Science.gov

Sample records for activity protein expression

  1. Protein inhibitor of activated STAT3 inhibits adipogenic gene expression

    SciTech Connect

    Deng Jianbei; Hua Kunjie; Caveney, Erica J.; Takahashi, Nobuyuki; Harp, Joyce B. . E-mail: jharp@unc.edu

    2006-01-20

    Protein inhibitor of activated STAT3 (PIAS3), a cytokine-induced repressor of signal transducer and activator of transcription 3 (STAT3) and a modulator of a broad array of nuclear proteins, is expressed in white adipose tissue, but its role in adipogenesis is not known. Here, we determined that PIAS3 was constitutively expressed in 3T3-L1 cells at all stages of adipogenesis. However, it translocated from the nucleus to the cytoplasm 4 days after induction of differentiation by isobutylmethylxanthine, dexamethasone, and insulin (MDI). In ob/ob mice, PIAS3 expression was increased in white adipose tissue depots compared to lean mice and was found in the cytoplasm of adipocytes. Overexpression of PIAS3 in differentiating preadipocytes, which localized primarily to the nucleus, inhibited mRNA level gene expression of adipogenic transcription factors C/EBP{alpha} and PPAR{gamma}, as well as their downstream target genes aP2 and adiponectin. PIAS3 also inhibited C/EBP{alpha} promoter activation mediated specifically by insulin, but not dexamethasone or isobutylmethylxanthine. Taken together, these data suggest that PIAS3 may play an inhibitory role in adipogenesis by modulating insulin-activated transcriptional activation events. Increased PIAS3 expression in adipose tissue may play a role in the metabolic disturbances of obesity.

  2. GILT expression in B cells diminishes cathepsin S steady-state protein expression and activity

    PubMed Central

    Phipps-Yonas, Hannah; Semik, Vikki; Hastings, Karen Taraszka

    2013-01-01

    MHC class II-restricted Ag processing requires protein degradation in the endocytic pathway for the activation of CD4+ T cells. Gamma-interferon-inducible lysosomal thiol reductase (GILT) facilitates Ag processing by reducing protein disulfide bonds in this compartment. Lysosomal cysteine protease cathepsin S (CatS) contains disulfide bonds and mediates essential steps in MHC class II-restricted processing, including proteolysis of large polypeptides and cleavage of the invariant chain. We sought to determine whether GILT’s reductase activity regulates CatS expression and function. Confocal microscopy confirmed that GILT and CatS colocalized within lysosomes of B cells. GILT expression posttranscriptionally decreased the steady-state protein expression of CatS in primary B cells and B-cell lines. GILT did not substantially alter the expression of other lysosomal proteins, including H2-M, H2-O, or CatL. GILT’s reductase active site was necessary for diminished CatS protein levels, and GILT expression decreased the half-life of CatS, suggesting that GILT-mediated reduction of protein disulfide bonds enhances CatS degradation. GILT expression decreased the proteolysis of a CatS selective substrate. This study illustrates a physiologic mechanism that regulates CatS and has implications for fine tuning MHC class II-restricted Ag processing and for the development of CatS inhibitors, which are under investigation for the treatment of autoimmune disease. PMID:23012103

  3. GTP cyclohydrolase I expression, protein, and activity determine intracellular tetrahydrobiopterin levels, independent of GTP cyclohydrolase feedback regulatory protein expression.

    PubMed

    Tatham, Amy L; Crabtree, Mark J; Warrick, Nicholas; Cai, Shijie; Alp, Nicholas J; Channon, Keith M

    2009-05-15

    GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r(2) = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r(2) = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression.

  4. Mycobacterium tuberculosis Rv1096 protein: gene cloning, protein expression, and peptidoglycan deacetylase activity

    PubMed Central

    2014-01-01

    Background Many bacteria modulate and evade the immune defenses of their hosts through peptidoglycan (PG) deacetylation. The PG deacetylases from Streptococcus pneumonia, Listeria monocytogenes and Lactococcus lactis have been characterized. However, thus far, the PG deacetylase of Mycobacterium tuberculosis has not been identified. Results In this study, we cloned the Rv1096 gene from the M. tuberculosis H37Rv strain and expressed Rv1096 protein in both Escherichia coli and M. smegmatis. The results showed that the purified Rv1096 protein possessed metallo-dependent PG deacetylase activity, which increased in the presence of Co2+. The kinetic parameters of the PG deacetylase towards M. smegmatis PG as a substrate were as follows: Km, 0.910 ± 0.007 mM; Vmax, 0.514 ± 0.038 μMmin-1; and Kcat = 0.099 ± 0.007 (S-1). Additionally, the viability of M. smegmatis in the presence of over-expressed Rv1096 protein was 109-fold higher than that of wild-type M. smegmatis after lysozyme treatment. Additionally, light microscopy and scanning electron microscopy showed that in the presence of over-expressed Rv1096 protein, M. smegmatis kept its regular shape, with an undamaged cell wall and smooth surface. These results indicate that Rv1096 caused deacetylation of cell wall PG, leading to lysozyme resistance in M. smegmatis. Conclusion We have determined that M. tuberculosis Rv1096 is a PG deacetylase. The PG deacetylase activity of Rv1096 contributed to lysozyme resistance in M. smegmatis. Our findings suggest that deacetylation of cell wall PG may be involved in evasion of host immune defenses by M. tuberculosis. PMID:24975018

  5. Expression of proteins and protein kinase activity during germination of aerial spores of Streptomyces granaticolor.

    PubMed

    Mikulík, Karel; Bobek, Jan; Bezousková, Silvia; Benada, Oldrich; Kofronová, Olga

    2002-11-29

    Dormant aerial spores of Streptomyces granaticolor contain pre-existing pool of mRNA and active ribosomes for rapid translation of proteins required for earlier steps of germination. Activated spores were labeled for 30 min with [35S]methionine/cysteine in the presence or absence of rifamycin (400 microg/ml) and resolved by two-dimensional electrophoresis. About 320 proteins were synthesized during the first 30 min of cultivation at the beginning of swelling, before the first DNA replication. Results from nine different experiments performed in the presence of rifamycin revealed 15 protein spots. Transition from dormant spores to swollen spores is not affected by the presence of rifamycin but further development of spores is stopped. To support existence of pre-existing pool of mRNA in spores, cell-free extract of spores (S30 fraction) was used for in vitro protein synthesis. These results indicate that RNA of spores possesses mRNA functionally competent and provides templates for protein synthesis. Cell-free extracts isolated from spores, activated spores, and during spore germination were further examined for in vitro protein phosphorylation. The analyses show that preparation from dormant spores catalyzes phosphorylation of only seven proteins. In the absence of phosphatase inhibitors, several proteins were partially dephosphorylated. The activation of spores leads to a reduction in phosphorylation activity. Results from in vitro phosphorylation reaction indicate that during germination phosphorylation/dephosphorylation of proteins is a complex function of developmental changes.

  6. Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins

    SciTech Connect

    Nayak, Jaladhi; Gastonguay, Adam J.; Talipov, Marat R.; Vakeel, Padmanabhan; Span, Elise A.; Kalous, Kelsey S.; Kutty, Raman G.; Jensen, Davin R.; Pokkuluri, Phani Raj; Sem, Daniel S.; Rathore, Rajendra; Ramchandran, Ramani

    2014-12-18

    Background: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function. We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.

  7. Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins

    DOE PAGES

    Nayak, Jaladhi; Gastonguay, Adam J.; Talipov, Marat R.; ...

    2014-12-18

    Background: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function.more » We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.« less

  8. Muscle contractile activity regulates Sirt3 protein expression in rat skeletal muscles.

    PubMed

    Hokari, Fumi; Kawasaki, Emi; Sakai, Atsushi; Koshinaka, Keiichi; Sakuma, Kunihiro; Kawanaka, Kentaro

    2010-08-01

    Sirt3, a member of the sirtuin family, is known to control cellular mitochondrial function. Furthermore, because sirtuins require NAD for their deacetylase activity, nicotinamide phosphoribosyltransferase (Nampt), which is a rate-limiting enzyme in the intracellular NAD biosynthetic pathway, influences their activity. We examined the effects of exercise training and normal postural contractile activity on Sirt3 and Nampt protein expression in rat skeletal muscles. Male rats were trained by treadmill running at 20 m/min, 60 min/day, 7 days/wk for 4 wk. This treadmill training program increased the Sirt3 protein expression in the soleus and plantaris muscles by 49% and 41%, respectively (P < 0.05). Moreover, a 4-wk voluntary wheel-running program also induced 66% and 95% increases in Sirt3 protein in the plantaris and triceps muscles of rats, respectively (P < 0.05). Treadmill-running and voluntary running training induced no significant changes in Nampt protein expression in skeletal muscles. In resting rats, the soleus muscle, which is recruited during normal postural activity, possessed the greatest expression levels of the Sirt3 and Nampt proteins, followed by the plantaris and triceps muscles. Furthermore, the Sirt3, but not Nampt, protein level was reduced in the soleus muscles from immobilized hindlimbs compared with that shown in the contralateral control muscle. These results demonstrated that 1) Sirt3 protein expression is upregulated by exercise training in skeletal muscles and 2) local postural contractile activity plays an important role in maintaining a high level of Sirt3 protein expression in postural muscle.

  9. Chemokines derived from soluble fusion proteins expressed in Escherichia coli are biologically active

    SciTech Connect

    Magistrelli, Giovanni; Gueneau, Franck; Muslmani, Machadiya; Ravn, Ulla; Kosco-Vilbois, Marie; Fischer, Nicolas . E-mail: nfischer@novimmune.com

    2005-08-26

    Chemokines are a class of low molecular weight proteins that are involved in leukocytes trafficking. Due to their involvement in recruiting immune cells to sites of inflammation, chemokines, and chemokine receptors have become an attractive class of therapeutic targets. However, when expressed in Escherichia coli chemokines are poorly soluble and accumulate in inclusion bodies. Several purification methods have been described but involve time-consuming refolding, buffer exchange, and purification steps that complicate expression of these proteins. Here, we describe a simple and reliable method to express chemokines as fusions to the protein NusA. The fusion proteins were largely found in the soluble fraction and could be readily purified in a single step. Proteolytic cleavage was used to obtain soluble recombinant chemokines that were found to be very active in a novel in vitro chemotaxis assays. This method could be applied to several {alpha} and {beta} human chemokines, suggesting that it is generally applicable to this class of proteins.

  10. Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

    PubMed

    Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

    1995-02-10

    Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain.

  11. Cloning and expression of a cDNA for the T-cell-activating protein TAP.

    PubMed Central

    Reiser, H; Coligan, J; Palmer, E; Benacerraf, B; Rock, K L

    1988-01-01

    The T-cell-activating protein TAP is a murine phosphatidylinositol-anchored glycoprotein whose expression is controlled by the Ly-6 locus. Previous studies have suggested an important role for this protein in physiological T-cell activation. Using oligonucleotide probes, we have now isolated a cDNA clone whose predicted sequence would encode a protein with an NH2-terminal sequence identical to that of the TAP molecule. Further analysis of the predicted protein sequence revealed a cysteine-rich protein with a hydrophobic domain at the COOH terminus and without N-linked glycosylation sites--all features consistent with our previous analysis of the TAP protein. In Southern blot analysis, the Ly-6.2 cDNA clone detects a multigene family and a restriction fragment length polymorphism that maps precisely to the Ly-6 locus. Expression of the cDNA clone in COS cells demonstrates that it codes for TAP and clarifies the relationship between the epitopes recognized by various alpha Ly-6 monoclonal antibodies. Finally, we have studied the expression of Ly-6 mRNA in a variety of cell lineages. Ly-6 transcripts were detected in all organs examined, including spleen, kidney, lung, brain, and heart. This demonstrates that the Ly-6 locus is transcriptionally active in a wide range of organs and suggests that the role of TAP or TAP-like proteins might extend to other tissues. Images PMID:2895473

  12. Enhanced calcium cycling and contractile function in transgenic hearts expressing constitutively active G alpha o* protein.

    PubMed

    Zhu, Ming; Gach, Agnieszka A; Liu, GongXin; Xu, Xiaomei; Lim, Chee Chew; Zhang, Julie X; Mao, Lan; Chuprun, Kurt; Koch, Walter J; Liao, Ronglih; Koren, Gideon; Blaxall, Burns C; Mende, Ulrike

    2008-03-01

    In contrast to the other heterotrimeric GTP-binding proteins (G proteins) Gs and Gi, the functional role of G o is still poorly defined. To investigate the role of G alpha o in the heart, we generated transgenic mice with cardiac-specific expression of a constitutively active form of G alpha o1* (G alpha o*), the predominant G alpha o isoform in the heart. G alpha o expression was increased 3- to 15-fold in mice from 5 independent lines, all of which had a normal life span and no gross cardiac morphological abnormalities. We demonstrate enhanced contractile function in G alpha o* transgenic mice in vivo, along with increased L-type Ca2+ channel current density, calcium transients, and cell shortening in ventricular G alpha o*-expressing myocytes compared with wild-type controls. These changes were evident at baseline and maintained after isoproterenol stimulation. Expression levels of all major Ca2+ handling proteins were largely unchanged, except for a modest reduction in Na+/Ca2+ exchanger in transgenic ventricles. In contrast, phosphorylation of the ryanodine receptor and phospholamban at known PKA sites was increased 1.6- and 1.9-fold, respectively, in G alpha o* ventricles. Density and affinity of beta-adrenoceptors, cAMP levels, and PKA activity were comparable in G alpha o* and wild-type myocytes, but protein phosphatase 1 activity was reduced upon G alpha o* expression, particularly in the vicinity of the ryanodine receptor. We conclude that G alpha o* exerts a positive effect on Ca2+ cycling and contractile function. Alterations in protein phosphatase 1 activity rather than PKA-mediated phosphorylation might be involved in hyperphosphorylation of key Ca2+ handling proteins in hearts with constitutive G alpha o activation.

  13. A 3 base pair deletion in TBX1 leads to reduced protein expression and transcriptional activity

    PubMed Central

    Xu, Yuejuan; Fang, Shaohai; Zhang, Erge; Pu, Tian; Cao, Ruixue; Fu, Qihua; Li, Fen; Chen, Sun; Sun, Kun; Xu, Rang

    2017-01-01

    Transcription factor TBX1 plays a pivotal role in heart development and has been implicated in 22q11.2 deletion syndrome. The structure of this protein has been elucidated, and several mutations have been identified that disrupt TBX1 localization, DNA/protein-binding, or mRNA expression. This study reports a mutation in the TBX1 gene that leads to significantly reduced expression of the mutant protein. A total of 773 conotruncal heart defect patients and 516 unrelated healthy control individuals were enrolled, none of which harbored a 22q11.2 deletion or duplication. We identified a mutation, c.303-305delGAA, located in the third exon of TBX1 that does not disrupt TBX1 mRNA expression or DNA binding activity, but results in decreased TBX1 protein levels and transcriptional activity. Through protein degradation studies we demonstrated that TBX1 is degraded primarily in proteasomes. Although the c.303-305delGAA mutation leads to low levels of the mutant protein, we found that increased protein degradation was not the cause, and we hypothesize that an alternate mechanism, such as translational inhibition, may be the cause. PMID:28272434

  14. Application of Protein Expression Profiling to Screen Chemicals for Androgenic Activity.

    EPA Science Inventory

    Protein expression changes can be used for detection of biomarkers that can be applied diagnostically to screen chemicals for endocrine modifying activity. In this study, Surface Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS) coupled with a s...

  15. Expression of T-cell-activating protein in peripheral lymphocyte subsets.

    PubMed Central

    Yeh, E T; Reiser, H; Benacerraf, B; Rock, K L

    1986-01-01

    T-cell-activating protein (TAP) is an allelic 12-kDa membrane protein that participates in T-cell activation. Soluble anti-TAP monoclonal antibodies can trigger antigen-specific, major histocompatibility complex-restricted T-cell hybridomas to produce interleukin 2 and are mitogenic for normal T cells and thymocytes. TAP is expressed on 10% of thymocytes, which are mainly cortisone-resistant and mature. In the periphery, TAP is expressed on 70% of resting T cells but not on resting B cells. In this report, we analyze in detail the nature of TAP expression on peripheral lymphocyte subsets by immunofluorescence techniques. We show that all inducer (L3T4+) T cells are TAP+. In contrast, only 50% of Lyt-2+ T cells express detectable TAP. Functional studies demonstrated that at least part of the heterogeneity of TAP expression is present in the Lyt-2+ cytolytic T-cell (CTL) subset. Unstimulated CTL precursors are TAP- but are induced to express TAP in the effector state. Furthermore, this reflects actual synthesis of TAP, as TAP is detectable on activated Lyt-2+ CTLs passaged in vitro under conditions where passive acquisition can be ruled out. To extend this observation, we have studied the expression of TAP on activated T and B cells. Upon activation, all T and B cells became TAP+. Furthermore, the TAP molecules on B and T cells are indistinguishable by NaDodSO4/polyacrylamide gel electrophoresis. This suggests that TAP expression defines further heterogeneity of lymphocytes, with activation being one parameter influencing its expression. Images PMID:3020545

  16. Expression of Active Fluorophore Proteins in the Milk of Transgenic Pigs Bypassing the Secretory Pathway

    PubMed Central

    Mukherjee, Ayan; Garrels, Wiebke; Talluri, Thirumala R.; Tiedemann, Daniela; Bősze, Zsuzsanna; Ivics, Zoltán; Kues, Wilfried A.

    2016-01-01

    We describe the expression of recombinant fluorescent proteins in the milk of two lines of transgenic pigs generated by Sleeping Beauty transposon-mediated genetic engineering. The Sleeping Beauty transposon consisted of an ubiquitously active CAGGS promoter driving a fluorophore cDNA, encoding either Venus or mCherry. Importantly, the fluorophore cDNAs did not encode for a signal peptide for the secretory pathway, and in previous studies of the transgenic animals a cytoplasmic localization of the fluorophore proteins was found. Unexpectedly, milk samples from lactating sows contained high levels of bioactive Venus or mCherry fluorophores. A detailed analysis suggested that exfoliated cells of the mammary epithelium carried the recombinant proteins passively into the milk. This is the first description of reporter fluorophore expression in the milk of livestock, and the findings may contribute to the development of an alternative concept for the production of bioactive recombinant proteins in the udder. PMID:27086548

  17. Reduced Duodenal Cytochrome P450 3A Protein Expression and Catalytic Activity in Patients with Cirrhosis

    PubMed Central

    McConn, Donavon J.; Lin, Yvonne S.; Mathisen, Terri L.; Blough, David K.; Xu, Yang; Hashizume, Takanori; Taylor, Shari L.; Thummel, Kenneth E.; Shuhart, Margaret C.

    2009-01-01

    The small intestine and liver express high levels of cytochrome P450 3A (CYP3A), an enzyme subfamily contributing significantly to drug metabolism. In patients with cirrhosis, reduced metabolism of drugs is typically attributed to decreased liver function, but it is unclear whether intestinal drug metabolism is also compromised. In this study, we compared CYP3A protein expression and in vitro midazolam hydroxylation in duodenal mucosal biopsies from subjects with normal liver function (controls; n=20) and subjects with varying severity of cirrhosis (n=23). Compared to samples from controls, duodenal CYP3A expression and total midazolam hydroxylation was reduced by 47% and 34%, respectively in samples from subjects with cirrhosis. Greater decreases in CYP3A expression were seen in subjects with increasing severity of cirrhosis. Thus, patients with advanced cirrhosis may have increased drug exposure following oral dosing as a result of both impaired liver function and decreased intestinal CYP3A expression and activity. PMID:19212316

  18. Tumor necrosis factor gene expression is mediated by protein kinase C following activation by ionizing radiation.

    SciTech Connect

    Hallahan, D. E.; Virudachalam, S.; Sherman, M. L.; Huberman, E.; Kufe, D. W.; Weichselbaum, R. R.; Univ. of Chicago; Dana-Farber Cancer Inst.; Univ. of Chicago

    1991-01-01

    Tumor necrosis factor (TNF) production following X-irradiation has been implicated in the biological response to ionizing radiation. Protein kinase C (PKC) is suggested to participate in TNF transcriptional induction and X-ray-mediated gene expression. We therefore studied radiation-mediated TNF expression in HL-60 cells with diminished PKC activity produced by either pretreatment with protein kinase inhibitors or prolonged 12-O-tetradecanoylphorbol-13-acetate treatment. Both treatments resulted in attenuation of radiation-mediated TNF induction. Consistent with these results, we found no detectable induction of TNF expression following X-irradiation in the HL-60 variant deficient in PKC-mediated signal transduction. The rapid activation of PKC following {gamma}-irradiation was established using an in vitro assay measuring phosphorylation of a PKC specific substrate. A 4.5-fold increase in PKC activity occurred 15 to 30 s following irradiation, which declined to baseline at 60 s. Two-dimensional gel electrophoresis of phosphoproteins extracted from irradiated cells demonstrated in vivo phosphorylation of the PKC specific substrate Mr 80,000 protein at 45 s following X-irradiation. These findings indicate that signal transduction via the PKC pathway is required for the induction of TNF gene expression by ionizing radiation.

  19. Mitogen-activated protein kinase in Pfiesteria piscicida and its growth rate-related expression.

    PubMed

    Lin, Senjie; Zhang, Huan

    2003-01-01

    A full-length cDNA (1,434 bp) of mitogen-activated protein kinase (MAPK), a key molecule of a signal transduction cascade, was isolated from the estuarine heterotrophic dinoflagellate Pfiesteria piscicida. This cDNA (Ppmapk1) encoded a protein (PpMAPK1) of 428 amino acid residues that shared about 30 to 40% amino acid similarity with MAPKs in other organisms. Phylogenetic analysis indicated that PpMAPK1 was tightly clustered with MAPK3 in protozoans. Using reverse transcription-PCR, expression of this gene was evaluated for P. piscicida cultures grown under different conditions. While salinity shock, heat shock, starvation, and a subsequent encounter with prey did not appear to affect expression of this gene, Ppmapk1 expression level was correlated with growth rate, suggesting involvement of this gene in the regulation of cell proliferation in the organism.

  20. Activity, Expression and Function of a Second Drosophila Protein Kinase a Catalytic Subunit Gene

    PubMed Central

    Melendez, A.; Li, W.; Kalderon, D.

    1995-01-01

    The DC2 gene was isolated previously on the basis of sequence similarity to DCO, the major Drosophila protein kinase A (PKA) catalytic subunit gene. We show here that the 67-kD Drosophila DC2 protein behaves as a PKA catalytic subunit in vitro. DC2 is transcribed in mesodermal anlagen of early embryos. This expression depends on dorsal but on neither twist nor snail activity. DC2 transcriptional fusions mimic this embryonic expression and are also expressed in subsets of cells in the optic lamina, wing disc and leg discs of third instar larvae. A saturation screen of a small deficiency interval containing DC2 for recessive lethal mutations yielded no DC2 alleles. We therefore isolated new deficiencies to generate deficiency trans-heterozygotes that lacked DC2 activity. These animals were viable and fertile. The absence of DC2 did not affect the viability or phenotype of imaginal disc cells lacking DC0 activity or embryonic hatching of animals with reduced DC0 activity. Furthermore, transgenes expressing DC2 from a DC0 promoter did not efficiently rescue a variety of DC0 mutant phenotypes. These observations indicate that DC2 is not an essential gene and is unlikely to be functionally redundant with DC0, which has multiple unique functions during development. PMID:8601490

  1. Sequential expression, activity and nuclear localization of prolyl oligopeptidase protein in the developing rat brain.

    PubMed

    Hannula, Mirva J; Männistö, Pekka T; Myöhänen, Timo T

    2011-01-01

    Prolyl oligopeptidase (POP) is a serine protease that hydrolyzes peptides shorter than 30-mer. Some evidence has recently been obtained that POP can generate protein-protein interactions and therefore participate in various physiological and pathological events. Several studies have reported that POP may be involved in neurogenesis since its activity increases during development and can be found in the nucleus of proliferating tissues. In cell cultures, POP has been shown to be localized in the nucleus, but only early in the development, since during maturation it is moved to the cytosol. We have now studied for the first time the expression of POP protein, its enzymatic activity and nuclear localization in vivo in the developing rat brain. We observed that enzymatic activity of POP is highest on embryonic day 18 while the protein amounts reach their peak at birth. Furthermore, POP is located in the nucleus only early in the development but is transferred to the cytosol already before parturition. Our in vivo results confirm the previous cell culture results supporting the role of POP in neurogenesis. A discordance of antenatal protein amounts and enzymatic activities is suggesting a tight regulation of POP activity and possibly even a nonhydrolytic role at that stage.

  2. The potential function of steroid sulphatase activity in steroid production and steroidogenic acute regulatory protein expression.

    PubMed Central

    Sugawara, Teruo; Fujimoto, Seiichiro

    2004-01-01

    The first step in the biosynthesis of steroid hormones is conversion of cholesterol into pregnenolone. StAR (steroidogenic acute regulatory) protein plays a crucial role in the intra-mitochondrial movement of cholesterol. STS (steroid sulphatase), which is present ubiquitously in mammalian tissues, including the placenta, adrenal gland, testis and ovary, desulphates a number of 3beta-hydroxysteroid sulphates, including cholesterol sulphate. The present study was designed to examine the effect of STS on StAR protein synthesis and steroidogenesis in cells. Steroidogenic activities of COS-1 cells that had been co-transfected with a vector for the cholesterol P450scc (cytochrome P450 side-chain-cleavage enzyme) system, named F2, a StAR expression vector (pStAR), and an STS expression vector (pSTS) were assayed. Whole-cell extracts were subjected to SDS/PAGE and then to Western blot analysis. pSTS co-expressed in COS-1 cells with F2 and pStAR increased pregnenolone synthesis 2-fold compared with that of co-expression with F2 and pStAR. Western blot analysis using COS-1 cells that had been co-transfected with pSTS, F2 and pStAR revealed that StAR protein levels increased, whereas STS and P450scc protein levels did not change. The amount of StAR protein translation products increased when pSTS was added to an in vitro transcription-translation reaction mixture. Pulse-chase experiments demonstrated that the 37 kDa StAR pre-protein disappeared significantly ( P <0.01) more slowly in COS-1 cells that had been transfected with pSTS than in COS-1 cells that had not been transfected with pSTS. The increase in StAR protein level is not a result of an increase in StAR gene expression, but is a result of both an increase in translation and a longer half-life of the 37 kDa pre-StAR protein. In conclusion, STS increases StAR protein expression level and stimulates steroid production. PMID:14969586

  3. [Nonstructural protein 1 of tick-borne encephalitis virus activates the expression of immunoproteasome subunits].

    PubMed

    Kuzmenko, Y V; Starodubova, E S; Karganova, G G; Timofeev, A V; Karpov, V L

    2016-01-01

    The interaction of viral proteins with host cell components plays an important role in antiviral immune response. One of the key steps of antiviral defense is the formation of immunoproteasomes. The effect of nonstructural protein 1 (NS1) of tick-borne encephalitis virus on the immunoproteasome formation was studied. It was shown that cell expression of NS1 does not reduce the efficacy of the immunoproteasome generation in response to interferon-γ stimulation and even increases the content of the immunoproteasome subunits without the interferon-γ treatment. Thus, NS1 of tick-borne encephalitis virus activates, rather than blocks the mechanisms of immune defense in the cell.

  4. Maternal age effects on myometrial expression of contractile proteins, uterine gene expression, and contractile activity during labor in the rat

    PubMed Central

    Elmes, Matthew; Szyszka, Alexandra; Pauliat, Caroline; Clifford, Bethan; Daniel, Zoe; Cheng, Zhangrui; Wathes, Claire; McMullen, Sarah

    2015-01-01

    Advanced maternal age of first time pregnant mothers is associated with prolonged and dysfunctional labor and significant risk of emergency cesarean section. We investigated the influence of maternal age on myometrial contractility, expression of contractile associated proteins (CAPs), and global gene expression in the parturient uterus. Female Wistar rats either 8 (YOUNG n = 10) or 24 (OLDER n = 10) weeks old were fed laboratory chow, mated, and killed during parturition. Myometrial strips were dissected to determine contractile activity, cholesterol (CHOL) and triglycerides (TAG) content, protein expression of connexin-43 (GJA1), prostaglandin-endoperoxide synthase 2 (PTGS2), and caveolin 1 (CAV-1). Maternal plasma concentrations of prostaglandins PGE2, PGF2α, and progesterone were determined by RIA. Global gene expression in uterine samples was compared using Affymetrix Genechip Gene 2.0 ST arrays and Ingenuity Pathway analysis (IPA). Spontaneous contractility in myometrium exhibited by YOUNG rats was threefold greater than OLDER animals (P < 0.027) but maternal age had no significant effect on myometrial CAP expression, lipid profiles, or pregnancy-related hormones. OLDER myometrium increased contractile activity in response to PGF2α, phenylephrine, and carbachol, a response absent in YOUNG rats (all P < 0.002). Microarray analysis identified that maternal age affected expression of genes related to immune and inflammatory responses, lipid transport and metabolism, steroid metabolism, tissue remodeling, and smooth muscle contraction. In conclusion YOUNG laboring rat myometrium seems primed to contract maximally, whereas activity is blunted in OLDER animals and requires stimulation to meet contractile potential. Further work investigating maternal age effects on myometrial function is required with focus on lipid metabolism and inflammatory pathways. PMID:25876907

  5. Cellular prion protein in the bovine mammary gland is selectively expressed in active lactocytes.

    PubMed

    Didier, Andrea; Dietrich, Richard; Steffl, Martin; Gareis, Manfred; Groschup, Martin H; Müller-Hellwig, Simone; Märtlbauer, Erwin; Amselgruber, Werner M

    2006-11-01

    The cellular prion protein (PrP(c)) is a highly conserved glycoprotein with a still enigmatic physiological function. It is mainly expressed in the central nervous system but accumulating data suggest that PrP(c) is also found in a broad spectrum of non-neuronal tissue. Here we investigated the cell-type-related PrP(c) expression in the bovine mammary gland by using immunohistochemistry (IHC), ELISA, Western blot, and real-time RT-PCR. Specific immunostaining of serial sections revealed that PrP(c) is selectively localized in mammary gland epithelial cells. Particularly strong expression was found at the basolateral surface of those cells showing active secretion. Results obtained by RT-PCR and ELISA complemented IHC findings. No correlation was found between the level of PrP(c) expression and other parameters such as age of the animals under study or stage of lactation.

  6. Activator protein-1 complex expressed by magnetism in cultured rat hippocampal neurons.

    PubMed

    Hirai, Takao; Nakamichi, Noritaka; Yoneda, Yukio

    2002-03-22

    Brief exposure for 15 min to static magnetic filed at 100 mT led to marked but transient potentiation of binding of a radiolabeled probe for activator protein-1 (AP1) in immature cultured rat hippocampal neurons with high expression of growth-associated protein-43. Immunoblotting and supershift analyses revealed that brief exposure to static magnetic field increased AP1 DNA binding through expression of Fra-2, c-Jun, and Jun-D proteins in immature cultured hippocampal neurons. Significantly less potent increases were seen in both intracellular free Ca(2+) concentration and AP1 binding following the addition of N-methyl-d-aspartate in these immature neurons exposed to magnetism 24 h before. These results suggest that brief exposure to weak static magnetic field may lead to desensitization of NMDA receptor channels through modulation of de novo synthesis of particular inducible target proteins at the level of gene transcription by the AP1 complex expressed in the nucleus of immature cultured rat hippocampal neurons.

  7. Mitogen-activated protein kinase phosphatase-1 expression in macrophages is controlled by lymphocytes during macrophage activation.

    PubMed

    Luo, Chong; Yang, Xiqiang; Yao, Lan; Jiang, Liping; Liu, Wei; Li, Xin; Wang, Lijia

    2012-01-01

    The viewpoints on the control of innate immune cells by the adaptive immune system during sepsis remain controversial. Mitogen-activated protein kinase phosphatase-1 (MKP-1) is essential to the negative control of innate immunity and suppresses the activation of macrophages by inhibiting activated mitogen-activated protein kinase (MAPK). The purpose of the current study was to observe inflammatory response and macrophage activation in mice with severe combined immunodeficiency (SCID) with endotoxemia and to determine the role of MKP-1 in the control of macrophage activation by the adaptive immune system. Endotoxemia was induced in wild-type and SCID mice by an intraperitoneal injection of lipopolysaccharide (LPS), and all of the SCID mice died. SCID mice produced more inflammatory cytokines than BALB/c mice systemically and locally. TNF-α mRNA expression was higher and MKP-1 mRNA expression was lower in peritoneal macrophages (PMa) from SCID mice compared to PMa from wild-type mice after and even before LPS injection. Thioglycollate-stimulated PMa from wild-type mice were stimulated with LPS in vitro in the presence or absence of pan-T cells. The levels of TNF-α and IL-6 were higher in the supernatants from PMa cultured alone compared to PMa co-cultured with pan-T cells, and PMa MKP-1 mRNA and protein expression were higher when PMa were co-cultured with pan-T cells. Therefore, pan-T cells can up-regulate MKP-1 expression in macrophages and inhibit the secretion of inflammatory cytokines secretion by macrophages. In SCID mice, lymphocyte deficiency, especially T cell deficiency, causes insufficient MKP-1 expression in macrophages, which can be responsible for the severe inflammation and bad prognosis of septic SCID mice. MKP-1 plays an important role in the control of macrophage activation by the adaptive immune system.

  8. Expression and purification of active recombinant human bone morphogenetic 7-2 dimer fusion protein.

    PubMed

    Dang, Jianli; Jing, Lei; Shi, Weiwei; Qin, Ping; Li, Yuyin; Diao, Aipo

    2015-11-01

    Bone morphogenetic proteins (BMPs) have been applied in bone regeneration therapy due to their significant osteogenic activity, however, the complicated processing and high cost in producing recombinant BMP have limited their use in the clinic. In this study, we have developed a simple method to prepare recombinant human BMP7-BMP2 fusion protein with a flexible peptide linker (rhBMP7-2). The rhBMP7-2 protein is expressed efficiently in Escherichia coli, and the denatured protein purified by anion exchange chromatography then refolded by dialysis. The yield was about 6.8 mg per gram of wet cell weight. The bioactivity of re-folded rhBMP7-2 was measured by alkaline phosphatase assay and alizarin red staining using both C2C12 and MC3T3-E1 cells, and also using the rat subcutaneous ectopic bone formation model. High level osteogenic activity was found in all the assays tested demonstrating the production of corrected folded and active rhBMP7-2 protein.

  9. Expression and purification of an active cecropin-like recombinant protein against multidrug resistance Escherichia coli.

    PubMed

    Téllez, Germán Alberto; Castaño-Osorio, Jhon Carlos

    2014-08-01

    Lucilin is a 36 residue cecropin antimicrobial peptide identified as a partial genetic sequence in Lucilia sericata maggots. The antimicrobial spectrum and toxicity profile of Lucilin is unknown. We first report the expression of Lucilin as an active recombinant fusion protein with a cysteine protease domain (CPD) tag. The fusion protein, GWLK-Lucilin-CPD-His8, showed maximum overexpression in Escherichia coli BL21 cells after 12h induction with 0.5mM IPTG (isopropyl beta-d-thiogalactoside) and growth conditions were 37 °C and 150 rpm shaking. The fusion protein was expressed as a soluble form and was purified by Ni-IMAC. The purified protein was active against E. coli ATCC 35218 with a MIC of 0.68 μM, and a clinical isolate of E. coli with extended spectrum beta-lactamase (ESBL) with a MIC of 0.8 μM. The recombinant GWLK-Lucilin-CPD-His8 was not toxic against human erythrocytes or Vero cells with a therapeutic index >63. The results suggest that GWLK-Lucilin-CPD-His8 represents a potential candidate for therapy against multidrug resistant Gram-negative bacteria.

  10. Epigallocatechin activates haem oxygenase-1 expression via protein kinase Cδ and Nrf2

    PubMed Central

    Ogborne, Richard M.; Rushworth, Stuart A.; O’Connell, Maria A.

    2008-01-01

    The Nrf2/anti-oxidant response element (ARE) pathway plays an important role in regulating cellular anti-oxidants, including haem oxygenase-1 (HO-1). Various kinases have been implicated in the pathways leading to Nrf2 activation. Here, we investigated the effect of epigallocatechin (EGC) on ARE-mediated gene expression in human monocytic cells. EGC time and dose dependently increased HO-1 mRNA and protein expression but had minimal effect on expression of other ARE-regulated genes, including NAD(P)H:quinone oxidoreductase 1, glutathione cysteine ligase and ferritin. siRNA knock down of Nrf2 significantly inhibited EGC-induced HO-1 expression. Furthermore, inhibition of PKC by Ro-31-8220 dose dependently decreased EGC-induced HO-1 mRNA expression, whereas MAP kinase and phosphatidylinositol-3-kinase pathway inhibitors had no significant effect. EGC stimulated phosphorylation of PKCαβ and δ in THP-1 cells. PKCδ inhibition significantly decreased EGC-induced HO-1 mRNA expression, whereas PKCα- and β-specific inhibitors had no significant effect. These results demonstrate for the first time that EGC-induced HO-1 expression occurs via PKCδ and Nrf2. PMID:18586007

  11. Fibroblast activation protein is expressed by rheumatoid myofibroblast-like synoviocytes

    PubMed Central

    Bauer, Stefan; Jendro, Michael C; Wadle, Andreas; Kleber, Sascha; Stenner, Frank; Dinser, Robert; Reich, Anja; Faccin, Erica; Gödde, Stefan; Dinges, Harald; Müller-Ladner, Ulf; Renner, Christoph

    2006-01-01

    Fibroblast activation protein (FAP), as described so far, is a type II cell surface serine protease expressed by fibroblastic cells in areas of active tissue remodelling such as tumour stroma or healing wounds. We investigated the expression of FAP by fibroblast-like synoviocytes (FLSs) and compared the synovial expression pattern in rheumatoid arthritis (RA) and osteoarthritis (OA) patients. Synovial tissue from diseased joints of 20 patients, 10 patients with refractory RA and 10 patients with end-stage OA, was collected during routine surgery. As a result, FLSs from intensively inflamed synovial tissues of refractory RA expressed FAP at high density. Moreover, FAP expression was co-localised with matrix metalloproteinases (MMP-1 and MMP-13) and CD44 splice variants v3 and v7/8 known to play a major role in the concert of extracellular matrix degradation. The pattern of signals appeared to constitute a characteristic feature of FLSs involved in rheumatoid arthritic joint-destructive processes. These FAP-expressing FLSs with a phenotype of smooth muscle actin-positive myofibroblasts were located in the lining layer of the synovium and differ distinctly from Thy-1-expressing and non-proliferating fibroblasts of the articular matrix. The intensity of FAP-specific staining in synovial tissue from patients with RA was found to be different when compared with end-stage OA. Because expression of FAP by RA FLSs has not been described before, the findings of this study highlight a novel element in cartilage and bone destruction of arthritic joints. Moreover, the specific expression pattern qualifies FAP as a therapeutic target for inhibiting the destructive potential of fibroblast-like synovial cells. PMID:17105646

  12. Prostaglandin F2α regulates the expression of uterine activation proteins via multiple signalling pathways.

    PubMed

    Xu, Chen; You, Xingji; Liu, Weina; Sun, Qianqian; Ding, Xiaoying; Huang, Ying; Ni, Xin

    2015-01-01

    Prostaglandin F2α (PGF2A) has multiple roles in the birth process in addition to its vital contractile role. Our previous study has demonstrated that PGF2A can modulate uterine activation proteins (UAPs) in cultured pregnant human myometrial smooth muscle cells (HMSMCs). The objective of this study was to define the signalling pathways responsible for PGF2A modulation of UAPs in myometrium. It was found that PGF2A stimulated the expression of (GJA1) connexin 43 (CX43), prostaglandin endoperoxide synthase 2 (PTGS2) and oxytocin receptor (OTR) in cultured HMSMCs. The inhibitors of phospholipase C (PLC) and protein kinase C (PKC) blocked PGF2A-stimulated expression of CX43. The inhibitors of ERK, P38 and NFκB also blocked the effect of PGF2A on CX43 expression, whereas PI3K and calcineurin/nuclear factor of activated T-cells (NFAT) pathway inhibitors did not reverse the effect of PGF2A on CX43. For PTGS2 and OTR, PLC, PI3K, P38 and calcineurin/NFAT signalling pathways were involved in PGF2A action, whereas PKC and NFκB signalling were not involved. In addition, PGF2A activated NFAT, PI3K, NFκB, ERK and P38 signalling pathways. Our data suggest that PGF2A stimulates CX43, PTGS2 and OTR through divergent signalling pathways.

  13. VIP1 response elements mediate mitogen-activated protein kinase 3-induced stress gene expression

    PubMed Central

    Pitzschke, Andrea; Djamei, Armin; Teige, Markus; Hirt, Heribert

    2009-01-01

    The plant pathogen Agrobacterium tumefaciens transforms plant cells by delivering its T-DNA into the plant cell nucleus where it integrates into the plant genome and causes tumor formation. A key role of VirE2-interacting protein 1 (VIP1) in the nuclear import of T-DNA during Agrobacterium-mediated plant transformation has been unravelled and VIP1 was shown to undergo nuclear localization upon phosphorylation by the mitogen-activated protein kinase MPK3. Here, we provide evidence that VIP1 encodes a functional bZIP transcription factor that stimulates stress-dependent gene expression by binding to VIP1 response elements (VREs), a DNA hexamer motif. VREs are overrepresented in promoters responding to activation of the MPK3 pathway such as Trxh8 and MYB44. Accordingly, plants overexpressing VIP1 accumulate high levels of Trxh8 and MYB44 transcripts, whereas stress-induced expression of these genes is impaired in mpk3 mutants. Trxh8 and MYB44 promoters are activated by VIP1 in a VRE-dependent manner. VIP1 strongly enhances expression from a synthetic promoter harboring multiple VRE copies and directly interacts with VREs in vitro and in vivo. Chromatin immunoprecipitation assays of the MYB44 promoter confirm that VIP1 binding to VREs is enhanced under conditions of MPK3 pathway stimulation. These results provide molecular insight into the cellular mechanism of target gene regulation by the MPK3 pathway. PMID:19820165

  14. [Overexpression of NHE1 suppresses ABCA1 protein expression via increasing calpain activity in RAW264.7 cells].

    PubMed

    Mo, Xiangang; Wang, Lan; Guo, Jing; Hong, Wei; Long, Shiqi; Zhang, Li; Xiang, Ning; Yang, Juan

    2017-01-01

    Objective To investigate the effect of over-expressed Na(+)/H(+) exchanger 1 (NHE1) on the protein expression of adenosine three phosphate binding cassette transporter A1 (ABCA1) in RAW264.7 cells. Methods RAW264.7 cells were infected with the adenoviral vector encoding NHE1-EGFP (AdNHE1). The infected RAW264.7 cells were subjected to Western blot analysis for NHE1-EGFP fusion protein. The subcellular localization of NHE1-EGFP fusion protein was observed by confocal laser scanning microscopy. NHE1 activity was measured by the method of pH recovery in response to an acute acid pulse. Furthermore, Western blotting was performed to determine ABCA1 protein levels and calpain activity in NHE1-overexpressing RAW264.7 cells. The effect of calpain inhibitor N-acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN) on ABCA1 protein levels in the presence of TO-901317 was examined by Western blotting. Results NHE1-EGFP fusion protein was highly expressed and localized in cytoplasm and cell membrane of RAW264.7 cells infected with AdNHE1. NHE1-EGFP fusion protein reduced ABCA1 protein expression and increased calpain activity. The calpain inhibitor ALLN blocked the decrease of ABCA1 protein expression. Conclusion Overexpressed NHE1 suppresses the expression of ABCA1 protein via increasing the calpain activity in RAW264.7 cells.

  15. Expression and activity analysis of a new fusion protein targeting ovarian cancer cells.

    PubMed

    Su, Manman; Chang, Weiqin; Wang, Dingding; Cui, Manhua; Lin, Yang; Wu, Shuying; Xu, Tianmin

    2015-09-01

    The aim of the present study was to develop a new therapeutic drug to improve the prognosis of ovarian cancer patients. Human urokinase-type plasminogen activator (uPA)17-34-kunitz-type protease inhibitor (KPI) eukaryotic expression vector was constructed and recombinant human uPA17-34-KPI (rhuPA17-34-KPI) in P. pastoris was expressed. In the present study, the DNA sequences that encode uPA 17-34 amino acids were created according to the native amino acids sequence and inserted into the KPI-pPICZαC vector, which was constructed. Then, uPA17‑34-KPI-pPICZαC was transformed into P. pastoris X-33, and rhuPA17-34-KPI was expressed by induction of methanol. The bioactivities of a recombinant fusion protein were detected with trypsin inhibition analysis, and the inhibitory effects on the growth of ovarian cancer cells were identified using the TUNEL assay, in vitro wound‑healing assay and Matrigel model analysis. The results of the DNA sequence analysis of the recombinant vector uPA17-34-KPI‑pPICZα demonstrated that the DNA‑encoding human uPA 17-34 amino acids, 285-288 amino acids of amyloid precursor protein (APP) and 1-57 amino acids of KPI were correctly inserted into the pPICZαC vector. Following induction by methonal, the fusion protein with a molecular weight of 8.8 kDa was observed using SDS-PAGE and western blot analysis. RhuPA17-34-KPI was expressed in P. pastoris with a yield of 50 mg/l in a 50-ml tube. The recombinant fusion protein was able to inhibit the activity of trypsin, inhibit growth and induce apoptosis of SKOV3 cells, and inhibit the invasion and metastasis of ovarian cancer cells. By considering uPA17-34 amino acid specific binding uPAR as the targeted part of fusion protein and utilizing the serine protease inhibitor activity of KPI, it was found that the recombinant fusion protein uPA17-34-KPI inhibited the invasion and metastasis of ovarian tumors, and may therefore be regarded as effective in targeted treatment.

  16. Mitogen-activated protein kinase signaling controls basal and oncostatin M-mediated JUNB gene expression.

    PubMed

    Hicks, Mellissa J; Hu, Qiuping; Macrae, Erin; DeWille, James

    2015-05-01

    The mitogen-activated protein kinase (MAPK) pathway is aberrantly activated in many human cancers, including breast cancer. Activation of MAPK signaling is associated with the increased expression of a wide range of genes that promote cell survival, proliferation, and migration. This report investigated the influence of MAPK signaling on the regulation and expression of JUNB in human breast cancer cell lines. JUNB has been associated with tumor suppressor and oncogenic functions, with most reports describing JUNB as an oncogene in breast cancer. Our results indicated that JUNB expression is elevated in MCF10A(met), SKBR3, and MDA-MB-231 human breast cancer cell lines compared to nontransformed MCF10A mammary epithelial cells. Increased RAS/MAPK signaling in MCF10A(met) cells correlates with the increased association of RNA polymerase II (Pol II) phosphorylated on serine 5 (Pol IIser5p) with the JUNB proximal promoter. Pol IIser5p is the "transcription initiating" form of Pol II. Treatment with U0126, a MAPK pathway inhibitor, reduces Pol IIser5p association with the JUNB proximal promoter and reduces JUNB expression. Oncostatin M (OSM) enhances MAPK and STAT3 signaling and significantly induces JUNB expression. U0126 treatment reduces OSM-induced Pol IIser5p binding to the JUNB proximal promoter and JUNB expression, but does not reduce pSTAT3 levels or the association of pSTAT3 with the JUNB proximal promoter. These results demonstrate that the MAPK pathway plays a primary role in the control of JUNB gene expression by promoting the association of Pol IIser5p with the JUNB proximal promoter.

  17. Tanshinone I increases CYP1A2 protein expression and enzyme activity in primary rat hepatocytes.

    PubMed

    Lee, Wayne Y W; Zhou, Xuelin; Or, Penelope M Y; Kwan, Yiu Wa; Yeung, John H K

    2012-01-15

    This study investigated the effects of Danshen and its active ingredients on the protein expression and enzymatic activity of CYP1A2 in primary rat hepatocytes. The ethanolic extract of Danshen roots (containing mainly tanshinones) inhibited CYP1A2-catalyzed phenacetin O-deethylation (IC(50)=24.6 μg/ml) in primary rat hepatocytes while the water extract containing mainly salvianolic acid B and danshenshu had no effect. Individual tanshinones such as cryptotanshinone, dihydrotanshinone, tanshinone IIA inhibited the CYP1A2-mediated metabolism with IC(50) values at 12.9, 17.4 and 31.9 μM, respectively. After 4-day treatment of the rat hepatocytes, the ethanolic extract of Danshen and tanshinone I increased rat CYP1A2 activity by 6.8- and 5.2-fold, respectively, with a concomitant up-regulation of CYP1A2 protein level by 13.5- and 6.5-fold, respectively. CYP1A2 induction correlated with the up-regulation of mRNA level of aryl hydrocarbon receptor (AhR), which suggested a positive feedback mechanism of tanshinone I-mediated CYP1A2 induction. A formulated Danshen pill (containing mainly danshensu and salvianolic acid B and the tanshinones) up-regulated CYP1A2 protein expression and enzyme activity, but danshensu and salvianolic acid B, when used individually, did not affect CYP1A2 activity. This study was the first report on the Janus action of the tanshinones on rat CYP1A2 activity.

  18. Cyclic AMP-receptor protein activates aerobactin receptor IutA expression in Vibrio vulnificus.

    PubMed

    Kim, Choon-Mee; Kim, Seong-Jung; Shin, Sung-Heui

    2012-04-01

    The ferrophilic bacterium Vibrio vulnificus can utilize the siderophore aerobactin of Escherichia coli for iron acquisition via its specific receptor IutA. This siderophore piracy by V. vulnificus may contribute to its survival and proliferation, especially in mixed bacterial environments. In this study, we examined the effects of glucose, cyclic AMP (cAMP), and cAMP-receptor protein (Crp) on iutA expression in V. vulnificus. Glucose dose-dependently repressed iutA expression. A mutation in cya encoding adenylate cyclase required for cAMP synthesis severely repressed iutA expression, and this change was recovered by in trans complementing cya or the addition of exogenous cAMP. Furthermore, a mutation in crp encoding Crp severely repressed iutA expression, and this change was recovered by complementing crp. Accordingly, glucose deprivation under iron-limited conditions is an environmental signal for iutA expression, and Crp functions as an activator that regulates iutA expression in response to glucose availability.

  19. Carnitine Palmitoyltransferase 1 Increases Lipolysis, UCP1 Protein Expression and Mitochondrial Activity in Brown Adipocytes

    PubMed Central

    Calderon-Dominguez, María; Sebastián, David; Fucho, Raquel; Weber, Minéia; Mir, Joan F.; García-Casarrubios, Ester; Obregón, María Jesús; Zorzano, Antonio; Valverde, Ángela M.; Serra, Dolors

    2016-01-01

    The discovery of active brown adipose tissue (BAT) in adult humans and the fact that it is reduced in obese and diabetic patients have put a spotlight on this tissue as a key player in obesity-induced metabolic disorders. BAT regulates energy expenditure through thermogenesis; therefore, harnessing its thermogenic fat-burning power is an attractive therapeutic approach. We aimed to enhance BAT thermogenesis by increasing its fatty acid oxidation (FAO) rate. Thus, we expressed carnitine palmitoyltransferase 1AM (CPT1AM), a permanently active mutant form of CPT1A (the rate-limiting enzyme in FAO), in a rat brown adipocyte (rBA) cell line through adenoviral infection. We found that CPT1AM-expressing rBA have increased FAO, lipolysis, UCP1 protein levels and mitochondrial activity. Additionally, enhanced FAO reduced the palmitate-induced increase in triglyceride content and the expression of obese and inflammatory markers. Thus, CPT1AM-expressing rBA had enhanced fat-burning capacity and improved lipid-induced derangements. This indicates that CPT1AM-mediated increase in brown adipocytes FAO may be a new approach to the treatment of obesity-induced disorders. PMID:27438137

  20. Bone morphogenetic proteins induce the expression of noggin, which limits their activity in cultured rat osteoblasts.

    PubMed Central

    Gazzerro, E; Gangji, V; Canalis, E

    1998-01-01

    Bone morphogenetic proteins (BMPs) induce the differentiation of cells of the osteoblastic lineage and enhance the function of the osteoblast. Growth factors are regulated by binding proteins, but there is no information about binding proteins for BMPs in skeletal cells. Noggin specifically binds BMPs, but its expression by cells of the osteoblastic lineage has not been reported. We tested for the expression of noggin and its induction by BMP-2 in cultures of osteoblast-enriched cells from 22-d-old fetal rat calvariae (Ob cells). BMP-2 caused a time- and dose-dependent increase in noggin mRNA and polypeptide levels, as determined by Northern and Western blot analyses. The effects of BMP-2 on noggin transcripts were dependent on protein, but independent of DNA synthesis. BMP-2 increased the rates of noggin transcription as determined by nuclear run-on assays. BMP-4, BMP-6, and TGF-beta1 increased noggin mRNA in Ob cells, but basic fibroblast growth factor, platelet- derived growth factor BB, and IGF-I did not. Noggin decreased the stimulatory effects of BMPs on DNA and collagen synthesis and alkaline phosphatase activity in Ob cells. In conclusion, BMPs induce noggin transcription in Ob cells, a probable mechanism to limit BMP action in osteoblasts. PMID:9854046

  1. Activity, expression and function of a second Drosophila protein kinase a catalytic subunit gene

    SciTech Connect

    Melendez, A.; Li, W.; Kalderon, D.

    1995-12-01

    The DC2 was isolated previously on the basis of sequence similarity to DC0, the major Drosophila protein kinase A (PKA) catalytic subunit gene. We show here that the 67-kD Drosophila DC2 protein behaves as a PKA catalytic subunit in vitro. DC2 is transcribed in mesodermal anlagen of early embryos. This expression depends on dorsal but on neither twist nor snail activity. DC2 transcriptional fusions mimic this embryonic expression and are also expressed in subsets of cells in the optic lamina, wing disc and leg discs of third instar larvae. A saturation screen of a small deficiency interval containing DC2 for recessive lethal mutations yielded no DC2 alleles. We therefore isolated new deficiencies to generate deficiency trans-heterozygotes that lacked DC2 activity. These animals were viable and fertile. The absence of DC2 promoter did not efficiently rescue a variety of DC0 mutant phenotypes. These observations indicate that DC2 is not an essential gene and is unlikely to be functionally redundant with DC0, which has multiple unique functions during development. 62 refs., 10 figs., 2 tabs.

  2. Prophenoloxidase activation and antimicrobial peptide expression induced by the recombinant microbe binding protein of Manduca sexta.

    PubMed

    Wang, Yang; Jiang, Haobo

    2017-04-01

    Manduca sexta microbe binding protein (MBP) is a member of the β-1,3-glucanase-related protein superfamily that includes Gram-negative bacteria-binding proteins (GNBPs), β-1,3-glucan recognition proteins (βGRPs), and β-1,3-glucanases. Our previous and current studies showed that the purified MBP from baculovirus-infected insect cells had stimulated prophenoloxidase (proPO) activation in the hemolymph of naïve and immune challenged larvae and that supplementation of the exogenous MBP and peptidoglycans (PGs) had caused synergistic increases in PO activity. To explore the underlying mechanism, we separated by SDS-PAGE naïve and induced larval plasma treated with buffer or MBP and detected on immunoblots changes in intensity and/or mobility of hemolymph (serine) proteases [HP14, HP21, HP6, HP8, proPO-activating proteases (PAPs) 1-3] and their homologs (SPH1, SPH2). In a nickel pull-down assay, we observed association of MBP with proHP14 (slightly), βGRP2, PG recognition protein-1 (PGRP1, indirectly), SPH1, SPH2, and proPO2. Further experiments indicated that diaminopimelic acid (DAP) or Lys PG, MBP, PGRP1, and proHP14 together trigger the proPO activation system in a Ca(2+)-dependent manner. Injection of the recombinant MBP into the 5th instar naïve larvae significantly induced the expression of several antimicrobial peptide genes, revealing a possible link between HP14 and immune signal transduction. Together, these results suggest that the recognition of Gram-negative or -positive bacteria via their PGs induces the melanization and Toll pathways in M. sexta.

  3. Heat Shock Protein-70 Expression in Vitiligo and its Relation to the Disease Activity

    PubMed Central

    Doss, Reham William; El-Rifaie, Abdel-Aziz A; Abdel-Wahab, Amr M; Gohary, Yasser M; Rashed, Laila A

    2016-01-01

    Background: Vitiligo is a progressive depigmenting disorder characterized by the loss of functional melanocytes from the epidermis. The etiopathogenesis of vitiligo is still unclear. Heat shock proteins (HSPs) are prime candidates to connect stress to the skin. HSPs were found to be implicated in autoimmune diseases such as rheumatoid arthritis and other skin disorders as psoriasis. Aim and Objectives: The aim of this study was to map the level of HSP-70 in vitiligo lesions to declare its role in the pathogenesis and activity of vitiligo. Materials and Methods: The study included thirty patients with vitiligo and 30 age- and sex-matched healthy controls. Vitiligo patients were divided as regards to the disease activity into highly active, moderately active, and inactive vitiligo groups. Skin biopsies were taken from the lesional and nonlesional skin of patients and from the normal skin of the controls. HSP-70 messenger RNA (mRNA) expression was estimated using quantitative real-time polymerase chain reaction. Results: Our analysis revealed a significantly higher expression of HSP-70 mRNA in lesional skin biopsies from vitiligo patients compared to nonlesional skin biopsies from vitiligo patients (P < 0.001) and compared to skin biopsies from healthy controls (P < 0.001). The level of HSP-70 was not found to be correlated with age, sex, or disease duration. The expression of HSP-70 was correlated with the disease activity and patients with active vitiligo showed higher mean HSP-70 level compared to those with inactive disease. Conclusions: HSP-70 plays a role in the pathogenesis of vitiligo and may enhance the immune response in active disease. PMID:27512186

  4. Leukocyte trafficking-associated vascular adhesion protein 1 is expressed and functionally active in atherosclerotic plaques

    PubMed Central

    Silvola, Johanna M. U.; Virtanen, Helena; Siitonen, Riikka; Hellberg, Sanna; Liljenbäck, Heidi; Metsälä, Olli; Ståhle, Mia; Saanijoki, Tiina; Käkelä, Meeri; Hakovirta, Harri; Ylä-Herttuala, Seppo; Saukko, Pekka; Jauhiainen, Matti; Veres, Tibor Z.; Jalkanen, Sirpa; Knuuti, Juhani; Saraste, Antti; Roivainen, Anne

    2016-01-01

    Given the important role of inflammation and the potential association of the leukocyte trafficking-associated adhesion molecule vascular adhesion protein 1 (VAP-1) with atherosclerosis, this study examined whether functional VAP-1 is expressed in atherosclerotic lesions and, if so, whether it could be targeted by positron emission tomography (PET). First, immunohistochemistry revealed that VAP-1 localized to endothelial cells of intra-plaque neovessels in human carotid endarterectomy samples from patients with recent ischemic symptoms. In low-density lipoprotein receptor-deficient mice expressing only apolipoprotein B100 (LDLR−/−ApoB100/100), VAP-1 was expressed on endothelial cells lining inflamed atherosclerotic lesions; normal vessel walls in aortas of C57BL/6N control mice were VAP-1-negative. Second, we discovered that the focal uptake of VAP-1 targeting sialic acid-binding immunoglobulin-like lectin 9 based PET tracer [68Ga]DOTA-Siglec-9 in atherosclerotic plaques was associated with the density of activated macrophages (r = 0.58, P = 0.022). As a final point, we found that the inhibition of VAP-1 activity with small molecule LJP1586 decreased the density of macrophages in inflamed atherosclerotic plaques in mice. Our results suggest for the first time VAP-1 as a potential imaging target for inflamed atherosclerotic plaques, and corroborate VAP-1 inhibition as a therapeutic approach in the treatment of atherosclerosis. PMID:27731409

  5. The roles of AMY1 copies and protein expression in human salivary α-amylase activity.

    PubMed

    Yang, Ze-Min; Lin, Jing; Chen, Long-Hui; Zhang, Min; Chen, Wei-Wen; Yang, Xiao-Rong

    2015-01-01

    Salivary α-amylase (sAA) activity has been extensively investigated in nutrition and psychology. But few studies were performed to assess the role played by sAA gene (AMY1) copies and protein expression in basal and stimulus-induced sAA activity. The sAA activity, amount and AMY1 copy number were determined from 184 saliva samples pre- and post-citric acid stimulation. Our findings showed that citric acid could induce significant increase in sAA activity, total sAA amount, and glycosylated sAA amount, among which the glycosylated sAA amount had the largest response. The correlation analysis showed that AMY1 copy number, total sAA amount and AMY1 copy number×total sAA amount had significantly positive and successively increasing correlations with sAA activity in unstimulated and stimulated saliva, respectively, and furthermore, we observed higher correlations in unstimulated saliva when compared with the corresponding correlations in stimulated saliva. We also observed significant correlations between glycosylated sAA amount and sAA activity in unstimulated and stimulated saliva, respectively. Interestingly, the correlations were higher in stimulated saliva than in unstimulated saliva, and the correlations between glycosylated sAA amount and sAA activity were higher than that of between total sAA amount and sAA activity in stimulated saliva. Moreover, total sAA amount ratio and glycosylated sAA amount ratio showed significantly positive correlation with sAA activity ratio. AMY1 copy number had no correlation with sAA activity ratio. These findings suggested that AMY1 copy number and sAA amount played crucial roles in sAA activity; however, the roles were attenuated after stimulation due to fortified release of glycosylated sAA.

  6. P-gp Protein Expression and Transport Activity in Rodent Seizure Models and Human Epilepsy.

    PubMed

    Hartz, Anika M S; Pekcec, Anton; Soldner, Emma L B; Zhong, Yu; Schlichtiger, Juli; Bauer, Bjoern

    2017-03-02

    A cure for epilepsy is currently not available, and seizure genesis, seizure recurrence, and resistance to antiseizure drugs remain serious clinical problems. Studies show that the blood-brain barrier is altered in animal models of epilepsy and in epileptic patients. In this regard, seizures increase expression of blood-brain barrier efflux transporters such as P-glycoprotein (P-gp), which is thought to reduce brain uptake of antiseizure drugs, and thus, contribute to antiseizure drug resistance. The goal of the current study was to assess the viability of combining in vivo and ex vivo preparations of isolated brain capillaries from animal models of seizures and epilepsy as well as from patients with epilepsy to study P-gp at the blood-brain barrier. Exposing isolated rat brain capillaries to glutamate ex vivo upregulated P-gp expression to levels that were similar to those in capillaries isolated from rats that had status epilepticus or chronic epilepsy. Moreover, the fold-increase in P-gp protein expression seen in animal models is consistent with the fold-increase in P-gp observed in human brain capillaries isolated from patients with epilepsy compared to age-matched control individuals. Overall, the in vivo/ex vivo approach presented here allows detailed analysis of the mechanisms underlying seizure-induced changes of P-gp expression and transport activity at the blood-brain barrier. This approach can be extended to other blood-brain barrier proteins that might contribute to drug-resistant epilepsy or other CNS disorders as well.

  7. p38 mitogen-activated protein kinase plays a key role in regulating MAPKAPK2 expression

    SciTech Connect

    Sudo, Tatsuhiko . E-mail: sudo@riken.jp; Kawai, Kayoko; Matsuzaki, Hiroshi; Osada, Hiroyuki

    2005-11-18

    One of three major families of the mitogen-activated kinases (MAPK), p38 as well as JNK, has been shown to transduce extracellular stress stimuli into cellular responses by phospho-relay cascades. Among p38 families, p38{alpha} is a widely characterized isoform and the biological phenomena are explained by its kinase activity regulating functions of its downstream substrates. However, its specific contributions to each phenomenon are yet not fully elucidated. For better understanding of the role of MAPKs, especially p38{alpha}, we utilized newly established mouse fibroblast cell lines originated from a p38{alpha} null mouse, namely, a parental cell line without p38{alpha} gene locus, knockout of p38{alpha} (KOP), Zeosin-resistant (ZKOP), revertant of p38{alpha} (RKOP), and Exip revertant (EKOP). EKOP is smaller in size but grows faster than the others. Although comparable amounts of ERK and JNK are expressed in each cell line, ERK is highly phosphorylated in EKOP even in normal culture conditions. Serum stimulation after serum starvation led to ERK phosphorylation in RKOP and ZKOP, but not in EKOP as much. On the contrary, relative phosphorylation level of JNK to total JNK in response to UV was low in RKOP. And its phosphorylation as well as total JNK is slightly lower in EKOP. RKOP is less sensitive to UV irradiation as judged by the survival rate. Stress response upon UV or sorbitol stimuli, leading to mitogen activate protein kinase activated kinase 2 (MAPKAPK2) phosphorylation, was only observed in RKOP. Further experiments reveal that MAPKAPK2 expression is largely suppressed in ZKOP and EKOP. Its expression was recovered by re-introduction of p38{alpha}. The loss of MAPKAPK2 expression accompanied by the defect of p38{alpha} is confirmed in an embryonic extract prepared from p38{alpha} null mice. These data demonstrate that p38 signal pathway is regulated not only by phosphorylation but also by modulation of the expression of its component. Together, we have

  8. Expression and activity of multidrug resistance proteins in mature endothelial cells and their precursors: A challenging correlation

    PubMed Central

    Bielawska-Pohl, Aleksandra; Wojtowicz, Karolina; Jura, Roksana; Paprocka, Maria; Wojdat, Elżbieta; Kozłowska, Urszula; Klimczak, Aleksandra; Grillon, Catherine; Kieda, Claudine; Duś, Danuta

    2017-01-01

    Active cellular transporters of harmful agents—multidrug resistance (mdr) proteins—are present in tumor, stem and endothelial cells, among others. While mdr proteins are broadly studied in tumor cells, their role in non-tumor cells and the significance of their action not connected with removal of harmful xenobiotics is less extensively documented. Proper assessment of mdr proteins expression is difficult. Mdr mRNA presence is most often evaluated but that does not necessarily correlate with the protein level. The protein expression itself is difficult to determine; usually cells with mdr overexpression are studied, not cells under physiological conditions, in which a low expression level of mdr protein is often insufficient for detection in vitro. Various methods are used to identify mdr mRNA and protein expression, together with functional tests demonstrating their biological drug transporting activities. Data comparing different methods of investigating expression of mdr mRNAs and their corresponding proteins are still scarce. In this article we present the results of a study concerning mdr mRNA and protein expression. Our goal was to search for the best method to investigate the expression level and functional activity of five selected mdr proteins—MDR1, BCRP, MRP1, MRP4 and MRP5—in established in vitro cell lines of human endothelial cells (ECs) and their progenitors. Endothelial cells demonstrated mdr presence at the mRNA level, which was not always confirmed at the protein level or in functional tests. Therefore, several different assays had to be applied for evaluation of mdr proteins expression and functions in endothelial cells. Among them functional tests seemed to be the most conclusive, although not very specific. PMID:28212450

  9. Protein expression-yeast.

    PubMed

    Nielsen, Klaus H

    2014-01-01

    Yeast is an excellent system for the expression of recombinant eukaryotic proteins. Both endogenous and heterologous proteins can be overexpressed in yeast (Phan et al., 2001; Ton and Rao, 2004). Because yeast is easy to manipulate genetically, a strain can be optimized for the expression of a specific protein. Many eukaryotic proteins contain posttranslational modifications that can be performed in yeast but not in bacterial expression systems. In comparison with mammalian cell culture expression systems, growing yeast is both faster and less expensive, and large-scale cultures can be performed using fermentation. While several different yeast expression systems exist, this chapter focuses on the budding yeast Saccharomyces cerevisiae and will briefly describe some options to consider when selecting vectors and tags to be used for protein expression. Throughout this chapter, the expression and purification of yeast eIF3 is shown as an example alongside a general scheme outline.

  10. Suppressed expression of mitogen-activated protein kinases in hyperthermia induced defective neural tube.

    PubMed

    Zhang, Tianliang; Leng, Zhaoting; Liu, Wenjing; Wang, Xia; Yan, Xue; Yu, Li

    2015-05-06

    Neural tube defects (NTDs) are common congenital malformations. Mitogen-activated protein kinases (MAPKs) pathway is involved in many physiological processes. HMGB1 has been showed closely associated with neurulation and NTDs induced by hyperthermia and could activate MAPKs pathway. Since hyperthermia caused increased activation of MAPKs in many systems, the present study aims to investigate whether HMGB1 contributes to hyperthermia induced NTDs through MAPKs pathway. The mRNA levels of MAPKs and HMGB1 between embryonic day 8.5 and 10 (E8.5-10) in hyperthermia induced defective neural tube were detected by real-time quantitative polymerase chain reaction (qPCR). By immunofluorescence and western blotting, the expressions of HMGB1 and phosphorylated MAPKs (ERK1/2, JNK and p38) in neural tubes after hyperthermia were studied. The mRNA levels of MAPKs and HMGB1, as well as the expressions of HMGB1 along with phosphorylated JNK, p38 and ERK, were downregulated in NTDs groups induced by hyperthermia compared with control. The findings suggested that HMGB1 may contribute to hyperthermia induced NTDs formation through decreased cell proliferation due to inhibited phosphorylated ERK1/2 MAPK.

