown to encode, by alternative splicing, two polypeptides, Go1 alpha and Go2 alpha, which differ only in their carboxyl-terminal part. To determine unambiguously whether the two Go alpha subunits detected in neuroblastoma cells were actually the products of different mRNAs, rabbit polyclonal antibodies were generated against synthetic peptides (amino acids 291-302) of both sequences. Specificity of the two affinity-purified antipeptide antibodies was assessed on Western blots by comparing their immunoreactivities with those of other G alpha antibodies. On a blotted mixture of purified brain guanine nucleotide-binding proteins, the anti-alpha o1 and anti-alpha o2 peptide antibodies only recognized the 39-kDa Go alpha subunit. Furthermore, the immunological recognition of brain membranes from 15-day-old mouse fetuses by antipeptide antibodies could be specifically blocked by addition of the corresponding antigen. When membrane proteins from differentiated neuroblastoma cells and mouse fetus brain were blotted after two-dimensional gel electrophoresis, the anti-alpha o1 and anti-alpha o2 peptide antibodies labeled a 39-kDa subunit focused at a pI value of 5.55 or 5.80, respectively. Study of the ontogenesis of both Go alpha subunits revealed the predominance of Go2 alpha in the frontal cortex at day 15 of gestation. Thereafter, there was a progressive decline of the Go2 alpha polypeptide to a very low level, concomitant with an increase in the Go1 alpha protein, which plateaued about 15 days after birth to a level 8 times higher than at gestational day 15. Similarly, on neuroblastoma cells, the Go2 alpha subunit was almost exclusively present in undifferentiated cells, and differentiation induced the appearance of the Go1 alpha subunit, with a reduction in the amount of Go2 alpha polypeptide. Thus, the evolution of the two Go alpha subunits during cell differentiation, unambiguously identified with specific antibodies, suggests that neuronal differentiation is responsible for the on/off switch of the expression of the Go alpha isoforms and indicates that Go1 alpha, rather than Go2 alpha, is involved in neurotransmission.

Expression and function of CD8 alpha/beta chains on rat and human mast cells.

PubMed

Kim, Mi-Sun; Kim, Sung-Hoon; Lee, Hye-Jung; Kim, Hyung-Min

2004-03-01

The expression and functional role of CD8 glycoprotein, a marker of cytotoxic/suppressor T lymphocytes and NK cells, were not studied on freshly isolated connective tissue type rat peritoneal mast cells, a rat mucosal type mast cell line (RBL 2H3), or human mast cell line (HMC-1). We used the reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, immunohistochemistry and enzyme-linked immunosorbent assay. RT-PCR and Western blot analysis identified the presence of CD8 alpha/beta chains on the mast cells, and immunohistochemistry confirmed CD8alpha expression on rat or human mast cells. Functional studies demonstrated that stimulation of CD8 alpha/beta chains on rat mast cells induced the secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), which are regarded as important mediators during infection. However, co-stimulation with stem cell factor had no effect on CD8-induced mediator secretion. Our findings demonstrate novel biological roles of CD8 molecules in mast cells.

Human Lung Cancer Risks from Radon – Part I - Influence from Bystander Effects - A Microdose Analysis

PubMed Central

Leonard, Bobby E.; Thompson, Richard E.; Beecher, Georgia C.

2010-01-01

Since the publication of the BEIR VI report in 1999 on health risks from radon, a significant amount of new data has been published showing various mechanisms that may affect the ultimate assessment of radon as a carcinogen, at low domestic and workplace radon levels, in particular the Bystander Effect (BE) and the Adaptive Response radio-protection (AR). We analyzed the microbeam and broadbeam alpha particle data of Miller et al. (1995, 1999), Zhou et al. (2001, 2003, 2004), Nagasawa and Little (1999, 2002), Hei et al. (1999), Sawant et al. (2001a) and found that the shape of the cellular response to alphas is relatively independent of cell species and LET of the alphas. The same alpha particle traversal dose response behavior should be true for human lung tissue exposure to radon progeny alpha particles. In the Bystander Damage Region of the alpha particle response, there is a variation of RBE from about 10 to 35. There is a transition region between the Bystander Damage Region and Direct Damage Region of between one and two microdose alpha particle traversals indicating that perhaps two alpha particle “hits” are necessary to produce the direct damage. Extrapolation of underground miners lung cancer risks to human risks at domestic and workplace levels may not be valid. PMID:21731539

Production of interleukin-1alpha by human endometrial stromal cells is triggered during menses and dysfunctional bleeding and is induced in culture by epithelial interleukin-1alpha released upon ovarian steroids withdrawal.

PubMed

Pretto, Chrystel M; Gaide Chevronnay, Héloïse P; Cornet, Patricia B; Galant, Christine; Delvaux, Denis; Courtoy, Pierre J; Marbaix, Etienne; Henriet, Patrick

2008-10-01

Endometrial breakdown during menstruation and dysfunctional bleeding is triggered by the abrupt expression of matrix metalloproteinases (MMPs), including interstitial collagenase (MMP-1). The paracrine induction of MMP-1 in stromal cells via epithelium-derived IL-1alpha is repressed by ovarian steroids. However, the control by estradiol (E) and progesterone (P) of endometrial IL-1alpha expression and bioactivity remains unknown. Variations of endometrial IL-1alpha mRNA and protein along the menstrual cycle and during dysfunctional bleeding were determined using RT-PCR, in situ hybridization, and immunolabeling. The mechanism of EP control was analyzed using culture of explants, laser capture microdissection, and purified cells. Data were compared with expression changes of IL-1beta and IL-1 receptor antagonist. IL-1alpha is synthesized by epithelial cells throughout the cycle but E and/or P prevents its release. In contrast, endometrial stromal cells produce IL-1alpha only at menses and during irregular bleeding in areas of tissue breakdown. Stromal expression of IL-1alpha, like that of MMP-1, is repressed by P (alone or with E) but triggered by epithelium-derived IL-1alpha released upon EP withdrawal. Our experiments in cultured endometrium suggest that IL-1alpha released by epithelial cells triggers the production of IL-1alpha by stromal cells in a paracrine amplification loop to induce MMP-1 expression during menstruation and dysfunctional bleeding. All three steps of this amplification cascade are repressed by EP.

Expression of the transforming growth factor alpha protooncogene in differentiating human promyelocytic leukemia (HL-60) cells.

PubMed

Walz, T M; Malm, C; Wasteson, A

1993-01-01

The process of myeloid differentiation in human promyelocytic leukemia cells (HL-60) is accompanied by the coordinate expression of numerous protooncogenes. To investigate the expression of transforming growth factor alpha (TGF-alpha) in myeloid differentiation, HL-60 cells were induced to differentiate into granulocytes with 1.25% dimethyl sulfoxide, 0.2 microM all-trans retinoic acid, or 500 microM N6,O2-dibutyryladenosine-3'5'-cyclic monophosphate or differentiated along the monocyte/macrophage pathway with 0.1 microM phorbol-12-myristate-13-acetate. Using Northern blot analyses, TGF-alpha transcripts were detected within 24 h of treatment in cells differentiating toward granulocytes; maximal levels of gene expression were reached after 3 days or later and remained essentially constant throughout the observation period. These cells released TGF-alpha protein, as demonstrated by analysis of the incubation medium. In contrast, no TGF-alpha RNA or protein was detectable in HL-60 cell cultures when induced with phorbol-12-myristate-13-acetate. Epidermal growth factor receptor transcripts could not be detected either in undifferentiated or in differentiated HL-60 cells; therefore it appears as if an autocrine loop involving TGF-alpha in HL-60 cells is unlikely. In conclusion, the results demonstrate, for the first time, the expression of TGF-alpha in human granulocyte precursor cells. Our findings may indicate novel regulatory pathways in hematopoiesis.

76 FR 45838 - Government-Owned Inventions; Availability for Licensing

Federal Register 2010, 2011, 2012, 2013, 2014

2011-08-01

...-effects, reducing their pain burden during treatment. IL13-receptor-alpha-2 (IL13-R[alpha]2) is a cell... kill only those cancer cells which express IL13-R[alpha]2. Our inventors previously constructed fusion... overexpressed IL13-R[alpha]2, as well as other types of diseased cells (asthma, pulmonary fibrosis) which...

Effects of alpha/beta-androstenediol immune regulating hormones on bone remodeling and apoptosis in osteoblasts.

PubMed

Urban, Nicole H; Chamberlin, Brett; Ramage, Samuel; Roberts, Zachary; Loria, Roger M; Beckman, Matthew J

2008-06-01

A large body of evidence suggests that the immune system directly impacts bone physiology. We tested whether immune regulating hormones (IRH), 17beta-androstenediol (beta-AED), 7beta,17beta-androstenetriol (beta-AET) or the 17alpha-androstenediol (alpha-AED), and 7alpha,17beta-androstenetriol (alpha-AET) metabolites could directly influence bone remodeling in vitro using human fetal osteoblasts (FOB-9). The impact on bone remodeling was examined by comparing the ratio of RANKL/OPG gene expression in response to AED and AET compounds. The alpha-AED was found to significantly increase in the ratio of RANKL/OPG gene expression and altering the morphology of RANKL stained FOB-9 cells. Cell viability was assessed using a Live/Dead assay. Again alpha-AED was unique in its ability to reduce the proportion of viable cells, and to induce mild apoptosis of FOB-9 cells. Treatment of FOB-9 cells with WY14643, an activator of PPAR-alpha and -gamma, also significantly elevated the percentage of dead cells. This increase was abolished by co-treatment with GW9962, a specific inhibitor of PPAR-gamma. Analysis of PPAR-gamma mRNA by Quantitative RT-PCR and its activation by DNA binding demonstrated that alpha-AED increased PPAR-gamma activation by 19%, while beta-AED conferred a 37% decrease in PPAR-gamma activation. In conclusion, alpha-AED opposed beta-AED by elevating a bone resorption scenario in osteoblast cells. The increase in RANKL/OPG is modulated by an activation of PPAR-gamma that in turn caused mild apoptosis of FOB-9 cells.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Cheng-hu; Cao, Guo-Fan; Jiang, Qin, E-mail: Jqin710@vip.sina.com

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-addedmore » 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.« less

Phosphorylation of MAP65-1 by Arabidopsis Aurora Kinases Is Required for Efficient Cell Cycle Progression1[OPEN

PubMed Central

Weimer, Annika K.; Stoppin-Mellet, Virginie; Kosetsu, Ken; Cedeño, Cesyen; Jaquinod, Michel; Njo, Maria; De Milde, Liesbeth; Tompa, Peter; Inzé, Dirk; Beeckman, Tom; Vantard, Marylin

2017-01-01

Aurora kinases are key effectors of mitosis. Plant Auroras are functionally divided into two clades. The alpha Auroras (Aurora1 and Aurora2) associate with the spindle and the cell plate and are implicated in controlling formative divisions throughout plant development. The beta Aurora (Aurora3) localizes to centromeres and likely functions in chromosome separation. In contrast to the wealth of data available on the role of Aurora in other kingdoms, knowledge on their function in plants is merely emerging. This is exemplified by the fact that only histone H3 and the plant homolog of TPX2 have been identified as Aurora substrates in plants. Here we provide biochemical, genetic, and cell biological evidence that the microtubule-bundling protein MAP65-1—a member of the MAP65/Ase1/PRC1 protein family, implicated in central spindle formation and cytokinesis in animals, yeasts, and plants—is a genuine substrate of alpha Aurora kinases. MAP65-1 interacts with Aurora1 in vivo and is phosphorylated on two residues at its unfolded tail domain. Its overexpression and down-regulation antagonistically affect the alpha Aurora double mutant phenotypes. Phospho-mutant analysis shows that Aurora contributes to the microtubule bundling capacity of MAP65-1 in concert with other mitotic kinases. PMID:27879390

Tumor necrosis factor alpha (TNF-alpha)-induced cell adhesion to human endothelial cells is under dominant control of one TNF receptor type, TNF-R55

PubMed Central

1993-01-01

Tumor necrosis factor alpha (TNF-alpha) is a pleiotropic cytokine triggering cell responses through two distinct membrane receptors. Stimulation of leukocyte adhesion to the endothelium is one of the many TNF-alpha activities and is explained by the upregulation of adhesion molecules on the endothelial cell surface. Human umbilical vein endothelial cells (HUVEC) were isolated, cultured, and demonstrated to express both TNF receptor types, TNF-R55 and TNF-R75. Cell adhesion to HUVEC was studied using the HL60, U937, and MOLT-4 cell lines. HUVEC were activated by either TNF-alpha, binding to both TNF-R55 and TNF- R75, and by receptor type-specific agonists, binding exclusively to TNF- R55 or to TNF-R75. The TNF-alpha-induced cell adhesion to HUVEC was found to be controlled almost exclusively by TNF-R55. This finding correlated with the exclusive activity of TNF-R55 in the TNF-alpha- dependent regulation of the expression of the intercellular adhesion molecule type 1 (ICAM-1), E-selectin, and vascular cell adhesion molecule type 1 (VCAM-1). The CD44 adhesion molecule in HUVEC was also found to be upregulated through TNF-R55. However, both TNF-R55 and TNF- R75 upregulate alpha 2 integrin expression in HUVEC. The predominant role of TNF-R55 in TNF-alpha-induced adhesion in HUVEC may correlate with its specific control of NF-kappa B activation, since kappa B elements are known to be present in ICAM-1, E-selectin, and VCAM-1 gene regulatory sequences. PMID:8386742

Progesterone biotransformation by plant cell suspension cultures.

PubMed Central

Yagen, B; Gallili, G E; Mateles, R I

1978-01-01

Progesterone was converted to 5alpha-pregnane-3alpha-ol-20-one, delta4-pregnene-20alpha-ol-3-one, delta4-pregnene-14alpha-ol-3,20-dione, delta4-pregnene-7beta,14alpha-diol-3,20-dione, and delta4-pregnene-6beta,11alpha-diol-3,20-dione by cell cultures of Lycopersicon esculentum. Cell cultures of Capsicum frutescens (green) metabolized progesterone to delta4-pregnene-20alpha-ol-3-one in very high yield, and Vinca rosea yielded delta4-pregnene-20beta-ol-3-one and delta4-pregnene-14alpha-ol-3,20-dione. A stereospecific reduction of the keto groups and a double bond and stereospecific introduction of hydroxyl groups at the 6, 11, and 14 positions have been observed. The mono- and dihydroxylated progesterones have not previously been reported as metabolic products of progesterone by plant cell systems and represent de novo hydroxylation of a nonglycosylated steroid. PMID:697360

Impact of lysosomal storage disorders on biology of mesenchymal stem cells: Evidences from in vitro silencing of glucocerebrosidase (GBA) and alpha-galactosidase A (GLA) enzymes.

PubMed

Squillaro, Tiziana; Antonucci, Ivana; Alessio, Nicola; Esposito, Anna; Cipollaro, Marilena; Melone, Mariarosa Anna Beatrice; Peluso, Gianfranco; Stuppia, Liborio; Galderisi, Umberto

2017-12-01

Lysosomal storage disorders (LDS) comprise a group of rare multisystemic diseases resulting from inherited gene mutations that impair lysosomal homeostasis. The most common LSDs, Gaucher disease (GD), and Fabry disease (FD) are caused by deficiencies in the lysosomal glucocerebrosidase (GBA) and alpha-galactosidase A (GLA) enzymes, respectively. Given the systemic nature of enzyme deficiency, we hypothesized that the stem cell compartment of GD and FD patients might be also affected. Among stem cells, mesenchymal stem cells (MSCs) are a commonly investigated population given their role in hematopoiesis and the homeostatic maintenance of many organs and tissues. Since the impairment of MSC functions could pose profound consequences on body physiology, we evaluated whether GBA and GLA silencing could affect the biology of MSCs isolated from bone marrow and amniotic fluid. Those cell populations were chosen given the former's key role in organ physiology and the latter's intriguing potential as an alternative stem cell model for human genetic disease. Our results revealed that GBA and GLA deficiencies prompted cell cycle arrest along with the impairment of autophagic flux and an increase of apoptotic and senescent cell percentages. Moreover, an increase in ataxia-telangiectasia-mutated staining 1 hr after oxidative stress induction and a return to basal level at 48 hr, along with persistent gamma-H2AX staining, indicated that MSCs properly activated DNA repair signaling, though some damages remained unrepaired. Our data therefore suggest that MSCs with reduced GBA or GLA activity are prone to apoptosis and senescence due to impaired autophagy and DNA repair capacity. © 2017 Wiley Periodicals, Inc.

alpha2-chimaerin, a Cdc42/Rac1 regulator, is selectively expressed in the rat embryonic nervous system and is involved in neuritogenesis in N1E-115 neuroblastoma cells.

PubMed

Hall, C; Michael, G J; Cann, N; Ferrari, G; Teo, M; Jacobs, T; Monfries, C; Lim, L

2001-07-15

Neuronal differentiation involves Rac and Cdc42 GTPases. alpha-Chimaerin, a Rac/Cdc42 regulator, occurs as alpha1- and alternatively spliced Src homology 2 (SH2) domain-containing alpha2-isoforms. alpha2-chimaerin mRNA was highly expressed in the rat embryonic nervous system, especially in early postmitotic neurons. alpha1-chimaerin mRNA was undetectable before embryonic day 16.5. Adult alpha2-chimaerin mRNA was restricted to neurons within specific brain regions, with highest expression in the entorhinal cortex. alpha2-chimaerin protein localized to neuronal perikarya, dendrites, and axons. The overall pattern of alpha2-chimaerin mRNA expression resembles that of cyclin-dependent kinase regulator p35 (CDK5/p35) which participates in neuronal differentiation and with which chimaerin interacts. To determine whether alpha2-chimaerin may have a role in neuronal differentiation and the relevance of the SH2 domain, the morphological effects of both chimaerin isoforms were investigated in N1E-115 neuroblastoma cells. When plated on poly-lysine, transient alpha2-chimaerin but not alpha1-chimaerin transfectants formed neurites. Permanent alpha2-chimaerin transfectants generated neurites whether or not they were stimulated by serum starvation, and many cells were enlarged. Permanent alpha1-chimaerin transfectants displayed numerous microspikes and contained F-actin clusters, a Cdc42-phenotype, but generated few neurites. In neuroblastoma cells, alpha2-chimaerin was predominantly soluble with some being membrane-associated, whereas alpha1-chimaerin was absent from the cytosol, being membrane- and cytoskeleton-associated, paralleling their subcellular distribution in brain. Transient transfection with alpha2-chimaerin mutated in the SH2 domain (N94H) generated an alpha1-chimaerin-like phenotype, protein partitioned in the particulate fraction, and in NGF-stimulated pheochromocytoma cell line 12 (PC12) cells, neurite formation was inhibited. These results indicate a role for alpha2-chimaerin in morphological differentiation for which its SH2 domain is vital.

Immune cell inflammatory cytokine responses differ between central and systemic compartments in response to acute exercise in mice.

PubMed

Pervaiz, Nabeel; Hoffman-Goetz, Laurie

2012-01-01

Exhaustive exercise induces apoptosis and oxidative stress in systemic organs and tissues and is associated with increased levels of pro-inflammatory cytokines. The effects of acute exercise on cytokine expression and apoptosis of immune cells in the central nervous system (CNS) have not been well characterized. We investigated the effects of a single bout of strenuous exercise on the expression of TNF-alpha, IL-6, and IL-beta, as well as the apoptotic status of cells in the hippocampus of healthy mice. To compare central vs. systemic differences, cytokine expression in the intestinal lymphocytes of a subset of mice were also assessed. Female C57BL/6 mice were divided into three groups: sedentary controls (NOTREAD) (n = 22), treadmill exercise with immediate sacrifice (TREAD-Imm) (n = 21), or treadmill exercise with sacrifice after 2 hours (TREAD-2h). TNF-alpha, IL-6, and IL-1beta expression in the hippocampus and intestinal lymphocytes were measured by Western blot analysis. Percentages of hippocampal cells undergoing apoptosis (Annexin+) or necrosis (Propidium Iodide+) were determined through flow cytometry. Plasma levels of 8-isoprostane and corticosterone were measured using commercially available EIA kits. Acute treadmill exercise led to significant decreases in TNF-alpha (p<0.05) and increases in IL-6 (p<0.05) expression in the hippocampus of healthy mice. No effects of acute exercise on the apoptotic status of hippocampal cells were observed. In intestinal lymphocytes, the exercise bout led to significant increases in TNF-alpha (p<0.05), IL-6 (p<0.05), and IL-1beta (p<0.05). Acute exercise was associated with a significant increase in both plasma 8-isoprostane (p<0.05) and corticosterone (p<0.05) levels. Acute exercise differentially affects the pattern ofpro-inflammatory cytokine expression in the hippocampus compared to intestinal lymphocytes and, further, does not induce apoptosis in hippocampal cells.

Alpha-adrenergic blocker mediated osteoblastic stem cell differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Yoon Jung; Lee, Jue Yeon; Research Center, Nano Intelligent Biomedical Engineering Corporation

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 clinicallymore » 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 according to our data doxazosin might be useful for application in the field of bone metabolism.« less

Rotaviruses induce an early membrane permeabilization of MA104 cells and do not require a low intracellular Ca2+ concentration to initiate their replication cycle.

PubMed Central

Cuadras, M A; Arias, C F; López, S

1997-01-01

In this work, we found that rotavirus infection induces an early membrane permeabilization of MA104 cells and promotes the coentry of toxins, such as alpha-sarcin, into the cell. This cell permeability was shown to depend on infectious virus and was also shown to be virus dose dependent, with 10 infectious particles per cell being sufficient to achieve maximum permeability; transient, lasting no more than 15 min after virus entry and probably occurring concomitantly with virus penetration; and specific, since cells that are poorly permissive for rotavirus were not permeabilized. The rotavirus-mediated coentry of toxins was not blocked by the endocytosis inhibitors dansylcadaverine and cytochalasin D or by the vacuolar proton-ATPase inhibitor bafilomycin A1, suggesting that neither endocytocis nor an intraendosomal acidic pH or a proton gradient is required for permeabilization of the cells. Compounds that raise the intracellular concentration of calcium ([Ca2+]i) by different mechanisms, such as the calcium ionophores A23187 and ionomycin and the endoplasmic reticulum calcium-ATPase inhibitor thapsigargin, did not block the coentry of alpha-sarcin or affect the onset of viral protein synthesis, suggesting that a low [Ca2+]i is not essential for the initial steps of the virus life cycle. Since the entry of alpha-sarcin correlates with virus penetration in all parameters tested, the assay for permeabilization to toxins might be a useful tool for studying and characterizing the route of entry and the mechanism used by rotaviruses to traverse the cell membrane and initiate a productive replication cycle. PMID:9371563

Transforming growth factor-alpha stimulates enterocyte proliferation and accelerates intestinal recovery following methotrexate-induced intestinal mucositis in a rat and a cell culture model.

PubMed

Sukhotnik, Igor; Shteinberg, Dan; Ben Lulu, Shani; Bashenko, Yulia; Mogilner, Jorge G; Ure, Benno M; Shaoul, Ron; Shamian, Benhoor; Coran, Arnold G

2008-12-01

Recent evidence suggests that transforming growth factor-alpha (TGF-alpha) enhances enterocyte proliferation and exerts a gut trophic effect. The purpose of the present study was to evaluate the effect of TGF-alpha on enterocyte proliferation and intestinal recovery following methotrexate (MTX)-induced intestinal mucositis in rats and in Caco-2 cells. Nonpretreated Caco-2 cells and those pretreated with MTX were incubated with increasing concentrations of TGF-alpha. Cell proliferation was determined by FACS cytometry. Adult rats were divided into three groups: control rats treated with vehicle, MTX rats treated with one dose (20 microg/kg) of MTX given intraperitoneally, and MTX-TGF-alpha rats treated with one dose of MTX followed by two doses of TGF-alpha (75 microg/kg a day). Three days after MTX injection, rats were sacrificed. Intestinal mucosal damage (Park's score), mucosal structural changes, and enterocyte proliferation were measured at sacrifice. Western blotting was used to determine the level of extracellular signal-related kinase (ERK) protein, a marker of cell proliferation. A nonparametric Kruskal-Wallis ANOVA test was used for statistical analysis with P value less than 0.05 considered statistically significant. The in vitro experiment demonstrated that treatment with TGF-alpha of Caco-2 cells resulted in a significant stimulation of cell proliferation in a dose-dependent manner. The in vivo experiment showed that treatment with TGF-alpha resulted in a significant increase in bowel and mucosal weight, DNA and protein content in jejunum and ileum, villus height in jejunum and ileum, crypt depth in ileum, and increased cell proliferation in jejunum and ileum compared to the MTX group. MTX-TGF-alpha rats also had a significantly lower intestinal injury score in ileum when compared to MTX animals. The increase in levels of cell proliferation in MTX-TGF-alpha rats corresponded with the increase in ERK protein levels in intestinal mucosa. Treatment with TGF-alpha prevents mucosal injury, enhances ERK-induced enterocyte proliferation, and improves intestinal recovery following MTX-induced intestinal mucositis in rats. These findings correlated with the observation that TGF-alpha also caused a significant stimulation of cell proliferation in a Caco-2 cell culture model treated with MTX. These observations may have significant implications for the treatment of patients on chemotherapy who develop severe mucositis.

IFN-{gamma} sensitizes MIN6N8 insulinoma cells to TNF-{alpha}-induced apoptosis by inhibiting NF-{kappa}B-mediated XIAP upregulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Hun Sik; Kim, Sunshin; Lee, Myung-Shik

2005-10-28

Although X-linked inhibitor of apoptosis protein (XIAP) is an important intracellular suppressor of apoptosis in a variety of cell types, its role in cytokine-induced pancreatic {beta}-cell apoptosis remains unclear. Here, we found that: (i) XIAP level was inversely correlated with tumor necrosis factor (TNF)-{alpha}-induced apoptosis in MIN6N8 insulinoma cells; (ii) adenoviral XIAP overexpression abrogated the TNF-{alpha}-induced apoptosis through inhibition of caspase activity; (iii) downregulation of XIAP by antisense oligonucleotide or Smac peptide sensitized MIN6N8 cells to TNF-{alpha}-induced apoptosis; (iv) XIAP expression was induced by TNF-{alpha} through a nuclear factor-{kappa}B (NF-{kappa}B)-dependent pathway, and interferon (IFN)-{gamma} prevented such an induction in amore » manner independent of NF-{kappa}B, which presents a potential mechanism underlying cytotoxic IFN-{gamma}/TNF-{alpha} synergism. Taken together, our results suggest that XIAP is an important modulator of TNF-{alpha}-induced apoptosis of MIN6N8 cells, and XIAP regulation in pancreatic {beta}-cells might play an important role in pancreatic {beta}-cell apoptosis and in the pathogenesis of type 1 diabetes.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Kai; Zhu, Fei; Zhang, Han-zhong

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 oralmore » 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-Right-Pointing-Pointer VCAM-1/VLA-4 mediated TNF-{alpha}-enhanced cell fusions.« less

Dexamethasone but not indomethacin inhibits human phagocyte nicotinamide adenine dinucleotide phosphate oxidase activity by down-regulating expression of genes encoding oxidase components.

PubMed

Condino-Neto, A; Whitney, C; Newburger, P E

1998-11-01

We investigated the effects of dexamethasone or indomethacin on the NADPH oxidase activity, cytochrome b558 content, and expression of genes encoding the components gp91-phox and p47-phox of the NADPH oxidase system in the human monocytic THP-1 cell line, differentiated with IFN-gamma and TNF-alpha, alone or in combination, for up to 7 days. IFN-gamma and TNF-alpha, alone or in combination, caused a significant up-regulation of the NADPH oxidase system as reflected by an enhancement of the PMA-stimulated superoxide release, cytochrome b558 content, and expression of gp91-phox and p47-phox genes on both days 2 and 7 of cell culture. Noteworthy was the tremendous synergism between IFN-gamma and TNF-alpha for all studied parameters. Dexamethasone down-regulated the NADPH oxidase system of cytokine-differentiated THP-1 cells as assessed by an inhibition on the PMA-stimulated superoxide release, cytochrome b558 content, and expression of the gp91-phox and p47-phox genes. The nuclear run-on assays indicated that dexamethasone down-regulated the NADPH oxidase system at least in part by inhibiting the transcription of gp91-phox and p47-phox genes. Indomethacin inhibited only the PMA-stimulated superoxide release of THP-1 cells differentiated with IFN-gamma and TNF-alpha during 7 days. None of the other parameters was affected by indomethacin. We conclude that dexamethasone down-regulates the NADPH oxidase system at least in part by inhibiting the expression of genes encoding the gp91-phox and p47-phox components of the NADPH oxidase system.

Expression of cell adhesion molecules in the normal and T3 blocked development of the tadpole's kidney of Bufo arenarum (Amphibian, Anuran, Bufonidae).

PubMed

Izaguirre, M F; García-Sancho, M N; Miranda, L A; Tomas, J; Casco, V H

2008-08-01

Cell adhesion molecules act as signal transducers from the extracellular environment to the cytoskeleton and the nucleus and consequently induce changes in the expression pattern of structural proteins. In this study, we showed the effect of thyroid hormone (TH) inhibition and arrest of metamorphosis on the expression of E-cadherin, beta-and alpha-catenin in the developing kidney of Bufo arenarum. Cell adhesion molecules have selective temporal and spatial expression during development suggesting a specific role in nephrogenesis. In order to study mechanisms controlling the expression of adhesion molecules during renal development, we blocked the B. arenarum metamorphosis with a goitrogenic substance that blocks TH synthesis. E-cadherin expression in the proximal tubules is independent of thyroid control. However, the blockage of TH synthesis causes up-regulation of E-cadherin in the collecting ducts, the distal tubules and the glomeruli. The expression of beta-and alpha-catenin in the collecting ducts, the distal tubules, the glomeruli and the mesonephric mesenchyme is independent of TH. TH blockage causes up-regulation of beta-and alpha-catenin in the proximal tubules. In contrast to E-cadherin, the expression of the desmosomal cadherin desmoglein 1 (Dsg-1) is absent in the control of the larvae kidney during metamorphosis and is expressed in some interstitial cells in the KClO4 treated larvae. According to this work, the Dsg-1 expression is down-regulated by TH. We demonstrated that the expression of E-cadherin, Dsg-1, beta-catenin and alpha-catenin are differentially affected by TH levels, suggesting a hormone-dependent role of these proteins in the B. arenarum renal metamorphosis.

Activation of peroxisome proliferator-activated receptor-{alpha} enhances fatty acid oxidation in human adipocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Joo-Young; Hashizaki, Hikari; Goto, Tsuyoshi

2011-04-22

Highlights: {yields} PPAR{alpha} activation increased mRNA expression levels of adipocyte differentiation marker genes and GPDH activity in human adipocytes. {yields} PPAR{alpha} activation also increased insulin-dependent glucose uptake in human adipocytes. {yields} PPAR{alpha} activation did not affect lipid accumulation in human adipocytes. {yields} PPAR{alpha} activation increased fatty acid oxidation through induction of fatty acid oxidation-related genes in human adipocytes. -- Abstract: Peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPAR{alpha} in adipocytes have been unclarified. We examined the functions of PPAR{alpha} using human multipotent adipose tissue-derived stem cells as a humanmore » adipocyte model. Activation of PPAR{alpha} by GW7647, a potent PPAR{alpha} agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPAR{gamma}, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPAR{alpha} activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPAR{gamma} is activated. On the other hand, PPAR{alpha} activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPAR{alpha}-dependent manner. Moreover, PPAR{alpha} activation increased the production of CO{sub 2} and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPAR{alpha} stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPAR{alpha} agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected effects of PPAR{alpha} activation are very valuable for managing diabetic conditions accompanied by obesity, because PPAR{gamma} agonists, usually used as antidiabetic drugs, induce excessive lipid accumulation in adipocytes in addition to improvement of insulin resistance.« less

Biology Based Lung Cancer Model for Chronic Low Radon Exposures

NASA Astrophysics Data System (ADS)

TruÅ£ǎ-Popa, Lucia-Adina; Hofmann, Werner; Fakir, Hatim; Cosma, Constantin

2008-08-01

Low dose effects of alpha particles at the tissue level are characterized by the interaction of single alpha particles, affecting only a small fraction of the cells within that tissue. Alpha particle intersections of bronchial target cells during a given exposure period were simulated by an initiation-promotion model, formulated in terms of cellular hits within the cycle time of the cell (dose-rate) and then integrated over the whole exposure period (dose). For a given average number of cellular hits during the lifetime of bronchial cells, the actual number of single and multiple hits was selected from a Poisson distribution. While oncogenic transformation is interpreted as the primary initiation step, stimulated mitosis by killing adjacent cells is assumed to be the primary radiological promotion event. Analytical initiation and promotion functions were derived from experimental in vitro data on oncogenic transformation and cellular survival. To investigate the shape of the lung cancer risk function at chronic, low level exposures in more detail, additional biological factors describing the tissue response and operating specifically at low doses were incorporated into the initiation-promotion model. These mechanisms modifying the initial response at the cellular level were: adaptive response, genomic instability, induction of apoptosis by surrounding cells, and detrimental as well as protective bystander mechanisms. To quantify the effects of these mechanisms as functions of dose, analytical functions were derived from the experimental evidence presently available. Predictions of lung cancer risk, including these mechanisms, exhibit a distinct sublinear dose-response relationship at low exposures, particularly for very low exposure rates.

Resolution of G(s)alpha and G(q)alpha/G(11)alpha proteins in membrane domains by two-dimensional electrophoresis: the effect of long-term agonist stimulation.

PubMed

Matousek, P; Novotný, J; Svoboda, P

2004-01-01

Low-density membrane-domain fractions were prepared from S49 lymphoma cells and clone e2m11 of HEK293 cells expressing a large number of thyrotropin-releasing hormone receptor (TRH-R) and G(11)alpha by flotation on sucrose density gradients. The intact cell structure was broken by detergent-extraction, alkaline-treatment or drastic homogenization. Three types of low-density membranes were resolved by two-dimensional electrophoresis and analyzed for G(s)alpha (S49) or G(q)alpha/G11) (e2m11) content. Four individual immunoblot signals of Gsalpha protein were identified in S49 lymphoma cells indicating complete resolution of the long G(s)alpha L+/-ser and short G(s)alpha S+/-ser variants of G(s)alpha. All these were diminished by prolonged agonist (isoprenaline) stimulation. In e2m11-HEK cells, five different immunoblot signals were detected indicating post-translational modification of G proteins of G(q)alpha/G(11)alpha family. The two major spots corresponding to exogenously (over)expressed G(11)alpha and endogenous G(q)alpha were reduced; the minor spots diminished by hormonal stimulation. Parallel analysis by silver staining of the total protein content indicated that no major changes in protein composition occurred under these conditions. Our data thus indicate that agonist-stimulation of target cells results in down-regulation of all different members of G(s) and G(q)/G(11) families. This agonist-specific effect may be demonstrated in crude membrane as well as domain/raft preparations and it is not accompanied by changes in overall protein composition.

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

PubMed

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

2010-07-01

Vitamin E, a micronutrient (comprising alpha-, beta-, gamma- and delta-tocopherols, alpha-, beta-, gamma- and delta-tocotrienols), has documented antioxidant and non-antioxidant effects, some of which inhibit inflammation and angiogenesis. We compared the abilities of alpha-, gamma- and delta-tocopherols to regulate human blood cytotoxicity (BEC) and lymphatic endothelial cytotoxicity (LEC), proliferation, invasiveness, permeability, capillary formation and suppression of TNF-alpha-induced VCAM-1 as in vitro models of inflammatory angiogenesis. alpha-, gamma- and delta-tocopherols were not toxic to either cell type up to 40 microM. In BEC, confluent cell density was decreased by all concentrations of delta- and gamma-tocopherol (10-40 microM) but not by alpha-tocopherol. LEC showed no change in cell density in response to tocopherols. delta-Tocopherol (40 microM), but not other isomers, decreased BEC invasiveness. In LEC, all doses of gamma-tocopherol, as well as the highest dose of alpha-tocopherol (40 microM), decreased cell invasiveness. delta-Tocopherol had no effect on LEC invasiveness at any molarity. delta-Tocopherol dose dependently increased cell permeability at 48 h in BEC and LEC; alpha- and gamma-tocopherols showed slight effects. Capillary tube formation was decreased by high dose (40 microM) concentrations of alpha-, gamma- and delta-tocopherol, but showed no effects with smaller doses (10-20 microM) in BEC. gamma-Tocopherol (10-20 microM) and alpha-tocopherol (10 microM), but not delta-tocopherol, increased LEC capillary tube formation. Lastly, in BEC, alpha-, gamma- and delta-tocopherol each dose-dependently reduced TNF-alpha-induced expression of VCAM-1. In LEC, there was no significant change to TNF-alpha-induced VCAM-1 expression with any concentration of alpha-, gamma- or delta-tocopherol. These data demonstrate that physiological levels (0-40 microM) of alpha-, gamma- and delta-tocopherols are nontoxic and dietary tocopherols, especially delta-tocopherol, can limit several BEC and LEC endothelial behaviors associated with angiogenesis. Tocopherols may therefore represent important nutrient-signals that limit cell behaviors related to inflammation/angiogenesis, which when deficient, may predispose individuals to risks associated with elevated angiogenesis such as inflammation and cancer; further differences seen from the tocopherols may be due to their blood or lymphatic cell origin. (c) 2010 Elsevier Inc. All rights reserved.

Adult neural stem cell cycling in vivo requires thyroid hormone and its alpha receptor.

PubMed

Lemkine, G F; Raj, A; Alfama, G; Turque, N; Hassani, Z; Alegria-Prévot, O; Samarut, J; Levi, G; Demeneix, B A

2005-05-01

Thyroid hormones (TH) are essential for brain development. However, information on if and how this key endocrine factor affects adult neurogenesis is fragmentary. We thus investigated the effects of TH on proliferation and apoptosis of stem cells in the subventricular zone (SVZ), as well as on migration of transgene-tagged neuroblasts out of the stem cell niche. Hypothyroidism significantly reduced all three of these processes, inhibiting generation of new cells. To determine the mechanisms relaying TH action in the SVZ, we analyzed which receptor was implicated and whether the effects were played out directly at the level of the stem cell population. The alpha TH receptor (TRalpha), but not TRbeta, was found to be expressed in nestin positive progenitor cells of the SVZ. Further, use of TRalpha mutant mice showed TRalpha to be required to maintain full proliferative activity. Finally, a direct TH transcriptional effect, not mediated through other cell populations, was revealed by targeted gene transfer to stem cells in vivo. Indeed, TH directly modulated transcription from the c-myc promoter reporter construct containing a functional TH response element containing TRE but not from a mutated TRE sequence. We conclude that liganded-TRalpha is critical for neurogenesis in the adult mammalian brain.

Tumor necrosis factor-alpha inhibits stem cell factor-induced proliferation of human bone marrow progenitor cells in vitro. Role of p55 and p75 tumor necrosis factor receptors.

PubMed Central

Rusten, L S; Smeland, E B; Jacobsen, F W; Lien, E; Lesslauer, W; Loetscher, H; Dubois, C M; Jacobsen, S E

1994-01-01

Stem cell factor (SCF), a key regulator of hematopoiesis, potently synergizes with a number of hematopoietic growth factors. However, little is known about growth factors capable of inhibiting the actions of SCF. TNF-alpha has been shown to act as a bidirectional regulator of myeloid cell proliferation and differentiation. This study was designed to examine interactions between TNF-alpha and SCF. Here, we demonstrate that TNF-alpha potently and directly inhibits SCF-stimulated proliferation of CD34+ hematopoietic progenitor cells. Furthermore, TNF-alpha blocked all colony formation stimulated by SCF in combination with granulocyte colony-stimulating factor (CSF) or CSF-1. The synergistic effect of SCF observed in combination with GM-CSF or IL-3 was also inhibited by TNF-alpha, resulting in colony numbers similar to those obtained in the absence of SCF. These effects of TNF-alpha were mediated through the p55 TNF receptor, whereas little or no inhibition was signaled through the p75 TNF receptor. Finally, TNF-alpha downregulated c-kit cell-surface expression on CD34+ bone marrow cells, and this was predominantly a p55 TNF receptor-mediated event as well. Images PMID:7518828

Acetylsalicylic acid regulates MMP-2 activity and inhibits colorectal invasion of murine B16F0 melanoma cells in C57BL/6J mice: effects of prostaglandin F(2)alpha.

PubMed

Tsai, Chin-Shaw Stella; Luo, Shue-Fen; Ning, Chung-Chu; Lin, Chien-Liang; Jiang, Ming-Chung; Liao, Ching-Fong

2009-08-01

Epidemiological studies indicate that acetylsalicylic acid may reduce the risk of mortality due to colon cancers. Metastasis is the major cause of cancer death. Matrix metalloproteinases (MMPs) play important roles in tumor invasion regulation, and prostaglandin F(2)alpha (PGF(2)alpha) is a key stimulator of MMP production. Thus, we investigated whether acetylsalicylic acid regulated MMP activity and the invasion of cancer cells and whether PGF(2)alpha attenuated acetylsalicylic acid-inhibited invasion of cancer cells. Gelatin-based zymography assays showed that acetylsalicylic acid inhibited the MMP-2 activity of B16F0 melanoma cells. Matrigel-based chemoinvasion assays showed that acetylsalicylic acid inhibited the invasion of B16F0 cells. Acetylsalicylic acid can inhibit PGF(2)alpha synthesis and PGF(2)alpha is a key stimulator of MMP-2 production. Our data showed that PGF(2)alpha treatment attenuated the acetylsalicylic acid-inhibited invasion of B16F0 cells. In animal experiments, acetylsalicylic acid reduced colorectal metastasis of B16F0 cells in C57BL/6J mice by 44%. Our results suggest that PGF(2)alpha is a therapeutic target for metastasis inhibition and acetylsalicylic acid may possess anti-metastasis ability.

Apoptosis of murine melanoma B16-BL6 cells induced by quercetin targeting mitochondria, inhibiting expression of PKC-alpha and translocating PKC-delta.

PubMed

Zhang, Xian-Ming; Chen, Jia; Xia, Yu-Gui; Xu, Qiang

2005-03-01

In our previous study, quercetin was found to induce apoptosis of murine melanoma B16-BL6 cells. The cellular and molecular mechanism of quercetin-induced apoptosis was investigated in the present study. Nuclear morphology was determined by fluorescence microscopy. DNA fragmentation was analyzed by electrophoresis and quantified by the diphenylamine method. The transmembrane potential of mitochondria was measured by flow cytometry. Bcl-2, Bcl-X(L), PKC-alpha, PKC-beta, and PKC-delta were detected by Western blotting. Caspase activity was determined spectrophotometrically. Quercetin induced the condensation of nuclei of B16-BL6 cells in a dose-dependent pattern as visualized by Hoechst 33258 and propidium iodide dying. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, significantly enhanced apoptosis induced by quercetin, while doxorubicin, a PKC inhibitor, markedly decreased it. Both PMA and doxorubicin showed a consistent effect on the fragmentation of nuclear DNA caused by various dosages of quercetin. Quercetin dose-dependently led to loss of the mitochondrial membrane potential, which was also significantly reinforced or antagonized by PMA and doxorubicin, respectively. Moreover, PMA showed reinforcement, while doxorubicin showed significant antagonization, of the quercetin-mediated decrease in the expression of Bcl-2. Quercetin promoted caspase-3 activity in a dose-dependent manner, which was also regulated by PMA and doxorubicin with a pattern similar to that seen in their effect on apoptosis, mitochondrial membrane potential and Bcl-2 expression, but none of these were directly affected by PMA and doxorubicin. Free fatty acid and chlorpromazine, a PKC activator and inhibitor, respectively, did not interfere with these effects of quercetin. B16-BL6 cells expressed PKC-alpha, PKC-beta, and PKC-delta. Quercetin dose-dependently inhibited the expression of PKC-alpha but not that of PKC-beta and PKC-delta. Doxorubicin almost completely blocked the effect of quercetin on the expression of PKC-alpha. Quercetin was also involved in the translocation of PKC-delta from the cytosol to the nucleus. PMA enhanced the effect of quercetin on the translocation of PKC-delta. These results indicate that quercetin induced apoptosis of murine melanoma B16-BL6 cells by injuring their mitochondria, increasing the activity of caspase-3, inhibiting the expression of Bcl-2 and PKC-alpha, and inducing the translocation of PKC-delta. Doxorubicin inhibited these effects of quercetin by blocking the decreased expression of PKC-alpha induced by quercetin while PMA increased these effects by enhancing the translocation of PKC-delta induced by quercetin.

Beta3 subunits promote expression and nicotine-induced up-regulation of human nicotinic alpha6* nicotinic acetylcholine receptors expressed in transfected cell lines.

PubMed

Tumkosit, Prem; Kuryatov, Alexander; Luo, Jie; Lindstrom, Jon

2006-10-01

Nicotinic acetylcholine receptors (AChRs) containing alpha6 subunits are typically found at aminergic nerve endings where they play important roles in nicotine addiction and Parkinson's disease. alpha6* AChRs usually contain beta3 subunits. beta3 subunits are presumed to assemble only in the accessory subunit position within AChRs where they do not participate in forming acetylcholine binding sites. Assembly of subunits in the accessory position may be a critical final step in assembly of mature AChRs. Human alpha6 AChRs subtypes were permanently transfected into human tsA201 human embryonic kidney (HEK) cell lines. alpha6beta2beta3 and alpha6beta4beta3 cell lines were found to express much larger amounts of AChRs and were more sensitive to nicotine-induced increase in the amount of AChRs than were alpha6beta2 or alpha6beta4 cell lines. The increased sensitivity to nicotine-induced up-regulation was due not to a beta3-induced increase in affinity for nicotine but probably to a direct effect on assembly of AChR subunits. HEK cells express only a small amount of mature alpha6beta2 AChRs, but many of these subunits are on the cell surface. This contrasts with Xenopus laevis oocytes, which express a large amount of incorrectly assembled alpha6beta2 subunits that bind cholinergic ligands but form large amorphous intracellular aggregates. Monoclonal antibodies (mAbs) were made to the alpha6 and beta3 subunits to aid in the characterization of these AChRs. The alpha6 mAbs bind to epitopes C-terminal of the extracellular domain. These data demonstrate that both cell type and the accessory subunit beta3 can play important roles in alpha6* AChR expression, stability, and up-regulation by nicotine.

Monte Carlo calculations of the cellular S-values for α-particle-emitting radionuclides incorporated into the nuclei of cancer cells of the MDA-MB231, MCF7 and PC3 lines.

PubMed

Rojas-Calderón, E L; Ávila, O; Ferro-Flores, G

2018-05-01

S-values (dose per unit of cumulated activity) for alpha particle-emitting radionuclides and monoenergetic alpha sources placed in the nuclei of three cancer cell models (MCF7, MDA-MB231 breast cancer cells and PC3 prostate cancer cells) were obtained by Monte Carlo simulation. The MCNPX code was used to calculate the fraction of energy deposited in the subcellular compartments due to the alpha sources in order to obtain the S-values. A comparison with internationally accepted S-values reported by the MIRD Cellular Committee for alpha sources in three sizes of spherical cells was also performed leading to an agreement within 4% when an alpha extended source uniformly distributed in the nucleus is simulated. This result allowed to apply the Monte Carlo Methodology to evaluate S-values for alpha particles in cancer cells. The calculation of S-values for nucleus, cytoplasm and membrane of cancer cells considering their particular geometry, distribution of the radionuclide source and chemical composition by means of Monte Carlo simulation provides a good approach for dosimetry assessment of alpha emitters inside cancer cells. Results from this work provide information and tools that may help researchers in the selection of appropriate radiopharmaceuticals in alpha-targeted cancer therapy and improve its dosimetry evaluation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Apolipoprotein CIII-induced THP-1 cell adhesion to endothelial cells involves pertussis toxin-sensitive G protein- and protein kinase C alpha-mediated nuclear factor-kappaB activation.

PubMed

Kawakami, Akio; Aikawa, Masanori; Nitta, Noriko; Yoshida, Masayuki; Libby, Peter; Sacks, Frank M

2007-01-01

Plasma apolipoprotein CIII (apoCIII) independently predicts risk for coronary heart disease (CHD). We recently reported that apoCIII directly enhances adhesion of human monocytes to endothelial cells (ECs), and identified the activation of PKC alpha as a necessary upstream event of enhanced monocyte adhesion. This study tested the hypothesis that apoCIII activates PKC alpha in human monocytic THP-1 cells, leading to NF-kappaB activation. Among inhibitors specific to PKC activators, phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609 limited apoCIII-induced PKC alpha activation and THP-1 cell adhesion. ApoCIII increased PC-PLC activity in THP-1 cells, resulting in PKC alpha activation. Pertussis toxin (PTX) inhibited apoCIII-induced PC-PLC activation and subsequent PKC alpha activation, implicating PTX-sensitive G protein pathway. ApoCIII further activated nuclear factor-kappaB (NF-kappaB) through PKC alpha in THP-1 cells and augmented beta1-integrin expression. The NF-kappaB inhibitor peptide SN50 partially inhibited apoCIII-induced beta1-integrin expression and THP-1 cell adhesion. ApoCIII-rich VLDL had similar effects to apoCIII alone. PTX-sensitive G protein pathway participates critically in PKC alpha stimulation in THP-1 cells exposed to apoCIII, activating NF-kappaB, and increasing beta1-integrin. This action causes monocytic cells to adhere to endothelial cells. Furthermore, because leukocyte NF-kappaB activation contributes to inflammatory aspects of atherogenesis, apoCIII may stimulate diverse inflammatory responses through monocyte activation.

Prostaglandin production by melanocytic cells and the effect of alpha-melanocyte stimulating hormone.

PubMed

Nicolaou, Anna; Estdale, Sian E; Tsatmali, Marina; Herrero, Daniel Pascual; Thody, Anthony J

2004-07-16

Prostaglandins are potent mediators of the inflammatory response and are also involved in cancer development. In this study, we show that human melanocytes and FM55 melanoma cells express cyclooxygenase-1 and -2 (COX-1 and -2) and thus have the capability to produce prostaglandins. The FM55 cells produced predominantly PGE2 and PGF2alpha, whereas the HaCaT keratinocyte cell line produced mainly PGE2. The anti-inflammatory peptide, alpha-melanocyte stimulating hormone (alpha-MSH), reduced prostaglandin production in FM55 and HaCaT cells and reversed the effect of the pro-inflammatory cytokine TNF-alpha in the former. These results indicate that melanocytes produce prostaglandins and that alpha-MSH, by inhibiting this response, may play an important role in regulating inflammatory responses in the skin.

