Severe hypoxia induces chemo-resistance in clinical cervical tumors through MVP over-expression

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Lara, Pedro C; Lloret, Marta; Clavo, Bernardino; Apolinario, Rosa M; Henríquez-Hernández, Luis Alberto; Bordón, Elisa; Fontes, Fausto; Rey, Agustín

2009-01-01

Oxygen molecule modulates tumour response to radiotherapy. Higher radiation doses are required under hypoxic conditions to induce cell death. Hypoxia may inhibit the non-homologous end-joining DNA repair through down regulating Ku70/80 expression. Hypoxia induces drug resistance in clinical tumours, although the mechanism is not clearly elucidated. Vaults are ribonucleoprotein particles with a hollow barrel-like structure composed of three proteins: major vault protein (MVP), vault poly(ADP-ribose) polymerase, and telomerase associated protein-1 and small untranslated RNA. Over-expression of MVP has been associated with chemotherapy resistance. Also, it has been related to poor outcome in patients treated with radiotherapy alone. The aim of the present study was to assess the relation of Major Vault Protein expression and tumor hypoxia in clinical cervical tumors. MVP, p53 and angiogenesis, together with tumor oxygenation, were determined in forty-three consecutive patients suffering from localized cervix carcinoma. High MVP expression was related to severe hypoxia compared to low MVP expressing tumors (p = 0.022). Tumors over-expressing MVP also showed increased angiogenesis (p = 0.003). Besides it, in this study we show for the first time that severe tumor hypoxia is associated with high MVP expression in clinical cervical tumors. Up-regulation of MVP by hypoxia is of critical relevance as chemotherapy is currently a standard treatment for those patients. From our results it could be suggested that hypoxia not only induces increased genetic instability, oncogenic properties and metastatization, but through the correlation observed with MVP expression, another pathway of chemo and radiation resistance could be developed

GPBAR1/TGR5 mediates bile acid-induced cytokine expression in murine Kupffer cells.

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Guiyu Lou

Full Text Available GPBAR1/TGR5 is a novel plasma membrane-bound G protein-coupled bile acid (BA receptor. BAs are known to induce the expression of inflammatory cytokines in the liver with unknown mechanism. Here we show that without other external stimuli, TGR5 activation alone induced the expression of interleukin 1β (IL-1β and tumor necrosis factor-α (TNF-α in murine macrophage cell line RAW264.7 or murine Kupffer cells. The TGR5-mediated increase of pro-inflammatory cytokine expression was suppressed by JNK inhibition. Moreover, the induced pro-inflammatory cytokine expression in mouse liver by 1% cholic acid (CA diet was blunted in JNK-/- mice. TGR5 activation by its ligands enhanced the phosphorylation levels, DNA-binding and trans-activities of c-Jun and ATF2 transcription factors. Finally, the induced pro-inflammatory cytokine expression in Kupffer cells by TGR5 activation correlated with the suppression of Cholesterol 7α-hydroxylase (Cyp7a1 expression in murine hepatocytes. These results suggest that TGR5 mediates the BA-induced pro-inflammatory cytokine production in murine Kupffer cells through JNK-dependent pathway. This novel role of TGR5 may correlate to the suppression of Cyp7a1 expression in hepatocytes and contribute to the delicate BA feedback regulation.

Nucleotide Pool Depletion Induces G-Quadruplex-Dependent Perturbation of Gene Expression

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Charikleia Papadopoulou

2015-12-01

Full Text Available Nucleotide pool imbalance has been proposed to drive genetic instability in cancer. Here, we show that slowing replication forks by depleting nucleotide pools with hydroxyurea (HU can also give rise to both transient and permanent epigenetic instability of a reporter locus, BU-1, in DT40 cells. HU induces stochastic formation of Bu-1low variants in dividing cells, which have lost the H3K4me3 present in untreated cells. This instability is potentiated by an intragenic G quadruplex, which also promotes local H2Ax phosphorylation and transient heterochromatinization. Genome-wide, gene expression changes induced by HU significantly overlap with those resulting from loss of the G4-helicases FANCJ, WRN, and BLM. Thus, the effects of global replication stress induced by nucleotide pool depletion can be focused by local replication impediments caused by G quadruplex formation to induce epigenetic instability and changes in gene expression, a mechanism that may contribute to selectable transcriptional changes in cancer.

RAE-1 expression is induced during experimental autoimmune encephalomyelitis and is correlated with microglia cell proliferation.

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Djelloul, Mehdi; Popa, Natalia; Pelletier, Florence; Raguénez, Gilda; Boucraut, José

2016-11-01

Retinoic acid early induced transcript-1 (RAE-1) glycoproteins are ligands of the activating immune receptor NKG2D. They are known as stress molecules induced in pathological conditions. We previously reported that progenitor cells express RAE-1 in physiological conditions and we described a correlation between RAE-1 expression and cell proliferation. In addition, we showed that Raet1 transcripts are induced in the spinal cord of experimental autoimmune encephalomyelitis (EAE) mice. EAE is a model for multiple sclerosis which is accompanied by microglia proliferation and activation, recruitment of immune cells and neurogenesis. We herein studied the time course expression of the two members of the Raet1 gene family present in C57BL/6 mice, namely Raet1d and Raet1e, in the spinal cord during EAE. We report that Raet1d and Raet1e genes are induced early upon EAE onset and reach a maximal expression at the peak of the pathology. We show that myeloid cells, i.e. macrophages as well as microglia, are cellular sources of Raet1 transcripts. We also demonstrate that only Raet1d expression is induced in microglia, whereas macrophages expressed both Raet1d and Raet1e. Furthermore, we investigated the dynamics of RAE-1 expression in microglia cultures. RAE-1 induction correlated with cell proliferation but not with M1/M2 phenotypic orientation. We finally demonstrate that macrophage colony-stimulating factor (M-CSF) is a major factor controlling RAE-1 expression in microglia. Copyright © 2016 Elsevier Inc. All rights reserved.

CAR expression and inducibility of CYP2B genes in liver of rats treated with PB-like inducers

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Pustylnyak, Vladimir O.; Gulyaeva, Lyudmila F.; Lyakhovich, Vyacheslav V.

2005-01-01

The expression of the CAR gene and inducibility of CYP2B protein in the liver of male Wistar rats treated with phenobarbital (PB) and triphenyldioxane (TPD) were investigated. To clarify the role of phosphorylation/dephosphorylation in these processes, rats were treated with inhibitors of Ca 2+ /calmodulin-dependent kinase II (W 7 ) or protein phosphatases PP1 and PP2A (OA) before induction. Constitutive expression of the CAR gene in livers of untreated rats was detected by multiplex RT-PCR. Treatment with W 7 resulted in a 2.8-fold induction of CAR gene expression, whereas OA led to a 2.4-fold decrease of the mRNA level. The same results were obtained for CYP2B genes expression, which were increased by W 7 treatment (two-fold) and decreased by OA (2.3-fold). PB-induction did not lead to significant alteration in the level of CAR gene expression, although CYP2B genes expression was enhanced two-fold over control values. TPD caused a two-fold increase of both CAR and CYP2B mRNA levels. Both inducers reduced the effects of inhibitors on CAR gene expression. Results of EMSA showed that PB, TPD or W 7 alone induced formation of complexes of NR1 with nuclear proteins. Appearance of the complexes correlated with an increase in CYP2B expression, and their intensities were modulated by the protein kinase inhibitors. Thus, our results demonstrate that constitutive expressions of CAR as well as CYP2B during induction are regulated by phosphorylation/dephosphorylation processes

Substrate Stiffness Influences Doxorubicin-Induced p53 Activation via ROCK2 Expression

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Takahiro Ebata

2017-01-01

Full Text Available The physical properties of the extracellular matrix (ECM, such as stiffness, are involved in the determination of the characteristics of cancer cells, including chemotherapy sensitivity. Resistance to chemotherapy is often linked to dysfunction of tumor suppressor p53; however, it remains elusive whether the ECM microenvironment interferes with p53 activation in cancer cells. Here, we show that, in MCF-7 breast cancer cells, extracellular stiffness influences p53 activation induced by the antitumor drug doxorubicin. Cell growth inhibition by doxorubicin was increased in response to ECM rigidity in a p53-dependent manner. The expression of Rho-associated coiled coil-containing protein kinase (ROCK 2, which induces the activation of myosin II, was significantly higher when cells were cultured on stiffer ECM substrates. Knockdown of ROCK2 expression or pharmacological inhibition of ROCK decreased doxorubicin-induced p53 activation. Our results suggest that a soft ECM causes downregulation of ROCK2 expression, which drives resistance to chemotherapy by repressing p53 activation.

Pregnancy-induced gene expression changes in vivo among women with rheumatoid arthritis: a pilot study.

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Goin, Dana E; Smed, Mette Kiel; Pachter, Lior; Purdom, Elizabeth; Nelson, J Lee; Kjærgaard, Hanne; Olsen, Jørn; Hetland, Merete Lund; Zoffmann, Vibeke; Ottesen, Bent; Jawaheer, Damini

2017-05-25

Little is known about gene expression changes induced by pregnancy in women with rheumatoid arthritis (RA) and healthy women because the few studies previously conducted did not have pre-pregnancy samples available as baseline. We have established a cohort of women with RA and healthy women followed prospectively from a pre-pregnancy baseline. In this study, we tested the hypothesis that pregnancy-induced changes in gene expression among women with RA who improve during pregnancy (pregDAS improved ) overlap substantially with changes observed among healthy women and differ from changes observed among women with RA who worsen during pregnancy (pregDAS worse ). Global gene expression profiles were generated by RNA sequencing (RNA-seq) from 11 women with RA and 5 healthy women before pregnancy (T0) and at the third trimester (T3). Among the women with RA, eight showed an improvement in disease activity by T3, whereas three worsened. Differential expression analysis was used to identify genes demonstrating significant changes in expression within each of the RA and healthy groups (T3 vs T0), as well as between the groups at each time point. Gene set enrichment was assessed in terms of Gene Ontology processes and protein networks. A total of 1296 genes were differentially expressed between T3 and T0 among the 8 pregDAS improved women, with 161 genes showing at least two-fold change (FC) in expression by T3. The majority (108 of 161 genes) were also differentially expressed among healthy women (qexpression between the pregDAS improved and pregDAS worse groups, all of which were inducible by type I interferon (IFN). These IFN-inducible genes were over-expressed at T3 compared to the T0 baseline among the pregDAS improved women. In our pilot RNA-seq dataset, increased pregnancy-induced expression of type I IFN-inducible genes was observed among women with RA who improved during pregnancy, but not among women who worsened. These findings warrant further investigation into

Bupivacaine-induced apoptosis independently of WDR35 expression in mouse neuroblastoma Neuro2a cells

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2012-01-01

Background Bupivacaine-induced neurotoxicity has been shown to occur through apoptosis. Recently, bupivacaine was shown to elicit reactive oxygen species (ROS) production and induce apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK) in a human neuroblastoma cell line. We have reported that WDR35, a WD40-repeat protein, may mediate apoptosis through caspase-3 activation. The present study was undertaken to test whether bupivacaine induces apoptosis in mouse neuroblastoma Neuro2a cells and to determine whether ROS, p38 MAPK, and WDR35 are involved. Results Our results showed that bupivacaine induced ROS generation and p38 MAPK activation in Neuro2a cells, resulting in apoptosis. Bupivacaine also increased WDR35 expression in a dose- and time-dependent manner. Hydrogen peroxide (H2O2) also increased WDR35 expression in Neuro2a cells. Antioxidant (EUK-8) and p38 MAPK inhibitor (SB202190) treatment attenuated the increase in caspase-3 activity, cell death and WDR35 expression induced by bupivacaine or H2O2. Although transfection of Neuro2a cells with WDR35 siRNA attenuated the bupivacaine- or H2O2-induced increase in expression of WDR35 mRNA and protein, in contrast to our previous studies, it did not inhibit the increase in caspase-3 activity in bupivacaine- or H2O2-treated cells. Conclusions In summary, our results indicated that bupivacaine induced apoptosis in Neuro2a cells. Bupivacaine induced ROS generation and p38 MAPK activation, resulting in an increase in WDR35 expression, in these cells. However, the increase in WDR35 expression may not be essential for the bupivacaine-induced apoptosis in Neuro2a cells. These results may suggest the existence of another mechanism of bupivacaine-induced apoptosis independent from WDR35 expression in Neuro2a cells. PMID:23227925

Wnt/β-catenin signaling changes C2C12 myoblast proliferation and differentiation by inducing Id3 expression

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Zhang, Long; Shi, Songting; Zhang, Juan; Zhou, Fangfang; Dijke, Peter ten

2012-01-01

Highlights: ► Expression of Id3 but not Id1 is induced by Wnt3a stimulation in C2C12 cells. ► Wnt3a induces Id3 expression via canonical Wnt/β-catenin pathway. ► Wnt3a-induced Id3 expression does not depend on BMP signaling activation. ► Induction of Id3 expression is critical determinant in Wnt3a-induced cell proliferation and differentiation. -- Abstract: Canonical Wnt signaling plays important roles in regulating cell proliferation and differentiation. In this study, we report that inhibitor of differentiation (Id)3 is a Wnt-inducible gene in mouse C2C12 myoblasts. Wnt3a induced Id3 expression in a β-catenin-dependent manner. Bone morphogenetic protein (BMP) also potently induced Id3 expression. However, Wnt-induced Id3 expression occurred independent of the BMP/Smad pathway. Functional studies showed that Id3 depletion in C2C12 cells impaired Wnt3a-induced cell proliferation and alkaline phosphatase activity, an early marker of osteoblast cells. Id3 depletion elevated myogenin induction during myogenic differentiation and partially impaired Wnt3a suppressed myogenin expression in C2C12 cells. These results suggest that Id3 is an important Wnt/β-catenin induced gene in myoblast cell fate determination.

Ethanol enhances arsenic-induced cyclooxygenase-2 expression via both NFAT and NF-κB signalings in colorectal cancer cells

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Wang, Lei; Hitron, John Andrew [Center for Research on Environmental Disease, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Toxicology and Cancer Biology, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Wise, James T.F. [Pharmacology and Nutritional Sciences, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Son, Young-Ok; Roy, Ram Vinod [Center for Research on Environmental Disease, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Toxicology and Cancer Biology, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Kim, Donghern; Dai, Jin [Toxicology and Cancer Biology, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Pratheeshkumar, Poyil [Center for Research on Environmental Disease, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Toxicology and Cancer Biology, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Zhang, Zhuo [Toxicology and Cancer Biology, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Xu, Mei; Luo, Jia [Pharmacology and Nutritional Sciences, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Shi, Xianglin, E-mail: xshi5@uky.edu [Center for Research on Environmental Disease, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Toxicology and Cancer Biology, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States)

2015-10-15

Arsenic is a known carcinogen to humans, and chronic exposure to environmental arsenic is a worldwide health concern. As a dietary factor, ethanol carries a well-established risk for malignancies, but the effects of co-exposure to arsenic and ethanol on tumor development are not well understood. In the present study, we hypothesized that ethanol would enhance the function of an environmental carcinogen such as arsenic through increase in COX-2 expression. Our in vitro results show that ethanol enhanced arsenic-induced COX-2 expression. We also show that the increased COX-2 expression associates with intracellular ROS generation, up-regulated AKT signaling, with activation of both NFAT and NF-κB pathways. We demonstrate that antioxidant enzymes have an inhibitory effect on arsenic/ethanol-induced COX-2 expression, indicating that the responsive signaling pathways from co-exposure to arsenic and ethanol relate to ROS generation. In vivo results also show that co-exposure to arsenic and ethanol increased COX-2 expression in mice. We conclude that ethanol enhances arsenic-induced COX-2 expression in colorectal cancer cells via both the NFAT and NF-κB pathways. These results imply that, as a common dietary factor, ethanol ingestion may be a compounding risk factor for arsenic-induced carcinogenesis/cancer development. - Highlights: • Arsenic is able to induce Cox-2 expression in colorectal cancer cells. • Ethanol, a diet nutritional factor, could enhance arsenic-induced Cox-2. • The up-regulation of Cox-2 via both NFAT and NF-κB activities.

Ethanol enhances arsenic-induced cyclooxygenase-2 expression via both NFAT and NF-κB signalings in colorectal cancer cells

International Nuclear Information System (INIS)

Wang, Lei; Hitron, John Andrew; Wise, James T.F.; Son, Young-Ok; Roy, Ram Vinod; Kim, Donghern; Dai, Jin; Pratheeshkumar, Poyil; Zhang, Zhuo; Xu, Mei; Luo, Jia; Shi, Xianglin

2015-01-01

Arsenic is a known carcinogen to humans, and chronic exposure to environmental arsenic is a worldwide health concern. As a dietary factor, ethanol carries a well-established risk for malignancies, but the effects of co-exposure to arsenic and ethanol on tumor development are not well understood. In the present study, we hypothesized that ethanol would enhance the function of an environmental carcinogen such as arsenic through increase in COX-2 expression. Our in vitro results show that ethanol enhanced arsenic-induced COX-2 expression. We also show that the increased COX-2 expression associates with intracellular ROS generation, up-regulated AKT signaling, with activation of both NFAT and NF-κB pathways. We demonstrate that antioxidant enzymes have an inhibitory effect on arsenic/ethanol-induced COX-2 expression, indicating that the responsive signaling pathways from co-exposure to arsenic and ethanol relate to ROS generation. In vivo results also show that co-exposure to arsenic and ethanol increased COX-2 expression in mice. We conclude that ethanol enhances arsenic-induced COX-2 expression in colorectal cancer cells via both the NFAT and NF-κB pathways. These results imply that, as a common dietary factor, ethanol ingestion may be a compounding risk factor for arsenic-induced carcinogenesis/cancer development. - Highlights: • Arsenic is able to induce Cox-2 expression in colorectal cancer cells. • Ethanol, a diet nutritional factor, could enhance arsenic-induced Cox-2. • The up-regulation of Cox-2 via both NFAT and NF-κB activities.

Regulatable Transgene Expression for Prevention of Chemotherapy-Induced Peripheral Neuropathy

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Daisuke Kawata

2017-09-01

Full Text Available Chemotherapy-induced peripheral neuropathy (CIPN is a debilitating complication associated with drug treatment of cancer for which there are no effective strategies of prevention or treatment. In this study, we examined the effect of intermittent expression of neurotophin-3 (NT-3 or interleukin-10 (IL-10 from replication-defective herpes simplex virus (HSV-based regulatable vectors delivered by subcutaneous inoculation to the dorsal root ganglion (DRG on the development of paclitaxel-induced peripheral neuropathy. We constructed two different tetracycline (tet-on-based regulatable HSV vectors, one expressing NT-3 and the other expressing IL-10, in which the transactivator expression in the tet-on system was under the control of HSV latency-associated promoter 2 (LAP-2, and expression of the transgene was controlled by doxycycline (DOX. We examined the therapeutic effect of intermittent expression of the transgene in animals with paclitaxel-induced peripheral neuropathy modeled by intraperitoneal injection of paclitaxel (16 mg/kg once a week for 5 weeks. Intermittent expression of either NT-3 or IL-10 3 days before and 1 day after paclitaxel administration protected animals against paclitaxel-induced peripheral neuropathy over the course of 5 weeks. These results suggest the potential of regulatable vectors for prevention of chemotherapy-induced peripheral neuropathy.

Regulatable Transgene Expression for Prevention of Chemotherapy-Induced Peripheral Neuropathy.

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Kawata, Daisuke; Wu, Zetang

2017-09-15

Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating complication associated with drug treatment of cancer for which there are no effective strategies of prevention or treatment. In this study, we examined the effect of intermittent expression of neurotophin-3 (NT-3) or interleukin-10 (IL-10) from replication-defective herpes simplex virus (HSV)-based regulatable vectors delivered by subcutaneous inoculation to the dorsal root ganglion (DRG) on the development of paclitaxel-induced peripheral neuropathy. We constructed two different tetracycline (tet)-on-based regulatable HSV vectors, one expressing NT-3 and the other expressing IL-10, in which the transactivator expression in the tet-on system was under the control of HSV latency-associated promoter 2 (LAP-2), and expression of the transgene was controlled by doxycycline (DOX). We examined the therapeutic effect of intermittent expression of the transgene in animals with paclitaxel-induced peripheral neuropathy modeled by intraperitoneal injection of paclitaxel (16 mg/kg) once a week for 5 weeks. Intermittent expression of either NT-3 or IL-10 3 days before and 1 day after paclitaxel administration protected animals against paclitaxel-induced peripheral neuropathy over the course of 5 weeks. These results suggest the potential of regulatable vectors for prevention of chemotherapy-induced peripheral neuropathy.

Ipomoea aquatica Extract Shows Protective Action Against Thioacetamide-Induced Hepatotoxicity

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A. Hamid A. Hadi

2012-05-01

Full Text Available In the Indian system of traditional medicine (Ayurveda it is recommended to consume Ipomoea aquatica to mitigate disorders like jaundice. In this study, the protective effects of ethanol extract of I. aquatica against liver damage were evaluated in thioacetamide (TAA-induced chronic hepatotoxicity in rats. There was no sign of toxicity in the acute toxicity study, in which Sprague-Dawley (SD rats were orally fed with I. aquatica (250 and 500 mg/kg for two months along with administration of TAA (i.p injection 200 mg/kg three times a week for two months. The results showed that the treatment of I. aquatica significantly lowered the TAA-induced serum levels of hepatic enzyme markers (ALP, ALT, AST, protein, albumin, bilirubin and prothrombin time. The hepatic content of activities and expressions SOD and CAT that were reduced by TAA were brought back to control levels by the plant extract supplement. Meanwhile, the rise in MDA level in the TAA receiving groups also were significantly reduced by I. aquatica treatment. Histopathology of hepatic tissues by H&E and Masson trichrome stains displayed that I. aquatica has reduced the incidence of liver lesions, including hepatic cells cloudy swelling, infiltration, hepatic necrosis, and fibrous connective tissue proliferation induced by TAA in rats. Therefore, the results of this study show that the protective effect of I. aquatica in TAA-induced liver damage might be contributed to its modulation on detoxification enzymes and its antioxidant and free radical scavenger effects. Moreover, it confirms a scientific basis for the traditional use of I. aquatica for the treatment of liver disorders.

Radiation induced expression of survivin in Ewing sarcoma cell-lines

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Sheikh-Mounessi, F.; Willich, N.; Greve, B.

2009-01-01

Full text: Introduction: Survivin belongs to the Inhibitor of Apoptosis Protein Family (IAP), is a protein of 16.5 kD and active as a homodimer. It is overexpressed in nearly all human tumors and has a vital function in cell division and apoptotic processes. Beside its role as a relevant prognostic and predictive factor it was described to be a molecular target to improve effectiveness of radiotherapy. We investigated the radiation induced survivin expression in Ewing sarcoma cell-lines. Methods: Ewing sarcoma cells were either irradiated with 10 Gy X-ray and harvested at different time points (0, 2, 4, 6, 10 and 24 h) or irradiated with different doses (0, 2, 5 and 10 Gy) and harvested 24 h later. Protein and mRNA expression was analysed by Westernblot or Real-Time PCR. Results: Directly after irradiation with 10 Gy X-ray survivin mRNA expression was increased in relation to the reference GAPDH. Protein expression was increased in a time dependent manner and reached a maximum after 24h. Three of four investigated cell-lines showed a significant dose dependent increase of survivin protein concentration 24h after irradiation. The same three cell-lines showed a LD50 of >30 Gy. The line with the lowest dose dependent survivin induction was investigated to be most radiosensitive (LD50 = 24 Gy). Discussion: Ewing sarcoma is a childhood tumor with relatively poor prognosis. This tumor often shows significant therapeutic resistance to chemo- and/or radiotherapy. It would be of high interest to find new therapeutic approaches for its treatment. We found a remarkable overexpression of survivin in untreated Ewing sarcoma and a time and dose dependent increase of survivin protein concentration after irradiation with X-ray. The cell-line with the lowest survivin induction showed the highest radiosensitivity. In conclusion, our results show that survivin is an inducible radioresistance factor in Ewing sarcoma. This may open new therapeutic options to treat this aggressive

Attenuation of doxorubicin-induced cardiotoxicity by esculetin through modulation of Bmi-1 expression.

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Xu, Fan; Li, Xiao; Liu, Lanfang; Xiao, Xu; Zhang, Li; Zhang, Shenglin; Lin, Pingping; Wang, Xiaojie; Wang, Yongwei; Li, Qingshan

2017-09-01

The protective effects and mechanisms of esculetin on doxorubicin (DOX)-induced injury of H9c2 cells were investigated. H9c2 cells were cultured and the logarithmic growth phase of the cells was divided into a control group, a DOX group and an esculetin + DOX group. Cell viability was detected by MTT assay. Annexin V-PI (AV-PI) double staining flow cytometry was carried out to detect cell apoptosis. Intracellular reactive oxygen species (ROS) were detected by flow cytometry. Transmission electron microscope (TEM) was used to evaluate cell ultrastructure. Cleaved caspase-3, cleaved PARP, Bcl-2, Bid and Bmi-1 proteins levels were investigated by western blot analysis. Bmi-1 siRNA was used to detect the role of Bmi-1 in the protective effects of esculetin against DOX-induced toxicity in H9c2 cells. The MTT and AV-PI double staining results showed that esculetin significantly increased H9c2 cell viability. Compared with the control group, the levels of cleaved caspase-3, cleaved PARP, Bid and ROS levels were significantly decreased, but the expression of Bcl-2 and Bmi-1 were significantly increased in the esculetin + DOX group. TEM showed that the cell structure of the mitochondria was protected by esculetin. The results of Bmi-1 siRNA showed that esculetin could protect DOX-induced cardiotoxicity by modulating Bmi-1 expression. Esculetin can protect DOX-induced cardiotoxicity and the effects may be attributable to modulation of Bmi-1 expression, provoking intracellular ROS accumulation, protecting the structure of mitochondria and reducing cell apoptosis.

SIRT1 sensitizes hepatocellular carcinoma cells expressing hepatitis B virus X protein to oxidative stress-induced apoptosis

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Srisuttee, Ratakorn; Koh, Sang Seok; Malilas, Waraporn; Moon, Jeong; Cho, Il-Rae; Jhun, Byung Hak; Horio, Yoshiyuki; Chung, Young-Hwa

2012-01-01

Highlights: ► Up-regulation of SIRT1 protein and activity sensitizes Hep3B-HBX cells to oxidative stress-induced apoptosis. ► Nuclear localization of SIRT1 is not required for oxidation-induced apoptosis. ► Ectopic expression and enhanced activity of SIRT1 attenuate JNK phosphorylation. ► Inhibition of SIRT1 activity restores resistance to oxidation-induced apoptosis through JNK activation. -- Abstract: We previously showed that SIRT1 deacetylase inhibits proliferation of hepatocellular carcinoma cells expressing hepatitis B virus (HBV) X protein (HBX), by destabilization of β-catenin. Here, we report another role for SIRT1 in HBX-mediated resistance to oxidative stress. Ectopic expression and enhanced activity of SIRT1 sensitize Hep3B cells stably expressing HBX to oxidative stress-induced apoptosis. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for sensitization of oxidation-mediated apoptosis. Furthermore, ectopic expression of SIRT1 and treatment with resveratrol (a SIRT1 activator) attenuated JNK phosphorylation, which is a prerequisite for resistance to oxidative stress-induced apoptosis. Conversely, suppression of SIRT1 activity with nicotinamide inhibited the effect of resveratrol on JNK phosphorylation, leading to restoration of resistance to oxidation-induced apoptosis. Taken together, these results suggest that up-regulation of SIRT1 under oxidative stress may be a therapeutic strategy for treatment of hepatocellular carcinoma cells related to HBV through inhibition of JNK activation.

SIRT1 sensitizes hepatocellular carcinoma cells expressing hepatitis B virus X protein to oxidative stress-induced apoptosis

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Srisuttee, Ratakorn [WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735 (Korea, Republic of); Koh, Sang Seok [Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, University of Science and Technology, Daejeon 305-333 (Korea, Republic of); Department of Functional Genomics, University of Science and Technology, Daejeon 305-333 (Korea, Republic of); Malilas, Waraporn; Moon, Jeong; Cho, Il-Rae [WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735 (Korea, Republic of); Jhun, Byung Hak [Department of Applied Nanoscience, Pusan National University, Busan 609-735 (Korea, Republic of); Horio, Yoshiyuki [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan); Chung, Young-Hwa, E-mail: younghc@pusan.ac.kr [WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735 (Korea, Republic of)

2012-12-07

Highlights: Black-Right-Pointing-Pointer Up-regulation of SIRT1 protein and activity sensitizes Hep3B-HBX cells to oxidative stress-induced apoptosis. Black-Right-Pointing-Pointer Nuclear localization of SIRT1 is not required for oxidation-induced apoptosis. Black-Right-Pointing-Pointer Ectopic expression and enhanced activity of SIRT1 attenuate JNK phosphorylation. Black-Right-Pointing-Pointer Inhibition of SIRT1 activity restores resistance to oxidation-induced apoptosis through JNK activation. -- Abstract: We previously showed that SIRT1 deacetylase inhibits proliferation of hepatocellular carcinoma cells expressing hepatitis B virus (HBV) X protein (HBX), by destabilization of {beta}-catenin. Here, we report another role for SIRT1 in HBX-mediated resistance to oxidative stress. Ectopic expression and enhanced activity of SIRT1 sensitize Hep3B cells stably expressing HBX to oxidative stress-induced apoptosis. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for sensitization of oxidation-mediated apoptosis. Furthermore, ectopic expression of SIRT1 and treatment with resveratrol (a SIRT1 activator) attenuated JNK phosphorylation, which is a prerequisite for resistance to oxidative stress-induced apoptosis. Conversely, suppression of SIRT1 activity with nicotinamide inhibited the effect of resveratrol on JNK phosphorylation, leading to restoration of resistance to oxidation-induced apoptosis. Taken together, these results suggest that up-regulation of SIRT1 under oxidative stress may be a therapeutic strategy for treatment of hepatocellular carcinoma cells related to HBV through inhibition of JNK activation.

Halobenzoquinone-Induced Alteration of Gene Expression Associated with Oxidative Stress Signaling Pathways.

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Li, Jinhua; Moe, Birget; Liu, Yanming; Li, Xing-Fang

2018-06-05

Halobenzoquinones (HBQs) are emerging disinfection byproducts (DBPs) that effectively induce reactive oxygen species and oxidative damage in vitro. However, the impacts of HBQs on oxidative-stress-related gene expression have not been investigated. In this study, we examined alterations in the expression of 44 genes related to oxidative-stress-induced signaling pathways in human uroepithelial cells (SV-HUC-1) upon exposure to six HBQs. The results show the structure-dependent effects of HBQs on the studied gene expression. After 2 h of exposure, the expression levels of 9 to 28 genes were altered, while after 8 h of exposure, the expression levels of 29 to 31 genes were altered. Four genes ( HMOX1, NQO1, PTGS2, and TXNRD1) were significantly upregulated by all six HBQs at both exposure time points. Ingenuity pathway analysis revealed that the Nrf2 pathway was significantly responsive to HBQ exposure. Other canonical pathways responsive to HBQ exposure included GSH redox reductions, superoxide radical degradation, and xenobiotic metabolism signaling. This study has demonstrated that HBQs significantly alter the gene expression of oxidative-stress-related signaling pathways and contributes to the understanding of HBQ-DBP-associated toxicity.

Mechanism of inhibitory effect of atorvastatin on resistin expression induced by tumor necrosis factor-α in macrophages

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Chua Su-Kiat

2009-05-01

Full Text Available Abstract Atorvastatin has been shown to reduce resistin expression in macrophages after pro-inflammatory stimulation. However, the mechanism of reducing resistin expression by atorvastatin is not known. Therefore, we sought to investigate the molecular mechanisms of atorvastatin for reducing resistin expression after proinflammatory cytokine, tumor necrosis factor-α (TNF-α stimulation in cultured macrophages. Cultured macrophages were obtained from human peripheral blood mononuclear cells. TNF-α stimulation increased resistin protein and mRNA expression and atorvastatin inhibited the induction of resistin by TNF-α. Addition of mevalonate induced resistin protein expression similar to TNF-α stimulation. However, atorvastatin did not have effect on resistin protein expression induced by mevalonate. SP600125 and JNK small interfering RNA (siRNA completely attenuated the resistin protein expression induced by TNF-α and mevalonate. TNF-α induced phosphorylation of Rac, while atorvastatin and Rac-1 inhibitor inhibited the phosphorylation of Rac induced by TNF-α. The gel shift and promoter activity assay showed that TNF-α increased AP-1-binding activity and resistin promoter activity, while SP600125 and atorvastatin inhibited the AP-1-binding activity and resistin promoter activity induced by TNF-α. Recombinant resistin and TNF-α significantly reduced glucose uptake in cultured macrophages, while atorvastatin reversed the reduced glucose uptake by TNF-α. In conclusion, JNK and Rac pathway mediates the inhibitory effect of atorvastatin on resistin expression induced by TNF-α.

Bacterial feeding, Leishmania infection and distinct infection routes induce differential defensin expression in Lutzomyia longipalpis.

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Telleria, Erich L; Sant'Anna, Maurício R Viana; Alkurbi, Mohammad O; Pitaluga, André N; Dillon, Rod J; Traub-Csekö, Yara M

2013-01-11

Phlebotomine insects harbor bacterial, viral and parasitic pathogens that can cause diseases of public health importance. Lutzomyia longipalpis is the main vector of visceral leishmaniasis in the New World. Insects can mount a powerful innate immune response to pathogens. Defensin peptides take part in this response and are known to be active against Gram-positive and Gram-negative bacteria, and some parasites. We studied the expression of a defensin gene from Lutzomyia longipalpis to understand its role in sand fly immune response. We identified, sequenced and evaluated the expression of a L. longipalpis defensin gene by semi-quantitative RT-PCR. The gene sequence was compared to other vectors defensins and expression was determined along developmental stages and after exposure of adult female L. longipalpis to bacteria and Leishmania. Phylogenetic analysis showed that the L. longipalpis defensin is closely related to a defensin from the Old World sand fly Phlebotomus duboscqi. Expression was high in late L4 larvae and pupae in comparison to early larval stages and newly emerged flies. Defensin expression was modulated by oral infection with bacteria. The Gram-positive Micrococcus luteus induced early high defensin expression, whilst the Gram-negative entomopathogenic Serratia marcescens induced a later response. Bacterial injection also induced defensin expression in adult insects. Female sand flies infected orally with Leishmania mexicana showed no significant difference in defensin expression compared to blood fed insects apart from a lower defensin expression 5 days post Leishmania infection. When Leishmania was introduced into the hemolymph by injection there was no induction of defensin expression until 72 h later. Our results suggest that L. longipalpis modulates defensin expression upon bacterial and Leishmania infection, with patterns of expression that are distinct among bacterial species and routes of infection.

Duct- and Acinar-Derived Pancreatic Ductal Adenocarcinomas Show Distinct Tumor Progression and Marker Expression

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Rute M.M. Ferreira

2017-10-01

Full Text Available The cell of origin of pancreatic ductal adenocarcinoma (PDAC has been controversial. Here, we show that identical oncogenic drivers trigger PDAC originating from both ductal and acinar cells with similar histology but with distinct pathophysiology and marker expression dependent on cell of origin. Whereas acinar-derived tumors exhibited low AGR2 expression and were preceded by pancreatic intraepithelial neoplasias (PanINs, duct-derived tumors displayed high AGR2 and developed independently of a PanIN stage via non-mucinous lesions. Using orthotopic transplantation and chimera experiments, we demonstrate that PanIN-like lesions can be induced by PDAC as bystanders in adjacent healthy tissues, explaining the co-existence of mucinous and non-mucinous lesions and highlighting the need to distinguish between true precursor PanINs and PanIN-like bystander lesions. Our results suggest AGR2 as a tool to stratify PDAC according to cell of origin, highlight that not all PanIN-like lesions are precursors of PDAC, and add an alternative progression route to the current model of PDAC development.

Involvement of mitogen-activated protein kinases and NFκB in LPS-induced CD40 expression on human monocytic cells

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Wu Weidong; Alexis, Neil E.; Chen Xian; Bromberg, Philip A.; Peden, David B.

2008-01-01

CD40 is a costimulatory molecule linking innate and adaptive immune responses to bacterial stimuli, as well as a critical regulator of functions of other costimulatory molecules. The mechanisms regulating lipopolysaccharide (LPS)-induced CD40 expression have not been adequately characterized in human monocytic cells. In this study we used a human monocytic cell line, THP-1, to investigate the possible mechanisms of CD40 expression following LPS exposure. Exposure to LPS resulted in a dose- and time-dependent increase in CD40 expression. Further studies using immunoblotting and pharmacological inhibitors revealed that mitogen-activated protein kinases (MAPKs) and NFκB were activated by LPS exposure and involved in LPS-induced CD40 expression. Activation of MAPKs was not responsible for LPS-induced NFκB activation. TLR4 was expressed on THP-1 cells and pretreatment of cells with a Toll-like receptor 4 (TLR4) neutralizing antibody (HTA125) significantly blunted LPS-induced MAPK and NFκB activation and ensuing CD40 expression. Additional studies with murine macrophages expressing wild type and mutated TLR4 showed that TLR4 was implicated in LPS-induced ERK and NFκB activation, and CD40 expression. Moreover, blockage of MAPK and NFκB activation inhibited LPS-induced TLR4 expression. In summary, LPS-induced CD40 expression in monocytic cells involves MAPKs and NFκB

Wolbachia-induced cytoplasmic incompatibility is associated with decreased Hira expression in male Drosophila.

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Ya Zheng

Full Text Available BACKGROUND: Wolbachia are obligate endosymbiotic bacteria that infect numerous species of arthropods and nematodes. Wolbachia can induce several reproductive phenotypes in their insect hosts including feminization, male-killing, parthenogenesis and cytoplasmic incompatibility (CI. CI is the most common phenotype and occurs when Wolbachia-infected males mate with uninfected females resulting in no or very low numbers of viable offspring. However, matings between males and females infected with the same strain of Wolbachia result in viable progeny. Despite substantial scientific effort, the molecular mechanisms underlying CI are currently unknown. METHODOLOGY/PRINCIPAL FINDINGS: Gene expression studies were undertaken in Drosophila melanogaster and D. simulans which display differential levels of CI using quantitative RT-PCR. We show that Hira expression is correlated with the induction of CI and occurs in a sex-specific manner. Hira expression is significantly lower in males which induce strong CI when compared to males inducing no CI or Wolbachia-uninfected males. A reduction in Hira expression is also observed in 1-day-old males that induce stronger CI compared to 5-day-old males that induce weak or no CI. In addition, Hira mutated D. melanogaster males mated to uninfected females result in significantly decreased hatch rates comparing with uninfected crosses. Interestingly, wMel-infected females may rescue the hatch rates. An obvious CI phenotype with chromatin bridges are observed in the early embryo resulting from Hira mutant fertilization, which strongly mimics the defects associated with CI. CONCLUSIONS/SIGNIFICANCE: Our results suggest Wolbachia-induced CI in Drosophila occurs due to a reduction in Hira expression in Wolbachia-infected males leading to detrimental effects on sperm fertility resulting in embryo lethality. These results may help determine the underlying mechanism of CI and provide further insight in to the important role

PPARα deficiency augments a ketogenic diet-induced circadian PAI-1 expression possibly through PPARγ activation in the liver

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Oishi, Katsutaka; Uchida, Daisuke; Ohkura, Naoki; Horie, Shuichi

2010-01-01

Research highlights: → PPARα deficiency augments a ketogenic diet-induced circadian PAI-1 expression. → Hepatic expressions of PPARγ and PCG-1α are induced by a ketogenic diet. → PPARγ antagonist attenuates a ketogenic diet-induced PAI-1 expression. → Ketogenic diet advances the phase of circadian clock in a PPARα-independent manner. -- Abstract: An increased level of plasminogen activator inhibitor-1 (PAI-1) is considered a risk factor for cardiovascular diseases, and PAI-1 gene expression is under the control of molecular circadian clocks in mammals. We recently showed that PAI-1 expression is augmented in a phase-advanced circadian manner in mice fed with a ketogenic diet (KD). To determine whether peroxisome proliferator-activated receptor α (PPARα) is involved in hypofibrinolytic status induced by a KD, we examined the expression profiles of PAI-1 and circadian clock genes in PPARα-null KD mice. Chronic administration of bezafibrate induced the PAI-1 gene expression in a PPARα-dependent manner. Feeding with a KD augmented the circadian expression of PAI-1 mRNA in the hearts and livers of wild-type (WT) mice as previously described. The KD-induced mRNA expression of typical PPARα target genes such as Cyp4A10 and FGF21 was damped in PPARα-null mice. However, plasma PAI-1 concentrations were significantly more elevated in PPARα-null KD mice in accordance with hepatic mRNA levels. These observations suggest that PPARα activation is dispensable for KD-induced PAI-1 expression. We also found that hyperlipidemia, fatty liver, and the hepatic expressions of PPARγ and its coactivator PCG-1α were more effectively induced in PPARα-null, than in WT mice on a KD. Furthermore, KD-induced hepatic PAI-1 expression was significantly suppressed by supplementation with bisphenol A diglycidyl ether, a PPARγ antagonist, in both WT and PPARα-null mice. PPARγ activation seems to be involved in KD-induced hypofibrinolysis by augmenting PAI-1 gene expression

Hormone-induced protection against mammary tumorigenesis is conserved in multiple rat strains and identifies a core gene expression signature induced by pregnancy.

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Blakely, Collin M; Stoddard, Alexander J; Belka, George K; Dugan, Katherine D; Notarfrancesco, Kathleen L; Moody, Susan E; D'Cruz, Celina M; Chodosh, Lewis A

2006-06-15

Women who have their first child early in life have a substantially lower lifetime risk of breast cancer. The mechanism for this is unknown. Similar to humans, rats exhibit parity-induced protection against mammary tumorigenesis. To explore the basis for this phenomenon, we identified persistent pregnancy-induced changes in mammary gene expression that are tightly associated with protection against tumorigenesis in multiple inbred rat strains. Four inbred rat strains that exhibit marked differences in their intrinsic susceptibilities to carcinogen-induced mammary tumorigenesis were each shown to display significant protection against methylnitrosourea-induced mammary tumorigenesis following treatment with pregnancy levels of estradiol and progesterone. Microarray expression profiling of parous and nulliparous mammary tissue from these four strains yielded a common 70-gene signature. Examination of the genes constituting this signature implicated alterations in transforming growth factor-beta signaling, the extracellular matrix, amphiregulin expression, and the growth hormone/insulin-like growth factor I axis in pregnancy-induced alterations in breast cancer risk. Notably, related molecular changes have been associated with decreased mammographic density, which itself is strongly associated with decreased breast cancer risk. Our findings show that hormone-induced protection against mammary tumorigenesis is widely conserved among divergent rat strains and define a gene expression signature that is tightly correlated with reduced mammary tumor susceptibility as a consequence of a normal developmental event. Given the conservation of this signature, these pathways may contribute to pregnancy-induced protection against breast cancer.

Celastrol inhibits TGF-β1-induced epithelial–mesenchymal transition by inhibiting Snail and regulating E-cadherin expression

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Kang, Hyereen; Lee, Minjae [Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul 138-736 (Korea, Republic of); Jang, Sung-Wuk, E-mail: swjang@amc.seoul.kr [Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul 138-736 (Korea, Republic of); Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul 138-736 (Korea, Republic of)

2013-08-09

Highlights: •We investigated the effects of celastrol on TGF-β1-induced EMT in epithelial cells. •Celastrol regulates TGF-β1-induced morphological changes and E-cadherin expression. •Celastrol inhibits TGF-β1-induced Snail expression. •Celastrol strongly suppresses TGF-β1-induced invasion in MDCK and A549 cells. -- Abstract: The epithelial–mesenchymal transition (EMT) is a pivotal event in the invasive and metastatic potentials of cancer progression. Celastrol inhibits the proliferation of a variety of tumor cells including leukemia, glioma, prostate, and breast cancer; however, the possible role of celastrol in the EMT is unclear. We investigated the effect of celastrol on the EMT. Transforming growth factor-beta 1 (TGF-β1) induced EMT-like morphologic changes and upregulation of Snail expression. The downregulation of E-cadherin expression and upregulation of Snail in Madin–Darby Canine Kidney (MDCK) and A549 cell lines show that TGF-β1-mediated the EMT in epithelial cells; however, celastrol markedly inhibited TGF-β1-induced morphologic changes, Snail upregulation, and E-cadherin expression. Migration and invasion assays revealed that celastrol completely inhibited TGF-β1-mediated cellular migration in both cell lines. These findings indicate that celastrol downregulates Snail expression, thereby inhibiting TGF-β1-induced EMT in MDCK and A549 cells. Thus, our findings provide new evidence that celastrol suppresses lung cancer invasion and migration by inhibiting TGF-β1-induced EMT.

Lung inflammation induces IL-1β expression in hypoglossal neurons in rat brainstem

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Jafri, Anjum; Belkadi, Abdelmadjid; Zaidi, Syed I. A.; Getsy, Paulina; Wilson, Christopher G.; Martin, Richard J.

2013-01-01

Perinatal inflammation is associated with respiratory morbidity. Immune modulation of brainstem respiratory control centers may provide a link for this pathobiology. We exposed 11-day old rats to intratracheal lipopolysaccharide (LPS, 0.5 µg/g) to test the hypothesis that intrapulmonary inflammation increases expression of the proinflammatory cytokine IL-1β within respiratory-related brainstem regions. Intratracheal LPS resulted in a 32% increase in IL-1β protein expression in the medulla oblongata. In situ hybridization showed increased intensity of IL-1β mRNA but no change in neuronal numbers. Co-localization experiments showed that hypoglossal neurons express IL-1β mRNA and immunostaining showed a 43% increase in IL-1β protein-expressing cells after LPS exposure. LPS treatment also significantly increased microglial cell numbers though they did not express IL-1β mRNA. LPS-induced brainstem expression of neuronal IL-1β mRNA and protein may have implications for our understanding of the vulnerability of neonatal respiratory control in response to a peripheral pro-inflammatory stimulus. PMID:23648475

Strong synergistic induction of CYP1A1 expression by andrographolide plus typical CYP1A inducers in mouse hepatocytes

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Jaruchotikamol, Atika [Department of Toxicology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Jarukamjorn, Kanokwan [Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002 (Thailand); Sirisangtrakul, Wanna [Department of Toxicology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002 (Thailand); Sakuma, Tsutomu; Kawasaki, Yuki [Department of Toxicology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Nemoto, Nobuo [Department of Toxicology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan)

2007-10-15

The effects of andrographolide, the major diterpenoid constituent of Andrographis paniculata, on the expression of cytochrome P450 superfamily 1 members, including CYP1A1, CYP1A2, and CYP1B1, as well as on aryl hydrocarbon receptor (AhR) expression in primary cultures of mouse hepatocytes were investigated in comparison with the effects of typical CYP1A inducers, including benz[a]anthracene, {beta}-naphthoflavone, and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Andrographolide significantly induced the expression of CYP1A1 and CYP1A2 mRNAs in a concentration-dependent manner, as did the typical CYP1A inducers, but did not induce that of CYP1B1 or AhR. Interestingly, andrographolide plus the typical CYP1A inducers synergistically induced CYP1A1 expression, and the synergism was blocked by an AhR antagonist, resveratrol. The CYP1A1 enzyme activity showed a similar pattern of induction. This is the first report that shows that andrographolide has a potency to induce CYP1A1 enzyme and indicates that andrographolide could be a very useful compound for investigating the regulatory mechanism of the CYP1A1 induction pathway. In addition, our findings suggest preparing advice for rational administration of A. paniculata, according to its ability to induce CYP1A1 expression.

Strong synergistic induction of CYP1A1 expression by andrographolide plus typical CYP1A inducers in mouse hepatocytes

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Jaruchotikamol, Atika; Jarukamjorn, Kanokwan; Sirisangtrakul, Wanna; Sakuma, Tsutomu; Kawasaki, Yuki; Nemoto, Nobuo

2007-01-01

The effects of andrographolide, the major diterpenoid constituent of Andrographis paniculata, on the expression of cytochrome P450 superfamily 1 members, including CYP1A1, CYP1A2, and CYP1B1, as well as on aryl hydrocarbon receptor (AhR) expression in primary cultures of mouse hepatocytes were investigated in comparison with the effects of typical CYP1A inducers, including benz[a]anthracene, β-naphthoflavone, and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Andrographolide significantly induced the expression of CYP1A1 and CYP1A2 mRNAs in a concentration-dependent manner, as did the typical CYP1A inducers, but did not induce that of CYP1B1 or AhR. Interestingly, andrographolide plus the typical CYP1A inducers synergistically induced CYP1A1 expression, and the synergism was blocked by an AhR antagonist, resveratrol. The CYP1A1 enzyme activity showed a similar pattern of induction. This is the first report that shows that andrographolide has a potency to induce CYP1A1 enzyme and indicates that andrographolide could be a very useful compound for investigating the regulatory mechanism of the CYP1A1 induction pathway. In addition, our findings suggest preparing advice for rational administration of A. paniculata, according to its ability to induce CYP1A1 expression

BMP-2 Induced Expression of Alx3 That Is a Positive Regulator of Osteoblast Differentiation.

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Takashi Matsumoto

Full Text Available Bone morphogenetic proteins (BMPs regulate many aspects of skeletal development, including osteoblast and chondrocyte differentiation, cartilage and bone formation, and cranial and limb development. Among them, BMP-2, one of the most potent osteogenic signaling molecules, stimulates osteoblast differentiation, while it inhibits myogenic differentiation in C2C12 cells. To evaluate genes involved in BMP-2-induced osteoblast differentiation, we performed cDNA microarray analyses to compare BMP-2-treated and -untreated C2C12 cells. We focused on Alx3 (aristaless-like homeobox 3 which was clearly induced during osteoblast differentiation. Alx3, a homeobox gene related to the Drosophilaaristaless gene, has been linked to developmental functions in craniofacial structures and limb development. However, little is known about its direct relationship with bone formation. In the present study, we focused on the mechanisms of Alx3 gene expression and function during osteoblast differentiation induced by BMP-2. In C2C12 cells, BMP-2 induced increase of Alx3 gene expression in both time- and dose-dependent manners through the BMP receptors-mediated SMAD signaling pathway. In addition, silencing of Alx3 by siRNA inhibited osteoblast differentiation induced by BMP-2, as showed by the expressions of alkaline phosphatase (Alp, Osteocalcin, and Osterix, while over-expression of Alx3 enhanced osteoblast differentiation induced by BMP-2. These results indicate that Alx3 expression is enhanced by BMP-2 via the BMP receptors mediated-Smad signaling and that Alx3 is a positive regulator of osteoblast differentiation induced by BMP-2.

Transgenic over-expression of YY1 induces pathologic cardiac hypertrophy in a sex-specific manner

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Stauffer, Brian L.; Dockstader, Karen; Russell, Gloria; Hijmans, Jamie; Walker, Lisa; Cecil, Mackenzie; Demos-Davies, Kimberly; Medway, Allen; McKinsey, Timothy A.; Sucharov, Carmen C.

2015-01-01

YY1 can activate or repress transcription of various genes. In cardiac myocytes in culture YY1 has been shown to regulate expression of several genes involved in myocyte pathology. YY1 can also acutely protect the heart against detrimental changes in gene expression. In this study we show that cardiac over-expression of YY1 induces pathologic cardiac hypertrophy in male mice, measured by changes in gene expression and lower ejection fraction/fractional shortening. In contrast, female animals are protected against pathologic gene expression changes and cardiac dysfunction. Furthermore, we show that YY1 regulates, in a sex-specific manner, the expression of mammalian enable (Mena), a factor that regulates cytoskeletal actin dynamics and whose expression is increased in several models of cardiac pathology, and that Mena expression in humans with heart failure is sex-dependent. Finally, we show that sex differences in YY1 expression are also observed in human heart failure. In summary, this is the first work to show that YY1 has a sex-specific effect in the regulation of cardiac pathology. PMID:25935483

Acute Vhl gene inactivation induces cardiac HIF-dependent erythropoietin gene expression.

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Marta Miró-Murillo

Full Text Available Von Hippel Lindau (Vhl gene inactivation results in embryonic lethality. The consequences of its inactivation in adult mice, and of the ensuing activation of the hypoxia-inducible factors (HIFs, have been explored mainly in a tissue-specific manner. This mid-gestation lethality can be also circumvented by using a floxed Vhl allele in combination with an ubiquitous tamoxifen-inducible recombinase Cre-ER(T2. Here, we characterize a widespread reduction in Vhl gene expression in Vhl(floxed-UBC-Cre-ER(T2 adult mice after dietary tamoxifen administration, a convenient route of administration that has yet to be fully characterized for global gene inactivation. Vhl gene inactivation rapidly resulted in a marked splenomegaly and skin erythema, accompanied by renal and hepatic induction of the erythropoietin (Epo gene, indicative of the in vivo activation of the oxygen sensing HIF pathway. We show that acute Vhl gene inactivation also induced Epo gene expression in the heart, revealing cardiac tissue to be an extra-renal source of EPO. Indeed, primary cardiomyocytes and HL-1 cardiac cells both induce Epo gene expression when exposed to low O(2 tension in a HIF-dependent manner. Thus, as well as demonstrating the potential of dietary tamoxifen administration for gene inactivation studies in UBC-Cre-ER(T2 mouse lines, this data provides evidence of a cardiac oxygen-sensing VHL/HIF/EPO pathway in adult mice.

Phaleria macrocarpa reduces glomerular growth factor expression in alloxan-induced diabetic rats

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Evy Sulistyoningrum

2013-08-01

Full Text Available Background Diabetic nephropathy (DN is the most serious complication of diabetes, causing end-stage renal disease throughout the world. Recent studies have reported a direct role of vascular endothelial growth factor (VEGF and transforming growth factor-â (TGF-â in DN pathogenesis. VEGF and TGF-â are expressed early in glomeruli in response to hyperglycemia. Active substances of Phaleria macrocarpa (PM pericarp are known to have nephroprotective effects. This study aimed to evaluate the effects of Phaleria macrocarpa (Scheff. Boerl pericarp extract on VEGF and TGF-â expression in alloxan-induced diabetic rats. Methods An experimental study was conducted on twenty five male albino (Sprague Dawley rats divided into five groups (of five each: normal control; diabetic; diabetic + metformin 100 mg/kgBW; diabetic + methanolic PM extract 250 mg/kgBW; and diabetic + aqueous PM extract 250 mg/kgBW. Diabetes was induced by alloxan monohydrate 150 mg/BW intraperitoneally. Treatment was given for 3 weeks. VEGF and TGF-â expression analysis was performed by means of immunohistochemical technique. Differences between groups were assessed by one-way ANOVA. Results VEGF expression in the PM extract group was significantly lower than that in the diabetic group and even metformin group (p<0.01. TGF-â expression in methanolic PM extract group was significantly lower than in diabetic and metformin group (p<0.01, but aqueous PM extract group only showed significancy when compared with diabetic group (p< 0.01. Conclusions Phaleria macrocarpa pericarp extract reduces glomerular expression of TGF-â and VEGF in alloxan-induced diabetic rats.

The murine homeobox gene Msx-3 shows highly restricted expression in the developing neural tube.

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Shimeld, S M; McKay, I J; Sharpe, P T

1996-04-01

The mouse homeobox-genes Msx-1 and Msx-2 are expressed in several areas of the developing embryo, including the neural tube, neural crest, facial processes and limb buds. Here we report the characterisation of a third mouse Msx gene, which we designate Msx-3. The embryonic expression of Msx-3 was found to differ from that of Msx-1 and -2 in that it was confined to the dorsal neural tube. In embryos with 5-8 somites a segmental pattern of expression was observed in the hindbrain, with rhombomeres 3 and 5 lacking Msx-3 while other rhombomeres expressed Msx-3. This pattern was transient, however, such that in embryos with 18 or more somites expression was continuous throughout the dorsal hindbrain and anterior dorsal spinal cord. Differentiation of dorsal cell types in the neural tube can be induced by addition of members of the Tgf-beta family. Additionally, Msx-1 and -2 have been shown to be activated by addition of the Tgf-beta family member Bmp-4. To determine if Bmp-4 could activate Msx-3, we incubated embryonic hindbrain explants with exogenous Bmp-4. The dorsal expression of Msx-3 was seen to expand into more ventral regions of the neurectoderm in Bmp-4-treated cultures, implying that Bmp-4 may be able to mimic an in vivo signal that induces Msx-3.

Hepatocyte growth factor limits autoimmune neuroinflammation via glucocorticoid-induced leucine zipper expression in dendritic cells.

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Benkhoucha, Mahdia; Molnarfi, Nicolas; Dunand-Sauthier, Isabelle; Merkler, Doron; Schneiter, Gregory; Bruscoli, Stefano; Riccardi, Carlo; Tabata, Yasuhiko; Funakoshi, Hiroshi; Nakamura, Toshikazu; Reith, Walter; Santiago-Raber, Marie-Laure; Lalive, Patrice H

2014-09-15

Autoimmune neuroinflammation, including multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), a prototype for T cell-mediated autoimmunity, is believed to result from immune tolerance dysfunction leading to demyelination and substantial neurodegeneration. We previously showed that CNS-restricted expression of hepatocyte growth factor (HGF), a potent neuroprotective factor, reduced CNS inflammation and clinical deficits associated with EAE. In this study, we demonstrate that systemic HGF treatment ameliorates EAE through the development of tolerogenic dendritic cells (DCs) with high expression levels of glucocorticoid-induced leucine zipper (GILZ), a transcriptional repressor of gene expression and a key endogenous regulator of the inflammatory response. RNA interference-directed neutralization of GILZ expression by DCs suppressed the induction of tolerance caused by HGF. Finally, adoptive transfer of HGF-treated DCs from wild-type but not GILZ gene-deficient mice potently mediated functional recovery in recipient mice with established EAE through effective modulation of autoaggressive T cell responses. Altogether, these results show that by inducing GILZ in DCs, HGF reproduces the mechanism of immune regulation induced by potent immunomodulatory factors such as IL-10, TGF-β1, and glucocorticoids and therefore that HGF therapy may have potential in the treatment of autoimmune dysfunctions. Copyright © 2014 by The American Association of Immunologists, Inc.

Effect of ethylene action inhibitors upon wound-induced gene expression in tomato pericarp

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Henstrand, J.M.; Handa, A.K.

1989-01-01

The contribution of wound-ethylene to wound-induced gene expression was investigated in unripe tomato pericarp using inhibitors of ethylene action. Wounded unripe tomato pericarp was treated with 2,5-norbornadiene or silver thiosulfate to inhibit specifically the induction of ethylene-dependent mRNA species. Poly(A) + RNAs isolated from these tissues after 12 hours of wounding were translated in vitro in a rabbit reticulocyte lysate system and [ 35 S]methionine-labeled polypeptides were compared to unwounded controls after separation by one and two-dimensional polyacrylamide gel electrophoresis. Results show that mechanical wounding induces a dramatic shift in gene expression (over 50 mRNA species) but expression of less than 15% of these genes is affected by the treatment with ethylene action inhibitors. A selective decrease in mRNAs coding for a 37 kilodalton doublet and 75 kilodalton polypeptides is observed in 2,5-norbornadiene and silver thiosulfate treated wounded pericarp. Levels of hydroxyproline-rich glycoprotein mRNAs induced in wounded tissue were not influenced by inhibitors of ethylene action

Sialoadhesin expressed on IFN-induced monocytes binds HIV-1 and enhances infectivity.

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Hans Rempel

2008-04-01

Full Text Available HIV-1 infection dysregulates the immune system and alters gene expression in circulating monocytes. Differential gene expression analysis of CD14(+ monocytes from subjects infected with HIV-1 revealed increased expression of sialoadhesin (Sn, CD169, Siglec 1, a cell adhesion molecule first described in a subset of macrophages activated in chronic inflammatory diseases.We analyzed sialoadhesin expression on CD14(+ monocytes by flow cytometry and found significantly higher expression in subjects with elevated viral loads compared to subjects with undetectable viral loads. In cultured CD14(+ monocytes isolated from healthy individuals, sialoadhesin expression was induced by interferon-alpha and interferon-gamma but not tumor necrosis factor-alpha. Using a stringent binding assay, sialoadhesin-expressing monocytes adsorbed HIV-1 through interaction with the sialic acid residues on the viral envelope glycoprotein gp120. Furthermore, monocytes expressing sialoadhesin facilitated HIV-1 trans infection of permissive cells, which occurred in the absence of monocyte self-infection.Increased sialoadhesin expression on CD14(+ monocytes occurred in response to HIV-1 infection with maximum expression associated with high viral load. We show that interferons induce sialoadhesin in primary CD14(+ monocytes, which is consistent with an antiviral response during viremia. Our findings suggest that circulating sialoadhesin-expressing monocytes are capable of binding HIV-1 and effectively delivering virus to target cells thereby enhancing the distribution of HIV-1. Sialoadhesin could disseminate HIV-1 to viral reservoirs during monocyte immunosurveillance or migration to sites of inflammation and then facilitate HIV-1 infection of permissive cells.

Irreversible inhibition of RANK expression as a possible mechanism for IL-3 inhibition of RANKL-induced osteoclastogenesis

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Khapli, Shruti M.; Tomar, Geetanjali B.; Barhanpurkar, Amruta P.; Gupta, Navita; Yogesha, S.D.; Pote, Satish T. [National Center for Cell Science, University of Pune Campus, Pune 411 007 (India); Wani, Mohan R., E-mail: mohanwani@nccs.res.in [National Center for Cell Science, University of Pune Campus, Pune 411 007 (India)

2010-09-03

Research highlights: {yields} IL-3 inhibits receptor activator of NF-{kappa}B ligand (RANKL)-induced osteoclastogenesis. {yields} IL-3 inhibits RANKL-induced JNK activation. {yields} IL-3 down-regulates expression of c-Fos and NFATc1 transcription factors. {yields} IL-3 down-regulates RANK expression posttranscriptionally and irreversibly. {yields} IL-3 inhibits in vivo RANK expression. -- Abstract: IL-3, a cytokine secreted by activated T lymphocytes, stimulates the proliferation, differentiation and survival of pluripotent hematopoietic stem cells. In this study, we investigated the mechanism of inhibitory action of IL-3 on osteoclast differentiation. We show here that IL-3 significantly inhibits receptor activator of NF-{kappa}B (RANK) ligand (RANKL)-induced activation of c-Jun N-terminal kinase (JNK). IL-3 down-regulates expression of c-Fos and nuclear factor of activated T cells (NFATc1) transcription factors. In addition, IL-3 down-regulates RANK expression posttranscriptionally in both purified osteoclast precursors and whole bone marrow cells. Furthermore, the inhibitory effect of IL-3 on RANK expression was irreversible. Interestingly, IL-3 inhibits in vivo RANK expression in mice. Thus, we provide the first evidence that IL-3 irreversibly inhibits RANK expression that results in inhibition of important signaling molecules induced by RANKL.