  11. Fragile X mental retardation protein controls ion channel expression and activity.

    PubMed

    Ferron, Laurent

    2016-10-15

    Fragile X-associated disorders are a family of genetic conditions resulting from the partial or complete loss of fragile X mental retardation protein (FMRP). Among these disorders is fragile X syndrome, the most common cause of inherited intellectual disability and autism. FMRP is an RNA-binding protein involved in the control of local translation, which has pleiotropic effects, in particular on synaptic function. Analysis of the brain FMRP transcriptome has revealed hundreds of potential mRNA targets encoding postsynaptic and presynaptic proteins, including a number of ion channels. FMRP has been confirmed to bind voltage-gated potassium channels (Kv 3.1 and Kv 4.2) mRNAs and regulates their expression in somatodendritic compartments of neurons. Recent studies have uncovered a number of additional roles for FMRP besides RNA regulation. FMRP was shown to directly interact with, and modulate, a number of ion channel complexes. The sodium-activated potassium (Slack) channel was the first ion channel shown to directly interact with FMRP; this interaction alters the single-channel properties of the Slack channel. FMRP was also shown to interact with the auxiliary β4 subunit of the calcium-activated potassium (BK) channel; this interaction increases calcium-dependent activation of the BK channel. More recently, FMRP was shown to directly interact with the voltage-gated calcium channel, Cav 2.2, and reduce its trafficking to the plasma membrane. Studies performed on animal models of fragile X syndrome have revealed links between modifications of ion channel activity and changes in neuronal excitability, suggesting that these modifications could contribute to the phenotypes observed in patients with fragile X-associated disorders.

  12. Spindle Assembly Checkpoint Protein Cdc20 Transcriptionally Activates Expression of Ubiquitin Carrier Protein UbcH10*

    PubMed Central

    Nath, Somsubhra; Banerjee, Taraswi; Sen, Debrup; Das, Tania; Roychoudhury, Susanta

    2011-01-01

    The spindle assembly checkpoint (SAC) ensures accurate segregation of chromosomes by monitoring kinetochore attachment of spindles during mitosis. Proper progression of mitosis depends on orderly ubiquitination and subsequent degradation of various mitotic inhibitors. At the molecular level, upon removal of SAC, Cdc20 activates E3 ubiquitin ligase anaphase-promoting complex/cyclosome that, along with E2 ubiquitin-conjugating enzyme UbcH10, executes this function. Both Cdc20 and UbcH10 are overexpressed in many cancer types and are associated with defective SAC function leading to chromosomal instability. The precise mechanism of correlated overexpression of these two proteins remains elusive. We show that Cdc20 transcriptionally up-regulates UbcH10 expression. The WD40 domain of Cdc20 is required for this activity. Physical interaction between Cdc20 and anaphase-promoting complex/cyclosome-CBP/p300 complex and its subsequent recruitment to the UBCH10 promoter are involved in this transactivation process. This transcriptional regulatory function of Cdc20 was observed to be cell cycle-specific. We hypothesize that this co-regulated overexpression of both proteins contributes to chromosomal instability. PMID:21454660

  13. Ultrabithorax protein is necessary but not sufficient for full activation of decapentaplegic expression in the visceral mesoderm.

    PubMed Central

    Sun, B; Hursh, D A; Jackson, D; Beachy, P A

    1995-01-01

    To elucidate the mechanisms by which homeotic selector (HOM) genes specify the unique features of Drosophila segments, we have analyzed the regulation of decapentaplegic (dpp), a transforming growth factor (TGF)-beta superfamily member, and have found that the Ultrabithorax (Ubx) HOM protein directly activates dpp expression in parasegment 7 (PS7) of the embryonic visceral mesoderm. Other factors are also required, including one that appears to act through homeodomain protein binding sites and may be encoded by extradenticle (exd). The exd protein binds in a highly co-operative manner to regulatory sequences mediating PS7-specific dpp expression, consistent with a genetic requirement for exd function in normal visceral mesoderm expression of dpp. A second mechanism contributing to PS7 expression of dpp appears not to require Ubx protein directly, and involves a general visceral mesoderm enhancer coupled to a spatially specific repression element. Thus, even in an apparently simple case where visceral mesoderm expression of the dpp target gene mirrors that of the Ubx HOM protein, full activation by Ubx protein requires at least one additional factor. In addition, a distinct regulatory mode not directly involving Ubx protein also appears to contribute to PS7-specific expression. Images PMID:7859741

  14. Design and construction of an in-plant activation cassette for transgene expression and recombinant protein production in plants.

    PubMed

    Dugdale, Benjamin; Mortimer, Cara L; Kato, Maiko; James, Tess A; Harding, Robert M; Dale, James L

    2014-05-01

    Virus-based transgene expression systems have become particularly valuable for recombinant protein production in plants. The dual-module in-plant activation (INPACT) expression platform consists of a uniquely designed split-gene cassette incorporating the cis replication elements of Tobacco yellow dwarf geminivirus (TYDV) and an ethanol-inducible activation cassette encoding the TYDV Rep and RepA replication-associated proteins. The INPACT system is essentially tailored for recombinant protein production in stably transformed plants and provides both inducible and high-level transient transgene expression with the potential to be adapted to diverse crop species. The construction of a novel split-gene cassette, the inducible nature of the system and the ability to amplify transgene expression via rolling-circle replication differentiates this system from other DNA- and RNA-based virus vector systems used for stable or transient recombinant protein production in plants. Here we provide a detailed protocol describing the design and construction of a split-gene INPACT cassette, and we highlight factors that may influence optimal activation and amplification of gene expression in transgenic plants. By using Nicotiana tabacum, the protocol takes 6-9 months to complete, and recombinant proteins expressed using INPACT can accumulate to up to 10% of the leaf total soluble protein.

  15. Inhibition of Protein Farnesylation Arrests Adipogenesis and Affects PPARγ Expression and Activation in Differentiating Mesenchymal Stem Cells

    PubMed Central

    Rivas, Daniel; Akter, Rahima; Duque, Gustavo

    2007-01-01

    Protein farnesylation is required for the activation of multiple proteins involved in cell differentiation and function. In white adipose tissue protein, farnesylation has shown to be essential for the successful differentiation of preadipocytes into adipocytes. We hypothesize that protein farnesylation is required for PPARγ2 expression and activation, and therefore for the differentiation of human mesenchymal stem cells (MSCs) into adipocytes. MSCs were plated and induced to differentiate into adipocytes for three weeks. Differentiating cells were treated with either an inhibitor of farnesylation (FTI-277) or vehicle alone. The effect of inhibition of farnesylation in differentiating adipocytes was determined by oil red O staining. Cell survival was quantified using MTS Formazan. Additionally, nuclear extracts were obtained and prelamin A, chaperon protein HDJ-2, PPARγ, and SREBP-1 were determined by western blot. Finally, DNA binding PPARγ activity was determined using an ELISA-based PPARγ activation quantification method. Treatment with an inhibitor of farnesylation (FTI-277) arrests adipogenesis without affecting cell survival. This effect was concomitant with lower levels of PPARγ expression and activity. Finally, accumulation of prelamin A induced an increased proportion of mature SREBP-1 which is known to affect PPARγ activity. In summary, inhibition of protein farnesylation arrests the adipogenic differentiation of MSCs and affects PPARγ expression and activity. PMID:18274630

  16. Inhibition of Protein Farnesylation Arrests Adipogenesis and Affects PPARgamma Expression and Activation in Differentiating Mesenchymal Stem Cells.

    PubMed

    Rivas, Daniel; Akter, Rahima; Duque, Gustavo

    2007-01-01

    Protein farnesylation is required for the activation of multiple proteins involved in cell differentiation and function. In white adipose tissue protein, farnesylation has shown to be essential for the successful differentiation of preadipocytes into adipocytes. We hypothesize that protein farnesylation is required for PPARgamma2 expression and activation, and therefore for the differentiation of human mesenchymal stem cells (MSCs) into adipocytes. MSCs were plated and induced to differentiate into adipocytes for three weeks. Differentiating cells were treated with either an inhibitor of farnesylation (FTI-277) or vehicle alone. The effect of inhibition of farnesylation in differentiating adipocytes was determined by oil red O staining. Cell survival was quantified using MTS Formazan. Additionally, nuclear extracts were obtained and prelamin A, chaperon protein HDJ-2, PPARgamma, and SREBP-1 were determined by western blot. Finally, DNA binding PPARgamma activity was determined using an ELISA-based PPARgamma activation quantification method. Treatment with an inhibitor of farnesylation (FTI-277) arrests adipogenesis without affecting cell survival. This effect was concomitant with lower levels of PPARgamma expression and activity. Finally, accumulation of prelamin A induced an increased proportion of mature SREBP-1 which is known to affect PPARgamma activity. In summary, inhibition of protein farnesylation arrests the adipogenic differentiation of MSCs and affects PPARgamma expression and activity.

  17. Transient changes of enzyme activities and expression of stress proteins in the small intestine of piglets after weaning.

    PubMed

    Tao, Xin; Xu, Ziwei; Men, Xiaoming

    2015-01-01

    To determine the transient effects of weaning on the small intestine, 16 piglets were slaughtered at days 0, 1, 4 and 7 after weaning. Jejunal samples were collected to examine different enzyme activities and mRNA expressions of two stress protein families, namely, heat-shock proteins (HSP) and trefoil factors (TFF). Results showed that the activities of ceruloplasmin, alkaline phosphatase and lactate dehydrogenase, were significantly changed at Day 1 and/or Day 4. The mRNA expressions of HSP10, HSP60 and HSP90 showed a pattern of increased expression with time after weaning. Expression significantly differed between Day 0 and Day 7 after weaning. The mRNA expression of HSP70 was significantly increased on Day 1 only. Similarly, the mRNA expressions of TFF1 and TFF2 were significantly increased on Day 7 compared with those on Day 0. Expression of TFF3 was not affected by time after weaning. In conclusion, the present study indicated that weaning induced transient injury to small intestinal morphology and function. Particularly it changed enzyme activities and gene expression of stress proteins in the small intestine of piglets. At first time, a change in the gene expression of HSP10 and a gene overexpression of TFF1 in the small intestine of piglets after weaning was found.

  18. Fasting alters protein expression of AMP-activated protein kinase in the hypothalamus of broiler chicks (Gallus gallus domesticus).

    PubMed

    Song, Zhigang; Liu, Lei; Yue, Yunshuang; Jiao, Hongchao; Lin, Hai; Sheikhahmadi, Ardashir; Everaert, Nadia; Decuypere, Eddy; Buyse, Johan

    2012-09-15

    An experiment was conducted to investigate the effects of fasting and re-feeding on hypothalamic 5'-AMP-activated protein kinase (AMPK) levels and (an)orexigenic neuropeptides. Male Arbor Acres chicks (7-day-old, n=160) were allocated to four equal treatment groups: control chicks (fed ad libitum for 48 h, C48), chicks that were fasted for 48 h (F48), chicks that were first fasted for 48 h and then re-fed for 24h (F48C24), and chicks that were fed ad libitum for 72h (C72). Fasting for 48 h significantly (P<0.05) increased the ratio of phosphorylated AMPKα to total AMPKα and phosphorylated LKB1 to total LKB1, whereas re-feeding for 24h reduced these ratios to that of the ad libitum fed C72 chicks. The gene expressions of agouti-related peptide (AgRP), neuropeptide Y (NPY), melanocortin receptor 4, melanin-concentrating hormone, prepro-orexins and carnitine palmitoyltransferase-1 were significantly (P<0.05) increased in the fasted chicks relative to the ad libitum fed C48 group. The gene expression of pro-opiomelanocortin (POMC), as well as cocaine- and amphetamine-regulated transcript (CART) was not affected by the nutritional status. Fasting significantly (P<0.05) decreased the mRNA levels of fatty acid synthase (FAS) and sterol regulatory element binding protein-1 (SREBP-1). The results suggest that the LKB1/AMPK signal pathway is involved in the energy homeostasis of fasted chicks, and its possible role in feed intake regulation might be mediated by the AgRP/NPY rather than the POMC/CART pathway.

  19. Changes of expression of stretch-activated potassium channel TREK-1 mRNA and protein in hypertrophic myocardium.

    PubMed

    Cheng, Longxian; Su, Fengcheng; Ripen, Nsenga; Fan, Hong; Huang, Kai; Wang, Min; Peng, Hongyu; Mei, Chunli; Zhao, Fang; Liao, Yuhua

    2006-01-01

    The expression of stretch-activated potassium channel TREK-1 mRNA and protein of hypertrophic myocardium was measured. Using a model of hypertrophy induced by coarctation of abdominal aorta in male Wistar rats, the expression of TREK-1 mRNA and protein was detected by using semi-quantitative RT PCR and Western blot respectively. At 4th and 8th week after constriction of the abdominal aorta, rats developed significant left ventricular hypertrophy. As compared to sham-operated group, stretch-activated potassium channel TREK-1 mRNA was strongly expressed and protein was up-regulated in operation groups (P < 0.05). It was concluded that the expression of TREK-1 was up-regulated in hypertrophic myocardium induced by chronic pressure overload in Wistar rats.

  20. AMP-activated protein kinase counteracted the inhibitory effect of glucose on the phosphoenolpyruvate carboxykinase gene expression in rat hepatocytes.

    PubMed

    Hubert, A; Husson, A; Chédeville, A; Lavoinne, A

    2000-09-22

    The effect of AMP-activated protein kinase (AMPK) in the regulation of the phosphoenolpyruvate carboxykinase (PEPCK) gene expression was studied in isolated rat hepatocytes. Activation of AMPK by AICAR counteracted the inhibitory effect of glucose on the PEPCK gene expression, both at the mRNA and the transcriptional levels. It is proposed that a target for AMPK is involved in the inhibitory effect of glucose on PEPCK gene transcription.

  1. A role for protein phosphatase 2A in regulating p38 mitogen activated protein kinase activation and tumor necrosis factor-alpha expression during influenza virus infection.

    PubMed

    Law, Anna H Y; Tam, Alex H M; Lee, Davy C W; Lau, Allan S Y

    2013-04-02

    Influenza viruses of avian origin continue to pose pandemic threats to human health. Some of the H5N1 and H9N2 virus subtypes induce markedly elevated cytokine levels when compared with the seasonal H1N1 virus. We previously showed that H5N1/97 hyperinduces tumor necrosis factor (TNF)-alpha through p38 mitogen activated protein kinase (MAPK). However, the detailed mechanisms of p38MAPK activation and TNF-alpha hyperinduction following influenza virus infections are not known. Negative feedback regulations of cytokine expression play important roles in avoiding overwhelming production of proinflammatory cytokines. Here we hypothesize that protein phosphatases are involved in the regulation of cytokine expressions during influenza virus infection. We investigated the roles of protein phosphatases including MAPK phosphatase-1 (MKP-1) and protein phosphatase type 2A (PP2A) in modulating p38MAPK activation and downstream TNF-alpha expressions in primary human monocyte-derived macrophages (PBMac) infected with H9N2/G1 or H1N1 influenza virus. We demonstrate that H9N2/G1 virus activated p38MAPK and hyperinduced TNF-alpha production in PBMac when compared with H1N1 virus. H9N2/G1 induced PP2A activity in PBMac and, with the treatment of a PP2A inhibitor, p38MAPK phosphorylation and TNF-alpha production were further increased in the virus-infected macrophages. However, H9N2/G1 did not induce the expression of PP2A indicating that the activation of PP2A is not mediated by p38MAPK in virus-infected PBMac. On the other hand, PP2A may not be the targets of H9N2/G1 in the upstream of p38MAPK signaling pathways since H1N1 also induced PP2A activation in primary macrophages. Our results may provide new insights into the control of cytokine dysregulation.

  2. Prostaglandin E2 stimulates S100A8 expression by activating protein kinase A and CCAAT/enhancer-binding-protein-beta in prostate cancer cells.

    PubMed

    Miao, Lin; Grebhardt, Sina; Shi, Jiandang; Peipe, Isabelle; Zhang, Ju; Mayer, Doris

    2012-11-01

    S100A8 and S100A9 are strongly expressed in epithelial cells of human prostate cancer. However, the regulation of their expression is unclear. Here we show that S100A8 and to a lesser extent S100A9 mRNA expression is induced by prostaglandin E2 in a dose and time-dependent manner in PC-3 prostate cancer cells as well as in BPH-1 benign prostatic epithelial cells. Prostanoid receptor EP2 antagonist AH6809 and EP4 antagonist AH23848, as well as protein kinase A inhibitor H89, inhibited prostaglandin E2 mediated increase in S100A8 mRNA expression as well as promoter activity. Sequence analysis detected a potential binding site of the transcription factor CCAAT/enhancer-binding-protein-beta within the proximal S100A8 promoter. CCAAT/enhancer-binding-protein-beta overexpression increased S100A8 mRNA and protein expression as well as its promoter activity. The latter was prevented by mutation of the potential CCAAT/enhancer-binding-protein-beta binding site within the S100A8 promoter. Chromatin immunoprecipitation revealed increased binding of CCAAT/enhancer-binding-protein-beta to the S100A8 promoter in prostaglandin E2 treated cells. Knockdown of CCAAT/enhancer-binding-protein-beta by siRNA blocked prostaglandin E2 mediated induction of S100A8 promoter activity and mRNA expression. Our results indicate that in prostate cancer cells, S100A8 expression is stimulated by prostaglandin E2 via EP2 and EP4 receptors through activation of the protein kinase A signaling pathway and subsequent stimulation of CCAAT/enhancer-binding-protein-beta binding to the S100A8 promoter.

  3. Expression of Fibroblast Activating Protein and Correlation with Histological Grade, Mitotic Index and Ki67 Expression in Canine Mast Cell Tumours.

    PubMed

    Giuliano, A; Dos Santos Horta, R; Constantino-Casas, F; Hoather, T; Dobson, J

    2017-01-01

    Fibroblast activating protein (FAP) is a membrane serine protease expressed by activated fibroblasts, particularly tumour associated fibroblasts (TAFs). FAP expression has not been reported in canine mast cell tumours (MCTs). The objective of this study was to evaluate the expression of FAP in TAFs and its correlation with histological grade, mitotic index and Ki67 expression in canine MCTs. FAP expression was evaluated by immunohistochemistry (IHC) in 30 canine MCTs. Twenty-eight (90%) of the MCTs expressed FAP in the stroma, 16 cases showed low to intermediate FAP score and 14 cases had a high FAP score. FAP was correlated positively with both Patnaik (P = 0.007) and Kiupel (P = 0.008) grading systems, mitotic index (P = 0.0008) and Ki67 expression (P = 0.009). High stromal FAP expression could be a potential negative prognostic factor in canine MCTs.

  4. Fetoprotective activity of breast cancer resistance protein (BCRP, ABCG2): expression and function throughout pregnancy.

    PubMed

    Hahnova-Cygalova, Lenka; Ceckova, Martina; Staud, Frantisek

    2011-02-01

    The medical treatment of pregnant women, as well as their fetuses, has become a common clinical practice in developed countries. Therefore, detailed knowledge of maternofetal pharmacokinetics, including the role of drug-efflux transporters in the fetoplacental unit, is crucial to optimize drug choice and dosage schemes and to avoid or exploit possible drug-drug interactions on placental transporters in order to assure appropriate drug levels in the mother and/or fetus. Breast cancer resistance protein (BCRP, ABCG2) is the most recent member of ATP-binding cassette drug-efflux transporters that has been associated with resistance in cancer chemotherapy. Importantly, ABCG2 has also been localized in various normal tissues, affecting the pharmacokinetics of several xenobiotics as well as a number of physiological substances. Extensive expression of ABCG2 in tissue barriers, such as the blood-brain barrier, intestine, testis, or placenta, suggests that ABCG2 plays an important role in the protection of sensitive tissues against toxins. In the placenta, ABCG2 has been experimentally evidenced to actively pump its substrates in the fetal-to-maternal direction and to play an important role in transplacental pharmacokinetics, fetal protection, and detoxication. Further, ABCG2 expression in embryonic and fetal membranes over the course of pregnancy helps ensure proper function of the fetoplacental unit. In this review, we summarize the current knowledge regarding expression and function of ABCG2 in the fetoplacental unit during the development of the fetus and overview the aspects of transplacental pharmacokinetics, ABCG2 regulation, and clinical significance of the transporter for pharmacotherapy in pregnancy.

  5. Early weaning increases intestinal permeability, alters expression of cytokine and tight junction proteins, and activates mitogen-activated protein kinases in pigs.

    PubMed

    Hu, C H; Xiao, K; Luan, Z S; Song, J

    2013-03-01

    Although weaning stress has been reported to impair intestinal barrier function, the mechanisms have not yet been elucidated. In the present study, the intestinal morphology and permeability and mRNA expressions of tight junction proteins and cytokines in the intestine of piglets during the 2 wk after weaning were assessed. The phosphorylated (activated) ratios of p38, c-Jun NH(2)-terminal kinase (JNK), and extracellular regulated kinases (ERK1/2) were determined to investigate whether mitogen-activated protein kinase (MAPK) signaling pathways are involved in the early weaning process. A shorter villus and deeper crypt were observed on d 3 and 7 postweaning. Although damaged intestinal morphology recovered to preweaning values on d 14 postweaning, the intestinal mucosal barrier, which was reflected by transepithelial electrical resistance (TER) and paracellular flux of dextran (4 kDa) in the Ussing chamber and tight junction protein expression, was not recovered. Compared with the preweaning stage (d 0), jejunal TER and mRNA expressions of occludin and claudin-1 on d 3, 7, and 14 postweaning and Zonula occludens-1 (ZO-1) mRNA on d 3 and 7 postweaning were reduced, and paracellular flux of dextran on d 3, 7, and 14 postweaning was increased. An increase (P < 0.05) of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) mRNA on d 3 and d 7 postweaning and an increase (P < 0.05) of interferon-γ (IFN-γ) mRNA on d 3 postweaning were observed compared with d 0. No significant increase of transforming growth factor β1 (TGF-β1) and interleukin-10 (IL-10) mRNA after weaning was observed. The phosphorylated (activated) ratios of JNK and p38 on d 3 and 7 postweaning and the phosphorylated ratio of ERK1/2 on d 3 postweaning were increased (P < 0.05) compared with d 0. The results indicated that early weaning induced sustained impairment in the intestinal barrier, decreased mRNA expression of tight junction proteins, and upregulated the expression of proinflammatory

  6. Bavachalcone-induced manganese superoxide dismutase expression through the AMP-activated protein kinase pathway in human endothelial cells.

    PubMed

    Dang, Yanqi; Ling, Shuang; Duan, Ju; Ma, Jing; Ni, Rongzhen; Xu, Jin-Wen

    2015-01-01

    Mitochondrial oxidative stress has been suggested as a major etiological factor in cardiovascular diseases. Manganese superoxide dismutase (MnSOD) is an essential antioxidant mitochondrial enzyme. Although polyphenols can induce MnSOD expression, their mechanism of action remains unclear. We examined the effect of bavachalcone, a bioactive compound isolated from Psoralea corylifolia, on MnSOD protein expression and explored whether this effect is mediated through the AMP-activated protein kinase (AMPK) signaling pathway. Our data showed that bavachalcone enhanced the luciferase activity of the MnSOD promoter and increased MnSOD mRNA and protein expressions. Moreover, bavachalcone suppressed the mitochondrial superoxide production in endothelial cells. Conversely, bavachalcone stimulated liver kinase B1 and AMPKα phosphorylation. mRNA interference by using short hairpin RNA (shRNA) of AMPK inhibited bavachalcone-induced MnSOD expression. A-769662, an AMPK activator, also stimulated AMPK activity and increased MnSOD expression. Furthermore, AMPK knockdown by shRNA-AMPK reversed the inhibitory effects of bavachalcone on mitochondrial superoxide production in endothelial cells. These findings indicate that bavachalcone can protect the endothelial function by increasing AMPK activity and MnSOD expression and reducing mitochondrial oxidative stress. .

  7. Nerve Growth Factor Promoter Activity Revealed in Mice Expressing Enhanced Green Fluorescent Protein

    PubMed Central

    Kawaja, Michael D.; Smithson, Laura J.; Elliott, Janet; Trinh, Gina; Crotty, Anne-Marie; Michalski, Bernadeta; Fahnestock, Margaret

    2012-01-01

    Nerve growth factor (NGF) and its precursor proNGF are perhaps the best described growth factors of the mammalian nervous system. There remains, however, a paucity of information regarding the precise cellular sites of proNGF/NGF synthesis. Here we report the generation of transgenic mice in which the NGF promoter controls the ectopic synthesis of enhanced green fluorescent protein (EGFP). These transgenic mice provide an unprecedented resolution of both neural cells (e.g., neocortical and hippocampal neurons) and non-neural cells (e.g., renal interstitial cells and thymic reticular cells) that display NGF promoter activity from postnatal development to adulthood. Moreover, the transgene is inducible by injury. At 2 days after sciatic nerve ligation, a robust population of EGFP-positive cells is seen in the proximal nerve stump. These transgenic mice offer novel insights into the cellular sites of NGF promoter activity and can be used as models for investigating the regulation of proNGF/NGF expression after injury. PMID:21456011

  8. Differential Regulation of Proinflammatory Cytokine Expression by Mitogen-Activated Protein Kinases in Macrophages in Response to Intestinal Parasite Infection

    PubMed Central

    Lim, Mei Xing; Png, Chin Wen; Tay, Crispina Yan Bing; Teo, Joshua Ding Wei; Jiao, Huipeng; Lehming, Norbert

    2014-01-01

    Blastocystis is a common enteric protistan parasite that can cause acute, as well as chronic, infection and is associated with irritable bowel syndrome (IBS). However, the pathogenic status of Blastocystis infection remains unclear. In this study, we found that Blastocystis antigens induced abundant expression of proinflammatory cytokines, including interleukin 1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α), in mouse intestinal explants, in mouse colitis colon, and in macrophages. Further investigation utilizing RAW264.7 murine macrophages showed that Blastocystis treatment in RAW264.7 macrophages induced the activation of ERK, JNK, and p38, the three major groups of mammalian mitogen-activated protein (MAP) kinases that play essential roles in the expression of proinflammatory cytokines. ERK inhibition in macrophages significantly suppressed both mRNA and protein expression of IL-6 and TNF-α and mRNA expression of IL-1β. On the other hand, JNK inhibition resulted in reductions in both c-Jun and ERK activation and significant suppression of all three proinflammatory cytokines at both the mRNA and protein levels. Inhibition of p38 suppressed only IL-6 protein expression with no effect on the expression of IL-1β and TNF-α. Furthermore, we found that serine proteases produced by Blastocystis play an important role in the induction of ERK activation and proinflammatory cytokine expression by macrophages. Our study thus demonstrated for the first time that Blastocystis could induce the expression of various proinflammatory cytokines via the activation of MAP kinases and that infection with Blastocystis may contribute to the pathogenesis of inflammatory intestinal diseases through the activation of inflammatory pathways in host immune cells, such as macrophages. PMID:25156742

  9. Genetic and epigenetic background and protein expression profiles in relation to telomerase activation in medullary thyroid carcinoma

    PubMed Central

    Wang, Na; Kjellin, Hanna; Sofiadis, Anastasios; Fotouhi, Omid; Juhlin, C. Christofer; Bäckdahl, Martin; Zedenius, Jan; Xu, Dawei; Lehtiö, Janne; Larsson, Catharina

    2016-01-01

    Medullary thyroid carcinomas (MTCs) exhibit telomerase activation in strong association with shorter patient survival. To understand the background of telomerase activation we quantified TERT copy numbers and TERT promoter methylation in 42 MTCs and normal thyroid references. Gain of TERT was demonstrated by quantitative PCR in 5/39 sporadic MTC. Increased methylation index (MetI) for CpG methylation at the TERT promoter was found in sporadic MTCs (P < 0.0001) and in MEN 2 associated MTCs (P = 0.011) vs. normal thyroid tissues. MetI correlated positively with TERT gene expression (r = 0.432, P = 0.006) and negatively with telomere length (r = −0.343, P = 0.032). MTC cases with MetI above the median of 52% had shorter survival as compared to cases with lower MetI (P = 0.005 for overall survival and P = 0.007 for disease-related survival). Protein expression profiles obtained by mass spectrometry were then studied in relation to telomerase activation in MTCs. Comparing protein levels between tumors defined by telomerase activity status, 240 proteins were associated with telomerase activity. Among telomerase activation positive cases a set of proteins was found to discriminate between MTCs with high and low TERT gene expression with enrichment for proteins involved in telomerase regulation. XRCC5 mRNA expression was found increased in MTCs vs. normal thyroid (P = 0.007). In conclusion the findings suggest a role for TERT copy number gain, TERT promoter methylation and XRCC5 expression in telomerase activation and telomere maintenance of MTC. PMID:26870890

  10. Dynamic expression of the Slit-Robo GTPase activating protein genes during development of the murine nervous system.

    PubMed

    Bacon, Claire; Endris, Volker; Rappold, Gudrun

    2009-03-10

    We investigated the expression of the three known Slit-Robo GTPase activating protein (srGAP) genes in the developing murine nervous system using in situ hybridization. The three genes are expressed during embryonic and early postnatal development in the murine nervous system, showing a distinct pattern of expression in the olfactory system, the eye, forebrain and midbrain structures, the cerebellum, the spinal cord, and dorsal root ganglia, which we discuss in relation to Slit-Robo expression patterns and signaling pathways. We also report srGAP2 expression in zones of neuronal differentiation and srGAP3 in ventricular zones of neurogenesis in many different tissues of the central nervous system (CNS). Compared to srGAP2 and srGAP3, the onset of srGAP1 expression is later in most CNS tissues. We propose that these differences in expression point to functional differences between these three genes in the development of neural tissues.

  11. Expression of peroxisome proliferator-activated receptor and CCAAT/enhancer binding protein transcription factors in cultured human sebocytes.

    PubMed

    Chen, WenChieh; Yang, Chao-Chun; Sheu, Hamm-Ming; Seltmann, Holger; Zouboulis, Christos C

    2003-09-01

    Lipid synthesis and accumulation represent a major step in sebocyte differentiation and it may be of importance for sebocytes to express two families of transcription factors, CCAAT/enhancer binding proteins (c/EBPs) and peroxisome proliferator-activated receptors (PPARs), which were found to play a crucial role in the differentiation of adipocytes. Using the immortalized human sebaceous gland cell line SZ95 we examined the expression of the molecules before and after treatment with testosterone, 5alpha-dihydrotestosterone, dexamethasone, 17beta-estradiol and genistein, at 6, 12, 24, and 48 h, respectively. Reverse transcription-PCR analysis showed expression of peroxisome proliferator-activated receptors -alpha, -delta, -gamma1, -gamma2 and CCAAT/enhancer binding proteins-alpha, -beta, -gamma-delta in native SZ95 sebocytes. In western blot studies, high levels of CCAAT/enhancer binding proteins-alpha and -beta, and peroxisome proliferator-activated receptors-gamma were expressed at 6, 24, and 12 h, respectively. Immunostaining of the cultured sebocytes showed the CCAAT/enhancer binding proteins-alpha and -beta mainly localized within nuclei, whereas peroxisome proliferator-activated receptors-gamma in the cytoplasm. Strong staining of sebocytes was immunohistochemically revealed in the basal layer of sebaceous glands in human scalp and sebaceous nevus. Genistein down-regulated the expression of CCAAT/enhancer binding proteins-alpha and -beta, and peroxisome proliferator-activated receptors-gamma on the protein level. Treatment with linoleic acid for 48 h induced further differentiation of sebocytes leading to abundant lipid synthesis.

  12. Expression and purification of active protein kinases from wheat germ extracts.

    PubMed

    Sonkoly, Boglárka; Bardóczy, Viola; Mészáros, Tamás

    2011-01-01

    In vitro functional studies of eukaryotic kinases are often constrained by the availability of pure and -enzymatically active kinase of interest. Though numerous proteins have been synthesized by cell-based systems, in vivo production of properly folded, eukaryotic proteins remains a challenging task. Current wheat-germ-based cell-free in vitro translation systems present a plausible alternative for protein synthesis since majority of eukaryotic proteins could be obtained in their native folded form with general protocols. The use of special in vitro translation vectors with ligation-independent cloning sites and cleavable affinity tags eliminates further bottlenecks of the protein producing procedure and makes this system a reasonable method for simultaneous generation of active kinases.

  13. p53 Degradation Activity, Expression, and Subcellular Localization of E6 Proteins from 29 Human Papillomavirus Genotypes

    PubMed Central

    Mesplède, Thibault; Gagnon, David; Bergeron-Labrecque, Fanny; Azar, Ibrahim; Sénéchal, Hélène; Coutlée, François

    2012-01-01

    Human papillomaviruses (HPVs) are the etiological agents of cervical cancer and other human malignancies. HPVs are classified into high- and low-risk genotypes according to their association with cancer. Host cell transformation by high-risk HPVs relies in part on the ability of the viral E6 protein to induce the degradation of p53. We report the development of a cellular assay that accurately quantifies the p53 degradation activity of E6 in vivo, based on the fusion of p53 to Renilla luciferase (RLuc-p53). This assay was used to measure the p53 degradation activities of E6 proteins from 29 prevalent HPV types and variants of HPV type 16 (HPV16) and HPV33 by determining the amount of E6 expression vector required to reduce by half the levels of RLuc-p53 (50% effective concentration [EC50]). These studies revealed an unexpected variability in the p53 degradation activities of different E6 proteins, even among active types whose EC50s span more than 2 log units. Differences in activity were greater between types than between variants and did not correlate with differences in the intracellular localization of E6, with most being predominantly nuclear. Protein and mRNA expression of the 29 E6 proteins was also examined. For 16 high-risk types, spliced transcripts that encode shorter E6*I proteins of variable sizes and abundances were detected. Mutation of the splice donor site in five different E6 proteins increased their p53 degradation activity, suggesting that mRNA splicing can limit the activity of some high-risk E6 types. The quantification of p53 degradation in vivo represents a novel tool to systematically compare the oncogenic potentials of E6 proteins from different HPV types and variants. PMID:22013048

  14. Slack sodium-activated potassium channel membrane expression requires p38 mitogen-activated protein kinase phosphorylation.

    PubMed

    Gururaj, Sushmitha; Fleites, John; Bhattacharjee, Arin

    2016-04-01

    p38 MAPK has long been understood as an inducible kinase under conditions of cellular stress, but there is now increasing evidence to support its role in the regulation of neuronal function. Several phosphorylation targets have been identified, an appreciable number of which are ion channels, implicating the possible involvement of p38 MAPK in neuronal excitability. The KNa channel Slack is an important protein to be studied as it is highly and ubiquitously expressed in DRG neurons and is important in the maintenance of their firing accommodation. We sought to examine if the Slack channel could be a substrate of p38 MAPK activity. First, we found that the Slack C-terminus contains two putative p38 MAPK phosphorylation sites that are highly conserved across species. Second, we show via electrophysiology experiments that KNa currents and further, Slack currents, are subject to tonic modulation by p38 MAPK. Third, biochemical approaches revealed that Slack channel regulation by p38 MAPK occurs through direct phosphorylation at the two putative sites of interaction, and mutating both sites prevented surface expression of Slack channels. Based on these results, we conclude that p38 MAPK is an obligate regulator of Slack channel function via the trafficking of channels into the membrane. The present study identifies Slack KNa channels as p38 MAPK substrates.

  15. Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

    SciTech Connect

    Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald

    2015-03-01

    Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.

  16. Chip, a widely expressed chromosomal protein required for segmentation and activity of a remote wing margin enhancer in Drosophila

    PubMed Central

    Morcillo, Patrick; Rosen, Christina; Baylies, Mary K.; Dorsett, Dale

    1997-01-01

    The mechanisms allowing remote enhancers to regulate promoters several kilobase pairs away are unknown but are blocked by the Drosophila suppressor of Hairy-wing protein (Suhw) that binds to gypsy retrovirus insertions between enhancers and promoters. Suhw bound to a gypsy insertion in the cut gene also appears to act interchromosomally to antagonize enhancer–promoter interactions on the homologous chromosome when activity of the Chip gene is reduced. This implicates Chip in enhancer–promoter communication. We cloned Chip and find that it encodes a homolog of the recently discovered mouse Nli/Ldb1/Clim-2 and Xenopus Xldb1 proteins that bind nuclear LIM domain proteins. Chip protein interacts with the LIM domains in the Apterous homeodomain protein, and Chip interacts genetically with apterous, showing that these interactions are important for Apterous function in vivo. Importantly, Chip also appears to have broad functions beyond interactions with LIM domain proteins. Chip is present in all nuclei examined and at numerous sites along the salivary gland polytene chromosomes. Embryos without Chip activity lack segments and show abnormal gap and pair–rule gene expression, although no LIM domain proteins are known to regulate segmentation. We conclude that Chip is a ubiquitous chromosomal factor required for normal expression of diverse genes at many stages of development. We suggest that Chip cooperates with different LIM domain proteins and other factors to structurally support remote enhancer–promoter interactions. PMID:9334334

  17. Expression of 5-lipoxygenase and 5-lipoxygenase-activating protein in human fetal membranes throughout pregnancy and at term.

    PubMed

    Brown, N L; Slater, D M; Alvi, S A; Elder, M G; Sullivan, M H; Bennett, P R

    1999-07-01

    Lipoxygenase metabolites may be involved in human parturition. 5-lipoxygenase (5-LOX) catalyses the first steps in the synthesis of leukotrienes from arachidonic acid, and its activity is dependent on 5-LOX activating protein (FLAP). The expression of 5-LOX and FLAP were investigated in fetal membranes to determine whether there are changes with gestational age or at term with the onset of labour. No significant differences were found in the expression of 5-LOX or FLAP mRNA in the amnion at different gestational ages or at term. In the chorion-decidua, 5-LOX mRNA expression was significantly higher in the first trimester of pregnancy than in the second and third trimesters. At term, there was a significant increase in both 5-LOX mRNA and protein expression in the chorion-decidua in the time after labour, compared with the time before labour. The expression of FLAP mRNA was also significantly higher in the chorion-decidua in the first trimester of pregnancy compared with the third trimester, and at term in the time after labour compared with the time before labour. Expression of FLAP protein was not studied, as an antibody is not currently available. These results are consistent with a role for 5-LOX and FLAP in the control of parturition at term, and also suggest an involvement earlier in pregnancy.

  18. Developmental Expression of CYP2B6: A Comprehensive Analysis of mRNA Expression, Protein Content and Bupropion Hydroxylase Activity and the Impact of Genetic Variation.

    PubMed

    Pearce, Robin E; Gaedigk, Roger; Twist, Greyson P; Dai, Hongying; Riffel, Amanda K; Leeder, J Steven; Gaedigk, Andrea

    2016-07-01

    Although CYP2B6 catalyzes the biotransformation of many drugs used clinically for children and adults, information regarding the effects of development on CYP2B6 expression and activity are scarce. Utilizing a large panel of human liver samples (201 donors: 24 fetal, 141 pediatric, and 36 adult), we quantified CYP2B6 mRNA and protein expression levels, characterized CYP2B6 (bupropion hydroxylase) activity in human liver microsomes (HLMs), and performed an extensive genotype analysis to differentiate CYP2B6 haplotypes such that the impact of genetic variation on these parameters could be assessed. Fetal livers contained extremely low levels of CYP2B6 mRNA relative to postnatal samples and fetal HLMs did not appear to catalyze bupropion hydroxylation; however, fetal CYP2B6 protein levels were not significantly different from postnatal levels. Considerable interindividual variation in CYP2B6 mRNA expression, protein levels, and activity was observed in postnatal HLMs (mRNA, ∼40,000-fold; protein, ∼300-fold; activity, ∼600-fold). The extremely wide range of interindividual variability in CYP2B6 expression and activity was significantly associated with age (P < 0.01) following log transformation of the data. Our data suggest that CYP2B6 activity appears as early as the first day of life, increases through infancy, and by 1 year of age, CYP2B6 levels and activity may approach those of adults. Surprisingly, CYP2B6 interindividual variability was not significantly associated with genetic variation in CYP2B6, nor was it associated with differences in gender or ethnicity, suggesting that factors other than these are largely responsible for the wide range of variability in CYP2B6 expression and activity observed among a large group of individuals/samples.

  19. Identification of positive-acting domains in GCN2 protein kinase required for translational activation of GCN4 expression

    SciTech Connect

    Wek, R.C.; Ramirez, M.; Jackson, B.M.; Hinnebusch, A.G. )

    1990-06-01

    GCN4 is a transcriptional activator of amino acid-biosynthetic genes in the yeast {ital Saccharomyces cerevisiae}. GCN2, a translational activator of {ital GCN4} expression, contains a domain homologous to the catalytic subunit of eukaryotic protein kinases. Substitution of a highly conserved lysine residue in the kinase domain abolished GCN2 regulatory function in vivo and its ability to autophosphorylate in vitro, indicating that GCN2 acts as a protein kinase in stimulating {ital GCN4} expression. Elevated {ital GCN2} gene dosage led to depression of {ital GCN4} under nonstarvation conditions; however, the authors found that {ital GCN2} mRNA and protein levels did not increase in wild-type cells in response to amino acid starvation. Therefore, it appears that GCN2 protein kinase function is stimulated postranslationally in amino acid-starved cells. Three dominant-constitutive {ital GCN2} point mutations were isolated that led to derepressed {ital GCN4} expression under nonstarvation conditions. Two of the {ital GCN2}(Con) mutations mapped in the kinase domain itself. The third mapped just downstream from a carboxyl-terminal segment homologous to histidyl-tRNA synthetase (HisRS), which the authors suggest might function to detect uncharged tRNA in amino acid-starved cells and activate the adjacent protein kinase moiety.

  20. Circadian expression of the presynaptic active zone protein Bruchpilot in the lamina of Drosophila melanogaster.

    PubMed

    Górska-Andrzejak, Jolanta; Makuch, Renata; Stefan, Joanna; Görlich, Alicja; Semik, Danuta; Pyza, Elzbieta

    2013-01-01

    In the fly's visual system, the morphology of cells and the number of synapses change during the day. In the present study we show that in the first optic neuropil (lamina) of Drosophila melanogaster, a presynaptic active zone protein Bruchpilot (BRP) exhibits a circadian rhythm in abundance. In day/night (or light/dark, LD) conditions the level of BRP increases two times, in the morning and in the evening. The same pattern of changes in the BRP level was detected in whole brain homogenates, thus indicating that the majority of synapses in the brain peaks twice during the day. However, these two peaks in BRP abundance, measured as the fluorescence intensity of immunolabeling, seem to be regulated differently. The peak in the morning is predominantly regulated by light and involves the transduction pathway in the retina photoreceptors. This peak is present neither in wild-type Canton-S flies in constant darkness (DD), nor in norpA(7) phototransduction mutant in LD. However, it also depends on the clock gene per, because it is abolished in the per(0) arrhythmic mutant. In turn, the peak of BRP in the evening is endogenously regulated by an input from the pacemaker located in the brain. This peak is present in Canton-S flies in DD, as well as in the norpA(7) mutant in LD, but is absent in per(01), tim,(01) and cry(01) mutants in LD. In addition both peaks seem to depend on clock gene-expressing photoreceptors and glial cells of the visual system.

  1. Sequence analysis, expression, and binding activity of recombinant major outer sheath protein (Msp) of Treponema denticola.

    PubMed Central

    Fenno, J C; Müller, K H; McBride, B C

    1996-01-01

    The gene encoding the major outer sheath protein (Msp) of the oral spirochete Treponema denticola ATCC 35405 was cloned, sequenced, and expressed in Escherichia coli. Preliminary sequence analysis showed that the 5' end of the msp gene was not present on the 5.5-kb cloned fragment described in a recent study (M. Haapasalo, K. H. Müller, V. J. Uitto, W. K. Leung, and B. C. McBride, Infect. Immun. 60:2058-2065,1992). The 5' end of msp was obtained by PCR amplification from a T. denticola genomic library, and an open reading frame of 1,629 bp was identified as the coding region for Msp by combining overlapping sequences. The deduced peptide consisted of 543 amino acids and had a molecular mass of 58,233 Da. The peptide had a typical prokaryotic signal sequence with a potential cleavage site for signal peptidase 1. Northern (RNA) blot analysis showing the msp transcript to be approximately 1.7 kb was consistent with the identification of a promoter consensus sequence located optimally upstream of msp and a transcription termination signal found downstream of the stop codon. The entire msp sequence was amplified from T. denticola genomic DNA and cloned in E. coli by using a tightly regulated T7 RNA polymerase vector system. Expression of Msp was toxic to E. coli when the entire msp gene was present. High levels of Msp were produced as inclusion bodies when the putative signal peptide sequence was deleted and replaced by a vector-encoded T7 peptide sequence. Recombinant Msp purified to homogeneity from a clone containing the full-length msp gene adhered to immobilized laminin and fibronectin but not to bovine serum albumin. Attachment of recombinant Msp was decreased in the presence of soluble substrate. Attachment of T. denticola to immobilized laminin and fibronectin was increased by pretreatment of the substrate with recombinant Msp. These studies lend further support to the hypothesis that Msp mediates the extracellular matrix binding activity of T. denticola. PMID

  2. Hansenula polymorpha expressed heat shock protein gp96 exerts potent T cell activation activity as an adjuvant.

    PubMed

    Li, Yang; Song, Haolei; Li, Jin; Wang, Yanzhong; Yan, Xiaoli; Zhao, Bao; Zhang, Xiaojun; Wang, Saifeng; Chen, Lizhao; Qiu, Bingsheng; Meng, Songdong

    2011-02-20

    Previous studies together with ours showed that heat shock protein gp96 as an adjuvant induces antigen specific T cell responses against cancer and infectious diseases. However, at present there is no efficient method to obtain high amount of full-length gp96 by in vitro expression. Here, we used the yeast Hansenula polymorpha as an efficient host for gp96 recombinant protein production. The transformant clones with highly expressed recombinant proteins were screened and selected by measuring the halo size which indicates enzymatic hydrolysis of starch in the medium. High-level production of gp96 (around 150mg/mL) was achieved by using high-cell density fed-batch cultivations. We showed that peptide binding of the recombinant protein has similar specificity and intrinsic binding parameters as that of the native gp96. We next examined the self-assembly properties and high-order structures of the recombinant protein. Moreover, the H. polymorpha expressed recombinant gp96 can effectively induce HBV-specific CTL response in immunized mice while Escherichia coli-expressed gp96 cannot. Our results therefore may provide bases for structure and functional studies of gp96 and thereby potentially expedite the development of gp96-based vaccines for immunotherapy of cancer or infectious diseases.

  3. Erectile Dysfunction Drugs Changed the Protein Expressions and Activities of Drug-Metabolising Enzymes in the Liver of Male Rats

    PubMed Central

    Hassan, Mostafa

    2016-01-01

    Erectile dysfunction (ED) is a major health problem and is mainly associated with the persistent inability of men to maintain sufficient erection for satisfactory sexual performance. Millions of men are using sildenafil, vardenafil, and/or tadalafil for ED treatment. Cytochrome P450s (CYPs) play a central role in the metabolism of a wide range of xenobiotics as well as endogenous compounds. Susceptibility of individuals to the adverse effects of different drugs is mainly dependent on the expression of CYPs proteins. Therefore, changes in activities of phase I drug-metabolising enzymes [arylhydrocarbon hydroxylase (AHH), dimethylnitrosamine N-demethylase (DMN-dI), 7-ethoxycoumarin-O-deethylase (ECOD), and ethoxyresorufin-O-deethylase ((EROD)] and the protein expression of different CYPs isozymes (CYP1A2, CYP2E1, CYP2B1/2, CYP3A4, CYP2C23, and CYP2C6) were determined after treatment of male rats with either low or high doses of sildenafil (Viagra), tadalafil (Cialis), and/or vardenafil (Levitra) for 3 weeks. The present study showed that low doses of tadalafil and vardenafil increased DMN-dI activity by 32 and 23%, respectively. On the other hand, high doses of tadalafil, vardenafil, and sildenafil decreased such activity by 50, 56, and 52%, respectively. In addition, low doses of tadalafil and vardenafil induced the protein expression of CYP2E1. On the other hand, high doses of either tadalafil or sildenafil were more potent inhibitors to CYP2E1 expression than vardenafil. Moreover, low doses of both vardenafil and sildenafil markedly increased AHH activity by 162 and 247%, respectively, whereas high doses of tadalafil, vardenafil, and sildenafil inhibited such activity by 36, 49, and 57% and inhibited the EROD activity by 39, 49, and 33%, respectively. Low and high doses of tadalafil, vardenafil, and sildenafil inhibited the activity of NADPH-cytochrome c reductase as well as its protein expression. In addition, such drugs inhibited the expression of CYP B1/2 along

  4. Gli2 protein expression level is a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

    PubMed

    Sugiyama, Y; Sasajima, J; Mizukami, Y; Koizumi, K; Kawamoto, T; Ono, Y; Karasaki, H; Tanabe, H; Fujiya, M; Kohgo, Y

    2016-06-01

    The hedgehog pathway is known to promote proliferation of pancreatic ductal adenocarcinoma (PDA) and has been shown to restrain tumor progression. To understand how hedgehog causes these effects, we sought to carefully examine protein expression of hedgehog signaling components during different tumor stages. Genetically engineered mice, Pdx1-Cre;LSL-KrasG12D and Pdx1-Cre;LSL-KrasG12D;p53lox/+, were utilized to model distinct phases of tumorigenesis, pancreatic intraepithelial neoplasm (PanIN) and PDA. Human pancreatic specimens of intraductal papillary mucinous neoplasm (IPMN) and PDA were also employed. PanIN and IPMN lesions highly express Sonic Hedgehog, at a level that is slightly higher than that observed in PDA. GLI2 protein is also expressed in both PanIN/IPMN and PDA. Although there was no difference in the nuclear staining, the cytoplasmic GLI2 level in PDA was modest in comparison to that in PanIN/IPMN. Hedgehog interacting protein was strongly expressed in the precursors, whereas the level in PDA was significantly attenuated. There were no differences in expression of Patched1 at early and late stages. Finally, a strong correlation between Sonic Hedgehog and GLI2 staining was found in both human and murine pancreatic tumors. The results indicate that the GLI2 protein level could serve as a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

  5. Expression and activity of the 5'-AMP-activated protein kinase pathway in selected tissues during chicken embryonic development.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 5’-AMP-activated protein kinase (AMPK) is a highly conserved serine/threonine protein kinase and a key part of a kinase signaling cascade that senses cellular energy status (AMP/ATP ratio) and acts to maintain energy homeostasis by coordinately regulating energy-consuming and energy-generating m...

  6. Expression of the GM2-activator protein in the methylotrophic yeast Pichia pastoris, purification, isotopic labeling, and biophysical characterization.

    PubMed

    Wendeler, Michaela; Hoernschemeyer, Joerg; John, Michael; Werth, Norbert; Schoeniger, Maike; Lemm, Thorsten; Hartmann, Rudolf; Kessler, Horst; Sandhoff, Konrad

    2004-03-01

    The GM2-activator protein (GM2AP) belongs to a group of five small, nonenzymatic proteins that are essential cofactors for the degradation of glycosphingolipids in the lysosome. It mediates the interaction between the water-soluble enzyme beta-hexosaminidase A and its membrane-embedded substrate, ganglioside GM2, at the lipid-water interphase. Inherited defects in the gene encoding this glycoprotein cause a fatal neurological storage disorder, the AB variant of GM2 gangliosidosis. With the aim to establish a convenient eukaryotic system that allows the efficient production of functionally folded, glycosylated GM2AP and offers the potential of cost-efficient isotopic labeling for structural studies by NMR spectroscopy, we established the expression of recombinant GM2AP in the methylotrophic yeast Pichia pastoris. For the construction of expression plasmids, either the full cDNA encoding human GM2AP preproprotein was cloned in the expression vector pPIC3.5K, or the cDNA encoding only the mature form of GM2AP was inserted in the vector pPIC9K under control of the alcohol oxidase 1 promoter. Both plasmids led to the successful secretory expression of active, glycosylated GM2AP, which could easily be purified by Ni-NTA chromatography due to the hexahistidine tag introduced at the C-terminus. Remarkably, the expression of this membrane-active protein in P. pastoris was accompanied by two peculiarities which were not encountered in other expression systems for GM2AP: First, a significant fraction of the secreted protein existed in the form of aggregates, and second, considerable amounts of noncovalently bound lipids were associated with the recombinant protein. A three-step purification scheme was therefore devised consisting of Ni-NTA, reversed phase, and gel filtration chromatography, which finally yielded 10-12 mg of purified, monomeric GM2AP per liter of expression supernatant. MALDI- and ESI-TOF mass spectrometry were employed to assess the processing, homogeneity

  7. Activation of extrasynaptic, but not synaptic, NMDA receptors modifies amyloid precursor protein expression pattern and increases amyloid-ß production.

    PubMed

    Bordji, Karim; Becerril-Ortega, Javier; Nicole, Olivier; Buisson, Alain

    2010-11-24

    Calcium is a key mediator controlling essential neuronal functions depending on electrical activity. Altered neuronal calcium homeostasis affects metabolism of amyloid precursor protein (APP), leading to increased production of β-amyloid (Aβ), and contributing to the initiation of Alzheimer's disease (AD). A linkage between excessive glutamate receptor activation and neuronal Aβ release was established, and recent reports suggest that synaptic and extrasynaptic NMDA receptor (NMDAR) activation may have distinct consequences in plasticity, gene regulation, and neuronal death. Here, we report for the first time that prolonged activation of extrasynaptic NMDAR, but not synaptic NMDAR, dramatically increased the neuronal production of Aβ. This effect was preceded by a shift from APP695 to Kunitz protease inhibitory domain (KPI) containing APPs (KPI-APPs), isoforms exhibiting an important amyloidogenic potential. Conversely, after synaptic NMDAR activation, we failed to detect any KPI-APP expression and neuronal Aβ production was not modified. Calcium imaging data showed that intracellular calcium concentration after extrasynaptic NMDAR stimulation was lower than after synaptic activation. This suggests distinct signaling pathways for each pool of receptors. We found that modification of neuronal APP expression pattern triggered by extrasynaptic NMDAR activation was regulated at an alternative splicing level involving calcium-/calmodulin-dependent protein kinase IV, but overall APP expression remained identical. Finally, memantine dose-dependently inhibited extrasynaptic NMDAR-induced KPI-APPs expression as well as neuronal Aβ release. Altogether, these data suggest that a chronic activation of extrasynaptic NMDAR promotes amyloidogenic KPI-APP expression leading to neuronal Aβ release, representing a causal risk factor for developing AD.

  8. Hypoxia-Inducible Factor 1 Is an Inductor of Transcription Factor Activating Protein 2 Epsilon Expression during Chondrogenic Differentiation.

    PubMed

    Niebler, Stephan; Angele, Peter; Kujat, Richard; Bosserhoff, Anja K

    2015-01-01

    The transcription factor AP-2ε (activating enhancer-binding protein epsilon) is expressed in cartilage of humans and mice. However, knowledge about regulatory mechanisms influencing AP-2ε expression is limited. Using quantitative real time PCR, we detected a significant increase in AP-2ε mRNA expression comparing initial and late stages of chondrogenic differentiation processes in vitro and in vivo. Interestingly, in these samples the expression pattern of the prominent hypoxia marker gene angiopoietin-like 4 (Angptl4) strongly correlated with that of AP-2ε suggesting that hypoxia might represent an external regulator of AP-2ε expression in mammals. In order to show this, experiments directly targeting the activity of hypoxia-inducible factor-1 (HIF1), the complex mediating responses to oxygen deprivation, were performed. While the HIF1-activating compounds 2,2'-dipyridyl and desferrioxamine resulted in significantly enhanced mRNA concentration of AP-2ε, siRNA against HIF1α led to a significantly reduced expression rate of AP-2ε. Additionally, we detected a significant upregulation of the AP-2ε mRNA level after oxygen deprivation. In sum, these different experimental approaches revealed a novel role for the HIF1 complex in the regulation of the AP-2ε gene in cartilaginous cells and underlined the important role of hypoxia as an important external regulatory stimulus during chondrogenic differentiation modulating the expression of downstream transcription factors.

  9. Motor skill learning enhances the expression of activity-regulated cytoskeleton-associated protein in the rat cerebellum.

    PubMed

    Wang, Dean-Chuan; Lin, Yu-Yi; Chen, Tsan-Ju; Lin, Hwai-Ting

    2014-11-01

    Motor skill learning is essential for environmental adaptations during everyday life. It has been shown that the cerebellum plays an important role in both the adaptation of eye movements and the motor skill learning. However, the neuronal substrates responsible for consolidation of complex motor skills rather than simple reflexes are still uncertain. Because the induction of immediate-early genes activity-regulated cytoskeleton-associated protein (Arc) and zinc finger binding protein clone 268 (Zif268) has been regarded as a marker for recent neuronal activity, therefore, in the present study, a rat paradigm of motor skill learning was used to investigate the protein expression of Arc and zif268 in the cerebellum after motor skill learning. Rats were trained to traverse the runway apparatus for 5 days. Protein samples were collected from the cerebellar cortices 1 hour after the training on days 1, 3, and 5, and analyzed by western blotting. The results showed that the expression of Arc, but not zif268, was significantly increased in the cerebellum following motor skill learning. These findings suggest that motor skill learning induces Arc expression in the cerebellum, which may play a role in acquiring complex motor skills.

  10. Brown Adipose YY1 Deficiency Activates Expression of Secreted Proteins Linked to Energy Expenditure and Prevents Diet-Induced Obesity

    PubMed Central

    Verdeguer, Francisco; Soustek, Meghan S.; Hatting, Maximilian; Blättler, Sharon M.; McDonald, Devin; Barrow, Joeva J.

    2015-01-01

    Mitochondrial oxidative and thermogenic functions in brown and beige adipose tissues modulate rates of energy expenditure. It is unclear, however, how beige or white adipose tissue contributes to brown fat thermogenic function or compensates for partial deficiencies in this tissue and protects against obesity. Here, we show that the transcription factor Yin Yang 1 (YY1) in brown adipose tissue activates the canonical thermogenic and uncoupling gene expression program. In contrast, YY1 represses a series of secreted proteins, including fibroblast growth factor 21 (FGF21), bone morphogenetic protein 8b (BMP8b), growth differentiation factor 15 (GDF15), angiopoietin-like 6 (Angptl6), neuromedin B, and nesfatin, linked to energy expenditure. Despite substantial decreases in mitochondrial thermogenic proteins in brown fat, mice lacking YY1 in this tissue are strongly protected against diet-induced obesity and exhibit increased energy expenditure and oxygen consumption in beige and white fat depots. The increased expression of secreted proteins correlates with elevation of energy expenditure and promotion of beige and white fat activation. These results indicate that YY1 in brown adipose tissue controls antagonistic gene expression programs associated with energy balance and maintenance of body weight. PMID:26503783

  11. Human Immunodeficiency Virus Tat-Activated Expression of Poliovirus Protein 2A Inhibits mRNA Translation

    NASA Astrophysics Data System (ADS)

    Sun, Xiao-Hong; Baltimore, David

    1989-04-01

    To study the effect of poliovirus protein 2A on cellular RNA translation, the tat control system of human immunodeficiency virus (HIV) was used. Protein 2A was expressed from a plasmid construct (pHIV/2A) incorporating the HIV long terminal repeat. Protein synthesis was measured by using chloramphenicol acetyltransferase as a reporter gene driven by the Rous sarcoma virus long terminal repeat. When HIV/2A was contransfected with the reporter, addition of a tat-producing plasmid caused at least a 50-fold drop in chloramphenicol acetyltransferase synthesis. A HeLa cell line carrying HIV/2A was established. In it, tat expression caused more than a 10-fold drop in chloramphenicol acetyltransferase synthesis from the reporter plasmid. Furthermore, 2A induction by tat caused cleavage of the cellular translation factor P220, a part of eukaryotic translation initiation factor 4F. Thus protein 2A can, by itself, carry out the inhibition of cellular protein synthesis characteristic of a poliovirus infection. Also, the HIV tat activation provides a very effective method to control gene expression in mammalian cells.

  12. Cloning, expression, purification, and activity assay of proteins related to D-lactic acid formation in Lactobacillus rhamnosus.

    PubMed

    Wang, Xiuwen; Zheng, Zhaojuan; Dou, Peipei; Qin, Jiayang; Wang, Xiaochen; Ma, Cuiqing; Tang, Hongzhi; Xu, Ping

    2010-08-01

    Two proteins that might be responsible for D-lactic acid (D-LA) formation were screened from the genome database of Lactobacillus rhamnosus GG. The coding genes of the two proteins in L. rhamnosus CASL, ldhD1 and ldhD2, were cloned and expressed in Escherichia coli Rosetta with an inducible expression vector pETDuet-1 (Novagen, Darmstadt, Germany), respectively. The two purified proteins, LdhD-1 and LdhD-2, migrated as a single protein band separately, both corresponding to an apparent molecular mass between 35 kDa and 45 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activities of LdhD-1 and LdhD-2 catalyzing pyruvate to LA were 0.02 U/mg and 0.21 U/mg, respectively. The configuration of LA converted from pyruvate was determined using high-performance liquid chromatography equipped with a chiral column. Only D-LA was detected when LdhD-1 and LdhD-2 were tested. In summary, the two proteins cloned and expressed in this study were most probably responsible for D-LA formation during fermentation of L. rhamnosus CASL.

  13. S-Adenosylmethionine Regulates Dual-Specificity Mitogen-Activated Protein Kinase Phosphatase Expression in Mouse and Human Hepatocytes

    PubMed Central

    Tomasi, Maria Lauda; Ramani, Komal; Lopitz-Otsoa, Fernando; Rodríguez, Manuel S.; Li, Tony W. H.; Ko, Kwangsuk; Yang, Heping; Bardag-Gorce, Fawzia; Iglesias-Ara, Ainhoa; Feo, Francesco; Pascale, Maria Rosa; Mato, José M.; Lu, Shelly C.

    2010-01-01

    Increased mitogen-activated protein kinase (MAPK) activity correlates with a more malignant hepatocellular carcinoma (HCC) phenotype. There is a reciprocal regulation between p44/42 MAPK (extracellular signal-regulated kinase [ERK]1/2) and the dual-specificity MAPK phosphatase MKP-1/DUSP1. ERK phosphorylates DUSP1, facilitating its proteasomal degradation, whereas DUSP1 inhibits ERK activity. Methionine adenosyltransferase 1a (Mat1a) knockout (KO) mice express hepatic S-adenosylmethionine (SAM) deficiency and increased ERK activity and develop HCC. The aim of this study was to examine whether DUSP1 expression is regulated by SAM and if so, elucidate the molecular mechanisms. Studies were conducted using Mat1a KO mice livers, cultured mouse and human hepatocytes, and 20S and 26S proteasomes. DUSP1 messenger RNA (mRNA) and protein levels were reduced markedly in livers of Mat1a KO mice and in cultured mouse and human hepatocytes with protein falling to lower levels than mRNA. SAM treatment protected against the fall in DUSP1 mRNA and protein levels in mouse and human hepatocytes. SAM increased DUSP1 transcription, p53 binding to DUSP1 promoter, and stability of its mRNA and protein. Proteasomal chymotrypsin-like and caspase-like activities were increased in Mat1a KO livers and cultured hepatocytes, which was blocked by SAM treatment. SAM inhibited chymotrypsin-like and caspase-like activities by 40% and 70%, respectively, in 20S proteasomes and caused rapid degradation of some of the 26S proteasomal subunits, which was blocked by the proteasome inhibitor MG132. SAM treatment in Mat1a KO mice for 7 days raised SAM, DUSP1, mRNA and protein levels and lowered proteosomal and ERK activities. Conclusion DUSP1 mRNA and protein levels are lower in Mat1a KO livers and fall rapidly in cultured hepatocytes. SAM treatment increases DUSP1 expression through multiple mechanisms, and this may suppress ERK activity and malignant degeneration. PMID:20196119

  14. Renal Dnase1 Enzyme Activity and Protein Expression Is Selectively Shut Down in Murine and Human Membranoproliferative Lupus Nephritis

    PubMed Central

    Rekvig, Ole Petter

    2010-01-01

    Background Deposition of chromatin-IgG complexes within glomerular membranes is a key event in the pathogenesis of lupus nephritis. We recently reported an acquired loss of renal Dnase1 expression linked to transformation from mild to severe membranoproliferative lupus nephritis in (NZBxNZW)F1 mice. As this may represent a basic mechanism in the progression of lupus nephritis, several aspects of Dnase1 expression in lupus nephritis were analyzed. Methodology/Principal Findings Total nuclease activity and Dnase1 expression and activity was evaluated using in situ and in vitro analyses of kidneys and sera from (NZBxNZW)F1 mice of different ages, and from age-matched healthy controls. Immunofluorescence staining for Dnase1 was performed on kidney biopsies from (NZBxNZW)F1 mice as well as from human SLE patients and controls. Reduced serum Dnase1 activity was observed in both mesangial and end-stage lupus nephritis. A selective reduction in renal Dnase1 activity was seen in mice with massive deposition of chromatin-containing immune complexes in glomerular capillary walls. Mice with mild mesangial nephritis showed normal renal Dnase1 activity. Similar differences were seen when comparing human kidneys with severe and mild lupus nephritis. Dnase1 was diffusely expressed within the kidney in normal and mildly affected kidneys, whereas upon progression towards end-stage renal disease, Dnase1 was down-regulated in all renal compartments. This demonstrates that the changes associated with development of severe nephritis in the murine model are also relevant to human lupus nephritis. Conclusions/Significance Reduction in renal Dnase1 expression and activity is limited to mice and SLE patients with signs of membranoproliferative nephritis, and may be a critical event in the development of severe forms of lupus nephritis. Reduced Dnase1 activity reflects loss in the expression of the protein and not inhibition of enzyme activity. PMID:20856893

  15. Magnesium chelatase subunit D from pea: characterization of the cDNA, heterologous expression of an enzymatically active protein and immunoassay of the native protein.

    PubMed

    Luo, M; Weinstein, J D; Walker, C J

    1999-12-01

    Mg-chelatase catalyzes the insertion of Mg into protoporphyrin and lies at the branchpoint of heme and (bacterio)chlorophyll synthesis. In prokaryotes, three genes--BchI, D and H--encode subunits for Mg-chelatase. In higher plants, homologous cDNAs for the I, D and H subunits have been characterized. Since the N-terminal half of the D subunit is homologous to the I subunit, the C-terminal portion of the pea D was used for antigen production. The antibody recognized the chloroplast D subunit and was used to demonstrate that this subunit associated with the membranes in the presence of MgCl2. The antibody immunoprecipitated the native protein and inhibited Mg-chelatase activity. Expression in Escherichia coli with a construct for the full-length protein (minus the putative transit peptide) resulted in induction of 24.5 kDa (major) and 89 kDa (minor) proteins which could only be solubilized in 6 M urea. However, when host cells were co-transformed with expression vectors for the full-length D subunit and for the 70 kDa HSP chaperonin protein, a substantial portion of the 89 kDa protein was expressed in a soluble form which was active in a Mg-chelatase reconstitution assay.

  16. Human Immunodeficiency Virus Integration Protein Expressed in Escherichia Coli Possesses Selective DNA Cleaving Activity

    NASA Astrophysics Data System (ADS)

    Sherman, Paula A.; Fyfe, James A.

    1990-07-01

    The human immunodeficiency virus (HIV) integration protein, a potential target for selective antiviral therapy, was expressed in Escherichia coli. The purified protein, free of detectable contaminating endonucleases, selectively cleaved double-stranded DNA oligonucleotides that mimic the U3 and the U5 termini of linear HIV DNA. Two nucleotides were removed from the 3' ends of both the U5 plus strand and the U3 minus strand; in both cases, cleavage was adjacent to a conserved CA dinucleotide. The reaction was metal-ion dependent, with a preference for Mn2+ over Mg2+. Reaction selectivity was further demonstrated by the lack of cleavage of an HIV U5 substrate on the complementary (minus) strand, an analogous substrate that mimics the U3 terminus of an avian retrovirus, and an HIV U5 substrate in which the conserved CA dinucleotide was replaced with a TA dinucleotide. Such an integration protein-mediated cleavage reaction is expected to occur as part of the integration event in the retroviral life cycle, in which a double-stranded DNA copy of the viral RNA genome is inserted into the host cell DNA.

  17. Basal expression of the cystic fibrosis transmembrane conductance regulator gene is dependent on protein kinase A activity.

    PubMed Central

    McDonald, R A; Matthews, R P; Idzerda, R L; McKnight, G S

    1995-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel that becomes activated after phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrate that PKA also plays a crucial role in maintaining basal expression of the CFTR gene in the human colon carcinoma cell line T84. Inhibition of PKA activity by expression of a dominant-negative regulatory subunit or treatment with the PKA-selective inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) caused a complete suppression of CFTR gene expression without affecting other constitutively active genes. Basal expression of a 2.2-kb region of the CFTR promoter linked to a luciferase reporter gene (CFTR-luc) exhibited the same dependence on PKA. The ability of cAMP to induce CFTR over basal levels is cell-type specific. In T84 cells, both the endogenous CFTR gene and CFTR-luc exhibited only a modest inducibility (approximately 2-fold), whereas in the human choriocarcinoma cell line JEG-3, CFTR-luc could be induced at least 4-fold. A variant cAMP-response element is present at position -48 to -41 in the CFTR promoter, and mutation of this sequence blocks basal expression. We conclude that cAMP, acting through PKA, is an essential regulator of basal CFTR gene expression and may mediate an induction of CFTR in responsive cell types. Images Fig. 1 Fig. 3 PMID:7543684

  18. Molecular cloning of amphioxus uncoupling protein and assessment of its uncoupling activity using a yeast heterologous expression system

    SciTech Connect

    Chen, Kun; Sun, Guoxun; Lv, Zhiyuan; Wang, Chen; Jiang, Xueyuan; Li, Donghai; Zhang, Chenyu

    2010-10-01

    Research highlights: {yields} Invertebrates, for example amphioxus, do express uncoupling proteins. {yields} Both the sequence and the uncoupling activity of amphioxus UCP resemble UCP2. {yields} UCP1 is the only UCP that can form dimer on yeast mitochondria. -- Abstract: The present study describes the molecular cloning of a novel cDNA fragment from amphioxus (Branchiostoma belcheri) encoding a 343-amino acid protein that is highly homologous to human uncoupling proteins (UCP), this protein is therefore named amphioxus UCP. This amphioxus UCP shares more homology with and is phylogenetically more related to mammalian UCP2 as compared with UCP1. To further assess the functional similarity of amphioxus UCP to mammalian UCP1 and -2, the amphioxus UCP, rat UCP1, and human UCP2 were separately expressed in Saccharomyces cerevisiae, and the recombinant yeast mitochondria were isolated and assayed for the state 4 respiration rate and proton leak, using pYES2 empty vector as the control. UCP1 increased the state 4 respiration rate by 2.8-fold, and the uncoupling activity was strongly inhibited by GDP, while UCP2 and amphioxus UCP only increased the state 4 respiration rate by 1.5-fold and 1.7-fold in a GDP-insensitive manner, moreover, the proton leak kinetics of amphioxus UCP was very similar to UCP2, but much different from UCP1. In conclusion, the amphioxus UCP has a mild, unregulated uncoupling activity in the yeast system, which resembles mammalian UCP2, but not UCP1.