Sickle Cell Trait and Fatal Exertional Heat Illness: Implications for Exercise-Related Death of Young Ddults

DTIC Science & Technology

2008-10-22

athletes who utilize ordinary weather reports? Alpha- Thalassemia Protects Against Exertional Mortality with Sickle Cell Trait • 30% of African...Americans have alpha- thalassemia (2-3 alpha genes instead of 4). In those with sickle cell trait the main effect is to lower the Hb S fraction below 36...expected 15 cases with alpha- thalassemia & ប% S • Two cases had Hb S < 36% , implying about a 7.5-fold protection for those with alpha thalassemia

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Shicheng; Yamauchi, Hitoshi

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 bandmore » 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.« less

Structural elucidation of novel phosphocholine-containing glycosylinositol-phosphoceramides in filamentous fungi and their induction of cell death of cultured rice cells.

PubMed Central

Aoki, Kazuhiro; Uchiyama, Ryosuke; Itonori, Saki; Sugita, Mutsumi; Che, Fang-Sik; Isogai, Akira; Hada, Noriyasu; Hada, Junko; Takeda, Tadahiro; Kumagai, Hidehiko; Yamamoto, Kenji

2004-01-01

Novel ZGLs (zwitterionic glycosphingolipids) have been found in and extracted from the mycelia of filamentous fungi ( Acremonium sp.) isolated from soil. Five ZGLs (ZGL1-ZGL5) were structurally elucidated by sugar compositional analysis, methylation analysis, periodate oxidation, matrix-assisted laser-desorption ionization-time-of-flight MS, (1)H-NMR spectroscopy and fast-atom bombardment MS. Their chemical structures were as follows: GlcN(alpha1-2)Ins1-P-1Cer (ZGL1), Man(alpha1-6)GlcN(alpha1-2)Ins1-P-1Cer (ZGL2), Man(alpha1-6)Man(alpha1-6)GlcN(alpha1-2)Ins1-P-1Cer (ZGL3), PC-->6Man(alpha1-6)GlcN(alpha1-2)Ins1- P -1Cer (ZGL4), and PC-->6Man(alpha1-6)Man(alpha1-6)GlcN(alpha1-2)Ins1-P-1Cer (ZGL5) (where Cer is ceramide and PC is phosphocholine). In addition, one acidic glycosphingolipid, which was the precursor of ZGLs, was also characterized as inositol-phosphoceramide. The core structure of the ZGLs, GlcN(alpha1-2)Ins1- P, is rather different from those found in other fungi, such as Man(alpha1-2)Ins1- P and Man(alpha1-6)Ins1- P. Interestingly, the terminal mannose residue of ZGL4 and ZGL5 was modified further with a PC group. The presence of PC-containing glycosylinositol-phosphoceramides has not been reported previously in any organism. The ceramide constituents of both ZGLs and acidic glycosphingolipid were essentially the same, and consisted of a 4-hydroxyoctadecasphinganine (phytosphingosine) as the sole sphingoid base and 2-hydroxytetracosanoic acid (>90%) as the major fatty acid. ZGLs were found to cause cell death in suspensions of cultured rice cells. The cell death-inducing activity of ZGLs is probably due to the characteristic glycan moiety of Man(alpha1-6)GlcN, and PC-containing ZGLs had high activity. This study is the first to demonstrate that fungal glycosylinositol-phosphoceramides induce cell death in cultured rice cells. PMID:14583095

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

PubMed

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

2003-12-01

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

Myofibroblast distribution in Dupuytren's cords: correlation with digital contracture.

PubMed

Verjee, Liaquat Suleman; Midwood, Kim; Davidson, Dominique; Essex, David; Sandison, Ann; Nanchahal, Jagdeep

2009-12-01

Dupuytren's tissue has typically been described as being composed of myofibroblast-rich palmar nodules and relatively acellular tendon-like cords. We aimed to determine myofibroblast distribution (alpha-smooth muscle actin [alpha-SMA] positive cells) within Dupuytren's tissue and to correlate histologically defined alpha-SMA-positive nodules with digital contracture and recurrent disease. One hundred and three digital Dupuytren's cords (72 fasciectomy, 31 dermofasciectomy) were stained with anti-alpha-SMA antibody. The presence of alpha-SMA-positive nodules, their surface area, and alpha-SMA-positive cells were quantified throughout excised Dupuytren's tissue. Clinical data on diathesis, flexion deformity, and previous surgeries were collected. Cords were nodular (66%) or non-nodular (34%). Nodular cords contained 1 (55%), 2 (33%), or 3 or more nodules (12%) composed of localized collections of cells. The mean total nodule surface area was 23 mm(2) (range, 1.3-105 mm(2)). Nodules contained the highest number of alpha-SMA-positive cells (mean 97%, 2374 cells/mm(2)) compared to peri-nodular areas (mean 32%, 763 cells/mm(2)), and more distant cord (mean 8%, 495 cells/mm(2)). Non-nodular cords contained 9% to 17% alpha-SMA-positive cells (mean 475-663 cells/mm(2)), with higher numbers distally. There was greater digital contracture in patients with non-nodular cords. Thirty-six of 38 proximal interphalangeal (PIP) joint-marked samples had a nodule that co-localized with the PIP joint. Nodule size did not correlate with flexion deformity or with primary or recurrent disease. We found that two thirds of digital cords were nodular. Nodules were hypercellular, the majority being alpha-SMA-positive cells. Nodules varied in size and co-localized with the PIP joint. Cord was relatively cellular throughout; a proportion of these cells were alpha-SMA-positive and cells aligned with collagen fibers. Non-nodular cords correlated with significantly greater digital flexion contracture. We propose that cells in nodular cords contract and deposit extracellular matrix components. The matrix is then remodeled in shortened configuration, and as fixed flexion deformity develops, stress shielding eventually leads to myofibroblast apoptosis, and cord becomes less cellular.

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

USDA-ARS?s Scientific Manuscript database

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...

Divergent effects of 17-{beta}-estradiol on human vascular smooth muscle and endothelial cell function diminishes TNF-{alpha}-induced neointima formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nintasen, Rungrat; Multidisciplinary Cardiovascular Research Center; Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University

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 protectivemore » 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 was no modulation by E2 in either cell-type. In conclusion, TNF-{alpha} induced SV neointima formation, increased SMC proliferation and migration, impaired SV-EC migration and increased expression of adhesion molecules. E2 exerted distinct cell-type and function-specific modulation, the mechanisms underlying which are worthy of further detailed study.« less

Tocopherol production in plant cell cultures.

PubMed

Caretto, Sofia; Nisi, Rossella; Paradiso, Annalisa; De Gara, Laura

2010-05-01

Tocopherols, collectively known as vitamin E, are lipophilic antioxidants, essential dietary components for mammals and exclusively synthesized by photosynthetic organisms. Of the four forms (alpha, beta, gamma and delta), alpha-tocopherol is the major vitamin E form present in green plant tissues, and has the highest vitamin E activity. Synthetic alpha-tocopherol, being a racemic mixture of eight different stereoisomers, always results less effective than the natural form (R,R,R) alpha-tocopherol. This raises interest in obtaining this molecule from natural sources, such as plant cell cultures. Plant cell and tissue cultures are able to produce and accumulate valuable metabolites that can be used as food additives, nutraceuticals and pharmaceuticals. Sunflower cell cultures, growing under heterotrophic conditions, were exploited to establish a suitable in vitro production system of natural alpha-tocopherol. Optimization of culture conditions, precursor feeding and elicitor application were used to improve the tocopherol yields of these cultures. Furthermore, these cell cultures were useful to investigate the relationship between alpha-tocopherol biosynthesis and photomixotrophic culture conditions, revealing the possibility to enhance tocopherol production by favouring sunflower cell photosynthetic properties. The modulation of alpha-tocopherol levels in plant cell cultures can provide useful hints for a regulatory impact on tocopherol metabolism.

Development of a transmission alpha particle dosimetry technique using A549 cells and a Ra-223 source for targeted alpha therapy.

PubMed

Al Darwish, R; Staudacher, A H; Li, Y; Brown, M P; Bezak, E

2016-11-01

In targeted radionuclide therapy, regional tumors are targeted with radionuclides delivering therapeutic radiation doses. Targeted alpha therapy (TAT) is of particular interest due to its ability to deliver alpha particles of high linear energy transfer within the confines of the tumor. However, there is a lack of data related to alpha particle distribution in TAT. These data are required to more accurately estimate the absorbed dose on a cellular level. As a result, there is a need for a dosimeter that can estimate, or better yet determine the absorbed dose deposited by alpha particles in cells. In this study, as an initial step, the authors present a transmission dosimetry design for alpha particles using A549 lung carcinoma cells, an external alpha particle emitting source (radium 223; Ra-223) and a Timepix pixelated semiconductor detector. The dose delivery to the A549 lung carcinoma cell line from a Ra-223 source, considered to be an attractive radionuclide for alpha therapy, was investigated in the current work. A549 cells were either unirradiated (control) or irradiated for 12, 1, 2, or 3 h with alpha particles emitted from a Ra-223 source positioned below a monolayer of A549 cells. The Timepix detector was used to determine the number of transmitted alpha particles passing through the A549 cells and DNA double strand breaks (DSBs) in the form of γ-H2AX foci were examined by fluorescence microscopy. The number of transmitted alpha particles was correlated with the observed DNA DSBs and the delivered radiation dose was estimated. Additionally, the dose deposited was calculated using Monte Carlo code SRIM. Approximately 20% of alpha particles were transmitted and detected by Timepix. The frequency and number of γ-H2AX foci increased significantly following alpha particle irradiation as compared to unirradiated controls. The equivalent dose delivered to A549 cells was estimated to be approximately 0.66, 1.32, 2.53, and 3.96 Gy after 12, 1, 2, and 3 h irradiation, respectively, considering a relative biological effectiveness of alpha particles of 5.5. The study confirmed that the Timepix detector can be used for transmission alpha particle dosimetry. If cross-calibrated using biological dosimetry, this method will give a good indication of the biological effects of alpha particles without the need for repeated biological dosimetry which is costly, time consuming, and not readily available.

Relative biological effectiveness (RBE) of alpha radiation in cultured porcine aortic endothelial cells.

PubMed

Thomas, Patricia; Tracy, Bliss; Ping, Tilly; Baweja, Anar; Wickstrom, Mark; Sidhu, Narinder; Hiebert, Linda

2007-03-01

Northern peoples can receive elevated radiation doses (1- 10 mSv/y) from transfer of polonium-210 (210Po) through the lichen-caribou-human food chain. Ingested 210Po is primarily blood-borne and thus many of its short range alpha particles irradiate the endothelial cells lining the blood vessels. The relative biological effectiveness (RBE) of alpha particles vs. x-rays was examined in porcine aortic endothelial cells as a surrogate for understanding what might happen to human endothelial cells in northern populations consuming traditional foods. Cultured porcine aortic endothelial cells were exposed to x-ray and 210Po alpha particle radiation. Alpha irradiation was applied to the cell cultures internally via the culture medium and externally, using thin-bottomed culture dishes. The results given here are based on the external irradiation method, which was found to be more reliable. Dose-response curves were compared for four lethal endpoints (cell viability, live cell fraction, release of lactate dehydrogenase [LDH] and clonogenic survival) to determine the relative biological effectiveness (RBE) of alpha radiation. The alpha RBE for porcine cells varied from 1.6-21, depending on the endpoint: 21.2+/-4.5 for cell viability, 12.9+/-2.7 for decrease in live cell number, 5.3+/-0.4 for LDH release to the medium but only 1.6 +/-0.1 for clonogenic survival. The low RBE of 1.6 was due to x-ray hypersensitivity of endothelial cells at low doses.

T1alpha/podoplanin shows raft-associated distribution in mouse lung alveolar epithelial E10 cells.

PubMed

Barth, Kathrin; Bläsche, Robert; Kasper, Michael

2010-01-01

T1alpha/(podoplanin) is abundantly expressed in the alveolar epithelial type I cells (ATI) of rodent and human lungs. Caveolin-1 is a classical primary structural protein of plasmalemal invaginations, so-called caveolae, which represent specialized lipid rafts, and which are particularly abundant in ATI cells. The biological functions of T1alpha in the alveolar epithelium are unknown. Here we report on the characteristics of raft domains in the microplicae/microvillar protrusions of ATI cells, which contain T1alpha. Detergent resistant membranes (DRMs) from cell lysates of the mouse epithelial ATI-like cell line E10 were prepared using different detergents followed by flotation in a sucrose gradient and tested by Western and dot blots with raft markers (caveolin-1, GM1) and nonraft markers (transferrin receptor, PDI and beta-Cop). Immunocytochemistry was employed for the localization of T1alpha in E10 cells and in situ in rat lungs. Our biochemical results showed that the solubility or insolubility of T1alpha and caveolin-1 differs in Triton X-100 and Lubrol WX, two distinct non-ionic detergents. Caveolin-1 was unsoluble in both detergents, whereas T1alpha was Triton X-100 soluble but Lubrol WX insoluble. Immunofluorescence double stainings revealed that both proteins were colocalized with GM1, while caveolin-1 and T1alpha were not colocalized in the plasma membrane. Cholesterol depletion modified the segregation of T1alpha in Lubrol WX DRMs. Cellular processes in ultrathin sections of cultured mouse E10 cells were immunogold positive. Immunoelectron microscopy (postembedding) of rat lung tissue revealed the preferential localization of T1alpha on apical microvillar protrusions of ATI cells. We conclude that T1alpha and caveolin-1 are located in distinct plasma membrane microdomains, which differ in their protein-lipid interactions. The raft-associated distribution of T1alpha may have an impact on a specific, not yet clarified function of this protein in the alveolar epithelium. 2010 S. Karger AG, Basel

A Novel Di-Leucine Motif at the N-Terminus of Human Organic Solute Transporter Beta Is Essential for Protein Association and Membrane Localization.

PubMed

Xu, Shuhua; Soroka, Carol J; Sun, An-Qiang; Backos, Donald S; Mennone, Albert; Suchy, Frederick J; Boyer, James L

2016-01-01

The heteromeric membrane protein Organic Solute Transporter alpha/beta is the major bile acid efflux transporter in the intestine. Physical association of its alpha and beta subunits is essential for their polarized basolateral membrane localization and function in the transport of bile acids and other organic solutes. We identified a highly conserved acidic dileucine motif (-EL20L21EE) at the extracellular amino-tail of organic solute transporter beta from multiple species. To characterize the role of this protein interacting domain in the association of the human beta and alpha subunits and in membrane localization of the transporter, Leu20 and Leu21 on the amino-tail of human organic solute transporter beta were replaced with alanines by site-directed mutagenesis. Co-immunoprecipitation study in HEK293 cells demonstrated that substitution of the leucine residues with alanines prevented the interaction of the human beta mutant with the alpha subunit. Membrane biotinylation demonstrated that the LL/AA mutant eliminated membrane expression of both subunits. Computational-based modelling of human organic solute transporter beta suggested that the LL/AA mutation substantially alters both the structure and lipophilicity of the surface, thereby not only affecting the interaction with the alpha subunit but also possibly impacting the capacity of the beta subunit to traffick through the cell and interact with the membrane. In summary, our findings indicate that the dileucine motif in the extracellular N-terminal region of human organic solute transporter beta subunit plays a critical role in the association with the alpha subunit and in its polarized plasma membrane localization.

Acquisition of T regulatory function in cathepsin L-inhibited T cells by eye-derived CTLA-2alpha during inflammatory conditions.

PubMed

Sugita, Sunao; Horie, Shintaro; Nakamura, Orie; Maruyama, Kazuichi; Takase, Hiroshi; Usui, Yoshihiko; Takeuchi, Masaru; Ishidoh, Kazumi; Koike, Masato; Uchiyama, Yasuo; Peters, Christoph; Yamamoto, Yoshimi; Mochizuki, Manabu

2009-10-15

Pigment epithelium isolated from the eye possesses immunosuppressive properties such as regulatory T (Treg) cell induction; e.g., cultured retinal pigment epithelium (RPE) converts CD4(+) T cells into Treg cells in vitro. RPE constitutively expresses a novel immunosuppressive factor, CTLA-2alpha, which is a cathepsin L (CathL) inhibitor, and this molecule acts via RPE to induce Treg cells. To clarify CTLA-2alpha's role in the T cell response to RPE in ocular inflammation, we used the experimental autoimmune uveitis (EAU) animal model to examine this new immunosuppressive property of RPE. In EAU models, TGF-beta, but not IFN-gamma inflammatory cytokines, promotes the up-regulation of the expression of CTLA-2alpha in RPE. Similarly, CTLA-2alpha via RPE was able to promote TGF-beta production by the CD4(+) T cells. The RPE-exposed T cells (RPE-induced Treg cells) greatly produced TGF-beta and suppressed bystander effector T cells. There was less expression of CathL by the RPE-exposed T cells, and CathL-inhibited T cells were able to acquire the Treg phenotype. Moreover, CathL-deficient mice spontaneously produced Treg cells, with the increase in T cells potentially providing protection against ocular inflammation. More importantly, CD4(+) T cells from EAU in CathL knockout mice or rCTLA-2alpha from EAU animals were found to contain a high population of forkhead box p3(+) T cells. In both EAU models, there was significant suppression of the ocular inflammation. These results indicate that RPE secretes CTLA-2alpha, thereby enabling the bystander T cells to be converted into Treg cells via TGF-beta promotion.

Pur-alpha regulates cytoplasmic stress granule dynamics and ameliorates FUS toxicity

PubMed Central

Daigle, J Gavin; Krishnamurthy, Karthik; Ramesh, Nandini; Casci, Ian; Monaghan, John; McAvoy, Kevin; Godfrey, Earl W; Daniel, Dianne C.; Johnson, Edward M.; Monahan, Zach; Shewmaker, Frank; Pasinelli, Piera; Pandey, Udai Bhan

2016-01-01

Amyotrophic lateral Sclerosis is characterized by progressive loss of motor neurons in the brain and spinal cord. Mutations in several genes, including FUS, TDP43, Matrin 3, hnRNPA2 and other RNA binding proteins, have been linked to ALS pathology. Recently, Pur-alpha a DNA/RNA binding protein was found to bind to C9orf72 repeat expansions and could possibly play a role in the pathogenesis of ALS. When overexpressed, Pur-alpha mitigates toxicities associated with Fragile X tumor ataxia syndrome (FXTAS) and C9orf72 repeat expansion diseases in Drosophila and mammalian cell culture models. However, the function of Pur-alpha in regulating ALS pathogenesis has not been fully understood. We identified Pur-alpha as a novel component of cytoplasmic stress granules (SGs) in ALS patient cells carrying disease-causing mutations in FUS. When cells were challenged with stress, we observed that Pur-alpha co-localized with mutant FUS in ALS patient cells and became trapped in constitutive SGs. We also found that FUS physically interacted with Pur-alpha in mammalian neuronal cells. Interestingly, shRNA mediated knock down of endogenous Pur-alpha significantly reduced formation of cytoplasmic stress granules in mammalian cells suggesting that Pur-alpha is essential for the formation of SGs. Furthermore, ectopic expression of Pur-alpha blocked cytoplasmic mislocalization of mutant FUS and strongly suppressed toxicity associated with mutant FUS expression in primary motor neurons. Our data emphasizes the importance of stress granules in ALS pathogenesis and identifies Pur-alpha as a novel regulator of SG dynamics. PMID:26728149

Ulex europaeus I lectin as a marker for vascular endothelium in human tissues.

PubMed

Holthöfer, H; Virtanen, I; Kariniemi, A L; Hormia, M; Linder, E; Miettinen, A

1982-07-01

Ulex europaeus I agglutinin, a lectin specific for some alpha-L-fucose-containing glycocompounds, was used in fluorescence microscopy to stain cryostat sections of human tissues. The endothelium of vessels of all sizes was stained ubiquitously in all tissues studied as judged by double staining with a known endothelial marker, antibodies against human clotting factor VIII. Cultured human umbilical vein endothelial cells, but not fibroblasts, also bound Ulex lectin. The staining was not affected by the blood group type of the tissue donor. In some tissues Ulex lectin presented additional binding to epithelial structures. Also, this was independent on the blood group or the ability of the tissue donor to secrete soluble blood group substances. Lotus tetragonolobus agglutinin, another lectin specific for some alpha-L-fucose-containing moieties failed to react with endothelial cells. Our results suggest that Ulex europaeus I agglutinin is a good histologic marker for endothelium in human tissues.

Effect of thalidomide on the expression of TNF-alpha m-RNA and synthesis of TNF-alpha in cells from leprosy patients with reversal reaction.

PubMed

Tadesse, Azeb; Abebe, Markos; Bizuneh, Elizabeth; Mulugeta, Wondwossen; Aseffa, Abraham; Shannon, E J

2006-01-01

Hypersensitivity reactions called reversal reaction (RR) and erythema nodosum leprosum (ENL) occur in leprosy. They are characterized by an increase in tumor necrosis factor-alpha (TNF-alpha). Thalidomide is an effective treatment for ENL but not RR. Its effectiveness in ENL is attributed to inhibition of TNF-alpha, and this does not explain its failure to treat RR. We assessed thalidomide's effect on TNF-alpha in RR. Mononuclear cells from RR and non-RR patients and healthy individuals were treated with thalidomide and M.leprae (AFB), a cytosol fraction of M. leprae or Dharmendra lepromin. Thalidomide suppressed TNF-alpha, but when some RR patients' cells were stimulated with AFB, it enhanced TNF-alpha.

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.

Human common acute lymphoblastic leukemia-derived cell lines are competent to recombine their T-cell receptor delta/alpha regions along a hierarchically ordered pathway.

PubMed

Hansen-Hagge, T E; Yokota, S; Reuter, H J; Schwarz, K; Bartram, C R

1992-11-01

Rearrangements of the T-cell receptor (TCR) delta locus are observed in the majority of human B-cell precursor acute lymphoblastic leukemias (ALL) with a striking predominance of V delta 2(D)D delta 3 recombinations in common ALL (cALL) patients. Recently, we and others showed that almost 20% of cALL cases are characterized by further recombination of V delta 2(D)D delta 3 segments to J alpha elements, thereby deleting the TCR delta locus in analogy to the delta Rec/psi J alpha pathway in differentiating alpha/beta-positive T cells. We report here that two human cALL-derived cell lines, REH and Nalm-6, are competent to recombine the TCR delta/alpha locus under standard tissue culture conditions. Analysis of different REH subclones obtained by limiting dilution of the initial culture showed a biased recombination of V delta 2D delta 3 to distinct J alpha elements. During prolonged tissue culture, a subclone acquired growth advantage and displaced parental cells as well as other subclones. Frequently, the DJ junctions of REH subclones contained extended stretches of palindromic sequences derived from modified D delta 3 coding elements. The other cell line, Nalm-6, started the TCR delta/alpha recombination with an unusual signal joint of a cryptic recombinase signal sequence (RSS) upstream of D delta 3 to the 3' RSS of D delta 3. The RSS dimer was subsequently rearranged in all investigated subclones to an identical J alpha element. Both cell lines might become valuable tools to unravel the complex regulation of TCR delta/alpha recombination pathways in malignant and normal lymphopoiesis.

Hepatocyte nuclear factor-4alpha induces transdifferentiation of hematopoietic cells into hepatocytes.

PubMed

Khurana, Satish; Jaiswal, Amit K; Mukhopadhyay, Asok

2010-02-12

Hematopoietic stem cells can directly transdifferentiate into hepatocytes because of cellular plasticity, but the molecular basis of transdifferentiation is not known. Here, we show the molecular basis using lineage-depleted oncostatin M receptor beta-expressing (Lin(-)OSMRbeta(+)) mouse bone marrow cells in a hepatic differentiation culture system. Differentiation of the cells was marked by the expression of albumin. Hepatocyte nuclear factor (HNF)-4alpha was expressed and translocated into the nuclei of the differentiating cells. Suppression of its activation in OSM-neutralized culture medium inhibited cellular differentiation. Ectopic expression of full-length HNF4alpha in 32D myeloid cells resulted in decreased myeloid colony-forming potential and increased expression of hepatocyte-specific genes and proteins. Nevertheless, the neohepatocytes produced in culture expressed active P450 enzyme. The obligatory role of HNF4alpha in hepatic differentiation was confirmed by transfecting Lin(-)OSMRbeta(+) cells with dominant negative HNF4alpha in the differentiation culture because its expression inhibited the transcription of the albumin and tyrosine aminotransferase genes. The loss and gain of functional activities strongly suggested that HNF4alpha plays a central role in the transdifferentiation process. For the first time, this report demonstrates the mechanism of transdifferentiation of hematopoietic cells into hepatocytes, in which HNF4alpha serves as a molecular switch.

Immunosuppressant effect of IDS 30, a stinging nettle leaf extract, on myeloid dendritic cells in vitro.

PubMed

Broer, Johanna; Behnke, Bert

2002-04-01

Dendritic cells are important antigen presenting cells that play a role in the initiation of rheumatoid arthritis (RA). The stinging nettle leaf extract IDS 30 (Hox alpha) has been recommended for adjuvant therapy of rheumatic diseases. We investigated the immunomodulating effect of IDS 30 extract on the maturation of hematopoietic dendritic cells. Human dendritic cells were generated from peripheral blood mononuclear cells cultured in granulocyte macrophage-colony stimulating factor and interleukin 4 (IL-4). Dendritic cell maturation was induced by keyhole limped hemocyanin (KLH). Dendritic cell phenotype was characterized by flow cytometric analysis; dendritic cell cytokine production was measured by ELISA. The ability of dendritic cells to activate naive autologous T cells was evaluated by mixed leukocyte reaction. IDS 30 prevented the maturation of dendritic cells, but did not affect their viability. IDS 30 reduced the expression of CD83 and CD86. It increased the expression of chemokine receptor 5 and CD36 in a dose dependent manner. The secretion of tumor necrosis factor-alpha was reduced. Application of IDS 30 to dendritic cells in culture caused a high endocytosis of dextran and a low capacity to stimulate T cell proliferation. Our in vitro results showed the suppressive effect of IDS 30 on the maturation of human myeloid dendritic cells, leading to reduced induction of primary T cell responses. This may contribute to the therapeutic effect of IDS 30 on T cell mediated inflammatory diseases like RA.

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

PubMed

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

2017-04-01

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

TNF{alpha} acting on TNFR1 promotes breast cancer growth via p42/P44 MAPK, JNK, Akt and NF-{kappa}B-dependent pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rivas, Martin A.; Carnevale, Romina P.; Proietti, Cecilia J.

2008-02-01

Tumor necrosis factor {alpha} (TNF{alpha}) enhances proliferation of chemically-induced mammary tumors and of T47D human cell line through not fully understood pathways. Here, we explored the intracellular signaling pathways triggered by TNF{alpha}, the participation of TNF{alpha} receptor (TNFR) 1 and TNFR2 and the molecular mechanism leading to breast cancer growth. We demonstrate that TNF{alpha} induced proliferation of C4HD murine mammary tumor cells and of T47D cells through the activation of p42/p44 MAPK, JNK, PI3-K/Akt pathways and nuclear factor-kappaB (NF-{kappa}B) transcriptional activation. A TNF{alpha}-specific mutein selectively binding to TNFR1 induced p42/p44 MAPK, JNK, Akt activation, NF-{kappa}B transcriptional activation and cell proliferation,more » just like wild-type TNF{alpha}, while a mutein selective for TNFR2 induced only p42/p44 MAPK activation. Interestingly, blockage of TNFR1 or TNFR2 with specific antibodies was enough to impair TNF{alpha} signaling and biological effect. Moreover, in vivo TNF{alpha} administration supported C4HD tumor growth. We also demonstrated, for the first time, that injection of a selective inhibitor of NF-{kappa}B activity, Bay 11-7082, resulted in regression of TNF{alpha}-promoted tumor. Bay 11-7082 blocked TNF{alpha} capacity to induce cell proliferation and up-regulation of cyclin D1 and of Bcl-x{sub L}in vivo and in vitro. Our results reveal evidence for TNF{alpha} as a breast tumor promoter, and provide novel data for a future therapeutic approach using TNF{alpha} antagonists and NF-{kappa}B pharmacological inhibitors in established breast cancer treatment.« less

Heterodimerization with beta2-adrenergic receptors promotes surface expression and functional activity of alpha1D-adrenergic receptors.

PubMed

Uberti, Michelle A; Hague, Chris; Oller, Heide; Minneman, Kenneth P; Hall, Randy A

2005-04-01

The alpha1D-adrenergic receptor (alpha1D-AR) is a G protein-coupled receptor (GPCR) that is poorly trafficked to the cell surface and largely nonfunctional when heterologously expressed by itself in a variety of cell types. We screened a library of approximately 30 other group I GPCRs in a quantitative luminometer assay for the ability to promote alpha1D-AR cell surface expression. Strikingly, these screens revealed only two receptors capable of inducing robust increases in the amount of alpha1D-AR at the cell surface: alpha1B-AR and beta2-AR. Confocal imaging confirmed that coexpression with beta2-AR resulted in translocation of alpha1D-AR from intracellular sites to the plasma membrane. Additionally, coimmunoprecipitation studies demonstrated that alpha1D-AR and beta2-AR specifically interact to form heterodimers when coexpressed in HEK-293 cells. Ligand binding studies revealed an increase in total alpha1D-AR binding sites upon coexpression with beta2-AR, but no apparent effect on the pharmacological properties of the receptors. In functional studies, coexpression with beta2-AR significantly enhanced the coupling of alpha1D-AR to norepinephrine-stimulated Ca2+ mobilization. Heterodimerization of beta2-AR with alpha1D-AR also conferred the ability of alpha1D-AR to cointernalize upon beta2-AR agonist stimulation, revealing a novel mechanism by which these different adrenergic receptor subtypes may regulate each other's activity. These findings demonstrate that the selective association of alpha1D-AR with other receptors is crucial for receptor surface expression and function and also shed light on a novel mechanism of cross talk between alpha1- and beta2-ARs that is mediated through heterodimerization and cross-internalization.

The mammalian iris-ciliary complex affects organization and synthesis of cytoskeletal proteins of organ and tissue cultured lens epithelial cells.

PubMed

Banerjee, A; Emanuel, K; Parafina, J; Bagchi, M

1992-10-01

A water soluble growth inhibitor was isolated from the mammalian ocular iris-ciliary complex. The molecular weight of this protein is 10 kD or lower as determined by ultrafiltration fractionation. The iris-ciliary (IC) complex water soluble protein(s) significantly inhibits synthesis of lower molecular weight proteins of the epithelial cells of the organ cultured mammalian ocular lens. It was also found that this inhibitory effect of IC is mediated via the structural organization of the lens. Monolayer cultures of the lens epithelial cells exposed to IC did not manifest any inhibition of their protein synthesis. Moreover, these tissue cultured lens epithelial (TCLE) cells showed a significant increase in their protein synthetic activities in response to the presence of IC factors in the culture medium. It is postulated that the IC activity is modulated via either the lens capsule, an extracellular matrix, or due to the specific organization of the intact lens. The specific effects of IC on the cytoskeletal organization and synthesis in the organ cultured lens epithelial (OCLE) and TCLE cells were also examined. Both groups, treated with IC factors, manifested significant alterations in their protein synthetic activities and cytoskeletal architecture. The 3H-leucine incorporation experiments showed that alpha-actin and alpha-tubulin synthesis is partially inhibited by IC factors in OCLE cells but vimentin synthesis is not, whereas in TCLE cells all of them showed increased synthesis in response to IC factors. Turnover rates of these proteins in both OCLE and TCLE cells were also computed. The immunofluorescence and microscopic evaluation of OCLE and TCLE cells exposed to IC factors illustrated significant alteration in the cytoarchitecture of the filaments. We demonstrate that an inhibitor(s) molecule of 10 kD or lower size isolated from IC inhibited protein synthesis of OCLE cells and stimulated protein synthesis in TCLE cells. The IC factor also affects the synthesis and organization of cytoskeletal filaments of both the OCLE and TCLE cells.

Silencing alpha-fetoprotein inhibits VEGF and MMP-2/9 production in human hepatocellular carcinoma cell.

PubMed

Meng, Wenbo; Li, Xun; Bai, Zhongtian; Li, Yan; Yuan, Jinqiu; Liu, Tao; Yan, Jun; Zhou, Wence; Zhu, Kexiang; Zhang, Hui; Li, Yumin

2014-01-01

Alpha-fetoprotein not only serves as a diagnostic marker for liver cancer, but also posses a variety of biological functions. However, the role of Alpha-fetoprotein on tumor angiogenesis and cell invasion remains incompletely understood. In this study, we aimed to evaluate if Alpha-fetoprotein can regulate the major angiogenic factors and matrix metalloproteinases in human liver cancer cells. Alpha-fetoprotein silencing was achieved by Stealth RNAi. Expression of Alpha-fetoprotein was examined by a full-automatic electrochemistry luminescence immunity analyzer. Expression of VEGF, VEGFR-2, MMP-9, and MMP-2 was examined by Western blot and immunocytochemistry. Apoptosis was detected by TUNEL assay. Angiogenesis was detected by in vitro angiogenesis assay kit. Silencing of Alpha-fetoprotein led to an increased apoptosis, which was associated with a decreased expression of vascular endothelial growth factor, vascular endothelial growth factor receptor 2, matrix metalloproteinases-2/9. These results suggest that Alpha-fetoprotein may play a regulatory role on angiogenesis and cell invasion during liver cancer development.

Mechanisms and regulation of polymorphonuclear leukocyte and eosinophil adherence to human airway epithelial cells.

PubMed

Jagels, M A; Daffern, P J; Zuraw, B L; Hugli, T E

1999-09-01

Polymorphonuclear leukocytes (PMN) and eosinophils (Eos) are important cellular participants in a variety of acute and chronic inflammatory reactions in the airway. Histologic evidence has implicated direct interactions between these two subsets of leukocytes and airway epithelial cells during inflammation. A comprehensive characterization and comparison of physiologic stimuli and adhesion molecule involvement in granulocyte-epithelial-cell interactions done with nontransformed human airway epithelial cells has not been reported. We therefore examined the regulation and biochemical mechanisms governing granulocyte-epithelial-cell adhesion, using either purified PMN or Eos and primary cultures of human bronchial epithelial cells (HBECs). We investigated the involvement of a number of proinflammatory signals associated with allergic and nonallergic airway inflammation, as well as the contribution of several epithelial and leukocyte adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and members of the beta(1), beta(2), and beta(7) integrin families. ICAM-1 was expressed at low levels on cultured HBECs and was markedly upregulated after stimulation with interferon (IFN)-gamma or, to a lesser extent, with tumor necrosis factor (TNF)-alpha or interleukin (IL)-1. VCAM-1 was not present on resting HBECs, and was not upregulated after stimulation with IFN-gamma, IL-1, IL-4, or TNF-alpha. PMN adhesion to HBECs could be induced either through activation of PMN with IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), or C5a, but not with IL-5 or by preactivation of HBECs with TNF-alpha or IFN-gamma. Blocking antibody studies indicated that PMN-HBEC adherence depended on beta(2) integrins, primarily alpha(M)beta(2) (Mac-1). Adherence of Eos to HBECs could be induced through activation of Eos with IL-5, GM-CSF, or C5a, but not with IL-8 or by prior activation of HBECs with TNF-alpha of IFN-gamma. Maximal adhesion of Eos and PMN required pretreatment of HBECs with either TNF-alpha or IFN-gamma in addition to leukocyte activation. Adherence of Eos to unstimulated HBECs was mediated through both beta(1) and beta(2) integrins, whereas adhesion of Eos to activated HBECs was dominated by beta(2) integrins. Adhesion of both Eos and PMN was inhibited by treatment of HBECs with blocking antibodies to ICAM-1. Differential utilization of beta(1) and beta(2) integrins by Eos, depending on the activation state of the epithelium, is a novel finding and may affect activation and/or recruitment of Eos in airway tissue. Mechanisms of adhesion of HBECs to Eos and PMN, as evidenced by the different responsiveness of the two latter types of cells to IL-8 and IL-5, may account for a prevalence of Eos over PMN in certain airway diseases.

A single mutation results in diploid gamete formation and parthenogenesis in a Drosophila yemanuclein-alpha meiosis I defective mutant

PubMed Central

2010-01-01

Background Sexual reproduction relies on two key events: formation of cells with a haploid genome (the gametes) and restoration of diploidy after fertilization. Therefore the underlying mechanisms must have been evolutionary linked and there is a need for evidence that could support such a model. Results We describe the identification and the characterization of yem1, the first yem-alpha mutant allele (V478E), which to some extent affects diploidy reduction and its restoration. Yem-alpha is a member of the Ubinuclein/HPC2 family of proteins that have recently been implicated in playing roles in chromatin remodeling in concert with HIRA histone chaperone. The yem1 mutant females exhibited disrupted chromosome behavior in the first meiotic division and produced very low numbers of viable progeny. Unexpectedly these progeny did not display paternal chromosome markers, suggesting that they developed from diploid gametes that underwent gynogenesis, a form of parthenogenesis that requires fertilization. Conclusions We focus here on the analysis of the meiotic defects exhibited by yem1 oocytes that could account for the formation of diploid gametes. Our results suggest that yem1 affects chromosome segregation presumably by affecting kinetochores function in the first meiotic division. This work paves the way to further investigations on the evolution of the mechanisms that support sexual reproduction. PMID:21080953

C/EBP beta regulation of the tumor necrosis factor alpha gene.

PubMed Central

Pope, R M; Leutz, A; Ness, S A

1994-01-01

Activated macrophages contribute to chronic inflammation by the secretion of cytokines and proteinases. Tumor necrosis factor alpha (TNF alpha) is particularly important in this process because of its ability to regulate other inflammatory mediators in an autocrine and paracrine fashion. The mechanism(s) responsible for the cell type-specific regulation of TNF alpha is not known. We present data to show that the expression of TNF alpha is regulated by the transcription factor C/EBP beta (NF-IL6). C/EBP beta activated the TNF alpha gene promoter in cotransfection assays and bound to it at a site which failed to bind the closely related protein C/EBP alpha. Finally, a dominant-negative version of C/EBP beta blocked TNF alpha promoter activation in myeloid cells. Our results implicate C/EBP beta as an important regulator of TNF alpha by myelomonocytic cells. Images PMID:7929820

Zinc in human health: effect of zinc on immune cells.

PubMed

Prasad, Ananda S

2008-01-01

Although the essentiality of zinc for plants and animals has been known for many decades, the essentiality of zinc for humans was recognized only 40 years ago in the Middle East. The zinc-deficient patients had severe immune dysfunctions, inasmuch as they died of intercurrent infections by the time they were 25 years of age. In our studies in an experimental human model of zinc deficiency, we documented decreased serum testosterone level, oligospermia, severe immune dysfunctions mainly affecting T helper cells, hyperammonemia, neurosensory disorders, and decreased lean body mass. It appears that zinc deficiency is prevalent in the developing world and as many as two billion subjects may be growth retarded due to zinc deficiency. Besides growth retardation and immune dysfunctions, cognitive impairment due to zinc deficiency also has been reported recently. Our studies in the cell culture models showed that the activation of many zinc-dependent enzymes and transcription factors were adversely affected due to zinc deficiency. In HUT-78 (T helper 0 [Th(0)] cell line), we showed that a decrease in gene expression of interleukin-2 (IL-2) and IL-2 receptor alpha(IL-2Ralpha) were due to decreased activation of nuclear factor-kappaB (NF-kappaB) in zinc deficient cells. Decreased NF-kappaB activation in HUT-78 due to zinc deficiency was due to decreased binding of NF-kappaB to DNA, decreased level of NF-kappaB p105 (the precursor of NF-kappaB p50) mRNA, decreased kappaB inhibitory protein (IkappaB) phosphorylation, and decreased Ikappa kappa. These effects of zinc were cell specific. Zinc also is an antioxidant and has anti-inflammatory actions. The therapeutic roles of zinc in acute infantile diarrhea, acrodermatitis enteropathica, prevention of blindness in patients with age-related macular degeneration, and treatment of common cold with zinc have been reported. In HL-60 cells (promyelocytic leukemia cell line), zinc enhances the up-regulation of A20 mRNA, which, via TRAF pathway, decreases NF-kappaB activation, leading to decreased gene expression and generation of tumor necrosis factor-alpha (TNF-alpha), IL-1beta, and IL-8. We have reported recently that in both young adults and elderly subjects, zinc supplementation decreased oxidative stress markers and generation of inflammatory cytokines.

Effect of proinflammatory cytokines on PIGA- hematopoiesis.

PubMed

Kulkarni, Shashikant; Bessler, Monica

2003-09-01

Blood cells from patients with paroxysmal nocturnal hemoglobinuria lack glycosyl phosphatidylinositol (GPI)-linked proteins, due to a somatic mutation in the X-linked PIGA gene. It is believed that clonal expansion of PIGA- blood cells is due to a survival advantage in the hostile marrow environment of aplastic anemia. Here we investigated the effects of inhibitory cytokines in mice genetically engineered to have blood cells deficient in GPI-linked proteins. The effect of inhibitory cytokines (tumor necrosis factor-alpha [TNF-alpha], interferon-gamma [IFN-gamma], macrophage inflammatory protein-1 alpha [MIP-1alpha], and transforming growth factor-beta1 [TGF-beta1]) was investigated, using clonogenic assays, competitive repopulation, and in vivo induction of proinflammatory cytokines by double-stranded RNA. The expression of Fas on progenitor cells and its up-regulation by inhibitory cytokines were analyzed by flow cytometry. TNF-alpha, IFN-gamma, MIP-1alpha, and TGF-beta1 suppressed colony formation in a dose-dependent fashion that was similar for PIGA+ and PIGA- blood bone marrow cells. Competitive repopulation of bone marrow cells cultured in IFN-gamma and TNF-alpha resulted in a comparable ability of PIGA+ and PIGA- hematopoietic stem cells to reconstitute hematopoiesis. Fas expression was minimal on PIGA+ and PIGA- progenitor cells and was up-regulated to the same extent in response to IFN-gamma and TNF-alpha as assessed by Fas antibody-mediated apoptosis. Similarly, in vivo induction of proinflammatory cytokines by double-stranded RNA had no effect on the proportion of circulating PIGA- blood cells. These results indicate that PIGA+ and PIGA- hematopoietic progenitor cells respond similarly to inhibitory cytokines, suggesting that other factors are responsible for the clonal expansion of paroxysmal nocturnal hemoglobinuria cells.

Network Oscillations Drive Correlated Spiking of ON and OFF Ganglion Cells in the rd1 Mouse Model of Retinal Degeneration

PubMed Central

Margolis, David J.; Gartland, Andrew J.; Singer, Joshua H.; Detwiler, Peter B.

2014-01-01

Following photoreceptor degeneration, ON and OFF retinal ganglion cells (RGCs) in the rd-1/rd-1 mouse receive rhythmic synaptic input that elicits bursts of action potentials at ∼10 Hz. To characterize the properties of this activity, RGCs were targeted for paired recording and morphological classification as either ON alpha, OFF alpha or non-alpha RGCs using two-photon imaging. Identified cell types exhibited rhythmic spike activity. Cross-correlation of spike trains recorded simultaneously from pairs of RGCs revealed that activity was correlated more strongly between alpha RGCs than between alpha and non-alpha cell pairs. Bursts of action potentials in alpha RGC pairs of the same type, i.e. two ON or two OFF cells, were in phase, while bursts in dissimilar alpha cell types, i.e. an ON and an OFF RGC, were 180 degrees out of phase. This result is consistent with RGC activity being driven by an input that provides correlated excitation to ON cells and inhibition to OFF cells. A2 amacrine cells were investigated as a candidate cellular mechanism and found to display 10 Hz oscillations in membrane voltage and current that persisted in the presence of antagonists of fast synaptic transmission and were eliminated by tetrodotoxin. Results support the conclusion that the rhythmic RGC activity originates in a presynaptic network of electrically coupled cells including A2s via a Na+-channel dependent mechanism. Network activity drives out of phase oscillations in ON and OFF cone bipolar cells, entraining similar frequency fluctuations in RGC spike activity over an area of retina that migrates with changes in the spatial locus of the cellular oscillator. PMID:24489706

CP-25, a Novel Anti-inflammatory and Immunomodulatory Drug, Inhibits the Functions of Activated Human B Cells through Regulating BAFF and TNF-alpha Signaling and Comparative Efficacy with Biological Agents.

PubMed

Zhang, Feng; Shu, Jin-Ling; Li, Ying; Wu, Yu-Jing; Zhang, Xian-Zheng; Han, Le; Tang, Xiao-Yu; Wang, Chen; Wang, Qing-Tong; Chen, Jing-Yu; Chang, Yan; Wu, Hua-Xun; Zhang, Ling-Ling; Wei, Wei

2017-01-01

Paeoniflorin-6'- O -benzene sulfonate (code: CP-25) was the chemistry structural modifications of Paeoniflorin (Pae). CP-25 inhibited B cells proliferation stimulated by B cell activating factor belonging to the TNF family (BAFF) or Tumor necrosis factor alpha (TNF-alpha). CP-25, Rituximab and Etanercept reduced the percentage and numbers of CD19 + B cells, CD19 + CD20 + B cells, CD19 + CD27 + B cells and CD19 + CD20 + CD27 + B cells induced by BAFF or TNF-alpha. There was significant difference between CP-25 and Rituximab or CP-25 and Etanercept. CP-25 down-regulated the high expression of BAFFR, BCMA, and TACI stimulated by BAFF or TNF-alpha. The effects of Rituximab and Etanercept on BAFFR or BCMA were stronger than that of CP-25. CP-25, Rituximab and Etanercept down-regulated significantly the expression of TNFR1 and TNFR2 on B cell stimulated by BAFF or TNF-alpha. CP-25, Rituximab and Etanercept down-regulated the expression of MKK3, P-p38, P-p65, TRAF2, and p52 in B cells stimulated by BAFF and the expression of TRAF2 and P-p65 in B cells stimulated by TNF-alpha. These results suggest that CP-25 regulated moderately activated B cells function by regulating the classical and alternative NF-κB signaling pathway mediated by BAFF and TNF-alpha-TRAF2-NF-κB signaling pathway. This study suggests that CP-25 may be a promising anti-inflammatory immune and soft regulation drug.

Potential late-onset Alzheimer's disease-associated mutations in the ADAM10 gene attenuate {alpha}-secretase activity.

PubMed

Kim, Minji; Suh, Jaehong; Romano, Donna; Truong, Mimy H; Mullin, Kristina; Hooli, Basavaraj; Norton, David; Tesco, Giuseppina; Elliott, Kathy; Wagner, Steven L; Moir, Robert D; Becker, K David; Tanzi, Rudolph E

2009-10-15

ADAM10, a member of a disintegrin and metalloprotease family, is an alpha-secretase capable of anti-amyloidogenic proteolysis of the amyloid precursor protein. Here, we present evidence for genetic association of ADAM10 with Alzheimer's disease (AD) as well as two rare potentially disease-associated non-synonymous mutations, Q170H and R181G, in the ADAM10 prodomain. These mutations were found in 11 of 16 affected individuals (average onset age 69.5 years) from seven late-onset AD families. Each mutation was also found in one unaffected subject implying incomplete penetrance. Functionally, both mutations significantly attenuated alpha-secretase activity of ADAM10 (>70% decrease), and elevated Abeta levels (1.5-3.5-fold) in cell-based studies. In summary, we provide the first evidence of ADAM10 as a candidate AD susceptibility gene, and report two potentially pathogenic mutations with incomplete penetrance for late-onset familial AD.

Role of Different Alpha-Synuclein Strains in Synucleinopathies, Similarities with other Neurodegenerative Diseases

PubMed Central

Melki, Ronald

2015-01-01

Abstract Misfolded protein aggregates are the hallmark of several neurodegenerative diseases in humans. The main protein constituent of these aggregates and the regions within the brain that are affected differ from one neurodegenerative disorder to another. A plethora of reports suggest that distinct diseases have in common the ability of protein aggregates to spread and amplify within the central nervous system. This review summarizes briefly what is known about the nature of the protein aggregates that are infectious and the reason they are toxic to cells. The chameleon property of polypeptides which aggregation into distinct high-molecular weight assemblies is associated to different diseases, in particular, that of alpha-synuclein which aggregation is the hallmark of distinct synucleinopathies, is discussed. Finally, strategies targeting the formation and propagation of structurally distinct alpha-synuclein assemblies associated to different synucleinopathies are presented and their therapeutic and diagnostic potential is discussed. PMID:25757830

PPAR{alpha} agonist fenofibrate protects the kidney from hypertensive injury in spontaneously hypertensive rats via inhibition of oxidative stress and MAPK activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hou, Xiaoyang; Division of Cardiothoracic Surgery, The Michael E. DeBakey Department of Surgery, Baylor College of Medicine, BCM 390, One Baylor Plaza, Houston, TX 77030; Shen, Ying H.

2010-04-09

Oxidative stress has been shown to play an important role in the development of hypertensive renal injury. Peroxisome proliferator-activated receptors {alpha} (PPAR{alpha}) has antioxidant effect. In this study, we demonstrated that fenofibrate significantly reduced proteinuria, inflammatory cell recruitment and extracellular matrix (ECM) proteins deposition in the kidney of SHRs without apparent effect on blood pressure. To investigate the mechanisms involved, we found that fenofibrate treatment markedly reduced oxidative stress accompanied by reduced activity of renal NAD(P)H oxidase, increased activity of Cu/Zn SOD, and decreased phosphorylation of p38MAPK and JNK in the kidney of SHRs. Taken together, fenofibrate treatment can protectmore » against hypertensive renal injury without affecting blood pressure by inhibiting inflammation and fibrosis via suppression of oxidative stress and MAPK activity.« less

Intracellular disposal of incompletely folded human alpha1-antitrypsin involves release from calnexin and post-translational trimming of asparagine-linked oligosaccharides.