Irreversible inhibition of RANK expression as a possible mechanism for IL-3 inhibition of RANKL-induced osteoclastogenesis

International Nuclear Information System (INIS)

Khapli, Shruti M.; Tomar, Geetanjali B.; Barhanpurkar, Amruta P.; Gupta, Navita; Yogesha, S.D.; Pote, Satish T.; Wani, Mohan R.

2010-01-01

Research highlights: → IL-3 inhibits receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis. → IL-3 inhibits RANKL-induced JNK activation. → IL-3 down-regulates expression of c-Fos and NFATc1 transcription factors. → IL-3 down-regulates RANK expression posttranscriptionally and irreversibly. → IL-3 inhibits in vivo RANK expression. -- Abstract: IL-3, a cytokine secreted by activated T lymphocytes, stimulates the proliferation, differentiation and survival of pluripotent hematopoietic stem cells. In this study, we investigated the mechanism of inhibitory action of IL-3 on osteoclast differentiation. We show here that IL-3 significantly inhibits receptor activator of NF-κB (RANK) ligand (RANKL)-induced activation of c-Jun N-terminal kinase (JNK). IL-3 down-regulates expression of c-Fos and nuclear factor of activated T cells (NFATc1) transcription factors. In addition, IL-3 down-regulates RANK expression posttranscriptionally in both purified osteoclast precursors and whole bone marrow cells. Furthermore, the inhibitory effect of IL-3 on RANK expression was irreversible. Interestingly, IL-3 inhibits in vivo RANK expression in mice. Thus, we provide the first evidence that IL-3 irreversibly inhibits RANK expression that results in inhibition of important signaling molecules induced by RANKL.

Expression of intracisternal A-particles in radiation-induced murine osteosarcomas

International Nuclear Information System (INIS)

Seidel, E.

1989-01-01

The expression of intracisternal A-type particles (IAPs) and the distribution of the corresponding type I and type II genes was investigated in radiation induced osteosarcomas of Balb/c, NMRI and CBA mice. The results can be summarized as follows: 1. Slot blot analysis showed that 9 out of 23 (i.e. 39%) of the examined tumours had enhanced IAP expression as compared to normal tissues. None of the CBA tumours were enhanced in IAP expression - only tumours of the strains Balb/c and NMRI expressed higher levels of IAP RNA. 2. Northern blot analysis of RNA from osteosarcomas revealed that the two IAP transcrips (7.2 kb and 5.4 kb) had the same mobility in these tissues as in normal tissue. Some tumours showed a higher level of expression of the 7.2 kb transcript as compared to normal tissues. This 7.2 kb transcript encodes the major structural protein p 73 of IAPs. 3. Hybridization of Southern blots of mouse DNA using the IAP type I specific probe revealed a unique fragment in three of the investigated Balb/c osteosarcomas. 4. Although it could not directly be shown that IAPs were involved in the genesis of osteosarcomas, the changes in IAP expression and the unique fragments suggest a possible correlation with tumour progression. In this light the level of IAP expression may serve as a marker for the degree of the malignancy of the tumour. (orig.) [de

Expression of HSF2 decreases in mitosis to enable stress-inducible transcription and cell survival

Science.gov (United States)

Elsing, Alexandra N.; Aspelin, Camilla; Björk, Johanna K.; Bergman, Heidi A.; Himanen, Samu V.; Kallio, Marko J.; Roos-Mattjus, Pia

2014-01-01

Unless mitigated, external and physiological stresses are detrimental for cells, especially in mitosis, resulting in chromosomal missegregation, aneuploidy, or apoptosis. Heat shock proteins (Hsps) maintain protein homeostasis and promote cell survival. Hsps are transcriptionally regulated by heat shock factors (HSFs). Of these, HSF1 is the master regulator and HSF2 modulates Hsp expression by interacting with HSF1. Due to global inhibition of transcription in mitosis, including HSF1-mediated expression of Hsps, mitotic cells are highly vulnerable to stress. Here, we show that cells can counteract transcriptional silencing and protect themselves against proteotoxicity in mitosis. We found that the condensed chromatin of HSF2-deficient cells is accessible for HSF1 and RNA polymerase II, allowing stress-inducible Hsp expression. Consequently, HSF2-deficient cells exposed to acute stress display diminished mitotic errors and have a survival advantage. We also show that HSF2 expression declines during mitosis in several but not all human cell lines, which corresponds to the Hsp70 induction and protection against stress-induced mitotic abnormalities and apoptosis. PMID:25202032

Obesity-induced endoplasmic reticulum stress suppresses nuclear factor-Y expression.

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Liu, Yulan; Zhang, Yuwei; Zhang, Yanjie; Zhang, Jinlong; Liu, Yin; Feng, Peiqun; Su, Zhiguang

2017-02-01

Nuclear transcription factor Y (NF-Y) is an evolutionarily conserved transcription factor composed of three subunits, NF-YA, NF-YB, and NF-YC. NF-Y plays crucial roles in pre-adipocyte maintenance and/or commitment to adipogenesis. NF-YA dysfunction in adipocyte resulted in an age-dependent progressive loss of adipose tissue associated with metabolic complications. Endoplasmic reticulum (ER) stress has emerged as an important mediator in the pathogenesis of obesity. However, it is not known if NF-YA is involved in the ER stress-mediated pathogenesis of obesity. We first examined the effects of ER stress on the NF-YA expression in cultured 3T3-L1 adipocytes; then in ob/ob genetic obesity mice, we tested the effect of chemical chaperones alleviating ER stress on the expression levels of NF-YA. Subsequently, we inhibited the new mRNA synthesis using actinomycin D in 3T3-L1 cells to explore the mechanism modulating NF-YA expression. Finally, we evaluated the involvement of PPARg in the regulation of NF-YA expression by ER stress. We demonstrated that both obesity- and chemical chaperone -induced ER stress suppressed NF-YA expression and alleviation of ER stress by chemical chaperone could recover NF-YA expression in ob/ob mice. Moreover, we showed that ER stress suppressed NF-YA mRNA transcription through the involvement of peroxisome proliferator-activated receptor gamma (PPARg). Activation of PPARg ameliorates the ER stress-induced NF-YA suppression. Our findings may point to a possible role of NF-YA in stress conditions that occur in chronic obesity, ER stress might be involved in the pathogenesis of obesity through NF-YA depletion.

Thymoquinone inhibits phorbol ester-induced activation of NF-κB and expression of COX-2, and induces expression of cytoprotective enzymes in mouse skin in vivo

International Nuclear Information System (INIS)

Kundu, Joydeb Kumar; Liu, Lijia; Shin, Jun-Wan; Surh, Young-Joon

2013-01-01

Highlights: •Thymoquinone inhibits phorbol ester-induced COX-2 expression in mouse skin. •Thymoquinone attenuates phosphorylation of IκBα and DNA binding of NF-κB in mouse skin. •Thymoquinone inhibits phosphorylation of p38 MAP kinase, JNK and Akt in mouse skin. •Thymoquinone induces the expression of cytoprotective proteins in mouse skin. -- Abstract: Thymoquinone (TQ), the active ingredient of Nigella sativa, has been reported to possess anti-inflammatory and chemopreventive properties. The present study was aimed at elucidating the molecular mechanisms of anti-inflammatory and antioxidative activities of thymoquinone in mouse skin. Pretreatment of female HR-1 hairless mouse skin with TQ attenuated 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced expression of cyclooxygenase-2 (COX-2). TQ diminished nuclear translocation and the DNA binding of nuclear factor-kappaB (NF-κB) via the blockade of phosphorylation and subsequent degradation of IκBα in TPA-treated mouse skin. Pretreatment with TQ attenuated the phosphorylation of Akt, c-Jun-N-terminal kinase and p38 mitogen-activated protein kinase, but not that of extracellular signal-regulated kinase-1/2. Moreover, topical application of TQ induced the expression of heme oxygenase-1, NAD(P)H-quinoneoxidoreductase-1, glutathione-S-transferase and glutamate cysteine ligase in mouse skin. Taken together, the inhibitory effects of TQ on TPA-induced COX-2 expression and NF-κB activation, and its ability to induce the expression of cytoprotective proteins provide a mechanistic basis of anti-inflammatory and antioxidative effects of TQ in hairless mouse skin

Ligand Binding Domain Protein in Tetracycline-Inducible Expression

African Journals Online (AJOL)

Purpose: To investigate tetracycline-inducible expression system for producing clinically usable, highquality liver X receptor ligand-binding domain recombinant protein. Methods: In this study, we have expressed and purified the recombinant liver X receptor β-ligand binding domain proteins in E. coli using a tetracycline ...

Data showing non-conventional HLA-B27 expression in axial joints and gut tissue from B27 transgenic rats, and in frozen and paraffin-fixed synovial SpA tissue

NARCIS (Netherlands)

Rysnik, Oliwia; McHugh, Kirsty; van Duivenvoorde, Leonie; van Tok, Melissa; Taurog, Joel; Kollnberger, Simon; Baeten, Dominique; Bowness, Paul

2016-01-01

Data is presented showing expression of non-conventional (NC) heavy chain forms of B27 in synovial tissues from SpA patients. Data is presented showing the expression patterns of NC-B27 in joint, gastrointestinal and lymphoid tissues from B27 transgenic (TG(1)) rats with M. tuberculosis-induced SpA.

Heme-Oxygenase-1 Expression Contributes to the Immunoregulation Induced by Fasciola hepatica and Promotes Infection

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Paula Carasi

2017-07-01

Full Text Available Fasciola hepatica, also known as the liver fluke, is a trematode that infects livestock and humans causing fasciolosis, a zoonotic disease of increasing importance due to its worldwide distribution and high economic losses. This parasite immunoregulates the host immune system by inducing a strong Th2 and regulatory T immune response by immunomodulating dendritic cell (DC maturation and alternative activation of macrophages. In this paper, we show that F. hepatica infection in mice induces the upregulation of heme-oxygenase-1 (HO-1, the rate-limiting enzyme in the catabolism of free heme that regulates the host inflammatory response. We show and characterize two different populations of antigen presenting cells that express HO-1 during infection in the peritoneum of infected animals. Cells that expressed high levels of HO-1 expressed intermediate levels of F4/80 but high expression of CD11c, CD38, TGFβ, and IL-10 suggesting that they correspond to regulatory DCs. On the other hand, cells expressing intermediate levels of HO-1 expressed high levels of F4/80, CD68, Ly6C, and FIZZ-1, indicating that they might correspond to alternatively activated macrophages. Furthermore, the pharmacological induction of HO-1 with the synthetic metalloporphyrin CoPP promoted F. hepatica infection increasing the clinical signs associated with the disease. In contrast, treatment with the HO-1 inhibitor SnPP protected mice from parasite infection, indicating that HO-1 plays an essential role during F. hepatica infection. Finally, HO-1 expression during F. hepatica infection was associated with TGFβ and IL-10 levels in liver and peritoneum, suggesting that HO-1 controls the expression of these immunoregulatory cytokines during infection favoring parasite survival in the host. These results contribute to the elucidation of the immunoregulatory mechanisms induced by F. hepatica in the host and provide alternative checkpoints to control fasciolosis.

Function and regulation of ATF 3 expression induced by ionizing radiation

International Nuclear Information System (INIS)

Fan, Feiyue; Wang, Yong; Du, Liqin; Zhan, Qimin

2008-01-01

Full text: Ionizing radiation results in a series of damages of mammalian cells as a genotoxic stress. There are some genes expressed after cells damaged, in which ATF 3, a member of ATF/CREB family of transcription factors, is one of them. In this report, we demonstrate that ATF 3 can be induced by ionizing radiation. The induction of ATF 3 protein requires normal status of p53 function in cells. There are some quantitative relationships between ATF 3 induction and dosages of radiation or time post-irradiation. In another word, ATF 3 expression induced by ionizing radiation present dose- and time-dependent. The regulation of ATF 3 expression refers to program of promoter and transcription. Radiation induces ATF 3 expression by activating the promoter and RNA transcription. In method of tetracycline-inducible system (tet-off), we have found that over-expression of ATF 3 protein brings caspase/PARP proteins into cleavage, which induces cell programmed death, and suppresses cell growth. Meanwhile, it was found that ATF 3 expression could slow down progression of cell from G 1 to S phase. It indicates ATF 3 protein might play a negative role in the control of cell cycle progression. It is very excited that expression of ATF 3 protein did not only suppress cell growth, but also demonstrated protecting effect of cell growth suppression resulting from ionizing radiation. It is suggested that ATF 3 protein might take part in the damage repair process of cells. (author)

Ketamine induces brain-derived neurotrophic factor expression via phosphorylation of histone deacetylase 5 in rats.

Science.gov (United States)

Choi, Miyeon; Lee, Seung Hoon; Park, Min Hyeop; Kim, Yong-Seok; Son, Hyeon

2017-08-05

Ketamine shows promise as a therapeutic agent for the treatment of depression. The increased expression of brain-derived neurotrophic factor (BDNF) has been associated with the antidepressant-like effects of ketamine, but the mechanism of BDNF induction is not well understood. In the current study, we demonstrate that the treatment of rats with ketamine results in the dose-dependent rapid upregulation of Bdnf promoter IV activity and expression of Bdnf exon IV mRNAs in rat hippocampal neurons. Transfection of histone deacetylase 5 (HDAC5) into rat hippocampal neurons similarly induces Bdnf mRNA expression in response to ketamine, whereas transfection of a HDAC5 phosphorylation-defective mutant (Ser259 and Ser498 replaced by Ala259 and Ala498), results in the suppression of ketamine-mediated BDNF promoter IV transcriptional activity. Viral-mediated hippocampal knockdown of HDAC5 induces Bdnf mRNA and protein expression, and blocks the enhancing effects of ketamine on BDNF expression in both unstressed and stressed rats, and thereby providing evidence for the role of HDAC5 in the regulation of Bdnf expression. Taken together, our findings implicate HDAC5 in the ketamine-induced transcriptional regulation of Bdnf, and suggest that the phosphorylation of HDAC5 regulates the therapeutic actions of ketamine. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression of p53, MDM2 in a mice hydradecarcinoma model induced by γ-ray irradiation

International Nuclear Information System (INIS)

Huang Yuecheng; Cai Jianming; Han Ling; Gao Fu; Sun Ding; Dong Zhitao; Zhe Wanli

2004-01-01

Objective: To investigate the role of the p53, MDM2 in carcinogenesis of mice hydradecarcinoma induced by γ-rays. Methods: A radiation-induced mice hydradecarcinoma model was established by γ-ray irradiation. Expression of MDM2 protein in hydradecarcinoma tissue, paracancerous tissue and normal control tissue was detected with Western blot. Immunoprecipitation (IP) was conducted to examine the phosphorylation level of MDM2 protein. PCR-SSCP was performed to detect p53 gene mutation. Results: Compared with the normal control tissue, the MDM2 protein expression and its phosphorylation level were significantly higher in hydradecarcinoma tissue. SSCP showed there were p53 gene mutations in hydradecarcinoma samples. Conclusion: p53/MDM2 pathway may be involved in the development and progression of hydradecarcinoma induced by γ-ray irradiation. The over-expression of MDM2 and hyperphosphorylation may be responsible for malignant transformation induced by irradiation by a possible mechanism of p53 inactivation. The gene mutation of p53 further supported the hypothesis that p53/MDM2 pathway played a central role in carcinogenesis of γray induced hydradecarcinoma. (authors)

Light-dependent expression of flg22-induced defense genes in Arabidopsis

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Satoshi eSano

2014-10-01

Full Text Available Chloroplasts have been reported to generate retrograde immune signals that activate defense gene expression in the nucleus. However, the roles of light and photosynthesis in plant immunity remain largely elusive. In this study, we evaluated the effects of light on the expression of defense genes induced by flg22, a peptide derived from bacterial flagellins which acts as a potent elicitor in plants. Whole-transcriptome analysis of flg22-treated Arabidopsis thaliana seedlings under light and dark conditions for 30 min revealed that a number of (30% genes strongly induced by flg22 (>4.0 require light for their rapid expression, whereas flg22-repressed genes include a significant number of genes that are down-regulated by light. Furthermore, light is responsible for the flg22-induced accumulation of salicylic acid, indicating that light is indispensable for basal defense responses in plants. To elucidate the role of photosynthesis in defense, we further examined flg22-induced defense gene expression in the presence of specific inhibitors of photosynthetic electron transport: 3-(3,4-dichlorophenyl-1,1-dimethylurea (DCMU and 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB. Light-dependent expression of defense genes was largely suppressed by DBMIB, but only partially suppressed by DCMU. These findings suggest that photosynthetic electron flow plays a role in controling the light-dependent expression of flg22-inducible defense genes.

Hedgehog signaling pathway is active in GBM with GLI1 mRNA expression showing a single continuous distribution rather than discrete high/low clusters.

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Chandra, Vikas; Das, Tapojyoti; Gulati, Puneet; Biswas, Nidhan K; Rote, Sarang; Chatterjee, Uttara; Ghosh, Samarendra N; Deb, Sumit; Saha, Suniti K; Chowdhury, Anup K; Ghosh, Subhashish; Rudin, Charles M; Mukherjee, Ankur; Basu, Analabha; Dhara, Surajit

2015-01-01

Hedgehog (Hh) signaling pathway is a valid therapeutic target in a wide range of malignancies. We focus here on glioblastoma multiforme (GBM), a lethal malignancy of the central nervous system (CNS). By analyzing RNA-sequencing based transcriptomics data on 149 clinical cases of TCGA-GBM database we show here a strong correlation (r = 0.7) between GLI1 and PTCH1 mRNA expression--as a hallmark of the canonical Hh-pathway activity in this malignancy. GLI1 mRNA expression varied in 3 orders of magnitude among the GBM patients of the same cohort showing a single continuous distribution-unlike the discrete high/low-GLI1 mRNA expressing clusters of medulloblastoma (MB). When compared with MB as a reference, the median GLI1 mRNA expression in GBM appeared 14.8 fold lower than that of the "high-Hh" cluster of MB but 5.6 fold higher than that of the "low-Hh" cluster of MB. Next, we demonstrated statistically significant up- and down-regulation of GLI1 mRNA expressions in GBM patient-derived low-passage neurospheres in vitro by sonic hedgehog ligand-enriched conditioned media (shh-CM) and by Hh-inhibitor drug vismodegib respectively. We also showed clinically achievable dose (50 μM) of vismodegib alone to be sufficient to induce apoptosis and cell cycle arrest in these low-passage GBM neurospheres in vitro. Vismodegib showed an effect on the neurospheres, both by down-regulating GLI1 mRNA expression and by inducing apoptosis/cell cycle arrest, irrespective of their relative endogenous levels of GLI1 mRNA expression. We conclude from our study that this single continuous distribution pattern of GLI1 mRNA expression technically puts almost all GBM patients in a single group rather than discrete high- or low-clusters in terms of Hh-pathway activity. That is suggestive of therapies with Hh-pathway inhibitor drugs in this malignancy without a need for further stratification of patients on the basis of relative levels of Hh-pathway activity among them.

(−-Epigallocatechin gallate inhibits endotoxin-induced expression of inflammatory cytokines in human cerebral microvascular endothelial cells

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Li Jieliang

2012-07-01

Full Text Available Abstract Background (−-Epigallocatechin gallate (EGCG is a major polyphenol component of green tea that has antioxidant activities. Lipopolysaccharide (LPS induces inflammatory cytokine production and impairs blood–brain barrier (BBB integrity. We examined the effect of EGCG on LPS-induced expression of the inflammatory cytokines in human cerebral microvascular endothelial cells (hCMECs and BBB permeability. Methods The expression of TNF-α, IL-1β and monocyte chemotactic protein-1 (MCP-1/CCL2 was determined by quantitative real time PCR (qRT-PCR and ELISA. Intercellular adhesion molecule 1 (ICAM-1 and vascular cell adhesion molecule (VCAM in hCMECs were examined by qRT-PCR and Western blotting. Monocytes that adhered to LPS-stimulated endothelial cells were measured by monocyte adhesion assay. Tight junctional factors were detected by qRT-PCR (Claudin 5 and Occludin and immunofluorescence staining (Claudin 5 and ZO-1. The permeability of the hCMEC monolayer was determined by fluorescence spectrophotometry of transmembrane fluorescin and transendothelial electrical resistance (TEER. NF-kB activation was measured by luciferase assay. Results EGCG significantly suppressed the LPS-induced expression of IL-1β and TNF-α in hCMECs. EGCG also inhibited the expression of MCP-1/CCL2, VCAM-1 and ICAM-1. Functional analysis showed that EGCG induced the expression of tight junction proteins (Occludin and Claudin-5 in hCMECs. Investigation of the mechanism showed that EGCG had the ability to inhibit LPS-mediated NF-κB activation. In addition, 67-kD laminin receptor was involved in the anti-inflammatory effect of EGCG. Conclusions Our results demonstrated that LPS induced inflammatory cytokine production in hCMECs, which could be attenuated by EGCG. These data indicate that EGCG has a therapeutic potential for endotoxin-mediated endothelial inflammation.

IP3-dependent intracellular Ca2+ release is required for cAMP-induced c-fos expression in hippocampal neurons

International Nuclear Information System (INIS)

Zhang, Wenting; Tingare, Asmita; Ng, David Chi-Heng; Johnson, Hong W.; Schell, Michael J.; Lord, Rebecca L.; Chawla, Sangeeta

2012-01-01

Highlights: ► cAMP-induced c-fos expression in hippocampal neurons requires a submembraneous Ca 2+ pool. ► The submembraneous Ca 2+ pool derives from intracellular ER stores. ► Expression of IP 3 -metabolizing enzymes inhibits cAMP-induced c-fos expression. ► SRE-mediated and CRE-mediated gene expression is sensitive to IP 3 -metabolizing enzymes. ► Intracellular Ca 2+ release is required for cAMP-induced nuclear translocation of TORC1. -- Abstract: Ca 2+ and cAMP are widely used in concert by neurons to relay signals from the synapse to the nucleus, where synaptic activity modulates gene expression required for synaptic plasticity. Neurons utilize different transcriptional regulators to integrate information encoded in the spatiotemporal dynamics and magnitude of Ca 2+ and cAMP signals, including some that are Ca 2+ -responsive, some that are cAMP-responsive and some that detect coincident Ca 2+ and cAMP signals. Because Ca 2+ and cAMP can influence each other’s amplitude and spatiotemporal characteristics, we investigated how cAMP acts to regulate gene expression when increases in intracellular Ca 2+ are buffered. We show here that cAMP-mobilizing stimuli are unable to induce expression of the immediate early gene c-fos in hippocampal neurons in the presence of the intracellular Ca 2+ buffer BAPTA-AM. Expression of enzymes that attenuate intracellular IP 3 levels also inhibited cAMP-dependent c-fos induction. Synaptic activity induces c-fos transcription through two cis regulatory DNA elements – the CRE and the SRE. We show here that in response to cAMP both CRE-mediated and SRE-mediated induction of a luciferase reporter gene is attenuated by IP 3 metabolizing enzymes. Furthermore, cAMP-induced nuclear translocation of the CREB coactivator TORC1 was inhibited by depletion of intracellular Ca 2+ stores. Our data indicate that Ca 2+ release from IP 3 -sensitive pools is required for cAMP-induced transcription in hippocampal neurons.

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

Energy Technology Data Exchange (ETDEWEB)

Zhong, Xia, E-mail: zhongxia1977@126.com [Department of Emergency, Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Li, Xiaonan; Liu, Fuli; Tan, Hui [Department of Emergency, Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Shang, Deya, E-mail: wenhuashenghuo1@163.com [Department of Emergency, Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China)

2012-08-24

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

Radiation and desiccation response motif mediates radiation induced gene expression in D. radiodurans

International Nuclear Information System (INIS)

Anaganti, Narasimha; Basu, Bhakti; Apte, Shree Kumar

2015-01-01

Deinococcus radiodurans is an extremophile that withstands lethal doses of several DNA damaging agents such as gamma irradiation, UV rays, desiccation and chemical mutagens. The organism responds to DNA damage by inducing expression of several DNA repair genes. At least 25 radiation inducible gene promoters harbour a 17 bp palindromic sequence known as radiation and desiccation response motif (RDRM) implicated in gamma radiation inducible gene expression. However, mechanistic details of gamma radiation-responsive up-regulation in gene expression remain enigmatic. The promoters of highly radiation induced genes ddrB (DR0070), gyrB (DR0906), gyrA (DR1913), a hypothetical gene (DR1143) and recA (DR2338) from D. radiodurans were cloned in a green fluorescence protein (GFP)-based promoter probe shuttle vector pKG and their promoter activity was assessed in both E. coli as well as in D. radiodurans. The gyrA, gyrB and DR1143 gene promoters were active in E. coli although ddrB and recA promoters showed very weak activity. In D. radiodurans, all the five promoters were induced several fold following 6 kGy gamma irradiation. Highest induction was observed for ddrB promoter (25 fold), followed by DR1143 promoter (15 fold). The induction in the activity of gyrB, gyrA and recA promoters was 5, 3 and 2 fold, respectively. To assess the role of RDRM, the 17 bp palindromic sequence was deleted from these promoters. The promoters devoid of RDRM sequence displayed increase in the basal expression activity, but the radiation-responsive induction in promoter activity was completely lost. The substitution of two conserved bases of RDRM sequence yielded decreased radiation induction of PDR0070 promoter. Deletion of 5 bases from 5'-end of PDR0070 RDRM increased basal promoter activity, but radiation induction was completely abolished. Replacement of RDRM with non specific sequence of PDR0070 resulted in loss of basal expression and radiation induction. The results demonstrate that

Kaempferol attenuates COX-2 expression in IL-6-induced macrophages and carrageenan-induced mouse paw edema by targeting STAT3 and NF-kB

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Anandita Basu

2017-10-01

Full Text Available Dietary polyphenols are reported to possess varied pharmacological activities, viz. antioxidant, anti-inflammatory, anti-cancer, anti-allergic actions. Here, we report the efficacy of Kaempferol (kae to attenuate expression of IL-6 induced cycloxygenase-2 (COX-2, an inducible isoform of cycloxygenase enzyme family that catalyzes synthesis of inflammatory mediators, prostanoids and prostaglandins. IL-6 is a pleiotropic cytokine involved in both acute and chronic inflammation. Our results showed that kae attenuated COX-2 expression at both mRNA and protein level in IL-6-induced THP1 macrophages. This attenuation of COX-2 expression by kae involved dose-dependent inhibition of phosphorylation of STAT3 (Tyr 705 and NF-kB p65 (Ser 536 leading to their deactivation and reduced nuclear localization in THP-1 macrophages. Moreover, kae modulates COX-2 expression as well as STAT3 and NF-kB activation in carrageenan-induced mouse paw edema model. RT-PCR and western blot analysis from paw tissues were harvested after kae injection (i.p followed by carrageenan-treatment in sub-plantar region of right hind paw. Results showed that kae attenuated COX-2 expression and STAT3 and NF-kB activation in carrageenan-induced mouse paw edema, suggesting that inhibition of both IL-6-STAT3-COX-2 and IL-6-NFkB-COX-2 axes by kae might be stimulus-independent. To understand binding affinity of kae with NF-kB and STAT3, docking analysis was performed using Patchdock server. From our findings, we observed strong binding affinity and transient interaction in both NF-kB/kae and STAT3/kae complexes. We noticed negative atomic contact energy and greater interface area for both the complexes. Selected complexes obtained from Patchdock were refined using Firedock online server which also suggested similar negative binding energy profile. It is plausible that kae attenuates COX-2 expression by directly binding to both STAT3 and NF-kB proteins and inhibiting their activation and

Anti-inflammatory effect of resveratrol on TNF-α-induced MCP-1 expression in adipocytes

International Nuclear Information System (INIS)

Zhu Jian; Yong Wei; Wu Xiaohong; Yu Ying; Lv Jinghuan; Liu Cuiping; Mao Xiaodong; Zhu Yunxia; Xu Kuanfeng; Han Xiao; Liu Chao

2008-01-01

Chronic low-grade inflammation characterized by adipose tissue macrophage accumulation and abnormal cytokine production is a key feature of obesity and type 2 diabetes. Adipose-tissue-derived monocyte chemoattractant protein (MCP)-1, induced by cytokines, has been shown to play an essential role in the early events during macrophage infiltration into adipose tissue. In this study we investigated the effects of resveratrol upon both tumor necrosis factor (TNF)-α-induced MCP-1 gene expression and its underlying signaling pathways in 3T3-L1 adipoctyes. Resveratrol was found to inhibit TNF-α-induced MCP-1 secretion and gene transcription, as well as promoter activity, which based on down-regulation of TNF-α-induced MCP-1 transcription. Nuclear factor (NF)-κB was determined to play a major role in the TNF-α-induced MCP-1 expression. Further analysis showed that resveratrol inhibited DNA binding activity of the NF-κB complex and subsequently suppressed NF-κB transcriptional activity in TNF-α-stimulated cells. Finally, the inhibition of MCP-1 may represent a novel mechanism of resveratrol in preventing obesity-related pathologies

RpoS induces expression of the Vibrio anguillarum quorum-sensing regulator VanT.

Science.gov (United States)

Weber, Barbara; Croxatto, Antony; Chen, Chang; Milton, Debra L

2008-03-01

In vibrios, regulation of the Vibrio harveyi-like LuxR transcriptional activators occurs post-transcriptionally via small regulatory RNAs (sRNAs) that destabilize the luxR mRNA at a low cell population, eliminating expression of LuxR. Expression of the sRNAs is modulated by the vibrio quorum-sensing phosphorelay systems. However, vanT mRNA, which encodes a LuxR homologue in Vibrio anguillarum, is abundant at low and high cell density, indicating that VanT expression may be regulated via additional mechanisms. In this study, Western analyses showed that VanT was expressed throughout growth with a peak of expression during late exponential growth. VanO induced partial destabilization of vanT mRNA via activation of at least one Qrr sRNA. Interestingly, the sigma factor RpoS significantly stabilized vanT mRNA and induced VanT expression during late exponential growth. This induction was in part due to RpoS repressing expression of Hfq, an RNA chaperone. RpoS is not part of the quorum-sensing regulatory cascade since RpoS did not regulate expression or activity of VanO, and RpoS was not regulated by VanO or VanT. VanT and RpoS were needed for survival following UV irradiation and for pigment and metalloprotease production, suggesting that RpoS works with the quorum-sensing systems to modulate expression of VanT, which regulates survival and stress responses.

Neuroinflammation induces glial aromatase expression in the uninjured songbird brain

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Saldanha Colin J

2011-07-01

Full Text Available Abstract Background Estrogens from peripheral sources as well as central aromatization are neuroprotective in the vertebrate brain. Under normal conditions, aromatase is only expressed in neurons, however following anoxic/ischemic or mechanical brain injury; aromatase is also found in astroglia. This increased glial aromatization and the consequent estrogen synthesis is neuroprotective and may promote neuronal survival and repair. While the effects of estradiol on neuroprotection are well studied, what induces glial aromatase expression remains unknown. Methods Adult male zebra finches (Taeniopygia guttata were given a penetrating injury to the entopallium. At several timepoints later, expression of aromatase, IL-1β-like, and IL-6-like were examined using immunohisotchemistry. A second set of zebra birds were exposed to phytohemagglutinin (PHA, an inflammatory agent, directly on the dorsal surface of the telencephalon without creating a penetrating injury. Expression of aromatase, IL-1β-like, and IL-6-like were examined using both quantitative real-time polymerase chain reaction to examine mRNA expression and immunohistochemistry to determine cellular expression. Statistical significance was determined using t-test or one-way analysis of variance followed by the Tukey Kramers post hoc test. Results Following injury in the zebra finch brain, cytokine expression occurs prior to aromatase expression. This temporal pattern suggests that cytokines may induce aromatase expression in the damaged zebra finch brain. Furthermore, evoking a neuroinflammatory response characterized by an increase in cytokine expression in the uninjured brain is sufficient to induce glial aromatase expression. Conclusions These studies are among the first to examine a neuroinflammatory response in the songbird brain following mechanical brain injury and to describe a novel neuroimmune signal to initiate aromatase expression in glia.

Neighboring Genes Show Correlated Evolution in Gene Expression

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Ghanbarian, Avazeh T.; Hurst, Laurence D.

2015-01-01

When considering the evolution of a gene’s expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking. PMID:25743543

Coffee induces breast cancer resistance protein expression in Caco-2 cells.

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Isshiki, Marina; Umezawa, Kazuo; Tamura, Hiroomi

2011-01-01

Coffee is a beverage that is consumed world-wide on a daily basis and is known to induce a series of metabolic and pharmacological effects, especially in the digestive tract. However, little is known concerning the effects of coffee on transporters in the gastrointestinal tract. To elucidate the effect of coffee on intestinal transporters, we investigated its effect on expression of the breast cancer resistance protein (BCRP/ABCG2) in a human colorectal cancer cell line, Caco-2. Coffee induced BCRP gene expression in Caco-2 cells in a coffee-dose dependent manner. Coffee treatment of Caco-2 cells also increased the level of BCRP protein, which corresponded to induction of gene expression, and also increased cellular efflux activity, as judged by Hoechst33342 accumulation. None of the major constituents of coffee tested could induce BCRP gene expression. The constituent of coffee that mediated this induction was extractable with ethyl acetate and was produced during the roasting process. Dehydromethylepoxyquinomicin (DHMEQ), an inhibitor of nuclear factor (NF)-κB, inhibited coffee-mediated induction of BCRP gene expression, suggesting involvement of NF-κB in this induction. Our data suggest that daily consumption of coffee might induce BCRP expression in the gastrointestinal tract and may affect the bioavailability of BCRP substrates.

Selective Inducible Nitric Oxide Synthase Inhibitor Reversed Zinc Chloride-Induced Spatial Memory Impairment via Increasing Cholinergic Marker Expression.

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Tabrizian, Kaveh; Azami, Kian; Belaran, Maryam; Soodi, Maliheh; Abdi, Khosrou; Fanoudi, Sahar; Sanati, Mehdi; Mottaghi Dastjerdi, Negar; Soltany Rezaee-Rad, Mohammad; Sharifzadeh, Mohammad

2016-10-01

Zinc, an essential micronutrient and biochemical element of the human body, plays structural, catalytic, and regulatory roles in numerous physiological functions. In the current study, the effects of a pretraining oral administration of zinc chloride (10, 25, and 50 mg/kg) for 14 consecutive days and post-training bilateral intra-hippocampal infusion of 1400W as a selective inducible nitric oxide synthase (iNOS) inhibitor (10, 50, and 100 μM/side), alone and in combination, on the spatial memory retention in Morris water maze (MWM) were investigated. Animals were trained for 4 days and tested 48 h after completion of training. Also, the molecular effects of these compounds on the expression of choline acetyltransferase (ChAT), as a cholinergic marker in the CA1 region of the hippocampus and medial septal area (MSA), were evaluated. Behavioral and molecular findings of this study showed that a 2-week oral administration of zinc chloride (50 mg/kg) impaired spatial memory retention in MWM and decreased ChAT expression. Immunohistochemical analysis of post-training bilateral intra-hippocampal infusion of 1400W revealed a significant increase in ChAT immunoreactivity. Furthermore, post-training bilateral intra-hippocampal infusion of 1400W into the CA1 region of the hippocampus reversed zinc chloride-induced spatial memory impairment in MWM and significantly increased ChAT expression in comparison with zinc chloride-treated animals. Taken together, these results emphasize the role of selective iNOS inhibitors in reversing zinc chloride-induced spatial memory deficits via modulation of cholinergic marker expression.

Inducible expression of trehalose synthase in Bacillus licheniformis.

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Li, Youran; Gu, Zhenghua; Zhang, Liang; Ding, Zhongyang; Shi, Guiyang

2017-02-01

Trehalose synthase (TreS) could transform maltose into trehalose via isomerization. It is a crucial enzyme in the process of trehalose enzymatical transformation. In this study, plasmid-based inducible expression systems were constructed to produce Thermomonospora curvata TreS in B. licheniformis. Xylose operons from B. subtilis, B. licheniformis and B. megaterium were introduced to regulate the expression of the gene encoding TreS. It was functionally expressed, and the BlsTs construct yielded the highest enzyme activity (12.1 U/mL). Furthermore, the effect of different cultural conditions on the inducible expression of BlsTs was investigated, and the optimal condition was as follows: 4% maltodextrin, 0.4% soybean powder, 1% xylose added after 10 h of growth and an induction time of 12 h at 37 °C. As a result, the maximal yield reached 24.7 U/mL. This study contributes to the industrial application of B. licheniformis, a GRAS workhorse for enzyme production. Copyright © 2016 Elsevier Inc. All rights reserved.

Caffeic acid phenethyl ester inhibits 3-MC-induced CYP1A1 expression through induction of hypoxia-inducible factor-1α

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Kim, Hyung Gyun [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Han, Eun Hee [Division of Life Science, Korea Basic Science Institute, Daejeon (Korea, Republic of); Im, Ji Hye; Lee, Eun Ji; Jin, Sun Woo [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.kr [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of)

2015-09-25

Caffeic acid phenethyl ester (CAPE), a natural component of propolis, is reported to have anticarcinogenic properties, although its precise chemopreventive mechanism remains unclear. In this study, we examined the effects of CAPE on 3-methylcholanthrene (3-MC)-induced CYP1A1 expression and activities. CAPE reduced the formation of the benzo[a]pyrene-DNA adduct. Moreover, CAPE inhibited 3-MC-induced CYP1A1 activity, mRNA expression, protein level, and promoter activity. CAPE treatment also decreased 3-MC-inducible xenobiotic-response element (XRE)-linked luciferase, aryl hydrocarbons receptor (AhR) transactivation and nuclear localization. CAPE induced hypoxia inducible factor-1α (HIF-1α) protein level and HIF-1α responsible element (HRE) transcriptional activity. CAPE-mediated HIF-1α reduced 3-MC-inducible CYP1A1 protein expression. Taken together, CAPE decreases 3-MC-mediated CYP1A1 expression, and this inhibitory response is associated with inhibition of AhR and HIF-1α induction. - Highlights: • CAPE reduced the formation of the benzo[a]pyrene-DNA adduct. • CAPE inhibited 3-MC-induced CYP1A1 expression. • CAPE induced HIF-1α induction. • CAPE-mediated HIF-1α reduced 3-MC-inducible CYP1A1 expression.

Monocrotophos induces the expression and activity of xenobiotic metabolizing enzymes in pre-sensitized cultured human brain cells.

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Vinay K Tripathi

Full Text Available The expression and metabolic profile of cytochrome P450s (CYPs is largely missing in human brain due to non-availability of brain tissue. We attempted to address the issue by using human brain neuronal (SH-SY5Y and glial (U373-MG cells. The expression and activity of CYP1A1, 2B6 and 2E1 were carried out in the cells exposed to CYP inducers viz., 3-methylcholanthrene (3-MC, cyclophosphamide (CPA, ethanol and known neurotoxicant- monocrotophos (MCP, a widely used organophosphorous pesticide. Both the cells show significant induction in the expression and CYP-specific activity against classical inducers and MCP. The induction level of CYPs was comparatively lower in MCP exposed cells than cells exposed to classical inducers. Pre-exposure (12 h of cells to classical inducers significantly added the MCP induced CYPs expression and activity. The findings were concurrent with protein ligand docking studies, which show a significant modulatory capacity of MCP by strong interaction with CYP regulators-CAR, PXR and AHR. Similarly, the known CYP inducers- 3-MC, CPA and ethanol have also shown significantly high docking scores with all the three studied CYP regulators. The expression of CYPs in neuronal and glial cells has suggested their possible association with the endogenous physiology of the brain. The findings also suggest the xenobiotic metabolizing capabilities of these cells against MCP, if received a pre-sensitization to trigger the xenobiotic metabolizing machinery. MCP induced CYP-specific activity in neuronal cells could help in explaining its effect on neurotransmission, as these CYPs are known to involve in the synthesis/transport of the neurotransmitters. The induction of CYPs in glial cells is also of significance as these cells are thought to be involved in protecting the neurons from environmental insults and safeguard them from toxicity. The data provide better understanding of the metabolizing capability of the human brain cells against

Expression of telomerase reverse transcriptase in radiation-induced chronic human skin ulcer

International Nuclear Information System (INIS)

Zhao Po; Li Zhijun; Lu Yali; Zhong Mei; Gu Qingyang; Wang Dewen

2001-01-01

Objective: To investigate the expression of the catalytic subunit of telomerase, telomerase reverse transcriptase (TRT) and the possible relationship between the TRT and cancer transformation or poor healing in radiation-induced chronic ulcer of human skin. Methods: Rabbit antibody against human TRT and SP immunohistochemical method were used to detect TRT expression in 24 cases of formalin-fixed, paraffin-embed human skin chronic ulcer tissues induced by radiation, 5 cases of normal skin, 2 of burned skin, and 8 of carcinoma. Results: The positive rate for TRT was 58.3%(14/24) in chronic radiation ulcers, of which the strongly positive rate was 41.7%(10/24) and the weakly positive 16.7%(4/24), 0% in normal (0/5) and burned skin (0/2), and 100% in carcinoma (8/8). The strongly positive expression of TRT was observed almost always in the cytoplasm and nucleus of squamous epithelial cells of proliferative epidermis but the negative and partly weakly positive expression in the smooth muscles, endothelia of small blood vessels and capillaries, and fibroblasts. Chronic inflammtory cells, plasmacytes and lymphocytes also showed weakly positive for TRT. Conclusion: TRT expression could be involved in the malignant transformation of chronic radiation ulcer into squamous carcinoma, and in the poor healing caused by sclerosis of small blood vessels and lack of granulation tissue consisting of capillaries and fibroblasts

Smad, but not MAPK, pathway mediates the expression of type I collagen in radiation induced fibrosis

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Yano, Hiroyuki; Hamanaka, Ryoji; Nakamura, Miki; Sumiyoshi, Hideaki; Matsuo, Noritaka; Yoshioka, Hidekatsu

2012-01-01

Highlights: ► We examine how radiation affects the expression level and signal pathway of collagen. ► TGF-β1 mRNA is elevated earlier than those of collagen genes after irradiation. ► Smad pathway mediates the expression of collagen in radiation induced fibrosis. ► 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. Real time RT-RCR showed that both α1and α2 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-β1 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-β 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 α2(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.

Smad, but not MAPK, pathway mediates the expression of type I collagen in radiation induced fibrosis

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Yano, Hiroyuki [Department of Matrix Medicine, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593 (Japan); Division of Radioisotope Research, Department of Research Support, Research Promotion Project, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593 (Japan); Hamanaka, Ryoji; Nakamura, Miki [Cell Biology, Faculty of Medicine, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593 (Japan); Sumiyoshi, Hideaki; Matsuo, Noritaka [Department of Matrix Medicine, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593 (Japan); Yoshioka, Hidekatsu, E-mail: hidey@oita-u.ac.jp [Department of Matrix Medicine, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593 (Japan)

2012-02-17

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. Real 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.

EGR-1 regulates Ho-1 expression induced by cigarette smoke

International Nuclear Information System (INIS)

Chen, Huaqun; Wang, Lijuan; Gong, Tao; Yu, Yang; Zhu, Chunhua; Li, Fen; Wang, Li; Li, Chaojun

2010-01-01

As an anti-oxidant molecule, heme oxygenase-1 (HO-1) has been implicated in the protection of lung injury by cigarette smoke (CS). The mechanisms regulating its expression have not been defined. In this report, the role of early growth response 1 (EGR-1) in the regulation of Ho-1 expression was investigated. In C57BL/6 mice with CS exposure, HO-1 was greatly increased in bronchial epithelial cells and alveolar inflammatory cells. In primary cultured mouse lung fibroblasts and RAW264.7 cells exposed to cigarette smoke water extract (CSE), an increase in HO-1 protein level was detected. In addition, CSE induced HO-1 expression was decreased in Egr-1 deficient mouse embryo fibroblasts (Egr-1 -/- MEFs). Nuclear localization of EGR-1 was examined in mouse lung fibroblasts after exposure to CSE. Luciferase reporter activity assays showed that the enhancer region of the Ho-1 gene containing a proposed EGR-1 binding site was responsible for the induction of HO-1. A higher increase of alveolar mean linear intercept (Lm) was observed in lung tissues, and a larger increase in the number of total cells and monocytes/macrophages from bronchial alveolar lavage fluid was found in CS-exposed mice by loss of function of EGR-1 treatment. In summary, the present data demonstrate that EGR-1 plays a critical role in HO-1 production induced by CS.

UVB-induced gene expression in the skin of Xiphophorus maculatus Jp 163 B☆

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Yang, Kuan; Boswell, Mikki; Walter, Dylan J.; Downs, Kevin P.; Gaston-Pravia, Kimberly; Garcia, Tzintzuni; Shen, Yingjia; Mitchell, David L.; Walter, Ronald B.

2014-01-01

Xiphophorus fish and interspecies hybrids represent long-standing models to study the genetics underlying spontaneous and induced tumorigenesis. The recent release of the Xiphophorus maculatus genome sequence will allow global genetic regulation studies of genes involved in the inherited susceptibility to UVB-induced melanoma within select backcross hybrids. As a first step toward this goal, we report results of an RNA-Seq approach to identify genes and pathways showing modulated transcription within the skin of X. maculatus Jp 163 B upon UVB exposure. X. maculatus Jp 163 B were exposed to various doses of UVB followed by RNA-Seq analysis at each dose to investigate overall gene expression in each sample. A total of 357 genes with a minimum expression change of 4-fold (p-adj fish skin to UVB exposure. PMID:24556253

LyGDI expression in HeLa cells increased its sensitivity to radiation-induced apoptosis

International Nuclear Information System (INIS)

Zhou Xinwen; Xu Yaxiang

2006-01-01

Objective: In order to confirm whether LyGDI has apoptotic signal transduction function and can increase the apoptotic rate of radiation-induced cell death, the lyGDI and mutant D19lyGDI gene, which constructed with the pCDNA3. 1 His A, were transfected into no-endogenous lyGDI HeLa cells. Methods Transient expressions of lyGDI and D19lyGDI in HeLa cells were analyzed by Western blot using anti-mono antibody of LyGDI and Xpress tag. Cell apoptosis was assayed with Annexin V-FITC apoptosis kit. To select stable clone, the transferred HeLa cells had been maintained in G418 medium for 3 weeks, then a cell line, which stably expressed LyGDI and mutant D19lyGDI, was selected. The selected cell line was irradiated with 12 Gy 60 Co y-rays. Caspase-3 activity of the cells was determined by Western blot and cell viability by clone-forming assay after 48 hours post-irradiation culture. Results: Western blot and Annexin V-FITC apoptotic analysis revealed that lyGDI and D19lyGDI transient expressions in HeLa cells induced apoptosis; Caspase-3 activity measurement and clone-forming assay showed that lyGDI increased sensitivity to radiation-induced cell apoptosis. Conclusions: lyGDI performs function in apoptosis signal transduction, its expression in HeLa cells can increase the sensitivity to radiation-induced cell apoptosis. (authors)

Root-Expressed Maize Lipoxygenase 3 Negatively Regulates Induced Systemic Resistance to Colletotrichum graminicola in Shoots

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Nasie eConstantino

2013-12-01

Full Text Available We have previously reported that disruption of a maize root-expressed 9-lipoxygenase (9-LOX gene, ZmLOX3, results in dramatic increase in resistance to diverse leaf and stalk pathogens. Despite evident economic significance of these findings, the mechanism behind this increased resistance remained elusive. In this study, we show that increased resistance of the lox3-4 mutants is due to constitutive activation of induced systemic resistance (ISR signaling. We showed that ZmLOX3 lacked expression in leaves in response to anthracnose leaf blight pathogen Colletotrichum graminicola, but was expressed constitutively in the roots, thus prompting our hypothesis: the roots of lox3-4 mutants are the source of increased resistance in leaves. Supporting this hypothesis, treatment of wild-type plants (WT with xylem sap of lox3-4 mutant induced resistance to C. graminicola to the levels comparable to those observed in lox3-4 mutant. Moreover, treating mutants with the sap collected from WT plants partially restored the susceptibility to C. graminicola. lox3-4 mutants showed primed defense responses upon infection, which included earlier and greater induction of defense-related PAL and GST genes compared to WT. In addition to the greater expression of the octadecanoid pathway genes, lox3-4 mutant responded earlier and with a greater accumulation of H2O2 in response to C. graminicola infection or treatment with alamethicin. These findings suggest that lox3-4 mutants display constitutive ISR-like signaling. In support of this idea, root colonization by Trichoderma virens strain GV29-8 induced the same level of disease resistance in WT as the treatment with the mutant sap, but had no additional resistance effect in lox3-4 mutant. While treatment with T. virens GV29 strongly and rapidly suppressed ZmLOX3 expression in hydroponically grown WT roots, T. virens Δsml mutant, which is deficient in ISR induction, was unable to suppress expression of ZmLOX3, thus

Epidermal growth factor protects squamous cell carcinoma against cisplatin-induced cytotoxicity through increased interleukin-1β expression.

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Shian-Chin Ko

Full Text Available The expression of cytokines, such as IL-1β, and the activation of the epidermal growth factor receptor (EGFR are crucial regulators in the process of carcinogenesis. The correlation between growth factor and activated cytokine signals in the control of tumor development is a critical issue to be clarified. In our study, we found that the IL-1β gene and protein expression were induced by EGF in squamous cell carcinoma. To clarify the mechanism involved in EGF-regulated IL-1β expression, we examined the transcriptional activity and mRNA stability of IL-1β in EGF-treated cells. We found that EGF induced the expression of IL-1β and was mediated through transcriptional activation, but not through mRNA stability. The involvement of Akt and NF-κB signaling pathways in the EGF-induced IL-1β gene expression was confirmed by knockdown of RelA and Akt in cells or treating cells with Akt and NF-κB inhibitors, LY294002 and parthenolide, respectively. The expression of dominant negative IκB also repressed the activation of NF-κB and inhibited EGF-induced IL-1β expression. Using immunofluorescence staining assay, the EGF-stimulated nuclear translocation of NF-κB (p65 was inhibited by pre-treating cells with LY294002 and parthenolide. Furthermore, EGF increased the binding of NF-κB to the NF-κB binding site of the IL-1β promoter through the activation of the Akt/NF-κB pathway, which resulted in activating IL-1β promoter activity. The expression and secretion of IL-1β induced by EGF considerably reduced chemotherapeutic drug cisplatin-induced cell death. These results showed that EGF enhanced the expression of IL-1β, which was mediated by the Akt/NF-κB pathway. The activation of EGF signaling and increase of IL-1β contributed to chemotherapeutic resistance of cancer cells, suggesting that the expression of IL-1β may be used as a biomarker to evaluate successful cancer treatment.

Phytoceramide Shows Neuroprotection and Ameliorates Scopolamine-Induced Memory Impairment

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Seikwan Oh

2011-10-01

Full Text Available The function and the role phytoceramide (PCER and phytosphingosine (PSO in the central nervous system has not been well studied. This study was aimed at investigating the possible roles of PCER and PSO in glutamate-induced neurotoxicity in cultured neuronal cells and memory function in mice. Phytoceramide showed neuro-protective activity in the glutamate-induced toxicity in cultured cortical neuronal cells. Neither phytosphingosine nor tetraacetylphytosphingosine (TAPS showed neuroproective effects in neuronal cells. PCER (50 mg/kg, p.o. recovered the scopolamine-induced reduction in step-through latency in the passive avoidance test; however, PSO did not modulate memory function on this task. The ameliorating effects of PCER on spatial memory were confirmed by the Morris water maze test. In conclusion, through behavioral and neurochemical experimental results, it was demonstrated that central administration of PCER produces amelioration of memory impairment. These results suggest that PCER plays an important role in neuroprotection and memory enhancement and PCER could be a potential new therapeutic agent for the treatment of neurodegenerative diseases such as Alzheimer’s disease.

Expression of hypoxia-induced factor-1 alpha in early-stage and in metastatic oral squamous cell carcinoma.

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Ribeiro, Maisa; Teixeira, Sarah R; Azevedo, Monarko N; Fraga, Ailton C; Gontijo, Antônio Pm; Vêncio, Eneida F

2017-04-01

To investigate hypoxia-induced factor-1 alpha expression in distinct oral squamous cell carcinoma subtypes and topographies and correlate with clinicopathological data. Hypoxia-induced factor-1 alpha expression was assessed by immunohistochemistry in 93 cases of OSCC. Clinical and histopathological data were reviewed from medical records. Hypoxia-induced factor-1 alpha status was distinct according to tumor location, subtype and topography affect. In superficial oral squamous cell carcinomas, most tumor cells overexpressed hypoxia-induced factor-1 alpha, whereas hypoxia-induced factor-1 alpha was restricted to the intratumoral region in conventional squamous cell carcinomas. All basaloid squamous cell carcinomas exhibited downregulation of hypoxia-induced factor-1 alpha. Interestingly, metastatic lymph nodes (91.7%, p = 0.001) and the intratumoral regions of corresponding primary tumors (58.3%, p = 0.142) showed hypoxia-induced factor-1 alpha-positive tumor cells. Overall survival was poor in patients with metastatic lymph nodes. Hypoxia-induced factor-1 alpha has distinct expression patterns in different oral squamous cell carcinoma subtypes and topographies, suggesting that low oxygen tension promotes the growth pattern of superficial and conventional squamous cell carcinoma, but not basaloid squamous cell carcinoma. Indeed, a hypoxic environment may facilitate regional metastasis, making it a useful diagnostic and prognostic marker in primary tumors.

Cellular stress induces cancer stem-like cells through expression of DNAJB8 by activation of heat shock factor 1.

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Kusumoto, Hiroki; Hirohashi, Yoshihiko; Nishizawa, Satoshi; Yamashita, Masamichi; Yasuda, Kazuyo; Murai, Aiko; Takaya, Akari; Mori, Takashi; Kubo, Terufumi; Nakatsugawa, Munehide; Kanaseki, Takayuki; Tsukahara, Tomohide; Kondo, Toru; Sato, Noriyuki; Hara, Isao; Torigoe, Toshihiko

2018-03-01

In a previous study, we found that DNAJB8, a heat shock protein (HSP) 40 family member is expressed in kidney cancer stem-like cells (CSC)/cancer-initiating cells (CIC) and that it has a role in the maintenance of kidney CSC/CIC. Heat shock factor (HSF) 1 is a key transcription factor for responses to stress including heat shock, and it induces HSP family expression through activation by phosphorylation. In the present study, we therefore examined whether heat shock (HS) induces CSC/CIC. We treated the human kidney cancer cell line ACHN with HS, and found that HS increased side population (SP) cells. Western blot analysis and qRT-PCR showed that HS increased the expression of DNAJB8 and SOX2. Gene knockdown experiments using siRNAs showed that the increase in SOX2 expression and SP cell ratio depends on DNAJB8 and that the increase in DNAJB8 and SOX2 depend on HSF1. Furthermore, treatment with a mammalian target of rapamycin (mTOR) inhibitor, temsirolimus, decreased the expression of DNAJB8 and SOX2 and the ratio of SP cells. Taken together, the results indicate that heat shock induces DNAJB8 by activation of HSF1 and induces cancer stem-like cells. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

A new maltose-inducible high-performance heterologous expression system in Bacillus subtilis.

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Yue, Jie; Fu, Gang; Zhang, Dawei; Wen, Jianping

2017-08-01

To improve heterologous proteins production, we constructed a maltose-inducible expression system in Bacillus subtilis. An expression system based on the promoter for maltose utilization constructed in B. subtilis. Successively, to improve the performance of the P malA -derived system, mutagenesis was employed by gradually shortening the length of P malA promoter and altering the spacing between the predicted MalR binding site and the -35 region. Furthermore, deletion of the maltose utilization genes (malL and yvdK) improved the P malA promoter activity. Finally, using this efficient maltose-inducible expression system, we enhanced the production of luciferase and D-aminoacylase, compared with the P hpaII system. A maltose-inducible expression system was constructed and evaluated. It could be used for high level expression of heterologous proteins production.

Hypoxia induced expression of endogenous markers in vitro is highly influenced by pH

International Nuclear Information System (INIS)

Sorensen, Brita Singers; Alsner, Jan; Overgaard, Jens; Horsman, Michael R.

2007-01-01

Background: Genes such as carbonic anhydrase IX (Ca9), glucose transporter 1 (Glut1), lactate dehydrogenase A (LDH-A), osteopontin (OPN) and lysyl oxidase (LOX) have been suggested as hypoxic markers, but inconsistent results suggest that factors other than oxygen influence their expression. The current study is a detailed investigation using a range of pH values from 6.3 to 7.5 in two human cell lines to establish the pH dependency of hypoxia induced gene expression. Methods: Human tumour cell lines (uterine cervix squamous cell carcinoma (SiHa) and pharyngeal squamous cell carcinoma [FaDu DD ]) were used. Hypoxia was induced by gassing cells in airtight chambers with various oxygen concentrations (21%, 1%, 0.1%, 0.01% and 0%) for up to 24 h. The media were titrated to a range of pH values (7.5, 7.0, 6.7, 6.5 and 6.3). Gene expression was determined by real-time PCR. Results: In both SiHa and FaDu DD cells Ca9 and LOX reached the highest level of expression at 1% oxygen. In FaDu DD cells, a pH of 6.5 had a medium suppression effect on the hypoxia induced expression of Ca9. pH 6.3 resulted in severe suppression of expression for Ca9 and LOX in both SiHa and FaDu DD . Glut1 and LDH-A had a similar expression pattern to each other, with a maximum expression at 0.01% oxygen, in both cell lines. For these genes pH 6.5 and 6.3 changed the expression pattern in SiHa cells. OPN was up regulated at low oxygen in SiHa cells, but was not induced by hypoxia in FaDu DD cells. Conclusion: As tumour hypoxia occurs in a deprived microenvironment, other environmental factors, for example low pH, might interact with the effect of low oxygen concentration on gene expression. This study shows that pH in two cell lines has a profound influence on the oxygen dependent induction of certain endogenous hypoxic markers

Apoptosis induced by radionuclide 153Sm and expression of relevant genes in three different cancer cells

International Nuclear Information System (INIS)

Zou Baomin; Duan Xiaoyi; Chen Wei; Hu Guoying

2003-01-01

To study apoptosis of PC-3, ER-75-30 and A549 cells induced by radionuclide 153 Sm and the expression of bcl-2, bax in apoptosis cells, MTT assay was used to detect the anti-tumor effect, light microscope, transmission electron microscope, flow cytometer were used to detect apoptosis, while image analysis was used to detect the expression of bcl-2 and bax. 153 Sm showed anti-tumor effect and could induce tumor cell apoptosis. Both bcl-2 and bax played an important role in apoptosis. Different kind of cells had different sensitivity to 153 Sm

SPBP is a sulforaphane induced transcriptional coactivator of NRF2 regulating expression of the autophagy receptor p62/SQSTM1.

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Sagar Ramesh Darvekar

Full Text Available Organisms exposed to oxidative stress respond by orchestrating a stress response to prevent further damage. Intracellular levels of antioxidant agents increase, and damaged components are removed by autophagy induction. The KEAP1-NRF2 signaling pathway is the main pathway responsible for cell defense against oxidative stress and for maintaining the cellular redox balance at physiological levels. Sulforaphane, an isothiocyanate derived from cruciferous vegetables, is a potent inducer of KEAP1-NRF2 signaling and antioxidant response element driven gene expression. In this study, we show that sulforaphane enhances the expression of the transcriptional coregulator SPBP. The expression curve peaks 6-8 hours post stimulation, and parallels the sulforaphane-induced expression of NRF2 and the autophagy receptor protein p62/SQSTM1. Reporter gene assays show that SPBP stimulates the expression of p62/SQSTM1 via ARE elements in the promoter region, and siRNA mediated knock down of SPBP significantly decreases the expression of p62/SQSTM1 and the formation of p62/SQSTM1 bodies in HeLa cells. Furthermore, SPBP siRNA reduces the sulforaphane induced expression of NRF2, and the expression of the autophagy marker protein LC3B. Both these proteins contain ARE-like elements in their promoter regions. Over-expressed SPBP and NRF2 acts synergistically on the p62/SQSTM1 promoter and colocalize in nuclear speckles in HeLa cells. Collectively, these results suggest that SPBP is a coactivator of NRF2, and hence may be important for securing enhanced and sustained expression of NRF2 induced genes such as proteins involved in selective autophagy.

Increased expression of interleukin-1β in triglyceride-induced macrophage cell death is mediated by p38 MAP kinase.

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Sung, Ho Joong; Son, Sin Jee; Yang, Seung-ju; Rhee, Ki-Jong; Kim, Yoon Suk

2012-07-01

Triglycerides (TG) are implicated in the development of atherosclerosis through formation of foam cells and induction of macrophage cell death. In this study, we report that addition of exogenous TG induced cell death in phorbol 12-myristate 13-acetate-differentiated THP-1 human macrophages. TG treatment induced a dramatic decrease in interleukin-1β (IL-1β) mRNA expression in a dose- and time-dependent manner. The expression of granulocyte macrophage colony-stimulating factor and platelet endothelial cell adhesion molecule remained unchanged. To identify signaling pathways involved in TG-induced downregulation of IL-1β, we added p38 MAPK, protein kinase C (PKC) or c-Raf1 specific inhibitors. We found that inhibition of p38 MAPK alleviated the TG-induced downregulation of IL-1β, whereas inhibition of PKC and c-Raf1 had no effect. This is the first report showing decreased IL-1β expression during TG-induced cell death in a human macrophage line. Our results suggest that downregulation of IL-1β expression by TG-treated macrophages may play a role during atherogenesis.

Nutrient Induced Type 2 and Chemical Induced Type 1 Experimental Diabetes Differently Modulate Gastric GLP-1 Receptor Expression

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Olga Bloch

2015-01-01

Full Text Available T2DM patients demonstrate reduced GLP-1 receptor (GLP-1R expression in their gastric glands. Whether induced T2DM and T1DM differently affect the gastric GLP-1R expression is not known. This study assessed extrapancreatic GLP-1R system in glandular stomach of rodents with different types of experimental diabetes. T2DM and T1DM were induced in Psammomys obesus (PO by high-energy (HE diet and by streptozotocin (STZ in Sprague Dawly (SD rats, respectively. GLP-1R expression was determined in glandular stomach by RT PCR and immunohistomorphological analysis. The mRNA expression and cellular association of the GLP-1R in principal glands were similar in control PO and SD rats. However, nutrient and chemical induced diabetes resulted in opposite alterations of glandular GLP-1R expression. Diabetic PO demonstrated increased GLP-1R mRNA expression, intensity of cellular GLP-1R immunostaining, and frequency of GLP-1R positive cells in the neck area of principal glands compared with controls. In contrast, SD diabetic rats demonstrated decreased GLP-1 mRNA, cellular GLP-1R immunoreactivity, and frequency of GLP-1R immunoreactive cells in the neck area compared with controls. In conclusion, nutrient and chemical induced experimental diabetes result in distinct opposite alterations of GLP-1R expression in glandular stomach. These results suggest that induced T1DM and T2DM may differently modulate GLP-1R system in enteropancreatic axis.

HIV-1 induces DCIR expression in CD4+ T cells.