  19. Involvement of p300 in constitutive and HIV-1 Tat-activated expression of glial fibrillary acidic protein in astrocytes

    PubMed Central

    Zou, Wei; Wang, Zhenyuan; Liu, Ying; Fan, Yan; Zhou, Betty Y.; Yang, X. Frank; He, Johnny J.

    2010-01-01

    HIV-1 Tat protein is an important pathogenic factor in HIV-1-associated neurological diseases. One hallmark of HIV-1 infection of the central nervous system (CNS) is astrocytosis, which is characterized by elevated GFAP expression in astrocytes. We have shown that Tat activates GFAP expression in astrocytes (Zhou, et al., Mol. Cell. Neurosci. 27:296, 2004) and that GFAP is an important regulator of Tat neurotoxicity (Zou, et. al., Am. J. Pathol. 171:1293, 2007). However, the underlying mechanisms for Tat-mediated GFAP up-regulation are not understood. In the current study, we reported concurrent up-regulation of adenovirus E1a-associated 300 kDa protein p300 and GFAP in Tat-expressing human astroytoma cells and primary astrocytes. We showed that p300 was indeed induced by Tat expression and HIV-1 infection and that the induction occurred at the transcriptional level through the cis-acting elements of early growth response 1 (Egr-1) within its promoter. Using siRNA, we further showed that p300 regulated both constitutive and Tat-mediated GFAP expression. Moreover, we showed that ectopic expression of p300 potentiated Tat transactivation activity and increased proliferation of HIV-1-infected astrocytes, but had little effect on HIV-1 replication in these cells. Taken together, these results demonstrate for the first time that Tat is a positive regulator of p300 expression, which in turn regulates GFAP expression, and suggest that the Tat-Egr-1-p300-GFAP axis likely contributes to Tat neurotoxicity and predisposes astrocytes to be an HIV-1 sanctuary in the CNS. PMID:20578042

  20. The novel Solanum tuberosum calcium dependent protein kinase, StCDPK3, is expressed in actively growing organs.

    PubMed

    Grandellis, Carolina; Giammaria, Verónica; Bialer, Magalí; Santin, Franco; Lin, Tian; Hannapel, David J; Ulloa, Rita M

    2012-12-01

    Calcium-dependent protein kinases (CDPKs) are key components of calcium regulated signaling cascades in plants. In this work, isoform StCDPK3 from Solanum tuberosum was studied and fully described. StCDPK3 encodes a 63 kDa protein with an N-terminal variable domain (NTV), rich in prolines and glutamines, which presents myristoylation and palmitoylation consensus sites and a PEST sequence indicative of rapid protein degradation. StCDPK3 gene (circa 11 kb) is localized in chromosome 3, shares the eight exons and seven introns structure with other isoforms from subgroup IIa and contains an additional intron in the 5'UTR region. StCDPK3 expression is ubiquitous being transcripts more abundant in early elongating stolons (ES), leaves and roots, however isoform specific antibodies only detected the protein in leaf particulate extracts. The recombinant 6xHis-StCDPK3 is an active kinase that differs in its kinetic parameters and calcium requirements from StCDPK1 and 2 isoforms. In vitro, StCDPK3 undergoes autophosphorylation regardless of the addition of calcium. The StCDPK3 promoter region (circa 1,800 bp) was subcloned by genome walking and fused to GUS. Light and ABRE responsive elements were identified in the promoter region as well as elements associated to expression in roots. StCDPK3 expression was enhanced by ABA while GA decreased it. Potato transgenic lines harboring StCDPK3 promoter∷GUS construct were generated by Agrobacterium tumefaciens mediated plant transformation. Promoter activity was detected in leaves, root tips and branching points, early ES, tuber eyes and developing sprouts indicating that StCDPK3 is expressed in actively growing organs.

  1. Diabetes and activation of peroxisome proliferator activated receptor alpha increases mitochondrial thioesterase I protein expression and activity in the heart

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mitochondrial thioesterase-I (MTE-I) catalyzes the de-esterification of fattyacyl-CoAs to fatty acid anions in the mitochondrial matrix, which are extruded to the cytosol, thus preventing the accumulation of toxic mitochondrial fattyacyl-CoAs. MTE-I mRNA expression in the heart is regulated by perox...

  2. Induced gene expression of the hypusine-containing protein eukaryotic initiation factor 5A in activated human T lymphocytes.

    PubMed Central

    Bevec, D; Klier, H; Holter, W; Tschachler, E; Valent, P; Lottspeich, F; Baumruker, T; Hauber, J

    1994-01-01

    The hypusine-containing protein eukaryotic initiation factor 5A (eIF-5A) is a cellular cofactor critically required for the function of the Rev transactivator protein of human immunodeficiency virus type 1 (HIV-1). eIF-5A localizes in the nuclear and cytoplasmic compartments of mammalian cells, suggesting possible activities on the level of regulated mRNA transport and/or protein translation. In this report we show that eIF-5A gene expression is constitutively low but inducible with T-lymphocyte-specific stimuli in human peripheral blood mononuclear cells (PBMCs) of healthy individuals. In contrast, eIF-5A is constitutively expressed at high levels in human cell lines as well as in various human organs. Comparison of eIF-5A levels in the PBMCs of uninfected and HIV-1-infected donors shows a significant upregulation of eIF-5A gene expression in the PBMCs of HIV-1 patients, compatible with a possible role of eIF-5A in HIV-1 replication during T-cell activation. Images PMID:7971969

  3. Effect of hyperosmotic conditions on flavin-containing monooxygenase activity, protein and mRNA expression in rat kidney

    PubMed Central

    Rodríguez-Fuentes, Gabriela; Coburn, Cary; Currás-Collazo, Margarita; Guillén, Gabriel; Schlenk, Daniel

    2010-01-01

    Flavin-containing monooxigenases (FMOs) are a polymorphic family of drug and pesticide metabolizing enzymes, found in the smooth endoplasmatic reticulum that catalyze the oxidation of soft nucleophilic heteroatom substances to their respective oxides. Previous studies in euryhaline fishes have indicated induction of FMO expression and activity in vivo under hyperosmotic conditions. In this study we evaluated the effect of hypersaline conditions in rat kidney. Male Sprague–Dawley rats were injected intraperitoneal with 3.5 M NaCl at a doses ranging from 0.3 cm3/100 g to 0.6 cm3/100 g in two separate treatments. Three hours after injection, FMO activities and FMO1 protein was examined in the first experiment, and the expression of FMO1 mRNA was measured in the second experiment from kidneys after treatment with NaCl. A positive significant correlation was found between FMO1 protein expression and plasma osmolarity (p < 0.05, r = 0.6193). Methyl-p-tolyl sulfide oxidase showed a statistically significant increase in FMO activity, and a positive correlation was observed between plasma osmolarity and production of FMO1-derived (R)-methyl-p-tolyl sulfoxide (p < 0.05, r = 0.6736). Expression of FMO1 mRNA was also positively correlated with plasma osmolality (p < 0.05, r = 0.8428). Similar to studies in fish, these results suggest that expression and activities of FMOs may be influenced by hyperosmotic conditions in the kidney of rats. PMID:19429252

  4. The Expression of Fibroblast Activation Protein in Clear Cell Renal Cell Carcinomas Is Associated with Synchronous Lymph Node Metastases

    PubMed Central

    Errarte, Peio; Guarch, Rosa; Pulido, Rafael; Blanco, Lorena; Nunes-Xavier, Caroline E.; Beitia, Maider; Gil, Javier; Angulo, Javier C.; López, José I.; Larrinaga, Gorka

    2016-01-01

    Clear cell renal cell carcinoma (CCRCC) is a heterogeneous and complex disease that frequently develops distant metastases. Fibroblast activation protein (FAP) is a serine peptidase the expression of which in cancer-associated fibroblasts has been associated with higher risk of metastases and poor survival. The objective of this study was to evaluate the role of FAP in metastatic CCRCC (mCCRCC). A series of 59 mCCRCC retrospectively collected was included in the study. Metastases developed either synchronous (n = 14) or metachronous to renal disease (n = 45). Tumor specimens were obtained from both primary lesion (n = 59) and metastases (n = 54) and FAP expression was immunohistochemically analyzed. FAP expression in fibroblasts from primary tumors correlated with FAP expression in the corresponding metastatic lesions. Also, primary and metastatic FAP expression was correlated with large tumor diameter (>7cm), high grade (G3/4), high stage (pT3/4), tumor necrosis and sarcomatoid transformation. The expression of FAP in primary tumors and in their metastases was associated both with synchronous metastases and also with metastases to the lymph nodes. FAP expression in the primary tumor was correlated with worse 10-year overall survival. Immunohistochemical detection of FAP in the stromal tumor fibroblasts could be a biomarker of early lymph node metastatic status and therefore could account for the poor prognosis of FAP positive CCRCC. PMID:28033421

  5. The selective protein kinase C inhibitor, Ro-31-8220, inhibits mitogen-activated protein kinase phosphatase-1 (MKP-1) expression, induces c-Jun expression, and activates Jun N-terminal kinase.

    PubMed

    Beltman, J; McCormick, F; Cook, S J

    1996-10-25

    The role of protein kinase C (PKC) in inflammation, mitogenesis, and differentiation has been deduced in part through the use of a variety of PKC inhibitors. Two widely used inhibitors are the structurally related compounds GF109203X and Ro-31-8220, both of which potently inhibit PKC activity and are believed to be highly selective. While using GF109203X and Ro-31-8220 to address the role of PKC in immediate early gene expression, we observed striking differential effects by each of these two compounds. Growth factors induce the expression of the immediate early gene products MAP kinase phosphatase-1 (MKP-1), c-Fos and c-Jun. Ro-31-8220 inhibits growth factor-stimulated expression of MKP-1 and c-Fos but strongly stimulated c-Jun expression, even in the absence of growth factors. GF109203X displays none of these properties. These data suggest that Ro-31-8220 may have other pharmacological actions in addition to PKC inhibition. Indeed, Ro-31-8220 strongly stimulates the stress-activated protein kinase, JNK1. Furthermore, Ro-31-8220 apparently activates JNK in a PKC-independent manner. Neither the down-regulation of PKC by phorbol esters nor the inhibition of PKC by GF109203X affected the ability of Ro-31-8220 to activate JNK1. These data suggest that, in addition to potently inhibiting PKC, Ro-31-8220 exhibits novel pharmacological properties which are independent of its ability to inhibit PKC.

  6. Cloning and expression of a transcription factor activator protein-1 (AP-1) member identified from manila clam Venerupis philippinarum.

    PubMed

    Wu, Luning; Zhang, Lei; Zhao, Jianmin; Ning, Xuanxuan; Mu, Changkao; Wang, Chunlin

    2015-02-15

    The transcription factor activator protein-1 (AP-1) proteins are implicated to play a major role in the regulation of numerous genes involved in the function and development of the immune system, cell differentiation, proliferation, apoptosis, etc. It can bind to promoter of its target genes in a sequence-specific manner to transactivate or repress them. In this study, the full-length cDNA of an AP-1 was identified from Venerupis philippinarum (denoted as VpAP-1) by EST analysis and RACE approaches. Phylogenetic analysis indicated that VpAP-1 had higher evolutional conservation to invertebrate than vertebrate counterparts and should be a new member of the AP-1 protein family. Spatial expression analysis found that VpAP-1 transcript was most abundantly expressed in the hemocytes and hepatopancreas, weakly expressed in the tissues of the gills, mantle and muscle. After Vibrio anguillarum challenge, the expression of VpAP-1 transcript in overall hemocyte population was up-regulated in the first 6h, and then decreased to 1.5-fold of the control group at 12h. As time progressed, a second peak of VpAP-1 expression was detected at 24h post-infection, which was 5-fold compared with that of the control group (P<0.01). After that, the expression level was sharply decreased and dropped to 0.5-fold of the control at 96h. The above results indicated that VpAP-1 was perhaps involved in the immune responses against microbe infection and might be contributed to the clearance of bacterial pathogens.

  7. Modeling Protein Expression and Protein Signaling Pathways

    PubMed Central

    Telesca, Donatello; Müller, Peter; Kornblau, Steven M.; Suchard, Marc A.; Ji, Yuan

    2015-01-01

    High-throughput functional proteomic technologies provide a way to quantify the expression of proteins of interest. Statistical inference centers on identifying the activation state of proteins and their patterns of molecular interaction formalized as dependence structure. Inference on dependence structure is particularly important when proteins are selected because they are part of a common molecular pathway. In that case, inference on dependence structure reveals properties of the underlying pathway. We propose a probability model that represents molecular interactions at the level of hidden binary latent variables that can be interpreted as indicators for active versus inactive states of the proteins. The proposed approach exploits available expert knowledge about the target pathway to define an informative prior on the hidden conditional dependence structure. An important feature of this prior is that it provides an instrument to explicitly anchor the model space to a set of interactions of interest, favoring a local search approach to model determination. We apply our model to reverse-phase protein array data from a study on acute myeloid leukemia. Our inference identifies relevant subpathways in relation to the unfolding of the biological process under study. PMID:26246646

  8. Genome-wide identification and transcriptional expression analysis of mitogen-activated protein kinase and mitogen-activated protein kinase kinase genes in Capsicum annuum

    PubMed Central

    Liu, Zhiqin; Shi, Lanping; Liu, Yanyan; Tang, Qian; Shen, Lei; Yang, Sheng; Cai, Jinsen; Yu, Huanxin; Wang, Rongzhang; Wen, Jiayu; Lin, Youquan; Hu, Jiong; Liu, Cailing; Zhang, Yangwen; Mou, Shaoliang; He, Shuilin

    2015-01-01

    The tripartite mitogen-activated protein kinase (MAPK) signaling cascades have been implicated in plant growth, development, and environment adaptation, but a comprehensive understanding of MAPK signaling at genome-wide level is limited in Capsicum annuum. Herein, genome-wide identification and transcriptional expression analysis of MAPK and MAPK kinase (MAPKK) were performed in pepper. A total of 19 pepper MAPK (CaMAPKs) genes and five MAPKK (CaMAPKKs) genes were identified. Phylogenetic analysis indicated that CaMAPKs and CaMAPKKs could be classified into four groups and each group contains similar exon-intron structures. However, significant divergences were also found. Notably, five members of the pepper MAPKK family were much less conserved than those found in Arabidopsis, and 9 Arabidopsis MAPKs did not have orthologs in pepper. Additionally, 7 MAPKs in Arabidopsis had either two or three orthologs in the pepper genome, and six pepper MAPKs and one MAPKK differing in sequence were found in three pepper varieties. Quantitative real-time RT-PCR analysis showed that the majority of MAPK and MAPKK genes were ubiquitously expressed and transcriptionally modified in pepper leaves after treatments with heat, salt, and Ralstonia solanacearum inoculation as well as exogenously applied salicylic acid, methyl jasmonate, ethephon, and abscisic acid. The MAPKK-MAPK interactome was tested by yeast two-hybrid assay, the results showed that one MAPKK might interact with multiple MAPKs, one MAPK might also interact with more than one MAPKKs, constituting MAPK signaling networks which may collaborate in transmitting upstream signals into appropriate downstream cellular responses and processes. These results will facilitate future functional characterization of MAPK cascades in pepper. PMID:26442088

  9. Visiting a forest, but not a city, increases human natural killer activity and expression of anti-cancer proteins.

    PubMed

    Li, Q; Morimoto, K; Kobayashi, M; Inagaki, H; Katsumata, M; Hirata, Y; Hirata, K; Suzuki, H; Li, Y J; Wakayama, Y; Kawada, T; Park, B J; Ohira, T; Matsui, N; Kagawa, T; Miyazaki, Y; Krensky, A M

    2008-01-01

    We previously reported that a forest bathing trip enhanced human NK activity, number of NK cells, and intracellular anti-cancer proteins in lymphocytes. In the present study, we investigated how long the increased NK activity lasts and compared the effect of a forest bathing trip on NK activity with a trip to places in a city without forests. Twelve healthy male subjects, age 35-56 years, were selected with informed consent. The subjects experienced a three-day/two-night trip to forest fields and to a city, in which activity levels during both trips were matched. On day 1, subjects walked for two hours in the afternoon in a forest field; and on day 2, they walked for two hours in the morning and afternoon, respectively, in two different forest fields; and on day 3, the subjects finished the trip and returned to Tokyo after drawing blood samples and completing the questionnaire. Blood and urine were sampled on the second and third days during the trips, and on days 7 and 30 after the trip, and NK activity, numbers of NK and T cells, and granulysin, perforin, and granzymes A/B-expressing lymphocytes in the blood samples, and the concentration of adrenaline in urine were measured. Similar measurements were made before the trips on a normal working day as the control. Phytoncide concentrations in forest and city air were measured. The forest bathing trip significantly increased NK activity and the numbers of NK, perforin, granulysin, and granzyme A/B-expressing cells and significantly decreased the concentration of adrenaline in urine. The increased NK activity lasted for more than 7 days after the trip. In contrast, a city tourist visit did not increase NK activity, numbers of NK cells, nor the expression of selected intracellular anti-cancer proteins, and did not decrease the concentration of adrenaline in urine. Phytoncides, such as alpha-pinene and beta-pinene were detected in forest air, but almost not in city air. These findings indicate that a forest bathing

  10. Melatonin alleviates myosin light chain kinase expression and activity via the mitogen-activated protein kinase pathway during atherosclerosis in rabbits

    PubMed Central

    CHENG, XIAOWEN; WAN, YUFENG; XU, YUANHONG; ZHOU, QING; WANG, YUAN; ZHU, HUAQING

    2015-01-01

    Melatonin (MLT) is an endogenous indole compound with numerous biological activities that has been associated with atherosclerosis (AS). In the present study, rabbits were used as an AS model in order to investigate whether MLT affects endothelial cell permeability, myosin light chain kinase (MLCK) activity and MLCK expression via the mitogen-activated protein kinase (MAPK) pathway. Expression and activity of MLCK were measured using western blot analysis, quantitative polymerase chain reaction, immunohistochemistry and γ-32P-adenosine triphosphate incorporation. Endothelial permeability was detected using rhodamine phalloidin fluorescence staining. The phosphorylation of extracellular regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 in endothelial cells were also analyzed using western blot analysis. Atheromatous plaques were formed in rabbits with a high cholesterol diet; however, following treatment with MLT, the number and areas of atheromatous plaques were significantly reduced. In addition, MLT treatment reversed the increase of MLCK activity and expression that occurred in rabbits with high cholesterol intake. Furthermore, levels of phosphorylated ERK, JNK and p38 decreased following MLT treatment. In conclusion, the results of the present study indicated that AS may be associated with increased MLCK expression and activity, which was reduced following treatment with MLT. The mechanism of action of MLT was thought to proceed via modulating MAPK pathway signal transduction; however, further studies are required in order to fully elucidate the exact regulatory mechanisms involved. PMID:25339116

  11. Preparation of biologically active platelet-derived growth factor type BB from a fusion protein expressed in Escherichia coli

    SciTech Connect

    Hoppe, J.; Weich, H.A.; Eichner, W. )

    1989-04-04

    Preparations of the mitogen platelet-derived growth factor (PDGF) from human platelets contain two related polypeptides termed A chain and B chain. PDGF-B is highly homologous to a portion of p28{sup v-sis}, the transforming protein of simian sarcoma virus. The authors have studied the mitogenic potential of a PDGF-BB-like homodimer by expressing the sequence coding for the mature part of PDGF-B in Escherichia coli. Expression was achieved as cro-{beta}-gal-PDGF-B fusion protein which was exclusively found in the inclusion bodies. A monomeric PDGF-B fragment shortened by 12 amino acid residues from the NH{sub 2} terminus was excised from the fusion protein by CNBr cleavage. After protection of thiols by S-sulfonation, this fragment was purified by gel permeation chromatography and reversed-phase high-performance liquid chromatography. This monomeric protein was dimerized in the presence of a mixture of reduced and oxidized glutathione to yield biologically active rPDGF-BB with an overall yield of {approx}0.7 mg of rPDGF-BB/L of culture. Escherichia coli rPDGF-BB stimulated ({sup 3}H)thymidine incorporation into AKR2B fibroblast at concentrations of about 1 ng/mL.

  12. Slit-Robo GTPase-activating proteins are differentially expressed in murine dorsal root ganglia: modulation by peripheral nerve injury.

    PubMed

    Chen, Zhi-Bing; Zhang, Hai-Ying; Zhao, Jiu-Hong; Zhao, Wei; Zhao, Dan; Zheng, Lin-Feng; Zhang, Xian-Fang; Liao, Xiao-Ping; Yi, Xi-Nan

    2012-04-01

    The Slit-Robo GTPase-activating proteins (srGAPs) play an important role in neurite outgrowth and axon guidance; however, little is known about its role in nerve regeneration after injury. Here, we studied the expression of srGAPs in mouse dorsal root ganglia (DRG) following sciatic nerve transection (SNT) using morphometric and immunohistochemical techniques. Reverse transcriptase polymerase chain reaction and Western blot analysis indicated that srGAP1 and srGAP3, but not srGAP2, were expressed in normal adult DRG. Following unilateral SNT, elevated mRNA and protein levels of srGAP1 and srGAP3 were detected in the ipsilateral relative to contralateral L(3-4) DRGs from day 3 to day 14. Immunohistochemical results showed that srGAP1 and srGAP3 were largely expressed in subpopulations of DRG neurons in naïve DRGs. However, after SNT, srGAP3 in neurons was significantly increased in the ipsilateral relative to contralateral DRGs, which peaked at day 7 to day 14. Interestingly, DRG neurons with strong srGAP3 labeling also coexpressed Robo2 after peripheral nerve injury. These results suggest that srGAPs are differentially expressed in murine DRG and srGAP3 are the predominant form. Moreover, srGAP3 may participate in Slit-Robo signaling in response to peripheral nerve injury or the course of nerve regeneration.

  13. Genome-Wide Identification and Expression Analysis of the Mitogen-Activated Protein Kinase Gene Family in Cassava

    PubMed Central

    Yan, Yan; Wang, Lianzhe; Ding, Zehong; Tie, Weiwei; Ding, Xupo; Zeng, Changying; Wei, Yunxie; Zhao, Hongliang; Peng, Ming; Hu, Wei

    2016-01-01

    Mitogen-activated protein kinases (MAPKs) play central roles in plant developmental processes, hormone signaling transduction, and responses to abiotic stress. However, no data are currently available about the MAPK family in cassava, an important tropical crop. Herein, 21 MeMAPK genes were identified from cassava. Phylogenetic analysis indicated that MeMAPKs could be classified into four subfamilies. Gene structure analysis demonstrated that the number of introns in MeMAPK genes ranged from 1 to 10, suggesting large variation among cassava MAPK genes. Conserved motif analysis indicated that all MeMAPKs had typical protein kinase domains. Transcriptomic analysis suggested that MeMAPK genes showed differential expression patterns in distinct tissues and in response to drought stress between wild subspecies and cultivated varieties. Interaction networks and co-expression analyses revealed that crucial pathways controlled by MeMAPK networks may be involved in the differential response to drought stress in different accessions of cassava. Expression of nine selected MAPK genes showed that these genes could comprehensively respond to osmotic, salt, cold, oxidative stressors, and abscisic acid (ABA) signaling. These findings yield new insights into the transcriptional control of MAPK gene expression, provide an improved understanding of abiotic stress responses and signaling transduction in cassava, and lead to potential applications in the genetic improvement of cassava cultivars. PMID:27625666

  14. Silicon Mitigates Salinity Stress by Regulating the Physiology, Antioxidant Enzyme Activities, and Protein Expression in Capsicum annuum 'Bugwang'.

    PubMed

    Manivannan, Abinaya; Soundararajan, Prabhakaran; Muneer, Sowbiya; Ko, Chung Ho; Jeong, Byoung Ryong

    2016-01-01

    Silicon- (Si-) induced salinity stress resistance was demonstrated at physiological and proteomic levels in Capsicum annuum for the first time. Seedlings of C. annuum were hydroponically treated with NaCl (50 mM) with or without Si (1.8 mM) for 15 days. The results illustrated that saline conditions significantly reduced plant growth and biomass and photosynthetic parameters and increased the electrolyte leakage potential, lipid peroxidation, and hydrogen peroxide level. However, supplementation of Si allowed the plants to recover from salinity stress by improving their physiology and photosynthesis. During salinity stress, Si prevented oxidative damage by increasing the activities of antioxidant enzymes. Furthermore, Si supplementation recovered the nutrient imbalance that had occurred during salinity stress. Additionally, proteomic analysis by two-dimensional gel electrophoresis (2DE) followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) revealed that Si treatment upregulated the accumulation of proteins involved in several metabolic processes, particularly those associated with nucleotide binding and transferase activity. Moreover, Si modulated the expression of vital proteins involved in ubiquitin-mediated nucleosome pathway and carbohydrate metabolism. Overall, the results illustrate that Si application induced resistance against salinity stress in C. annuum by regulating the physiology, antioxidant metabolism, and protein expression.

  15. Epigenetic Activation of μ-Opioid Receptor Gene via Increased Expression and Function of Mitogen- and Stress-Activated Protein Kinase 1.

    PubMed

    Wagley, Yadav; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H

    2017-04-01

    Since the discovery of μ-opioid receptor (MOR) gene two decades ago, various regulatory factors have been shown to interact with the MOR promoter and modulate transcript levels. However, the majority of early transcriptional studies on MOR gene have not addressed how intracellular signaling pathways mediate extracellular modulators. In this study, we demonstrate that MOR epigenetic regulation requires multiple coordinated signals converging at the MOR promoter, involving mitogen-activated protein kinase (MAPK) activation and mitogen- and stress-activated protein kinase 1 (MSK1)-ranges of intracellular signaling pathways similar to those activated by opioid agonists. Inhibiting p38 MAPK or extracellular signal-regulated kinase (ERK) 1/2 MAPK (upstream activators of MSK1) reduced MOR expression levels; accordingly, the functional role of MSK1, but not MSK2, was demonstrated using genetic approaches. However, for maximal MSK1 effect, an open chromatin configuration was required, because in vitro CpG methylation of the MOR promoter abolished MSK1 activity. Finally, endogenous MSK1 levels concomitantly increased to regulate MOR gene expression during neuronal differentiation of P19 cells, suggesting a conserved role of this kinase in the epigenic activation of MOR in neurons. Taken together, our findings indicate that the expression of MOR gene requires the activity of intracellular signaling pathways that have been implicated in the behavioral outcomes of opioid drugs, which suggests that an autoregulatory mechanism may function in opioid systems.

  16. Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

    SciTech Connect

    Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho; Jung, Young Do

    2012-03-01

    Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H{sub 2}O{sub 2}) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H{sub 2}O{sub 2} increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells

  17. Jasmonic acid affects plant morphology and calcium-dependent protein kinase expression and activity in Solanum tuberosum.

    PubMed

    Ulloa, Rita M; Raíces, Marcela; MacIntosh, Gustavo C; Maldonado, Sara; Téllez-Iñón, María T

    2002-07-01

    The effect of jasmonic acid (JA) on plant growth and on calcium-dependent protein kinase (CDPK) activity and expression was studied in non-photoperiodic potato plants, Solanum tuberosum L. var. Spunta, grown in vitro. Stem cuttings were grown for 45 days (long treatment, LT) in MS medium with increasing concentrations of JA. For short treatments (ST) adult plants grown in MS were transferred for 1, 4 and 20 h to JA containing media. During the LT, low concentrations of JA promoted cell expansion and shoot elongation while higher concentrations caused growth inhibition. Under these conditions, treated plants showed root shortening and tuber formation was not induced. Morphological and histochemical studies using light microscopy and TEM analysis of leaves from treated plants revealed that JA also affected subcellular organelles of mesophyll cells. Peroxisomes increased in size and number, and an autophagic process was triggered in response to high concentrations of the hormone. CDPK activity, determined in crude extracts of treated plants (LT), was inhibited (up to 80%). Plant growth and CDPK inhibition were reverted upon transfer of the plants to hormone-free medium. Soluble CDPK activity decreased in response to JA short treatment. Concomitantly, a decline in the steady state levels of StCDPK2 mRNA, a potato CDPK isoform that is expressed in leaves, was observed. These data suggest that the phytohormone down-regulated the expression and activity of the kinase.

  18. The Fe-type nitrile hydratase from Comamonas testosteroni Ni1 does not require an activator accessory protein for expression in Escherichia coli

    SciTech Connect

    Kuhn, Misty L.; Martinez, Salette; Gumataotao, Natalie; Bornscheuer, Uwe; Liu, Dali; Holz, Richard C.

    2012-10-10

    We report herein the functional expression of an Fe-type nitrile hydratase (NHase) without the co-expression of an activator protein or the Escherichia coli chaperone proteins GroES/EL. Soluble protein was obtained when the {alpha}- and {beta}-subunit genes of the Fe-type NHase Comamonas testosteroni Ni1 (CtNHase) were synthesized with optimized E. coli codon usage and co-expressed. As a control, the Fe-type NHase from Rhodococcus equi TG328-2 (ReNHase) was expressed with (ReNHase{sup +Act}) and without (ReNHase{sup -Act}) its activator protein, establishing that expression of a fully functional, metallated ReNHase enzyme requires the co-expression of its activator protein, similar to all other Fe-type NHase enzymes reported to date, whereas the CtNHase does not. The X-ray crystal structure of CtNHase was determined to 2.4 {angstrom} resolution revealing an {alpha}{beta} heterodimer, similar to other Fe-type NHase enzymes, except for two important differences. First, two His residues reside in the CtNHase active site that are not observed in other Fe-type NHase enzymes and second, the active site Fe(III) ion resides at the bottom of a wide solvent exposed channel. The solvent exposed active site, along with the two active site histidine residues, are hypothesized to play a role in iron incorporation in the absence of an activator protein.

  19. The Fe-type nitrile hydratase from Comamonas testosteroni Ni1 does not require an activator accessory protein for expression in Escherichia coli.

    PubMed

    Kuhn, Misty L; Martinez, Salette; Gumataotao, Natalie; Bornscheuer, Uwe; Liu, Dali; Holz, Richard C

    2012-08-03

    We report herein the functional expression of an Fe-type nitrile hydratase (NHase) without the co-expression of an activator protein or the Escherichia coli chaperone proteins GroES/EL. Soluble protein was obtained when the α- and β-subunit genes of the Fe-type NHase Comamonas testosteroni Ni1 (CtNHase) were synthesized with optimized E. coli codon usage and co-expressed. As a control, the Fe-type NHase from Rhodococcus equi TG328-2 (ReNHase) was expressed with (ReNHase(+Act)) and without (ReNHase(-Act)) its activator protein, establishing that expression of a fully functional, metallated ReNHase enzyme requires the co-expression of its activator protein, similar to all other Fe-type NHase enzymes reported to date, whereas the CtNHase does not. The X-ray crystal structure of CtNHase was determined to 2.4Å resolution revealing an αβ heterodimer, similar to other Fe-type NHase enzymes, except for two important differences. First, two His residues reside in the CtNHase active site that are not observed in other Fe-type NHase enzymes and second, the active site Fe(III) ion resides at the bottom of a wide solvent exposed channel. The solvent exposed active site, along with the two active site histidine residues, are hypothesized to play a role in iron incorporation in the absence of an activator protein.

  20. Bid, a Widely Expressed Proapoptotic Protein of the Bcl-2 Family, Displays Lipid Transfer Activity

    PubMed Central

    Esposti, Mauro Degli; Erler, Janine T.; Hickman, John A.; Dive, Caroline

    2001-01-01

    Bid is an abundant proapoptotic protein of the Bcl-2 family that is crucial for the induction of death receptor-mediated apoptosis in primary tissues such as liver. Bid action has been proposed to involve the relocation of its truncated form, tBid, to mitochondria to facilitate the release of apoptogenic cytochrome c. The mechanism of Bid relocation to mitochondria was unclear. We report here novel biochemical evidence indicating that Bid has lipid transfer activity between mitochondria and other intracellular membranes, thereby explaining its dynamic relocation to mitochondria. First, physiological concentrations of phospholipids such as phosphatidic acid and phosphatidylgycerol induced an accumulation of full-length Bid in mitochondria when incubated with light membranes enriched in endoplasmic reticulum. Secondly, native and recombinant Bid, as well as tBid, displayed lipid transfer activity under the same conditions and at the same nanomolar concentrations leading to mitochondrial relocation and release of cytochrome c. Thus, Bid is likely to be involved in the transport and recycling of mitochondrial phospholipids. We discuss how this new role of Bid may relate to its proapoptotic action. PMID:11585909

  1. Aldosterone regulates cellular turnover and mitogen-activated protein kinase family expression in the neonatal rat kidney.

    PubMed

    Yim, Hyung Eun; Yoo, Kee Hwan; Bae, In Sun; Jang, Gi Young; Hong, Young Sook; Lee, Joo Won

    2009-06-01

    Growing evidence indicates that aldosterone is a potent mitogenic signal regulating genes involved in antiapoptosis, cell proliferation and growth. We investigated the role of endogenous aldosterone in renal development, cell proliferation and apoptosis, and mitogen-activated protein kinase (MAPK) family expression. Newborn rats were treated with either spironolactone (200 mg/kg/d) in olive oil or only olive oil for 7 days. TUNEL assay and proliferating cell nuclear antigen (PCNA) stain were performed on kidney sections. Immunoblots, immunohistochemical (IHC) stain, and reverse transcriptase-PCR for MAPKs were performed. PCNA-positive proliferating cells decreased and apoptotic cells increased significantly with spironolactone (P < 0.05). In the spironolactone-treated group, c-jun N-terminal kinase (JNK)-2 expression increased, whereas extracellular signal regulated kinase (ERK)-2 and p38 expressions decreased in immunoblots (P < 0.05) and IHC stain. ERK-2 and p38 mRNA expressions increased in the spironolactone-treated group (P < 0.05). This study demonstrates that aldosterone blockade in the developing kidney decreases cellular proliferation, increases apoptosis, and modulates the expressions of JNK-2, ERK-2, and p38. Aldosterone possibly participates in renal development and MAPK family may serve as, in part, the signaling intermediate through the mineralocorticoid receptor (MR) in the developing kidney. J. Cell. Physiol. 219: 724-733, 2009. (c) 2009 Wiley-Liss, Inc.

  2. Resveratrol Inhibits Expression and Binding Activity of the Monocyte Chemotactic Protein-1 Receptor, CCR2, on THP-1 Monocytes

    PubMed Central

    Cullen, John P.; Morrow, David; Jin, Ying; von Offenberg Sweeney, Nicholas; Sitzmann, James V.; Cahill, Paul A.; Redmond, Eileen M.

    2007-01-01

    Monocyte chemotactic protein-1 and its receptor, CCR2, play a key role in atherosclerosis. We determined the effect of the polyphenol, resveratrol, on CCR2 and the mechanisms involved. Resveratrol treatment inhibited 125I-MCP-1 binding to THP-1 cells; 31%, 56%, 84% decrease for 10, 50 and 100 µM resveratrol, in the absence of any effect on receptor affinity. The inhibitory effect of resveratrol on 125I-MCP-1 binding to THP-1 cells and on CCR2 protein expression determined by FACS analysis was attenuated by treatment with L-NAME (NOS inhibitor), PD98059 (MAPK inhibitor) and LY294002 (PI3K inhibitor), whereas neither X/XO (reactive oxygen species generator) nor ICI182780 (estrogen receptor antagonist) had any effect. Concomitant with a decrease in CCR2 protein expression, resveratrol inhibited THP-1 CCR2 mRNA levels, in the absence of any effect on its stability; 26% and 45% inhibition at 10 and 50 µM resveratrol, respectively. This effect was not altered by co-treatment with L-NAME, PD98059 or ICI182780, but was potentiated by LY294002 and X/XO. Conclusions: Resveratrol inhibits monocyte CCR2 binding activity in an NO-, MAPK- and PI3K-dependent manner, whereas it inhibits CCR2 mRNA in an NO- and MAPK-independent, PI3K-dependent manner. These inhibitory effects of resveratrol on chemokine receptor binding and expression may contribute, in part, to its cardiovascular protective activity in vivo. PMID:17499741

  3. Cystatin D Locates in the Nucleus at Sites of Active Transcription and Modulates Gene and Protein Expression*

    PubMed Central

    Ferrer-Mayorga, Gemma; Alvarez-Díaz, Silvia; Valle, Noelia; De Las Rivas, Javier; Mendes, Marta; Barderas, Rodrigo; Canals, Francesc; Tapia, Olga; Casal, J. Ignacio; Lafarga, Miguel; Muñoz, Alberto

    2015-01-01

    Cystatin D is an inhibitor of lysosomal and secreted cysteine proteases. Strikingly, cystatin D has been found to inhibit proliferation, migration, and invasion of colon carcinoma cells indicating tumor suppressor activity that is unrelated to protease inhibition. Here, we demonstrate that a proportion of cystatin D locates within the cell nucleus at specific transcriptionally active chromatin sites. Consistently, transcriptomic analysis show that cystatin D alters gene expression, including that of genes encoding transcription factors such as RUNX1, RUNX2, and MEF2C in HCT116 cells. In concordance with transcriptomic data, quantitative proteomic analysis identified 292 proteins differentially expressed in cystatin D-expressing cells involved in cell adhesion, cytoskeleton, and RNA synthesis and processing. Furthermore, using cytokine arrays we found that cystatin D reduces the secretion of several protumor cytokines such as fibroblast growth factor-4, CX3CL1/fractalkine, neurotrophin 4 oncostatin-M, pulmonary and activation-regulated chemokine/CCL18, and transforming growth factor B3. These results support an unanticipated role of cystatin D in the cell nucleus, controlling the transcription of specific genes involved in crucial cellular functions, which may mediate its protective action in colon cancer. PMID:26364852

  4. Functional cooperation between Smad proteins and activator protein-1 regulates transforming growth factor-beta-mediated induction of endothelin-1 expression.

    PubMed

    Rodríguez-Pascual, Fernando; Redondo-Horcajo, Mariano; Lamas, Santiago

    2003-06-27

    Endothelin-1 (ET-1) is a 21-amino-acid potent vasoconstrictor peptide that is mainly produced by vascular endothelial cells. Expression of the ET-1 gene is subject to complex regulation by numerous factors, among which transforming growth factor-beta (TGF-beta) is one of the most important. It has been widely documented that TGF-beta increases ET-1 mRNA and peptide levels. We have explored the mechanism by which TGF-beta upregulates ET-1 expression in endothelial cells. Transcriptional activation of the ET-1 promoter accounted for the TGF-beta-induced increase in ET-1 mRNA levels. We have identified within the ET-1 promoter two DNA elements indispensable for TGF-beta-mediated induction of ET-1: an activator protein-1 (AP-1) site at -108/-102, known to be important for constitutive and induced expression, and a novel regulatory sequence located at -193/-171, which constitutes a specific binding site for Smad transcription factors. Mutation of both elements abolished TGF-beta responsiveness. Binding of Smad3/Smad4 and c-Jun to their corresponding DNA elements was evidenced by electrophoretic mobility shift assays. Furthermore, the coactivator CREB-binding protein (CBP)/p300 was found to play an essential role in the induction of the gene. The simultaneous requirement for two distinct and independent DNA elements suggests that Smads and activator protein-1 functionally cooperate through CBP/p300 to mediate TGF-beta-induced transcriptional activation of the ET-1 gene.

  5. Receptor for Activated Protein Kinase C: Requirement for Efficient MicroRNA Function and Reduced Expression in Hepatocellular Carcinoma

    PubMed Central

    Otsuka, Motoyuki; Takata, Akemi; Yoshikawa, Takeshi; Kojima, Kentaro; Kishikawa, Takahiro; Shibata, Chikako; Takekawa, Mutsuhiro; Yoshida, Haruhiko; Omata, Masao; Koike, Kazuhiko

    2011-01-01

    MicroRNAs (miRNAs) are important regulators of gene expression that control physiological and pathological processes. A global reduction in miRNA abundance and function is a general trait of human cancers, playing a causal role in the transformed phenotype. Here, we sought to newly identify genes involved in the regulation of miRNA function by performing a genetic screen using reporter constructs that measure miRNA function and retrovirus-based random gene disruption. Of the six genes identified, RACK1, which encodes “receptor for activated protein kinase C” (RACK1), was confirmed to be necessary for full miRNA function. RACK1 binds to KH-type splicing regulatory protein (KSRP), a member of the Dicer complex, and is required for the recruitment of mature miRNAs to the RNA-induced silencing complex (RISC). In addition, RACK1 expression was frequently found to be reduced in hepatocellular carcinoma. These findings suggest the involvement of RACK1 in miRNA function and indicate that reduced miRNA function, due to decreased expression of RACK1, may have pathologically relevant roles in liver cancers. PMID:21935400

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

    SciTech Connect

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

    2006-11-15

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

  7. Strain differences in cytochrome P450 mRNA and protein expression, and enzymatic activity among Sprague Dawley, Wistar, Brown Norway and Dark Agouti rats.

    PubMed

    Nishiyama, Yoshihiro; Nakayama, Shouta M M; Watanabe, Kensuke P; Kawai, Yusuke K; Ohno, Marumi; Ikenaka, Yoshinori; Ishizuka, Mayumi

    2016-05-03

    Rat cytochrome P450 (CYP) exhibits inter-strain differences, but their analysis has been scattered across studies under different conditions. To identify these strain differences in CYP more comprehensively, mRNA expression, protein expression and metabolic activity among Wistar (WI), Sprague Dawley (SD), Dark Agouti (DA) and Brown Norway (BN) rats were compared. The mRNA level and enzymatic activity of CYP1A1 were highest in SD rats. The rank order of Cyp3a2 mRNA expression mirrored its protein expression, i.e., DA>BN>SD>WI, and was similar to the CYP3A2-dependent warfarin metabolic activity, i.e., DA>SD>BN>WI. These results suggest that the strain differences in CYP3A2 enzymatic activity are caused by differences in mRNA expression. Cyp2b1 mRNA levels, which were higher in DA rats, did not correlate with its protein expression or enzymatic activity. This suggests that the strain differences in enzymatic activity are not related to Cyp2b1 mRNA expression. In conclusion, WI rats tended to have the lowest CYP1A1, 2B1 and 3A2 mRNA expression, protein expression and enzymatic activity among the strains. In addition, SD rats had the highest CYP1A1 mRNA expression and activity, while DA rats had higher CYP2B1 and CYP3A2 mRNA and protein expression. These inter-strain differences in CYP could influence pharmacokinetic considerations in preclinical toxicological studies.

  8. Nicotine stimulates adhesion molecular expression via calcium influx and mitogen-activated protein kinases in human endothelial cells.

    PubMed

    Wang, Yajing; Wang, Zhaoxia; Zhou, Ying; Liu, Liming; Zhao, Yangxing; Yao, Chenjiang; Wang, Lianyun; Qiao, Zhongdong

    2006-02-01

    To evaluate the effect of nicotine on endothelium dysfunction and development of vascular diseases, we investigated the influence on adhesion molecular expression mediated by nicotine and the mechanism of this effect in human umbilical vein endothelial cells (HUVECs). The result showed that nicotine could induce surface/soluble vascular cell adhesion molecule (VCAM-1) and endothelial selectin (E-selectin) expression in a time-response decline manner and the peak appeared at 15 min. This action could be mediated by mitogen-activated protein kinase/extracellular signal regulated kinase 1/2 (MAPK/ERK1/2) and MAPK/p38 because their activation could be distinctly blocked by MAPK inhibitors, PD098059 or SB203580. Mecamylamine (non-selective nicotinic receptor inhibitor), alpha-bungarotoxin (alpha7 nicotinic receptor inhibitor) could block Ca2+ accumulation, and then, prevented the phosphorylation on ERK1/2 and p38. They also inhibited the surface/soluble VCAM-1, E-selectin production of HUVECs modulated by nicotine. Therefore, we concluded that: (i) nicotine obviously up-regulates VCAM-1 and E-selectin expression at 15 min in HUVECs, (ii) nicotine activates HUVECs triggered by the ERK1/2 and p38 phosphorylation with an involvement of intracellular calcium mobilization chiefly mediated by alpha7 nicotinic receptor, (iii) intracellular Ca2+ activates a sequential pathway from alpha7 nicotinic receptor to the phosphorylation of ERK1/2, p38. These elucidate that nicotine activates HUVECs through fast signal transduction pathway and arguments their capacity of adhesion molecular production. Further more nicotine may contribute its influence to the progression of vascular disease such as atherosclerotic lesion.

  9. A WRKY Transcription Factor Recruits the SYG1-Like Protein SHB1 to Activate Gene Expression and Seed Cavity Enlargement

    PubMed Central

    Kang, Xiaojun; Li, Wei; Zhou, Yun; Ni, Min

    2013-01-01

    Seed development in Arabidopsis and in many dicots involves an early proliferation of the endosperm to form a large embryo sac or seed cavity close to the size of the mature seed, followed by a second phase during which the embryo grows and replaces the endosperm. SHORT HYPOCOTYL UNDER BLUE1 (SHB1) is a member of the SYG1 protein family in fungi, Caenorhabditis elegans, flies, and mammals. SHB1 gain-of-function enhances endosperm proliferation, increases seed size, and up-regulates the expression of the WRKY transcription factor gene MINISEED3 (MINI3) and the LRR receptor kinase gene HAIKU2 (IKU2). Mutations in either IKU2 or MINI3 retard endosperm proliferation and reduce seed size. However, the molecular mechanisms underlying the establishment of the seed cavity and hence the seed size remain largely unknown. Here, we show that the expression of MINI3 and IKU2 is repressed before fertilization and after 4 days after pollination (DAP), but is activated by SHB1 from 2 to 4 DAP prior to the formation of the seed cavity. SHB1 associates with their promoters but without a recognizable DNA binding motif, and this association is abolished in mini3 mutant. MINI3 binds to W-boxes in, and recruits SHB1 to, its own and IKU2 promoters. Interestingly, SHB1, but not MINI3, activates transcription of pMINI3::GUS or pIKU2::GUS. We reveal a critical developmental switch through the activation of MINI3 expression by SHB1. The recruitment of SHB1 by MINI3 to its own and IKU2 promoters represents a novel two-step amplification to counter the low expression level of IKU2, which is a trigger for endosperm proliferation and seed cavity enlargement. PMID:23505389

  10. AMP-activated protein kinase mediates T cell activation-induced expression of FasL and COX-2 via protein kinase C theta-dependent pathway in human Jurkat T leukemia cells.

    PubMed

    Lee, Jung Yeon; Choi, A-Young; Oh, Young Taek; Choe, Wonchae; Yeo, Eui-Ju; Ha, Joohun; Kang, Insug

    2012-06-01

    AMP-activated protein kinase (AMPK), an important regulator of energy homeostasis, is known to be activated during T cell activation. T cell activation by T cell receptor (TCR) engagement or its pharmacological mimics, PMA plus ionomycin (PMA/Io), induces immunomodulatory FasL and cyclooxygenase-2 (COX-2) expression. In this study, we examined the role and mechanisms of AMPK in PMA/Io-induced expression of FasL and COX-2 in Jurkat T human leukemic cells. Inhibition of AMPK by a pharmacological agent, compound C, or AMPKα1 siRNA suppressed expression of FasL and COX-2 mRNAs and proteins in PMA/Io-activated Jurkat cells. It also reduced secretion of FasL protein and prostaglandin E2, a main product of COX-2, in Jurkat cells and peripheral blood lymphocytes activated with PMA/Io or monoclonal anti-CD3 plus anti-CD28. Consistently, inhibition of AMPK blocked promoter activities of FasL and COX-2 in activated Jurkat cells. As protein kinase C theta (PKCθ) is a central molecule for TCR signaling, we examined any possible cross-talk between AMPK and PKCθ in activated T cells. Of particular importance, we found that inhibition of AMPK blocked phosphorylation and activation of PKCθ, suggesting that AMPK is an upstream kinase of PKCθ. Moreover, we showed that AMPK was directly associated with PKCθ and phosphorylated Thr538 of PKCθ in PMA/Io-stimulated Jurkat cells. We also showed that inhibition of PKCθ by rottlerin or dominant negative PKCθ reduced AMPK-mediated transcriptional activation of NF-AT and AP-1 in activated Jurkat cells. Taken together, these results suggest that AMPK regulates expression of FasL and COX-2 via the PKCθ and NF-AT and AP-1 pathways in activated Jurkat cells.

  11. Leptospira Protein Expression During Infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We are characterizing protein expression in vivo during experimental leptospirosis using immunofluorescence microscopy. Coding regions for several proteins were identified through analysis of Leptospira interrogans serovar Copenhageni and L. borgpetersenii serovar Hardjo genomes. In addition, codi...

  12. Angiotensin II regulates brain (pro)renin receptor expression through activation of cAMP response element-binding protein.

    PubMed

    Li, Wencheng; Liu, Jiao; Hammond, Sean L; Tjalkens, Ronald B; Saifudeen, Zubaida; Feng, Yumei

    2015-07-15

    We reported that brain (pro)renin receptor (PRR) expression levels are elevated in DOCA-salt-induced hypertension; however, the underlying mechanism remained unknown. To address whether ANG II type 1 receptor (AT1R) signaling is involved in this regulation, we implanted a DOCA pellet and supplied 0.9% saline as the drinking solution to C57BL/6J mice. Sham pellet-implanted mice that were provided regular drinking water served as controls. Concurrently, mice were intracerebroventricularly infused with the AT1R blocker losartan, angiotensin-converting-enzyme inhibitor captopril, or artificial cerebrospinal fluid for 3 wk. Intracerebroventricular infusion of losartan or captopril attenuated DOCA-salt-induced PRR mRNA elevation in the paraventricular nucleus of the hypothalamus, suggesting a role for ANG II/AT1R signaling in regulating PRR expression during DOCA-salt hypertension. To test which ANG II/AT1R downstream transcription factors were involved in PRR regulation, we treated Neuro-2A cells with ANG II with or without CREB (cAMP response element-binding protein) or AP-1 (activator protein-1) inhibitors, or CREB siRNA. CREB and AP-1 inhibitors, as well as CREB knockdown abolished ANG II-induced increases in PRR levels. ANG II also induced PRR upregulation in primary cultured neurons. Chromatin immunoprecipitation assays revealed that ANG II treatment increased CREB binding to the endogenous PRR promoter in both cultured neurons and hypothalamic tissues of DOCA-salt hypertensive mice. This increase in CREB activity was reversed by AT1R blockade. Collectively, these findings indicate that ANG II acts via AT1R to upregulate PRR expression both in cultured cells and in DOCA-salt hypertensive mice by increasing CREB binding to the PRR promoter.

  13. Sodium-pump gene-expression, protein abundance and enzyme activity in isolated nephron segments of the aging rat kidney

    PubMed Central

    Scherzer, Pnina; Gal-Moscovici, Anca; Sheikh-Hamad, David; Popovtzer, Mordecai M

    2015-01-01

    Aging is associated with alteration in renal tubular functions, including sodium handling and concentrating ability. Na-K-ATPase plays a key role in driving tubular transport, and we hypothesized that decreased concentrating ability of the aging kidney is due in part to downregulation of Na-K-ATPase. In this study, we evaluated Na and K balance, aldosterone levels, and Na-K-ATPase gene expression, protein abundance, and activity in aging rat kidney. Na-K-ATPase activity (assayed microfluorometrically), mRNA (RT-PCR), and protein abundance (immunoblotting) were quantitated in the following isolated nephron segments: PCT, PST, MTAL, DCT, and CCD from 2, 8, 15, and 24 month-old-rats. In the course of aging, creatinine clearance decreased from 0.48 ± 0.02 mL/min/100 g BW to 0.28 ± 0.06 (P < 0.001) and aldosterone decreased from 23.6 ± 0.8 ng/dL to 13.2 ± 0.6 (P < 0.001). Serum Na+ and K+ increased by 4.0% and 22.5%, respectively. Na-K-ATPase activity, mRNA, and protein abundance of the α1 subunit displayed similar trends in all assayed segments; increasing in PCT and PST; decreasing in MTAL and DCT; increasing in CCD: in PCT they increased by 40%, 75%, and 250%, respectively; while in PST they increased by 80%, 50%, and 100%, respectively (P < 0.001). In MTAL they declined by 36%, 24%, and 34%, respectively, and in DCT by 38%, 59%, and 60%, respectively (P < 0.001). They were higher in CCD by 110%, 115%, and 246%, respectively (P < 0.001). Rats maintained Na/K balance; however with a steady state elevated serum K+. These results reveal quantitative changes in axial distribution of Na-K-ATPase at the level of gene expression, protein abundance, and activity in the nephrons of aging animals and may explain, in part, the pathophysiology of the senescent kidney. PMID:26056060

  14. MDCK cells expressing constitutively active Yes-associated protein (YAP) undergo apical extrusion depending on neighboring cell status

    PubMed Central

    Chiba, Takanori; Ishihara, Erika; Miyamura, Norio; Narumi, Rika; Kajita, Mihoko; Fujita, Yasuyuki; Suzuki, Akira; Ogawa, Yoshihiro; Nishina, Hiroshi

    2016-01-01

    Cell competition is a cell-cell interaction by which a cell compares its fitness to that of neighboring cells. The cell with the relatively lower fitness level is the “loser” and actively eliminated, while the cell with the relatively higher fitness level is the “winner” and survives. Recent studies have shown that cells with high Yes-associated protein (YAP) activity win cell competitions but the mechanism is unknown. Here, we report the unexpected finding that cells overexpressing constitutively active YAP undergo apical extrusion and are losers, rather than winners, in competitions with normal mammalian epithelial cells. Inhibitors of metabolism-related proteins such as phosphoinositide-3-kinase (PI3K), mammalian target of rapamycin (mTOR), or p70S6 kinase (p70S6K) suppressed this apical extrusion, as did knockdown of vimentin or filamin in neighboring cells. Interestingly, YAP-overexpressing cells switched from losers to winners when co-cultured with cells expressing K-Ras (G12V) or v-Src. Thus, the role of YAP in deciding cell competitions depends on metabolic factors and the status of neighboring cells. PMID:27324860

  15. The Saccharomyces cerevisiae Leu3 protein activates expression of GDH1, a key gene in nitrogen assimilation.

    PubMed

    Hu, Y; Cooper, T G; Kohlhaw, G B

    1995-01-01

    The Leu3 protein of Saccharomyces cerevisiae has been shown to be a transcriptional regulator of genes encoding enzymes of the branched-chain amino acid biosynthetic pathways. Leu3 binds to upstream activating sequences (UASLEU) found in the promoters of LEU1, LEU2, LEU4, ILV2, and ILV5. In vivo and in vitro studies have shown that activation by Leu3 requires the presence of alpha-isopropylmalate. In at least one case (LEU2), Leu3 actually represses basal-level transcription when alpha-isopropylmalate is absent. Following identification of a UASLEU-homologous sequence in the promoter of GDH1, the gene encoding NADP(+)-dependent glutamate dehydrogenase, we demonstrate that Leu3 specifically interacts with this UASLEU element. We then show that Leu3 is required for full activation of the GDH1 gene. First, the expression of a GDH1-lacZ fusion gene is three- to sixfold lower in a strain lacking the LEU3 gene than in an isogenic LEU3+ strain. Expression is restored to near-normal levels when the leu3 deletion cells are transformed with a LEU3-bearing plasmid. Second, a significant decrease in GDH1-lacZ expression is also seen when the UASLEU of the GDH1-lacZ construct is made nonfunctional by mutation. Third, the steady-state level of GDH1 mRNA decreases about threefold in leu3 null cells. The decrease in GDH1 expression in leu3 null cells is reflected in a diminished specific activity of NADP(+)-dependent glutamate dehydrogenase. We also demonstrate that the level of GDH1-lacZ expression correlates with the cells' ability to generate alpha-isopropylmalate and is lowest in cells unable to produce alpha-isopropylmalate. We conclude that GDH1, which plays an important role in the assimilation of ammonia in yeast cells, is, in part, activated by a Leu3-alpha-isopropylmalate complex. This conclusion suggests that Leu3 participates in transcriptional regulation beyond the branched-chain amino acid biosynthetic pathways.

  16. Myricetin Increases Hepatic Peroxisome Proliferator-Activated Receptor α Protein Expression and Decreases Plasma Lipids and Adiposity in Rats

    PubMed Central

    Chang, Chia Ju; Tzeng, Thing-Fong; Liou, Shorong-Shii; Chang, Yuan-Shiun; Liu, I-Min

    2012-01-01

    The aim of this study was to investigate the antiobesity and antihyperlipidaemic effects of myricetin. Myricetin exhibited a significant concentration-dependent decrease in the intracellular accumulation of triglyceride in 3T3-L1 adipocytes. The high-fat diet (HFD)-fed rats were dosed orally with myricetin or fenofibrate, once daily for eight weeks. Myricetin (300 mg kg−1 per day) displayed similar characteristics to fenofibrate (100 mg kg−1 per day) in reducing lowered body weight (BW) gain, visceral fat-pad weights and plasma lipid levels of HFD-fed rats. Myricetin also reduced the hepatic triglyceride and cholesterol contents, as well as lowered hepatic lipid droplets accumulation and epididymal adipocyte size in HFD-fed rats. Myricetin and fenofibrate reversed the HFD-induced down-regulation of the hepatic peroxisome proliferator activated receptor (PPAR)α. HFD-induced decreases of the hepatic protein level of acyl-CoA oxidase and cytochrome P450 isoform 4A1 were up-regulated by myricetin and fenofibrate. The elevated expressions of hepatic sterol regulatory element binding proteins (SREBPs) of HFD-fed rats were lowered by myricetin and fenofibrate. These results suggest that myricetin suppressed BW gain and body fat accumulation by increasing the fatty acid oxidation, which was likely mediated via up-regulation of PPARα and down-regulation of SREBP expressions in the liver of HFD-fed rats. PMID:22474525

  17. Yin Yang 1 cooperates with activator protein 2 to stimulate ERBB2 gene expression in mammary cancer cells.

    PubMed

    Begon, Dominique Y; Delacroix, Laurence; Vernimmen, Douglas; Jackers, Pascale; Winkler, Rosita

    2005-07-01

    Overexpression of the ERBB2 oncogene is observed in about 30% of breast cancers and is generally correlated with a poor prognosis. Previous results from our and other laboratories indicated that elevated transcriptional activity contributes significantly to the overexpression of ERBB2 mRNA in mammary adenocarcinoma cell lines. Activator protein 2 (AP-2) transcription factors account for this overexpression through two recognition sequences located 215 and 500 bp upstream from the transcription start site. Furthermore, AP-2 transcription factors are highly expressed in cancer cell lines overexpressing ERBB2. In this report, we examined the cooperative effect of Yin Yang 1 (YY1) on AP-2-induced activation of ERBB2 promoter activity. We detected high levels of YY1 transcription factor in mammary cancer cell lines. Notably, we showed that YY1 enhances AP-2alpha transcriptional activation of the ERBB2 promoter through an AP-2 site both in HepG2 and in HCT116 cells, whereas a carboxyl-terminal-truncated form of YY1 cannot. Moreover, we demonstrated the interaction between endogenous AP-2 and YY1 factors in the BT-474 mammary adenocarcinoma cell line. In addition, inhibition of endogenous YY1 protein by an antisense decreased the transcription of an AP-2-responsive ERBB2 reporter plasmid in BT-474 breast cancer cells. Finally, we detected in vivo AP-2 and YY1 occupancy of the ERBB2 proximal promoter in chromatin immunoprecipitation assays. Our data thus provide evidence that YY1 cooperates with AP-2 to stimulate ERBB2 promoter activity through the AP-2 binding sites.

  18. Human peroxisome proliferator-activated receptor mRNA and protein expression during development

    EPA Science Inventory

    The peroxisome proliferator-activated receptors (PPAR) are nuclear hormone receptors that regulate lipid and glucose homeostasis and are important in reproduction and development. PPARs are targets ofpharmaceuticals and are also activated by environmental contaminants, including ...

  19. Surface protein Esp enhances pro-inflammatory cytokine expression through NF-κB activation during enterococcal infection.

    PubMed

    Zou, Jun; Shankar, Nathan

    2016-01-01

    Enterococcal surface protein (Esp) is encoded on a pathogenicity island in Enterococcus faecalis and E. faecium and is involved in biofilm formation and binding to epithelial cells. In this study, using Esp-expressing E. faecalis MMH594 and its isogenic Esp-deficient strain, as well as purified Esp, we show that Esp is sufficient for activation of NF-κB and the subsequent production of pro-inflammatory cytokines IL-1β and TNF-α in macrophages in vitro. In a mouse peritonitis model, we also show that mice infected with Esp-expressing E. faecalis showed comparatively higher levels of cytokines TNF-α, IL-1β and IL-6 in peritoneal fluid, and IL-6 in serum. Moreover, neutrophil infiltration and tissue damage in the liver was higher in the mice infected with the Esp-expressing strain compared with mice infected with the Esp-deficient mutant. These results add Esp to the growing list of enterococcal virulence factors that can modulate inflammation during infection and has implications for enterococcal pathogenesis.

  20. Quercetin activates AMP-activated protein kinase by reducing PP2C expression protecting old mouse brain against high cholesterol-induced neurotoxicity.

    PubMed

    Lu, Jun; Wu, Dong-Mei; Zheng, Yuan-Lin; Hu, Bin; Zhang, Zi-Feng; Shan, Qun; Zheng, Zi-Hui; Liu, Chan-Min; Wang, Yong-Jian

    2010-10-01

    It is known that a high-cholesterol diet induces oxidative stress, inflammatory response, and beta-amyloid (Abeta) accumulation in mouse brain, resulting in neurodegenerative changes. Quercetin, a naturally occurring flavonoid, has been reported to possess numerous biological activities beneficial to health. Our previous studies have demonstrated that quercetin protects mouse brain against D-galactose-induced oxidative damage. Against this background, we evaluated the effect of quercetin on high-cholesterol-induced neurotoxicity in old mice and explored its potential mechanism. Our results showed that oral administration of quercetin significantly improved the behavioural performance of high-cholesterol-fed old mice in both a step-through test and the Morris water maze task. This is at least in part caused by decreasing ROS and protein carbonyl levels and restoring Cu--Zn superoxide dismutase (Cu, Zn-SOD) activity. Furthermore, quercetin also significantly activated the AMP-activated protein kinase (AMPK) via down-regulation of protein phosphatase 2C (PP2C), which reduced the integral optical density (IOD) of activated microglia cells and CD11b expression, down-regulated iNOS and cyclooxygenase-2 (COX-2) expression, and decreased IL-1beta, IL-6, and TNF-alpha expression in the brains of high-cholesterol-fed old mice through the suppression of NF-kappaB p65 nuclear translocation. Moreover, AMPK activation significantly increased 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and acetyl-CoA carboxylase (ACC) phosphorylation and reduced fatty acid synthase (FAS) expression in the brains of high-cholesterol-fed old mice, which reduced cholesterol levels, down-regulated cholesterol 24-hydroxylase (CYP46A1) and beta-amyloid converting enzyme 1 (BACE1) expression, decreased eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation, and lowered Abeta deposits. However, the neuroprotective effect of quercetin was weakened by intraperitoneal

  1. The mammalian AMP-activated protein kinase complex mediates glucose regulation of gene expression in the yeast Saccharomyces cerevisiae.

    PubMed

    Ye, Tian; Bendrioua, Loubna; Carmena, David; García-Salcedo, Raúl; Dahl, Peter; Carling, David; Hohmann, Stefan

    2014-06-05

    The AMP-activated protein kinase (AMPK) controls energy homeostasis in eukaryotic cells. Here we expressed hetero-trimeric mammalian AMPK complexes in a Saccharomyces cerevisiae mutant lacking all five genes encoding yeast AMPK/SNF1 components. Certain mammalian complexes complemented the growth defect of the yeast mutant on non-fermentable carbon sources. Phosphorylation of the AMPK α1-subunit was glucose-regulated, albeit not by the Glc7-Reg1/2 phosphatase, which performs this function on yeast AMPK/SNF1. AMPK could take over SNF1 function in glucose derepression. While indirectly acting anti-diabetic drugs had no effect on AMPK in yeast, compound 991 stimulated α1-subunit phosphorylation. Our results demonstrate a remarkable functional conservation of AMPK and that glucose regulation of AMPK may not be mediated by regulatory features of a specific phosphatase.

  2. Whole genome expression profiling associates activation of unfolded protein response with impaired production and release of epinephrine after recurrent hypoglycemia

    PubMed Central

    Kim, Juhye Lena; La Gamma, Edmund F.; Estabrook, Todd; Kudrick, Necla

    2017-01-01

    Recurrent hypoglycemia can occur as a major complication of insulin replacement therapy, limiting the long-term health benefits of intense glycemic control in type 1 and advanced type 2 diabetic patients. It impairs the normal counter-regulatory hormonal and behavioral responses to glucose deprivation, a phenomenon known as hypoglycemia associated autonomic failure (HAAF). The molecular mechanisms leading to defective counter-regulation are not completely understood. We hypothesized that both neuronal (excessive cholinergic signaling between the splanchnic nerve fibers and the adrenal medulla) and humoral factors contribute to the impaired epinephrine production and release in HAAF. To gain further insight into the molecular mechanism(s) mediating the blunted epinephrine responses following recurrent hypoglycemia, we utilized a global gene expression profiling approach. We characterized the transcriptomes during recurrent (defective counter-regulation model) and acute hypoglycemia (normal counter-regulation group) in the adrenal medulla of normal Sprague-Dawley rats. Based on comparison analysis of differentially expressed genes, a set of unique genes that are activated only at specific time points after recurrent hypoglycemia were revealed. A complementary bioinformatics analysis of the functional category, pathway, and integrated network indicated activation of the unfolded protein response. Furthermore, at least three additional pathways/interaction networks altered in the adrenal medulla following recurrent hypoglycemia were identified, which may contribute to the impaired epinephrine secretion in HAAF: greatly increased neuropeptide signaling (proenkephalin, neuropeptide Y, galanin); altered ion homeostasis (Na+, K+, Ca2+) and downregulation of genes involved in Ca2+-dependent exocytosis of secretory vesicles. Given the pleiotropic effects of the unfolded protein response in different organs, involved in maintaining glucose homeostasis, these findings uncover

  3. Hepatic expression of inflammatory genes and microRNAs in pigs with high "cholesteryl ester transfer protein" (CETP) activity.

    PubMed

    Cirera, Susanna; Tørsleff, Benedicte C Juul; Ritz, Christian; Fredholm, Merete; Heegaard, Peter M H; Skovgaard, Kerstin

    2016-10-01

    Human obesity and obesity-related diseases (ORD) are growing health problems worldwide and represent a major public health challenge. Most of these diseases are complex conditions, influenced by many genes (including microRNAs) and environmental factors. Many metabolic perturbations are associated with obesity; e.g., low levels of high-density lipoproteins (HDL) are high risk factors of cardiovascular events. A number of genetic, lifestyle, and environmental factors have been shown to contribute to the lowering of HDL-cholesterol. One of these factors is cholesteryl ester transfer protein (CETP) promoting the redistribution of cholesteryl esters, triglycerides, and phospholipids between plasma proteins. Moreover, obesity and ORD are often linked with chronic low-grade inflammation leading to insulin resistance and endothelial and microvascular dysfunctions. The aim of this study was to detect differences in the hepatic expression of genes involved in low-grade inflammation and of obesity- and cholesterol-related microRNAs in two mixed breed populations of pigs (Yorkshire-Göttingen minipig, YM and Duroc-Göttingen minipig, DM) including males and females, with extreme phenotypes for CETP activity levels (designated as CETP-high and CETP-low, respectively). Furthermore, breed and gender differences were also investigated. We found significant difference (P < 0.05) in hepatic expression levels of several mRNAs and microRNAs between the CETP-high and -low groups (C5, IL1RN, IL18, and miR-223-5p); between the two mixed breeds (IL1RAP and miR-140-5p); and between gender (APOA1, IL1RN, and FBLN1). Furthermore, when taking breed into account we show that the transcriptional levels of TNF, miR20a, miR33b, and miR130a differed between the two CETP groups. We conclude that increased CETP activity is accompanied by a modest differential hepatic expression of several microRNAs and inflammatory-related genes. Furthermore, our study demonstrates that when modeling the analysis

  4. Functional constraints on SoxE proteins in neural crest development: The importance of differential expression for evolution of protein activity.