PubMed

Liu, Y; Choudhury, P; Cabral, C M; Sifers, R N

1997-03-21

Protection of lung elastin fibers from proteolytic destruction is compromised by inefficient secretion of incompletely folded allelic variants of human alpha1-antitrypsin from hepatocytes. Pulse-chase radiolabeling with [35S]methionine and sucrose gradient sedimentation and coimmunoprecipitation techniques were employed to investigate quality control of human alpha1-antitrypsin secretion from stably transfected mouse hepatoma cells. The secretion-incompetent variant null(Hong Kong) (Sifers, R. N., Brashears-Macatee, S., Kidd, V. J., Muensch, H., and Woo, S. L. C. (1988) J. Biol. Chem. 263, 7330-7335) cannot fold into a functional conformation and was quantitatively associated with the molecular chaperone calnexin following biosynthesis. Assembly with calnexin required cotranslational trimming of glucose from asparagine-linked oligosaccharides. Intracellular disposal of pulse-radiolabeled molecules coincided with their release from calnexin. Released monomers and intracellular disposal were nonexistent in cells chased with cycloheximide, an inhibitor of protein synthesis. Post-translational trimming of asparagine-linked oligosaccharides and intracellular disposal were abrogated by 1-deoxymannojirimycin, an inhibitor of alpha-mannosidase activity, without affecting the monomer population. The data are consistent with a recently proposed quality control model (Hammond, C., Braakman, I., and Helenius, A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 913-917) in which intracellular disposal requires dissociation from calnexin and post-translational trimming of mannose from asparagine-linked oligosaccharides.

Presynaptic congenital myasthenic syndrome with a homozygous sequence variant in LAMA5 combines myopia, facial tics, and failure of neuromuscular transmission.

PubMed

Maselli, Ricardo A; Arredondo, Juan; Vázquez, Jessica; Chong, Jessica X; Bamshad, Michael J; Nickerson, Deborah A; Lara, Marian; Ng, Fiona; Lo, Victoria L; Pytel, Peter; McDonald, Craig M

2017-08-01

Defects in genes encoding the isoforms of the laminin alpha subunit have been linked to various phenotypic manifestations, including brain malformations, muscular dystrophy, ocular defects, cardiomyopathy, and skin abnormalities. We report here a severe defect of neuromuscular transmission in a consanguineous patient with a homozygous variant in the laminin alpha-5 subunit gene (LAMA5). The variant c.8046C>T (p.Arg2659Trp) is rare and has a predicted deleterious effect. The affected individual, who also carries a rare homozygous sequence variant in LAMA1, had muscle weakness, myopia, and facial tics. Magnetic resonance imaging of brain showed mild volume loss and periventricular T2 prolongation. Repetitive nerve stimulation revealed 50% decrement of compound muscle action potential amplitudes and 250% facilitation immediately after exercise, Endplate studies identified a profound reduction of the endplate potential quantal content and endplates with normal postsynaptic folding that were denuded or partially occupied by small nerve terminals. Expression studies revealed that p.Arg2659Trp caused decreased binding of laminin alpha-5 to SV2A and impaired laminin-521 cell-adhesion and cell projection support in primary neuronal cultures. In summary, this report describing severe neuromuscular transmission failure in a patient with a LAMA5 mutation expands the list of phenotypes associated with defects in genes encoding alpha-laminins. © 2017 Wiley Periodicals, Inc.

c-erbA and v-erbA modulate growth and gene expression of a mouse glial precursor cell line.

PubMed

Iglesias, T; Llanos, S; López-Barahona, M; Pérez-Aranda, A; Rodríguez-Peña, A; Bernal, J; Höhne, A; Seliger, B; Muñoz, A

1994-07-01

The c-erbA alpha protooncogene coding for the thyroid hormone (T3) receptor (TR alpha 1) and the viral, mutated v-erbA oncogene were expressed in an immortal mouse glial cell line (B3.1) using retroviral vectors. c-erbA alpha expression led to a decrease in cell proliferation in high and low serum conditions, both in the presence and in the absence of T3. In serum-free medium, c-erbA-expressing cells (B3.1 + TR alpha 1) were completely arrested, whereas cells expressing v-erbA (B3.1 + v-erbA) showed a higher DNA synthesis rate than normal B3.1 cells. Although proliferation of all three cell types was stimulated by platelet-derived growth factor and basic fibroblast growth factor, differences were also observed in the response to these agents. B3.1 + TR alpha 1 cells were more sensitive to platelet-derived growth factor than B3.1 and B3.1 + v-erbA cells. In contrast, B3.1 cells responded to basic fibroblast growth factor better than B3.1 + TR alpha 1 or B3.1 + v-erbA cells. Insulin-like growth factor I potentiated the action of platelet-derived growth factor and basic fibroblast growth factor. Again, different responses to treatment with insulin-like growth factor I alone were observed; B3.1 + TR alpha 1 cells did not respond to it, whereas B3.1 + v-erbA cells showed a dramatic stimulation by this agent. Interestingly, in the presence of T3, the blockade in B3.1 + TR alpha 1 cell proliferation was accompanied by the down-regulation of the typical astrocytic genes, glial fibrillary acidic protein and vimentin. These hormone effects were not found in v-erbA-expressing cells. In addition, v-erbA inhibited the basal expression of the cyclic nucleotide phosphodiesterase gene, an oligodendrocytic marker.(ABSTRACT TRUNCATED AT 250 WORDS)

A conjugate of camptothecin and a somatostatin analog against prostate cancer cell invasion via a possible signaling pathway involving PI3K/Akt, alphaVbeta3/alphaVbeta5 and MMP-2/-9.

PubMed

Sun, Li-Chun; Luo, Jing; Mackey, L Vienna; Fuselier, Joseph A; Coy, David H

2007-02-08

Camptothecin (CPT) was conjugated to the N-terminal of a somatostatin analog (SSA) directly via a carbamate group and a basic N-terminal linking motif, D-Lys-D-Tyr-Lys-D-Tyr-D-Lys. This new CPT-SSA conjugate termed JF-10-81 was evaluated as a receptor-specific delivery system for its anti-invasive and anti-angiogenic activities. It was found that, in addition to blocking migration and invasion of highly invasive prostate cancer PC-3 cells, this conjugate also inhibited in vitro capillary-like tube formation of endothelial cells and in vivo angiogenesis in C57B1/6N female mice. JF-10-81 was found to block PC-3 cell attachment to various extracellular matrix components, mainly to vitronectin, the ligand of cell surface receptors integrin alphaVbeta3 and alphaVbeta5. Additionally, JF-10-81 reduced expression of integrins alphaVbeta3 and alphaVbeta5 on PC-3 cell surfaces, without effects on beta1 or any alphabeta1 heterodimers. This conjugate also inactivated phosphorylation of protein kinase B (PKB/Akt), down-regulated the expression of latent matrix metalloproteinase (MMP) -2 and MMP-9, but had little effect on MMP-3/-10. Meanwhile, membrane type-1 matrix metalloproteinase (MT1-MMP) and the tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) were not detectable in PC-3 cells. alphaVbeta3/alphaVbeta5 and MMP-2/-9 are known to be highly expressed in many tumor cells and play an important role in tumor progression. Our results support that this conjugate could possibly inhibit prostate cancer PC-3 cell invasion through a signaling pathway involving PI3K/Akt, alphaVbeta3/alphaVbeta5 and MMP-2/-9, and this SSA could be used as an efficient vector to deliver CPT or other cytotoxic agents to target sites for cancer therapy.

Bombesin and thrombin affect discrete pools of intracellular calcium through different G-proteins.

PubMed

Wang, J L; Kalyanaraman, S; Vivo, M D; Gautam, N

1996-11-15

In mouse NIH 3T3 cells, the mitogens bombesin and thrombin induced Ca2+ release from intracellular stores. Ca2+ release induced by bombesin was inhibited by the Ca(2+)-ATPase inhibitor thapsigargin, while Ca2+ release induced by thrombin was unaffected by this agent. The Ca(2+)-release response to bombesin was not affected by pertussis toxin, but the response to thrombin was abolished by the toxin. Stable transfectants overexpressing the G-protein subunit type alpha 9 showed an accentuated response to bombesin, indicating that the bombesin receptor was coupled to a Gq-like G-protein. Together, these results show that the two mitogenic receptors are coupled to distinct G-proteins that affect functionally different pools of Ca2+. Organization of signalling pathways in this manner may allow cells to differentially encode information from different signals.

Optimization of the temporal pattern of radiation: An IMRT based study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Altman, Michael B.; Chmura, Steven J.; Deasy, Joseph O.

Purpose: To investigate how the temporal pattern of dose applied during a single-intensity modulated radiation therapy (IMRT) fraction can be arranged to maximize or minimize cell kill. Methods and Materials: Using the linear-quadratic repair-time model and a simplified IMRT delivery pattern model, the surviving fraction of cells for a single fraction was calculated for all permutations of the dose delivery pattern for an array of clinically based IMRT cases. Maximization of cell kill was achieved by concentrating the highest doses in the middle of a fraction, while minimization was achieved by spreading the highest doses between the beginning and end.more » The percent difference between maximum and minimum cell kill (%Diff{sub min/max}) and the difference between maximum and minimum total doses normalized to 2 Gy/fx ({delta}NTD{sub 2Gy}) was calculated for varying fraction durations (T), {alpha}/{beta} ratios, and doses/fx. Results: %Diff{sub min/max} and {delta}NTD{sub 2Gy} both increased with increasing T and with decreasing {alpha}/{beta}. The largest increases occurred with dose/fx. With {alpha}/{beta} = 3 Gy and 30 min/fx, %Diff{sub min/max} ranged from 2.7-5.3% for 2 Gy/fx to 48.6-74.1% for 10 Gy/fx, whereas {delta}NTD{sub 2Gy} ranged from 1.2 Gy-2.4 Gy for 30 fractions of 2 Gy/fx to 2.3-4.8 Gy for 2 fractions of 10.84 Gy/fx. Using {alpha}/{beta} = 1.5 Gy, an analysis of prostate hypofractionation schemes yielded differences in clinical outcome based on the pattern of applied dose ranging from 3.2%-6.1% of the treated population. Conclusions: Rearrangement of the temporal pattern of dose for a single IMRT fraction could be used to optimize cell kill and to directly, though modestly, affect treatment outcome.« less

UBXD4, a UBX-containing protein, regulates the cell surface number and stability of alpha3-containing nicotinic acetylcholine receptors.

PubMed

Rezvani, Khosrow; Teng, Yanfen; Pan, Yaping; Dani, John A; Lindstrom, Jon; García Gras, Eduardo A; McIntosh, J Michael; De Biasi, Mariella

2009-05-27

Adaptor proteins are likely to modulate spatially and temporally the trafficking of a number of membrane proteins, including neuronal nicotinic acetylcholine receptors (nAChRs). A yeast two-hybrid screen identified a novel UBX-containing protein, UBXD4, as one of the cytosolic proteins that interact directly with the alpha3 and alpha4 nAChR subunits. The function of UBX-containing proteins is largely unknown. Immunoprecipitation and confocal microscopy confirmed the interaction of UBXD4 with alpha3-containing nAChRs (alpha3* nAChRs) expressed in HEK293 cells, PC12 cells, and rat cortical neurons. Overexpression of UBXD4 in differentiated PC12 cells (dPC12) increased nAChR cell surface expression, especially that of the alpha3beta2 subtype. These findings were corroborated by electrophysiology, immunofluorescent staining, and biotinylation of surface receptors. Silencing of UBXD4 led to a significant reduction of alpha3* nAChRs in rat cortical neurons and dPC12 cells. Biochemical and immunofluorescence studies of endogenous UBXD4 showed that the protein is located in both the ER and cis-Golgi compartments. Our investigations also showed that the alpha3 subunit is ubiquitinated and that UBXD4 can interfere with its ubiquitination and consequent degradation by the proteasome. Our data suggest that UBXD4 modulates the distribution of alpha3* nAChRs between specialized intracellular compartments and the plasma membrane. This effect is achieved by controlling the stability of the alpha3 subunit and, consequently, the number of receptors at the cell surface.

DOE Office of Scientific and Technical Information (OSTI.GOV)

He, Xiao-dong; Tobo, Masayuki; Mogi, Chihiro

Highlights: Black-Right-Pointing-Pointer Glucocorticoid (GC) induced the expression of proton-sensing TDAG8 in macrophages. Black-Right-Pointing-Pointer GC enhanced acidic pH-induced cAMP accumulation and inhibition of TNF-{alpha} production. Black-Right-Pointing-Pointer The enhancement of the GC-induced actions was lost by TDAG8 deficiency. Black-Right-Pointing-Pointer GC-induced anti-inflammatory actions are partly mediated by TDAG8 expression. -- Abstract: Dexamethasone (DEX), a potent glucocorticoid, increased the expression of T-cell death associated gene 8 (TDAG8), a proton-sensing G protein-coupled receptor, which is associated with the enhancement of acidic pH-induced cAMP accumulation, in peritoneal macrophages. We explored the role of increased TDAG8 expression in the anti-inflammatory actions of DEX. The treatment of macrophagesmore » with either DEX or acidic pH induced the cell death of macrophages; however, the cell death was not affected by TDAG8 deficiency. While DEX inhibited lipopolysaccharide-induced production of tumor necrosis factor-{alpha}, an inflammatory cytokine, which was independent of TDAG8, at neutral pH, the glucocorticoid enhanced the acidic pH-induced inhibition of tumor necrosis factor-{alpha} production in a manner dependent on TDAG8. In conclusion, the DEX-induced increase in TDAG8 expression is in part involved in the glucocorticoid-induced anti-inflammatory actions through the inhibition of inflammatory cytokine production under the acidic pH environment. On the other hand, the role of TDAG8 in the DEX-induced cell death is questionable.« less

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

PubMed

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

1996-11-01

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

Evaluation of pGL1-TNF-alpha therapy in combination with radiation

NASA Technical Reports Server (NTRS)

Li, J.; Andres, M. L.; Fodor, I.; Nelson, G. A.; Gridley, D. S.

1998-01-01

Long-term control of high-grade brain tumors is rarely achieved with current therapeutic regimens. In this study a new plasmid-based human tumor necrosis factor-alpha (TNF-alpha) expression vector was synthesized (pGL1-TNF-alpha) and evaluated together with radiation in the aggressive, rapidly growing C6 rat glioma model. pGL1-TNF-alpha was successfully transfected into C6 cells in vitro using a cationic polyamine method. Expression was detected up to 7 days and averaged 0.4 ng of TNF-alpha in the culture medium from 1x10(5) cells. The expressed protein was biologically functional, as evidenced by growth inhibition of L929, a TNF-alpha-susceptible cell line. Using fluorescence-labeled monoclonal antibodies and laser scanning cytometry, we confirmed that both the P55 and P75 receptors for TNF-alpha were present on the C6 cell membrane. However, the receptors were present at low density and P55 was expressed more than the P75 receptor. These findings were in contrast to results obtained with TNF-alpha-susceptible L929 cells. Tests in athymic mice showed that pGL1-TNF-alpha administered intratumorally 16-18 h before radiation (each modality given three times) significantly inhibited C6 tumor progression (P<0.05). This effect was more than additive, because pGL1-TNF-alpha alone did not slow tumor growth and radiation alone had little effect on tumor growth. These results indicate that pGL1-TNF-alpha has potential to augment the antitumor effects of radiation against a tumor type that is virtually incurable.

Immunolocalization of bone-resorptive cytokines in rat pulp and periapical lesions following surgical pulp exposure.

PubMed

Tani-Ishii, N; Wang, C Y; Stashenko, P

1995-08-01

The bone-resorptive cytokines interleukin 1 (IL-1) and tumor necrosis factor (TNF) have been implicated in the pathogenesis of many chronic inflammatory diseases, including pulpitis and apical periodontitis.To further elucidate their role in these disorders, we have identified cells that express IL-1 alpha and TNF alpha in infected pulps and in developing rat periapical lesions after surgical pulp exposure. As detected by immunohistochemistry, IL-1 alpha- and TNF alpha-positive cells were present as early as 2 days after pulp exposure in both the pulp and periapical region. The numbers of cytokine-expressing cells increased up to day 4 in the pulp and up to day 30 in the periapex. In contrast, cells expressing IL-1 beta and TNF beta, the homologous forms of these mediators, were not found in pulp or periapical lesions during this period. Cells expressing IL-1 alpha and TNF alpha were identified primarily as macrophages and fibroblasts, with occasional staining of polymorphonuclear leukocytes. Osteoblasts and osteoclasts were also positive, whereas lymphocytes were negative. In general, cytokine-expressing cells were located proximal to abscesses and the root apex. These findings demonstrate that cells that express bone-resorptive cytokines IL-1 alpha and TNF alpha are present immediately after pulp exposure in this model, which supports the hypothesis that these mediators play a key role in pulpal and periapical pathogenesis, including the concomitant bone destruction. They also indicate that both resident connective tissue cells as well as infiltrating cells express bone-resorptive cytokines in response to infection in these lesions.

Antiangiogenic activity of semisynthetic biotechnological heparins: low-molecular-weight-sulfated Escherichia coli K5 polysaccharide derivatives as fibroblast growth factor antagonists.

PubMed

Presta, Marco; Oreste, Pasqua; Zoppetti, Giorgio; Belleri, Mirella; Tanghetti, Elena; Leali, Daria; Urbinati, Chiara; Bugatti, Antonella; Ronca, Roberto; Nicoli, Stefania; Moroni, Emanuela; Stabile, Helena; Camozzi, Maura; Hernandez, German Andrés; Mitola, Stefania; Dell'Era, Patrizia; Rusnati, Marco; Ribatti, Domenico

2005-01-01

Low-molecular-weight heparin (LMWH) exerts antitumor activity in clinical trials. The K5 polysaccharide from Escherichia coli has the same structure as the heparin precursor. Chemical and enzymatic modifications of K5 polysaccharide lead to the production of biotechnological heparin-like compounds. We investigated the fibroblast growth factor-2 (FGF2) antagonist and antiangiogenic activity of a series of LMW N,O-sulfated K5 derivatives. Surface plasmon resonance analysis showed that LMW-K5 derivatives bind FGF2, thus inhibiting its interaction with heparin immobilized to a BIAcore sensor chip. Interaction of FGF2 with tyrosine-kinase receptors (FGFRs), heparan sulfate proteoglycans (HSPGs), and alpha(v)beta3 integrin is required for biological response in endothelial cells. Similar to LMWH, LMW-K5 derivatives abrogate the formation of HSPG/FGF2/FGFR ternary complexes by preventing FGF2-mediated attachment of FGFR1-overexpressing cells to HSPG-bearing cells and inhibit FGF2-mediated endothelial cell proliferation. However, LMW-K5 derivatives, but not LMWH, also inhibit FGF2/alpha(v)beta3 integrin interaction and consequent FGF2-mediated endothelial cell sprouting in vitro and angiogenesis in vivo in the chick embryo chorioallantoic membrane. LMW N,O-sulfated K5 derivatives affect both HSPG/FGF2/FGFR and FGF2/alpha(v)beta3 interactions and are endowed with FGF2 antagonist and antiangiogenic activity. These compounds may provide the basis for the design of novel LMW heparin-like angiostatic compounds.

Partial agonist clonidine mediates alpha(2)-AR subtypes specific regulation of cAMP accumulation in adenylyl cyclase II transfected DDT1-MF2 cells.

PubMed

Limon-Boulez, I; Bouet-Alard, R; Gettys, T W; Lanier, S M; Maltier, J P; Legrand, C

2001-02-01

alpha2-Adrenergic receptor (alpha(2)-AR) activation in the pregnant rat myometrium at midterm potentiates beta(2)-AR stimulation of adenylyl cyclase (AC) via Gbetagamma regulation of the type II isoform of adenylyl cyclase. However, at term, alpha(2)-AR activation inhibits beta(2)-AR stimulation of AC. This phenomenon is associated with changes in alpha(2)-AR subtype expression (midterm alpha(2A/D)-AR > alpha(2B)-AR; term alpha(2B) >or =alpha(2A/D)-AR), without any change in ACII mRNA, suggesting that alpha(2A/D)- and alpha(2B)-AR differentially regulate beta(2)-cAMP production. To address this issue, we have stably expressed the same density of alpha(2A/D)- or alpha(2B)-AR with AC II in DDT1-MF2 cells. Clonidine (partial agonist) increased beta(2)-AR-stimulated cAMP production in alpha(2A/D)-AR-ACII transfectants but inhibited it in alpha(2B)-AR-ACII transfectants. In contrast, epinephrine (full agonist) enhanced beta(2)-stimulated ACII in both alpha(2A)- and alpha(2B)-ACII clonal cell lines. 4-Azidoanilido-[alpha-(32)P]GTP-labeling of activated G proteins indicated that, in alpha(2B)-AR transfectants, clonidine activated only Gi(2), whereas epinephrine, the full agonist, effectively coupled to Gi(2) and Gi(3). Thus, partial and full agonists selectively activate G proteins that lead to drug specific effects on effectors. Moreover, these data indicate that Gi(3) activation is required for potentiation of beta(2)-AR stimulation of AC by alpha(2A/D) and alpha(2B)-AR in DDT1-MF2 cells. This may reflect an issue of the amount of Gbetagamma released upon receptor activation and/or betagamma composition of Gi(3) versus Gi(2).

MicroRNA-7 facilitates the degradation of alpha-synuclein and its aggregates by promoting autophagy.

PubMed

Choi, Doo Chul; Yoo, Myungsik; Kabaria, Savan; Junn, Eunsung

2018-05-05

Alpha-Synuclein (α-Syn) is an important protein in the pathogenesis of Parkinson disease (PD) as it accumulates as fibrillar inclusions in affected brain regions including dopaminergic neurons in the substantia nigra. Elevated levels of α-Syn seem to be crucial in mediating its toxicity. Thus, detailed information regarding the regulatory mechanism of α-Syn expression in several layers such as transcription, post-transcription and post-translation is needed in order to devise therapeutic interventions for PD. Previously, we reported that expression of α-Syn is repressed by microRNA-7 (miR-7) through its effect on the 3'-untranslated region (UTR) of α-Syn mRNA. Here, we show that miR-7 also accelerates the clearance of α-Syn and its aggregates by promoting autophagy in differentiated ReNcell VM cells. Further, miR-7 facilitates the degradation of pre-formed fibrils of α-Syn transported from outside the cells. This additional mechanism for reducing α-Syn levels show miR-7 to be an important molecular target for PD and other alpha-synucleinopathies. Copyright © 2018 Elsevier B.V. All rights reserved.

Cow's milk increases the activities of human nuclear receptors peroxisome proliferator-activated receptors alpha and delta and retinoid X receptor alpha involved in the regulation of energy homeostasis, obesity, and inflammation.

PubMed

Suhara, W; Koide, H; Okuzawa, T; Hayashi, D; Hashimoto, T; Kojo, H

2009-09-01

The nuclear peroxisome proliferator-activated receptors (PPAR) have been shown to play crucial roles in regulating energy homeostasis including lipid and carbohydrate metabolism, inflammatory responses, and cell proliferation, differentiation, and survival. Because PPAR agonists have the potential to prevent or ameliorate diseases such as hyperlipidemia, diabetes, atherosclerosis, and obesity, we have explored new natural agonists for PPAR. For this purpose, cow's milk was tested for agonistic activity toward human PPAR subtypes using a reporter gene assay. Milk increased human PPARalpha activity in a dose-dependent manner with a 3.2-fold increase at 0.5% (vol/vol). It also enhanced human PPARdelta activity in a dose-dependent manner with an 11.5-fold increase at 0.5%. However, it only slightly affected human PPARgamma activity. Ice cream, butter, and yogurt also increased the activities of PPARalpha and PPARdelta, whereas vegetable cream affected activity of PPARdelta but not PPARalpha. Skim milk enhanced the activity of PPAR to a lesser degree than regular milk. Milk and fresh cream increased the activity of human retinoid X receptor (RXR)alpha as well as PPARalpha and PPARdelta, whereas neither affected vitamin D3 receptor, estrogen receptors alpha and beta, or thyroid receptors alpha and beta. Both milk and fresh cream were shown by quantitative real-time PCR to increase the quantity of mRNA for uncoupling protein 2 (UCP2), an energy expenditure gene, in a dose-dependent manner. The increase in UCP2 mRNA was found to be reduced by treatment with PPARdelta-short interfering (si)RNA. This study unambiguously clarified at the cellular level that cow's milk increased the activities of human PPARalpha, PPARdelta, and RXRalpha. The possible role in enhancing the activities of PPARalpha, PPARdelta, and RXRalpha, and the health benefits of cow's milk were discussed.

Urocortin 3 Marks Mature Human Primary and Embryonic Stem Cell-Derived Pancreatic Alpha and Beta Cells

PubMed Central

van der Meulen, Talitha; Xie, Ruiyu; Kelly, Olivia G.; Vale, Wylie W.; Sander, Maike; Huising, Mark O.

2012-01-01

The peptide hormone Urocortin 3 (Ucn 3) is abundantly and exclusively expressed in mouse pancreatic beta cells where it regulates insulin secretion. Here we demonstrate that Ucn 3 first appears at embryonic day (E) 17.5 and, from approximately postnatal day (p) 7 and onwards throughout adult life, becomes a unifying and exclusive feature of mouse beta cells. These observations identify Ucn 3 as a potential beta cell maturation marker. To determine whether Ucn 3 is similarly restricted to beta cells in humans, we conducted comprehensive immunohistochemistry and gene expression experiments on macaque and human pancreas and sorted primary human islet cells. This revealed that Ucn 3 is not restricted to the beta cell lineage in primates, but is also expressed in alpha cells. To substantiate these findings, we analyzed human embryonic stem cell (hESC)-derived pancreatic endoderm that differentiates into mature endocrine cells upon engraftment in mice. Ucn 3 expression in hESC-derived grafts increased robustly upon differentiation into mature endocrine cells and localized to both alpha and beta cells. Collectively, these observations confirm that Ucn 3 is expressed in adult beta cells in both mouse and human and appears late in beta cell differentiation. Expression of Pdx1, Nkx6.1 and PC1/3 in hESC-derived Ucn 3+ beta cells supports this. However, the expression of Ucn 3 in primary and hESC-derived alpha cells demonstrates that human Ucn 3 is not exclusive to the beta cell lineage but is a general marker for both the alpha and beta cell lineages. Ucn 3+ hESC-derived alpha cells do not express Nkx6.1, Pdx1 or PC1/3 in agreement with the presence of a separate population of Ucn 3+ alpha cells. Our study highlights important species differences in Ucn 3 expression, which have implications for its utility as a marker to identify mature beta cells in (re)programming strategies. PMID:23251699

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

PubMed

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

2006-01-01

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

The influence of surface integrin binding patterns on specific biomaterial-cell interactions

NASA Astrophysics Data System (ADS)

Beranek, Maggi Marie

As the future of biomaterials progresses toward bioactivity, the biomaterial surface must control non-specific protein adsorption and encourage selective protein and cell adsorption. Integrins alphavbeta3, alpha 1beta1, alpha5beta1 and alpha Mbeta2 are expressed on cells involved in endothelialization, inflammation, and intimal hyperplasia. These cellular events play a vital role in biomaterial biocompatibility, especially in the vascular environment. The overall hypothesis of these studies is that biomaterial surfaces exhibit selective integrin binding, which then specifies differential cell binding. To test this hypothesis, four specific aims were developed. The first aim was designed to determine whether metal and polymeric biomaterials exhibit selective integrin binding. The tested materials included 316L stainless steel, nitinol, gold, Elgiloy RTM, poly(D, L-lactide-co-glycolide), polycarbonate urethane and expanded polytetrafluoroethylene. Discrete integrin binding patterns were detected microscopically using integrin specific fluorescent antibodies. Stainless steel exhibited high level integrin alpha1beta 1 and low level integrin alphaMbeta2 binding pattern. This suggests that this metal surface should selectively encourage endothelial cell to inflammatory cell binding. In contrast, gold bound ten times the amount of integrin alphaMbeta2 compared to integrin alpha1beta1, which should encourage inflammatory cell adhesion. The 65/35 poly(D, L-lactide-co-glycolide) was the only polymeric biomaterial tested that had integrin binding levels comparable to metal biomaterials. Based on these observations, a combinational biomaterial with a surface pattern of 65/35 poly(D, L-lactide-co-glycolide) dots on a 316L stainless steel background was created. A pattern of high level integrin alpha1beta1 binding and low level integrin alpha Mbeta2 binding on this combinational surface indicates that this surface should selectively favor endothelial cell binding. In the second aim, the response of surface-bound integrins to flow-related shear stress was examined. Based on fluorescent analysis, total alphavbeta 3, alpha1beta1, and alpha5beta 1 appeared to increase on stainless steel after 90-minute low shear stress exposure, whereas only alpha5beta1 appeared to increase when exposed to high shear. 65/35 poly(D, L-lactide-co-glycolide) exhibited increased total binding of alpha5beta1 and alphaMbeta2, when exposed to either shear stress level. Exposure to either shear stress regimen appeared to increase binding of all integrins on the combinational surface. These responses to shear stress suggest differential integrin binding affinity compared to stainless steel. Using antibodies specific to the integrin subunits, the apparent increase in surface-bound integrins was found to be related to a surface disassociation of alpha and beta subunits. The third aim evaluated human aortic endothelial cells and acute monocytic leukemia cells (THP-1) cell binding to the tested biomaterial surfaces under both static and flow conditions. Both stainless steel and the combinational surface had increased endothelial cell binding compared to monocyte attachment. Pre-incubation of the surface with the specific integrins significantly inhibited human aortic endothelial cell binding. Aim four was designed to investigate the influence of surface bound integrins on human aortic endothelial cell migration under shear stress. If biomaterial surface integrin binding patterns are specific, then pre-bound surface integrins should competitively inhibit binding of cellular integrins to the surface. Cell migration distance on to alphavbeta3, alpha 1beta1, and alpha5beta1 pre-incubated stainless steel was decreased ten-fold, and decreased by three-fold on both 65/35 poly(D, L-lactide-coglycolide) and combinational surfaces compared to the respective bare surfaces. In contrast, migration distance on to alphaMbeta2 pre-coated stainless steel and combinational surface was decreased by only sixty percent and only fifty percent on alphaMbeta2 precoated 65/35 poly(D, L -lactide-co-glycolide). These results suggested that surface binding sites are selective and critical in governing endothelial cell migration. In conclusion, these results support the hypothesis that a surface that encourages specific integrin binding would promote differential cell binding. The novel integrin binding model used in this investigation may be a methodology that can be employed to evaluate potential vascular biomaterials.

Inhibition of 5-alpha-reductase activity induces stromal remodeling and smooth muscle de-differentiation in adult gerbil ventral prostate.

PubMed

Corradi, Lara S; Góes, Rejane M; Carvalho, Hernandes F; Taboga, Sebastião R

2004-06-01

Prostatic differentiation during embryogenesis and its further homeostatic state maintenance during adult life depend on androgens. Dihydrotestosterone, which is synthesized from testosterone by 5 alpha-reductase (5 alpha-r), is the active molecule triggering androgen action within the prostate. In the present work, we examined the effects of 5 alpha-reductase inhibition by finasteride in the ventral prostate (VP) of the adult gerbil, employing histochemical and electron microscopy techniques to demonstrate the morphological and organizational changes of the organ. After 10 days of finasteride treatment at a dose of 100 mg/kg/day, the prostatic complex (VP and dorsolateral prostate) absolute weight was reduced to about 18%. The epithelial cells became short and cuboidal, with less secretory blebs and reduced acid phosphatase activity. The luminal sectional area diminished, suggestive of decreased secretory activity. The stromal/epithelial ratio increased, the stroma becoming thicker but less cellular. There was a striking accumulation of collagen fibrils, which was accompanied by an increase in deposits of amorphous granular material adjacent to the basal lamina and in the clefts between smooth muscle cells (SMC). Additionally, the periacinar smooth muscle became loosely packed. Some SMC were atrophic and showed a denser array of the cytoskeleton, whereas other SMC had a highly irregular outline with numerous spine-like projections. The present data indicate that 5 alpha-r inhibition causes epithelial and stromal changes by affecting intra-prostatic hormone levels. These alterations are probably the result of an imbalance of the homeostatic interaction between the epithelium and the underlying stroma.

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).

Differential retention of alpha-vitamin E is correlated with its transporter gene expression and growth inhibition efficacy in prostate cancer cells.

PubMed

Ni, Jing; Pang, See-Too; Yeh, Shuyuan

2007-04-01

Epidemiological studies showed Vit E has protective effects against prostate cancer (PCa). Interestingly, different prostate cancer cells have different sensitivity to alpha-Vit E or VES treatment. The goal of this study is to determine whether cellular Vit E bioavailability and its transport proteins are important contributing factors. alpha-Vit E and its ester form, VES, were used to treat prostate cancer LNCaP, PC3, and DU145 cells, and their growth rates were determined by MTT assay. Cellular levels of Vit E were quantified using HPLC as the index of bioavailability. The expression levels of Vit E transport proteins were determined by real-time PCR. Among these PCa cells, only LNCaP cells were sensitive to 20 microM alpha-Vit E treatment, while both LNCaP and PC3 cells were sensitive to 20 microM VES treatment. Coordinately, cellular levels of alpha-Vit E and VES positively correlated to their inhibitory effects. Further study found expression levels of Vit E transport proteins, including tocopherol associated protein (TAP), scavenger receptor class B type I (SR-BI), alpha-tocopherol transfer protein (TTP), and ATP binding cassette transporter A1 (ABCA1), were different in various PCa cells, which may contribute to cellular Vit E bioavailability. This notion is further supported by the findings that overexpression or knockdown of TTP could coordinately alter cellular alpha-Vit E levels in PCa cells. Antiproliferative efficacy of alpha-Vit E is correlated with its cellular bioavailability in PCa cells. Modulating the expression of the efflux or influx transporters could sensitize the growth inhibition efficacy of Vit E in prostate cancer cells.

Autophagy modulators sensitize prostate epithelial cancer cell lines to TNF-alpha-dependent apoptosis.

PubMed

Giampietri, Claudia; Petrungaro, Simonetta; Padula, Fabrizio; D'Alessio, Alessio; Marini, Elettra Sara; Facchiano, Antonio; Filippini, Antonio; Ziparo, Elio

2012-11-01

TNF-alpha levels in prostate cancer correlate with the extent of disease and are significantly elevated in the metastatic stage. TNF receptor superfamily controls two distinct signalling cascades, leading to opposite effects, i.e. apoptosis and survival; in prostate cancer TNF-alpha-mediated signalling induces cell survival and resistance to therapy. The apoptosis of prostate epithelial cancer cells LNCaP and PC3 was investigated upon treatment with the autophagy inhibitor 3-methyladenine and the autophagy inducer rapamycin, in combination with TNF-alpha. Cells were exposed to these molecules for 18, 24 and 48 h. Autophagy was assessed via LC3 Western blot analysis; propidium iodide and TUNEL stainings followed by flow cytometry or caspase-8 and caspase-3 activation assays were performed to evaluate apoptosis. TNF-alpha-induced apoptosis was potentiated by 3-methyladenine in the androgen-responsive LNCaP cells, whereas no effect was observed in the androgen-insensitive PC3 cells. Interestingly such pro-apoptosis effect in LNCaP cells was associated with reduced c-Flip levels through proteasomal degradation via increased reactive oxygen species production and p38 activation; such c-Flip reduction was reversed in the presence of either the proteasome inhibitor MG132 or the reactive oxygen species scavenger N-acetyl-cysteine. Conversely in PC3 but not in LNCaP cells, rapamycin stimulated TNF-alpha-dependent apoptosis; such effect was associated with reduced c-Flip promoter activity and FoxO3a activation. We conclude that TNF-alpha-induced apoptosis may be potentiated, in prostate cancer epithelial cells, through autophagy modulators. Increased sensitivity to TNF-alpha-dependent apoptosis correlates with reduced c-Flip levels which are consequent to a post-transcriptional and a transcriptional mechanism in LNCaP and PC3 cells respectively.

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

PubMed

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

1995-08-01

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)

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

PubMed

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

2017-09-01

The use of high-linear energy transfer charged particles is gaining attention as a medical tool because of the emission of radiations with an efficient cell-killing ability. Considerable interest has developed in the use of targeted alpha-particle therapy for the treatment of micrometastases. Moreover, the use of helium beams is gaining momentum, especially for treating pediatric tumors. We analyzed the effects of alpha particles on bone marrow mesenchymal stromal cells (MSCs), which have a subpopulation of stem cells capable of generating adipocytes, chondrocytes, and osteocytes. Further, these cells contribute toward maintenance of homeostasis in the body. MSCs were irradiated with low and high doses of alpha particles or X-rays and a comparative biological analysis was performed. At a low dose (40 mGy), alpha particles exhibited a limited negative effect on the biology of MSCs compared with X-rays. No significant perturbation of cell cycle was observed, and a minimal increase in apoptosis or senescence was detected. Self-renewal was preserved as revealed by the CFU assay. On the contrary, with 2000 mGy alpha particles, we observed adverse effects on the vitality, functionality, and stemness of MSCs. These results are the consequence of different proportion of cells targeted by alpha particles or X-rays and the quality of induced DNA damage. The present study suggests that radiotherapy with alpha particles may spare healthy stem cells more efficaciously than X-ray treatments, an observation that should be taken into consideration by physicians while planning irradiation of tumor areas close to stem cell niches, such as bone marrow. J. Cell. Biochem. 118: 2993-3002, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Interferon-alpha and interferon-gamma modulate Fas-mediated apoptosis in mitomycin-C-resistant human Tenon's fibroblasts.

PubMed

Wang, Xiao Yang; Crowston, Jonathan G; White, Andrew J R; Zoellner, Hans; Healey, Paul R

2014-08-01

The aim of the study was to investigate, using a native mitomycin-C-resistant human Tenon's fibroblast cell line, the possibility that interferon-alpha and gamma could be used with Fas agonists as an alternative anti-fibrotic strategy to mitomycin-C in trabeculectomy. A clinically resistant and in vitro verified mitomycin-C-resistant human Tenon's fibroblast cell line was pretreated with interferon-alpha and interferon-gamma for 48 h before stimulation with an agonistic Fas antibody (CH11) for 2 days to induce cell death. Cell death assays were undertaken. Changes in apoptosis-related proteins were determined by flow cytometry and Western blot. Pretreatment with interferon-alpha or interferon-gamma for 48 h increased Fas, Fas-associated protein with death domain and caspase-8 expression. Protein expression was further increased by combined exposure to interferon-alpha and gamma. Pretreatment with cytokines had no effect on Fas-L and Bcl-2. Interferon-alpha alone did not change the rate of induced cell death. A combination of interferon-alpha and gamma synergistically increased the sensitivity of mitomycin-C-resistant human Tenon's fibroblast cell line to induced cell death. An antagonistic anti-Fas antibody (ZB4) completely blocked induced cell death. Broad caspase inhibitors specific for caspases-8 and -3 reduced induced deaths in interferon pretreated mitomycin-C-resistant human Tenon's fibroblast cell line in a dose-dependent manner. Interferon-alpha and interferon-gamma render mitomycin-C-resistant human Tenon's fibroblast cell line sensitive to Fas-mediated apoptosis. The mechanism involves increased death-inducing signalling complex formation by upregulation of Fas, Fas-associated protein with death domain and caspase-8 expression. © 2013 Royal Australian and New Zealand College of Ophthalmologists.

Ex vivo delivered stromal cell-derived factor-1alpha promotes stem cell homing and induces angiomyogenesis in the infarcted myocardium.

PubMed

Elmadbouh, I; Haider, Husnain Kh; Jiang, Shujia; Idris, Niagara Muhammad; Lu, Gang; Ashraf, Muhammad

2007-04-01

We aimed to optimize non-viral transfection of human stromal cell derived factor (SDF-1alpha) gene into skeletal myoblasts (SkM) and, transplant these cells to establish transient SDF-1alpha gradient to favor extra-cardiac stem cell translocation into infarcted heart. Optimized conditions for transfection of SDF-1alpha gene into syngenic SkM were achieved using FuGene6/phSDF-1alpha (3:2v/w, 4 h transfection) with 125 microM ZnCl(2) (p<0.001). After characterization for transgene overexpression by immunostaining, ELISA and PCR, the cells were transplanted in female rat model of myocardial infarction. Thirty-six rats were grouped (n=12/group) to receive 70 microl DMEM without cells (group-1) or containing 1.5 x 10(6) non-transfected (group-2) or SDF-1alpha transfected SkM (group-3). On day 4 post-transplantation (in 4 animals/group), marked expression of SDF-1alpha/sry-gene (p=0.003), total Akt, phospho-Akt and Bcl2 was observed in group-3. The number of CD31(+), C-kit(+) and CD34(+) cells was highest in group-3 hearts (p<0.01). Blood vessel density in group-3 was higher in both scar and peri-scar regions (p<0.001) as compared with other groups. Echocardiography showed improved indices of left ventricle contractile function and remodeling in group-3 (p<0.05) as compared with groups-1 and -2. We conclude that ex vivo SDF-1alpha transgene delivery promotes stem and progenitor cell migration to the heart, activates cell survival signaling and enhances angiomyogenesis in the infarcted heart.

Inhibitory effects of clotrimazole on TNF-alpha-induced adhesion molecule expression and angiogenesis.

PubMed

Thapa, Dinesh; Lee, Jong Suk; Park, Min-A; Cho, Mi-Yeon; Park, Young-Joon; Choi, Han Gon; Jeong, Tae Cheon; Kim, Jung-Ae

2009-04-01

Cell adhesion molecules play a pivotal role in chronic inflammation and pathological angiogenesis. In the present study, we investigated the inhibitory effects of clotrimazole (CLT) on tumor necrosis factor (TNF)-alpha-induced changes in adhesion molecule expression. CLT dose-dependently inhibited monocyte chemoattractant protein-1 (MCP-1), intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) expressions in TNF-alpha-stimulated HT29 colonic epithelial cells. This inhibitory action of CLT correlated with a significant reduction in TNF-alpha-induced adhesion of monocytes to HT29 cells, which was comparable to the inhibitory effects of anti-ICAM-1 and VCAM-1 monoclonal antibodies on monocyte-epithelial adhesion. These inhibitory actions of CLT were, at least in part, attributable to the inhibition of redox sensitive NF-kappaB activation, as CLT inhibited TNF-alpha-induced ROS generation as well as NF-kappaB nuclear translocation and activation in HT29 cells. Furthermore, the inhibition of TNF-alpha-induced monocyte adhesion was also mimicked by the specific NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC). Inflammatory mediators including TNF-alpha have known to promote angiogenesis, which in turn further contributes to inflammatory pathology. Therefore, we additionally evaluated whether CLT modulates TNF-alpha-induced angiogenesis using in vivo chick chorioallantoic membrane (CAM) assay. The CAM assay showed that CLT dose-dependently attenuated TNF-alpha-induced angiogenesis, and the effect was correlated with decreased inflammation of the CAM tissue. In conclusion, our results suggest that CLT can inhibit TNF-alpha-triggered expression of adhesion molecules, ICAM-1 and VCAM-1, and angiogenesis during inflammation.

Tumor necrosis factor-alpha stimulates the production of squamous cell carcinoma antigen in normal squamous cells.

PubMed

Numa, F; Takeda, O; Nakata, M; Nawata, S; Tsunaga, N; Hirabayashi, K; Suminami, Y; Kato, H; Hamanaka, S

1996-01-01

Squamous cell carcinoma (SCC) antigen, a tumor marker of squamous cell carcinoma, is also increased in several nonmalignant skin lesions, e.g. pemphigus. The aim of the present investigation was to determine if tumor necrosis factor-alpha (TNF-alpha), one of the important environmental factors, stimulated the production of SCC antigen in the normal squamous cells. The exposure of normal human epidermal keratinocytes to TNF-alpha (100 IU/ml) for 72 h greatly increased the SCC antigen production. The stimulatory effect of TNF-alpha (1,000 IU/ml) on the production of SCC antigen was also observed in the normal squamous epithelium tissue. These results would be helpful for understanding the increase of SCC antigen in several nonmalignant skin disorders.

OPC-13013, a cyclic nucleotide phosphodiesterase type III, inhibitor, inhibits cell proliferation and transdifferentiation of cultured rat hepatic stellate cells.

PubMed

Shimizu, E; Kobayashi, Y; Oki, Y; Kawasaki, T; Yoshimi, T; Nakamura, H

1999-01-01

Activated hepatic stellate cells (HSC; lipocytes; Ito cells) proliferate and are responsible for extracellular matrix synthesis during hepatic fibrogenesis. During activation, HSC undergo transdifferentiation into myofibroblasts expressing alpha-smooth muscle actin (alpha-SMA). Adenosine 3', 5'-cyclic monophosphate (cyclic AMP) is an ubiquitous intracellular signaling molecule, and is upregulated by the activation of adenylate cyclase and downregulated via hydrolysis by cyclic nucleotide phosphodiesterases (PDEs). Recently, increased intracellular cyclic AMP has been shown to inhibit HSC activation. The aim of the current study was to determine the effects of inhibition of PDEs on cell proliferation and transdifferentiation in cultured rat HSC. Cell proliferation was determined by [3H]thymidine incorporation, and Western blot analysis was performed for detection of alpha-SMA, a phenotypic marker of transdifferentiation into myofibroblast. When the cells were exposed to 3-isobutyl-1-methylxanthine (IBMX; 50-1000 microM), a nonselective PDE inhibitor, serum-stimulated [3H]thymidine incorporation was suppressed in a dose-dependent manner with a maximum inhibition of 66% at a concentration of 500 microM OPC-13013 (1-60 microM), a selective PDE III isoenzyme inhibitor, induced a dose-dependent inhibitory effect on serum-stimulated DNA synthesis that reached a maximum inhibition of 95% at a concentration of 60 microM, while neither 8-methoxymethyl-3-isobutyl-1-methylxanthine (8-MMX), a PDE I isoenzyme inhibitor, nor Ro-20-1724, a PDE IV isoenzyme inhibitor, had an inhibitory effect. Western blot analysis revealed that IBMX or OPC-13013 decreased alpha-SMA expression, while other selective PDE isoenzyme inhibitors did not have a suppressive effect. IBMX, OPC-13013 or Ro-20-1724, but not 8-MMX augmented forskolin-induced increase in intracellular cyclic AMP levels although cyclic AMP levels were not affected by treatment with any of these PDE inhibitors alone. These data indicate that inhibition of PDEs, especially PDE III isoenzyme, can produce an inhibitory effect on HSC activation. The PDE III isoenzyme may contribute to the regulation of HSC activation during fibrogenesis. In addition, OPC-13013 may have the potential to inhibit initiation and progression of hepatic fibrosis by interfering with HSC activation.

Toxicity of trichlorotoluene isomers: a 28-day feeding study in the rat.

PubMed

Chu, I; Shen, S Y; Villeneuve, D C; Secours, V E; Valli, V E

1984-03-01

Groups of 10 male and 10 female rats were fed alpha,alpha,alpha-, alpha,2,6- or 2,3,6- trichlorotoluene (TCT) in their diet at 0, 0.5, 5.0, 50 or 500 ppm for 28 days. Growth rate and food consumption were not affected by treatment. No deaths occurred. Significant increases in liver weights were observed in male rats fed 5.0 and 500 ppm 2,3,6-TCT. Mild serum biochemical changes occurred in male rats. These included increased SDH activities in the groups fed 5.0 and 50 ppm alpha, alpha, alpha-TCT, and 5.0 ppm 2,3,6-TCT. Alpha, alpha, alpha-TCT at 500 ppm caused elevated LDH activities in male rats. Hepatic microsomal aminopyrine N-demethylase activities were increased in male rats fed 500 ppm alpha,2,6-TCT. Hematological parameters were not affected by treatment. Mild histological changes were seen in the liver, kidney and thyroid of treated rats. Data presented here suggest that alpha, alpha, alpha-, alpha,2,6- and 2,3,6-TCT possess a low order of oral toxicity in the rat.

Localization of alpha 1-adrenoceptors in rat and human hearts by immunocytochemistry.

PubMed

Schulze, W; Fu, M L

1996-01-01

The localization of the alpha 1 adrenoceptors (alpha 1-AR) in the heart tissues from rat and human and in the cultured heart cells from neonatal rats was studied by indirect immunofluorescence and postembedding electronmicroscopical immuno-gold technique. With antipeptide antibodies directed against the second extracellular loop of the human alpha 1-AR (AS sequence 192-218), this receptor was found to be localized along the sarcolemma in both human and rat hearts. Similar localization sites were detected in cultivated rat neonatal cardiomyocytes. Beside the localization in cardiomyocytes, alpha 1-AR were identified in endothelial cells of capillaries and smooth muscle cells of coronary vessels, in neuronal endings, in mast cells of cultivated heart cells but not, or in less amount in fibroblasts. Interestingly, in the right atrium of rat heart the localization of alpha 1-AR was found to be near or on atrial natriuretic factor (ANF) granules, providing the basis for the alpha-adrenergic influence on ANF release. The immunocytochemical studies further confirm and complete the findings known by using autoradiographic binding studies with specific ligands.

Isolation of mouse pancreatic alpha, beta, duct and acinar populations with cell surface markers.