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Alexandra A Lambert

2010-11-01

Full Text Available The C-type lectin receptor DCIR, which has been shown very recently to act as an attachment factor for HIV-1 in dendritic cells, is expressed predominantly on antigen-presenting cells. However, this concept was recently challenged by the discovery that DCIR can also be detected in CD4(+ T cells found in the synovial tissue from rheumatoid arthritis (RA patients. Given that RA and HIV-1 infections share common features such as a chronic inflammatory condition and polyclonal immune hyperactivation status, we hypothesized that HIV-1 could promote DCIR expression in CD4(+ T cells. We report here that HIV-1 drives DCIR expression in human primary CD4(+ T cells isolated from patients (from both aviremic/treated and viremic/treatment naive persons and cells acutely infected in vitro (seen in both virus-infected and uninfected cells. Soluble factors produced by virus-infected cells are responsible for the noticed DCIR up-regulation on uninfected cells. Infection studies with Vpr- or Nef-deleted viruses revealed that these two viral genes are not contributing to the mechanism of DCIR induction that is seen following acute infection of CD4(+ T cells with HIV-1. Moreover, we report that DCIR is linked to caspase-dependent (induced by a mitochondria-mediated generation of free radicals and -independent intrinsic apoptotic pathways (involving the death effector AIF. Finally, we demonstrate that the higher surface expression of DCIR in CD4(+ T cells is accompanied by an enhancement of virus attachment/entry, replication and transfer. This study shows for the first time that HIV-1 induces DCIR membrane expression in CD4(+ T cells, a process that might promote virus dissemination throughout the infected organism.

PPAR{alpha} deficiency augments a ketogenic diet-induced circadian PAI-1 expression possibly through PPAR{gamma} activation in the liver

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Oishi, Katsutaka, E-mail: k-ooishi@aist.go.jp [Biological Clock Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki (Japan); Uchida, Daisuke [Biological Clock Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki (Japan); Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki (Japan); Ohkura, Naoki [Department of Clinical Molecular Biology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamihara, Kanagawa (Japan); Horie, Shuichi [Department of Clinical Biochemistry, Kagawa Nutrition University, Sakado, Saitama (Japan)

2010-10-15

Research highlights: {yields} PPAR{alpha} deficiency augments a ketogenic diet-induced circadian PAI-1 expression. {yields} Hepatic expressions of PPAR{gamma} and PCG-1{alpha} are induced by a ketogenic diet. {yields} PPAR{gamma} antagonist attenuates a ketogenic diet-induced PAI-1 expression. {yields} Ketogenic diet advances the phase of circadian clock in a PPAR{alpha}-independent manner. -- Abstract: An increased level of plasminogen activator inhibitor-1 (PAI-1) is considered a risk factor for cardiovascular diseases, and PAI-1 gene expression is under the control of molecular circadian clocks in mammals. We recently showed that PAI-1 expression is augmented in a phase-advanced circadian manner in mice fed with a ketogenic diet (KD). To determine whether peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) is involved in hypofibrinolytic status induced by a KD, we examined the expression profiles of PAI-1 and circadian clock genes in PPAR{alpha}-null KD mice. Chronic administration of bezafibrate induced the PAI-1 gene expression in a PPAR{alpha}-dependent manner. Feeding with a KD augmented the circadian expression of PAI-1 mRNA in the hearts and livers of wild-type (WT) mice as previously described. The KD-induced mRNA expression of typical PPAR{alpha} target genes such as Cyp4A10 and FGF21 was damped in PPAR{alpha}-null mice. However, plasma PAI-1 concentrations were significantly more elevated in PPAR{alpha}-null KD mice in accordance with hepatic mRNA levels. These observations suggest that PPAR{alpha} activation is dispensable for KD-induced PAI-1 expression. We also found that hyperlipidemia, fatty liver, and the hepatic expressions of PPAR{gamma} and its coactivator PCG-1{alpha} were more effectively induced in PPAR{alpha}-null, than in WT mice on a KD. Furthermore, KD-induced hepatic PAI-1 expression was significantly suppressed by supplementation with bisphenol A diglycidyl ether, a PPAR{gamma} antagonist, in both WT and PPAR

Alpha-phellandrene-induced DNA damage and affect DNA repair protein expression in WEHI-3 murine leukemia cells in vitro.

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Lin, Jen-Jyh; Wu, Chih-Chung; Hsu, Shu-Chun; Weng, Shu-Wen; Ma, Yi-Shih; Huang, Yi-Ping; Lin, Jaung-Geng; Chung, Jing-Gung

2015-11-01

Although there are few reports regarding α-phellandrene (α-PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α-PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α-PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α-PA on total cell viability and the results indicated that α-PA induced cell death. Comet assay and 4,6-diamidino-2-phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α-PA induced DNA damage and condensation in a concentration-dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α-PA induced DNA damage in WEHI-3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α-PA increased p-p53, p-H2A.X, 14-3-3-σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA-PK, and BRCA-1. © 2014 Wiley Periodicals, Inc.

Drug-induced regulation of target expression

DEFF Research Database (Denmark)

Iskar, Murat; Campillos, Monica; Kuhn, Michael

2010-01-01

Drug perturbations of human cells lead to complex responses upon target binding. One of the known mechanisms is a (positive or negative) feedback loop that adjusts the expression level of the respective target protein. To quantify this mechanism systems-wide in an unbiased way, drug......-induced differential expression of drug target mRNA was examined in three cell lines using the Connectivity Map. To overcome various biases in this valuable resource, we have developed a computational normalization and scoring procedure that is applicable to gene expression recording upon heterogeneous drug treatments....... In 1290 drug-target relations, corresponding to 466 drugs acting on 167 drug targets studied, 8% of the targets are subject to regulation at the mRNA level. We confirmed systematically that in particular G-protein coupled receptors, when serving as known targets, are regulated upon drug treatment. We...

Cell expression patterns of CD147 in N-diethylnitrosamine/phenobarbital-induced mouse hepatocellular carcinoma.

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Lu, Meng; Wu, Jiao; He, Feng; Wang, Xi-Long; Li, Can; Chen, Zhi-Nan; Bian, Huijie

2015-02-01

Overexpression of CD147/basigin in hepatic cells promotes the progression of hepatocellular carcinoma (HCC). Whether CD147 also expressed in liver non-parenchymal cells and associated with HCC development was unknown. The aim of the study was to explore time-dependent cell expression patterns of CD147 in a widely accepted N-diethylnitrosamine/phenobarbital (DEN/PB)-induced HCC mouse model. Liver samples collected at month 1-12 of post-DEN/PB administration were assessed the localization of CD147 in hepatocytes, endothelial cells, hepatic stellate cells, and macrophages. Immunohistochemistry analysis showed that CD147 was upregulated in liver tumors during month 1-8 of DEN/PB induction. Expression of CD147 was positively correlated with cytokeratin 18, a hepatocyte marker (r = 0.7857, P = 0.0279), CD31 (r = 0.9048, P = 0.0046), an endothelial cell marker, and CD68, a macrophage marker (r = 0.7619, P = 0.0368). A significant correlation was also observed between CD147 and alpha-smooth muscle actin (r = 0.8857, P = 0.0333) at DEN/PB initiation and early stage of tumor formation. Immunofluorescence and fluorescence in situ hybridization showed that CD147 co-expressed with cytokeratin 18, CD31, alpha-smooth muscle actin, and CD68. Moreover, there existed positive correlations between CD147 and microvessel density (r = 0.7857, P = 0.0279), CD147 and Ki-67 (r = 0.9341, P = 0.0022) in the development of DEN/PB-induced HCC. In conclusion, our results demonstrated that CD147 was upregulated in the liver parenchymal and mesenchymal cells and involved in angiogenesis and tumor cell proliferation in the development of DEN/PB-induced HCC.

Andrographolide inhibits hypoxia-induced hypoxia-inducible factor 1α and endothelin 1 expression through the heme oxygenase 1/CO/cGMP/MKP-5 pathways in EA.hy926 cells.

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Lin, Hung-Chih; Su, Shih-Li; Lin, Wan-Chun; Lin, Ai-Hsuan; Yang, Ya-Chen; Lii, Chong-Kuei; Chen, Haw-Wen

2018-03-01

Andrographolide is a potent anti-inflammatory agent found in Andrographis paniculata. Endothelin 1 (ET-1) is an endothelium-derived vasoconstrictor with pro-inflammatory properties secreted in response to hypoxia. Mitogen-activated protein kinase phosphatase 5 (MKP-5) is a dual-specificity phosphatase that dephosphorylates threonine and tyrosine residues of MAPKs. We showed previously that hypoxia-induced HIF-1α expression and ET-1 secretion are dependent on p38 MAPK in EA.hy926 cells. Here, we investigate what role MKP-5 plays in andrographolide's inhibition of hypoxia-induced expression of HIF-1α and ET-1. Hypoxic conditions were created using the hypoxia-mimetic agent CoCl 2 . Andrographolide enhanced HO-1 and MKP-5 expression and cellular cGMP content in addition to inhibiting hypoxia-induced ROS generation. Concomitantly, the HO-1 byproduct CO and the cGMP analogue 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) increased MKP-5 expression, and pretreatment with CO and 8-Br-cGMP inhibited hypoxia-induced HIF-1α and ET-1 expression. Transfection of HO-1 siRNA or pretreatment with the HO-1 inhibitor ZnPP-9 or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a specific inhibitor of soluble guanylate cyclase, reduced andrographolide-induced MKP-5 expression. Moreover, silencing MKP-5 or treatment with the phosphatase inhibitor vanadate abrogated andrographolide's suppressing hypoxia-induced p38 MAPK activation and HIF-1α expression. The inhibition of hypoxia-induced HIF-1α and ET-1 expression by andrographolide is likely associated with HO-1/CO/cGMP/MKP-5 pathways, which is involved in inhibiting hypoxia-induced p38 MAPK activation. © 2017 Wiley Periodicals, Inc.

High-level intracellular expression of heterologous proteins in Brevibacillus choshinensis SP3 under the control of a xylose inducible promoter

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D’Urzo Nunzia

2013-02-01

Full Text Available Abstract Background In past years research has focused on the development of alternative Gram positive bacterial expression systems to produce industrially relevant proteins. Brevibacillus choshinensis is an easy to handle non-sporulating bacterium, lacking extracellular proteases, that has been already shown to provide a high level of recombinant protein expression. One major drawback, limiting the applicability of the Brevibacillus expression system, is the absence of expression vectors based on inducible promoters. Here we used the PxylA inducible promoter, commonly employed in other Bacillae expression systems, in Brevibacillus. Results Using GFP, α-amylase and TcdA-GT as model proteins, high level of intracellular protein expression (up to 250 mg/L for the GFP was achieved in Brevibacillus, using the pHis1522 vector carrying the B. megaterium xylose-inducible promoter (PxylA. The GFP expression yields were more than 25 fold higher than those reported for B. megaterium carrying the same vector. All the tested proteins show significant increment in their expression levels (2-10 folds than those obtained using the available plasmids based on the P2 constitutive promoter. Conclusion Combining the components of two different commercially available Gram positive expression systems, such as Brevibacillus (from Takara Bio and B. megaterium (from Mobitec, we demonstrate that vectors based on the B. megaterium PxylA xylose inducible promoter can be successfully used to induce high level of intracellular expression of heterologous proteins in Brevibacillus.

Differentially expressed genes in iron-induced prion protein conversion

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Kim, Minsun; Kim, Eun-hee; Choi, Bo-Ran; Woo, Hee-Jong

2016-01-01

The conversion of the cellular prion protein (PrP C ) to the protease-resistant isoform is the key event in chronic neurodegenerative diseases, including transmissible spongiform encephalopathies (TSEs). Increased iron in prion-related disease has been observed due to the prion protein-ferritin complex. Additionally, the accumulation and conversion of recombinant PrP (rPrP) is specifically derived from Fe(III) but not Fe(II). Fe(III)-mediated PK-resistant PrP (PrP res ) conversion occurs within a complex cellular environment rather than via direct contact between rPrP and Fe(III). In this study, differentially expressed genes correlated with prion degeneration by Fe(III) were identified using Affymetrix microarrays. Following Fe(III) treatment, 97 genes were differentially expressed, including 85 upregulated genes and 12 downregulated genes (≥1.5-fold change in expression). However, Fe(II) treatment produced moderate alterations in gene expression without inducing dramatic alterations in gene expression profiles. Moreover, functional grouping of identified genes indicated that the differentially regulated genes were highly associated with cell growth, cell maintenance, and intra- and extracellular transport. These findings showed that Fe(III) may influence the expression of genes involved in PrP folding by redox mechanisms. The identification of genes with altered expression patterns in neural cells may provide insights into PrP conversion mechanisms during the development and progression of prion-related diseases. - Highlights: • Differential genes correlated with prion degeneration by Fe(III) were identified. • Genes were identified in cell proliferation and intra- and extracellular transport. • In PrP degeneration, redox related genes were suggested. • Cbr2, Rsad2, Slc40a1, Amph and Mvd were expressed significantly.

Expression of FSH receptor in ovary tissue of rats with letrozole-induced polycystic ovary syndrome

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Guo Hongsheng; An Changxin; Chen Dong

2009-01-01

Objective: To investigate the expressions of FSH receptor mRNA and protein in ovary tissue in rats with letrozole-induced polycystic ovary syndrome (PCOS), and to provide experimental data for the model application. Methods: Forty rats were randomly divided into two groups (n=20), in PCOS model group letrozole was administered once daily during 21 d, and in control group without any treatment. The gonadal hormone concentrations in serum were determined by radioimmunoassay, the histologic changes in ovaries were observed by HE staining, the expression of FSH receptor gene in ovary tissue was detected by realtime -PCR, Western blotting and immunohistochemistry. Results: Compared with control group, estradiol (E 2 ) and progesterone in model group showed a considerable reduction (P 0.05). Compared with control group, the ovaries from model group showed high incidence of subcapsular ovarian cyst and capsular thickening and decreased number of corpora lute a. The expressions of FSH receptor mRNA and protein were significantly higher in model group than those in control group (P<0.05). Conclusion: The expression of FSH receptor gene in letrozole-induced polycystic ovaries is similar with that of PCOS women, the rat model is proved to be an ideal PCOS animal model to study the pathophysiology of PCOS. (authors)

p53-Dependent radiation-induced apoptosis in vivo: relationship to Bcl-2 and Bax expression

International Nuclear Information System (INIS)

Hasegawa, Masatoshi; Suzuki, Yoshiyuki; Furuta, Masaya; Yamakawa, Michitaka; Maebayashi, Katsuya; Hayakawa, Kayoko; Saito, Yoshihiro; Mitsuhashi, Norio; Niibe, Hideo

1997-01-01

Purpose: A close correlation between p53 protein expression and radiation-induced apoptosis has already been reported, however, Bcl-2 and Bax expression and the ratio of Bcl-2 to Bax have been also suggested to play an important role in the regulation of apoptotic cell death. In this study, we investigated the relationship between p53-dependent radiation-induced apoptosis and expression of Bcl-2 and Bax by using human tumors transplanted into nude mice. Materials and Methods: Three human tumors (an ependymoblastoma, a glioblastoma, and a small cell lung cancer) were subcutaneously transplanted into nude mice and irradiated with single doses of 1, 2, 5, or 10 Gy. The tumors were excised 1, 3, 6, 12, 24, and 48 hours after irradiation, fixed in 10% formalin for 24 hours, and embedded in paraffin. Slides were stained with hematoxylin and eosin for morphologic examination. Immunohistochemical studies were performed with mouse monoclonal antibodies to demonstrate p53, p21 (WAF-1), Bcl-2, and Bax expression. TdT-mediated dUTP-biotin nick-end labeling (TUNEL) and electron microscopic studies were performed to identify apoptosis, and PCR-SSCP analysis was used to evaluate p53 gene mutation. Results: All of the tumors showed only a few cells undergoing apoptosis before irradiation. Beginning several hours after irradiation, only the ependymoblastoma showed a large increase in the number of cells undergoing apoptosis, peaking at 6 hours after irradiation, and there was a clear dose-effect relationship. In contrast, the other tumors showed much less change following irradiation, and the dose-effect relationship was not as clear as in the ependymoblastoma. Immunohistochemically, the non-irradiated ependymoblastoma was negative for p53, p21, Bcl-2, and Bax. Following irradiation, however, many of the tumor cells became positive for p53 and p21, and a few cells became positive for bcl-2. In contrast, the glioblastoma and the small cell lung cancer were positive for p53 and Bcl-2

Fetuin-A induces cytokine expression and suppresses adiponectin production.

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Anita M Hennige

Full Text Available BACKGROUND: The secreted liver protein fetuin-A (AHSG is up-regulated in hepatic steatosis and the metabolic syndrome. These states are strongly associated with low-grade inflammation and hypoadiponectinemia. We, therefore, hypothesized that fetuin-A may play a role in the regulation of cytokine expression, the modulation of adipose tissue expression and plasma concentration of the insulin-sensitizing and atheroprotective adipokine adiponectin. METHODOLOGY AND PRINCIPAL FINDINGS: Human monocytic THP1 cells and human in vitro differenttiated adipocytes as well as C57BL/6 mice were treated with fetuin-A. mRNA expression of the genes encoding inflammatory cytokines and the adipokine adiponectin (ADIPOQ was assessed by real-time RT-PCR. In 122 subjects, plasma levels of fetuin-A, adiponectin and, in a subgroup, the multimeric forms of adiponectin were determined. Fetuin-A treatment induced TNF and IL1B mRNA expression in THP1 cells (p<0.05. Treatment of mice with fetuin-A, analogously, resulted in a marked increase in adipose tissue Tnf mRNA as well as Il6 expression (27- and 174-fold, respectively. These effects were accompanied by a decrease in adipose tissue Adipoq mRNA expression and lower circulating adiponectin levels (p<0.05, both. Furthermore, fetuin-A repressed ADIPOQ mRNA expression of human in vitro differentiated adipocytes (p<0.02 and induced inflammatory cytokine expression. In humans in plasma, fetuin-A correlated positively with high-sensitivity C-reactive protein, a marker of subclinical inflammation (r = 0.26, p = 0.01, and negatively with total- (r = -0.28, p = 0.02 and, particularly, high molecular weight adiponectin (r = -0.36, p = 0.01. CONCLUSIONS AND SIGNIFICANCE: We provide novel evidence that the secreted liver protein fetuin-A induces low-grade inflammation and represses adiponectin production in animals and in humans. These data suggest an important role of fatty liver in the pathophysiology of insulin resistance and

Zingiber officinale, Piper retrofractum and Combination Induced Apoptosis and p53 Expression in Myeloma and WiDr Cell Lines

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HENY EKOWATI

2012-09-01

Full Text Available In previous studies, Zingiber officinale, Piper retrofractum, and the combination showed cytotoxic activity, induced apoptosis, and p53 expression of HeLa, T47D, and MCF-7 cell lines. This study was conducted to investigate the cytotoxic and apoptotic activity of Zingiber officinale (ZO, Piper retrofractum (PR, and the combination as well as their effect to p53 expression on Myeloma and WiDr cells. The powder of ZO, PR, and ZO + PR combination (1:1 were macerated with 96% ethanol for 3 x 24 hours. MTT cytotoxic assay was performed on Myeloma and WiDr cell lines. Apoptotic cells were stained with ethidium bromide and acridine orange. Imunohistochemical expression of p53 was examined on Myeloma and WiDr cell lines. Doxorubicin was used as positive control in all assays. Results showed that ZO, PR, and ZO + PR combination had cytotoxic activity on Myeloma cells with IC50 of 28, 36, and 55 mg/ml respectively and WiDr cell lines with IC50 of 74, 158, and 64 mg/ml respectively, induced apoptotic activity, and increased p53 expression on Myeloma and WiDr cells. These results suggest that ZO, PR, and their combination induced Myeloma and WiDr cells in apoptosis through p53 expression.

Gene expression of cyclin-dependent kinase inhibitors and effect of heparin on their expression in mice with hypoxia-induced pulmonary hypertension

International Nuclear Information System (INIS)

Yu Lunyin; Quinn, Deborah A.; Garg, Hari G.; Hales, Charles A.

2006-01-01

The balance between cell proliferation and cell quiescence is regulated delicately by a variety of mediators, in which cyclin-dependent kinases (CDK) and CDK inhibitors (CDKI) play a very important role. Heparin which inhibits pulmonary artery smooth muscle cell (PASMC) proliferation increases the levels of two CDKIs, p21 and p27, although only p27 is important in inhibition of PASMC growth in vitro and in vivo. In the present study we investigated the expression profile of all the cell cycle regulating genes, including all seven CDKIs (p21, p27, p57, p15, p16, p18, and p19), in the lungs of mice with hypoxia-induced pulmonary hypertension. A cell cycle pathway specific gene microarray was used to profile the 96 genes involved in cell cycle regulation. We also observed the effect of heparin on gene expression. We found that (a) hypoxic exposure for two weeks significantly inhibited p27 expression and stimulated p18 activity, showing a 98% decrease in p27 and 81% increase in p18; (b) other CDKIs, p21, p57, p15, p16, and p19 were not affected significantly in response to hypoxia; (c) heparin treatment restored p27 expression, but did not influence p18; (d) ERK1/2 and p38 were mediators in heparin upregulation of p27. This study provides an expression profile of cell cycle regulating genes under hypoxia in mice with hypoxia-induced pulmonary hypertension and strengthens the previous finding that p27 is the only CDKI involved in heparin regulation of PASMC proliferation and hypoxia-induced pulmonary hypertension

Microbiological profile and calprotectin expression in naturally occurring and experimentally induced gingivitis.

Science.gov (United States)

Farina, Roberto; Guarnelli, Maria Elena; Figuero, Elena; Herrera, David; Sanz, Mariano; Trombelli, Leonardo

2012-10-01

This study was performed to evaluate the microbiological profile and the calprotectin expression in gingival crevicular fluid (GCF) in spontaneous and experimentally induced gingival inflammation. Thirty-seven periodontally healthy subjects were evaluated in real life conditions (N-O gingivitis) as well as after 21 days of experimental gingivitis trial (E-I gingivitis). During the experimental gingivitis trial, in one maxillary quadrant (test quadrant), gingival inflammation was induced by oral hygiene abstention, while in the contralateral (control) quadrant, oral hygiene was routinely continued. The results of the study showed that (1) the microbiological profile of quadrants where gingival inflammation was experimentally induced (i.e., E-I test quadrants) differed significantly from that of either quadrants where gingival inflammation was controlled by proper plaque control (i.e., E-I control quadrants) or quadrants with N-O gingivitis, and (2) GCF calprotectin was significantly higher at E-I test quadrants compared to either E-I control quadrants or quadrants with N-O gingivitis. A positive intrasubject correlation was found between GCF concentration of calprotectin at sites presenting N-O and E-I gingivitis. N-O and E-I gingivitis showed a different microbiological profile of the subgingival environment. GCF calprotectin is a reliable marker of gingival inflammation, and its concentration in N-O gingivitis is correlated with its expression in E-I gingivitis. The modality of plaque accumulation seems to affect the subgingival microbiological profile associated with a gingivitis condition. Calprotectin levels in GCF may be regarded as a promising marker of the individual susceptibility to develop gingival inflammation in response to experimentally induced plaque accumulation.

Expression of extracellular matrix metalloproteinase inducer in odontogenic cysts.

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Ali, Mohammad Abdulhadi Abbas

2008-08-01

Extracellular matrix metalloproteinase inducer (EMMPRIN) is known to induce matrix metalloproteinase (MMP) production. The expression of EMMPRIN in odontogenic cysts has not been previously studied. This study was done to determine the presence and the variability of EMMPRIN expression in various types of odontogenic cysts. An immunohistochemical study using a polyclonal anti-EMMPRIN antibody was done using 48 odontogenic cyst cases: 13 odontogenic keratocysts (OKCs), 18 dentigerous cysts (DCs), and 17 periapical cysts (PAs). Twelve cases of normal dental follicles (DFs) were also included in this study for comparison. EMMPRIN immunoreactivity was detected in all of the cysts and DFs studied. In odontogenic cysts, EMMPRIN immunoreactivity was generally higher in basal cells than in suprabasal cells. The overall EMMPRIN expression in the epithelial lining of the 3 different types of odontogenic cyst was significantly higher than in the DFs. Overall EMMPRIN expression was also found to be significantly higher in the epithelial lining of OKCs than in the other types of cysts. This study confirmed that EMMPRIN is present in odontogenic cysts and DFs. The higher EMMPRIN expression in OKCs suggests that it may be involved in the aggressive behavior of this type of cyst.

Src Induces Podoplanin Expression to Promote Cell Migration*

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Shen, Yongquan; Chen, Chen-Shan; Ichikawa, Hitoshi; Goldberg, Gary S.

2010-01-01

Nontransformed cells can force tumor cells to assume a normal morphology and phenotype by the process of contact normalization. Transformed cells must escape this process to become invasive and malignant. However, mechanisms underlying contact normalization have not been elucidated. Here, we have identified genes that are affected by contact normalization of Src-transformed cells. Tumor cells must migrate to become invasive and malignant. Src must phosphorylate the adaptor protein Cas (Crk-associated substrate) to promote tumor cell motility. We report here that Src utilizes Cas to induce podoplanin (Pdpn) expression to promote tumor cell migration. Pdpn is a membrane-bound extracellular glycoprotein that associates with endogenous ligands to promote tumor cell migration leading to cancer invasion and metastasis. In fact, Pdpn expression accounted for a major part of the increased migration seen in Src-transformed cells. Moreover, nontransformed cells suppressed Pdpn expression in adjacent Src-transformed cells. Of >39,000 genes, Pdpn was one of only 23 genes found to be induced by transforming Src activity and suppressed by contact normalization of Src-transformed cells. In addition, we found 16 genes suppressed by Src and induced by contact normalization. These genes encode growth factor receptors, adaptor proteins, and products that have not yet been annotated and may play important roles in tumor cell growth and migration. PMID:20123990

Andrographolide suppresses high glucose-induced fibronectin expression in mesangial cells via inhibiting the AP-1 pathway.

Science.gov (United States)

Lan, Tian; Wu, Teng; Gou, Hongju; Zhang, Qianqian; Li, Jiangchao; Qi, Cuiling; He, Xiaodong; Wu, Pingxiang; Wang, Lijing

2013-11-01

Mesangial cells (MCs) proliferation and accumulation of glomerular matrix proteins such as fibronectin (FN) are the early features of diabetic nephropathy, with MCs known to upregulate matrix protein synthesis in response to high glucose. Recently, it has been found that andrographolide has renoprotective effects on diabetic nephropathy. However, the molecular mechanism underlying these effects remains unclear. Cell viability and proliferation was evaluated by MTT. FN expression was examined by immunofluorescence and immunoblotting. Activator protein-1 (AP-1) activation was assessed by immunoblotting, luciferase reporter and electrophoretic mobility shift assays. Andrographolide significantly decreased high glucose-induced cell proliferation and FN expression in MCs. Exposure of MCs to high glucose markedly stimulated the expression of phosphorylated c-jun, whereas the stimulation was inhibited by andrographolide. Plasmid pAP-1-Luc luciferase reporter assay showed that andrographolide blocked high glucose-induced AP-1 transcriptional activity. EMSA assay demonstrated that increased AP-1 binding to an AP-1 binding site at -1,029 in the FN gene promoter upon high glucose stimulation, and the binding were disrupted by andrographolide treatment. These data indicate that andrographolide suppresses high glucose-induced FN expression by inhibiting AP-1-mediated pathway. © 2013 Wiley Periodicals, Inc.

Drug-loaded nanoparticles induce gene expression in human pluripotent stem cell derivatives

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Gajbhiye, Virendra; Escalante, Leah; Chen, Guojun; Laperle, Alex; Zheng, Qifeng; Steyer, Benjamin; Gong, Shaoqin; Saha, Krishanu

2013-12-01

Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained higher fibroblast proliferation levels and MMP activity. The results demonstrate that the PEG-H40-DXC nanoparticle system provides an effective tool to controlling gene expression in human stem cell derivatives.Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained

Fluoride or/and aluminum induced toxicity in guinea pig teeth with the low expression of dentine phosphoprotein.

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Han, Tianlong; Wang, Min; Cao, Chunfang; Chen, Huacheng; Zhang, Guanghe; Wang, Liping; Wang, Jundong

2017-08-01

This study investigated the damage and expression of dentine phosphoprotein (DPP) in guinea pig teeth by the administration of fluoride (F) or/and aluminum (Al). Fifty-two guinea pigs were divided randomly into four groups (control, F, Al, and F+Al). F (150 mg NaF/L) or/and Al (300 mg AlCl 3 /L) were added in their drinking water for 90 days. The levels of F ion, dentine sialophosphoprotein (DSPP) gene, and DPP protein in incisor and molar were determined, respectively. The results showed that the concentrations of F ion in F and F+Al groups were increased significantly. F induced the mottled enamel and irregular abrasion of teeth, which might occur as a consequence of depressed DSPP mRNA and DPP protein expression. Both the gene and protein expressions showed obvious decrease induced by Al, especially by F. There were no synergistic effects between F and Al, instead, Al inhibited the toxicity of F. © 2017 Wiley Periodicals, Inc.

Differential expression of ozone-induced gene during exposures to ...

African Journals Online (AJOL)

Differential expression of ozone-induced gene during exposures to salt stress in Polygonum sibiricum Laxm leaves, stem and underground stem. ... PcOZI-1 mRNA in untreated plants was detected at low levels in underground stem, leaves and at higher levels in stem. PcOZI-1 mRNA accumulation was transiently induced ...

CREB activity in dopamine D1 receptor expressing neurons regulates cocaine-induced behavioral effects

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Bilbao, Ainhoa; Rieker, Claus; Cannella, Nazzareno; Parlato, Rosanna; Golda, Slawomir; Piechota, Marcin; Korostynski, Michal; Engblom, David; Przewlocki, Ryszard; Schütz, Günther; Spanagel, Rainer; Parkitna, Jan R.

2014-01-01

It is suggested that striatal cAMP responsive element binding protein (CREB) regulates sensitivity to psychostimulants. To test the cell-specificity of this hypothesis we examined the effects of a dominant-negative CREB protein variant expressed in dopamine receptor D1 (D1R) neurons on cocaine-induced behaviors. A transgenic mouse strain was generated by pronuclear injection of a BAC-derived transgene harboring the A-CREB sequence under the control of the D1R gene promoter. Compared to wild-type, drug-naïve mutants showed moderate alterations in gene expression, especially a reduction in basal levels of activity-regulated transcripts such as Arc and Egr2. The behavioral responses to cocaine were elevated in mutant mice. Locomotor activity after acute treatment, psychomotor sensitization after intermittent drug injections and the conditioned locomotion after saline treatment were increased compared to wild-type littermates. Transgenic mice had significantly higher cocaine conditioned place preference, displayed normal extinction of the conditioned preference, but showed an augmented cocaine-seeking response following priming-induced reinstatement. This enhanced cocaine-seeking response was associated with increased levels of activity-regulated transcripts and prodynorphin. The primary reinforcing effects of cocaine were not altered in the mutant mice as they did not differ from wild-type in cocaine self-administration under a fixed ratio schedule at the training dose. Collectively, our data indicate that expression of a dominant-negative CREB variant exclusively in neurons expressing D1R is sufficient to recapitulate the previously reported behavioral phenotypes associated with virally expressed dominant-negative CREB. PMID:24966820

Hypoxia-inducible factor 1-alpha up-regulates the expression of phospholipase D2 in colon cancer cells under hypoxic conditions.

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Liu, Maoxi; Du, Kunli; Fu, Zhongxue; Zhang, Shouru; Wu, Xingye

2015-01-01

Hypoxia is a common characteristic of solid tumors. Recent studies confirmed that phospholipase D2 (PLD2) plays significant roles in cancer progression. In this study, correlation between the expression of PLD2 and the change in the protein level of hypoxia-inducible factor 1-alpha (HIF1-α) was studied. Thirty human colon cancer tissues were examined for the expression of HIF1-α and PLD2 protein, and mRNA levels. SW480 and SW620 cells were exposed to normoxia (20 %) or hypoxia (Hypoxic stress induced PLD2 mRNA and protein expression in SW480 and SW620 cells. Cells transfected with HIF1-α siRNA showed attenuation of hypoxia stress-induced PLD2 expression. In vivo growth decreased in response to HIF1-α and PLD2 inhibition. These results suggest that PLD2 expression in colon cancer cells is up-regulated via HIF1-α in response to hypoxic stress and underscores the crucial role of HIF1-α-induced PLD2 in tumor growth.

Glutamine deprivation induces interleukin-8 expression in ataxia telangiectasia fibroblasts.

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Kim, Min-Hyun; Kim, Aryung; Yu, Ji Hoon; Lim, Joo Weon; Kim, Hyeyoung

2014-05-01

cultured without glutamine showed higher levels of ROS and IL-8 than those transfected with negative control siRNA; increased levels of ROS and IL-8 were suppressed by the treatment of glutamine. Glutamine deprivation induces ROS production, NF-κB activation, and IL-8 expression as well as a reduction in GSH in A-T fibroblasts, all of which are attenuated by glutamine supplementation.

The Effects of IGF-1 on Trk Expressing DRG Neurons with HIV-gp120- Induced Neurotoxicity.

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Li, Hao; Liu, Zhen; Chi, Heng; Bi, Yanwen; Song, Lijun; Liu, Huaxiang

2016-01-01

HIV envelope glycoprotein gp120 is the main protein that causes HIVassociated sensory neuropathy. However, the underlying mechanisms of gp120-induced neurotoxicity are still unclear. There are lack effective treatments for relieving HIV-related neuropathic symptoms caused by gp120-induced neurotoxicity. In the present study, tyrosine kinase receptor (Trk)A, TrkB, and TrkC expression in primary cultured dorsal root ganglion (DRG) neurons with gp120-induced neurotoxicity was investigated. The effects of IGF-1 on distinct Trk-positive DRG neurons with gp120-induced neurotoxicity were also determined. The results showed that gp120 not only dose-dependently induced DRG neuronal apoptosis and inhibited neuronal survival and neurite outgrowth, but also decreased distinct Trk expression levels. IGF-1 rescued DRG neurons from apoptosis and improved neuronal survival of gp120 neurotoxic DRG neurons in vitro. IGF-1 also improved TrkA and TrkB, but not TrkC, expression in gp120 neurotoxic conditions. The effects of IGF-1 could be blocked by preincubation with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. These results suggested that gp120 may have a wide range of neurotoxicity on different subpopulations of DRG neurons, while IGF-1 might only relieve some subpopulations of DRG neurons with gp120-induced neurotoxicity. These data provide novel information of mechanisms of gp120 neurotoxicity on primary sensory neurons and the potential therapeutic effects of IGF-1 on gp120-induced neurotoxicity.

Lithium ions induce prestalk-associated gene expression and inhibit prespore gene expression in Dictyostelium discoideum

NARCIS (Netherlands)

Peters, Dorien J.M.; Lookeren Campagne, Michiel M. van; Haastert, Peter J.M. van; Spek, Wouter; Schaap, Pauline

1989-01-01

We investigated the effect of Li+ on two types of cyclic AMP-regulated gene expression and on basal and cyclic AMP-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) levels. Li+ effectively inhibits cyclic AMP-induced prespore gene expression, half-maximal inhibition occurring at about 2mM-LiCl.

Direct evidence that radiation induced micronuclei of early embryos require a mitosis for expression

International Nuclear Information System (INIS)

Mueller, W.U.; Schlusen, I.; Streffer, C.

1991-01-01

The naturally synchronous development of early mouse embryos was exploited to address the question, whether micronuclei require a mitosis for expression or whether they can be expressed in the same cell cycle, in which exposure to X-rays or caffeine took place. Experiments with 2-cell and with 4-cell embryos showed that micronulcei are expressed only if a mitosis is completed. There was no indication, even after doses up to 20 Gy, that micronuclei can be expressed before the mitosis was reached, which followed exposure. Furthermore, no nuclear fragmentation pointing to apoptosis could be detected in the cycle, in which cells were exposed. The same results were obtained when caffeine (5 mM) was used as micronucleus inducing agent. (orig.)

Changes in gene expression linked to methamphetamine-induced dopaminergic neurotoxicity.

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Xie, Tao; Tong, Liqiong; Barrett, Tanya; Yuan, Jie; Hatzidimitriou, George; McCann, Una D; Becker, Kevin G; Donovan, David M; Ricaurte, George A

2002-01-01

The purpose of these studies was to examine the role of gene expression in methamphetamine (METH)-induced dopamine (DA) neurotoxicity. First, the effects of the mRNA synthesis inhibitor, actinomycin-D, and the protein synthesis inhibitor, cycloheximide, were examined. Both agents afforded complete protection against METH-induced DA neurotoxicity and did so independently of effects on core temperature, DA transporter function, or METH brain levels, suggesting that gene transcription and mRNA translation play a role in METH neurotoxicity. Next, microarray technology, in combination with an experimental approach designed to facilitate recognition of relevant gene expression patterns, was used to identify gene products linked to METH-induced DA neurotoxicity. This led to the identification of several genes in the ventral midbrain associated with the neurotoxic process, including genes for energy metabolism [cytochrome c oxidase subunit 1 (COX1), reduced nicotinamide adenine dinucleotide ubiquinone oxidoreductase chain 2, and phosphoglycerate mutase B], ion regulation (members of sodium/hydrogen exchanger and sodium/bile acid cotransporter family), signal transduction (adenylyl cyclase III), and cell differentiation and degeneration (N-myc downstream-regulated gene 3 and tau protein). Of these differentially expressed genes, we elected to further examine the increase in COX1 expression, because of data implicating energy utilization in METH neurotoxicity and the known role of COX1 in energy metabolism. On the basis of time course studies, Northern blot analyses, in situ hybridization results, and temperature studies, we now report that increased COX1 expression in the ventral midbrain is linked to METH-induced DA neuronal injury. The precise role of COX1 and other genes in METH neurotoxicity remains to be elucidated.

GATA-dependent regulation of TPO-induced c-mpl gene expression during megakaryopoiesis.

Science.gov (United States)

Sunohara, Masataka; Morikawa, Shigeru; Fuse, Akira; Sato, Iwao

2014-01-01

Thrombopoietin (TPO) and its receptor, c-Mpl, play the crucial role during megakaryocytopoiesis. Previously, we have shown that the promoter activity of c-mpl induced by TPO is modulated by transcription through a PKC-dependent pathway and that GATA(-77) is involved as a positive regulatory element in TPO-induced c-mpl gene expression in the megakaryoblastic CMK cells. In this research, to examine participating possibility of GATA promoter element in TPO- induced c-mpl gene expression through a PKC-independent pathway, the promoter activity of site-directed mutagenesis and the effect of potein kinase C modulator were measured by a transient transfection assay system. Together with our previous results on the TPO-induced c-mpl promoter, this study indicates destruction of -77GATA in c-mpl promoter decreased the activity by 47.3% under existence of GF109203. These results suggest that GATA promoter element plays significant role in TPO-induced c-mpl gene expression through a PKC-independent pathway.

Protective effects of L-selenomethionine on space radiation induced changes in gene expression.

Science.gov (United States)

Stewart, J; Ko, Y-H; Kennedy, A R

2007-06-01

Ionizing radiation can produce adverse biological effects in astronauts during space travel. Of particular concern are the types of radiation from highly energetic, heavy, charged particles known as HZE particles. The aims of our studies are to characterize HZE particle radiation induced biological effects and evaluate the effects of L-selenomethionine (SeM) on these adverse biological effects. In this study, microarray technology was used to measure HZE radiation induced changes in gene expression, as well as to evaluate modulation of these changes by SeM. Human thyroid epithelial cells (HTori-3) were irradiated (1 GeV/n iron ions) in the presence or in the absence of 5 microM SeM. At 6 h post-irradiation, all cells were harvested for RNA isolation. Gene Chip U133Av2 from Affymetrix was used for the analysis of gene expression, and ANOVA and EASE were used for a determination of the genes and biological processes whose differential expression is statistically significant. Results of this microarray study indicate that exposure to small doses of radiation from HZE particles, 10 and 20 cGy from iron ions, induces statistically significant differential expression of 196 and 610 genes, respectively. In the presence of SeM, differential expression of 77 out of 196 genes (exposure to 10 cGy) and 336 out of 610 genes (exposure to 20 cGy) is abolished. In the presence or in the absence of SeM, radiation from HZE particles induces differential expression of genes whose products have roles in the induction of G1/S arrest during the mitotic cell cycle, as well as heat shock proteins. Some of the genes, whose expressions were affected by radiation from HZE particles and were unchanged in irradiated cells treated with SeM, have been shown to have altered expression levels in cancer cells. The conclusions of this report are that radiation from HZE particles can induce differential expression of many genes, some of which are known to play roles in the same processes that have

Transgenic mice for a tamoxifen-induced, conditional expression of the Cre recombinase in osteoclasts.

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Maria Arantzazu Sanchez-Fernandez

Full Text Available BACKGROUND: Studies on osteoclasts, the bone resorbing cells, have remained limited due to the lack of transgenic mice allowing the conditional knockout of genes in osteoclasts at any time during development or adulthood. METHODOLOGY/PRINCIPAL FINDING: We report here on the generation of transgenic mice which specifically express a tamoxifen-inducible Cre recombinase in osteoclasts. These mice, generated on C57BL/6 and FVB background, express a fusion Cre recombinase-ERT2 protein whose expression is driven by the promoter of cathepsin K (CtsK, a gene highly expressed in osteoclasts. We tested the cellular specificity of Cre activity in CtsKCreERT2 strains by breeding with Rosa26LacZ reporter mice. PCR and histological analyses of the CtsKCreERT2LacZ positive adult mice and E17.5 embryos show that Cre activity is restricted largely to bone tissue. In vitro, primary osteoclasts derived from the bone marrow of CtsKCreERT2+/-LacZ+/- adult mice show a Cre-dependent β-galactosidase activity after tamoxifen stimulation. CONCLUSIONS/SIGNIFICANCE: We have generated transgenic lines that enable the tamoxifen-induced, conditional deletion of loxP-flanked genes in osteoclasts, thus circumventing embryonic and postnatal gene lethality and avoiding gene deletion in other cell types. Such CtsKCreERT2 mice provide a convenient tool to study in vivo the different facets of osteoclast function in bone physiology during different developmental stages and adulthood of mice.

Fractalkine expression induces endothelial progenitor cell lysis by natural killer cells.

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Dilyana Todorova

Full Text Available BACKGROUND: Circulating CD34(+ cells, a population that includes endothelial progenitors, participate in the maintenance of endothelial integrity. Better understanding of the mechanisms that regulate their survival is crucial to improve their regenerative activity in cardiovascular and renal diseases. Chemokine-receptor cross talk is critical in regulating cell homeostasis. We hypothesized that cell surface expression of the chemokine fractalkine (FKN could target progenitor cell injury by Natural Killer (NK cells, thereby limiting their availability for vascular repair. METHODOLOGY/PRINCIPAL FINDINGS: We show that CD34(+-derived Endothelial Colony Forming Cells (ECFC can express FKN in response to TNF-α and IFN-γ inflammatory cytokines and that FKN expression by ECFC stimulates NK cell adhesion, NK cell-mediated ECFC lysis and microparticles release in vitro. The specific involvement of membrane FKN in these processes was demonstrated using FKN-transfected ECFC and anti-FKN blocking antibody. FKN expression was also evidenced on circulating CD34(+ progenitor cells and was detected at higher frequency in kidney transplant recipients, when compared to healthy controls. The proportion of CD34(+ cells expressing FKN was identified as an independent variable inversely correlated to CD34(+ progenitor cell count. We further showed that treatment of CD34(+ circulating cells isolated from adult blood donors with transplant serum or TNF-α/IFN-γ can induce FKN expression. CONCLUSIONS: Our data highlights a novel mechanism by which FKN expression on CD34(+ progenitor cells may target their NK cell mediated killing and participate to their immune depletion in transplant recipients. Considering the numerous diseased contexts shown to promote FKN expression, our data identify FKN as a hallmark of altered progenitor cell homeostasis with potential implications in better evaluation of vascular repair in patients.

Resveratrol reverses morphine-induced neuroinflammation in morphine-tolerant rats by reversal HDAC1 expression

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Ru-Yin Tsai

2016-06-01

Conclusion: Resveratrol restores the antinociceptive effect of morphine by reversing morphine infusion-induced spinal cord neuroinflammation and increase in TNFR1 expression. The reversal of the morphine-induced increase in TNFR1 expression by resveratrol is partially due to reversal of the morphine infusion-induced increase in HDAC1 expression. Resveratrol pretreatment can be used as an adjuvant in clinical pain management for patients who need long-term morphine treatment or with neuropathic pain.

Cimetidine attenuates vinorelbine-induced phlebitis in mice by militating E-selectin expression.

Science.gov (United States)

Wang, Zhuo; Ma, Lijuan; Wang, Xuebin; Cai, Heping; Huang, Jin; Liu, Jiyong; Hu, Jinhong; Su, Dingfeng

2014-08-01

We investigated E-selectin expression in mice and rabbits with vinorelbine-induced phlebitis and the effect of cimetidine. To find the relationship between E-selectin expression and vinorelbine-induced phlebitis. Mouse and rabbit model of vinorelbine-induced phlebitis was established by intravenous infusion of vinorelbine. Pathological observation, molecular-biological determination of E-selectin and protein function of it was evaluated. Grossly, we observed swelling, edema and cord-like vessel changes in mice receiving vinorelbine but only mild edema in mice pretreated with cimetidine. Pathological scoring yielded a total score of 37 for vinorelbine-treated mice and 17 for mice pretreated with cimetidine (P phlebitis in mice probably by suppressing increased expression of E-selectin.

Arginine induces GH gene expression by activating NOS/NO signaling in rat isolated hemi-pituitaries

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S.C.F. Olinto

2012-11-01

Full Text Available The amino acid arginine (Arg is a recognized secretagogue of growth hormone (GH, and has been shown to induce GH gene expression. Arg is the natural precursor of nitric oxide (NO, which is known to mediate many of the effects of Arg, such as GH secretion. Arg was also shown to increase calcium influx in pituitary cells, which might contribute to its effects on GH secretion. Although the mechanisms involved in the effects of Arg on GH secretion are well established, little is known about them regarding the control of GH gene expression. We investigated whether the NO pathway and/or calcium are involved in the effects of Arg on GH gene expression in rat isolated pituitaries. To this end, pituitaries from approximately 170 male Wistar rats (~250 g were removed, divided into two halves, pooled (three hemi-pituitaries and incubated or not with Arg, as well as with different pharmacological agents. Arg (71 mM, the NO donor sodium nitroprusside (SNP, 1 and 0.1 mM and a cyclic guanosine monophosphate (cGMP analogue (8-Br-cGMP, 1 mM increased GH mRNA expression 60 min later. The NO acceptor hemoglobin (0.3 µM blunted the effect of SNP, and the combined treatment with Arg and L-NAME (a NO synthase (NOS inhibitor, 55 mM abolished the stimulatory effect of Arg on GH gene expression. The calcium channel inhibitor nifedipine (3 µM also abolished Arg-induced GH gene expression. The present study shows that Arg directly induces GH gene expression in hemi-pituitaries isolated from rats, excluding interference from somatostatinergic neurons, which are supposed to be inhibited by Arg. Moreover, the data demonstrate that the NOS/NO signaling pathway and calcium mediate the Arg effects on GH gene expression.

Arginine induces GH gene expression by activating NOS/NO signaling in rat isolated hemi-pituitaries

Energy Technology Data Exchange (ETDEWEB)

Olinto, S.C.F. [Faculdade de Ciências Integradas do Pontal, Universidade Federal de Uberlândia, Ituiutaba, MG (Brazil); Adrião, M.G. [Departamento de Morfologia e Fisiologia, Universidade Federal Rural de Pernambuco, Recife, PE (Brazil); Castro-Barbosa, T.; Goulart-Silva, F.; Nunes, M.T. [Departamento de Fisiologia e Biofísica, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP (Brazil)

2012-06-01

The amino acid arginine (Arg) is a recognized secretagogue of growth hormone (GH), and has been shown to induce GH gene expression. Arg is the natural precursor of nitric oxide (NO), which is known to mediate many of the effects of Arg, such as GH secretion. Arg was also shown to increase calcium influx in pituitary cells, which might contribute to its effects on GH secretion. Although the mechanisms involved in the effects of Arg on GH secretion are well established, little is known about them regarding the control of GH gene expression. We investigated whether the NO pathway and/or calcium are involved in the effects of Arg on GH gene expression in rat isolated pituitaries. To this end, pituitaries from approximately 170 male Wistar rats (∼250 g) were removed, divided into two halves, pooled (three hemi-pituitaries) and incubated or not with Arg, as well as with different pharmacological agents. Arg (71 mM), the NO donor sodium nitroprusside (SNP, 1 and 0.1 mM) and a cyclic guanosine monophosphate (cGMP) analogue (8-Br-cGMP, 1 mM) increased GH mRNA expression 60 min later. The NO acceptor hemoglobin (0.3 µM) blunted the effect of SNP, and the combined treatment with Arg and L-NAME (an NO synthase (NOS) inhibitor, 55 mM) abolished the stimulatory effect of Arg on GH gene expression. The calcium channel inhibitor nifedipine (3 µM) also abolished Arg-induced GH gene expression. The present study shows that Arg directly induces GH gene expression in hemi-pituitaries isolated from rats, excluding interference from somatostatinergic neurons, which are supposed to be inhibited by Arg. Moreover, the data demonstrate that the NOS/NO signaling pathway and calcium mediate the Arg effects on GH gene expression.

Arginine induces GH gene expression by activating NOS/NO signaling in rat isolated hemi-pituitaries

International Nuclear Information System (INIS)

Olinto, S.C.F.; Adrião, M.G.; Castro-Barbosa, T.; Goulart-Silva, F.; Nunes, M.T.

2012-01-01

The amino acid arginine (Arg) is a recognized secretagogue of growth hormone (GH), and has been shown to induce GH gene expression. Arg is the natural precursor of nitric oxide (NO), which is known to mediate many of the effects of Arg, such as GH secretion. Arg was also shown to increase calcium influx in pituitary cells, which might contribute to its effects on GH secretion. Although the mechanisms involved in the effects of Arg on GH secretion are well established, little is known about them regarding the control of GH gene expression. We investigated whether the NO pathway and/or calcium are involved in the effects of Arg on GH gene expression in rat isolated pituitaries. To this end, pituitaries from approximately 170 male Wistar rats (∼250 g) were removed, divided into two halves, pooled (three hemi-pituitaries) and incubated or not with Arg, as well as with different pharmacological agents. Arg (71 mM), the NO donor sodium nitroprusside (SNP, 1 and 0.1 mM) and a cyclic guanosine monophosphate (cGMP) analogue (8-Br-cGMP, 1 mM) increased GH mRNA expression 60 min later. The NO acceptor hemoglobin (0.3 µM) blunted the effect of SNP, and the combined treatment with Arg and L-NAME (an NO synthase (NOS) inhibitor, 55 mM) abolished the stimulatory effect of Arg on GH gene expression. The calcium channel inhibitor nifedipine (3 µM) also abolished Arg-induced GH gene expression. The present study shows that Arg directly induces GH gene expression in hemi-pituitaries isolated from rats, excluding interference from somatostatinergic neurons, which are supposed to be inhibited by Arg. Moreover, the data demonstrate that the NOS/NO signaling pathway and calcium mediate the Arg effects on GH gene expression

Decitabine induces delayed reactive oxygen species (ROS) accumulation in leukemia cells and induces the expression of ROS generating enzymes.

Science.gov (United States)

Fandy, Tamer E; Jiemjit, Anchalee; Thakar, Manjusha; Rhoden, Paulette; Suarez, Lauren; Gore, Steven D

2014-03-01

Azanucleoside DNA methyltransferase (DNMT) inhibitors are currently approved by the U.S. Food and Drug Administration for treatment of myelodysplastic syndrome. The relative contributions of DNMT inhibition and other off-target effects to their clinical efficacy remain unclear. Data correlating DNA methylation reversal and clinical response have been conflicting. Consequently, it is necessary to investigate so-called off-target effects and their impact on cell survival and differentiation. Flow cytometry was used for cell cycle, apoptosis, and reactive oxygen species (ROS) accumulation analysis. Gene expression analysis was performed using real-time PCR. DNA methylation was detected by methylation-specific PCR. Mitochondrial membrane potential was analyzed using JC-1 dye staining. Western blotting was used for quantitative protein expression analysis. 5-Aza-2'-deoxycytidine (DAC) induced cell-cycle arrest and apoptosis in leukemia cells. p53 expression was dispensable for DAC-induced apoptosis. DAC induced delayed ROS accumulation in leukemia cells but not in solid tumor cells and p53 expression was dispensable for ROS increase. ROS increase was deoxycytidine kinase dependent, indicating that incorporation of DAC into nuclear DNA is required for ROS generation. ROS accumulation by DAC was caspase-independent and mediated the dissipation of the mitochondrial membrane potential. Concordantly, ROS scavengers diminished DAC-induced apoptosis. DAC induced the expression of different NADPH oxidase isoforms and upregulated Nox4 protein expression in an ATM-dependent manner, indicating the involvement of DNA damage signaling in Nox4 upregulation. These data highlight the importance of mechanisms other than DNA cytosine demethylation in modulating gene expression and suggest investigating the relevance of ROS accumulation to the clinical activity of DAC. ©2014 AACR

An attack of the plant parasite Cuscuta reflexa induces the expression of attAGP, an attachment protein of the host tomato.

Science.gov (United States)

Albert, Markus; Belastegui-Macadam, Xana; Kaldenhoff, Ralf

2006-11-01

Dodder or Cuscutaceae are holoparasitic plants subsisting on other dicotyledonous plants. The infection process is initiated by adherence of Cuscuta prehaustoria to the host surface, followed by penetration attempts by hyphae. In the case of a successful infection, these organs connect the parasite's vascular tissue to that of the host. Here we show that contact of Cuscuta reflexa prehaustoria to tomato induces the expression of a new arabinogalactan protein (AGP), attAGP, in the tomato precisely at the site of dodder attack. We show that attAGP is a plasma membrane-bound cell wall-localized protein. Using the RNAi technique and attAGP-targeted virus-induced gene silencing, we observed a correlation between attAGP expression level and force of attachment of the parasite to host tomatoes. If the expression level of attAGP was reduced, the C. reflexa attachment capability was significantly reduced, too. We conclude that C. reflexa infection induced a signal in the host leading to expression of tomato attAGP, which promotes the parasite's adherence.

Enhancement of ATRA-induced differentiation of neuroblastoma cells with LOX/COX inhibitors: an expression profiling study.

Science.gov (United States)

Chlapek, Petr; Redova, Martina; Zitterbart, Karel; Hermanova, Marketa; Sterba, Jaroslav; Veselska, Renata

2010-05-11

We performed expression profiling of two neuroblastoma cell lines, SK-N-BE(2) and SH-SY5Y, after combined treatment with all-trans retinoic acid (ATRA) and inhibitors of lipoxygenases (LOX) and cyclooxygenases (COX). This study is a continuation of our previous work confirming the possibility of enhancing ATRA-induced cell differentiation in these cell lines by the application of LOX/COX inhibitors and brings more detailed information concerning the mechanisms of the enhancement of ATRA-induced differentiation of neuroblastoma cells. Caffeic acid, as an inhibitor of 5-lipoxygenase, and celecoxib, as an inhibitor on cyclooxygenase-2, were used in this study. Expression profiling was performed using Human Cancer Oligo GEArray membranes that cover 440 cancer-related genes. Cluster analyses of the changes in gene expression showed the concentration-dependent increase in genes known to be involved in the process of retinoid-induced neuronal differentiation, especially in cytoskeleton remodeling. These changes were detected in both cell lines, and they were independent of the type of specific inhibitors, suggesting a common mechanism of ATRA-induced differentiation enhancement. Furthermore, we also found overexpression of some genes in the same cell line (SK-N-BE(2) or SH-SY5Y) after combined treatment with both ATRA and CA, or ATRA and CX. Finally, we also detected that gene expression was changed after treatment with the same inhibitor (CA or CX) in combination with ATRA in both cell lines. Obtained results confirmed our initial hypothesis of the common mechanism of enhancement in ATRA-induced cell differentiation via inhibition of arachidonic acid metabolic pathway.

Repeated electroacupuncture attenuating of apelin expression and function in the rostral ventrolateral medulla in stress-induced hypertensive rats.

Science.gov (United States)

Zhang, Cheng-Rong; Xia, Chun-Mei; Jiang, Mei-Yan; Zhu, Min-Xia; Zhu, Ji-Min; Du, Dong-Shu; Liu, Min; Wang, Jin; Zhu, Da-Nian

2013-08-01

Studies have revealed that apelin is a novel multifunctional peptide implicated both in blood pressure (BP) regulation and cardiac function control. Evidence shows that apelin and its receptor (APJ) in the rostral ventrolateral medulla (RVLM) may play an important role in central BP regulation; however, its role is controversial and very few reports have shown the relationship between acupuncture and apelin. Our study aims to both investigate the apelinergic system role in stress-induced hypertension (SIH) and determine whether acupuncture therapy effects on hypertension involve the apelinergic system in the RVLM. We established the stress-induced hypertensive rat (SIHR) model using electric foot-shock stressors with noise interventions. The expression of both apelin and the APJ receptor in the RVLM neurons was examined by immunohistochemical staining and Western blots. The results showed apelin expression increased remarkably in SIHR while APJ receptor expression showed no significant difference between control and SIHR groups. Microinjection of apelin-13 into the RVLM of control rats or SIHR produced pressor and tachycardic effects. Furthermore, effects induced by apelin-13 in SIHR were significantly greater than those of control rats. In addition, repetitive electroacupuncture (EA) stimulation at the Zusanli (ST-36) acupoint attenuated hypertension and apelin expression in the RVLM in SIHR; it also attenuated the pressor effect elicited by exogenous apelin-13 microinjection in SIHR. The results suggest that augmented apelin in the RVLM was part of the manifestations of SIH; the antihypertensive effects of EA might be associated with the attenuation of apelin expression and function in the RVLM, which might be a novel role for EA in SIH setting. Copyright © 2013 Elsevier Inc. All rights reserved.

Stat3 induces oncogenic Skp2 expression in human cervical carcinoma cells

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Huang, Hanhui [Shanghai Medical College of Fudan University, Shanghai 200032 (China); Zhao, Wenrong [Department of Gynecology, Obstetrics and Gynecology Hospital of Fudan University, Shanghai 200011 (China); Yang, Dan, E-mail: yangdandr@gmail.com [Department of Gynecology, Shanghai First Maternity and Infant Hospital, Tongji University, Shanghai 200040 (China)

2012-02-03

Highlights: Black-Right-Pointing-Pointer Upregulation of Skp2 by IL-6 or Stat3 activation. Black-Right-Pointing-Pointer Stat3 activates Skp2 expression through bound to its promoter region. Black-Right-Pointing-Pointer Stat3 activates Skp2 expression through recruitment of P300. Black-Right-Pointing-Pointer Stat3 activation decreases the P27 stability. -- Abstract: Dysregulated Skp2 function promotes cell proliferation, which is consistent with observations of Skp2 over-expression in many types of human cancers, including cervical carcinoma (CC). However, the molecular mechanisms underlying elevated Skp2 expression have not been fully explored. Interleukin-6 (IL-6) induced Stat3 activation is viewed as crucial for multiple tumor growth and metastasis. Here, we demonstrate that Skp2 is a direct transcriptional target of Stat3 in the human cervical carcinoma cells. Our data show that IL-6 administration or transfection of a constitutively activated Stat3 in HeLa cells activates Skp2 mRNA transcription. Using luciferase reporter and ChIP assays, we show that Stat3 binds to the promoter region of Skp2 and promotes its activity through recruiting P300. As a result of the increase of Skp2 expression, endogenous p27 protein levels are markedly decreased. Thus, our results suggest a previously unknown Stat3-Skp2 molecular network controlling cervical carcinoma development.

Ectopic AP4 expression induces cellular senescence via activation of p53 in long-term confluent retinal pigment epithelial cells.

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Wang, Yiping; Wong, Matthew Man-Kin; Zhang, Xiaojian; Chiu, Sung-Kay

2015-11-15

When cells are grown to confluence, cell-cell contact inhibition occurs and drives the cells to enter reversible quiescence rather than senescence. Confluent retinal pigment epithelial (RPE) cells exhibiting contact inhibition was used as a model in this study to examine the role of overexpression of transcription factor AP4, a highly expressed transcription factor in many types of cancer, in these cells during long-term culture. We generated stable inducible RPE cell clones expressing AP4 or AP4 without the DNA binding domain (DN-AP4) and observed that, when cultured for 24 days, RPE cells with a high level of AP4 exhibit a large, flattened morphology and even cease proliferating; these changes were not observed in DN-AP4-expressing cells or non-induced cells. In addition, AP4-expressing cells exhibited senescence-associated β-galactosidase activity and the senescence-associated secretory phenotype. We demonstrated that the induced cellular senescence was mediated by enhanced p53 expression and that AP4 regulates the p53 gene by binding directly to two of the three E-boxes present on the promoter of the p53 gene. Moreover, we showed that serum is essential for AP4 in inducing p53-associated cellular senescence. Collectively, we showed that overexpression of AP4 mediates cellular senescence involving in activation of p53 in long-term post-confluent RPE cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Calcium-mediated signaling and calmodulin-dependent kinase regulate hepatocyte-inducible nitric oxide synthase expression.

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Zhang, Baochun; Crankshaw, Will; Nesemeier, Ryan; Patel, Jay; Nweze, Ikenna; Lakshmanan, Jaganathan; Harbrecht, Brian G

2015-02-01

Induced nitric oxide synthase (iNOS) is induced in hepatocytes by shock and inflammatory stimuli. Excessive NO from iNOS mediates shock-induced hepatic injury and death, so understanding the regulation of iNOS will help elucidate the pathophysiology of septic shock. In vitro, cytokines induce iNOS expression through activation of signaling pathways including mitogen-activated protein kinases and nuclear factor κB. Cytokines also induce calcium (Ca(2+)) mobilization and activate calcium-mediated intracellular signaling pathways, typically through activation of calmodulin-dependent kinases (CaMK). Calcium regulates NO production in macrophages but the role of calcium and calcium-mediated signaling in hepatocyte iNOS expression has not been defined. Primary rat hepatocytes were isolated, cultured, and induced to produce NO with proinflammatory cytokines. Calcium mobilization and Ca(2+)-mediated signaling were altered with ionophore, Ca(2+) channel blockers, and inhibitors of CaMK. The Ca(2+) ionophore A23187 suppressed cytokine-stimulated NO production, whereas Ethylene glycol tetraacetic acid and nifedipine increased NO production, iNOS messenger RNA, and iNOS protein expression. Inhibition of CaMK with KN93 and CBD increased NO production but the calcineurin inhibitor FK 506 decreased iNOS expression. These data demonstrate that calcium-mediated signaling regulates hepatocyte iNOS expression and does so through a mechanism independent of calcineurin. Changes in intracellular calcium levels may regulate iNOS expression during hepatic inflammation induced by proinflammatory cytokines. Copyright © 2015 Elsevier Inc. All rights reserved.