    PubMed

    Lee, Eric M; Yuan, Tian; Ballim, Reyna D; Nguyen, Kristy; Kelsh, Robert N; Medeiros, Daniel M; McCauley, David W

    2016-10-01

    Vertebrate SoxE genes (Sox8, 9, and 10) are key regulators of neural crest cell (NCC) development. These genes arose by duplication from a single SoxE gene in the vertebrate ancestor. Although SoxE paralogs are coexpressed early in NCC development, later, Sox9 is restricted to skeletogenic lineages in the head, and Sox10 to non-skeletogenic NCC in the trunk and head. When this subfunctionalization evolved and its possible role in the evolution of the neural crest are unknown. Sea lampreys are basal vertebrates that also possess three SoxE genes, while only a single SoxE is present in the cephalochordate amphioxus. In order to address the functional divergence of SoxE genes, and to determine if differences in their biochemical functions may be linked to changes in neural crest developmental potential, we examined the ability of lamprey and amphioxus SoxE genes to regulate differentiation of NCC derivatives in zebrafish colourless (cls) mutants lacking expression of sox10. Our findings suggest that the proto-vertebrate SoxE gene possessed both melanogenic and neurogenic capabilities prior to SoxE gene duplication. Following the agnathan-gnathostome split, lamprey SoxE1 and SoxE3 largely lost their melanogenic and/or enteric neurogenic properties, while gnathostome SoxE paralogs have retained functional conservation. We posit that this difference in protein subfunctionalization is a direct consequence of the independent regulation of SoxE paralog expression between the two lineages. Specifically, we propose that the overlapping expression of gnathostome SoxE paralogs in early neural crest largely constrained the function of gnathostome SoxE proteins. In contrast, the largely non-overlapping expression of lamprey SoxE paralogs allowed them to specialize with regard to their DNA-binding and/or protein interaction properties. Restriction of developmental potential among cranial and trunk neural crest in lampreys may be related to constraints on SoxE activity among

  5. Modulation of Brahma expression by the mitogen-activated protein kinase/extracellular signal regulated kinase pathway is associated with changes in melanoma proliferation.

    PubMed

    Mehrotra, Aanchal; Saladi, Srinivas Vinod; Trivedi, Archit R; Aras, Shweta; Qi, Huiling; Jayanthy, Ashika; Setaluri, Vijayasaradhi; de la Serna, Ivana L

    2014-12-01

    Brahma (BRM) and Brahma-related gene 1(BRG1) are catalytic subunits of SWItch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complexes. BRM is epigenetically silenced in a wide-range of tumors. Mutations in the v-raf murine sarcoma viral oncogene homolog B1 (BRAF) gene occur frequently in melanoma and lead to constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK1/2) pathway. We tested the hypothesis that BRM expression is modulated by oncogenic BRAF and phosphorylation of ERK1/2 in melanocytes and melanoma cells. Expression of oncogenic BRAF in melanocytes and melanoma cells that are wild-type for BRAF decreased BRM expression and increased BRG1 expression. Inhibition of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) or selective inhibition of BRAF in melanoma cells that harbor oncogenic BRAF increased BRM expression and decreased BRG1 expression. Increased BRM expression was associated with increased histone acetylation on the BRM promoter. Over-expression of BRM in melanoma cells that harbor oncogenic BRAF promoted changes in cell cycle progression and apoptosis consistent with a tumor suppressive role. Upon inhibition of BRAF(V600E) with PLX4032, BRM promoted survival. PLX4032 induced changes in BRM function were correlated with increased acetylation of the BRM protein. This study provides insights into the epigenetic consequences of inhibiting oncogenic BRAF in melanoma through modulation of SWI/SNF subunit expression and function.

  6. Peroxisome Proliferator Activated Receptors Alpha, Beta, and Gamma mRNA and protein expression in human fetal tissues

    EPA Science Inventory

    Peroxisome proliferator-activated receptors (PPARs) regulate lipid and glucose homeostasis, are targets of pharmaceuticals, and are also activated by environmental contaminants. Almost nothing is known about expression of PPARs during human fetal development. This study examine...

  7. Involvement of mitogen-activated protein kinases and NF{kappa}B in LPS-induced CD40 expression on human monocytic cells

    SciTech Connect

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

    2008-04-15

    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 NF{kappa}B were activated by LPS exposure and involved in LPS-induced CD40 expression. Activation of MAPKs was not responsible for LPS-induced NF{kappa}B 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 NF{kappa}B 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 NF{kappa}B activation, and CD40 expression. Moreover, blockage of MAPK and NF{kappa}B activation inhibited LPS-induced TLR4 expression. In summary, LPS-induced CD40 expression in monocytic cells involves MAPKs and NF{kappa}B.

  8. Bamboo Vinegar Decreases Inflammatory Mediator Expression and NLRP3 Inflammasome Activation by Inhibiting Reactive Oxygen Species Generation and Protein Kinase C-α/δ Activation

    PubMed Central

    Ka, Shuk-Man; Chen, Ann; Tasi, Yu-Ling; Liu, May-Lan; Chiu, Yi-Chich; Hua, Kuo-Feng

    2013-01-01

    Bamboo vinegar (BV), a natural liquid derived from the condensation produced during bamboo charcoal production, has been used in agriculture and as a food additive, but its application to immune modulation has not been reported. Here, we demonstrated that BV has anti-inflammatory activities both in vitro and in vivo. BV reduced inducible nitric oxide synthase expression and nitric oxide levels in, and interleukin-6 secretion by, lipopolysaccharide-activated macrophages without affecting tumor necrosis factor-α secretion and cyclooxygenase-2 expression. The mechanism for the anti-inflammatory effect of BV involved decreased reactive oxygen species production and protein kinase C-α/δ activation. Furthermore, creosol (2-methoxy-4-methylphenol) was indentified as the major anti-inflammatory compound in BV. Impaired cytokine expression and NLR family, pyrin domain-containing 3 (NLRP3) inflammasome activation was seen in mice treated with creosol. These findings provide insights into how BV regulates inflammation and suggest that it may be a new source for the development of anti-inflammatory agents or a healthy supplement for preventing and ameliorating inflammation- and NLRP3 inflammasome-related diseases, including metabolic syndrome. PMID:24124509

  9. Nintedanib modulates surfactant protein-D expression in A549 human lung epithelial cells via the c-Jun N-terminal kinase-activator protein-1 pathway.

    PubMed

    Kamio, Koichiro; Usuki, Jiro; Azuma, Arata; Matsuda, Kuniko; Ishii, Takeo; Inomata, Minoru; Hayashi, Hiroki; Kokuho, Nariaki; Fujita, Kazue; Saito, Yoshinobu; Miya, Toshimichi; Gemma, Akihiko

    2015-06-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive disease with a high mortality rate. Signalling pathways activated by several tyrosine kinase receptors are known to be involved in lung fibrosis, and this knowledge has led to the development of the triple tyrosine kinase inhibitor nintedanib, an inhibitor of vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR), for the treatment of IPF. Pulmonary surfactant protein D (SP-D), an important biomarker of IPF, reportedly attenuates bleomycin-induced pulmonary fibrosis in mice. In this study, we investigated whether nintedanib modulates SP-D expression in human lung epithelial (A549) cells using quantitative real-time reverse transcriptase polymerase chain reaction and western blotting. To investigate the mechanisms underlying the effects of nintedanib, we evaluated the phosphorylation of c-Jun N-terminal kinase (JNK) and its downstream target c-Jun. The effect of the JNK inhibitor SP600125 on c-Jun phosphorylation was also tested. Activation of activator protein-1 (AP-1) was examined using an enzyme-linked immunosorbent assay-based test, and cell proliferation assays were performed to estimate the effect of nintedanib on cell proliferation. Furthermore, we treated mice with nintedanib to examine its in vivo effect on SP-D levels in lungs. These experiments showed that nintedanib up-regulated SP-D messenger RNA expression in a dose-dependent manner at concentrations up to 5 μM, with significant SP-D induction observed at concentrations of 3 μM and 5 μM, in comparison with that observed in vehicle controls. Nintedanib stimulated a rapid increase in phosphorylated JNK in A549 cells within 30 min of treatment and stimulated c-Jun phosphorylation, which was inhibited by the JNK inhibitor SP600125. Additionally, nintedanib was found to activate AP-1. A549 cell proliferation was not affected by nintedanib at any of the tested

  10. Effects of stoichiometry and temperature perturbations on beech litter decomposition, enzyme activities and protein expression

    NASA Astrophysics Data System (ADS)

    Keiblinger, K. M.; Schneider, T.; Roschitzki, B.; Schmid, E.; Eberl, L.; Hämmerle, I.; Leitner, S.; Richter, A.; Wanek, W.; Riedel, K.; Zechmeister-Boltenstern, S.

    2011-12-01

    Microbes are major players in leaf litter decomposition and therefore advances in the understanding of their control on element cycling are of paramount importance. Our aim was to investigate the influence of leaf litter stoichiometry in terms of carbon (C) : nitrogen (N) : phosphorus (P) on the decomposition process, and to follow changes in microbial community structure and function in response to temperature-stress treatments. To elucidate how the stoichiometry of beech litter (Fagus sylvatica L.) and stress treatments interactively affect the decomposition processes, a terrestrial microcosm experiment was conducted. Beech litter from different Austrian sites covering C:N ratios from 39 to 61 and C:P ratios from 666 to 1729 were incubated at 15 °C and 60% moisture for six months. Part of the microcosms were then subjected to severe changes in temperature (+30 °C and -15 °C) to monitor the influence of temperature stress. Extracellular enzyme activities were assayed and respiratory activities measured. A semi-quantitative metaproteomics approach (1D-SDS PAGE combined with liquid chromatography and tandem mass-spectrometry; unique spectral counting) was employed to investigate the impact of the applied stress treatments in dependency of litter stoichiometry on structure and function of the decomposing community. In litter with narrow C:nutrient ratios microbial decomposers were most abundant. Cellulase, chitinase, phosphatase and protease activity decreased after heat and frost treatments. Decomposer communities and specific functions varied with site i.e. stoichiometry. The applied stress evoked strong changes of enzyme activities, dissolved organic nitrogen and litter pH. Freeze treatments resulted in a decline in residual plant litter material, and increased fungal abundance indicating slightly accelerated decomposition. Overall, we could detect a strong effect of litter stoichiometry on microbial community structure as well as function. Temperature

  11. Immune Activation in the Female Genital Tract: Expression Profiles of Soluble Proteins in Women at High Risk for HIV Infection

    PubMed Central

    Francis, Suzanna C.; Hou, Yanwen; Baisley, Kathy; van de Wijgert, Janneke; Watson-Jones, Deborah; Ao, Trong T.; Herrera, Carolina; Maganja, Kaballa; Andreasen, Aura; Kapiga, Saidi; Coulton, Gary R.; Hayes, Richard J.; Shattock, Robin J.

    2016-01-01

    Soluble cervicovaginal biomarkers of inflammation, immune activation and risk of HIV acquisition are needed to reliably assess the safety of new biomedical prevention strategies including vaccines and microbicides. However, a fuller understanding of expression profiles in women at high risk for HIV infection is crucial to the effective use of these potential biomarkers in Phase 3 trial settings. We have measured 45 soluble proteins and peptides in cervicovaginal lavage samples from 100 HIV negative women at high risk for HIV infection. Women were followed over one menstrual cycle to investigate modulation by hormonal contraception, menstrual cycle phase, recent sexual exposure and intravaginal practices. Women using injectable DMPA had increased concentration of several soluble proteins of the innate and adaptive immune system, including IL-1α, IL-1β, IL-2, MIP-1β, IP-10, IL-8, TGF-β, HBD4, IgA, IgG1, and IgG2. Women using combined oral contraceptives had a similar signature. There were differences in concentrations among samples from post-ovulation compared to pre-ovulation, notably increased immunoglobulins. Increased prostate-specific antigen, indicative of recent sexual exposure, was correlated with increased IL-6, MCP-1, and SLPI, and decreased GM-CSF and HBD3. The identified signature profiles may prove critical in evaluating the potential safety and impact on risk of HIV acquisition of different biomedical intervention strategies. PMID:26814891

  12. Pentoxifylline Regulates Plasminogen Activator Inhibitor-1 Expression and Protein Kinase A Phosphorylation in Radiation-Induced Lung Fibrosis

    PubMed Central

    Bae, Chang-Hwan; Jin, Young-Woo; Lee, Seung-Sook

    2017-01-01

    Purpose. Radiation-induced lung fibrosis (RILF) is a serious late complication of radiotherapy. In vitro studies have demonstrated that pentoxifylline (PTX) has suppressing effects in extracellular matrix production in fibroblasts, while the antifibrotic action of PTX alone using clinical dose is yet unexplored. Materials and Methods. We used micro-computed tomography (micro-CT) and histopathological analysis to evaluate the antifibrotic effects of PTX in a rat model of RILF. Results. Micro-CT findings showed that lung density, volume loss, and mediastinal shift are significantly increased at 16 weeks after irradiation. Simultaneously, histological analysis demonstrated thickening of alveolar walls, destruction of alveolar structures, and excessive collagen deposition in the irradiated lung. PTX treatment effectively attenuated the fibrotic changes based on both micro-CT and histopathological analyses. Western analysis also revealed increased levels of plasminogen activator inhibitor- (PAI-) 1 and fibronectin (FN) and PTX treatment reduced expression of PAI-1 and FN by restoring protein kinase A (PKA) phosphorylation but not TGF-β/Smad in both irradiated lung tissues and epithelial cells. Conclusions. Our results demonstrate the antifibrotic effect of PTX on radiation-induced lung fibrosis and its effect on modulation of PKA and PAI-1 expression as possible antifibrotic mechanisms. PMID:28337441

  13. Recombinant protein expression in Nicotiana.

    PubMed

    Matoba, Nobuyuki; Davis, Keith R; Palmer, Kenneth E

    2011-01-01

    Recombinant protein pharmaceuticals are now widely used in treatment of chronic diseases, and several recombinant protein subunit vaccines are approved for human and veterinary use. With growing demand for complex protein pharmaceuticals, such as monoclonal antibodies, manufacturing capacity is becoming limited. There is increasing need for safe, scalable, and economical alternatives to mammalian cell culture-based manufacturing systems, which require substantial capital investment for new manufacturing facilities. Since a seminal paper reporting immunoglobulin expression in transgenic plants was published in 1989, there have been many technological advances in plant expression systems to the present time where production of proteins in leaf tissues of nonfood crops such as Nicotiana species is considered a viable alternative. In particular, transient expression systems derived from recombinant plant viral vectors offer opportunities for rapid expression screening, construct optimization, and expression scale-up. Extraction of recombinant proteins from Nicotiana leaf tissues can be achieved by collection of secreted protein fractions, or from a total protein extract after grinding the leaves with buffer. After separation from solids, the major purification challenge is contamination with elements of the photosynthetic complex, which can be solved by application of a variety of facile and proven strategies. In conclusion, the technologies required for safe, efficient, scalable manufacture of recombinant proteins in Nicotiana leaf tissues have matured to the point where several products have already been tested in phase I clinical trials and will soon be followed by a rich pipeline of recombinant vaccines, microbicides, and therapeutic proteins.

  14. Effects of stoichiometry and temperature perturbations on beech leaf litter decomposition, enzyme activities and protein expression

    NASA Astrophysics Data System (ADS)

    Keiblinger, K. M.; Schneider, T.; Roschitzki, B.; Schmid, E.; Eberl, L.; Hämmerle, I.; Leitner, S.; Richter, A.; Wanek, W.; Riedel, K.; Zechmeister-Boltenstern, S.

    2012-11-01

    Microbes are major players in leaf litter decomposition and therefore advances in the understanding of their control on element cycling are of paramount importance. Our aim was to investigate the influence of leaf litter stoichiometry in terms of carbon (C) : nitrogen (N) : phosphorus (P) ratios on the decomposition processes and to track changes in microbial community structures and functions in response to temperature stress treatments. To elucidate how the stoichiometry of beech leaf litter (Fagus sylvatica L.) and stress treatments interactively affect the microbial decomposition processes, a terrestrial microcosm experiment was conducted. Beech litter from different Austrian sites covering C:N ratios from 39 to 61 and C:P ratios from 666 to 1729 were incubated at 15 °C and 60% moisture for six months. Part of the microcosms were then subjected to severe changes in temperature (+30 °C and -15 °C) to monitor the influence of temperature stress. Extracellular enzyme activities were assayed and respiratory activities measured. A semi-quantitative metaproteomics approach (1D-SDS PAGE combined with liquid chromatography and tandem mass spectrometry; unique spectral counting) was employed to investigate the impact of the applied stress treatments in dependency of litter stoichiometry on structure and function of the decomposing community. In litter with narrow C:nutrient (C:N, C:P) ratios, microbial decomposers were most abundant. Cellulase, chitinase, phosphatase and protease activity decreased after heat and freezing treatments. Decomposer communities and specific functions varied with site, i.e. stoichiometry. The applied stress combined with the respective time of sampling evoked changes of enzyme activities and litter pH. Freezing treatments resulted in a decline in residual plant litter material and increased fungal abundance, indicating slightly accelerated decomposition. Overall, a strong effect of litter stoichiometry on microbial community structures and

  15. Activation of AMP-activated protein kinase induce expression of FoxO1, FoxO3a, and myostatin after exercise-induced muscle damage.

    PubMed

    Lee, Kihyuk; Ochi, Eisuke; Song, Hongsun; Nakazato, Koichi

    2015-10-23

    AMP-activated protein kinase (AMPK) has been shown to regulate protein metabolism in skeletal muscle. We previously found that levels of Forkhead box proteins, FoxO1 and FoxO3a, and myostatin in rat gastrocnemius increased after exercise-induced muscle damage (EIMD). Eccentric muscle contractions (ECs), defined as elongation of muscle under tension, were used for inducing EIMD. The objective of this study was to clarify whether AMPK participates in activation and expression of FoxO proteins and myostatin in rat gastrocnemius muscle after EIMD. Wistar rats were randomly assigned into the following three groups; CON (n = 6), 180ECs group (ankle angular velocity, 180°/s; n = 6), and 30ECs group (ankle angular velocity, 30°/s; n = 6). 20 ECs were conducted with percutaneous electrical stimulation of gastrocnemius and simultaneous forced dorsiflexion of ankle joint (from 0° to 45°). To evaluate activation of AMPK, we measured the phosphorylated states of AMPK and acetyl CoA carboxylase. For evaluation of the direct relationships of AMPK and other proteins, we also examined contents of FoxOs and myostatin with stimulation of L6 myotube with AMPK agonist, 5 -aminoimidazole -4 -carboxamide -1-β-d-ribofuranoside (AICAR) (0.1, 0.5, 1, 1.5, and 2 mM). Western blotting was employed for protein analysis. Significant torque deficit was only observed in the 180ECs, suggesting EIMD. We also observed that phosphorylated AMPKα was induced in response to 180ECs (p < 0.01 vs. CON). Additionally, the level of phosphorylated acetyl CoA carboxylase was significantly higher in response to 180ECs and 30ECs. The phosphorylated states of FoxO1, FoxO3a, and myostatin expression were increased significantly in response to 180ECs. Furthermore, treatment of L6 myotubes with AICAR showed similar tendencies to that observed in in vivo gastrocnemius muscle treated with 180ECs. Therefore, we conclude that activation of AMPK plays a key role in increasing the level of FoxO1, FoxO3a

  16. A stimulus-specific role for CREB-binding protein (CBP) in T cell receptor-activated tumor necrosis factor gene expression

    NASA Astrophysics Data System (ADS)

    Falvo, James V.; Brinkman, Brigitta M. N.; Tsytsykova, Alla V.; Tsai, Eunice Y.; Yao, Tso-Pang; Kung, Andrew L.; Goldfeld, Anne E.

    2000-04-01

    The cAMP response element binding protein (CREB)-binding protein (CBP)/p300 family of coactivator proteins regulates gene transcription through the integration of multiple signal transduction pathways. Here, we show that induction of tumor necrosis factor (TNF-) gene expression in T cells stimulated by engagement of the T cell receptor (TCR) or by virus infection requires CBP/p300. Strikingly, in mice lacking one copy of the CBP gene, TNF- gene induction by TCR activation is inhibited, whereas virus induction of the TNF- gene is not affected. Consistent with these findings, the transcriptional activity of CBP is strongly potentiated by TCR activation but not by virus infection of T cells. Thus, CBP gene dosage and transcriptional activity are critical in TCR-dependent TNF-α gene expression, demonstrating a stimulus-specific requirement for CBP in the regulation of a specific gene.

  17. Heterologous Expression of MeLEA3: A 10 kDa Late Embryogenesis Abundant Protein of Cassava, Confers Tolerance to Abiotic Stress in Escherichia coli with Recombinant Protein Showing In Vitro Chaperone Activity.

    PubMed

    Barros, Nicolle L F; da Silva, Diehgo T; Marques, Deyvid N; de Brito, Fabiano M; dos Reis, Savio P; de Souza, Claudia R B

    2015-01-01

    Late embryogenesis abundant (LEA) proteins are small molecular weight proteins involved in acquisition of tolerance to drought, salinity, high temperature, cold, and freezing stress in many plants. Previous studies revealed a cDNA sequence coding for a 10 kDa atypical LEA protein, named MeLEA3, predicted to be located into mitochondria with potential role in salt stress response of cassava (Manihot esculenta Crantz). Here we aimed to produce the recombinant MeLEA3 protein by heterologous expression in Escherichia coli and evaluate the tolerance of bacteria expressing this protein under abiotic stress. Our result revealed that the recombinant MeLEA3 protein conferred a protective function against heat and salt stress in bacterial cells. Also, the recombinant MeLEA3 protein showed in vitro chaperone activity by protection of NdeI restriction enzyme activity under heat stress.

  18. Improvement of two-way active avoidance memory requires protein kinase a activation and brain-derived neurotrophic factor expression in the dorsal hippocampus.

    PubMed

    Datta, Subimal; Siwek, Donald F; Huang, Max P

    2009-07-01

    Previous studies have shown that two-way active avoidance (TWAA) memory processing involves a functional interaction between the pontine wave (P wave) generator and the CA3 region of the dorsal hippocampus (DH-CA3). The present experiments examined whether the interaction between P wave generator activity and the DH-CA3 involves the intracellular protein kinase A (PKA) signaling system. In the first series of experiments, rats were subjected to a session of TWAA training followed immediately by bilateral microinjection of either the PKA activation inhibitor (KT-5720) or vehicle control into the DH-CA3 and tested for TWAA memory 24 h later. The results indicated that immediate KT-5720 infusion impaired improvement of TWAA performance. Additional experiments showed that KT-5720 infusion also blocked TWAA training-induced BDNF expression in the DH-CA3. Together, these findings suggest that the PKA activation and BDNF expression in the DH-CA3 is essential for the improvement of TWAA memory.

  19. Involvement of adenosine monophosphate-activated protein kinase in the influence of timed high-fat evening diet on the hepatic clock and lipogenic gene expression in mice.

    PubMed

    Huang, Yan; Zhu, Zengyan; Xie, Meilin; Xue, Jie

    2015-09-01

    A high-fat diet may result in changes in hepatic clock gene expression, but potential mechanisms are not yet elucidated. Adenosine monophosphate-activated protein kinase (AMPK) is a serine/threonine protein kinase that is recognized as a key regulator of energy metabolism and certain clock genes. Therefore, we hypothesized that AMPK may be involved in the alteration of hepatic clock gene expression under a high-fat environment. This study aimed to examine the effects of timed high-fat evening diet on the activity of hepatic AMPK, clock genes, and lipogenic genes. Mice with hyperlipidemic fatty livers were induced by orally administering high-fat milk via gavage every evening (19:00-20:00) for 6 weeks. Results showed that timed high-fat diet in the evening not only decreased the hepatic AMPK protein expression and activity but also disturbed its circadian rhythm. Accordingly, the hepatic clock genes, including clock, brain-muscle-Arnt-like 1, cryptochrome 2, and period 2, exhibited prominent changes in their expression rhythms and/or amplitudes. The diurnal rhythms of the messenger RNA expression of peroxisome proliferator-activated receptorα, acetyl-CoA carboxylase 1α, and carnitine palmitoyltransferase 1 were also disrupted; the amplitude of peroxisome proliferator-activated receptorγcoactivator 1α was significantly decreased at 3 time points, and fatty liver was observed. These findings demonstrate that timed high-fat diet at night can change hepatic AMPK protein levels, activity, and circadian rhythm, which may subsequently alter the circadian expression of several hepatic clock genes and finally result in the disorder of hepatic lipogenic gene expression and the formation of fatty liver.

  20. Ghrelin Increases Beta-Catenin Level through Protein Kinase A Activation and Regulates OPG Expression in Rat Primary Osteoblasts

    PubMed Central

    Mrak, Emanuela; Casati, Lavinia; Pagani, Francesca; Rubinacci, Alessandro; Zarattini, Guido; Sibilia, Valeria

    2015-01-01

    Ghrelin, by binding growth hormone secretagogue receptor (GHS-R), promotes osteoblast proliferation but the signaling mechanism of GHS-R on these cells remains unclear. Since canonical Wnt/β-catenin pathway is critically associated with bone homeostasis, we investigated its involvement in mediating ghrelin effects in osteoblasts and in osteoblast-osteoclast cross talk. Ghrelin (10−10M) significantly increased β-catenin levels in rat osteoblasts (rOB). This stimulatory action on β-catenin involves a specific interaction with GHS-R1a, as it is prevented by the selective GHS-R1a antagonist, D-Lys3-GHRP-6 (10−7M). The effect of ghrelin on β-catenin involves the phosphorylation and inactivation of GSK-3β via protein kinase A (PKA). Inhibition of PKA activity reduces the facilitatory action of ghrelin on β-catenin stabilization. Ghrelin treatment of rOB significantly increases the expression of osteoprotegerin (OPG), which plays an important role in the regulation of osteoclastogenesis, and this effect is blocked by D-Lys3-GHRP-6. Furthermore, ghrelin reduced RANKL/OPG ratio thus contrasting osteoclastogenesis. Accordingly, conditioned media from rOB treated with ghrelin decreased the number of multinucleated TRAcP+ cells as compared with the conditioned media from untreated-control rOB. Our data suggest new roles for ghrelin in modulating bone homeostasis via a specific interaction with GHSR-1a in osteoblasts with subsequent enhancement of both β-catenin levels and OPG expression. PMID:25866509

  1. Over-expression of rice leucine-rich repeat protein results in activation of defense response, thereby enhancing resistance to bacterial soft rot in Chinese cabbage.

    PubMed

    Park, Young Ho; Choi, Changhyun; Park, Eun Mi; Kim, Hyo Sun; Park, Hong Jae; Bae, Shin Cheol; Ahn, Ilpyung; Kim, Min Gab; Park, Sang Ryeol; Hwang, Duk-Ju

    2012-10-01

    Pectobacterium carotovorum subsp. carotovorum causes soft rot disease in various plants, including Chinese cabbage. The simple extracellular leucine-rich repeat (eLRR) domain proteins have been implicated in disease resistance. Rice leucine-rich repeat protein (OsLRP), a rice simple eLRR domain protein, is induced by pathogens, phytohormones, and salt. To see whether OsLRP enhances disease resistance to bacterial soft rot, OsLRP was introduced into Chinese cabbage by Agrobacterium-mediated transformation. Two independent transgenic lines over-expressing OsLRP were generated and further analyzed. Transgenic lines over-expressing OsLRP showed enhanced disease resistance to bacterial soft rot compared to non-transgenic control. Bacterial growth was retarded in transgenic lines over-expressing OsLRP compared to non-transgenic controls. We propose that OsLRP confers enhanced resistance to bacterial soft rot. Monitoring expression of defense-associated genes in transgenic lines over-expressing OsLRP, two different glucanases and Brassica rapa polygalacturonase inhibiting protein 2, PDF1 were constitutively activated in transgenic lines compared to non-transgenic control. Taken together, heterologous expression of OsLRP results in the activation of defense response and enhanced resistance to bacterial soft rot.

  2. Brain Region–Specific Decrease in the Activity and Expression of Protein Kinase A in the Frontal Cortex of Regressive Autism

    PubMed Central

    Ji, Lina; Chauhan, Ved; Flory, Michael J.; Chauhan, Abha

    2011-01-01

    Autism is a severe neurodevelopmental disorder that is characterized by impaired language, communication, and social skills. In regressive autism, affected children first show signs of normal social and language development but eventually lose these skills and develop autistic behavior. Protein kinases are essential in G-protein-coupled, receptor-mediated signal transduction and are involved in neuronal functions, gene expression, memory, and cell differentiation. We studied the activity and expression of protein kinase A (PKA), a cyclic AMP–dependent protein kinase, in postmortem brain tissue samples from the frontal, temporal, parietal, and occipital cortices, and the cerebellum of individuals with regressive autism; autistic subjects without a clinical history of regression; and age-matched developmentally normal control subjects. The activity of PKA and the expression of PKA (C-α), a catalytic subunit of PKA, were significantly decreased in the frontal cortex of individuals with regressive autism compared to control subjects and individuals with non-regressive autism. Such changes were not observed in the cerebellum, or the cortices from the temporal, parietal, and occipital regions of the brain in subjects with regressive autism. In addition, there was no significant difference in PKA activity or expression of PKA (C-α) between non-regressive autism and control groups. These results suggest that regression in autism may be associated, in part, with decreased PKA-mediated phosphorylation of proteins and abnormalities in cellular signaling. PMID:21909354

  3. Macrophages in T cell/histiocyte rich large B cell lymphoma strongly express metal-binding proteins and show a bi-activated phenotype.

    PubMed

    Hartmann, Sylvia; Tousseyn, Thomas; Döring, Claudia; Flüchter, Patricia; Hackstein, Holger; Herreman, An; Ponzoni, Maurilio; de Wolf-Peeters, Chris; Facchetti, Fabio; Gascoyne, Randy D; Küppers, Ralf; Steidl, Christian; Hansmann, Martin-Leo

    2013-12-01

    Abundant macrophage infiltration in tumors often correlates with a poor prognosis. T cell/histiocyte rich large B cell lymphoma (THRLBCL) is a distinct aggressive B cell lymphoma entity showing a high macrophage content. To further elucidate the role of tumor-associated macrophages in THRLBCL, we performed gene expression profiling of microdissected histiocyte subsets of THRLBCL, nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), Piringer lymphadenitis, sarcoidosis, nonspecific lymphadenitis and monocytes from peripheral blood. In a supervised principal component analysis, histiocytes from THRLBCL were most closely related to epithelioid cells from NLPHL, with both types of cells expressing genes related to proinflammatory and regulatory macrophage activity. Moreover, histiocytes from THRLBCL strongly expressed metal-binding proteins like MT2A, by which histiocytes of THRLBCL can be distinguished from the other histiocyte subsets investigated. Interestingly, the validation at the protein level showed a strong expression of TXN, CXCL9, MT2A and SOD2 not only in macrophages of THRLBCL but also in the tumor cells of NLPHL and classical Hodgkin lymphoma (cHL). Overall, the present findings indicate that macrophages in the microenvironment of THRLBCL have acquired a distinct gene expression pattern that is characterized by a mixed M1/M2 phenotype and a strong expression of several metal binding proteins. The microenvironments in NLPHL and THRLBCL appear to have a similar influence on the macrophage phenotype. The high expression of metal binding proteins in histiocytes of THRLBCL may be diagnostically useful, but a potential pathophysiological role remains to be identified.

  4. Suppression of the lipopolysaccharide-induced expression of MARCKS-related protein (MRP) affects transmigration in activated RAW264.7 cells.

    PubMed

    Chun, Kwang-Rok; Bae, Eun Mi; Kim, Jae-Kwan; Suk, Kyoungho; Lee, Won-Ha

    2009-01-01

    The molecular action mechanism of MRP, one of the protein kinase C (PKC) substrates, has been under intense investigation, but reports on its role in macrophage function remain controversial. The treatment of macrophage cell lines with bacterial lipopolysaccharide (LPS) induced a high level of MRP expression suggesting that MRP plays a role in the function of activated macrophages. In order to investigate the role of MRP in activated RAW264.7 cells, we stably transfected MRP-specific shRNA expression constructs and tested for alterations in macrophage-related functions. The down-regulation of MRP expression resulted in a marked reduction in chemotaxis toward MCP-1 or extracellular matrix proteins. Furthermore, pharmacological inhibitors of PKC significantly inhibited the chemotaxis in RAW264.7 cells. These data reveals the pivotal role of MRP in the transmigration of activated RAW264.7 cells.

  5. What is critical for plant thermogenesis? Differences in mitochondrial activity and protein expression between thermogenic and non-thermogenic skunk cabbages.

    PubMed

    Ito-Inaba, Yasuko; Hida, Yamato; Inaba, Takehito

    2009-12-01

    Thermogenesis during the blooming of inflorescence is found in several but not all aroids. To understand what is critical for thermogenesis, we investigated the difference between thermogenic and non-thermogenic skunk cabbages (Symplocarpus renifolius and Lysichiton camtschatcensis), which are closely related in morphology and phylogeny. Critical parameters of mitochondrial biogenesis, including density, respiratory activity, and protein expression were compared between these two species. Mitochondrial density, respiratory activity, and the amount of alternative oxidase (AOX) in L. camtschatcensis spadix mitochondria were lower than in S. renifolius spadix mitochondria, while the level of uncoupling protein (UCP) was higher. AOX and UCP mRNAs in L. camtschatcensis were constitutively expressed in various tissues, such as the spadix, the spathe, the stalk, and the leaves. cDNA encoding two putative thermogenic proteins, AOX and UCP were isolated from L. camtschatcensis, and their primary structure was analyzed by multiple alignment and phylogenetic tree reconstruction. AOX and UCP protein of two the skunk cabbage species are closely related in structure, compared with other isoforms in thermogenic plants. Our results suggest that mitochondrial density, respiratory activity, and protein expression, rather than the primary structure of AOX or UCP proteins, may play critical roles in thermogenesis in plants.

  6. Epstein-Barr virus latent membrane protein-1 activates CD25 expression in lymphoma cells involving the NFkappaB pathway.

    PubMed

    Vockerodt, M; Tesch, H; Kube, D

    2001-12-01

    Epstein-Barr virus (EBV) is associated with several human malignancies including Burkitt's lymphoma (BL), Hodgkin's disease (HD) and nasopharyngeal carcinoma. A variety of cytokines and receptors have been described to be activated by EBV. Here we show that the IL-2 receptor (IL-2R) alpha-chain, which is weakly expressed on normal resting lymphoid cells, is activated by EBV. Comparison of EBV-negative BL cell lines and their EBV convertants showed an enhanced CD25 expression in EBV-positive BL cells. Transient expression of the oncogenic virus protein latent membrane protein-1 (LMP1) in L428 Hodgkin's lymphoma cells and in Burkitt's lymphoma cells (BL2, BL41, BL30) cells leads to enhanced CD25 expression. Both C-terminal activating regions (CTARs) of LMP1 are involved in CD25 activation. Inhibition of LMP1-mediated NFkappaB enhancement by a constitutive repressive form of IkappaB-alpha resulted in decreased CD25 surface expression, indicating that NFkappaB is involved in CD25 gene regulation. Furthermore, LMP1-mediated CD25 activation was associated with enhanced levels of the soluble form of CD25 (sCD25) in L428 Hodgkin's lymphoma cells but not in BL cells. LMP1 associated enhanced expression of membrane CD25 and soluble CD25 may have immunomodulatory functions and could be involved in biology of EBV-associated diseases.

  7. Modified C-reactive protein is expressed by stroke neovessels and is a potent activator of angiogenesis in vitro.

    PubMed

    Slevin, Mark; Matou-Nasri, Sabine; Turu, Marta; Luque, Ana; Rovira, Norma; Badimon, Lina; Boluda, Susana; Potempa, Lawrence; Sanfeliu, Coral; de Vera, Nuria; Krupinski, Jerzy

    2010-01-01

    Native C-reactive protein (nCRP) is a pentameric oligo-protein and an acute phase reactant whose serum expression is increased in patients with inflammatory disease. We have identified by immunohistochemistry, significant expression of a tissue-binding insoluble modified version or monomeric form of CRP (mCRP) associated with angiogenic microvessels in peri-infarcted regions of patients studied with acute ischaemic stroke. mCRP, but not nCRP was expressed in the cytoplasm and nucleus of damaged neurons. mCRP co-localized with CD105, a marker of angiogenesis in regions of revascularisation. In vitro investigations demonstrated that mCRP was preferentially expressed in human brain microvessel endothelial cells following oxygen-glucose deprivation and mCRP (but not column purified nCRP) associated with the endothelial cell surface, and was angiogenic to vascular endothelial cells, stimulating migration and tube formation in matrigel more strongly than fibroblast growth factor-2. The mechanism of signal transduction was not through the CD16 receptor. Western blotting showed that mCRP stimulated phosphorylation of the key down-stream mitogenic signalling protein ERK1/2. Pharmacological inhibition of ERK1/2 phosphorylation blocked the angiogenic effects of mCRP. We propose that mCRP may contribute to the neovascularization process and because of its abundant presence, be important in modulating angiogenesis in both acute stroke and later during neuro-recovery.

  8. High glucose enhances microRNA-26a to activate mTORC1 for mesangial cell hypertrophy and matrix protein expression

    PubMed Central

    Dey, Nirmalya; Bera, Amit; Das, Falguni; Ghosh-Choudhury, Nandini; Kasinath, Balakuntalam S.; Choudhury, Goutam Ghosh

    2015-01-01

    High glucose milieu inhibits PTEN expression to activate Akt kinase and induces glomerular mesangial cell hypertrophy and matrix protein expression in diabetic nephropathy. Specific mechanism by which high glucose inhibits PTEN expression is not clear. We found that high glucose increased the expression of the microRNA-26a (miR-26a) in mesangial cells. Using a sensor plasmid with 3’UTR-driven luciferase, we showed PTEN as a target of miR-26a in response to high glucose. Overexpression of miR-26a reduced the PTEN protein levels resulting in increased Akt kinase activity similar to high glucose treatment. In contrast, anti-miR-26a reversed high glucose-induced suppression of PTEN with concomitant inhibition of Akt kinase activity. Akt-mediated phosphorylation of tuberin and PRAS40 regulates mTORC1, which is necessary for mesangial cell hypertrophy and matrix protein expression. Inhibition of high glucose-induced miR-26a blocked phosphorylation of tuberin and PRAS40, which lead to suppression of phosphorylation of S6 kinase and 4EBP-1, two substrates of mTORC1. Furthermore, we show that expression of miR-26a induced mesangial cell hypertrophy and increased fibronectin and collagen I (α2) expression similar to that observed with the cells incubated with high glucose. Anti-miR-26a inhibited these phenomena in response to high glucose. Together our results provide the first evidence for the involvement of miR-26a in high glucose-induced mesangial cell hypertrophy and matrix protein expression. These data indicate the potential therapeutic utility of anti-miR-26a for the complications of diabetic kidney disease. PMID:25797045

  9. Identification of the expressed protein and the impact of change in ascorbate peroxidase activity related to endodormancy breaking in Pyrus pyrifolia.

    PubMed

    Takemura, Yoshihiro; Kuroki, Katsuou; Jiang, Mingfeng; Matsumoto, Kazuhiro; Tamura, Fumio

    2015-01-01

    Endodormancy is an important feature of perennial deciduous fruit trees that survive in the extreme climates brought about by seasonal variation. To acquire a comprehensive knowledge of the biochemical processes occurring just before endodormancy breaking, the buds collected in the pre-breaking period (PP) phase were used as samples to identify the proteins related to the breaking of endodormancy in the Japanese pear (Pyrus pyrifolia Nakai). Using nano-ESI-LC-MS/MS analysis, 96 proteins were overlapped by analyses of three times and identified as expressed proteins at the PP stage. Among these proteins, dehydrin, several classes of heat shock proteins (HSP), auxin-binding protein, and auxin-induced protein were identified in the floral bud in the PP stage. The majority of these proteins were involved primarily in the oxidation-reduction process. We focused on catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) as enzymes regulating the levels of hydrogen peroxide (H2O2) in the bud. From measurements taken during the deepest period (DP), PP, mid-breaking period (MP), and late-breaking period (LP) of endodormancy, CAT activity decreased gradually, while APX activity also decreased from DP to MP, but then increased rapidly during LP. Protein data for PP and the rapid increase in APX activity observed in LP provided knowledge of the biochemical processes that regulate the consecutive transition from endodormancy breaking to ecodormancy induction in the Japanese pear.

  10. The Expression of a Xylanase Targeted to ER-Protein Bodies Provides a Simple Strategy to Produce Active Insoluble Enzyme Polymers in Tobacco Plants

    PubMed Central

    Llop-Tous, Immaculada; Ortiz, Miriam; Torrent, Margarita; Ludevid, M. Dolors

    2011-01-01

    Background Xylanases deserve particular attention due to their potential application in the feed, pulp bleaching and paper industries. We have developed here an efficient system for the production of an active xylanase in tobacco plants fused to a proline-rich domain (Zera) of the maize storage protein γ-zein. Zera is a self-assembling domain able to form protein aggregates in vivo packed in newly formed endoplasmic reticulum-derived organelles known as protein bodies (PBs). Methodology/Principal Findings Tobacco leaves were transiently transformed with a binary vector containing the Zera-xylanase coding region, which was optimized for plant expression, under the control of the 35S CaMV promoter. The fusion protein was efficiently expressed and stored in dense PBs, resulting in yields of up to 9% of total protein. Zera-xylanase was post-translationally modified with high-mannose-type glycans. Xylanase fused to Zera was biologically active not only when solubilized from PBs but also in its insoluble form. The resistance of insoluble Zera-xylanase to trypsin digestion demonstrated that the correct folding of xylanase in PBs was not impaired by Zera oligomerization. The activity of insoluble Zera-xylanase was enhanced when substrate accessibility was facilitated by physical treatments such as ultrasound. Moreover, we found that the thermostability of the enzyme was improved when Zera was fused to the C-terminus of xylanase. Conclusion/Significance In the present work we have successfully produced an active insoluble aggregate of xylanase fused to Zera in plants. Zera-xylanase chimeric protein accumulates within ER-derived protein bodies as active aggregates that can easily be recovered by a simple density-based downstream process. The production of insoluble active Zera-xylanase protein in tobacco outlines the potential of Zera as a fusion partner for producing enzymes of biotechnological relevance. Zera-PBs could thus become efficient and low-cost bioreactors for

  11. RNA helicase activity of the plum pox potyvirus CI protein expressed in Escherichia coli. Mapping of an RNA binding domain.

    PubMed Central

    Fernández, A; Laín, S; García, J A

    1995-01-01

    The plum pox potyvirus (PPV) cylindrical inclusion (CI) protein fused to the maltose binding protein (MBP) has been synthesized in Escherichia coli and purified by affinity chromatography in amylose resin. In the absence of any other viral factors, the fusion product had NTPase, RNA binding and RNA helicase activities. These in vitro activities were not affected by removal of the last 103 amino acids of the CI protein. However, other deletions in the C-terminal part of the protein, although leaving intact all the region conserved in RNA helicases, drastically impaired the ability to unwind dsRNA and to hydrolyze NTPs. A mutant protein lacking the last 225 residues retained the competence to interact with RNA. Further deletions mapped boundaries of the RNA binding domain within residues 350 and 402 of the PPV CI protein. This region includes the arginine-rich motif VI, the most carboxy terminal conserved domain of RNA helicases of the superfamily SF2. These results indicate that NTP hydrolysis is not an essential component for RNA binding of the PPV CI protein. Images PMID:7538661

  12. Quantitative expression patterns of peroxisome proliferator-activated receptor-{beta}/{delta} (PPAR{beta}/{delta}) protein in mice

    SciTech Connect

    Girroir, Elizabeth E.; Hollingshead, Holly E.; He Pengfei; Zhu Bokai; Perdew, Gary H.; Peters, Jeffrey M.

    2008-07-04

    The expression patterns of PPAR{beta}/{delta} have been described, but the majority of these data are based on mRNA data. To date, there are no reports that have quantitatively examined the expression of PPAR{beta}/{delta} protein in mouse tissues. In the present study, a highly specific PPAR{beta}/{delta} antibody was developed, characterized, and used to examine tissue expression patterns of PPAR{beta}/{delta}. As compared to commercially available anti-PPAR{beta}/{delta} antibodies, one of six polyclonal anti-PPAR{beta}/{delta} antibodies developed was significantly more effective for immunoprecipitation of in vitro-translated PPAR{beta}/{delta}. This antibody was used for quantitative Western blot analysis using radioactive detection methods. Expression of PPAR{beta}/{delta} was highest in colon, small intestine, liver, and keratinocytes as compared to other tissues including heart, spleen, skeletal muscle, lung, brain, and thymus. Interestingly, PPAR{beta}/{delta} expression was localized in the nucleus and RXR{alpha} can be co-immunoprecipitated with nuclear PPAR{beta}/{delta}. Results from these studies demonstrate that PPAR{beta}/{delta} expression is highest in intestinal epithelium, liver, and keratinocytes, consistent with significant biological roles in these tissues.

  13. Baculovirus-expressed vitamin D-binding protein-macrophage activating factor (DBP-maf) activates osteoclasts and binding of 25-hydroxyvitamin D(3) does not influence this activity.

    PubMed

    Swamy, N; Ghosh, S; Schneider, G B; Ray, R

    2001-01-01

    Vitamin D-binding protein (DBP) is a multi-functional serum protein that is converted to vitamin D-binding protein-macrophage activating factor (DBP-maf) by post-translational modification. DBP-maf is a new cytokine that mediates bone resorption by activating osteoclasts, which are responsible for resorption of bone. Defective osteoclast activation leads to disorders like osteopetrosis, characterized by excessive accumulation of bone mass. Previous studies demonstrated that two nonallelic mutations in the rat with osteopetrosis have independent defects in the cascade involved in the conversion of DBP to DBP-maf. The skeletal defects associated with osteopetrosis are corrected in these mutants with in vivo DBP-maf treatment. This study evaluates the effects of various forms of DBP-maf (native, recombinant, and 25-hydroxyvitamin D(3) bound) on osteoclast function in vitro in order to determine some of the structural requirements of this protein that relate to bone resorbing activities. Osteoclast activity was determined by evaluating pit formation using osteoclasts, isolated from the long bones of newborn rats, incubated on calcium phosphate coated, thin film, Ostologic MultiTest Slides. Incubation of osteoclasts with ex vivo generated native DBP-maf resulted in a dose dependent, statistically significant, activation of the osteoclasts. The activation was similar whether or not the vitamin D binding site of the DBP-maf was occupied. The level of activity in response to DBP-maf was greater than that elicited by optimal doses of other known stimulators (PTH and 1,25(OH(2)D(3)) of osteoclast function. Furthermore, another potent macrophage activating factor, interferon--gamma, had no effect on osteoclast activity. The activated form of a full length recombinant DBP, expressed in E. coli showed no activity in the in vitro assay. Contrary to this finding, baculovirus-expressed recombinant DBP-maf demonstrated significant osteoclast activating activity. The normal

  14. Mitogen-activated protein kinase kinase 5 (MKK5)-mediated signalling cascade regulates expression of iron superoxide dismutase gene in Arabidopsis under salinity stress.

    PubMed

    Xing, Yu; Chen, Wei-hua; Jia, Wensuo; Zhang, Jianhua

    2015-09-01

    Superoxide dismutases (SODs) are involved in plant adaptive responses to biotic and abiotic stresses but the upstream signalling process that modulates their expression is not clear. Expression of two iron SODs, FSD2 and FSD3, was significantly increased in Arabidopsis in response to NaCl treatment but blocked in transgenic MKK5-RNAi plant, mkk5. Using an assay system for transient expression in protoplasts, it was found that mitogen-activated protein kinase kinase 5 (MKK5) was also activated in response to salt stress. Overexpression of MKK5 in wild-type plants enhanced their tolerance to salt treatments, while mkk5 mutant exhibited hypersensitivity to salt stress in germination on salt-containing media. Moreover, another kinase, MPK6, was also involved in the MKK5-mediated iron superoxide dismutase (FSD) signalling pathway in salt stress. The kinase activity of MPK6 was totally turned off in mkk5, whereas the activity of MPK3 was only partially blocked. MKK5 interacted with the MEKK1 protein that was also involved in the salt-induced FSD signalling pathway. These data suggest that salt-induced FSD2 and FSD3 expressions are influenced by MEKK1 via MKK5-MPK6-coupled signalling. This MAP kinase cascade (MEKK1, MKK5, and MPK6) mediates the salt-induced expression of iron superoxide dismutases.

  15. Resemblance and Dissemblance of Arabidopsis Type II Peroxiredoxins: Similar Sequences for Divergent Gene Expression, Protein Localization, and Activity1

    PubMed Central

    Bréhélin, Claire; Meyer, Etienne H.; de Souris, Jean-Paul; Bonnard, Géraldine; Meyer, Yves

    2003-01-01

    The Arabidopsis type II peroxiredoxin (PRXII) family is composed of six different genes, five of which are expressed. On the basis of the nucleotide and protein sequences, we were able to define three subgroups among the PRXII family. The first subgroup is composed of AtPRXII-B, -C, and -D, which are highly similar and localized in the cytosol. AtPRXII-B is ubiquitously expressed. More striking is the specific expression of AtPRXII-C and AtPRXII-D localized in pollen. The second subgroup comprises the mitochondrial AtPRXII-F, the corresponding gene of which is expressed constitutively. We show that AtPRXII-E, belonging to the last subgroup, is expressed mostly in reproductive tissues and that its product is addressed to the plastid. By in vitro enzymatic experiments, we demonstrate that glutaredoxin is the electron donor of recombinant AtPRXII-B for peroxidase reaction, but the donors of AtPRXII-E and AtPRXII-F have still to be identified. PMID:12913160

  16. Ursolic Acid Inhibits Na+/K+-ATPase Activity and Prevents TNF-α-Induced Gene Expression by Blocking Amino Acid Transport and Cellular Protein Synthesis

    PubMed Central

    Yokomichi, Tomonobu; Morimoto, Kyoko; Oshima, Nana; Yamada, Yuriko; Fu, Liwei; Taketani, Shigeru; Ando, Masayoshi; Kataoka, Takao

    2011-01-01

    Pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, induce the expression of a wide variety of genes, including intercellular adhesion molecule-1 (ICAM-1). Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid) was identified to inhibit the cell-surface ICAM-1 expression induced by pro-inflammatory cytokines in human lung carcinoma A549 cells. Ursolic acid was found to inhibit the TNF-α-induced ICAM-1 protein expression almost completely, whereas the TNF-α-induced ICAM-1 mRNA expression and NF-κB signaling pathway were decreased only partially by ursolic acid. In line with these findings, ursolic acid prevented cellular protein synthesis as well as amino acid uptake, but did not obviously affect nucleoside uptake and the subsequent DNA/RNA syntheses. This inhibitory profile of ursolic acid was similar to that of the Na+/K+-ATPase inhibitor, ouabain, but not the translation inhibitor, cycloheximide. Consistent with this notion, ursolic acid was found to inhibit the catalytic activity of Na+/K+-ATPase. Thus, our present study reveals a novel molecular mechanism in which ursolic acid inhibits Na+/K+-ATPase activity and prevents the TNF-α-induced gene expression by blocking amino acid transport and cellular protein synthesis. PMID:24970122

  17. Effect of tacrolimus on activity and expression of P-glycoprotein and ATP-binding cassette transporter A5 (ABCA5) proteins in hematoencephalic barrier cells.

    PubMed

    Quezada, Claudia Andrea; Garrido, Wallys Ximena; González-Oyarzún, Mauricio Alejandro; Rauch, María Cecilia; Salas, Mónica Roxana; San Martín, Rody Enrique; Claude, Alejandro Andrés; Yañez, Alejandro Javier; Slebe, Juan Carlos; Cárcamo, Juan Guillermo

    2008-10-01

    Tacrolimus is an agent used in clinical immunosuppressive drug therapies. A wide spectrum of adverse effects has been reported in association with this immunosuppressor, including neurotoxic effect. The upper limit of therapeutic blood concentrations of tacrolimus has been described as 30 ng/ml in immunosuppressed patients. We investigated the effect of this therapeutic dose of tacrolimus on the expression and activity of the multidrug resistance protein 1 (MDR1 or Pgp, P-glycoprotein) and ATP-binding cassette transporters A5 (ABCA5) in human brain microvascular endothelial cells (HBMEC), derived from Blood-Brain Barrier (BBB) endothelium, these being the most predominantly expressed transcripts in these cells. The expression and activity of MDR1 transporter decreased with 30 ng/ml tacrolimus. The cell viability was not changed with the therapeutic dose used. By contrast, ABCA5 transcripts, of unknown role as yet, increased their expression at this concentration. We propose that the secondary cytotoxic effects of this immunosuppressor on CSN, besides the functional blockade related to multidrug resistance proteins, such as MDR1, and probably ABCA5, could be linked to variations in the expression levels of these proteins at the BBB.

  18. Reactive Oxygen Species-Dependent c-Fos/Activator Protein 1 Induction Upregulates Heme Oxygenase-1 Expression by Bradykinin in Brain Astrocytes.

    PubMed

    Hsieh, Hsi-Lung; Wang, Hui-Hsin; Wu, Cheng-Ying; Yang, Chuen-Mao

    2010-12-15

    Heme oxygenase-1 (HO-1) plays a crucial role in tissue pathological changes such as brain injuries. Our previous studies have demonstrated that bradykinin (BK) induces the expression of several inflammatory proteins, including matrix metalloproteinase-9 and COX-2, via mitogen-activated protein kinases and nuclear factor-κB (NF-κB) in rat brain astrocytes (RBA-1). However, the molecular mechanisms underlying BK-induced HO-1 expression in RBA-1 cells remain poorly defined. Here we demonstrated that BK induced HO-1 expression and enzymatic activity via a B(2) BK receptor-activated reactive oxygen species (ROS)-dependent signaling pathway. NADPH oxidase (Nox)-dependent ROS generation led to activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun-N-terminal kinase (JNK) and then activated the downstream molecules NF-κB and c-Jun, respectively. The c-Fos, an activator protein 1 (AP-1) subunit, was upregulated by activation of NF-κB and c-Jun, which bound to HO-1 promoter and thereby turned on transcription of HO-1 gene. The rat HO-1 promoter containing a putative AP-1 cis-binding site was identified as a crucial domain linking to BK action. Taken together, these results suggested that in RBA-1 cells, activation of ERK/NF-κB and JNK/c-Jun cascades by a Nox/ROS-dependent event enhancing c-Fos/AP-1 activity is essential for HO-1 upregulation and activation induced by BK. Moreover, ROS-dependent NF-E2-related factor 2 activation also contributes to HO-1 induction by BK in astrocytes.

  19. Protein arginine methyltransferase 5 (Prmt5) promotes gene expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) and its target genes during adipogenesis.

    PubMed

    LeBlanc, Scott E; Konda, Silvana; Wu, Qiong; Hu, Yu-Jie; Oslowski, Christine M; Sif, Saïd; Imbalzano, Anthony N

    2012-04-01

    Regulation of adipose tissue formation by adipogenic-regulatory proteins has long been a topic of interest given the ever-increasing health concerns of obesity and type 2 diabetes in the general population. Differentiation of precursor cells into adipocytes involves a complex network of cofactors that facilitate the functions of transcriptional regulators from the CCATT/enhancer binding protein, and the peroxisome proliferator-activated receptor (PPAR) families. Many of these cofactors are enzymes that modulate the structure of chromatin by altering histone-DNA contacts in an ATP-dependent manner or by posttranslationally modifying the histone proteins. Here we report that inhibition of protein arginine methyltransferase 5 (Prmt5) expression in multiple cell culture models for adipogenesis prevented the activation of adipogenic genes. In contrast, overexpression of Prmt5 enhanced adipogenic gene expression and differentiation. Chromatin immunoprecipitation experiments indicated that Prmt5 binds to and dimethylates histones at adipogenic promoters. Furthermore, the presence of Prmt5 promoted the binding of ATP-dependent chromatin-remodeling enzymes and was required for the binding of PPARγ2 at PPARγ2-regulated promoters. The data indicate that Prmt5 acts as a coactivator for the activation of adipogenic gene expression and promotes adipogenic differentiation.

  20. ICAM-1-induced expression of proinflammatory cytokines in astrocytes: involvement of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways.

    PubMed

    Lee, S J; Drabik, K; Van Wagoner, N J; Lee, S; Choi, C; Dong, Y; Benveniste, E N

    2000-10-15

    ICAM-1 is a transmembrane glycoprotein of the Ig superfamily involved in cell adhesion. ICAM-1 is aberrantly expressed by astrocytes in CNS pathologies such as multiple sclerosis, experimental allergic encephalomyelitis, and Alzheimer's disease, suggesting a possible role for ICAM-1 in these disorders. ICAM-1 has been shown to be important for leukocyte diapedesis through brain microvessels and subsequent binding to astrocytes. However, other functional roles for ICAM-1 expression on astrocytes have not been well elucidated. Therefore, we investigated the intracellular signals generated upon ICAM-1 engagement on astrocytes. ICAM-1 ligation by a mAb to rat ICAM-1 induced mRNA expression of proinflammatory cytokines such as IL-1alpha, IL-1beta, IL-6, and TNF-alpha. Examination of cytokine protein production revealed that ICAM-1 ligation results in IL-6 secretion by astrocytes, whereas IL-1beta and IL-1alpha protein is expressed intracellularly in astrocytes. The involvement of mitogen-activated protein kinases (MAPKs) in ICAM-1-mediated cytokine expression in astrocytes was tested, as the MAPK extracellular signal-regulated kinase (ERK) was previously shown to be activated upon ICAM-1 engagement. Our results indicate that ERK1/ERK2, as well as p38 MAPK, are activated upon ligation of ICAM-1. Studies using pharmacological inhibitors demonstrate that both p38 MAPK and ERK1/2 are involved in ICAM-1-induced IL-6 expression, whereas only ERK1/2 is important for IL-1alpha and IL-1beta expression. Our data support the role of ICAM-1 on astrocytes as an inflammatory mediator in the CNS and also uncover a novel signal transduction pathway through p38 MAPK upon ICAM-1 ligation.

  1. Secretory fluorescent protein, a secretion green fluorescent fusion protein with alkaline phosphatase activity as a sensitive and traceable reporter in baculovirus expression system.

    PubMed

    Teng, Chao-Yi; Wu, Tzong-Yuan

    2007-07-01

    The advantages of using traceable fluorescent protein (enhanced green fluorescent protein; EGFP) and a secretory alkaline phosphatase (SEAP) have been used to generate a reporter gene: the secretory fluorescent protein (SEFP). Sf21 cells, infected with the recombinant baculovirus containing the SEFP gene, revealed both traceable fluorescence and easily detectable alkaline phosphatase activity in the culture medium. The distribution of SEFP within the cells revealed that it was excluded from the nucleus, implying that the accumulation of SEFP in a secretory pathway, similar to that of the secretion signal-tagged FPs. Furthermore, the time- and dose-dependent release from the blockage of brefeldin A (BFA) confirmed that the secretion of SEFP was mediated by the secretion pathway and excluded leakage from viral infection. This SEFP reporter gene with traceable fluorescence and alkaline phosphatase activity may become a useful tool for studies on secretory protein production.

  2. Inhibition of microsomal triglyceride transfer protein expression and apolipoprotein B100 secretion by the citrus flavonoid naringenin and by insulin involves activation of the mitogen-activated protein kinase pathway in hepatocytes.

    PubMed

    Allister, Emma M; Borradaile, Nica M; Edwards, Jane Y; Huff, Murray W

    2005-06-01

    Microsomal triglyceride transfer protein (MTP) is necessary for hepatocyte assembly and secretion of apolipoprotein (apo)B100-containing lipoproteins. The citrus flavonoid naringenin, like insulin, decreased MTP expression in HepG2 cells, resulting in inhibition of apoB100 secretion; however, the mechanism for naringenin is independent of insulin receptor substrate-1/2. Recently, it was reported that insulin decreased MTP expression in HepG2 cells via the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) (MAPK(erk)) pathway. We hypothesized that naringenin acts via a similar mechanism. Inhibition of MAPK kinase (MEK) 1/2 in HepG2 cells significantly attenuated the naringenin- and insulin-induced reduction in MTP expression. Both naringenin and insulin increased ERK1/2 phosphorylation, which was completely inhibited by MEK1/2 inhibition and enhanced by inhibition of MAPK(p38), a negative regulator of MAPK(erk) activity. Inhibition of MEK1/2 significantly attenuated both the naringenin- and insulin-induced decrease in apoB100 secretion demonstrating a direct link between MAPK(erk) activation and apoB100 secretion. Furthermore, both compounds increased MAPK(p38) activation, and therefore inhibition of MAPK(p38) amplified thenaringenin- and insulin-induced decrease in apoB100 secretion. We conclude that MAPK(erk) signaling in hepatocytes is critical for inhibition of apoB100 secretion by naringenin and insulin. Therefore, naringenin may prove useful for activating insulin-signaling pathways important for regulation of hepatocyte lipid homeostasis.

  3. Reduced Expression of the Retinoblastoma Protein Shows That the Related Signaling Pathway Is Essential for Mediating the Antineoplastic Activity of Erufosine

    PubMed Central

    Zaharieva, Maya M.; Kirilov, Milen; Chai, Minquang; Berger, Stefan M.; Konstantinov, Spiro; Berger, Martin R.

    2014-01-01

    Erufosine is a new antineoplastic agent of the group of alkylphosphocholines, which interferes with signal transduction and induces apoptosis in various leukemic and tumor cell lines. The present study was designed to examine for the first time the mechanism of resistance to erufosine in malignant cells with permanently reduced expression of the retinoblastoma (Rb) protein. Bearing in mind the high number of malignancies with reduced level of this tumor-suppressor, this investigation was deemed important for using erufosine, alone or in combination, in patients with compromised RB1 gene expression. For this purpose, clones of the leukemic T-cell line SKW-3 were used, which had been engineered to constantly express differently low Rb levels. The alkylphosphocholine induced apoptosis, stimulated the expression of the cyclin dependent kinase inhibitor p27Kip1 and inhibited the synthesis of cyclin D3, thereby causing a G2 phase cell cycle arrest and death of cells with wild type Rb expression. In contrast, Rb-deficiency impeded the changes induced by eru-fosine in the expression of these proteins and abrogated the induction of G2 arrest, which was correlated with reduced antiproliferative and anticlonogenic activities of the compound. In conclusion, analysis of our results showed for the first time that the Rb signaling pathway is essential for mediating the antineoplastic activity of erufosine and its efficacy in patients with malignant diseases may be predicted by determining the Rb status. PMID:24987858

  4. Degradation of Activated Protein Kinases by Ubiquitination

    PubMed Central

    Lu, Zhimin; Hunter, Tony

    2009-01-01

    Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular functions. Once a protein kinase is activated, its activity is subsequently downregulated through a variety of mechanisms. Accumulating evidence indicates that the activation of protein kinases commonly initiates their downregulation via the ubiquitin/proteasome pathway. Failure to regulate protein kinase activity or expression levels can cause human diseases. PMID:19489726

  5. Unique role of SRSF2 in transcription activation and diverse functions of the SR and hnRNP proteins in gene expression regulation.

    PubMed

    Mo, Sudong; Ji, Xiong; Fu, Xiang-Dong

    2013-01-01

    Transcription pause release from gene promoters has been recognized to be a critical point for transcriptional regulation in higher eukaryotes. Recent studies suggest that regulatory RNAs are extensively involved in transcriptional control, which may enlist various RNA binding proteins. We recently showed a key role of SRSF2, a member of the SR family of splicing regulators, in binding to promoter-associated small RNA to mediate transcription pause release, a regulatory strategy akin to the function of the HIV Tat protein via binding to the TAR element in nascent RNA to activate transcription. In this report, we further dissect the structural requirement for SRSF2 to function as a transcription activator and extend the analysis to multiple SR and hnRNP proteins by using the MS2 tethering strategy. Our results reveal that SRSF2 is a unique SR protein that activates transcription in a position-dependent manner while three other SR proteins enhance translation in a position-independent fashion. In contrast, multiple hnRNP proteins appear to negatively influence mRNA levels, especially when tethered in the gene body. These findings suggest broad participation of RNA binding proteins in diverse aspects of regulated gene expression at both the transcriptional and posttranscriptional levels in mammalian cells.

  6. Expression of adenosine 5'-monophosphate-Activated protein kinase (AMPK) in ovine testis (Ovis aries): In vivo regulation by nutritional state.

    PubMed

    Taibi, N; Dupont, J; Bouguermouh, Z; Froment, P; Ramé, C; Anane, A; Amirat, Z; Khammar, F

    2017-03-01

    In the present study, we identified AMPK and investigated its potential role in steroidogenesis in vivo in the ovine testis in response to variation in nutritional status (fed control vs. restricted). We performed immunoblotting to show that both active and non-active forms of AMPK exist in ovine testis and liver. In testis, we confirmed these results by immunohistochemistry. We found a correlation between ATP (Adenosine-Triphosphate) levels and the expression of AMPK in liver. Also, low and high caloric diets induce isoform-dependent AMPK expression, with an increase in α2, ß1ß2 and γ1 activity levels. Although the restricted group exhibited an increase in lipid balance, only the triglyceride and HC-VLDL (Cholesterol-Very low density lipoprotein) fractions showed significant differences between groups, suggesting an adaptive mechanism. Moreover, the relatively low rate of non-esterified fatty acid released into the circulation implies re-esterification to compensate for the physiological need. In the fed control group, AMPK activates the production of testosterone in Leydig cells; this is, in turn, associated with an increase in the expression of 3ß-HSD (3 beta hydroxy steroid deshydrogenase), p450scc (Cholesterol side-chain cleavage enzyme) and StAR (Steroidogenic acute regulatory protein) proteins induced by decreased MAPK ERK½ (Extracellular signal-regulated kinase -Mitogen-activated protein kinase) phosphorylation. In contrast, in the restricted group, testosterone secretion was reduced but intracellular cholesterol concentration was not. Furthermore, the combination of high levels of lipoproteins and emergence of the p38 MAP kinase pathway suggest the involvement of pro-inflammatory cytokines, as confirmed by transcriptional repression of the StAR protein. Taken together, these results suggest that AMPK expression is tissue dependent.

  7. Early inhibition of MMP activity in ischemic rat brain promotes expression of tight junction proteins and angiogenesis during recovery.

    PubMed

    Yang, Yi; Thompson, Jeffrey F; Taheri, Saeid; Salayandia, Victor M; McAvoy, Thera A; Hill, Jeff W; Yang, Yirong; Estrada, Eduardo Y; Rosenberg, Gary A

    2013-07-01

    In cerebral ischemia, matrix metalloproteinases (MMPs) have a dual role by acutely disrupting tight junction proteins (TJPs) in the blood-brain barrier (BBB) and chronically promoting angiogenesis. Since TJP remodeling of the neurovascular unit (NVU) is important in recovery and early inhibition of MMPs is neuroprotective, we hypothesized that short-term MMP inhibition would reduce infarct size and promote angiogenesis after ischemia. Adult spontaneously hypertensive rats had a transient middle cerebral artery occlusion with reperfusion. At the onset of ischemia, they received a single dose of the MMP inhibitor, GM6001. They were studied at multiple times up to 4 weeks with immunohistochemistry, biochemistry, and magnetic resonance imaging (MRI). We observed newly formed vessels in peri-infarct regions at 3 weeks after reperfusion. Dynamic contrast-enhanced MRI showed BBB opening in new vessels. Along with the new vessels, pericytes expressed zonula occludens-1 (ZO-1) and MMP-3, astrocytes expressed ZO-1, occludin, and MMP-2, while endothelial cells expressed claudin-5. The GM6001, which reduced tissue loss at 3 to 4 weeks, significantly increased new vessel formation with expression of TJPs and MMPs. Our results show that pericytes and astrocytes act spatiotemporally, contributing to extraendothelial TJP formation, and that MMPs are involved in BBB restoration during recovery. Early MMP inhibition benefits neurovascular remodeling after stroke.

  8. Early inhibition of MMP activity in ischemic rat brain promotes expression of tight junction proteins and angiogenesis during recovery

    PubMed Central

    Yang, Yi; Thompson, Jeffrey F; Taheri, Saeid; Salayandia, Victor M; McAvoy, Thera A; Hill, Jeff W; Yang, Yirong; Estrada, Eduardo Y; Rosenberg, Gary A

    2013-01-01

    In cerebral ischemia, matrix metalloproteinases (MMPs) have a dual role by acutely disrupting tight junction proteins (TJPs) in the blood–brain barrier (BBB) and chronically promoting angiogenesis. Since TJP remodeling of the neurovascular unit (NVU) is important in recovery and early inhibition of MMPs is neuroprotective, we hypothesized that short-term MMP inhibition would reduce infarct size and promote angiogenesis after ischemia. Adult spontaneously hypertensive rats had a transient middle cerebral artery occlusion with reperfusion. At the onset of ischemia, they received a single dose of the MMP inhibitor, GM6001. They were studied at multiple times up to 4 weeks with immunohistochemistry, biochemistry, and magnetic resonance imaging (MRI). We observed newly formed vessels in peri-infarct regions at 3 weeks after reperfusion. Dynamic contrast-enhanced MRI showed BBB opening in new vessels. Along with the new vessels, pericytes expressed zonula occludens-1 (ZO-1) and MMP-3, astrocytes expressed ZO-1, occludin, and MMP-2, while endothelial cells expressed claudin-5. The GM6001, which reduced tissue loss at 3 to 4 weeks, significantly increased new vessel formation with expression of TJPs and MMPs. Our results show that pericytes and astrocytes act spatiotemporally, contributing to extraendothelial TJP formation, and that MMPs are involved in BBB restoration during recovery. Early MMP inhibition benefits neurovascular remodeling after stroke. PMID:23571276

  9. Protein kinase A and regulation of neonatal Nav1.5 expression in human breast cancer cells: activity-dependent positive feedback and cellular migration.