PubMed

Dorrell, Craig; Grompe, Maria T; Pan, Fong Cheng; Zhong, Yongping; Canaday, Pamela S; Shultz, Leonard D; Greiner, Dale L; Wright, Chris V; Streeter, Philip R; Grompe, Markus

2011-06-06

Tools permitting the isolation of live pancreatic cell subsets for culture and/or molecular analysis are limited. To address this, we developed a collection of monoclonal antibodies with selective surface labeling of endocrine and exocrine pancreatic cell types. Cell type labeling specificity and cell surface reactivity were validated on mouse pancreatic sections and by gene expression analysis of cells isolated using FACS. Five antibodies which marked populations of particular interest were used to isolate and study viable populations of purified pancreatic ducts, acinar cells, and subsets of acinar cells from whole pancreatic tissue or of alpha or beta cells from isolated mouse islets. Gene expression analysis showed the presence of known endocrine markers in alpha and beta cell populations and revealed that TTR and DPPIV are primarily expressed in alpha cells whereas DGKB and GPM6A have a beta cell specific expression profile. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Estrogen-related receptor {alpha} modulates the expression of adipogenesis-related genes during adipocyte differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ijichi, Nobuhiro; Ikeda, Kazuhiro; Horie-Inoue, Kuniko

2007-07-06

Estrogen-related receptor {alpha} (ERR{alpha}) is an orphan nuclear receptor that regulates cellular energy metabolism by modulating gene expression involved in fatty acid oxidation and mitochondrial biogenesis in brown adipose tissue. However, the physiological role of ERR{alpha} in adipogenesis and white adipose tissue development has not been well studied. Here, we show that ERR{alpha} and ERR{alpha}-related transcriptional coactivators, peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) coactivator-1{alpha} (PGC-1{alpha}) and PGC-1{beta}, can be up-regulated in 3T3-L1 preadipocytes at mRNA levels under the adipogenic differentiation condition including the inducer of cAMP, glucocorticoid, and insulin. Gene knockdown by ERR{alpha}-specific siRNA results in mRNA down-regulation of fatty acidmore » binding protein 4, PPAR{gamma}, and PGC-1{alpha} in 3T3-L1 cells in the adipogenesis medium. ERR{alpha} and PGC-1{beta} mRNA expression can be also up-regulated in another preadipocyte lineage DFAT-D1 cells and a pluripotent mesenchymal cell line C3H10T1/2 under the differentiation condition. Furthermore, stable expression of ERR{alpha} in 3T3-L1 cells up-regulates adipogenic marker genes and promotes triglyceride accumulation during 3T3-L1 differentiation. These results suggest that ERR{alpha} may play a critical role in adipocyte differentiation by modulating the expression of various adipogenesis-related genes.« less

Cleavage of the actin-capping protein alpha -adducin at Asp-Asp-Ser-Asp633-Ala by caspase-3 is preceded by its phosphorylation on serine 726 in cisplatin-induced apoptosis of renal epithelial cells.

PubMed

van de Water, B; Tijdens, I B; Verbrugge, A; Huigsloot, M; Dihal, A A; Stevens, J L; Jaken, S; Mulder, G J

2000-08-18

Decreased phosphorylation of focal adhesion kinase and paxillin is associated with loss of focal adhesions and stress fibers and precedes the onset of apoptosis (van de Water, B., Nagelkerke, J. F., and Stevens, J. L. (1999) J. Biol. Chem. 274, 13328-13337). The cortical actin cytoskeletal network is also lost during apoptosis, yet little is known about the temporal relationship between altered phosphorylation of proteins that are critical in the regulation of this network and their potential cleavage by caspases during apoptosis. Adducins are central in the cortical actin network organization. Cisplatin caused apoptosis of renal proximal tubular epithelial cells, which was associated with the cleavage of alpha-adducin into a 74-kDa fragment; this was blocked by a general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk). Hemagglutinin-tagged human alpha-adducin was cleaved into a similar 74-kDa fragment by caspase-3 in vitro but not by caspase-6 or -7. Asp-Arg-Val-Asp(29)-Glu, Asp-Ile-Val-Asp(208)-Arg, and Asp-Asp-Ser-Asp(633)-Ala were identified as the principal caspase-3 cleavage sites; Asp-Asp-Ser-Asp(633)-Ala was key in the formation of the 74-kDa fragment. Cisplatin also caused an increased phosphorylation of alpha-adducin and gamma-adducin in the MARCKS domain that preceded alpha-adducin cleavage and was associated with loss of adducins from adherens junctions; this was not affected by z-VAD-fmk. In conclusion, the data support a model in which increased phosphorylation of alpha-adducin due to cisplatin leads to dissociation from the cytoskeleton, a situation rendered irreversible by caspase-3-mediated cleavage of alpha-adducin at Asp-Asp-Ser-Asp(633)-Ala.

Porcine arterivirus activates the NF-{kappa}B pathway through I{kappa}B degradation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Sang-Myeong; Kleiboeker, Steven B.

2005-11-10

Nuclear factor-kappaB (NF-{kappa}B) is a critical regulator of innate and adaptive immune function as well as cell proliferation and survival. The present study demonstrated for the first time that a virus belonging to the Arteriviridae family activates NF-{kappa}B in MARC-145 cells and alveolar macrophages. In porcine reproductive and respiratory syndrome virus (PRRSV)-infected cells, NF-{kappa}B activation was characterized by translocation of NF-{kappa}B from the cytoplasm to the nucleus, increased DNA binding activity, and NF-{kappa}B-regulated gene expression. NF-{kappa}B activation was increased as PRRSV infection progressed and in a viral dose-dependent manner. UV-inactivation of PRRSV significantly reduced the level of NF-{kappa}B activation. Degradationmore » of I{kappa}B protein was detected late in PRRSV infection, and overexpression of the dominant negative form of I{kappa}B{alpha} (I{kappa}B{alpha}DN) significantly suppressed NF-{kappa}B activation induced by PRRSV. However, I{kappa}B{alpha}DN did not affect viral replication and viral cytopathic effect. PRRSV infection induced oxidative stress in cells by generating reactive oxygen species (ROS), and antioxidants inhibited NF-{kappa}B DNA binding activity in PRRSV-infected cells, suggesting ROS as a mechanism by which NF-{kappa}B was activated by PRRSV infection. Moreover, NF-{kappa}B-dependent expression of matrix metalloproteinase (MMP)-2 and MMP-9 was observed in PRRSV-infected cells, an observation which implies that NF-{kappa}B activation is a biologically significant aspect of PRRSV pathogenesis. The results presented here provide a basis for understanding molecular pathways of pathology and immune evasion associated with disease caused by PRRSV.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ewan, Kenneth B.R.; Oketch-Rabah, Hellen A.; Ravani, Shraddha A.

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}more » 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.« less

Pheromone induction of agglutination in Saccharomyces cerevisiae a cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Terrance, K.; Lipke, P.N.

1987-10-01

a-Agglutinin, the cell surface sexual agglutinin of yeast a cells, was assayed by its ability to bind its complementary agglutinin, ..cap alpha..-agglutinin. The specific binding of /sup 125/I-..cap alpha..-agglutinin to a cells treated with the sex pheromone ..cap alpha..-factor was 2 to 2.5 times that of binding to a cells not treated with ..cap alpha..-factor. Competition with unlabeled ..cap alpha..-agglutinin revealed that the increased binding was due to increased cell surface expression of a-agglutinin, with no apparent change in the binding constant. The increase in site number was similar to the increase in cellular agglutinability. Increased expression of a-agglutinin followedmore » the same kinetics as the increase in cellular agglutinability, with a 10-min lag followed by a 15- to 20-min response time. Induction kinetics were similar in cells in phases G1 and G2 of the cell cycle. Maximal expression levels were similar in cells treated with excess pheromone and in cells exposed to pheromone after destruction of constitutively expressed a-agglutinin.« less

Novel function of STAT1beta in B cells: induction of cell death by a mechanism different from that of STAT1alpha.

PubMed

Najjar, Imen; Schischmanoff, Pierre Olivier; Baran-Marszak, Fanny; Deglesne, Pierre-Antoine; Youlyouz-Marfak, Ibtissam; Pampin, Mathieu; Feuillard, Jean; Bornkamm, Georg W; Chelbi-Alix, Mounira K; Fagard, Remi

2008-12-01

Alternate splicing of STAT1 produces two isoforms: alpha, known as the active form, and beta, previously shown to act as a dominant-negative factor. Most studies have dealt with STAT1alpha, showing its involvement in cell growth control and cell death. To examine the specific function of either isoform in cell death, a naturally STAT1-deficient human B cell line was transfected to express STAT1alpha or STAT1beta. STAT1alpha, expressed alone, enhanced cell death, potentiated the fludarabine-induced apoptosis, and enhanced the nuclear location, the phosphorylation, and the transcriptional activity of p53. Unexpectedly, STAT1beta, expressed alone, induced cell death through a mechanism that was independent of the nuclear function of p53. Indeed, in STAT1beta-expressing B cells, p53 was strictly cytoplasmic where it formed clusters, and there was no induction of the transcriptional activity of p53. These data reveal a novel role of STAT1beta in programmed cell death, which is independent of p53.

Withaferin A inhibits tumor necrosis factor-alpha-induced expression of cell adhesion molecules by inactivation of Akt and NF-kappaB in human pulmonary epithelial cells.

PubMed

Oh, Jung Hwa; Kwon, Taeg Kyu

2009-05-01

We here investigated the functional effect of withaferin A on airway inflammation and its action mechanism. Withaferin A inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in human lung epithelial A549 cells stimulated with tumor necrosis factor-alpha (TNF-alpha), resulting in the suppression of leukocyte adhesion to lung epithelial A549 cells. In addition, withaferin A inhibited TNF-alpha-induced expression of adhesion molecules (ICAM-1 and VCAM-1) protein and mRNA in a dose-dependent manner. Withaferin A prevented DNA binding activity of nuclear factor-kappaB (NF-kappaB) and nuclear translocation of NF-kappaB. It also inhibited phosphorylation of Akt and extracellular signal-regulated kinase (ERK), which are upstream in the regulation of adhesion molecules by TNF-alpha. Furthermore, withaferin A inhibited U937 monocyte adhesion to A549 cells stimulated by TNF-alpha, suggesting that it may inhibit the binding of these cells by regulating the expression of critical adhesion molecules by TNF-alpha. Taken together, these results suggest that withaferin A inhibits cell adhesion through inhibition of ICAM-1 and VCAM-1 expression, at least in part, by blocking Akt and down-regulating NF-kappaB activity.

The sodium pump alpha1 subunit as a potential target to combat apoptosis-resistant glioblastomas.

PubMed

Lefranc, Florence; Kiss, Robert

2008-03-01

To review the involvement of the ion transporter Na+/K+-ATPase (NaK) in the migration and proliferation of glioma cells. Preliminary studies indicate that NaK alpha1 subunits seem to be upregulated in a proportion of glioblastomas but not in normal brain tissues. The present review focuses on (1) the natural resistance of migrating malignant glioma cells to apoptosis, (2) autophagic cell death as an alternative to combat malignant gliomas, (3) the fact that reducing the levels of malignant glioma cell motility can restore proapoptotic drug sensitivity,and (4) on the observation that inhibiting the NaK activity reduces both glioma cell proliferation and migration. The natural ligands of the NaK are the cardiotonic steroids. A hemisynthetic derivative of 2"-oxovoruscharin (UNBS1450), a novel cardenolide, displays unique structural features, making its binding affinity to NaK alpha subunits (including alpha1) 10 to 100 times higher than that of other cardenolides. UNBS1450 markedly decreases intracellular ATP concentration in glioma cells, disorganizes the actin cytoskeleton, and leads to autophagic cell death in NaK alpha1 over-expressing glioma cells. Glioblastoma patients who do not respond to chemotherapy and whose tumors over-express NaK alpha1 subunits could benefit from a treatment using ligands with marked binding affinity for the NaK alpha1 subunit.

Leptin acts on neoplastic behavior and expression levels of genes related to hypoxia, angiogenesis, and invasiveness in oral squamous cell carcinoma.

PubMed

Sobrinho Santos, Eliane Macedo; Guimarães, Talita Antunes; Santos, Hércules Otacílio; Cangussu, Lilian Mendes Borborema; de Jesus, Sabrina Ferreira; Fraga, Carlos Alberto de Carvalho; Cardoso, Claudio Marcelo; Santos, Sérgio Henrique Souza; de Paula, Alfredo Maurício Batista; Gomez, Ricardo Santiago; Guimarães, André Luiz Sena; Farias, Lucyana Conceição

2017-05-01

Leptin, one of the main hormones controlling energy homeostasis, has been associated with different cancer types. In oral cancer, its effect is not well understood. We investigated, through in vitro and in vivo assays, whether leptin can affect the neoplastic behavior of oral squamous cell carcinoma. Expression of genes possibly linked to the leptin pathway was assessed in leptin-treated oral squamous cell carcinoma cells and also in tissue samples of oral squamous cell carcinoma and oral mucosa, including leptin, leptin receptor, hypoxia-inducible factor 1-alpha, E-cadherin, matrix metalloproteinase-2, matrix metalloproteinase-9, Col1A1, Ki67, and mir-210. Leptin treatment favored higher rates of cell proliferation and migration, and reduced apoptosis. Accordingly, leptin-treated oral squamous cell carcinoma cells show decreased messenger RNA caspase-3 expression, and increased levels of E-cadherin, Col1A1, matrix metalloproteinase-2, matrix metalloproteinase-9, and mir-210. In tissue samples, hypoxia-inducible factor 1-alpha messenger RNA and protein expression of leptin and leptin receptor were high in oral squamous cell carcinoma cases. Serum leptin levels were increased in first clinical stages of the disease. In animal model, oral squamous cell carcinoma-induced mice show higher leptin receptor expression, and serum leptin level was increased in dysplasia group. Our findings suggest that leptin seems to exert an effect on oral squamous cell carcinoma cells behavior and also on molecular markers related to cell proliferation, migration, and tumor angiogenesis.

A kinetic comparison of the processing and secretion of the alpha beta dimer and the uncombined alpha and beta subunits of chorionic gonadotropin synthesized by human choriocarcinoma cells.

PubMed

Peters, B P; Krzesicki, R F; Hartle, R J; Perini, F; Ruddon, R W

1984-12-25

Human choriocarcinoma cells (JAR) synthesize the alpha and beta subunits of the glycoprotein hormone chorionic gonadotropin (hCG) (R.W. Ruddon, C.A. Hanson, A. H. Bryan, G.J. Putterman, E.L. White, F. Perini, K. S. Meade, and P.H. Aldenderfer (1980) J. Biol. Chem. 255, 1000-1007). In addition to the hCG dimer (alpha beta), JAR cells secrete uncombined alpha and beta subunits into the culture medium (L.A. Cole, R.J. Hartle, J.A. Laferla, and R.W. Ruddon (1983) Endocrinology 113, 1176-1178). Pulse-chase studies with [35S]methionine or [3H]mannose were carried out in order to compare free alpha, free beta, and the alpha beta dimer with regard to the kinetics of synthesis, N-linked oligosaccharide processing, and secretion and to determine the kinetics of alpha-beta subunit combination. A panel of three antisera was used to immunoprecipitate directly the free subunits and the alpha beta dimer sequentially from the same cell lysates and culture media. The alpha subunit of hCG was synthesized in a slight molar excess (1.2-1.5-fold) over the beta subunit, and alpha beta dimer was rapidly formed by combination of the intracellular alpha and beta precursors. Dimer formation was already apparent in JAR cells following a 10-min biosynthetic labeling incubation with [35S]methionine. The combination of subunits ceased by 30 min of chase even though 51% of alpha and 44% of beta remained free within the cells. Combination of the alpha and beta precursors had occurred before their N-linked oligosaccharides were processed beyond the Man8GlcNAc2 structure. The initial trimming of glucosyl and mannosyl units from the high-mannose oligosaccharides of the hCG precursors occurred more rapidly for free alpha and CG-alpha than for free beta and CG-beta. JAR cells accumulated alpha precursors bearing mostly Man8GlcNAc2 units and beta precursors bearing Man8GlcNAc2 units that represent the substrates of the rate-limiting step in the secretory pathway. In spite of the fact that their N-linked oligosaccharides were trimmed at different rates, free alpha, free beta, and alpha beta dimer were all secreted into the medium at the same rate, with a half-time of 35 min. The secreted hCG forms were stable in the chase medium between 4 and 8h, indicating that extracellular degradation, combination of free subunits to form dimer, or dissociation of dimer to form free subunits did not occur.(ABSTRACT TRUNCATED AT 400 WORDS)

Assessment of carbon nanoparticle exposure on murine macrophage function

NASA Astrophysics Data System (ADS)

Suro-Maldonado, Raquel M.

There is growing concern about the potential cytotoxicity of nanoparticles. Exposure to respirable ultrafine particles (2.5uM) can adversely affect human health and have been implicated with episodes of increased respiratory diseases such as asthma and allergies. Nanoparticles are of particular interest because of their ability to penetrate into the lung and potentially elicit health effects triggering immune responses. Nanoparticles are structures and devises with length scales in the 1 to 100-nanometer range. Black carbon (BC) nanoparticles have been observed to be products of combustion, especially flame combustion and multi-walled carbon nanotubes (MWCNT) have been shown to be found in both indoor and outdoor air. Furthermore, asbestos, which have been known to cause mesothelioma as well as lung cancer, have been shown to be structurally identical to MWCNTs. The aims of these studies were to examine the effects of carbon nanoparticles on murine macrophage function and clearance mechanisms. Macrophages are immune cells that function as the first line of defense against invading pathogens and are likely to be amongst the first cells affected by nanoparticles. Our research focused on two manufactured nanoparticles, MWCNT and BC. The two were tested against murine-derived macrophages in a chronic contact model. We hypothesized that long-term chronic exposure to carbon nanoparticles would decrease macrophages ability to effectively respond to immunological challenge. Production of nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), cell surface macrophage; activation markers, reactive oxygen species formation (ROS), and antigen processing and presentation were examined in response to lipopolysaccharide (LPS) following a 144hr exposure to the particulates. Data demonstrated an increase in TNF-alpha, and NO production; a decrease in phagocytosis and antigen processing and presentation; and a decrease in the expression levels of cell surface macrophage activation markers. The data suggests that carbon nanoparticle exposure alters macrophage responses to LPS marked by increased inflammation while potentially limiting clearance and ability to interact with effector T-cells. Thus, physiological exposure to carbon nanoparticles could potentially lead to ineffective pulmonary immunity.

The ability of walnut extract and fatty acids to protect against the deleterious effects of oxidative stress and inflammation in hippocampal cells.

PubMed

Carey, Amanda N; Fisher, Derek R; Joseph, James A; Shukitt-Hale, Barbara

2013-01-01

Previous research from our lab has demonstrated that dietary walnut supplementation protects against age-related cognitive declines in rats; however, the cellular mechanisms by which walnuts and polyunsaturated fatty acids (PUFAs) may affect neuronal health and functioning in aging are undetermined. We assessed if pretreatment of primary hippocampal neurons with walnut extract or PUFAs would protect cells against dopamine- and lipopolysaccharide-mediated cell death and calcium dysregulation. Rat primary hippocampal neurons were pretreated with varying concentrations of walnut extract, linoleic acid, alpha-linolenic acid, eicosapentaenoic acid, or docosahexaenoic acid prior to exposure to either dopamine or lipopolysaccharide. Viability was assessed using the Live/Dead Cellular Viability/Cytotoxicity Kit. Also, the ability of the cells to return to baseline calcium levels after depolarization was measured with fluorescent imaging. Results indicated that walnut extract, alpha-linolenic acid, and docosahexaenoic acid provided significant protection against cell death and calcium dysregulation; the effects were pretreatment concentration dependent and stressor dependent. Linoleic acid and eicosapentaenoic acid were not as effective at protecting hippocampal cells from these insults. Walnut extract and omega-3 fatty acids may protect against age-related cellular dysfunction, but not all PUFAs are equivalent in their beneficial effects.

A new strategy to identify hepatitis B virus entry inhibitors by AlphaScreen technology targeting the envelope-receptor interaction.

PubMed

Saso, Wakana; Tsukuda, Senko; Ohashi, Hirofumi; Fukano, Kento; Morishita, Ryo; Matsunaga, Satoko; Ohki, Mio; Ryo, Akihide; Park, Sam-Yong; Suzuki, Ryosuke; Aizaki, Hideki; Muramatsu, Masamichi; Sureau, Camille; Wakita, Takaji; Matano, Tetsuro; Watashi, Koichi

2018-06-22

Current anti-hepatitis B virus (HBV) agents have limited effect in curing HBV infection, and thus novel anti-HBV agents with different modes of action are in demand. In this study, we applied AlphaScreen assay to high-throughput screening of small molecules inhibiting the interaction between HBV large surface antigen (LHBs) and the HBV entry receptor, sodium taurocholate cotransporting polypeptide (NTCP). From the chemical screening, we identified that rapamycin, an immunosuppressant, strongly inhibited the LHBs-NTCP interaction. Rapamycin inhibited hepatocyte infection with HBV without significant cytotoxicity. This activity was due to impaired attachment of the LHBs preS1 domain to cell surface. Pretreatment of target cells with rapamycin remarkably reduced their susceptibility to preS1 attachment, while rapamycin pretreatment to preS1 did not affect its attachment activity, suggesting that rapamycin targets the host side. In support of this, a surface plasmon resonance analysis showed a direct interaction of rapamycin with NTCP. Consistently, rapamycin also prevented hepatitis D virus infection, whose entry into cells is also mediated by NTCP. We also identified two rapamycin derivatives, everolimus and temsirolimus, which possessed higher anti-HBV potencies than rapamycin. Thus, this is the first report for application of AlphaScreen technology that monitors a viral envelope-receptor interaction to identify viral entry inhibitors. Copyright © 2018 Elsevier Inc. All rights reserved.

Pertussis toxin-catalyzed ADP-ribosylation of a G protein in mouse oocytes, eggs, and preimplantation embryos: Developmental changes and possible functional roles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jones, J.; Schultz, R.M.

1990-06-01

G proteins, which in many somatic cells serve as mediators of signal transduction, were identified in preimplantation mouse embryos by their capacity to undergo pertussis toxin-catalyzed ADP-ribosylation. Two pertussis toxin (PT) substrates with Mr = 38,000 and 39,000 (alpha 38 and alpha 39) are present in approximately equal amounts. Relative to the amount in freshly isolated germinal vesicle (GV)-intact oocytes, the amount of PT-catalyzed ADP-ribosylation of alpha 38-39 falls during oocyte maturation, rises between the one- and two-cell stages, falls by the eight-cell and morula stages, and increases again by the blastocyst stage. The decrease in PT-catalyzed ADP-ribosylation of alphamore » 38-39 that occurs during oocyte maturation, however, does not require germinal vesicle breakdown (GVBD), since inhibiting GVBD with 3-isobutyl-1-methyl xanthine (IBMX) does not prevent the decrease in the extent of PT-catalyzed ADP-ribosylation. A biologically active phorbol diester (12-O-tetradecanoyl phorbol 13-acetate), but not an inactive one (4 alpha-phorbol 12,13-didecanoate, 4 alpha-PDD), totally inhibits the increase in PT-catalyzed ADP-ribosylation of alpha 38-39 that occurs between the one- and two-cell stage; TPA inhibits cleavage, but not transcriptional activation, which occurs in the two-cell embryo. In contrast, cytochalasin D, genistein, or aphidicolin, each of which inhibits cleavage of one-cell embryos, or alpha-amanitin or H8, each of which inhibits transcriptional activation but not cleavage of one-cell embryos, have little or inhibitory effects on the increase in PT-catalyzed ADP-ribosylation of alpha 38-39. Results of immunoblotting experiments using an antibody that is highly specific for alpha il-3 reveal the presence of a cross-reactive species of Mr = 38,000 (alpha 38) in the GV-intact oocyte, metaphase II-arrested egg, and one-, two-cell embryos.« less

Strychnine, but not PMBA, inhibits neuronal nicotinic acetylcholine receptors expressed by rabbit retinal ganglion cells.

PubMed

Renna, J M; Strang, C E; Amthor, F R; Keyser, K T

2007-01-01

Strychnine is considered a selective competitive antagonist of glycine gated Cl- channels (Saitoh et al., 1994) and studies have used strychnine at low micromolar concentrations to study the role of glycine in rabbit retina (Linn, 1998; Protti et al., 2005). However, other studies have shown that strychnine, in the concentrations commonly used, is also a potent competitive antagonist of alpha7 nicotinic acetylcholine receptors (nAChRs; Matsubayashi et al., 1998). We tested the effects of low micromolar concentrations of strychnine and 3-[2'-phosphonomethyl[1,1'-biphenyl]-3-yl] alanine (PMBA), a specific glycine receptor blocker (Saitoh et al., 1994; Hosie et al., 1999) on the activation of both alpha7 nAChRs on retinal ganglion cells and on ganglion cell responses to a light flash. Extracellular recordings were obtained from ganglion cells in an isolated retina/choroid preparation and 500 microM choline was used as an alpha7 agonist (Alkondon et al., 1997). We recorded from brisk sustained and brisk transient OFF cells, many of which have been previously shown to have alpha7 receptors (Strang et al., 2005). Further, we tested the effect of strychnine, PMBA and alpha-bungarotoxin on the binding of tetramethylrhodamine alpha-bungarotoxin in the inner plexiform layer. Our data indicates that strychnine, at doses as low as 1.0 microM, can inhibit the alpha7 nAChR-mediated response to choline, but PMBA at concentrations as high as 0.4 microM does not. Binding studies show strychnine and alpha-bungarotoxin inhibit binding of labeled alpha-bungarotoxin in the IPL. Thus, the effects of strychnine application may be to inhibit glycine receptors expressed by ganglion cell or to inhibit amacrine cell alpha7 nAChRs, both of which would result in an increase in the ganglion cell responses. Further research will be required to disentangle the effects of strychnine previously believed to be caused by a single mechanism of glycine receptor inhibition.

Chondrogenic differentiation of growth factor-stimulated precursor cells in cartilage repair tissue is associated with increased HIF-1alpha activity.

PubMed

Gelse, K; Mühle, C; Knaup, K; Swoboda, B; Wiesener, M; Hennig, F; Olk, A; Schneider, H

2008-12-01

To investigate the chondrogenic potential of growth factor-stimulated periosteal cells with respect to the activity of Hypoxia-inducible Factor 1alpha (HIF-1alpha). Scaffold-bound autologous periosteal cells, which had been activated by Insulin-like Growth Factor 1 (IGF-1) or Bone Morphogenetic Protein 2 (BMP-2) gene transfer using both adeno-associated virus (AAV) and adenoviral (Ad) vectors, were applied to chondral lesions in the knee joints of miniature pigs. Six weeks after transplantation, the repair tissues were investigated for collagen type I and type II content as well as for HIF-1alpha expression. The functional role of phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling on BMP-2/IGF-1-induced HIF-1alpha expression was assessed in vitro by employing specific inhibitors. Unstimulated periosteal cells formed a fibrous extracellular matrix in the superficial zone and a fibrocartilaginous matrix in deep zones of the repair tissue. This zonal difference was reflected by the absence of HIF-1alpha staining in superficial areas, but moderate HIF-1alpha expression in deep zones. In contrast, Ad/AAVBMP-2-stimulated periosteal cells, and to a lesser degree Ad/AAVIGF-1-infected cells, adopted a chondrocyte-like phenotype with strong intracellular HIF-1alpha staining throughout all zones of the repair tissue and formed a hyaline-like matrix. In vitro, BMP-2 and IGF-1 supplementation increased HIF-1alpha protein levels in periosteal cells, which was based on posttranscriptional mechanisms rather than de novo mRNA synthesis, involving predominantly the MEK/ERK pathway. This pilot experimental study on a relatively small number of animals indicated that chondrogenesis by precursor cells is facilitated in deeper hypoxic zones of cartilage repair tissue and is stimulated by growth factors which enhance HIF-1alpha activity.

N-acetylcysteine attenuates TNF-alpha-induced p38 MAP kinase activation and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells.

PubMed

Hashimoto, S; Gon, Y; Matsumoto, K; Takeshita, I; Horie, T

2001-01-01

1. We have previously shown that tumour necrosis factor-alpha (TNF-alpha) activates p38 mitogen-activated protein (MAP) kinase to produce interleukin-8 (IL-8) by human pulmonary vascular endothelial cells. Reactive oxygen species (ROS) including H(2)O(2) generated by TNF-alpha can act as signalling intermediates for cytokine induction; therefore, scavenging ROS by anti-oxidants is important for the regulation of cytokine production. However, the effect of N-acetylcysteine (NAC), which acts as a precursor of glutathione (GSH) synthesis, on TNF-alpha-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells has not been determined. To clarify these issues, we examined the effect of NAC on TNF-alpha-induced activation of p38 MAP kinase, MAP kinase kinase (MKK) 3 and MKK6 which are upstream regulators of p38 MAP kinase, and p38 MAP kinase-mediated IL-8 production. 2. Human pulmonary vascular endothelial cells that had been preincubated with NAC were stimulated with TNF-alpha and then the activation of p38 MAP kinase and MKK3/MKK6 in the cells and IL-8 concentrations in the culture supernatants were determined. 3. Intracellular GSH levels increased in NAC-treated cells. 4. NAC attenuated TNF-alpha-induced activation of p38 MAP kinase and MKK3/MKK6. 5. NAC attenuated p38 MAP kinase-mediated IL-8 production by TNF-alpha-stimulated cells. 6. These results indicate that the cellular reduction and oxidation (redox) regulated by intracellular GSH is critical for TNF-alpha-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells, and we emphasize that anti-oxidant therapy is an important strategy for the treatment of acute lung injury.

Adoptive immunotherapy mediated by ex vivo expanded natural killer T cells against CD1d-expressing lymphoid neoplasms.

PubMed

Bagnara, Davide; Ibatici, Adalberto; Corselli, Mirko; Sessarego, Nadia; Tenca, Claudya; De Santanna, Amleto; Mazzarello, Andrea; Daga, Antonio; Corvò, Renzo; De Rossi, Giulio; Frassoni, Francesco; Ciccone, Ermanno; Fais, Franco

2009-07-01

CD1d is a monomorphic antigen presentation molecule expressed in several hematologic malignancies. Alpha-galactosylceramide (alpha-GalCer) is a glycolipid that can be presented to cytotoxic CD1d-restricted T cells. These reagents represent a potentially powerful tool for cell mediated immunotherapy. We set up an experimental model to evaluate the use of adoptively transferred cytotoxic CD1d-restricted T cells and alpha-GalCer in the treatment of mice engrafted with CD1d(+) lymphoid neoplastic cells. To this end the C1R cell line was transfected with CD1c or CD1d molecules. In addition, upon retroviral infection firefly luciferase was expressed on C1R transfected cell lines allowing the evaluation of tumor growth in xenografted immunodeficient NOD/SCID mice. The C1R-CD1d cell line was highly susceptible to specific CD1d-restricted T cell cytotoxicity in the presence alpha-GalCer in vitro. After adoptive transfer of CD1d-restricted T cells and alpha-GalCer to mice engrafted with both C1R-CD1c and C1R-CD1d, a reduction in tumor growth was observed only in CD1d(+) masses. In addition, CD1d-restricted T-cell treatment plus alpha-GalCer eradicated small C1R-CD1d(+) nodules. Immunohistochemical analysis revealed that infiltrating NKT cells were mainly observed in CD1d nodules. Our results indicate that ex vivo expanded cytotoxic CD1d-restricted T cells and alpha-GalCer may represent a new immunotherapeutic tool for treatment of CD1d(+) hematologic malignancies.

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

DNA-binding activity of TNF-{alpha} inducing protein from Helicobacter pylori

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuzuhara, T.; Suganuma, M.; Oka, K.

2007-11-03

Tumor necrosis factor-{alpha} (TNF-{alpha}) inducing protein (Tip{alpha}) is a carcinogenic factor secreted from Helicobacter pylori (H. pylori), mediated through both enhanced expression of TNF-{alpha} and chemokine genes and activation of nuclear factor-{kappa}B. Since Tip{alpha} enters gastric cancer cells, the Tip{alpha} binding molecules in the cells should be investigated. The direct DNA-binding activity of Tip{alpha} was observed by pull down assay using single- and double-stranded genomic DNA cellulose. The surface plasmon resonance assay, indicating an association between Tip{alpha} and DNA, revealed that the affinity of Tip{alpha} for (dGdC)10 is 2400 times stronger than that of del-Tip{alpha}, an inactive Tip{alpha}. This suggestsmore » a strong correlation between DNA-binding activity and carcinogenic activity of Tip{alpha}. And the DNA-binding activity of Tip{alpha} was first demonstrated with a molecule secreted from H. pylori.« less

Type-I interferon receptor expression: its circadian rhythm and downregulation after interferon-alpha administration in peripheral blood cells from renal cancer patients.

PubMed

Shiba, Masahiro; Nonomura, Norio; Nakai, Yasutomo; Nakayama, Masashi; Takayama, Hitoshi; Inoue, Hitoshi; Tsujimura, Akira; Nishimura, Kazuo; Okuyama, Akihiko

2009-04-01

To investigate the regulation of interferon-alpha (IFN-alpha) receptor expression in metastatic renal cell carcinoma (RCC) after IFN-alpha administration. Blood sampling was carried out in eight patients with metastatic RCC and six healthy volunteers. Flow-cytometric analysis using a monoclonal antibody against the active subunit of the type-I IFN-alpha receptor (IFNAR2) was carried out to examine the circadian rhythm of IFNAR2 expression in peripheral blood mononuclear cells (PBMC) as well as its downregulation after IFN-alpha administration. According to its circadian rhythm IFNAR2 in PBMC had a peak expression at night. Once IFN-alpha is administered, IFNAR2 levels in PBMC showed downregulation within 48 h and recovered within another 48 h. Our findings might support the establishment of an optimal schedule for IFN-alpha administration.

HPV-18 confers resistance to TNF-{alpha} in organotypic cultures of human keratinocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boccardo, Enrique; Noya, Francisco; Broker, Thomas R.

2004-10-25

The proinflammatory cytokine tumor necrosis factor-alpha (TNF-{alpha}) inhibits normal keratinocytes proliferation. However, many human papillomavirus (HPV)-immortalized or transformed cell lines are resistant to TNF-{alpha} antiproliferative effect. The present study analyzes the effects of TNF-{alpha} on organotypic cultures of primary human keratinocytes (PHKs) that express HPV-18 oncogenes. Raft cultures prepared with PHKs acutely transfected with HPV-18 whole genome or infected with recombinant retroviruses containing only E6/E7 or E7 were treated with 2 nM TNF-{alpha}. While BrdU incorporation into basal/parabasal cells of normal PHKs cultures was markedly inhibited by TNF-{alpha} cultures transfected with HPV-18 whole genome showed proliferation in all cell strata.more » Furthermore, BrdU incorporation into cultures expressing E6/E7 or E7 was not significantly reduced, indicating that E7 alone confers partial resistance to TNF-{alpha}. Besides, TNF-{alpha} treatment did not alter p16{sup ink4a}, p21{sup cip1}, p27{sup kip1}, or cyclin E levels, but did reduce cyclin A and PCNA levels in sensitive cells.« less

[Aromatase activities of endometrial carcinomas and both basic and clinical analyses of endometrial hyperplasia as a premalignant disease].

PubMed

Sasaki, H

1993-08-01

Paraffin-embedded materials obtained from 117 cases of endometrial hyperplasia and 84 cases of carcinoma were used for measurement of both ki-ras and p53 gene mutation and aromatase (ARO) and TGF-alpha immunostaining. The overall incidence of ki-ras mutations in the hyperplasia specimens (16%) was similar to the incidence detected in carcinomas (18%). None of 117 endometrial hyperplasias were found to have mutations in the p53 gene, whereas mutations were seen in 3 (13.3%) endometrial carcinomas. The intensity of both ARO and TGF-alpha immunostaining was increased in glands of both hyperplasia and carcinoma, and also in the interstitium of carcinoma. The positive sites of both ARO and TGF-alpha were almost the same, with an incidence below 40% in both hyperplasias and carcinomas. The cultured cells of endometrial carcinoma showed aromatase activity below MCF-7 cells, because testosterone was converted to estradiol (E2). TGF-alpha induced cell growth with at an optimal concentration. In HEC-59 cells, TGF-alpha increased both ARO-activity and mRNA. Some promoters on ARO-exon 1 in HEC-59 cells were different from those in BeWo cells. Progesterone inhibited the E2-induced excretion of pre TGF-alpha in endometrial carcinoma cells. These findings suggest that endometrial hyperplasia can be a premalignant condition of carcinoma, and can be initiated by both ki-ras codon 12 mutation and abnormal activity of ARO induced by TGF-alpha. In addition, HEC-59 cells may possess autocrine/paracrine properties involving ARO, E2 and TGF-alpha.

Expression of {beta}{sub 1} integrins in human endometrial stromal and decidual cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shiokawa, Shigetatsu; Yoshimura, Yasunori; Nakamura, Yukio

The present study was undertaken to investigate the expression of {beta}{sub 1} integrins in human endometrium and decidua using flow cytometry, immunohistochemistry, and immunoprecipitation. Fluorescence-activated flow cytometry demonstrated the greater expression of the {beta}{sub 1}, {alpha}{sub 1}, {alpha}{sub 2}, and {alpha}{sub 5} subunits of the {beta}{sub 1} integrin family in cultured stromal cells from the midsecretory phase, than in those of the early proliferative phase. The addition of estradiol (E{sub 2}) and progesterone (P) to cultured stromal cells in the early proliferative phase increased the expression of {beta}{sub 1} integrins in vitro. Flow cytometry also demonstrated the expression of themore » {beta}{sub 1}, {alpha}{sub 1}, {alpha}{sub 2}, {alpha}{sub 3}, {alpha}{sub 5}, and {alpha}{sub 6} subunits of {beta}{sub 1} integrin family in cultured decidual cells, and the enriched-fraction of prolactin (PRL)-producing decidual cells isolated by Percoll gradients showed high levels of {beta}{sub 1} integrins expression. Immunohistochemistry confirmed the {beta}{sub 1} integrin cell surface phenotypes in cultured decidual cells observed by flow cytometry. In summary, the present study demonstrated that endometrial stromal and decidual cells expressed {beta}{sub 1} integrin subunits at their surfaces. The expression exhibited a variability throughout the menstrual cycles, being predominantly detected in the secretory phase, and was maintained highly in the decidua. Thus, {beta}{sub 1} integrins in human endometrium and decidua may be important in mediating the organization of extracellular matrix proteins derived from embryos during the early stage of implantation. 43 refs., 7 figs., 2 tabs.« less

Impact of inflammation on male fertility.

PubMed

Sarkar, Oli; Bahrainwala, Jamila; Chandrasekaran, Sambamurthy; Kothari, Shiva; Mathur, Premendu P; Agarwal, Ashok

2011-01-01

The male uro-genital tract is susceptible to gram-negative bacterial infections that produce a state of inflammation, particularly in the testis and epididymis. Development of germline stem cells into motile spermatozoa takes place in these organs and thus any impairment therein has a direct effect on male fertility. A number of factors are known to impair male fertility including environmental and chemical factors, lifestyle, and infections. The last is a little-known and poorly understood cause of male sub-/infertility. The presence of the pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF- alpha), interleukin-1alpha (IL-1alpha) and interleukin-1beta (IL-1beta) in the male uro-genital tract following bacterial infections suggests that such infections could have cytokine-mediated anti-fertility effects. Furthermore, inflammation has been associated with elevated levels of reactive oxygen species and oxidative stress both of which affect male fertility. The present article summarizes the effects of inflammation on the testis, epididymis and spermatozoa. We review the correlations between inflammation and oxidative stress vis-à-vis spermatogenesis and discuss the implications of infections on male fertility/infertility and assisted reproductive technologies for the male.

Successive Intramuscular Boosting with IFN-Alpha Protects Mycobacterium bovis BCG-Vaccinated Mice against M. lepraemurium Infection

PubMed Central

Guerrero, G. G.; Rangel-Moreno, J.; Islas-Trujillo, S.; Rojas-Espinosa, Ó.

2015-01-01

Leprosy caused by Mycobacterium leprae primarily affects the skin and peripheral nerves. As a human infectious disease, it is still a significant health and economic burden on developing countries. Although multidrug therapy is reducing the number of active cases to approximately 0.5 million, the number of cases per year is not declining. Therefore, alternative host-directed strategies should be addressed to improve treatment efficacy and outcome. In this work, using murine leprosy as a model, a very similar granulomatous skin lesion to human leprosy, we have found that successive IFN-alpha boosting protects BCG-vaccinated mice against M. lepraemurium infection. No difference in the seric isotype and all IgG subclasses measured, neither in the TH1 nor in the TH2 type cytokine production, was seen. However, an enhanced iNOS/NO production in BCG-vaccinated/i.m. IFN-alpha boosted mice was observed. The data provided in this study suggest a promising use for IFN-alpha boosting as a new prophylactic alternative to be explored in human leprosy by targeting host innate cell response. PMID:26484351

Activation of mouse and human peroxisome proliferator-activated receptor-alpha (PPARα) by perfluoroalkyl acids (PFAAs) of 5, 7, 8, 11, and 12 carbon chain lengths in C05-1 cells.

EPA Science Inventory

PFAAs are surfactants that have been found globally in the environment and in tissues of humans and wildlife. They adversely affect perinatal survival and development in rodents and PPARα is involved in inducing these effects. Our previous study demonstrated that some PFAAs activ...

beta subunits influence the biophysical and pharmacological differences between P- and Q-type calcium currents expressed in a mammalian cell line.

PubMed

Moreno, H; Rudy, B; Llinás, R

1997-12-09

Human epithelial kidney cells (HEK) were prepared to coexpress alpha1A, alpha2delta with different beta calcium channel subunits and green fluorescence protein. To compare the calcium currents observed in these cells with the native neuronal currents, electrophysiological and pharmacological tools were used conjointly. Whole-cell current recordings of human epithelial kidney alpha1A-transfected cells showed small inactivating currents in 80 mM Ba2+ that were relatively insensitive to calcium blockers. Coexpression of alpha1A, betaIb, and alpha2delta produced a robust inactivating current detected in 10 mM Ba2+, reversibly blockable with low concentration of omega-agatoxin IVA (omega-Aga IVA) or synthetic funnel-web spider toxin (sFTX). Barium currents were also supported by alpha1A, beta2a, alpha2delta subunits, which demonstrated the slowest inactivation and were relatively insensitive to omega-Aga IVA and sFTX. Coexpression of beta3 with the same combination as above produced inactivating currents also insensitive to low concentration of omega-Aga IVA and sFTX. These data indicate that the combination alpha1A, betaIb, alpha2delta best resembles P-type channels given the rate of inactivation and the high sensitivity to omega-Aga IVA and sFTX. More importantly, the specificity of the channel blocker is highly influenced by the beta subunit associated with the alpha1A subunit.

Effect of transforming growth factor-alpha on enterocyte apoptosis is correlated with EGF receptor expression along the villus-crypt axis during methotrexate-induced intestinal mucositis in a rat.

PubMed

Sukhotnik, Igor; Shteinberg, Dan; Ben Lulu, Shani; Bashenko, Yulia; Mogilner, Jorge G; Ure, Benno M; Shaoul, Ron; Coran, Arnold G

2008-11-01

The purpose of the present study was to evaluate the effect of transforming-growth factor-alpha (TGF-alpha) on enterocyte apoptosis following methotrexate (MTX) induced intestinal mucositis in a rat and in Caco-2 cells. Non-pretreated and pretreated with MTX Caco-2 cells were incubated with increasing concentrations of TGF-alpha. Cell apoptosis was determined by FACS cytometry. Adult rats were divided into four groups: Control, Control-TGF-alpha, MTX, and MTX- TGF-alpha rats. Three days later rats were sacrificed. Enterocyte apoptosis were measured at sacrifice. RT-PCR and Western Blotting was used to determine the level of Bax and Bcl-2 mRNA and protein. Real time PCR was used to measure epidermal growth factor receptor (EGFr) expression along the villus-crypt axis. The in vitro experiment has shown that treatment with TGF-alpha of Caco-2 cells results in a significant inhibition of cell apoptosis in a dose-dependent manner. In vivo experiment, a decreased levels of apoptosis in MTX- TGF-alpha rats corresponded with the decrease in Bax and with the increase in Bcl-2 at both mRNA and protein levels. The inhibiting effect of TGF-alpha on enterocyte apoptosis was strongly correlated with EGFr expression along the villus-crypt axis. In conclusion, treatment with TGF-alpha inhibits enterocyte apoptosis following MTX- injury in the rat.

IgG1 antimycobacterial antibodies can reverse the inhibitory effect of pentoxifylline on tumour necrosis factor alpha (TNF-alpha) secreted by mycobacterial antigen-stimulated adherent cells.

PubMed

Thakurdas, S M; Hasan, Z; Hussain, R

2004-05-01

Chronic inflammation associated with cachexia, weight loss, fever and arthralgia is the hallmark of advanced mycobacterial diseases. These symptoms are attributed to the chronic stimulation of tumour necrosis factor (TNF)-alpha. Mycobacterial components directly stimulate adherent cells to secrete TNF-alpha. We have shown recently that IgG1 antimycobacterial antibodies play a role in augmenting TNF-alpha in purified protein derivative (PPD)-stimulated adherent cells from non-BCG-vaccinated donors. We now show that IgG1 antibodies can also augment TNF-alpha expression in stimulated adherent cells obtained from BCG-vaccinated donors and this augmentation is not linked to interleukin (IL)-10 secretion. In addition IgG1 antimycobacterial antibodies can reverse the effect of TNF-alpha blockers such as pentoxifylline and thalidomide. These studies therefore have clinical implications for anti-inflammatory drug treatments which are used increasingly to alleviate symptoms associated with chronic inflammation.

Antisense oligodeoxynucleotide inhibition of a swelling-activated cation channel in osteoblast-like osteosarcoma cells

NASA Technical Reports Server (NTRS)

Duncan, R. L.; Kizer, N.; Barry, E. L.; Friedman, P. A.; Hruska, K. A.

1996-01-01

By patch-clamp analysis, we have shown that chronic, intermittent mechanical strain (CMS) increases the activity of stretch-activated cation channels of osteoblast-like UMR-106.01 cells. CMS also produces a swelling-activated whole-cell conductance (Gm) regulated by varying strain levels. We questioned whether the swelling-activated conductance was produced by stretch-activated cation channel activity. We have identified a gene involved in the increase in conductance by using antisense oligodeoxynucleotides (ODN) derived from the alpha 1-subunit genes of calcium channels found in UMR-106.01 cells (alpha1S, alpha1C, and alpha1D). We demonstrate that alpha 1C antisense ODNs abolish the increase in Gm in response to hypotonic swelling following CMS. Antisense ODNs to alpha1S and alpha1D, sense ODNs to alpha1C, and sham permeabilization had no effect on the conductance increase. In addition, during cell-attached patch-clamp studies, antisense ODNs to alpha1c completely blocked the swelling-activated and stretch-activated nonselective cation channel response to strain. Antisense ODNs to alpha1S treatment produced no effect on either swelling-activated or stretch-activated cation channel activity. There were differences in the stretch-activated and swelling-activated cation channel activity, but whether they represent different channels could not be determined from our data. Our data indicate that the alpha1C gene product is involved in the Gm and the activation of the swelling-activated cation channels induced by CMS. The possibility that swelling-activated cation channel genes are members of the calcium channel superfamily exists, but if alpha1c is not the swelling-activated cation channel itself, then its expression is required for induction of swelling-activated cation channel activity by CMS.

Inhibition of TNF-alpha-induced NF-kappaB activation and IL-8 release in A549 cells with the proteasome inhibitor MG-132.

PubMed

Fiedler, M A; Wernke-Dollries, K; Stark, J M

1998-08-01

The working hypothesis of the studies described herein was that inhibition of proteasome-mediated IkappaB degradation would inhibit TNF-alpha-induced nuclear factor-kappaB (NF-kappaB) activation, interleukin-8 (IL-8) gene transcription, and IL-8 protein release in A549 cells. Mutational analysis of the 5' flanking region of the IL-8 gene confirmed that an intact NF-kappaB site is necessary for TNF-alpha-induced IL-8 gene transcription. The addition of TNF-alpha to A549 cells resulted in rapid loss of IkappaB from the cytoplasm of cells, associated with a corresponding increase in NF-kappaB-binding activity in nuclear extracts from the cells. However, pretreatment of the cells with the proteasome inhibitor N-cbz-Leu-Leu-leucinal (MG-132, 10 microM) reversed the effects of TNF-alpha on IL-8 release from A549 cells (as determined with an enzyme-linked immunosorbent assay [ELISA]) and on IL-8 gene transcription (as determined with reporter-gene assays). MG-132 reversed the effects of TNF-alpha on IkappaB degradation as determined by Western blot analysis. IkappaB phosphorylation and ubiquination were not altered by MG-132, which implies that the effects of MG-132 were secondary to proteasome inhibition. MG-132 also reversed the increase in NF-kappaB binding in nuclear extracts from TNF-alpha-treated cells. These studies show that inhibition of proteasome-mediated IkappaB degradation results in inhibition of TNF-alpha induced IL-8 production in A549 cells by limiting NF-kappaB-mediated gene transcription.

Control of proliferating potential of myeloid leukemia cells during long-term treatment with vitamin D3 analogues and other differentiation inducers in combination with antileukemic drugs: in vitro and in vivo studies.

PubMed

Kasukabe, T; Honma, Y; Hozumi, M; Suda, T; Nishii, Y

1987-01-15

Growth inhibition of murine and human myeloid leukemia cells by differentiation inducers during long-term culture was examined to improve the strategy for therapy of myeloid leukemia by differentiation inducers. When the effect of 1 alpha,25-dihydroxyvitamin D3, a typical differentiation inducer, on proliferation of mouse myeloid leukemia M1 cells was examined at a constant product of time and concentration (480 nM in 20 days), the continuous treatment with 24 nM 1 alpha,25-dihydroxyvitamin D3 was the most effective for inhibition of cell proliferation. After 20 days, the cumulative cell number was reduced about 3 X 10(5) times by continuous treatment with 24 nM 1 alpha,25-dihydroxyvitamin D3. Similar results were obtained when M1 cells were treated continuously with dexamethasone. M1 cells resistant to 1 alpha,25-dihydroxyvitamin D3 appeared about 25 days after the start of continuous treatment with 24 nM 1 alpha,25-dihydroxyvitamin D3. On the other hand, when M1 cells were treated continuously with 1 alpha,25-dihydroxyvitamin D3 and noncytotoxic doses of antileukemic drugs such as 1-beta-D-arabinofuranosylcytosine and daunomycin, resistant cells did not appear for at least 35 days. A similar effect of 1 alpha,25-dihydroxyvitamin D3 and antileukemic drugs on cell proliferation was observed with the human monoblast-like cell line U937. The survival of syngeneic SL mice inoculated with M1 cells was prolonged more by treatment with both 1 alpha-hydroxyvitamin D3 and daunomycin than by treatment with either drug alone. These results suggest that continuous treatment with both differentiation inducers and certain antileukemic drugs may be more effective therapeutically than treatment with a differentiation inducer alone.