Gene expression profile and genomic alterations in colonic tumours induced by 1,2-dimethylhydrazine (DMH) in rats

International Nuclear Information System (INIS)

Femia, Angelo Pietro; Luceri, Cristina; Toti, Simona; Giannini, Augusto; Dolara, Piero; Caderni, Giovanna

2010-01-01

Azoxymethane (AOM) or 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in rats shares many phenotypical similarities with human sporadic colon cancer and is a reliable model for identifying chemopreventive agents. Genetic mutations relevant to human colon cancer have been described in this model, but comprehensive gene expression and genomic analysis have not been reported so far. Therefore, we applied genome-wide technologies to study variations in gene expression and genomic alterations in DMH-induced colon cancer in F344 rats. For gene expression analysis, 9 tumours (TUM) and their paired normal mucosa (NM) were hybridized on 4 × 44K Whole rat arrays (Agilent) and selected genes were validated by semi-quantitative RT-PCR. Functional analysis on microarray data was performed by GenMAPP/MappFinder analysis. Array-comparative genomic hybridization (a-CGH) was performed on 10 paired TUM-NM samples hybridized on Rat genome arrays 2 × 105K (Agilent) and the results were analyzed by CGH Analytics (Agilent). Microarray gene expression analysis showed that Defcr4, Igfbp5, Mmp7, Nos2, S100A8 and S100A9 were among the most up-regulated genes in tumours (Fold Change (FC) compared with NM: 183, 48, 39, 38, 36 and 32, respectively), while Slc26a3, Mptx, Retlna and Muc2 were strongly down-regulated (FC: -500; -376, -167, -79, respectively). Functional analysis showed that pathways controlling cell cycle, protein synthesis, matrix metalloproteinases, TNFα/NFkB, and inflammatory responses were up-regulated in tumours, while Krebs cycle, the electron transport chain, and fatty acid beta oxidation were down-regulated. a-CGH analysis showed that four TUM out of ten had one or two chromosomal aberrations. Importantly, one sample showed a deletion on chromosome 18 including Apc. The results showed complex gene expression alterations in adenocarcinomas encompassing many altered pathways. While a-CGH analysis showed a low degree of genomic imbalance, it is interesting to

DNA damage and defence gene expression after oxidative stress induced by x-rays and diesel exhaust particles

Energy Technology Data Exchange (ETDEWEB)

Risom, Lotte

2004-07-01

Particulate air pollution is one the most important environmental health factors for people living in cities. Especially the exhaust particles from traffic are possible causes for cancer and cardiopulmonary diseases. The aim of this thesis was to characterize the health effects of diesel exhaust particles (DEP) by inducing oxidative stress and analyse the underlying mechanisms. Methods for determining oxidative stress, DNA damage, and gene expression were validated and calibrated in lung tissue by studying the dose response relations after ionizing radiation. The study showed the feasibility of partial-body x-ray irradiation as an in vivo model for induction and repair of oxidative DNA damage, of DNA repair enzymes expression, and antioxidant defense genes. A 'nose-only' mouse model for inhalation of ultra-fine particles showed that particles induce oxidative DNA damage in lung tissue and in bronchoalveolar lavage cells. The exposure increased the expression of HO-1 mRNA and oxoguanine DNA glycosylase OGG1 mRNA. The levels of 8-oxodG and OGG1 mRNA were mirror images. Colon and liver were analysed after administration of DEP in the diet with or without increasing doses of sucrose. This study indicated that DEP induces DNA adducts and oxidative stress through formation of DNA strand breaks, DNA repair enzyme expression, apoptosis, and protein oxidisation in colon and liver at relatively low exposure doses. The thesis is based on four published journal articles. (ln)

DNA damage and defence gene expression after oxidative stress induced by x-rays and diesel exhaust particles

International Nuclear Information System (INIS)

Risom, Lotte

2004-01-01

Particulate air pollution is one the most important environmental health factors for people living in cities. Especially the exhaust particles from traffic are possible causes for cancer and cardiopulmonary diseases. The aim of this thesis was to characterize the health effects of diesel exhaust particles (DEP) by inducing oxidative stress and analyse the underlying mechanisms. Methods for determining oxidative stress, DNA damage, and gene expression were validated and calibrated in lung tissue by studying the dose response relations after ionizing radiation. The study showed the feasibility of partial-body x-ray irradiation as an in vivo model for induction and repair of oxidative DNA damage, of DNA repair enzymes expression, and antioxidant defense genes. A 'nose-only' mouse model for inhalation of ultra-fine particles showed that particles induce oxidative DNA damage in lung tissue and in bronchoalveolar lavage cells. The exposure increased the expression of HO-1 mRNA and oxoguanine DNA glycosylase OGG1 mRNA. The levels of 8-oxodG and OGG1 mRNA were mirror images. Colon and liver were analysed after administration of DEP in the diet with or without increasing doses of sucrose. This study indicated that DEP induces DNA adducts and oxidative stress through formation of DNA strand breaks, DNA repair enzyme expression, apoptosis, and protein oxidisation in colon and liver at relatively low exposure doses. The thesis is based on four published journal articles. (ln)

Nuclear Factor-Kappa B Activity Regulates Brain Expression of P-Glycoprotein in the Kainic Acid-Induced Seizure Rats

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Nian Yu

2011-01-01

Full Text Available This study was aimed to investigate the effect of NF-κB activity on the seizure susceptibility, brain damage, and P-gp expression in kainic acid- (KA- induced seizure rats. Male SD rats were divided into saline control group (NS group, KA induced epilepsy group (EP group, and epilepsy group intervened with NF-κB inhibitor-pyrrolidine dithiocarbamate salt (PDTC group or with dexamethasone (DEX group. No seizures were observed in the rats of NS group. Compared with NS group, increased P-gp expression and NF-κB activation in the rat brain of the EP group were observed after KA micro-injection. Both PDTC and DEX pre-treatment significantly increased the latency to grade III or V seizure onset compared to EP group but failed to show neuron-protective effect as the number of survival neurons didn't significantly differ from that in EP group. Furthermore, PDTC pre-treatment significantly decreased P-gp expression along with NF-κB activation in the hippocampus CA3 area and amygdala complex of rats compared with the EP group, implying that NF-κB activation involved in the seizure susceptibility and seizure induced brain P-gp over-expression. Additionally, DEX pre-treatment only decreased P-gp expression level without inhibition of NF-κB activation, suggesting NF-κB independent pathway may also participate in regulating seizure induced P-gp over-expression.

Construction and Development of a Cardiac Tissue-Specific and Hypoxia-Inducible Expression Vector

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Shahrooz Ghaderi

2018-03-01

Full Text Available Purpose: Cardiovascular gene therapy is a sophisticated approach, thanks to the safety of vectors, stable transgene expression, delivery method, and different layers of the heart. To date, numerous expression vectors have been introduced in biotechnology and biopharmacy industries in relation to genetic manipulation. Despite the rapid growth of these modalities, they must be intelligently designed, addressing the cardiac-specific transgene expression and less side effects. Herein, we conducted a pilot project aiming to design a cardiac-specific hypoxia-inducible expression cassette. Methods: We explored a new approach to design an expression cassette containing cardiac specific enhancer, hypoxia response elements (HRE, cardiac specific promoter, internal ribosome entry site (IRES, and beta globin poly A sequence to elicit specific and inducible expression of the gene of interest. Enhanced green fluorescent protein (eGFP was sub-cloned by BglII and NotI into the cassette. The specificity and inducible expression of the cassette was determined in both mouse myoblast C2C12 and mammary glandular tumor 4T1 as ‘twin’ cells. eGFP expression was evaluated by immunofluorescence microscope and flow cytometry at 520 nm emission peak. Results: Our data revealed that the designed expression cassette provided tissue specific and hypoxia inducible (O2<1% transgene expression. Conclusion: It is suggested that cardiac-specific enhancer combined with cardiac-specific promoter are efficient for myoblast specific gene expression. As well, this is for the first time that HRE are derived from three well known hypoxia-regulated promoters. Therefore, there is no longer need to overlap PCR process for one repeated sequence just in one promoter.

Estrogen induced {beta}-1,4-galactosyltransferase 1 expression regulates proliferation of human breast cancer MCF-7 cells

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Choi, Hee-Jung [Department of Biological Sciences, College of Natural Sciences, Pusan National University, Busan (Korea, Republic of); Division of Applied Medicine, School of Korean Medicine, Pusan National University, Yangsan-city, Gyeongsangnam-do (Korea, Republic of); Chung, Tae-Wook; Kim, Cheorl-Ho [Department of Molecular and Cellular Glycobiology, College of Natural Science, Sungkyunkwan University, Suwon, Kyungki-do (Korea, Republic of); Jeong, Han-Sol; Joo, Myungsoo [Division of Applied Medicine, School of Korean Medicine, Pusan National University, Yangsan-city, Gyeongsangnam-do (Korea, Republic of); Youn, BuHyun, E-mail: bhyoun72@pusan.ac.kr [Department of Biological Sciences, College of Natural Sciences, Pusan National University, Busan (Korea, Republic of); Ha, Ki-Tae, E-mail: hagis@pusan.ac.kr [Division of Applied Medicine, School of Korean Medicine, Pusan National University, Yangsan-city, Gyeongsangnam-do (Korea, Republic of)

2012-10-05

Highlights: Black-Right-Pointing-Pointer We examined the regulation and biological functions of B4GALT1 expression induced by estrogen. Black-Right-Pointing-Pointer Estrogen-induced B4GALT1 expression through the direct binding of ER-{alpha} to ERE in MCF-7 cells. Black-Right-Pointing-Pointer B4GALT1 expression activates the proliferation of MCF-7 cells via its receptor function. Black-Right-Pointing-Pointer Thus, we suggest B4GALT1 as a molecular target for inhibiting breast cancer proliferation. -- Abstract: Beta 1,4-galactosyltransferase 1 (B4GALT1) synthesizes galactose {beta}-1,4-N-acetylglucosamine (Gal{beta}1-4GlcNAc) groups on N-linked sugar chains of glycoproteins, which play important roles in many biological events, including the proliferation and migration of cancer cells. A previous microarray study reported that this gene is expressed by estrogen treatment in breast cancer. In this study, we examined the regulatory mechanisms and biological functions of estrogen-induced B4GALT1 expression. Our data showed that estrogen-induced expression of B4GALT1 is localized in intracellular compartments and in the plasma membrane. In addition, B4GALT1 has an enzyme activity involved in the production of the Gal{beta}1-4GlcNAc structure. The result from a promoter assay and chromatin immunoprecipitation revealed that 3 different estrogen response elements (EREs) in the B4GALT1 promoter are critical for responsiveness to estrogen. In addition, the estrogen antagonists ICI 182,780 and ER-{alpha}-ERE binding blocker TPBM inhibit the expression of estrogen-induced B4GALT1. However, the inhibition of signal molecules relating to the extra-nuclear pathway, including the G-protein coupled receptors, Ras, and mitogen-activated protein kinases, had no inhibitory effects on B4GALT1 expression. The knock-down of the B4GALT1 gene and the inhibition of membrane B4GALT1 function resulted in the significant inhibition of estrogen-induced proliferation of MCF-7 cells. Considering

Estrogen induced β-1,4-galactosyltransferase 1 expression regulates proliferation of human breast cancer MCF-7 cells

International Nuclear Information System (INIS)

Choi, Hee-Jung; Chung, Tae-Wook; Kim, Cheorl-Ho; Jeong, Han-Sol; Joo, Myungsoo; Youn, BuHyun; Ha, Ki-Tae

2012-01-01

Highlights: ► We examined the regulation and biological functions of B4GALT1 expression induced by estrogen. ► Estrogen-induced B4GALT1 expression through the direct binding of ER-α to ERE in MCF-7 cells. ► B4GALT1 expression activates the proliferation of MCF-7 cells via its receptor function. ► Thus, we suggest B4GALT1 as a molecular target for inhibiting breast cancer proliferation. -- Abstract: Beta 1,4-galactosyltransferase 1 (B4GALT1) synthesizes galactose β-1,4-N-acetylglucosamine (Galβ1-4GlcNAc) groups on N-linked sugar chains of glycoproteins, which play important roles in many biological events, including the proliferation and migration of cancer cells. A previous microarray study reported that this gene is expressed by estrogen treatment in breast cancer. In this study, we examined the regulatory mechanisms and biological functions of estrogen-induced B4GALT1 expression. Our data showed that estrogen-induced expression of B4GALT1 is localized in intracellular compartments and in the plasma membrane. In addition, B4GALT1 has an enzyme activity involved in the production of the Galβ1-4GlcNAc structure. The result from a promoter assay and chromatin immunoprecipitation revealed that 3 different estrogen response elements (EREs) in the B4GALT1 promoter are critical for responsiveness to estrogen. In addition, the estrogen antagonists ICI 182,780 and ER-α-ERE binding blocker TPBM inhibit the expression of estrogen-induced B4GALT1. However, the inhibition of signal molecules relating to the extra-nuclear pathway, including the G-protein coupled receptors, Ras, and mitogen-activated protein kinases, had no inhibitory effects on B4GALT1 expression. The knock-down of the B4GALT1 gene and the inhibition of membrane B4GALT1 function resulted in the significant inhibition of estrogen-induced proliferation of MCF-7 cells. Considering these results, we propose that estrogen regulates the expression of B4GALT1 through the direct binding of ER-α to ERE and

Characterization of chemically induced liver injuries using gene co-expression modules.

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Gregory J Tawa

Full Text Available Liver injuries due to ingestion or exposure to chemicals and industrial toxicants pose a serious health risk that may be hard to assess due to a lack of non-invasive diagnostic tests. Mapping chemical injuries to organ-specific damage and clinical outcomes via biomarkers or biomarker panels will provide the foundation for highly specific and robust diagnostic tests. Here, we have used DrugMatrix, a toxicogenomics database containing organ-specific gene expression data matched to dose-dependent chemical exposures and adverse clinical pathology assessments in Sprague Dawley rats, to identify groups of co-expressed genes (modules specific to injury endpoints in the liver. We identified 78 such gene co-expression modules associated with 25 diverse injury endpoints categorized from clinical pathology, organ weight changes, and histopathology. Using gene expression data associated with an injury condition, we showed that these modules exhibited different patterns of activation characteristic of each injury. We further showed that specific module genes mapped to 1 known biochemical pathways associated with liver injuries and 2 clinically used diagnostic tests for liver fibrosis. As such, the gene modules have characteristics of both generalized and specific toxic response pathways. Using these results, we proposed three gene signature sets characteristic of liver fibrosis, steatosis, and general liver injury based on genes from the co-expression modules. Out of all 92 identified genes, 18 (20% genes have well-documented relationships with liver disease, whereas the rest are novel and have not previously been associated with liver disease. In conclusion, identifying gene co-expression modules associated with chemically induced liver injuries aids in generating testable hypotheses and has the potential to identify putative biomarkers of adverse health effects.

Engineering an efficient and tight D-amino acid-inducible gene expression system in Rhodosporidium/Rhodotorula species.

Science.gov (United States)

Liu, Yanbin; Koh, Chong Mei John; Ngoh, Si Te; Ji, Lianghui

2015-10-26

Rhodosporidium and Rhodotorula are two genera of oleaginous red yeast with great potential for industrial biotechnology. To date, there is no effective method for inducible expression of proteins and RNAs in these hosts. We have developed a luciferase gene reporter assay based on a new codon-optimized LUC2 reporter gene (RtLUC2), which is flanked with CAR2 homology arms and can be integrated into the CAR2 locus in the nuclear genome at >90 % efficiency. We characterized the upstream DNA sequence of a D-amino acid oxidase gene (DAO1) from R. toruloides ATCC 10657 by nested deletions. By comparing the upstream DNA sequences of several putative DAO1 homologs of Basidiomycetous fungi, we identified a conserved DNA motif with a consensus sequence of AGGXXGXAGX11GAXGAXGG within a 0.2 kb region from the mRNA translation initiation site. Deletion of this motif led to strong mRNA transcription under non-inducing conditions. Interestingly, DAO1 promoter activity was enhanced about fivefold when the 108 bp intron 1 was included in the reporter construct. We identified a conserved CT-rich motif in the intron with a consensus sequence of TYTCCCYCTCCYCCCCACWYCCGA, deletion or point mutations of which drastically reduced promoter strength under both inducing and non-inducing conditions. Additionally, we created a selection marker-free DAO1-null mutant (∆dao1e) which displayed greatly improved inducible gene expression, particularly when both glucose and nitrogen were present in high levels. To avoid adding unwanted peptide to proteins to be expressed, we converted the original translation initiation codon to ATC and re-created a translation initiation codon at the start of exon 2. This promoter, named P DAO1-in1m1 , showed very similar luciferase activity to the wild-type promoter upon induction with D-alanine. The inducible system was tunable by adjusting the levels of inducers, carbon source and nitrogen source. The intron 1-containing DAO1 promoters coupled with a DAO1 null

Undervaluing Gratitude: Expressers Misunderstand the Consequences of Showing Appreciation.

Science.gov (United States)

Kumar, Amit; Epley, Nicholas

2018-06-01

Expressing gratitude improves well-being for both expressers and recipients, but we suggest that an egocentric bias may lead expressers to systematically undervalue its positive impact on recipients in a way that could keep people from expressing gratitude more often in everyday life. Participants in three experiments wrote gratitude letters and then predicted how surprised, happy, and awkward recipients would feel. Recipients then reported how receiving an expression of gratitude actually made them feel. Expressers significantly underestimated how surprised recipients would be about why expressers were grateful, overestimated how awkward recipients would feel, and underestimated how positive recipients would feel. Expected awkwardness and mood were both correlated with participants' willingness to express gratitude. Wise decisions are guided by an accurate assessment of the expected value of action. Underestimating the value of prosocial actions, such as expressing gratitude, may keep people from engaging in behavior that would maximize their own-and others'-well-being.

Transforming growth factor β1 induces the expression of collagen type I by DNA methylation in cardiac fibroblasts.

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Xiaodong Pan

Full Text Available Transforming growth factor-beta (TGF-β, a key mediator of cardiac fibroblast activation, has a major influence on collagen type I production. However, the epigenetic mechanisms by which TGF-β induces collagen type I alpha 1 (COL1A1 expression are not fully understood. This study was designed to examine whether or not DNA methylation is involved in TGF-β-induced COL1A1 expression in cardiac fibroblasts. Cells isolated from neonatal Sprague-Dawley rats were cultured and stimulated with TGF-β1. The mRNA levels of COL1A1 and DNA methyltransferases (DNMTs were determined via quantitative polymerase chain reaction and the protein levels of collagen type I were determined via Western blot as well as enzyme-linked immunosorbent assay. The quantitative methylation of the COL1A1 promoter region was analyzed using the MassARRAY platform of Sequenom. Results showed that TGF-β1 upregulated the mRNA expression of COL1A1 and induced the synthesis of cell-associated and secreted collagen type I in cardiac fibroblasts. DNMT1 and DNMT3a expressions were significantly downregulated and the global DNMT activity was inhibited when treated with 10 ng/mL of TGF-β1 for 48 h. TGF-β1 treatment resulted in a significant reduction of the DNA methylation percentage across multiple CpG sites in the rat COL1A1 promoter. Thus, TGF-β1 can induce collagen type I expression through the inhibition of DNMT1 and DNMT3a expressions as well as global DNMT activity, thereby resulting in DNA demethylation of the COL1A1 promoter. These findings suggested that the DNMT-mediated DNA methylation is an important mechanism in regulating the TGF-β1-induced COL1A1 gene expression.

Metformin-induced inhibition of the mitochondrial respiratory chain increases FGF21 expression via ATF4 activation

International Nuclear Information System (INIS)

Kim, Kook Hwan; Jeong, Yeon Taek; Kim, Seong Hun; Jung, Hye Seung; Park, Kyong Soo; Lee, Hae-Youn; Lee, Myung-Shik

2013-01-01

Highlights: •Metformin induces FGF21 expression in an AMPK independent manner. •Metformin enhances FGF21 expression by inhibiting mitochondrial complex I activity. •The PERK-eIF2α-ATF4 axis is required for metformin-induced FGF21 expression. •Metformin activates the ATF4-FGF21 axis in the liver of mouse. •Metformin increases serum FGF21 level in diabetic human subjects. -- Abstract: Fibroblast growth factor 21 (FGF21) is an endocrine hormone that exhibits anti-obesity and anti-diabetes effects. Because metformin is widely used as a glucose-lowering agent in patients with type 2 diabetes (T2D), we investigated whether metformin modulates FGF21 expression in cell lines, and in mice or human subjects. We found that metformin increased the expression and release of FGF21 in a diverse set of cell types, including rat hepatoma FaO, primary mouse hepatocytes, and mouse embryonic fibroblasts (MEFs). Intriguingly, AMP-activated protein kinase (AMPK) was dispensable for the induction of FGF21 by metformin. Mammalian target of rapamycin complex 1 (mTORC1) and peroxisome proliferator-activated receptor α (PPARα), which are additional targets of metformin, were not involved in metformin-induced FGF21 expression. Importantly, inhibition of mitochondrial complex I activity by metformin resulted in FGF21 induction through PKR-like ER kinase (PERK)-eukaryotic translation factor 2α (eIF2α)-activating transcription factor 4 (ATF4). We showed that metformin activated ATF4 and increased FGF21 expression in the livers of mice, which led to increased serum levels of FGF21. We also found that serum FGF21 level was increased in human subjects with T2D after metformin therapy for 6 months. In conclusion, our results indicate that metformin induced expression of FGF21 through an ATF4-dependent mechanism by inhibiting mitochondrial respiration independently of AMPK. Therefore, FGF21 induction by metformin might explain a portion of the beneficial metabolic effects of metformin

Metformin-induced inhibition of the mitochondrial respiratory chain increases FGF21 expression via ATF4 activation

Energy Technology Data Exchange (ETDEWEB)

Kim, Kook Hwan [Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong Gangnam-gu, Seoul 135-710 (Korea, Republic of); Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University School of Medicine, 50 Irwon-dong Gangnam-gu, Seoul 135-710 (Korea, Republic of); Jeong, Yeon Taek [Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong Gangnam-gu, Seoul 135-710 (Korea, Republic of); Kim, Seong Hun [Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University School of Medicine, 50 Irwon-dong Gangnam-gu, Seoul 135-710 (Korea, Republic of); Jung, Hye Seung; Park, Kyong Soo [Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-dong Chongno-gu, Seoul 110-744 (Korea, Republic of); Lee, Hae-Youn [Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong Gangnam-gu, Seoul 135-710 (Korea, Republic of); Lee, Myung-Shik, E-mail: mslee0923@skku.edu [Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong Gangnam-gu, Seoul 135-710 (Korea, Republic of); Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University School of Medicine, 50 Irwon-dong Gangnam-gu, Seoul 135-710 (Korea, Republic of)

2013-10-11

Highlights: •Metformin induces FGF21 expression in an AMPK independent manner. •Metformin enhances FGF21 expression by inhibiting mitochondrial complex I activity. •The PERK-eIF2α-ATF4 axis is required for metformin-induced FGF21 expression. •Metformin activates the ATF4-FGF21 axis in the liver of mouse. •Metformin increases serum FGF21 level in diabetic human subjects. -- Abstract: Fibroblast growth factor 21 (FGF21) is an endocrine hormone that exhibits anti-obesity and anti-diabetes effects. Because metformin is widely used as a glucose-lowering agent in patients with type 2 diabetes (T2D), we investigated whether metformin modulates FGF21 expression in cell lines, and in mice or human subjects. We found that metformin increased the expression and release of FGF21 in a diverse set of cell types, including rat hepatoma FaO, primary mouse hepatocytes, and mouse embryonic fibroblasts (MEFs). Intriguingly, AMP-activated protein kinase (AMPK) was dispensable for the induction of FGF21 by metformin. Mammalian target of rapamycin complex 1 (mTORC1) and peroxisome proliferator-activated receptor α (PPARα), which are additional targets of metformin, were not involved in metformin-induced FGF21 expression. Importantly, inhibition of mitochondrial complex I activity by metformin resulted in FGF21 induction through PKR-like ER kinase (PERK)-eukaryotic translation factor 2α (eIF2α)-activating transcription factor 4 (ATF4). We showed that metformin activated ATF4 and increased FGF21 expression in the livers of mice, which led to increased serum levels of FGF21. We also found that serum FGF21 level was increased in human subjects with T2D after metformin therapy for 6 months. In conclusion, our results indicate that metformin induced expression of FGF21 through an ATF4-dependent mechanism by inhibiting mitochondrial respiration independently of AMPK. Therefore, FGF21 induction by metformin might explain a portion of the beneficial metabolic effects of metformin.

YB-1 facilitates basal and 5-fluorouracil-inducible expression of the human major vault protein (MVP) gene.

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Stein, Ulrike; Bergmann, Stephan; Scheffer, George L; Scheper, Rik J; Royer, Hans-Dieter; Schlag, Peter M; Walther, Wolfgang

2005-05-19

Vaults have been suggested to play a direct role in multidrug resistance (MDR) to anticancer drugs. The human major vault protein (MVP) also known as lung resistance-related protein (LRP) represents the predominant component of vaults that may be involved in the defense against xenobiotics. Here, we demonstrate that besides MDR-related cytostatics, also the non-MDR-related drug 5-fluorouracil (5-FU) was able to induce MVP mRNA and protein expression. Treatment with 5-FU amplified the binding activity and interaction of the transcription factor Y-box binding protein-1 (YB-1) with the Y-box of the human MVP gene promoter in a time-dependent manner. 5-FU also induced reporter expressions driven by a panel of newly generated MVP promoter deletion mutants. Interestingly, stably YB-1 overexpressing cell clones showed enhanced binding of YB-1 to the Y-box motif, associated with enhanced basal as well as 5-FU-inducible MVP promoter-driven reporter expressions. Moreover, transduction of YB-1 cDNA led to increased expression of endogenous MVP protein. Under physiological conditions, we observed a strong coexpression of MVP and YB-1 in human colon carcinoma specimen. In summary, our data demonstrate a direct involvement of YB-1 in controlling basal and 5-FU-induced MVP promoter activity. Therefore, YB-1 is directly linked to MVP-mediated drug resistance.

Retinoid-induced expression and activity of an immediate early tumor suppressor gene in vascular smooth muscle cells.

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Jeffrey W Streb

2011-04-01

Full Text Available Retinoids are used clinically to treat a number of hyper-proliferative disorders and have been shown in experimental animals to attenuate vascular occlusive diseases, presumably through nuclear receptors bound to retinoic acid response elements (RARE located in target genes. Here, we show that natural or synthetic retinoids rapidly induce mRNA and protein expression of a specific isoform of A-Kinase Anchoring Protein 12 (AKAP12β in cultured smooth muscle cells (SMC as well as the intact vessel wall. Expression kinetics and actinomycin D studies indicate Akap12β is a retinoid-induced, immediate-early gene. Akap12β promoter analyses reveal a conserved RARE mildly induced with atRA in a region that exhibits hyper-acetylation. Immunofluorescence microscopy and protein kinase A (PKA regulatory subunit overlay assays in SMC suggest a physical association between AKAP12β and PKA following retinoid treatment. Consistent with its designation as a tumor suppressor, inducible expression of AKAP12β attenuates SMC growth in vitro. Further, immunohistochemistry studies establish marked decreases in AKAP12 expression in experimentally-injured vessels of mice as well as atheromatous lesions in humans. Collectively, these results demonstrate a novel role for retinoids in the induction of an AKAP tumor suppressor that blocks vascular SMC growth thus providing new molecular insight into how retiniods may exert their anti-proliferative effects in the injured vessel wall.

Transforming growth factor β-induced expression of chondroitin sulfate proteoglycans is mediated through non-Smad signaling pathways.

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Jahan, Naima; Hannila, Sari S

2015-01-01

The expression of chondroitin sulfate proteoglycans (CSPGs) by reactive astrocytes is a major factor contributing to glial scarring and regenerative failure after spinal cord injury, but the molecular mechanisms underlying CSPG expression remain largely undefined. One contributing factor is transforming growth factor β (TGFβ), which is upregulated after injury and has been shown to induce expression of CSPGs in vitro. TGFβ typically mediates its effects through the Smad2/3 signaling pathway, and it has been suggested that this pathway is responsible for CSPG expression. However, there is evidence that TGFβ can also activate non-Smad signaling pathways. In this study, we report that TGFβ-induced expression of three different CSPGs--neurocan, brevican, and aggrecan--is mediated through non-Smad signaling pathways. We observed significant increases in TGFβ-induced expression of neurocan, brevican, and aggrecan following siRNA knockdown of Smad2 or Smad4, which indicates that Smad signaling is not required for the expression of these CSPGs. In addition, we show that neurocan, aggrecan, and brevican levels are significantly reduced when TGFβ is administered in the presence of either the PI3K inhibitor LY294002 or the mTOR inhibitor rapamycin, but not the MEK1/2 inhibitor U0126. This suggests that TGFβ mediates this effect through non-Smad-dependent activation of the PI3K-Akt-mTOR signaling pathway, and targeting this pathway may therefore be an effective means of reducing CSPG expression in the injured CNS. Copyright © 2014 Elsevier Inc. All rights reserved.

FORMALDEHYDE-INDUCED GENE EXPRESSION IN F344 RAT NASAL RESPIRATORY EPITHELIUM.

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Formaldehyde-induced gene expression in F344 rat nasal respiratory epithelium ABSTRACTFormaldehyde, an occupational and environmental toxicant used extensively in the manufacturing of many household and personal use products, is known to induce squamous cell carci...

VsENOD5, VsENOD12 and VsENOD40 expression during Rhizobium-induced nodule formation on Vicia sativa roots

DEFF Research Database (Denmark)

Vijn, I; Yang, W C; Pallisgård, N

1995-01-01

We isolated ENOD5, ENOD12 and ENOD40 homologues from Vicia sativa and studied their expression pattern during Rhizobium-induced nodule formation. Comparison of the VsENOD40 nucleotide sequence with the pea, soybean and alfalfa ENOD40 sequences showed that the sequences contain two conserved regions...... the expression pattern of VsENOD5, VsENOD12 and VsENOD40 during Rhizobium-induced nodule formation. Although the expression of these genes is largely similar to that of the pea counterparts, differences where found for the expression of VsENOD12 and VsENOD40 in Vicia. VsENOD12 is expressed in the whole...... prefixation zone II, whereas in pea ENOD12 is only expressed in the distal part of this zone. VsENOD40 is expressed in the uninfected cells of interzone II-III, while in pea ENOD40 is expressed in both the uninfected and infected cells of this zone. Udgivelsesdato: 1995-Sep...

Mucosal Progranulin expression is induced by H. pylori, but independent of Secretory Leukocyte Protease Inhibitor (SLPI expression

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Treiber Gerhard

2011-05-01

Full Text Available Abstract Background Mucosal levels of Secretory Leukocyte Protease Inhibitor (SLPI are specifically reduced in relation to H. pylori-induced gastritis. Progranulin is an epithelial growth factor that is proteolytically degraded into fragments by elastase (the main target of SLPI. Considering the role of SLPI for regulating the activity of elastase, we studied whether the H. pylori-induced reduction of SLPI and the resulting increase of elastase-derived activity would reduce the Progranulin protein levels both ex vivo and in vitro. Methods The expression of Progranulin was studied in biopsies of H. pylori-positive, -negative and -eradicated subjects as well as in the gastric tumor cell line AGS by ELISA, immunohistochemistry and real-time RT-PCR. Results H. pylori-infected subjects had about 2-fold increased antral Progranulin expression compared to H. pylori-negative and -eradicated subjects (P H. pylori infection; both epithelial and infiltrating immune cells contributed to the higher Progranulin expression levels. The H. pylori-induced upregulation of Progranulin was verified in AGS cells infected by H. pylori. The down-regulation of endogenous SLPI expression in AGS cells by siRNA methodology did not affect the Progranulin expression independent of the infection by H. pylori. Conclusions Taken together, Progranulin was identified as novel molecule that is upregulated in context to H. pylori infection. In contrast to other diseases, SLPI seems not to have a regulatory role for Progranulin in H. pylori-mediated gastritis.

Mucosal Progranulin expression is induced by H. pylori, but independent of Secretory Leukocyte Protease Inhibitor (SLPI) expression.

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Wex, Thomas; Kuester, Doerthe; Schönberg, Cornelius; Schindele, Daniel; Treiber, Gerhard; Malfertheiner, Peter

2011-05-26

Mucosal levels of Secretory Leukocyte Protease Inhibitor (SLPI) are specifically reduced in relation to H. pylori-induced gastritis. Progranulin is an epithelial growth factor that is proteolytically degraded into fragments by elastase (the main target of SLPI). Considering the role of SLPI for regulating the activity of elastase, we studied whether the H. pylori-induced reduction of SLPI and the resulting increase of elastase-derived activity would reduce the Progranulin protein levels both ex vivo and in vitro. The expression of Progranulin was studied in biopsies of H. pylori-positive, -negative and -eradicated subjects as well as in the gastric tumor cell line AGS by ELISA, immunohistochemistry and real-time RT-PCR. H. pylori-infected subjects had about 2-fold increased antral Progranulin expression compared to H. pylori-negative and -eradicated subjects (P Progranulin and SLPI levels were identified. Immunohistochemical analysis confirmed the upregulation of Progranulin in relation to H. pylori infection; both epithelial and infiltrating immune cells contributed to the higher Progranulin expression levels. The H. pylori-induced upregulation of Progranulin was verified in AGS cells infected by H. pylori. The down-regulation of endogenous SLPI expression in AGS cells by siRNA methodology did not affect the Progranulin expression independent of the infection by H. pylori. Taken together, Progranulin was identified as novel molecule that is upregulated in context to H. pylori infection. In contrast to other diseases, SLPI seems not to have a regulatory role for Progranulin in H. pylori-mediated gastritis.

Changes in expression of cytokines in polyhexamethylene guanidine-induced lung fibrosis in mice: Comparison of bleomycin-induced lung fibrosis.

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Kim, Min-Seok; Kim, Sung-Hwan; Jeon, Doin; Kim, Hyeon-Young; Lee, Kyuhong

2018-01-15

Inhalation of polyhexamethylene guanidine (PHMG) causes irreversible pulmonary injury, such as pulmonary fibrosis. However, the mechanism underlying PHMG-induced lung injury is unclear. In this study, we compared the difference in time-dependent lung injury between PHMG- and bleomycin (BLM)-treated mice and determined cytokines involved in inducing lung injury by performing cytokine antibody array analysis. Mice were treated once with 1.8mg/kg BLM or 1.2mg/kg PHMG through intratracheal instillation and were sacrificed on days 7 and 28. Bronchoalveolar lavage fluid (BALF) analysis showed that the number of neutrophils was significantly higher in PHMG-treated mice than in BLM-treated mice on day 7. Histopathological analysis showed inflammatory cell infiltration and fibrosis mainly in the terminal bronchioles and alveoli in the lungs of PHMG- and BLM-treated mice. However, continuous macrophage infiltration in the alveolar space and bronchioloalveolar epithelial hyperplasia (BEH) were only observed in PHMG-treated mice. Cytokine antibody array analysis showed that 15 and eight cytokines were upregulated in PHMG- and BLM-treated mice, respectively, on day 7. On day 28, 13 and five cytokines were upregulated in PHMG and BLM-treated mice, respectively. In addition, the expressed cytokines between days 7 and 28 in BLM-treated mice were clearly different, but were similar in PHMG-treated mice. Consequently, between PHMG- and BLM-treated mice, we observed differences in the expression patterns and types of cytokines. These differences are considered to be a result of the inflammatory processes induced by both substances, which may mainly involve macrophage infiltration. Therefore, continuous induction of the inflammatory response by PHMG may play an important role in the development of pulmonary fibrosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of C-phycocyanin and Spirulina on Salicylate-Induced Tinnitus, Expression of NMDA Receptor and Inflammatory Genes

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Hwang, Juen-Haur; Chen, Jin-Cherng; Chan, Yin-Ching

2013-01-01

Effects of C-phycocyanin (C-PC), the active component of Spirulina platensis water extract on the expressions of N-methyl D-aspartate receptor subunit 2B (NR2B), tumor necrosis factor–α (TNF-α), interleukin-1β (IL-1β), and cyclooxygenase type 2 (COX-2) genes in the cochlea and inferior colliculus (IC) of mice were evaluated after tinnitus was induced by intraperitoneal injection of salicylate. The results showed that 4-day salicylate treatment (unlike 4-day saline treatment) caused a significant increase in NR2B, TNF-α, and IL-1β mRNAs expression in the cochlea and IC. On the other hand, dietary supplementation with C-PC or Spirulina platensis water extract significantly reduced the salicylate-induced tinnitus and down-regulated the mRNAs expression of NR2B, TNF-α, IL-1β mRNAs, and COX-2 genes in the cochlea and IC of mice. The changes of protein expression levels were generally correlated with those of mRNAs expression levels in the IC for above genes. PMID:23533584

Effects of C-phycocyanin and Spirulina on salicylate-induced tinnitus, expression of NMDA receptor and inflammatory genes.

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Juen-Haur Hwang

Full Text Available Effects of C-phycocyanin (C-PC, the active component of Spirulina platensis water extract on the expressions of N-methyl D-aspartate receptor subunit 2B (NR2B, tumor necrosis factor-α (TNF-α, interleukin-1β (IL-1β, and cyclooxygenase type 2 (COX-2 genes in the cochlea and inferior colliculus (IC of mice were evaluated after tinnitus was induced by intraperitoneal injection of salicylate. The results showed that 4-day salicylate treatment (unlike 4-day saline treatment caused a significant increase in NR2B, TNF-α, and IL-1β mRNAs expression in the cochlea and IC. On the other hand, dietary supplementation with C-PC or Spirulina platensis water extract significantly reduced the salicylate-induced tinnitus and down-regulated the mRNAs expression of NR2B, TNF-α, IL-1β mRNAs, and COX-2 genes in the cochlea and IC of mice. The changes of protein expression levels were generally correlated with those of mRNAs expression levels in the IC for above genes.

Kaempferol induces DNA damage and inhibits DNA repair associated protein expressions in human promyelocytic leukemia HL-60 cells.

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Wu, Lung-Yuan; Lu, Hsu-Feng; Chou, Yu-Cheng; Shih, Yung-Luen; Bau, Da-Tian; Chen, Jaw-Chyun; Hsu, Shu-Chun; Chung, Jing-Gung

2015-01-01

Numerous evidences have shown that plant flavonoids (naturally occurring substances) have been reported to have chemopreventive activities and protect against experimental carcinogenesis. Kaempferol, one of the flavonoids, is widely distributed in fruits and vegetables, and may have cancer chemopreventive properties. However, the precise underlying mechanism regarding induced DNA damage and suppressed DNA repair system are poorly understood. In this study, we investigated whether kaempferol induced DNA damage and affected DNA repair associated protein expression in human leukemia HL-60 cells in vitro. Percentages of viable cells were measured via a flow cytometry assay. DNA damage was examined by Comet assay and DAPI staining. DNA fragmentation (ladder) was examined by DNA gel electrophoresis. The changes of protein levels associated with DNA repair were examined by Western blotting. Results showed that kaempferol dose-dependently decreased the viable cells. Comet assay indicated that kaempferol induced DNA damage (Comet tail) in a dose-dependent manner and DAPI staining also showed increased doses of kaempferol which led to increased DNA condensation, these effects are all of dose-dependent manners. Western blotting indicated that kaempferol-decreased protein expression associated with DNA repair system, such as phosphate-ataxia-telangiectasia mutated (p-ATM), phosphate-ataxia-telangiectasia and Rad3-related (p-ATR), 14-3-3 proteins sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK), O(6)-methylguanine-DNA methyltransferase (MGMT), p53 and MDC1 protein expressions, but increased the protein expression of p-p53 and p-H2AX. Protein translocation was examined by confocal laser microscopy, and we found that kaempferol increased the levels of p-H2AX and p-p53 in HL-60 cells. Taken together, in the present study, we found that kaempferol induced DNA damage and suppressed DNA repair and inhibited DNA repair associated protein expression in HL-60

Retinal neuroprotection by hypoxic preconditioning is independent of hypoxia-inducible factor-1 alpha expression in photoreceptors.

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Thiersch, Markus; Lange, Christina; Joly, Sandrine; Heynen, Severin; Le, Yun Zheng; Samardzija, Marijana; Grimm, Christian

2009-06-01

Hypoxic preconditioning stabilizes hypoxia-inducible factor (HIF) 1 alpha in the retina and protects photoreceptors against light-induced cell death. HIF-1 alpha is one of the major transcription factors responding to low oxygen tension and can differentially regulate a large number of target genes. To analyse whether photoreceptor-specific expression of HIF-1 alpha is essential to protect photoreceptors by hypoxic preconditioning, we knocked down expression of HIF-1 alpha specifically in photoreceptor cells, using the cyclization recombinase (Cre)-lox system. The Cre-mediated knockdown caused a 20-fold reduced expression of Hif-1 alpha in the photoreceptor cell layer. In the total retina, RNA expression was reduced by 65%, and hypoxic preconditioning led to only a small increase in HIF-1 alpha protein levels. Accordingly, HIF-1 target gene expression after hypoxia was significantly diminished. Retinas of Hif-1 alpha knockdown animals did not show any pathological alterations, and tolerated hypoxic exposure in a comparable way to wild-type retinas. Importantly, the strong neuroprotective effect of hypoxic preconditioning against light-induced photoreceptor degeneration persisted in knockdown mice, suggesting that hypoxia-mediated survival of light exposure does not depend on an autocrine action of HIF-1 alpha in photoreceptor cells. Hypoxia-mediated stabilization of HIF-2 alpha and phosphorylation of signal transducer and activator of transcription 3 (STAT 3) were not affected in the retinas of Hif-1 alpha knockdown mice. Thus, these factors are candidates for regulating the resistance of photoreceptors to light damage after hypoxic preconditioning, along with several potentially neuroprotective genes that were similarly induced in hypoxic knockdown and control mice.

Endogenous α-crystallin inhibits expression of caspase-3 induced by hypoxia in retinal neurons.

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Ying, Xi; Peng, Yanli; Zhang, Jiaping; Wang, Xingli; Wu, Nan; Zeng, Yuxiao; Wang, Yi

2014-08-28

To investigate the expression of endogenous, hypoxic stress-induced α-crystallin and caspase-3 in rat retinal neurons in vitro. Retinal neurons were cultured from Long-Evans rats. The expression of endogenous α-crystallin was analyzed by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). Furthermore, hypoxic exposure was performed in cultured cells, and the expression of endogenous α-crystallin and caspase-3 was assayed by Western blotting. Positive α-crystallin staining was observed in cultured retinal neurons, and expression of endogenous α-crystallin mRNA peaked 3-5d after inoculation (Pendogenous, hypoxic stress-induced α-crystallin expression increased gradually, peaking 6h after hypoxia. The expression was more abundant compared to the control (Pendogenous α-crystallin in retinal neurons, especially over-expression induced by hypoxic stress, results in the down regulation of caspase-3. The data suggest that endogenous α-crystallin may act as an endogenous neuroprotective factor in retinal neurons. Copyright © 2014 Elsevier Inc. All rights reserved.

Enhancement of ATRA-induced differentiation of neuroblastoma cells with LOX/COX inhibitors: an expression profiling study

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Hermanova Marketa

2010-05-01

Full Text Available Abstract Background We performed expression profiling of two neuroblastoma cell lines, SK-N-BE(2 and SH-SY5Y, after combined treatment with all-trans retinoic acid (ATRA and inhibitors of lipoxygenases (LOX and cyclooxygenases (COX. This study is a continuation of our previous work confirming the possibility of enhancing ATRA-induced cell differentiation in these cell lines by the application of LOX/COX inhibitors and brings more detailed information concerning the mechanisms of the enhancement of ATRA-induced differentiation of neuroblastoma cells. Methods Caffeic acid, as an inhibitor of 5-lipoxygenase, and celecoxib, as an inhibitor on cyclooxygenase-2, were used in this study. Expression profiling was performed using Human Cancer Oligo GEArray membranes that cover 440 cancer-related genes. Results Cluster analyses of the changes in gene expression showed the concentration-dependent increase in genes known to be involved in the process of retinoid-induced neuronal differentiation, especially in cytoskeleton remodeling. These changes were detected in both cell lines, and they were independent of the type of specific inhibitors, suggesting a common mechanism of ATRA-induced differentiation enhancement. Furthermore, we also found overexpression of some genes in the same cell line (SK-N-BE(2 or SH-SY5Y after combined treatment with both ATRA and CA, or ATRA and CX. Finally, we also detected that gene expression was changed after treatment with the same inhibitor (CA or CX in combination with ATRA in both cell lines. Conclusions Obtained results confirmed our initial hypothesis of the common mechanism of enhancement in ATRA-induced cell differentiation via inhibition of arachidonic acid metabolic pathway.

Salicylic acid inhibits UV- and Cis-Pt-induced human immunodeficiency virus expression

International Nuclear Information System (INIS)

Woloschak, G.E.; Panozzo, J.; Libertin, C.R.; Schreck, S.; South Carolina Univ., Columbia, SC

1994-01-01

Previous studies have shown that exposure of HeLa cells stably transfected with a human immunodeficiency virus-long terminal repeat-chloramphenicol acetyl transferase (HIV-LTR-CAT) construct to UV light-induced expression from the HIV LTR. By culturing the cells with salicylic acid we demonstrated dose-dependent repression of this induced HIV expression. Repression was evident if salicylic acid was administered 2 h before, at the same time as, or up to 6 h after exposure to the DNA-damaging agent. The kinetics were similar for UV- and for cis-Pt-induced HIV expression, and induction was dependent on the UV dose or cis-Pt concentration added to the culture. These results suggest a role for the prostaglandins or the cyclooxygenase pathway or both in HIV induction mediated by DNA-damaging agents

Genistein-induced alterations of radiation-responsive gene expression

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Grace, M.B. [Armed Forces Radiobiology Research Institute, 8901 Wisconsin Avenue, Bethesda, MD 20889-5603 (United States)], E-mail: grace@afrri.usuhs.mil; Blakely, W.F.; Landauer, M.R. [Armed Forces Radiobiology Research Institute, 8901 Wisconsin Avenue, Bethesda, MD 20889-5603 (United States)

2007-07-15

In order to clarify the molecular mechanism of radioprotection and understand biological dosimetry in the presence of medical countermeasure-radioprotectants, their effects on ionizing radiation (IR)-responsive molecular biomarkers must be examined. We used genistein in a radiation model system and measured gene expression by multiplex QRT-PCR assay in drug-treated healthy human blood cultures. Genistein has been demonstrated to be a radiosensitizer of malignant cells and a radioprotector against IR-induced lethality in a mouse model. Whole-blood cultures were supplemented with 50, 100, and 200{mu}M concentrations of genistein, 16 h prior to receiving a 2-Gy ({sup 60}Co-{gamma} rays, 10 cGy/min) dose of IR. Total RNA was isolated from whole blood 24 h postirradiation for assessments. Combination treatments of genistein and IR resulted in no significant genistein effects on ddb2 and bax downstream transcripts to p53, or proliferating cell-nuclear antigen, pcna, necessary for DNA synthesis and cell-cycle progression. Use of these radiation-responsive targets would be recommended for dose-assessment applications. We also observed decreased expression of pro-survival transcript, bcl-2. Genistein and IR-increased expression of cdkn1a and gadd45a, showing that genistein also stimulates p53 transcriptional activity. These results confirm published molecular signatures for genistein in numerous in vitro models. Evaluation of gene biomarkers may be further exploited for devising novel radiation countermeasure and/or therapeutic strategies.

Water avoidance stress induces frequency through cyclooxygenase-2 expression: a bladder rat model.

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Yamamoto, Keisuke; Takao, Tetsuya; Nakayama, Jiro; Kiuchi, Hiroshi; Okuda, Hidenobu; Fukuhara, Shinichiro; Yoshioka, Iwao; Matsuoka, Yasuhiro; Miyagawa, Yasushi; Tsujimura, Akira; Nonomura, Norio

2012-02-01

Water avoidance stress is a potent psychological stressor and it is associated with visceral hyperalgesia, which shows degeneration of the urothelial layer mimicking interstitial cystitis. Cyclooxygenase-2 inhibitors have been recognized to ameliorate frequency both in clinical and experimental settings. We investigated the voiding pattern and cyclooxygenase-2 expression in a rat bladder model of water avoidance stress. After being subjected to water avoidance stress or a sham procedure, rats underwent metabolic cage analysis and cystometrography. Real time reverse transcription polymerase chain reaction was carried out to examine cyclooxygenase-2 messenger ribonucleic acid in bladders of rats. Protein expression of cyclooxygenase-2 was analyzed with immunohistochemistry and western blotting. Furthermore, the effects of the cyclooxygenase-2 inhibitor, etodolac, were investigated by carrying out cystometrography, immunohistochemistry and western blotting. Metabolic cage analysis and cystometrography showed significantly shorter intervals and less volume of voiding in water avoidance stress rats. Significantly higher expression of cyclooxygenase-2 messenger ribonucleic acid was verified by reverse transcription polymerase chain reaction. Immunohistochemistry and western blotting showed significantly higher cyclooxygenase-2 protein levels in water avoidance stress bladders. Furthermore, immunohistochemistry showed high cyclooxygenase-2 expression exclusively in smooth muscle cells. All water avoidance stress-induced changes were reduced by cyclooxygenase-2 inhibitor pretreatment. Chronic stress might cause frequency through cyclooxygenase-2 gene upregulation in bladder smooth muscle cells. Further study of cyclooxygenase-2 in the water avoidance stress bladder might provide novel therapeutic modalities for interstitial cystitis. © 2011 The Japanese Urological Association.

Heavy metal-induced gene expression in fish and fish cell lines

International Nuclear Information System (INIS)

Price-Haughey, J.; Bonham, K.; Gedamu, L.

1986-01-01

Two isoforms of metallothionein (MT) have been isolated from rainbow trout livers following CdCl 2 injections. These MTs have been identified by standard procedures and appear to be similar to mammalian MTs. Total RNA from such induced livers was shown to contain high levels of MT-mRNA activity when translated in cell free systems. This activity was demonstrated to be in the 8 to 10S region of a sucrose gradient. The RNA fractions also showed homology to a mouse MT-I cDNA probe. The exposure of rainbow trout hepatoma (RTH) cells to various concentrations of CdCl 2 and ZnCl 2 induced the expression of MT and MT-mRNA. Exposure of Chinook salmon embryonic (CHSE) cells to these metals, however, did not result in MT synthesis, suggesting that the MT genes have not become committed to transcription. Instead, an unknown low molecular weight (MW = 14 kDa) protein was induced. This metal-inducible protein (MIP) was capable of binding 109 Cd and was stable to heating, while the binding of the metal to this protein was not. These characteristics have been reported for a protein induced in rainbow trout liver following environmental exposure to cadmium

Expanding the molecular toolbox for Lactococcus lactis: construction of an inducible thioredoxin gene fusion expression system

LENUS (Irish Health Repository)

Douillard, Francois P

2011-08-09

Abstract Background The development of the Nisin Inducible Controlled Expression (NICE) system in the food-grade bacterium Lactococcus lactis subsp. cremoris represents a cornerstone in the use of Gram-positive bacterial expression systems for biotechnological purposes. However, proteins that are subjected to such over-expression in L. lactis may suffer from improper folding, inclusion body formation and\\/or protein degradation, thereby significantly reducing the yield of soluble target protein. Although such drawbacks are not specific to L. lactis, no molecular tools have been developed to prevent or circumvent these recurrent problems of protein expression in L. lactis. Results Mimicking thioredoxin gene fusion systems available for E. coli, two nisin-inducible expression vectors were constructed to over-produce various proteins in L. lactis as thioredoxin fusion proteins. In this study, we demonstrate that our novel L. lactis fusion partner expression vectors allow high-level expression of soluble heterologous proteins Tuc2009 ORF40, Bbr_0140 and Tuc2009 BppU\\/BppL that were previously insoluble or not expressed using existing L. lactis expression vectors. Over-expressed proteins were subsequently purified by Ni-TED affinity chromatography. Intact heterologous proteins were detected by immunoblotting analyses. We also show that the thioredoxin moiety of the purified fusion protein was specifically and efficiently cleaved off by enterokinase treatment. Conclusions This study is the first description of a thioredoxin gene fusion expression system, purposely developed to circumvent problems associated with protein over-expression in L. lactis. It was shown to prevent protein insolubility and degradation, allowing sufficient production of soluble proteins for further structural and functional characterization.

Induced ICER Iγ down-regulates cyclin A expression and cell proliferation in insulin-producing β cells

International Nuclear Information System (INIS)

Inada, Akari; Weir, Gordon C.; Bonner-Weir, Susan

2005-01-01

We have previously found that cyclin A expression is markedly reduced in pancreatic β-cells by cell-specific overexpression of repressor inducible cyclic AMP early repressor (ICER Iγ) in transgenic mice. Here we further examined regulatory effects of ICER Iγ on cyclin A gene expression using Min6 cells, an insulin-producing cell line. The cyclin A promoter luciferase assay showed that ICER Iγ directly repressed cyclin A gene transcription. In addition, upon ICER Iγ overexpression, cyclin A mRNA levels markedly decreased, thereby confirming an inhibitory effect of ICER Iγ on cyclin A expression. Suppression of cyclin A results in inhibition of BrdU incorporation. Under normal culture conditions endogenous cyclin A is abundant in these cells, whereas ICER is hardly detectable. However, serum starvation of Min6 cells induces ICER Iγ expression with a concomitant very low expression level of cyclin A. Cyclin A protein is not expressed unless the cells are in active DNA replication. These results indicate a potentially important anti-proliferative effect of ICER Iγ in pancreatic β cells. Since ICER Iγ is greatly increased in diabetes as well as in FFA- or high glucose-treated islets, this effect may in part exacerbate diabetes by limiting β-cell proliferation

The relationship between radiation-induced apoptosis and the expression of cytokines in the rat's liver

International Nuclear Information System (INIS)

An, Eun Joo; Lee, Kyung Ja; Rhee, Chung Sik

2000-01-01

To determine the role of cytokines in the apoptosis of rat's liver following irradiation. Sprague-Dawley rats were irradiated to entire body with a single dose of 8 Gy. The rats were divided into 5 groups according to the sacrifice day after irradiation. The liver and blood after 1, 3, 5, 7, and 14 days irradiation were sampled for evaluation of mechanism of apoptosis and role of cytokine in relation to radiation-induced tissue damage. The study was composed of microscopic evaluation of liver tissue, in situ detection method for apoptosis, immunohistochemical stain of IL-1, IL-4, IL-6 and TNF, bioassay and radioimmunoassay of IL-6 in liver tissue and blood. Radiation-induced liver damage was noted from first day of radiation, and most severe parenchymal damage associated with infiltration of chronic inflammatory cells was seen in the groups of 5 days after radiation. A number of apoptosis were observed 1 day after radiation on both light microscope and in situ method. Afterwards, the number of apoptosis was gradually diminished. On immunohistochemical study, IL-1 and TNF were expressed 1, 3 days after radiation, but not expressed after that. IL-4 was not expressed in the entire groups. IL-6 was expressed with strong positivity in 1, 3 days after radiation. Bioassay and RIA of IL-6 in liver tissue and blood showed the highest value in 1 day after radiation, and the value is diminished after then. Apoptosis seemed to be the important mechanism of radiation-induced liver damage, and is possibly induced by the release of cytokines, such as IL-1, IL-6, TNF in view the simultaneously increased appearance of apoptosis and cytokines

Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its expected roles in the bovine endometrium during gestation.

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Mishra, B; Kizaki, K; Koshi, K; Ushizawa, K; Takahashi, T; Hosoe, M; Sato, T; Ito, A; Hashizume, K

2012-02-01

Extracellular matrix metalloproteinase inducer (EMMPRIN) and its induced matrix metalloproteinases (MMPs) play a crucial role in tissue remodeling during the peri-implantation period. However, the role of EMMPRIN in the bovine placenta is still unclear. We have postulated that EMMPRIN might play a regulatory role in trophoblastic cell functions during gestation by itself or through the regulation of MMP expression. In this study, EMMPRIN mRNA was detected in the bovine placentome and interplacentome throughout gestation, and its expression was significantly higher in the cotyledon during late gestation. In situ hybridization showed that EMMPRIN mRNA was expressed in the caruncular epithelium and the cotyledonary epithelium, including binucleate cells. Western blot analysis detected a band representing a protein of approximately 65 kDa in the caruncular and cotyledonary tissues, and the intensity of its expression was increased in both of these tissues during late gestation. The expression levels of MMP-2 and MMP-14 in the bovine placenta were higher during late gestation, as was observed for EMMPRIN. Therefore, EMMPRIN might regulate trophoblastic cell functions, especially those of binucleate cells, through MMP expression in the bovine placenta. Copyright © 2012 Elsevier Inc. All rights reserved.

Protective effects of the exopolysaccharide Lasiodiplodan against DNA damage and inflammation induced by doxorubicin in rats: Cytogenetic and gene expression assays

International Nuclear Information System (INIS)

Mello, M.B.; Machado, C.S.; Ribeiro, D.L.; Aissa, A.F.; Burim, R.V.; Alves da Cunha, M.A.; Barcelos, G.R.M.

2017-01-01

The lasiodiplodan (LS) is a β-(1 → 6)-D-glucan produced by the fungus Lasiodiplodia theobromae and some of the biological activities of LS were reported as hypoglycemic, anticoagulant, anti-proliferative and anticancer action; however, its effects on DNA instability and modulation of gene expression are still unclear. Aims of study were investigate the genotoxic effects of lasiodiplodan, and its protective activity against DNA damage induced by doxorubicin (DXR) and its impact on the expression of genes associated with DNA damage and inflammatory response pathways. Therefore, Wistar rats were treated (15 days) orally with LS (5.0; 10 and 20 mg/kg bw) alone and in combination with DXR (15 mg/kg bw; administrated intraperitoneally on 14th day) as well as their respective controls: distilled water and DXR. Monitoring of DNA damage was assessed by comet and micronucleus (MN) assays and gene expression was evaluated by PCR-Arrays. Treatments with LS alone did not induce disturbances on DNA; when LS was given in combination with DXR, comet and MN formations were reduced to those found in the respective controls. Moreover, LS was able to reduce the disturbances on gene expressions induced by DXR treatment, since the animals that receive LS associated with DXR showed no alteration in the expression of genes related to DNA damage response. Also, DXR induced several up- and down-regulation of several genes associated to inflammatory process, while the animals that received LS + DXR had their gene expression patterns similar to those found in the control group. In conclusion, our results showed that LS did not induce disturbances on DNA stability and significantly reduce the DNA damage and inflammation caused by DXR exposure. In addition, we give further information concerning the molecular mechanisms associated to LS protective effects which seems to be a promising nutraceutical with chemopreventive potential.

Modulation of iridovirus-induced apoptosis by endocytosis, early expression, JNK, and apical caspase

International Nuclear Information System (INIS)

Chitnis, Nilesh S.; D'Costa, Susan M.; Paul, Eric R.; Bilimoria, Shaen L.

2008-01-01

Chilo iridescent virus (CIV) is the type species for the family Iridoviridae, which are large, isometric, cytoplasmic dsDNA viruses. We examined the mechanism of apoptosis induction by CIV. High CIV doses (CIV XS ; 400 μg/ml), UV-irradiated virus (CIV UV ; 10 μg/ml) and CVPE (CIV protein extract; 10 μg/ml) induced apoptosis in 60% of treated Choristoneura fumiferana (IPRI-CF-124T) cells. Normal doses of infectious CIV (10 μg/ml) induced apoptosis in only 10% of C. fumiferana (CF) cells. Apoptosis was inhibited by Z-IETD-FMK, an apical caspase inhibitor, indicating that CIV-induced apoptosis requires caspase activity. The putative caspase in CF cells was designated Cf-caspase-i. CIV UV or CVPE enhanced Cf-caspase-i activity by 80% at 24 h relative to mock-treated cells. Since the MAP kinase pathway induces or inhibits apoptosis depending on the context, we used JNK inhibitor SP600125 and demonstrated drastic suppression of CVPE-induced apoptosis. Thus, the JNK signaling pathway is significant for apoptosis in this system. Virus interaction with the cell surface was not sufficient for apoptosis since CIV UV particles bound to polysterene beads failed to induce apoptosis. Endocytosis inhibitors (bafilomycin or ammonium chloride) negated apoptosis induction by CIV UV , CIV XS or CVPE indicating that entry through this mode is required. Given the weak apoptotic response to infectious CIV, we postulated that viral gene expression inhibited apoptosis. CIV infection of cells pretreated with cycloheximide induced apoptosis in 69% of the cells compared to 10% in normal infections. Furthermore, blocking viral DNA replication with aphidicolin or phosphonoacetic acid suppressed apoptosis and Cf-caspase-i activity, indicating that early viral expression is necessary for inhibition of apoptosis, and de novo synthesis of viral proteins is not required for induction. We show for the first time that, in a member of the family Iridoviridae, apoptosis: (i) requires entry and

Experimentally nonylphenol-polluted diet induces the expression of silent genes VTG and ERα in the liver of male lizard Podarcis sicula

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Verderame, Mariailaria; Prisco, Marina; Andreuccetti, Piero; Aniello, Francesco; Limatola, Ermelinda

2011-01-01

Endocrine Disruptor Chemicals (EDCs) with estrogen-like properties i.e nonylphenol (NP) induce vitellogenin (VTG) synthesis in males of aquatic and semi-aquatic specie. In the oviparous species VTG is a female-specific oestrogen dependent protein. Males are unable to synthesize VTG except after E 2 treatment. This study aimed to verify if NP, administered via food and water, is able to induce the expression of VTG even in males of vertebrates with a terrestrial habitat such as the lizard Podarcis. By means of ICC, ISH, W/B and ELISA we demonstrated that NP induces the presence of VTG in the plasma and its expression in the liver. VTG, undetectable in untreated males, reaches the value of 4.34 μg/μl in the experimental ones. Expression analysis and ISH in the liver showed that an NP-polluted diet also elicits the expression of ERα in the liver which is known to be related to VTG synthesis in Podarcis. - Highlights: → Nonylphenol (NP) polluted diet induces VTG synthesis in a terrestrial vertebrate. → VTG and ERα genes are unexpressed in the liver of untreated male lizards Podarcis. → In the liver cells of NP-treated males the expression of both VTG and ERα occurs. → In treated males VTG synthesis is coupled with ERα expression as in breeding females. - NP-polluted diet induces the expression of ERα and VTG in the liver.

Proanthocyanidins Prevent High Glucose-Induced Eye Malformation by Restoring Pax6 Expression in Chick Embryo

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Rui-Rong Tan

2015-08-01

Full Text Available Gestational diabetes mellitus (GDM is one of the leading causes of offspring malformations, in which eye malformation is an important disease. It has raised demand for therapy to improve fetal outcomes. In this study, we used chick embryo to establish a GDM model to study the protective effects of proanthocyanidins on eye development. Chick embryos were exposed to high glucose (0.2 mmol/egg on embryo development day (EDD 1. Proanthocyanidins (1 and 10 nmol/egg were injected into the air sac on EDD 0. Results showed that both dosages of proanthocyanidins could prevent the eye malformation and rescue the high glucose-induced oxidative stress significantly, which the similar effects were showed in edaravone. However, proanthocyanidins could not decrease the glucose concentration of embryo eye. Moreover, the key genes regulating eye development, Pax6, was down-regulated by high glucose. Proanthocyanidins could restore the suppressed expression of Pax6. These results indicated proanthocyanidins might be a promising natural agent to prevent high glucose-induced eye malformation by restoring Pax6 expression.