    PubMed

    Chioni, Athina-Myrto; Shao, Dongmin; Grose, Richard; Djamgoz, Mustafa B A

    2010-02-01

    Voltage-gated Na(+) channels (VGSCs) are expressed in excitable cells (e.g. neurons and muscles), as well as in some classically 'non-excitable' cells (e.g. fibroblasts), and in carcinomas. In general, functional expression of VGSCs in plasma membrane (PM) is hierarchical and dynamic. Previously, we have shown that an activity-dependent positive feedback mechanism involving cAMP-dependent protein kinase A (PKA) plays a significant role in upregulation of VGSCs in strongly metastatic rat prostate cancer Mat-LyLu cells expressing Nav1.7. Here, we investigated the possible role of PKA in VGSC regulation and its functional consequences in strongly metastatic human breast cancer (BCa) MDA-MB-231 cells, where the neonatal splice form of Nav1.5 (nNav1.5) is the predominant VGSC present. Treatment with the PKA activator forskolin for 24h increased mRNA and PM protein levels of nNav1.5, without changing the total VGSC protein level. Opposite effects were obtained by application of the PKA inhibitor KT5720 or the highly specific VGSC blocker tetrodotoxin (TTX), the latter implying activity-dependent upregulation. We tested the possibility, therefore, that the activity dependence of VGSC (nNav1.5) expression involved PKA. Indeed, TTX pretreatment reduced the level of phosphorylated PKA and eliminated basal and PKA-stimulated cellular migration. These data suggested that activity-dependent positive feedback mediated by PKA plays an important role in the functional expression of nNav1.5 in BCa, and in turn, this enhances the cells' metastatic potential.

  10. [Expression of GST-3B fusion protein of Escherichia coli of Ee strain producing SLT-IIe toxin and study on its biological activities and immunogenicity].

    PubMed

    Liu, Guo-ping; Wu, Bin; Lin, Yi-yuan; Jin, Mei-lin; Chen, Huan-chun

    2007-08-01

    Three copies of DNA fragment encoding the truncated SLT-IIeB of Ee strain which was responsible for the edema disease in piglets in Hubei province were fused to the downstream of glutathione S-transferase (GST) of pGEX-KG expression vector, resulting in the fusion expression plasmid pK3 B. After transformed into E. coli BL21 (DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-3B fusion protein was expressed in high level. Western blot was performed to confirm that the expressed fusion protein could specifically react with antiserum against diseases of edema of swine. The fusion protein was further purified and used as an antigen for receptor-binding inhibition assay. The receptor-binding inhibition assay showed GST-3B fusion protein had more strong biological activities than GST-B. The fusion protein of GST-3B or GST-B was purified and emulsified with Freund' s incomplete adjuvant in equal volumes to get subunit bacterin. Groups of SPF KM mice were vaccinated subcutaneously at 0 week with 25 micrograms and at 2 weeks with 25 micrograms of purified GST-3B or GST-B and challenged intraperitoneally with volume of 5 x OD50 Ee strain. Serological tests were performed one week interval with ELISA. The IgG titres against SLT-IIeB in the sera collected at the same period from the Group GST-3B were higher than in the Group GST-B and the immune protection rate against Ee strain was respectively 60% and 40%. These results show the fusion protein GST-3B had more strong biological activities, immunogenicity and better protection against Ee strain, which built a good foundation for the further research of high efficacy vaccine against porcine edema disease.

  11. Nitric oxide induces heat-shock protein 70 expression in vascular smooth muscle cells via activation of heat shock factor 1.

    PubMed Central

    Xu, Q; Hu, Y; Kleindienst, R; Wick, G

    1997-01-01

    Current data suggest that nitric oxide (NO) is a double-edged sword that could result in relaxation and/or cytotoxicity of vascular smooth muscle cells (SMCs) via cGMP- dependent or -independent signal pathways. Stress or heat shock proteins (hsps) have been shown to be augmented in arterial SMCs during acute hypertension and atherosclerosis, both conditions that are believed to correlate with disturbed NO production. In the present study, we demonstrate that NO generated from sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine, and spermine/nitric oxide complex leads to hsp70 induction in cultured SMCs. Western blot analysis demonstrated that hsp70 protein expression peaked between 6 and 12 h after treatment with SNP, and elevated protein levels were preceded by induction of hsp70 mRNA within 3 h. Induction of hsp70 mRNA was associated with the activation of heat shock transcription factor 1 (HSF1), suggesting that the response was regulated at the transcriptional level. HSF1 activation was completely blocked by hemoglobin, dithiothreitol, and cycloheximide, suggesting that the protein damage and nascent polypeptide formation induced by NO may initiate this activation. Furthermore, SMCs pretreated with heat shock (42 degrees C) for 30 min were significantly protected from death induced by NO. Thus, we provide evidence that NO induces hsp70 expression in SMCs via HSF1 activation. Induction of hsp70 could be important in protecting SMCs from injury resulting from NO stimulation. PMID:9276725

  12. RAS–Mitogen-Activated Protein Kinase Signal Is Required for Enhanced PD-L1 Expression in Human Lung Cancers

    PubMed Central

    Sumimoto, Hidetoshi; Takano, Atsushi; Teramoto, Koji; Daigo, Yataro

    2016-01-01

    Ectopic programmed cell death ligand 1 (PD-L1) expression in non-small cell lung cancers (NSCLCs) is related to immune evasion by cancer, and it is a molecular target of immune checkpoint therapies. Although some altered signals in NSCLCs are responsible for ectopic PD-L1 expression, the precise mechanisms remain obscure. Because we found a higher frequency of EGFR/KRAS mutations in NSCLC cell lines with high PD-L1 expression (p < 0.001), we evaluated the relationships between downstream signals and PD-L1 expression, particularly in three KRAS-mutant adenocarcinoma cell lines. The MEK inhibitor U0126 (20 μM) significantly decreased the surface PD-L1 levels by 50–60% compared with dimethyl sulfoxide (p < 0.0001). Phorbol 12-myristate 13-acetate stimulation (100 nM, 15 min) increased (p < 0.05) and two ERK2 siRNAs as well as KRAS siRNAs decreased (p < 0.05) PD-L1 expression. The transcriptional activity of the potential AP-1 site (+4785 to +5056 from the transcription start site) in the PD-L1 gene was demonstrated by luciferase assays, which was inhibited by U0126. The chromatin immunoprecipitation assay demonstrated the binding of cJUN to the AP-1 site. Two STAT3 siRNAs decreased PD-L1 expression by 10–32% in two of the three KRAS-mutant lung adenocarcinoma cell lines (p < 0.05), while the PI3K inhibitor LY294002 (40 μM) did not change the expression level. Supervised cluster analysis and gene set enrichment analysis between the PD-L1-high and -low NSCLCs revealed a correlation between PD-L1 expression and genes/pathways related to cell motility/adhesion. These results indicate that MAPK signaling is the dominant downstream signal responsible for ectopic PD-L1 expression, in which STAT3 is also involved to some extent. Furthermore, MAPK signaling may control the expression of PD-L1 and several genes related to enhanced cell motility. Our findings suggest that MAPK, along with STAT3, is important for determining PD-L1 expression, which could be useful for

  13. Co-active receptor tyrosine kinases mitigate the effect of FGFR inhibitors in FGFR1-amplified lung cancers with low FGFR1 protein expression.

    PubMed

    Kotani, H; Ebi, H; Kitai, H; Nanjo, S; Kita, K; Huynh, T G; Ooi, A; Faber, A C; Mino-Kenudson, M; Yano, S

    2016-07-07

    Targeted therapies are effective in subsets of lung cancers with EGFR mutations and anaplastic lymphoma kinase (ALK) translocations. Large-scale genomics have recently expanded the lung cancer landscape with FGFR1 amplification found in 10-20% of squamous cell carcinomas (SCCs). However, the response rates have been low for biomarker-directed fibroblast growth factor receptor (FGFR) inhibitor therapy in SCC, which contrasts to the relatively high rates of response seen in EGFR mutant and ALK-translocated lung cancers treated with epidermal growth factor receptor (EGFR) inhibitors and ALK inhibitors, respectively. In order to better understand the low response rates of FGFR1-amplified lung cancers to FGFR inhibitors, relationships between gene copy number, mRNA expression and protein expression of FGFR1 were assessed in cell lines, tumor specimens and data from The Cancer Genome Atlas. The importance of these factors for the sensitivity to FGFR inhibitors was determined by analyzing drug screen data and conducting in vitro and in vivo experiments. We report that there was a discrepancy between FGFR1 amplification level and FGFR1 protein expression in a number of these cell lines, and the cancers with unexpectedly low FGFR1 expression were uniformly resistant to the different FGFR inhibitors. Further interrogation of the receptor tyrosine kinase activity in these discordant cell lines revealed co-activation of HER2 and platelet-derived growth factor receptor-α (PDGFRα) caused by gene amplification or ligand overexpression maintained phosphoinositide 3-kinase (PI3K) and MEK/ERK signaling even in the presence of FGFR inhibitor. Accordingly, co-inhibition of FGFR1 and HER2 or PDGFRα led to enhanced drug responses. In contrast, FGFR1-amplified high FGFR1 protein-expressing lung cancers are sensitive to FGFR inhibitor monotherapy by downregulating ERK signaling. Addition of a PI3K inhibitor to these high FGFR1 protein-expressing cancers further sensitized them to FGFR

  14. The SNF5 protein of Saccharomyces cerevisiae is a glutamine- and proline-rich transcriptional activator that affects expression of a broad spectrum of genes.

    PubMed Central

    Laurent, B C; Treitel, M A; Carlson, M

    1990-01-01

    The Saccharomyces cerevisiae SNF5 gene affects expression of both glucose- and phosphate-regulated genes and appears to function in transcription. We report the nucleotide sequence, which predicts that SNF5 encodes a 102,536-dalton protein. The N-terminal third of the protein is extremely rich in glutamine and proline. Mutants carrying a deletion of the coding sequence were viable but grew slowly, indicating that the SNF5 gene is important but not essential. Evidence that SNF5 affects expression of the cell type-specific genes MF alpha 1 and BAR1 at the RNA level extends the known range of SNF5 function. SNF5 is apparently required for expression of a wide variety of differently regulated genes. A bifunctional SNF5-beta-galactosidase fusion protein was localized in the nucleus by immunofluorescence. No DNA-binding activity was detected for SNF5. A LexA-SNF5 fusion protein, when bound to a lexA operator, functioned as a transcriptional activator. Images PMID:2233708

  15. Protein expression of small conductance calcium-activated potassium channels is altered in inferior colliculus neurons of the genetically epilepsy-prone rat

    PubMed Central

    N’Gouemo, Prosper; Yasuda, Robert P.; Faingold, Carl L.

    2009-01-01

    The genetically epilepsy-prone rat (GEPR) exhibits inherited predisposition to sound stimuli-induced generalized tonic-clonic seizures (audiogenic reflex seizures) and is a valid model to study the physiopathology of epilepsy. In this model, the inferior colliculus (IC) exhibits enhanced neuronal firing that is critical in the initiation of reflex audiogenic seizures. The mechanisms underlying IC neuronal hyperexcitability that leads to seizure susceptibility are not as yet fully understood. The present report shows that the levels of protein expression of SK1 and SK3 subtypes of the small conductance Ca2+-activated K+ channels were significantly decreased, while SK2 channel proteins were increased in IC neurons of seizure-naive GEPR-3s (SN-GEPR-3), as compared to control Sprague-Dawley rats. No significant change was found in the expression of BK channel proteins in IC neurons of SN-GEPR-3s. Single episode of reflex audiogenic seizures in the GEPR-3s did not significantly alter the protein expression of SK1-3 and BK channels in IC neurons compared to SN-GEPR-3s. Thus, downregulation of SK1 and SK3 channels and upregulation of SK2 channels provide direct evidence that these Ca2+-activated K+ channels play important roles in IC neuronal hyperexcitability that leads to inherited seizure susceptibility in the GEPR. PMID:19254702

  16. Expression of Six Proteins Causes Reprogramming of Porcine Fibroblasts Into Induced Pluripotent Stem Cells With Both Active X Chromosomes.

    PubMed

    Fukuda, Tomokazu; Tani, Tetsuya; Haraguchi, Seiki; Donai, Kenichiro; Nakajima, Nobuyoshi; Uenishi, Hirohide; Eitsuka, Takahiro; Miyagawa, Makoto; Song, Sanghoun; Onuma, Manabu; Hoshino, Yumi; Sato, Eimei; Honda, Arata

    2017-03-01

    In this study, we created porcine-induced pluripotent stem (iPS) cells with the expression of six reprogramming factors (Oct3/4, Klf4, Sox2, c-Myc, Lin28, and Nanog). The resulting cells showed growth dependent on LIF (leukemia inhibitory factor) and expression of multiple stem cell markers. Furthermore, the iPS cells caused teratoma formation with three layers of differentiation and had both active X chromosomes (XaXa). Our iPS cells satisfied the both of important characteristics of stem cells: teratoma formation and activation of both X chromosomes. Injection of these iPS cells into morula stage embryos showed that these cells participate in the early stage of porcine embryogenesis. Furthermore, the RNA-Seq analysis detected that expression levels of endogenous pluripotent related genes, NANOG, SOX2, ZFP42, OCT3/4, ESRRB, and ERAS were much higher in iPS with six factors than that with four reprogramming factors. We can conclude that the expression of six reprogramming factors enables the creation of porcine iPS cells, which is partially close to naive iPS state. J. Cell. Biochem. 118: 537-553, 2017. © 2016 Wiley Periodicals, Inc.

  17. Hindbrain A2 noradrenergic neuron adenosine 5'-monophosphate-activated protein kinase activation, upstream kinase/phosphorylase protein expression, and receptivity to hormone and fuel reporters of short-term food deprivation are regulated by estradiol.

    PubMed

    Briski, Karen P; Alenazi, Fahaad S H; Shakya, Manita; Sylvester, Paul W

    2016-09-12

    Estradiol (E) mitigates acute and postacute adverse effects of 12 hr-food deprivation (FD) on energy balance. Hindbrain 5'-monophosphate-activated protein kinase (AMPK) regulates hyperphagic and hypothalamic metabolic neuropeptide and norepinephrine responses to FD in an E-dependent manner. Energy-state information from AMPK-expressing hindbrain A2 noradrenergic neurons shapes neural responses to metabolic imbalance. Here we investigate the hypothesis that FD causes divergent changes in A2 AMPK activity in E- vs. oil (O)-implanted ovariectomized female rats, alongside dissimilar adjustments in circulating metabolic fuel (glucose, free fatty acids [FFA]) and energy deficit-sensitive hormone (corticosterone, glucagon, leptin) levels. FD decreased blood glucose in oil (O)- but not E-implanted ovariectomized female rats and elevated and reduced glucagon levels in O and E, respectively. FD decreased circulating leptin in O and E, but increased corticosterone and FFA concentrations in E only. Western blot analysis of laser-microdissected A2 neurons showed that glucocorticoid receptor type II and very-long-chain acyl-CoA synthetase 3 protein profiles were amplified in FD/E vs. FD/O. A2 total AMPK protein was elevated without change in activity in FD/O, whereas FD/E exhibited increased AMPK activation along with decreased upstream phosphatase expression. The catecholamine biosynthetic enzyme dopamine-β-hydroxylase (DβH) was increased in FD/O but not FD/E A2 cells. The data show discordance between A2 AMPK activation and glycemic responses to FD; sensor activity was refractory to glucose decrements in FD/O but augmented in FD/E despite stabilized glucose and elevated FFA levels. E-dependent amplification of AMPK activity may reflect adaptive conversion to fatty acid oxidation and/or glucocorticoid stimulation. FD augmentation of A2 DβH protein profiles in FD/O but not FD/E animals suggests that FD may correspondingly regulate NE synthesis vs. metabolism/release in the

  18. Cyclic AMP-responsive expression of the surfactant protein-A gene is mediated by increased DNA binding and transcriptional activity of thyroid transcription factor-1.

    PubMed

    Li, J; Gao, E; Mendelson, C R

    1998-02-20

    Surfactant protein (SP)-A gene transcription is stimulated by factors that increase cyclic AMP. In the present study, we observed that three thyroid transcription factor-1 (TTF-1) binding elements (TBEs) located within a 255 base pair region flanking the 5'-end of the baboon SP-A2 (bSP-A2) gene are required for maximal cyclic AMP induction of bSP-A2 promoter activity. We found that TTF-1 DNA binding activity was increased in nuclear extracts of pulmonary type II cells cultured in the presence of cyclic AMP. By contrast, the levels of immunoreactive TTF-1 protein were similar in nuclear extracts of control and cyclic AMP-treated type II cells. The incorporation of [32P]orthophosphate into immunoprecipitated TTF-1 protein also was markedly increased by cyclic AMP treatment. Moreover, exposure of nuclear extracts from cyclic AMP-treated type II cells either to potato acid phosphatase or alkaline phosphatase abolished the cyclic AMP-induced increase in TTF-1 DNA-binding activity. Interestingly, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), known to activate protein kinase C, also enhanced incorporation of [32P]orthophosphate into TTF-1 protein; however, the DNA binding activity of TTF-1 was decreased in nuclear extracts of TPA-treated type II cells. Expression vectors encoding TTF-1 and the catalytic subunit of protein kinase A (PKA-cat) were cotransfected into A549 lung adenocarcinoma cells together with an SPA:human growth hormone fusion gene (255 base pairs of 5'-flanking DNA from the baboon SP-A2 gene linked to human growth hormone, as reporter) containing TBEs, or with a reporter gene construct containing three tandem TBEs fused upstream of the bSP-A2 gene TATA box and the transcription initiation site. Coexpression of TTF-1 and PKA-cat increased fusion gene expression 3-4-fold as compared with expression of TTF-1 in the absence of PKA-cat. Moreover, the transcriptional activity of TTF-1 was suppressed by cotransfection of a dominant negative form

  19. Gene Expression Regulation by the Curli Activator CsgD Protein: Modulation of Cellulose Biosynthesis and Control of Negative Determinants for Microbial Adhesion

    PubMed Central

    Brombacher, Eva; Baratto, Andrea; Dorel, Corinne; Landini, Paolo

    2006-01-01

    Curli fibers, encoded by the csgBAC genes, promote biofilm formation in Escherichia coli and other enterobacteria. Curli production is dependent on the CsgD transcription activator, which also promotes cellulose biosynthesis. In this study, we investigated the effects of CsgD expression from a weak constitutive promoter in the biofilm formation-deficient PHL565 strain of E. coli. We found that despite its function as a transcription activator, the CsgD protein is localized in the cytoplasmic membrane. Constitutive CsgD expression promotes biofilm formation by PHL565 and activates transcription from the csgBAC promoter; however, csgBAC expression remains dependent on temperature and the growth medium. Constitutive expression of the CsgD protein results in altered transcription patterns for at least 24 novel genes, in addition to the previously identified CsgD-dependent genes. The cspA and fecR genes, encoding regulatory proteins responding to cold shock and to iron, respectively, and yoaD, encoding a putative negative regulator of cellulose biosynthesis, were found to be some of the novel CsgD-regulated genes. Consistent with the predicted functional role, increased expression of the yoaD gene negatively affects cell aggregation, while yoaD inactivation results in stimulation of cell aggregation and leads to increased cellulose production. Inactivation of fecR results in significant increases in both cell aggregation and biofilm formation, while the effects of cspA are not as strong in the conditions tested. Our results indicate that CsgD can modulate cellulose biosynthesis through activation of the yoaD gene. In addition, the positive effect of CsgD on biofilm formation might be enhanced by repression of the fecR gene. PMID:16513732

  20. Gene expression regulation by the Curli activator CsgD protein: modulation of cellulose biosynthesis and control of negative determinants for microbial adhesion.

    PubMed

    Brombacher, Eva; Baratto, Andrea; Dorel, Corinne; Landini, Paolo

    2006-03-01

    Curli fibers, encoded by the csgBAC genes, promote biofilm formation in Escherichia coli and other enterobacteria. Curli production is dependent on the CsgD transcription activator, which also promotes cellulose biosynthesis. In this study, we investigated the effects of CsgD expression from a weak constitutive promoter in the biofilm formation-deficient PHL565 strain of E. coli. We found that despite its function as a transcription activator, the CsgD protein is localized in the cytoplasmic membrane. Constitutive CsgD expression promotes biofilm formation by PHL565 and activates transcription from the csgBAC promoter; however, csgBAC expression remains dependent on temperature and the growth medium. Constitutive expression of the CsgD protein results in altered transcription patterns for at least 24 novel genes, in addition to the previously identified CsgD-dependent genes. The cspA and fecR genes, encoding regulatory proteins responding to cold shock and to iron, respectively, and yoaD, encoding a putative negative regulator of cellulose biosynthesis, were found to be some of the novel CsgD-regulated genes. Consistent with the predicted functional role, increased expression of the yoaD gene negatively affects cell aggregation, while yoaD inactivation results in stimulation of cell aggregation and leads to increased cellulose production. Inactivation of fecR results in significant increases in both cell aggregation and biofilm formation, while the effects of cspA are not as strong in the conditions tested. Our results indicate that CsgD can modulate cellulose biosynthesis through activation of the yoaD gene. In addition, the positive effect of CsgD on biofilm formation might be enhanced by repression of the fecR gene.

  1. The cAMP Response Element Binding protein (CREB) is activated by Insulin-like Growth Factor-1 (IGF-1) and regulates myostatin gene expression in skeletal myoblast

    SciTech Connect

    Zuloaga, R.; Fuentes, E.N.; Molina, A.; Valdés, J.A.

    2013-10-18

    Highlights: •IGF-1 induces the activation of CREB via IGF-1R/PI3K/PLC signaling pathway. •Calcium dependent signaling pathways regulate myostatin gene expression. •IGF-1 regulates myostatin gene expression via CREB transcription in skeletal myoblast. -- Abstract: Myostatin, a member of the Transforming Growth Factor beta (TGF-β) superfamily, plays an important role as a negative regulator of skeletal muscle growth and differentiation. We have previously reported that IGF-1 induces a transient myostatin mRNA expression, through the activation of the Nuclear Factor of Activated T cells (NFAT) in an IP{sub 3}/calcium-dependent manner. Here we examined the activation of CREB transcription factor as downstream targets of IGF-1 during myoblast differentiation and its role as a regulator of myostatin gene expression. In cultured skeletal myoblast, IGF-1 induced the phosphorylation and transcriptional activation of CREB via IGF-1 Receptor/Phosphatidylinositol 3-Kinase (PI3K)/Phospholipase C gamma (PLC γ), signaling pathways. Also, IGF-1 induced calcium-dependent molecules such as Calmodulin Kinase II (CaMK II), Extracellular signal-regulated Kinases (ERK), Protein Kinase C (PKC). Additionally, we examined myostatin mRNA levels and myostatin promoter activity in differentiated myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents of myostatin and its reporter activity after treatment with IGF-1. The expression of myostatin in differentiated myoblast was downregulated by the transfection of siRNA–CREB and by pharmacological inhibitors of the signaling pathways involved in CREB activation. By using pharmacological and genetic approaches together these data demonstrate that IGF-1 regulates the myostatin gene expression via CREB transcription factor during muscle cell differentiation.

  2. Lipid peroxidation, antioxidant activities and stress protein (HSP72/73, GRP94) expression in kidney and liver of rats under lithium treatment.

    PubMed

    Nciri, Riadh; Allagui, Mohamed Salah; Bourogaa, Ezzedine; Saoudi, Monji; Murat, Jean-Claude; Croute, Françoise; Elfeki, Abdelfettah

    2012-03-01

    The present work was aimed at studying the effects of a subchronic lithium treatment on rat liver and kidneys, paying attention to the relationship between lithium toxicity, oxidative stress, and stress protein expression. Male rats were submitted to lithium treatment by adding 2 g of lithium carbonate/kg of food for different durations up to 1 month. This treatment led to serum concentrations ranging from 0.5 mM (day 7) to 1.34 mM (day 28) and renal insufficiency highlighted by an increase of blood creatinine and urea levels and a decrease of urea excretion. Lithium treatment was found to trigger an oxidative stress both in kidney and liver, leading to an increase of lipid peroxidation level (TBARS) and of superoxide dismutase and catalase activities. Conversely, glutathione peroxidase activity was reduced. Constitutive HSP73 (heat shock protein 73) expression was not modified by lithium treatment, whereas inducible HSP72 was down-regulated in kidney. GRP94 (glucose regulated protein 94) appeared as two isoforms of 92 and 98 kDa: the 98-kDa protein being overexpressed in kidney by lithium treatment whereas 92-kDa protein was underexpressed both in kidney and liver.

  3. Regulation of kinase cascade activation and heat shock protein expression by poly(ADP-ribose) polymerase inhibition in doxorubicin-induced heart failure.

    PubMed

    Bartha, Eva; Solti, Izabella; Szabo, Aliz; Olah, Gabor; Magyar, Klara; Szabados, Eszter; Kalai, Tamas; Hideg, Kalman; Toth, Kalman; Gero, Domokos; Szabo, Csaba; Sumegi, Balazs; Halmosi, Robert

    2011-10-01

    Cardiomyopathy is one of the most severe side effects of the chemotherapeutic agent doxorubicin (DOX). The formation of reactive oxygen species plays a critical role in the development of cardiomyopathies, and the pathophysiological cascade activates nuclear enzyme poly(ADP-ribose) polymerase (PARP), and kinase pathways. We characterized the effects of the PARP-inhibitor and kinase-modulator compound L-2286 in DOX-induced cardiac injury models. We studied the effect of the established superoxide dismutase-mimic Tempol and compared the effects of this agent with those of the PARP inhibitor. In the rat H9C2 cardiomyocytes, in which DOX-induced poly(ADP-ribosyl)ation, L-2286 protected them from the DOX-induced injury in a concentration-dependent manner. In the in vivo studies, mice were pretreated (for 1 week) with L-2286 or Tempol before the DOX treatment. Both the agents improved the activation of cytoprotective kinases, Akt, phospho-specific protein kinase C ϵ, ζ/λ and suppressed the activity of cell death promoting kinases glycogen synthase kinase-3β, JNK, and p38 mitogen-activated protein kinase, but the effect of PARP inhibitor was more pronounced and improved the survival as well. L-2286 activated the phosphorylation of proapoptotic transcription factor FKHR1 and promoted the expression of Hsp72 and Hsp90. These data suggest that the mode of the cytoprotective action of the PARP inhibitor may include the modulation of kinase pathways and heat shock protein expression.

  4. Celastrol can inhibit proteasome activity and upregulate the expression of heat shock protein genes, hsp30 and hsp70, in Xenopus laevis A6 cells.

    PubMed

    Walcott, Shantel E; Heikkila, John J

    2010-06-01

    In eukaryotes, the ubiquitin-proteasome system (UPS) is responsible for the degradation of most proteins. Proteasome inhibition, which has been associated with various diseases, can cause alterations in various intracellular processes including the expression of heat shock protein (hsp) genes. In this study, we show that celastrol, a quinone methide triterpene and anti-inflammatory agent, inhibited proteasome activity and enhanced HSP accumulation in Xenopus laevis A6 kidney epithelial cells. Treatment of cells with celastrol induced the accumulation of ubiquitinated protein and inhibited chymotrypsin-like activity. This was accompanied by a dose- and time-dependent accumulation of HSP30 and HSP70. Celastrol-induced HSP accumulation was mediated by HSF1-DNA binding activity since this response was inhibited by the HSF1 activation inhibitor, KNK437. Simultaneous exposure of cells with celastrol plus either mild heat shock or the proteasome inhibitor, MG132, produced an enhanced accumulation of HSP30 that was greater than the sum of the individual stressors alone. Immunocytochemical analysis revealed that celastrol-induced HSP30 accumulation occurred in the cytoplasm in a granular pattern supplemented with larger circular HSP30 staining structures. HSP30 was also noted in the nucleus with less staining in the nucleolus. In some cells, celastrol induced the collapse of the actin cytoskeleton and conversion to a rounder morphology. In conclusion, this study has shown that celastrol inhibited proteasome activity and induced HSF1-mediated expression of hsp genes in amphibian cells.

  5. Generating controlled reducing environments in aerobic recombinant Escherichia coli fermentations: effects on cell growth, oxygen uptake, heat shock protein expression, and in vivo CAT activity.

    PubMed

    Gill, R T; Cha, H J; Jain, A; Rao, G; Bentley, W E

    1998-07-20

    The independent control of culture redox potential (CRP) by the regulated addition of a reducing agent, dithiothreitol (DTT) was demonstrated in aerated recombinant Escherichia coli fermentations. Moderate levels of DTT addition resulted in minimal changes to specific oxygen uptake, growth rate, and dissolved oxygen. Excessive levels of DTT addition were toxic to the cells resulting in cessation of growth. Chloramphenicol acetyltransferase (CAT) activity (nmoles/microgram total protein min.) decreased in batch fermentation experiments with respect to increasing levels of DTT addition. To further investigate the mechanisms affecting CAT activity, experiments were performed to assay heat shock protein expression and specific CAT activity (nmoles/microgram CAT min.). Expression of such molecular chaperones as GroEL and DnaK were found to increase after addition of DTT. Additionally, sigma factor 32 (sigma32) and several proteases were seen to increase dramatically during addition of DTT. Specific CAT activity (nmoles/microgram CAT min. ) varied greatly as DTT was added, however, a minimum in activity was found at the highest level of DTT addition in E. coli strains RR1 [pBR329] and JM105 [pROEX-CAT]. In conjunction, cellular stress was found to reach a maximum at the same levels of DTT. Although DTT addition has the potential for directly affecting intracellular protein folding, the effects felt from the increased stress within the cell are likely the dominant effector. That the effects of DTT were measured within the cytoplasm of the cell suggests that the periplasmic redox potential was also altered. The changes in specific CAT activity, molecular chaperones, and other heat shock proteins, in the presence of minimal growth rate and oxygen uptake alterations, suggest that the ex vivo control of redox potential provides a new process for affecting the yield and conformation of heterologous proteins in aerated E. coli fermentations.

  6. Kruppel-like factor 4 protein regulates isoproterenol-induced cardiac hypertrophy by modulating myocardin expression and activity.

    PubMed

    Yoshida, Tadashi; Yamashita, Maho; Horimai, Chihiro; Hayashi, Matsuhiko

    2014-09-19

    Kruppel-like factor 4 (KLF4) plays an important role in vascular diseases, including atherosclerosis and vascular injury. Although KLF4 is expressed in the heart in addition to vascular cells, the role of KLF4 in cardiac disease has not been fully determined. The goals of this study were to investigate the role of KLF4 in cardiac hypertrophy and to determine the underlying mechanisms. Cardiomyocyte-specific Klf4 knockout (CM Klf4 KO) mice were generated by the Cre/LoxP technique. Cardiac hypertrophy was induced by chronic infusion of the β-adrenoreceptor agonist isoproterenol (ISO). Results showed that ISO-induced cardiac hypertrophy was enhanced in CM Klf4 KO mice compared with control mice. Accelerated cardiac hypertrophy in CM Klf4 KO mice was accompanied by the augmented cellular enlargement of cardiomyocytes as well as the exaggerated expression of fetal cardiac genes, including atrial natriuretic factor (Nppa). Additionally, induction of myocardin, a transcriptional cofactor regulating fetal cardiac genes, was enhanced in CM Klf4 KO mice. Interestingly, KLF4 regulated Nppa expression by modulating the expression and activity of myocardin, providing a mechanical basis for accelerated cardiac hypertrophy in CM Klf4 KO mice. Moreover, we showed that KLF4 mediated the antihypertrophic effect of trichostatin A, a histone deacetylase inhibitor, because ISO-induced cardiac hypertrophy in CM Klf4 KO mice was attenuated by olmesartan, an angiotensin II type 1 antagonist, but not by trichostatin A. These results provide novel evidence that KLF4 is a regulator of cardiac hypertrophy by modulating the expression and the activity of myocardin.

  7. Skeletal muscle Heat shock protein 60 increases after endurance training and induces peroxisome proliferator-activated receptor gamma coactivator 1 α1 expression

    PubMed Central

    Barone, Rosario; Macaluso, Filippo; Sangiorgi, Claudia; Campanella, Claudia; Marino Gammazza, Antonella; Moresi, Viviana; Coletti, Dario; Conway de Macario, Everly; Macario, Alberto JL; Cappello, Francesco; Adamo, Sergio; Farina, Felicia; Zummo, Giovanni; Di Felice, Valentina

    2016-01-01

    Heat shock protein 60 (Hsp60) is a chaperone localizing in skeletal muscle mitochondria, whose role is poorly understood. In the present study, the levels of Hsp60 in fibres of the entire posterior group of hindlimb muscles (gastrocnemius, soleus, and plantaris) were evaluated in mice after completing a 6-week endurance training program. The correlation between Hsp60 levels and the expression of four isoforms of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α) were investigated only in soleus. Short-term overexpression of hsp60, achieved by in vitro plasmid transfection, was then performed to determine whether this chaperone could have a role in the activation of the expression levels of PGC1α isoforms. The levels of Hsp60 protein were fibre-type specific in the posterior muscles and endurance training increased its content in type I muscle fibers. Concomitantly with the increased levels of Hsp60 released in the blood stream of trained mice, mitochondrial copy number and the expression of three isoforms of PGC1α increased. Overexpressing hsp60 in cultured myoblasts induced only the expression of PGC1 1α, suggesting a correlation between Hsp60 overexpression and PGC1 1 α activation. PMID:26812922

  8. Regulation of p53, nuclear factor {kappa}B and cyclooxygenase-2 expression by bromelain through targeting mitogen-activated protein kinase pathway in mouse skin

    SciTech Connect

    Kalra, Neetu; Bhui, Kulpreet; Roy, Preeti; Srivastava, Smita; George, Jasmine; Prasad, Sahdeo; Shukla, Yogeshwer

    2008-01-01

    Bromelain is a pharmacologically active compound, present in stems and immature fruits of pineapples (Ananas cosmosus), which has been shown to have anti-edematous, anti-inflammatory, anti-thrombotic and anti-metastatic properties. In the present study, antitumorigenic activity of bromelain was recorded in 7,12-dimethylbenz(a)anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted 2-stage mouse skin model. Results showed that bromelain application delayed the onset of tumorigenesis and reduced the cumulative number of tumors, tumor volume and the average number of tumors/mouse. To establish a cause and effect relationship, we targeted the proteins involved in the cell death pathway. Bromelain treatment resulted in upregulation of p53 and Bax and subsequent activation of caspase 3 and caspase 9 with concomitant decrease in antiapoptotic protein Bcl-2 in mouse skin. Since persistent induction of cyclooxygenase-2 (Cox-2) is frequently implicated in tumorigenesis and is regulated by nuclear factor-kappa B (NF-{kappa}B), we also investigated the effect of bromelain on Cox-2 and NF-{kappa}B expression. Results showed that bromelain application significantly inhibited Cox-2 and inactivated NF-{kappa}B by blocking phosphorylation and subsequent degradation of I{kappa}B{alpha}. In addition, bromelain treatment attenuated DMBA-TPA-induced phosphorylation of extracellular signal-regulated protein kinase (ERK1/2), mitogen-activated protein kinase (MAPK) and Akt. Taken together, we conclude that bromelain induces apoptosis-related proteins along with inhibition of NF-{kappa}B-driven Cox-2 expression by blocking the MAPK and Akt/protein kinase B signaling in DMBA-TPA-induced mouse skin tumors, which may account for its anti-tumorigenic effects.

  9. Regulation of p53, nuclear factor kappaB and cyclooxygenase-2 expression by bromelain through targeting mitogen-activated protein kinase pathway in mouse skin.

    PubMed

    Kalra, Neetu; Bhui, Kulpreet; Roy, Preeti; Srivastava, Smita; George, Jasmine; Prasad, Sahdeo; Shukla, Yogeshwer

    2008-01-01

    Bromelain is a pharmacologically active compound, present in stems and immature fruits of pineapples (Ananas cosmosus), which has been shown to have anti-edematous, anti-inflammatory, anti-thrombotic and anti-metastatic properties. In the present study, antitumorigenic activity of bromelain was recorded in 7,12-dimethylbenz(a)anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted 2-stage mouse skin model. Results showed that bromelain application delayed the onset of tumorigenesis and reduced the cumulative number of tumors, tumor volume and the average number of tumors/mouse. To establish a cause and effect relationship, we targeted the proteins involved in the cell death pathway. Bromelain treatment resulted in upregulation of p53 and Bax and subsequent activation of caspase 3 and caspase 9 with concomitant decrease in antiapoptotic protein Bcl-2 in mouse skin. Since persistent induction of cyclooxygenase-2 (Cox-2) is frequently implicated in tumorigenesis and is regulated by nuclear factor-kappa B (NF-kappaB), we also investigated the effect of bromelain on Cox-2 and NF-kappaB expression. Results showed that bromelain application significantly inhibited Cox-2 and inactivated NF-kappaB by blocking phosphorylation and subsequent degradation of IkappaBalpha. In addition, bromelain treatment attenuated DMBA-TPA-induced phosphorylation of extracellular signal-regulated protein kinase (ERK1/2), mitogen-activated protein kinase (MAPK) and Akt. Taken together, we conclude that bromelain induces apoptosis-related proteins along with inhibition of NF-kappaB-driven Cox-2 expression by blocking the MAPK and Akt/protein kinase B signaling in DMBA-TPA-induced mouse skin tumors, which may account for its anti-tumorigenic effects.

  10. Constitutive expression of pathogenesis-related proteins and antioxydant enzyme activities triggers maize resistance towards Fusarium verticillioides.

    PubMed

    Maschietto, Valentina; Lanubile, Alessandra; Leonardis, Silvana De; Marocco, Adriano; Paciolla, Costantino

    2016-08-01

    Fusarium verticillioides is a fungal pathogen of maize that causes ear rot and contaminates the grains with fumonisin mycotoxins. Breeding for resistance to Fusarium emerged as the most economic and environmentally safe strategy; therefore the discovery of resistant sources and effective molecular markers are a priority. Ears of resistant (CO441 and CO433) and susceptible (CO354 and CO389) maize lines were inoculated with F. verticillioides and the expression of pathogenesis-related (PR) genes (PR1, PR5, PRm3, PRm6) and genes that protect from oxidative stress (peroxidase, catalase, superoxide dismutase and ascorbate peroxidase) were evaluated in the kernels at 72h post inoculation. In addition, the oxidation level and the enzymatic activity of ascorbate-glutathione cycle, catalase, superoxide dismutase and cytosolic and wall peroxidases were investigated. The uninoculated kernels of the resistant lines showed higher gene expression and enzymatic activities, highlighting the key role of constitutive resistance in limiting pathogen attack. In contrast, the susceptible lines activated defensive genes only after pathogen inoculation, resulting in increased levels of H2O2 and lipid peroxidation, as well as lower enzymatic activities. The constitutive defenses observed in this study from seed could be profitably exploited to develop markers to speed up conventional breeding programs in the selection of resistant genotypes.

  11. Increased Expression of CCN2, Epithelial Membrane Antigen, and Fibroblast Activation Protein in Hepatocellular Carcinoma with Fibrous Stroma Showing Aggressive Behavior

    PubMed Central

    Yoo, Jeong Eun; Ko, Jung Eun; Lee, Jee San; Kim, Hyunki; Choi, Jin Sub; Park, Young Nyun

    2014-01-01

    Tumor behavior is affected by the tumor microenvironment, composed of cancer-associated fibroblasts (CAFs). Meanwhile, hepatocellular carcinomas (HCC) with fibrous stroma reportedly exhibit aggressive behavior suggestive of tumor-stroma interaction. However, evidence of the crosstalk remains unclear. In this study, CCN2, epithelial membrane antigen (EMA), fibroblast activation protein (FAP), and keratin 19 (K19) expression was studied in 314 HCCs (cohort 1), 42 scirrhous HCCs (cohort 2), and 36 chronic hepatitis/cirrhosis specimens by immunohistochemistry. Clinicopathological parameters were analyzed according to the expressions of these markers. In tumor epithelial cells from cohort 1, CCN2 and EMA were expressed in 15.3% and 17.2%, respectively, and their expressions were more frequent in HCCs with fibrous stroma (≥5% of tumor area) than those without (P<0.05 for all); CCN2 expression was well correlated with K19 and EMA expression. In tumor stromal cells, FAP expression was found in 6.7%. In cohort 2, CCN2, EMA, and FAP expression was noted in 40.5%, 40.5%, and 66.7%, respectively, which was more frequent than that in cohort 1 (P<0.05 for all). Additionally, EMA expression was associated with the expression of K19, CCN2, and FAP (P<0.05 for all); EMA expressing tumor epithelial cells showed a topographic closeness to FAP-expressing CAFs. Analysis of disease-free survival revealed CCN2 expression to be a worse prognostic factor in both cohort 1 (P = 0.005) and cohort 2 (P = 0.023), as well as EMA as a worse prognostic factor in cohort 2 (P = 0.048). In conclusion, expression of CCN2, EMA, and FAP may be involved in the activation of CAFs in HCC, giving rise to aggressive behavior. Significant correlation between EMA-expressing tumor cells and FAP-expressing CAFs and their topographic closeness suggests possible cross-talk between tumor epithelial cells and stromal cells in the tumor microenvironment of HCC. PMID:25126747

  12. Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast

    DOEpatents

    Mayfield, Stephen P

    2015-01-13

    Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery of proteins/peptides, especially gut active proteins, without purification is disclosed.

  13. Robust expression of a bioactive mammalian protein in chlamydomonas chloroplast

    DOEpatents

    Mayfield, Stephen P.

    2010-03-16

    Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery or proteins/peptides, especially gut active proteins, without purification is disclosed.

  14. Modulation of protein expression and activity by radiation: Relevance to intracoronary radiation for the prevention of restenosis

    SciTech Connect

    Vodovotz, Yoram; Mitchell, James B.; Lucia, M. Scott; McKinney, Leslie; Kollum, Marc; Cottin, Yves; Chan, Rosanna C.; Barcellos-Hoff, Mary Helen; Waksman, Ron

    2001-08-25

    Restenosis is a common complication of percutaneous transluminal coronary angioplasty. Recent studies have demonstrated a striking reduction in the neointimal hyperplasia characteristic of restenosis following intracoronary radiation (IR), but the mechanisms by which radiation reduces neointima formation following balloon overstretch injury are not elucidated fully. In addition to direct antimitotic effects mediated via oxygen free radicals, ionizing radiation can induce the expression of numerous genes and thereby mediate indirect effects. Additionally, IR prevents restenosis at the cost of decreased healing and increased thrombosis, and we suggest that these adverse reactions can be modulated by adjunct pharmacology or gene-based strategies. This review discusses several genes and proteins modulated by radiation in the context of arterial injury, and their possible therapeutic relevance.

  15. Expression profiles of two small heat shock proteins and antioxidant enzyme activity in Mytilus galloprovincialis exposed to cadmium at environmentally relevant concentrations

    NASA Astrophysics Data System (ADS)

    You, Liping; Ning, Xuanxuan; Chen, Leilei; Zhang, Linbao; Zhao, Jianmin; Liu, Xiaoli; Wu, Huifeng

    2014-03-01

    Small heat shock proteins encompass a widespread but diverse class of proteins, which play key roles in protecting organisms from various stressors. In the present study, the full-length cDNAs of two small heat shock proteins (MgsHSP22 and MgsHSP24.1) were cloned from Mytilus galloprovincialis, which encoded peptides of 181 and 247 amino acids, respectively. Both MgsHSP22 and MgsHSP24.1 were detected in all tissues examined by real-time PCR, with the highest expression being observed in muscle and gonad tissues. The real-time PCR results revealed that Cd significantly inhibited MgsHSP22 expression at 24 h and MgsHSP24.1 at 24 and 48 h under 5 μg/L Cd 2+ exposure. MgsHSP24.1 expression was also significantly inhibited after 50 μg/L Cd2+ exposure for 48 h. With regard to antioxidant enzymes, increased GPx and CAT activity were detected under Cd2+ stress (5 and 50 μg/L), while no significant difference in SOD activity was observed throughout the experiment. Overall, both MgsHsps and antioxidant enzymes revealed their potential as Cd stress biomarkers in M. galloprovincialis.

  16. Wheat germ systems for cell-free protein expression.

    PubMed

    Harbers, Matthias

    2014-08-25

    Cell-free protein expression plays an important role in biochemical research. However, only recent developments led to new methods to rapidly synthesize preparative amounts of protein that make cell-free protein expression an attractive alternative to cell-based methods. In particular the wheat germ system provides the highest translation efficiency among eukaryotic cell-free protein expression approaches and has a very high success rate for the expression of soluble proteins of good quality. As an open in vitro method, the wheat germ system is a preferable choice for many applications in protein research including options for protein labeling and the expression of difficult-to-express proteins like membrane proteins and multiple protein complexes. Here I describe wheat germ cell-free protein expression systems and give examples how they have been used in genome-wide expression studies, preparation of labeled proteins for structural genomics and protein mass spectroscopy, automated protein synthesis, and screening of enzymatic activities. Future directions for the use of cell-free expression methods are discussed.

  17. Systematic expression profiling analysis mines dys-regulated modules in active tuberculosis based on re-weighted protein-protein interaction network and attract algorithm.

    PubMed

    Sun, Ying; Weng, Yan; Zhang, Ying; Yan, Xiang; Guo, Lei; Wang, Jia; Song, Xin; Yuan, Ying; Chang, Fu-Ye; Wang, Chun-Ling

    2017-03-18

    About 90% of tuberculosis (TB) patients latently infected with Mycobacterium tuberculosis (Mtb) show no symptoms, yet have a 10% chance in lifetime to progress active TB. Nevertheless, current diagnosis approaches need improvement in efficiency and sensitivity. The objective of this work was to detect potential signatures for active TB to further improve the understanding of the biological roles of functional modules involved in this disease. First, targeted networks of active TB and control groups were established via re-weighting protein-protein interaction (PPI) networks using Pearson's correlation coefficient (PCC). Candidate modules were detected from the targeted networks, and the modules with Jaccard score >0.7 were defined as attractors. After that, identification of dys-regulated modules was conducted from the attractors using attract method, Subsequently, gene oncology (GO) enrichment analyses were implemented for genes in the dys-regulated modules. We obtained 33 and 65 candidate modules from the targeted networks of control and active TB groups, respectively. Overall, 13 attractors were identified. Using the cut-off criteria of false discovery rate <0.05, there were 4 dys-regulated modules (Module 1, 2, 3, and 4). Based on the GO annotation results, genes in Modules 1, 2 and 4 were only involved in translation. Most genes in Module 1, 2 and 4 were associated with ribosomes. Accordingly, these dys-regulated modules might serve as potential biomarkers of active TB, facilitating the development for a more efficient, and sensitive diagnostic assay for active TB.

  18. Protein phosphatase 2A is essential to maintain active Wnt signaling and its Aβ tumor suppressor subunit is not expressed in colon cancer cells.

    PubMed

    Carmen Figueroa-Aldariz, M; Castañeda-Patlán, M Cristina; Santoyo-Ramos, Paula; Zentella, Alejandro; Robles-Flores, Martha

    2015-11-01

    Canonical Wnt signaling is altered in most cases of colorectal cancer. Experimental evidence indicates that protein phosphatase 2A (PP2A) may play either positive or negative roles in Wnt signaling but its precise in vivo functions remain elusive. In this work, using colon cultured cell lines we showed that basal PP2A activity is markedly reduced in malignant cells compared to non-malignant counterparts. We found that whereas normal or cancer cells displaying not altered Wnt signaling express mRNAs coding for PP2A-A scaffold α and β isoforms, cancer cells which have altered Wnt signaling do not express the Aβ isoform mRNA. Remarkably, we found that the Aβ protein levels are lost in all colon cancer cells, and in patients' tumor biopsies. In addition, all cancer cells exhibit higher levels of RalA activity, compared to non-malignant cells. Rescue experiments to restore Aβ expression in malignant RKO cells, diminished the RalGTPase activation and cell proliferation, indicating that the Aβ isoform acts as tumor suppressor in colon cancer cells. Reciprocal co-immunoprecipitation and immunofluorescence studies showed that the PP2A-C and -Aα subunits, expressed in all colon cells, interact in vivo with β-catenin only in malignant cells. Selective inhibition of PP2A did not significantly affect cellular apoptosis but induced dose-dependent negative effects in β-catenin-mediated transcriptional activity and in cell proliferation of malignant cells, indicating that the residual PP2A activity found in malignant cells, mediated by -C and Aα core subunits, is essential to maintain active Wnt signaling and cell proliferation in colon cancer cells.

  19. Estrogen regulates energy metabolic pathway and upstream adenosine 5'-monophosphate-activated protein kinase and phosphatase enzyme expression in dorsal vagal complex metabolosensory neurons during glucostasis and hypoglycemia.

    PubMed

    Tamrakar, Pratistha; Ibrahim, Baher A; Gujar, Amit D; Briski, Karen P

    2015-02-01

    The ability of estrogen to shield the brain from the bioenergetic insult hypoglycemia is unclear. Estradiol (E) prevents hypoglycemic activation of the energy deficit sensor adenosine 5'-monophosphate-activated protein kinase (AMPK) in hindbrain metabolosensory A2 noradrenergic neurons. This study investigates the hypothesis that estrogen regulates A2 AMPK through control of fuel metabolism and/or upstream protein kinase/phosphatase enzyme expression. A2 cells were harvested by laser microdissection after insulin or vehicle (V) injection of E- or oil (O)-implanted ovariectomized female rats. Cell lysates were evaluated by immunoblot for glycolytic, tricarboxylic acid cycle, respiratory chain, and acetyl-CoA-malonyl-CoA pathway enzymes. A2 phosphofructokinase (PFKL), isocitrate dehydrogenase, pyruvate dehydrogenase, and ATP synthase subunit profiles were elevated in E/V vs. O/V; hypoglycemia augmented PFKL and α-ketoglutarate dehydrogenase expression in E only. Hypoglycemia increased A2 Ca(2+) /calmodulin-dependent protein kinase-β in O and reduced protein phosphatase in both groups. A2 phospho-AMPK levels were equivalent in O/V vs. E/V but elevated during hypoglycemia in O only. These results implicate E in compensatory upregulation of substrate catabolism and corresponding maintenance of energy stability of A2 metabolosensory neurons during hypoglycemia, outcomes that support the potential viability of molecular substrates for hormone action as targets for therapies alleviating hypoglycemic brain injury.

  20. Expression of coxsackievirus and adenovirus receptor (CAR)-Fc fusion protein in Pichia pastoris and characterization of its anti-coxsackievirus activity.

    PubMed

    Zhang, Kebin; Yu, Hua; Xie, Wei; Xu, Zihui; Zhou, Shiwen; Huang, Chunji; Sheng, Halei; He, Xiaomei; Xiong, Junzhi; Qian, Guisheng

    2013-04-15

    Coxsackievirus and adenovirus receptors (CARs) are the common cellular receptors which mediate coxsackievirus or adenovirus infection. Receptor trap therapy, which uses soluble viral receptors to block the attachment and internalization of virus, has been developed for the inhibition of virus infection. In this study, we have constructed a pPIC3.5K/CAR-Fc expression plasmid for the economical and scale-up production of CAR-Fc fusion protein in Pichia pastoris. The coding sequence of the fusion protein was optimized according to the host codon usage bias. The amount of the CAR-Fc protein to total cell protein was up to 10% by 1% methanol induction for 96h and the purity was up to 96% after protein purification. Next, the virus pull-down assay demonstrated the binding activity of the CAR-Fc to coxsackievirus. The analyses of MTT assay, immunofluorescence staining and quantitative real-time PCR after virus neutralization assay revealed that CAR-Fc could significantly block coxsackievirus B3 infection in vitro. In coxsackievirus B3 infected mouse models, CAR-Fc treatment reduced mortality, myocardial edema, viral loads and inflammation, suggesting the significant virus blocking effect in vivo. Our results indicated that the P. pastoris expression system could be used to produce large quantities of bioactive CAR-Fc for further clinical purpose.

  1. Leucine residues in conserved region of 33K protein of bovine adenovirus - 3 are important for binding to major late promoter and activation of late gene expression.

    PubMed

    Kulshreshtha, Vikas; Islam, Azharul; Ayalew, Lisanework E; Tikoo, Suresh K

    2015-09-01

    The L6 region of bovine adenovirus 3 (BAdV-3) encode 33K (spliced) and 22K (unspliced) proteins. Earlier, anti-33K serum detected five major and three minor proteins in BAdV-3 infected cells. Here, we demonstrate that anti-sera raised against L6-22K protein detected two proteins of 42 and 37 kDa in BAdV-3 infected cells and one protein of 42 kDa in transfected cells expressing splice-site variant 22K protein (pC.22K containing substituted splice acceptor/donor sequence). Unlike 22K, 33K stimulated the transcription from the major late promoter (MLP) by binding to the downstream sequence elements (DE). Analysis of the variant proteins demonstrated that amino acids 201-240 of the conserved C-terminus of 33K containing the potential leucine zipper and RS repeat are required for the activation of MLP. Furthermore, amino acid substitution analysis demonstrated that unlike arginine residues of RS repeat, the leucine residues (217, 224, 232 and 240) of the conserved leucine zipper appear required for the binding of 33K to the MLP.

  2. Commensal Bacteria-induced Interleukin 1β (IL-1β) Secreted by Macrophages Up-regulates Hepcidin Expression in Hepatocytes by Activating the Bone Morphogenetic Protein Signaling Pathway.

    PubMed

    Shanmugam, Nanda Kumar N; Chen, Kejie; Cherayil, Bobby J

    2015-12-18

    The liver hormone hepcidin is the central regulator of systemic iron metabolism. Its increased expression in inflammatory states leads to hypoferremia and anemia. Elucidation of the mechanisms that up-regulate hepcidin during inflammation is essential for developing rational therapies for this anemia. Using mouse models of inflammatory bowel disease, we have shown previously that colitis-associated hepcidin induction is influenced by intestinal microbiota composition. Here we investigate how two commensal bacteria, Bifidobacterium longum and Bacteroides fragilis, representative members of the gut microbiota, affect hepcidin expression. We found that supernatants of a human macrophage cell line infected with either of the bacteria up-regulated hepcidin when added to a human hepatocyte cell line. This activity was abrogated by neutralization of IL-1β. Moreover, purified IL-1β increased hepcidin expression when added to the hepatocyte line or primary human hepatocytes and when injected into mice. IL-1β activated the bone morphogenetic protein (BMP) signaling pathway in hepatocytes and in mouse liver, as indicated by increased phosphorylation of small mothers against decapentaplegic proteins. Activation of BMP signaling correlated with IL-1β-induced expression of BMP2 in human hepatocytes and activin B in mouse liver. Treatment of hepatocytes with two different chemical inhibitors of BMP signaling or with a neutralizing antibody to BMP2 prevented IL-1β-induced up-regulation of hepcidin. Our results clarify how commensal bacteria affect hepcidin expression and reveal a novel connection between IL-1β and activation of BMP signaling. They also suggest that there may be differences between mice and humans with respect to the mechanism by which IL-1β up-regulates hepcidin.

  3. Genome-Wide Identification, Evolution, and Co-expression Network Analysis of Mitogen-Activated Protein Kinase Kinase Kinases in Brachypodium distachyon

    PubMed Central

    Feng, Kewei; Liu, Fuyan; Zou, Jinwei; Xing, Guangwei; Deng, Pingchuan; Song, Weining; Tong, Wei; Nie, Xiaojun

    2016-01-01

    Mitogen-activated protein kinase (MAPK) cascades are the conserved and universal signal transduction modules in all eukaryotes, which play the vital roles in plant growth, development, and in response to multiple stresses. In this study, we used bioinformatics methods to identify 86 MAPKKK protein encoded by 73 MAPKKK genes in Brachypodium. Phylogenetic analysis of MAPKKK family from Arabidopsis, rice, and Brachypodium has classified them into three subfamilies, of which 28 belonged to MEKK, 52 to Raf, and 6 to ZIK subfamily, respectively. Conserved protein motif, exon-intron organization, and splicing intron phase in kinase domains supported the evolutionary relationships inferred from the phylogenetic analysis. And gene duplication analysis suggested the chromosomal segment duplication happened before the divergence of the rice and Brachypodium, while all of three tandem duplicated gene pairs happened after their divergence. We further demonstrated that the MAPKKKs have evolved under strong purifying selection, implying the conservation of them. The splicing transcripts expression analysis showed that the splicesome translating longest protein tended to be adopted. Furthermore, the expression analysis of BdMAPKKKs in different organs and development stages as well as heat, virus and drought stresses revealed that the MAPKKK genes were involved in various signaling pathways. And the circadian analysis suggested there were 41 MAPKKK genes in Brachypodium showing cycled expression in at least one condition, of which seven MAPKKK genes expressed in all conditions and the promoter analysis indicated these genes possessed many cis-acting regulatory elements involved in circadian and light response. Finally, the co-expression network of MAPK, MAPKK, and MAPKKK in Brachypodium was constructed using 144 microarray and RNA-seq datasets, and ten potential MAPK cascades pathway were predicted. To conclude, our study provided the important information for evolutionary and

  4. Adrenergic activation of steroid 5alpha-reductase gene expression in rat C6 glioma cells: involvement of cyclic amp/protein kinase A-mediated signaling pathway.

    PubMed

    Morita, Kyoji; Arimochi, Hideki; Tsuruo, Yoshihiro

    2004-01-01

    Steroid 5alpha-reductase (5alpha-R) is well known as the enzyme converting progesterone and other steroid hormones to their 5alpha-reduced metabolites and has been reported to be localized in both neuronal and glial cells in the brain. Previously, the enzyme activity in glial cells has been shown to be enhanced either by coculturing with neuronal cells or by adding the conditioned medium of neuronal cells, suggesting a possible implication of neuro-glial interactions in the regulation of neurosteroid metabolism in the brain. In the present studies, the effects of adrenergic agonists on 5alpha-R mRNA and protein levels in rat C6 glioma cells were examined as one of the model experiments for investigating the influence of neuronal activity on the expression of 5alpha-R gene in the glial cell. The direct challenge of beta-adrenergic agonists to glioma cells resulted in the rapid and transient elevation of 5alpha-R mRNA levels through the activation of the cyclic AMP (cAMP)/protein kinase A-mediated signaling pathway. Further studies showed that cAMP-induced 5alpha-R mRNA expression was completely abolished by pretreatment of cells with actinomycin D and also indicated that the elevation of 5alpha-R mRNA levels was accompanied by an increase in enzyme protein in the cells. These findings provide strong evidence that the stimulation of beta-adrenergic receptors might induce the transcriptional activation of 5alpha-R gene expression in glial cells, proposing the possibility that neuronal activity might be involved in the production of neuroactive 5alpha-reduced steroids in the brain.

  5. Escherichia coli maltose-binding protein (MBP) directly induces mouse Th1 activation through upregulating TLR2 and downregulating TLR4 expressions.

    PubMed

    Wang, Fang; Ni, Weihua; Liu, Guomu; Wang, Juan; Xie, Fei; Yuan, Hongyan; Guo, Yingying; Zhai, RuiPing; Chen, Tanxiu; Li, Qiongshu; Tai, Guixiang

    2015-06-01

    Maltose-binding protein (MBP), a component of the maltose transport system of Escherichia coli, has been commonly thought to have minimal bioactivity. Our previous studies demonstrated that MBP could significantly enhance Bacillus Calmette-Guerin (BCG)-induced T helper 1 (Th1) cell activation in mice. In the present study, we analyzed the effect of MBP on mouse T cells and found that MBP promoted the proliferation and IFN-γ production of CD4(+) T cells, suggesting that MBP directly induces Th1 activation. To explore the mechanism of Th1 activation, the expression of Toll-like receptor 2/4 (TLR2/4) on purified mouse CD4(+) T cells was detected. The results showed that MBP up-regulated TLR2 while down-regulated TLR4 expression, accompanied by a clear increase in MyD88 expression and IκB phosphorylation. Notably, the addition of anti-TLR2 antibody abrogated the MBP-induced CD4(+) T cells proliferation, IFN-γ secretion and MyD88 expression, whereas the addition of anti-TLR4 antibody exhibited a contradictive effect. Besides, the block of either TLR2 or TLR4 both reduced IκB phosphorylation. These results above suggest that TLR2-mediated MyD88-dependent pathway contributes to MBP-induced Th1 activation, while TLR4 appears to counteract this effect via MyD88-independent pathway.

  6. Expression, purification, and evaluation for anticancer activity of ribosomal protein L31 gene (RPL31) from the giant panda (Ailuropoda melanoleuca).

    PubMed

    Su, Xiu-Lan; Hou, Yi-Ling; Yan, Xiang-Hui; Ding, Xiang; Hou, Wan-Ru; Sun, Bing; Zhang, Si-Nan

    2012-09-01

    Ribosomal protein L31 gene is a component of the 60S large ribosomal subunit encoded by RPL31 gene, while ribosomal protein L31 (RPL31) is an important constituent of peptidyltransferase center. In our research, the cDNA and the genomic sequence of RPL31 were cloned successfully from the giant panda (Ailuropoda melanoleuca) using RT-PCR technology respectively, following sequencing and analyzing preliminarily. We constructed a recombinant expression vector contained RPL31 cDNA and over-expressed it in Escherichia coli using pET28a plasmids. The expression product was purified to obtain recombinant protein of RPL31 from the giant panda. Recombinant protein of RPL31 obtained from the experiment acted on human laryngeal carcinoma Hep-2 and human hepatoma HepG-2 cells for study of its anti-cancer activity by MTT [3-(4, 5-dimehyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide] method. Then observe these cells growth depressive effect. The result indicated that the cDNA fragment of the RPL31 cloned from the giant panda is 419 bp in size, containing an open reading frame of 378 bp, and deduced protein was composed of 125 amino acids with an estimated molecular weight of 14.46-kDa and PI of 11.21. The length of the genomic sequence is 8,091 bp, which was found to possess four exons and three introns. The RPL31 gene can be readily expressed in E.coli, expecting 18-kDa polypeptide that formed inclusion bodies. Recombinant protein RPL31 from the giant panda consists of 157 amino acids with an estimated molecular weight of 17.86 kDa and PI of 10.77. The outcomes showed that the cell growth inhibition rate in a time- and dose-dependent on recombinant protein RPL31. And also indicated that the effect at low concentrations was better than high concentrations on Hep-2 cells, and the concentration of 0.33 μg/mL had the best rate of growth inhibition, 44 %. Consequently, our study aimed at revealing the recombinant protein RPL31 anti-cancer function from the giant panda

  7. Selection for low or high primary dormancy in Lolium rigidum Gaud seeds results in constitutive differences in stress protein expression and peroxidase activity

    PubMed Central

    Goggin, Danica E.; Powles, Stephen B.; Steadman, Kathryn J.

    2011-01-01

    Seed dormancy in wild Lolium rigidum Gaud (annual ryegrass) populations is highly variable and not well characterized at the biochemical level. To identify some of the determinants of dormancy level in these seeds, the proteomes of subpopulations selected for low and high levels of primary dormancy were compared by two-dimensional polyacrylamide gel electrophoresis of extracts from mature, dry seeds. High-dormancy seeds showed higher expression of small heat shock proteins, enolase, and glyoxalase I than the low-dormancy seeds. The functional relevance of these differences in protein expression was confirmed by the fact that high-dormancy seeds were more tolerant to high temperatures imposed at imbibition and had consistently higher glyoxalase I activity over 0–42 d dark stratification. Higher expression of a putative glutathione peroxidase in low-dormancy seeds was not accompanied by higher activity, but these seeds had a slightly more oxidized glutathione pool and higher total peroxidase activity. Overall, these biochemical and physiological differences suggest that L. rigidum seeds selected for low dormancy are more prepared for rapid germination via peroxidase-mediated cell wall weakening, whilst seeds selected for high dormancy are constitutively prepared to survive environmental stresses, even in the absence of stress during seed development. PMID:20974739

  8. Role of an expansin-like molecule in Dictyostelium morphogenesis and regulation of its gene expression by the signal transducer and activator of transcription protein Dd-STATa.

    PubMed

    Ogasawara, Shun; Shimada, Nao; Kawata, Takefumi

    2009-02-01

    Expansins are proteins involved in plant morphogenesis, exerting their effects on cellulose to extend cell walls. Dictyostelium is an organism that possesses expansin-like molecules, but their functions are not known. In this study, we analyzed the expL7 (expansin-like 7) gene, which has been identified as a putative target of Dd-STATa, a Dictyostelium homolog of the metazoan signal transducer and activator of transcription (STAT) proteins. Promoter fragments of the expL7 were fused to a lacZ reporter and the expression patterns determined. As expected from the behavior of the endogenous expL7 gene, the expL7/lacZ fusion gene was downregulated in Dd-STATa null slugs. In the parental strain, the expL7 promoter was activated in the anterior tip region. Mutational analysis of the promoter identified a sequence that was necessary for expression in tip cells. In addition, an activator sequence for pstAB cells was identified. These sequences act in combination with the repressor region to prevent ectopic expL7 expression in the prespore and prestalk regions of the slug and culminant. Although the expL7 null mutant showed no phenotypic change, the expL7 overexpressor showed aberrant stalk formation. These results indicate that the expansin-like molecule is important for morphogenesis in Dictyostelium.

  9. Activated expression of an Arabidopsis HD-START protein confers drought tolerance with improved root system and reduced stomatal density.

    PubMed

    Yu, Hong; Chen, Xi; Hong, Yuan-Yuan; Wang, Yao; Xu, Ping; Ke, Sheng-Dong; Liu, Hai-Yan; Zhu, Jian-Kang; Oliver, David J; Xiang, Cheng-Bin

    2008-04-01

    Drought is one of the most important environmental constraints limiting plant growth and agricultural productivity. To understand the underlying mechanism of drought tolerance and to identify genes for improving this important trait, we conducted a gain-of-function genetic screen for improved drought tolerance in Arabidopsis thaliana. One mutant with improved drought tolerance was isolated and designated as enhanced drought tolerance1. The mutant has a more extensive root system than the wild type, with deeper roots and more lateral roots, and shows a reduced leaf stomatal density. The mutant had higher levels of abscisic acid and Pro than the wild type and demonstrated an increased resistance to oxidative stress and high levels of superoxide dismutase. Molecular genetic analysis and recapitulation experiments showed that the enhanced drought tolerance is caused by the activated expression of a T-DNA tagged gene that encodes a putative homeodomain-START transcription factor. Moreover, overexpressing the cDNA of the transcription factor in transgenic tobacco also conferred drought tolerance associated with improved root architecture and reduced leaf stomatal density. Therefore, we have revealed functions of the homeodomain-START factor that were gained upon altering its expression pattern by activation tagging and provide a key regulator that may be used to improve drought tolerance in plants.

  10. Blunted activation of NF-{kappa}B and NF-{kappa}B-dependent gene expression by geranylgeranylacetone: Involvement of unfolded protein response

    SciTech Connect

    Hayakawa, Kunihiro; Hiramatsu, Nobuhiko; Okamura, Maro; Yao, Jian; Paton, Adrienne W.; Paton, James C.; Kitamura, Masanori

    2008-01-04

    Geranylgeranylacetone (GGA), an anti-ulcer agent, has anti-inflammatory potential against experimental colitis and ischemia-induced renal inflammation. However, molecular mechanisms involved in its anti-inflammatory effects are largely unknown. We found that, in glomerular mesangial cells, GGA blocked activation of nuclear factor-{kappa}B and consequent induction of monocyte chemoattractant protein 1 (MCP-1) by inflammatory cytokines. It was inversely correlated with induction of unfolded protein response (UPR) evidenced by expression of 78 kDa glucose-regulated protein (GRP78) and suppression of endoplasmic reticulum stress-responsive alkaline phosphatase. Various inducers of UPR including tunicamycin, thapsigargin, A23187, 2-deoxyglucose, dithiothreitol, and AB{sub 5} subtilase cytotoxin reproduced the suppressive effects of GGA. Furthermore, attenuation of UPR by stable transfection with GRP78 diminished the anti-inflammatory effects of GGA. These results disclosed a novel, UPR-dependent mechanism underlying the anti-inflammatory potential of GGA.

  11. Cotton plants expressing a hemipteran-active Bacillus thuringiensis crystal protein impact the development and survival of Lygus hesperus (Hemiptera: Miridae) nymphs.

    PubMed

    Baum, James A; Sukuru, Uma R; Penn, Stephen R; Meyer, Steven E; Subbarao, Shubha; Shi, Xiaohong; Flasinski, Stanislaw; Heck, Gregory R; Brown, Robert S; Clark, Thomas L

    2012-04-01

    The plant bugs Lygus hesperus Knight (Hemiptera: Miridae) and L. lineolaris (Palisot de Beauvois) have emerged as economic pests of cotton in the United States. These hemipteran species are refractory to the insect control traits found in genetically modified commercial varieties of cotton. In this article, we report the isolation and characterization of a 35 kDa crystal protein from Bacillus thuringiensis, designated TIC807, which causes reduced mass gain and mortality of L. hesperus and L. lineolaris nymphs when presented in an artificial diet feeding assay. Cotton plants expressing the TIC807 protein were observed to impact the survival and development of L. hesperus nymphs in a concentration-dependent manner. These results, demonstrating in planta activity of a Lygus insecticidal protein, represent an important milestone in the development of cotton varieties protected from Lygus feeding damage.