Radioresistance in murine solid tumors induced by interleukin-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Braunschweiger, P.G.; Basrur, V.; Santos, O.

1996-02-01

Interleukin-1 (IL-1) has radioprotective activity in hematopoietic lineages and in other normal cell renewal systems, but little is known about the effects of IL-1{alpha} on the radiosensitivity of tumor cell populations. The present studies were conducted to investigate the effects of IL-1{alpha} on the radiosensitivity of clonogenic cells in RIF-1 and SCC-7 tumors. Radioresistance was detected within 2-4 h after administration of IL-1{alpha} (0.5 {mu}g/mouse, ip) and characterized by increases in D{sub 0}, D{sub q}, {alpha}/{Beta} and SF2. This radioresistance was similar to that seen in tumors rendered totally hypoxic before X irradiation. Tirapazamine, a hypoxic cell cytotoxin, and IL-1{alpha}more » had synergistic schedule-dependent antitumor activity in vivo, suggesting that IL-1-induced radioresistance in vivo is due to hypoxia. Radioresistance induced by IL-1{alpha} was transient, and the data suggested reoxygenation within 12 h. In vitro, IL-1{alpha} had no direct effect on the radiosensitivity of SCC-7 cells in tissue culture under aerobic conditions. However, an increase in D{sub 0}, {alpha}/{Beta} and SF2 was seen in clonogenic tumor cells from primary cultures treated with IL-1{alpha} under aerobic conditions. Superoxide dismutase and catalase prevented the induction of radioresistance by IL-1{alpha} in vitro, suggesting that oxidative responses from tumor macrophages after administration of IL-1{alpha} may be responsible for induced radioresistance by IL-1 in vitro. Although oxidant stress induced by IL-1 may play an important role in the activity of IL-1{alpha} both in vivo and in vitro in our models, the mechanisms by which such responses modulate tumor radiosensitivity in vivo and in vitro are likely quite different. 32 refs., 6 figs., 1 tab.« less

Peroxisome proliferator-activated receptor gamma coactivator-1 alpha acts as a tumor suppressor in hepatocellular carcinoma.

PubMed

Liu, Rui; Zhang, Haiyang; Zhang, Yan; Li, Shuang; Wang, Xinyi; Wang, Xia; Wang, Cheng; Liu, Bin; Zen, Ke; Zhang, Chen-Yu; Zhang, Chunni; Ba, Yi

2017-04-01

Peroxisome proliferator-activated receptor gamma coactivator-1 alpha plays a crucial role in regulating the biosynthesis of mitochondria, which is closely linked to the energy metabolism in various tumors. This study investigated the regulatory role of peroxisome proliferator-activated receptor gamma coactivator-1 alpha in the pathogenesis of hepatocellular carcinoma. In this study, the changes of peroxisome proliferator-activated receptor gamma coactivator-1 alpha messenger RNA levels between normal human liver and hepatocellular carcinoma tissue were examined by quantitative reverse transcription polymerase chain reaction. Knockdown of peroxisome proliferator-activated receptor gamma coactivator-1 alpha was conducted by RNA interference in the human liver cell line L02, while overexpression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha was conducted by adenovirus encoding peroxisome proliferator-activated receptor gamma coactivator-1 alpha complementary DNA in the human hepatocarcinoma cell line HepG2. Cellular morphological changes were observed via optical and electron microscopy. Cellular apoptosis was determined by Hoechst 33258 staining. In addition, the expression levels of 21,400 genes in tissues and cells were detected by microarray. It was shown that peroxisome proliferator-activated receptor gamma coactivator-1 alpha expression was significantly downregulated in hepatocellular carcinoma compared with normal liver tissues. After knockdown of peroxisome proliferator-activated receptor gamma coactivator-1 alpha expression in L02 cells, cells reverted to immature and dedifferentiated morphology exhibiting cancerous tendency. Apoptosis occurred in the HepG2 cells after transfection by adenovirus encoding peroxisome proliferator-activated receptor gamma coactivator-1 alpha. Microarray analysis showed consistent results. The results suggest that peroxisome proliferator-activated receptor gamma coactivator-1 alpha acts as a tumor suppressor in the formation and development of hepatocellular carcinoma and that peroxisome proliferator-activated receptor gamma coactivator-1 alpha may be a potential therapeutic target for hepatocellular carcinoma.

Transient maintenance in bioreactor improves health of neuronal cells.

PubMed

Di Loreto, Silvia; Sebastiani, Pierluigi; Benedetti, Elisabetta; Zimmitti, Vincenzo; Caracciolo, Valentina; Amicarelli, Fernanda; Cimini, Annamaria; Adorno, Domenico

2006-01-01

To examine whether a neuronal cell suspension can be held in vitro for a relatively short period without compromising survival rates and functionality, we have set up an experimental protocol planning 24 h of suspension culture in a rotary wall vessel (RWV) bioreactor before plating in a conventional adherent system. Apoptosis measurement and activated caspase-8, -9, and -3 detection have demonstrated that survey of the cells was not affected. The activity of major antioxidant enzymes (AOE), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), was significantly decreased in RWV-maintained cells. A significant decrease of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) is coupled with a level of activated nuclear factor-kappaB (NF-kappaB) protein significantly lower in RVW cells than in the control. On the contrary, the level of IL-6 expression did not change between the test and the control. A significant up-regulation of growth-associated protein-43 (GAP-43), peroxisome proliferator-activated receptor-beta/delta (PPARbeta/delta), and acyl-CoA synthetase 2 (ACS2) in RWV cells has been detected. We provide the evidence that primary neuronal cells, at an early stage of development, can be maintained in a suspension condition before adherent plating. This experimental environment does not induce detrimental effects but may have an activator role, leading cells to development and maturation in a tridimensional state.

Mutations in the estrogen receptor alpha hormone binding domain promote stem cell phenotype through notch activation in breast cancer cell lines.

PubMed

Gelsomino, L; Panza, S; Giordano, C; Barone, I; Gu, G; Spina, E; Catalano, S; Fuqua, S; Andò, S

2018-04-24

The detection of recurrent mutations affecting the hormone binding domain (HBD) of estrogen receptor alpha (ERα/ESR1) in endocrine therapy-resistant and metastatic breast cancers has prompted interest in functional characterization of these genetic alterations. Here, we explored the role of HBD-ESR1 mutations in influencing the behavior of breast cancer stem cells (BCSCs), using various BC cell lines stably expressing wild-type or mutant (Y537 N, Y537S, D538G) ERα. Compared to WT-ERα clones, mutant cells showed increased CD44 + /CD24 - ratio, mRNA levels of stemness genes, Mammosphere Forming Efficiency (MFE), Self-Renewal and migratory capabilities. Mutant clones exhibited high expression of NOTCH receptors/ligands/target genes and blockade of NOTCH signaling reduced MFE and migratory potential. Mutant BCSC activity was dependent on ERα phosphorylation at serine 118, since its inhibition decreased MFE and NOTCH4 activation only in mutant cells. Collectively, we demonstrate that the expression of HBD-ESR1 mutations may drive BC cells to acquire stem cell traits through ER/NOTCH4 interplay. We propose the early detection of HBD-ESR1 mutations as a challenge in precision medicine strategy, suggesting the development of tailored-approaches (i.e. NOTCH inhibitors) to prevent disease development and metastatic spread in BC mutant-positive patients. Copyright © 2018 Elsevier B.V. All rights reserved.

Anti-fibrotic effects of thalidomide on hepatic stellate cells and dimethylnitrosamine-intoxicated rats.

PubMed

Chong, Lee-Won; Hsu, Yi-Chao; Chiu, Yung-Tsung; Yang, Kuo-Ching; Huang, Yi-Tsau

2006-05-01

Tumor necrosis factor-alpha (TNF-alpha) plays a central role in cellular necrosis, apoptosis, organ failure, tissue damage, inflammation and fibrosis. These processes, occurring in liver injury, may lead to cirrhosis. Thalidomide, alpha-N-phthalidoglutarimide, (C(13)H(10)N(2))(4), has been shown to have immunomodulatory and anti-inflammatory properties, possibly mediated through its anti-TNF-alpha effect. In this study, we investigated the in vitro and in vivo effects of thalidomide on hepatic fibrosis. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with transforming growth factor-beta1 (TGF-beta1) or TNF-alpha. The inhibitory effects of thalidomide on the NFkappaB signaling cascade and fibrosis markers including alpha-smooth muscle actin (alpha-SMA) and collagen, were assessed. An in vivo therapeutic study was conducted in dimethylnitrosamine (DMN)-treated rats, which were randomly assigned to 1 of 4 groups: vehicle (0.7% carboxyl methyl cellulose, CMC), thalidomide (40 mg/kg), thalidomide (200 mg/kg), or silymarin (50 mg/kg), each given by gavage twice daily for 3 weeks starting after 1 week of DMN administration. Thalidomide (100-800 nM) concentration-dependently inhibited NFkappaB transcriptional activity induced by TNF-alpha, including IKKalpha expression and IkappaBalpha phosphorylation in HSC-T6 cells. In addition, thalidomide also suppressed TGF-beta1-induced alpha-SMA expression and collagen deposition in HSC-T6 cells. Fibrosis scores of livers from DMN-treated rats receiving high dose of thalidomide (0.89 +/- 0.20) were significantly reduced in comparison with those of DMN-treated rats receiving vehicle (1.56 +/- 0.18). Hepatic collagen contents of DMN rats were also significantly reduced by either thalidomide or silymarin treatment. Immunohistochemical double staining results showed that alpha-SMA- and NFkappaB-positive cells were decreased in the livers from DMN rats receiving either thalidomide or silymarin treatment. In addition, real-time PCR analysis indicated that hepatic mRNA expressions of TGF-beta1, alpha-SMA, collagen 1alpha2, TNF-alpha and iNOS genes were attenuated by thalidomide treatment. In conclusion, our results showed that thalidomide inhibited activation of HSC-T6 cells by TNF-alpha and ameliorated liver fibrosis in DMN-intoxicated rats.

Impaired plasmacytoid dendritic cell (PDC)-NK cell activity in viremic human immunodeficiency virus infection attributable to impairments in both PDC and NK cell function.

PubMed

Conry, Sara J; Milkovich, Kimberly A; Yonkers, Nicole L; Rodriguez, Benigno; Bernstein, Helene B; Asaad, Robert; Heinzel, Frederick P; Tary-Lehmann, Magdalena; Lederman, Michael M; Anthony, Donald D

2009-11-01

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections impair plasmacytoid dendritic cell (PDC) and natural killer (NK) cell subset numbers and functions, though little is known about PDC-NK cell interactions during these infections. We evaluated PDC-dependent NK cell killing and gamma interferon (IFN-gamma) and granzyme B production, using peripheral blood mononuclear cell (PBMC)-based and purified cell assays of samples from HCV- and HIV-infected subjects. CpG-enhanced PBMC killing and IFN-gamma and granzyme B activity (dependent on PDC and NK cells) were impaired in viremic HIV infection. In purified PDC-NK cell culture experiments, CpG-enhanced, PDC-dependent NK cell activity was cell contact and IFN-alpha dependent, and this activity was impaired in viremic HIV infection but not in HCV infection. In heterologous PDC-NK cell assays, impaired PDC-NK cell killing activity was largely attributable to an NK cell defect, while impaired PDC-NK cell IFN-gamma-producing activity was attributable to both PDC and NK cell defects. Additionally, the response of NK cells to direct IFN-alpha stimulation was defective in viremic HIV infection, and this defect was not attributable to diminished IFN-alpha receptor expression, though IFN-alpha receptor and NKP30 expression was closely associated with killer activity in viremic HIV infection but not in healthy controls. These data indicate that during uncontrolled HIV infection, PDC-dependent NK cell function is impaired, which is in large part attributable to defective IFN-alpha-induced NK cell activity and not to altered IFN-alpha receptor, NKP30, NKP44, NKP46, or NKG2D expression.

NMDA receptor dependent PGC-1alpha up-regulation protects the cortical neuron against oxygen-glucose deprivation/reperfusion injury.

PubMed

Luo, Yun; Zhu, Wenjing; Jia, Jia; Zhang, Chenyu; Xu, Yun

2009-09-01

The peroxisome proliferator activated receptor coactivator 1 alpha (PGC-1alpha) is a nuclear transcriptional coactivator that is widely expressed in the brain areas. Over-expression of PGC-1alpha can protect neuronal cells from oxidant-induced injury. The purpose of the current study is to investigate the role of PGC-1alpha in the oxygen (anoxia) deprivation (OGD) neurons. The PGC-1alpha mRNA and protein level between control and OGD neurons were examined by real-time PCR and Western blot. More PGC-1alpha expression was found in the OGD neurons compared with the normal group. Over-expression of PGC-1alpha suppressed cell apoptosis while inhibition of the PGC-1alpha expression induced cell apoptosis in OGD neurons. Furthermore, increase of PGC-1alpha resulted in activation of N-methyl-D-aspartate (NMDA) receptor, p38, and ERK mitogen-activated protein kinase (MAPK) pathway. The blocking of the NMDA receptor by its antagonists MK-801 reduced PGC-1alpha mRNA expression in OGD neurons, while NMDA itself can directly induce the expression of PGC-1alpha in neuronal cells. At the same time, PD98059 (ERK MAPK inhibitor) and SB203580 (P38 MAPK inhibitor) also prevented the up-regulation of PGC-1alpha in OGD neurons and MK801 can inhibit the expression of P38 and ERK MAPK. These data suggested that the expression of PGC-1alpha was up-regulated in OGD mice cortical neurons, which protected the neurons against OGD injury. Moreover, this effect was correlated to the NMDA receptor and the ERK and P38 MAPK pathway. The protective effect of PGC-1alpha on OGD cortical neurons may be useful for stroke therapy.

Functional assessment of recombinant human alpha(2)-adrenoceptor subtypes with cytosensor microphysiometry.

PubMed

Pihlavisto, M; Scheinin, M

1999-12-03

We applied the Cytosensor Microphysiometry system to study the three human alpha(2)-adrenoceptor subtypes, alpha(2A), alpha(2B) and alpha(2C), expressed in Chinese hamster ovary (CHO) cells, and assessed its potential in the quantitative monitoring of agonist activity. The natural full agonist, (-)-noradrenaline, was used to define agonist efficacy. The imidazole derivative dexmedetomidine was a potent full agonist of all three receptor subtypes. The imidazolines clonidine and UK 14,304 (5-bromo-N-(4, 5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine) appeared to be partial agonists at alpha(2B)-adrenoceptors (E(max) approximately 60% of (-)-noradrenaline) but full agonists at alpha(2A)- and alpha(2C)-adrenoceptors. The responses mediated by all three alpha(2)-adrenoceptor subtypes were partly inhibited by the sodium-hydrogen (Na(+)/H(+)) exchange inhibitor, MIA (5-(N-methyl-N-isobutyl)-amiloride). The agonist responses were totally abolished by pretreatment with pertussis toxin in cells with alpha(2A)- and alpha(2C)-adrenoceptors, and partly abolished in cells with alpha(2B)-adrenoceptors. The residual signal in alpha(2B)-cells was sensitive to the intracellular Ca(2+)chelator, BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester). Cholera toxin (which acts on G(s)-proteins) had no effect on the agonist responses. The results suggest that the extracellular acidification responses mediated by all three human alpha(2)-adrenoceptor subtypes are dependent on Na(+)/H(+)exchange and G(i/o) pathways, and that alpha(2B)-adrenoceptors are capable of coupling to another, G(i/o)-independent and Ca(2+)-dependent signaling pathway.

Vitamin D 1alpha-hydroxylase (CYP1alpha) polymorphism in Graves' disease, Hashimoto's thyroiditis and type 1 diabetes mellitus.

PubMed

Pani, Michael A; Regulla, Karoline; Segni, Maria; Krause, Maren; Hofmann, Stefan; Hufner, Michael; Herwig, Jurgen; Pasquino, Anna Maria; Usadel, Klaus-H; Badenhoop, Klaus

2002-06-01

The vitamin D endocrine system plays a role in the regulation of (auto)immunity and cell proliferation. Vitamin D 1alpha-hydroxylase (CYP1alpha) is one of the key enzymes regulating both systemic and tissue levels of 1,25-dihyroxyvitamin D(3) (1,25(OH)(2)D(3)). Administration of 1,25(OH)(2)D(3), whose serum levels were found to be reduced in type 1 diabetes and thyroid autoimmunity, prevents these diseases in animal models. We therefore investigated a recently reported CYP1alpha polymorphism for an association with type 1 diabetes mellitus, Graves' disease and Hashimoto's thyroiditis. Four hundred and seven Caucasian pedigrees with one offspring affected by either type 1 diabetes (209 families), Graves' disease (92 families) or Hashimoto's thyroiditis (106 families) were genotyped for a C/T polymorphism in intron 6 of the CYP1alpha gene on chromosome 12q13.1-13.3 and transmission disequilibrium testing (TDT) was performed. Subsets of affected offspring stratified for HLA-DQ haplotype were compared using chi(2) testing. There was no deviation from the expected transmission frequency in either type 1 diabetes mellitus (P=0.825), Graves' disease (P=0.909) or Hashimoto's thyroiditis (P=0.204). However, in Hashimoto's thyroiditis the CYP1alpha C allele was significantly more often transmitted to HLA-DQ2(-) patients (27 transmitted vs 14 not transmitted; TDT: P=0.042) than expected. The C allele was less often transmitted to HLA-DQ2(+) patients (9 transmitted vs 12 not transmitted; TDT: P=0.513), although the difference was not significant (chi(2) test: P=0.143). A similar difference was observed in type 1 diabetes between offspring with high and low risk HLA-DQ haplotypes (chi(2) test: P=0.095). The CYP1alpha intron 6 polymorphism appears not to be associated with type 1 diabetes mellitus, Graves' disease and Hashimoto's thyroiditis. A potential association in subsets of patients with type 1 diabetes and Hashimoto's thyroiditis should be further investigated as well as its functional implications.

[Cloning of Chinese Banna minipig inbred-line alpha1,3-galactosyltransferase gene and construction of its recombinant eukaryotic expression vector].

PubMed

Zhu, Shengming; Wang, Yanping; Zheng, Hong; Cheng, Jingqiu; Lu, Yanrong; Zeng, Yangzhi; Wang, Yu; Wang, Zhu

2009-04-01

This study sought to clone Chinese Banna minipig inbred-line (BMI) alpha1,3-galactosyltransferase (alpha1,3-GT) gene and construct its recombinant eukaryotic expression vector. Total RNA was isolated from BMI liver. Full length cDNA of alpha1,3-GT gene was amplified by RT-PCR and cloned into pMD18-T vector to sequence. Subsequently, alpha1,3-GT gene was inserted into pEGFP-N1 to construct eukaryotic expression vector pEGFP-N1-GT. Then the reconstructed plasmid pEGFP-N1-GT was transiently transfected into human lung cancer cell line A549. The expression of alpha1,3-GT mRNA in transfected cells was detected by RT-PCR. FITC-BS-IB4 lectin was used in the direct immunofluorescence method, which was performed to observe the alpha-Gal synthesis function of BMI alpha1,3-GT in transfected cells. The results showed that full length of BMI alpha1,3-GT cDNA was 1116 bp. BMI alpha1,3-GT cDNA sequence was highly homogenous with those of mouse and bovine, and was exactly the same as the complete sequence of those of swine, pEGFP-N1-GT was confirmed by enzyme digestion and PCR. The expression of alpha1,3-GT mRNA was detected in A549 cells transfected by pEGFP-N1-GT. The expression of alpha-Gal was observed on the membrane of A549 cells transfected by pEGFP-N1-GT. Successful cloning of BMI alpha1,3-GT cDNA and construction of its eukaryotic expression vector have established a foundation for further research and application of BMI alpha1,3-GT in the fields of xenotransplantation and immunological therapy of cancer.

Immune defects in families and patients with xeroderma pigmentosum and trichothiodystrophy.

PubMed Central

Mariani, E; Facchini, A; Honorati, M C; Lalli, E; Berardesca, E; Ghetti, P; Marinoni, S; Nuzzo, F; Astaldi Ricotti, G C; Stefanini, M

1992-01-01

Xeroderma pigmentosum (XP) is a rare autosomal recessive disease characterized by photosensitivity, a high incidence of cancer in sun-exposed portions of the skin and a reduced capacity to repair the u.v.-induced DNA damage. One of the XP mutations (XP-D) has also been identified in patients affected by trichothiodystrophy (TTD), a rare autosomal recessive disease characterized by brittle hair, mental and physical retardation, peculiar face and ichthyosis. However, in these patients there is no evidence of increased skin tumour incidence. Since an impairment of cell-mediated immunity has been proposed as a co-factor in the cancer proneness of XP patients, we investigated the involvement of immune defect(s) in five XP patients, five TTD patients, their parents, and 24 TTD relatives. We evaluated the phenotype of circulating lymphocytes, natural killer (NK) cell lytic activity, target cell binding of NK cells at single cell level and the effect of interferons (IFN) alpha and beta on NK cell activity. The relative proportion of CD3+ and CD4+ circulating lymphocytes was reduced in XP but not in TTD patients. NK cell lytic activity was decreased in XP patients and their mothers, but their fathers showed normal lytic activity. NK activity varied among TTD families: four out of five patients and their relatives presented low NK cell activity, and one family was normal. In TTD family members, NK activity increased after incubation with IFN-alpha or IFN-beta, but never reached normal values. In contrast, in XP patients and their mothers, the defect was almost completely corrected after in vitro incubation with IFN-alpha or IFN-beta. Our study indicates impaired NK lytic activity in the majority of TTD and XP patients and that this defect is present also in members of their families. In addition, XP patients present a low number of circulating T cells. These multiple abnormalities, together with DNA repair defects, could be related to the increased cancer risk in XP patients. PMID:1535035

Vascular endothelial cells express isoforms of protein kinase A inhibitor.

PubMed

Lum, Hazel; Hao, Zengping; Gayle, Dave; Kumar, Priyadarsini; Patterson, Carolyn E; Uhler, Michael D

2002-01-01

The expression and function of the endogenous inhibitor of cAMP-dependent protein kinase (PKI) in endothelial cells are unknown. In this study, overexpression of rabbit muscle PKI gene into endothelial cells inhibited the cAMP-mediated increase and exacerbated thrombin-induced decrease in endothelial barrier function. We investigated PKI expression in human pulmonary artery (HPAECs), foreskin microvessel (HMECs), and brain microvessel endothelial cells (HBMECs). RT-PCR using specific primers for human PKI alpha, human PKI gamma, and mouse PKI beta sequences detected PKI alpha and PKI gamma mRNA in all three cell types. Sequencing and BLAST analysis indicated that forward and reverse DNA strands for PKI alpha and PKI gamma were of >96% identity with database sequences. RNase protection assays showed protection of the 542 nucleotides in HBMEC and HPAEC PKI alpha mRNA and 240 nucleotides in HBMEC, HPAEC, and HMEC PKI gamma mRNA. Western blot analysis indicated that PKI gamma protein was detected in all three cell types, whereas PKI alpha was found in HBMECs. In summary, endothelial cells from three different vascular beds express PKI alpha and PKI gamma, which may be physiologically important in endothelial barrier function.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chan, R.K.; Otte, C.A.

Eight independently isolated mutants which are supersensitive (Sst/sup -/) to the G1 arrest induced by the tridecapeptide pheromone ..cap alpha.. factor were identified by screening mutagenized Saccharomyces cerevisiae MATa cells on solid medium for increased growth inhibition by ..cap alpha.. factor. These mutants carries lesions in two complementation groups, sst1 and sst2. Mutations at the sst1 locus were mating type specific: MATa sst1 cells were supersensitive to ..cap alpha.. factor, but MAT..cap alpha.. sst1 cells were not supersensitive to a factor. In contrast, mutations at the sst2 locus conferred supersensitivity to the pheromones of the opposite mating type on bothmore » MATa and MAT..cap alpha.. cells. Even in the absence of added ..cap alpha.. pheromone, about 10% of the cells in exponentially growing cultures of MATa strains carrying any of three different alleles of sst2 (including the ochre mutation sst2-4) had the aberrant morphology (''shmoo'' shape) that normally develops only after MATa cells are exposed to ..cap alpha.. factor. This ''self-shmooing'' phenotype was genetically linked to the sst2 mutations, although the leakiest allele isolated (sst2-3) did not display this characteristic. Normal MATa/MAT..cap alpha.. diploids do not respond to pheromones; diploids homozygous for an sst2 mutation (MATa/MAT..cap alpha.. sst2-1/sst2-1) were still insensitive to ..cap alpha.. factor. The sst1 gene was mapped to within 6.9 centimorgans of his6 on chromosome IX. The sst2 gene was unlinked to sst1, was not centromere linked, and was shown to be neither linked nor centromere distal to MAT on the right arm of chromosome III.« less

Biotin deficiency up-regulates TNF-alpha production in murine macrophages.

PubMed

Kuroishi, Toshinobu; Endo, Yasuo; Muramoto, Koji; Sugawara, Shunji

2008-04-01

Biotin, a water-soluble vitamin of the B complex, functions as a cofactor of carboxylases that catalyze an indispensable cellular metabolism. Although significant decreases in serum biotin levels have been reported in patients with chronic inflammatory diseases, the biological roles of biotin in inflammatory responses are unclear. In this study, we investigated the effects of biotin deficiency on TNF-alpha production. Mice were fed a basal diet or a biotin-deficient diet for 8 weeks. Serum biotin levels were significantly lower in biotin-deficient mice than biotin-sufficient mice. After i.v. administration of LPS, serum TNF-alpha levels were significantly higher in biotin-deficient mice than biotin-sufficient mice. A murine macrophage-like cell line, J774.1, was cultured in a biotin-sufficient or -deficient medium for 4 weeks. Cell proliferation and biotinylation of intracellular proteins were decreased significantly in biotin-deficient cells compared with biotin-sufficient cells. Significantly higher production and mRNA expression of TNF-alpha were detected in biotin-deficient J774.1 cells than biotin-sufficient cells in response to LPS and even without LPS stimulation. Intracellular TNF-alpha expression was inhibited by actinomycin D, indicating that biotin deficiency up-regulates TNF-alpha production at the transcriptional level. However, the expression levels of TNF receptors, CD14, and TLR4/myeloid differentiation protein 2 complex were similar between biotin-sufficient and -deficient cells. No differences were detected in the activities of the NF-kappaB family or AP-1. The TNF-alpha induction by biotin deficiency was down-regulated by biotin supplementation in vitro and in vivo. These results indicate that biotin deficiency may up-regulate TNF-alpha production or that biotin excess down-regulates TNF-alpha production, suggesting that biotin status may influence inflammatory diseases.

Carcinoembryonic antigen induces signal transduction in Kupffer cells.

PubMed

Gangopadhyay, A; Lazure, D A; Thomas, P

1997-09-16

Carcinoembryonic antigen (CEA), an intercellular adhesion molecule and a mediator of hepatic metastasis, is processed by an 80 kDa receptor on murine and human Kupffer cells in the liver. Activation of rat Kupffer cells in vitro by CEA via the 80 kDa receptor produced cytokines IL-1alpha and TNF-alpha which involved tyrosine phosphorylation. The peak response of TNF-alpha was 5.6 times greater than the corresponding IL-1alpha response and was associated with enhanced tyrosine phosphorylation of 108 and 125 kDa proteins. Lipopolysaccharide (LPS) treatment, on the other hand, phosphorylated two major proteins with MW of 93 and 119 kDa associated with the loss of phosphorylation from a 125 kDa protein. Results demonstrate that CEA-induced IL-1alpha and TNF-alpha production involves tyrosine phosphorylation and the signaling in CEA treated cells is different than that seen with LPS stimulation.

Expression of feline interferon-alpha subtypes in Esherichia coli, and their antiviral activity and animal species specificity.

PubMed

Taira, Osamu; Suzuki, Makoto; Takeuchi, Yuko; Aramaki, Yoshitaka; Sakurai, Itsuki; Watanabe, Takao; Motokawa, Kenji; Arai, Setsuo; Sato, Hisaaki; Maehara, Nobutoshi

2005-05-01

Two kinds of FeIFN-alpha consisting of 166 amino acids (aa) and 171 aa were expressed in Escherichia coli, and the purified proteins were tested for antiviral activity on homologous and heterologous animal cells. Crude FeIFN induced in feline cells revealed antiviral activity on both homologous and heterologous animal cells. In contrast, both types of recombinant FeIFN-alpha revealed antiviral activity only on the feline cells. All of the FeIFN-alpha subtypes showed high activity to vesicular stomatitis virus, and the three species of feline viruses belonging to different families.

Glutamine, glutamate, and other possible regulators of alpha-ketoglutarate and malate uptake by synaptic terminals.

PubMed

Shank, R P; Campbell, G L

1984-04-01

The uptake of alpha-ketoglutarate and malate by rat brain synaptosomal preparations was found to be affected by a variety of substances at physiologically relevant concentrations. Glutamine altered the uptake of alpha-ketoglutarate by causing an apparent reduction in the substrate-carrier affinity and an increase in Vmax. In contrast, glutamine did not appear to affect the Vmax of malate uptake, but it did increase markedly the uptake velocity at low concentrations of malate. L-Glutamate and L-aspartate were comparatively strong inhibitors of alpha-ketoglutarate and malate uptake. N-Acetylaspartate was a weak inhibitor of alpha-ketoglutarate uptake, a finding that contrasts with our previous observation that this compound potently inhibited alpha-ketoglutarate uptake by synaptosomes obtained from the cerebellum of 8- to 14-day-old mice. Ca2+ exhibited a variable effect but usually enhanced the uptake of alpha-ketoglutarate. The addition of small amounts of postmicrosomal supernatant to the incubation medium enhanced the uptake of alpha-ketoglutarate by low-density synaptosomes. By comparison, the uptake of glutamate, glutamine, gamma-aminobutyric acid, and several other amino acids was not affected. The enhancement of alpha-ketoglutarate uptake by the supernatant was due to a heat labile substance that was retained by dialysis tubing (MW cutoff = 8,000) and Amicon filter cones (CF 25), and was precipitated by ammonium sulfate at 60% saturation. In experiments in which the metabolic conversion of [U-14C] alpha-ketoglutarate to glutamate, aspartate, glutamine, and gamma-aminobutyric acid was determined, the presence of glutamine and glutamate in the incubation medium did not affect the pattern of labelling appreciably.

Equol an isoflavonoid: potential for improved prostate health, in vitro and in vivo evidence

PubMed Central

2011-01-01

Background To determine: in vitro binding affinity of equol for 5alpha-dihydrotestosterone (5alpha-DHT), in vitro effects of equol treatment in human prostate cancer (LNCap) cells, and in vivo effects of equol on rat prostate weight and circulating levels of sex steroid hormones. Methods First, in vitro equol binding affinity for 5alpha-DHT was determined using 14C5alpha-DHT combined with cold 5alpha-DHT (3.0 nM in all samples). These steroids were incubated with increasing concentrations of equol (0-2,000 nM) and analyzed by Sephadex LH-20 column chromatography. 14C5alpha-DHT peak/profiles were determined by scintillation counting of column fractions. Using the 14C5alpha-DHT peak (0 nM equol) as a reference standard, a binding curve was generated by quantifying shifts in the 14C5alpha-DHT peaks as equol concentrations increased. Second, equol's in vitro effects on LNCap cells were determined by culturing cells (48 hours) in the presence of increasing concentrations of dimethyl sulfoxide (DMSO) (vehicle-control), 5alpha-DHT, equol or 5alpha-DHT+equol. Following culture, prostate specific antigen (PSA) levels were quantified via ELISA. Finally, the in vivo effects of equol were tested in sixteen male Long-Evans rats fed a low isoflavone diet. From 190-215 days, animals received 0.1cc s.c. injections of either DMSO-control vehicle (n = 8) or 1.0 mg/kg (body weight) of equol (in DMSO) (n = 8). At 215 days, body and prostate weights were recorded, trunk blood was collected and serum assayed for luteinizing hormone (LH), 5alpha-DHT, testosterone and 17beta-estradiol levels. Results Maximum and half maximal equol binding to 5alpha-DHT occurred at approximately 100 nM and 4.8 nM respectively. LNCap cells cultured in the presence of 5alpha-DHT significantly increased PSA levels. However, in the presence of 5alpha-DHT+equol, equol blocked the significant increases in PSA levels from LNCap cells. In vivo equol treatment significantly decreased rat prostate weights and serum 5alpha-DHT levels but did not alter LH, testosterone, and estradiol levels. Conclusions Equol administration appears to have potential beneficial effects for prostate health and other 5alpha-DHT mediated disorders. Equol administration: reduces PSA levels from LNCap cells under 5alpha-DHT stimulation, decreases rat prostate size, decreases serum 5alpha-DHT levels and androgen hormone action, while not altering other circulating sex steroids or LH levels. PMID:21232127

Decorin causes autophagy in endothelial cells via Peg3

PubMed Central

Buraschi, Simone; Neill, Thomas; Goyal, Atul; Poluzzi, Chiara; Smythies, James; Owens, Rick T.; Schaefer, Liliana; Torres, Annabel; Iozzo, Renato V.

2013-01-01

Soluble decorin affects the biology of several receptor tyrosine kinases by triggering receptor internalization and degradation. We found that decorin induced paternally expressed gene 3 (Peg3), an imprinted tumor suppressor gene, and that Peg3 relocated into autophagosomes labeled by Beclin 1 and microtubule-associated light chain 3. Decorin evoked Peg3-dependent autophagy in both microvascular and macrovascular endothelial cells leading to suppression of angiogenesis. Peg3 coimmunoprecipitated with Beclin 1 and LC3 and was required for maintaining basal levels of Beclin 1. Decorin, via Peg3, induced transcription of Beclin 1 and microtubule-associated protein 1 light chain 3 alpha genes, thereby leading to a protracted autophagic program. Mechanistically, decorin interacted with VEGF receptor 2 (VEGFR2) in a region overlapping with its natural ligand VEGFA, and VEGFR2 was required for decorin-evoked Beclin 1 and microtubule-associated protein 1 light chain 3 alpha expression as well as for Peg3 induction in endothelial cells. Moreover, decorin induced VEGFR2-dependent mitochondrial fragmentation and loss of mitochondrial membrane potential. Thus, we have unveiled a mechanism for a secreted proteoglycan in inducing Peg3, a master regulator of macroautophagy in endothelial cells. PMID:23798385

Receptor-mediated cytotoxicity of alpha-MSH fragments containing melphalan in a human melanoma cell line.

PubMed

Morandini, R; Süli-Vargha, H; Libert, A; Loir, B; Botyánszki, J; Medzihradszky, K; Ghanem, G

1994-01-02

Four alpha-MSH drug conjugates have been synthesized, 2 C-terminal (Pep 3 and 4) and 2 central fragments (Pep 1 and 2), the latter being the 4-10 sequence known to be the main alpha-MSH-receptor-recognition site. Melphalan was introduced into each sequence at different locations. Their ability to recognize alpha-MSH receptors as well as their cytotoxic effects were compared in 3 cell lines: melanoma, carcinoma and fibroblast cells. Pep 1 and 2 were able to specifically bind to MSH receptors on melanoma cells by displacing labelled alpha-MSH from its binding sites at concentrations similar to the 4-10 heptapeptide sequence known to contain the main receptor-recognition site. They subsequently penetrate the cell, most probably by a receptor internalization mechanism, since about half of their effect could be inhibited by competition at the receptor level. Significant and selective cytotoxic effects to melanoma cells could be observed after only 2 hr exposure to the drug conjugates. Interestingly, these 2 conjugates, differing only in melphalan position, showed completely different cytotoxicity in terms of IC50 values, Pep 1 being 24 times more toxic to all cells; but the 2 were equally specific to melanoma cells. However, they both were less toxic to all cells than melphalan itself. Furthermore, Pep 1 and 2 were able to block the receptor and, unlike Pep 3 and 4, their cytotoxic effect could be significantly inhibited by an alpha-MSH agonist. Pep 3 and 4 were 5 to 10 times less toxic than melphalan to melanoma and carcinoma cells and 50 times less to fibroblast cells, and did not show any cell-type selectivity. They were less toxic than Pep 1 to melanoma and carcinoma cells by a factor of 2, but equally toxic to fibroblasts. In contrast, they were more toxic than Pep 2 to fibroblasts, melanoma and carcinoma by a factor of 3, 10 and 24 respectively. Our data strongly suggest a receptor-mediated cytotoxicity mechanism occurring with alpha-MSH central fragments in human melanoma cells due to the presence of alpha-MSH-specific receptors. This mechanism appeared to be both peptide- and cell-type-specific.

Inhibition of laminin alpha 1-chain expression leads to alteration of basement membrane assembly and cell differentiation

PubMed Central

1996-01-01

The expression of the constituent alpha 1 chain of laminin-1, a major component of basement membranes, is markedly regulated during development and differentiation. We have designed an antisense RNA strategy to analyze the direct involvement of the alpha 1 chain in laminin assembly, basement membrane formation, and cell differentiation. We report that the absence of alpha 1-chain expression, resulting from the stable transfection of the human colonic cancer Caco2 cells with an eukaryotic expression vector comprising a cDNA fragment of the alpha 1 chain inserted in an antisense orientation, led to (a) an incorrect secretion of the two other constituent chains of laminin-1, the beta 1/gamma 1 chains, (b) the lack of basement membrane assembly when Caco2-deficient cells were cultured on top of fibroblasts, assessed by the absence of collagen IV and nidogen deposition, and (c) changes in the structural polarity of cells accompanied by the inhibition of an apical digestive enzyme, sucrase-isomaltase. The results demonstrate that the alpha 1 chain is required for secretion of laminin-1 and for the assembly of basement membrane network. Furthermore, expression of the laminin alpha 1-chain gene may be a regulatory element in determining cell differentiation. PMID:8609173

Thalidomide inhibits tumor necrosis factor-alpha production and antigen presentation by Langerhans cells.

PubMed

Deng, Liang; Ding, Wanhong; Granstein, Richard D

2003-11-01

Thalidomide is an effective treatment for several inflammatory and autoimmune disorders including erythema nodosum leprosum, Behcet's syndrome, discoid lupus erythematosus, and Crohn's disease. Thalidomide is believed to exert its anti-inflammatory effects, at least in part, by inhibiting tumor necrosis factor-alpha (TNF-alpha) production by monocytes. We studied the effects of thalidomide on epidermal Langerhans cells (LC). LCs are epidermal antigen-presenting dendritic cells that play important roles in skin immune responses. Using the murine epidermis-derived dendritic cell lines, XS106A from A/J mice and XS52 from BALB/c mice as surrogates for LC, we found that thalidomide inhibited TNF-alpha production in a concentration-dependent manner. Northern blot analysis revealed that thalidomide significantly decreased the peak-induced mRNA level of TNF-alpha in XS106A cells and XS52 cells. We then examined the effect of thalidomide on fresh LC enriched to approximately 98% using positive selection of Ia+ cells with antibodies conjugated to magnetic microspheres. TNF-alpha production was reduced by 67.7% at a thalidomide concentration of 200 microg per mL. Thalidomide also had a profound inhibitory effect on the ability of LC to present antigen to a responsive TH1 clone. Thalidomide inhibits TNF-alpha production and the antigen-presenting ability of epidermal LCs. These mechanisms may contribute to the therapeutic effects observed with this agent.

Electrical characteristics of mammalian cells on porous supports

NASA Astrophysics Data System (ADS)

Chen, Guo

2003-10-01

The quantification of epithelial barrier functions by measuring the trans-epithelial electrical resistance (TER) and using the Electric Cell-substrate Impedance Sensing (ECIS) has been complicated by the current flowing inside the narrow space underneath cells. This thesis work, by examining the electrical characteristics of epithelial cells on porous supports, is aimed to tackle this problem. A mathematical model has been constructed to quantify the impedance from the various sources within a cell/electrode system. This model presents three cell-related parameters, alpha, Rb and Cm: alpha stands for the impedance contribution from the above-mentioned current underneath cells, Rb is an equivalent representation of epithelial barrier functions and Cm denotes the capacitive impedance of cell membranes. Analysis of the three parameters as well as the electrode impedance (Z e) has revealed two experimental approaches to reduce or eliminate the complication of alpha to the deduction of Rb: lowering alpha down to zero or lowering both Ze and alpha. The experimental realization of the first approach has been studied by examining the electrical characteristics of the African green monkey kidney (BS-C-1) and Madin-Darby canine kidney (MDCK-II) cells on porous filters of mixed esters of cellulose or nitrocellulose. A unique setup featuring a plastic/filter/plastic triple-layer structure was constructed to measure the impedance of cells on filters. With the extremely low alpha, all the electrical characteristics can be explained by using an equivalent circuit and Rb can be directly obtained from the resistance difference in the low frequency range. The second approach has been experimentally investigated by examining the electrical characteristics of BS-C-1 cells on porous/rough electrodes, i.e. the gold ECIS electrodes electrochemically coated with conducting polypyrrole/heparin composites or platinum black. Ze and alpha, especially the former, were found to be significantly lowered, which greatly reduces the effect of alpha and yields many new impedance features. Rb can be also directly obtained in a different way from that for the solely lowered alpha on the non-conducting porous filters.

Regulation of neurosteroid allopregnanolone biosynthesis in the rat spinal cord by glycine and the alkaloidal analogs strychnine and gelsemine.

PubMed

Venard, C; Boujedaini, N; Belon, P; Mensah-Nyagan, A G; Patte-Mensah, C

2008-04-22

The neurosteroid allopregnanolone (3alpha,5alpha-THP) is well characterized as a potentially therapeutic molecule which exerts important neurobiological actions including neuroprotective, antidepressant, anxiolytic, anesthetic and analgesic effects. We have recently observed that neurons and glial cells of the rat spinal cord (SC) contain various key steroidogenic enzymes such as 5alpha-reductase and 3alpha-hydroxysteroid oxido-reductase which are crucial for 3alpha,5alpha-THP biosynthesis. Furthermore, we demonstrated that the rat SC actively produces 3alpha,5alpha-THP. As the key factors regulating neurosteroid production by nerve cells are unknown and because glycine is one of the pivotal inhibitory neurotransmitters in the SC, we investigated glycine effects on 3alpha,5alpha-THP biosynthesis in the rat SC. Glycine markedly stimulated [(3)H]-progesterone conversion into [(3)H]3alpha,5alpha-THP by SC slices. The alkaloid strychnine, well-known as a glycine receptor (Gly-R) antagonist, blocked glycine stimulatory effect on 3alpha,5alpha-THP formation. Gelsemine, another alkaloid containing the same functional groups as strychnine, increased 3alpha,5alpha-THP synthesis. The stimulatory effects of glycine and gelsemine on 3alpha,5alpha-THP production were additive when the two drugs were combined. These results demonstrate that glycine and gelsemine, acting via Gly-R, upregulate 3alpha,5alpha-THP biosynthesis in the SC. The data also revealed a structure-activity relationship of the analogs strychnine and gelsemine on neurosteroidogenesis. Possibilities are opened for glycinergic agents and gelsemine utilization to stimulate selectively 3alpha,5alpha-THP biosynthetic pathways in diseases evoked by a decreased neurosteroidogenic activity of nerve cells.

A new mechanism for DNA alterations induced by alpha particles such as those emitted by radon and radon progeny.

PubMed Central

Lehnert, B E; Goodwin, E H

1997-01-01

The mechanism(s) by which alpha (alpha) particles like those emitted from inhaled radon and radon progeny cause their carcinogenic effects in the lung remains unclear. Although direct nuclear traversals by alpha-particles may be involved in mediating these outcomes, increasing evidence indicates that a particles can cause alterations in DNA in the absence of direct hits to cell nuclei. Using the occurrence of excessive sister chromatid exchanges (SCE) as an index of DNA damage in human lung fibroblasts, we investigated the hypothesis that alpha-particles may induce DNA damage through the generation of extracellular factors. We have found that a relatively low dose of alpha-particles can result in the generation of extracellular factors, which, upon transfer to unexposed normal human cells, can cause excessive SCE to an extent equivalent to that observed when the cells are directly irradiated with the same irradiation dose. A short-lived, SCE-inducing factor(s) is generated in alpha-irradiated culture medium containing serum in the absence of cells. A more persistent SCE-inducing factor(s), which can survive freeze-thaw and is heat labile is produced by fibroblasts after exposure to the alpha-particles. These results indicate that the initiating target for alpha-particle-induced genetic changes can be larger than a cell's nucleus or even a whole cell. How transmissible factors like those observed here in vitro may extend to the in vivo condition in the context of a-particle-induced carcinogenesis in the respiratory tract remains to be determined. PMID:9400706

Overexpression of IL-7R alpha provides a competitive advantage during early T-cell development.

PubMed

Laouar, Yasmina; Crispe, I Nicholas; Flavell, Richard A

2004-03-15

Critical checkpoints controlling early thymic T-cell development and homeostasis are set by the proper signaling function of the interleukin 7 receptor (IL-7R) and the pre-T-cell antigen receptor. Although alpha beta T-cell development is observed in IL-7- and IL-7R alpha-deficient mice, the number of thymocytes is significantly reduced, implying a role for the IL-7R in controlling the size of the thymic T-cell compartment. Here, we report the overexpression of IL-7R alpha that occurs in the early T-cell compartment from AKR/J mice, animals that are highly susceptible to the spontaneous development of thymoma. Increased IL-7R alpha was revealed by surface staining, and increased IL-7R alpha mRNA was documented by using reverse transcriptase-polymerase chain reaction (RT-PCR). This resulted in increased survival of AKR/J early thymocytes, shown by the decreased frequency of TUNEL(+) (terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate [dUTP]-fluorescein nick end labeling) cells. In an in vivo thymocyte repopulation model, AKR/J thymocytes had a selective advantage over healthy thymocytes. This advantage occurred at early stages of T-cell development. Our findings support the model that overexpression of growth factor receptors can contribute to proliferation and malignancy.

Bombesin and thrombin affect discrete pools of intracellular calcium through different G-proteins.

PubMed Central

Wang, J L; Kalyanaraman, S; Vivo, M D; Gautam, N

1996-01-01

In mouse NIH 3T3 cells, the mitogens bombesin and thrombin induced Ca2+ release from intracellular stores. Ca2+ release induced by bombesin was inhibited by the Ca(2+)-ATPase inhibitor thapsigargin, while Ca2+ release induced by thrombin was unaffected by this agent. The Ca(2+)-release response to bombesin was not affected by pertussis toxin, but the response to thrombin was abolished by the toxin. Stable transfectants overexpressing the G-protein subunit type alpha 9 showed an accentuated response to bombesin, indicating that the bombesin receptor was coupled to a Gq-like G-protein. Together, these results show that the two mitogenic receptors are coupled to distinct G-proteins that affect functionally different pools of Ca2+. Organization of signalling pathways in this manner may allow cells to differentially encode information from different signals. PMID:8947471

Identification of a novel cyclosporin-sensitive element in the human tumor necrosis factor alpha gene promoter

PubMed Central

1993-01-01

Tumor necrosis factor alpha (TNF-alpha), a cytokine with pleiotropic biological effects, is produced by a variety of cell types in response to induction by diverse stimuli. In this paper, TNF-alpha mRNA is shown to be highly induced in a murine T cell clone by stimulation with T cell receptor (TCR) ligands or by calcium ionophores alone. Induction is rapid, does not require de novo protein synthesis, and is completely blocked by the immunosuppressant cyclosporin A (CsA). We have identified a human TNF-alpha promoter element, kappa 3, which plays a key role in the calcium-mediated inducibility and CsA sensitivity of the gene. In electrophoretic mobility shift assays, an oligonucleotide containing kappa 3 forms two DNA protein complexes with proteins that are present in extracts from unstimulated T cells. These complexes appear in nuclear extracts only after T cell stimulation. Induction of the inducible nuclear complexes is rapid, independent of protein synthesis, and blocked by CsA, and thus, exactly parallels the induction of TNF-alpha mRNA by TCR ligands or by calcium ionophore. Our studies indicate that the kappa 3 binding factor resembles the preexisting component of nuclear factor of activated T cells. Thus, the TNF-alpha gene is an immediate early gene in activated T cells and provides a new model system in which to study CsA-sensitive gene induction in activated T cells. PMID:8376940

alpha-Tocopheryl succinate promotes selective cell death induced by vitamin K3 in combination with ascorbate.

PubMed

Tomasetti, M; Strafella, E; Staffolani, S; Santarelli, L; Neuzil, J; Guerrieri, R

2010-04-13

A strategy to reduce the secondary effects of anti-cancer agents is to potentiate the therapeutic effect by their combination. A combination of vitamin K3 (VK3) and ascorbic acid (AA) exhibited an anti-cancer synergistic effect, associated with extracellular production of H(2)O(2) that promoted cell death. The redox-silent vitamin E analogue alpha-tocopheryl succinate (alpha-TOS) was used in combination with VK3 and AA to evaluate their effect on prostate cancer cells. Prostate cancer cells were sensitive to alpha-TOS and VK3 treatment, but resistant to AA upto 3.2 mM. When combined, a synergistic effect was found for VK3-AA, whereas alpha-TOS-VK3 and alpha-TOS-AA combination showed an antagonist and additive effect, respectively. However, sub-lethal doses of AA-VK3 combination combined with a sub-toxic dose of alpha-TOS showed to induce efficient cell death that resembles autoschizis. Associated with this cell demise, lipid peroxidation, DNA damage, cytoskeleton alteration, lysosomal-mitochondrial perturbation, and release of cytochrome c without caspase activation were observed. Inhibition of lysosomal proteases did not attenuate cell death induced by the combined agents. Furthermore, cell deaths by apoptosis and autoschizis were detected. These finding support the emerging idea that synergistic combinations of some agents can overcome toxicity and other side-effects associated with high doses of single drugs creating the opportunity for therapeutically relevant selectivity.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chan, R.K.; Otte, C.A.