Dragon (repulsive guidance molecule RGMb) inhibits E-cadherin expression and induces apoptosis in renal tubular epithelial cells.

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Liu, Wenjing; Li, Xiaoling; Zhao, Yueshui; Meng, Xiao-Ming; Wan, Chao; Yang, Baoxue; Lan, Hui-Yao; Lin, Herbert Y; Xia, Yin

2013-11-01

Dragon is one of the three members of the repulsive guidance molecule (RGM) family, i.e. RGMa, RGMb (Dragon), and RGMc (hemojuvelin). We previously identified the RGM members as bone morphogenetic protein (BMP) co-receptors that enhance BMP signaling. Our previous studies found that Dragon is highly expressed in the tubular epithelial cells of mouse kidneys. However, the roles of Dragon in renal epithelial cells are yet to be defined. We now show that overexpression of Dragon increased cell death induced by hypoxia in association with increased cleaved poly(ADP-ribose) polymerase and cleaved caspase-3 levels in mouse inner medullary collecting duct (IMCD3) cells. Dragon also inhibited E-cadherin expression but did not affect epithelial-to-mesenchymal transition induced by TGF-β in IMCD3 cells. Previous studies suggest that the three RGM members can function as ligands for the receptor neogenin. Interestingly, our present study demonstrates that the Dragon actions on apoptosis and E-cadherin expression in IMCD3 cells were mediated by the neogenin receptor but not through the BMP pathway. Dragon expression in the kidney was up-regulated by unilateral ureteral obstruction in mice. Compared with wild-type mice, heterozygous Dragon knock-out mice exhibited 45-66% reduction in Dragon mRNA expression, decreased epithelial apoptosis, and increased tubular E-cadherin expression and had attenuated tubular injury after unilateral ureteral obstruction. Our results suggest that Dragon may impair tubular epithelial integrity and induce epithelial apoptosis both in vitro and in vivo.

Stress-related hormone norepinephrine induces interleukin-6 expression in GES-1 cells

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Yang, R.; Lin, Q.; Gao, H.B.; Zhang, P.

2014-01-01

In the current literature, there is evidence that psychological factors can affect the incidence and progression of some cancers. Interleukin 6 (IL-6) is known to be elevated in individuals experiencing chronic stress and is also involved in oncogenesis and cancer progression. However, the precise mechanism of IL-6 induction by the stress-related hormone norepinephrine (NE) is not clear, and, furthermore, there are no reports about the effect of NE on IL-6 expression in gastric epithelial cells. In this study, we examined the effect of NE on IL-6 expression in immortalized human gastric epithelial cells (GES-1 cells). Using real-time PCR and enzyme-linked immunoassay, we demonstrated that NE can induce IL-6 mRNA and protein expression in GES-1 cells. The induction is through the β-adrenergic receptor-cAMP-protein kinase A pathway and mainly at the transcriptional level. Progressive 5′-deletions and site-directed mutagenesis of the parental construct show that, although activating-protein-1 (AP-1), cAMP-responsive element binding protein (CREB), CCAAT-enhancer binding protein-β (C/EBP-β), and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) binding sites are all required in the basal transcription of IL-6, only AP-1 and CREB binding sites in the IL-6 promoter are required in NE-induced IL-6 expression. The results suggest that chronic stress may increase IL-6 secretion of human gastric epithelial cells, at least in part, by the stress-associated hormone norepinephrine, and provides basic data on stress and gastric cancer progression

Stress-related hormone norepinephrine induces interleukin-6 expression in GES-1 cells

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Yang, R.; Lin, Q.; Gao, H.B.; Zhang, P. [Department of Biochemistry and Molecular Cell Biology, School of Medicine, Shanghai Jiao Tong University, Shanghai, China, Department of Biochemistry and Molecular Cell Biology, School of Medicine, Shanghai Jiao Tong University, Shanghai (China)

2014-02-17

In the current literature, there is evidence that psychological factors can affect the incidence and progression of some cancers. Interleukin 6 (IL-6) is known to be elevated in individuals experiencing chronic stress and is also involved in oncogenesis and cancer progression. However, the precise mechanism of IL-6 induction by the stress-related hormone norepinephrine (NE) is not clear, and, furthermore, there are no reports about the effect of NE on IL-6 expression in gastric epithelial cells. In this study, we examined the effect of NE on IL-6 expression in immortalized human gastric epithelial cells (GES-1 cells). Using real-time PCR and enzyme-linked immunoassay, we demonstrated that NE can induce IL-6 mRNA and protein expression in GES-1 cells. The induction is through the β-adrenergic receptor-cAMP-protein kinase A pathway and mainly at the transcriptional level. Progressive 5′-deletions and site-directed mutagenesis of the parental construct show that, although activating-protein-1 (AP-1), cAMP-responsive element binding protein (CREB), CCAAT-enhancer binding protein-β (C/EBP-β), and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) binding sites are all required in the basal transcription of IL-6, only AP-1 and CREB binding sites in the IL-6 promoter are required in NE-induced IL-6 expression. The results suggest that chronic stress may increase IL-6 secretion of human gastric epithelial cells, at least in part, by the stress-associated hormone norepinephrine, and provides basic data on stress and gastric cancer progression.

A case showing effective radiotherapy for a radiation-induced glioblastoma

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Fukui, Kimiko; Inamura, Takanori; Nakamizo, Akira; Ikezaki, Kiyonobu; Inoha, Satoshi; Nakamura, Kazumasa; Matsuzaki, Akinobu; Fukui, Masashi [Kyushu Univ., Fukuoka (Japan). Graduate School of Medical Sciences

2001-07-01

Radiation-induced glioblastoma is usually resistant to all treatments. We report a case with radiation-induced glioblastoma, in which radiotherapy was remarkably effective. A 14-year-old female with a history of acute lymphoblastic leukemia, at the age of 7, underwent 15 Gy of radiotherapy to the whole brain. She was admitted to our department due to the development of headache and nausea. Magnetic resonance imaging showed an irregularly enhanced mass in the left frontal lobe. Partial removal of the mass was performed and histological examination showed it to be glioblastoma with a high MIB-1 index. The patient underwent 40 Gy of local radiotherapy and chemotherapy with ACNU and Interferon-{beta} for 2 years. The residual tumor disappeared after the radiotherapy, and her status is still ''complete remission'', 29 months after the onset. (author)

Expression of a pathogen-induced cysteine protease (AdCP) in tapetum results in male sterility in transgenic tobacco.

Science.gov (United States)

Shukla, Pawan; Singh, Naveen Kumar; Kumar, Dilip; Vijayan, Sambasivam; Ahmed, Israr; Kirti, Pulugurtha Bharadwaja

2014-06-01

Usable male sterility systems have immense potential in developing hybrid varieties in crop plants, which can also be used as a biological safety containment to prevent horizontal transgene flow. Barnase-Barstar system developed earlier was the first approach to engineer male sterility in plants. In an analogous situation, we have evolved a system of inducing pollen abortion and male sterility in transgenic tobacco by expressing a plant gene coding for a protein with known developmental function in contrast to the Barnase-Barstar system, which deploys genes of prokaryotic origin, i.e., from Bacillus amyloliquefaciens. We have used a plant pathogen-induced gene, cysteine protease for inducing male sterility. This gene was identified in the wild peanut, Arachis diogoi differentially expressed when it was challenged with the late leaf spot pathogen, Phaeoisariopsis personata. Arachis diogoi cysteine protease (AdCP) was expressed under the strong tapetum-specific promoter (TA29) and tobacco transformants were generated. Morphological and histological analysis of AdCP transgenic plants showed ablated tapetum and complete pollen abortion in three transgenic lines. Furthermore, transcript analysis displayed the expression of cysteine protease in these male sterile lines and the expression of the protein was identified in western blot analysis using its polyclonal antibody raised in the rabbit system.

Ghrelin knockout mice show decreased voluntary alcohol consumption and reduced ethanol-induced conditioned place preference.

Science.gov (United States)

Bahi, Amine; Tolle, Virginie; Fehrentz, Jean-Alain; Brunel, Luc; Martinez, Jean; Tomasetto, Catherine-Laure; Karam, Sherif M

2013-05-01

Recent work suggests that stomach-derived hormone ghrelin receptor (GHS-R1A) antagonism may reduce motivational aspects of ethanol intake. In the current study we hypothesized that the endogenous GHS-R1A agonist ghrelin modulates alcohol reward mechanisms. For this purpose ethanol-induced conditioned place preference (CPP), ethanol-induced locomotor stimulation and voluntary ethanol consumption in a two-bottle choice drinking paradigm were examined under conditions where ghrelin and its receptor were blocked, either using ghrelin knockout (KO) mice or the specific ghrelin receptor (GHS-R1A) antagonist "JMV2959". We showed that ghrelin KO mice displayed lower ethanol-induced CPP than their wild-type (WT) littermates. Consistently, when injected during CPP-acquisition, JMV2959 reduced CPP-expression in C57BL/6 mice. In addition, ethanol-induced locomotor stimulation was lower in ghrelin KO mice. Moreover, GHS-R1A blockade, using JMV2959, reduced alcohol-stimulated locomotion only in WT but not in ghrelin KO mice. When alcohol consumption and preference were assessed using the two-bottle choice test, both genetic deletion of ghrelin and pharmacological antagonism of the GHS-R1A (JMV2959) reduced voluntary alcohol consumption and preference. Finally, JMV2959-induced reduction of alcohol intake was only observed in WT but not in ghrelin KO mice. Taken together, these results suggest that ghrelin neurotransmission is necessary for the stimulatory effect of ethanol to occur, whereas lack of ghrelin leads to changes that reduce the voluntary intake as well as conditioned reward by ethanol. Our findings reveal a major, novel role for ghrelin in mediating ethanol behavior, and add to growing evidence that ghrelin is a key mediator of the effects of multiple abused drugs. Copyright © 2013 Elsevier Inc. All rights reserved.

Effect of Bcl-2/Bax gene expression on apoptosis of spermatogenic cells of mouse testes induced by low dose radiation

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Liu Guangwei; Wang Chunyan; Lu Zhe; Liu Shunchun; Gong Shouliang

2003-01-01

The different kinds of spermatogenic cells were separated using density gradient centrifugation and their apoptosis and Bcl-2 and Bax protein expression were measured with flow cytometry and immunohistochemical method, respectively. The results showed the apoptosis in all kinds of spermatogenic cells induced by low dose radiation (LDR) had a obvious regularity. When the doses were 0.025 and 0.05 Gy, spermatogonia apoptosis was dominant. With the increase of irradiation dose (0.075-0.2 Gy), spermatocytes also showed an apoptotic change, but the apoptotic percentage of spermatogonia was significantly higher than that of spermatocytes. Moreover, the apoptosis of spermatids and spermatozoa scarcely occurred after LDR. Bax protein was primarily expressed in spermatogonia and spermatocytes, and the former was significantly higher than that of the latter after LDR. With the increase of irradiation dose, Bax protein expression showed a upgrading tendency, but that of spermatids and spermatozoa scarcely occurred. Bcl-2 protein was primarily expressed in spermatids and spermatozoa, but the Bcl-2 protein expressions of spermatogonia and spermatocytes scarcely occurred after LDR. These results imply that the interacting regulation of Bcl-2 and Bax gene expression might be involved in selective apoptosis of spermatogenic cells induced by LDR, which provided an experimental evidence for further exploring the apoptotic mechanism of adaptive response of spermatogenic cells by LDR

Amphiregulin mediates hCG-induced StAR expression and progesterone production in human granulosa cells.

Science.gov (United States)

Fang, Lanlan; Yu, Yiping; Zhang, Ruizhe; He, Jingyan; Sun, Ying-Pu

2016-04-26

Progesterone plays critical roles in maintaining a successful pregnancy at the early embryonic stage. Human chorionic gonadotropin (hCG) rapidly induces amphiregulin (AREG) expression. However, it remains unknown whether AREG mediates hCG-induced progesterone production. Thus, the objective of this study was to investigate the role of AREG in hCG-induced progesterone production and the underlying molecular mechanism in human granulosa cells; primary cells were used as the experimental model. We demonstrated that the inhibition of EGFR and the knockdown of AREG abolished hCG-induced steroidogenic acute regulatory protein (StAR) expression and progesterone production. Importantly, follicular fluid AREG levels were positively correlated with progesterone levels in the follicular fluid and serum. Treatment with AREG increased StAR expression and progesterone production, and these stimulatory effects were abolished by EGFR inhibition. Moreover, activation of ERK1/2, but not PI3K/Akt, signaling was required for the AREG-induced up-regulation of StAR expression and progesterone production. Our results demonstrate that AREG mediates hCG-induced StAR expression and progesterone production in human granulosa cells, providing novel evidence for the role of AREG in the regulation of steroidogenesis.

Behavioral experience induces zif268 expression in mature granule cells but suppresses its expression in immature granule cells

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Kylie A. Huckleberry

2015-08-01

Full Text Available Thousands of neurons are born each day in the dentate gyrus (DG, but many of these cells die before reaching maturity. Both death and survival of adult-born neurons are regulated by neuronal activity in DG. The immediate-early gene (IEG zif268 is an important mediator of these effects, as its expression is induced by neural activity and knockout of zif268 impairs survival of adult-born neurons (Veyrac et al., 2013. Despite the apparent importance of zif268 for adult neurogenesis, its behavior-induced expression has not been fully characterized in adult-born neurons. Here we characterize behavior-evoked expression of zif268 in mature and newborn dentate granule cells (DGCs. In the general granule cell population, zif268 expression peaked 1 hour after novel environment exposure and returned to baseline by 8 hours post-exposure. However, in the doublecortin-positive (DCX+ immature neurons, zif268 expression was suppressed relative to home cage for at least 8 hours post-exposure. We next determined that exposure to water maze training, an enriched environment, or a novel environment caused approximately equal suppression of zif268 expression in DCX+ cells and approximately equal activation of zif268 in the general DGC population and in 6-week-old adult-born neurons. Finally, we asked whether zif268 suppression varied as a function of age within the DCX+ population, which ranges in age from 0 to approximately 4 weeks. Novel environment exposure had no significant effect on zif268 expression in 2- or 4-week-old BrdU-labeled neurons, but it significantly suppressed zif268 expression in 3-week-old neurons. In summary, behavioral experience transiently activated expression of zif268 in mature DGCs but caused a more long-lasting suppression of zif268 expression in immature, adult-born DGCs. We hypothesize that zif268 suppression inhibits memory-related synaptic plasticity in immature DGCs or mediates learning-induced apoptosis of immature adult

Robust heat-inducible gene expression by two endogenous hsp70-derived promoters in transgenic Aedes aegypti

Science.gov (United States)

Carpenetti, Tiffany L. G.; Aryan, Azadeh; Myles, Kevin M.; Adelman, Zach N.

2011-01-01

Aedes aegypti is an important vector of the viruses that cause dengue fever, dengue hemorrhagic fever, and yellow fever. Reverse genetic approaches to the study of gene function in this mosquito have been limited by the lack of a robust inducible promoter to allow precise temporal control over a protein-encoding or hairpin RNA transgene. Likewise, investigations into the molecular and biochemical basis of vector competence would benefit from the ability to activate an anti-pathogen molecule at specific times during infection. We have characterized the ability of genomic sequences derived from two Ae. aegypti hsp70 genes to drive heat-inducible expression of a reporter in both transient and germline transformation contexts. AaHsp70-luciferase transcripts accumulated specifically after heat shock, and displayed a pattern of rapid induction and decay similar to endogenous AaHsp70 genes. Luciferase expression in transgenic Ae. aegypti increased by ∼25-50 fold in whole adults by four hours after heat-shock, with significant activity (∼20 fold) remaining at 24 hr. Heat-induced expression was even more dramatic in midgut tissues, with one strain showing a ∼2500-fold increase in luciferase activity. The AaHsp70 promoters described could be valuable for gene function studies as well as for the precise timing of the expression of anti-pathogen molecules. PMID:22142225

Inhibition of carcinogen induced c-Ha-ras and c-fos proto-oncogenes expression by dietary curcumin

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Lipigorngoson Suwiwek

2001-01-01

Full Text Available Abstract Background We investigated the chemopreventive action of dietary curcumin on 7,12-dimethylbenz(aanthracene (DMBA-initiated and 12,0-tetradecanoylphorbol-13-acetate (TPA-promoted skin tumor formation in Swiss albino mice. Curcumin, a yellow coloring matter isolated from roots of Curcuma longa Linn, is a phenolic compound possessing antioxidant, free radical scavenger, and antiinflammatory properties. It has been shown by previously reported work that TPA-induced skin tumors were inhibited by topical application of curcumin, and curcumin has been shown to inhibit a variety of biological activities of TPA. Topical application of curcumin was reported to inhibit TPA-induced c-fos, c-jun and c-myc gene expression in mouse skin. This paper reports the effects of orally administered curcumin, which was consumed as a dietary component at concentrations of 0.2 % or 1 %, in ad libitum feeding. Results Animals in which tumors had been initiated with DMBA and promoted with TPA experienced significantly fewer tumors and less tumor volume if they ingested either 0.2% or 1% curcumin diets. Also, the dietary consumption of curcumin resulted in a significantly decreased expression of ras and fos proto-oncogenes in the tumorous skin, as measured by enhanced chemiluminesence Western blotting detection system (Amersham. Conclusions Whereas earlier work demonstrated that topical application of curcumin to mouse skin inhibited TPA-induced expression of c-fos, c-jun and c-myc oncogenes, our results are the first to show that orally consumed curcumin significantly inhibited DMBA- and TPA-induced ras and fos gene expression in mouse skin.

Inhibition of carcinogen induced c-Ha-ras and c-fos proto-oncogenes expression by dietary curcumin

International Nuclear Information System (INIS)

Limtrakul, Porn-ngarm; Anuchapreeda, Songyot; Lipigorngoson, Suwiwek; Dunn, Floyd W

2001-01-01

We investigated the chemopreventive action of dietary curcumin on 7,12-dimethylbenz(a)anthracene (DMBA)-initiated and 12,0-tetradecanoylphorbol-13-acetate (TPA)-promoted skin tumor formation in Swiss albino mice. Curcumin, a yellow coloring matter isolated from roots of Curcuma longa Linn, is a phenolic compound possessing antioxidant, free radical scavenger, and antiinflammatory properties. It has been shown by previously reported work that TPA-induced skin tumors were inhibited by topical application of curcumin, and curcumin has been shown to inhibit a variety of biological activities of TPA. Topical application of curcumin was reported to inhibit TPA-induced c-fos, c-jun and c-myc gene expression in mouse skin. This paper reports the effects of orally administered curcumin, which was consumed as a dietary component at concentrations of 0.2 % or 1 %, in ad libitum feeding. Animals in which tumors had been initiated with DMBA and promoted with TPA experienced significantly fewer tumors and less tumor volume if they ingested either 0.2% or 1% curcumin diets. Also, the dietary consumption of curcumin resulted in a significantly decreased expression of ras and fos proto-oncogenes in the tumorous skin, as measured by enhanced chemiluminesence Western blotting detection system (Amersham). Whereas earlier work demonstrated that topical application of curcumin to mouse skin inhibited TPA-induced expression of c-fos, c-jun and c-myc oncogenes, our results are the first to show that orally consumed curcumin significantly inhibited DMBA- and TPA-induced ras and fos gene expression in mouse skin

Choline supplementation alleviates fluoride-induced testicular toxicity by restoring the NGF and MEK expression in mice

International Nuclear Information System (INIS)

Zhang, Jianhai; Zhang, Yufang; Liang, Chen; Wang, Nasui; Zheng, Heping; Wang, Jundong

2016-01-01

Fluoride is known to cause male reproductive toxicity, and the elucidation of its underlying mechanisms is an ongoing research focus in reproductive toxicology and epidemiology. Choline, an essential nutrient, has been extensively studied for its benefits in nervous system yet was rarely discussed for its prospective effect in male reproductive system. This study aims to explore the potential protective role of choline against NaF-induced male reproductive toxicity via MAPK pathway. The male mice were administrated by 150 mg/L NaF in drinking water, 5.75 g/kg choline in diet, and their combination respectively from maternal gestation to postnatal 15 weeks. The results showed that fluoride exposure reduced body weight growth, lowered sperm count and survival percentages, altered testicular histology, down-regulated the mRNA expressions of NGF, Ras, Raf, and MEK genes in testes, as well as significantly decreased the expressions of both NGF and phosphor-MEK proteins in testes. Examination of data from choline-treated mice revealed that choline supplementation ameliorated these fluoride-induced changes. Taken together, our findings suggest that choline supplementation alleviates fluoride-induced testicular toxicity by restoring the NGF and phosphor-MEK expression. The suitable dosage and supplementation periods of choline await further exploration. - Highlights: • Fluoride exposure altered the growth and development, sperm count and sperm survival percentages, testicular histology • Fluoride exposure decreased NGF, Ras, and Mek mRNA and NGF and p-MEK protein expressions in testis of mice. • Choline supplementation diminishes fluoride-induced testicular toxicity.

Choline supplementation alleviates fluoride-induced testicular toxicity by restoring the NGF and MEK expression in mice

Energy Technology Data Exchange (ETDEWEB)

Zhang, Jianhai [Shanxi Key Laboratory of Ecological Animal Science and Environmental Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi 030801 (China); Zhang, Yufang [Shanxi Key Laboratory of Ecological Animal Science and Environmental Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi 030801 (China); Veterinary Station in Chen Villages of Lin Country, Linxian, Shanxi 033200 (China); Liang, Chen [Shanxi Key Laboratory of Ecological Animal Science and Environmental Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi 030801 (China); Wang, Nasui [Division of Endocrinology and Metabolism, Department of Medicine, University of Virginia, Charlottesville, VA 22908 (United States); Division of Endocrinology and Metabolism, Department of Medicine, The First Affiliated Hospital of Shantou University Medical College, Shantou (China); Zheng, Heping [Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908 (United States); Wang, Jundong, E-mail: wangjd53@outlook.com [Shanxi Key Laboratory of Ecological Animal Science and Environmental Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi 030801 (China)

2016-11-01

Fluoride is known to cause male reproductive toxicity, and the elucidation of its underlying mechanisms is an ongoing research focus in reproductive toxicology and epidemiology. Choline, an essential nutrient, has been extensively studied for its benefits in nervous system yet was rarely discussed for its prospective effect in male reproductive system. This study aims to explore the potential protective role of choline against NaF-induced male reproductive toxicity via MAPK pathway. The male mice were administrated by 150 mg/L NaF in drinking water, 5.75 g/kg choline in diet, and their combination respectively from maternal gestation to postnatal 15 weeks. The results showed that fluoride exposure reduced body weight growth, lowered sperm count and survival percentages, altered testicular histology, down-regulated the mRNA expressions of NGF, Ras, Raf, and MEK genes in testes, as well as significantly decreased the expressions of both NGF and phosphor-MEK proteins in testes. Examination of data from choline-treated mice revealed that choline supplementation ameliorated these fluoride-induced changes. Taken together, our findings suggest that choline supplementation alleviates fluoride-induced testicular toxicity by restoring the NGF and phosphor-MEK expression. The suitable dosage and supplementation periods of choline await further exploration. - Highlights: • Fluoride exposure altered the growth and development, sperm count and sperm survival percentages, testicular histology • Fluoride exposure decreased NGF, Ras, and Mek mRNA and NGF and p-MEK protein expressions in testis of mice. • Choline supplementation diminishes fluoride-induced testicular toxicity.

Egr-1 Upregulates Siva-1 Expression and Induces Cardiac Fibroblast Apoptosis

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Karin Zins

2014-01-01

Full Text Available The early growth response transcription factor Egr-1 controls cell specific responses to proliferation, differentiation and apoptosis. Expression of Egr-1 and downstream transcription is closely controlled and cell specific upregulation induced by processes such as hypoxia and ischemia has been previously linked to multiple aspects of cardiovascular injury. In this study, we showed constitutive expression of Egr-1 in cultured human ventricular cardiac fibroblasts, used adenoviral mediated gene transfer to study the effects of continuous Egr-1 overexpression and studied downstream transcription by Western blotting, immunohistochemistry and siRNA transfection. Apoptosis was assessed by fluorescence microscopy and flow cytometry in the presence of caspase inhibitors. Overexpression of Egr-1 directly induced apoptosis associated with caspase activation in human cardiac fibroblast cultures in vitro assessed by fluorescence microscopy and flow cytometry. Apoptotic induction was associated with a caspase activation associated loss of mitochondrial membrane potential and transient downstream transcriptional up-regulation of the pro-apoptotic gene product Siva-1. Suppression of Siva-1 induction by siRNA partially reversed Egr-1 mediated loss of cell viability. These findings suggest a previously unknown role for Egr-1 and transcriptional regulation of Siva-1 in the control of cardiac accessory cell death.

IBMX protects human proximal tubular epithelial cells from hypoxic stress through suppressing hypoxia-inducible factor-1α expression.

Science.gov (United States)

Hasan, Arif Ul; Kittikulsuth, Wararat; Yamaguchi, Fuminori; Musarrat Ansary, Tuba; Rahman, Asadur; Shibayama, Yuki; Nakano, Daisuke; Hitomi, Hirofumi; Tokuda, Masaaki; Nishiyama, Akira

2017-09-15

Hypoxia predisposes renal fibrosis. This study was conducted to identify novel approaches to ameliorate the pathogenic effect of hypoxia. Using human proximal tubular epithelial cells we showed that a pan-phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX) dose and time dependently downregulated hypoxia-inducible factor 1α (HIF-1α) mRNA expression, which was further augmented by addition of a transcriptional inhibitor, actinomycin D. IBMX also increased the cellular cyclic adenosine monophosphate (cAMP) level. Luciferase assay showed that blocking of protein kinase A (PKA) using H89 reduced, while 8-Br-cAMP agonized the repression of HIF-1α promoter activity in hypoxic condition. Deletion of cAMP response element binding sites from the HIF-1α promoter abrogated the effect of IBMX. Western blot and immunofluorescent study confirmed that the CoCl 2 induced increased HIF-1α protein in whole cell lysate and in nucleus was reduced by the IBMX. Through this process, IBMX attenuated both CoCl 2 and hypoxia induced mRNA expressions of two pro-fibrogenic factors, platelet-derived growth factor B and lysyl oxidase. Moreover, IBMX reduced production of a mesenchymal transformation factor, β-catenin; as well as protected against hypoxia induced cell-death. Taken together, our study showed novel evidence that the PDE inhibitor IBMX can downregulate the transcription of HIF-1α, and thus may attenuate hypoxia induced renal fibrosis. Copyright © 2017 Elsevier Inc. All rights reserved.

Tetracycline-inducible gene expression system in Leishmania mexicana

Czech Academy of Sciences Publication Activity Database

Kraeva, N.; Ishemgulova, A.; Lukeš, Julius; Yurchenko, Vyacheslav

2014-01-01

Roč. 198, č. 1 (2014), s. 11-13 ISSN 0166-6851 R&D Projects: GA MŠk(CZ) EE2.3.30.0032 Institutional support: RVO:60077344 Keywords : Leishmania mexicana * Gene expression * Tet-inducible system Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.787, year: 2014

c-Jun N-terminal kinase mediates AML1-ETO protein-induced connexin-43 expression

International Nuclear Information System (INIS)

Gao Fenghou; Wang Qiong; Wu Yingli; Li Xi; Zhao Kewen; Chen Guoqiang

2007-01-01

AML1-ETO fusion protein, a product of leukemia-related chromosomal translocation t(8;21), was reported to upregulate expression of connexin-43 (Cx43), a member of gap junction-constituted connexin family. However, its mechanism(s) remains unclear. By bioinformatic analysis, here we showed that there are two putative AML1-binding consensus sequences followed by two activated protein (AP)1 sites in the 5'-flanking region upstream to Cx43 gene. AML1-ETO could directly bind to these two AML1-binding sites in electrophoretic mobility shift assay, but luciferase reporter assay revealed that the AML1 binding sites were not indispensable for Cx43 induction by AML1-ETO protein. Conversely, AP1 sites exerted an important role in this event. In agreement, AML1-ETO overexpression in leukemic U937 cells activated c-Jun N-terminal kinase (JNK), while its specific inhibitor SP600125 effectively abrogated AML1-ETO-induced Cx43 expression, indicating that JNK signaling pathway contributes to AML1-ETO induced Cx43 expression. These results would shed new insights for understanding mechanisms of AML1-ETO-associated leukemogenesis

Microarray-based screening of differentially expressed genes in glucocorticoid-induced avascular necrosis

Science.gov (United States)

Huang, Gangyong; Wei, Yibing; Zhao, Guanglei; Xia, Jun; Wang, Siqun; Wu, Jianguo; Chen, Feiyan; Chen, Jie; Shi, Jingshen

2017-01-01

The underlying mechanisms of glucocorticoid (GC)-induced avascular necrosis of the femoral head (ANFH) have yet to be fully understood, in particular the mechanisms associated with the change of gene expression pattern. The present study aimed to identify key genes with a differential expression pattern in GC-induced ANFH. E-MEXP-2751 microarray data were downloaded from the ArrayExpress database. Differentially expressed genes (DEGs) were identified in 5 femoral head samples of steroid-induced ANFH rats compared with 5 placebo-treated rat samples. Gene Ontology (GO) and pathway enrichment analyses were performed upon these DEGs. A total 93 DEGs (46 upregulated and 47 downregulated genes) were identified in GC-induced ANFH samples. These DEGs were enriched in different GO terms and pathways, including chondrocyte differentiation and detection of chemical stimuli. The enrichment map revealed that skeletal system development was interconnected with several other GO terms by gene overlap. The literature mined network analysis revealed that 5 upregulated genes were associated with femoral necrosis, including parathyroid hormone receptor 1 (PTHR1), vitamin D (1,25-Dihydroxyvitamin D3) receptor (VDR), collagen, type II, α1, proprotein convertase subtilisin/kexin type 6 and zinc finger protein 354C (ZFP354C). In addition, ZFP354C and VDR were identified to transcription factors. Furthermore, PTHR1 was revealed to interact with VDR, and α-2-macroglobulin (A2M) interacted with fibronectin 1 (FN1) in the PPI network. PTHR1 may be involved in GC-induced ANFH via interacting with VDR. A2M may also be involved in the development of GC-induced ANFH through interacting with FN1. An improved understanding of the molecular mechanisms underlying GC-induced ANFH may provide novel targets for diagnostics and therapeutic treatment. PMID:28393228

Microarray‑based screening of differentially expressed genes in glucocorticoid‑induced avascular necrosis.

Science.gov (United States)

Huang, Gangyong; Wei, Yibing; Zhao, Guanglei; Xia, Jun; Wang, Siqun; Wu, Jianguo; Chen, Feiyan; Chen, Jie; Shi, Jingshen

2017-06-01

The underlying mechanisms of glucocorticoid (GC)‑induced avascular necrosis of the femoral head (ANFH) have yet to be fully understood, in particular the mechanisms associated with the change of gene expression pattern. The present study aimed to identify key genes with a differential expression pattern in GC‑induced ANFH. E‑MEXP‑2751 microarray data were downloaded from the ArrayExpress database. Differentially expressed genes (DEGs) were identified in 5 femoral head samples of steroid‑induced ANFH rats compared with 5 placebo‑treated rat samples. Gene Ontology (GO) and pathway enrichment analyses were performed upon these DEGs. A total 93 DEGs (46 upregulated and 47 downregulated genes) were identified in GC‑induced ANFH samples. These DEGs were enriched in different GO terms and pathways, including chondrocyte differentiation and detection of chemical stimuli. The enrichment map revealed that skeletal system development was interconnected with several other GO terms by gene overlap. The literature mined network analysis revealed that 5 upregulated genes were associated with femoral necrosis, including parathyroid hormone receptor 1 (PTHR1), vitamin D (1,25‑Dihydroxyvitamin D3) receptor (VDR), collagen, type II, α1, proprotein convertase subtilisin/kexin type 6 and zinc finger protein 354C (ZFP354C). In addition, ZFP354C and VDR were identified to transcription factors. Furthermore, PTHR1 was revealed to interact with VDR, and α‑2‑macroglobulin (A2M) interacted with fibronectin 1 (FN1) in the PPI network. PTHR1 may be involved in GC‑induced ANFH via interacting with VDR. A2M may also be involved in the development of GC‑induced ANFH through interacting with FN1. An improved understanding of the molecular mechanisms underlying GC‑induced ANFH may provide novel targets for diagnostics and therapeutic treatment.

Activated human mast cells induce LOX-1-specific scavenger receptor expression in human monocyte-derived macrophages.

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Mervi Alanne-Kinnunen

Full Text Available Activated mast cells in atherosclerotic lesions degranulate and release bioactive compounds capable of regulating atherogenesis. Here we examined the ability of activated human primary mast cells to regulate the expression of the major scavenger receptors in cultured human primary monocyte-derived macrophages (HMDMs.Components released by immunologically activated human primary mast cells induced a transient expression of lectin-like oxidized LDL receptor (LOX-1 mRNA in HMDMs, while the expression of two other scavenger receptors, MSR1 and CD36, remained unaffected. The LOX-1-inducing secretory components were identified as histamine, tumor necrosis factor alpha (TNF-α, and transforming growth factor beta (TGF-β1, which exhibited a synergistic effect on LOX-1 mRNA expression. Histamine induced a transient expression of LOX-1 protein. Mast cell -induced increase in LOX-1 expression was not associated with increased uptake of oxidized LDL by the macrophages.Mast cell-derived histamine, TNF-α, and TGF-β1 act in concert to induce a transient increase in LOX-1 expression in human primary monocyte-derived macrophages. The LOX-1-inducing activity potentially endows mast cells a hitherto unrecognized role in the regulation of innate immune reactions in atherogenesis.

Inflammatory Cytokines Induce Podoplanin Expression at the Tumor Invasive Front.

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Kunita, Akiko; Baeriswyl, Vanessa; Meda, Claudia; Cabuy, Erik; Takeshita, Kimiko; Giraudo, Enrico; Wicki, Andreas; Fukayama, Masashi; Christofori, Gerhard

2018-05-01

Tumor invasion is a critical first step in the organismic dissemination of cancer cells and the formation of metastasis in distant organs, the most important prognostic factor and the actual cause of death in most of the cancer patients. We report herein that the cell surface protein podoplanin (PDPN), a potent inducer of cancer cell invasion, is conspicuously expressed by the invasive front of squamous cell carcinomas (SCCs) of the cervix in patients and in the transgenic human papillomavirus/estrogen mouse model of cervical cancer. Laser capture microscopy combined with gene expression profiling reveals that the expression of interferon-responsive genes is up-regulated in PDPN-expressing cells at the tumor invasive front, which are exposed to CD45-positive inflammatory cells. Indeed, PDPN expression can be induced in cultured SCC cell lines by single or combined treatments with interferon-γ, transforming growth factor-β, and/or tumor necrosis factor-α. Notably, shRNA-mediated ablation of either PDPN or STAT1 in A431 SCC cells repressed cancer cell invasion on s.c. transplantation into immunodeficient mice. The results highlight the induction of tumor cell invasion by the inflammatory cytokine-stimulated expression of PDPN in the outermost cell layers of cervical SCC. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Isorhamnetin protects against oxidative stress by activating Nrf2 and inducing the expression of its target genes

Energy Technology Data Exchange (ETDEWEB)

Yang, Ji Hye; Shin, Bo Yeon; Han, Jae Yun; Kim, Mi Gwang; Wi, Ji Eun [College of Pharmacy, Chosun University, Gwangju, 501-759 (Korea, Republic of); Kim, Young Woo; Cho, Il Je; Kim, Sang Chan [Medical Research Center for Globalization of Herbal Formulation, College of Korean Medicine, Daegu Haany University, Gyeongsan 712-715 (Korea, Republic of); Shin, Sang Mi [College of Pharmacy, Chosun University, Gwangju, 501-759 (Korea, Republic of); Ki, Sung Hwan, E-mail: shki@chosun.ac.kr [College of Pharmacy, Chosun University, Gwangju, 501-759 (Korea, Republic of)

2014-01-15

Isorhamentin is a 3′-O-methylated metabolite of quercetin, and has been reported to have anti-inflammatory and anti-proliferative effects. However, the effects of isorhamnetin on Nrf2 activation and on the expressions of its downstream genes in hepatocytes have not been elucidated. Here, we investigated whether isorhamnetin has the ability to activate Nrf2 and induce phase II antioxidant enzyme expression, and to determine the protective role of isorhamnetin on oxidative injury in hepatocytes. In HepG2 cells, isorhamnetin increased the nuclear translocation of Nrf2 in a dose- and time-dependent manner, and consistently, increased antioxidant response element (ARE) reporter gene activity and the protein levels of hemeoxygenase (HO-1) and of glutamate cysteine ligase (GCL), which resulted in intracellular GSH level increases. The specific role of Nrf2 in isorhamnetin-induced Nrf2 target gene expression was verified using an ARE-deletion mutant plasmid and Nrf2-knockout MEF cells. Deletion of the ARE in the promoter region of the sestrin2 gene, which is recently identified as the Nrf2 target gene by us, abolished the ability of isorhamnetin to increase luciferase activity. In addition, Nrf2 deficiency completely blocked the ability of isorhamnetin to induce HO-1 and GCL. Furthermore, isorhamnetin pretreatment blocked t-BHP-induced ROS production and reversed GSH depletion by t-BHP and consequently, due to reduced ROS levels, decreased t-BHP-induced cell death. In addition isorhamnetin increased ERK1/2, PKCδ and AMPK phosphorylation. Finally, we showed that Nrf2 deficiency blocked the ability of isorhamnetin to protect cells from injury induced by t-BHP. Taken together, our results demonstrate that isorhamnetin is efficacious in protecting hepatocytes against oxidative stress by Nrf2 activation and in inducing the expressions of its downstream genes. - Highlights: • We investigated the effect of isorhamnetin on Nrf2 activation. • Isorhamnetin increased Nrf2

Arsenite induced oxidative damage in mouse liver is associated with increased cytokeratin 18 expression

Energy Technology Data Exchange (ETDEWEB)

Gonsebatt, M.E. [UNAM, Ciudad Universitaria, Dept. Medicina Genomica y Toxicologia Ambiental, Instituto de Investigaciones Biomedicas, Mexico (Mexico); Razo, L.M. del; Sanchez-Pena, L.C. [Seccion de Toxicologia, CINVESTAV, Mexico (Mexico); Cerbon, M.A. [Facultad de Quimica, UNAM, Departamento de Biologia, Mexico (Mexico); Zuniga, O.; Ramirez, P. [Facultad de Estudios Superiores Cuautitlan, UNAM, Laboratorio de Toxicologia Celular, Coordinacion General de Estudios de Posgrado e Investigacion, Cuautitlan Izcalli, Estado de Mexico (Mexico)

2007-09-15

Cytokeratins (CK) constitute a family of cytoskeletal intermediate filament proteins that are typically expressed in epithelial cells. An abnormal structure and function are effects that are clearly related to liver diseases as non-alcoholic steatohepatitis, cirrhosis and hepatocellular carcinoma. We have previously observed that sodium arsenite (SA) induced the synthesis of CK18 protein and promotes a dose-related disruption of cytoplasmic CK18 filaments in a human hepatic cell line. Both abnormal gene expression and disturbance of structural organization are toxic effects that are likely to cause liver disease by interfering with normal hepatocyte function. To investigate if a disruption in the CK18 expression pattern is associated with arsenite liver damage, we investigated CK18 mRNA and protein levels in liver slices treated with low levels of SA. Organotypic cultures were incubated with 0.01, 1 and 10 {mu}M of SA in the absence and presence of N-acetyl cysteine (NAC). Cell viability and inorganic arsenic metabolism were determined. Increased expression of CK18 was observed after exposure to SA. The addition of NAC impeded the oxidative effects of SA exposure, decreasing the production of thiobarbituric acid-reactive substances and significantly diminishing the up regulation of CK18 mRNA and protein. Liver arsenic levels correlated with increased levels of mRNA. Mice treated with intragastric single doses of 2.5 and 5 mg/kg of SA showed an increased expression of CK18. Results suggest that CK18 expression may be a sensible early biomarker of oxidative stress and damage induced by arsenite in vitro and in vivo. Then, during SA exposure, altered CK expression may compromise liver function. (orig.)

Amniotic Fluid Cells Show Higher Pluripotency-Related Gene Expression Than Allantoic Fluid Cells.

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Kehl, Debora; Generali, Melanie; Görtz, Sabrina; Geering, Diego; Slamecka, Jaroslav; Hoerstrup, Simon P; Bleul, Ulrich; Weber, Benedikt

2017-10-01

Amniotic fluid represents an abundant source of multipotent stem cells, referred as broadly multipotent given their differentiation potential and expression of pluripotency-related genes. However, the origin of this broadly multipotent cellular fraction is not fully understood. Several sources have been proposed so far, including embryonic and extraembryonic tissues. In this regard, the ovine developmental model uniquely allows for direct comparison of fetal fluid-derived cells from two separate fetal fluid cavities, the allantois and the amnion, over the entire duration of gestation. As allantoic fluid mainly collects fetal urine, cells originating from the efferent urinary tract can directly be compared with cells deriving from the extraembryonic amniotic tissues and the fetus. This study shows isolation of cells from the amniotic [ovine amniotic fluid cells (oAFCs)] and allantoic fluid [ovine allantoic fluid cells (oALCs)] in a strictly paired fashion with oAFCs and oALCs derived from the same fetus. Both cell types showed cellular phenotypes comparable to standard mesenchymal stem cells (MSCs), with trilineage differentiation potential, and expression of common ovine MSC markers. However, the expression of MSC markers per single cell was higher in oAFCs as measured by flow cytometry. oAFCs exhibited higher proliferative capacities and showed significantly higher expression of pluripotency-related genes OCT4, STAT3, NANOG, and REX1 by quantitative real-time polymerase chain reaction compared with paired oALCs. No significant decrease of pluripotency-related gene expression was noted over gestation, implying that cells with high differentiation potential may be isolated at the end of pregnancy. In conclusion, this study suggests that cells with highest stem cell characteristics may originate from the fetus itself or the amniotic fetal adnexa rather than from the efferent urinary tract or the allantoic fetal adnexa.

Cannabidivarin (CBDV suppresses pentylenetetrazole (PTZ-induced increases in epilepsy-related gene expression

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Naoki Amada

2013-11-01

Full Text Available To date, anticonvulsant effects of the plant cannabinoid, cannabidivarin (CBDV, have been reported in several animal models of seizure. However, these behaviourally observed anticonvulsant effects have not been confirmed at the molecular level. To examine changes to epilepsy-related gene expression following chemical convulsant treatment and their subsequent control by phytocannabinoid administration, we behaviourally evaluated effects of CBDV (400 mg/kg, p.o. on acute, pentylenetetrazole (PTZ: 95 mg/kg, i.p.-induced seizures, quantified expression levels of several epilepsy-related genes (Fos, Casp 3, Ccl3, Ccl4, Npy, Arc, Penk, Camk2a, Bdnf and Egr1 by qPCR using hippocampal, neocortical and prefrontal cortical tissue samples before examining correlations between expression changes and seizure severity. PTZ treatment alone produced generalised seizures (median: 5.00 and significantly increased expression of Fos, Egr1, Arc, Ccl4 and Bdnf. Consistent with previous findings, CBDV significantly decreased PTZ-induced seizure severity (median: 3.25 and increased latency to the first sign of seizure. Furthermore, there were correlations between reductions of seizure severity and mRNA expression of Fos, Egr1, Arc, Ccl4 and Bdnf in the majority of brain regions in the CBDV+PTZ treated group. When CBDV treated animals were grouped into CBDV responders (criterion: seizure severity ≤3.25 and non-responders (criterion: seizure severity >3.25, PTZ-induced increases of Fos, Egr1, Arc, Ccl4 and Bdnf expression were suppressed in CBDV responders. These results provide the first molecular confirmation of behaviourally observed effects of the non-psychoactive, anticonvulsant cannabinoid, CBDV, upon chemically-induced seizures and serve to underscore its suitability for clinical development.

Dynamic Blue Light-Inducible T7 RNA Polymerases (Opto-T7RNAPs) for Precise Spatiotemporal Gene Expression Control.

Science.gov (United States)

Baumschlager, Armin; Aoki, Stephanie K; Khammash, Mustafa

2017-11-17

Light has emerged as a control input for biological systems due to its precise spatiotemporal resolution. The limited toolset for light control in bacteria motivated us to develop a light-inducible transcription system that is independent from cellular regulation through the use of an orthogonal RNA polymerase. Here, we present our engineered blue light-responsive T7 RNA polymerases (Opto-T7RNAPs) that show properties such as low leakiness of gene expression in the dark state, high expression strength when induced with blue light, and an inducible range of more than 300-fold. Following optimization of the system to reduce expression variability, we created a variant that returns to the inactive dark state within minutes once the blue light is turned off. This allows for precise dynamic control of gene expression, which is a key aspect for most applications using optogenetic regulation. The regulators, which only require blue light from ordinary light-emitting diodes for induction, were developed and tested in the bacterium Escherichia coli, which is a crucial cell factory for biotechnology due to its fast and inexpensive cultivation and well understood physiology and genetics. Opto-T7RNAP, with minor alterations, should be extendable to other bacterial species as well as eukaryotes such as mammalian cells and yeast in which the T7 RNA polymerase and the light-inducible Vivid regulator have been shown to be functional. We anticipate that our approach will expand the applicability of using light as an inducer for gene expression independent from cellular regulation and allow for a more reliable dynamic control of synthetic and natural gene networks.

Testosterone-induced adult neurosphere growth is mediated by sexually-dimorphic aromatase expression

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Mark Ian Ransome

2015-07-01

Full Text Available We derived adult neural stem/progenitor cells (NSPCs from the sub-ventricular zone of male and female mice to examine direct responses to principal sex hormones. In the presence of epidermal growth factor (EGF and fibroblast growth factor-2 (FGF2 NSPCs of both sexes expressed nestin and sox2 and could be maintained as neurospheres without addition of any sex hormones. The reverse was not observed; neither testosterone (T, 17β-oestradiol (E2 nor progesterone (P4 was able to support neurosphere growth in the absence of EGF and FGF2. 10nM T, E2 or P4 induced nestin(+ cell proliferation within 20 minutes and enhanced neurosphere growth over 7 days irrespective of sex, which was abolished by Erk inhibition with 20M U0126. Maintaining neurospheres with each sex hormone did not affect subsequent neuronal differentiation. However, 10nM T, E2 or P4 added during differentiation increased III tubulin(+ neuron production with E2 being more potent compared to T and P4 in both sexes. Androgen receptor (AR inhibition with 20M flutamide but not aromatase inhibition with 10M letrozole reduced basal and T-induced neurosphere growth in females, while only concurrent inhibition of AR and aromatase produced the same effect in males. This sex-specific effect was supported by higher aromatase expression in male neurospheres compared to females measured by Western blot and green fluorescent protein reporter. 10M menadione induced oxidative stress, impaired neurosphere growth and up-regulated aromatase expression in both sexes. However, under oxidative stress letrozole significantly exacerbated impaired neurosphere growth in males only. While both E2 and T could prevent oxidative stress-induced growth reduction in both sexes, the effects of T were dependent on innate aromatase activity. We show for the first time that intrinsic androgen and estrogen signalling may impact the capacity of NSPCs to produce neural progenitors under pathological conditions of

Oxidative stress induces the decline of brain EPO expression in aging rats.

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Li, Xu; Chen, Yubao; Shao, Siying; Tang, Qing; Chen, Weihai; Chen, Yi; Xu, Xiaoyu

2016-10-01

Brain Erythropoietin (EPO), an important neurotrophic factor and neuroprotective factor, was found to be associated with aging. Studies found EPO expression was significantly decreased in the hippocampus of aging rat compared with that of the youth. But mechanisms of the decline of the brain EPO during aging remain unclear. The present study utilized a d-galactose (d-gal)-induced aging model in which the inducement of aging was mainly oxidative injury, to explore underlying mechanisms for the decline of brain EPO in aging rats. d-gal-induced aging rats (2months) were simulated by subcutaneously injecting with d-gal at doses of 50mg·kg(-1), 150mg·kg(-1) and 250mg·kg(-1) daily for 8weeks while the control group received vehicle only. These groups were all compared with the aging rats (24months) which had received no other treatment. The cognitive impairment was assessed using Morris water maze (MWM) in the prepared models, and the amount of β-galactosidase, the lipid peroxidation product malondialdehyde (MDA) level and the superoxide dismutase (SOD) activity in the hippocampus was examined by assay kits. The levels of EPO, EPOR, p-JAK2 and hypoxia-inducible factor-2α (HIF-2α) in the hippocampus were detected by western blot. Additionally, the correlation coefficient between EPO/EPOR expression and MDA level was analyzed. The MWM test showed that compared to control group, the escape latency was significantly extended and the times of crossing the platform was decreased at the doses of 150mg·kg(-1) and 250mg·kg(-1) (paging rats, the expressions of EPO, EPOR, p-JAK2, and HIF-2αin the brain of d-gal-treated rats were significantly decreased (paging could result in the decline of EPO in the hippocampus and oxidative stress might be the main reason for the decline of brain EPO in aging rats, involved with the decrease of HIF-2α stability. Copyright © 2016 Elsevier Inc. All rights reserved.

Data showing non-conventional HLA-B27 expression in axial joints and gut tissue from B27 transgenic rats, and in frozen and paraffin-fixed synovial SpA tissue

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Oliwia Rysnik

2016-12-01

Full Text Available Data is presented showing expression of non-conventional (NC heavy chain forms of B27 in synovial tissues from SpA patients. Data is presented showing the expression patterns of NC-B27 in joint, gastrointestinal and lymphoid tissues from B27 transgenic (TG1 rats with M. tuberculosis-induced SpA. Expression of NC-B27 was determined by immunohistochemistry and flow cytometry using HC10 and HD6 antibodies. These data are the extension of the data presented and discussed in “Non-conventional forms of HLA-B27 are expressed in Spondyloarthritis joints and gut tissue” (O. Rysnik, K. McHugh, L. M. van Duivenvoorde, M. N. van Tok, G. Guggino, J. D. Taurog, S. Kollnberger, F. Ciccia, D. L. Baeten, P. Bowness, 2016 [1].

The microRNA machinery regulates fasting-induced changes in gene expression and longevity in Caenorhabditis elegans.

Science.gov (United States)

Kogure, Akiko; Uno, Masaharu; Ikeda, Takako; Nishida, Eisuke

2017-07-07

Intermittent fasting (IF) is a dietary restriction regimen that extends the lifespans of Caenorhabditis elegans and mammals by inducing changes in gene expression. However, how IF induces these changes and promotes longevity remains unclear. One proposed mechanism involves gene regulation by microRNAs (miRNAs), small non-coding RNAs (∼22 nucleotides) that repress gene expression and whose expression can be altered by fasting. To test this proposition, we examined the role of the miRNA machinery in fasting-induced transcriptional changes and longevity in C. elegans We revealed that fasting up-regulated the expression of the miRNA-induced silencing complex (miRISC) components, including Argonaute and GW182, and the miRNA-processing enzyme DRSH-1 (the ortholog of the Drosophila Drosha enzyme). Our lifespan measurements demonstrated that IF-induced longevity was suppressed by knock-out or knockdown of miRISC components and was completely inhibited by drsh-1 ablation. Remarkably, drsh-1 ablation inhibited the fasting-induced changes in the expression of the target genes of DAF-16, the insulin/IGF-1 signaling effector in C. elegans Fasting-induced transcriptome alterations were substantially and modestly suppressed in the drsh-1 null mutant and the null mutant of ain-1 , a gene encoding GW182, respectively. Moreover, miRNA array analyses revealed that the expression levels of numerous miRNAs changed after 2 days of fasting. These results indicate that components of the miRNA machinery, especially the miRNA-processing enzyme DRSH-1, play an important role in mediating IF-induced longevity via the regulation of fasting-induced changes in gene expression. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Inhibition of EMMPRIN and MMP-9 Expression by Epigallocatechin-3-Gallate through 67-kDa Laminin Receptor in PMA-Induced Macrophages.

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Wang, Qi-Ming; Wang, Hao; Li, Ya-Fei; Xie, Zhi-Yong; Ma, Yao; Yan, Jian-Jun; Gao, Yi Fan Wei; Wang, Ze-Mu; Wang, Lian-Sheng

2016-01-01

It is well documented that overexpression of EMMPRIN (extracellular matrix metalloproteinase inducer) and MMPs (matrix metalloproteinases) by monocytes/macrophages plays an important role in atherosclerotic plaque rupture. Green tea polyphenol epigallocatechin-3-gallate (EGCG) has a variety of pharmacological properties and exerts cardiovascular protective effects. Recently, the 67-kD laminin receptor (67LR) has been identified as a cell surface receptor of EGCG. The aim of the present study was to evaluate the effects of EGCG on the expression of EMMPRIN and MMP-9 in PMA-induced macrophages, and the potential mechanisms underlying its effects. Human monocytic THP-1 cells were induced to differentiate into macrophages with phorbol 12-myristate 13-acetate (PMA). Protein expression and MMP-9 activity were assayed by Western blot and Gelatin zymography, respectively. Real-time PCR was used to examine EMMPRIN and MMP-9 mRNA expression. We showed that EGCG (10-50µmol/L) significantly inhibited the expression of EMMPRIN and MMP-9 and activation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 and c-Jun N-terminal kinase (JNK) in PMA-induced macrophages. Downregulation of EMMPRIN by gene silencing hindered PMA-induced MMP-9 secretion and expression, indicating an important role of EMMPRIN in the inhibition of MMP-9 by EGCG. Moreover, 67LR was involved in EGCG-mediated suppression of EMMPRIN and MMP-9 expression. Anti-67LR antibody treatment led to abrogation of the inhibitory action of EGCG on the expression of EMMPRIN and MMP-9 and activation of ERK1/2, p38, and JNK. Our results indicate that EGCG restrains EMMPRIN and MMP-9 expression via 67LR in PMA-induced macrophages, which also suggests that EGCG may be a possible therapeutic agent for stabilizing atherosclerotic plaque. © 2016 The Author(s) Published by S. Karger AG, Basel.

Inhibition of EMMPRIN and MMP-9 Expression by Epigallocatechin-3-Gallate through 67-kDa Laminin Receptor in PMA-Induced Macrophages

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Qi-Ming Wang

2016-11-01

Full Text Available Background/Aims: It is well documented that overexpression of EMMPRIN (extracellular matrix metalloproteinase inducer and MMPs (matrix metalloproteinases by monocytes/macrophages plays an important role in atherosclerotic plaque rupture. Green tea polyphenol epigallocatechin-3-gallate (EGCG has a variety of pharmacological properties and exerts cardiovascular protective effects. Recently, the 67-kD laminin receptor (67LR has been identified as a cell surface receptor of EGCG. The aim of the present study was to evaluate the effects of EGCG on the expression of EMMPRIN and MMP-9 in PMA-induced macrophages, and the potential mechanisms underlying its effects. Methods: Human monocytic THP-1 cells were induced to differentiate into macrophages with phorbol 12-myristate 13-acetate (PMA. Protein expression and MMP-9 activity were assayed by Western blot and Gelatin zymography, respectively. Real-time PCR was used to examine EMMPRIN and MMP-9 mRNA expression. Results: We showed that EGCG (10-50µmol/L significantly inhibited the expression of EMMPRIN and MMP-9 and activation of extracellular signal-regulated kinase 1/2 (ERK1/2, p38 and c-Jun N-terminal kinase (JNK in PMA-induced macrophages. Downregulation of EMMPRIN by gene silencing hindered PMA-induced MMP-9 secretion and expression, indicating an important role of EMMPRIN in the inhibition of MMP-9 by EGCG. Moreover, 67LR was involved in EGCG-mediated suppression of EMMPRIN and MMP-9 expression. Anti-67LR antibody treatment led to abrogation of the inhibitory action of EGCG on the expression of EMMPRIN and MMP-9 and activation of ERK1/2, p38, and JNK. Conclusion: Our results indicate that EGCG restrains EMMPRIN and MMP-9 expression via 67LR in PMA-induced macrophages, which also suggests that EGCG may be a possible therapeutic agent for stabilizing atherosclerotic plaque.

Aberrant neuronal activity-induced signaling and gene expression in a mouse model of RASopathy.

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Franziska Altmüller

2017-03-01

Full Text Available Noonan syndrome (NS is characterized by reduced growth, craniofacial abnormalities, congenital heart defects, and variable cognitive deficits. NS belongs to the RASopathies, genetic conditions linked to mutations in components and regulators of the Ras signaling pathway. Approximately 50% of NS cases are caused by mutations in PTPN11. However, the molecular mechanisms underlying cognitive impairments in NS patients are still poorly understood. Here, we report the generation and characterization of a new conditional mouse strain that expresses the overactive Ptpn11D61Y allele only in the forebrain. Unlike mice with a global expression of this mutation, this strain is viable and without severe systemic phenotype, but shows lower exploratory activity and reduced memory specificity, which is in line with a causal role of disturbed neuronal Ptpn11 signaling in the development of NS-linked cognitive deficits. To explore the underlying mechanisms we investigated the neuronal activity-regulated Ras signaling in brains and neuronal cultures derived from this model. We observed an altered surface expression and trafficking of synaptic glutamate receptors, which are crucial for hippocampal neuronal plasticity. Furthermore, we show that the neuronal activity-induced ERK signaling, as well as the consecutive regulation of gene expression are strongly perturbed. Microarray-based hippocampal gene expression profiling revealed profound differences in the basal state and upon stimulation of neuronal activity. The neuronal activity-dependent gene regulation was strongly attenuated in Ptpn11D61Y neurons. In silico analysis of functional networks revealed changes in the cellular signaling beyond the dysregulation of Ras/MAPK signaling that is nearly exclusively discussed in the context of NS at present. Importantly, changes in PI3K/AKT/mTOR and JAK/STAT signaling were experimentally confirmed. In summary, this study uncovers aberrant neuronal activity-induced

Fascaplysin sensitizes cells to TRAIL-induced apoptosis through upregulating DR5 expression

Science.gov (United States)

Wang, Feng; Chen, Haimin; Yan, Xiaojun; Zheng, Yanling

2013-05-01

This study investigated the molecular mechanism of anti-tumor effect of fascaplysin, a nitrogenous red pigment firstly isolated from a marine sponge. Microarray analysis show that the TNF and TNF receptor superfamily in human umbilical vein endothelial cells (HUVEC) and human hepatocarcinoma cells (BEL-7402) were significantly regulated by fascaplysin. Western Blot results reveal that fascaplysin increased the expression of cleaved caspase-9, active caspase-3, and decreased the level of procaspase-8 and Bid. Flow cytometry and cytotoxicity tests indicate that fascaplysin sensitized cells to tumor necrosis-related apoptosisinducing ligand-(TRAIL) induced apoptosis, which was markedly blocked by TRAIL R2/Fc chimera, a dominant negative form of TRAIL receptor DR5. Therefore, our results demonstrate that fascaplysin promotes apoptosis through the activation of TRAIL signaling pathway by upregulating DR5 expression.

TGF-β induces the expression of the adaptor Ndfip1 to silence IL-4 production during iTreg cell differentiation.

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Beal, Allison M; Ramos-Hernández, Natalia; Riling, Chris R; Nowelsky, Erin A; Oliver, Paula M

2011-11-13

Mice deficient in the adaptor Ndfip1 develop inflammation at sites of environmental antigen exposure. We show here that such mice had fewer inducible regulatory T cells (iT(reg) cells). In vitro, Ndfip1-deficient T cells expressed normal amounts of the transcription factor Foxp3 during the first 48 h of iT(reg) cell differentiation; however, this expression was not sustained. Abortive Foxp3 expression was caused by production of interleukin 4 (IL-4) by Ndfip1(-/-) cells. We found that Ndfip1 expression was transiently upregulated during iT(reg) cell differentiation in a manner dependent on transforming growth factor-β (TGF-β). Once expressed, Ndfip1 promoted degradation of the transcription factor JunB mediated by the E3 ubiquitin ligase Itch, thus preventing IL-4 production. On the basis of our data, we propose that TGF-β signaling induces Ndfip1 expression to silence IL-4 production, thus permitting iT(reg) cell differentiation.

Identification of salt-stress induced differentially expressed genes in ...

African Journals Online (AJOL)

Identification of salt-stress induced differentially expressed genes in barley leaves using the annealingcontrol- primer-based GeneFishing technique. S Lee, K Lee, K Kim, GJ Choi, SH Yoon, HC Ji, S Seo, YC Lim, N Ahsan ...

Increased Expression of CCN2 in the Red Flashing Light-Induced Myopia in Guinea Pigs

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Hong Wang

2013-01-01

Full Text Available Visual environment plays an important role in the occurrence of myopia. We previously showed that the different flashing lights could result in distinct effects on the ocular growth and development of myopia. CCN2 has been reported to regulate various cellular functions and biological processes. However, whether CCN2 signaling was involved in the red flashing light-induced myopia still remains unknown. In the present study, we investigated the effects of the red flashing lights exposure on the refraction and axial length of the eyes in vivo and then evaluated their effects on the expression of CCN2 and TGF-β in sclera tissues. Our data showed that the eyes exposed to the red flashing light became more myopic with a significant increase of the axial length and decrease of the refraction. Both CCN2 and TGF-β, as well as p38 MAPK and PI3K, were highly expressed in the sclera tissues exposed to the red flashing light. Both CCN2 and TGF-β were found to have the same gene expression profile in vivo. In conclusion, our findings found that CCN2 signaling pathway plays an important role in the red flashing light-induced myopia in vivo. Moreover, our study establishes a useful animal model for experimental myopia research.

DHA down-regulates phenobarbital-induced cytochrome P450 2B1 gene expression in rat primary hepatocytes by attenuating CAR translocation

International Nuclear Information System (INIS)

Li, C.-C.; Lii, C.-K.; Liu, K.-L.; Yang, J.-J.; Chen, H.-W.