  12. Phosphatase activities analyzed by in vivo expressions.

    PubMed

    Schweighofer, Alois; Ayatollahi, Zahra; Meskiene, Irute

    2009-01-01

    Protein phosphatases act to reverse phosphorylation-related modifications induced by protein kinases. Type 2C protein phosphatases (PP2C) are monomeric Ser/Thr phosphatases that require a metal for their activity and are abundant in prokaryotes and eukaryotes. In plants, such as Medicago and Arabidopsis PP2Cs control several essential processes, including ABA signaling, development, and wound-induced mitogen-activated protein kinase (MAPK) pathways. In vitro assays with recombinant proteins and yeast two-hybrid systems usually provide initial information about putative PP2C substrates; however, these observations have to be verified in vivo. Therefore, a method for transient expression in isolated Arabidopsis suspension cell protoplasts was developed to assay PP2C action in living cells. This system has proven to be very useful in producing active enzymes and their substrates and in performing enzymatic reactions in vivo. Transient gene expression in isolated cells enabled assembly of functional protein kinase cascades and the creation of phosphorylated targets for PP2Cs. The method is based on the co-transformation and transient co-expression of different PP2C proteins with MAPK. It shows that epitope-tagged PP2C and MAPK proteins exhibit high enzymatic activities and produce substantial protein amounts easily monitored by Western blot analysis. Additionally, PP2C phosphatase activities can be directly tested in protein extracts from protoplasts, suggesting a possibility for analysis of activities of new PP2C family members.

  13. Activation of latent HIV-1 expression by protein kinase C agonists. A novel therapeutic approach to eradicate HIV-1 reservoirs.

    PubMed

    Sánchez-Duffhues, Gonzalo; Vo, Minh Q; Pérez, Moisés; Calzado, Marco A; Moreno, Santiago; Appendino, Giovanni; Muñoz, Eduardo

    2011-03-01

    The persistence of latent HIV-infected cellular reservoirs represents the major hurdle to virus eradication in patients treated with highly active antiretroviral therapy. The molecular mechanisms by which integrated HIV-1 is repressed during latency have been partially identified in different models of HIV-1 latency, and the involvement of multiple processes has been demonstrated. Therefore, several molecular targets amenable to pharmacological manipulation have emerged to antagonize HIV-1 latency in the viral reservoirs. In this context, it has been suggested that successful depletion of such latent reservoirs will require a combination of therapeutic agents that can specifically and efficiently act on cells harbouring latent HIV-1 provirus. HIV-1 reactivation therapy is a potential therapeutic option to purge the viral reservoirs. The goal of this therapy is to enhance the transcriptional activity of the latent HIV-1 without inducing the polyclonal activation of non-infected cells. In this sense natural or semisynthetic protein kinase C agonists lacking tumour-promoter activities clearly fulfil this criterion, thereby opening new research avenues to purge HIV-1 reservoirs. In this review article, we have succinctly summarized the known effects of "natural products", focusing on phorboids like prostratin and ingenols, macrolides like bryostatin 1, and macrocyclic polyesters like ingols and jatrophanes. A comprehensive view on the molecular mechanisms underlying the principle of HIV-1 reactivation from latency is provided, discussing the combination of "natural products" with other experimental or conventional therapeutics.

  14. Expression of peptide fragments from proADM and involvement of mitogen-activated protein kinase signaling pathways in pulmonary remodeling induced by high pulmonary blood flow.

    PubMed

    Li, Wei; Guo, Aili; Wang, Lijuan; Kong, Qingyu; Wang, Rong; Han, Li; Zhao, Cuifen

    2016-01-01

    Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive pulmonary arterial remodeling and right ventricular failure. Despite recent advances in pathophysiological mechanism exploration and new therapeutic approaches, PAH remains a challenging condition. In this study, we investigated the roles of the peptide fragments from proadrenomedullin (proADM) such as adrenomedullin (ADM), adrenotensin (ADT), and proadrenomedullin N-terminal 20 peptide (PAMP) during pulmonary remodeling caused by high pulmonary blood flow, and probed the possible involvement of mitogen-activated protein kinase (MAPK) signal transduction pathways. Sixteen rat models of PAH were artificially established by surgically connecting the left common carotid artery to the external jugular vein. We subcutaneously injected an extracellular signal-regulated protein kinase (ERK1/2) inhibitor, PD98059, in eight rats, treated another eight rats with an equal volume of saline. Eight rats without connections served as the control group. We observed that mRNA expression levels of ADM, stress-activated protein kinase (SAPK), and ERK1/2 were significantly elevated in the shunted rats; furthermore, ERK1/2 levels were significantly inhibited by PD98059. Protein levels of ADM, PAMP, p-SAPK, and p-ERK1/2 were significantly higher ADT was lower, and p-p38 remained unchanged in the rat models compared with the controls. However, the protein expression of both ADM and p-ERK1/2 was significantly inhibited by PD98059. Our results suggest that levels of ADM, ADT, and PAMP respond to pulmonary remodeling, and that activation of the SAPK and ERK1/2 signaling pathways is involved in pulmonary hypertension and artery remodeling caused by high pulmonary blood flow.

  15. Transforming growth factor-{beta} inhibits CCAAT/enhancer-binding protein expression and PPAR{gamma} activity in unloaded bone marrow stromal cells

    SciTech Connect

    Ahdjoudj, S.; Kaabeche, K.; Holy, X.; Fromigue, O.; Modrowski, D.; Zerath, E.; Marie, P.J. . E-mail: pierre.marie@larib.inserm.fr

    2005-02-01

    The molecular mechanisms regulating the adipogenic differentiation of bone marrow stromal cells in vivo remain largely unknown. In this study, we investigated the regulatory effects of transforming growth factor beta-2 (TGF-{beta}2) on transcription factors involved in adipogenic differentiation induced by hind limb suspension in rat bone marrow stromal cells in vivo. Time course real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis of gene expression showed that skeletal unloading progressively increases the expression of CCAAT/enhancer-binding protein (C/EBP){alpha} and C/EBP{beta} {alpha} at 5 days in bone marrow stromal cells resulting in increased peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}2) transcripts at 7 days. TGF-{beta}2 administration in unloaded rats corrected the rise in C/EBP{alpha} and C/EBP{beta} transcripts induced by unloading in bone marrow stromal cells. This resulted in inhibition of PPAR{gamma}2 expression that was associated with increased Runx2 expression. Additionally, the inhibition of C/EBP{alpha} and C/EBP{beta} expression by TGF-{beta}2 was associated with increased PPAR{gamma} serine phosphorylation in bone marrow stromal cells, a mechanism that inhibits PPAR{gamma} transactivating activity. The sequential inhibitory effect of TGF-{beta}2 on C/EBP{alpha}, C/EBP{beta}, and PPAR{gamma}2 resulted in reduced LPL expression and abolition of bone marrow stromal cell adipogenic differentiation, which contributed to prevent bone loss induced by skeletal unloading. We conclude that TGF-{beta}2 inhibits the excessive adipogenic differentiation of bone marrow stromal cells induced by skeletal unloading by inhibiting C/EBP{alpha}, C/EBP{beta}, and PPAR{gamma} expression and activity, which provides a sequential mechanism by which TGF-{beta}2 regulates adipogenic differentiation of bone marrow stromal cells in vivo.

  16. Activation of protein kinase Czeta mediates luteinizing hormone- or forskolin-induced NGFI-B expression in preovulatory granulosa cells of rat ovary.

    PubMed

    Park, Jae-Il; Kim, Sun-Gyun; Chun, Jang-Soo; Seo, You-Mi; Jeon, Mi-Jin; Ohba, Motoi; Kim, Hyun-Jin; Chun, Sang-Young

    2007-05-30

    We have previously demonstrated that luteinizing hormone (LH) induces a rapid and transient expression of NGFI-B in the ovary. In this report, we investigated the signaling pathway for LH- and forskolin-induced NGFI-B expression in cultured rat granulosa cells of preovulatory follicles. LH- or forskolin-induced NGFI-B expression was suppressed by high dose of protein kinase C (PKC) inhibitor RO 31-8220 (10 microM), but not by low doses RO 31-8220 (0.1-1.0 microM) or adenylate cyclase inhibitor MDL-12,300A, implicating the involvement of atypical PKCs. Kinase assay revealed that LH treatment of granulosa cells resulted in a rapid stimulation of atypical PKCzeta activity. Interestingly, like LH, forskolin was also able to activate PKCzeta. Treatment with the cell-permeable PKCzeta-specific inhibitor pseudosubstrate peptide inhibited LH-or forskolin-induced NGFI-B expression, indicating the essential role of PKCzeta. Consistent with this promise, in granulosa cells depleted of diacylglycerol sensitive PKCs by prolonged treatment with tetradecanoylphobol-13-acetate, LH or forskolin could still induce NGFI-B expression, and RO 31-8220 or the PKCzeta pseudosubstrate peptide inhibited LH- or forskolin-induced NGFI-B expression. Furthermore, overexpression of dominant-negative PKCzeta in primary granulosa cells using a replication-defective adenovirus vector resulted in the suppression of LH- or forskolin-induced NGFI-B expression. Our findings demonstrate that PKCzeta, which is activated by LH or forskolin, contributes to the induction of NGFI-B in granulosa cells of preovulatory follicles.

  17. Fluorescent visualisation of the hypothalamic oxytocin neurones activated by cholecystokinin-8 in rats expressing c-fos-enhanced green fluorescent protein and oxytocin-monomeric red fluorescent protein 1 fusion transgenes.

    PubMed

    Katoh, A; Shoguchi, K; Matsuoka, H; Yoshimura, M; Ohkubo, J-I; Matsuura, T; Maruyama, T; Ishikura, T; Aritomi, T; Fujihara, H; Hashimoto, H; Suzuki, H; Murphy, D; Ueta, Y

    2014-05-01

    The up-regulation of c-fos gene expression is widely used as a marker of neuronal activation elicited by various stimuli. Anatomically precise observation of c-fos gene products can be achieved at the RNA level by in situ hybridisation or at the protein level by immunocytochemistry. Both of these methods are time and labour intensive. We have developed a novel transgenic rat system that enables the trivial visualisation of c-fos expression using an enhanced green fluorescent protein (eGFP) tag. These rats express a transgene consisting of c-fos gene regulatory sequences that drive the expression of a c-fos-eGFP fusion protein. In c-fos-eGFP transgenic rats, robust nuclear eGFP fluorescence was observed in osmosensitive brain regions 90 min after i.p. administration of hypertonic saline. Nuclear eGFP fluorescence was also observed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) 90 min after i.p. administration of cholecystokinin (CCK)-8, which selectively activates oxytocin (OXT)-secreting neurones in the hypothalamus. In double transgenic rats that express c-fos-eGFP and an OXT-monomeric red fluorescent protein 1 (mRFP1) fusion gene, almost all mRFP1-positive neurones in the SON and PVN expressed nuclear eGFP fluorescence 90 min after i.p. administration of CCK-8. It is possible that not only a plane image, but also three-dimensional reconstruction image may identify cytoplasmic vesicles in an activated neurone at the same time.

  18. Statin induction of liver fatty acid-binding protein (L-FABP) gene expression is peroxisome proliferator-activated receptor-alpha-dependent.

    PubMed

    Landrier, Jean-François; Thomas, Charles; Grober, Jacques; Duez, Hélène; Percevault, Frédéric; Souidi, Maâmar; Linard, Christine; Staels, Bart; Besnard, Philippe

    2004-10-29

    Statins are drugs widely used in humans to treat hypercholesterolemia. Statins act by inhibiting cholesterol synthesis resulting in the activation of the transcription factor sterol-responsive element-binding protein-2 that controls the expression of genes involved in cholesterol homeostasis. Statin therapy also decreases plasma triglyceride and non-esterified fatty acid levels, but the mechanism behind this effect remains more elusive. Liver fatty acid-binding protein (L-FABP) plays a role in the influx of long-chain fatty acids into hepatocytes. Here we show that L-FABP is a target for statins. In rat hepatocytes, simvastatin treatment induced L-FABP mRNA levels in a dose-dependent manner. Moreover, L-FABP promoter activity was induced by statin treatment. Progressive 5'-deletion analysis revealed that the peroxisome proliferator-activated receptor (PPAR)-responsive element located at position -67/-55 was responsible for the statin-mediated transactivation of the rat L-FABP promoter. Moreover, treatment with simvastatin and the PPARalpha agonist Wy14,649 resulted in a synergistic induction of L-FABP expression (mRNA and protein) in rat Fao hepatoma cells. This effect was also observed in vivo in wild-type mice but not in PPARalpha-null animals demonstrating the direct implication of PPARalpha in L-FABP regulation by statin treatment. Statin treatment resulted in a rise in PPARalpha mRNA levels both in vitro and in vivo and activated the mouse PPARalpha promoter in a reporter assay. Altogether, these data demonstrate that L-FABP expression is up-regulated by statins through a mechanism involving PPARalpha. Moreover, PPARalpha might be a statin target gene. These effects might contribute to the triglyceride/non-esterified fatty acid-lowering properties of statins.

  19. The time course of activity-regulated cytoskeletal (ARC) gene and protein expression in the whisker-barrel circuit using two paradigms of whisker stimulation.

    PubMed

    Khodadad, Aida; Adelson, P David; Lifshitz, Jonathan; Thomas, Theresa Currier

    2015-05-01

    Immediate early genes have previously demonstrated a rapid increase in gene expression after various behavioral paradigms. The main focus of this article is to identify a molecular marker of circuit activation after manual whisker stimulation or exploration of a novel environment. To this end, we investigated the dynamics of ARC transcription in adult male rats during whisker somatosensation throughout the whisker barrel circuit. At various time points, tissue was biopsied from the ventral posterior medial nucleus (VPM) of the thalamus, primary somatosensory barrel field (S1BF) cortex and hippocampus for quantification using real-time PCR and western blot. Our results show that there were no significant differences in ARC gene or protein expression in the VPM after both types of stimulation. However, manual whisker stimulation resulted in increased ARC gene expression at 15, 30, 60 and 300 min in the S1BF, and 15 min in the hippocampus (p<0.05). Also, exploration of a novel environment resulted in increased ARC mRNA expression at 15 and 30 min in the S1BF and at 15 min in the hippocampus (p<0.05). The type of stimulation (manual versus exploration of a novel environment) influenced the magnitude of ARC gene expression in the S1BF (p<0.05). These data are the first to demonstrate that ARC is a specific, quantifiable and input dependent molecular marker of circuit activation which can serve to quantify the impact of brain injury and subsequent rehabilitation on whisker sensation.

  20. Characterization of functionally active interleukin-18/eGFP fusion protein expression during cell cycle phases in recombinant chicken DF1 Cells.

    PubMed

    Wu, Hsing Chieh; Chen, Yu San; Shien, Jui Hung; Shen, Pin Chun; Lee, Long Huw

    2016-05-01

    The dependence of foreign gene expression on cell cycle phases in mammalian cells has been described. In this study, a DF1/chIL-18a cell line that stably expresses the fusion protein chIL-18 was constructed and the enhanced green fluorescence protein connected through a (G4 S)3 linker sequence investigated the relationship between cell cycle phases and fusion protein production. DF1/chIL-18a cells (1 × 10(5) ) were inoculated in 60-mm culture dishes containing 5 mL of media to achieve 50%-60% confluence and were cultured in the presence of the cycle-specific inhibitors 10058-F4, aphidicolin, and colchicine for 24 and 48 h. The percentage of cell density and mean fluorescence intensity in each cell cycle phase were assessed using flow cytometry. The inhibitors effectively arrested cell growth. The fusion protein production rate was higher in the S phase than in the G0/G1 and G2/M phases. When cell cycle progression was blocked in the G0/G1, S, and G2/M phases by the addition of 10058-F4, aphidicolin, and colchicine, respectively, the aphidicolin-induced single cells showed higher fusion protein levels than did the 10058-F4- or colchicine-induced phase cells and the uninduced control cells. Although the cells did not proliferate after the drug additions, the amount of total fusion protein accumulated in aphidicolin-treated cells was similar to that in the untreated cultures. Fusion protein is biologically active because it induces IFN-γ production in splenocyte cultures of chicken. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:581-591, 2016.

  1. Silver nanoparticles and dissolved silver activate contrasting immune responses and stress-induced heat shock protein expression in sea urchin.

    PubMed

    Magesky, Adriano; de Oliveira Ribeiro, Ciro A; Beaulieu, Lucie; Pelletier, Émilien

    2016-12-12

    Using immune cells of sea urchin Strongylocentrotus droebachiensis in early development as a model, the cellular protective mechanisms against ionic and poly(allylamine)-coated silver nanoparticle (AgNPs; 14 ± 6 nm) treatments at 100 μg L(-1) were investigated. Oxidative stress, heat shock protein expression, and pigment production by spherulocytes were determined as well as AgNP translocation pathways and their multiple effects on circulating coelomocytes. Sea urchins showed an increasing resilience to Ag over time because ionic Ag is accumulated in a steady way, although nanoAg levels dropped between 48 h and 96 h. A clotting reaction emerged on tissues injured by dissolved Ag (present as chloro-complexes in seawater) between 12 h and 48 h. Silver contamination and nutritional state influenced the production of reactive oxygen species. After passing through coelomic sinuses and gut, AgNPs were found in coelomocytes. Inside blood vessels, apoptosis-like processes appeared in coelomocytes highly contaminated by poly(allylamine)-coated AgNPs. Increasing levels of Ag accumulated by urchins once exposed to AgNPs pointed to a Trojan-horse mechanism operating over 12-d exposure. However, under short-term treatments, physical interactions of poly(allylamine)-coated AgNPs with cell structures might be, at some point, predominant and responsible for the highest levels of stress-related proteins detected. The present study is the first report detailing nano-translocation in a marine organism and multiple mechanisms by which sea urchin cells can deal with toxic AgNPs. Environ Toxicol Chem 2016;9999:1-15. © 2016 SETAC.

  2. Galectin-3 Protein Modulates Cell Surface Expression and Activation of Vascular Endothelial Growth Factor Receptor 2 in Human Endothelial Cells*

    PubMed Central

    Markowska, Anna I.; Jefferies, Kevin C.; Panjwani, Noorjahan

    2011-01-01

    Angiogenesis is heavily influenced by VEGF-A and its family of receptors, particularly VEGF receptor 2 (VEGF-R2). Like most cell surface proteins, VEGF-R2 is glycosylated, although the function of VEGF-R2 with respect to its glycosylation pattern is poorly characterized. Galectin-3, a glycan binding protein, interacts with the EGF and TGFβ receptors, retaining them on the plasma membrane and altering their signal transduction. Because VEGF-R2 is glycosylated and both galectin-3 and VEGF-R2 are involved with angiogenesis, we hypothesized that galectin-3 binds VEGF-R2 and modulates its signal transduction as well. Employing a Western blot analysis approach, we found that galectin-3 induces phosphorylation of VEGF-R2 in endothelial cells. Knockdown of galectin-3 and Mgat5, an enzyme that synthesizes high-affinity glycan ligands of galectin-3, reduced VEGF-A mediated angiogenesis in vitro. A direct interaction on the plasma membrane was detected between galectin-3 and VEGF-R2, and this interaction was dependent on the expression of Mgat5. Using immunofluorescence and cell surface labeling, we found an increase in the level of internalized VEGF-R2 in both Mgat5 and galectin-3 knockdown cells, suggesting that galectin-3 retains the receptor on the plasma membrane. Finally, we observed reduced suture-induced neovascularization in the corneas of Gal3−/− and Mgat5−/− mice. These findings are consistent with the hypothesis that, like its role with the EGF and TGFβ receptors, galectin-3 contributes to the plasma membrane retention and proangiogenic function of VEGF-R2. PMID:21715322

  3. Expressing a fusion protein with protease and chitinase activities increases the virulence of the insect pathogen Beauveria bassiana.

    PubMed

    Fang, Weiguo; Feng, Jin; Fan, Yanhua; Zhang, Yongjun; Bidochka, Michael J; Leger, Raymond J St; Pei, Yan

    2009-10-01

    Entomopathogenic fungi, such as Beauveria bassiana and Metarhizium anisopliae are being developed as alternatives to chemical insecticides. They infect insects by direct penetration of the cuticle using a combination of physical pressure and extracellular hydrolytic enzymes such as proteases and chitinases. Previously we found that overexpression of a subtilisin-like protease (Pr1A) or a chitinase (Bbchit1) resulted in increased virulence of M. anisopliae and B. bassiana, respectively. In this study, we found that a mixture of the B. bassiana Pr1A homolog (CDEP1) and Bbchit1 degraded insect cuticle in vitro more efficiently than either CDEP1 or Bbchit1 alone. Based on this we produced three plasmid constructs; (1) Bbchit1, (2) CDEP1, and (3) a fusion gene of Bbchit1 linked to CDEP1 each under the control of the constitutive gpd promoter from Aspergillus nidulans. B. bassiana transformants secreting the fusion protein (CDEP1:Bbchit1) penetrated the cuticle significantly faster than the wild type or transformants overexpressing either Bbchit1 or CDEP1. Compared to the wild type, the transformant overexpressing CDEP1 showed a 12.5% reduction in LT(50), without a reduction in LC(50). The LT(50) of the transformant expressing CDEP1:Bbchit1 was reduced by 24.9%. Strikingly, expression of CDEP1:Bbchit1 resulted in a 60.5% reduction in LC(50), more than twice the reduction obtained by overexpression of Bbchit1 (28.5%). This work represents a significant step towards the development of hypervirulent insect pathogens for effective pest control.

  4. Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen

    PubMed Central

    Rawlings, Andrea E.; Levdikov, Vladimir M.; Blagova, Elena; Colledge, Vicki L.; Mas, Philippe J.; Tunaley, James; Vavrova, Ludmila; Wilson, Keith S.; Barak, Imrich; Hart, Darren J.; Wilkinson, Anthony J.

    2010-01-01

    SpoIIE is a dual function protein that plays important roles during sporulation in Bacillus subtilis. It binds to the tubulin-like protein FtsZ causing the cell division septum to relocate from mid-cell to the cell pole, and it dephosphorylates SpoIIAA phosphate leading to establishment of differential gene expression in the two compartments following the asymmetric septation. Its 872 residue polypeptide contains a multiple-membrane spanning sequence at the N-terminus and a PP2C phosphatase domain at the C-terminus. The central segment that binds to FtsZ is unlike domains of known structure or function, moreover the domain boundaries are poorly defined and this has hampered the expression of soluble fragments of SpoIIE at the levels required for structural studies. Here we have screened over 9000 genetic constructs of spoIIE using a random incremental truncation library approach, ESPRIT, to identify a number of soluble C-terminal fragments of SpoIIE that were aligned with the protein sequence to map putative domains and domain boundaries. The expression and purification of three fragments were optimised, yielding multimilligram quantities of the PP2C phosphatase domain, the putative FtsZ-binding domain and a larger fragment encompassing both these domains. All three fragments are monomeric and the PP2C domain-containing fragments have phosphatase activity. PMID:20817757

  5. Hydrogen peroxide stimulates ubiquitin-conjugating activity and expression of genes for specific E2 and E3 proteins in skeletal muscle myotubes

    NASA Technical Reports Server (NTRS)

    Li, Yi-Ping; Chen, Yuling; Li, Andrew S.; Reid, Michael B.

    2003-01-01

    Reactive oxygen species (ROS) are thought to promote muscle atrophy in chronic wasting diseases, but the underlying mechanism has not been determined. Here we show that H2O2 stimulates ubiquitin conjugation to muscle proteins through transcriptional regulation of the enzymes (E2 and E3 proteins) that conjugate ubiquitin to muscle proteins. Incubation of C2C12 myotubes with 100 microM H2O2 increased the rate of 125I-labeled ubiquitin conjugation to muscle proteins in whole cell extracts. This response required at least 4-h exposure to H2O2 and persisted for at least 24 h. Preincubating myotubes with cycloheximide or actinomycin D blocked H2O2 stimulation of ubiquitin-conjugating activity, suggesting that gene transcription is required. Northern blot analyses revealed that H2O2 upregulates expression of specific E3 and E2 proteins that are thought to regulate muscle catabolism, including atrogin1/MAFbx, MuRF1, and E214k. These results suggest that ROS stimulate protein catabolism in skeletal muscle by upregulating the ubiquitin conjugation system.

  6. Angiotensin II-induced protein kinase D activates the ATF/CREB family of transcription factors and promotes StAR mRNA expression.

    PubMed

    Olala, Lawrence O; Choudhary, Vivek; Johnson, Maribeth H; Bollag, Wendy B

    2014-07-01

    Aldosterone synthesis is initiated upon the transport of cholesterol from the outer to the inner mitochondrial membrane, where the cholesterol is hydrolyzed to pregnenolone. This process is the rate-limiting step in acute aldosterone production and is mediated by the steroidogenic acute regulatory (StAR) protein. We have previously shown that angiotensin II (AngII) activation of the serine/threonine protein kinase D (PKD) promotes acute aldosterone production in bovine adrenal glomerulosa cells, but the mechanism remains unclear. Thus, the purpose of this study was to determine the downstream signaling effectors of AngII-stimulated PKD activity. Our results demonstrate that overexpression of the constitutively active serine-to-glutamate PKD mutant enhances, whereas the dominant-negative serine-to-alanine PKD mutant inhibits, AngII-induced StAR mRNA expression relative to the vector control. PKD has been shown to phosphorylate members of the activating transcription factor (ATF)/cAMP response element binding protein (CREB) family of leucine zipper transcription factors, which have been shown previously to bind the StAR proximal promoter and induce StAR mRNA expression. In primary glomerulosa cells, AngII induces ATF-2 and CREB phosphorylation in a time-dependent manner. Furthermore, overexpression of the constitutively active PKD mutant enhances the AngII-elicited phosphorylation of ATF-2 and CREB, and the dominant-negative mutant inhibits this response. Furthermore, the constitutively active PKD mutant increases the binding of phosphorylated CREB to the StAR promoter. Thus, these data provide insight into the previously reported role of PKD in AngII-induced acute aldosterone production, providing a mechanism by which PKD may be mediating steroidogenesis in primary bovine adrenal glomerulosa cells.

  7. Fraxetin Induces Heme Oxygenase-1 Expression by Activation of Akt/Nrf2 or AMP-activated Protein Kinase α/Nrf2 Pathway in HaCaT Cells

    PubMed Central

    Kundu, Juthika; Chae, In Gyeong; Chun, Kyung-Soo

    2016-01-01

    Background Fraxetin (7,8-dihydroxy-6-methoxy coumarin), a coumarin derivative, has been reported to possess antioxidative, anti-inflammatory and neuroprotective effects. A number of recent observations suggest that the induction of heme oxygenase-1 (HO-1) inhibits inflammation and tumorigenesis. In the present study, we determined the effect of fraxetin on HO-1 expression in HaCaT human keratinocytes and investigated its underlying molecular mechanisms. Methods Reverse transcriptase-PCR and Western blot analysis were performed to detect HO-1 mRNA and protein expression, respectively. Cell viability was measured by the MTS test. The induction of intracellular reactive oxygen species (ROS) by fraxetin was evaluated by 2′,7′-dichlorofluorescin diacetate staining. Results Fraxetin upregulated mRNA and protein expression of HO-1. Incubation with fraxetin induced the localization of nuclear factor-erythroid-2-related factor-2 (Nrf2) in the nucleus and increased the antioxidant response element-reporter gene activity. Fraxetin also induced the phosphorylation of Akt and AMP-activated protein kinase (AMPK)α and diminished the expression of phosphatase and tensin homolog, a negative regulator of Akt. Pharmacological inhibition of Akt and AMPKα abrogated fraxetin-induced expression of HO-1 and nuclear localization of Nrf2. Furthermore, fraxetin generated ROS in a concentration-dependent manner. Conclusions Fraxetin induces HO-1 expression through activation of Akt/Nrf2 or AMPKα/Nrf2 pathway in HaCaT cells. PMID:27722139

  8. Parathyroid hormone-related protein inhibits DKK1 expression through c-Jun-mediated inhibition of β-catenin activation of the DKK1 promoter in prostate cancer.

    PubMed

    Zhang, H; Yu, C; Dai, J; Keller, J M; Hua, A; Sottnik, J L; Shelley, G; Hall, C L; Park, S I; Yao, Z; Zhang, J; McCauley, L K; Keller, E T

    2014-05-08

    Prostate cancer (PCa)bone metastases are unique in that majority of them induce excessive mineralized bone matrix, through undefined mechanisms, as opposed to most other cancers that induce bone resorption. Parathyroid hormone-related protein (PTHrP) is produced by PCa cells and intermittent PTHrP exposure has bone anabolic effects, suggesting that PTHrP could contribute to the excess bone mineralization. Wnts are bone-productive factors produced by PCa cells, and the Wnt inhibitor Dickkopfs-1 (DKK1) has been shown to promote PCa progression. These findings, in conjunction with the observation that PTHrP expression increases and DKK1 expression decreases as PCa progresses, led to the hypothesis that PTHrP could be a negative regulator of DKK1 expression in PCa cells and, hence, allow the osteoblastic activity of Wnts to be realized. To test this, we first demonstrated that PTHrP downregulated DKK1 mRNA and protein expression. We then found through multiple mutated DKK1 promoter assays that PTHrP, through c-Jun activation, downregulated the DKK1 promoter through a transcription factor (TCF) response element site. Furthermore, chromatin immunoprecipitation (ChIP) and re-ChIP assays revealed that PTHrP mediated this effect through inducing c-Jun to bind to a transcriptional activator complex consisting of β-catenin, which binds the most proximal DKK1 promoter, the TCF response element. Together, these results demonstrate a novel signaling linkage between PTHrP and Wnt signaling pathways that results in downregulation of a Wnt inhibitor allowing for Wnt activity that could contribute the osteoblastic nature of PCa.

  9. Parathyroid hormone-related protein inhibits DKK1 expression through c-Jun-mediated inhibition of β-Catenin activation of the DKK1 promoter in prostate cancer

    PubMed Central

    Zhang, H.; Yu, C.; Dai, J.; Keller, JM.; Hua, A.; Sottnik, JL.; Shelley, G.; Hall, CL.; Park, SI.; Yao, Z.; Zhang, J.; McCauley, LK.; Keller, ET.

    2014-01-01

    Prostate cancer bone metastases are unique in that that majority of them induce excessive mineralized bone matrix, through undefined mechanisms, as opposed to most other cancers that induce bone resorption. Parathyroid hormone-related protein (PTHrP) is produced by prostate cancer cells and intermittent PTHrP exposure has bone anabolic effects suggesting PTHrP could contribute to the excess bone mineralization. Wnts are bone productive factors produced by prostate cancer cells and the Wnt inhibitor DKK1 has been shown to promote prostate cancer progression. These findings, in conjunction with the observation that PTHrP expression increases and DKK1 expression decreases as prostate cancer progresses led to the hypothesis that PTHrP could be a negative regulator of DKK1 expression in prostate cancer cells, and hence allow the osteoblastic activity of Wnts to be realized. To test this, we first demonstrated that PTHrP downregulated DKK1 mRNA and protein expression. We then found through multiple mutated DKK1 promoter assays that PTHrP, through c-Jun activation, downregulated the DKK1 promoter through a TCF-response element site. Furthermore, chromatin immunoprecipitation (ChIP) and reChIP assays revealed that PTHrP-mediated this effect through inducing c-Jun to bind to a transcriptional activator complex consisting of β-catenin that binds the most proximal DKK1 promoter TCF-response element. Together, these results demonstrate a novel signaling linkage between PTHrP and Wnt signaling pathways that results in downregulation of a Wnt inhibitor allowing for Wnt activity that could contribute the osteoblastic nature of prostate cancer. PMID:23752183

  10. Alpha1-chimaerin, a Rac1 GTPase-activating protein, is expressed at reduced mRNA levels in the brain of Alzheimer's disease patients

    PubMed Central

    Kato, Tomoko; Konishi, Yoshihiro; Shimohama, Shun; Beach, Thomas G.; Akatsu, Hiroyasu; Tooyama, Ikuo

    2015-01-01

    Alpha1-chimaerin is a GTPase-activating protein (GAP) for Rac1, a member of the Rho small GTPase family, whose action leads to the inactivation of Rac1. Rac1 activity is upregulated in Alzheimer's disease, but little is known about the role of α1-chimaerin. In this study, we investigated the expression and localization of α1-chimaerin mRNA in postmortem human brains from patients with Alzheimer's disease and control subjects. In situ hybridization studies demonstrated that α1-chimaerin was expressed by neurons in the neo-cortex of the temporal lobe and the hippocampus of both controls and Alzheimer's disease cases, with the signal intensity dramatically decreased in patients with Alzheimer's disease. Real-time PCR analysis confirmed a significant reduction of α1-chimaerin mRNA expression in the temporal cortex of Alzheimer's disease cases. In contrast, α2-chimaerin mRNA levels showed no significant difference between the groups. The present study showed reduced α1-chimaerin expression in the brain of Alzheimer's disease cases, suggesting a role in the upregulation of Rac1 activity during the disease process. PMID:25676811

  11. C-terminal binding protein (CtBP) activates the expression of E-box clock genes with CLOCK/CYCLE in Drosophila.

    PubMed

    Itoh, Taichi Q; Matsumoto, Akira; Tanimura, Teiichi

    2013-01-01

    In Drosophila, CLOCK/CYCLE heterodimer (CLK/CYC) is the primary activator of circadian clock genes that contain the E-box sequence in their promoter regions (hereafter referred to as "E-box clock genes"). Although extensive studies have investigated the feedback regulation of clock genes, little is known regarding other factors acting with CLK/CYC. Here we show that Drosophila C-terminal binding protein (dCtBP), a transcriptional co-factor, is involved in the regulation of the E-box clock genes. In vivo overexpression of dCtBP in clock cells lengthened or abolished circadian locomotor rhythm with up-regulation of a subset of the E-box clock genes, period (per), vrille (vri), and PAR domain protein 1ε (Pdp1ε). Co-expression of dCtBP with CLK in vitro also increased the promoter activity of per, vri, Pdp1ε and cwo depending on the amount of dCtBP expression, whereas no effect was observed without CLK. The activation of these clock genes in vitro was not observed when we used mutated dCtBP which carries amino acid substitutions in NAD+ domain. These results suggest that dCtBP generally acts as a putative co-activator of CLK/CYC through the E-box sequence.

  12. Piperine activates human pregnane X receptor to induce the expression of cytochrome P450 3A4 and multidrug resistance protein 1

    SciTech Connect

    Wang, Yue-Ming; Lin, Wenwei; Chai, Sergio C.; Wu, Jing; Ong, Su Sien; Schuetz, Erin G.; Chen, Taosheng

    2013-10-01

    Activation of the pregnane X receptor (PXR) and subsequently its target genes, including those encoding drug transporters and metabolizing enzymes, while playing substantial roles in xenobiotic detoxification, might cause undesired drug-drug interactions. Recently, an increased awareness has been given to dietary components for potential induction of diet–drug interactions through activation of PXR. Here, we studied, whether piperine (PIP), a major component extracted from the widely-used daily spice black pepper, could induce PXR-mediated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1). Our results showed that PIP activated human PXR (hPXR)-mediated CYP3A4 and MDR1 expression in human hepatocytes, intestine cells, and a mouse model; PIP activated hPXR by recruiting its coactivator SRC-1 in both cellular and cell-free systems; PIP bound to the hPXR ligand binding domain in a competitive ligand binding assay in vitro. The dichotomous effects of PIP on induction of CYP3A4 and MDR1 expression observed here and inhibition of their activity reported elsewhere challenges the potential use of PIP as a bioavailability enhancer and suggests that caution should be taken in PIP consumption during drug treatment in patients, particularly those who favor daily pepper spice or rely on certain pepper remedies. - Highlights: • Piperine induces PXR-mediated CYP3A4 and MDR1 expression. • Piperine activates PXR by binding to PXR and recruiting coactivator SRC-1. • Piperine induces PXR activation in vivo. • Caution should be taken in piperine consumption during drug treatment.

  13. Engineering the cellular protein secretory pathway for enhancement of recombinant tissue plasminogen activator expression in Chinese hamster ovary cells: effects of CERT and XBP1s genes.

    PubMed

    Rahimpour, Azam; Vaziri, Behrouz; Moazzami, Reza; Nematollahi, Leila; Barkhordari, Farzaneh; Kokabee, Leila; Adeli, Ahmad; Mahboudi, Fereidoun

    2013-08-01

    Cell line development is the most critical and also the most time-consuming step in the production of recombinant therapeutic proteins. In this regard, a variety of vector and cell engineering strategies have been developed for generating high-producing mammalian cells; however, the cell line engineering approach seems to show various results on different recombinant protein producer cells. In order to improve the secretory capacity of a recombinant tissue plasminogen activator (t-PA)-producing Chinese hamster ovary (CHO) cell line, we developed cell line engineering approaches based on the ceramide transfer protein (CERT) and X-box binding protein 1 (XBP1) genes. For this purpose, CERT S132A, a mutant form of CERT that is resistant to phosphorylation, and XBP1s were overexpressed in a recombinant t-PA-producing CHO cell line. Overexpression of CERT S132A increased the specific productivity of t-PA-producing CHO cells up to 35%. In contrast, the heterologous expression of XBP1s did not affect the t-PA expression rate. Our results suggest that CERTS132A- based secretion engineering could be an effective strategy for enhancing recombinant t- PA production in CHO cells.

  14. Expression of a soluble form of iodotyrosine deiodinase for active site characterization by engineering the native membrane protein from Mus musculus

    SciTech Connect

    Buss, Jennifer M.; McTamney, Patrick M.; Rokita, Steven E.

    2012-06-27

    Reductive deiodination is critical for thyroid function and represents an unusual exception to the more common oxidative and hydrolytic mechanisms of dehalogenation in mammals. Studies on the reductive processes have been limited by a lack of convenient methods for heterologous expression of the appropriate proteins in large scale. The enzyme responsible for iodide salvage in the thyroid, iodotyrosine deodinase, is now readily generated after engineering its gene from Mus musculus. High expression of a truncated derivative lacking the membrane domain at its N-terminal was observed in Sf9 cells, whereas expression in Pichia pastoris remained low despite codon optimization. Ultimately, the desired expression in Escherichia coli was achieved after replacing the two conserved Cys residues of the deiodinase with Ala and fusing the resulting protein to thioredoxin. This final construct provided abundant enzyme for crystallography and mutagenesis. Utility of the E. coli system was demonstrated by examining a set of active site residues critical for binding to the zwitterionic portion of substrate.

  15. A milk protein gene promoter directs the expression of human tissue plasminogen activator cDNA to the mammary gland in transgenic mice

    SciTech Connect

    Pittius, C.W.; Hennighausen, L.; Lee, E.; Westphal, H.; Nicols, E.; Vitale, J.; Gordon, K. )

    1988-08-01

    Whey acidic protein (WAP) is a major whey protein in mouse milk. Its gene is expressed in the lactating mammary gland and is inducible by steroid and peptide hormones. A series of transgenic mice containing a hybrid gene in which human tissue plasminogen activator (tPA) cDNA is under the control of the murine WAP gene promoter had previously been generated. In this study, 21 tissues from lactating and virgin transgenic female mice containing the WAP-tPA hybrid gene were screened for the distribution of murine WAP and human tPA transcripts. Like the endogenous WAP RNA, WAP-tPA RNA was expressed predominantly in mammary gland tissue and appeared to be inducible by lactation. Whereas WAP transcripts were not detected in 22 tissues of virgin mice, low levels of WAP-tPA RNA, which were not modulated during lactation, were found in tongue, kidney, and sublingual gland. These studies demonstrate that the WAP gene promoter can target the expression of a transgene to the mammary gland and that this expression is inducible during lactation.

  16. Loss of the repressor REST in uterine fibroids promotes aberrant G protein-coupled receptor 10 expression and activates mammalian target of rapamycin pathway

    PubMed Central

    Varghese, Binny V.; Koohestani, Faezeh; McWilliams, Michelle; Colvin, Arlene; Gunewardena, Sumedha; Kinsey, William H.; Nowak, Romana A.; Nothnick, Warren B.; Chennathukuzhi, Vargheese M.

    2013-01-01

    Uterine fibroids (leiomyomas) are the most common tumors of the female reproductive tract, occurring in up to 77% of reproductive-aged women, yet molecular pathogenesis remains poorly understood. A role for atypically activated mammalian target of rapamycin (mTOR) pathway in the pathogenesis of uterine fibroids has been suggested in several studies. We identified that G protein-coupled receptor 10 [GPR10, a putative signaling protein upstream of the phosphoinositide 3-kinase–protein kinase B/AKT–mammalian target of rapamycin (PI3K/AKT–mTOR) pathway] is aberrantly expressed in uterine fibroids. The activation of GPR10 by its cognate ligand, prolactin releasing peptide, promotes PI3K–AKT–mTOR pathways and cell proliferation specifically in cultured primary leiomyoma cells. Additionally, we report that RE1 suppressing transcription factor/neuron-restrictive silencing factor (REST/NRSF), a known tumor suppressor, transcriptionally represses GPR10 in the normal myometrium, and that the loss of REST in fibroids permits GPR10 expression. Importantly, mice overexpressing human GPR10 in the myometrium develop myometrial hyperplasia with excessive extracellular matrix deposition, a hallmark of uterine fibroids. We demonstrate previously unrecognized roles for GPR10 and its upstream regulator REST in the pathogenesis of uterine fibroids. Importantly, we report a unique genetically modified mouse model for a gene that is misexpressed in uterine fibroids. PMID:23284171

  17. A Saccharomyces Cerevisiae Rad52 Allele Expressing a C-Terminal Truncation Protein: Activities and Intragenic Complementation of Missense Mutations

    PubMed Central

    Boundy-Mills, K. L.; Livingston, D. M.

    1993-01-01

    A nonsense allele of the yeast RAD52 gene, rad52-327, which expresses the N-terminal 65% of the protein was compared to two missense alleles, rad52-1 and rad52-2, and to a deletion allele. While the rad52-1 and the deletion mutants have severe defects in DNA repair, recombination and sporulation, the rad52-327 and rad52-2 mutants retain either partial or complete capabilities in repair and recombination. These two mutants behave similarly in most tests of repair and recombination during mitotic growth. One difference between these two alleles is that a homozygous rad52-2 diploid fails to sporulate, whereas the homozygous rad52-327 diploid sporulates weakly. The low level of sporulation by the rad52-327 diploid is accompanied by a low percentage of spore viability. Among these viable spores the frequency of crossing over for markers along chromosome VII is the same as that found in wild-type spores. rad52-327 complements rad52-2 for repair and sporulation. Weaker intragenic complementation occurs between rad52-327 and rad52-1. PMID:8417987

  18. Streamlined expressed protein ligation using split inteins.

    PubMed

    Vila-Perelló, Miquel; Liu, Zhihua; Shah, Neel H; Willis, John A; Idoyaga, Juliana; Muir, Tom W

    2013-01-09

    Chemically modified proteins are invaluable tools for studying the molecular details of biological processes, and they also hold great potential as new therapeutic agents. Several methods have been developed for the site-specific modification of proteins, one of the most widely used being expressed protein ligation (EPL) in which a recombinant α-thioester is ligated to an N-terminal Cys-containing peptide. Despite the widespread use of EPL, the generation and isolation of the required recombinant protein α-thioesters remain challenging. We describe here a new method for the preparation and purification of recombinant protein α-thioesters using engineered versions of naturally split DnaE inteins. This family of autoprocessing enzymes is closely related to the inteins currently used for protein α-thioester generation, but they feature faster kinetics and are split into two inactive polypeptides that need to associate to become active. Taking advantage of the strong affinity between the two split intein fragments, we devised a streamlined procedure for the purification and generation of protein α-thioesters from cell lysates and applied this strategy for the semisynthesis of a variety of proteins including an acetylated histone and a site-specifically modified monoclonal antibody.

  19. Herpes simplex virus type 1 ICP0 protein mediates activation of adeno-associated virus type 2 rep gene expression from a latent integrated form.

    PubMed

    Geoffroy, Marie-Claude; Epstein, Alberto L; Toublanc, Estelle; Moullier, Philippe; Salvetti, Anna

    2004-10-01

    Adeno-associated virus type 2 (AAV-2) is a human parvovirus that requires the presence of a helper virus, such as the herpes simplex virus type 1 (HSV-1) to accomplish a complete productive cycle. In the absence of helper virus, AAV-2 can establish a latent infection that is characterized by the absence of expression of viral genes. So far, four HSV-1 early genes, UL5/8/52 (helicase primase complex) and UL29 (single-stranded DNA-binding protein), were defined as sufficient for AAV replication when cells were transfected with a plasmid carrying the wild-type AAV-2 genome. However, none of these viral products was shown to behave as a transcriptional factor able to activate AAV gene expression. Our study provides the first evidence that the immediate-early HSV-1 protein ICP0 can promote rep gene expression in cells latently infected with wild-type AAV-2. This ICP0-mediated effect occurs at the transcriptional level and involves the ubiquitin-proteasome pathway. Furthermore, using deletion mutants, we demonstrate that the localization of ICP0 to ND10 and their disruption is not required for the activation of the rep promoter, whereas binding of ICP0 to the ubiquitin-specific protease HAUSP makes a significant contribution to this effect.

  20. Pb2+ induces gastrin gene expression by extracellular signal-regulated kinases 1/2 and transcription factor activator protein 1 in human gastric carcinoma cells.

    PubMed

    Chan, Chien-Pin; Tsai, Yao-Ting; Chen, Yao-Li; Hsu, Yu-Wen; Tseng, Joseph T; Chuang, Hung-Yi; Shiurba, Robert; Lee, Mei-Hsien; Wang, Jaw-Yuan; Chang, Wei-Chiao

    2015-02-01

    Divalent lead ions (Pb(2+) ) are toxic environmental pollutants known to cause serious health problems in humans and animals. Absorption of Pb(2+) from air, water, and food takes place in the respiratory and digestive tracts. The ways in which absorbed Pb(2+) affects cell physiology are just beginning to be understood at the molecular level. Here, we used reverse transcription PCR and Western blotting to analyze cultures of human gastric carcinoma cells exposed to 10 μM lead nitrate. We found that Pb(2+) induces gastrin hormone gene transcription and translation in a time-dependent manner. Promoter deletion analysis revealed that activator protein 1 (AP1) was necessary for gastrin gene transcription in cells exposed to Pb(2+) . MitogIen-activated protein kinase (MAPK)/ERK kinase inhibitor PD98059 suppressed the Pb(2+) -induced increase in messenger RNA. Epidermal growth factor receptor (EGFR) inhibitors AG1478 and PD153035 reduced both transcription and phosphorylation by extracellular signal-regulated kinase (ERK1/2). Cells exposed to Pb(2+) also increased production of c-Jun protein, a component of AP1, and over-expression of c-Jun enhanced activation of the gastrin promoter. In sum, the findings suggest the EGFR-ERK1/2-AP1 pathway mediates the effects of Pb(2+) on gastrin gene activity in cell culture.

  1. Upregulation of AKT1 protein expression in forskolin-stimulated macrophage: evidence from ChIP analysis that CREB binds to and activates the AKT1 promoter.

    PubMed

    Misra, Uma Kant; Pizzo, Salvatore Vincent

    2007-03-01

    Recently, we reported that silencing CREB gene expression by RNAi significantly attenuates forskolin-induced activation of Akt1. We now provide evidence that forskolin-treatment causes transcriptional and translational upregulation of Akt1 in macrophages. Akt synthesis was demonstrated by [(14)C]leucine or [(35)S] incorporation into newly synthesized Akt1 protein. Akt protein levels increased by approximately 1.5-fold after only a 5 min exposure of macrophages to forskolin. Akt1 levels thereafter rapidly returned to basal values (t(1/2) approximately 15 min). Maximal upregulation of Akt1 occurred in cells treated with 10 microM forskolin. Forskolin-dependent Akt1 synthesis was abolished by pretreating the cells with CREB-directed dsRNA as demonstrated at both the message and protein level, as well as by determining the synthesis of [(35)S]-labeled Akt1 protein. The PKA inhibitor H-89, greatly attenuated forskolin-induced Akt1 synthesis. Transcriptional and translational inhibitors also greatly reduced Akt1 synthesis in forskolin-stimulated [(14)C]leucine-labeled macrophages. Using a chromatin immunoprecipitation assay, we demonstrate that CREB binds to a CRE binding domain of the Akt1 gene promoter. In conclusion, we show here for the first time transcriptional upregulation of Akt1 by CREB, based upon Akt1 protein synthesis and its modulation by transitional and translational inhibitors in forskolin-stimulated cells, Akt1 protein. and mRNA levels upon silencing CREB gene expression, and binding of CREB to the Akt1 gene promoter.

  2. Transgenic tobacco plants expressing siRNA targeted against the Mungbean yellow mosaic virus transcriptional activator protein gene efficiently block the viral DNA accumulation.

    PubMed

    Shanmugapriya, Gnanasekaran; Das, Sudhanshu Sekhar; Veluthambi, Karuppannan

    2015-06-01

    Mungbean yellow mosaic virus (MYMV) is a bipartite begomovirus that infects many pulse crops such as blackgram, mungbean, mothbean, Frenchbean, and soybean. We tested the efficacy of the transgenically expressed intron-spliced hairpin RNA gene of the transcriptional activator protein (hpTrAP) in reducing MYMV DNA accumulation. Tobacco plants transformed with the MYMV hpTrAP gene accumulated 21-22 nt siRNA. Leaf discs of the transgenic plants, agroinoculated with the partial dimers of MYMV, displayed pronounced reduction in MYMV DNA accumulation. Thus, silencing of the TrAP gene, a suppressor of gene silencing, emerged as an effective strategy to control MYMV.

  3. Dual Activation of TRIF and MyD88 Adaptor Proteins by Angiotensin II Evokes Opposing Effects on Pressure, Cardiac Hypertrophy and Inflammatory Gene Expression

    PubMed Central

    Singh, Madhu V.; Cicha, Michael Z.; Meyerholz, David K.; Chapleau, Mark W.; Abboud, François M.

    2015-01-01

    Hypertension is recognized as an immune disorder whereby immune cells play a defining role in the genesis and progression of the disease. The innate immune system and its component toll-like receptors (TLRs) are key determinants of the immunological outcome through their pro-inflammatory response. TLR activated signaling pathways utilize several adaptor proteins of which adaptor proteins MyD88 and TRIF define two major inflammatory pathways. In this study, we compared the contributions of MyD88 and TRIF adaptor proteins to angiotensin II (Ang II)-induced hypertension and cardiac hypertrophy in mice. Deletion of MyD88 did not prevent cardiac hypertrophy and the pressor response to Ang II tended to increase. Moreover, the increase in inflammatory gene expression (Tnfa, Nox4 and Agtr1a) was significantly greater in the heart and kidney of MyD88-deficient mice compared with wild type mice. Thus, pathways involving MyD88 may actually restrain the inflammatory responses. On the other hand, in mice with non-functional TRIF (Trifmut mice), Ang II induced hypertension and cardiac hypertrophy were abrogated, and pro-inflammatory gene expression in heart and kidneys was unchanged or decreased. Our results indicate that Ang II induces activation of a pro-inflammatory innate immune response, causing hypertension, and cardiac hypertrophy. These effects require functional adaptor protein TRIF-mediated pathways. However, the common MyD88 dependent signaling pathway, which is also activated simultaneously by Ang II, paradoxically exerts a negative regulatory influence on these responses. PMID:26195481

  4. Temporal expression of the human alcohol dehydrogenase gene family during liver development correlates with differential promoter activation by hepatocyte nuclear factor 1, CCAAT/enhancer-binding protein alpha, liver activator protein, and D-element-binding protein.

    PubMed Central

    van Ooij, C; Snyder, R C; Paeper, B W; Duester, G

    1992-01-01

    The human class I alcohol dehydrogenase (ADH) gene family consists of ADH1, ADH2, and ADH3, which are sequentially activated in early fetal, late fetal, and postnatal liver, respectively. Analysis of ADH promoters revealed differential activation by several factors previously shown to control liver transcription. In cotransfection assays, the ADH1 promoter, but not the ADH2 or ADH3 promoter, was shown to respond to hepatocyte nuclear factor 1 (HNF-1), which has previously been shown to regulate transcription in early liver development. The ADH2 promoter, but not the ADH1 or ADH3 promoter, was shown to respond to CCAAT/enhancer-binding protein alpha (C/EBP alpha), a transcription factor particularly active during late fetal liver and early postnatal liver development. The ADH1, ADH2, and ADH3 promoters all responded to the liver transcription factors liver activator protein (LAP) and D-element-binding protein (DBP), which are most active in postnatal liver. For all three promoters, the activation by LAP or DBP was higher than that seen by HNF-1 or C/EBP alpha, and a significant synergism between C/EBP alpha and LAP was noticed for the ADH2 and ADH3 promoters when both factors were simultaneously cotransfected. A hierarchy of ADH promoter responsiveness to C/EBP alpha and LAP homo- and heterodimers is suggested. In all three ADH genes, LAP bound to the same four sites previously reported for C/EBP alpha (i.e., -160, -120, -40, and -20 bp), but DBP bound strongly only to the site located at -40 bp relative to the transcriptional start. Mutational analysis of ADH2 indicated that the -40 bp element accounts for most of the promoter regulation by the bZIP factors analyzed. These studies suggest that HNF-1 and C/EBP alpha help establish ADH gene family transcription in fetal liver and that LAP and DBP help maintain high-level ADH gene family transcription in postnatal liver. Images PMID:1620113

  5. HSP70 binding protein 1 (HspBP1) suppresses HIV-1 replication by inhibiting NF-κB mediated activation of viral gene expression

    PubMed Central

    Chaudhary, Priyanka; Khan, Sohrab Zafar; Rawat, Pratima; Augustine, Tracy; Raynes, Deborah A.; Guerriero, Vince; Mitra, Debashis

    2016-01-01

    HIV-1 efficiently hijacks host cellular machinery and exploits a plethora of host–viral interactions for its successful survival. Identifying host factors that affect susceptibility or resistance to HIV-1 may offer a promising therapeutic strategy against HIV-1. Previously, we have reported that heat shock proteins, HSP40 and HSP70 reciprocally regulate HIV-1 gene-expression and replication. In the present study, we have identified HSP70 binding protein 1 (HspBP1) as a host-intrinsic inhibitor of HIV-1. HspBP1 level was found to be significantly down modulated during HIV-1 infection and virus production inversely co-related with HspBP1 expression. Our results further demonstrate that HspBP1 inhibits HIV-1 long terminal repeat (LTR) promoter activity. Gel shift and chromatin immunoprecipitation assays revealed that HspBP1 was recruited on HIV-1 LTR at NF-κB enhancer region (κB sites). The binding of HspBP1 to κB sites obliterates the binding of NF-κB hetero-dimer (p50/p65) to the same region, leading to repression in NF-κB mediated activation of LTR-driven gene-expression. HspBP1 also plays an inhibitory role in the reactivation of latently infected cells, corroborating its repressive effect on NF-κB pathway. Thus, our results clearly show that HspBP1 acts as an endogenous negative regulator of HIV-1 gene-expression and replication by suppressing NF-κB-mediated activation of viral transcription. PMID:26538602

  6. Combined analysis of eIF4E and 4E-binding protein expression predicts breast cancer survival and estimates eIF4E activity.

    PubMed

    Coleman, L J; Peter, M B; Teall, T J; Brannan, R A; Hanby, A M; Honarpisheh, H; Shaaban, A M; Smith, L; Speirs, V; Verghese, E T; McElwaine, J N; Hughes, T A

    2009-05-05

    Increased eukaryotic translation initiation factor 4E (eIF4E) expression occurs in many cancers, and makes fundamental contributions to carcinogenesis by stimulating the expression of cancer-related genes at post-transcriptional levels. This key role is highlighted by the facts that eIF4E levels can predict prognosis, and that eIF4E is an established therapeutic target. However, eIF4E activity is a complex function of expression levels and phosphorylation statuses of eIF4E and eIF4E-binding proteins (4E-BPs). Our hypothesis was that the combined analyses of these pathway components would allow insights into eIF4E activity and its influence on cancer. We have determined expression levels of eIF4E, 4E-BP1, 4E-BP2 and phosphorylated 4E-BP1 within 424 breast tumours, and have carried out analyses to combine these and relate the product to patient survival, in order to estimate eIF4E activity. We show that this analysis gives greater prognostic insights than that of eIF4E alone. We show that eIF4E and 4E-BP expression are positively associated, and that 4E-BP2 has a stronger influence on cancer behaviour than 4E-BP1. Finally, we examine eIF4E, estimated eIF4E activity, and phosphorylated 4E-BP1 as potential predictive biomarkers for eIF4E-targeted therapies, and show that each determines selection of different patient groups. We conclude that eIF4E's influence on cancer survival is modulated substantially by 4E-BPs, and that combined pathway analyses can estimate functional eIF4E.

  7. Susceptibility of juvenile and adult blood–brain barrier to endothelin-1: regulation of P-glycoprotein and breast cancer resistance protein expression and transport activity

    PubMed Central

    2012-01-01

    Background P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) play a critical role in keeping neurotoxic substances from entering the brain. We and others have previously reported an impact of inflammation on the regulation of adult blood–brain barrier (BBB) efflux transporters. However, studies in children have not been done. From the pediatric clinical perspective, it is important to understand how the central nervous system (CNS) and BBB drug efflux transporters differ in childhood from those of adults under normal and inflammatory conditions. Therefore, we examined and compared the regulation of P-gp and BCRP expression and transport activity in young and adult BBB and investigated the molecular mechanisms underlying inflammatory responses. Methods Rats at postnatal day (P) P21 and P84, corresponding to the juvenile and adult stages of human brain maturation, respectively, were treated with endothelin-1 (ET-1) given by the intracerebroventricular (icv) route. Twenty-four hours later, we measured P-gp and BCRP protein expression in isolated brain capillary by immunoblotting as well as by transport activity in vivo by measuring the unbound drug partitioning coefficient of the brain (Kp,uu,brain) of known efflux transporter substrates administered intravenously. Glial activation was measured by immunohistochemistry. The release of cytokines/chemokines (interleukins-1α, 1-β (IL-1β), -6 (IL-6), -10 (IL-10), monocyte chemoattractant protein (MCP-1/CCL2), fractalkine and tissue inhibitor of metalloproteinases-1 (TIMP-1)) were simultaneously measured in brain and serum samples using the Agilent Technology cytokine microarray. Results We found that juvenile and adult BBBs exhibited similar P-gp and BCRP transport activities in the normal physiological conditions. However, long-term exposure of the juvenile brain to low-dose of ET-1 did not change BBB P-gp transport activity but tended to decrease BCRP transport activity in the juvenile brain, while a

  8. Expression of human papilloma virus type 16 E5 protein in amelanotic melanoma cells regulates endo-cellular pH and restores tyrosinase activity

    PubMed Central

    Di Domenico, Fabio; Foppoli, Cesira; Blarzino, Carla; Perluigi, Marzia; Paolini, Francesca; Morici, Salvatrice; Coccia, Raffaella; Cini, Chiara; De Marco, Federico

    2009-01-01

    Background Melanin synthesis, the elective trait of melanocytes, is regulated by tyrosinase activity. In tyrosinase-positive amelanotic melanomas this rate limiting enzyme is inactive because of acidic endo-melanosomal pH. The E5 oncogene of the Human Papillomavirus Type 16 is a small transmembrane protein with a weak transforming activity and a role during the early steps of viral infections. E5 has been shown to interact with 16 kDa subunit C of the trans-membrane Vacuolar ATPase proton pump ultimately resulting in its functional suppressions. However, the cellular effects of such an interaction are still under debate. With this work we intended to explore whether the HPV16 E5 oncoprotein does indeed interact with the vacuolar ATPase proton pump once expressed in intact human cells and whether this interaction has functional consequences on cell metabolism and phenotype. Methods The expression of the HPV16-E5 oncoproteins was induced in two Tyrosinase-positive amelanotic melanomas (the cell lines FRM and M14) by a retroviral expression construct. Modulation of the intracellular pH was measured with Acridine orange and fluorescence microscopy. Expression of tyrosinase and its activity was followed by RT-PCR, Western Blot and enzyme assay. The anchorage-independence growth and the metabolic activity of E5 expressing cells were also monitored. Results We provide evidence that in the E5 expressing cells interaction between E5 and V-ATPase determines an increase of endo-cellular pH. The cellular alkalinisation in turn leads to the post-translational activation of tyrosinase, melanin synthesis and phenotype modulation. These effects are associated with an increased activation of tyrosine analogue anti-blastic drugs. Conclusion Once expressed within intact human cells the HPV16-E5 oncoprotein does actually interact with the vacuolar V-ATPase proton pump and this interaction induces a number of functional effects. In amelanotic melanomas these effects can modulate the

  9. Vitamin D Binding Protein-Macrophage Activating Factor Directly Inhibits Proliferation, Migration, and uPAR Expression of Prostate Cancer Cells

    PubMed Central

    Bielenberg, Diane R.; Dridi, Sami; Wu, Jason; Jiang, Weihua; Huang, Bin; Pirie-Shepherd, Steven; Fannon, Michael

    2010-01-01

    Background Vitamin D binding protein-macrophage activating factor (DBP-maf) is a potent inhibitor of tumor growth. Its activity, however, has been attributed to indirect mechanisms such as boosting the immune response by activating macrophages and inhibiting the blood vessel growth necessary for the growth of tumors. Methods and Findings In this study we show for the first time that DBP-maf exhibits a direct and potent effect on prostate tumor cells in the absence of macrophages. DBP-maf demonstrated inhibitory activity in proliferation studies of both LNCaP and PC3 prostate cancer cell lines as well as metastatic clones of these cells. Flow cytometry studies with annexin V and propidium iodide showed that this inhibitory activity is not due to apoptosis or cell death. DBP-maf also had the ability to inhibit migration of prostate cancer cells in vitro. Finally, DBP-maf was shown to cause a reduction in urokinase plasminogen activator receptor (uPAR) expression in prostate tumor cells. There is evidence that activation of this receptor correlates with tumor metastasis. Conclusions These studies show strong inhibitory activity of DBP-maf on prostate tumor cells independent of its macrophage activation. PMID:20976141

  10. The ENTPD1 promoter polymorphism -860 A > G (rs3814159) is associated with increased gene transcription, protein expression, CD39/NTPDase1 enzymatic activity, and thromboembolism risk.

    PubMed

    Maloney, James P; Branchford, Brian R; Brodsky, Gary L; Cosmic, Maxwell S; Calabrese, David W; Aquilante, Christina L; Maloney, Kelly W; Gonzalez, Joseph R; Zhang, Weiming; Moreau, Kerrie L; Wiggins, Kerri L; Smith, Nicholas L; Broeckel, Ulrich; Di Paola, Jorge

    2017-03-16

    Ectonucleoside triphosphate diphosphohydrolase 1 (NTPDase1) degrades the purines ATP and ADP that are key regulators of inflammation and clotting. We hypothesized that NTPDase1 polymorphisms exist and that they regulate this pathway. We sequenced the ENTPD1 gene (encoding NTPDase1) in 216 subjects then assessed genotypes in 2 cohorts comprising 2213 humans to identify ENTPD1 polymorphisms associated with venous thromboembolism (VTE). The G allele of the intron 1 polymorphism rs3176891 was more common in VTE vs. controls (odds ratio 1.26-1.9); it did not affect RNA splicing, but it was in strong linkage disequilibrium with the G allele of the promoter polymorphism rs3814159, which increased transcriptional activity by 8-fold. Oligonucleotides containing the G allele of this promoter region bound nuclear extracts more avidly. Carriers of rs3176891 G had endothelial cells with increased NTPDase1 activity and protein expression, and had platelets with enhanced aggregation. Thus, the G allele of rs3176891 marks a haplotype associated with increased clotting and platelet aggregation attributable to a promoter variant associated with increased transcription, expression, and activity of NTPDase1. We term this gain-of-function phenotype observed with rs3814159 G "CD39 Denver."-Maloney, J. P., Branchford, B. R., Brodsky, G. L., Cosmic, M. S., Calabrese, D. W., Aquilante, C. L., Maloney, K. W., Gonzalez, J. R., Zhang, W., Moreau, K. L., Wiggins, K. L., Smith, N. L., Broeckel, U., Di Paola, J. The ENTPD1 promoter polymorphism -860 A > G (rs3814159) is associated with increased gene transcription, protein expression, CD39/NTPDase1 enzymatic activity, and thromboembolism risk.

  11. Dexmedetomidine increases the activity of excitatory amino acid transporter type 3 expressed in Xenopus oocytes: the involvement of protein kinase C and phosphatidylinositol 3-kinase.

    PubMed

    Do, Sang-Hwan; Park, Seong-Joo; Shin, Hyun-Jung; Paik, Hye-Sun; Zuo, Zhiyi; Yoon, Hea-Jo; Ryu, Jung-Hee

    2014-09-05

    Dexmedetomidine, an α2 adrenergic agonist, has neuroprotective and anticonvulsant properties in addition to its sedative and anxiolytic effects. We hypothesized that dexmedetomidine would increase the activity of excitatory amino acid transporter type 3 (EAAT3) and that this effect would involve protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K), two protein kinases known to regulate EAAT3 activity. EAAT3 was expressed in Xenopus oocytes by injecting its mRNA. Two-electrode voltage clamping was used to record membrane currents before, during, and after application of 30 μM l-glutamate in the presence of 0.1-30 nM dexmedetomidine. Dexmedetomidine-treated oocytes were also exposed to a PKC activator (phorbol-12-myristate-13-acetate [PMA]), PKC inhibitors (chelerythrine, staurosporine, and calphostin C), and PI3K inhibitors (wortmannin and LY294002) before current measurement. Dexmedetomidine application resulted in a concentration-dependent increase in the EAAT3 activity in response to l-glutamate. The kinetic study showed that dexmedetomidine significantly increased the Vmax without changing Km. Treatment of oocytes with PMA significantly increased transporter currents compared with controls, but treatment with dexmedetomidine plus PMA did not further increase the response compared with PMA or dexmedetomidine alone. In addition, pre-treatment of oocytes with PKC inhibitors and PI3K inhibitors significantly abolished the dexmedetomidine-enhanced EAAT3 activity. These results suggest that dexmedetomidine increases the activity of EAAT3 expressed in Xenopus oocytes. PKC and PI3K seem to mediate this effect. These findings may explain the neuroprotective and anticonvulsant effects of dexmedetomidine.

  12. Molecular characterization and expression of AMP-activated protein kinase in response to low-salinity stress in the Pacific white shrimp Litopenaeus vannamei.

    PubMed

    Xu, Chang; Li, Erchao; Xu, Zhixin; Wang, Shifeng; Chen, Ke; Wang, Xiaodan; Li, Tongyu; Qin, Jian G; Chen, Liqiao

    2016-08-01

    AMP-activated protein kinase (AMPK) serves as a major regulator of cellular energy metabolism by activating ATP production pathways and blocking ATP consumption. However, information on AMPK genes in aquatic animals is limited. In this study, three subunits of AMPK were cloned from the Pacific white shrimp Litopenaeus vannamei. The full-length cDNAs of the α, β and γ subunits were 1617, 1243 and 3467bp long, respectively, with open reading frames of 1566, 873 and 2988bp encoding for 521, 290 and 996 amino acids, respectively. Amino acid sequence alignments of the three subunits showed that the functional domains in the L. vannamei proteins retained the highest similarity with those of other animals, at 89%, 58%, and 75%, respectively. The expression levels of the three subunits were higher in the muscle and gills than in the eyestalk and hepatopancreas. The mRNA levels of AMPK-α and AMPK-β were up-regulated in the hepatopancreas and muscle after acute low-salinity stress at 3psu for 6h compared with control salinity at 20psu. After 8-week salinity stress at 3psu, AMPK-α and AMPK-β mRNA levels in the hepatopancreas were significantly higher than those of the control at 30psu. However, in the muscle only AMPK-γ mRNA was significantly up-regulated at low salinity relative to controls. Muscle and hepatopancreas showed increases in AMPK protein after 6h exposure to low salinity, but there were no differences seen after long term acclimation. The change patterns of protein were slightly differing from the mRNA patterns due to the distinguishing function of individual subunits of AMPK. These findings confirm that three AMPK subunits are present in L. vannamei and that all encode proteins with conserved functional domains. The three AMPK subunits are all regulated at the transcriptional and protein levels to manage excess energy expenditure during salinity stress.

  13. Production of a urokinase plasminogen activator-IgG fusion protein (uPA-IgG) in the baculovirus expression system.

    PubMed

    Kost, T A; Ignar, D M; Clay, W C; Andrews, J; Leray, J D; Overton, L; Hoffman, C R; Kilpatrick, K E; Ellis, B; Emerson, D L

    1997-04-29

    Numerous studies have demonstrated the importance of urokinase plasminogen activator (uPA) and its receptor, uPAR, in the processes of tumor progression and metastasis. Thus, the uPA/uPAR interaction may represent an important target for inhibiting metastatic disease. The baculovirus expression system was used to produce high levels of a secreted uPA-Immunoglobulin G fusion protein (uPA-IgG) which could then be used for displacing uPA from the surface of tumor cells. The recombinant uPA-IgG fusion protein was placed under the control of either the viral polyhedrin promoter or a copy of the viral basic protein promoter. Recombinant viruses were then used to infect Sf9 and BTI-Tn-5B1-4 cells. Infection of both cell types resulted in the production of secreted uPA-IgG. The molecular mass of the secreted protein as determined by SDS-PAGE was approximately 40 kDa. The highest level of secreted uPA-IgG, 444 microg/ml, was found in the culture medium of BTI-Tn-5B1-4 cells 72 h post-infection with the basic protein promoter-uPA-IgG virus. In the case of Sf9 cells, the highest level of secreted protein was 195 microg/ml. The amount of cell-associated uPA-IgG in infected BTI-Tn-5B1-4 cells was significantly less than that of infected Sf9 cells, reflecting the superior secretory capability of the BTI-Tn-5B1-4 cells. The uPA-IgG was readily purified using a combination of zinc chelate and sephacryl S-100 column chromatography. Routinely, greater than 100 mg of greater than 95% pure protein could be obtained per liter of culture medium collected at 72 h post-infection of BTI-Tn-5B1-4 cells with the basic protein promoter virus. BIAcore analysis and competition binding assays using LOX human malignant melanoma cells expressing uPAR indicated that the purified recombinant protein possessed similar ligand binding characteristics to that of human uPA.