Saccharomyces cerevisiae MATa cells carrying mutations in either sst1 or sst2 are supersensitive to the G1 arrest induced by ..cap alpha.. factor pheromone. When sst1 mutants were mixed with normal SST/sup +/ cells, the entire population recovered together from ..cap alpha.. factor arrest, suggesting that SST/sup +/ cells helped sst1 mutants to recover. Complementation tests and linkage analysis showed that sst1 and bar1, a mutation which eliminates the ability of MATa cells to act as a ''barrier'' to the diffusion of ..cap alpha.. factor, were lesions in the same genes. These findings suggest that sst1 mutants are defective in recoverymore » from ..cap alpha.. factor arrest because they are unable to degrade the pheromone. In contrast, recovery of sst2 mutants was not potentiated by the presence of SST/sup +/ cells in mixing experiments. When either normal MATa cells or mutant cells carrying defects in sst1 or sst2 were exposed to ..cap alpha.. factor for 1 h and then washed free of the pheromone, the sst2 cells subsequently remained arrested in the absence of ..cap alpha.. factor for a much longer time than SST/sup +/ or sst1 cells. These observations suggest that the defect in sst2 mutants is intrinsic to the cell and is involved in the mechanism of ..cap alpha.. factor action at some step after the initial interaction of the pheromone with the cell. The presence of an sst2 mutation appears to cause a growth debility, since repeated serial subculture of haploid sst2-1 strains led to the accumulation of faster-growing revertants that were pheromone resistant and were mating defective (''sterile'').« less

Induction of IgA B cell differentiation of bone marrow-derived B cells by Peyer's patch autoreactive helper T cells.

PubMed

Kihira, T; Kawanishi, H

1995-08-01

The objective of this study was to demonstrate in vitro that bone marrow-derived pro/pre-B cells bearing mu mRNA can switch their Ig heavy-chain isotype to that of alpha mRNA-expressing B cells after contact with Peyer's patches-derived activated autoreactive CD4+ T cells. Bone marrow-derived pro/pre-B cells and activated autoreactive Peyer's patch, mesenteric lymph node, or spleen CD4+ T cells were co-cultured in the presence of recombinant (r) IL-2, rIL-7, and Con A for 3 days. The mixed cultured cells were isolated for preparation of total RNA. Dot/slot hybridization, using murine C mu (pu3741) and C alpha (P alpha J558) Ig heavy-chain cDNA probes, detected C mu and C alpha Ig heavy-chain mRNA transcripts. The magnitude of each mRNA expression was measured demsitometrically. In addition, the secreted class-specific Ig contents from the co-cultured supernatants were measured. The results indicate that activated autoreactive Peyer's patch and mesenteric lymph node CD4+ T cells provide a specific Ig heavy-chain switch from mu to alpha (Peyer's patch CD4+ T cells > mesenteric lymph node CD4+ T cells) in bone marrow-derived pro/pre-B cells and also assist to develop IgA-secreting plasma cells. The alpha heavy-chain switch and IgA production do not occur in the presence of activated autoreactive spleen CD4+ T cells. These results support the view that autoreactive gut Peyer's patch CD4+ T cells, at least, regulate IgA B cell heavy-chain switching and terminal differentiation during gut mucosal B cell development.

SM22{alpha}-induced activation of p16{sup INK4a}/retinoblastoma pathway promotes cellular senescence caused by a subclinical dose of {gamma}-radiation and doxorubicin in HepG2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Tae Rim; Lee, Hee Min; Lee, So Yong

Research highlights: {yields} SM22{alpha} overexpression in HepG2 cells leads cells to a growth arrest state, and the treatment of a subclinical dose of {gamma}-radiation or doxorubicin promotes cellular senescence. {yields} SM22{alpha} overexpression elevates p16{sup INK4a} followed by pRB activation, but there are no effects on p53/p21{sup WAF1/Cip1} pathway. {yields} SM22{alpha}-induced MT-1G activates p16{sup INK4a}/pRB pathway, which promotes cellular senescence by damaging agents. -- Abstract: Smooth muscle protein 22-alpha (SM22{alpha}) is known as a transformation- and shape change-sensitive actin cross-linking protein found in smooth muscle tissue and fibroblasts; however, its functional role remains uncertain. We reported previously that SM22{alpha} overexpression confersmore » resistance against anti-cancer drugs or radiation via induction of metallothionein (MT) isozymes in HepG2 cells. In this study, we demonstrate that SM22{alpha} overexpression leads cells to a growth arrest state and promotes cellular senescence caused by treatment with a subclinical dose of {gamma}-radiation (0.05 and 0.1 Gy) or doxorubicin (0.01 and 0.05 {mu}g/ml), compared to control cells. Senescence growth arrest is known to be controlled by p53 phosphorylation/p21{sup WAF1/Cip1} induction or p16{sup INK4a}/retinoblastoma protein (pRB) activation. SM22{alpha} overexpression in HepG2 cells elevated p16{sup INK4a} followed by pRB activation, but did not activate the p53/p21{sup WAF1/Cip1} pathway. Moreover, MT-1G, which is induced by SM22{alpha} overexpression, was involved in the activation of the p16{sup INK4a}/pRB pathway, which led to a growth arrest state and promoted cellular senescence caused by damaging agents. Our findings provide the first demonstration that SM22{alpha} modulates cellular senescence caused by damaging agents via regulation of the p16{sup INK4a}/pRB pathway in HepG2 cells and that these effects of SM22{alpha} are partially mediated by MT-1G.« less

Chimeras of the integrin beta subunit mid-region reveal regions required for heterodimer formation and for activation.

PubMed

Hyland, R H; Douglass, W A; Tan, S M; Law, S K

2001-01-01

A central region of the beta2 integrin subunit, RN (residues D300 to C459), was replaced by the equivalent sequences from beta1 and beta7 to give the chimeras beta2RN1 and beta2RN7. Whilst the former construct failed to form heterodimer at the cell surface with alphaL, the later of these could be expressed together with the alphaL subunit to form a variant LFA-1. Based on recent modelling work, the RN region consists of two parts, one is the C-terminal end of the putative A-domain (RB, residues D300 to A359), and the other the mid-region (BN, residues Y360 to C459). Chimeras exchanging the two component regions were made. Of the four resultant chimeras, only the beta2RB1 chimera failed to support LFA-1 expression. Thus the beta1 specific residues of this region affect the interaction with the alphaL subunit. Whereas the alphaL/beta2RB7 LFA-1 variant is wildtype like with respect to ICAM-1 adhesion, the alphaLbeta2BN1 and alphaLbeta2BN7, as well as the alphaLbeta2RN7, variants are more adhesive than the wildtype. These results suggest that an authentic beta2 mid-region is, in part, required for maintaining the LFA-1 in a resting state.

Anchorage mediated by integrin alpha6beta4 to laminin 5 (epiligrin) regulates tyrosine phosphorylation of a membrane-associated 80-kD protein

PubMed Central

1996-01-01

Detachment of basal keratinocytes from basement membrane signals a differentiation cascade. Two integrin receptors alpha6beta4 and alpha3beta1 mediate adhesion to laminin 5 (epiligrin), a major extracellular matrix protein in the basement membrane of epidermis. By establishing a low temperature adhesion system at 4 degrees C, we were able to examine the exclusive role of alpha6beta4 in adhesion of human foreskin keratinocyte (HFK) and the colon carcinoma cell LS123. We identified a novel 80-kD membrane-associated protein (p80) that is tyrosine phosphorylated in response to dissociation of alpha6beta4 from laminin 5. The specificity of p80 phosphorylation for laminin 5 and alpha6beta4 was illustrated by the lack of regulation of p80 phosphorylation on collagen, fibronectin, or poly-L-lysine surfaces. We showed that blocking of alpha3beta1 function using inhibitory mAbs, low temperature, or cytochalasin D diminished tyrosine phosphorylation of focal adhesion kinase but not p80 phosphorylation. Therefore, under our assay conditions, p80 phosphorylation is regulated by alpha6beta4, while motility via alpha3beta1 causes phosphorylation of focal adhesion kinase. Consistent with a linkage between p80 dephosphorylation and alpha6beta4 anchorage to laminin 5, we found that phosphatase inhibitor sodium vanadate, which blocked the p80 dephosphorylation, prevented the alpha6beta4-dependent cell anchorage to laminin 5 at 4degreesC. In contrast, adhesion at 37 degrees C via alpha3beta1 was unaffected. Furthermore, by in vitro kinase assay, we identified a kinase activity for p80 phosphorylation in suspended HFKs but not in attached cells. The kinase activity, alpha6beta4, and its associated adhesion structure stable anchoring contacts were all cofractionated in the Triton- insoluble cell fraction that lacks alpha3beta1. Thus, regulation of p80 phosphorylation, through the activities of p80 kinase and phosphatase, correlates with alpha6beta4-SAC anchorage to laminin 5 at 4 degrees C in epithelial cells of the skin and intestine. Transmembrane signaling through p80 is an early tyrosine phosphorylation event responsive to and possibly required for anchorage to laminin 5 by HFK and LS123 epithelial cells. PMID:8647901

[Effect of Yiguan Decoction on differentiation of bone marrow mesenchymal stem cells into hepatocyte-like cells: an experimental research].

PubMed

Ping, Jian; Chen, Hong-Yun; Yang, Zhou; Yang, Cheng; Xu, Lie-Ming

2014-03-01

To observe the effect of Yiguan Decoction (YGD) on differentiation of bone marrow mesenchymal stem cells (BMSCs) into hepatocyte-like cells in vitro. Rat BMSCs were isolated using whole bone marrow adherent method. The properties of BMSCs were identified by analyzing the expression of surface cytokines by flow cytometry. The third passage cells were differentiated into fat cells to identify their features. BMSCs were incubated with hepatocyte growth factor (HGF) plus fibroblast growth factor 4 (FGF4) or YGD containing serum YGD for 21 days. The mRNA expression of alpha-fetoprotein (alphaAFP), albumin (Alb), and hepatocyte nuclear factor 4alpha (HNF4alpha) were detected by real time PCR. Expression of AFP and cytokeratin 18 (CK18) protein was detected by cell immunofluorescence. Glycogen synthesis was observed using periodic acid-Schiff stain (PAS). CK18, Wnt 3alpha, and alphacatenin protein expressions were detected by Western blot. High expression of CD90, CD29, and CD44, and low expression of CD34 and CD11b were observed in BMSCs isolated by whole bone mar- row adherent method, and numerous lipid droplets were observed in BMSCs using oil red O staining. Both YGD containing serum and growth factor stimulated the expression levels of Alb, AFP, HNF4alpha mRNA and CK18 protein. The down-regulated expression of Wnt 3alpha and beta-catenin could be detected at 21 days after induction. The synthesized glycogen granule could be seen. Down-regulated Wnt 3alpha and beta-catenin expression could also be observed. YGD could induce the differentiation of rat BMSCs into hepatocyte-like cells, which was related to down-regulating Wnt/beta-catenin signal pathway.

Interferon-alpha and interferon-gamma sensitize human tenon fibroblasts to mitomycin-C.

PubMed

Wang, Xiao Yang; Crowston, Jonathan G; Zoellner, Hans; Healey, Paul R

2007-08-01

To investigate the effect of interferon (IFN)-alpha and IFN-gamma pretreatment on mitomycin C (MMC)-induced cell death in human Tenon fibroblasts (HTFs) and the mechanisms by which IFN-alpha and IFN-gamma modulate the susceptibility of HTFs to MMC. HTFs were pretreated with IFN-alpha and IFN-gamma for 48 hours before 5-minute application of 0.4 mg/mL MMC. Cell death after 48 hours was determined by Annexin V/propidium iodide (PI) staining and lactate dehydrogenase (LDH) release assay. Fas, Fas-ligand, and Bcl-2 expression were determined by flow cytometry. Fas associated death domain (FADD), Bax, cytochrome c, and caspase expression were determined by Western blot analysis and immunofluorescence staining. MMC treatment increased cell death and upregulated Fas and FADD expression, but had no effect on Fas-Ligand, Bax, Bcl-2, or cytochrome c. Neither IFN-alpha nor IFN-gamma alone induced HTF death, but each increased cell death 2 days after MMC treatment in a dose-dependent fashion. Combination IFN-alpha and IFN-gamma had a synergistic effect. IFN-alpha and IFN-gamma pretreatment increased Fas expression. Fas upregulation was associated with increased sensitivity to MMC. IFN pretreatment increased procaspase-8, procaspase-9, and procaspase-3 expression, and caspase-3 activation. Caspase-8, caspase-3, and broad caspase inhibitors, but not caspase-9 inhibitor, inhibited MMC-induced cell death in nonpretreated and IFN-pretreated cells. IFN-alpha and IFN-gamma enhance the susceptibility of HTFs to MMC-induced cell death through a Fas-mediated and a caspase-3-dependent pathway. Pretreatment with IFN primed HTFs to MMC, providing a potential means for initially slowing the healing response with IFN and subsequently terminating fibroblast activity through MMC-induced cell death.

Comparison of prostaglandin F2alpha, bimatoprost (prostamide), and butaprost (EP2 agonist) on Cyr61 and connective tissue growth factor gene expression.

PubMed

Liang, Yanbin; Li, Chen; Guzman, Victor M; Evinger, Albert J; Protzman, Charles E; Krauss, Achim H-P; Woodward, David F

2003-07-18

Connective tissue growth factor (CTGF) and Cyr61 (cysteine-rich angiogenic protein 61) are members of the CCN gene family that encode multifunctional, extracellular matrix-associated signaling proteins. Because the mechanism of action of certain anti-glaucoma drugs involves extracellular matrix remodeling of ocular ciliary muscle, with a resultant increase in drainage of aqueous humor from the eye, we compared the effects of three pharmacologically distinct ocular hypotensive agents on Cyr61 and CTGF gene expression. Thus, prostaglandin F2alpha (PGF2alpha) (FP receptor agonist), Butaprost (EP2 receptor agonist), and Bimatoprost (a prostamide) were compared. Using Affymetrix gene chip technology, we first identified that PGF2alpha dramatically up-regulated Cyr61 and CTGF mRNA expression in HEK 293/EBNA cells (hFP-HEK 293/EBNA). Northern blot further confirmed the Cyr61 and CTGF up-regulation is in a dose- and time-dependent manner. PGF2alpha-induced up-regulation of Cyr61 appeared to exclusively involve the Rho pathway, and up-regulation of CTGF was via multiple intracellular pathways. Because prostamide receptors are, to date, defined only at the pharmacological level, Bimatoprost effects on Cyr61 and CTGF were studied in the isolated feline iris sphincter preparation, a tissue highly responsive to prostamides. Both PGF2alpha and Bimatoprost up-regulated Cyr61 mRNA expression in the cat iris tissue. Only PGF2alpha up-regulated CTGF mRNA expression in the cat iris. Therefore, PGF2alpha and Bimatoprost appear to interact with different receptors populations in the cat iris, according to their markedly different effects on CTGF. Activation of prostaglandin EP2 receptors (Gs-coupled) also up-regulated Cyr61 but not CTGF mRNA expression in the isolated cat iris. Similar data were observed in human primary ciliary smooth muscle cells. Thus, despite quite different signal transduction pathways, FP receptor stimulation up-regulates CTGF and Cyr61. The prostamide analog Bimatoprost and an EP2-selective agonist affects only Cyr61.

Antitumor activity of type I and type III interferons in BNL hepatoma model.

PubMed

Abushahba, Walid; Balan, Murugabaskar; Castaneda, Ismael; Yuan, Yao; Reuhl, Kenneth; Raveche, Elizabeth; de la Torre, Andrew; Lasfar, Ahmed; Kotenko, Sergei V

2010-07-01

Hepatocellular carcinoma (HCC) occurs most commonly secondary to cirrhosis due to chronic hepatitis C or B virus (HCV/HBV) infections. Type I interferon (IFN-alpha) treatment of chronic HCV/HBV infections reduces the incidence of HCC in cirrhotic patients. However, IFN-alpha toxicity limits its tolerability and efficacy highlighting a need for better therapeutic treatments. A recently discovered type III IFN (IFN-lambda) has been shown to possess antiviral properties against HCV and HBV in vitro. In phase I clinical trials, IFN-lambda treatment did not cause significant adverse reactions. Using a gene therapy approach, we compared the antitumor properties of IFN-alpha and IFN-lambda in a transplantable hepatoma model of HCC. BALB/c mice were inoculated with syngeneic BNL hepatoma cells, or BNL cells expressing IFN-lambda (BNL.IFN-lambda cells) or IFN-alpha (BNL.IFN-alpha cells). Despite the lack of antiproliferative activity of IFNs on BNL cells, both BNL.IFN-lambda and BNL.IFN-alpha cells displayed retarded growth kinetics in vivo. Depletion of NK cells from splenocytes inhibited splenocyte-mediated cytotoxicity, demonstrating that NK cells play a role in IFN-induced antitumor responses. However, isolated NK cells did not respond directly to IFN-lambda. There was also a marked NK cell infiltration in IFN-lambda producing tumors. In addition, IFN-lambda and, to a lesser extent, IFN-alpha enhanced immunocytotoxicity of splenocytes primed with irradiated BNL cells. Splenocyte cytotoxicity against BNL cells was dependent on IL-12 and IFN-gamma, and mediated by dendritic cells. In contrast to NK cells, isolated from spleen CD11c+ and mPDCA+ dendritic cells responded directly to IFN-lambda. The antitumor activities of IFN-lambda against hepatoma, in combination with HCV and HBV antiviral activities warrant further investigation into the clinical use of IFN-lambda to prevent HCC in HCV/HBV-infected cirrhotic patients, as well as to treat liver cancer.

Vesicle-associated membrane protein 2 mediates trafficking of {alpha}5{beta}1 integrin to the plasma membrane

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hasan, Nazarul; Hu, Chuan, E-mail: chuan.hu@louisville.edu

2010-01-01

Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of {alpha}5{beta}1 integrin. VAMP2 was present on vesicles containing endocytosed {beta}1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cellmore » surface {alpha}5{beta}1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of {alpha}5{beta}1, without altering cell surface expression of {alpha}2{beta}1 integrin or {alpha}3{beta}1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of {alpha}5{beta}1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.« less

The influence of interferon alpha on the rat liver injured by chronic administration of carbon tetrachloride.

PubMed

Madro, Agnieszka; Słomka, Maria; Celiński, Krzysztof; Chibowski, Daniel; Czechowska, Grazyna; Kleinrok, Zdzisław; Karpińska, Agnieszka

2002-01-01

Due to their complex and not fully known etiopathogenesis as well as difficulties in treatment, chronic hepatitis and cirrhosis still remain one of the main problems of hepatologists. Nowadays, the use of IFN alpha is considered the most effective method of treatment in chronic hepatitis. Recently, a new property of IFN, i.e. its effects on the reduction of fibrosis, has been discovered. The aim of the paper was to examine the effects of IFN alpha on biochemical parameters (AlAt and AspAt activities), on the metabolic function of the liver and its morphologic picture observed under the light and electron microscope after the 3- and 6-week CCl4-induced damage. The experiments were carried out in Wistar male rats. To evaluate the liver function, the test of aminophenazone elimination in the isolated perfused rat livers was used according to Miller modified by Hafte. Additionally, AspAt and AlAt activities were determined. The liver specimens were analysed under the light and electron microscope and using immunohistochemical methods. The findings show that after the 3-week CCl4-induced liver damage, IFN alpha does not significantly affect AlAt and AspAt activities, irrespective of the dose used. IFN alpha administered after the 6-week damage significantly changes those activities when the doses used are high. It was found that carbon tetrachloride does not result in evident cirrhotic changes, however it activates Ito cells, causes focal retraction of the stroma and fibrosis. The increased number of Ito cells in Disse's space observed in immunohistochemical and ultrastructural examinations is indicative of the activation of liver fibrotic processes following CCl4 administration in both variants used. IFN alpha substantially weakens fibrogenesis of the CCl4-damaged liver which is visible in the decreased number of Ito cells and weaker expression of the stroma retraction. Moreover, IFN alpha administered to the experimental animals after the CCl4-induced injury of the liver increases aminophenazone clearance, especially when used in higher doses. Positive effects of IFN confirmed in the studies suggest that the drug may be used in patients with chronic hepatitis and early cirrhosis since it is likely not only to eliminate the virus but also to improve the liver function and reduce fibrosis.

Defective Wound-healing in Aging Gingival Tissue.

PubMed

Cáceres, M; Oyarzun, A; Smith, P C

2014-07-01

Aging may negatively affect gingival wound-healing. However, little is known about the mechanisms underlying this phenomenon. The present study examined the cellular responses associated with gingival wound-healing in aging. Primary cultures of human gingival fibroblasts were obtained from healthy young and aged donors for the analysis of cell proliferation, cell invasion, myofibroblastic differentiation, and collagen gel remodeling. Serum from young and old rats was used to stimulate cell migration. Gingival repair was evaluated in Sprague-Dawley rats of different ages. Data were analyzed by the Mann-Whitney and Kruskal-Wallis tests, with a p value of .05. Fibroblasts from aged donors showed a significant decrease in cell proliferation, migration, Rac activation, and collagen remodeling when compared with young fibroblasts. Serum from young rats induced higher cell migration when compared with serum from old rats. After TGF-beta1 stimulation, both young and old fibroblasts demonstrated increased levels of alpha-SMA. However, alpha-SMA was incorporated into actin stress fibers in young but not in old fibroblasts. After 7 days of repair, a significant delay in gingival wound-healing was observed in old rats. The present study suggests that cell migration, myofibroblastic differentiation, collagen gel remodeling, and proliferation are decreased in aged fibroblasts. In addition, altered cell migration in wound-healing may be attributable not only to cellular defects but also to changes in serum factors associated with the senescence process. © International & American Associations for Dental Research.

Angiotensin II type 1 receptor blockers prevent tumor necrosis factor-alpha-mediated endothelial nitric oxide synthase reduction and superoxide production in human umbilical vein endothelial cells.

PubMed

Kataoka, Hiroki; Murakami, Ryuichiro; Numaguchi, Yasushi; Okumura, Kenji; Murohara, Toyoaki

2010-06-25

Decrease in endothelial nitric oxide synthase (eNOS) expression is one of the adverse outcomes of endothelial dysfunction. Tumor necrosis factor-alpha (TNF-alpha) is known to decrease eNOS expression and is an important mediator of endothelial dysfunction. We hypothesized that an angiotensin II type 1 (AT1) receptor blocker would improve endothelial function via not only inhibition of the angiotensin II signaling but also inhibition of the TNF-alpha-mediated signaling. Therefore we investigated whether an AT1 receptor blocker would restore the TNF-alpha-induced decrease in eNOS expression in cultured human umbilical vein endothelial cells (HUVEC). Pretreatment of HUVEC with an antioxidant (superoxide dismutase, alpha-tocopherol) or AT1 receptor blockers (olmesartan or candesartan) restored the TNF-alpha-dependent reduction of eNOS. The AT1 receptor blocker decreased the TNF-alpha-dependent increase of 8-isoprostane. The superoxide dismutase activities in HUVEC were stable during AT1 receptor blocker treatment, and the AT1 receptor blocker did not scavenge superoxide directly. The AT1 receptor blocker also decreased TNF-alpha-induced phosphorylation of I kappaB alpha and cell death. These results suggest that AT1 receptor blockers are able to ameliorate TNF-alpha-dependent eNOS reduction or cell injury by inhibiting superoxide production or nuclear factor-kappaB activation. (c) 2010 Elsevier B.V. All rights reserved.

An evaluation of the results of alluvial groundwater sampling from 1987--1990 at the Durango disposal site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1991-04-01

This evaluation was conducted to determine if surface discharges of contaminated water from a retention pond and seepage of tailings pore water from the disposal cell have affected ground I water quality in the alluvial deposits east and northeast of the Bodo Canyon disposal cell. The question of whether corrective remedial action is needed for the alluvial groundwater downgradient of the disposal cell is also addressed. Maximum observed concentrations of seven hazardous constituents equalled or exceeded proposed US Environmental Protection Agency (EPA) maximum concentration limits (MCLs) in the alluvial groundwater downgradient of the disposal cell. These constituents include chromium, lead,more » molybdenum, net gross alpha, radium-226 and -228, selenium, and uranium. Concentrations greater than MCLs for molybdenum, net gross alpha, and radium-226 and -228 may be naturally occurring in the alluvial groundwater. There is no statistical evidence that these hazardous constituents are groundwater contaminants with concentrations that exceed the MCLs in alluvial groundwater. However, the median selenium concentration in monitor well 608 exceeds the MCL. Therefore, selenium contamination in the alluvial groundwater in the area of monitor well 608 is possible. Selenium concentrations show no definite increasing or decreasing trend. Since groundwater contamination by selenium is possible in one monitor well, but concentrations are not increasing, corrective action is not warranted at this time. Alluvial groundwater quality will continue to be monitored quarterly and the discharge from the retention pond should be sampled after treatment to ascertain its potential affects on groundwater quality.« less

Persisting mild hypothermia suppresses hypoxia-inducible factor-1alpha protein synthesis and hypoxia-inducible factor-1-mediated gene expression.

PubMed

Tanaka, Tomoharu; Wakamatsu, Takuhiko; Daijo, Hiroki; Oda, Seiko; Kai, Shinichi; Adachi, Takehiko; Kizaka-Kondoh, Shinae; Fukuda, Kazuhiko; Hirota, Kiichi

2010-03-01

The transcription factor hypoxia-inducible factor-1 (HIF-1) plays an essential role in regulating gene expression in response to hypoxia-ischemia. Ischemia causes the tissue not only to be hypoxic but also to be hypothermic because of the hypoperfusion under certain circumstances. On the other hand, the induced hypothermia is one of the most common therapeutic modalities to extend tolerance to hypoxia. Although hypoxia elicits a variety of cellular and systemic responses at different organizational levels in the body, little is known about how hypoxia-induced responses are affected by low temperature. We examined the influence of mild hypothermic conditions (28-32 degrees C) on HIF-1 in both in vitro and in vivo settings. In vitro experiments adopting cultured cells elucidated that hypoxia-induced HIF-1 activation was resistant to 4-h exposure to the low temperature. In contrast, exposure to the low temperature as long as 24 h suppressed HIF-1 activation and the subsequent upregulation of HIF-1 target genes such as VEGF or GLUT-1. HIF-1alpha protein stability in the cell was not affected by hypothermic treatment. Furthermore, intracellular ATP content was reduced under 1% O(2) conditions but was not largely affected by hypothermic treatment. The evidence indicates that reduction of oxygen consumption is not largely involved in suppression of HIF-1. In addition, we demonstrated that HIF-1 DNA-binding activity and HIF-1-dependent gene expressions induced under 10% O(2) atmosphere in mouse brain were not influenced by treatment under 3-h hypothermic temperature but were inhibited under 5-h treatment. On the other hand, we indicated that warming ischemic legs of mice for 24 h preserved HIF-1 activity. In this report we describe for the first time that persisting low temperature significantly reduced HIF-1alpha neosynthesis under hypoxic conditions, leading to a decrease in gene expression for adaptation to hypoxia in both in vitro and in vivo settings.

Differential effects of phthalates on the testis and the liver.

PubMed

Bhattacharya, Nandini; Dufour, Jannette M; Vo, My-Nuong; Okita, Janice; Okita, Richard; Kim, Kwan Hee

2005-03-01

Phthalates have been shown to elicit contrasting effects on the testis and the liver, causing testicular degeneration and promoting abnormal hepatocyte proliferation and carcinogenesis. In the present study, we compared the effects of phthalates on testicular and liver cells to better understand the mechanisms by which phthalates cause testicular degeneration. In vivo treatment of rats with di-(2-ethylhexyl) phthalate (DEHP) caused a threefold increase of germ cell apoptosis in the testis, whereas apoptosis was not changed significantly in livers from the same animals. Western blot analyses revealed that peroxisome proliferator-activated receptor (PPAR) alpha is equally abundant in the liver and the testis, whereas PPAR gamma and retinoic acid receptor (RAR) alpha are expressed more in the testis. To determine whether the principal metabolite of DEHP, mono-(2-ethylhexyl) phthalate (MEHP), or a strong peroxisome proliferator, 4-chloro-6(2,3-xylindino)-2-pyrimidinylthioacetic acid (Wy-14,643), have a differential effect in Sertoli and liver cells by altering the function of RAR alpha and PPARs, their nuclear trafficking patterns were compared in Sertoli and liver cells after treatment. Both MEHP and Wy-14,643 increased the nuclear localization of PPAR alpha and PPAR gamma in Sertoli cells, but they decreased the nuclear localization of RAR alpha, as previously shown. Both PPAR alpha and PPAR gamma were in the nucleus and cytoplasm of liver cells, but RAR alpha was predominant in the cytoplasm, regardless of the treatment. At the molecular level, MEHP and Wy-14,643 reduced the amount of phosphorylated mitogen-activated protein kinase (activated MAPK) in Sertoli cells. In comparison, both MEHP and Wy-14,643 increased phosphorylated MAPK in liver cells. These results suggest that phthalates may cause contrasting effects on the testis and the liver by differential activation of the MAPK pathway, RAR alpha, PPAR alpha, and PPAR gamma in these organs.

C-terminal cleavage of DeltaNp63alpha is associated with TSA-induced apoptosis in immortalized corneal epithelial cells.

PubMed

Robertson, Danielle M; Ho, Su-Inn; Cavanagh, H Dwight

2010-08-01

In the central human corneal epithelium, loss of DeltaNp63 occurs in all surface epithelial cells preparing to undergo desquamation, suggesting a potential role for DeltaNp63 isoforms in mediating surface cell apoptotic shedding. In this study, the authors investigated a role for DeltaNp63 isoforms in caspase-mediated apoptosis in a telomerase-immortalized corneal epithelial cell line. For in vitro studies, hTCEpi cells were cultured in KGM-2 serum-free culture media containing 0.15 mM calcium. To assess dynamic protein interactions among individual DeltaNp63 isoforms, DeltaNp63-EGFP expression plasmids were transiently expressed in hTCEpi cells and evaluated by FRAP. Trichostatin-A (TSA; 3.31 muM) was used to induce cell death as measured by caspase activity. Cleavage and loss of endogenous DeltaNp63alpha, DeltaNp63-EGFP expression plasmids, and p53 were assessed after treatment with TSA and siRNA. Transient expression of DeltaNp63-EGFP alpha and beta isoforms resulted in the formation of a smaller isoform similar in size to DeltaNp63gamma-EGFP. FRAP demonstrated that DeltaNp63alpha-EGFP has greater immobile fraction than beta or gamma. TSA induced caspase-mediated apoptotic pathways; caspase induction was accompanied by a decrease in endogenous DeltaNp63alpha and p53. TSA upregulated DeltaNp63-EGFP plasmid expression; this was accompanied by a selective increase in cleavage of DeltaNp63alpha-EGFP. siRNA knockdown of DeltaNp63alpha correlated with a reduction in p53 independently of TSA. DeltaNp63alpha is the dominant active isoform in corneal epithelial cell nuclei. Loss of DeltaNp63alpha occurs during apoptotic signaling by cleavage at the C terminus. The corresponding loss of p53 suggests that a significant relationship appears to exist between these two regulatory proteins.

Dynamic interactions between Pit-1 and C/EBPalpha in the pituitary cell nucleus.

PubMed

Demarco, Ignacio A; Voss, Ty C; Booker, Cynthia F; Day, Richard N

2006-11-01

The homeodomain (HD) transcription factors are a structurally conserved family of proteins that, through networks of interactions with other nuclear proteins, control patterns of gene expression during development. For example, the network interactions of the pituitary-specific HD protein Pit-1 control the development of anterior pituitary cells and regulate the expression of the hormone products in the adult cells. Inactivating mutations in Pit-1 disrupt these processes, giving rise to the syndrome of combined pituitary hormone deficiency. Pit-1 interacts with CCAAT/enhancer-binding protein alpha (C/EBPalpha) to regulate prolactin transcription. Here, we used the combination of biochemical analysis and live-cell microscopy to show that two different point mutations in Pit-1, which disrupted distinct activities, affected the dynamic interactions between Pit-1 and C/EBPalpha in different ways. The results showed that the first alpha-helix of the POU-S domain is critical for the assembly of Pit-1 with C/EBPalpha, and they showed that DNA-binding activity conferred by the HD is critical for the final intranuclear positioning of the metastable complex. This likely reflects more general mechanisms that govern cell-type-specific transcriptional control, and the results from the analysis of the point mutations could indicate an important link between the mislocalization of transcriptional complexes and disease processes.

Transient dwarfism and hypogonadism in mice lacking Otx1 reveal prepubescent stage-specific control of pituitary levels of GH, FSH and LH.

PubMed

Acampora, D; Mazan, S; Tuorto, F; Avantaggiato, V; Tremblay, J J; Lazzaro, D; di Carlo, A; Mariano, A; Macchia, P E; Corte, G; Macchia, V; Drouin, J; Brûlet, P; Simeone, A

1998-04-01

Genetic and molecular approaches have enabled the identification of regulatory genes critically involved in determining cell types in the pituitary gland and/or in the hypothalamus. Here we report that Otx1, a homeobox-containing gene of the Otx gene family, is postnatally transcribed and translated in the pituitary gland. Cell culture experiments indicate that Otx1 may activate transcription of the growth hormone (GH), follicle-stimulating hormone (betaFSH), luteinizing hormone (betaLH) and alpha-glycoprotein subunit (alphaGSU) genes. Analysis of Otx1 null mice indicates that, at the prepubescent stage, they exhibit transient dwarfism and hypogonadism due to low levels of pituitary GH, FSH and LH hormones which, in turn, dramatically affect downstream molecular and organ targets. Nevertheless, Otx1-/- mice gradually recover from most of these abnormalities, showing normal levels of pituitary hormones with restored growth and gonadal function at 4 months of age. Expression patterns of related hypothalamic and pituitary cell type restricted genes, growth hormone releasing hormone (GRH), gonadotropin releasing hormone (GnRH) and their pituitary receptors (GRHR and GnRHR) suggest that, in Otx1-/- mice, hypothalamic and pituitary cells of the somatotropic and gonadotropic lineages appear unaltered and that the ability to synthesize GH, FSH and LH, rather than the number of cells producing these hormones, is affected. Our data indicate that Otx1 is a new pituitary transcription factor involved at the prepubescent stage in the control of GH, FSH and LH hormone levels and suggest that a complex regulatory mechanism might exist to control the physiological need for pituitary hormones at specific postnatal stages.

Radiation resistant PIDECα cell using photon intermediate direct energy conversion and a 210Po source.

PubMed

Weaver, Charles L; Schott, Robert J; Prelas, Mark A; Wisniewski, Denis A; Rothenberger, Jason B; Lukosi, Eric D; Oh, Kyuhak

2018-02-01

Radiation damage is a significant concern with both alphavoltaic and betavoltaic cells because their performance degrades, especially with high-energy - (>200keV) beta and alpha particles. Indirect excitation methods, such as the Photon Intermediate Direct Energy Conversion (PIDEC) framework, can protect the transducer from radiation. A nuclear battery using a 90 Sr beta source was constructed by the author's research group, which demonstrated the radiation resistance of a PIDEC cell driven by beta particles (PIDECβ cell). Use of alpha sources to drive nuclear batteries would appear to be much more attractive than beta sources due to higher potential power density. However, they are also subject to higher rates of radiation damage. This paper describes the successful incorporation of alpha particles into the PIDEC framework using the alpha emitter 210 Po to form a PIDECα cell. The PIDECα cell transducer was exposed to alpha particles for over one year without experiencing adverse effects from radiation damage. Copyright © 2017 Elsevier Ltd. All rights reserved.

The effect of interferon on the receptor sites to rabies virus on mouse neuroblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Briggs, D.J.

1989-01-01

The binding of rabies virus to mouse neuroblastoma cells (MNA) primed with alpha interferon (IFN-{alpha}), beta interferon (IFN-{beta}), or alpha bungarotoxin (BTX) was examined. A saturable number of receptor sites to rabies virus was calculated by increasing the amount of {sup 3}H-CVS added to a constant number of untreated MNA cells. MNA cells were then exposed to 20 I.U. of IFN-{alpha}, IFN-{beta}, or 1 {mu}g of BTX and assayed to determine if these treatments had an effect on the number of receptor sites to rabies virus. Total amount of {sup 3}H-CVS bound to MNA cells was determined during a threemore » hour incubation period. Cold competition assays using 1,000 fold excess unlabeled CVS were used to determine non-specific binding for each treatment. Specific binding was then calculated by subtracting non-specific binding from the total amount of CVS bound to MNA cells. A similar amount of total viral protein bound to untreated and IFN-{beta}, and BTX treated cells after 180 minutes of incubation. The bound protein varied by only 0.07 {mu}g. However, the amount of specific and non-specific binding varied a great deal between treatments. BTX caused an increase in non-specific and a decrease in specific binding of rabies virus. IFN-{beta} produced variable results in non-specific and specific binding while IFN-{alpha} caused mainly specific binding to occur. The most significant change brought about by IFN-{alpha} was an increase in the rate of viral attachment. At 30 minutes post-infection, IFN-{alpha} treated cells had bound 90% of the total amount of virus bound to untreated cells after 180 minutes. The increased binding rate did not cause a productive infection of rabies virus. No viral production was evident after an incubation period of 48 hours in either IFN-{alpha} or IFN-{beta} treated cells.« less

BRCA1-IRIS regulates cyclin D1 expression in breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakuci, Enkeleda; Mahner, Sven; DiRenzo, James

2006-10-01

The regulator of cell cycle progression, cyclin D1, is up-regulated in breast cancer cells; its expression is, in part, dependent on ER{alpha} signaling. However, many ER{alpha}-negative tumors and tumor cell lines (e.g., SKBR3) also show over-expression of cyclin D1. This suggests that, in addition to ER{alpha} signaling, cyclin D1 expression is under the control of other signaling pathways; these pathways may even be over-expressed in the ER{alpha}-negative cells. We previously noticed that both ER{alpha}-positive and -negative cell lines over-express BRCA1-IRIS mRNA and protein. Furthermore, the level of over-expression of BRCA1-IRIS in ER{alpha}-negative cell lines even exceeded its over-expression level inmore » ER{alpha}-positive cell lines. In this study, we show that: (1) BRCA1-IRIS forms complex with two of the nuclear receptor co-activators, namely, SRC1 and SRC3 (AIB1) in an ER{alpha}-independent manner. (2) BRCA1-IRIS alone, or in connection with co-activators, is recruited to the cyclin D1 promoter through its binding to c-Jun/AP1 complex; this binding activates the cyclin D1 expression. (3) Over-expression of BRCA1-IRIS in breast cells over-activates JNK/c-Jun; this leads to the induction of cyclin D1 expression and cellular proliferation. (4) BRCA1-IRIS activation of JNK/c-Jun/AP1 appears to account for this, because in cells that were depleted from BRCA1-IRIS, JNK remained inactive. However, depletion of SRC1 or SRC3 instead reduced c-Jun expression. Our data suggest that this novel signaling pathway links BRCA1-IRIS to cellular proliferation through c-Jun/AP1 nuclear pathway; finally, this culminates in the increased expression of the cyclin D1 gene.« less

Cloning, cell-type specificity, and regulatory function of the mouse alpha(1B)-adrenergic receptor promoter.

PubMed

Zuscik, M J; Piascik, M T; Perez, D M

1999-12-01

The functionality of a 3422-base pair promoter fragment from the mouse alpha(1B)-adrenergic receptor (alpha(1B)AR) gene was examined. This fragment, cloned from a mouse genomic library, was found to have significant sequence homology to the known human and rat alpha(1B)AR promoters. However, the consensus motif of several key cis-acting elements is not conserved among the rat, human, and mouse genes, suggesting species specificity. Confirming fidelity of the murine promoter, robust in vitro expression of a chloramphenicol acetyltransferase (CAT) reporter was detected in known alpha(1B)AR-expressing BC(3)H1, NB41A3, and DDT(1)MF-2 cells transiently transfected with a promoter-CAT construct. Conversely, minimal CAT expression was detected in known alpha(1B)AR-negative RAT-1 and R3T3 cells. These findings were extended by transfecting the same promoter-CAT construct into various primary cell types. In support of the hypothesis that alpha(1)ARs are differentially expressed in the smooth muscle of the vasculature, primary cultures of superior mesenteric and renal artery vascular smooth muscle cells showed significantly stronger CAT expression than did vascular smooth muscle cells derived from pulmonary, femoral, and iliac arteries. Primary osteoblastic bone-forming cells, which are known to be alpha(1B)AR negative, showed minimal CAT expression. Indicating regulatory function through cis-acting elements, RAT-1, R3T3, NB41A3, BC(3)H1, and DDT(1)MF2 cells transfected with the promoter-CAT construct all showed increased CAT production when challenged with forskolin or hypoxic conditions. Additionally, tissue-specific regulation of the promoter was observed when cells were simultaneously challenged with both forskolin and hypoxia. These results collectively demonstrate that a 3.4-kb PvuII fragment of the murine alpha(1B)AR gene promoter can: 1) drive tissue-specific production of a CAT reporter in both clonal and primary cell lines; and 2) confer tissue-specific regulation of that CAT reporter when induced by challenge with forskolin and/or hypoxic conditions.

The insecticide fipronil and its metabolite fipronil sulphone inhibit the rat alpha1beta2gamma2L GABA(A) receptor.

PubMed

Li, P; Akk, G

2008-11-01

Fipronil is the active ingredient in a number of widely used insecticides. Human exposure to fipronil leads to symptoms (headache, nausea and seizures) typically associated with the antagonism of GABA(A) receptors in the brain. In this study, we have examined the modulation of the common brain GABA(A) receptor subtype by fipronil and its major metabolite, fipronil sulphone. Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing rat alpha1beta2gamma2L GABA(A) receptors. The major effect of fipronil was to increase the rate of current decay in macroscopic recordings. In single-channel recordings, the presence of fipronil resulted in shorter cluster durations without affecting the intracluster open and closed time distributions or the single-channel conductance. The alpha1V256S mutation, previously shown alleviate channel inhibition by inhibitory steroids and several insecticides, had a relatively small effect on channel block by fipronil. The mode of action of fipronil sulphone was similar to that of its parent compound but the metabolite was less potent at inhibiting the alpha1beta2gamma2L receptor. We conclude that exposure to fipronil induces accumulation of receptors in a novel, long-lived blocked state. This process proceeds in parallel with and independently of, channel desensitization. The lower potency of fipronil sulphone indicates that the conversion serves as a detoxifying process in mammalian brain.

Incensole acetate, a novel anti-inflammatory compound isolated from Boswellia resin, inhibits nuclear factor-kappa B activation.

PubMed

Moussaieff, Arieh; Shohami, Esther; Kashman, Yoel; Fride, Ester; Schmitz, M Lienhard; Renner, Florian; Fiebich, Bernd L; Munoz, Eduardo; Ben-Neriah, Yinon; Mechoulam, Raphael

2007-12-01

Boswellia resin is a major anti-inflammatory agent in herbal medical tradition, as well as a common food supplement. Its anti-inflammatory activity has been attributed to boswellic acid and its derivatives. Here, we re-examined the anti-inflammatory effect of the resin, using inhibitor of nuclear factor-kappaB alpha (IkappaB alpha) degradation in tumor necrosis factor (TNF) alpha-stimulated HeLa cells for a bioassay-guided fractionation. We thus isolated two novel nuclear factor-kappaB (NF-kappaB) inhibitors from the resin, their structures elucidated as incensole acetate (IA) and its nonacetylated form, incensole (IN). IA inhibited TAK/TAB-mediated IkappaB kinase (IKK) activation loop phosphorylation, resulting in the inhibition of cytokine and lipopolysaccharide-mediated NF-kappaB activation. It had no effect on IKK activity in vitro, and it did not suppress IkappaB alpha phosphorylation in costimulated T-cells, indicating that the kinase inhibition is neither direct nor does it affect all NF-kappaB activation pathways. The inhibitory effect seems specific; IA did not interfere with TNFalpha-induced activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase. IA treatment had a robust anti-inflammatory effect in a mouse inflamed paw model. Cembrenoid diterpenoids, specifically IA and its derivatives, may thus constitute a potential novel group of NF-kappaB inhibitors, originating from an ancient anti-inflammatory herbal remedy.

The adaptor protein alpha-syntrophin regulates adipocyte lipid droplet growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eisinger, Kristina; Rein-Fischboeck, Lisa; Pohl, Rebekka

The scaffold protein alpha-syntrophin (SNTA) regulates lipolysis indicating a role in lipid homeostasis. Adipocytes are the main lipid storage cells in the body, and here, the function of SNTA has been analyzed in 3T3-L1 cells. SNTA is expressed in preadipocytes and is induced early during adipogenesis. Knock-down of SNTA in preadipocytes increases their proliferation. Proteins which are induced during adipogenesis like adiponectin and caveolin-1, and the inflammatory cytokine IL-6 are at normal levels in the mature cells differentiated from preadipocytes with low SNTA. This suggests that SNTA does neither affect differentiation nor inflammation. Expression of proteins with a role inmore » cholesterol and triglyceride homeostasis is unchanged. Consequently, basal and epinephrine induced lipolysis as well as insulin stimulated phosphorylation of Akt and ERK1/2 are normal. Importantly, adipocytes with low SNTA form smaller lipid droplets and store less triglycerides. Stearoyl-CoA reductase and MnSOD are reduced upon SNTA knock-down but do not contribute to lower lipid levels. Oleate uptake is even increased in cells with SNTA knock-down. In summary, current data show that SNTA is involved in the expansion of lipid droplets independent of adipogenesis. Enhanced preadipocyte proliferation and capacity to store surplus fatty acids may protect adipocytes with low SNTA from lipotoxicity in obesity. - Highlights: • Alpha-syntrophin (SNTA) is expressed in 3T3-L1adipocytes. • SNTA knock-down in preadipocytes has no effect on adipogenesis. • Mature 3T3-L1 differentiated from cells with low SNTA form small lipid droplets. • SCD1 and MnSOD are reduced in adipocytes with low SNTA. • SCD1 knock-down does not alter triglyceride levels.« less

SU-E-J-03: A Comprehensive Comparison Between Alpha and Beta Emitters for Cancer Radioimmunotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, C.Y.; Guatelli, S; Oborn, B

2014-06-01

Purpose: The purpose of this study is to perform a comprehensive comparison of the therapeutic efficacy and cytotoxicity of alpha and beta emitters for Radioimmunotherapy (RIT). For each stage of cancer development, specific models were built for the separate objectives of RIT to be addressed:a) kill isolated cancer cells in transit in the lymphatic and vascular circulation,b) regress avascular cell clusters,c) regress tumor vasculature and tumors. Methods: Because of the nature of short range, high LET alpha and long energy beta radiation and heterogeneous antigen expression among cancer cells, the microdosimetric approach is essential for the RIT assessment. Geant4 basedmore » microdosimetric models are developed for the three different stages of cancer progression: cancer cells, cell clusters and tumors. The energy deposition, specific energy resulted from different source distribution in the three models was calculated separately for 4 alpha emitting radioisotopes ({sup 211}At, {sup 213}Bi, {sup 223}Ra and {sup 225}Ac) and 6 beta emitters ({sup 32}P, {sup 33}P, {sup 67}Cu, {sup 90}Y, {sup 131}I and {sup 177}Lu). The cell survival, therapeutic efficacy and cytotoxicity are determined and compared between alpha and beta emitters. Results: We show that internal targeted alpha radiation has advantages over beta radiation for killing isolated cancer cells, regressing small cell clusters and also solid tumors. Alpha particles have much higher dose specificity and potency than beta particles. They can deposit 3 logs more dose than beta emitters to single cells and solid tumor. Tumor control probability relies on deep penetration of radioisotopes to cancer cell clusters and solid tumors. Conclusion: The results of this study provide a quantitative understanding of the efficacy and cytotoxicity of RIT for each stage of cancer development.« less

Concise review: Inner ear stem cells--an oxymoron, but why?

PubMed

Ronaghi, Mohammad; Nasr, Marjan; Heller, Stefan

2012-01-01

Hearing loss, caused by irreversible loss of cochlear sensory hair cells, affects millions of patients worldwide. In this concise review, we examine the conundrum of inner ear stem cells, which obviously are present in the inner ear sensory epithelia of nonmammalian vertebrates, giving these ears the ability to functionally recover even from repetitive ototoxic insults. Despite the inability of the mammalian inner ear to regenerate lost hair cells, there is evidence for cells with regenerative capacity because stem cells can be isolated from vestibular sensory epithelia and from the neonatal cochlea. Challenges and recent progress toward identification of the intrinsic and extrinsic signaling pathways that could be used to re-establish stemness in the mammalian organ of Corti are discussed. Copyright © 2011 AlphaMed Press.

Molecular and functional expression of voltage-operated calcium channels during osteogenic differentiation of human mesenchymal stem cells.

PubMed

Zahanich, Ihor; Graf, Eva M; Heubach, Jürgen F; Hempel, Ute; Boxberger, Sabine; Ravens, Ursula

2005-09-01

We used the patch-clamp technique and RT-PCR to study the molecular and functional expression of VOCCs in undifferentiated hMSCs and in cells undergoing osteogenic differentiation. L-type Ca2+ channel blocker nifedipine did not influence alkaline phosphatase activity, calcium, and phosphate accumulation of hMSCs during osteogenic differentiation. This study suggests that osteogenic differentiation of hMSCs does not require L-type Ca2+ channel function. During osteogenic differentiation, mesenchymal stem cells from human bone marrow (hMSCs) must adopt the calcium handling of terminally differentiated osteoblasts. There is evidence that voltage-operated calcium channels (VOCCs), including L-type calcium channels, are involved in regulation of osteoblast function. We therefore studied whether VOCCs play a critical role during osteogenic differentiation of hMSCs. Osteogenic differentiation was induced in hMSCs cultured in maintenance medium (MM) by addition of ascorbate, beta-glycerophosphate, and dexamethasone (ODM) and was assessed by measuring alkaline phosphatase activity, expression of osteopontin, osteoprotegerin, RANKL, and mineralization. Expression of Ca2+ channel alpha1 subunits was shown by semiquantitative or single cell RT-PCR. Voltage-activated calcium currents of hMSCs were measured with the whole cell voltage-clamp technique. mRNA for the pore-forming alpha1C and alpha1G subunits of the L-type and T-type Ca2+ channels, respectively, was found in comparable amounts in cells cultured in MM or ODM. The limitation of L-type Ca2+ currents to a subpopulation of hMSCs was confirmed by single cell RT-PCR, where mRNA for the alpha1C subunits was detectable in only 50% of the cells cultured in MM. Dihydropyridine-sensitive L-type Ca2+ currents were found in 13% of cells cultured in MM and in 12% of the cells cultured in ODM. Under MM and ODM culture conditions, the cells positive for L-type Ca2+ currents were significantly larger than cells without Ca2+ currents as deduced from membrane capacitance; thus, current densities were comparable. Addition of the L-type Ca2+ channel blocker nifedipine to the culture media did not influence alkaline phosphatase activity and the extent of mineralization. These results suggest that, in the majority of hMSCs, Ca2+ entry through the plasma membrane is mediated by some channels other than VOCCs, and blockade of the L-type Ca2+ channels does not affect early osteogenic differentiation of hMSCs.