2007-01-01

The constitutive androstane receptor (CAR) plays an important role in regulating the expression of detoxifying enzymes, including cytochrome P450 2B (CYP 2B). Phenobarbital (PB) induction of human CYP 2B6 and mouse CYP 2b10 has been shown to be mediated by CAR. Our previous study showed that PB-induced CYP 2B1 expression in rat primary hepatocytes is down-regulated by both n-6 and n-3 polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA); however, the mechanism for this down-regulation by DHA was previously unknown. The objective of the present study was to determine whether change in CAR translocation is involved in the down-regulation by n-6 and n-3 PUFAs of PB-induced CYP 2B1 expression in rat primary hepatocytes. We used 100 μM arachidonic acid, linoleic acid, eicosapentaenoic acid, and DHA to test this hypothesis. PB triggered the translocation of CAR from the cytosol into the nucleus in a dose-dependent and time-dependent manner in our hepatocyte system, and the CAR distribution in rat primary hepatocytes was significantly affected by DHA. DHA treatment decreased PB-inducible accumulation of CAR in the nuclear fraction and increased it in the cytosolic fraction in a dose-dependent manner. The down-regulation of CYP 2B1 expression by DHA occurred in a dose-dependent manner, and a similar pattern was found for the nuclear accumulation of CAR. The results of immunoprecipitation showed a CAR/RXR heterodimer bound to nuclear receptor binding site 1 (NR-1) of the PB-responsive enhancer module (PBREM) of the CYP 2B1gene. The EMSA results showed that PB-induced CAR binding to NR-1 was attenuated by DHA. Taken together, these results suggest that attenuation of CAR translocation and decreased subsequent binding to NR-1 are involved in DHA's down-regulation of PB-induced CYP 2B1 expression

Exercise-induced liver chemokine CXCL-1 expression is linked to muscle-derived interleukin-6 expression

DEFF Research Database (Denmark)

Pedersen, Line; Pilegaard, Henriette; Hansen, Jakob

2011-01-01

interleukin-6 (IL-6) and muscle IL-6 mRNA. In contrast, exercise-induced regulation of liver CXCL-1 mRNA expression was completely blunted in IL-6 knockout mice. Based on these findings, we examined the possible existence of a muscle-to-liver axis by overexpressing IL-6 in muscles. This resulted in increases...... in serum CXCL-1 (5-fold) and liver CXCL-1 mRNA expression (24-fold) compared with control. Because IL-6 expression and release are known to be augmented during exercise in glycogen-depleted animals, CXCL-1 and IL-6 expression were examined after exercise in overnight-fasted mice.We found that fasting...... significantly augmented serum CXCL-1, and CXCL-1 expression in liver and muscle. Taken together, these data indicate that liver is the main source of serum CXCL-1 during exercise in mice, and that the CXCL-1 expression in the liver is regulated by muscle-derived IL-6....

Oncogenic tyrosine kinase NPM-ALK induces expression of the growth-promoting receptor ICOS

DEFF Research Database (Denmark)

Zhang, Qian; Wang, HongYi; Kantekure, Kanchan

2011-01-01

Here we report that T-cell lymphoma cells carrying the NPM-ALK fusion protein (ALK(+) TCL) frequently express the cell-stimulatory receptor ICOS. ICOS expression in ALK(+) TCL is moderate and strictly dependent on the expression and enzymatic activity of NPM-ALK. NPM-ALK induces ICOS expression v...

Modulation of macrophage Ia expression by lipopolysaccharide: Stem cell requirements, accessory lymphocyte involvement, and IA-inducing factor production

International Nuclear Information System (INIS)

Wentworth, P.A.; Ziegler, H.K.

1989-01-01

The mechanism of induction of murine macrophage Ia expression by lipopolysaccharide (LPS) was studied. Intraperitoneal injection of 1 microgram of LPS resulted in a 3- to 10-fold increase in the number of IA-positive peritoneal macrophages (flow cytometry and immunofluorescence) and a 6-to 16-fold increase by radioimmunoassay. The isolated lipid A moiety of LPS was a potent inducer of macrophage Ia expression. Ia induction required a functional myelopoietic system as indicated by the finding that the response to LPS was eliminated in irradiated (900 rads) mice and reinstated by reconstitution with bone marrow cells. Comparison of LPS-induced Ia expression in normal and LPS-primed mice revealed a faster secondary response to LPS. The memory response could be adoptively transferred to normal mice with nonadherent spleen cells prepared 60 days after LPS injection. Spleen cells prepared 5 days after LPS injection caused Ia induction in LPS-nonresponder mice; such induction was not observed in irradiated (900 rads) recipients. The cell responsible for this phenomenon was identified as a Thy-1+, immunoglobulin-negative nonadherent cell. The biosynthesis and expression of Ia were not increased by direct exposure of macrophages to LPS in vitro. Small amounts of LPS inhibited Ia induction by gamma interferon. LPS showed positive regulatory effects on Ia expression by delaying the loss of Ia expression on cultured macrophages and by stimulating the production of Ia-inducing factors. Supernatants from cultured spleen cells stimulated with LPS in vitro contained antiviral and Ia-inducing activity that was acid labile, indicating that the active factor is gamma interferon. We conclude that induction of Ia expression by LPS in vivo is a bone-marrow-dependent, radiation-sensitive process which involves the stimulation of a gamma interferon-producing accessory lymphocyte and a delay in Ia turnover

Agave tequilana MADS genes show novel expression patterns in meristems, developing bulbils and floral organs.

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Delgado Sandoval, Silvia del Carmen; Abraham Juárez, María Jazmín; Simpson, June

2012-03-01

Agave tequilana is a monocarpic perennial species that flowers after 5-8 years of vegetative growth signaling the end of the plant's life cycle. When fertilization is unsuccessful, vegetative bulbils are induced on the umbels of the inflorescence near the bracteoles from newly formed meristems. Although the regulation of inflorescence and flower development has been described in detail for monocarpic annuals and polycarpic species, little is known at the molecular level for these processes in monocarpic perennials, and few studies have been carried out on bulbils. Histological samples revealed the early induction of umbel meristems soon after the initiation of the vegetative to inflorescence transition in A. tequilana. To identify candidate genes involved in the regulation of floral induction, a search for MADS-box transcription factor ESTs was conducted using an A. tequilana transcriptome database. Seven different MIKC MADS genes classified into 6 different types were identified based on previously characterized A. thaliana and O. sativa MADS genes and sequences from non-grass monocotyledons. Quantitative real-time PCR analysis of the seven candidate MADS genes in vegetative, inflorescence, bulbil and floral tissues uncovered novel patterns of expression for some of the genes in comparison with orthologous genes characterized in other species. In situ hybridization studies using two different genes showed expression in specific tissues of vegetative meristems and floral buds. Distinct MADS gene regulatory patterns in A. tequilana may be related to the specific reproductive strategies employed by this species.

Expression of miRNA-122 Induced by Liver Toxicants in Zebrafish

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Hyun-Sik Nam

2016-01-01

Full Text Available MicroRNA-122 (miRNA-122, also known as liver-specific miRNA, has recently been shown to be a potent biomarker in response to liver injury in mammals. The objective of this study was to examine its expression in response to toxicant treatment and acute liver damage, using the zebrafish system as an alternative model organism. For the hepatotoxicity assay, larval zebrafish were arrayed in 24-well plates. Adult zebrafish were also tested and arrayed in 200 mL cages. Animals were exposed to liver toxicants (tamoxifen or acetaminophen at various doses, and miRNA-122 expression levels were analyzed using qRT-PCR in dissected liver, brain, heart, and intestine, separately. Our results showed no significant changes in miRNA-122 expression level in tamoxifen-treated larvae; however, miRNA-122 expression was highly induced in tamoxifen-treated adults in a tissue-specific manner. In addition, we observed a histological change in adult liver (0.5 μM and cell death in larval liver (5 μM at different doses of tamoxifen. These results indicated that miRNA-122 may be utilized as a liver-specific biomarker for acute liver toxicity in zebrafish.

Arsenite enhances tumor necrosis factor-α-induced expression of vascular cell adhesion molecule-1

International Nuclear Information System (INIS)

Tsou, T.-C.; Yeh, Szu Ching; Tsai, E.-M.; Tsai, F.-Y.; Chao, H.-R.; Chang, Louis W.

2005-01-01

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

Experimentally nonylphenol-polluted diet induces the expression of silent genes VTG and ER{alpha} in the liver of male lizard Podarcis sicula

Energy Technology Data Exchange (ETDEWEB)

Verderame, Mariailaria; Prisco, Marina; Andreuccetti, Piero [Department of Biological Sciences, Evolutionary and Comparative Biology Division, University Federico II of Naples, Via Mezzocannone 8, 80134 Naples (Italy); Aniello, Francesco [Department of Biological Sciences, Genetic and Molecular Biology Division, University Federico II of Naples, Via Mezzocannone 8, 80134 Naples (Italy); Limatola, Ermelinda, E-mail: limatola@unina.it [Department of Biological Sciences, Evolutionary and Comparative Biology Division, University Federico II of Naples, Via Mezzocannone 8, 80134 Naples (Italy)

2011-05-15

Endocrine Disruptor Chemicals (EDCs) with estrogen-like properties i.e nonylphenol (NP) induce vitellogenin (VTG) synthesis in males of aquatic and semi-aquatic specie. In the oviparous species VTG is a female-specific oestrogen dependent protein. Males are unable to synthesize VTG except after E{sub 2} treatment. This study aimed to verify if NP, administered via food and water, is able to induce the expression of VTG even in males of vertebrates with a terrestrial habitat such as the lizard Podarcis. By means of ICC, ISH, W/B and ELISA we demonstrated that NP induces the presence of VTG in the plasma and its expression in the liver. VTG, undetectable in untreated males, reaches the value of 4.34 {mu}g/{mu}l in the experimental ones. Expression analysis and ISH in the liver showed that an NP-polluted diet also elicits the expression of ER{alpha} in the liver which is known to be related to VTG synthesis in Podarcis. - Highlights: > Nonylphenol (NP) polluted diet induces VTG synthesis in a terrestrial vertebrate. > VTG and ER{alpha} genes are unexpressed in the liver of untreated male lizards Podarcis. > In the liver cells of NP-treated males the expression of both VTG and ER{alpha} occurs. > In treated males VTG synthesis is coupled with ER{alpha} expression as in breeding females. - NP-polluted diet induces the expression of ER{alpha} and VTG in the liver.

Alpha particles induce expression of immunogenic markers on tumour cells

International Nuclear Information System (INIS)

Gorin, J.B.; Gouard, S.; Cherel, M.; Davodeau, F.; Gaschet, J.; Morgenstern, A.; Bruchertseifer, F.

2013-01-01

The full text of the publication follows. Radioimmunotherapy (RIT) is an approach aiming at targeting the radioelements to tumours, usually through the use of antibodies specific for tumour antigens. The radiations emitted by the radioelements then induce direct killing of the targeted cells as well as indirect killing through bystander effect. Interestingly, it has been shown that ionizing radiations, in some settings of external radiotherapy, can foster an immune response directed against tumour cells. Our research team is dedicated to the development of alpha RIT, i.e RIT using alpha particle emitters, we therefore decided to study the effects of such particles on tumour cells in regards to their immunogenicity. First, we studied the effects of bismuth 213, an alpha emitter, on cellular death and autophagy in six different tumour cell lines. Then, we measured the expression of 'danger' signals and MHC molecules at the cell surface to determine whether irradiation with 213 Bi could cause the tumour cells to be recognized by the immune system. Finally a co-culture of dendritic cells with irradiated tumour cells was performed to test whether it would induce dendritic cells to mature. No apoptosis was detected within 48 hours after irradiation in any cell line, however half of them exhibited signs of autophagy. No increase in membrane expression of 'danger' signals was observed after treatment with 213 Bi, but we showed an increase in expression of MHC class I and II for some cell lines. Moreover, the co-culture experiment indicated that the immunogenicity of a human adenocarcinoma cell line (LS 174T) was enhanced in vitro after irradiation with alpha rays. These preliminary data suggest that alpha particles could be of interest in raising an immune response associated to RIT. (authors)

Lipopolysaccharide induces autotaxin expression in human monocytic THP-1 cells

International Nuclear Information System (INIS)

Li Song; Zhang Junjie

2009-01-01

Autotaxin (ATX) is a secreted enzyme with lysophospholipase D (lysoPLD) activity, which converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), a bioactive phospholipid involved in numerous biological activities, including cell proliferation, differentiation, and migration. In the present study, we found that bacterial lipopolysaccharide (LPS), a well-known initiator of the inflammatory response, induced ATX expression in monocytic THP-1 cells. The activation of PKR, JNK, and p38 MAPK was required for the ATX induction. The LPS-induced ATX in THP-1 cells was characterized as the β isoform. In the presence of LPC, ATX could promote the migrations of THP-1 and Jurkat cells, which was inhibited by pertussis toxin (PTX), an inhibitor of Gi-mediated LPA receptor signaling. In summary, LPS induces ATX expression in THP-1 cells via a PKR, JNK and p38 MAPK-mediated mechanism, and the ATX induction is likely to enhance immune cell migration in proinflammatory response by regulating LPA levels in the microenvironment.

Alpha5 nicotinic acetylcholine receptor mediates nicotine-induced HIF-1α and VEGF expression in non-small cell lung cancer

Energy Technology Data Exchange (ETDEWEB)

Ma, Xiaoli; Jia, Yanfei; Zu, Shanshan [Central Laboratory, Jinan Central Hospital Affiliated to Shandong University, Jinan 250013 (China); Li, Ruisheng [Institute of Infectious Diseases, 302 Military Hospital, Beijing 100039 (China); Jia, Ying; Zhao, Yun; Xiao, Dongjie [Central Laboratory, Jinan Central Hospital Affiliated to Shandong University, Jinan 250013 (China); Dang, Ningning [Department of Dermatology, Jinan Central Hospital Affiliated to Shandong University, Jinan 250013 (China); Wang, Yunshan [Central Laboratory, Jinan Central Hospital Affiliated to Shandong University, Jinan 250013 (China)

2014-07-15

By binding to nicotinic acetylcholine receptors (nAChRs), nicotine induces the proliferation and apoptosis of non-small cell lung cancer (NSCLC). Previous studies have indicated that α5-nAChR is highly associated with lung cancer risk and nicotine dependence. However, the mechanisms through which α5-nAChRs may influence lung carcinogenesis are far from clear. In the present study, we investigated the roles of α5-nAChR in the nicotine-induced expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF). Immunohistochemistry was used to detect the expression of α5-nAChR and HIF-1α in 60 specimens of lung cancer and para-carcinoma tissue. The correlations between the expression levels of α5-nAChR and HIF-1α and other clinicopathological data were analyzed. In a cell line that highly expressed α5-nAChR, the loss of α5-nAChR function by siRNA was used to study whether α5-nAChR is involved in the nicotine-induced expression of HIF-1α and VEGF through the activation of the ERK1/2 and PI3K/Akt signaling pathways. Cell growth was detected using the cell counting kit-8 (CCK-8). α5-nAChR (78.3%) and HIF-1α (88.3%) were both overexpressed in NSCLC, and their expression levels were found to be correlated with each other (P < 0.05). In the A549 cell line, α5-nAChR and HIF-1α were found to be expressed under normal conditions, and their expression levels were significantly increased in response to nicotine treatment. The silencing of α5-nAChR significantly inhibited the nicotine-induced cell proliferation compared with the control group and attenuated the nicotine-induced upregulation of HIF-1α and VEGF, and these effects required the cooperation of the ERK1/2 and PI3K/Akt signaling pathways. These results show that the α5-nAChR/HIF-1α/VEGF axis is involved in nicotine-induced tumor cell proliferation, which suggests that α5-nAChR may serve as a potential anticancer target in nicotine-associated lung cancer. - Highlights

Alpha5 nicotinic acetylcholine receptor mediates nicotine-induced HIF-1α and VEGF expression in non-small cell lung cancer

International Nuclear Information System (INIS)

Ma, Xiaoli; Jia, Yanfei; Zu, Shanshan; Li, Ruisheng; Jia, Ying; Zhao, Yun; Xiao, Dongjie; Dang, Ningning; Wang, Yunshan

2014-01-01

By binding to nicotinic acetylcholine receptors (nAChRs), nicotine induces the proliferation and apoptosis of non-small cell lung cancer (NSCLC). Previous studies have indicated that α5-nAChR is highly associated with lung cancer risk and nicotine dependence. However, the mechanisms through which α5-nAChRs may influence lung carcinogenesis are far from clear. In the present study, we investigated the roles of α5-nAChR in the nicotine-induced expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF). Immunohistochemistry was used to detect the expression of α5-nAChR and HIF-1α in 60 specimens of lung cancer and para-carcinoma tissue. The correlations between the expression levels of α5-nAChR and HIF-1α and other clinicopathological data were analyzed. In a cell line that highly expressed α5-nAChR, the loss of α5-nAChR function by siRNA was used to study whether α5-nAChR is involved in the nicotine-induced expression of HIF-1α and VEGF through the activation of the ERK1/2 and PI3K/Akt signaling pathways. Cell growth was detected using the cell counting kit-8 (CCK-8). α5-nAChR (78.3%) and HIF-1α (88.3%) were both overexpressed in NSCLC, and their expression levels were found to be correlated with each other (P < 0.05). In the A549 cell line, α5-nAChR and HIF-1α were found to be expressed under normal conditions, and their expression levels were significantly increased in response to nicotine treatment. The silencing of α5-nAChR significantly inhibited the nicotine-induced cell proliferation compared with the control group and attenuated the nicotine-induced upregulation of HIF-1α and VEGF, and these effects required the cooperation of the ERK1/2 and PI3K/Akt signaling pathways. These results show that the α5-nAChR/HIF-1α/VEGF axis is involved in nicotine-induced tumor cell proliferation, which suggests that α5-nAChR may serve as a potential anticancer target in nicotine-associated lung cancer. - Highlights

Expression of transforming growth factor alpha and epidermal growth factor receptor in rat lung neoplasms induced by plutonium-239

International Nuclear Information System (INIS)

Stegelmeier, B.L.; Gillett, N.A.; Hahn, F.F.; Kelly, G.; Rebar, A.H.

1994-01-01

Ninety-two rat lung proliferative lesions and neoplasms induced by inhaled 239 PuO 2 were evaluated for aberrant expression of transforming growth factor alpha (TGF-α) and epidermal growth factor receptor (EGFR). Expression of TGF-α protein, measured by immunohistochemistry, was higher in 94% of the squamous cell carcinomas and 87% of the foci of alveolar epithelial squamous metaplasia than that exhibited by the normal-appearing, adjacent lung parenchyma. In contrast, only 20% of adenocarcinomas and foci of epithelial hyperplasia expressed elevated levels of TGF-α. Many neoplasms expressing TGF-α also expressed excessive levels of EGFR mRNA. Southern and DNA slot blot analyses showed that the elevated EGFR expression was not due to amplification of the EGFR gene. These data suggest that increased amounts of TGF-α were early alterations in the progression of plutonium-induced squamous cell carcinoma, and these increases may occur in parallel with overexpression of the receptor for this growth factor. Together, these alterations create a potential autocrine loop for sustaining clonal expansion of cells initiated by high-LET radiation. 44 refs., 4 figs., 1 tab

Dragon (Repulsive Guidance Molecule RGMb) Inhibits E-cadherin Expression and Induces Apoptosis in Renal Tubular Epithelial Cells*

Science.gov (United States)

Liu, Wenjing; Li, Xiaoling; Zhao, Yueshui; Meng, Xiao-Ming; Wan, Chao; Yang, Baoxue; Lan, Hui-Yao; Lin, Herbert Y.; Xia, Yin

2013-01-01

Dragon is one of the three members of the repulsive guidance molecule (RGM) family, i.e. RGMa, RGMb (Dragon), and RGMc (hemojuvelin). We previously identified the RGM members as bone morphogenetic protein (BMP) co-receptors that enhance BMP signaling. Our previous studies found that Dragon is highly expressed in the tubular epithelial cells of mouse kidneys. However, the roles of Dragon in renal epithelial cells are yet to be defined. We now show that overexpression of Dragon increased cell death induced by hypoxia in association with increased cleaved poly(ADP-ribose) polymerase and cleaved caspase-3 levels in mouse inner medullary collecting duct (IMCD3) cells. Dragon also inhibited E-cadherin expression but did not affect epithelial-to-mesenchymal transition induced by TGF-β in IMCD3 cells. Previous studies suggest that the three RGM members can function as ligands for the receptor neogenin. Interestingly, our present study demonstrates that the Dragon actions on apoptosis and E-cadherin expression in IMCD3 cells were mediated by the neogenin receptor but not through the BMP pathway. Dragon expression in the kidney was up-regulated by unilateral ureteral obstruction in mice. Compared with wild-type mice, heterozygous Dragon knock-out mice exhibited 45–66% reduction in Dragon mRNA expression, decreased epithelial apoptosis, and increased tubular E-cadherin expression and had attenuated tubular injury after unilateral ureteral obstruction. Our results suggest that Dragon may impair tubular epithelial integrity and induce epithelial apoptosis both in vitro and in vivo. PMID:24052264

Interferon-γ induces expression of MHC class II on intestinal epithelial cells and protects mice from colitis.

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Christoph Thelemann

Full Text Available Immune responses against intestinal microbiota contribute to the pathogenesis of inflammatory bowel diseases (IBD and involve CD4(+ T cells, which are activated by major histocompatibility complex class II (MHCII molecules on antigen-presenting cells (APCs. However, it is largely unexplored how inflammation-induced MHCII expression by intestinal epithelial cells (IEC affects CD4(+ T cell-mediated immunity or tolerance induction in vivo. Here, we investigated how epithelial MHCII expression is induced and how a deficiency in inducible epithelial MHCII expression alters susceptibility to colitis and the outcome of colon-specific immune responses. Colitis was induced in mice that lacked inducible expression of MHCII molecules on all nonhematopoietic cells, or specifically on IECs, by continuous infection with Helicobacter hepaticus and administration of interleukin (IL-10 receptor-blocking antibodies (anti-IL10R mAb. To assess the role of interferon (IFN-γ in inducing epithelial MHCII expression, the T cell adoptive transfer model of colitis was used. Abrogation of MHCII expression by nonhematopoietic cells or IECs induces colitis associated with increased colonic frequencies of innate immune cells and expression of proinflammatory cytokines. CD4(+ T-helper type (Th1 cells - but not group 3 innate lymphoid cells (ILCs or Th17 cells - are elevated, resulting in an unfavourably altered ratio between CD4(+ T cells and forkhead box P3 (FoxP3(+ regulatory T (Treg cells. IFN-γ produced mainly by CD4(+ T cells is required to upregulate MHCII expression by IECs. These results suggest that, in addition to its proinflammatory roles, IFN-γ exerts a critical anti-inflammatory function in the intestine which protects against colitis by inducing MHCII expression on IECs. This may explain the failure of anti-IFN-γ treatment to induce remission in IBD patients, despite the association of elevated IFN-γ and IBD.

Behavioral experience induces zif268 expression in mature granule cells but suppresses its expression in immature granule cells

Science.gov (United States)

Huckleberry, Kylie A.; Kane, Gary A.; Mathis, Rita J.; Cook, Sarah G.; Clutton, Jonathan E.; Drew, Michael R.

2015-01-01

Thousands of neurons are born each day in the dentate gyrus (DG), but many of these cells die before reaching maturity. Both death and survival of adult-born neurons are regulated by neuronal activity in the DG. The immediate-early gene (IEG) zif268 appears to be an important mediator of these effects, as its expression can be induced by neural activity and knockout of zif268 impairs survival of adult-born neurons (Richardson et al., 1992; Veyrac et al., 2013). Despite the apparent importance of zif268 for adult neurogenesis, its behavior-induced expression has not been fully characterized in adult-born neurons. Here we characterize behavior-evoked expression of zif268 in mature and newborn dentate granule cells (DGCs). We first quantified zif268 expression in doublecortin-positive (DCX+) immature neurons and in the general granule cell population after brief exposure to a novel environment (NE). In the general granule cell population, zif268 expression peaked 1 h after NE exposure and returned to baseline by 8 h post-exposure. However, in the DCX+ cells, zif268 expression was suppressed relative to home cage for at least 8 h post-exposure. We next asked whether suppression of zif268 in DCX+ immature cells occurs in other behavioral paradigms that recruit the hippocampus. Exposure to Morris water maze (MWM) training, an enriched environment, or a NE caused approximately equal suppression of zif268 expression in DCX+ cells and approximately equal activation of zif268 expression among the general granule cell population. The same behavioral procedures activated zif268 expression in 6-week-old BrdU-labeled adult-born neurons, indicating that zif268 suppression is specific to immature neurons. Finally, we asked whether zif268 suppression varied as a function of age within the DCX+ population, which ranges in age from 0 to approximately 4 weeks. NE exposure had no significant effect on zif268 expression in 2- or 4-week-old BrdU-labeled neurons, but it significantly

IL-1β upregulates Muc5ac expression via NF-κB-induced HIF-1α in asthma.

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Wu, Shouzhen; Li, Hailong; Yu, Lijuan; Wang, Ning; Li, Xu; Chen, Wei

2017-12-01

The manifest and important feature in respiratory diseases, including asthma and COPD (chronic obstructive pulmonary disease), is the increased numbers and hypersecretion of goblet cells and overexpression of mucins, especially Muc5ac. Many proinflammatory cytokines play important roles in goblet cell metaplasia and overproduction of Muc5ac. However, the effect of IL-1β on Muc5ac expression in asthma remains unknown. Here, we detected the correlation between IL-1β and Muc5ac in asthma patients and further explored the mechanism of IL-1β-induced Muc5ac overexpression. Our results showed that Muc5ac and IL-1β were up-regulated in 41 patients with asthma and that Muc5ac overexpression was related with IL-1β in asthma (R 2 =0.668, p≪0.001). Furthermore, the correlation between IL-1β and Muc5ac is higher in severe group than that in moderate group. In vitro experiments with normal human bronchial epithelial cells (NHBECs) showed that IL-1β up-regulated Muc5ac expression in NHBEC in a time- and dosage-dependent manner. Hypoxia-induced HIF-1α was responsible for Muc5ac expression mediated by IL-1β. Knocking down HIF-1α by siRNA decreased Muc5ac expression under hypoxia even in IL-1β-treated NHBEC cells. Luciferase reporter assay showed that HIF-1α enhanced Muc5ac promoter activity in HEK293T cells. HIF-1α could specifically bind to the promoter of Muc5ac by EMSA. The correlation among IL-1β, HIF-1α and Muc5ac was observed in patients with asthma. Mechanically, NF-κB activation was essential to IL-1β-induced HIF-1α upregulation via the canonical pathway of NF-κB. The level of nuclear p65, a subunit of NF-κB, was obviously increased in NHBEC cells under IL-1β treatment. IL-1β did not change either HIF-1α or Muc5ac expression when inhibiting NF-κB signaling with Bay11-7082, an inhibitor of NF-κB. Collectively, we concluded that IL-1β up-regulated Muc5ac expression via NF-κB-induced HIF-1α in asthma and provided a potential therapeutic target for

Expression of transcription factors Slug in the lens epithelial cells undergoing epithelial-mesenchymal transition induced by connective tissue growth factor

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Ying-Na Wang

2015-10-01

Full Text Available AIM:To investigate the expression of transcription factors Slug in human lens epithelial cells (HLECs undergoing epithelial-mesenchymal transition (EMT induced by connective tissue growth factor (CTGF.METHODS: HLECs were treated with CTGF of different concentrations (20, 50 and 100 ng/mL or without CTGF (control for 24h. The morphological changes of HLECs were analysed by microscopy. The expression and cellular localization of Slug was evaluated by immumo-fluorescence. Expressions of Slug, E-cadherin and alpha smooth muscle actin (α-SMA were further determined by Western blot analysis. RESULTS: HLECs showed spidle fibrolasts-like characteristics and loosely connected each other after CTGF treatment. The immuno-fluorescence staining indicated that Slug was localized in the nuclei and its expression was induced by CTGF. The relative expressions of Slug protein were 1.64±0.11, 1.96 ±0.03, 3.12 ±0.10, and 4.08±0.14, respectively, in response to control group and treatment with CTGF of 20, 50 and 100 ng/mL (F=443.86, PCONCLUSION: Transcription factor Slug may be involved in EMT of HLECs induced by CTGF in vitro.

Choline supplementation alleviates fluoride-induced testicular toxicity by restoring the NGF and MEK expression in mice.

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Zhang, Jianhai; Zhang, Yufang; Liang, Chen; Wang, Nasui; Zheng, Heping; Wang, Jundong

2016-11-01

Fluoride is known to cause male reproductive toxicity, and the elucidation of its underlying mechanisms is an ongoing research focus in reproductive toxicology and epidemiology. Choline, an essential nutrient, has been extensively studied for its benefits in nervous system yet was rarely discussed for its prospective effect in male reproductive system. This study aims to explore the potential protective role of choline against NaF-induced male reproductive toxicity via MAPK pathway. The male mice were administrated by 150mg/L NaF in drinking water, 5.75g/kg choline in diet, and their combination respectively from maternal gestation to postnatal 15weeks. The results showed that fluoride exposure reduced body weight growth, lowered sperm count and survival percentages, altered testicular histology, down-regulated the mRNA expressions of NGF, Ras, Raf, and MEK genes in testes, as well as significantly decreased the expressions of both NGF and phosphor-MEK proteins in testes. Examination of data from choline-treated mice revealed that choline supplementation ameliorated these fluoride-induced changes. Taken together, our findings suggest that choline supplementation alleviates fluoride-induced testicular toxicity by restoring the NGF and phosphor-MEK expression. The suitable dosage and supplementation periods of choline await further exploration. Copyright © 2016 Elsevier Inc. All rights reserved.

Oleocanthal Modulates Estradiol-Induced Gene Expression Involving Estrogen Receptor α.

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Keiler, Annekathrin Martina; Djiogue, Sefirin; Ehrhardt, Tino; Zierau, Oliver; Skaltsounis, Leandros; Halabalaki, Maria; Vollmer, Günter

2015-09-01

Oleocanthal is a bioactive compound from olive oil. It has attracted considerable attention as it is anti-inflammatory, antiproliferative, and has been shown to possess neuroprotective properties in vitro and in vivo. Delineated from its polyphenolic structure, the aim of this study was to characterize oleocanthal towards estrogenic properties. This might contribute to partly explain the beneficial effects described for the Mediterranean diet. Estrogenic properties of oleocanthal were assessed by different methods: a) stimulation of reporter gene activity in MVLN or RNDA cells either expressing estrogen receptor α or β, b) stimulation of luciferase reporter gene activity in U2OS osteosarcoma cells expressing estrogen receptor α or β, and c) elucidation of the impact on estradiol-induced gene expression in U2OS cells transduced with both estrogen receptors. Depending on the cell line origin, oleocanthal inhibited luciferase activity (MVLN, U2OS-estrogen receptor β) or weakly induced reporter gene activity at 10 µM in U2OS-estrogen receptor α cells. However, oleocanthal inhibited stimulation of luciferase activity by estradiol from both estrogen receptors. Oleocanthal, if given alone, did not stimulate gene expression in U2OS cells, but it significantly modulated the response of estradiol. Oleocanthal enhanced the effect of estradiol on the regulation of those genes, which are believed to be regulated through heterodimeric estrogen receptors. As the estrogenic response pattern of oleocanthal is rather unique, we compared the results obtained with oleacein. Oleocanthal binds to both estrogen receptors inducing estradiol-agonistic or antiagonistic effects depending on the cell line. Regarding regulation of gene expression in U2OS-estrogen receptor α/β cells, oleocanthal and oleacein enhanced estradiol-mediated regulation of heterodimer-regulated genes. Georg Thieme Verlag KG Stuttgart · New York.

Activation of p44/42 in Human Natural Killer Cells Decreases Cell-surface Protein Expression: Relationship to Tributyltin-induced alterations of protein expression

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Dudimah, Fred D.; Abraha, Abraham; Wang, Xiaofei; Whalen, Margaret M.

2010-01-01

Tributyltin (TBT) activates the mitogen activated protein kinase (MAPK), p44/42 in human natural killer (NK) cells. TBT also reduces NK cytotoxic function and decreases the expression of several NK-cell proteins. To understand the role that p44/42 activation plays in TBT-induced loss of NK cell function, we have investigated how selective activation of p44/42 by phorbol 12-myristate 13-acetate (PMA) affects NK cells. Previously we showed that PMA caused losses of lytic function similar to those seen with TBT exposures. Here we examined activation of p44/42 in the regulation of NK-cell protein expression and how this regulation may explain the protein expression changes seen with TBT exposures. NK cells exposed to PMA were examined for levels of cell-surface proteins, granzyme mRNA, and perforin mRNA expression. The expression of CD11a, CD16, CD18, and CD56 were reduced, perforin mRNA levels were unchanged and granzyme mRNA levels were increased. To verify that activation of p44/42 was responsible for the alterations seen in CD11a, CD16, CD18, and CD56 with PMA, NK cells were treated with the p44/42 pathway inhibitor (PD98059) prior to PMA exposures. In the presence of PD98059, PMA caused no decreases in the expression of the cell-surface proteins. Results of these studies indicate that the activation of p44/42 may lead to the loss of NK cell cytotoxic function by decreasing the expression of CD11a, CD16, CD18, and CD56. Further, activation of p44/42 appears to be at least in part responsible for the TBT-induced decreases in expression of CD16, CD18, and CD56. PMID:20883105

The HPV-16 E7 oncoprotein induces centriole multiplication through deregulation of Polo-like kinase 4 expression

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Duensing Stefan

2011-05-01

Full Text Available Abstract Background Infection with high-risk human papillomaviruses (HPVs such as HPV-16 is intimately associated with squamous cell carcinomas (SCCs of the anogenital tract and a subset of oropharyngeal carcinomas. Such lesions, including pre-invasive precursors, frequently show multipolar mitoses and aneuploidy. The high-risk HPV-16-encoded E7 oncoprotein has been shown to rapidly induce centrosome abnormalities thereby causing the formation of supernumerary mitotic spindle poles and increasing the risk for chromosome missegregation. HPV-16 E7 has been found to rapidly induce centriole overduplication, in part, through the simultaneous formation of more than one daughter centriole at single maternal centrioles (centriole multiplication. The precise molecular mechanism that underlies HPV-16 E7-induced centriole multiplication, however, remains poorly understood. Findings Here, we show that human keratinocytes engineered to stably express the HPV-16 E7 oncoprotein exhibit aberrant Polo-like kinase 4 (PLK4 protein expression at maternal centrioles. Real-time quantitative reverse transcriptase (qRT-PCR analysis of these cells revealed an increase of PLK4 mRNA levels compared to control cells. Importantly, the ability of the HPV-16 E7 oncoprotein to induce centriole multiplication was found to correlate with its ability to activate the PLK4 promoter and to up-regulate PLK4 mRNA. Conclusions These results highlight the critical role of PLK4 transcriptional deregulation in centriole multiplication in HPV-16 E7-expressing cells. Our findings encourage further experiments to test transcriptional inhibitors or small molecules targeting PLK4 to prevent centriole abnormalities, mitotic infidelity and malignant progression in HPV-associated neoplasms and other tumors in which PLK4 regulation is disrupted.

Expression of the sweetpotato R2R3-type IbMYB1a gene induces anthocyanin accumulation in Arabidopsis.

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Chu, Hyosub; Jeong, Jae Cheol; Kim, Wook-Jin; Chung, Dong Min; Jeon, Hyo Kon; Ahn, Young Ock; Kim, Sun Ha; Lee, Haeng-Soon; Kwak, Sang-Soo; Kim, Cha Young

2013-06-01

R2R3-type MYB transcription factors (TFs) play important roles in transcriptional regulation of anthocyanins. The R2R3-type IbMYB1 is known to be a key regulator of anthocyanin biosynthesis in the storage roots of sweetpotato. We previously showed that transient expression of IbMYB1a led to anthocyanin pigmentation in tobacco leaves. In this article, we generated transgenic Arabidopsis plants expressing the IbMYB1a gene under the control of CaMV 35S promoter, and the sweetpotato SPO and SWPA2 promoters. Overexpression of IbMYBa in transgenic Arabidopsis produced strong anthocyanin pigmentation in seedlings and generated a deep purple color in leaves, stems and seeds. Reverse transcription-polymerase chain reaction analysis showed that IbMYB1a expression induced upregulation of several structural genes in the anthocyanin biosynthetic pathway, including 4CL, CHI, F3'H, DFR, AGT, AAT and GST. Furthermore, overexpression of IbMYB1a led to enhanced expression of the AtTT8 (bHLH) and PAP1/AtMYB75 genes. high-performance liquid chromatography analysis revealed that IbMYB1a expression led to the production of cyanidin as a major core molecule of anthocyanidins in Arabidopsis, as occurs in the purple leaves of sweetpotato (cv. Sinzami). This result shows that the IbMYB1a TF is sufficient to induce anthocyanin accumulation in seedlings, leaves, stems and seeds of Arabidopsis plants. Copyright © Physiologia Plantarum 2012.

Expression of Egr1 and p53 in human carotid plaques and apoptosis induced by 7-oxysterol or p53.

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Miah, Sayem; Zadeh, Shahram Nour Mohammad; Yuan, Xi-Ming; Li, Wei

2013-07-01

Egr-1 and p53 are involved in pathology of both atherosclerosis and cancer. However, it is unknown whether p53 and Egr1 are interactively involved in apoptosis in atherosclerosis. We found that in human carotid plaques, the expression of p53 was inversely correlated with Egr1. In U937 cells, 7β-hydroxycholesterol and 7-ketocholesterol induced production of reactive oxygen species (ROS), transient up-regulation of Egr1 followed by late induction of p53 and apoptosis. Cells with nuclear fragmentation induced by 7-oxysterol or p53 showed increased levels of p53, but decreased levels of Egr1. In conclusion, ROS induced by 7-oxysterols may function as an early initiator of Egr1 expression. The late induced p53 by 7-oxysterols contributes to apoptotic cell death and is linked to the reduction of Egr1 levels, which resembles the differential expression of p53 and Egr1 in human atheroma progression. Copyright © 2012 Elsevier GmbH. All rights reserved.

Hydrogen sulfide inhibits high glucose-induced NADPH oxidase 4 expression and matrix increase by recruiting inducible nitric oxide synthase in kidney proximal tubular epithelial cells.

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Lee, Hak Joo; Lee, Doug Yoon; Mariappan, Meenalakshmi M; Feliers, Denis; Ghosh-Choudhury, Goutam; Abboud, Hanna E; Gorin, Yves; Kasinath, Balakuntalam S

2017-04-07

High-glucose increases NADPH oxidase 4 (NOX4) expression, reactive oxygen species generation, and matrix protein synthesis by inhibiting AMP-activated protein kinase (AMPK) in renal cells. Because hydrogen sulfide (H 2 S) inhibits high glucose-induced matrix protein increase by activating AMPK in renal cells, we examined whether H 2 S inhibits high glucose-induced expression of NOX4 and matrix protein and whether H 2 S and NO pathways are integrated. High glucose increased NOX4 expression and activity at 24 h in renal proximal tubular epithelial cells, which was inhibited by sodium hydrosulfide (NaHS), a source of H 2 S. High glucose decreased AMPK phosphorylation and activity, which was restored by NaHS. Compound C, an AMPK inhibitor, prevented NaHS inhibition of high glucose-induced NOX4 expression. NaHS inhibition of high glucose-induced NOX4 expression was abrogated by N (ω)-nitro-l-arginine methyl ester, an inhibitor of NOS. NaHS unexpectedly augmented the expression of inducible NOS (iNOS) but not endothelial NOS. iNOS siRNA and 1400W, a selective iNOS inhibitor, abolished the ameliorative effects of NaHS on high glucose-induced NOX4 expression, reactive oxygen species generation, and, matrix laminin expression. Thus, H 2 S recruits iNOS to generate NO to inhibit high glucose-induced NOX4 expression, oxidative stress, and matrix protein accumulation in renal epithelial cells; the two gasotransmitters H 2 S and NO and their interaction may serve as therapeutic targets in diabetic kidney disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of genes showing differential expression profile ...

Indian Academy of Sciences (India)

3Department of Natural Sciences, International Christian University, Mitaka, Tokyo 181-8585, Japan ... the changes of expression predicted from gene function suggested association ... ate School of Science and Technology, Niigata University.

Berberine attenuates CCN2-induced IL-1β expression and prevents cartilage degradation in a rat model of osteoarthritis

Energy Technology Data Exchange (ETDEWEB)

Liu, Shan-Chi [Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China); Lee, Hsiang-Ping [Graduate Institute of Chinese Medicine, China Medical University, Taichung, Taiwan (China); Department of Chinese Medicine, China Medical University Hospital, Taichung, Taiwan (China); Hung, Chun-Yin [Department of Orthopaedic Surgery, China Medical University Beigang Hospital, Yun-Lin County, Taiwan (China); Tsai, Chun-Hao [Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan (China); Department of Orthopedic Surgery, China Medical University Hospital, Taichung, Taiwan (China); Li, Te-Mao [School of Chinese Medicine, China Medical University, Taichung, Taiwan (China); Tang, Chih-Hsin, E-mail: chtang@mail.cmu.edu.tw [Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China); Department of Pharmacology, School of Medicine, China Medical University, Taichung, Taiwan (China); Department of Biotechnology, College of Health Science, Asia University, Taichung, Taiwan (China)

2015-11-15

Connective tissue growth factor (CTGF; also known as CCN2) is an inflammatory mediator that is abundantly expressed in osteoarthritis (OA). Interleukin-1β (IL-1β) plays a pivotal role in OA pathogenesis. Berberine exhibits an anti-inflammatory effect, but the mechanisms by which it modulates CCN2-induced IL-1β expression in OA synovial fibroblasts (OASFs) remain unknown. We showed that CCN2-induced IL-1β expression is mediated by the activation of α{sub v}β{sub 3}/α{sub v}β{sub 5} integrin-dependent reactive oxygen species (ROS) generation, and subsequent activation of apoptosis signal-regulating kinase 1 (ASK1), p38/JNK, and nuclear factor-κB (NF-κB) signaling pathways. This IL-1β expression in OASFs is attenuated by N-acetylcysteine (NAC), inhibitors of ASK1, p38, or JNK, or treatment with berberine. Furthermore, berberine also reverses cartilage damage in an experimental model of collagenase-induced OA (CIOA). We observed that CCN2 increased IL-1β expression via α{sub v}β{sub 3}/α{sub v}β{sub 5} integrins, ROS, and ASK1, p38/JNK, and NF-κB signaling pathways. Berberine was found to inhibit these signaling components in OASFs in vitro and prevent cartilage degradation in vivo. We suggest a novel therapeutic strategy of using berberine for managing OA. - Highlights: • CCN2 induce IL-1β production via αvβ3/αvβ5 integrin, ROS, ASK1, p38/JNK, and NF-κB. • Berberine attenuates CCN2-induced IL-1β expression in vitro and in OA rat model. • Berberine as natural drug of choice for anti-inflammatory effect to ameliorates OA.

Glial cell line-derived neurotrophic factor protects against high-fat diet-induced hepatic steatosis by suppressing hepatic PPAR-γ expression.

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Mwangi, Simon Musyoka; Peng, Sophia; Nezami, Behtash Ghazi; Thorn, Natalie; Farris, Alton B; Jain, Sanjay; Laroui, Hamed; Merlin, Didier; Anania, Frank; Srinivasan, Shanthi

2016-01-15

Glial cell line-derived neurotrophic factor (GDNF) protects against high-fat diet (HFD)-induced hepatic steatosis in mice, however, the mechanisms involved are not known. In this study we investigated the effects of GDNF overexpression and nanoparticle delivery of GDNF in mice on hepatic steatosis and fibrosis and the expression of genes involved in the regulation of hepatic lipid uptake and de novo lipogenesis. Transgenic overexpression of GDNF in liver and other metabolically active tissues was protective against HFD-induced hepatic steatosis. Mice overexpressing GDNF had significantly reduced P62/sequestosome 1 protein levels suggestive of accelerated autophagic clearance. They also had significantly reduced peroxisome proliferator-activated receptor-γ (PPAR-γ) and CD36 gene expression and protein levels, and lower expression of mRNA coding for enzymes involved in de novo lipogenesis. GDNF-loaded nanoparticles were protective against short-term HFD-induced hepatic steatosis and attenuated liver fibrosis in mice with long-standing HFD-induced hepatic steatosis. They also suppressed the liver expression of steatosis-associated genes. In vitro, GDNF suppressed triglyceride accumulation in Hep G2 cells through enhanced p38 mitogen-activated protein kinase-dependent signaling and inhibition of PPAR-γ gene promoter activity. These results show that GDNF acts directly in the liver to protect against HFD-induced cellular stress and that GDNF may have a role in the treatment of nonalcoholic fatty liver disease.

Dynamic, mating-induced gene expression changes in female head and brain tissues of Drosophila melanogaster

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Stirling Emma J

2010-10-01

Full Text Available Abstract Background Drosophila melanogaster females show changes in behavior and physiology after mating that are thought to maximize the number of progeny resulting from the most recent copulation. Sperm and seminal fluid proteins induce post-mating changes in females, however, very little is known about the resulting gene expression changes in female head and central nervous system tissues that contribute to the post-mating response. Results We determined the temporal gene expression changes in female head tissues 0-2, 24, 48 and 72 hours after mating. Females from each time point had a unique post-mating gene expression response, with 72 hours post-mating having the largest number of genes with significant changes in expression. At most time points, genes expressed in the head fat body that encode products involved in metabolism showed a marked change in expression. Additional analysis of gene expression changes in dissected brain tissues 24 hours post-mating revealed changes in transcript abundance of many genes, notably, the reduced transcript abundance of genes that encode ion channels. Conclusions Substantial changes occur in the regulation of many genes in female head tissues after mating, which might underlie aspects of the female post-mating response. These results provide new insights into the physiological and metabolic changes that accompany changes in female behaviors.

The hepatic Raldh1 expression is elevated in Zucker fatty rats and its over-expression introduced the retinal-induced Srebp-1c expression in INS-1 cells.

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Yang Li

Full Text Available The roles of vitamin A (VA in the development of metabolic diseases remain unanswered. We have reported that retinoids synergized with insulin to induce the expression of sterol-regulatory element-binding protein 1c gene (Srebp-1c expression in primary rat hepatocytes. Additionally, the hepatic Srebp-1c expression is elevated in Zucker fatty (ZF rats, and reduced in those fed a VA deficient diet. VA is metabolized to retinoic acid (RA for regulating gene expression. We hypothesized that the expression of RA production enzymes contributes to the regulation of the hepatic Srebp-1c expression. Therefore, we analyzed their expression levels in Zucker lean (ZL and ZF rats. The mRNA levels of retinaldehyde dehydrogenase family 1 gene (Raldh1 were found to be higher in the isolated and cultured primary hepatocytes from ZF rats than that from ZL rats. The RALDH1 protein level was elevated in the liver of ZF rats. Retinol and retinal dose- and time-dependently induced the expression of RA responsive Cyp26a1 gene in hepatocytes and hepatoma cells. INS-1 cells were identified as an ideal tool to study the effects of RA production on the regulation of gene expression because only RA, but not retinal, induced Srebp-1c mRNA expression in them. Recombinant adenovirus containing rat Raldh1 cDNA was made and used to infect INS-1 cells. The over-expression of RALDH1 introduced the retinal-mediated induction of Srebp-1c expression in INS-1 cells. We conclude that the expression levels of the enzymes for RA production may contribute to the regulation of RA responsive genes, and determine the responses of the cells to retinoid treatments. The elevated hepatic expression of Raldh1 in ZF rats may cause the excessive RA production from retinol, and in turn, result in higher Srebp-1c expression. This excessive RA production may be one of the factors contributing to the elevated lipogenesis in the liver of ZF rats.

Ethylene-induced senescence-related gene expression requires protein synthesis

International Nuclear Information System (INIS)

Lawton, K.A.; Raghothama, K.G.; Woodson, W.R.

1990-01-01

We have investigated the effects of inhibiting protein synthesis on the ethylene-induced expression of 3 carnation senescence-related genes, pSR5, pSR8, and pSR12. Treatment of preclimacteric carnation petal discs with 1μg/ml of cycloheximide, a cytoplasmic protein synthesis inhibitor, for 3h inhibited protein synthesis by >80% as quantitated by the incorporation of [35S]methionine into protein. Pre-treatment of petal discs with cycloheximide prevented ethylene-induced SR transcript accumulation. Cycloheximide treatment of petal discs held in air did not result in increased levels of SR mRNA. These results indicate that ethylene does not interact with pre-formed factors but rather that the activation of SR gene expression by ethylene is mediated by labile protein factor(s) synthesized on cytoplasmic ribosomes. Experiments are currently underway to determine if cycloheximide exerts its effect at the transcriptional or post-transcriptional level

Exercise-induced metallothionein expression in human skeletal muscle fibres

DEFF Research Database (Denmark)

Penkowa, Milena; Keller, Pernille; Keller, Charlotte

2005-01-01

in both type I and II muscle fibres. This is the first report demonstrating that MT-I + II are significantly induced in human skeletal muscle fibres following exercise. As MT-I + II are antioxidant factors that protect various tissues during pathological conditions, the MT-I + II increases post exercise......Exercise induces free oxygen radicals that cause oxidative stress, and metallothioneins (MTs) are increased in states of oxidative stress and possess anti-apoptotic effects. We therefore studied expression of the antioxidant factors metallothionein I and II (MT-I + II) in muscle biopsies obtained...... in response to 3 h of bicycle exercise performed by healthy men and in resting controls. Both MT-I + II proteins and MT-II mRNA expression increased significantly in both type I and II muscle fibres after exercise. Moreover, 24 h after exercise the levels of MT-II mRNA and MT-I + II proteins were still highly...

HIF1 Contributes to Hypoxia-Induced Pancreatic Cancer Cells Invasion via Promoting QSOX1 Expression

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Chen-Ye Shi

2013-08-01

Full Text Available Background: Quiescin sulfhydryl oxidase 1 (QSOX1, which oxidizes sulfhydryl groups to form disulfide bonds in proteins, is found to be over-expressed in various pancreatic cancer cell lines and patients. QSOX1 promotes invasion of pancreatic cancer cells by activating MMP-2 and MMP-9. However, its regulatory mechanism remains largely undefined. Methods: Real-time PCR and Western blot were employed to detect the expression of QSOX1 in human pancreatic cancer cell lines under hypoxic condition. Luciferase reporter and ChIP assays were used to assess the regulation of QSOX1 by hypoxia-inducible factor 1 (HIF-1. Small interfering RNA (siRNA was applied to knock down endogenous expression of QSOX1. Matrigel-coated invasion chamber essays were conducted to detect the invasion capacity of QSOX1-depleted cells. Results: Both hypoxia and hypoxia mimicking reagent up-regulated the expression of QSOX1 in human pancreatic cancer cell lines. Knockdown of HIF-1α eliminated hypoxia induced QSOX1 expression. HIF-1α was found directly bound to two hypoxia-response elements (HRE of QSOX1 gene, both of which were required for HIF-1 induced QSOX1 expression. Moreover, QSOX1 silencing blocked hypoxia-induced pancreatic cancer cells invasion. Conclusion: QSOX1 is a direct target of HIF-1 and may contribute to hypoxia-induced pancreatic cancer cells invasion.

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

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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

Expression of manganese superoxide dismutase in rat blood, heart and brain during induced systemic hypoxia

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Septelia I. Wanandi

2011-02-01

Full Text Available Background: Hypoxia results in an increased generation of ROS. Until now, little is known about the role of MnSOD - a major endogenous antioxidant enzyme - on the cell adaptation response against hypoxia. The aim of this study was to  determine the MnSOD mRNA expression and levels of specific activity in blood, heart and brain of rats during induced systemic hypoxia.Methods: Twenty-five male Sprague Dawley rats were subjected to systemic hypoxia in an hypoxic chamber (at 8-10% O2 for 0, 1, 7, 14 and 21 days, respectively. The mRNA relative expression of MnSOD was analyzed using Real Time RT-PCR. MnSOD specific activity was determined using xanthine oxidase inhibition assay.Results: The MnSOD mRNA relative expression in rat blood and heart was decreased during early induced systemic hypoxia (day 1 and increased as hypoxia continued, whereas the mRNA expression in brain was increased since day 1 and reached its maximum level at day 7. The result of MnSOD specific activity during early systemic hypoxia was similar to the mRNA expression. Under very late hypoxic condition (day 21, MnSOD specific activity in blood, heart and brain was significantly decreased. We demonstrate a positive correlation between MnSOD mRNA expression and specific activity in these 3 tissues during day 0-14 of induced systemic hypoxia. Furthermore, mRNA expression and specific activity levels in heart strongly correlate with those in blood.Conclusion: The MnSOD expression at early and late phases of induced systemic hypoxia is distinctly regulated. The MnSOD expression in brain differs from that in blood and heart revealing that brain tissue can  possibly survive better from induced systemic hypoxia than heart and blood. The determination of MnSOD expression in blood can be used to describe its expression in heart under systemic hypoxic condition. (Med J Indones 2011; 20:27-33Keywords: MnSOD, mRNA expression, ROS, specific activity, systemic hypoxia

Expression profiles of miRNAs from bovine mammary glands in response to Streptococcus agalactiae-induced mastitis.

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Pu, Junhua; Li, Rui; Zhang, Chenglong; Chen, Dan; Liao, Xiangxiang; Zhu, Yihui; Geng, Xiaohan; Ji, Dejun; Mao, Yongjiang; Gong, Yunchen; Yang, Zhangping

2017-08-01

This study aimed to describe the expression profiles of microRNAs (miRNAs) from mammary gland tissues collected from dairy cows with Streptococcus agalactiae-induced mastitis and to identify differentially expressed miRNAs related to mastitis. The mammary glands of Chinese Holstein cows were challenged with Streptococcus agalactiae to induce mastitis. Small RNAs were isolated from the mammary tissues of the test and control groups and then sequenced using the Solexa sequencing technology to construct two small RNA libraries. Potential target genes of these differentially expressed miRNAs were predicted using the RNAhybrid software, and KEGG pathways associated with these genes were analysed. A total of 18 555 913 and 20 847 000 effective reads were obtained from the test and control groups, respectively. In total, 373 known and 399 novel miRNAs were detected in the test group, and 358 known and 232 novel miRNAs were uncovered in the control group. A total of 35 differentially expressed miRNAs were identified in the test group compared to the control group, including 10 up-regulated miRNAs and 25 down-regulated miRNAs. Of these miRNAs, miR-223 exhibited the highest degree of up-regulation with an approximately 3-fold increase in expression, whereas miR-26a exhibited the most decreased expression level (more than 2-fold). The RNAhybrid software predicted 18 801 genes as potential targets of these 35 miRNAs. Furthermore, several immune response and signal transduction pathways, including the RIG-I-like receptor signalling pathway, cytosolic DNA sensing pathway and Notch signal pathway, were enriched in these predicted targets. In summary, this study provided experimental evidence for the mechanism underlying the regulation of bovine mastitis by miRNAs and showed that miRNAs might be involved in signal pathways during S. agalactiae-induced mastitis.

[Expression of aquaporin-4 during brain edema in rats with thioacetamide-induced acute encephalopathy].

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Wang, Li-Qing; Zhu, Sheng-Mei; Zhou, Heng-Jun; Pan, Cai-Fei

2011-09-27

To investigate the expression of aquaporin-4 (AQP4) during brain edema in rats with thioacetamide-induced acute liver failure and encephalopathy. The rat model of acute hepatic failure and encephalopathy was induced by intraperitoneal injection of thioacetamide (TAA) at a 24-hour interval for 2 consecutive days. Thirty-two SD rats were randomly divided into the model group (n = 24) and the control group (normal saline, n = 8). And then the model group was further divided into 3 subgroups by the timepoint of decapitation: 24 h (n = 8), 48 h (n = 8) and 60 h (n = 8). Then we observed their clinical symptoms and stages of HE, indices of liver function and ammonia, liver histology and brain water content. The expression of AQP4 protein in brain tissues was measured with Western blot and the expression of AQP4mRNA with RT-PCR (reverse transcription-polymerase chain reaction). Typical clinical manifestations of hepatic encephalopathy occurred in all TAA-administrated rats. The model rats showed the higher indices of ALT (alanine aminotransferase), AST (aspartate aminotransferase), TBIL (total bilirubin) and ammonia than the control rats (P liver failure and encephalopathy plays a significant role during brain edema. AQP4 is one of the molecular mechanisms for the occurrence of brain edema in hepatic encephalopathy.

Sporadic on/off switching of HTLV-1 Tax expression is crucial to maintain the whole population of virus-induced leukemic cells.

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Mahgoub, Mohamed; Yasunaga, Jun-Ichirou; Iwami, Shingo; Nakaoka, Shinji; Koizumi, Yoshiki; Shimura, Kazuya; Matsuoka, Masao

2018-02-06

Viruses causing chronic infection artfully manipulate infected cells to enable viral persistence in vivo under the pressure of immunity. Human T-cell leukemia virus type 1 (HTLV-1) establishes persistent infection mainly in CD4+ T cells in vivo and induces leukemia in this subset. HTLV-1-encoded Tax is a critical transactivator of viral replication and a potent oncoprotein, but its significance in pathogenesis remains obscure due to its very low level of expression in vivo. Here, we show that Tax is expressed in a minor fraction of leukemic cells at any given time, and importantly, its expression spontaneously switches between on and off states. Live cell imaging revealed that the average duration of one episode of Tax expression is ∼19 hours. Knockdown of Tax rapidly induced apoptosis in most cells, indicating that Tax is critical for maintaining the population, even if its short-term expression is limited to a small subpopulation. Single-cell analysis and computational simulation suggest that transient Tax expression triggers antiapoptotic machinery, and this effect continues even after Tax expression is diminished; this activation of the antiapoptotic machinery is the critical event for maintaining the population. In addition, Tax is induced by various cytotoxic stresses and also promotes HTLV-1 replication. Thus, it seems that Tax protects infected cells from apoptosis and increases the chance of viral transmission at a critical moment. Keeping the expression of Tax minimal but inducible on demand is, therefore, a fundamental strategy of HTLV-1 to promote persistent infection and leukemogenesis. Copyright © 2018 the Author(s). Published by PNAS.

Inhibition of interleukin-6 decreases atrogene expression and ameliorates tail suspension-induced skeletal muscle atrophy

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Yakabe, Mitsutaka; Ota, Hidetaka; Iijima, Katsuya; Eto, Masato; Ouchi, Yasuyoshi; Akishita, Masahiro

2018-01-01

Background Interleukin-6 (IL-6) is an inflammatory cytokine. Whether systemic IL-6 affects atrogene expression and disuse-induced skeletal muscle atrophy is unclear. Methods Tail-suspended mice were used as a disuse-induced muscle atrophy model. We administered anti-mouse IL-6 receptor antibody, beta-hydroxy-beta-methylbutyrate (HMB) and vitamin D to the mice and examined the effects on atrogene expression and muscle atrophy. Results Serum IL-6 levels were elevated in the mice. Inhibition of IL-6 receptor suppressed muscle RING finger 1 (MuRF1) expression and prevented muscle atrophy. HMB and vitamin D inhibited the serum IL-6 surge, downregulated the expression of MuRF1 and atrogin-1 in the soleus muscle, and ameliorated atrophy in the mice. Conclusion Systemic IL-6 affects MuRF1 expression and disuse-induced muscle atrophy. PMID:29351340

Inhibition of interleukin-6 decreases atrogene expression and ameliorates tail suspension-induced skeletal muscle atrophy.

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Mitsutaka Yakabe

Full Text Available Interleukin-6 (IL-6 is an inflammatory cytokine. Whether systemic IL-6 affects atrogene expression and disuse-induced skeletal muscle atrophy is unclear.Tail-suspended mice were used as a disuse-induced muscle atrophy model. We administered anti-mouse IL-6 receptor antibody, beta-hydroxy-beta-methylbutyrate (HMB and vitamin D to the mice and examined the effects on atrogene expression and muscle atrophy.Serum IL-6 levels were elevated in the mice. Inhibition of IL-6 receptor suppressed muscle RING finger 1 (MuRF1 expression and prevented muscle atrophy. HMB and vitamin D inhibited the serum IL-6 surge, downregulated the expression of MuRF1 and atrogin-1 in the soleus muscle, and ameliorated atrophy in the mice.Systemic IL-6 affects MuRF1 expression and disuse-induced muscle atrophy.

Cortical Astrocytes Acutely Exposed to the Monomethylarsonous Acid (MMAIII) Show Increased Pro-inflammatory Cytokines Gene Expression that is Consistent with APP and BACE-1: Over-expression.

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Escudero-Lourdes, C; Uresti-Rivera, E E; Oliva-González, C; Torres-Ramos, M A; Aguirre-Bañuelos, P; Gandolfi, A J

2016-10-01

Long-term exposure to inorganic arsenic (iAs) through drinking water has been associated with cognitive impairment in children and adults; however, the related pathogenic mechanisms have not been completely described. Increased or chronic inflammation in the brain is linked to impaired cognition and neurodegeneration; iAs induces strong inflammatory responses in several cells, but this effect has been poorly evaluated in central nervous system (CNS) cells. Because astrocytes are the most abundant cells in the CNS and play a critical role in brain homeostasis, including regulation of the inflammatory response, any functional impairment in them can be deleterious for the brain. We propose that iAs could induce cognitive impairment through inflammatory response activation in astrocytes. In the present work, rat cortical astrocytes were acutely exposed in vitro to the monomethylated metabolite of iAs (MMA III ), which accumulates in glial cells without compromising cell viability. MMA III LD 50 in astrocytes was 10.52 μM, however, exposure to sub-toxic MMA III concentrations (50-1000 nM) significantly increased IL-1β, IL-6, TNF-α, COX-2, and MIF-1 gene expression. These effects were consistent with amyloid precursor protein (APP) and β-secretase (BACE-1) increased gene expression, mainly for those MMA III concentrations that also induced TNF-α over-expression. Other effects of MMA III on cortical astrocytes included increased proliferative and metabolic activity. All tested MMA III concentrations led to an inhibition of intracellular lactate dehydrogenase (LDH) activity. Results suggest that MMA III induces important metabolic and functional changes in astrocytes that may affect brain homeostasis and that inflammation may play a major role in cognitive impairment-related pathogenicity in As-exposed populations.

Enhancement of high glucose-induced PINK1 expression by melatonin stimulates neuronal cell survival: Involvement of MT2 /Akt/NF-κB pathway.