  14. Expression of nuclear factor of activated T cells (NFAT) and downstream muscle-specific proteins in ground squirrel skeletal and heart muscle during hibernation.

    PubMed

    Zhang, Yichi; Storey, Kenneth B

    2016-01-01

    The thirteen-lined ground squirrel (Ictidomys tridecemlineatus) undergoes remarkable adaptive changes during hibernation. Interestingly, skeletal muscle remodelling occurs during the torpor-arousal cycle of hibernation to prevent net muscle loss despite inactivity. Reversible cardiomyocyte hypertrophy occurs in cardiac muscle, allowing the heart to preserve cardiac output during hibernation, while avoiding chronic maladaptive hypertrophy post-hibernation. We propose that calcium signalling proteins [calcineurin (Cn), calmodulin (CaM), and calpain], the nuclear factor of activated T cell (NFAT) family of transcription factors, and the NFAT targets myoferlin and myomaker contribute significantly to adaptations taking place in skeletal and cardiac muscle during hibernation. Protein-level analyses were performed over several conditions: euthermic room temperature (ER), euthermic cold room (EC), entrance into (EN), early (ET), and late torpor (LT) time points, in addition to early (EA), interbout (IA), and late arousal (LA) time points using immunoblotting and DNA-protein interaction (DPI) enzyme-linked immunosorbent assay (ELISAs). In skeletal and cardiac muscle, NFATc2 protein levels were elevated during torpor. NFATc4 increased throughout the torpor-arousal cycle in both tissues, and NFATc1 showed this trend in cardiac muscle only. NFATc3 showed an elevation in DNA-binding activity but not expression during torpor. Myoferlin protein levels dramatically increased during torpor in both skeletal and cardiac muscle. Myomaker levels also increased significantly in cardiac muscle during torpor. Cardiac Cn levels remained stable, whereas CaM and calpain decreased throughout the torpor-arousal cycle. Activation and/or upregulation of NFATc2, c3, myoferlin, and myomaker at torpor could be part of a stress-response mechanism to preserve skeletal muscle mass, whereas CaM and calpain appear to initiate the rapid reversal of cardiac hypertrophy during arousal through

  15. Theaflavins enhance intestinal barrier of Caco-2 Cell monolayers through the expression of AMP-activated protein kinase-mediated Occludin, Claudin-1, and ZO-1.

    PubMed

    Park, Ha-Young; Kunitake, Yuri; Hirasaki, Naoto; Tanaka, Mitsuru; Matsui, Toshiro

    2015-01-01

    We investigated the effect of theaflavins (TFs) on membrane barrier of Caco-2 cells. For fluorescein-transport experiments, the apparent permeability (Papp) of fluorescein in Caco-2 cells pretreated with 20 μM TFs were significantly decreased compared with that in untreated cells. Although the respective monomeric catechins did not show any Papp reduction, purpurogallin pretreatment resulted in a significant Papp reduction similar to that of TF-3'-O-gallate (TF3'G) pretreatment. This indicates that the benzotropolone moiety may play a crucial role in the Papp reduction or tight junction (TJ)-closing effect induced by TFs. In TF-3'-O-gallate-pretreated Caco-2 cells, fluorescein transport was completely restored by compound C (AMPK inhibitor). In addition, TF3'G significantly increased both the mRNA and protein expression of TJ-related proteins (occludin, claudin-1, and ZO-1) as well as the phosphorylation of AMPK. It was, thus, concluded that TFs could enhance intestinal barrier function by increasing the expression of TJ-related proteins through the activation of AMPK in Caco-2 cells.

  16. Effects of doxepin on brain-derived neurotrophic factor, tumor necrosis factor alpha, mitogen-activated protein kinase 14, and AKT1 genes expression in rat hippocampus

    PubMed Central

    Eidelkhani, Nastaran; Radahmadi, Maryam; Kazemi, Mohammad; Rafiee, Laleh; Alaei, Hojjatallah; Reisi, Parham

    2015-01-01

    Background: It has been suggested that doxepin in addition to enhancement of noradrenaline and serotonin levels may have neuroprotective effects. Therefore, this study investigated the effect of doxepin on gene expression of brain-derived neurotrophic factor (BDNF), tumor necrosis factor alpha (TNF-α), mitogen-activated protein kinase 14 (MAPK14), and serine-threonine protein kinase AKT1 in rat hippocampus. Materials and Methods: Male rats were divided randomly into three groups: Control, doxepin 1 mg/kg, and doxepin 5 mg/kg. Rats received an i.p injection of doxepin for 21 days. Then the hippocampi were dissected for the measurement of the expression of BDNF, TNF-α, MAPK14, and AKT1 genes. Results: Our results showed no significant effects of doxepin on gene expression of BDNF, TNF-α, MAPK14, and AKT1 genes in the hippocampus. Conclusions: These results did not show significant effects of doxepin on the genes that affect the neuronal survival in intact animals. However, more studies need to be done, especially in models associated with neuronal damage. PMID:26601091

  17. Effects of intravenous anesthetics on the activity of glutamate transporter EAAT3 expressed in Xenopus oocytes: evidence for protein kinase C involvement.

    PubMed

    Yun, Jung-Yeon; Kim, Jin-Hee; Kim, Hae-Kyoung; Lim, Young-Jin; Do, Sang-Hwan; Zuo, Zhiyi

    2006-02-15

    We investigated the effects of the intravenous anesthetics, thiopental, etomidate and ketamine, on the activity of one type of glutamate transporters, EAAT3 (excitatory amino acid transporter type 3). Rat EAAT3 was expressed in Xenopus oocytes by injection of its mRNA. Using two-electrode voltage clamp, membrane currents were recorded after the application of L-glutamate (30 microM) in the presence or absence of various concentrations of the anesthetics. Thiopental (0.3-30 microM) and ketamine (3-1000 microM) did not affect EAAT3 activity. Etomidate decreased EAAT3 activity in a concentration-dependent manner (0.10-10 microM). Etomidate at 1 microM significantly decreased the Vmax, but not the Km of EAAT3 for glutamate. Chelerythrine, a protein kinase C (PKC) inhibitor, significantly decreased EAAT3 activity, however, there were no statistical differences among the chelerythrine, etomidate or chelerythrine plus etomidate groups. Likewise, the combination of staurosporine, another PKC inhibitor, and etomidate did not decrease the responses further compared with staurosporine or etomidate alone. Phorbol-12-myrisate-13-acetate, a PKC activator, abolished etomidate-induced decrease in EAAT3 activity. Since our results showed that thiopental and ketamine did not affect EAAT3 activity significantly, EAAT3 may not be a target for their anesthetic effects. Our results also suggest that etomidate, possibly via PKC, decreased EAAT3 activity at clinically relevant concentrations.

  18. Regulation of the expression and activity of glucose and lactic acid metabolism-related genes by protein kinase C in skeletal muscle cells.

    PubMed

    Otake, Sho; Kobayashi, Masaki; Narumi, Katsuya; Sasaki, Shotaro; Kikutani, Yurika; Furugen, Ayako; Watanabe, Meguho; Takahashi, Natsuko; Ogura, Jiro; Yamaguchi, Hiroaki; Iseki, Ken

    2013-01-01

    Protein kinase C (PKC) modulators are very attractive therapeutic targets in cancer. Since most cancer cells display increased glycolysis, elucidations of the effects of PKC activation on glycolysis is necessary for the development of effective medicine. In the present study, to clarify the role of PKC in the regulation of glycolysis, we examined the effect of phorbol 12-myristate 13-acetate (PMA), a PKC activator, on the expression and activity of glucose and lactic acid metabolism-related genes in human rhabdomyosarcoma cells (RD cells). In parallel to increases in glucose uptake and mRNA levels of glucose transporters (GLUTs) induced by PMA treatment for 6 h, the hexokinase (HK) mRNA level and activity were also significantly increased in RD cells. On the other hand, a significant increase in lactate dehydrogenase (LDH) mRNA level and activity was seen when the cells were incubated with PMA for 24 h, but not for 6 or 12 h, and was associated with lactic acid production. These effects by PMA treatment were markedly suppressed by Bisindolylmaleimide (BIM), a PKC inhibitor. Furthermore, chetomin, a hypoxia-inducible factor 1 (HIF-1) inhibitor, completely abrogated the increment of LDH mRNA level and activity as well as monocarboxylate transporter (MCT) 4, a lactic acid efflux transporter. In conclusion, we found that HK and LDH activity induced by PKC activation was associated with the glucose uptake and lactic acid level and that LDH and MCT4 are modulated by a common factor, HIF-1.

  19. Glucose elevates NITRATE TRANSPORTER2.1 protein levels and nitrate transport activity independently of its HEXOKINASE1-mediated stimulation of NITRATE TRANSPORTER2.1 expression.

    PubMed

    de Jong, Femke; Thodey, Kate; Lejay, Laurence V; Bevan, Michael W

    2014-01-01

    Mineral nutrient uptake and assimilation is closely coordinated with the production of photosynthate to supply nutrients for growth. In Arabidopsis (Arabidopsis thaliana), nitrate uptake from the soil is mediated by genes encoding high- and low-affinity transporters that are transcriptionally regulated by both nitrate and photosynthate availability. In this study, we have studied the interactions of nitrate and glucose (Glc) on gene expression, nitrate transport, and growth using glucose-insensitive2-1 (gin2-1), which is defective in sugar responses. We confirm and extend previous work by showing that HEXOKINASE1-mediated oxidative pentose phosphate pathway (OPPP) metabolism is required for Glc-mediated NITRATE TRANSPORTER2.1 (NRT2.1) expression. Treatment with pyruvate and shikimate, two products derived from intermediates of the OPPP that are destined for amino acid production, restores wild-type levels of NRT2.1 expression, suggesting that metabolites derived from OPPP metabolism can, together with Glc, directly stimulate high levels of NRT2.1 expression. Nitrate-mediated NRT2.1 expression is not influenced by gin2-1, showing that Glc does not influence NRT2.1 expression through nitrate-mediated mechanisms. We also show that Glc stimulates NRT2.1 protein levels and transport activity independently of its HEXOKINASE1-mediated stimulation of NRT2.1 expression, demonstrating another possible posttranscriptional mechanism influencing nitrate uptake. In gin2-1 plants, nitrate-responsive biomass growth was strongly reduced, showing that the supply of OPPP metabolites is essential for assimilating nitrate for growth.

  20. Expression of constitutively active Akt/protein kinase B signals GLUT4 translocation in the absence of an intact actin cytoskeleton.

    PubMed

    Eyster, Craig A; Duggins, Quwanza S; Olson, Ann Louise

    2005-05-06

    The actin cytoskeleton has been shown to be required for insulin-dependent GLUT4 translocation; however, the role that the actin network plays is unknown. Actin may play a role in formation of an active signaling complex, or actin may be required for movement of vesicles to the plasma membrane surface. To distinguish between these possibilities, we examined the ability of myr-Akt, a constitutively active form of Akt that signals GLUT4 translocation to the plasma membrane in the absence of insulin, to signal translocation of an HA-GLUT4-GFP reporter protein in the presence or absence of an intact cytoskeleton in 3T3-L1 adipocytes. Expression of myr-Akt signaled the redistribution of the GLUT4 reporter protein to the cell surface in the absence or presence of 10 microm latrunculin B, a concentration sufficient to completely inhibit insulin-dependent redistribution of the GLUT4 reporter to the cell surface. These data suggest that the actin network plays a primary role in organization of the insulin-signaling complex. To further support this conclusion, we measured the activation of known signaling proteins using a saturating concentration of insulin in cells pretreated without or with 10 microm latrunculin B. We found that latrunculin treatment did not affect insulin-dependent tyrosine phosphorylation of the insulin receptor beta-subunit and IRS-1 but completely inhibited activation of Akt/PKB enzymatic activity. Phosphorylation of Akt/PKB at Ser-473 and Thr-308 was inhibited by latrunculin B treatment, indicating that the defect in signaling lies prior to Akt/PKB activation. In summary, our data support the hypothesis that the actin network plays a role in organization of the insulin-signaling complex but is not required for vesicle trafficking and/or fusion.

  1. Connective tissue-activating peptide III (CTAP-III): cloning the synthetic gene and characterization of the protein expressed in E. coli

    SciTech Connect

    Johnson, P.H.; Castor, C.W.; Walz, D.A.

    1986-05-01

    CTAP-III, an ..cap alpha..-granule protein secreted by human platelets, is known to stimulate mitogenesis, extracellular matrix synthesis, and plasminogen activator synthesis in human fibroblast cultures. From its primary sequence, a synthetic gene was constructed to code for a methionine-free derivative (Leu substituted for Met-21), then cloned and expressed in E. coli using a new expression vector containing regulatory elements of the colicin E1 operon. Partially purified recombinant CTAP-III showed a line of identity with CTAP-III by immunodiffusion against rabbit antibody to platelet-derived CTAP-III. Immunodetection of the reduced protein after SDS-PAGE electrophoresis showed a molecular weight (mobility) in agreement with the natural form. Biologic activity of rCTAP-III eluted from an antiCTAP-III immunoaffinity column was measured in human synovial cell bioassay systems. rCTAP-III stimulated synovial cell synthesis of /sup 14/C-hyaluronic acid approximately 13-fold; significant (P < 0.001) mitogenesis was also observed. These studies indicate that a sufficient quantity of bioactive peptide can be obtained for a more comprehensive study of its biologic properties.

  2. Protein and messenger RNA expression of interleukin 1 system members in bovine ovarian follicles and effects of interleukin 1β on primordial follicle activation and survival in vitro.

    PubMed

    Passos, J R S; Costa, J J N; da Cunha, E V; Silva, A W B; Ribeiro, R P; de Souza, G B; Barroso, P A A; Dau, A M P; Saraiva, M V A; Gonçalves, P B D; van den Hurk, R; Silva, J R V

    2016-01-01

    This study aimed to investigate the expression of interleukin 1 (IL-1) system members (proteins and messenger RNA of ligands and receptors) and its distribution in ovarian follicles of cyclic cows and to evaluate the effects of IL-1β on the survival and activation of primordial follicles in vitro. The ovaries were processed for localization of IL-1 system in preantral and antral follicles by immunohistochemical, real-time polymerase chain reaction, and Western blot analysis. For in vitro studies, ovarian fragments were cultured in α-MEM(+) supplemented with IL-1β (0, 1, 10, 50, or 100 ng/mL), and after 6 d, the cultured tissues were processed for histologic analysis. Immunohistochemical results showed that the IL-1 system proteins IL-1β, IL-1RA, IL-1RI, and IL-1RII were detected in the cytoplasm of oocytes and granulosa cells from all follicular categories and theca cells of antral follicles. Variable levels of messenger RNA for the IL-1 system members were observed at different stages of development. After 6 d of culture, the presence of IL-1β (10 or 50 ng/mL) was effective in maintaining the percentage of normal follicles and in promoting primordial follicle activation. In conclusion, IL-1 system members are differentially expressed in ovarian follicles according to their stage of development. Moreover, IL-1β promotes the development of primordial follicles. These results indicate an important role of the IL-1 system in the regulation of bovine folliculogenesis.

  3. Disorders in barrier protein mRNA expression and placenta secretory activity under the influence of polychlorinated biphenyls in vitro.

    PubMed

    Wojciechowska, A; Mlynarczuk, J; Kotwica, J

    2017-02-01

    Pregnancy disorders are often correlated with the presence of organic pollutants in the tissues of living bodies. The aim of this study was to investigate the effects (over 24 and 48 hours) of polychlorinated biphenyls (PCBs) 153, 126, and 77 at doses of 1, 10, and 100 ng/mL on barrier function and secretory activity in cow placentome sections collected during the second trimester of pregnancy. None of the PCBs affected the viability of the sections (P > 0.05). Polychlorinated biphenyl 153 decreased (P < 0.05) connexin 26 (Cx 26) mRNA expression, and all three PCBs reduced (P < 0.05) Cx 43 mRNA expression. Cx 32 mRNA expression showed a downward trend (P > 0.05) under the influence of PCBs 126 and 77. Moreover, PCBs 153 and 126 increased keratin 8 (KRT8) mRNA expression, whereas all PCBs decreased (P < 0.05) placenta specific protein 1 (PLAC-1) mRNA expression without changing (P > 0.05) hypoxia inducible factor 1α (HIF1α) mRNA expression. Concomitantly, PCBs 153 and 126 stimulated (P < 0.05) cyclooxygenase 2 (COX-2) mRNA expression, all PCBs increased (P < 0.05) prostaglandin E2 synthase (PGES) mRNA expression, and PCBs 126 and 77 increased prostaglandin E2 (PGE2) secretion. All three PCBs decreased (P < 0.05) prostaglandin F2α synthase (PGFS) mRNA expression and prostaglandin F2α (PGF2α) secretion. In addition, all three PCBs increased (P < 0.05) neurophysin I/oxytocin (NP-I/OT) mRNA expression and OT secretion but did not affect peptidyl-glycine-α-amidating monooxygenase (PGA) mRNA expression (P > 0.05). Moreover, the PCBs increased (P < 0.05) estradiol (E2) secretion, whereas progesterone (P4) secretion remained unchanged (P > 0.05). These changes could affect trophoblast invasion and uterine contractility and thus impact the course of gestation and/or fetal development in the cow.

  4. Metformin inhibits advanced glycation end products (AGEs)-induced growth and VEGF expression in MCF-7 breast cancer cells by suppressing AGEs receptor expression via AMP-activated protein kinase.

    PubMed

    Ishibashi, Y; Matsui, T; Takeuchi, M; Yamagishi, S

    2013-05-01

    Metformin use has been reported to decrease breast cancer incidence and mortality in diabetic patients. We have previously shown that advanced glycation end products (AGEs) and their receptor (RAGE) interaction stimulate growth and/or migration of pancreatic cancer and melanoma cells. However, effects of metformin on AGEs-RAGE axis in breast cancers remain unknown. We examined here whether and how metformin could block the AGEs-induced growth and vascular endothelial growth factor (VEGF) expression in MCF-7 breast cancer cells. Cell proliferation was measured with an electron coupling reagent WST-1 based colorimetric assay. Gene expression level was evaluated by real-time reverse-transcription polymerase chain reactions. AGEs significantly increased cell proliferation of MCF-7 cells, which was completely prevented by the treatment with 0.01 or 0.1 mM metformin or anti-RAGE antibodies. Furthermore, metformin at 0.01 mM completely suppressed the AGEs-induced upregulation of RAGE and VEGF mRNA levels in MCF-7 cells. An inhibitor of AMP-activated protein kinase, compound C significantly blocked the growth-inhibitory and RAGE and VEGF suppressing effects of metformin in AGEs-exposed MCF-7 cells. Our present study suggests that metformin could inhibit the AGEs-induced growth and VEGF expression in MCF-7 breast cancer cells by suppressing RAGE gene expression via AMP-activated protein kinase pathway. Metformin may protect against breast cancer expansion in diabetic patients by blocking the AGEs-RAGE axis.

  5. [Effects of arsenic trioxide or retinoic acid on mRNA and protein expression of tissue factor and thrombomodulin and procoagulant activity in NB4 cells].

    PubMed

    Zhang, Xiao-Hui; Hu, Yu; Hong, Mei; Xia, Ling-Hui; Guo, Tao; Shen, Guan-Xin; Wei, Wen-Ning; Song, Shan-Jun

    2007-04-01

    To investigate the effect of arsenic trioxide (As(2)O(3)) or all-trans retinoic acid (ATRA) on the mRNA and protein expression of tissue factor (TF) and thrombomodulin (TM) and procoagulant activity (PCA) in NB4 cells. The NB4 cells were cultured in vitro and treated with As(2)O(3) or ATRA, expression of TF and TM antigen, and PCA change of treated NB4 cells were detected with ELISA, TF and TM mRNA transcription on the NB4 cells was assayed with reversed transcription polymerase chain reaction (RT-PCR). The results showed that 1 micromol/L As(2)O(3) and 1 micromol/L ATRA both gradually downregulated the expression of TF antigen and mRNA on NB4 cells, a human promyelocytic leukemia cell line, in time-dependent manner, as compared with control. The levels of TF antigen expression in AS(2)O(3) group were 13.3 +/- 1.8, 8.6 +/- 1.9, 10.8 +/- 1.5, 2.0 +/- 0.6 and 2.6 +/- 0.9 ng/10(7) respectively; while the levels of TF antigen expression in ATRA group were 12.4 +/- 1.1, 11.3 +/- 1.8, 5.7 +/- 1.7, 2.8 +/- 0.8 and 2.0 +/- 0.6 ng/10(7) at 24, 48, 72, 96 and 120 hours respectively (P<0.05). The procoagulant activity (PCA) of NB4 cells was decreased, blood coagulation times were 123.5 +/- 10.5, 156.3 +/- 11.6, 179.3 +/- 15.3, 248.9 +/- 20.1, 312.0 +/- 29.8 seconds in As(2)O(3) groups, respectively; 76.4 +/- 5.6, 146.8 +/- 10.9, 198.2 +/- 15.6, 265.8 +/- 20.6 and 363.8 +/- 31.9 seconds in ATRA groups respectively at 24, 48, 72, 96 and 120 hours (P<0.05). ATRA upregulated TM antigen expression on NB4 cells. It is concluded that the As(2)O(3) and ATRA decrease mRNA transcription of TF, downregulate expression of TF and reduce procoagulant activity in NB4 cells. The TM transcription and expression upregulated by ATRA may alleviate dysfunction of coagulation in APL.

  6. Activation of AMP-activated protein kinase by kainic acid mediates brain-derived neurotrophic factor expression through a NF-kappaB dependent mechanism in C6 glioma cells

    SciTech Connect

    Yoon, Hana; Oh, Young Taek; Lee, Jung Yeon; Choi, Ji Hyun; Lee, Ju Hie; Baik, Hyung Hwan; Kim, Sung Soo; Choe, Wonchae; Yoon, Kyung-Sik; Ha, Joohun; Kang, Insug

    2008-07-04

    AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis. Kainic acid (KA), a prototype excitotoxin is known to induce brain-derived neurotrophic factor (BDNF) in brain. In this study, we examined the role of AMPK in KA-induced BDNF expression in C6 glioma cells. We showed that KA and KA receptor agonist induced activation of AMPK and KA-induced AMPK activation was blocked by inhibition of Ca{sup 2+}/calmodulin-dependent protein kinase kinase (CaMKK) {beta}. We then showed that inhibition of AMPK by compound C, a selective inhibitor of AMPK, or small interfering RNA of AMPK{alpha}1 blocked KA-induced BDNF mRNA and protein expression. Inhibition of AMPK blocked KA-induced phosphorylation of CaMKII and I kappaB kinase (IKK) in C6 cells. Finally, we showed that inhibition of AMPK reduced DNA binding and transcriptional activation of nuclear factor-kappaB (NF-{kappa}B) in KA-treated cells. These results suggest that AMPK mediates KA-induced BDNF expression by regulating NF-{kappa}B activation.

  7. Complexes containing activating transcription factor (ATF)/cAMP-responsive-element-binding protein (CREB) interact with the CCAAT/enhancer-binding protein (C/EBP)-ATF composite site to regulate Gadd153 expression during the stress response.

    PubMed Central

    Fawcett, T W; Martindale, J L; Guyton, K Z; Hai, T; Holbrook, N J

    1999-01-01

    Gadd153, also known as chop, encodes a member of the CCAAT/enhancer-binding protein (C/EBP) transcription factor family and is transcriptionally activated by cellular stress signals. We recently demonstrated that arsenite treatment of rat pheochromocytoma PC12 cells results in the biphasic induction of Gadd153 mRNA expression, controlled in part through binding of C/EBPbeta and two uncharacterized protein complexes to the C/EBP-ATF (activating transcription factor) composite site in the Gadd153 promoter. In this report, we identified components of these additional complexes as two ATF/CREB (cAMP-responsive-element-binding protein) transcription factors having differential binding activities dependent upon the time of arsenite exposure. During arsenite treatment of PC12 cells, we observed enhanced binding of ATF4 to the C/EBP-ATF site at 2 h as Gadd153 mRNA levels increased, and enhanced binding of ATF3 complexes at 6 h as Gadd153 expression declined. We further demonstrated that ATF4 activates, while ATF3 represses, Gadd153 promoter activity through the C/EBP-ATF site. ATF3 also repressed ATF4-mediated transactivation and arsenite-induced activation of the Gadd153 promoter. Our results suggest that numerous members of the ATF/CREB family are involved in the cellular stress response, and that regulation of stress-induced biphasic Gadd153 expression in PC12 cells involves the ordered, sequential binding of multiple transcription factor complexes to the C/EBP-ATF composite site. PMID:10085237

  8. The human CFTR protein expressed in CHO cells activates aquaporin-3 in a cAMP-dependent pathway: study by digital holographic microscopy.

    PubMed

    Jourdain, Pascal; Becq, Frédéric; Lengacher, Sylvain; Boinot, Clément; Magistretti, Pierre J; Marquet, Pierre

    2014-02-01

    The transmembrane water movements during cellular processes and their relationship to ionic channel activity remain largely unknown. As an example, in epithelial cells it was proposed that the movement of water could be directly linked to cystic fibrosis transmembrane conductance regulator (CFTR) protein activity through a cAMP-stimulated aqueous pore, or be dependent on aquaporin. Here, we used digital holographic microscopy (DHM) an interferometric technique to quantify in situ the transmembrane water fluxes during the activity of the epithelial chloride channel, CFTR, measured by patch-clamp and iodide efflux techniques. We showed that the water transport measured by DHM is fully inhibited by the selective CFTR blocker CFTRinh172 and is absent in cells lacking CFTR. Of note, in cells expressing the mutated version of CFTR (F508del-CFTR), which mimics the most common genetic alteration encountered in cystic fibrosis, we also show that the water movement is profoundly altered but restored by pharmacological manipulation of F508del-CFTR-defective trafficking. Importantly, whereas activation of this endogenous water channel required a cAMP-dependent stimulation of CFTR, activation of CFTR or F508del-CFTR by two cAMP-independent CFTR activators, genistein and MPB91, failed to trigger water movements. Finally, using a specific small-interfering RNA against the endogenous aquaporin AQP3, the water transport accompanying CFTR activity decreased. We conclude that water fluxes accompanying CFTR activity are linked to AQP3 but not to a cAMP-stimulated aqueous pore in the CFTR protein.

  9. Heterologous expression of surface-active proteins from barley and filamentous fungi in Pichia pastoris and characterization of their contribution to beer gushing.

    PubMed

    Lutterschmid, Georg; Muranyi, Monika; Stübner, Matthias; Vogel, Rudi F; Niessen, Ludwig

    2011-05-14

    The spontaneous over-foaming of beer upon opening, i.e. beer gushing, is an unwanted phenomenon for the brewing industry. Currently, surface-active proteins from filamentous fungi and non-specific lipid transfer proteins (nsLTP1) from barley are discussed as gushing inducers. In our study the class I hydrophobin FcHyd3p from Fusarium culmorum, the class II hydrophobin Hfb2 from Trichoderma reesei, the alkaline foam protein A (AfpA) from F. graminearum and nsLTP1 from Hordeum vulgare cv. Marnie (barley) were heterologously expressed in Pichia pastoris and used in gushing tests. The class I hydrophobin FcHyd3p was unable to induce gushing in beer. The class II hydrophobin Hfb2 was able to induce gushing in beer, but proved to be inhibited by heat treatment as well as by the presence of enriched hop compounds. Both resulted in a reduced gushing potential. AfpA and nsLTP1 exhibited no gushing-inducing potential at the amounts added to beer. Addition of these proteins to beer or carbonated water previously treated with class II hydrophobins revealed a gushing reducing character.

  10. Estrogen secreting adrenal adenocarcinoma in an 18-month-old boy: aromatase activity, protein expression, mRNA and utilization of gonadal type promoter.

    PubMed

    Watanabe, T; Yasuda, T; Noda, H; Wada, K; Kazukawa, I; Someya, T; Minamitani, K; Minagawa, M; Wataki, K; Matsunaga, T; Ohnuma, N; Kohno, Y; Harada, N

    2000-12-01

    We examined clinical, endocrinological and molecular biological aspects of an estrogen-secreting adrenal carcinoma in an 18-month-old male to clarify the pathogenesis of this condition. An 18-month-old boy was referred for evaluation of progressive bilateral gynecomastia and appearance of pubic hair. The patient had elevated plasma estradiol (349 pg/ml) and testosterone (260 ng/dl) levels that completely suppressed FSH and LH levels, and was subsequently diagnosed with an adrenal tumor on the right side. After removal of a 300-g adenocarcinoma, gynecomastia regressed and essentially normal hormone levels were restored. Aromatase activity in the tumor tissue determined by the 3H-water method was 71.0-104.4 pmol/min/mg protein. High levels of aromatase protein and mRNA in the tumor tissue were also demonstrated, while neither aromatase activity nor protein was detected in normal adrenal glands. To investigate the regulation of aromatase expression in the adrenal carcinoma, we examined the usage of alternate promoters responsible for aromatase gene transcription. In the present case, the amounts of aromatase mRNA utilizing gonadal types of exon 1c (1.3) and 1d (II) were significantly higher than those that using other exon 1s. This result suggested that the utilization of a gonadal-type exon 1 might be involved in the over-production of aromatase in estrogen-secreting adrenal carcinoma.

  11. Consequences of heat shock protein 72 (Hsp72) expression and activity on stress-induced apoptosis in CD30+ NPM-ALK+ anaplastic large-cell lymphomas.

    PubMed

    Bonvini, P; Zorzi, E; Mussolin, L; Pillon, M; Romualdi, C; Peron, M; d'Amore, E S G; Lamant, L; Rosolen, A

    2012-06-01

    Understanding the mechanisms that control stress-induced apoptosis is critical to explain how tumours respond to treatment, as cancer cells frequently escape drug toxicity by regulating stress response through heat shock protein (HSP) expression. The overexpression of Hsp72, in particular, results in increased incidence of cell transformation, and correlates with poor prognosis in a wide range of cancers. We have shown that Hsp72 assists folding of oncogenic NPM-ALK kinase in anaplastic large-cell lymphomas (ALCLs), but its role in the maintenance of the malignant phenotype remains uncertain. Therefore, we assessed Hsp72 expression in ALCLs, investigating more in detail the mechanisms that regulate its status and activity. We found that Hsp72 is unique among the HSPs involved in tumourigenesis to be overexpressed in ALK(+) tumours and cell lines and to be induced by stress. Different from other HSPs, Hsp72 prevents cell injury, Bax activation and death by apoptosis in ALK(+) cells, acting both upstream and downstream of mitochondria. Conversely, Hsp72 is underexpressed in ALK(-) ALCL cells, and it is unable to protect cells from apoptosis under stress. Moreover, when Hsp72 expression is reduced following NPM-ALK inhibition, lymphoma cells undergo apoptosis, demonstrating the importance of Hsp72 in regulating ALCL stress response and drug sensitivity.

  12. Expression of multiple proteins in transgenic plants

    DOEpatents

    Vierstra, Richard D.; Walker, Joseph M.

    2002-01-01

    A method is disclosed for the production of multiple proteins in transgenic plants. A DNA construct for introduction into plants includes a provision to express a fusion protein of two proteins of interest joined by a linking domain including plant ubiquitin. When the fusion protein is produced in the cells of a transgenic plant transformed with the DNA construction, native enzymes present in plant cells cleave the fusion protein to release both proteins of interest into the cells of the transgenic plant. Since the proteins are produced from the same fusion protein, the initial quantities of the proteins in the cells of the plant are approximately equal.

  13. Pancreatic islet cells express a family of inwardly rectifying K+ channel subunits which interact to form G-protein-activated channels.

    PubMed

    Ferrer, J; Nichols, C G; Makhina, E N; Salkoff, L; Bernstein, J; Gerhard, D; Wasson, J; Ramanadham, S; Permutt, A

    1995-11-03

    Insulin secretion is associated with changes in pancreatic beta-cell K+ permeability. A degenerate polymerase chain reaction strategy based on the conserved features of known inwardly rectifying K+ (KIR) channel genes was used to identify members of this family expressed in human pancreatic islets and insulinoma. Three related human KIR transcript sequences were found: CIR (also known as cardiac KATP-1), GIRK1, and GIRK2 (KATP-2). The pancreatic islet CIR and GIRK2 full-length cDNAs were cloned, and their genes were localized to human chromosomes 11q23-ter and 21, respectively. Northern blot analysis detected CIR mRNA at similar levels in human islets and exocrine pancreas, while the abundance of GIRK2 mRNA in the two tissues was insufficient for detection by this method. Using competitive reverse-transcription polymerase chain reaction, CIR was found to be present at higher levels than GIRK2 mRNA in native purified beta-cells. Xenopus oocytes injected with M2 muscarinic receptor (M2) plus either GIRK2 or CIR cRNA expressed only very small carbachol-induced currents, while co-injection of CIR plus GIRK2 along with M2 resulted in expression of carbachol-activated strong inwardly rectifying currents. Activators of KATP channels failed to elicit currents in the presence or absence of co-expressed sulfonylurea receptor. These results show that two components of islet cell KIR channels, CIR and GIRK2, may interact to form heteromeric G-protein-activated inwardly rectifying K+ channels that do not possess the typical properties of KATP channels.

  14. Thyroid hormones directly activate the expression of the human and mouse uncoupling protein-3 genes through a thyroid response element in the proximal promoter region

    PubMed Central

    2004-01-01

    The transcription of the human UCP3 (uncoupling protein-3) gene in skeletal muscle is tightly regulated by metabolic signals related to fatty acid availability. However, changes in thyroid status also modulate UCP3 gene expression, albeit by unknown mechanisms. We created transgenic mice bearing the entire human UCP3 gene to investigate the effect of thyroid hormones on human UCP3 gene expression. Treatment of human UCP3 transgenic mice with thyroid hormones induced the expression of the human gene in skeletal muscle. In addition, transient transfection experiments demonstrate that thyroid hormones activate the transcription of the human UCP3 gene promoter when MyoD and the TR (thyroid hormone receptor) were co-transfected. The action of thyroid hormones on UCP3 gene transcription is mediated by the binding of the TR to a proximal region in the UCP3 gene promoter that contains a direct repeat structure. An intact DNA sequence of this site is required for thyroid hormone responsiveness and TR binding. Chromatin immunoprecipitation assays revealed that the TR binds this element in vivo. The murine Ucp3 gene promoter was also dependent on MyoD and responsive to thyroid hormone in transient transfection assays. However, it was much less sensitive to thyroid hormone than the human UCP3 promoter. In summary, UCP3 gene transcription is activated by thyroid hormone treatment in vivo, and this activation is mediated by a TRE (thyroid hormone response element) in the proximal promoter region. Such regulation suggests a link between UCP3 gene expression and the effects of thyroid hormone on mitochondrial function in skeletal muscle. PMID:15496137

  15. FLZ protects dopaminergic neuron through activating protein kinase B/mammalian target of rapamycin pathway and inhibiting RTP801 expression in Parkinson's disease models.

    PubMed

    Bao, X-Q; Kong, X-C; Qian, C; Zhang, D

    2012-01-27

    The pathogenesis of Parkinson's disease is characterized by progressive degeneration of dopaminergic neurons in substantia nigra (SNpc). FLZ, a novel synthetic squamosamide derivative from a Chinese herb, has been shown to have neuroprotective effects in experimental Parkinson's disease (PD) models. However, it is still unclear whether FLZ protects against PD through regulating the function of dopaminergic system. In this study, we carried out a set of in vitro and in vivo experiments to address these questions. Oral administration of FLZ significantly improved motor dysfunction of mice challenged by MPTP. The beneficial effects of FLZ on motor behavior attributed to the elevation of dopamine level in striatum, tyrosine hydroxylase (TH)-positive cells, and TH activity in the middle brain of mouse. Mechanism study showed that treatment of FLZ increased the phosphorylation of activating protein kinase B (Akt) and mammalian target of rapamycin (mTOR). Using LY294002 to block phosphoinositide 3-kinases (PI3K)/Akt signaling pathway prevented the phosphorylation of mTOR and attenuated the neuroprotection of FLZ in MN9D cells challenged by MPP(+). In addition, FLZ reduced the expression of RTP801, an important protein in PD, in mice and cells intoxicated by MPTP/MPP(+). Taken together, these results revealed a novel role that FLZ elevated TH expression and activity in dopaminergic neuron through activation of Akt/mTOR survival pathway and inhibition of RTP801 in MPTP/MPP(+)-induced PD models. The data also provided evidence that FLZ had potent neuroprotecive effects and might become a new promising anti-PD drug.

  16. mRNA Expression and activity of ion-transporting proteins in gills of the blue crab Callinectes sapidus: effects of waterborne copper.

    PubMed

    Martins, Camila M G; Almeida, Daniela Volcan; Marins, Luis Fernando Fernandes; Bianchini, Adalto

    2011-01-01

    Waterborne Cu effects on the transcription of genes encoding ion-transporting proteins and the activities of these proteins were evaluated in gills of the blue crab Callinectes sapidus acclimated to diluted (2‰) and full (30‰) seawater. Crabs were exposed (96 h) to an environmentally relevant concentration of dissolved Cu (0.78 µM) and had their posterior (osmoregulating) gills dissected for enzymatic and molecular analysis. Endpoints analyzed were the activity of key enzymes involved in crab osmoregulation (sodium-potassium adenosine triphosphatase [Na(+)/K(+)-ATPase], hydrogen adenosine triphosphatase [H(+)-ATPase], and carbonic anhydrase [CA]) and the mRNA expression of genes encoding these enzymes and the sodium-potassium-chloride (Na(+)/K(+)/2Cl⁻) cotransporter. Copper effects were observed only in crabs acclimated to diluted seawater (hyperosmoregulating crabs) and were associated with an inhibition of the expression of mRNA of genes encoding the Na(+)/K(+)-ATPase and the Na(+)/K(+)/2Cl⁻ cotransporter. However, Cu did not affect Na(+)/K(+)-ATPase activity, indicating that the gene transcription is downregulated before a significant inhibition of the enzyme activity can be observed. This also suggests the existence of a compensatory response of this enzyme to prevent osmoregulatory disturbances after short-term exposure to environmentally relevant Cu concentrations. These findings suggest that Cu is a potential ionoregulatory toxicant in blue crabs C. sapidus acclimated to low salinity. The lack of Cu effect on blue crabs acclimated to full seawater would be due to the reduced ion uptake needed for the regulation of the hemolymph osmotic concentration in full seawater (30‰). Also, this could be explained considering the lower bioavailability of toxic Cu (free ion) associated with the higher ionic content and dissolved organic matter concentration in high salinity (30‰) than in diluted seawater (2‰).

  17. First molluscan transcription factor activator protein-1 (Ap-1) member from disk abalone and its expression profiling against immune challenge and tissue injury.

    PubMed

    De Zoysa, Mahanama; Nikapitiya, Chamilani; Lee, Youngdeuk; Lee, Sukkyoung; Oh, Chulhong; Whang, Ilson; Yeo, Sang-Yeop; Choi, Cheol Young; Lee, Jehee

    2010-12-01

    The regulation of transcriptional activation is an essential and critical point in gene expression. In this study, we describe a novel transcription factor activator protein-1 (Ap-1) gene from disk abalone Haliotis discus discus (AbAp-1) for the first time in mollusk. It was identified by homology screening of an abalone normalized cDNA library. The cloned AbAp-1 consists of a 945 bp coding region that encodes a putative protein containing 315 amino acids. The AbAp-1 gene is composed of a characteristic Jun transcription factor domain and a highly conserved basic leucine zipper (bZIP) signature similar to known Ap-1 genes. The AbAp-1 shares 46, 43 and, 40% amino acid identities with fish (Takifugu rubripes), human and insect (Ixodes scapularis) Ap-1, respectively. Quantitative real time RT-PCR analysis confirmed that AbAp-1 gene expression is constitutive in all selected tissues. AbAp-1 was upregulated in gills after bacteria (Vibrio alginolyticus, Vibrio parahemolyticus and Lysteria monocytogenes) challenge; and, upregulated in hemocytes and gills by viral hemorrhagic septicemia virus (VHSV) challenge. Shell damage and tissue injury also increased the transcriptional level of Ap-1 in mantle together with other transcription factors (NF-kB, LITAF) and pro-inflammatory TNF-α. All results considered, identification and gene expression data demonstrate that abalone Ap-1 is an important regulator in innate immune response against bacteria and virus, as well as in the inflammatory response during tissue injury. In addition, stimulation of Ap-1 under different external stimuli could be useful to understand the Ap-1 biology and its downstream target genes, especially in abalone-like mollusks.

  18. High Estradiol Concentrations Induce Heat Shock Protein 70 Expression and Suppress Nuclear Factor Kappa B Activation in Human Endometrial Epithelial Cells.

    PubMed

    Chen, Chin-Der; Chen, Shee-Uan; Chou, Chia-Hung; Chen, Mei-Jou; Wen, Wen-Fen; Wu, Szu-Yuan; Yang, Yu-Shih; Yang, Jehn-Hsiahn

    2016-09-07

    The high serum estradiol (E2) concentrations induced during in vitro fertilization are detrimental to endometrial receptivity and may result in lower embryo implantation rates. We have previously found that high E2 concentrations inhibit the activation of nuclear factor kappa B (NF-kappa B), which led to endometrial epithelial cells (EECs) apoptosis. The objective of this study is to investigate the signaling pathways through which high E2 results in NF-kappa B downregulation in EECs. Isolated human EECs were cultured in different concentrations of E2 (10(-10), 10(-9), 10(-8), 10(-7) M). The expression of heat shock protein 70 (Hsp70) and heat shock factor 1 (HSF-1) were upregulated under supraphysiological E2 (10(-7) M) concentration, whereas phosphorylated inhibitory kappa B-alpha (pI kappa B-alpha) and NF-kappa B p65 subunits were downregulated. Immunohistochemistry of C57BL/6 mouse EECs, that were exposed in vivo to high serum E2 from the administration of 20 IUs of equine chorionic gonadotropin, also demonstrated the same increase in HSF-1 and Hsp70 expression, and decrease in NF-kappa B. Immunoprecipitation of the induced Hsp70 proteins was achieved with the addition of inhibitory kappa B kinase gamma (IKK-gamma) antibodies, and elimination of this reaction occurred after addition of hsp70 siRNA. In conclusion, high E2 concentrations enhance HSF-1 and Hsp70 expression in EECs. The induced Hsp70 forms a complex with IKK-gamma and inhibits pI kappa B-alpha, which consequently suppresses NF-kappa B activation.

  19. Effects of Glycated Whey Protein Concentrate on Pro-inflammatory Cytokine Expression and Phagocytic Activity in RAW264.7 Macrophages.

    PubMed

    Chun, Su-Hyun; Lee, Hyun Ah; Lee, Keon Bong; Kim, Sae Hun; Park, Kun-Young; Lee, Kwang-Won

    2016-01-01

    The aim of this study was to determine the stimulatory effects of Maillard reaction, a non-enzymatic browning reaction on the expression of pro-inflammatory cytokines and phagocytic activity induced by whey protein concentrate (WPC). Glycated WPC (G-WPC) was prepared by a reaction between WPC and the lactose it contained. The fluorescence intensity of G-WPC dramatically increased after one day, and high molecular weight complexes formed via the Maillard reaction were also observed in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles. G-WPC demonstrated immunomodulatory effects, including stimulation of increased nitric oxide production and cytokine expressions (i.e., tumor necrosis factor-α, interleukin (IL)-1β, and IL-6), compared to WPC. Furthermore, the phagocytic activity of RAW264.7 cells was significantly increased upon treatment with G-WPC, compared to WPC. Therefore, we suggest that G-WPC can be utilized as an improved dietary source for providing immune modulating activity.

  20. Differential Expression of the Activator Protein 1 Transcription Factor Regulates Interleukin-1ß Induction of Interleukin 6 in the Developing Enterocyte

    PubMed Central

    Cahill, Catherine M.; Tam, Bosco; Rajanala, Susruthi; Rogers, Jack T.; Walker, W. Allan

    2016-01-01

    The innate immune response is characterized by activation of transcription factors, nuclear factor kappa B and activator protein-1 and their downstream targets, the pro-inflammatory cytokines including interleukin 1β and interleukin 6. Normal development of this response in the intestine is critical to survival of the human neonate and delays can cause the onset of devastating inflammatory diseases such as necrotizing enterocolitis. Previous studies have addressed the role of nuclear factor kappa B in the development of the innate immune response in the enterocyte, however despite its central role in the control of multiple pro-inflammatory cytokine genes, little is known on the role of Activator Protein 1 in this response in the enterocyte. Here we show that the canonical Activator Protein 1 members, cJun and cFos and their upstream kinases JNK and p38 play an essential role in the regulation of interleukin 6 in the immature enterocyte. Our data supports a model whereby the cFos/cJun heterodimer and the more potent cJun homodimer downstream of JNK are replaced by less efficient JunD containing dimers, contributing to the decreased responsiveness to interleukin 1β and decreased interleukin 6 secretion observed in the mature enterocyte. The tissue specific expression of JunB in colonocytes and colon derived tissues together with its ability to repress Interleukin-1β induction of an Interleukin-6 gene reporter in the NCM-460 colonocyte suggests that induction of JunB containing dimers may offer an attractive therapeutic strategy for the control of IL-6 secretion during inflammatory episodes in this area of the intestine PMID:26799482

  1. Role of Exchange Protein Directly Activated by Cyclic AMP Isoform 1 in Energy Homeostasis: Regulation of Leptin Expression and Secretion in White Adipose Tissue.

    PubMed

    Hu, Yaohua; Robichaux, William G; Mei, Fang C; Kim, Eun Ran; Wang, Hui; Tong, Qingchun; Jin, Jianping; Xu, Mingxuan; Chen, Ju; Cheng, Xiaodong

    2016-10-01

    Epacs (exchange proteins directly activated by cyclic AMP [cAMP]) act as downstream effectors of cAMP and play important roles in energy balance and glucose homeostasis. While global deletion of Epac1 in mice leads to heightened leptin sensitivity in the hypothalamus and partial protection against high-fat diet (HFD)-induced obesity, the physiological functions of Epac1 in white adipose tissue (WAT) has not been explored. Here, we report that adipose tissue-specific Epac1 knockout (AEKO) mice are more prone to HFD-induced obesity, with increased food intake, reduced energy expenditure, and impaired glucose tolerance. Despite the fact that AEKO mice on HFD display increased body weight, these mice have decreased circulating leptin levels compared to their wild-type littermates. In vivo and in vitro analyses further reveal that suppression of Epac1 in WAT decreases leptin mRNA expression and secretion by inhibiting cAMP response element binding (CREB) protein and AKT phosphorylation, respectively. Taken together, our results demonstrate that Epac1 plays an important role in regulating energy balance and glucose homeostasis by promoting leptin expression and secretion in WAT.

  2. Heterologous expression of newly identified galectin-8 from sea urchin embryos produces recombinant protein with lactose binding specificity and anti-adhesive activity

    PubMed Central

    Karakostis, Kostantinos; Costa, Caterina; Zito, Francesca; Matranga, Valeria

    2015-01-01

    Galectin family members specifically bind beta-galactoside derivatives and are involved in different cellular events, including cell communication, signalling, apoptosis, and immune responses. Here, we report a tandem-repeat type galectin from the Paracentrotus lividus sea urchin embryo, referred to as Pl-GAL-8. The 933nt sequence encodes a protein of 34.73 kDa, containing the conserved HFNPRF and WGxExR motifs in the two highly similar carbohydrate-recognition domains (CRD). The three-dimensional protein structure model of the N-CRD confirms the high evolutionary conservation of carbohydrate binding sites. The temporal gene expression is regulated during development and transcripts localize at the tip of the archenteron at gastrula stage, in a subset of the secondary mesenchyme cells that differentiate into blastocoelar (immune) cells. Functional studies using a recombinant Pl-GAL-8 expressed in bacteria demonstrate its hemo-agglutinating activity on human red blood cells through the binding to lactose, as well as its ability in inhibiting the adhesion of human Hep-G2 cells to the substrate. The recent implications in autoimmune diseases and inflammatory disorders make Gal-8 an attractive candidate for therapeutic purposes. Our results offer a solid basis for addressing the use of the new Pl-GAL-8 in functional and applicative studies, respectively in the developmental and biomedical fields. PMID:26640155

  3. Community proteogenomics highlights microbial strain-variant protein expression within activated sludge performing enhanced biological phosphorus removal.

    SciTech Connect

    Wilmes, P; Andersson, Anders F.; Lefsrud, Mark G; Wexler, Margaret; Shah, Manesh B; Zhang, B; Hettich, Robert {Bob} L; Bond, P. L.; Verberkmoes, Nathan C; Banfield, Jillian F.

    2008-01-01

    Enhanced biological phosphorus removal (EBPR) selects for polyphosphate accumulating organisms to achieve phosphate removal from wastewater. We used highresolution community proteomics to identify key metabolic pathways in "Candidatus Accumulibacter phosphatis"-mediated EBPR and to evaluate the contributions of co- 5 existing strains within the dominant population. Results highlight the importance of denitrification, fatty acid cycling and the glyoxylate bypass in EBPR. Despite overall strong similarity in protein profiles under anaerobic and aerobic conditions, fatty acid degradation proteins were more abundant during the anaerobic phase. By comprehensive genome-wide alignment of orthologous proteins, we uncovered strong 10 functional partitioning for enzyme variants involved in both core-metabolism and EBPR-specific pathways among the dominant strains. These findings emphasize the importance of genetic diversity in maintaining the stable performance of EBPR systems and demonstrate the power of integrated cultivation-independent genomics and proteomics for analysis of complex biotechnological systems.

  4. p21(Cip-1/SDI-1/WAF-1) expression via the mitogen-activated protein kinase signaling pathway in insulin-induced chondrogenic differentiation of ATDC5 cells.

    PubMed

    Nakajima, Masahiro; Negishi, Yoichi; Tanaka, Hiroyasu; Kawashima, Kohtaro

    2004-08-06

    The embryonal carcinoma-derived cell line, ATDC5, differentiates into chondrocytes in response to insulin or insulin-like growth factor-I stimulation. In this study, we investigated the roles of mitogen-activated protein (MAP) kinases in insulin-induced chondrogenic differentiation of ATDC5 cells. Insulin-induced accumulation of glycosaminoglycan and expression of chondrogenic differentiation markers, type II collagen, type X collagen, and aggrecan mRNA were inhibited by the MEK1/2 inhibitor (U0126) and the p38 MAP kinase inhibitor (SB203580). Conversely, the JNK inhibitor (SP600125) enhanced the synthesis of glycosaminoglycan and expression of chondrogenic differentiation markers. Insulin-induced phosphorylation of ERK1/2 and JNK but not that of p38 MAP kinase. We have previously clarified that the induction of the cyclin-dependent kinase inhibitor, p21(Cip-1/SDI-1/WAF-1), is essential for chondrogenic differentiation of ATDC5 cells. To assess the relationship between the induction of p21 and MAP kinase activity, we investigated the effect of these inhibitors on insulin-induced p21 expression in ATDC5 cells. Insulin-induced accumulation of p21 mRNA and protein was inhibited by the addition of U0126 and SB203580. In contrast, SP600125 enhanced it. Inhibitory effects of U0126 or stimulatory effects of SP600125 on insulin-induced chondrogenic differentiation were observed when these inhibitors exist in the early phase of differentiation, suggesting that MEK/ERK and JNK act on early phase differentiation. SB202580, however, is necessary not only for early phase but also for late phase differentiation, indicating that p38 MAP kinase stimulates differentiation by acting during the entire period of cultivation. These results for the first time demonstrate that up-regulation of p21 expression by ERK1/2 and p38 MAP kinase is required for chondrogenesis, and that JNK acts as a suppressor of chondrogenesis by down-regulating p21 expression.

  5. Osmostress-induced gene expression--a model to understand how stress-activated protein kinases (SAPKs) regulate transcription.

    PubMed

    de Nadal, Eulàlia; Posas, Francesc

    2015-09-01

    Adaptation is essential for maximizing cell survival and for cell fitness in response to sudden changes in the environment. Several aspects of cell physiology change during adaptation. Major changes in gene expression are associated with cell exposure to environmental changes, and several aspects of mRNA biogenesis appear to be targeted by signaling pathways upon stress. Exhaustive reviews have been written regarding adaptation to stress and regulation of gene expression. In this review, using osmostress in yeast as a prototypical case study, we highlight those aspects of regulation of gene induction that are general to various environmental stresses as well as mechanistic aspects that are potentially conserved from yeast to mammals.

  6. Stable enhanced green fluorescent protein expression after differentiation and transplantation of reporter human induced pluripotent stem cells generated by AAVS1 transcription activator-like effector nucleases.

    PubMed

    Luo, Yongquan; Liu, Chengyu; Cerbini, Trevor; San, Hong; Lin, Yongshun; Chen, Guokai; Rao, Mahendra S; Zou, Jizhong

    2014-07-01

    Human induced pluripotent stem (hiPS) cell lines with tissue-specific or ubiquitous reporter genes are extremely useful for optimizing in vitro differentiation conditions as well as for monitoring transplanted cells in vivo. The adeno-associated virus integration site 1 (AAVS1) locus has been used as a "safe harbor" locus for inserting transgenes because of its open chromatin structure, which permits transgene expression without insertional mutagenesis. However, it is not clear whether targeted transgene expression at the AAVS1 locus is always protected from silencing when driven by various promoters, especially after differentiation and transplantation from hiPS cells. In this paper, we describe a pair of transcription activator-like effector nucleases (TALENs) that enable more efficient genome editing than the commercially available zinc finger nuclease at the AAVS1 site. Using these TALENs for targeted gene addition, we find that the cytomegalovirus-immediate early enhancer/chicken β-actin/rabbit β-globin (CAG) promoter is better than cytomegalovirus 7 and elongation factor 1α short promoters in driving strong expression of the transgene. The two independent AAVS1, CAG, and enhanced green fluorescent protein (EGFP) hiPS cell reporter lines that we have developed do not show silencing of EGFP either in undifferentiated hiPS cells or in randomly and lineage-specifically differentiated cells or in teratomas. Transplanting cardiomyocytes from an engineered AAVS1-CAG-EGFP hiPS cell line in a myocardial infarcted mouse model showed persistent expression of the transgene for at least 7 weeks in vivo. Our results show that high-efficiency targeting can be obtained with open-source TALENs and that careful optimization of the reporter and transgene constructs results in stable and persistent expression in vitro and in vivo.

  7. A species-specific nucleotide sequence of Mycobacterium tuberculosis encodes a protein that exhibits hemolytic activity when expressed in Escherichia coli.

    PubMed Central

    Leão, S C; Rocha, C L; Murillo, L A; Parra, C A; Patarroyo, M E

    1995-01-01

    Species-specific proteins may be implicated in the unique pathogenic mechanisms characteristic of Mycobacterium tuberculosis. In previous studies, a 3.0-kb species-specific DNA fragment of M. tuberculosis was identified (C. A. Parra, L. P. Londoño, P. del Portillo, and M. E. Patarroyo, Immun. 59:3411-3417, 1991). The nucleotide sequence of this 3.0-kb fragment has been obtained. This sequence was shown to contain two open reading frames (ORFs) whose putative gene products share 68.9% identity between each other. The major ORF shows 57.8% similarity with PLC-N and 53.2% similarity with PLC-H, two phospholipase C enzymes from Pseudomonas aeruginosa. The major ORF was amplified by PCR and cloned into the pGEX-5T expression vector. Cell extracts of Escherichia coli overexpressing this glutathione S-transferase fusion protein were shown to produce beta-hemolysis suggestive of phospholipase activity. Since phospholipase C enzymes have been reported as virulence factors of P. aeruginosa and also of the intracellular pathogen Listeria monocytogenes, it is possible that the proteins identified in this study could also play a role in sustaining tuberculosis infection in humans. PMID:7591062

  8. Regulation of cAMP-induced arylalkylamine N-acetyltransferase, Period1, and MKP-1 gene expression by mitogen-activated protein kinases in the rat pineal gland.

    PubMed

    Chansard, Mathieu; Iwahana, Eiko; Liang, Jian; Fukuhara, Chiaki

    2005-10-03

    In rodent pineal glands, sympathetic innervation, which leads to norepinephrine release, is a key process in the circadian regulation of physiology and certain gene expressions. It has been shown that gene expression of the rate-limiting enzyme in the melatonin synthesis arylalkylamine N-acetyltransferase (Aa-Nat), circadian clock gene Period1, and mitogen-activated protein kinase (MAPK) phosphtase-1 (MKP-1), is controlled mainly by a norepinephrine-beta-adrenergic receptor-cAMP signaling cascade in the rat pineal gland. To further dissect the signaling cascades that regulate those gene expressions, we examined whether MAPKs are involved in cAMP-induced gene expression. Western blot and immunohistochemical analyses showed that one of the three MAPKs, c-Jun N-terminal kinase (JNK), was expressed in the pineal, and was phosphorylated by cAMP analogue stimulation with a peak 20 min after start of the stimulation, in vitro. A specific JNK inhibitor SP600125 (Anthra[1,9-cd]pyrazol-6(2H)-one1,9-pyrazoloanthrone), but not its negative control (N1-Methyl-1,9-pyrazoloanthrone), significantly reduced cAMP-stimulated Aa-Nat, Period1, and MKP-1 mRNA levels. Although another MAPK, p38(MAPK), has also been shown to be activated by cAMP stimulation, a p38(MAPK) inhibitor, SB203580 (4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole, HCl), showed no effect on cAMP-induced Aa-Nat and Period1 mRNA levels; whereas SB203580, but not its negative analogue SB202474 (4-Ethyl-2(p-methoxyphenyl)-5-(4'-pyridyl)-IH-imidazole, DiHCl), significantly reduced cAMP-induced MKP-1 mRNA levels. Taken together, our data suggest that cAMP-induced Aa-Nat and Period1 are likely to be mediated by activation of JNK, whereas MKP-1 may be mediated by both p38(MAPK) and JNK activations.

  9. Coevolution of gene expression among interacting proteins

    SciTech Connect

    Fraser, Hunter B.; Hirsh, Aaron E.; Wall, Dennis P.; Eisen,Michael B.

    2004-03-01

    Physically interacting proteins or parts of proteins are expected to evolve in a coordinated manner that preserves proper interactions. Such coevolution at the amino acid-sequence level is well documented and has been used to predict interacting proteins, domains, and amino acids. Interacting proteins are also often precisely coexpressed with one another, presumably to maintain proper stoichiometry among interacting components. Here, we show that the expression levels of physically interacting proteins coevolve. We estimate average expression levels of genes from four closely related fungi of the genus Saccharomyces using the codon adaptation index and show that expression levels of interacting proteins exhibit coordinated changes in these different species. We find that this coevolution of expression is a more powerful predictor of physical interaction than is coevolution of amino acid sequence. These results demonstrate previously uncharacterized coevolution of gene expression, adding a different dimension to the study of the coevolution of interacting proteins and underscoring the importance of maintaining coexpression of interacting proteins over evolutionary time. Our results also suggest that expression coevolution can be used for computational prediction of protein protein interactions.

  10. Cell Surface Expression of the Major Amyloid-β Peptide (Aβ)-degrading Enzyme, Neprilysin, Depends on Phosphorylation by Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase (MEK) and Dephosphorylation by Protein Phosphatase 1a*

    PubMed Central

    Kakiya, Naomasa; Saito, Takashi; Nilsson, Per; Matsuba, Yukio; Tsubuki, Satoshi; Takei, Nobuyuki; Nawa, Hiroyuki; Saido, Takaomi C.

    2012-01-01

    Neprilysin is one of the major amyloid-β peptide (Aβ)-degrading enzymes, the expression of which declines in the brain during aging. The decrease in neprilysin leads to a metabolic Aβ imbalance, which can induce the amyloidosis underlying Alzheimer disease. Pharmacological activation of neprilysin during aging therefore represents a potential strategy to prevent the development of Alzheimer disease. However, the regulatory mechanisms mediating neprilysin activity in the brain remain unclear. To address this issue, we screened for pharmacological regulators of neprilysin activity and found that the neurotrophic factors brain-derived neurotrophic factor, nerve growth factor, and neurotrophins 3 and 4 reduce cell surface neprilysin activity. This decrease was mediated by MEK/ERK signaling, which enhanced phosphorylation at serine 6 in the neprilysin intracellular domain (S6-NEP-ICD). Increased phosphorylation of S6-NEP-ICD in primary neurons reduced the levels of cell surface neprilysin and led to a subsequent increase in extracellular Aβ levels. Furthermore, a specific inhibitor of protein phosphatase-1a, tautomycetin, induced extensive phosphorylation of the S6-NEP-ICD, resulting in reduced cell surface neprilysin activity. In contrast, activation of protein phosphatase-1a increased cell surface neprilysin activity and lowered Aβ levels. Taken together, these results indicate that the phosphorylation status of S6-NEP-ICD influences the localization of neprilysin and affects extracellular Aβ levels. Therefore, maintaining S6-NEP-ICD in a dephosphorylated state, either by inhibition of protein kinases involved in its phosphorylation or by activation of phosphatases catalyzing its dephosphorylation, may represent a new approach to prevent reduction of cell surface neprilysin activity during aging and to maintain physiological levels of Aβ in the brain. PMID:22767595

  11. Characterization of Mitogen-Activated Protein Kinase Expression in Nucleus Accumbens and Hippocampus of Rats Subjected to Food Selection in the Cafeteria Diet Protocol.

    PubMed

    Sarro-Ramírez, Andrea; Sánchez, Daniel; Tejeda-Padrón, Alma; Buenfil-Canto, Linda Vianey; Valladares-García, Jorge; Pacheco-Pantoja, Elda; Arias-Carrión, Oscar; Murillo-Rodríguez, Eric

    2016-01-01

    Obesity is a world-wide health problem that requires different experimental perspectives to understand the onset of this disease, including the neurobiological basis of food selection. From a molecular perspective, obesity has been related with activity of several endogenous molecules, including the mitogenactivated protein kinases (MAP-K). The aim of this study was to characterize MAP-K expression in hedonic and learning and memory brain-associated areas such as nucleus accumbens (AcbC) and hippocampus (HIPP) after food selection. We show that animals fed with cafeteria diet during 14 days displayed an increase in p38 MAP-K activity in AcbC if chose cheese. Conversely, a diminution was observed in animals that preferred chocolate in AcbC. Also, a decrease of p38 MAP-K phosphorylation was found in HIPP in rats that selected either cheese or chocolate. Our data demonstrate a putative role of MAP-K expression in food selection. These findings advance our understanding of neuromolecular basis engaged in obesity.

  12. Gonadotropin-releasing hormone treatment improves locomotor activity, urinary function and neurofilament protein expression after spinal cord injury in ovariectomized rats.

    PubMed

    Calderón-Vallejo, Denisse; Quintanar, J Luis

    2012-05-02

    It was reported that the hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH) produces neurotrophic effects and that the spinal cord possesses GnRH receptors. The aim of the present study was to determine whether administration of GnRH improves locomotor activity, urinary function and neurofilament (NFs) protein expression after spinal cord injury (SCI) in ovariectomized rats. SCI was induced by balloon inflation model resulting in paraplegia. Locomotion was evaluated according to the Basso, Beattie, and Bresnahan Scale. Rats were subjected to bladder compression, twice daily until bladder reflex was established. NFs of 68, 160 and 200 kDa from spinal cords were analyzed by electrophoresis. GnRH (60 μg/kg) or physiologic NaCl solution was administered at 1 day after SCI and then daily for 15 days and the functional evaluation was realized for 5 weeks. Our results indicate that locomotor activity, restoration of urinary dysfunction and NFs expression of 160 and 200 kDa were improved in SCI animals given GnRH compared to those without treatment. These findings suggest that GnRH acts as a neurotrophic factor and may be used as a potential therapeutic agent for treatment of SCI.

  13. [Changes of activity and expression of protein phosphatase type 2A during the apoptosis of NB4 and MR2 cells induced by arsenic trioxide].

    PubMed

    Xu, Xi-Hui; Ouyang, Jian; Xie, Pin-Hao; Chen, Jun-Hao

    2008-10-01

    This study was aimed to investigate the change of expression and activity of protein phosphatases type 2A (PP2A) during the apoptosis of NB4 and MR2 cells induced by Arsenic trioxide (ATO). NB4 and MR2 cells were incubated with Okadaic acid (OKA) (0.5 nmol/L), ATO (0.5 - 2.0 micromol/L), and the combination of OKA and ATO at the same doses as in the single-agent treatment respectively. Then the proliferation of NB4 and MR2 cells was determined by MTT assay, the morphologic changes of cells were evaluated by Wright's staining, the apoptosis rates were detected by flow cytometry. At last, the activities of PP2A were evaluated by the serine/threonine phosphatase assay system, and the levels of PP2A subunits were detected by Western blot analysis. The results showed that ATO inhibited proliferation of NB4 and MR2 cells, and the inhibition rates of ATO on the two cells significantly increased after the addition of OKA. OKA could augment the apoptosis of NB4 and MR2 cells induced by ATO. During the apoptosis of NB4 and MR2 cells, the activity of PP2A decreased with increasing concentration of ATO, and OKA augmented the inhibitory effect of ATO on the activity. The level of PP2A structural subunit (PP2A-A) decreased during ATO-induced apoptosis of NB4 and MR2 cells, that expressions of B and C subunits of PP2A were relatively unaltered. It is concluded that the activity of PP2A decreases with increasing concentration of ATO during the apoptosis of NB4 and MR2 cells, and the decrease of the activity of PP2A maybe is related to the repression of expression of PP2A -A subunit; the inhibition of the activity of PP2A can promote the ATO induced apoptosis of NB4 and MRL cells.

  14. In Planta Expression Screens of Phytophthora infestans RXLR Effectors Reveal Diverse Phenotypes, Including Activation of the Solanum bulbocastanum Disease Resistance Protein Rpi-blb2[W

    PubMed Central

    Oh, Sang-Keun; Young, Carolyn; Lee, Minkyoung; Oliva, Ricardo; Bozkurt, Tolga O.; Cano, Liliana M.; Win, Joe; Bos, Jorunn I.B.; Liu, Hsin-Yin; van Damme, Mireille; Morgan, William; Choi, Doil; Van der Vossen, Edwin A.G.; Vleeshouwers, Vivianne G.A.A.; Kamoun, Sophien

    2009-01-01

    The Irish potato famine pathogen Phytophthora infestans is predicted to secrete hundreds of effector proteins. To address the challenge of assigning biological functions to computationally predicted effector genes, we combined allele mining with high-throughput in planta expression. We developed a library of 62 infection-ready P. infestans RXLR effector clones, obtained using primer pairs corresponding to 32 genes and assigned activities to several of these genes. This approach revealed that 16 of the 62 examined effectors cause phenotypes when expressed inside plant cells. Besides the well-studied AVR3a effector, two additional effectors, PexRD8 and PexRD3645-1, suppressed the hypersensitive cell death triggered by the elicitin INF1, another secreted protein of P. infestans. One effector, PexRD2, promoted cell death in Nicotiana benthamiana and other solanaceous plants. Finally, two families of effectors induced hypersensitive cell death specifically in the presence of the Solanum bulbocastanum late blight resistance genes Rpi-blb1 and Rpi-blb2, thereby exhibiting the activities expected for Avrblb1 and Avrblb2. The AVRblb2 family was then studied in more detail and found to be highly variable and under diversifying selection in P. infestans. Structure-function experiments indicated that a 34–amino acid region in the C-terminal half of AVRblb2 is sufficient for triggering Rpi-blb2 hypersensitivity and that a single positively selected AVRblb2 residue is critical for recognition by Rpi-blb2. PMID:19794118

  15. Reconstitution of mammalian excision repair activity with mutant cell-free extracts and XPAC and ERCC1 proteins expressed in Escherichia coli.

    PubMed Central

    Park, C H; Sancar, A

    1993-01-01

    Nucleotide excision repair in humans involves the coordinated actions of 8-10 proteins. To understand the roles of each of these proteins in excision it is necessary to develop an in vitro excision repair system reconstituted entirely from purified proteins. Towards this goal we have expressed in E. coli two of the 8 genes known to be essential for the excision reaction. XPAC and ERCC1 were expressed as fusion proteins with the Escherichia coli maltose binding protein (MBP) and purified to > 80% homogeneity by affinity chromatography. The purified proteins either as fusions or after cleavage from the MBP were able to complement the CFE of cells with mutations in the corresponding genes in an excision assay with thymine dimer containing substrate. Images PMID:8255764

  16. Expression of recombinant AccMRJP1 protein from royal jelly of Chinese honeybee in Pichia pastoris and its proliferation activity in an insect cell line

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Main royal jelly protein 1 (MRJP1) is the most abundant member of the main royal jelly protein (MRJP) family among honeybees. Mature MRJP1 cDNA of the Chinese honeybee (Apis cerana cerana MRJP1, or AccMRJP1) was expressed in Pichia pastoris. SDS-PAGE showed that recombinant AccMRJP1 was identical in...

  17. Effect of Potato Virus Y on the NADP-Malic Enzyme from Nicotiana tabacum L.: mRNA, Expressed Protein and Activity

    PubMed Central

    Doubnerová, Veronika; Müller, Karel; Čeřovská, Noemi; Synková, Helena; Spoustová, Petra; Ryšlavá, Helena

    2009-01-01

    The effect of biotic stress induced by viral infection (Potato virus Y, strain NTN and O) on NADP-malic enzyme (EC 1.1.1.40) in tobacco plants (Nicotiana tabacum L., cv. Petit Havana, SR1) was tested at the transcriptional, translational and activity level. The increase of enzyme activity in infected leaves was correlated with the increased amount of expressed protein and with mRNA of cytosolic NADP-ME isoform. Transcription of the chloroplastic enzyme was not influenced by viral infection. The increase of the enzyme activity was also detected in stems and roots of infected plants. The effect of viral infection induced by Potato virus Y, NTN strain, causing more severe symptoms, was compared with the effect induced by milder strain PVYO. The observed increase in NADP-malic enzyme activity in all parts of the studied plants was higher in the case of PVYNTN strain than in the case of strain PVYO. The relevance of NADP-malic enzyme in plants under stress conditions was discussed. PMID:20111689

  18. Effects of propofol on the activity of rat glutamate transporter type 3 expressed in Xenopus oocytes: the role of protein kinase C.