Schindler disease: the molecular lesion in the alpha-N-acetylgalactosaminidase gene that causes an infantile neuroaxonal dystrophy.

PubMed Central

Wang, A M; Schindler, D; Desnick, R

1990-01-01

Schindler disease is a recently recognized infantile neuroaxonal dystrophy resulting from the deficient activity of the lysosomal hydrolase, alpha-N-acetylgalctosaminidase (alpha-GalNAc). The recent isolation and expression of the full-length cDNA encoding alpha-GalNAc facilitated the identification of the molecular lesions in the affected brothers from family D, the first cases described with this autosomal recessive disease. Southern and Northern hybridization analyses of DNA and RNA from the affected homozygotes revealed a grossly normal alpha-GalNAc gene structure and normal transcript sizes and amounts. Therefore, the alpha-GalNAc transcript from an affected homozygote was reverse-transcribed, amplified by the polymerase chain reaction (PCR), and sequenced. A single G to A transition at nucleotide 973 was detected in multiple subclones containing the PCR products. This point mutation resulted in a glutamic acid to lysine substitution in residue 325 (E325K) of the alpha-GalNAc polypeptide. The base substitution was confirmed by dot blot hybridization analyses of PCR-amplified genomic DNA from family members with allele-specific oligonucleotides. Furthermore, transient expression of an alpha-GalNAc construct containing the E325K mutation resulted in the expression of an immunoreactive polypeptide which had no detectable alpha-GalNAc activity. Images PMID:2243144

Biological effects of THC and a lipophilic cannabis extract on normal and insulin resistant 3T3-L1 adipocytes.

PubMed

Gallant, M; Odei-Addo, F; Frost, C L; Levendal, R-A

2009-10-01

Type 2 diabetes, a chronic disease, affects about 150 million people world wide. It is characterized by insulin resistance of peripheral tissues such as liver, skeletal muscle, and fat. Insulin resistance is associated with elevated levels of tumor necrosis factor alpha (TNF-alpha), which in turn inhibits insulin receptor tyrosine kinase autophosphorylation. It has been reported that cannabis is used in the treatment of diabetes. A few reports indicate that smoking cannabis can lower blood glucose in diabetics. Delta(9)-tetrahydrocannabinol (THC) is the primary psychoactive component of cannabis. This study aimed to determine the effect of a lipophilic cannabis extract on adipogenesis, using 3T3-L1 cells, and to measure its effect on insulin sensitivity in insulin resistant adipocytes. Cells were cultured in Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) and differentiated over a 3 day period for all studies. In the adipogenesis studies, differentiated cells were exposed to the extract in the presence and absence of insulin. Lipid content and glucose uptake was subsequently measured. Insulin-induced glucose uptake increased, while the rate of adipogenesis decreased with increasing THC concentration. Insulin-resistance was induced using TNF-alpha, exposed to the extract and insulin-induced glucose uptake measured. Insulin-induced glucose was increased in these cells after exposure to the extract. Semiquantitative real time polymerase chain reaction (RT-PCR) was performed after ribonucleic acid (RNA) extraction to evaluate the effects of the extract on glucose transporter isotype 4 (GLUT-4), insulin receptor substrate-1 (IRS-1) and IRS-2 gene expression.

Radiation-Induce Immune Modulation in Prostate Cancer

DTIC Science & Technology

2005-01-01

Prostate-specific antigen Prostate carcinoma Mammoglobin-A Breast carcinoma Overexpressed Alpha - fetoprotein Hepatocellular carcinoma and yolk-sac tumors...Interleukin-3 cooperates with tumor necrosis factor alpha for the development of human dendritic/Langerhans cells from cord blood CD34+ hematopoietic progenitor...additional subsets, e.g. Langerhans cells of the epidermis, and dermal or interstitial DC. PDC are the major interferon- alpha (IFNca) producing cells

Method for characterizing the upset response of CMOS circuits using alpha-particle sensitive test circuits

NASA Technical Reports Server (NTRS)

Buehler, Martin G. (Inventor); Nixon, Robert H. (Inventor); Soli, George A. (Inventor); Blaes, Brent R. (Inventor)

1995-01-01

A method 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. A technique utilizing test structures to quickly and inexpensively characterize the SEU sensitivity of standard cell latches intended for use in a space environment. This bench-level approach utilizes alpha particles to induce upsets in a low LET sensitive 4-k bit test SRAM. This SRAM consists of cells that employ an offset voltage to adjust their upset sensitivity and an enlarged sensitive drain junction to enhance the cell's upset rate.

Gene-specific changes in alpha-tubulin transcript accumulation in developing cotton fibers.

PubMed

Whittaker, D J; Triplett, B A

1999-09-01

The fibers of cotton (Gossypium hirsutum) are single-cell trichomes that undergo rapid and synchronous elongation. Cortical microtubules provide spatial information necessary for the alignment of cellulose microfibrils that confine and regulate cell elongation. We used gene-specific probes to investigate alpha-tubulin transcript levels in elongating cotton fibers. Two discrete patterns of transcript accumulation were observed. Whereas transcripts of alpha-tubulin genes GhTua2/3 and GhTua4 increased in abundance from 10 to 20 d post anthesis (DPA), GhTua1 and GhTua5 transcripts were abundant only through to 14 DPA, and dropped significantly at 16 DPA with the onset of secondary wall synthesis. This is the first report, to our knowledge, of gene-specific changes in tubulin transcript levels during the development of a terminally differentiated plant cell. The decrease in abundance of GhTua1 and GhTua5 transcripts was correlated with pronounced changes in cell wall structure, suggesting that alpha-tubulin isoforms may be functionally distinct in elongating fiber cells. Although total alpha-tubulin transcript levels were much higher in fiber than several other tissues, including the hypocotyl and pollen, none of the alpha-tubulins was specific to fiber cells.

Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.

PubMed

Uzé, G; Lutfalla, G; Bandu, M T; Proudhon, D; Mogensen, K E

1992-05-15

A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes.

Alpha oscillations and their impairment in affective and post-traumatic stress disorders.

PubMed

Eidelman-Rothman, Moranne; Levy, Jonathan; Feldman, Ruth

2016-09-01

Affective and anxiety disorders are debilitating conditions characterized by impairments in cognitive and social functioning. Elucidating their neural underpinnings may assist in improving diagnosis and developing targeted interventions. Neural oscillations are fundamental for brain functioning. Specifically, oscillations in the alpha frequency range (alpha rhythms) are prevalent in the awake, conscious brain and play an important role in supporting perceptual, cognitive, and social processes. We review studies utilizing various alpha power measurements to assess abnormalities in brain functioning in affective and anxiety disorders as well as obsessive compulsive and post-traumatic stress disorders. Despite some inconsistencies, studies demonstrate associations between aberrant alpha patterns and these disorders both in response to specific cognitive and emotional tasks and during a resting state. We conclude by discussing methodological considerations and future directions, and underscore the need for much further research on the role of alpha functionality in social contexts. As social dysfunction accompanies most psychiatric conditions, research on alpha's involvement in social processes may provide a unique window into the neural mechanisms underlying these disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

Role of hypoxia-inducible factor-{alpha} in hepatitis-B-virus X protein-mediated MDR1 activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Hyo-Kyung; Han, Chang Yeob; Cheon, Eun-Pa

2007-06-01

The transition from chemotherapy-responsive cancer cells to chemotherapy-resistant cancer cells is mainly accompanied by the increased expression of multi-drug resistance 1 (MDR1). We found that hepatitis-B-virus X protein (HBx) increases the transcriptional activity and protein level of MDR1 in a hepatoma cell line, H4IIE. In addition, HBx overexpression made H4IIE cells more resistant to verapamil-uptake. HBx stabilized hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) and induced the nuclear translocation of C/EBP{beta}. Reporter gene analyses showed that HBx increased the reporter activity in the cells transfected with the reporter containing MDR1 gene promoter. Moreover, the luciferase reporter gene activity was significantly inhibited by HIF-1{alpha} siRNAmore » but not by overexpression of C/EBP dominant negative mutant. These results imply that HBx increases the MDR1 transporter activity through the transcriptional activation of the MDR1 gene with HIF-1{alpha} activation, and suggest HIF-1{alpha} for the therapeutic target of HBV-mediated chemoresistance.« less

Alpha-tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II.

PubMed

Dong, L-F; Low, P; Dyason, J C; Wang, X-F; Prochazka, L; Witting, P K; Freeman, R; Swettenham, E; Valis, K; Liu, J; Zobalova, R; Turanek, J; Spitz, D R; Domann, F E; Scheffler, I E; Ralph, S J; Neuzil, J

2008-07-17

Alpha-tocopheryl succinate (alpha-TOS) is a selective inducer of apoptosis in cancer cells, which involves the accumulation of reactive oxygen species (ROS). The molecular target of alpha-TOS has not been identified. Here, we show that alpha-TOS inhibits succinate dehydrogenase (SDH) activity of complex II (CII) by interacting with the proximal and distal ubiquinone (UbQ)-binding site (Q(P) and Q(D), respectively). This is based on biochemical analyses and molecular modelling, revealing similar or stronger interaction energy of alpha-TOS compared to that of UbQ for the Q(P) and Q(D) sites, respectively. CybL-mutant cells with dysfunctional CII failed to accumulate ROS and underwent apoptosis in the presence of alpha-TOS. Similar resistance was observed when CybL was knocked down with siRNA. Reconstitution of functional CII rendered CybL-mutant cells susceptible to alpha-TOS. We propose that alpha-TOS displaces UbQ in CII causing electrons generated by SDH to recombine with molecular oxygen to yield ROS. Our data highlight CII, a known tumour suppressor, as a novel target for cancer therapy.

Functional characterization of an alpha-factor-like Sordaria macrospora peptide pheromone and analysis of its interaction with its cognate receptor in Saccharomyces cerevisiae.

PubMed

Mayrhofer, Severine; Pöggeler, Stefanie

2005-04-01

The homothallic filamentous ascomycete Sordaria macrospora possesses genes which are thought to encode two pheromone precursors and two seven-transmembrane pheromone receptors. The pheromone precursor genes are termed ppg1 and ppg2. The putative products derived from the gene sequence show structural similarity to the alpha-factor precursors and a-factor precursors of the yeast Saccharomyces cerevisiae. Likewise, sequence similarity has been found between the putative products of the pheromone receptor genes pre2 and pre1 and the S. cerevisiae Ste2p alpha-factor receptor and Ste3p a-factor receptor, respectively. To investigate whether the alpha-factor-like pheromone-receptor pair of S. macrospora is functional, a heterologous yeast assay was used. Our results show that the S. macrospora alpha-factor-like pheromone precursor PPG1 is processed into an active pheromone by yeast MATalpha cells. The S. macrospora PRE2 protein was demonstrated to be a peptide pheromone receptor. In yeast MATa cells lacking the endogenous Ste2p receptor, the S. macrospora PRE2 receptor facilitated all aspects of the pheromone response. Using a synthetic peptide, we can now predict the sequence of one active form of the S. macrospora peptide pheromone. We proved that S. macrospora wild-type strains secrete an active pheromone into the culture medium and that disruption of the ppg1 gene in S. macrospora prevents pheromone production. However, loss of the ppg1 gene does not affect vegetative growth or fertility. Finally, we established the yeast assay as an easy and useful system for analyzing pheromone production in developmental mutants of S. macrospora.

The N-end rule pathway catalyzes a major fraction of the protein degradation in skeletal muscle

NASA Technical Reports Server (NTRS)

Solomon, V.; Lecker, S. H.; Goldberg, A. L.

1998-01-01

In skeletal muscle, overall protein degradation involves the ubiquitin-proteasome system. One property of a protein that leads to rapid ubiquitin-dependent degradation is the presence of a basic, acidic, or bulky hydrophobic residue at its N terminus. However, in normal cells, substrates for this N-end rule pathway, which involves ubiquitin carrier protein (E2) E214k and ubiquitin-protein ligase (E3) E3alpha, have remained unclear. Surprisingly, in soluble extracts of rabbit muscle, we found that competitive inhibitors of E3alpha markedly inhibited the 125I-ubiquitin conjugation and ATP-dependent degradation of endogenous proteins. These inhibitors appear to selectively inhibit E3alpha, since they blocked degradation of 125I-lysozyme, a model N-end rule substrate, but did not affect the degradation of proteins whose ubiquitination involved other E3s. The addition of several E2s or E3alpha to the muscle extracts stimulated overall proteolysis and ubiquitination, but only the stimulation by E3alpha or E214k was sensitive to these inhibitors. A similar general inhibition of ubiquitin conjugation to endogenous proteins was observed with a dominant negative inhibitor of E214k. Certain substrates of the N-end rule pathway are degraded after their tRNA-dependent arginylation. We found that adding RNase A to muscle extracts reduced the ATP-dependent proteolysis of endogenous proteins, and supplying tRNA partially restored this process. Finally, although in muscle extracts the N-end rule pathway catalyzes most ubiquitin conjugation, it makes only a minor contribution to overall protein ubiquitination in HeLa cell extracts.

Sodium 4-phenylbutyrate upregulates ENaC and sodium absorption in T84 cells.

PubMed

Iordache, Claudiu; Duszyk, Marek

2007-01-15

Butyrate and other short-chain fatty acids (SCFA), produced by colonic bacterial flora, affect numerous epithelial cell functions. To better understand how SCFA regulate ion transport, we investigated the effects of 4-phenylbutyrate (4-PBA) on Na(+) absorption in T84 cells. Under standard cell culture conditions, the short circuit current did not display any amiloride-sensitive Na(+) absorption and was wholly representative of Cl(-) secretion. However, when T84 cells were grown in the presence of 5 mM 4-PBA, a gradual appearance of amiloride-sensitive Na(+) channel (ENaC) activity was observed that reached a plateau after 24 h. Quantitative RT-PCR and Western blot studies of ENaC subunit expression indicated that 4-PBA stimulated alpha and gamma subunits. Trichostatin A, an inhibitor of histone deacetylase, mimicked the effects of 4-PBA, suggesting that 4-PBA affects ENaC expression by inhibiting deacetylases. 4-PBA had no effect on ENaC expression in airway epithelial cells indicating tissue-specific effect. We conclude that butyrate plays an important role in regulating colonic Na(+) absorption by increasing ENaC transcription and activity.

Effects of interferon-alpha subtypes on the TH1/TH2 balance in peripheral blood mononuclear cells from patients with hepatitis virus infection-associated liver disorders.

PubMed

Ariyasu, Toshio; Tanaka, Takeshi; Fujioka, Noboru; Yanai, Yoshiaki; Yamamoto, Shigeto; Yamauchi, Hiroshi; Ikegami, Hakuo; Ikeda, Masao; Kurimoto, Masashi

2005-01-01

Interferon-alpha (IFN-alpha) has recently been shown to modulate in vitro T helper (Th) 1-driven responses in the peripheral blood mononuclear cells (PBMC) of patients with hepatitis B virus or C virus infection. In this study, we examined the in vitro effects of IFN-alpha subtypes (IFN-alpha1, -alpha2, -alpha5, -alpha8, and -alpha10) on the Th1/Th2 balance in PBMC obtained from patients with hepatitis virus infection-associated liver disorders and chronic hepatitis (CH), in comparison with the effect on healthy control volunteer PBMC. The Th1-type cell percentages and Th1/Th2 ratios were significantly higher in the PBMC of patients when compared with controls both before and after cultivation in vitro, with the IFN-alpha subtypes. The IFNalpha-5 induced an increase in the Th2-type cell percentages in both control and patient PBMC, resulting in that IFN-alpha5 lowered the Th1/Th2 ratio in patients with CH. Furthermore, statistical analysis revealed that IFN-alpha8 significantly promoted an increase in the Th1/Th2 ratios of PBMC from patients with CH and liver cirrhosis (LC) but not that of PBMC from patients with LC-hepatocellular carcinoma (HCC) and HCC. These findings imply that hepatitis virus infection and its disease status modify the effects of IFN-alpha subtypes on Th1 and Th2 immune balance in patients. Our findings should help to elucidate the mechanisms underlying successful IFN therapy for hepatitis virus infection and prevention of hepatocellular carcinogenesis.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gulec, Cagri, E-mail: cagri.gulec@gmail.com; Coban, Neslihan, E-mail: neslic@istanbul.edu.tr; Ozsait-Selcuk, Bilge, E-mail: ozsaitb@istanbul.edu.tr

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

De-phosphorylation of TR{alpha}-1 by p44/42 MAPK inhibition enhances T{sub 3}-mediated GLUT5 gene expression in the intestinal cell line Caco-2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mochizuki, Kazuki; Sakaguchi, Naomi; Takabe, Satsuki

2007-08-10

Thyroid hormone and p44/42 MAPK inactivation are important in intestinal differentiation. We demonstrated not only that treatment with p44/42 MAPK inhibitor U0126 in intestinal cell line Caco-2 cells reduced the phosphorylation of serine and threonine residues of TR{alpha}-1, but also that T{sub 3} and U0126 synergistically induced GLUT5 gene expression. EMSA demonstrated that the binding activity of TR{alpha}-1-RXR heterodimer on GLUT5-TRE in nuclear proteins of Caco-2 cells was synergistically enhanced by co-incubation in vitro with T{sub 3} and CIAP, which strongly de-phosphorylates proteins. ChIP and transfection assays revealed that co-treatment of T{sub 3} and U0126 induces TR{alpha}-1-RXR binding to GLUT5-TREmore » on the human GLUT5 enhancer region, and recruitment of the transcriptional complex in cells. These results suggest that inactivation of p44/42 MAPK enhances T{sub 3}-induced GLUT5 gene expression in Caco-2 cells through increasing TR{alpha}-1 transactivity and binding activity to the GLUT5-TRE, probably due to de-phosphorylation of TR{alpha}-1.« less

Documentation of normal and leukemic myelopoietic progenitor cells with high-resolution phase-contrast time-lapse cinematography.

PubMed

Boll, I T

2001-08-01

The high-resolution phase-contrast, time-lapse cinematography using oil immersion lenses and 16-mm film demonstrates the kinetic cell events as maturation, locomotion, mitosis, and apoptosis of cells cultivated at 37 degrees C for up to 10 days. 0.5 v/v frozen-thawed sera with presumably high cytokine concentrations were added to the plasma or agar clot. Vital progenitor cells from human bone marrow and blood have a large, bright, unstructured nucleus with a large nucleolus and a narrow rim of cytoplasm (nuclear/cytoplasmic volume ratio = 0.7). Their nuclei are 6-14 micrometer in diameter and double their volume within 8 h. Many (70%) move at a mean speed of 2 micrometer/min, and many (30%) multiply with alpha-2alpha mitoses, generating progenitor cell families. Various disturbances during the course of mitosis lead to the formation of polyploid cells, thereby yielding the megakaryocytic cell line. Some of the progenitor cells undergo asymmetric alpha-alphan mitoses: One of the two initially identical daughter cells remains a progenitor cell in the morphological sense, whereas the other daughter cell - depending on the size of its mother cell - matures in the same culture medium to form a granulocytopoietic, monocytopoietic or erythrocytopoietic cell line. - In acute myeloid leukemias (AML), the blasts and their nuclei are slightly larger than the corresponding progenitor cells and move faster (5 micrometer/min). Symmetric alpha-2alpha mitoses permit unlimited multiplication of the leukemic blasts if contact with cytotoxic lymphocytes does not render them apoptotic. This results in more stromal cells than normal. Granulocytopenia, monocytopenia, and anemia occur due to the genetic impairment of signaling control for asymmetric alpha-alphan mitoses, and thrombocytopenia occurs due to the reduction in polyploidization. Copyright 2001 S. Karger GmbH, Freiburg

Selective Cytotoxicity and Combined Effects of Camptothecin or Paclitaxel with Sodium-R-Alpha Lipoate on A549 Human Non-Small Cell Lung Cancer Cells

PubMed Central

Ibrahim, Sherif; Gao, Dayuan; Sinko, Patrick J.

2013-01-01

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and remains the deadliest form of cancer in the US and worldwide. New therapies are highly sought after to improve outcome. The effect of sodium-R-alpha lipoate on camptothecin- and paclitaxel-induced cytotoxicity was evaluated on A549 NSCLC and BEAS-2B ‘normal’ lung epithelial cells. Combination indices (CI) and dose reduction indices (DRI) were investigated by studying the cytotoxicity of sodium-R-alpha lipoate (0–16 mM), camptothecin (0–25 nM) and paclitaxel (0–0.06 nM) alone and in combination. 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium-bromide (MTT) was used to assess cytotoxicity. The combinational cytotoxic effects of sodium-R-alpha lipoate with camptothecin or paclitaxel were analyzed using a simulation of dose effects (CompuSyn®3.01). The effects of sodium-R-alpha lipoate on camptothecin- and paclitaxel-induced cytotoxicity varied based on concentrations and treatment times. It was found that sodium-R-alpha lipoate wasn’t cytotoxic towards BEAS-2B cells at any of the concentrations tested. For A549 cells, CIs [(additive (CI=1); synergistic (CI<1); antagonistic (CI>1)] were lower and DRIs were higher for the camptothecin/sodium-R-alpha-lipoate combination (CI=~0.17–1.5; DRI=~2.2–22.6) than the paclitaxel/sodium-R-alpha-lipoate combination (CI=~0.8–9.9; DRI=~0.10–5.8) suggesting that the camptothecin regimen was synergistic and that the addition of sodium-R-alpha lipoate was important for reducing the camptothecin dose and potential for adverse effects. PMID:24063429

Insulin-like growth factor-binding protein 7 alters the sensitivity to interferon-based anticancer therapy in hepatocellular carcinoma cells.

PubMed

Tomimaru, Y; Eguchi, H; Wada, H; Noda, T; Murakami, M; Kobayashi, S; Marubashi, S; Takeda, Y; Tanemura, M; Umeshita, K; Doki, Y; Mori, M; Nagano, H

2010-05-11

A striking efficiency of interferon (IFN)-based anticancer therapy for advanced hepatocellular carcinoma (HCC) has been reported. Because its clinical efficiency greatly depends on each patient's local response, prediction of local response is crucial. Continuous exposure of IFN-alpha to parental PLC/PRF/5 cells (PLC-P) and a limiting dilution method resulted in the establishment of IFN-resistant cell clones (PLC-Rs). Microarray analyses of PLC-P and PLC-Rs identified insulin-like growth factor-binding protein 7 (IGFBP7) as one of the most significantly downregulated genes in PLC-Rs. Changes in anticancer effects of IFN-alpha were examined in HCC cells after genetic manipulation of IGFBP7 expression. The correlation between immunohistochemically determined IGFBP7 expression and the response to IFN-alpha/5-fluorouracil (5-FU) therapy was investigated in surgically resected HCC specimens. PLC-R cells showed a remarkable downregulation of IGFBP7 and resistance to IFN-alpha, compared with PLC-P. Parental PLC/PRF/5 cells transfected with short hairpin RNA against IGFBP7 showed a significant resistance to IFN-alpha relative to control cells (IC(50) fold increase=14.38 times). Insulin-like growth factor-binding protein 7 transfection into PLC-R restored sensitivity to IFN-alpha. In resected specimens, IGFBP7 expression significantly correlated with the response to IFN-alpha/5-FU therapy. IGFBP7 could be a useful predictor of the response to IFN-based therapy in advanced HCC.

The metabolism of testosterone by dermal papilla cells cultured from human pubic and axillary hair follicles concurs with hair growth in 5 alpha-reductase deficiency.

PubMed

Hamada, K; Thornton, M J; Laing, I; Messenger, A G; Randall, V A

1996-05-01

Androgens regulate the growth of many human hair follicles, but only pubic, axillary, and scalp hair growth occur in men with 5 alpha-reductase deficiency. This suggests that 5 alpha-dihydrotestosterone is the active intracellular androgen in androgen-dependent follicles, except in the axilla and pubis. Since the dermal papilla plays a major regulatory role in hair follicles and may be the site of androgen action, we have investigated androgen metabolism in six primary lines of cultured dermal papilla cells from pubic and axillary hair follicles; previous studies have shown that beard cells take up and metabolize testosterone, retaining and secreting 5 alpha-dihydrotestosterone. After 24 h preincubation in serum-free Eagle's medium 199, 100-mm dishes of confluent cells were incubated for 2 h with 5 nM [1,2,6,7-3H]testosterone. Media were collected and the cells washed with phosphate-buffered saline and extracted with chloroform: methanol (2:1). After the addition of unlabeled and 14C-labeled marker steroids, the extracts were analyzed by a two-step thin-layer chromatography system; steroid identity was confirmed by recrystallization to a constant 3H/14C ratio. Beard and pubic dermal papilla cells were also incubated for 24 h, and the medium was analyzed at various times. The results from pubic and axillary primary cell lines were similar. In both cells and media the major steroid identified was testosterone, but significant amounts of androstenedione were present, indicating 17 beta-hydroxysteroid dehydrogenase activity; androstenedione was also identified within the cells, but a small amount of 5 alpha-dihydrotestosterone was only identified in one pubic cell line. Beard dermal papilla cells secreted large amounts of 5 alpha-dihydrotestosterone into the medium over 24 h in contrast to pubic cells, which produced only very small amounts. The pubic and axillary cell results contrasts with the observations of pronounced 5 alpha-dihydrotestosterone in beard cells and confirm that androgen metabolism in cultured dermal papilla cells reflects the parent follicle's ability to respond to androgen in the absence of 5 alpha-reductase type II in vivo. This supports our hypothesis that androgen acts on hair follicles via the dermal papilla and suggests that cultured dermal papilla cells may offer an important model system for studies of androgen action.

Graves' ophthalmopathy: low-dose dexamethasone reduces retinoic acid receptor-alpha gene expression in orbital fibroblasts.

PubMed

Cury, Sarah Santiloni; Oliveira, Miriane; Síbio, Maria Teresa; Clara, Sueli; Luvizotto, Renata De Azevedo Melo; Conde, Sandro; Jorge, Edson Nacib; Nunes, Vania Dos Santos; Nogueira, Célia Regina; Mazeto, Gláucia Maria Ferreira da Silva

2018-05-17

Graves' ophthalmopathy (GO) is an autoimmune disease that leads to ocular proptosis caused by fat accumulation and inflammation, and the main treatment is corticosteroid therapy. Retinoid acid receptor-alpha (RARα) seems to be associated with inflammation and adipocyte differentiation. This study aimed to assess the effect of glucocorticoid treatment on orbital fibroblasts of GO patient treated or not with different glucocorticoid doses. Orbital fibroblasts collected during orbital decompression of a female patient with moderately severe/severe GO were cultivated and treated with 10 nM and 100 nM dexamethasone (Dex). rRARα gene expression in the treated and untreated cells was then compared. Fibroblast RARα expression was not affected by 100 nM Dex. On the other hand, RARα expression was 24% lower in cells treated with 10 nM Dex (p < 0.05). Orbital fibroblasts from a GO patient expressed the RARα gene, which was unaffected by higher, but decreased with lower doses of glucocorticoid.

Tumor necrosis factor (cachectin) is an endogenous pyrogen and induces production of interleukin 1.

PubMed

Dinarello, C A; Cannon, J G; Wolff, S M; Bernheim, H A; Beutler, B; Cerami, A; Figari, I S; Palladino, M A; O'Connor, J V

1986-06-01

Recombinant human tumor necrosis factor (rTNF alpha) injected intravenously into rabbits produces a rapid-onset, monophasic fever indistinguishable from the fever produced by rIL-1. On a weight basis (1 microgram/kg) rTNF alpha and rIL-1 produce the same amount of fever and induce comparable levels of PGE2 in rabbit hypothalamic cells in vitro; like IL-1, TNF fever is blocked by drugs that inhibit cyclooxygenase. At higher doses (10 micrograms/kg) rTNF alpha produces biphasic fevers. The first fever reaches peak elevation 45-55 min after bolus injection and likely represents a direct action on the thermoregulatory center. During the second fever peak (3 h later), a circulating endogenous pyrogen can be shown present using passive transfer of plasma into fresh rabbits. This likely represents the in vivo induction of IL-1. In vitro, rTNF alpha induces the release of IL-1 activity from human mononuclear cells with maximal production observed at 50-100 ng/ml of rTNF alpha. In addition, rTNF alpha and rIFN-gamma have a synergistic effect on IL-1 production. The biological activity of rTNF alpha could be distinguished from IL-1 in three ways: the monophasic pyrogenic activity of rIL-1 was destroyed at 70 degrees C, whereas rTNF alpha remained active; anti-IL-1 neutralized IL-1 but did recognize rTNF alpha or natural cachectin nor neutralize its cytotoxic effect; and unlike IL-1, rTNF alpha was not active in the mitogen-stimulated T cell proliferation assay. The possibility that endotoxin was responsible for rTNF alpha fever and/or the induction of IL-1 was ruled-out in several studies: rTNF alpha produced fever in the endotoxin-resistant C3H/HeJ mice; the IL-1-inducing property of rTNF alpha was destroyed either by heat (70 degrees C) or trypsinization, and was unaffected by polymyxin B; pyrogenic tolerance to daily injections of rTNF alpha did not occur; levels of endotoxin, as determined in the Limulus amebocyte lysate, were below the minimum rabbit pyrogen dose; and these levels of endotoxin were confirmed by gas chromatography/mass spectrometry analysis for the presence of beta-hydroxymyristic acid. Although rTNF alpha is not active in T cell proliferation assays, it may mimic IL-1 in a T cell assay, since high concentrations of rTNF alpha induced IL-1 from epithelial or macrophagic cells in the thymocyte preparations. These studies show that TNF (cachectin) is another endogenous pyrogen which, like IL-1 and IFN-alpha, directly stimulate hypothalamic PGE2 synthesis. In addition, rTNF alpha is an endogenous inducer of IL-1.(ABSTRACT TRUNCATED AT 400 WORDS)

How actin crosslinking and bundling proteins cooperate to generate an enhanced cell mechanical response

NASA Technical Reports Server (NTRS)

Tseng, Yiider; Kole, Thomas P.; Lee, Jerry S H.; Fedorov, Elena; Almo, Steven C.; Schafer, Benjamin W.; Wirtz, Denis

2005-01-01

Actin-crosslinking proteins organize actin filaments into dynamic and complex subcellular scaffolds that orchestrate important mechanical functions, including cell motility and adhesion. Recent mutation studies have shown that individual crosslinking proteins often play seemingly non-essential roles, leading to the hypothesis that they have considerable redundancy in function. We report live-cell, in vitro, and theoretical studies testing the mechanical role of the two ubiquitous actin-crosslinking proteins, alpha-actinin and fascin, which co-localize to stress fibers and the basis of filopodia. Using live-cell particle tracking microrheology, we show that the addition of alpha-actinin and fascin elicits a cell mechanical response that is significantly greater than that originated by alpha-actinin or fascin alone. These live-cell measurements are supported by quantitative rheological measurements with reconstituted actin filament networks containing pure proteins that show that alpha-actinin and fascin can work in concert to generate enhanced cell stiffness. Computational simulations using finite element modeling qualitatively reproduce and explain the functional synergy of alpha-actinin and fascin. These findings highlight the cooperative activity of fascin and alpha-actinin and provide a strong rationale that an evolutionary advantage might be conferred by the cooperative action of multiple actin-crosslinking proteins with overlapping but non-identical biochemical properties. Thus the combination of structural proteins with similar function can provide the cell with unique properties that are required for biologically optimal responses.

Characterization of iron uptake from transferrin by murine endothelial cells.

PubMed

Hallmann, R; Savigni, D L; Morgan, E H; Baker, E

2000-01-01

Iron is required by the brain for normal function, however, the mechanisms by which it crosses the blood-brain barrier (BBB) are poorly understood. The uptake and efflux of transferrin (Tf) and Fe by murine brain-derived (bEND3) and lymph node-derived (m1END1) endothelial cell lines was compared. The effects of iron chelators, metabolic inhibitors and the cellular activators, lipopolysaccharide (LPS) and tumour necrosis factor-alpha (TNF-alpha), on Tf and Fe uptake were investigated. Cells were incubated with 59Fe-125I-Tf; Fe uptake was shown to increase linearly over time for both cell lines, while Tf uptake reached a plateau within 2 h. Both Tf and Fe uptake were saturable. bEND3 cells were shown to have half as many Tf receptors as m1END1 cells, but the mean cycling times of a Tf molecule were the same. Tf and Fe efflux from the cells were measured over time, revealing that after 2 h only 25% of the Tf but 80% of the Fe remained associated with the cells. Of 7 iron chelators, only deferriprone (L1) markedly decreased Tf uptake. However, Fe uptake was reduced by more than 50% by L1, pyridoxal isonicotinoyl hydrazone (PIH) and desferrithiocin (DFT). The cellular activators TNF-alpha or LPS had little effect on Tf turnover, but they accelerated Fe uptake in both endothelial cell types. Phenylarsenoxide (PhAsO) and N-ethyl maleimide (NEM), inhibitors of Tf endocytosis, reduced both Tf and Fe uptake in both cell lines, while bafilomycin A1, an inhibitor of endosomal acidification, reduced Fe uptake but did not affect Tf uptake. The results suggest that Tf and Fe uptake by both bEND3 and m1END1 is via receptor-mediated endocytosis with release of Fe from Tf within the cell and recycling of apo-Tf. On the basis of Tf- and Fe-metabolism both cell lines are similar and therefore well suited for use in in vitro models for Fe transport across the BBB.

Growth-related gene product {alpha}: A chemotactic cytokine for neutrophils in rheumatoid arthritis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koch, A.E.; Pope, R.M.; Shah, M.R.

Leukocyte recruitment is critical in the inflammation seen in rheumatoid arthritis (RA). To determine whether the chemokine growth-related gene product {alpha} (gro{alpha}) plays a role in this process, we examined synovial tissue (ST), synovial fluid (SF), and plasma samples from 102 patients with arthritis. RA SF contained more antigenic gro{alpha} (mean 5.3 {+-} 1.9 ng/ml) than did SFs from either osteoarthritis (OA) or other forms of arthritis (mean 0.1 ng/ml) (p < 0.05). RA plasma contained more gro{alpha} (mean 4.3 {+-} 1.8 ng/ml) than normal plasma (mean 0.1 ng/ml) (p < 0.05). RA ST fibroblasts (1.2 x 10{sup 5}/cells/ml RPMImore » 1640/24 h) produced antigenic gro{alpha} (mean 0.2 {+-} 0.1 ng/ml), and this production was increased significantly upon incubation with TNF-{alpha} (mean 1.3 {+-} 0.3 ng/ml) or IL-1{beta} (mean 2.3 {+-} 0.6 ng/ml) (p < 0.05). Cells from RA SF also produced gro{alpha}: neutrophils (PMNs) (10{sup 7} cells/ml/24 h) produced 3.7 {+-} 0.7 ng/ml. RA SF mononuclear cells produced gro{alpha}, particularly upon incubation with LPS or PHA. Immunoreactive ST gro{alpha} was found in greater numbers of RA compared with either OA or normal lining cells, as well as in RA compared with OA subsynovial macrophages (p < 0.05). IL-8 accounted for a mean of 36% of the RA SF chemotactic activity for PMNs, while epithelial neutrophil-activating peptide-78 accounted for 34%, and gro{alpha} for 28%, of this activity. Combined neutralization of all three chemokines in RA SFs resulted in a mean decrease of 50% of the chemotactic activity for PMNs present in the RA SFs. These results indicate that gro{alpha} plays an important role in the ingress of PMNs into the RA joint. 54 refs., 6 figs., 1 tab.« less

Estrogen-related receptor {alpha} is essential for the expression of antioxidant protection genes and mitochondrial function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rangwala, Shamina M.; Li, Xiaoyan; Lindsley, Loren

2007-05-25

Estrogen-related receptor {alpha} (ERR{alpha}) is an important mediator of mitochondrial biogenesis and function. To investigate the transcriptional network controlling these phenomena, we investigated mitochondrial gene expression in embryonic fibroblasts isolated from ERR{alpha} null mice. Peroxisome proliferator-activated receptor {gamma} coactivator-1{alpha} (PGC-1{alpha}) stimulated mitochondrial gene expression program in control cells, but not in the ERR{alpha} null cells. Interestingly, the induction of levels of mitochondrial oxidative stress protection genes in response to increased PGC-1{alpha} levels was dependent on ERR{alpha}. Furthermore, we found that the PGC-1{alpha}-mediated induction of estrogen-related receptor {gamma} and nuclear respiratory factor 2 (NRF-2), was dependent on the presence of ERR{alpha}.more » Basal levels of NRF-2 were decreased in the absence of ERR{alpha}. The absence of ERR{alpha} resulted in a decrease in citrate synthase enzyme activity in response to PGC-1{alpha} overexpression. Our results indicate an essential role for ERR{alpha} as a key regulator of oxidative metabolism.« less

Prostaglandin F2alpha-F-prostanoid receptor signaling promotes neutrophil chemotaxis via chemokine (C-X-C motif) ligand 1 in endometrial adenocarcinoma.

PubMed

Wallace, Alison E; Sales, Kurt J; Catalano, Roberto D; Anderson, Richard A; Williams, Alistair R W; Wilson, Martin R; Schwarze, Jurgen; Wang, Hongwei; Rossi, Adriano G; Jabbour, Henry N

2009-07-15

The prostaglandin F(2alpha) (PGF(2alpha)) receptor (FP) is elevated in endometrial adenocarcinoma. This study found that PGF(2alpha) signaling via FP regulates expression of chemokine (C-X-C motif) ligand 1 (CXCL1) in endometrial adenocarcinoma cells. Expression of CXCL1 and its receptor, CXCR2, are elevated in cancer tissue compared with normal endometrium and localized to glandular epithelium, endothelium, and stroma. Treatment of Ishikawa cells stably transfected with the FP receptor (FPS cells) with 100 nmol/L PGF(2alpha) increased CXCL1 promoter activity, mRNA, and protein expression, and these effects were abolished by cotreatment of cells with FP antagonist or chemical inhibitors of Gq, epidermal growth factor receptor, and extracellular signal-regulated kinase. Similarly, CXCL1 was elevated in response to 100 nmol/L PGF(2alpha) in endometrial adenocarcinoma explant tissue. CXCL1 is a potent neutrophil chemoattractant. The expression of CXCR2 colocalized to neutrophils in endometrial adenocarcinoma and increased neutrophils were present in endometrial adenocarcinoma compared with normal endometrium. Conditioned media from PGF(2alpha)-treated FPS cells stimulated neutrophil chemotaxis, which could be abolished by CXCL1 protein immunoneutralization of the conditioned media or antagonism of CXCR2. Finally, xenograft tumors in nude mice arising from inoculation with FPS cells showed increased neutrophil infiltration compared with tumors arising from wild-type cells or following treatment of mice bearing FPS tumors with CXCL1-neutralizing antibody. In conclusion, our results show a novel PGF(2alpha)-FP pathway that may regulate the inflammatory microenvironment in endometrial adenocarcinoma via neutrophil chemotaxis.

A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans.

PubMed

MacDonald, Patrick E; De Marinis, Yang Zhang; Ramracheya, Reshma; Salehi, Albert; Ma, Xiaosong; Johnson, Paul R V; Cox, Roger; Eliasson, Lena; Rorsman, Patrik

2007-06-01

Glucagon, secreted from pancreatic islet alpha cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the alpha cells themselves. We examined hormone secretion and Ca(2+) responses of alpha and beta cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn(2+) signalling was blocked, but was reversed by low concentrations (1-20 muM) of the ATP-sensitive K(+) (KATP) channel opener diazoxide, which had no effect on insulin release or beta cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 muM). Higher diazoxide concentrations (>/=30 muM) decreased glucagon and insulin secretion, and alpha- and beta-cell Ca(2+) responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 muM) stimulated glucagon secretion, whereas high concentrations (>10 muM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na(+) (TTX) and N-type Ca(2+) channels (omega-conotoxin), but not L-type Ca(2+) channels (nifedipine), prevented glucagon secretion. Both the N-type Ca(2+) channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an alpha-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.

Liposomal formulation of {alpha}-tocopheryl maleamide: In vitro and in vivo toxicological profile and anticancer effect against spontaneous breast carcinomas in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Turanek, Jaroslav; Wang Xiufang; Knoetigova, Pavlina

2009-06-15

The vitamin E analogue {alpha}-tocopheryl succinate ({alpha}-TOS) is an efficient anti-cancer drug. Improved efficacy was achieved through the synthesis of {alpha}-tocopheryl maleamide ({alpha}-TAM), an esterase-resistant analogue of {alpha}-tocopheryl maleate. In vitro tests demonstrated significantly higher cytotoxicity of {alpha}-TAM towards cancer cells (MCF-7, B16F10) compared to {alpha}-TOS and other analogues prone to esterase-catalyzed hydrolysis. However, in vitro models demonstrated that {alpha}-TAM was cytotoxic to non-malignant cells (e.g. lymphocytes and bone marrow progenitors). Thus we developed lyophilized liposomal formulations of both {alpha}-TOS and {alpha}-TAM to solve the problem with cytotoxicity of free {alpha}-TAM (neurotoxicity and anaphylaxis), as well as the low solubilitymore » of both drugs. Remarkably, neither acute toxicity nor immunotoxicity implicated by in vitro tests was detected in vivo after application of liposomal {alpha}-TAM, which significantly reduced the growth of cancer cells in hollow fiber implants. Moreover, liposomal formulation of {alpha}-TAM and {alpha}-TOS each prevented the growth of tumours in transgenic FVB/N c-neu mice bearing spontaneous breast carcinomas. Liposomal formulation of {alpha}-TAM demonstrated anti-cancer activity at levels 10-fold lower than those of {alpha}-TOS. Thus, the liposomal formulation of {alpha}-TAM preserved its strong anti-cancer efficacy while eliminating the in vivo toxicity found of the free drug applied in DMSO. Liposome-based targeted delivery systems for analogues of vitamin E are of interest for further development of efficient and safe drug formulations for clinical trials.« less

Functional integrins from normal and glycosylation-deficient baby hamster kidney cells. Terminal processing of asparagine-linked oligosaccharides is not correlated with fibronectin-binding activity.

PubMed

Koyama, T; Hughes, R C

1992-12-25

We have examined the properties of the alpha 5 beta 1 integrin of baby hamster kidney (BHK) cells, a ricin-resistant variant Ric14 lacking N-acetylglucosaminyl transferase I, and hence unable to complete assembly of hybrid- or complex-type N-glycans, and BHK cells treated with 1-deoxymannojirimycin (dMM), an inhibitor of Golgi mannosidases involved in the initial processing of N-glycan precursors. Comparable amounts of alpha 5 beta 1 integrin were isolated from these cells by chromatography of detergent extracts on a fibronectin cell-binding fragment affinity column and elution with EDTA. The alpha 5 beta 1 integrin obtained from normal BHK cells by fibronectin affinity chromatography contained mainly endoglycosidase H-resistant oligosaccharides, whereas in RicR14 cells or dMM-treated BHK cells these were entirely endoglycosidase H-sensitive. Analysis of lactoperoxidase labeled or long term biosynthetically 35S-labeled proteins from cultures of normal or glycosylation deficient cells showed similar steady state levels of alpha 5 beta 1 integrin and expression at the cell surface. Pulse-chase experiments in normal BHK cells showed rapid conversion of the alpha 5 subunit into a mature form containing oligosaccharides resistant to endoglycosidase H and slower maturation of a precursor beta 1 subunit, as in other cell types. In Ric14 cells the precursor beta 1 subunit was found to carry glycans larger than the fully processed Man5GlcNAc2 glycan of the mature subunit, indicating that the bulk precursor pool had not been translocated into the cis-Golgi compartment containing mannosidase I. We conclude that in BHK cells terminal oligosaccharide processing of alpha 5 beta 1 integrin subunits is not required for dimer formation, surface expression, and fibronectin binding, and that expression of the glycosylation defect of Ric14 cells on the alpha 5 beta 1 integrin does not account for the reduced adhesiveness of these cells on fibronectin compared with normal and dMM-treated BHK cells.

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

PubMed

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

2013-12-31

Type I interferons (IFNs) exert an array of important biological functions on the innate immune response and has become a useful tool in the treatment of various diseases. An increasing demand in the usage of recombinant IFNs, mainly due to the treatment of chronic hepatitis C infection, augmented the need of quality control for this biopharmaceutical. A traditional bioassay for IFN potency assessment is the cytopathic effect reduction antiviral assay where a given cell line is preserved by IFN from a lytic virus activity using the cell viability as a frequent measure of end point. However, type I IFNs induce other biological effects such as cell-cycle arrest and apoptosis that can influence directly on viability of many cell lines. Here, we standardized a cytopathic effect reduction antiviral assay using Hep-2C cell/mengovirus combination and studied a possible impact of cell viability variations caused by IFN-alpha 2b on responses generated on the antiviral assay. Using the four-parameter logistic model, we observed less correlation and less linearity on antiviral assay when responses from IFN-alpha 2b 1000 IU/ml were considered in the analysis. Cell viability tests with MTT revealed a clear cell growth inhibition of Hep-2C cells under stimulation with IFN-alpha 2b. Flow cytometric cell-cycle analysis and apoptosis assessment showed an increase of S+G2 phase and higher levels of apoptotic cells after treatment with IFN-alpha 2b 1000 IU/ml under our standardized antiviral assay procedure. Considering our studied dose range, we also observed strong STAT1 activation on Hep-2C cells after stimulation with the higher doses of IFN-alpha 2b. Our findings showed that the reduction of cell viability driven by IFN-alpha can cause a negative impact on antiviral assays. We assume that the cell death induction and the cell growth inhibition effect of IFNs should also be considered while employing antiviral assay protocols in a quality control routine and emphasizes the importance of new approaches for IFN potency determination. Copyright © 2013 Elsevier B.V. All rights reserved.

Integrin distributions in renal cell carcinomas of various grades of malignancy.

PubMed Central

Korhonen, M.; Laitinen, L.; Ylänne, J.; Koukoulis, G. K.; Quaranta, V.; Juusela, H.; Gould, V. E.; Virtanen, I.

1992-01-01

We studied 41 renal cell carcinomas, classified according to histologic grades G1 through G3, by indirect immunofluorescence microscopy using a panel of monoclonal antibodies (MAb) against various integrin subunits, and the basement membrane (BM) components laminin and collagen type IV. Selected cases also were immunostained using the avidin-biotin-complex method. The alpha 3 and beta 1 integrin subunits were detected in tumor cells of all the carcinomas. All G1 carcinomas, like normal tubular epithelial cells, expressed the alpha 6 subunit, whereas it was lacking in 20% and 40% of G2 and G3 carcinomas, respectively. Furthermore, when alpha 6 was expressed, a lack of basally polarized organization of the subunit, coupled with disorganization of the BM components, correlated with histologic grade. Another feature that appeared to characterize the more anaplastic tumors was their high level (80%) of the alpha v subunit expression as compared with its absence in the G1 carcinomas. Stromal myofibroblasts, identified by double-labeling with anti-myosin, were often characterized by the expression of the alpha 1, alpha 3, alpha 5 and beta 1 subunits. These results indicate that changes in integrin expression in renal cell carcinomas may be correlated with their degree of histologic malignancy. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:1443050

Neisseria meningitidis Opc invasin binds to the cytoskeletal protein alpha-actinin.

PubMed

Sa E Cunha, Claudia; Griffiths, Natalie J; Murillo, Isabel; Virji, Mumtaz

2009-03-01

Neisseria meningitidis Opc protein is an effective invasin for human endothelial cells. We have investigated novel human endothelial receptors targeted by Opc and observed that Opc-expressing bacteria interacted with a 100 kDa protein in whole-cell lysates of human endothelial and epithelial cells. The identity of the protein was established as alpha-actinin by mass spectrometry. Opc expression was essential for the recognition of alpha-actinin whether provided in a purified form or in cell extracts. The interaction of the two proteins did not involve intermediate molecules. As there was no demonstrable expression of alpha-actinin on the surfaces of any of the eight cell lines studied, the likelihood of the interactions after meningococcal internalization was examined. Confocal imaging demonstrated considerable colocalization of N. meningitidis with alpha-actinin especially after a prolonged period of internalization. This may imply that bacteria and alpha-actinin initially occur in separate compartments and co-compartmentalization occurs progressively over the 8 h infection period used. In conclusion, these studies have identified a novel and an intracellular target for the N. meningitidis Opc invasin. Since alpha-actinin is a modulator of a variety of signalling pathways and of cytoskeletal functions, its targeting by Opc may enable bacteria to survive/translocate across endothelial barriers.

The differentiation of hepatocyte-like cells from monkey embryonic stem cells.

PubMed

Ma, Xiaocui; Duan, Yuyou; Jung, Christine J; Wu, Jian; VandeVoort, Catherine A; Zern, Mark A

2008-12-01

Embryonic stem cells (ESC) hold great potential for the treatment of liver diseases. Here, we report the differentiation of rhesus macaque ESC along a hepatocyte lineage. The undifferentiated monkey ESC line, ORMES-6, was cultured in an optimal culture condition in an effort to differentiate them into hepatocyte-like cells in vitro. The functional efficacy of the differentiated hepatic cells was evaluated using RT-PCR for the expression of hepatocyte specific genes, and Western blot analysis and immunocytochemistry for hepatic proteins such as alpha-fetoprotein (AFP), albumin and alpha1-antitrypsin (alpha1-AT). Functional assays were performed using the periodic acid schiff (PAS) reaction and ELISA. The final yield of ESC-derived hepatocyte-like cells was measured by flow cytometry for cells that were transduced with a liver-specific lentivirus vector containing the alpha1-AT promoter driving the expression of green fluorescence protein (GFP). The treatment of monkey ESC with an optimal culture condition yielded hepatocyte-like cells that expressed albumin, alpha1-AT, AFP, hepatocyte nuclear factor 3beta, glucose-6-phophatase, and cytochrome P450 genes and proteins as determined by RT-PCR and Western blot analysis. Immunofluorescent staining showed the cells positive for albumin, AFP, and alpha1-AT. PAS staining demonstrated that the differentiated cells showed hepatocyte functional activity. Albumin could be detected in the medium after 20 days of differentiation. Flow cytometry data showed that 6.5 +/- 1.0% of the total differentiated cells were positive for GFP. These results suggest that by using a specific, empirically determined, culture condition, we were able to direct monkey ESC toward a hepatocyte lineage.