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Onphachanh, Xaykham; Lee, Hyun Jik; Lim, Jae Ryong; Jung, Young Hyun; Kim, Jun Sung; Chae, Chang Woo; Lee, Sei-Jung; Gabr, Amr Ahmed; Han, Ho Jae

2017-09-01

Hyperglycemia is a representative hallmark and risk factor for diabetes mellitus (DM) and is closely linked to DM-associated neuronal cell death. Previous investigators reported on a genome-wide association study and showed relationships between DM and melatonin receptor (MT), highlighting the role of MT signaling by assessing melatonin in DM. However, the role of MT signaling in DM pathogenesis is unclear. Therefore, we investigated the role of mitophagy regulators in high glucose-induced neuronal cell death and the effect of melatonin against high glucose-induced mitophagy regulators in neuronal cells. In our results, high glucose significantly increased PTEN-induced putative kinase 1 (PINK1) and LC-3B expressions; as well it decreased cytochrome c oxidase subunit 4 expression and Mitotracker™ fluorescence intensity. Silencing of PINK1 induced mitochondrial reactive oxygen species (ROS) accumulation and mitochondrial membrane potential impairment, increased expressions of cleaved caspases, and increased the number of annexin V-positive cells. In addition, high glucose-stimulated melatonin receptor 1B (MTNR1B) mRNA and PINK1 expressions were reversed by ROS scavenger N-acetyl cysteine pretreatment. Upregulation of PINK1 expression in neuronal cells is suppressed by pretreatment with MT 2 receptor-specific inhibitor 4-P-PDOT. We further showed melatonin stimulated Akt phosphorylation, which was followed by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) phosphorylation and nuclear translocation. Silencing of PINK1 expression abolished melatonin-regulated mitochondrial ROS production, cleaved caspase-3 and caspase-9 expressions, and the number of annexin V-positive cells. In conclusion, we have demonstrated the melatonin stimulates PINK1 expression via an MT 2 /Akt/NF-κB pathway, and such stimulation is important for the prevention of neuronal cell apoptosis under high glucose conditions. © 2017 The Authors. Journal of Pineal Research

Construction of Egr1-mediated human truncated apoptosis inducing factor expression vector and its expression regularity induced by radiation in breast cancer MCF-7 cells

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Wang Jianfeng; Gong Shouliang; Wang Zhicheng; Fang Fang; Liu Yang; Wu Jiahui

2012-01-01

Objective: To clone human truncated apoptosis inducing factor (AIF) cDNA sequence, and to construct early growth response 1 (Egr1)-mediated recombinant expression vector pcDNA 3.1-Egr1-AIF Δ1-480 (pEgr1-AIFΔ 1-480 ), and to observe its regularity induced by radiation in human breast cancer MCF-7 cells. Methods: The total mRNA extracted from human leukemia Jurkat cells used as template, and the human AIFΔ 1-480 was acquired by RT-PCR, and it was linked to pMD18T vector for sequencing. Egr1 fragment was digested from pMD19T-Egr1 by restrictive enzyme, and the Egr1-mediated expression plasmid pEgr1-AIFΔ 1-480 was constructed by gene recombination. There were control group, pcDNA3.1 group, pAIFΔ 1-480 group and pEgr1-AIFΔ 1-480 group in the experiment. After the plasmids in various groups were transfected into human breast cancer MCF-7 cells, the AIF and AIFΔ 1-480 protein expression time-effect (0, 2, 4, 12, 24 and 48 h after 2.0 Gy irradiation) and dose-effect (24 h after 0, 0.2, 0.5, 1.0, 2.0 and 5.0 Gy irradiation) regularity were measured by Western blotting method. Results: The sequencing results showed that the AIFΔ 1-480 acquired by RT-PCR was consistent with the sequence expected, the pEgr-AIFΔ 1-480 was confirmed by PCR and restrictive enzyme digestion. After 0-48 h the MCF-7 cells were irradiated by 2.0 Gy, and the AIF protein expressed in the cells in each group, and it increased significantly from 4 h and the AIF expressions in 4, 12, 24 and 48 h groups were higher than that in 0 h group (P<0.05), and it reached to maximum value at 48 h. But the AIFΔ 1-480 protein expressed in the cells in pAIFΔ 1-480 and pEgr1-AIFΔ 1-480 groups from 2 h (P<0.05), and it reached to peak value at 24 h. The AIFΔ 1-480 expressions in pEgr1-AIFΔ 1-480 group were higher than those in pAIFΔ 1-480 group at and 48 h (P<0.05). After the MCF-7 cells were irradiated by 0-5 Gy for 24 h, the AIF protein expressed in the cells in each group, but the AIFΔ 1-480 protein

Metformin reduces the endotoxin-induced down-regulation of apolipoprotein E gene expression in macrophages

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Stavri, Simona; Trusca, Violeta G.; Simionescu, Maya; Gafencu, Anca V., E-mail: anca.gafencu@icbp.ro

2015-05-29

The atheroprotective role of macrophage-derived apolipoprotein E (apoE) is well known. Our previous reports demonstrated that inflammatory stress down-regulates apoE expression in macrophages, aggravating atherogenesis. Metformin, extensively used as an anti-diabetic drug, has also anti-inflammatory properties, and thus confers vascular protection. In this study, we questioned whether metformin could have an effect on apoE expression in macrophages in normal conditions or under lipopolysaccharide (LPS)-induced stress. The results showed that metformin slightly increases the apoE expression only at high doses (5–10 mM). Low doses of metformin (1–3 mM) significantly reduce the LPS down-regulatory effect on apoE expression in macrophages. Our experiments demonstrated that LPS-induced NF-κB binds to the macrophage-specific distal regulatory element of apoE gene, namely to the multienhancer 2 (ME.2) and its 5′-deletion fragments. The NF-κB binding on ME.2 and apoE promoter has a down-regulatory effect. In addition, data revealed that metformin impairs NF-κB nuclear translocation, and thus, improves the apoE levels in macrophages under inflammatory stress. The positive effect of metformin in the inflammatory states, its clinical safety and low cost, make this drug a potential adjuvant in the therapeutic strategies for atherosclerosis. - Highlights: • High doses of metformin slightly increase apoE expression in macrophages. • Low doses of metformin up-regulate apoE gene in endotoxin-stressed macrophages. • Metformin reduces the negative effect of LPS on apoE expression by NF-κB inhibition.

Metformin reduces the endotoxin-induced down-regulation of apolipoprotein E gene expression in macrophages

International Nuclear Information System (INIS)

Stavri, Simona; Trusca, Violeta G.; Simionescu, Maya; Gafencu, Anca V.

2015-01-01

The atheroprotective role of macrophage-derived apolipoprotein E (apoE) is well known. Our previous reports demonstrated that inflammatory stress down-regulates apoE expression in macrophages, aggravating atherogenesis. Metformin, extensively used as an anti-diabetic drug, has also anti-inflammatory properties, and thus confers vascular protection. In this study, we questioned whether metformin could have an effect on apoE expression in macrophages in normal conditions or under lipopolysaccharide (LPS)-induced stress. The results showed that metformin slightly increases the apoE expression only at high doses (5–10 mM). Low doses of metformin (1–3 mM) significantly reduce the LPS down-regulatory effect on apoE expression in macrophages. Our experiments demonstrated that LPS-induced NF-κB binds to the macrophage-specific distal regulatory element of apoE gene, namely to the multienhancer 2 (ME.2) and its 5′-deletion fragments. The NF-κB binding on ME.2 and apoE promoter has a down-regulatory effect. In addition, data revealed that metformin impairs NF-κB nuclear translocation, and thus, improves the apoE levels in macrophages under inflammatory stress. The positive effect of metformin in the inflammatory states, its clinical safety and low cost, make this drug a potential adjuvant in the therapeutic strategies for atherosclerosis. - Highlights: • High doses of metformin slightly increase apoE expression in macrophages. • Low doses of metformin up-regulate apoE gene in endotoxin-stressed macrophages. • Metformin reduces the negative effect of LPS on apoE expression by NF-κB inhibition

STAT3 and NF-κB are common targets for kaempferol-mediated attenuation of COX-2 expression in IL-6-induced macrophages and carrageenan-induced mouse paw edema

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Anandita Basu

2017-12-01

Full Text Available Cycloxygenase-2 (COX-2 is the inducible isoform of cycloxygenase enzyme family that catalyzes synthesis of inflammatory mediators, prostanoids and prostaglandins, and therefore, can be targeted by anti-inflammatory drugs. Here, we showed a plant polyphenol, kaempferol, attenuated IL-6-induced COX-2 expression in human monocytic THP-1 cells suggesting its beneficial role in chronic inflammation. Kaempferol deactivated and prevented nuclear localization of two major transcription factors STAT3 and NF-κB, mutually responsible for COX-2 induction in response to IL-6. Moreover, STAT3 and NF-κB were simultaneously deactivated by kaempferol in acute inflammation, as shown by carrageenan-induced mouse paw edema model. The concomitant reduction in COX-2 expression in paw tissues suggested kaempferol’s role in mitigation of inflammation by targeting STAT3 and NF-κB.

Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood

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N.E. Gomes

2010-09-01

Full Text Available Lipopolysaccharide (LPS activates neutrophils and monocytes, inducing a wide array of biological activities. LPS rough (R and smooth (S forms signal through Toll-like receptor 4 (TLR4, but differ in their requirement for CD14. Since the R-form LPS can interact with TLR4 independent of CD14 and the differential expression of CD14 on neutrophils and monocytes, we used the S-form LPS from Salmonella abortus equi and the R-form LPS from Salmonella minnesota mutants to evaluate LPS-induced activation of human neutrophils and monocytes in whole blood from healthy volunteers. Expression of cell surface receptors and reactive oxygen species (ROS and nitric oxide (NO generation were measured by flow cytometry in whole blood monocytes and neutrophils. The oxidative burst was quantified by measuring the oxidation of 2',7'-dichlorofluorescein diacetate and the NO production was quantified by measuring the oxidation of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. A small increase of TLR4 expression by monocytes was observed after 6 h of LPS stimulation. Monocyte CD14 modulation by LPS was biphasic, with an initial 30% increase followed by a 40% decrease in expression after 6 h of incubation. Expression of CD11b was rapidly up-regulated, doubling after 5 min on monocytes, while down-regulation of CXCR2 was observed on neutrophils, reaching a 50% reduction after 6 h. LPS induced low production of ROS and NO. This study shows a complex LPS-induced cell surface receptor modulation on human monocytes and neutrophils, with up- and down-regulation depending on the receptor. R- and S-form LPS activate human neutrophils similarly, despite the low CD14 expression, if the stimulation occurs in whole blood.

Poly(I:C) induces expressions of MMP-1, -2, and -3 through various signaling pathways including IRF3 in human skin fibroblasts.

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Yao, Cheng; Lee, Dong Hun; Oh, Jang-Hee; Kim, Min-Kyoung; Kim, Kyu Han; Park, Chi-Hyun; Chung, Jin Ho

2015-10-01

Ultraviolet (UV) irradiation can result in premature skin aging (photoaging) which is characterized by decreased expression of collagen and increased expression of matrix metalloproteinases (MMPs). Double-stranded RNAs (dsRNAs) can be generated at various conditions including virally infected cells or UV-damaged skin cells. Recent studies have shown that a synthetic dsRNA, polyinosinic-polycytidylic acid (poly(I:C)), can reduce procollagen expression in human skin fibroblasts. However, little is known about the effect of poly(I:C) on the expression of MMPs in skin fibroblasts and its underlying mechanisms. We examined the effect of poly(I:C) on MMP-1, -2, and -3 expressions in human skin fibroblasts. Then, we further explored the underlying signaling pathways involved in the processes. Human skin fibroblasts were treated with poly(I:C) for the indicated times in the presence or the absence of various chemical inhibitors or small interfering RNAs (siRNAs) at the indicated concentrations. Protein and mRNA levels of various target molecules were examined by Western blotting and quantitative real-time PCR, respectively. Poly(I:C) induced MMP-1, -2, and -3 expressions, which were dependent on TLR3. Poly(I:C) also induced activations of the mitogen-activated protein kinases (MAPKs), the nuclear factor-kappaB (NF-κB) and the interferon regulatory factor 3 (IRF3) pathways. By using specific inhibitors, we found that poly(I:C)-induced expressions of MMP-1, -2, and -3 were differentially regulated by these signaling pathways. In particular, we found that the inhibition of IRF3 signaling pathways attenuated poly(I:C)-induced expressions of all the three MMPs. Our data show that the expressions of MMP-1, -2, and -3 are induced by poly(I:C) through various signaling pathways in human skin fibroblasts and suggest that TLR3 and/or IRF3 may be good targets for regulating the expressions of MMP-1, -2, and -3 induced by dsRNAs. Copyright © 2015 Elsevier Ireland Ltd. All rights

SIRT-1 regulates TGF-β-induced dermal fibroblast migration via modulation of Cyr61 expression.

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Kwon, Eun-Jeong; Park, Eun-Jung; Yu, Hyeran; Huh, Jung-Sik; Kim, Jinseok; Cho, Moonjae

2018-05-01

SIRT1 is a NAD-dependent protein deacetylase that participates in cellular regulation. The increased migration of fibroblasts is an important phenotype in fibroblast activation. The role of SIRT1 in cell migration remains controversial as to whether SIRT1 acts as an activator or suppressor of cell migration. Therefore, we have established the role of SIRT1 in the migration of human dermal fibroblasts and explored targets of SIRT1 during dermal fibroblast migration. SIRT1 and Cyr61 were expressed in human dermal fibroblasts and the stimulation with TGF-β further induced their expression. Treatment with resveratrol (RSV), a SIRT1 agonist, or overexpression of SIRT1 also promoted the expression Cyr61 in human dermal fibroblasts, whereas the inhibition of SIRT1 activity by nicotinamide or knockdown of SIRT1 decreased the level of Cyr61, as well as TGF-β or RSV-induced Cyr61 expression. Blocking of ERK signaling by PD98509 reduced the expression of Cyr61 induced by TGF-β or RSV. TGF-β, RSV, or SIRT1 overexpression enhanced β-catenin as well as Cyr61 expression. This stimulation was reduced by the Wnt inhibitor XAV939. RSV increased migration and nicotinamide attenuated RSV-induced migration of human dermal fibroblasts. Furthermore, SIRT1 overexpression promoted cell migration, whereas blocking Cyr61 attenuated SIRT1-stimulated migration of human dermal fibroblasts. SIRT1 increased cell migration by stimulating Cyr61 expression and the ERK and Wnt/β-catenin signaling. SIRT1-induced Cyr61 activity is very important for human dermal fibroblasts migration.

[The effect of diethylstilbestrol on inducing abdominal cryptorchidism and relevant genetic expression in rats].

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Zhang, Lin; Zheng, Xin-min; Zheng, Hang; Yang, Zhi-wei; Li, Shi-wen

2009-05-01

To study the effect of diethylstilbestrol (DES) at different doses on transabdominal testicular descent in rats and the expression of INSL3 in the testis and HOXA10 in the gubernaculum. Fifty E13.5 (embryonic day 13.5) pregnant female SD rats were randomly divided into five groups that received a subcutaneous injection of DMSO, 2.5, 5.0, 10.0 and 20.0 mg/kg DES (group A, B, C, D and E), respectively. Male offspring were killed at E19.5, and then fetal mortality, the degree of transabdominal testicular ascent (DTA) was determined by a stereomicroscope. The mRNA expressions of INSL3 in the testis and HOXA10 in the gubernaculum were determined by RT-PCR. The expression of INSL3 protein was determined by Western blotting. Male fetal mortality in group A, B, C, D, and E were 3.57%, 6.90%, 12.00%, 19.23% and 36.36%, respectively, which showed a dose-effect relationship between DES and the male fatal mortality (r=0.999, P0.05), those in other groups were down-regulated significantly (q=12.4304, 17.2477 and 20.2789, respectively, Pdescent dose-dependently via down-regulating the expression of INSL3. HOXA10 may play no role in low-dosage DES induced intra-abdominal cryptorchidism, but down-regulated HOXA10 mRNA was involved in high-dosage DES induced ones.

Expression of immediate-early genes in the dorsal cochlear nucleus in salicylate-induced tinnitus.

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Hu, Shou-Sen; Mei, Ling; Chen, Jian-Yong; Huang, Zhi-Wu; Wu, Hao

2016-02-01

Spontaneous neuronal activity in dorsal cochlear nucleus (DCN) may be involved in the physiological processes underlying salicylate-induced tinnitus. As a neuronal activity marker, immediate-early gene (IEG) expression, especially activity-dependent cytoskeletal protein (Arc/Arg3.1) and the early growth response gene-1 (Egr-1), appears to be highly correlated with sensory-evoked neuronal activity. However, their relationships with tinnitus induced by salicylate have rarely been reported in the DCN. In this study, we assessed the effect of acute and chronic salicylate treatment on the expression of N-methyl D-aspartate receptor subunit 2B (NR2B), Arg3.1, and Egr-1. We also observed ultrastructural alterations in the DCN synapses in an animal model of tinnitus. Levels of mRNA and protein expression of NR2B and Arg3.1 were increased in rats that were chronically administered salicylate (200 mg/kg, twice daily for 3, 7, or 14 days). These levels returned to baseline 14 days after cessation of treatment. However, no significant changes were observed in Egr-1 gene expression in any groups. Furthermore, rats subjected to long-term salicylate administration showed more presynaptic vesicles, thicker and longer postsynaptic densities, and increased synaptic interface curvature. Alterations of Arg3.1 and NR2B may be responsible for the changes in the synaptic ultrastructure. These changes confirm that salicylate can cause neural plasticity changes at the DCN level.

Angiotensin II modulates interleukin-1β-induced inflammatory gene expression in vascular smooth muscle cells via interfering with ERK-NF-κB crosstalk

International Nuclear Information System (INIS)

Xu, Shanqin; Zhi, Hui; Hou, Xiuyun; Jiang, Bingbing

2011-01-01

Highlights: → We examine how angiotensin II modulates ERK-NF-κB crosstalk and gene expression. → Angiotensin II suppresses IL-1β-induced prolonged ERK and NF-κB activation. → ERK-RSK1 signaling is required for IL-1β-induced prolonged NF-κB activation. → Angiotensin II modulates NF-κB responsive genes via regulating ERK-NF-κB crosstalk. → ERK-NF-κB crosstalk is a novel mechanism regulating inflammatory gene expression. -- Abstract: Angiotensin II is implicated in cardiovascular diseases, which is associated with a role in increasing vascular inflammation. The present study investigated how angiotensin II modulates vascular inflammatory signaling and expression of inducible nitric oxide synthase (iNOS) and vascular cell adhesion molecule (VCAM)-1. In cultured rat aortic vascular smooth muscle cells (VSMCs), angiotensin II suppressed interleukin-1β-induced prolonged phosphorylation of extracellular signal-regulated kinase (ERK) and ribosomal S6 kinase (RSK)-1, and nuclear translocation of nuclear factor (NF)-κB, leading to decreased iNOS but enhanced VCAM-1 expression, associated with an up-regulation of mitogen-activated protein kinase phosphatase-1 expression. Knock-down of RSK1 selectively down regulated interleukin-1β-induced iNOS expression without influencing VCAM-1 expression. In vivo experiments showed that interleukin-1β, iNOS, and VCAM-1 expression were detectable in the aortic arches of both wild-type and apolipoprotein E-deficient (ApoE -/- ) mice. VCAM-1 and iNOS expression were higher in ApoE -/- than in wild type mouse aortic arches. Angiotensin II infusion (3.2 mg/kg/day, for 6 days, via subcutaneous osmotic pump) in ApoE -/- mice enhanced endothelial and adventitial VCAM-1 and iNOS expression, but reduced medial smooth muscle iNOS expression associated with reduced phosphorylation of ERK and RSK-1. These results indicate that angiotensin II can differentially modulate inflammatory gene expression in aortic smooth muscle cells

Retinal and choroidal expression of BMP-2 in lens-induced myopia and recovery from myopia in guinea pigs.

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Li, Honghui; Wu, Juan; Cui, Dongmei; Zeng, Junwen

2016-03-01

The present study investigated the retinal and choroidal expression of bone morphogenetic protein-2 (BMP-2) in myopia and in myopia recovery in a guinea pig model. For this investigation, two groups of guinea pigs, lens‑induced myopia and recovery from myopia, were used, and defocused myopia was induced the guinea pigs wearing ‑4.00 D lenses on the right eyes for 3 weeks, with the left eyes serving as the contralateral. In the following week, the lenses of the guinea pigs in the recovery group were removed, and the refractive power and axial length were measured. The expression of BMP‑2 in the eyeballs was observed using immunohistochemistry and analyzed using Western blot analysis. After 3 weeks, the eyes acquired relative myopia and longer axial lengths in the two groups of guinea pigs. After 1 week without lenses in the recovery group, the myopia and axial lengths regressed. Immunofluorescence staining showed that BMP‑2 was expressed in the posterior retina, RPE, choroid and sclera. The expression of BMP‑2 decreased in the myopic retina of the guinea pigs. Following the regression of myopia in the recovery group, no difference in the expression of BMP‑2 was observed between the recovered treated eyes and the contralateral eyes. The choroidal expression level of BMP‑2 in the treated eyes showed no significant changes in either group. Therefore, BMP‑2 may be involved in the development of myopia, however, it does not have a primary role in the retinal and choroidal signals regulating scleral remodeling.

TNF-α blockade induces IL-10 expression in human CD4+ T cells

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Evans, Hayley G.; Roostalu, Urmas; Walter, Gina J.; Gullick, Nicola J.; Frederiksen, Klaus S.; Roberts, Ceri A.; Sumner, Jonathan; Baeten, Dominique L.; Gerwien, Jens G.; Cope, Andrew P.; Geissmann, Frederic; Kirkham, Bruce W.; Taams, Leonie S.

2014-02-01

IL-17+ CD4+ T (Th17) cells contribute to the pathogenesis of several human inflammatory diseases. Here we demonstrate that TNF inhibitor (TNFi) drugs induce the anti-inflammatory cytokine IL-10 in CD4+ T cells including IL-17+ CD4+ T cells. TNFi-mediated induction of IL-10 in IL-17+ CD4+ T cells is Treg-/Foxp3-independent, requires IL-10 and is overcome by IL-1β. TNFi-exposed IL-17+ CD4+ T cells are molecularly and functionally distinct, with a unique gene signature characterized by expression of IL10 and IKZF3 (encoding Aiolos). We show that Aiolos binds conserved regions in the IL10 locus in IL-17+ CD4+ T cells. Furthermore, IKZF3 and IL10 expression levels correlate in primary CD4+ T cells and Aiolos overexpression is sufficient to drive IL10 in these cells. Our data demonstrate that TNF-α blockade induces IL-10 in CD4+ T cells including Th17 cells and suggest a role for the transcription factor Aiolos in the regulation of IL-10 in CD4+ T cells.

Low dose radiation induced protein and its effect on expression of CD25 molecule in lymphocytes

International Nuclear Information System (INIS)

Lu Duicai; Su Liaoyuan

2001-01-01

Objective: To find the substantial basis for effects of low dose radiation, on development, extraction, and the biogical activity of the low-dose radiation-induced proteins, and the effects of LDR induced proteins on CD25 molecule expression of human lymphocytes. Methods: 1. Healthy Kumning male mice exposed to radiation of 226 Ra γ-rays at 5, 10 and 15 cGy respectively. The mice were killed 2 hours after exposure, the spleen cells were broken with ultrasonic energy and then ultra-centrifugalized at low temperature (4 degree C). The LDR-induced proteins were obtained in the supernatant solution. Then the changes of CD25 molecule was measured by flow cytometry (FCM) with immunofluorescence technique, which was used to reflect the effect of LDR induced proteins on CD25 molecule expression of human lymphocytes. Results: LDR induced proteins were obtained from spleen cells in mice exposed to 5-15 cGy whole body radiation. Conclusion: The expression of CD25 molecule of lymphocytes was increased significantly after use of LDR induced proteins. LDR induced proteins can enhance expression of CD25 molecule of lymphocytes slightly

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

International Nuclear Information System (INIS)

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

2012-01-01

Highlights: ► TNF-α induces MMP-9 expression and secretion to promote RPE cell migration. ► MAPK activation is not critical for TNF-α-induced MMP-9 expression. ► Akt and mTORC1 signaling mediate TNF-α-induced MMP-9 expression. ► SIN1 knockdown showed no significant effect on MMP-9 expression by TNF-α. -- Abstract: Tumor necrosis factor-alpha (TNF-α) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-α 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-α-induced in vitro RPE cell migration. Reversely, exogenously-added active MMP-9 promoted RPE cell migration. Suppression Akt/mTOR complex 1(mTORC1) activation by LY 294002 and rapamycin inhibited TNF-α-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-α promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.

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

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Wang, Cheng-hu; Cao, Guo-Fan [The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029 (China); Jiang, Qin, E-mail: Jqin710@vip.sina.com [The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029 (China); Yao, Jin, E-mail: dryaojin@yahoo.com [The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029 (China)

2012-08-17

Highlights: Black-Right-Pointing-Pointer TNF-{alpha} induces MMP-9 expression and secretion to promote RPE cell migration. Black-Right-Pointing-Pointer MAPK activation is not critical for TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer Akt and mTORC1 signaling mediate TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer SIN1 knockdown showed no significant effect on MMP-9 expression by TNF-{alpha}. -- Abstract: Tumor necrosis factor-alpha (TNF-{alpha}) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-{alpha} promotes human RPE cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression. Inhibition of MMP-9 by its inhibitor or its neutralizing antibody inhibited TNF-{alpha}-induced in vitro RPE cell migration. Reversely, exogenously-added active MMP-9 promoted RPE cell migration. Suppression Akt/mTOR complex 1(mTORC1) activation by LY 294002 and rapamycin inhibited TNF-{alpha}-mediated MMP-9 expression. To introduce a constitutively active Akt (CA-Akt) in cultured RPE cells increased MMP-9 expression, and to block mTORC1 activation by rapamycin inhibited its effect. RNA interference (RNAi)-mediated silencing of SIN1, a key component of mTOR complex 2 (mTORC2), had no effect on MMP-9 expression or secretion. In conclusion, this study suggest that TNF-{alpha} promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.

Matrix rigidity induces osteolytic gene expression of metastatic breast cancer cells.

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Nazanin S Ruppender

Full Text Available Nearly 70% of breast cancer patients with advanced disease will develop bone metastases. Once established in bone, tumor cells produce factors that cause changes in normal bone remodeling, such as parathyroid hormone-related protein (PTHrP. While enhanced expression of PTHrP is known to stimulate osteoclasts to resorb bone, the environmental factors driving tumor cells to express PTHrP in the early stages of development of metastatic bone disease are unknown. In this study, we have shown that tumor cells known to metastasize to bone respond to 2D substrates with rigidities comparable to that of the bone microenvironment by increasing expression and production of PTHrP. The cellular response is regulated by Rho-dependent actomyosin contractility mediated by TGF-ß signaling. Inhibition of Rho-associated kinase (ROCK using both pharmacological and genetic approaches decreased PTHrP expression. Furthermore, cells expressing a dominant negative form of the TGF-ß receptor did not respond to substrate rigidity, and inhibition of ROCK decreased PTHrP expression induced by exogenous TGF-ß. These observations suggest a role for the differential rigidity of the mineralized bone microenvironment in early stages of tumor-induced osteolysis, which is especially important in metastatic cancer since many cancers (such as those of the breast and lung preferentially metastasize to bone.

[The mechanisms of p21WAF1/Cip-1 expression in MOLT-4 cell line induced by TSA].

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Song, Yi; Liu, Mei-Ju; Zhao, Guo-Wei; Qian, Jun-Jie; Dong, Yan; Liu, Hua; Sun, Guo-Jing; Mei, Zhu-Zhong; Liu, Bin; Tian, Bao-Lei; Sun, Zhi-Xian

2005-04-01

To investigate the function and molecular mechanism of p21(WAF1/Cip-1) expression in MOLT-4 cells induced by HDAC inhibitor TSA, the expression pattern of p21(WAF1/Cip-1) and the distribution of cell cycle in TSA treated cells were analyzed. The results showed that TSA could effectively induce G(2)/M arrest and apoptosis of MOLT-4 cells. Kinetic experiments demonstrated that p21(WAF1/Cip-1) were upregulated quickly before cell arrested in G(2)/M and began decreasing at the early stage of apoptosis. Meanwhile, the proteasome inhibitor MG-132 could inhibit the decrease of p21(WAF1/Cip-1) at the early stage of apoptosis, which showed that proteasome pathway involved in p21(WAF1/Cip-1) degradation during the TSA induced G(2)/M arrest and apoptosis responses. This study also identified that the protein level of p21(WAF1/Cip-1) was highly associated with the cell cycle change induced by TSA. Compared to cells treated by TSA only, exposure MOLT-4 cells to TSA meanwhile treatment with MG-132 increased the protein level of p21(WAF1/Cip-1) and increased the numbers of cell in G(2)/M-phase, whereas the cell apoptosis were delayed. It is concluded that p21(WAF1/Cip-1) plays a significant role in G(2)/M arrest and apoptosis signaling induced by TSA in MOLT-4 cells.

Irinotecan (CPT-11)-induced elevation of bile acids potentiates suppression of IL-10 expression

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Fang, Zhong-Ze; Zhang, Dunfang; Cao, Yun-Feng; Xie, Cen; Lu, Dan; Sun, Dong-Xue; Tanaka, Naoki; Jiang, Changtao; Chen, Qianming; Chen, Yu; Wang, Haina; Gonzalez, Frank J.

2016-01-01

Irinotecan (CPT-11) is a first-line anti-colon cancer drug, however; CPT-11-induced toxicity remains a key factor limiting its clinical application. To search for clues to the mechanism of CPT-11-induced toxicity, metabolomics was applied using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry. Intraperitoneal injection of 50 mg/kg of CPT-11 induced loss of body weight, and intestine toxicity. Changes in gallbladder morphology suggested alterations in bile acid metabolism, as revealed at the molecular level by analysis of the liver, bile, and ileum metabolomes between the vehicle-treated control group and the CPT-11-treated group. Analysis of immune cell populations further showed that CPT-11 treatment significantly decreased the IL-10-producing CD4 T cell frequency in intestinal lamina propria lymphocytes, but not in spleen or mesenteric lymph nodes. In vitro cell culture studies showed that the addition of bile acids deoxycholic acid and taurodeoxycholic acid accelerated the CPT-11-induced suppression of IL-10 secretion by activated CD4 + naive T cells isolated from mouse splenocytes. These results showed that CPT-11 treatment caused metabolic changes in the composition of bile acids that altered CPT-11-induced suppression of IL-10 expression. - Highlights: • CPT-11 is an effective anticancer drug, but induced toxicity limits its application in the clinic. • CPT-11 decreased IL-10-producing CD4 T cell frequency in intestinal lamina propria lymphocytes. • CPT-11 altered the composition of bile acid metabolites, notably DCA and TDCA in liver, bile and intestine. • DCA and TDCA potentiated CPT-11-induced suppression of IL-10 secretion by active CD4 + naive T cells.

Irinotecan (CPT-11)-induced elevation of bile acids potentiates suppression of IL-10 expression

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Fang, Zhong-Ze [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD (United States); Department of Toxicology, School of Public Health, Tianjin Medical University, Tianjin (China); Joint Center for Translational Medicine, Dalian Institute of Chemical Physics, Chinese Academy of Sciences and First Affiliated Hospital of Liaoning Medical University, Dalian (China); Zhang, Dunfang [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu (China); Cao, Yun-Feng [Joint Center for Translational Medicine, Dalian Institute of Chemical Physics, Chinese Academy of Sciences and First Affiliated Hospital of Liaoning Medical University, Dalian (China); Xie, Cen [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD (United States); Lu, Dan [Department of Immunology, Tianjin Key Laboratory of Cellular and Molecular Immunology, Tianjin Medical University, Tianjin (China); Sun, Dong-Xue; Tanaka, Naoki; Jiang, Changtao [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD (United States); Chen, Qianming; Chen, Yu [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu (China); Wang, Haina [School of Pharmaceutical Sciences, Shandong University, Jinan (China); Gonzalez, Frank J., E-mail: gonzalef@mail.nih.gov [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD (United States)

2016-01-15

Irinotecan (CPT-11) is a first-line anti-colon cancer drug, however; CPT-11-induced toxicity remains a key factor limiting its clinical application. To search for clues to the mechanism of CPT-11-induced toxicity, metabolomics was applied using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry. Intraperitoneal injection of 50 mg/kg of CPT-11 induced loss of body weight, and intestine toxicity. Changes in gallbladder morphology suggested alterations in bile acid metabolism, as revealed at the molecular level by analysis of the liver, bile, and ileum metabolomes between the vehicle-treated control group and the CPT-11-treated group. Analysis of immune cell populations further showed that CPT-11 treatment significantly decreased the IL-10-producing CD4 T cell frequency in intestinal lamina propria lymphocytes, but not in spleen or mesenteric lymph nodes. In vitro cell culture studies showed that the addition of bile acids deoxycholic acid and taurodeoxycholic acid accelerated the CPT-11-induced suppression of IL-10 secretion by activated CD4{sup +} naive T cells isolated from mouse splenocytes. These results showed that CPT-11 treatment caused metabolic changes in the composition of bile acids that altered CPT-11-induced suppression of IL-10 expression. - Highlights: • CPT-11 is an effective anticancer drug, but induced toxicity limits its application in the clinic. • CPT-11 decreased IL-10-producing CD4 T cell frequency in intestinal lamina propria lymphocytes. • CPT-11 altered the composition of bile acid metabolites, notably DCA and TDCA in liver, bile and intestine. • DCA and TDCA potentiated CPT-11-induced suppression of IL-10 secretion by active CD4{sup +} naive T cells.

Curcumin modulates dopaminergic receptor, CREB and phospholipase c gene expression in the cerebral cortex and cerebellum of streptozotocin induced diabetic rats

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George Naijil

2010-05-01

Full Text Available Abstract Curcumin, an active principle component in rhizome of Curcuma longa, has proved its merit for diabetes through its anti-oxidative and anti-inflammatory properties. This study aims at evaluating the effect of curcumin in modulating the altered dopaminergic receptors, CREB and phospholipase C in the cerebral cortex and cerebellum of STZ induced diabetic rats. Radioreceptor binding assays and gene expression was done in the cerebral cortex and cerebellum of male Wistar rats using specific ligands and probes. Total dopaminergic receptor binding parameter, Bmax showed an increase in cerebral cortex and decrease in the cerebellum of diabetic rats. Gene expression studies using real time PCR showed an increased expression of dopamine D1 and D2 receptor in the cerebral cortex of diabetic rats. In cerebellum dopamine D1 receptor was down regulated and D2 receptor showed an up regulation. Transcription factor CREB and phospholipase C showed a significant down regulation in cerebral cortex and cerebellum of diabetic rats. We report that curcumin supplementation reduces diabetes induced alteration of dopamine D1, D2 receptors, transcription factor CREB and phospholipase C to near control. Our results indicate that curcumin has a potential to regulate diabetes induced malfunctions of dopaminergic signalling, CREB and Phospholipase C expression in cerebral cortex and cerebellum and thereby improving the cognitive and emotional functions associated with these regions. Furthermore, in line with these studies an interaction between curcumin and dopaminergic receptors, CREB and phospholipase C is suggested, which attenuates the cortical and cerebellar dysfunction in diabetes. These results suggest that curcumin holds promise as an agent to prevent or treat CNS complications in diabetes.

Curcumin modulates dopaminergic receptor, CREB and phospholipase C gene expression in the cerebral cortex and cerebellum of streptozotocin induced diabetic rats.

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Kumar, T Peeyush; Antony, Sherin; Gireesh, G; George, Naijil; Paulose, C S

2010-05-31

Curcumin, an active principle component in rhizome of Curcuma longa, has proved its merit for diabetes through its anti-oxidative and anti-inflammatory properties. This study aims at evaluating the effect of curcumin in modulating the altered dopaminergic receptors, CREB and phospholipase C in the cerebral cortex and cerebellum of STZ induced diabetic rats. Radioreceptor binding assays and gene expression was done in the cerebral cortex and cerebellum of male Wistar rats using specific ligands and probes. Total dopaminergic receptor binding parameter, B(max) showed an increase in cerebral cortex and decrease in the cerebellum of diabetic rats. Gene expression studies using real time PCR showed an increased expression of dopamine D1 and D2 receptor in the cerebral cortex of diabetic rats. In cerebellum dopamine D1 receptor was down regulated and D2 receptor showed an up regulation. Transcription factor CREB and phospholipase C showed a significant down regulation in cerebral cortex and cerebellum of diabetic rats. We report that curcumin supplementation reduces diabetes induced alteration of dopamine D1, D2 receptors, transcription factor CREB and phospholipase C to near control. Our results indicate that curcumin has a potential to regulate diabetes induced malfunctions of dopaminergic signalling, CREB and Phospholipase C expression in cerebral cortex and cerebellum and thereby improving the cognitive and emotional functions associated with these regions. Furthermore, in line with these studies an interaction between curcumin and dopaminergic receptors, CREB and phospholipase C is suggested, which attenuates the cortical and cerebellar dysfunction in diabetes. These results suggest that curcumin holds promise as an agent to prevent or treat CNS complications in diabetes.

Fisetin Induces Apoptosis Through p53-Mediated Up-Regulation of DR5 Expression in Human Renal Carcinoma Caki Cells.

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Min, Kyoung-Jin; Nam, Ju-Ock; Kwon, Taeg Kyu

2017-08-02

Fisetin is a natural compound found in fruits and vegetables such as strawberries, apples, cucumbers, and onions. Since fisetin can elicit anti-cancer effects, including anti-proliferation and anti-migration, we investigated whether fisetin induced apoptosis in human renal carcinoma (Caki) cells. Fisetin markedly induced sub-G1 population and cleavage of poly (ADP-ribose) polymerase (PARP), which is a marker of apoptosis, and increased caspase activation. We found that pan-caspase inhibitor (z-VAD-fmk) inhibited fisetin-induced apoptosis. In addition, fisetin induced death receptor 5 (DR5) expression at the transcriptional level, and down-regulation of DR5 by siRNA blocked fisetin-induced apoptosis. Furthermore, fisetin induced p53 protein expression through up-regulation of protein stability, whereas down-regulation of p53 by siRNA markedly inhibited fisetin-induced DR5 expression. In contrast, fisetin induced up-regulation of CHOP expression and reactive oxygen species production, which had no effect on fisetin-induced apoptosis. Taken together, our study demonstrates that fisetin induced apoptosis through p53 mediated up-regulation of DR5 expression at the transcriptional level.

Low dose decitabine treatment induces CD80 expression in cancer cells and stimulates tumor specific cytotoxic T lymphocyte responses.

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Li-Xin Wang

Full Text Available Lack of immunogenicity of cancer cells has been considered a major reason for their failure in induction of a tumor specific T cell response. In this paper, we present evidence that decitabine (DAC, a DNA methylation inhibitor that is currently used for the treatment of myelodysplastic syndrome (MDS, acute myeloid leukemia (AML and other malignant neoplasms, is capable of eliciting an anti-tumor cytotoxic T lymphocyte (CTL response in mouse EL4 tumor model. C57BL/6 mice with established EL4 tumors were treated with DAC (1.0 mg/kg body weight once daily for 5 days. We found that DAC treatment resulted in infiltration of IFN-γ producing T lymphocytes into tumors and caused tumor rejection. Depletion of CD8(+, but not CD4(+ T cells resumed tumor growth. DAC-induced CTL response appeared to be elicited by the induction of CD80 expression on tumor cells. Epigenetic evidence suggests that DAC induces CD80 expression in EL4 cells via demethylation of CpG dinucleotide sites in the promoter of CD80 gene. In addition, we also showed that a transient, low-dose DAC treatment can induce CD80 gene expression in a variety of human cancer cells. This study provides the first evidence that epigenetic modulation can induce the expression of a major T cell co-stimulatory molecule on cancer cells, which can overcome immune tolerance, and induce an efficient anti-tumor CTL response. The results have important implications in designing DAC-based cancer immunotherapy.

Low Dose Decitabine Treatment Induces CD80 Expression in Cancer Cells and Stimulates Tumor Specific Cytotoxic T Lymphocyte Responses

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Zhou, Ji-Hao; Yao, Yu-Shi; Li, Yong-Hui; Xu, Yi-Han; Li, Jing-Xin; Gao, Xiao-Ning; Zhou, Min-Hang; Jiang, Meng-Meng; Gao, Li; Ding, Yi; Lu, Xue-Chun; Shi, Jin-Long; Luo, Xu-Feng; Wang, Jia; Wang, Li-Li; Qu, Chunfeng; Bai, Xue-Feng; Yu, Li

2013-01-01

Lack of immunogenicity of cancer cells has been considered a major reason for their failure in induction of a tumor specific T cell response. In this paper, we present evidence that decitabine (DAC), a DNA methylation inhibitor that is currently used for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and other malignant neoplasms, is capable of eliciting an anti-tumor cytotoxic T lymphocyte (CTL) response in mouse EL4 tumor model. C57BL/6 mice with established EL4 tumors were treated with DAC (1.0 mg/kg body weight) once daily for 5 days. We found that DAC treatment resulted in infiltration of IFN-γ producing T lymphocytes into tumors and caused tumor rejection. Depletion of CD8+, but not CD4+ T cells resumed tumor growth. DAC-induced CTL response appeared to be elicited by the induction of CD80 expression on tumor cells. Epigenetic evidence suggests that DAC induces CD80 expression in EL4 cells via demethylation of CpG dinucleotide sites in the promoter of CD80 gene. In addition, we also showed that a transient, low-dose DAC treatment can induce CD80 gene expression in a variety of human cancer cells. This study provides the first evidence that epigenetic modulation can induce the expression of a major T cell co-stimulatory molecule on cancer cells, which can overcome immune tolerance, and induce an efficient anti-tumor CTL response. The results have important implications in designing DAC-based cancer immunotherapy. PMID:23671644

Low dose decitabine treatment induces CD80 expression in cancer cells and stimulates tumor specific cytotoxic T lymphocyte responses.

Science.gov (United States)

Wang, Li-Xin; Mei, Zhen-Yang; Zhou, Ji-Hao; Yao, Yu-Shi; Li, Yong-Hui; Xu, Yi-Han; Li, Jing-Xin; Gao, Xiao-Ning; Zhou, Min-Hang; Jiang, Meng-Meng; Gao, Li; Ding, Yi; Lu, Xue-Chun; Shi, Jin-Long; Luo, Xu-Feng; Wang, Jia; Wang, Li-Li; Qu, Chunfeng; Bai, Xue-Feng; Yu, Li

2013-01-01

Lack of immunogenicity of cancer cells has been considered a major reason for their failure in induction of a tumor specific T cell response. In this paper, we present evidence that decitabine (DAC), a DNA methylation inhibitor that is currently used for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and other malignant neoplasms, is capable of eliciting an anti-tumor cytotoxic T lymphocyte (CTL) response in mouse EL4 tumor model. C57BL/6 mice with established EL4 tumors were treated with DAC (1.0 mg/kg body weight) once daily for 5 days. We found that DAC treatment resulted in infiltration of IFN-γ producing T lymphocytes into tumors and caused tumor rejection. Depletion of CD8(+), but not CD4(+) T cells resumed tumor growth. DAC-induced CTL response appeared to be elicited by the induction of CD80 expression on tumor cells. Epigenetic evidence suggests that DAC induces CD80 expression in EL4 cells via demethylation of CpG dinucleotide sites in the promoter of CD80 gene. In addition, we also showed that a transient, low-dose DAC treatment can induce CD80 gene expression in a variety of human cancer cells. This study provides the first evidence that epigenetic modulation can induce the expression of a major T cell co-stimulatory molecule on cancer cells, which can overcome immune tolerance, and induce an efficient anti-tumor CTL response. The results have important implications in designing DAC-based cancer immunotherapy.

Hypoxic stress up-regulates the expression of Toll-like receptor 4 in macrophages via hypoxia-inducible factor.

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Kim, So Young; Choi, Yong Jun; Joung, Sun Myung; Lee, Byung Ho; Jung, Yi-Sook; Lee, Joo Young

2010-04-01

Toll-like receptors (TLRs) are germline-encoded innate immune receptors that recognize invading micro-organisms and induce immune and inflammatory responses. Deregulation of TLRs is known to be closely linked to various immune disorders and inflammatory diseases. Cells at sites of inflammation are exposed to hypoxic stress, which further aggravates inflammatory processes. We have examined if hypoxic stress modulates the TLR activity of macrophages. Hypoxia and CoCl(2) (a hypoxia mimetic) enhanced the expression of TLR4 messenger RNA and protein in macrophages (RAW264.7 cells), whereas the messenger RNA of other TLRs was not increased. To determine the underlying mechanism, we investigated the role of hypoxia-inducible factor 1 (HIF-1) in the regulation of TLR4 expression. Knockdown of HIF-1alpha expression by small interfering RNA inhibited hypoxia-induced and CoCl(2)-induced TLR4 expression in macrophages, while over-expression of HIF-1alpha potentiated TLR4 expression. Chromatin immunoprecipitation assays revealed that HIF-1alpha binds to the TLR4 promoter region under hypoxic conditions. In addition, deletion or mutation of a putative HIF-1-binding motif in the TLR4 promoter greatly attenuated HIF-1alpha-induced TLR4 promoter reporter expression. Up-regulation of TLR4 expression by hypoxic stress enhanced the response of macrophages to lipopolysaccharide, resulting in increased expression of cyclooxygenase-2, interleukin-6, regulated on activation normal T cell expressed and secreted, and interferon-inducible protein-10. These results demonstrate that TLR4 expression in macrophages is up-regulated via HIF-1 in response to hypoxic stress, suggesting that hypoxic stress at sites of inflammation enhances susceptibility to subsequent infection and inflammatory signals by up-regulating TLR4.

IL-6 Promotes FSH-Induced VEGF Expression Through JAK/STAT3 Signaling Pathway in Bovine Granulosa Cells

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Meng Yang

2017-11-01

Full Text Available Background/Aims: Vascular endothelial growth factor (VEGF has been demonstrated to play a pivotal role in the regulation of angiogenesis in ovarian follicular development, particularly during the preovulatory period. Although numerous studies have shown that interleukin-6 (IL-6 is one of the major inducing factors that regulate the expression of VEGF in non-ovarian cells, whether it involved in regulating the expression of VEGF in normal ovarian granulosa cells is still unknown. The aim of this study was to elucidate the mechanisms underlying the effect of IL-6 on FSH-induced VEGF expression in bovine granulosa cells derived from large follicles. Methods: VEGF mRNA expression in granulosa cells after IL-6 with/without inhibitors treatment was analyzed by RT-qPCR. Phosphorylation levels of ERK1/2 and STAT3 proteins induced by IL-6 were analyzed by western blotting. The protein levels produced by granulosa cells were detected by ELISA. Results: High concentration of IL-6 (10ng/ml can significantly up-regulate FSH-induced VEGF gene and protein expression levels in granulosa cells, and also promote the VEGF upstream regulators HIF-1α and COX2 mRNA expression. VEGF expression levels were significantly decreased after specifically blocking HIF-1α and COX2 by using inhibitors. The up-regulation effect of IL-6 on FSH-induced VEGF expression in granulosa cells mainly through activating the JAK/STAT3 signaling pathway, which can be impaired by JAK inhibitors. Conclusion: IL-6 can promote FSH-induced VEGF expression in granulosa cells, which is mainly achieved by increasing the expression of HIF-1α and COX2.This promoting effect is mediated by activating the JAK/STAT3 pathway. Moreover, there may be a synergistic relationship between FSH and IL-6 in the regulation of VEGF expression.

The mGlu2/3 Receptor Agonists LY354740 and LY379268 Differentially Regulate Restraint-Stress-Induced Expression of c-Fos in Rat Cerebral Cortex

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M. M. Menezes

2013-01-01

Full Text Available Metabotropic glutamate 2/3 (mGlu2/3 receptors have emerged as potential therapeutic targets due to the ability of mGlu2/3 receptor agonists to modulate excitatory transmission at specific synapses. LY354740 and LY379268 are selective and potent mGlu2/3 receptor agonists that show both anxiolytic- and antipsychotic-like effects in animal models. We compared the efficacy of LY354740 and LY379268 in attenuating restraint-stress-induced expression of the immediate early gene c-Fos in the rat prelimbic (PrL and infralimbic (IL cortex. LY354740 (10 and 30 mg/kg, i.p. showed statistically significant and dose-related attenuation of stress-induced increase in c-Fos expression, in the rat cortex. By contrast, LY379268 had no effect on restraint-stress-induced c-Fos upregulation (0.3–10 mg/kg, i.p.. Because both compounds inhibit serotonin 2A receptor (5-HT2AR-induced c-Fos expression, we hypothesize that LY354740 and LY379268 have different in vivo properties and that 5-HT2AR activation and restraint stress induce c-Fos through distinct mechanisms.

Differential Gene Expression Patterns in Chicken Cardiomyocytes during Hydrogen Peroxide-Induced Apoptosis.

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Wan, Chunyun; Xiang, Jinmei; Li, Youwen; Guo, Dingzong

2016-01-01

Hydrogen peroxide (H2O2) is both an exogenous and endogenous cytotoxic agent that can reliably induce apoptosis in numerous cell types for studies on apoptosis signaling pathways. However, little is known of these apoptotic processes in myocardial cells of chicken, a species prone to progressive heart failure. Sequencing of mRNA transcripts (RNA-Seq) allows for the identification of differentially expressed genes under various physiological and pathological conditions to elucidate the molecular pathways involved, including cellular responses to exogenous and endogenous toxins. We used RNA-seq to examine genes differentially expressed during H2O2-induced apoptosis in primary cultures of embryonic chicken cardiomyocytes. Following control or H2O2 treatment, RNA was extracted and sequencing performed to identify novel transcripts up- or downregulated in the H2O2 treatment group and construct protein-protein interaction networks. Of the 19,268 known and 2,160 novel transcripts identified in both control and H2O2 treatment groups, 4,650 showed significant differential expression. Among them, 55.63% were upregulated and 44.37% downregulated. Initiation of apoptosis by H2O2 was associated with upregulation of caspase-8, caspase-9, and caspase-3, and downregulation of anti-apoptotic genes API5 and TRIA1. Many other differentially expressed genes were associated with metabolic pathways (including 'Fatty acid metabolism', 'Alanine, aspartate, and glutamate metabolism', and 'Biosynthesis of unsaturated fatty acids') and cell signaling pathways (including 'PPAR signaling pathway', 'Adipocytokine signaling pathway', 'TGF-beta signaling pathway', 'MAPK signaling pathway', and 'p53 signaling pathway'). In chicken cardiomyocytes, H2O2 alters the expression of numerous genes linked to cell signaling and metabolism as well as genes directly associated with apoptosis. In particular, H2O2 also affects the biosynthesis and processing of proteins and unsaturated fatty acids. These

Abnormal hippocampal BDNF and miR-16 expression is associated with depression-like behaviors induced by stress during early life.

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Mei Bai

Full Text Available Some environmental stressors lead to the onset of depression via inhibiting hippocampal BDNF expression, but other environmental stressors-induced depression exhibits no change in BDNF expression. The underlying mechanisms behind the divergence remain unknown. In this study, depression-like behaviors were induced in rats by maternal deprivation (MD and chronic unpredictable stress (CUPS. Depression-like behaviors were tested by open field test, forced swimming test, and sucrose consumption test. BDNF and miR-16 expressions in the hippocampus were examined by real-time PCR. MD and CUPS rats crawled less distance, exhibited decreased vertical activity, and produced more fecal pellets than control rats in the open field test. However, MD rats crawled less distance and produced significantly less fecal pellets than CUPS rats. In the forced swimming and sucrose consumption tests, CUPS and MD rats exhibited longer floating time and consumed less sucrose than control rats, but MD rats exhibited shorter floating time and consumed less sucrose than CUPS rats. MD but not CUPS rats showed lower BDNF mRNA and higher miR-16 expression than control rats. In MD rats, BDNF mRNA expression negatively correlated with the expression of miR-16. BDNF expression positively correlated with the total distance rats crawled and vertical activity in the open field test while miR-16 expression negatively correlated the two behaviors. BDNF positively correlated with sucrose preference rate while miR-16 negatively correlated with sucrose preference rate of the sucrose consumption test. Our study suggests that MD and CUPS induced different depression-like behaviors in rats. Depression induced by MD but not CUPS was significantly associated with upregulation of miR-16 and possibly subsequent downregulation of BDNF in hippocampus.

Mapping photothermally induced gene expression in living cells and tissues by nanorod-locked nucleic acid complexes.

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Riahi, Reza; Wang, Shue; Long, Min; Li, Na; Chiou, Pei-Yu; Zhang, Donna D; Wong, Pak Kin

2014-04-22

The photothermal effect of plasmonic nanostructures has numerous applications, such as cancer therapy, photonic gene circuit, large cargo delivery, and nanostructure-enhanced laser tweezers. The photothermal operation can also induce unwanted physical and biochemical effects, which potentially alter the cell behaviors. However, there is a lack of techniques for characterizing the dynamic cell responses near the site of photothermal operation with high spatiotemporal resolution. In this work, we show that the incorporation of locked nucleic acid probes with gold nanorods allows photothermal manipulation and real-time monitoring of gene expression near the area of irradiation in living cells and animal tissues. The multimodal gold nanorod serves as an endocytic delivery reagent to transport the probes into the cells, a fluorescence quencher and a binding competitor to detect intracellular mRNA, and a plasmonic photothermal transducer to induce cell ablation. We demonstrate the ability of the gold nanorod-locked nucleic acid complex for detecting the spatiotemporal gene expression in viable cells and tissues and inducing photothermal ablation of single cells. Using the gold nanorod-locked nucleic acid complex, we systematically characterize the dynamic cellular heat shock responses near the site of photothermal operation. The gold nanorod-locked nucleic acid complex enables mapping of intracellular gene expressions and analyzes the photothermal effects of nanostructures toward various biomedical applications.

Probucol Attenuates Cyclophosphamide-induced Oxidative Apoptosis, p53 and Bax Signal Expression in Rat Cardiac Tissues

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Yousif A. Asiri

2010-01-01

Full Text Available Cyclophosphamide (CP is a widely used drug in cancer chemotherapy and immunosuppression, which could cause toxicity of the normal cells due to its toxic metabolites. Probucol, a cholesterol-lowering drug, acts as potential inhibitor of DNA damage and shows to protect against doxorubicin-induced cardiomyopathy by enhancing the endogenous antioxidant system including glutathione peroxidase, catalase and superoxide dismutase. This study examined the possible protective effects of probucol, a lipid-lowering compound with strong antioxidant properties, against CPinduced cardiotoxicity. This objective could be achieved through studying the gene expression-based on the possible protective effects of probucol against CP-induced cardiac failure in rats. Adult male Wistar albino rats were assigned into four treatment groups: Animals in the first (control and second (probucol groups were injected intraperitoneally with corn oil and probucol (61 mg/kg/day, respectively, for two weeks. Animals in the third (CP and fourth (probucol plus CP groups were injected with the same doses of corn oil and probucol (61 mg/kg/day, respectively, for one week before and one week after a single dose of CP (200 mg/kg, I.P.. The p53, Bax, Bcl2 and oxidative genes signal expression were measured by real time PCR. CP-induced cardiotoxicity was clearly observed by a significant increase in serum creatine phosphokinase isoenzyme (CK-MB (117%, lactate dehydrogenase (LDH (64%, free (69% and esterified cholesterol (42% and triglyceride (69% compared to control group. In cardiac tissues, CP significantly increases the mRNA expression levels of apoptotic genes, p53 with two-fold and Bax with 1.6-fold, and decreases the anti-apoptotic gene Bcl2 with 0.5-fold. Moreover, CP caused downregulation of antioxidant genes, glutathione peroxidase, catalase, and superoxide dismutase and increased the lipid peroxidation and decreased adenosine triphosphate (ATP (40% and ATP/ADP (44% in cardiac

Aloe vera downregulates LPS-induced inflammatory cytokine production and expression of NLRP3 inflammasome in human macrophages.

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Budai, Marietta M; Varga, Aliz; Milesz, Sándor; Tőzsér, József; Benkő, Szilvia

2013-12-01

Aloe vera has been used in traditional herbal medicine as an immunomodulatory agent inducing anti-inflammatory effects. However, its role on the IL-1β inflammatory cytokine production has not been studied. IL-1β production is strictly regulated both at transcriptional and posttranslational levels through the activity of Nlrp3 inflammasome. In this study we aimed to determine the effect of Aloe vera on the molecular mechanisms of Nlrp3 inflammasome-mediated IL-1β production in LPS-activated human THP-1 cells and monocyte-derived macrophages. Our results show that Aloe vera significantly reduced IL-8, TNFα, IL-6 and IL-1β cytokine production in a dose dependent manner. The inhibitory effect was substantially more pronounced in the primary cells. We found that Aloe vera inhibited the expression of pro-IL-1β, Nlrp3, caspase-1 as well as that of the P2X7 receptor in the LPS-induced primary macrophages. Furthermore, LPS-induced activation of signaling pathways like NF-κB, p38, JNK and ERK were inhibited by Aloe vera in these cells. Altogether, we show for the first time that Aloe vera-mediated strong reduction of IL-1β appears to be the consequence of the reduced expression of both pro-IL-1β as well as Nlrp3 inflammasome components via suppressing specific signal transduction pathways. Furthermore, we show that the expression of the ATP sensor P2X7 receptor is also downregulated by Aloe vera that could also contribute to the attenuated IL-1β cytokine secretion. These results may provide a new therapeutic approach to regulate inflammasome-mediated responses. Copyright © 2013 Elsevier Ltd. All rights reserved.

Transcriptome Changes Associated with Delayed Flower Senescence on Transgenic Petunia by Inducing Expression of etr1-1, a Mutant Ethylene Receptor

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Lin, Jing; Liu, Gang; Zhang, Zhen; Chang, Youhong; Reid, Michael S.; Jiang, Cai-Zhong

2013-01-01

Flowers of ethylene-sensitive ornamental plants transformed with ethylene-insensitive 1-1(etr1-1), a mutant ethylene receptor first isolated from Arabidopsis, are known to have longer shelf lives. We have generated petunia plants in which the etr1-1 gene was over-expressed under the control of a chemically-inducible promoter, which would allow expression of etr1-1 to be initiated at the desired time and stage of development. Here, we showed that transgenic plants grew and developed normally without a chemical inducer. Semi-quantitative RT-PCR demonstrated that the abundance of transcripts of Arabidopsis etr1-1 gene was substantially induced in flowers with 30 μM dexamethasone (DEX). Consequently, t he life of the flowers was almost doubled and the peak of ethylene production was delayed. We compared gene expression changes of petals with DEX to those without DEX at 24 h and 48 h by microarray. Our results indicated that transcripts of many putative genes encoding transcription factors were down-regulated by etr1-1 induced expression at the early stage. In addition, putative genes involved in gibberellin biosynthesis, response to jasmonic acid/gibberellins stimulus, cell wall modification, ethylene biosynthesis, and cell death were down-regulated associating with etr1-1 induced expression. We investigated time-course gene expression profiles and found two profiles which displayed totally opposite expression patterns under these two treatments. In these profiles, ‘the regulation of transcription’ was predominant in GO categories. Taking all results together, we concluded those transcription factors down-regulated at early stage might exert a major role in regulating the senescence process which were consequently characterized by cell wall modification and cell death. PMID:23874385

Transcriptome changes associated with delayed flower senescence on transgenic petunia by inducing expression of etr1-1, a mutant ethylene receptor.

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Hong Wang

Full Text Available Flowers of ethylene-sensitive ornamental plants transformed with ethylene-insensitive 1-1(etr1-1, a mutant ethylene receptor first isolated from Arabidopsis, are known to have longer shelf lives. We have generated petunia plants in which the etr1-1 gene was over-expressed under the control of a chemically-inducible promoter, which would allow expression of etr1-1 to be initiated at the desired time and stage of development. Here, we showed that transgenic plants grew and developed normally without a chemical inducer. Semi-quantitative RT-PCR demonstrated that the abundance of transcripts of Arabidopsis etr1-1 gene was substantially induced in flowers with 30 μM dexamethasone (DEX. Consequently, t he life of the flowers was almost doubled and the peak of ethylene production was delayed. We compared gene expression changes of petals with DEX to those without DEX at 24 h and 48 h by microarray. Our results indicated that transcripts of many putative genes encoding transcription factors were down-regulated by etr1-1 induced expression at the early stage. In addition, putative genes involved in gibberellin biosynthesis, response to jasmonic acid/gibberellins stimulus, cell wall modification, ethylene biosynthesis, and cell death were down-regulated associating with etr1-1 induced expression. We investigated time-course gene expression profiles and found two profiles which displayed totally opposite expression patterns under these two treatments. In these profiles, 'the regulation of transcription' was predominant in GO categories. Taking all results together, we concluded those transcription factors down-regulated at early stage might exert a major role in regulating the senescence process which were consequently characterized by cell wall modification and cell death.

The STAT3 inhibitor pimozide impedes cell proliferation and induces ROS generation in human osteosarcoma by suppressing catalase expression.

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Cai, Nan; Zhou, Wei; Ye, Lan-Lan; Chen, Jun; Liang, Qiu-Ni; Chang, Gang; Chen, Jia-Jie

2017-01-01

Currently, there is a considerable need to develop new treatments for osteosarcoma (OS), a very aggressive bone cancer. The activation of STAT3 signaling is positively associated with poor prognosis and aggressive progression in OS patients. Our previous study reported that the FDA-approved antipsychotic drug pimozide had anti-tumor activity against hepatocellular carcinoma and prostate cancer cells by suppressing STAT3 activity. Therefore, the aim of this study was to investigate the specific effect of pimozide on OS cells and the underlying molecular mechanism. Pimozide inhibited cell proliferation, colony formation, and sphere formation capacities of the OS cells in a dose-dependent manner, inducing G0/G1 phase cell cycle arrest. Pimozide reduced the percentage of side population cells representing cancer stem-like cells and enhanced the sensitivity of OS cells to 5-FU induced proliferative inhibition. In addition, pimozide induced apoptosis of U2OS cells, which showed increased expression of cleaved-PARP, a marker of programmed cell death. Moreover, pimozide suppressed Erk signaling in OS cells. Importantly, pimozide induced ROS generation by downregulating the expression of the antioxidant enzyme catalase (CAT). NAC treatment partially reversed the ROS generation and cytotoxic effects induced by pimozide. CAT treatment attenuated the pimozide-induced proliferation inhibition. The decrease of CAT expression induced by pimozide was potentially mediated through the suppression of cellular STAT3 activity in OS cells. Thus, pimozide may be a novel STAT3 inhibitor that suppresses cellular STAT3 activity to inhibit OS cells or stem-like cells and is a novel potential anti-cancer agent in OS treatment.

Endogenous brain IL-1 mediates LPS-induced anorexia and hypothalamic cytokine expression.

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Layé, S; Gheusi, G; Cremona, S; Combe, C; Kelley, K; Dantzer, R; Parnet, P

2000-07-01

The present study was designed to determine the role of endogenous brain interleukin (IL)-1 in the anorexic response to lipopolysaccharide (LPS). Intraperitoneal administration of LPS (5-10 microgram/mouse) induced a dramatic, but transient, decrease in food intake, associated with an enhanced expression of proinflammatory cytokine mRNA (IL-1beta, IL-6, and tumor necrosis factor-alpha) in the hypothalamus. This dose of LPS also increased plasma levels of IL-1beta. Intracerebroventricular pretreatment with IL-1 receptor antagonist (4 microgram/mouse) attenuated LPS-induced depression of food intake and totally blocked the LPS-induced enhanced expression of proinflammatory cytokine mRNA measured in the hypothalamus 1 h after treatment. In contrast, LPS-induced increases in plasma levels of IL-1beta were not altered. These findings indicate that endogenous brain IL-1 plays a pivotal role in the development of the hypothalamic cytokine response to a systemic inflammatory stimulus.

Chemical memory reactions induced bursting dynamics in gene expression.