Ovarian Sertoli-Leydig cell tumor with heterologous elements of gastrointestinal type associated with elevated serum alpha-fetoprotein level: an unusual case and literature review

PubMed Central

Horta, Mariana; Cunha, Teresa Margarida; Marques, Rita Canas; Félix, Ana

2014-01-01

Here we describe the case of a 19-year-old woman with a poorly differentiated ovarian Sertoli-Leydig cell tumor and an elevated serum alpha-fetoprotein level. The patient presented with diffuse abdominal pain and bloating. Physical examination, ultrasound, and magnetic resonance imaging revealed a right ovarian tumor that was histopathologically diagnosed as a poorly differentiated Sertoli-Leydig cell tumor with heterologous elements. Her alpha-fetoprotein serum level was undetectable after tumor resection. Sertoli-Leydig cell tumors are rare sex cord-stromal tumors that account for 0.5% of all ovarian neoplasms. Sertoli-Leydig cell tumors tend to be unilateral and occur in women under 30 years of age. Although they are the most common virilizing tumor of the ovary, about 60% are endocrine-inactive tumors. Elevated serum levels of alpha-fetoprotein are rarely associated with Sertoli-Leydig cell tumors, with only approximately 30 such cases previously reported in the literature. The differential diagnosis should include common alpha-fetoprotein-producing ovarian entities such as germ cell tumors, as well as other non-germ cell tumors that have been rarely reported to produce this tumor marker. PMID:25926909

Ovarian Sertoli-Leydig cell tumor with heterologous elements of gastrointestinal type associated with elevated serum alpha-fetoprotein level: an unusual case and literature review.

PubMed

Horta, Mariana; Cunha, Teresa Margarida; Marques, Rita Canas; Félix, Ana

2014-11-01

Here we describe the case of a 19-year-old woman with a poorly differentiated ovarian Sertoli-Leydig cell tumor and an elevated serum alpha-fetoprotein level. The patient presented with diffuse abdominal pain and bloating. Physical examination, ultrasound, and magnetic resonance imaging revealed a right ovarian tumor that was histopathologically diagnosed as a poorly differentiated Sertoli-Leydig cell tumor with heterologous elements. Her alpha-fetoprotein serum level was undetectable after tumor resection. Sertoli-Leydig cell tumors are rare sex cord-stromal tumors that account for 0.5% of all ovarian neoplasms. Sertoli-Leydig cell tumors tend to be unilateral and occur in women under 30 years of age. Although they are the most common virilizing tumor of the ovary, about 60% are endocrine-inactive tumors. Elevated serum levels of alpha-fetoprotein are rarely associated with Sertoli-Leydig cell tumors, with only approximately 30 such cases previously reported in the literature. The differential diagnosis should include common alpha-fetoprotein-producing ovarian entities such as germ cell tumors, as well as other non-germ cell tumors that have been rarely reported to produce this tumor marker.

Variation in interferon sensitivity and induction between Usutu and West Nile (lineages 1 and 2) viruses.

PubMed

Cacciotti, Giulia; Caputo, Beniamino; Selvaggi, Carla; la Sala, Andrea; Vitiello, Laura; Diallo, Diawo; Ceianu, Cornelia; Antonelli, Guido; Nowotny, Norbert; Scagnolari, Carolina

2015-11-01

Given the pivotal role of monocyte-derived dendritic cells (DCs) in determining the magnitude of the antiviral innate immune response, we sought to determine whether Usutu virus (USUV) and West Nile virus (WNV) lineages (L)1 and L2 can infect DCs and affect the rate of type I interferon (IFN) activation. The sensitivity of these viruses to types I and III IFNs was also compared. We found that USUV can infect DCs, induce higher antiviral activities, IFN alpha subtypes and the IFN stimulated gene (ISG)15 pathway, and is more sensitive to types I and III IFNs than WNVs. In contrast, we confirmed that IFN alpha/beta subtypes were more effective against WNV L2 than WNV L1. However, the replication kinetics, induction of IFN alpha subtypes and ISGs in DCs and the sensitivity to IFN lambda 1-3 did not differ between WNV L1 and L2. Copyright © 2015 Elsevier Inc. All rights reserved.

The thalidomide analogue CC-3052 inhibits HIV-1 and tumour necrosis factor-alpha (TNF-alpha) expression in acutely and chronically infected cells in vitro.

PubMed

La Maestra, L; Zaninoni, A; Marriott, J B; Lazzarin, A; Dalgleish, A G; Barcellini, W

2000-01-01

We investigated the in vitro effect of the water-soluble, highly stable thalidomide analogue CC-3052 on HIV-1 expression and TNF-alpha production in latently infected promonocytic U1 cells, acutely infected T cells and monocyte-derived human macrophages (MDM), and in mitogen-stimulated ex vivo cultures from patients with primary acute HIV-1 infection. HIV-1 expression was assessed by Northern blot analysis of RNAs, and ELISA for p24 antigen release and reverse transcriptase (RT) activity. TNF-alpha expression was evaluated by RT-polymerase chain reaction (PCR)-ELISA for mRNA and ELISA for protein secretion. We demonstrated that CC-3052 is able to inhibit HIV-1 expression, as evaluated by mRNA, p24 release and RT activity, in phorbol myristate acetate (PMA)- and cytokine-stimulated U1 cells. Furthermore, CC-3052 inhibited HIV-1 expression, as evaluated by p24 and RT activity, in acutely infected MDM and T cells. As far as TNF-alpha is concerned, CC-3052 significantly reduced TNF-alpha mRNA and protein secretion in PMA-stimulated U937 and U1 cells, and in PMA-stimulated uninfected and acutely infected MDM. Consistently, the addition of CC-3052 reduced TNF-alpha production in phytohaemagglutinin (PHA) and lipopolysaccharide (LPS)-stimulated whole blood cultures from patients during the primary acute phase of HIV-1 infection. Since TNF-alpha is among the most potent enhancers of HIV-1 expression, the effect of CC-3052 on TNF-alpha may account for its inhibitory activity on HIV-1 expression. Given the well documented immunopathological role of TNF-alpha and its correlation with viral load, advanced disease and poor prognosis, CC-3052 could be an interesting drug for the design of therapeutic strategies in association with anti-retroviral agents.

Thalidomide suppressed IL-1beta while enhancing TNF-alpha and IL-10, when cells in whole blood were stimulated with lipopolysaccharide.

PubMed

Shannon, Edward; Noveck, Robert; Sandoval, Felipe; Kamath, Burde

2008-01-01

Thalidomide is used to treat erythema nodosum leprosum (ENL). The events that precipitate this inflammatory reaction, which may occur in multibacillary leprosy patients, and the mechanism by which thalidomide arrest ENL, are not known. Thalidomide's ability to inhibit tumor necrosis factor alpha (TNF-alpha) in vitro has been proposed as a partial explanation of its effective treatment of ENL. In in vitro assays, thalidomide can enhance or suppress TNF-alpha. This is dependent on the stimulant used to evoke TNF-alpha; the procedure used to isolate the mononuclear cells from blood, and the predominant mononuclear cell type in the culture. To avoid artifacts that may occur during isolation of mononuclear cells from blood, we stimulated normal human blood with LPS and evaluated the effect of thalidomide and dexamethasone on TNF-alpha, and other inflammatory cytokines and biomarkers. Thalidomide suppressed interleukin 1 beta (IL-1beta) (p = 0.007), and it enhanced TNF-alpha (p = 0.007) and interleukin 10 (IL-10) (p = 0.031). Dexamethasone enhanced IL-10 (p = 0.013) and suppressed IL-1beta, TNF-alpha, interleukin 6 (IL-6), and interleukin 8 (IL-8) (p = 0.013). The two drugs did not suppress: C-reactive protein (CRP), Ig-superfamily cell-adhesion molecule 1 (ICAM 1), tumor necrosis factor receptor 1 (TNFR1), tumor necrosis factor receptor 2 (TNFR2), or amyloid A. In vitro and in vivo evidence is accumulating that TNF-alpha is not the primary cytokine targeted by thalidomide in ENL and other inflammatory conditions.

Smad, but not MAPK, pathway mediates the expression of type I collagen in radiation induced fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yano, Hiroyuki; Division of Radioisotope Research, Department of Research Support, Research Promotion Project, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593; Hamanaka, Ryoji

Highlights: Black-Right-Pointing-Pointer We examine how radiation affects the expression level and signal pathway of collagen. Black-Right-Pointing-Pointer TGF-{beta}1 mRNA is elevated earlier than those of collagen genes after irradiation. Black-Right-Pointing-Pointer Smad pathway mediates the expression of collagen in radiation induced fibrosis. Black-Right-Pointing-Pointer MAPK pathways are not affected in the expression of collagen after irradiation. -- Abstract: Radiation induced fibrosis occurs following a therapeutic or accidental radiation exposure in normal tissues. Tissue fibrosis is the excessive accumulation of collagen and other extracellular matrix components. This study investigated how ionizing radiation affects the expression level and signal pathway of type I collagen. Realmore » time RT-RCR showed that both {alpha}1and {alpha}2 chain of type I collagen mRNA were elevated from 48 h after irradiation with 10 Gy in NIH3T3 cells. The relative luciferase activities of both genes and type I collagen marker were elevated at 72 h. TGF-{beta}1 mRNA was elevated earlier than those of type I collagen genes. A Western blot analysis showed the elevation of Smad phosphorylation at 72 h. Conversely, treatment with TGF-{beta} receptor inhibitor inhibited the mRNA and relative luciferase activity of type I collagen. The phosphorylation of Smad was repressed with the inhibitor, and the luciferase activity was cancelled using a mutant construct of Smad binding site of {alpha}2(I) collagen gene. However, the MAPK pathways, p38, ERK1/2 and JNK, were not affected with specific inhibitors or siRNA. The data showed that the Smad pathway mediated the expression of type I collagen in radiation induced fibrosis.« less

Molecular Signatures of Peripheral Blood Mononuclear Cells during Chronic Interferon-alpha Treatment: Relationship with Depression and Fatigue

PubMed Central

Felger, Jennifer C.; Cole, Steve W.; Pace, Thaddeus W. W.; Hu, Fang; Woolwine, Bobbi J.; Doho, Gregory H.; Raison, Charles L.; Miller, Andrew H.

2012-01-01

Background Interferon (IFN)-alpha treatment for infectious disease and cancer causes high rates of depression and fatigue, and has been used to investigate the impact of inflammatory cytokines on brain and behavior. However, little is known about the transcriptional impact of chronic IFN-alpha on immune cells in vivo and its relationship to IFN-alpha-induced behavioral changes. Methods Genome-wide transcriptional profiling was performed on peripheral blood mononuclear cells from 21 patients with chronic hepatitis C either awaiting IFN-alpha therapy (n=10) or at 12 weeks of IFN-alpha treatment (n=11). Results Significance analysis of microarray data identified 252 up-regulated and 116 down-regulated gene transcripts. Of up-regulated genes, 2'-5'-oligoadenylate synthetase 2 (OAS2), a gene linked to chronic fatigue syndrome (CFS), was the only gene that was differentially expressed in patients with IFN-alpha-induced depression/fatigue, and correlated with depression and fatigue scores at 12 weeks (r=0.80, p=0.003 and r=0.70, p=0.017, respectively). Promoter-based bioinformatic analyses linked IFN-alpha-related transcriptional alterations to transcription factors involved in myeloid differentiation, IFN-alpha signaling, AP1 and CREB/ATF pathways, which were derived primarily from monocytes and plasmacytoid dendritic cells. IFN-alpha-treated patients with high depression/fatigue scores demonstrated up-regulation of genes bearing promoter motifs for transcription factors involved in myeloid differentiation, IFN-alpha and AP1 signaling, and reduced prevalence of motifs for CREB/ATF, which has been implicated in major depression. Conclusions Depression and fatigue during chronic IFN-alpha administration were associated with alterations in the expression (OAS2) and transcriptional control (CREB/ATF) of genes linked to behavioral disorders including CFS and major depression, further supporting an immune contribution to these diseases. PMID:22152193

Promotion of mouse fibroblast collagen gene expression by mast cells stimulated via the Fc epsilon RI. Role for mast cell-derived transforming growth factor beta and tumor necrosis factor alpha

PubMed Central

1994-01-01

Chronic allergic diseases and other disorders associated with mast cell activation can also be associated with tissue fibrosis, but a direct link between mast cell mediator release and fibroblast collagen gene expression has not been established. Using in situ hybridization, we show that the elicitation of an IgE-dependent passive cutaneous anaphylaxis (PCA) reaction in mice results in a transient, but marked augmentation of steady state levels of type alpha-1 (I) collagen mRNA in the dermis. While peak levels of collagen mRNA expression in the skin are observed 16-24 h after mast cell activation, substantial numbers of dermal cells are strongly positive for collagen mRNA at 1 and 2 h after antigen challenge, before circulating inflammatory cells are recruited into the tissues. Furthermore, experiments in mast cell- reconstituted or genetically mast cell-deficient WBB6F1-W/Wv mice demonstrate that the increased expression of collagen mRNA at sites of PCA reactions is entirely mast cell dependent. In vitro studies show that the supernatants of mouse serosal mast cells activated via the Fc epsilon RI markedly increase type alpha-1 (I) collagen mRNA levels in mouse embryonic skin fibroblasts, and also upregulate collagen secretion by these cells. The ability of mast cell supernatants to induce increased steady state levels of collagen mRNA in mouse skin fibroblasts is markedly diminished by absorption with antibodies specific for either of two mast cell-derived cytokines, transforming growth factor beta (TGF-beta 1) or tumor necrosis factor alpha (TNF- alpha), and is eliminated entirely by absorption with antibodies against both cytokines. Taken together, these findings demonstrate that IgE-dependent mouse mast cell activation can induce a transient and marked increase in steady state levels of type alpha-1 (I) collagen mRNA in dermal fibroblasts and that mast cell-derived TGF-beta 1 and TNF-alpha importantly contribute to this effect. PMID:7964480

Beta1-integrin and IL-1alpha expression as bystander effect of medium from irradiated cells: the pilot study.

PubMed

Osterreicher, Jan; Skopek, Jirí; Jahns, Juta; Hildebrandt, Guido; Psutka, Jan; Vilasová, Zdenka; Tanner, Judith Maria; Vogt, Jürgen; Butz, Tilman

2003-01-01

Bystander effects have been proposed as a third action pathway of ionising radiation besides direct and indirect effects. The purpose of the study was to investigate whether expression of interleukin-1alpha (IL-1alpha) and beta1-integrin is elevated in bystander cells as a marker for bystander effects in comparison with classical markers such as the clonogenic assay, apoptosis and the presence of micronuclei. The hybrid cell line E.A. hy.926 obtained by fusion of HUVEC cells with the epithelial cell line A 459 was irradiated with 0-5 Gy. Bystander effects were established via medium transfer at 45 min and 4 h after irradiation from irradiated to nonirradiated cell populations. In order to exclude effects of the irradiated medium itself, irradiated medium only was also used for transfer to nonirradiated cells. Then, cells were fixed at 1, 2, 6, and 24 h after irradiation or medium transport and IL-1alpha and beta1-integrin were detected and evaluated. A higher number of beta1-integrin-positive cells was observed in both irradiated and bystander cell populations than in the control group at 1 and 24 h after irradiation with 1 Gy or medium transfer. Significantly higher numbers of IL-1alpha-positive cells were found at 1, 2, and 6 h after irradiation with 1 Gy or medium transfer as well as at 2 and 6 h after irradiation with 5 Gy or medium transfer. Clonogenic survival decreased dependently on the dose in irradiated cells but did not show any significant difference between the bystander cell populations and sham-irradiated cells. The irradiated medium itself did not have any effect. It is concluded that beta1-integrin and IL-1alpha expression may serve as more sensitive markers of post-irradiation responses in bystander cell populations than the classical radiobiological markers. Moreover, overexpression of beta1-integrin and IL-1alpha may induce increased susceptibility to inflammation of bystander cells.

Leptin potentiates Prevotella intermedia lipopolysaccharide-induced production of TNF-alpha in monocyte-derived macrophages.

PubMed

Kim, Sung-Jo

2010-06-01

In addition to regulating body weight, leptin is also recognized for its role in the regulation of immune function and inflammation. The purpose of this study was to investigate the effect of leptin on Prevotella (P.) intermedia lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-alpha production in differentiated THP-1 cells, a human monocytic cell line. LPS from P. intermedia ATCC 25611 was prepared by the standard hot phenol-water method. THP-1 cells were incubated in the medium supplemented with phorbol myristate acetate to induce differentiation into macrophage-like cells. The amount of TNF-alpha and interleukin-8 secreted into the culture medium was determined by enzyme-linked immunosorbent assay (ELISA). TNF-alpha and Ob-R mRNA expression levels were determined by semi-quantitative reverse transcription-polymerase chain reaction analysis. Leptin enhanced P. intermedia LPS-induced TNF-alpha production in a dose-dependent manner. Leptin modulated P. intermedia LPS-induced TNF-alpha expression predominantly at the transcriptional level. Effect of leptin on P. intermedia LPS-induced TNF-alpha production was not mediated by the leptin receptor. The ability of leptin to enhance P. intermedia LPS-induced TNF-alpha production may be important in the establishment of chronic lesion accompanied by osseous tissue destruction observed in inflammatory periodontal disease.

Characterization of inhibin forms and their measurement by an inhibin alpha-subunit ELISA in serum from postmenopausal women with ovarian cancer.

PubMed

Robertson, D M; Stephenson, T; Pruysers, E; McCloud, P; Tsigos, A; Groome, N; Mamers, P; Burger, H G

2002-02-01

The aim of this study was to characterize the molecular wt forms of inhibins A and B and its free alpha-subunit present in serum from women with ovarian cancer as a basis for developing improved monoclonal antibody-based inhibin assays for monitoring ovarian cancer. Three new inhibin alpha-subunit (alphaC) ELISAs were developed using monoclonal antibodies directed to three nonoverlapping peptide regions of the alphaC region of the inhibin alpha-subunit. To characterize serum inhibin molecular wt forms present in women with ovarian cancer, existing inhibin immunoassays (inhibin A, inhibin B, and pro-alphaC) and the new alphaC ELISAs were applied to sera from women with granulosa cell tumors and mucinous carcinomas previously fractionated using a combined immunoaffinity chromatography, preparative SDS-PAGE, and electroelution procedure. The distribution and molecular size of dimeric inhibins and alpha-subunit detected were consistent with known mol wt forms of inhibins A and B and inhibin alpha-subunit and their precursor forms present in serum and follicular fluid from healthy women. The alphaC ELISAs recognized all known forms of inhibin and the free inhibin alpha-subunit, although differences between alphaC ELISAs were observed in their ability to detect high mol wt forms. To assess which of the alphaC ELISAs was preferred in application to ovarian cancer, the alphaC ELISAs were applied to serum from a range of normal postmenopausal women (n = 61) and postmenopausal women (n = 152) with ovarian (serous, mucinous, endometrioid, clear cell carcinomas, and granulosa cell tumors) and nonovarian (breast and colon) cancers. Despite differences in their ability to detect high mol wt forms of inhibin, the alphaC ELISAs showed similar sensitivity (i.e. proportion of cancer patients correctly detected) and specificity (proportion of controls correctly detected) indexes in the detection of mucinous carcinomas (84% and 95%) and granulosa cell tumors (100% and 95%) compared with earlier inhibin RIA or polyclonal antibody-based immunofluorometric assays. A combination of the alphaC ELISAs with the CA125 assay, an ovarian tumor marker that has a high sensitivity and specificity for other ovarian cancers (serous, clear cell, and endometrioid), resulted in an increase in sensitivity/specificity indexes (95% and 95%) for the all ovarian cancer group. These new monoclonal antibody-based inhibin alphaC ELISAs now provide practical and sensitive assays suitable for evaluation as diagnostic tests for monitoring ovarian cancers.

Affective and cognitive effects of global deletion of alpha3-containing gamma-aminobutyric acid-A receptors.

PubMed

Fiorelli, Roberto; Rudolph, Uwe; Straub, Carolin J; Feldon, Joram; Yee, Benjamin K

2008-09-01

Gamma-aminobutyric acid (GABA)A receptors characterized by the presence of the alpha3 subunit are the major GABAA receptor subtype expressed in brain stem monoaminergic nuclei. These alpha3-GABAA receptors are therefore in a unique position to regulate monoaminergic functions. To characterize the functional properties of alpha3-GABAA receptors, we present a preliminary assessment of the expression of affective and cognitive behaviour in male mice with a targeted deletion of the Gabra3 gene encoding the alpha3 subunit [alpha3 knockout (KO) mice] on a C57BL/6Jx129X1/SvJ F1 hybrid genetic background. The alpha3 KO mice did not exhibit any gross change of anxiety-like behaviour or spontaneous locomotor behaviour. In the Porsolt forced swim test for potential antidepressant activity, alpha3 KO mice exhibited reduced floating and enhanced swimming behaviour relative to wild-type controls. Performance on a two-choice sucrose preference test, however, revealed no evidence for an increase in sucrose preference in the alpha3 KO mice that would have substantiated a potential phenotype for depression-related behaviour. In contrast, a suggestion of an enhanced negative contrast effect was revealed in a one-bottle sucrose consumption test across different sucrose concentrations. These affective phenotypes were accompanied by alterations in the balance between conditioned responding to the discrete conditioned stimulus and to the context, and a suggestion of faster extinction, in the Pavlovian conditioned freezing paradigm. Spatial learning in the water maze reference memory test, however, was largely unchanged in the alpha3 KO mice, except for a trend of preservation during reversal learning. The novel phenotypes following global deletion of the GABAA receptor alpha3 subunit identified here provided relevant insights, in addition to our earlier study, into the potential behavioural relevance of this specific receptor subtypes in the modulation of both affective and cognitive functions.

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

PubMed

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

2017-09-29

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

Imbalance of tumor necrosis factor receptors during progression in bovine leukemia virus infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Konnai, Satoru; Usui, Tatsufumi; Ikeda, Manabu

2005-09-01

Previously, we found an up-regulation of tumor necrosis factor alpha (TNF)-{alpha} and an imbalance of TNF receptors in sheep experimentally infected with bovine leukemia virus (BLV). In order to investigate the different TNF-{alpha}-induced responses, in this study we examined the TNF-{alpha}-induced proliferative response and the expression levels of two distinct TNF receptors on peripheral blood mononuclear cells (PBMC) derived from BLV-uninfected cattle and BLV-infected cattle that were aleukemic (AL) or had persistent lymphocytosis (PL). The proliferative response of PBMC isolated from those cattle with PL in the presence of recombinant bovine TNF-{alpha} (rTNF-{alpha}) was significantly higher than those from ALmore » cattle and uninfected cattle and the cells from PL cattle expressed significantly higher mRNA levels of TNF receptor type II (TNF-RII) than those from AL and BLV-uninfected cattle. No difference was found in TNF-RI mRNA levels. Most cells expressing TNF-RII in PL cattle were CD5{sup +} or sIgM{sup +} cells and these cells showed resistance to TNF-{alpha}-induced apoptosis. Additionally, there were significant positive correlations between the changes in provirus load and TNF-RII mRNA levels, and TNF-{alpha}-induced proliferation and TNF-RII mRNA levels. These data suggest that imbalance in the expression of TNF receptors could at least in part contribute to the progression of lymphocytosis in BLV infection.« less

Differentiation of human skeletal muscle cells in culture: maturation as indicated by titin and desmin striation.

PubMed

van der Ven, P F; Schaart, G; Jap, P H; Sengers, R C; Stadhouders, A M; Ramaekers, F C

1992-10-01

This report describes a phenotyping study of differentiating human skeletal muscle cells in tissue culture. Satellite cells (adult myoblasts), isolated from biopsy material, showed a proliferative behaviour in high-nutrition medium, but fused to form myotubes when grown in low-nutrition medium. The expression and structural organization of the intermediate filament proteins desmin and vimentin as well as the sarcomeric constituents alpha-actin, alpha-actinin, nebulin, myosin and especially titin during myofibrillogenesis in vitro, were studied by means of indirect immunofluorescence assays. The proliferating myoblasts contained both desmin and vimentin, alpha-actinin and the filamentous form of actin. Shortly after the change of medium, expression of titin, sarcomeric myosin and skeletal muscle alpha-actin was found in mononuclear cells in a diffuse, filamentous (titin, myosin, alpha-actin) or punctate (titin, myosin) pattern. Four to 10 days after the medium change, mature myotubes showed desmin, titin, alpha-actinin, nebulin, sarcomeric myosin and actin cross-striations, while vimentin was no longer detected. We conclude that human skeletal muscle cell cultures are an appropriate model system to study the molecular basis of myofibrillogenesis. Especially the presence of desmin in a striated fashion points to a high degree of maturation of the muscle cell cultures.

Glucagon-Secreting Alpha Cell Selective Two-Photon Fluorescent Probe TP-α: For Live Pancreatic Islet Imaging.

PubMed

Agrawalla, Bikram Keshari; Chandran, Yogeswari; Phue, Wut-Hmone; Lee, Sung-Chan; Jeong, Yun-Mi; Wan, Si Yan Diana; Kang, Nam-Young; Chang, Young-Tae

2015-04-29

Two-photon (TP) microscopy has an advantage for live tissue imaging which allows a deeper tissue penetration up to 1 mm comparing to one-photon (OP) microscopy. While there are several OP fluorescence probes in use for pancreatic islet imaging, TP imaging of selective cells in live islet still remains a challenge. Herein, we report the discovery of first TP live pancreatic islet imaging probe; TP-α (Two Photon-alpha) which can selectively stain glucagon secreting alpha cells. Through fluorescent image based screening using three pancreatic cell lines, we discovered TP-α from a TP fluorescent dye library TPG (TP-Green). In vitro fluorescence test showed that TP-α have direct interaction and appear glucagon with a significant fluorescence increase, but not with insulin or other hormones/analytes. Finally, TP-α was successfully applied for 3D imaging of live islets by staining alpha cell directly. The newly developed TP-α can be a practical tool to evaluate and identify live alpha cells in terms of localization, distribution and availability in the intact islets.

Adenoviral receptor expression of normal bladder and transitional cell carcinoma of the bladder.

PubMed

Buscarini, Maurizio; Quek, Marcus L; Gilliam-Hegarich, Susan; Kasahara, Nori; Bochner, Bernard

2007-01-01

The insertion of absent or underexpressed genes into cancer cells to alter their malignant phenotype is an important potential application of available gene therapy technology. One of the more common viral vector systems that has been extensively studied for this purpose are the replication-deficient adenoviruses (Ad). Adenoviral infection of cells is mediated through a complex pathway, initiated following viral-cell attachment. Adenoviral-cell attachment occurs following interactions with a 46-kDa transmembrane protein with high affinity for both the Coxsackie and adenovirus, designated the CAR (Coxsackie and adenoviral receptor). Additional important cell-viral interactions that occur involve the alpha(v)-based integrins, specifically alpha(v)beta3 and alpha(v)beta5. The purpose of the present study was to determine the extent of expression and localization of the known Ad receptor proteins (CAR, alpha(v)beta3, and alpha(v)beta5) in normal and cancerous human bladders. Frozen tissue samples of normal bladder and invasive transitional cell cancers of the bladder were evaluated. Tissue blocks containing muscle-invasive transitional cell carcinoma (TCC) were obtained following radical cystectomy, which were performed at our institution. Thirty-two invasive transitional cell bladder tumors were evaluated, each with a matched sample of histologically normal-appearing bladder used as a control. Four additional samples of normal bladder were obtained from patients with no evidence of disease of the bladder and served as further controls. Three additional cases of invasive bladder cancer with no matching normal tissue were also evaluated. Identification of the CAR receptor was performed using the anti-CAR mouse monoclonal antibody designated RmBC. The integrins alpha(v)beta3 and alpha(v)beta5 were identified using the mouse monoclonal antibodies designated LM609 and P1F6 respectively. All slides were evaluated by two of the authors (M.B., B.B.) without knowledge of the clinical and pathological data. Normal bladder: Normal bladder mucosa demonstrated a marked positivity for CAR in 29/35 (82.8%) cases. In contrast, normal transitional epithelial cells were uniformly negative when tested for the integrins alpha(v)beta3 and alpha(v)beta5. Subepithelial tissues, specifically the connective tissue components of the lamina propria and deep muscle wall of the bladder, were positive for alpha(v)beta3 and for alpha(v)beta5 in 61 and 75% of samples, respectively. Endothelial cells associated with the various layers throughout the bladder uniformly expressed both integrins and served as a consistent internal control for both antibodies. An almost identical staining pattern of the endothelium was observed using LM609 and P1F6 in all samples tested. Bladder transitional cell carcinoma: CAR immunoreactivity against TCC cells was uniformly decreased compared to normal transitional cells. Nine tumors exhibited a weak positivity for CAR while the remaining samples were negative. In some cases, the absence of CAR positivity was associated with histological evidence of carcinoma in situ. In 6 cases, it led to the identification of small regions of carcinoma in situ that were not noted on primary pathological evaluation. Peritumoral connective tissue expressed both integrins in the majority of cases, similar to the pattern described above for normal bladder. Transitional cell cancers demonstrated a similar pattern of expression of alpha(v)beta5, in which all tumor cells exhibited minimal or no staining. The success of all viral-mediated gene therapy strategies relies on the ability of the vector to efficiently deliver its genetic material to a target cell population. In the current study, we demonstrate that the bladder epithelial layer consistently expresses high levels of CAR. Deeper layers of the epithelium also express CAR, including the basal layer cells. A decrease in the expression of CAR appears as an early event in bladder carcinogenesis. We observed that both alpha(v)beta3 and alpha(v)beta5 are strongly expressed in muscle cells surrounding the neoplastic cells, as well as within the peritumoral connective tissue. In cases of invasive bladder cancer that have lost CAR expression, an adenoviral vector may still be utilized through the less efficient interactions with the integrins. Bladder tumor tissue may be less susceptible to an adenoviral-mediated gene therapy approach in which a significant percentage of tumor cells require transduction. Adenoviral uptake by tumor or peritumoral cells with subsequent gene transfer could be predicted by the level of CAR and alpha(v)-based integrin expression. This would enhance our ability to identify those patients whose tumors would be more susceptible to Ad-mediated gene delivery as part of an antitumor treatment. 2007 S. Karger AG, Basel

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.

Gene transfer mediated by alpha2-macroglobulin.

PubMed Central

Schneider, H; Huse, K; Birkenmeier, G; Otto, A; Scholz, G H

1996-01-01

alpha2-Macroglobulin covalently linked to poly(L)-lysine can be used as a vehicle for receptor-mediated gene transfer. This modified alpha2-macroglobulin maintains its ability to bind to the alpha2-macroglobulin receptor, and was shown to introduce a luciferase reporter gene plasmid into HepG2 human hepatoma cells in vitro. The alpha2-macroglobulin receptor is a very large and multifunctional cell surface receptor, whose rapid and efficient internalization rate makes it attractive for gene therapy, e.g. for hepatic gene targeting via injection into the portal vein. PMID:8871570

Tumor necrosis factor-{alpha} induces MMP-9 expression via p42/p44 MAPK, JNK, and nuclear factor-{kappa}B in A549 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, C.-C.; Tseng, Hsiao-Wei; Hsieh, Hsi-Lung

2008-06-15

Matrix metalloproteinases (MMPs), in particular MMP-9, have been shown to be induced by cytokines including tumor necrosis factor-{alpha} (TNF-{alpha}) and contributes to airway inflammation. However, the mechanisms underlying MMP-9 expression induced by TNF-{alpha} in human A549 cells remain unclear. Here, we showed that TNF-{alpha} induced production of MMP-9 protein and mRNA is determined by zymographic, Western blotting, RT-PCR and ELISA assay, which were attenuated by inhibitors of MEK1/2 (U0126), JNK (SP600125), and NF-{kappa}B (helenalin), and transfection with dominant negative mutants of ERK2 ({delta}ERK) and JNK ({delta}JNK), and siRNAs for MEK1, p42 and JNK2. TNF-{alpha}-stimulated phosphorylation of p42/p44 MAPK and JNKmore » were attenuated by pretreatment with the inhibitors U0126 and SP600125 or transfection with dominant negative mutants of {delta}ERK and {delta}JNK. Furthermore, the involvement of NF-{kappa}B in TNF-{alpha}-induced MMP-9 production was consistent with that TNF-{alpha}-stimulated degradation of I{kappa}B-{alpha} and translocation of NF-{kappa}B into the nucleus which were blocked by helenalin, but not by U0126 and SP600125, revealed by immunofluorescence staining. The regulation of MMP-9 gene transcription by MAPKs and NF-{kappa}B was further confirmed by gene luciferase activity assay. MMP-9 promoter activity was enhanced by TNF-{alpha} in A549 cells transfected with wild-type MMP-9-Luc, which was inhibited by helenalin, U0126, or SP600125. In contrast, TNF-{alpha}-stimulated MMP-9 luciferase activity was totally lost in cells transfected with mutant-NF-{kappa}B MMP-9-luc. Moreover, pretreatment with actinomycin D and cycloheximide attenuated TNF-{alpha}-induced MMP-9 expression. These results suggest that in A549 cells, phosphorylation of p42/p44 MAPK, JNK, and transactivation of NF-{kappa}B are essential for TNF-{alpha}-induced MMP-9 gene expression.« less

Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.

PubMed Central

Uzé, G; Lutfalla, G; Bandu, M T; Proudhon, D; Mogensen, K E

1992-01-01

A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes. Images PMID:1533935

Production of interferon-alpha in high cell density cultures of recombinant Escherichia coli and its single step purification from refolded inclusion body proteins.

PubMed

Babu, K R; Swaminathan, S; Marten, S; Khanna, N; Rinas, U

2000-06-01

Escherichia coli TG1 transformed with a temperature-regulated interferon-alpha expression vector was grown to high cell density in defined medium containing glucose as the sole carbon and energy source, utilizing a simple fed-batch process. Feeding was carried out to achieve an exponential increase in biomass at growth rates which minimized acetate production. Thermal induction of such high cell density cultures resulted in the production of approximately 4 g interferon-alpha/l culture broth. Interferon-alpha was produced exclusively in the form of insoluble inclusion bodies and was solubilized under denaturing conditions, refolded in the presence of arginine and purified to near homogeneity, utilizing single-step ion-exchange chromatography on Q-Sepharose. The yield of purified interferon-alpha was approximately 300 mg/l with respect to the original high cell density culture broth (overall yield of approximately 7.5% active interferon-alpha). The purified recombinant interferon-alpha was found by different criteria to be predominantly monomeric and possessed a specific bioactivity of approximately 2.5 x 10(8) IU/mg based on viral cytopathic assay.

Production of alpha 1,3-galactosyltransferase gene-deficient pigs by somatic cell nuclear transfer: a novel selection method for gal alpha 1,3-Gal antigen-deficient cells.

PubMed

Fujimura, Tatsuya; Takahagi, Yoichi; Shigehisa, Tamotsu; Nagashima, Hiroshi; Miyagawa, Shuji; Shirakura, Ryota; Murakami, Hiroshi

2008-09-01

The objective of the present study was to isolate alpha 1,3-galactosyltransferase (GalGT)-gene double knockout (DKO) cells using a novel simple method of cell selection method. To obtain GalGT-DKO cells, GalGT-gene single knockout (SKO) fetal fibroblast cells were cultured for three to nine passages and GalGT-null cells were separated using a biotin-labeled IB4 lectin attached to streptavidin-coated magnetic beads. After 15-17 days of additional cultivation, seven GalGT-DKO cell colonies were obtained from a total of 2.5 x 10(7) GalGT-SKO cells. A total of 926 somatic nuclear transferred embryos reconstructed with the DKO cells were transferred into eight recipient pigs, producing four farrowed, three liveborns, and six stillborns. Absence of GalGT gene in the cloned pigs was confirmed by PCR and Southern blotting. Flow cytometric analysis revealed that alphaGal antigens were not present in the cells of the cloned DKO pigs.

Intratumoral IL-12 and TNF-alpha-loaded microspheres lead to regression of breast cancer and systemic antitumor immunity.

PubMed

Sabel, Michael S; Skitzki, Joseph; Stoolman, Lloyd; Egilmez, Nejat K; Mathiowitz, Edith; Bailey, Nicola; Chang, Wen-Jian; Chang, Alfred E

2004-02-01

Local, sustained delivery of cytokines at a tumor can enhance induction of antitumor immunity and may be a feasible neoadjuvant immunotherapy for breast cancer. We evaluated the ability of intratumoral poly-lactic-acid-encapsulated microspheres (PLAM) containing interleukin 12 (IL-12), tumor necrosis factor alpha (TNF-alpha), and granulocyte-macrophage colony stimulating factor (GM-CSF) in a murine model of breast cancer to generate a specific antitumor response. BALB/c mice with established MT-901 tumors underwent resection or treatment with a single intratumoral injection of PLAM containing IL-12, TNF-alpha, or GM-CSF, alone or in combination. Two weeks later, lymph nodes and spleens were harvested, activated with anti-CD3 monoclonal antibodies (mAb) and rhIL-2, and assessed for antitumor reactivity by an interferon gamma (IFNgamma) release assay. Tumor-infiltrating lymphocyte (TIL) analysis was performed on days 2 and 5 after treatment by mechanically processing the tumors to create a single cell suspension, followed by three-color fluorescence-activated cell sorter (FACS) analysis. Intratumoral injection of cytokine-loaded PLAM significantly suppressed tumor growth, with the combination of IL-12 and TNF-alpha leading to increased infiltration by polymorphonuclear cells and CD8+ T-cells in comparison with controls. The induction of tumor-specific reactive T-cells in the nodes and spleens, as measured by IFN-gamma production, was highest with IL-12 and TNF-alpha. This treatment resulted in resistance to tumor rechallenge. A single intratumoral injection of IL-12 and TNF-alpha-loaded PLAM into a breast tumor leads to infiltration by polymorphonuclear cells and CD8+ T-cells with subsequent tumor regression. In addition, this local therapy induces specific antitumor T-cells in the lymph nodes and spleens, resulting in memory immune response.

Fluid shear stress inhibits TNF-alpha-induced apoptosis in osteoblasts: a role for fluid shear stress-induced activation of PI3-kinase and inhibition of caspase-3

NASA Technical Reports Server (NTRS)

Pavalko, Fredrick M.; Gerard, Rita L.; Ponik, Suzanne M.; Gallagher, Patricia J.; Jin, Yijun; Norvell, Suzanne M.

2003-01-01

In bone, a large proportion of osteoblasts, the cells responsible for deposition of new bone, normally undergo programmed cell death (apoptosis). Because mechanical loading of bone increases the rate of new bone formation, we hypothesized that mechanical stimulation of osteoblasts might increase their survival. To test this hypothesis, we investigated the effects of fluid shear stress (FSS) on osteoblast apoptosis using three osteoblast cell types: primary rat calvarial osteoblasts (RCOB), MC3T3-E1 osteoblastic cells, and UMR106 osteosarcoma cells. Cells were treated with TNF-alpha in the presence of cyclohexamide (CHX) to rapidly induce apoptosis. Osteoblasts showed significant signs of apoptosis within 4-6 h of exposure to TNF-alpha and CHX, and application of FSS (12 dyne/cm(2)) significantly attenuated this TNF-alpha-induced apoptosis. FSS activated PI3-kinase signaling, induced phosphorylation of Akt, and inhibited TNF-alpha-induced activation of caspase-3. Inhibition of PI3-kinase, using LY294002, blocked the ability of FSS to rescue osteoblasts from TNF-alpha-induced apoptosis and blocked FSS-induced inhibition of caspase-3 activation in osteoblasts treated with TNF-alpha. LY294002 did not, however, prevent FSS-induced phosphorylation of Akt suggesting that activation of Akt alone is not sufficient to rescue cells from apoptosis. This result also suggests that FSS can activate Akt via a PI3-kinase-independent pathway. These studies demonstrate for the first time that application of FSS to osteoblasts in vitro results in inhibition of TNF-alpha-induced apoptosis through a mechanism involving activation of PI3-kinase signaling and inhibition of caspases. FSS-induced activation of PI3-kinase may promote cell survival through a mechanism that is distinct from the Akt-mediated survival pathway. Copyright 2002 Wiley-Liss, Inc.

Phenotypic and functional characterization of T cells from patients with myasthenia gravis.

PubMed Central

Mokhtarian, F; Pino, M; Ofosu-Appiah, W; Grob, D

1990-01-01

A study of cell surface phenotypes of PBL of myasthenia gravis (MG) patients showed that their T cells had a significantly higher percentage of 4B4+ T cells (the helper/inducer subset) than age- and sex-matched controls. The PBL of MG patients proliferated significantly higher than those of normal subjects (NS) in response to the purified alpha chain of the acetylcholine receptor (AChR). Anti-AChR antibody was present in sera of 88% of MG and none of the NS. The PBL B cells from MG only, when cultured with autologous T cells and stimulated with either pokeweed mitogen (69%), or AChR-alpha chain (38%), secreted antibody to AChR-alpha chain, whereas T and B cells alone secreted no antibody. T cells from PBL of MG patients were more readily cloned than T cells of NS, by limiting dilution, in the presence of recombinant IL-2 and in the absence of AChR-alpha chain. About 50% of T cell clones from MG patients, compared to none from NS, proliferated to AChR-alpha chain. This response was HLA-DR restricted. MG T cell clones did not display significant cytotoxic activity, as compared to control T cell clones. Our results indicate that in MG, 4B4+ regulatory T cells play their role in the pathogenesis of MG, not by cytotoxicity, but more likely by their ability to stimulate specific antibody production by B cells. Images PMID:1979338

Integrin expression by human osteoblasts cultured on degradable polymeric materials applicable for tissue engineered bone.

PubMed

El-Amin, Saadiq F; Attawia, Mohamed; Lu, Helen H; Shah, Asist K; Chang, Richard; Hickok, Noreen J; Tuan, Rocky S; Laurencin, Cato T

2002-01-01

The use of biodegradable polymers in the field of orthopaedic surgery has gained increased popularity, as surgical pins and screws, and as potential biological scaffolds for repairing cartilage and bone defects. One such group of polymers that has gained considerable attention are the polyesters, poly(lactide-co-glycolide) (PLAGA) and polylactic acid (PLA), because of their minimal tissue inflammatory response, favorable biocompatibility and degradation characteristics. The objective of this study was to evaluate human osteoblastic cell adherence and growth on PLAGA and PLA scaffolds by examining integrin receptor (alpha2, alpha3, alpha4, alpha5, alpha6 and beta1) expression. Primary human osteoblastic cells isolated from trabecular bone adhered efficiently to both PLAGA and PLA, with the rate of adherence on PLAGA comparable to that of control tissue culture polystyrene (TCPS), and significantly higher than on PLA polymers at 3, 6 and 12 h. Human osteoblastic phenotypic expression, alkaline phosphatase (ALP) activity was positive on both degradable matrices, whereas osteocalcin levels were significantly higher on cells grown on PLAGA than on PLA composites. Interestingly, the integrin subunits, alpha2, alpha3, alpha4, alpha5, alpha6 and beta1 were all expressed at higher levels by osteoblasts cultured on PLAGA than those on PLA as analyzed by westerns blots and by flow cytometry. Among the integrins, alpha2, beta5 and beta1 showed the greatest difference in levels between the two surfaces. Thus, both PLA and PLAGA support osteoblastic adhesion and its accompanying engagement of integrin receptor and expression of osteocalcin and ALP. However PLAGA consistently appeared to be a better substrate for osteoblastic cells based on these parameters. This study is one of the first to investigate the ability of primary human osteoblastic cells isolated from trabecular bone to adhere to the biodegradable polymers PLAGA and PLA, and to examine the expression of their key adhesion receptors (integrins) on these substrates.

Circulating natural killer T cells in patients with asthma.

PubMed

Ikegami, Yasuhiko; Yokoyama, Akihito; Haruta, Yoshinori; Hiyama, Keiko; Kohno, Nobuoki

2004-01-01

Recent studies suggest that therapies targeted at depletion or limiting of natural killer (NK) T cells may be a possible strategy for the treatment of asthma. In the present study, we measured the number of circulating V alpha24+ NKT cells in 32 asthmatic patients and compared these patients with 29 nonatopic healthy controls. We investigated the relationships between NKT cell number and clinical variables such as the number of eosinophils, the circulating level of IgE, and the severity of asthma. In addition, we also investigated the ability of NKT cells to proliferate in response to alpha-galactosyl ceramide (alpha-GalCer) in vitro. The V alpha24+ NKT cell counts of asthmatic patients were significantly lower than those of healthy controls. There were no significant differences observed in asthmatic patients among the subgroups in terms of atopic status and severity. There was no significant correlation between the number of NKT cells and clinical variables. The proliferative response to alpha-GalCer of the patients and controls was not significantly different, indicating no intrinsic proliferative defect of NKT cells in asthma. These results suggest that the number of circulating NKT cells was already decreased in patients with asthma. Further study, such as the evaluation of lung NKT cells, will be needed to determine the role of NKT cells in patients with asthma.

Hsp105 family proteins suppress staurosporine-induced apoptosis by inhibiting the translocation of Bax to mitochondria in HeLa cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamagishi, Nobuyuki; Ishihara, Keiichi; Saito, Youhei

2006-10-15

Hsp105 (Hsp105{alpha} and Hsp105{beta}), major heat shock proteins in mammalian cells, belong to a subgroup of the HSP70 family, HSP105/110. Previously, we have shown that Hsp105{alpha} has completely different effects on stress-induced apoptosis depending on cell type. However, the molecular mechanisms by which Hsp105{alpha} regulates stress-induced apoptosis are not fully understood. Here, we established HeLa cells that overexpress either Hsp105{alpha} or Hsp105{beta} by removing doxycycline and examined how Hsp105 modifies staurosporine (STS)-induced apoptosis in HeLa cells. Apoptotic features such as the externalization of phosphatidylserine on the plasma membrane and nuclear morphological changes were induced by the treatment with STS, andmore » the STS-induced apoptosis was suppressed by overexpression of Hsp105{alpha} or Hsp105{beta}. In addition, we found that overexpression of Hsp105{alpha} or Hsp105{beta} suppressed the activation of caspase-3 and caspase-9 by preventing the release of cytochrome c from mitochondria. Furthermore, the translocation of Bax to mitochondria, which results in the release of cytochrome c from the mitochondria, was also suppressed by the overexpression of Hsp105{alpha} or Hsp105{beta}. Thus, it is suggested that Hsp105 suppresses the stress-induced apoptosis at its initial step, the translocation of Bax to mitochondria in HeLa cells.« less

Site directed mutagenesis of the heme axial ligands of cytochrome b559 affects the stability of the photosystem II complex.

PubMed Central

Pakrasi, H B; De Ciechi, P; Whitmarsh, J

1991-01-01

Cytochrome (cyt) b559, an integral membrane protein, is an essential component of the photosystem II (PSII) complex in the thylakoid membranes of oxygenic photosynthetic organisms. Cyt b559 has two subunits, alpha and beta, each with one predicted membrane spanning alpha-helical domain. The heme cofactor of this cytochrome is coordinated between two histidine residues. Each of the two subunit polypeptides of cyt b559 has one His residue. To investigate the influence of these His residues on the structure of cyt b559 and the PSII complex, we used a site directed mutagenesis approach to replace each His residue with a Leu residue. Introduction of these missense mutations in the transformable unicellular cyanobacterium, Synechocystis 6803, resulted in complete loss of PSII activity. Northern blot analysis showed that these mutations did not affect the stability of the polycistronic mRNA that encompasses both the psbE and the psbF genes, encoding the alpha and the beta subunits, respectively. Moreover, both of the single His mutants showed the presence of the alpha subunit which was 1.5 kd smaller than the same polypeptide in wild type cells. A secondary effect of such a structural change was that D1 and D2, two proteins that form the catalytic core (reaction center) of PSII, were also destabilized. Our results demonstrate that proper axial coordination of the heme cofactor in cyt b559 is important for the structural integrity of the reaction center of PSII. Images PMID:1904816

Human Beta Cells Produce and Release Serotonin to Inhibit Glucagon Secretion from Alpha Cells.

PubMed

Almaça, Joana; Molina, Judith; Menegaz, Danusa; Pronin, Alexey N; Tamayo, Alejandro; Slepak, Vladlen; Berggren, Per-Olof; Caicedo, Alejandro

2016-12-20

In the pancreatic islet, serotonin is an autocrine signal increasing beta cell mass during metabolic challenges such as those associated with pregnancy or high-fat diet. It is still unclear whether serotonin is relevant for regular islet physiology and hormone secretion. Here, we show that human beta cells produce and secrete serotonin when stimulated with increases in glucose concentration. Serotonin secretion from beta cells decreases cyclic AMP (cAMP) levels in neighboring alpha cells via 5-HT 1F receptors and inhibits glucagon secretion. Without serotonergic input, alpha cells lose their ability to regulate glucagon secretion in response to changes in glucose concentration, suggesting that diminished serotonergic control of alpha cells can cause glucose blindness and the uncontrolled glucagon secretion associated with diabetes. Supporting this model, pharmacological activation of 5-HT 1F receptors reduces glucagon secretion and has hypoglycemic effects in diabetic mice. Thus, modulation of serotonin signaling in the islet represents a drug intervention opportunity. Published by Elsevier Inc.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wong, Helen Pui Shan; Yu Le; Lam, Emily Kai Yee

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 proliferationmore » 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.« less