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Tian, Tianhai

2013-01-01

Memory is a ubiquitous phenomenon in biological systems in which the present system state is not entirely determined by the current conditions but also depends on the time evolutionary path of the system. Specifically, many memorial phenomena are characterized by chemical memory reactions that may fire under particular system conditions. These conditional chemical reactions contradict to the extant stochastic approaches for modeling chemical kinetics and have increasingly posed significant challenges to mathematical modeling and computer simulation. To tackle the challenge, I proposed a novel theory consisting of the memory chemical master equations and memory stochastic simulation algorithm. A stochastic model for single-gene expression was proposed to illustrate the key function of memory reactions in inducing bursting dynamics of gene expression that has been observed in experiments recently. The importance of memory reactions has been further validated by the stochastic model of the p53-MDM2 core module. Simulations showed that memory reactions is a major mechanism for realizing both sustained oscillations of p53 protein numbers in single cells and damped oscillations over a population of cells. These successful applications of the memory modeling framework suggested that this innovative theory is an effective and powerful tool to study memory process and conditional chemical reactions in a wide range of complex biological systems.

Redeployment of germ layers related TFs shows regionalized expression during two non-embryonic developments.

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Ricci, Lorenzo; Cabrera, Fabien; Lotito, Sonia; Tiozzo, Stefano

2016-08-01

In all non-vertebrate metazoan phyla, species that evolved non-embryonic developmental pathways as means of propagation or regeneration can be found. In this context, new bodies arise through asexual reproduction processes (such as budding) or whole body regeneration, that lack the familiar temporal and spatial cues classically associated with embryogenesis, like maternal determinants, or gastrulation. The molecular mechanisms underlying those non-embryonic developments (i.e., regeneration and asexual reproduction), and their relationship to those deployed during embryogenesis are poorly understood. We have addressed this question in the colonial ascidian Botryllus schlosseri, which undergoes an asexual reproductive process via palleal budding (PB), as well as a whole body regeneration by vascular budding (VB). We identified early regenerative structures during VB and then followed the fate of differentiating tissues during both non-embryonic developments (PB and VB) by monitoring the expression of genes known to play key functions in germ layer specification with well conserved expression patterns in solitary ascidian embryogenesis. The expression patterns of FoxA1, GATAa, GATAb, Otx, Bra, Gsc and Tbx2/3 were analysed during both PB and VB. We found that the majority of these transcription factors were expressed during both non-embryonic developmental processes, revealing a regionalization of the palleal and vascular buds. Knockdown of GATAa by siRNA in palleal buds confirmed that preventing the correct development of one of these regions blocks further tissue specification. Our results indicate that during both normal and injury-induced budding, a similar alternative developmental program operates via early commitment of epithelial regions. Copyright © 2016. Published by Elsevier Inc.

Oxidative stress suppression by luteolin-induced heme oxygenase-1 expression

International Nuclear Information System (INIS)

Sun, Gui-bo; Sun, Xiao; Wang, Min; Ye, Jing-xue; Si, Jian-yong; Xu, Hui-bo; Meng, Xiang-bao; Qin, Meng; Sun, Jing; Wang, Hong-wei; Sun, Xiao-bo

2012-01-01

Luteolin, a flavonoid that exhibits antioxidative properties, exerts myocardial protection effects. However, the underlying molecular mechanisms are not yet fully understood. To investigate the effects of luteolin on myocardial injury protection and its possible mechanisms, a myocardial injury model was established with intragastric administration of 4 mg/kg isoproterenol (ISO) to male Sprague–Dawley rats (200–220 g) daily for 2 days. We found that pretreatment of luteolin (160, 80 and 40 mg/kg, i.g., respectively) daily for 15 days can prevent ISO-induced myocardial damage, including decrease of serum cardiac enzymes, improvement electrocardiography and heart vacuolation. Luteolin also improved the free radical scavenging and antioxidant potential, suggesting one possible mechanism of luteolin-induced cardio-protection is mediated by blocking the oxidative stress. To clarify the mechanisms, we performed the in vitro study by hydrogen peroxide (H 2 O 2 )-induced cytotoxicty model in H9c2 cells. We found that luteolin pretreatment prevented apoptosis, increased the expression of heme oxygenase-1 (HO-1), and enhanced the binding of Nrf2 to the antioxidant response element, providing an adaptive survival response against H 2 O 2 -derived oxidative cytotoxicity. The addition of Znpp, a selective HO-1 competitive inhibitor, reduced the cytoprotective ability of luteolin, indicating the vital role of HO-1 on these effects. Luteolin also activated Akt and ERK, whereas the addition of LY294002 and U0126, the pharmacologic inhibitors of PI3K and ERK, attenuated luteolin-induced HO-1 expression and cytoprotective effect. Taken together, the above findings suggest that luteolin protects against myocardial injury and enhances cellular antioxidant defense capacity through the activation of Akt and ERK signal pathways that leads to Nrf2 activation, and subsequently HO-1 induction. -- Highlights: ► Luteolin prevents isoproterenol-induced myocardial damage. ► Luteolin

Oxidative stress suppression by luteolin-induced heme oxygenase-1 expression

Energy Technology Data Exchange (ETDEWEB)

Sun, Gui-bo; Sun, Xiao; Wang, Min [Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100193 (China); Ye, Jing-xue [Jilin Agricultural University, No.2888, Xincheng Street, Changchun, 130021, Jilin (China); Si, Jian-yong [Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100193 (China); Xu, Hui-bo [Academy of Chinese Medical Sciences of Jilin Province, Gongnongda road 1745, Changchun, 130021, Jiblin (China); Meng, Xiang-bao; Qin, Meng; Sun, Jing [Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100193 (China); Wang, Hong-wei, E-mail: hwang@nju.edu.cn [Center for Translational Medicine and Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing 210093 (China); Sun, Xiao-bo, E-mail: sunsubmit@163.com [Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100193 (China)

2012-12-01

Luteolin, a flavonoid that exhibits antioxidative properties, exerts myocardial protection effects. However, the underlying molecular mechanisms are not yet fully understood. To investigate the effects of luteolin on myocardial injury protection and its possible mechanisms, a myocardial injury model was established with intragastric administration of 4 mg/kg isoproterenol (ISO) to male Sprague–Dawley rats (200–220 g) daily for 2 days. We found that pretreatment of luteolin (160, 80 and 40 mg/kg, i.g., respectively) daily for 15 days can prevent ISO-induced myocardial damage, including decrease of serum cardiac enzymes, improvement electrocardiography and heart vacuolation. Luteolin also improved the free radical scavenging and antioxidant potential, suggesting one possible mechanism of luteolin-induced cardio-protection is mediated by blocking the oxidative stress. To clarify the mechanisms, we performed the in vitro study by hydrogen peroxide (H{sub 2}O{sub 2})-induced cytotoxicty model in H9c2 cells. We found that luteolin pretreatment prevented apoptosis, increased the expression of heme oxygenase-1 (HO-1), and enhanced the binding of Nrf2 to the antioxidant response element, providing an adaptive survival response against H{sub 2}O{sub 2}-derived oxidative cytotoxicity. The addition of Znpp, a selective HO-1 competitive inhibitor, reduced the cytoprotective ability of luteolin, indicating the vital role of HO-1 on these effects. Luteolin also activated Akt and ERK, whereas the addition of LY294002 and U0126, the pharmacologic inhibitors of PI3K and ERK, attenuated luteolin-induced HO-1 expression and cytoprotective effect. Taken together, the above findings suggest that luteolin protects against myocardial injury and enhances cellular antioxidant defense capacity through the activation of Akt and ERK signal pathways that leads to Nrf2 activation, and subsequently HO-1 induction. -- Highlights: ► Luteolin prevents isoproterenol-induced myocardial damage. �

1,25-Dihydroxyvitamin-D3 Induces Avian β-Defensin Gene Expression in Chickens.

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Long Zhang

Full Text Available Host defense peptides (HDPs play a critical role in innate immunity. Specific modulation of endogenous HDP synthesis by dietary compounds has been regarded as a novel approach to boost immunity and disease resistance in animal production. 1,25-dihydroxy vitamin D3 (1,25D3 is well known as a powerful HDP inducer in humans, but limited information about the effect of 1,25D3 on HDPs in poultry is available. Here, we sought to examine whether 1,25D3 could stimulate avian β-defensin (AvBD expression in chickens. We used chicken embryo intestinal epithelial cells (CEIEPCs and peripheral blood mononuclear cells (PBMCs to study the effect of 1,25D3 on the expression of AvBDs. We observed that 1,25D3 is able to up-regulate the expression of several AvBDs in CEIEPCs and PBMCs, whereas it increased the amounts of AvBD4 mRNA in CEIEPCs only in the presence of lipopolysaccharide (LPS. On the other hand, LPS treatment not only inhibited the expression of CYP24A1 but also altered the expression pattern of VDR in CEIEPCs. Furthermore, AvBDs were not directly regulated by 1,25D3, as cycloheximide completely blocked 1,25D3-induced expression of AvBDs. Our observations suggest that 1,25D3 is capable of inducing AvBD gene expression and is a potential antibiotic alternative through augmentation of host innate immunity as well as disease control in chickens.

Decreased inducibility of TNF expression in lipid-loaded macrophages

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Kallin Bengt

2002-10-01

Full Text Available Abstract Background Inflammation and immune responses are considered to be very important in the pathogenesis of atherosclerosis. Lipid accumulation in macrophages of the arterial intima is a characteristic feature of atherosclerosis which can influence the inflammatory potential of macrophages. We studied the effects of lipid loading on the regulation of TNF expression in human monocyte-derived macrophages. Results In macrophages incubated with acetylated low density lipoprotein (ac-LDL for 2 days, mRNA expression of TNF in cells stimulated with TNF decreased by 75%. In cell cultures stimulated over night with IL-1β, lipid loading decreased secretion of TNF into culture medium by 48%. These results suggest that lipid accumulation in macrophages makes them less responsive to inflammatory stimuli. Decreased basal activity and inducibility of transcription factor AP-1 was observed in lipid-loaded cells, suggesting a mechanism for the suppression of cytokine expression. NF-κB binding activity and inducibility were only marginally affected by ac-LDL. LDL and ac-LDL did not activate PPARγ. In contrast, oxidized LDL stimulated AP-1 and PPARγ but inhibited NF-κB, indicating that the effects of lipid loading with ac-LDL were not due to oxidation of lipids. Conclusions Accumulation of lipid, mainly cholesterol, results in down-regulation of TNF expression in macrophages. Since monocytes are known to be activated by cell adhesion, these results suggest that foam cells in atherosclerotic plaques may contribute less potently to an inflammatory reaction than newly arrived monocytes/macrophages.

Effect of enhanced expression of connexin 43 on sunitinib-induced cytotoxicity in mesothelioma cells

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Miaki Uzu

2015-05-01

Full Text Available Connexin (Cx makes up a type of intercellular channel called gap junction (GJ. GJ plays a regulatory role in cellular physiology. The Cx expression level is often decreased in cancer cells compared to that in healthy ones, and the restoration of its expression has been shown to exert antiproliferative effects. This work aims to evaluate the effect of the restoration of connexin 43 (Cx43 (the most ubiquitous Cx subtype expression on sunitinib (SU-induced cytotoxicity in malignant mesothelioma (MM cells. Increased Cx43 expression in an MM cell line (H28 improved the ability of SU to inhibit receptor tyrosine kinase (RTK signaling. Moreover, higher Cx43 expression promoted SU-induced apoptosis. The cell viability test revealed that Cx43 enhanced the cytotoxic effect of SU in a GJ-independent manner. The effect of Cx43 on a proapoptotic factor, Bax, was then investigated. The interaction between Cx43 and Bax was confirmed by immunoprecipitation. Furthermore, higher Cx43 expression increased the production of a cleaved (active form of Bax during SU-induced apoptosis with no alteration in total Bax expression. These findings indicate that Cx43 most likely increases sensitivity to SU in H28 through direct interaction with Bax. In conclusion, we found that Cx43 overcame the chemoresistance of MM cells.

Effects of lactose as an inducer on expression of Helicobacter pylori rUreB and rHpaA,and Escherichia colirLTKA63 and rLTB

Institute of Scientific and Technical Information of China (English)

Jie Yan; Shou-Feng Zhao; Ya-Fei Mao; Yi-Hui Luo

2004-01-01

AIM: To demonstrate the effect of lactose as an inducer on expression of the recombinant proteins encoded by Helicobacter pylori ureB and hpaA, and Escherichia coli LTB and LTKA63 genes and to determine the optimal expression parameters.METHODS: By using SDS-PAGE and BIO-RAD gel image analysis system, the outputs of the target recombinant proteins expressed by pET32a-ureB-E. coliBL21, pET32ahpaA-E. coliBL21, pET32a-L TKA63-E. coliBL21 and pET32aLTB-E.coliBL21 were measured when using lactose as inducer at different dosages, original bacterial concentrations,various inducing temperatures and times. The results of the target protein expression induced by lactose were compared to those by isopropyl-β-D-thiogalactoside (IPTG).The proteins were expressed in E.coli.RESULTS: Lactose showed higher efficiency of inducing the expression of rHpaA, rUreB, rLTB and rLTKA63 than IPTG. The expression outputs of the target recombinant proteins induced at 37 ℃ were remarkably higher than those at 28 ℃. Other optimal expression parameters for the original bacterial concentrations, dosages of lactose and inducing time were 0.8, 50 g/L and 4 h for rHpaA; 0.8, 100 g/L and 4 h for rLTKA63; 1.2, 100 g/Land 5 h for both rUreB and rLTB, respectively.CONCLUSION: Lactose, a sugar with non-toxicity and low cost, is able to induce the recombinant genes to express the target proteins with higher efficiency than IPTG. The results in this study establish a beneficial foundation for industrial production of H pylori genetic engineering vaccine.

Minocycline attenuates experimental colitis in mice by blocking expression of inducible nitric oxide synthase and matrix metalloproteinases

International Nuclear Information System (INIS)

Huang, T.-Y.; Chu, H.-C.; Lin, Y.-L.; Lin, C.-K.; Hsieh, T.-Y.; Chang, W.-K.; Chao, Y.-C.; Liao, C.-L.

2009-01-01

In addition to its antimicrobial activity, minocycline exerts anti-inflammatory effects in several disease models. However, whether minocycline affects the pathogenesis of inflammatory bowel disease has not been determined. We investigated the effects of minocycline on experimental colitis and its underlying mechanisms. Acute and chronic colitis were induced in mice by treatment with dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid (TNBS), and the effect of minocycline on colonic injury was assessed clinically and histologically. Prophylactic and therapeutic treatment of mice with minocycline significantly diminished mortality rate and attenuated the severity of DSS-induced acute colitis. Mechanistically, minocycline administration suppressed inducible nitric oxide synthase (iNOS) expression and nitrotyrosine production, inhibited proinflammatory cytokine expression, repressed the elevated mRNA expression of matrix metalloproteinases (MMPs) 2, 3, 9, and 13, diminished the apoptotic index in colonic tissues, and inhibited nitric oxide production in the serum of mice with DSS-induced acute colitis. In DSS-induced chronic colitis, minocycline treatment also reduced body weight loss, improved colonic histology, and blocked expression of iNOS, proinflammatory cytokines, and MMPs from colonic tissues. Similarly, minocycline could ameliorate the severity of TNBS-induced acute colitis in mice by decreasing mortality rate and inhibiting proinflammatory cytokine expression in colonic tissues. These results demonstrate that minocycline protects mice against DSS- and TNBS-induced colitis, probably via inhibition of iNOS and MMP expression in intestinal tissues. Therefore, minocycline is a potential remedy for human inflammatory bowel diseases.

Insulin receptor substrate 1 expression enhances the sensitivity of 32D cells to chemotherapy-induced cell death

International Nuclear Information System (INIS)

Porter, Holly A.; Carey, Gregory B.; Keegan, Achsah D.

2012-01-01

The adapters IRS1 and IRS2 link growth factor receptors to downstream signaling pathways that regulate proliferation and survival. Both suppress factor-withdrawal-induced apoptosis and have been implicated in cancer progression. However, recent studies suggest IRS1 and IRS2 mediate differential functions in cancer pathogenesis. IRS1 promoted breast cancer proliferation, while IRS2 promoted metastasis. The role of IRS1 and IRS2 in controlling cell responses to chemotherapy is unknown. To determine the role of IRS1 and IRS2 in the sensitivity of cells to chemotherapy, we treated 32D cells lacking or expressing IRS proteins with various concentrations of chemotherapeutic agents. We found that expression of IRS1, in contrast to IRS2, enhanced the sensitivity of 32D cells to chemotherapy-induced apoptosis. When IRS2 was expressed with IRS1, the cells no longer showed enhanced sensitivity. Expression of IRS1 did not alter the expression of pro- and anti-apoptotic proteins; however, 32D-IRS1 cells expressed higher levels of Annexin A2. In 32D-IRS1 cells, IRS1 and Annexin A2 were both located in cytoplasmic and membrane fractions. We also found that IRS1 coprecipitated with Annexin A2, while IRS2 did not. Decreasing Annexin A2 levels reduced 32D-IRS1 cell sensitivity to chemotherapy. These results suggest IRS1 enhances sensitivity to chemotherapy in part through Annexin A2. -- Highlights: ► IRS1 enhanced the sensitivity of 32D cells to chemotherapy-induced apoptosis. ► This sensitivity is abrogated by the expression of IRS2. ► Expressing IRS1 in 32D cells increased levels of Annexin A2. ► Both IRS1 and Annexin A2 were located in cytoplasmic and membrane fractions. ► Decreasing Annexin A2 in 32D-IRS1 cells abated their sensitivity to chemotherapy.

LY294002 inhibits glucocorticoid-induced COX-2 gene expression in cardiomyocytes through a phosphatidylinositol 3 kinase-independent mechanism

International Nuclear Information System (INIS)

Sun Haipeng; Xu Beibei; Sheveleva, Elena; Chen, Qin M.

2008-01-01

Glucocorticoids induce COX-2 expression in rat cardiomyocytes. While investigating whether phosphatidylinositol 3 kinase (PI3K) plays a role in corticosterone (CT)-induced COX-2, we found that LY294002 (LY29) but not wortmannin (WM) attenuates CT from inducing COX-2 gene expression. Expression of a dominant-negative mutant of p85 subunit of PI3K failed to inhibit CT from inducing COX-2 expression. CT did not activate PI3K/AKT signaling pathway whereas LY29 and WM decreased the activity of PI3K. LY303511 (LY30), a structural analogue and a negative control for PI3K inhibitory activity of LY29, also suppressed COX-2 induction. These data suggest PI3K-independent mechanisms in regulating CT-induced COX-2 expression. LY29 and LY30 do not inhibit glucocorticoid receptor transactivity. Both compounds have been reported to inhibit Casein Kinase 2 activity and modulate potassium and calcium levels independent of PI3K, while LY29 has been reported to inhibit mammalian Target of Rapamycin (mTOR), and DNA-dependent Protein Kinase (DNA-PK). Inhibitor of Casein Kinase 2 (CK2), mTOR or DNA-PK failed to prevent CT from inducing COX-2 expression. Tetraethylammonium (TEA), a potassium channel blocker, and nimodipine, a calcium channel blocker, both attenuated CT from inducing COX-2 gene expression. CT was found to increase intracellular Ca 2+ concentration, which can be inhibited by LY29, TEA or nimodipine. These data suggest a possible role of calcium instead of PI3K in CT-induced COX-2 expression in cardiomyocytes

VCC-1 over-expression inhibits cisplatin-induced apoptosis in HepG2 cells

International Nuclear Information System (INIS)

Zhou, Zhitao; Lu, Xiao; Zhu, Ping; Zhu, Wei; Mu, Xia; Qu, Rongmei; Li, Ming

2012-01-01

Highlights: ► VCC-1 is hypothesized to be associated with carcinogenesis. ► Levels of VCC-1 are increased significantly in HCC. ► Over-expression of VCC-1 could promotes cellular proliferation rate. ► Over-expression of VCC-1 inhibit the cisplatin-provoked apoptosis in HepG2 cells. ► VCC-1 plays an important role in control the tumor growth and apoptosis. -- Abstract: Vascular endothelial growth factor-correlated chemokine 1 (VCC-1), a recently described chemokine, is hypothesized to be associated with carcinogenesis. However, the molecular mechanisms by which aberrant VCC-1 expression determines poor outcomes of cancers are unknown. In this study, we found that VCC-1 was highly expressed in hepatocellular carcinoma (HCC) tissue. It was also associated with proliferation of HepG2 cells, and inhibition of cisplatin-induced apoptosis of HepG2 cells. Conversely, down-regulation of VCC-1 in HepG2 cells increased cisplatin-induced apoptosis of HepG2 cells. In summary, these results suggest that VCC-1 is involved in cisplatin-induced apoptosis of HepG2 cells, and also provides some evidence for VCC-1 as a potential cellular target for chemotherapy.

Transcript-specific effects of adrenalectomy on seizure-induced BDNF expression in rat hippocampus

DEFF Research Database (Denmark)

Lauterborn, J C; Poulsen, F R; Stinis, C T

1998-01-01

Activity-induced brain-derived neurotrophic factor (BDNF) expression is negatively modulated by circulating adrenal steroids. The rat BDNF gene gives rise to four major transcript forms that each contain a unique 5' exon (I-IV) and a common 3' exon (V) that codes for BDNF protein. Exon-specific i......Activity-induced brain-derived neurotrophic factor (BDNF) expression is negatively modulated by circulating adrenal steroids. The rat BDNF gene gives rise to four major transcript forms that each contain a unique 5' exon (I-IV) and a common 3' exon (V) that codes for BDNF protein. Exon...... and in exon II-containing mRNA with 30-days survival. In the dentate gyrus granule cells, adrenalectomy markedly potentiated increases in exon I and II cRNA labeling, but not increases in exon III and IV cRNA labeling, elicited by one hippocampal afterdischarge. Similarly, for the granule cells and CA1...... no effect on exon IV-containing mRNA content. These results demonstrate that the negative effects of adrenal hormones on activity-induced BDNF expression are by far the greatest for transcripts containing exons I and II. Together with evidence for region-specific transcript expression, these results suggest...

Fisetin Induces Apoptosis Through p53-Mediated Up-Regulation of DR5 Expression in Human Renal Carcinoma Caki Cells

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Kyoung-jin Min

2017-08-01

Full Text Available Fisetin is a natural compound found in fruits and vegetables such as strawberries, apples, cucumbers, and onions. Since fisetin can elicit anti-cancer effects, including anti-proliferation and anti-migration, we investigated whether fisetin induced apoptosis in human renal carcinoma (Caki cells. Fisetin markedly induced sub-G1 population and cleavage of poly (ADP-ribose polymerase (PARP, which is a marker of apoptosis, and increased caspase activation. We found that pan-caspase inhibitor (z-VAD-fmk inhibited fisetin-induced apoptosis. In addition, fisetin induced death receptor 5 (DR5 expression at the transcriptional level, and down-regulation of DR5 by siRNA blocked fisetin-induced apoptosis. Furthermore, fisetin induced p53 protein expression through up-regulation of protein stability, whereas down-regulation of p53 by siRNA markedly inhibited fisetin-induced DR5 expression. In contrast, fisetin induced up-regulation of CHOP expression and reactive oxygen species production, which had no effect on fisetin-induced apoptosis. Taken together, our study demonstrates that fisetin induced apoptosis through p53 mediated up-regulation of DR5 expression at the transcriptional level.

Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression

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Makino, Yuichi; Cao, Renhai; Svensson, Kristian; Bertilsson, Göran; Asman, Mikael; Tanaka, Hirotoshi; Cao, Yihai; Berkenstam, Anders; Poellinger, Lorenz

2001-11-01

Alteration of gene expression is a crucial component of adaptive responses to hypoxia. These responses are mediated by hypoxia-inducible transcription factors (HIFs). Here we describe an inhibitory PAS (Per/Arnt/Sim) domain protein, IPAS, which is a basic helix-loop-helix (bHLH)/PAS protein structurally related to HIFs. IPAS contains no endogenous transactivation function but demonstrates dominant negative regulation of HIF-mediated control of gene expression. Ectopic expression of IPAS in hepatoma cells selectively impairs induction of genes involved in adaptation to a hypoxic environment, notably the vascular endothelial growth factor (VEGF) gene, and results in retarded tumour growth and tumour vascular density in vivo. In mice, IPAS was predominantly expressed in Purkinje cells of the cerebellum and in corneal epithelium of the eye. Expression of IPAS in the cornea correlates with low levels of expression of the VEGF gene under hypoxic conditions. Application of an IPAS antisense oligonucleotide to the mouse cornea induced angiogenesis under normal oxygen conditions, and demonstrated hypoxia-dependent induction of VEGF gene expression in hypoxic corneal cells. These results indicate a previously unknown mechanism for negative regulation of angiogenesis and maintenance of an avascular phenotype.

Differential gene expression of two extreme honey bee (Apis mellifera) colonies showing varroa tolerance and susceptibility.

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Jiang, S; Robertson, T; Mostajeran, M; Robertson, A J; Qiu, X

2016-06-01

Varroa destructor, an ectoparasitic mite of honey bees (Apis mellifera), is the most serious pest threatening the apiculture industry. In our honey bee breeding programme, two honey bee colonies showing extreme phenotypes for varroa tolerance/resistance (S88) and susceptibility (G4) were identified by natural selection from a large gene pool over a 6-year period. To investigate potential defence mechanisms for honey bee tolerance to varroa infestation, we employed DNA microarray and real time quantitative (PCR) analyses to identify differentially expressed genes in the tolerant and susceptible colonies at pupa and adult stages. Our results showed that more differentially expressed genes were identified in the tolerant bees than in bees from the susceptible colony, indicating that the tolerant colony showed an increased genetic capacity to respond to varroa mite infestation. In both colonies, there were more differentially expressed genes identified at the pupa stage than at the adult stage, indicating that pupa bees are more responsive to varroa infestation than adult bees. Genes showing differential expression in the colony phenotypes were categorized into several groups based on their molecular functions, such as olfactory signalling, detoxification processes, exoskeleton formation, protein degradation and long-chain fatty acid metabolism, suggesting that these biological processes play roles in conferring varroa tolerance to naturally selected colonies. Identification of differentially expressed genes between the two colony phenotypes provides potential molecular markers for selecting and breeding varroa-tolerant honey bees. © 2016 The Royal Entomological Society.

Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells

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Susilowati, Heni [Department of Oral Microbiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8504 (Japan); Okamura, Hirohiko [Department of Histology and Oral Histology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8504 (Japan); Hirota, Katsuhiko, E-mail: hirota@dent.tokushima-u.ac.jp [Department of Oral Microbiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8504 (Japan); Shono, Masayuki [Support Center for Advanced Medical Sciences, The University of Tokushima, Tokushima 770-8504 (Japan); Yoshida, Kaya [Department of Fundamental Oral Health Science, School of Oral Health and Welfare, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8504 (Japan); Murakami, Keiji [Department of Oral Microbiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8504 (Japan); Tabata, Atsushi; Nagamune, Hideaki [Department of Biological Science and Technology, Life System, Institute of Technology and Science, The University of Tokushima Graduate School, Tokushima 770-8506 (Japan); Haneji, Tatsuji [Department of Histology and Oral Histology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8504 (Japan); Miyake, Yoichiro [Department of Oral Microbiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8504 (Japan)

2011-01-07

Research highlights: {yields} ILY leads to the accumulation of [Ca{sup 2+}]i in the nucleus in HuCCT1 cells. {yields} ILY induced activation of NFAT1 through a calcineurin-dependent pathway. {yields} Calcineuri/NFAT pathway is involved in EGR-1 expression in response to ILY treatment. -- Abstract: Intermedilysin (ILY) is a cholesterol-dependent cytolysin produced by Streptococcus intermedius, which is associated with human brain and liver abscesses. Although intrahepatic bile duct cells play a valuable role in the pathogenesis of liver abscess, the molecular mechanism of ILY-treated intrahepatic bile duct cells remains unknown. In this study, we report that ILY induced a nuclear accumulation of intracellular calcium ([Ca{sup 2+}]i) in human cholangiocellular cells HuCCT1. We also demonstrate that 10 ng/ml ILY induced NFAT1 dephosphorylation and its nuclear translocation in HuCCT1 cells. In contrast to the result that ILY induced NF-{kappa}B translocation in human hepatic HepG2 cells, ILY did not affect NF-{kappa}B localization in HuCCT1 cells. Dephosphorylation and nuclear translocation of NFAT1 caused by ILY were prevented by [Ca{sup 2+}]i calcium chelator, BAPTA/AM, and calcineurin inhibitors, cyclosporine A and tacrolimus. ILY induced early growth response-1 (EGR-1) expression and it was inhibited by the pre-treatment with cyclosporine A, indicating that the calcineurin/NFAT pathway was involved in EGR-1 expression in response to ILY. ILY-induced calcineurin/NFAT1 activation and sequential EGR-1 expression might be related to the pathogenesis of S. intermedius in human bile duct cells.

Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells

International Nuclear Information System (INIS)

Susilowati, Heni; Okamura, Hirohiko; Hirota, Katsuhiko; Shono, Masayuki; Yoshida, Kaya; Murakami, Keiji; Tabata, Atsushi; Nagamune, Hideaki; Haneji, Tatsuji; Miyake, Yoichiro

2011-01-01

Research highlights: → ILY leads to the accumulation of [Ca 2+ ]i in the nucleus in HuCCT1 cells. → ILY induced activation of NFAT1 through a calcineurin-dependent pathway. → Calcineuri/NFAT pathway is involved in EGR-1 expression in response to ILY treatment. -- Abstract: Intermedilysin (ILY) is a cholesterol-dependent cytolysin produced by Streptococcus intermedius, which is associated with human brain and liver abscesses. Although intrahepatic bile duct cells play a valuable role in the pathogenesis of liver abscess, the molecular mechanism of ILY-treated intrahepatic bile duct cells remains unknown. In this study, we report that ILY induced a nuclear accumulation of intracellular calcium ([Ca 2+ ]i) in human cholangiocellular cells HuCCT1. We also demonstrate that 10 ng/ml ILY induced NFAT1 dephosphorylation and its nuclear translocation in HuCCT1 cells. In contrast to the result that ILY induced NF-κB translocation in human hepatic HepG2 cells, ILY did not affect NF-κB localization in HuCCT1 cells. Dephosphorylation and nuclear translocation of NFAT1 caused by ILY were prevented by [Ca 2+ ]i calcium chelator, BAPTA/AM, and calcineurin inhibitors, cyclosporine A and tacrolimus. ILY induced early growth response-1 (EGR-1) expression and it was inhibited by the pre-treatment with cyclosporine A, indicating that the calcineurin/NFAT pathway was involved in EGR-1 expression in response to ILY. ILY-induced calcineurin/NFAT1 activation and sequential EGR-1 expression might be related to the pathogenesis of S. intermedius in human bile duct cells.

Tamoxifen induces the expression of maspin through estrogen receptor-alpha.

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Liu, Zesheng; Shi, Heidi Y; Nawaz, Zafar; Zhang, Ming

2004-06-08

Maspin (mammary serine protease inhibitor) is a tumor suppressor gene that plays an important role in inhibiting tumor growth, invasion and metastasis. Maspin expression is down regulated at transcription level in primary and metastatic breast tumor cells. Previous studies on hormonal regulation of maspin prompt us to test whether an estrogen antagonist tamoxifen (TAM) can exert its anti-tumor function by up regulating maspin gene expression. For this purpose, we first tested whether maspin promoter could be activated in normal and several breast tumor cells. We then carried out a series of promoter analysis in which estrogen receptors and TAM were reconstituted in an in vitro cell culture system. Here we report our new finding that tumor suppresser gene maspin is one of the TAM target genes. TAM induces a maspin/luciferase reporter in cell culture and this induction requires the presence of (estrogen receptor alpha) ERalpha but not estrogen receptor-beta (ERbeta). Maspin promoter deletion and mutation analysis showed that the cis element(s) within a region between -90and+87 bp but not the HRE site (-272 bp) was involved in TAM induction of maspin expression. TAM bound ERalpha may directly control maspin gene expression through the interaction with cofactor (s). Analysis using several ERalpha mutants showed that the N-terminal A/B motif (AF-1) was critical for maspin basal level transcription activation. An ERalpha mutant with point mutations at DNA binding domain abolished estrogen induction of an ERE-luciferase reporter but was still active in activating maspin promoter by TAM. LBD-AF2 domain was required for ERalpha-dependent TAM induction. Deletion of LBD-AF2 or a point mutation in the ERalpha LBD-AF2 region (LBDmtL539A) completely abolished the activation of maspin promoter, suggesting that TAM induction of maspin involves the recruitment of cofactor(s) by ERalpha to the maspin promoter region. This finding indicates that one of the pathways for cancer

[Gene Expression Profile of Apoptosis in Leukemia Cells Induced by Hsp90 Selective inhibitor 17-AAG].

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Wang, Na-Na; Li, Zhi-Heng; Tao, Yan-Fang; Xu, Li-Xiao; Pan, Jian; Hu, Shao-Yan

2016-06-01

To investigate the apoptotic effects of Hsp90 selective inhibitor 17-AAG on human leukemia HL-60 and NB4 cells and analyse its possible mechanism. CCK-8 assay was used to quantify the growth inhibition of cells after exposure to 17-AAG for 24 hours. Flow cytometrve with annexin V/propidium iodide staining was used to detect apoptosis of leukemia cells. Then Western blot was used to detect the activation of apoptosis related protein caspase-3 and PARP level. Gene expression profile of NB4 cells treated with 17-AAG was analyzed with real-time PCR arrays. The inhibition of leukemia cell proliferation displayed a dose-dependent manner. Annexin V assay, cell cycle analysis and activation of PARP demonstrate that 17-AAG induced apoptosis leukemia cells. Real-time PCR array analysis showed that expression of 56 genes significantly up-regulated and expression of 23 genes were significantly down-regulated after 17-AAG treatment. The 17-AAG can inhibit the proliferation and induce the apoptosis of leukemia cells. After leukemia cells are treated with 17-AAG, the significant changes of apoptosis-related genes occured, and the cell apoptosis occurs via activating apoptosis related signaling pathway.

Expression of Peroxisome Proferator-Activated Receptor γ (PPARγ in Human Transitional Bladder Cancer and its Role in Inducing Cell Death

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You-Fei Guan

1999-10-01

Full Text Available The present study examined the expression and role of the thiazolidinedione (TZD-activated transcription factor, peroxisome proliferator-activated receptor γ (PPARγ, in human bladder cancers. In situ hybridization shows that PPARγ mRNA is highly expressed in all human transitional epithelial cell cancers (TCCa's studied (n=11. PPARγ was also expressed in five TCCa cell lines as determined by RNase protection assays and immunoblot. Retinoid X receptor α (RXRα, a 9-cis-retinoic acid stimulated (9-cis-RA heterodimeric partner of PPARγ, was also co-expressed in all TCCa tissues and cell lines. Treatment of the T24 bladder cancer cells with the TZD PPARγ agonist troglitazone, dramatically inhibited 3H-thymidine incorporation and induced cell death. Addition of the RXRα ligands, 9-cis-RA or LG100268, sensitized T24 bladder cancer cells to the lethal effect of troglitazone and two other PPARγ activators, ciglitazone and 15-deoxy-Δ12,14-PGJ2 (15dPGJ2. Troglitazone treatment increased expression of two cyclin-dependent kinase inhibitors, p21wAF1/CIP1 and p16INK4, reduced cyclin D1 expression, consistent with G1 arrest. Troglitazone also induced an endogenous PPARγ target gene in T24 cells, adipocyte-type fatty acid binding protein (A-FABP, the expression of which correlates with bladder cancer differentiation. In situ hybridization shows that A-FABP expression is localized to normal uroepithelial cells as well as some TCCa's. Taken together, these results demonstrate that PPARγ is expressed in human TCCa where it may play a role in regulating TCCa differentiation and survival, thereby providing a potential target for therapy of uroepithelial cancers.

Monocrotophos Induces the Expression of Xenobiotic Metabolizing Cytochrome P450s (CYP2C8 and CYP3A4) and Neurotoxicity in Human Brain Cells.

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Tripathi, Vinay Kumar; Kumar, Vivek; Pandey, Ankita; Vatsa, Pankhi; Dhasmana, Anupam; Singh, Rajat Pratap; Appikonda, Sri Hari Chandan; Hwang, Inho; Lohani, Mohtashim

2017-07-01

Expression of various cytochrome P450s (CYPs) in mammalian brain cells is well documented. However, such studies are hampered in neural/glial cells of human origin due to nonavailability of human brain cells. To address this issue, we investigated the expression and inducibility of CYP2C8 and CYP3A4 and their responsiveness against cyclophosphamide (CPA) and organophosphorus pesticide monocrotophos (MCP), a known developmental neurotoxicant in human neural (SH-SY5Y) and glial (U373-MG) cell lines. CPA induced significant expression of CYP2C8 and CYP3A4 in both types of cells in a time-dependent manner. Neural cell line exhibited relatively higher constitutive and inducible expression of CYPs than the glial cell line. MCP exposure alone could not induce the significant expression of CYPs, whereas the cells preexposed to CPA showed a significant response to MCP. Similar to the case of CPA induced expressions, neural cells were found to be more vulnerable than glial cells. Our data indicate differential expressions of CYPs in cultured human neural and glial cell lines. The findings were synchronized with protein ligand docking studies, which showed a significant modulatory capacity of MCP by strong interaction with CYP regulators-CAR and PXR. Similarly, the known CYP inducer CPA has also shown significant high docking scores with the two studied CYP regulators. We also observed a significant induction in reactive oxygen species (ROS), lipid peroxides (LPO), micronucleus (MN), chromosomal aberration (CA), and reduction in reduced glutathione (GSH) and catalase following the exposure of MCP. Moreover, the expressions of apoptotic markers such as caspase-3, caspase-9, Bax, and p53 were significantly upregulated, whereas the levels of antiapoptotic marker, Bcl2, was downregulated after the exposure of MCP in both cell lines. These findings confirm the involvement of ROS-mediated oxidative stress, which subsequently triggers apoptosis pathways in both human neural (SH-SY5Y

Low-dose Norfloxacin-treated leptospires induce less IL-1β release in J774A.1 cells following discrepant leptospiral gene expression.

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Cao, Yongguo; Xie, Xufeng; Zhang, Wenlong; Wu, Dianjun; Tu, Changchun

2018-06-01

Currently, accumulating evidence is challenging subtherapeutic therapy. Low-dose Norfloxacin (Nor) has been reported to suppress the immune response and worsen leptospirosis. In this study, we investigated the influence of low-dose Nor (0.03 μg/ml, 0.06 μg/ml, 0.125 μg/ml) on leptospiral gene expression and analyzed the immunomodulatory effects of low-dose Nor-treated leptospires in J774A.1 cells. To study the expression profiles of low-dose Nor-treated leptospires, we chose LipL71/LipL21 as reference genes determined by the geNorm applet in this experiment. The results showed that low-dose Nor up-regulated the expression of FlaB and inhibited the expression of 16S rRNA, LipL32, LipL41, Loa22, KdpA, and KdpB compared with the untreated leptospires. These results indicated that low-dose Nor could regulate leptospiral gene expression. Using RT-PCR, the gene expression of IL-1β and TNF-α in J774A.1 cells was detected. Nor-treated leptospires induced higher expression levels of both IL-1β and TNF-α. However, when analyzed by ELISA, the release of mature IL-1β was reduced compared with that observed in cells induced with no Nor-treated leptospires, although the TNF-α protein level showed no significant change. Our study indicated that the gene expression of leptospires could be modulated by low-dose Nor, which induced less IL-1β release in J774A.1 cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

A robust approach to identifying tissue-specific gene expression regulatory variants using personalized human induced pluripotent stem cells.

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Je-Hyuk Lee

2009-11-01

Full Text Available Normal variation in gene expression due to regulatory polymorphisms is often masked by biological and experimental noise. In addition, some regulatory polymorphisms may become apparent only in specific tissues. We derived human induced pluripotent stem (iPS cells from adult skin primary fibroblasts and attempted to detect tissue-specific cis-regulatory variants using in vitro cell differentiation. We used padlock probes and high-throughput sequencing for digital RNA allelotyping and measured allele-specific gene expression in primary fibroblasts, lymphoblastoid cells, iPS cells, and their differentiated derivatives. We show that allele-specific expression is both cell type and genotype-dependent, but the majority of detectable allele-specific expression loci remains consistent despite large changes in the cell type or the experimental condition following iPS reprogramming, except on the X-chromosome. We show that our approach to mapping cis-regulatory variants reduces in vitro experimental noise and reveals additional tissue-specific variants using skin-derived human iPS cells.

Gene expression analysis reveals new possible mechanisms of vancomycin-induced nephrotoxicity and identifies gene markers candidates.

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Dieterich, Christine; Puey, Angela; Lin, Sylvia; Lyn, Sylvia; Swezey, Robert; Furimsky, Anna; Fairchild, David; Mirsalis, Jon C; Ng, Hanna H

2009-01-01

Vancomycin, one of few effective treatments against methicillin-resistant Staphylococcus aureus, is nephrotoxic. The goals of this study were to (1) gain insights into molecular mechanisms of nephrotoxicity at the genomic level, (2) evaluate gene markers of vancomycin-induced kidney injury, and (3) compare gene expression responses after iv and ip administration. Groups of six female BALB/c mice were treated with seven daily iv or ip doses of vancomycin (50, 200, and 400 mg/kg) or saline, and sacrificed on day 8. Clinical chemistry and histopathology demonstrated kidney injury at 400 mg/kg only. Hierarchical clustering analysis revealed that kidney gene expression profiles of all mice treated at 400 mg/kg clustered with those of mice administered 200 mg/kg iv. Transcriptional profiling might thus be more sensitive than current clinical markers for detecting kidney damage, though the profiles can differ with the route of administration. Analysis of transcripts whose expression was changed by at least twofold compared with vehicle saline after high iv and ip doses of vancomycin suggested the possibility of oxidative stress and mitochondrial damage in vancomycin-induced toxicity. In addition, our data showed changes in expression of several transcripts from the complement and inflammatory pathways. Such expression changes were confirmed by relative real-time reverse transcription-polymerase chain reaction. Finally, our results further substantiate the use of gene markers of kidney toxicity such as KIM-1/Havcr1, as indicators of renal injury.

The new pLAI (lux regulon based auto-inducible expression system for recombinant protein production in Escherichia coli

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Nocadello Salvatore

2012-01-01

Full Text Available Abstract Background After many years of intensive research, it is generally assumed that no universal expression system can exist for high-level production of a given recombinant protein. Among the different expression systems, the inducible systems are the most popular for their tight regulation. However, induction is in many cases less favorable due to the high cost and/or toxicity of inducers, incompatibilities with industrial scale-up or detrimental growth conditions. Expression systems using autoinduction (or self-induction prove to be extremely versatile allowing growth and induction of recombinant proteins without the need to monitor cell density or add inducer. Unfortunately, almost all the actual auto inducible expression systems need endogenous or induced metabolic changes during the growth to trigger induction, both frequently linked to detrimental condition to cell growth. In this context, we use a simple modular approach for a cell density-based genetic regulation in order to assemble an autoinducible recombinant protein expression system in E. coli. Result The newly designed pLAI expression system places the expression of recombinant proteins in Escherichia coli under control of the regulatory genes of the lux regulon of Vibrio fischeri's Quorum Sensing (QS system. The pLAI system allows a tight regulation of the recombinant gene allowing a negligible basal expression and expression only at high cell density. Sequence optimization of regulative genes of QS of V. fischeri for expression in E. coli upgraded the system to high level expression. Moreover, partition of regulative genes between the plasmid and the host genome and introduction of a molecular safety lock permitted tighter control of gene expression. Conclusion Coupling gene expression to cell density using cell-to-cell communication provides a promising approach for recombinant protein production. The system allows the control of expression of the target recombinant gene

Modulation of haloperidol-induced patterns of the transcription factor Nur77 and Nor-1 expression by serotonergic and adrenergic drugs in the mouse brain

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Maheux, Jérôme; Vuillier, Laura; Mahfouz, Mylène; Rouillard, Claude; Lévesque, Daniel

2015-01-01

Different patterns of expression of the transcription factors of Nur77 and Nor-1 are induced following acute administration of typical and atypical antipsychotic drugs. The pharmacological profile of atypical antipsychotics suggests that serotonergic and/or adrenergic receptors might contribute to these reported differences. In order to test this possibility, we examined the abilities of serotonin 5-HT1A and 5-HT2A/2C, and α1- and α2-adrenergic receptor drugs to modify the pattern of Nur77 (NR4A1) and Nor-1 (NR4A3) mRNA expression induced by haloperidol. Various groups of mice were treated with either saline, DOI, a 5-HT2A/2C agonist, MDL11939, a 5-HT2A antagonist, 8-OH-DPAT, a 5-HT1A agonist, prazosin, an α1-adrenergic antagonist and idazoxan, an α2-adrenergic antagonist, alone or in combination with haloperidol. The 5-HT2A/2C agonist DOI alone significantly increased Nur77 expression in the medial striatum and nucleus accumbens. DOI reduced Nor-1 expression, while MDL11939 increased the expression of this transcript in the cortex. Prazosin reduced Nur77 expression in the dorsal striatum and nucleus accumbens. Interestingly, 8-OH-DPAT and MDL11939 partially prevented haloperidol-induced Nur77 up-regulation, while MDL11939 completely abolished Nor-1 expression in the striatum. In addition, MDL11939 decreased haloperidol-induced Nur77 and Nor-1 mRNA levels in the ventral tegmental area. On the contrary, idazoxan (α2 antagonist) consistently potentiated haloperidol-induced Nur77, but not Nor-1 mRNA levels in the striatum, whereas prazosin (α1 antagonist) remained without effect. Taken together, these results show the ability of a 5-HT1A agonist or a 5-HT2A antagonist to reduce haloperidol-induced Nur77 and Nor-1 striatal expression, suggesting that these serotonin receptor subtypes participate in the differential pattern of gene expression induced by typical and atypical antipsychotic drugs. PMID:21524335

Differential Expression of microRNAs in the Ovaries from Letrozole-Induced Rat Model of Polycystic Ovary Syndrome.

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Li, Dandan; Li, Chunjin; Xu, Ying; Xu, Duo; Li, Hongjiao; Gao, Liwei; Chen, Shuxiong; Fu, Lulu; Xu, Xin; Liu, Yongzheng; Zhang, Xueying; Zhang, Jingshun; Ming, Hao; Zheng, Lianwen

2016-04-01

Polycystic ovary syndrome (PCOS) is a complex and heterogeneous endocrine disorder. To understand the pathogenesis of PCOS, we established rat models of PCOS induced by letrozole and employed deep sequencing to screen the differential expression of microRNAs (miRNAs) in PCOS rats and control rats. We observed vaginal smear and detected ovarian pathological alteration and hormone level changes in PCOS rats. Deep sequencing showed that a total of 129 miRNAs were differentially expressed in the ovaries from letrozole-induced rat model compared with the control, including 49 miRNAs upregulated and 80 miRNAs downregulated. Furthermore, the differential expression of miR-201-5p, miR-34b-5p, miR-141-3p, and miR-200a-3p were confirmed by real-time polymerase chain reaction. Bioinformatic analysis revealed that these four miRNAs were predicted to target a large set of genes with different functions. Pathway analysis supported that the miRNAs regulate oocyte meiosis, mitogen-activated protein kinase (MAPK) signaling, phosphoinositide 3-kinase/Akt (PI3K-Akt) signaling, Rap1 signaling, and Notch signaling. These data indicate that miRNAs are differentially expressed in rat PCOS model and the differentially expressed miRNA are involved in the etiology and pathophysiology of PCOS. Our findings will help identify miRNAs as novel diagnostic markers and therapeutic targets for PCOS.

Cadmium induces matrix metalloproteinase-9 expression via ROS-dependent EGFR, NF-kB, and AP-1 pathways in human endothelial cells

International Nuclear Information System (INIS)

Lian, Sen; Xia, Yong; Khoi, Pham Ngoc; Ung, Trong Thuan; Yoon, Hyun Joong; Kim, Nam Ho; Kim, Kyung Keun; Jung, Young Do

2015-01-01

Highlights: • Cadmium induces MMP-9 expression through NADPH oxidase-derived ROS. • Cadmium induces MMP-9 through EGFR-mediated Akt, Erk1/2 and JNK1/2 signaling pathways. • Akt, MAPKs (Erk1/2 and JNK1/2) functioned as upstream signals of NF-kB and AP-1 respectively, in cadmium-induced MMP-9 in endothelial cells. • ROS production by NADPH oxidase is the furthest upstream signal in MMP-9 expression in ECV304 cells. - Abstract: Cadmium (Cd), a widespread cumulative pollutant, is a known human carcinogen, associated with inflammation and tumors. Matrix metalloproteinase-9 (MMP-9) plays a pivotal role in tumor metastasis; however, the mechanisms underlying the MMP-9 expression induced by Cd remain obscure in human endothelial cells. Here, Cd elevated MMP-9 expression in dose- and time-dependent manners in human endothelial cells. Cd increased ROS production and the ROS-producing NADPH oxidase. Cd translocates p47 phox , a key subunit of NADPH oxidase, to the cell membrane. Cd also activated the phosphorylation of EGFR, Akt, Erk1/2, and JNK1/2 in addition to promoting NF-kB and AP-1 binding activities. Specific inhibitor and mutagenesis studies showed that EGFR, Akt, Erk1/2, JNK1/2 and transcription factors NF-κB and AP-1 were related to Cd-induced MMP-9 expression in endothelial cells. Akt, Erk1/2, and JNK1/2 functioned as upstream signals in the activation of NF-κB and AP-1, respectively. In addition, N-acetyl-L-cystein (NAC), diphenyleneiodonium chloride (DPI) and apocynin (APO) inhibited the Cd-induced activation of EGFR, Akt, Erk1/2, JNK1/2, and p38 MAPK, indicating that ROS production by NADPH oxidase is the furthest upstream signal in MMP-9 expression. At present, it states that Cd displayed marked invasiveness in ECV304 cells, which was partially abrogated by MMP-9 neutralizing antibodies. These results demonstrated that Cd induces MMP-9 expression via ROS-dependent EGFR- > Erk1/2, JNK1/2- > AP-1 and EGFR- > Akt- > NF-κB signaling pathways and, in turn

Eicosapentaenoic acid inhibits TNF-α-induced matrix metalloproteinase-9 expression in human keratinocytes, HaCaT cells

International Nuclear Information System (INIS)

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

2008-01-01

Eicosapentaenoic acid (EPA) is an omega-3 (ω-3) polyunsaturated fatty acid (PUFA), which has anti-inflammatory and anti-cancer properties. Some reports have demonstrated that EPA inhibits NF-κB activation induced by tumor necrosis factor (TNF)-α 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-α-induced matrix metalloproteinases (MMP)-9 in human immortalized keratinocytes (HaCaT). TNF-α induced MMP-9 expression by NF-κB-dependent pathway. Pretreatment of EPA inhibited TNF-α-induced MMP-9 expression and p65 phosphorylation. However, EPA could not affect IκB-α phosphorylation, nuclear translocation of p65, and DNA binding activity of NF-κB. EPA inhibited TNF-α-induced p65 phosphorylation through p38 and Akt inhibition and this inhibition was IKKα-dependent event. Taken together, we demonstrate that EPA inhibits TNF-α-induced MMP-9 expression through inhibition of p38 and Akt activation

Insulin receptor substrate 1 expression enhances the sensitivity of 32D cells to chemotherapy-induced cell death

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Porter, Holly A.; Carey, Gregory B.; Keegan, Achsah D.

2012-01-01

The adaptors IRS1 and IRS2 link growth factor receptors to downstream signaling pathways that regulate proliferation and survival. Both suppress factor-withdrawal-induced apoptosis and have been implicated in cancer progression. However, recent studies suggest IRS1 and IRS2 mediate differential functions in cancer pathogenesis. IRS1 promoted breast cancer proliferation, while IRS2 promoted metastasis. The role of IRS1 and IRS2 in controlling cell responses to chemotherapy is unknown. To determine the role of IRS1 and IRS2 in the sensitivity of cells to chemotherapy, we treated 32D cells lacking or expressing IRS proteins with various concentrations of chemotherapeutic agents. We found that expression of IRS1, in contrast to IRS2, enhanced the sensitivity of 32D cells to chemotherapy-induced apoptosis. When IRS2 was expressed with IRS1, the cells no longer showed enhanced sensitivity. Expression of IRS1 did not alter the expression of pro- and anti-apoptotic proteins; however, 32D-IRS1 cells expressed higher levels of Annexin A2. In 32D-IRS1 cells, IRS1 and Annexin A2 were both located in cytoplasmic and membrane fractions. We also found that IRS1 coprecipitated with Annexin A2, while IRS2 did not. Decreasing Annexin A2 levels reduced 32D-IRS1 cell sensitivity to chemotherapy. These results suggest IRS1 enhances sensitivity to chemotherapy in part through Annexin A2. PMID:22652453

Bifunctional effects of fucoidan on the expression of inducible nitric oxide synthase

International Nuclear Information System (INIS)

Yang, Jin Won; Yoon, Se Young; Oh, Soo Jin; Kim, Sang Kyum; Kang, Keon Wook

2006-01-01

Algal fucoidan is a marine sulfated polysaccharide with a wide variety of biological activities including anti-thrombotic and anti-inflammatory effects. This study evaluated the effect of fucoidan on the expression of inducible nitric oxide synthase (iNOS) in a macrophage cell line, RAW264.7. Low concentration range of fucoidan (10 μg/ml) increased the basal expression level of iNOS in quiescent macrophages. However, we found for the first time that fucoidan inhibited the release of nitric oxide (NO) in RAW264.7 cells stimulated with lipopolysaccharide (LPS). Western blot analysis revealed that fucoidan suppressed the LPS-induced expression of the inducible nitric oxide synthase (iNOS) gene. Moreover, the activation of both nuclear factor-κB (NF-κB) and activator protein 1 (AP-1) are key steps in the transcriptional activation of the iNOS gene. Here, it was revealed that fucoidan selectively suppressed AP-1 activation, and that the activation of AP-1 appears to be essential for the induction of iNOS in activated macrophages. This inhibitory effect on AP-1 activation by fucoidan might be associated with its NO blocking and anti-inflammatory effects

Myofibril-Inducing RNA (MIR is essential for tropomyosin expression and myofibrillogenesis in axolotl hearts

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Lemanski Sharon L

2009-09-01

Full Text Available Abstract The Mexican axolotl, Ambystoma mexicanum, carries the naturally-occurring recessive mutant gene 'c' that results in a failure of homozygous (c/c embryos to form hearts that beat because of an absence of organized myofibrils. Our previous studies have shown that a noncoding RNA, Myofibril-Inducing RNA (MIR, is capable of promoting myofibrillogenesis and heart beating in the mutant (c/c axolotls. The present study demonstrates that the MIR gene is essential for tropomyosin (TM expression in axolotl hearts during development. Gene expression studies show that mRNA expression of various tropomyosin isoforms in untreated mutant hearts and in normal hearts knocked down with double-stranded MIR (dsMIR are similar to untreated normal. However, at the protein level, selected tropomyosin isoforms are significantly reduced in mutant and dsMIR treated normal hearts. These results suggest that MIR is involved in controlling the translation or post-translation of various TM isoforms and subsequently of regulating cardiac contractility.

Interleukin-1β-induced iNOS expression in human lung carcinoma A549 cells: involvement of STAT and MAPK pathways

International Nuclear Information System (INIS)

Ravichandran, Kameswaran; Tyagi, Alpna; Deep, Gagan; Agarwal, Chapla; Agarwal, Rajesh

2011-01-01

For understanding of signaling molecules important in lung cancer growth and progression, IL-1β effect was analyzed on iNOS expression and key signaling molecules in human lung carcinoma A549 cells and established the role of specific signaling molecules by using specific chemical inhibitors. IL-1β exposure (10 ng/ml) induced strong iNOS expression in serum starved A549 cells. Detailed molecular analyses showed that IL-1β increased expression of phosphorylated STAT1 (Tyr701 and Ser727) and STAT3 (Tyr705 and Ser727) both in total cell lysates and nuclear lysates. Further, IL-1β exposure strongly activated MAPKs (ERK1/2, JNK1/2 and p38) and Akt as well as increased nuclear levels of NF-κB and HIF-1α in A549 cells. Use of specific chemical inhibitors for JAK1 kinase (piceatannol), JAK2 kinase (AG-490), MEK1/2 (PD98059) and JNK1/2 (SP600125) revealed that IL-1β-induced iNOS expression involved signaling pathways in addition to JAKSTAT and ERK1/2-JNK1/2 activation. Overall, these results suggested that instead of specific pharmacological inhibitors, use of chemopreventive agents with broad spectrum efficacy to inhibit IL-1β-induced signaling cascades and iNOS expression would be a better strategy towards lung cancer prevention and/or treatment. (author)

Estrogen induced concentration dependent differential gene expression in human breast cancer (MCF7) cells: Role of transcription factors

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Chandrasekharan, Sabarinath; Kandasamy, Krishna Kumar; Dayalan, Pavithra; Ramamurthy, Viraragavan

2013-01-01

Highlights: •Estradiol (E2) at low dose induced cell proliferation in breast cancer cells. •E2 at high concentration induced cell stress in breast cancer cells. •Estrogen receptor physically interacts only with a few transcription factors. •Differential expression of genes with Oct-1 binding sites increased under stress. •Transcription factor binding sites showed distinct spatial distribution on genes. -- Abstract: Background: Breast cancer cells respond to estrogen in a concentration dependent fashion, resulting in proliferation or apoptosis. The mechanism of this concentration dependent differential outcome is not well understood yet. Methodology: Meta-analysis of the expression data of MCF7 cells treated with low (1 nM) or high (100 nM) dose of estradiol (E2) was performed. We identified genes differentially expressed at the low or the high dose, and examined the nature of regulatory elements in the vicinity of these genes. Specifically, we looked for the difference in the presence, abundance and spatial distribution of binding sites for estrogen receptor (ER) and selected transcription factors (TFs) in the genomic region up to 25 kb upstream and downstream from the transcription start site (TSS) of these genes. Results: It was observed that at high dose E2 induced the expression of stress responsive genes, while at low dose, genes involved in cell cycle were induced. We found that the occurrence of transcription factor binding regions (TFBRs) for certain factors such as Sp1 and SREBP1 were higher on regulatory regions of genes expressed at low dose. At high concentration of E2, genes with a higher frequency of Oct-1 binding regions were predominantly involved. In addition, there were differences in the spatial distribution pattern of the TFBRs in the genomic regions among the two sets of genes. Discussion: E2 induced predominantly proliferative/metabolic response at low concentrations; but at high concentration, stress–rescue responses were induced

Estrogen induced concentration dependent differential gene expression in human breast cancer (MCF7) cells: Role of transcription factors

Energy Technology Data Exchange (ETDEWEB)

Chandrasekharan, Sabarinath, E-mail: csab@bio.psgtech.ac.in [Department of Biotechnology, PSG College of Technology, Coimbatore 641004 (India); Kandasamy, Krishna Kumar [Max Planck Institute for Biology of Ageing, Cologne (Germany); Dayalan, Pavithra; Ramamurthy, Viraragavan [Department of Biotechnology, PSG College of Technology, Coimbatore 641004 (India)

2013-08-02

Highlights: •Estradiol (E2) at low dose induced cell proliferation in breast cancer cells. •E2 at high concentration induced cell stress in breast cancer cells. •Estrogen receptor physically interacts only with a few transcription factors. •Differential expression of genes with Oct-1 binding sites increased under stress. •Transcription factor binding sites showed distinct spatial distribution on genes. -- Abstract: Background: Breast cancer cells respond to estrogen in a concentration dependent fashion, resulting in proliferation or apoptosis. The mechanism of this concentration dependent differential outcome is not well understood yet. Methodology: Meta-analysis of the expression data of MCF7 cells treated with low (1 nM) or high (100 nM) dose of estradiol (E2) was performed. We identified genes differentially expressed at the low or the high dose, and examined the nature of regulatory elements in the vicinity of these genes. Specifically, we looked for the difference in the presence, abundance and spatial distribution of binding sites for estrogen receptor (ER) and selected transcription factors (TFs) in the genomic region up to 25 kb upstream and downstream from the transcription start site (TSS) of these genes. Results: It was observed that at high dose E2 induced the expression of stress responsive genes, while at low dose, genes involved in cell cycle were induced. We found that the occurrence of transcription factor binding regions (TFBRs) for certain factors such as Sp1 and SREBP1 were higher on regulatory regions of genes expressed at low dose. At high concentration of E2, genes with a higher frequency of Oct-1 binding regions were predominantly involved. In addition, there were differences in the spatial distribution pattern of the TFBRs in the genomic regions among the two sets of genes. Discussion: E2 induced predominantly proliferative/metabolic response at low concentrations; but at high concentration, stress–rescue responses were induced

Xenotropic type C virus expression in murine thymomas induced by radiation or 3-methylcholanthrene

International Nuclear Information System (INIS)

Mayer, A.; Duran-Reynals, M.L.

1981-01-01

Thymic lymphoma incidence and thymic expression of MuLV with xenotropic infectivity was monitored in AKR, RF, and reciprocal F 1 mice of the AKR X RF cross after treatment with either γ radiation or the chemical carcinogen 3-methylcholanthrene (MCA). These two inbred strains and the F 1 hybrids developed similary high incidences of thymoma, and lymphomatous cells from AKR mice and (ARK] X RF∫)F 1 mice were observed to be expressing MuLV with xenotropic host range. However, lymphoma cells from RF mice and (RF] X AKR∫)F 1 mice did not shed xenotropic MuLV. Thymic xenotropic virus expression was therefore not correlated with a high incidence of radiation or chemically induced thymoma, but rather appeared to be a phenotype genetically transmitted by AKR mice to F 1 mice of the AKR X RF cross as a dominant trait in induced thymomas. In addition, a maternal effect on thymic xenotropic virus expression in induced thymomas was observed by the comparison of reciprocal F 1 hybrids in this cross

Four inducible promoters for controlled gene expression in the oleaginous yeast Rhodotorula toruloides

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Alexander Michael Bedford Johns

2016-10-01

Full Text Available Rhodotorula (Rhodosporidium toruloides is an oleaginous yeast with great biotechnological potential, capable of accumulating lipid up to 70 % of its dry biomass, and of carotenoid biosynthesis. However, few molecular genetic tools are available for manipulation of this basidiomycete yeast and its high genomic GC content can make routine cloning difficult. We have developed plasmid vectors for transformation of R. toruloides which include elements for Saccharomyces cerevisiae in-yeast assembly; this method is robust to the assembly of GC-rich DNA and of large plasmids. Using such vectors we screened for controllable promoters, and identified inducible promoters from the genes NAR1, ICL1, CTR3 and MET16. These four promoters have independent induction/repression conditions and exhibit different levels and rates of induction in R. toruloides, making them appropriate for controllable transgene expression in different experimental situations. Nested deletions were used to identify regulatory regions in the four promoters, and to delimit the minimal inducible promoters, which are as small as 200 bp for the NAR1 promoter. The NAR1 promoter shows very tight regulation under repressed conditions as determined both by an EGFP reporter gene and by conditional rescue of a leu2 mutant. These new tools facilitate molecular genetic manipulation and controllable gene expression in R. toruloides.