    PubMed

    Do, Sang-Hwan; Ham, Byung-Moon; Zuo, Zhiyi

    2003-06-05

    We investigated the effects of propofol on one type of glutamate transporter, excitatory amino acid transporter 3 (EAAT3) and the role of protein kinase C (PKC) in mediating these effects. Rat EAAT3 was expressed in Xenopus oocytes. L-glutamate (30 microM)-induced membrane currents were measured. Propofol increased glutamate-induced inward currents significantly at two tested concentrations (30 and 100 microM) but not at other concentrations. Propofol (30 microM) significantly increased V(max), but not K(m) of EAAT3 for glutamate. The combination of phorbol-12-myrisate-13-acetate (PMA, a PKC activator) and propofol did not increase the responses further compared with PMA or propofol alone. Three PKC inhibitors (staurosporine, calphostin C, and chelerythrine) did not affect basal EAAT3 activity but significantly inhibited the propofol-enhanced EAAT3 activity. Our results suggest that propofol enhances EAAT3 activity at clinically relevant concentrations and PKC may mediate these effects.

  19. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, William C.; Brown, Christopher S.

    1994-01-01

    Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional sodium doedocyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

  20. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, W. C.; Brown, C. S.

    1995-01-01

    Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional SDS PAGE and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

  1. Oncogenic activation of the PI3K/Akt pathway promotes cellular glucose uptake by downregulating the expression of thioredoxin-interacting protein.

    PubMed

    Hong, Shin Yee; Yu, Fa-Xing; Luo, Yan; Hagen, Thilo

    2016-05-01

    Oncogenic activation of the PI3K/Akt pathway is known to play an important role to promote glucose metabolism in cancer cells. However, the molecular mechanism through which the PI3K/Akt signalling pathway promotes glucose utilisation in cancer cells is still not well understood. It has recently been shown that the oncogenic activation of the PI3K/Akt/mTOR signalling in lung adenocarcinoma is important in promoting the localisation of glucose transporter 1 (GLUT1) at the plasma membrane. We thus hypothesised that the effect of constitutive activation of the PI3K/AKT signalling on glucose metabolism is mediated by thioredoxin interacting protein (TXNIP), a known regulator of the GLUT1 plasma membrane localisation. Consistent with previous studies, inhibition of the PI3K/Akt pathway decreased cellular glucose uptake. Furthermore, inhibition of PI3K/Akt signalling in non-small cell lung cancer (NSCLC) cell lines using clinically used tyrosine kinase inhibitors (TKIs) resulted in a decrease in GLUT1 membrane localisation. We also observed that inhibition of the PI3K/Akt pathway in various cell lines, including NSCLC cells, resulted in an increase in TXNIP expression. Importantly, knockdown of TXNIP using siRNA in the NSCLC cells promoted GLUT1 to be localised at the plasma membrane and reversed the effect of PI3K/Akt inhibitors. Together, our results suggest that the oncogenic activation of PI3K/Akt signalling promotes cellular glucose uptake, at least in part, through the regulation of TXNIP expression. This mechanism may contribute to the Warburg effect in cancer cells.

  2. Sesamin suppresses STZ induced INS-1 cell apoptosis through inhibition of NF-κB activation and regulation of Bcl-2 family protein expression.

    PubMed

    Zheng, Shuguo; Zhao, Mengqiu; Ren, Younan; Wu, Yuanjie; Yang, Jieren

    2015-03-05

    Diverse risk factors for diabetes can induce oxidative stress, leading to pancreatic beta cell damage and insulin secretion dysfunction. In the present study, we evaluated the effect of sesamin on streptozotocin (STZ) induced apoptosis in INS-1 cells and the possible mechanisms implicated. After preincubation with indicated concentrations of sesamin (0.1, 1.0 and 10.0μmol/l) for 24h, INS-1 cells were exposed to STZ (3mmol/l) for 12h. Sesamin effectively improved STZ induced cell damage as determined by MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide] assay and insulin secretion capacity, and suppressed STZ induced cell apoptosis as evaluated by flow cytometry using annexin V and propidium iodide double staining. Western blot analysis demonstrated that sesamin markedly suppressed STZ induced nuclear factor kappa B (NF-κB) activation, with Bax protein down-regulated and Bcl-2 protein up-regulated significantly. Preincubation with sesamin resulted in an evident enhancement of total antioxidant capacity in INS-1 cells, accompanied by a significant reduction of intracellular reactive oxygen species and malondialdehyde, an end product of lipid peroxidation. Taken together, these findings suggested that sesamin was capable of suppressing STZ induced INS-1 cell apoptosis, which might be ascribed, at least partly, to the inhibition of NF-κB activation and subsequent regulation of Bcl-2 family protein expression. This study would provide a potential target for treatment of diabetes with sesamin as well as other antioxidants.

  3. Impaired Homocysteine Transmethylation and Protein-Methyltransferase Activity Reduce Expression of Selenoprotein P: Implications for Obesity and Metabolic Syndrome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Obesity causes Metabolic Syndrome and Type-II Diabetes, disrupting hepatic function, methionine (Met)/homocysteine (Hcy) transmethylation and methyltransferase (PRMT) activities. Selenoprotein P (SEPP1), exported from the liver, is the predominate form of plasma selenium (Se) and the physiological S...

  4. Anisomycin uses multiple mechanisms to stimulate mitogen-activated protein kinases and gene expression and to inhibit neuronal differentiation in PC12 phaeochromocytoma cells.

    PubMed

    Törocsik, B; Szeberényi, J

    2000-02-01

    Treatment of PC12 cells with nerve growth factor (NGF) stimulates extracellular signal-regulated kinases (ERKs), as well as stress-activated c-Jun N-terminal kinases (JNKs) and p38 kinase, and induces neuronal differentiation. While the pivotal role of ERKs in NGF-induced morphological differentiation is well established, the contribution of JNK- and p38-pathways is less clear. The role of the JNK- and p38-pathway in PC12 cells was analysed by using anisomycin, a protein synthesis inhibitor that activates JNKs and p38. Non-toxic concentrations of anisomycin were found to stimulate these enzyme activities as well as the expression of the early response genes c-jun, c-fos and zif268, and to inhibit NGF-induced neurite formation. These effects of anisomycin appear to be mediated by the generation of reactive oxygen species (ROS), which in turn act through both TrkA/Ras-dependent and -independent signalling pathways. In addition, cross-talk between the p38- and ERK-pathways appears to play a role in the action of anisomycin.

  5. Ribonucleases in HIV type 1 inhibition: effect of recombinant RNases on infection of primary T cells and immune activation-induced RNase gene and protein expression.

    PubMed

    Bedoya, Victoria I; Boasso, Adriano; Hardy, Andrew W; Rybak, Susanna; Shearer, Gene M; Rugeles, Maria T

    2006-09-01

    Ribonucleases (RNases) have therapeutic potential against cancer and viral diseases and have been reported to inhibit replication of the human immunodeficiency virus type 1 (HIV-1) in chronically infected cell lines. The ribonuclease eosinophil-derived neurotoxin (EDN) is responsible for the anti-HIV-1 activity of a soluble factor produced in response to human alloantigens (ASF). Four recombinant RNases (EDN; a four amino acid extension of the N-terminus EDN, -4EDN; RNase A; and angiogenin) were tested for inhibition of HIV-1 replication in PHA blasts. All RNases showed anti-HIV-1 activity, irrespective of whether the RNases were added before, during, or 2 h after infection. Polyclonal antibodies against the four RNases blocked the antiviral activity. ASF inhibited HIV-1 replication in vitro if added up to 4 h after infection. We demonstrated that allostimulation induced EDN, RNase A, and angiogenin mRNA expression in peripheral blood mononuclear cells (PBMCs), although only EDN protein was detected. We identified monocytes and dendritic cells, but not macrophages or T cells, as EDN-producing cells. These findings raise the possibilities that multiple naturally occurring RNases may contribute to protection against HIV-1 infection and could be considered for utilization in HIV-1 therapy.

  6. Molecular insight into the activity of LasR protein from Pseudomonas aeruginosa in the regulation of virulence gene expression by this organism.

    PubMed

    Chowdhury, Nilkanta; Bagchi, Angshuman

    2016-04-10

    Pseudomonas aeruginosa is an opportunistic human pathogen. This organism attacks human patients suffering from diseases like AIDS, cancer, cystic fibrosis, etc. One of the important virulent factors produced by this organism is Hydrogen Cyanide. This is expressed from the genes encoded by the hcnABC operon. The expressions of the genes encoded by hcnABC operon are mediated mainly by the interactions of LasR protein with the corresponding promoter region of the hcnABC operon. The LasR protein acts as a dimer and binds to the promoter DNA with the help of an autoinducer ligand. However, till date the detailed molecular mechanism of how the LasR protein interacts with the promoter DNA is not clearly known. Therefore, in this work, an attempt has been made to analyze the mode of interactions of the LasR protein with the promoter DNA region of the hcnABC operon. We analyzed the three dimensional structure of the LasR protein from Pseudomonas aeruginosa and docked the protein with the autoinducer ligand. We then docked the ligand-bound-LasR-protein as well the LasR-protein-without-the-autoinducer-ligand on to the promoter DNA region of hcnABC operon. We analyzed the details of the interaction profiles of LasR protein with the autoinducer ligand. We also deciphered the details of the LasR promoter-DNA interactions. We compared the modes of DNA bindings by the LasR protein in presence and absence of the autoinducer ligand and tried to analyze the molecular details of the binding of LasR protein with the promoter DNA region of hcnABC operon during hcnABC gene expression. This study may therefore pave the pathway for future experiments to determine the relative effects of the amino acid residues of LasR protein in DNA binding during the transcription of hcnABC operon.

  7. Mitogen-activated protein kinase 6 mediates nuclear translocation of ORE3 to promote ORE9 gene expression in methyl jasmonate-induced leaf senescence.

    PubMed

    Zhang, Yushan; Liu, Jian; Chai, Jinyu; Xing, Da

    2016-01-01

    Methyl jasmonate (MeJA) is a potent promoter of plant senescence. ORESARA3 (ORE3)/ETHYLENE INSENSITIVE2 (EIN2), a protein similar to the members of the disease-related Nramp metal transporter family, is involved in cross-talk among several senescence processes related to abscisic acid, ethylene, MeJA, age and darkness. Nevertheless, the mechanism involved in the regulation of ORE3/EIN2 in exogenous MeJA-induced leaf senescence remains unclear. The C-terminal end of ORE3/EIN2 (CEND) was cleaved from ORE3/EIN2 located in the endoplasmic reticulum and then transferred to the nucleus during MeJA-induced senescence. Further analyses showed that mitogen-activated protein kinase 6 (MPK6) promoted CEND cleavage and nuclear translocation. Nuclear CEND accumulated ETHYLENE INSENSITIVE3 (EIN3), a transcription factor that accelerates MeJA-induced leaf senescence wherein ORESARA9 (ORE9) expression was suppressed in ein3, ore3, and mpk6 mutant plants. ChIP experiments revealed that EIN3 bound directly to the ORE9 promoter and this binding was enhanced in MeJA-induced leaf senescence. This study revealed the effect of the signalling pathway involving MPK6-ORE3-EIN3-ORE9 on regulating leaf senescence and provided insights into the mechanism of MeJA in promoting leaf senescence in Arabidopsis thaliana.

  8. Quantitative expression profiling of G-protein-coupled receptors (GPCRs) in metastatic melanoma: the constitutively active orphan GPCR GPR18 as novel drug target.

    PubMed

    Qin, Y; Verdegaal, E M E; Siderius, M; Bebelman, J P; Smit, M J; Leurs, R; Willemze, R; Tensen, C P; Osanto, S

    2011-02-01

    G-protein-coupled receptors (GPCRs) have been implicated in the tumorigenesis and metastasis of human cancers and are considered amongst the most desirable targets for drug development. Utilizing a robust quantitative PCR array, we quantified expression of 94 human GPCRs, including 75 orphan GPCRs and 19 chemokine receptors, and 36 chemokine ligands, in 40 melanoma metastases from different individuals and benign nevi. Inter-metastatic site comparison revealed that orphan GPR174 and CCL28 are statistically significantly overexpressed in subcutaneous metastases, while P2RY5 is overexpressed in brain metastases. Comparison between metastases (all three metastatic sites) and benign nevi revealed that 16 genes, including six orphan receptors (GPR18, GPR34, GPR119, GPR160, GPR183 and P2RY10) and chemokine receptors CCR5, CXCR4, and CXCR6, were statistically significantly differentially expressed. Subsequent functional experiments in yeast and melanoma cells indicate that GPR18, the most abundantly overexpressed orphan GPCR in all melanoma metastases, is constitutively active and inhibits apoptosis, indicating an important role for GPR18 in tumor cell survival. GPR18 and five other orphan GPCRs with yet unknown biological function may be considered potential novel anticancer targets in metastatic melanoma.

  9. Molecular cloning, expression of orange-spotted grouper goose-type lysozyme cDNA, and lytic activity of its recombinant protein.

    PubMed

    Yin, Zhi-Xin; He, Jian-Guo; Deng, Wei-Xin; Chan, Siu-Ming

    2003-07-08

    Lysozyme acts as a non-specific innate-immunity molecule against the invasion of bacteria pathogens. A leukocyte cDNA library of orange-spotted grouper Epinephelus coioides was constructed and the goose-type (g-type) lysozyme cDNA was isolated. The complete cDNA consists of an open reading frame of 585 bp encoding a protein of 194 amino acids. This protein shows a 72.2% amino acid sequence identity with the flounder g-type lysozyme. Similar to most other species, the glu catalytic residue in g-type lysozymes of the grouper is conserved. Furthermore, like the flounder and carp, the 4 conserved cysteine residues identified in avian and mammalian g-type lysozymes were also absent from the grouper. Northern blot analysis indicated that the g-type lysozyme was expressed in intestine, liver, spleen, anterior kidney, posterior kidney, heart, gill, muscle and leukocytes. In addition, RT-PCR analysis detected the g-type lysozyme transcripts in the stomach, brain and ovary. When an orange-spotted grouper was injected with Vibrio alginolyticus, the number of lysozyme mRNA transcripts detected in the stomach, spleen, anterior kidney, posterior kidney, heart, brain and leucocytes increased 72 h after injection. Recombinant grouper g-type lysozyme produced in the Escherichia coli expression system showed lytic activity against Micrococcus lysodeikticus, V. alginolyticus from Epinephelus fario, V. vulnificus from culture water, Aeromonas hydrophila from soft-shell turtle, A. hydrophila from goldfish and V. parahaemolyticus, Pseudomonas fluorescens and V. fluvialis from culture water.

  10. The activity, protein, and mRNA expression of CYP2E1 and CYP3A1 in rats after exposure to acute and chronic high altitude hypoxia.

    PubMed

    Li, Xiangyang; Wang, Xuejun; Li, Yongping; Zhu, Junbo; Su, Xiaodong; Yao, Xingchen; Fan, Xueru; Duan, Yabin

    2014-12-01

    The effects of exposure to acute and chronic high altitude hypoxia on the activity and expression of CYP2E1 and CYP3A1 were examined in rats. Rats were divided into low altitude (LA, 400 m), acute moderate altitude hypoxia (AMH, 2800 m), chronic moderate altitude hypoxia (CMH, 2800 m), acute high altitude hypoxia (AHH, 4300 m), and chronic high altitude hypoxia groups (CHH, 4300 m). Probe drugs were administrated orally to all five groups. Then the serum concentration of probe drug and its metabolite was determined by RP-HPLC. The activity of CYP2E1 and CYP3A1 was evaluated using the ratio of the metabolite to chlorzoxazone and testosterone, respectively. ELISA and real-time PCR were used to analyze the protein and mRNA expression of CYP2E1 and CYP3A1 in liver microsomes, respectively. Chronic high altitude hypoxia caused significant decreases in the activity and protein and mRNA expression of rat CYP2E1 and CYP3A1 in vivo. Acute high altitude hypoxia was not found to change the activity, protein or mRNA expression of rat CYP2E1 or CYP3A1. This study showed significant changes in the activity and protein and mRNA expression of CYP2E1 or CYP3A1 in rats after exposure to chronic high altitude hypoxia.

  11. Mitogen-Activated Protein Kinase Phosphatase-1 Is a Key Regulator of Hypoxia-Induced Vascular Endothelial Growth Factor Expression and Vessel Density in Lung

    PubMed Central

    Shields, Kristin M.; Panzhinskiy, Evgeniy; Burns, Nana; Zawada, W. Michael; Das, Mita

    2011-01-01

    Although mitogen-activated protein kinase phosphatase-1 (MKP-1) is a key deactivator of MAP kinases, known effectors of lung vessel formation, whether it plays a role in the expression of proangiogenic vascular endothelial growth factor (VEGF) in hypoxic lung is unknown. We therefore hypothesized that MKP-1 is a crucial modulator of hypoxia-stimulated vessel development by regulating lung VEGF levels. Wild-type MKP-1+/+, heterozygous MKP-1+/−, and deficient MKP-1−/− mice were exposed to sea level (SL), Denver altitude (DA) (1609 m [5280 feet]), and severe high altitude (HYP) (∼5182 m [∼17,000 feet]) for 6 weeks. Hypoxia enhanced phosphorylation of p38 MAP kinase, a substrate of MKP-1, as well as α smooth muscle actin (αSMA) expression in vessels, respiratory epithelium, and interstitium of phosphatase-deficient lung. αSMA-positive vessel (<50 μm outside diameter) densities were markedly reduced, whereas vessel wall thickness was increased in hypoxic MKP-1−/− lung. Mouse embryonic fibroblasts (MEFs) of all three genotypes were isolated to pinpoint the mechanism involved in hypoxia-induced vascular abnormalities of MKP-1−/− lung. Sustained phosphorylation of p38 MAP kinase was observed in MKP-1-null MEFs in response to hypoxia exposure. Although hypoxia up-regulated VEGF levels in MKP-1+/+ MEFs eightfold, only a 70% increase in VEGF expression was observed in MKP-1-deficient cells. Therefore, our data strongly suggest that MKP-1 might be the key regulator of vascular densities through the regulation of VEGF levels in hypoxic lung. PMID:21224048

  12. Expression analysis of genes encoding mitogen-activated protein kinases in maize provides a key link between abiotic stress signaling and plant reproduction.

    PubMed

    Sun, Wei; Chen, Hao; Wang, Juan; Sun, Hong Wei; Yang, Shu Ke; Sang, Ya Lin; Lu, Xing Bo; Xu, Xiao Hui

    2015-01-01

    Mitogen-activated protein kinases (MAPKs) play important roles in stress responses and development in plants. Maize (Zea mays), an important cereal crop, is a model plant species for molecular studies. In the last decade, several MAPKs have been identified in maize; however, their functions have not been studied extensively. Genome-wide identification and expression analysis of maize MAPK genes could provide valuable information for understanding their functions. In this study, 20 non-redundant maize MAPK genes (ZmMPKs) were identified via a genome-wide survey. Phylogenetic analysis of MAPKs from maize, rice (Oryza sativa), Arabidopsis (Arabidopsis thaliana), poplar (Populus trichocarpa), and tomato (Solanum lycopersicum) classified them into four major classes. ZmMPKs in the same class had similar domains, motifs, and genomic structures. Gene duplication investigations suggested that segmental duplications made a large contribution to the expansion of ZmMPKs. A number of cis-acting elements related to plant development and response to stress and hormones were identified in the promoter regions of ZmMPKs. Furthermore, transcript profile analysis in eight tissues and organs at various developmental stages demonstrated that most ZmMPKs were preferentially expressed in reproductive tissues and organs. The transcript abundance of most ZmMPKs changed significantly under salt, drought, cold, or abscisic acid (ABA) treatments, implying that they might participate in abiotic stress and ABA signaling. These expression analyses indicated that ZmMPKs might serve as linkers between abiotic stress signaling and plant reproduction. Our data will deepen our understanding of the complexity of the maize MAPK gene family and provide new clues to investigate their functions.

  13. Ketamine inhibits tumor necrosis factor-{alpha} and interleukin-6 gene expressions in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation

    SciTech Connect

    Wu, G.-J.; Chen, T.-L.; Ueng, Y.-F.; Chen, R.-M.

    2008-04-01

    Our previous study showed that ketamine, an intravenous anesthetic agent, has anti-inflammatory effects. In this study, we further evaluated the effects of ketamine on the regulation of tumor necrosis factor-{alpha} (TNF-{alpha}) and interlukin-6 (IL-6) gene expressions and its possible signal-transducing mechanisms in lipopolysaccharide (LPS)-activated macrophages. Exposure of macrophages to 1, 10, and 100 {mu}M ketamine, 100 ng/ml LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. A concentration of 1000 {mu}M of ketamine alone or in combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF-{alpha} and IL-6 protein levels in concentration- and time-dependent manners. Meanwhile, treatment with ketamine concentration- and time-dependently alleviated the enhanced effects. LPS induced TNF-{alpha} and IL-6 mRNA syntheses. Administration of ketamine at a therapeutic concentration (100 {mu}M) significantly inhibited LPS-induced TNF-{alpha} and IL-6 mRNA expressions. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA into macrophages decreased cellular TLR4 levels. Co-treatment of macrophages with ketamine and TLR4 siRNA decreased the LPS-induced TNF-{alpha} and IL-6 productions more than alone administration of TLR4 siRNA. LPS stimulated phosphorylation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos from the cytoplasm to nuclei. However, administration of ketamine significantly decreased LPS-induced activation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos. LPS increased the binding of nuclear extracts to activator protein-1 consensus DNA oligonucleotides. Administration of ketamine significantly ameliorated LPS-induced DNA binding activity of activator protein-1. Therefore, a clinically relevant concentration of ketamine can inhibit TNF-{alpha} and IL-6 gene expressions in LPS-activated macrophages. The suppressive mechanisms

  14. Ketamine inhibits tumor necrosis factor-alpha and interleukin-6 gene expressions in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation.

    PubMed

    Wu, Gone-Jhe; Chen, Ta-Liang; Ueng, Yune-Fang; Chen, Ruei-Ming

    2008-04-01

    Our previous study showed that ketamine, an intravenous anesthetic agent, has anti-inflammatory effects. In this study, we further evaluated the effects of ketamine on the regulation of tumor necrosis factor-alpha (TNF-alpha) and interlukin-6 (IL-6) gene expressions and its possible signal-transducing mechanisms in lipopolysaccharide (LPS)-activated macrophages. Exposure of macrophages to 1, 10, and 100 microM ketamine, 100 ng/ml LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. A concentration of 1000 microM of ketamine alone or in combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF-alpha and IL-6 protein levels in concentration- and time-dependent manners. Meanwhile, treatment with ketamine concentration- and time-dependently alleviated the enhanced effects. LPS induced TNF-alpha and IL-6 mRNA syntheses. Administration of ketamine at a therapeutic concentration (100 microM) significantly inhibited LPS-induced TNF-alpha and IL-6 mRNA expressions. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA into macrophages decreased cellular TLR4 levels. Co-treatment of macrophages with ketamine and TLR4 siRNA decreased the LPS-induced TNF-alpha and IL-6 productions more than alone administration of TLR4 siRNA. LPS stimulated phosphorylation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos from the cytoplasm to nuclei. However, administration of ketamine significantly decreased LPS-induced activation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos. LPS increased the binding of nuclear extracts to activator protein-1 consensus DNA oligonucleotides. Administration of ketamine significantly ameliorated LPS-induced DNA binding activity of activator protein-1. Therefore, a clinically relevant concentration of ketamine can inhibit TNF-alpha and IL-6 gene expressions in LPS-activated macrophages. The suppressive mechanisms occur through

  15. Nitric oxide-proton stimulation of trigeminal ganglion neurons increases mitogen-activated protein kinase and phosphatase expression in neurons and satellite glial cells.

    PubMed

    Freeman, S E; Patil, V V; Durham, P L

    2008-12-02

    Elevated nitric oxide (NO) and proton levels in synovial fluid are implicated in joint pathology. However, signaling pathways stimulated by these molecules that mediate inflammation and pain in the temporomandibular joint (TMJ) have not been investigated. The goal of this study was to determine the effect of NO-proton stimulation of rat trigeminal neurons on the in vivo expression of mitogen-activated protein kinases (MAPKs) and phosphatases (MKPs) in trigeminal ganglion neurons and satellite glial cells. Low levels of the active MAPKs extracellular signal-regulated kinase (ERK), Jun amino-terminal kinase (JNK), and p38 were localized in the cytosol of neurons and satellite glial cells in unstimulated animals. However, increased levels of active ERK and p38, but not JNK, were detected in the cytosol and nucleus of V3 neurons and satellite glial cells 15 min and 2 h following bilateral TMJ injections of an NO donor diluted in pH 5.5 medium. While ERK levels returned to near basal levels 24 h after stimulation, p38 levels remained significantly elevated. In contrast to MKP-2 and MKP-3 levels that were barely detectable in neurons or satellite glial cells, MKP-1 staining was readily observed in satellite glial cells in ganglia from unstimulated animals. However, neuronal and satellite glial cell staining for MKP-1, MKP-2, and MKP-3 was significantly increased in response to NO-protons. Increased active ERK and p38 levels as well as elevated MKP levels were also detected in neurons and satellite glial cells located in V2 and V1 regions of the ganglion. Our data provide evidence that NO-proton stimulation of V3 neurons results in temporal and spatial changes in expression of active ERK and p38 and MKPs in all regions of the ganglion. We propose that in trigeminal ganglia these cellular events, which are involved in peripheral sensitization as well as control of inflammatory and nociceptive responses, may play a role in TMJ pathology.

  16. The influence of cis-acting P1 protein and translational elements on the expression of Potato virus Y helper-component proteinase (HCPro) in heterologous systems and its suppression of silencing activity.

    PubMed

    Tena Fernández, Fátima; González, Inmaculada; Doblas, Paula; Rodríguez, César; Sahana, Nandita; Kaur, Harpreet; Tenllado, Francisco; Praveen, Shelly; Canto, Tomas

    2013-06-01

    In the Potyvirus genus, the P1 protein is the first N-terminal product processed from the viral polyprotein, followed by the helper-component proteinase (HCPro). In silencing suppression patch assays, we found that Potato virus Y (PVY) HCPro expressed from a P1-HCPro sequence increased the accumulation of a reporter gene, whereas protein expressed from an HCPro sequence did not, even with P1 supplied in trans. This enhancing effect of P1 has been noted in other potyviruses, but has remained unexplained. We analysed the accumulation of PVY HCPro in infiltrated tissues and found that it was higher when expressed from P1-HCPro than from HCPro sequences. Co-expression of heterologous suppressors increased the steady-state level of mRNA expressed from the HCPro sequence, but not that of protein. This suggests that, in the absence of P1 upstream, either HCPro acquires a conformation that affects negatively its activity or stability, or that its translation is reduced. To test these options, we purified HCPro expressed in the presence or absence of upstream P1, and found no difference in purification pattern and final soluble state. By contrast, alteration of the Kozak context in the HCPro mRNA sequence to favour translation increased partially suppressor accumulation and activity. Furthermore, protein activity was not lower than in protein expressed from P1-HCPro sequences. Thus, a direct role for P1 on HCPro suppressor activity or stability, by influencing its conformation during translation, can be excluded. However, P1 could still have an indirect effect favouring HCPro accumulation. Our data highlight the relevance of cis-acting translational elements in the heterologous expression of HCPro.

  17. Vitamin D Transport Proteins Megalin and Disabled-2 Are Expressed in Prostate and Colon Epithelial Cells and Are Induced and Activated by All-Trans-Retinoic Acid

    PubMed Central

    Ternes, Shantel B.; Rowling, Matthew J.

    2014-01-01

    Megalin and disabled-2 (Dab2) are essential for uptake of the 25-hydroxycholecalciferol (25D3)-vitamin D binding protein (DBP) complex in tissues. In the kidney, this mechanism regulates serum 25D3 levels and production of 1,25-dihydroxycholecalciferol (1,25D3) by CYP27B1 for systemic use. Previously, we showed that mammary epithelial cells expressing CYP27B1 express megalin and Dab2 and internalize DBP by endocytosis, indicating 25D3 was accessible for conversion to 1,25D3 in extra-renal tissues. Moreover, induction of megalin and Dab2 (protein and mRNA abundance) by all-trans-retinoic acid (RA) enhanced DBP uptake. This suggests megalin and Dab2 play a central role in uptake of vitamin D and may predict actions of vitamin D in extra-renal tissues. Here, we characterized megalin and Dab2 expression and uptake of DBP in transformed human prostate and colon epithelial cells. Megalin and Dab2 were expressed in prostate and colon epithelial cells, which was markedly enhanced following treatment with RA. Furthermore, DBP uptake was stimulated by low-dose RA supplementation in LNCaP, PC-3, and Caco-2 cells. Taken together, these are the first studies to our knowledge that have demonstrated modulated expression of megalin and Dab2, as well as an association between increased expression of endocytic proteins with DBP uptake in prostate and colon cells. PMID:23909735

  18. The pan-neural bHLH proteins DEADPAN and ASENSE regulate mitotic activity and cdk inhibitor dacapo expression in the Drosophila larval optic lobes.

    PubMed

    Wallace, K; Liu, T H; Vaessin, H

    2000-01-01

    Developmental regulators and cell cycle regulators have to interface in order to ensure appropriate cell proliferation during organogenesis. Our analysis of the roles of the pan-neural genes deadpan and asense defines critical roles for these genes in regulation of mitotic activities in the larval optic lobes. Loss of deadpan results in reduced cell proliferation, while ectopic deadpan expression causes over-proliferation. In contrast, loss of asense results in increased proliferation, while ectopic asense expression causes reduced proliferation. Consistent with these observations endogenous Deadpan is expressed in mitotic areas of the optic lobes, and endogenous Asense is expressed in cells that will become quiescent. Altered Deadpan or Asense expression results in altered expression of the cyclin dependent kinase inhibitor gene dacapo. Thus, regulation of mitotic activity during optic lobe development may, at least in part, involve deadpan and asense mediated regulation of the cyclin dependent kinase inhibitor gene dacapo. genesis 26:77-85, 2000.

  19. A plant cell-expressed recombinant anti-TNF fusion protein is biologically active in the gut and alleviates immune-mediated hepatitis and colitis.

    PubMed

    Ilan, Yaron; Gingis-Velitski, Svetlana; Ben Ya'aco, Ami; Shabbat, Yehudit; Zolotarov, Lidya; Almon, Einat; Shaaltiel, Yoseph

    2017-03-01

    The orally administered BY-2 plant cell-expressed recombinant anti-TNF fusion protein (PRX-106) (n=6) consists of the soluble form of the human TNF receptor (TNFR) fused to the Fc component of a human antibody IgG1 domain.

  20. Genome-Wide Survey and Expression Profile Analysis of the Mitogen-Activated Protein Kinase (MAPK) Gene Family in Brassica rapa.

    PubMed

    Lu, Kun; Guo, Wenjin; Lu, Junxing; Yu, Hao; Qu, Cunmin; Tang, Zhanglin; Li, Jiana; Chai, Yourong; Liang, Ying

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are fundamental signal transduction modules in plants, controlling cell division, development, hormone signaling, and biotic and abiotic stress responses. Although MAPKs have been investigated in several plant species, a comprehensive analysis of the MAPK gene family has hitherto not been performed in Brassica rapa. In this study, we identified 32 MAPKs in the B. rapa genome by conducting BLASTP and syntenic block analyses, and screening for the essential signature motif (TDY or TEY) of plant MAPK proteins. Of the 32 BraMAPK genes retrieved from the Brassica Database, 13 exhibited exon splicing errors, excessive splicing of the 5' sequence, excessive retention of the 5' sequence, and sequencing errors of the 3' end. Phylogenetic trees of the 32 corrected MAPKs from B. rapa and of MAPKs from other plants generated by the neighbor-joining and maximum likelihood methods suggested that BraMAPKs could be divided into four groups (groups A, B, C, and D). Gene number expansion was observed for BraMAPK genes in groups A and D, which may have been caused by the tandem duplication and genome triplication of the ancestral genome of the Brassica progenitor. Except for five members of the BraMAPK10 subfamily, the identified BraMAPKs were expressed in most of the tissues examined, including callus, root, stem, leaf, flower, and silique. Quantitative real-time PCR demonstrated that at least six and five BraMAPKs were induced or repressed by various abiotic stresses and hormone treatments, respectively, suggesting their potential roles in the abiotic stress response and various hormone signal transduction pathways in B. rapa. This study provides valuable insight into the putative physiological and biochemical functions of MAPK genes in B. rapa.

  1. Genome-Wide Survey and Expression Profile Analysis of the Mitogen-Activated Protein Kinase (MAPK) Gene Family in Brassica rapa

    PubMed Central

    Yu, Hao; Qu, Cunmin; Tang, Zhanglin; Li, Jiana; Chai, Yourong; Liang, Ying

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are fundamental signal transduction modules in plants, controlling cell division, development, hormone signaling, and biotic and abiotic stress responses. Although MAPKs have been investigated in several plant species, a comprehensive analysis of the MAPK gene family has hitherto not been performed in Brassica rapa. In this study, we identified 32 MAPKs in the B. rapa genome by conducting BLASTP and syntenic block analyses, and screening for the essential signature motif (TDY or TEY) of plant MAPK proteins. Of the 32 BraMAPK genes retrieved from the Brassica Database, 13 exhibited exon splicing errors, excessive splicing of the 5' sequence, excessive retention of the 5' sequence, and sequencing errors of the 3' end. Phylogenetic trees of the 32 corrected MAPKs from B. rapa and of MAPKs from other plants generated by the neighbor-joining and maximum likelihood methods suggested that BraMAPKs could be divided into four groups (groups A, B, C, and D). Gene number expansion was observed for BraMAPK genes in groups A and D, which may have been caused by the tandem duplication and genome triplication of the ancestral genome of the Brassica progenitor. Except for five members of the BraMAPK10 subfamily, the identified BraMAPKs were expressed in most of the tissues examined, including callus, root, stem, leaf, flower, and silique. Quantitative real-time PCR demonstrated that at least six and five BraMAPKs were induced or repressed by various abiotic stresses and hormone treatments, respectively, suggesting their potential roles in the abiotic stress response and various hormone signal transduction pathways in B. rapa. This study provides valuable insight into the putative physiological and biochemical functions of MAPK genes in B. rapa. PMID:26173020

  2. The fruit of Acanthopanax senticosus (Rupr. et Maxim.) Harms improves insulin resistance and hepatic lipid accumulation by modulation of liver adenosine monophosphate-activated protein kinase activity and lipogenic gene expression in high-fat diet-fed obese mice.

    PubMed

    Saito, Tetsuo; Nishida, Miyako; Saito, Masafumi; Tanabe, Akari; Eitsuka, Takahiro; Yuan, Shi-Hua; Ikekawa, Nobuo; Nishida, Hiroshi

    2016-10-01

    Obesity-associated insulin resistance is a major risk factor for most metabolic diseases, including dyslipidemia and type 2 diabetes. Acanthopanax senticosus (Rupr. et Maxim.) Harms (Goka) root has been used in traditional Chinese medicine for treatment of diabetes and other conditions; however, little is known about the effects of Goka fruit (GF). Goka fruit is rich in anthocyanin, which has beneficial effects on obesity and insulin resistance via activation of adenosine monophosphate-activated protein kinase (AMPK). We hypothesized that GF can improve obesity-associated insulin resistance. The aim of the present study was to investigate whether GF improves insulin resistance in high-fat diet (HFD)-induced obese mice. High-fat diet mice treated with GF (500 and 1000 mg/kg) for 12 weeks showed an improved glucose tolerance and insulin sensitivity, as well as reduced plasma insulin and liver lipid accumulation. Moreover, GF administration to HFD mice resulted in down-regulation of fatty acid synthase expression and up-regulation of cholesterol 7-alpha-hydroxylase expression in the liver. Notably, AMPK phosphorylation in the liver increased after GF administration. In summary, GF supplementation improved obesity-associated insulin resistance and hepatic lipid accumulation through modulation of AMPK activity and lipid metabolism-associated gene expression.

  3. Expression and activation of platelet-derived growth factor β receptor, mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) in canine mammary tumours.

    PubMed

    Altamura, Gennaro; Uberti, Barbara Degli; Galiero, Giorgio; Martano, Manuela; Pirro, Antonella; Russo, Marco; Borzacchiello, Giuseppe

    2017-02-01

    Canine mammary tumours are frequent neoplasms mostly affecting intact female dogs, for which no 100% efficient therapy is available. Platelet derived growth factor β receptor (PDGFβR) is a tyrosine kinase receptor (TKR) with a potential role in human breast cancer and a series of canine tumours. In this study we demonstrated, for the first time, expression of PDGFβR and its downstream transduction molecules, mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) and extracellular signal-regulated kinase (ERK), as well as their activated forms in canine mammary tumours by both biochemical analysis and immunohistochemistry. PDGFβR was expressed and hyperphosphorylated in the majority of tumour samples and tumour derived cell lines. Additionally, both MEK and ERK were expressed and activated in cell lines as well as biopsies. TKR inhibitors (TKRi) are currently under investigation as possible therapy in human breast and several canine tumours, thus our in vivo and in vitro findings pave the way for future studies aimed at establishing a potential therapeutic employment of TKRi for the treatment of canine mammary cancer.

  4. Transient Protein Expression by Agroinfiltration in Lettuce.

    PubMed

    Chen, Qiang; Dent, Matthew; Hurtado, Jonathan; Stahnke, Jake; McNulty, Alyssa; Leuzinger, Kahlin; Lai, Huafang

    2016-01-01

    Current systems of recombinant protein production include bacterial, insect, and mammalian cell culture. However, these platforms are expensive to build and operate at commercial scales and/or have limited abilities to produce complex proteins. In recent years, plant-based expression systems have become top candidates for the production of recombinant proteins as they are highly scalable, robust, safe, and can produce complex proteins due to having a eukaryotic endomembrane system. Newly developed "deconstructed" viral vectors delivered via Agrobacterium tumefaciens (agroinfiltration) have enabled robust plant-based production of proteins with a wide range of applications. The leafy Lactuca sativa (lettuce) plant with its strong foundation in agriculture is an excellent host for pharmaceutical protein production. Here, we describe a method for agroinfiltration of lettuce that can rapidly produce high levels of recombinant proteins in a matter of days and has the potential to be scaled up to an agricultural level.

  5. Integral Membrane Protein Expression in Saccharomyces cerevisiae.

    PubMed

    Boswell-Casteel, Rebba C; Johnson, Jennifer M; Stroud, Robert M; Hays, Franklin A

    2016-01-01

    Eukaryotic integral membrane proteins are challenging targets for crystallography or functional characterization in a purified state. Since expression is often a limiting factor when studying this difficult class of biological macromolecules, the intent of this chapter is to focus on the expression of eukaryotic integral membrane proteins (IMPs) using the model organism Saccharomyces cerevisiae. S. cerevisiae is a prime candidate for the expression of eukaryotic IMPs because it offers the convenience of using episomal expression plasmids, selection of positive transformants, posttranslational modifications, and it can properly fold and target IMPs. Here we present a generalized protocol and insights based on our collective knowledge as an aid to overcoming the challenges faced when expressing eukaryotic IMPs in S. cerevisiae.

  6. Transferrin receptor 2 and HFE regulate furin expression via mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/Erk) signaling. Implications for transferrin-dependent hepcidin regulation

    PubMed Central

    Poli, Maura; Luscieti, Sara; Gandini, Valentina; Maccarinelli, Federica; Finazzi, Dario; Silvestri, Laura; Roetto, Antonella; Arosio, Paolo

    2010-01-01

    Background Impaired regulation of hepcidin in response to iron is the cause of genetic hemochromatosis associated with defects of HFE and transferrin receptor 2. However, the role of these proteins in the regulation of hepcidin expression is unclear. Design and Methods Hepcidin expression, SMAD and extracellular signal-regulated kinase (Erk) phosphorylation and furin expression were analyzed in hepatic HepG2 cells in which HFE and transferrin receptor 2 were down-regulated or expressed, or furin activity specifically inhibited. Furin expression was also analyzed in the liver of transferrin receptor 2 null mice. Results We showed that the silencing of HFE and transferrin receptor 2 reduced both Erk phosphorylation and furin expression, that the exogenous expression of the two enhanced the induction of phosphoErk1/2 and furin by holotransferrin, but that this did not occur when the pathogenic HFE mutant C282Y was expressed. Furin, phosphoErk1/2 and phosphoSMAD1/5/8 were down-regulated also in transferrin receptor 2-null mice. Treatment of HepG2 cells with an inhibitor of furin activity caused a strong suppression of hepcidin mRNA, probably due to the inhibition of bone morphogenic protein maturation. Conclusions The data indicate that transferrin receptor 2 and HFE are involved in holotransferrin-dependent signaling for the regulation of furin which involved Erk phosphorylation. Furin in turn may control hepcidin expression. PMID:20634490

  7. Promoter-dependent and -independent activation of insulin-like growth factor binding protein-5 gene expression by prostaglandin E2 in primary rat osteoblasts

    NASA Technical Reports Server (NTRS)

    McCarthy, T. L.; Casinghino, S.; Mittanck, D. W.; Ji, C. H.; Centrella, M.; Rotwein, P.

    1996-01-01

    Insulin-like growth factor (IGF) action is mediated by high affinity cell surface IGF receptors and modulated by a family of secreted IGF binding proteins (IGFBPs). IGFBP-5, the most conserved of six IGFBPs characterized to date, uniquely potentiates the anabolic actions of IGF-I for skeletal cells. In osteoblasts, IGFBP-5 production is stimulated by prostaglandin E2 (PGE2), a local factor that mediates certain effects induced by parathyroid hormone, cytokines such as interleukin-1 and transforming growth factor-beta, and mechanical strain. In this study, we show that transcriptional and post-transcriptional events initiated by PGE2 collaborate to enhance IGFBP-5 gene expression in primary fetal rat osteoblast cultures. PGE2 treatment stimulated up to a 7-fold rise in steady-state levels of IGFBP-5 mRNA throughout 32 h of incubation. Analysis of nascent IGFBP-5 mRNA suggested that PGE2 had only a modest stimulatory effect on IGFBP-5 gene transcription, and transient transfection studies with IGFBP-5 promoter-reporter genes confirmed that PGE2 enhanced promoter activity by approximately 2-fold. Similar stimulatory effects were seen with forskolin. A DNA fragment with only 51 base pairs of the 5'-flanking sequence retained hormonal responsiveness, which may be mediated by a binding site for transcription factor AP-2 located at positions -44 to -36 in the proximal IGFBP-5 promoter. Incubation of osteoblasts with the mRNA transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that PGE2 enhanced IGFBP-5 mRNA stability by 2-fold, increasing the t1/2 from 9 to 18 h. The effects of PGE2 on steady-state IGFBP-5 transcripts were abrogated by preincubating cells with cycloheximide, indicating that the effects of PGE2 on both gene transcription and mRNA stability required ongoing protein synthesis. Therefore, both promoter-dependent and -independent pathways converge to enhance IGFBP-5 gene expression in response to PGE2 in osteoblasts.

  8. Leucine modulates the effect of Walker factor, a proteolysis-inducing factor-like protein from Walker tumours, on gene expression and cellular activity in C2C12 myotubes.

    PubMed

    Gonçalves, Estela Maria; Salomão, Emilianne Miguel; Gomes-Marcondes, Maria Cristina Cintra

    2013-10-01

    Cancer-cachexia causes severe weight loss, particularly from the wasting of skeletal muscle, which occurs due to increased protein catabolism and/or decreased protein synthesis. The muscle protein degradation observed in cancer patients is mediated by a specific cytokine, proteolysis-inducing factor (PIF), which is produced by the tumour. This protein increases the ubiquitin-proteasome pathway activity, and the synthesis of muscle protein in these patients can be affected by several factors, including nutrient-related signalling. Some nutrients, such as leucine, can decrease the ubiquitin-proteasome pathway activity and increase the skeletal muscle protein content in cachectic animals. In this study, we investigated the effects of leucine on cell viability, morphology, functional proteasome activity, enzymatic activity, and protein synthesis and degradation in C2C12 myotubes exposed to the proteolysis-inducing factor (PIF)-like protein purified from Walker tumour-bearing rats. Walker factor (WF) had no cytotoxic effects on myotube cells and morphological characteristics were not altered in the presence of WF and/or leucine. However, increased alkaline phosphatase activity was observed. At higher WF concentrations, chymotrypsin-like activity, cathepsin B activity and 20S proteasome gene expression increased. Treating myotubes with leucine before exposure to WF causes leads to a decrease in proteasome activity as well as the activity of chymotrypsin and cathepsin enzymes. Total protein synthesis decreased in WF-treated cells concomitantly as protein degradation increased. After leucine exposure, the observed effects of WF were minimal or even reverted in some cases. Taken together, these results suggest an important modulatory effect for leucine on the effects of WF in C2C12 myotube cells.

  9. Biotechnology Protein Expression and Purification Facility

    NASA Technical Reports Server (NTRS)

    2003-01-01

    The purpose of the Project Scientist Core Facility is to provide purified proteins, both recombinant and natural, to the Biotechnology Science Team Project Scientists and the NRA-Structural Biology Test Investigators. Having a core facility for this purpose obviates the need for each scientist to develop the necessary expertise and equipment for molecular biology, protein expression, and protein purification. Because of this, they are able to focus their energies as well as their funding on the crystallization and structure determination of their target proteins.

  10. Constitutive Activation of the G-Protein Subunit G[alpha]s within Forebrain Neurons Causes PKA-Dependent Alterations in Fear Conditioning and Cortical "Arc" mRNA Expression

    ERIC Educational Resources Information Center

    Kelly, Michele P.; Cheung, York-Fong; Favilla, Christopher; Siegel, Steven J.; Kanes, Stephen J.; Houslay, Miles D.; Abel, Ted

    2008-01-01

    Memory formation requires cAMP signaling; thus, this cascade has been of great interest in the search for cognitive enhancers. Given that medications are administered long-term, we determined the effects of chronically increasing cAMP synthesis in the brain by expressing a constitutively active isoform of the G-protein subunit G[alpha]s…

  11. Separating proteins with activated carbon.

    PubMed

    Stone, Matthew T; Kozlov, Mikhail

    2014-07-15

    Activated carbon is applied to separate proteins based on differences in their size and effective charge. Three guidelines are suggested for the efficient separation of proteins with activated carbon. (1) Activated carbon can be used to efficiently remove smaller proteinaceous impurities from larger proteins. (2) Smaller proteinaceous impurities are most efficiently removed at a solution pH close to the impurity's isoelectric point, where they have a minimal effective charge. (3) The most efficient recovery of a small protein from activated carbon occurs at a solution pH further away from the protein's isoelectric point, where it is strongly charged. Studies measuring the binding capacities of individual polymers and proteins were used to develop these three guidelines, and they were then applied to the separation of several different protein mixtures. The ability of activated carbon to separate proteins was demonstrated to be broadly applicable with three different types of activated carbon by both static treatment and by flowing through a packed column of activated carbon.

  12. Preserved Expression of mRNA Coding von Willebrand Factor–Cleaving Protease ADAMTS13 by Selenite and Activated Protein C

    PubMed Central

    Ekaney, Michael L; Bockmeyer, Clemens L; Sossdorf, Maik; Reuken, Philipp A; Conradi, Florian; Schuerholz, Tobias; Blaess, Markus F; Friedman, Scott L; Lösche, Wolfgang; Bauer, Michael; Claus, Ralf A

    2015-01-01

    In sepsis, the severity-dependent decrease of von Willebrand factor (VWF)–inactivating protease, a disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13), results in platelet aggregation and consumption, leading to sepsis-associated thrombotic microangiopathy (TMA) and organ failure. Previous reports assessing its functional deficiency have pinpointed involvement of autoantibodies or mutations to propagate thrombotic thrombocytopenic purpura (TTP). However, mechanisms of acquired ADAMTS13 deficiency during host response remain unclear. To enhance understanding of ADAMTS13 deficiency in sepsis, we evaluated changes in expression of mRNA coding ADAMTS13 during septic conditions using primary cellular sources of the protease. We hypothesized that proinflammatory cytokines and constituents of serum from septic patients affect the transcriptional level of ADAMTS13 in vitro, and previously recommended therapeutic agents as adjunctive therapy for sepsis interact therewith. Cultured hepatic stellate cells (HSCs), endothelial cells (HMEC) and human precision-cut liver slices as an ex vivo model were stimulated with sepsis prototypic cytokines, bacterial endotoxin and pooled serum obtained from septic patients. Stimulation resulted in a significant decrease in ADAMTS13 mRNA between 10% and 80% of basal transcriptional rates. Costimulation of selenite or recombinant activated protein C (APC) with serum prevented ADAMTS13 decrease in HSCs and increased ADAMTS13 transcripts in HMEC. In archived clinical samples, the activity of ADAMTS13 in septic patients treated with APC (n = 5) increased with an accompanying decrease in VWF propeptide as surrogate for improved endothelial function. In conclusion, proinflammatory conditions of sepsis repress mRNA coding ADAMTS13 and the ameliorating effect by selenite and APC may support the concept for identification of beneficial mechanisms triggered by these drugs at a molecular level. PMID:25860876

  13. Preserved Expression of mRNA Coding von Willebrand Factor-Cleaving Protease ADAMTS13 by Selenite and Activated Protein C.

    PubMed

    Ekaney, Michael L; Bockmeyer, Clemens L; Sossdorf, Maik; Reuken, Philipp A; Conradi, Florian; Schuerholz, Tobias; Blaess, Markus F; Friedman, Scott L; Lösche, Wolfgang; Bauer, Michael; Claus, Ralf A

    2015-04-03

    In sepsis, the severity-dependent decrease of von Willebrand factor (VWF)-inactivating protease, a disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13), results in platelet aggregation and consumption, leading to sepsis-associated thrombotic microangiopathy (TMA) and organ failure. Previous reports assessing its functional deficiency have pinpointed involvement of autoantibodies or mutations to propagate thrombotic thrombocytopenic purpura (TTP). However, mechanisms of acquired ADAMTS13 deficiency during host response remain unclear. To enhance understanding of ADAMTS13 deficiency in sepsis, we evaluated changes in expression of mRNA coding ADAMTS13 during septic conditions using primary cellular sources of the protease. We hypothesized that proinflammatory cytokines and constituents of serum from septic patients affect the transcriptional level of ADAMTS13 in vitro, and previously recommended therapeutic agents as adjunctive therapy for sepsis interact therewith. Cultured hepatic stellate cells (HSCs), endothelial cells (HMEC) and human precision-cut liver slices as an ex vivo model were stimulated with sepsis prototypic cytokines, bacterial endotoxin and pooled serum obtained from septic patients. Stimulation resulted in a significant decrease in ADAMTS13 mRNA between 10% and 80% of basal transcriptional rates. Costimulation of selenite or recombinant activated protein C (APC) with serum prevented ADAMTS13 decrease in HSCs and increased ADAMTS13 transcripts in HMEC. In archived clinical samples, the activity of ADAMTS13 in septic patients treated with APC (n = 5) increased with an accompanying decrease in VWF propeptide as surrogate for improved endothelial function. In conclusion, proinflammatory conditions of sepsis repress mRNA coding ADAMTS13 and the ameliorating effect by selenite and APC may support the concept for identification of beneficial mechanisms triggered by these drugs at a molecular level.

  14. Enhanced membrane protein expression by engineering increased intracellular membrane production

    PubMed Central

    2013-01-01

    Background Membrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different expression systems, like mammalian cells, insect cells, bacteria and yeast are being used, but very few research efforts have been directed towards specific host cell customization for enhanced expression of membrane proteins. Here we show that by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis, enhanced expression of membrane proteins in yeast is achieved. Results We engineered the oleotrophic yeast, Yarrowia lipolytica, by deleting the phosphatidic acid phosphatase, PAH1, which led to massive proliferation of endoplasmic reticulum (ER) membranes. For all eight tested representatives of different integral membrane protein families, we obtained enhanced protein accumulation levels and in some cases enhanced proteolytic integrity in the ∆pah1 strain. We analysed the adenosine A2AR G-protein coupled receptor case in more detail and found that concomitant induction of the unfolded protein response in the ∆pah1 strain enhanced the specific ligand binding activity of the receptor. These data indicate an improved quality control mechanism for membrane proteins accumulating in yeast cells with proliferated ER. Conclusions We conclude that redirecting the metabolic flux of fatty acids away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis by PAH1 gene inactivation, provides a valuable approach to enhance eukaryotic membrane protein production. Complementary to this improvement in membrane protein quantity, UPR co-induction further enhances the quality of the membrane protein in terms of its proper folding and biological activity. Importantly, since these pathways are conserved in all eukaryotes, it will be of interest to investigate similar engineering approaches in other cell types of

  15. Mitogen-activated protein kinases and phosphatidylinositol 3-kinase are involved in Prevotella intermedia-induced proinflammatory cytokines expression in human periodontal ligament cells.

    PubMed

    Guan, Su-Min; Zhang, Ming; He, Jian-Jun; Wu, Jun-Zheng

    2009-08-28

    Chronic periodontitis is an inflammatory disease affecting periodontal connective tissues and alveolar bone. Proinflammatory mediators induced by periodontal pathogens play vital roles in the initiation and progression of the disease. In this study, we examined whether Prevotella intermedia induces proinflammatory cytokines expression in human periodontal ligament cells (hPDLs). The mRNA expression and protein production were determined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbant assay (ELISA) respectively. P. intermedia treatment dose- and time-dependently increased IL-6, IL-8 and M-CSF, but not IL-1beta and TNF-alpha mRNA expression and protein secretion. Preincubation of hPDLs with extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 kinase and phosphatidylinositol 3-kinase (PI3K) inhibitors PD98059, SP600125, SB203580 and LY294002 resulted in significant reduction in P. intermedia-induced IL-6, IL-8 and M-CSF expression. Blocking the synthesis of prostaglandin E(2) (PGE(2)) by indomethacin also abolished the stimulatory effects of P. intermedia on cytokines expression. Our results indicate that P. intermedia induces proinflammatory cytokines through MAPKs and PI3K signaling pathways, and PGE(2) is involved in the P. intermedia-induced proinflammatory cytokines upregulation.

  16. Expression and biochemical characterization of recombinant human epididymis protein 4.

    PubMed

    Hua, Ling; Liu, Yunhui; Zhen, Shuai; Wan, Deyou; Cao, Jiyue; Gao, Xin

    2014-10-01

    Whey acidic proteins (WAP) belong to a large gene family of antibacterial peptides that perform critical immune system functions. The function of human epididymis protein 4 (HE4), a 124-amino acid long polypeptide that has two whey acidic protein four-disulfide core (WFDC) domains, is not well studied. Here, a fusion gene encoding the HE4 protein fused to an IgG1 Fc domain was constructed. The recombinant HE4 protein was expressed as a secretory protein in Pichia pastoris and mammalian HEK293-F cells and was subsequently purified. Our data suggested that the HE4 protein produced by these two expression systems bound to both gram-negative and gram-positive bacteria, but demonstrated slightly inhibitory activity towards the growth of Staphylococcus aureus. Moreover, HE4 exhibited proteinase inhibitory activity towards trypsin, elastase, matrix metallopeptidase 9, and the secretory proteinases from Bacillus subtilis. The effects of glycosylation on the biochemical characterization of HE4 were also investigated. LC-ESI-MS glycosylation analysis showed that the high-mannose glycosylated form of HE4 expressed by P. pastoris has lower biological activity when compared to its complex-glycosylated form produced from HEK293-F cells. The implications of this are discussed, which may be provide theoretical basis for its important role in the development of cancer and innate immune system.

  17. Grape seed extract inhibits VEGF expression via reducing HIF-1α protein expression

    PubMed Central

    Lu, Jianming; Zhang, Keqiang; Chen, Shiuan; Wen, Wei

    2009-01-01

    Grape seed extract (GSE) is a widely consumed dietary supplement that has antitumor activity. Here, we have investigated the inhibitory effect of GSE on the expression of vascular endothelial growth factor (VEGF) and the mechanism underlying this action. We found that GSE inhibited VEGF messenger RNA (mRNA) and protein expression in U251 human glioma cells and MDA-MB-231 human breast cancer cells. GSE inhibited transcriptional activation of the VEGF gene through reducing protein but not mRNA expression of hypoxia-inducible factor (HIF) 1α. The inhibitory effect of GSE on HIF-1α expression was mainly through inhibiting HIF-1α protein synthesis rather than promoting protein degradation. Consistent with this result, GSE-suppressed phosphorylation of several important components involved in HIF-1α protein synthesis, such as Akt, S6 kinase and S6 protein. Furthermore, in the MDA-MB-231 tumor, we found that GSE treatment inhibited the expression of VEGF and HIF-1α and the phosphorylation of S6 kinase without altering the subcellular localization of HIF-1α, correlating with reduced vessel density and tumor size. Depletion of polyphenol with polyvinylpyrrolidone abolished the inhibitory activity of GSE, suggesting a water-soluble fraction of polyphenol in GSE is responsible for the inhibitory activity. Taken together, our results indicate that GSE inhibits VEGF expression by reducing HIF-1α protein synthesis through blocking Akt activation. This finding provides new insight into the mechanisms of anticancer activity of GSE and reveals a novel molecular mechanism underlying the antiangiogenic action of GSE. PMID:19131542

  18. Activation of NFAT-Dependent Gene Expression by Nef: Conservation among Divergent Nef Alleles, Dependence on SH3 Binding and Membrane Association, and Cooperation with Protein Kinase C-θ

    PubMed Central

    Manninen, Aki; Huotari, Päivi; Hiipakka, Marita; Renkema, G. Herma; Saksela, Kalle

    2001-01-01

    Here we show that the potential to regulate NFAT is a conserved property of different Nef alleles and that Nef residues involved in membrane targeting and SH3 binding are critical for this function. Cotransfection of an activated protein kinase C-θ (PKC-θ) with Nef implicated PKC-θ as a possible physiological cofactor of Nef in promoting NFAT-dependent gene expression and T-cell activation. PMID:11222731

  19. miR-155 activates cytokine gene expression in Th17 cells by regulating the DNA-binding protein Jarid2 to relieve polycomb-mediated repression.

    PubMed

    Escobar, Thelma M; Kanellopoulou, Chrysi; Kugler, David G; Kilaru, Gokhul; Nguyen, Cuong K; Nagarajan, Vijayaraj; Bhairavabhotla, Ravikiran K; Northrup, Daniel; Zahr, Rami; Burr, Patrick; Liu, Xiuhuai; Zhao, Keji; Sher, Alan; Jankovic, Dragana; Zhu, Jinfang; Muljo, Stefan A

    2014-06-19

    Specification of the T helper 17 (Th17) cell lineage requires a well-defined set of transcription factors, but how these integrate with posttranscriptional and epigenetic programs to regulate gene expression is poorly understood. Here we found defective Th17 cell cytokine expression in miR-155-deficient CD4+ T cells in vitro and in vivo. Mir155 was bound by Th17 cell transcription factors and was highly expressed during Th17 cell differentiation. miR-155-deficient Th17 and T regulatory (Treg) cells expressed increased amounts of Jarid2, a DNA-binding protein that recruits the Polycomb Repressive Complex 2 (PRC2) to chromatin. PRC2 binding to chromatin and H3K27 histone methylation was increased in miR-155-deficient cells, coinciding with failure to express Il22, Il10, Il9, and Atf3. Defects in Th17 cell cytokine expression and Treg cell homeostasis in the absence of Mir155 could be partially suppressed by Jarid2 deletion. Thus, miR-155 contributes to Th17 cell function by suppressing the inhibitory effects of Jarid2.

  20. miR-155 activates cytokine gene expression in Th17 cells by regulating the DNA-binding protein Jarid2 to relieve Polycomb-mediated repression

    PubMed Central

    Escobar, Thelma M.; Kanellopoulou, Chrysi; Kugler, David G.; Kilaru, Gokhul; Nguyen, Cuong K.; Nagarajan, Vijayaraj; Bhairavabhotla, Ravikiran K.; Northrup, Daniel; Zahr, Rami; Burr, Patrick; Liu, Xiuhuai; Zhao, Keji; Sher, Alan; Jankovic, Dragana; Zhu, Jinfang; Muljo, Stefan A.

    2014-01-01

    Specification of the T helper 17 (Th17) cell lineage requires a well defined set of transcription factors, but how these integrate with post-transcriptional and epigenetic programs to regulate gene expression is poorly understood. Here we found defective Th17 cell cytokine expression in miR-155-deficient CD4+ T cells in vitro and in vivo. Mir155 was bound by Th17 cell transcription factors and was highly expressed during Th17 cell differentiation. miR-155-deficient-Th17 and -T regulatory (Treg) cells expressed increased amounts of Jarid2, a DNA-binding protein that recruits the Polycomb Repressive Complex 2 (PRC2) to chromatin. PRC2 binding to chromatin and H3K27 histone methylation was increased in miR-155-deficient cells, coinciding with failure to express Il22, Il10, Il9 and Atf3. Defects in Th17 cell cytokine expression and Treg cell homeostasis in the absence of Mir155 could be partially suppressed by Jarid2 deletion. Thus, miR-155 contributes to Th17 cell function by suppressing the inhibitory effects of Jarid2. PMID:24856900

  1. Alpha 2-chimerin, an SH2-containing GTPase-activating protein for the ras-related protein p21rac derived by alternate splicing of the human n-chimerin gene, is selectively expressed in brain regions and testes.

    PubMed Central

    Hall, C; Sin, W C; Teo, M; Michael, G J; Smith, P; Dong, J M; Lim, H H; Manser, E; Spurr, N K; Jones, T A

    1993-01-01

    n-Chimerin (alpha 1-chimerin) is a brain GTPase-activating protein (GAP) for the ras-related p21rac. We now report the occurrence of another form of chimerin, termed alpha 2-chimerin. This is the product of an alternately spliced transcript of the human n-chimerin gene encoding an N-terminal SH2 (src homology 2) domain in addition to the phorbol ester receptor and GAP domains. alpha 1- and alpha 2-chimerin mRNAs were expressed differently. In the rat brain, only alpha 1-chimerin mRNA was expressed in cerebellar Purkinje cells, although both alpha 1- and alpha 2-chimerin mRNAs occurred in neurons in the cerebral cortex, hippocampus, and thalamus. Only alpha 2-chimerin RNA was expressed in rat testes, in early pachytene spermatocytes. A 45-kDa SH2-containing chimerin corresponding to the alpha 2 form was purified from rat brain. As with Escherichia coli 45-kDa recombinant alpha 2-chimerin, purified brain alpha 2-chimerin exhibited racGAP activity which was stimulated by phosphatidylserine. The recombinant SH2 domain bound several 32P-labelled phosphoproteins of PC12 cells, whose phosphorylation increased in response to trophic factors, including nerve growth factor. To examine the relationships of alpha 1- and alpha 2-chimerin transcripts, human genomic DNA clones were characterized. In alpha 2-chimerin mRNA, a 3' splice acceptor site within exon 1 of alpha 1-chimerin mRNA was used, replacing its 5' untranslated region and N-terminal coding sequence. The single human n-chimerin gene was mapped to chromosome 2q31-q32.1, colocalizing with the CRE-BP1 transcription factor gene (2q32). It contained several splice junctions conserved with the sequence-related protein kinase C and bcr genes. alpha 2-Chimerin is only the second SH2-containing GAP and the first example of an SH2 domain generated by alternate splicing. Images PMID:8336731

  2. ApoER2 and Reelin are expressed in regenerating peripheral nerve and regulate Schwann cell migration by activating the Rac1 GEF protein, Tiam1.

    PubMed

    Pasten, Consuelo; Cerda, Joaquín; Jausoro, Ignacio; Court, Felipe A; Cáceres, Alfredo; Marzolo, Maria-Paz

    2015-11-01

    ApoER2 and its ligand Reelin participate in neuronal migration during development. Upon receptor binding, Reelin induces the proteolytic processing of ApoER2 as well as the activation of signaling pathway, including small Rho GTPases. Besides its presence in the central nervous system (CNS), Reelin is also secreted by Schwann cells (SCs), the glial cells of the peripheral nervous system (PNS). Reelin deficient mice (reeler) show decreased axonal regeneration in the PNS; however neither the presence of ApoER2 nor the role of the Reelin signaling pathway in the PNS have been evaluated. Interestingly SC migration occurs during PNS development and during injury-induced regeneration and involves activation of small Rho GTPases. Thus, Reelin-ApoER2 might regulate SC migration during axon regeneration in the PNS. Here we demonstrate the presence of ApoER2 in PNS. After sciatic nerve injury Reelin was induced and its receptor ApoER2 was proteolytically processed. In vitro, SCs express both Reelin and ApoER2 and Reelin induces SC migration. To elucidate the molecular mechanism underlying Reelin-dependent SC migration, we examined the involvement of Rac1, a conspicuous small GTPase family member. FRET experiments revealed that Reelin activates Rac1 at the leading edge of SCs. In addition, Tiam1, a major Rac1-specific GEF was required for Reelin-induced SC migration. Moreover, Reelin-induced SC migration was decreased after suppression of the polarity protein PAR3, consistent with its association to Tiam1. Even more interesting, we demonstrated that PAR3 binds preferentially to the full-length cytoplasmic tail of ApoER2 corresponding to the splice-variant containing the exon 19 that encodes a proline-rich insert and that ApoER2 was required for SC migration. Our study reveals a novel function for Reelin/ApoER2 in PNS, inducing cell migration of SCs, a process relevant for PNS development and regeneration.

  3. Constitutive expression and enzymatic activity of Tan protein in brain and epidermis of Ceratitis capitata and of Drosophila melanogaster wild-type and tan mutants.

    PubMed

    Pérez, M M; Sabio, G; Badaracco, A; Quesada-Allué, L A

    2011-09-01

    The present report shows a partial biochemical characterization and life cycle expression of N-β-alanyldopamine hydrolase (Tan protein) in Ceratitis capitata and Drosophila melanogaster. This enzyme catalyzes the hydrolysis of N-β-alanyldopamine (NBAD), the main tanning precursor of insect brown cuticles. It also plays an important role in the metabolism of brain neurotransmitters, recycling dopamine and histamine. In contrast to NBAD-synthase, Tan is expressed constitutively in epidermis and does not respond directly to microbial challenge. Immunodetection experiments showed the novel localization of NBAD-hydrolase in the embryo central neural system and in different regions of the adult brain, in addition to optic lobes. We sequenced and characterized Drosophila mutants tan¹ and tan³. The latter appears to be a mutant with normal expression in neural tissue but weak one in epidermis.

  4. Enhanced expression of heat shock protein 70 (hsp70) and heat shock factor 1 (HSF1) activation in rheumatoid arthritis synovial tissue. Differential regulation of hsp70 expression and hsf1 activation in synovial fibroblasts by proinflammatory cytokines, shear stress, and antiinflammatory drugs.

    PubMed Central

    Schett, G; Redlich, K; Xu, Q; Bizan, P; Gröger, M; Tohidast-Akrad, M; Kiener, H; Smolen, J; Steiner, G

    1998-01-01

    Heat shock proteins (hsp) have been repeatedly implicated to participate in the pathogenesis of rheumatoid arthritis (RA). Herein, we investigated the regulation of synovial hsp70 expression by analyzing the DNA-binding activity of heat shock transcription factor 1 (HSF1) as well as inducible hsp70 expression. Experiments were performed both on synovial tissue and on synovial fibroblast-like cells (SFC). Gel mobility shift analysis revealed increased HSF1 activation, and Western blotting and immunohistochemistry revealed increased hsp70 expression in RA synovial tissue, but not in synovial tissue derived from patients with osteoarthritis. Proinflammatory cytokines (TNF-alpha, IL-1alpha, IL-6), but not IFN-gamma or TGF-beta, induced activation of HSF1-DNA binding and hsp70 expression in cultivated SFC. Activation of HSF1 in SFC was accompanied by hyperphosphorylation and nuclear translocation of HSF1. Furthermore, shear stress also induced a complete heat shock response in cultivated synovial cells. In contrast, nonsteroidal antiinflammatory drugs triggered only an incomplete heat shock response, with HSF1 activation but not hsp70 induction, whereas steroids and immunosuppressive drugs did not affect the heat shock response at all. In summary, these data suggest that induction of hsp70 expression in rheumatoid synovial tissue is based on transcriptional activation of HSF1 due to the presence of proinflammatory cytokines (and possibly also shear stress). PMID:9664071

  5. Expression of airway remodeling proteins in mast cell activated by TGF-β released in OVA-induced allergic responses and their inhibition by low-dose irradiation or 8-oxo-dG.

    PubMed

    Hong, Gwan Ui; Kim, Nam Goo; Ro, Jai Youl

    2014-04-01

    Allergic asthma is characterized by chronic airway remodeling, which is associated with the expression of extracellular matrix proteins (ECM) by TGF-β. However, to date there are no reports demonstrating that structural proteins are directly expressed in mast cells. This study aimed to investigate whether ECM proteins are expressed in mast cells activated with antigen/antibody reaction, and whether the resolution effects of irradiation or 8-oxo-dG may contribute to allergic asthma prevention. Bone marrow-derived mast cells (BMMCs) were activated with DNP-HSA/anti-DNP IgE antibody (act-BMMCs). C57BL/6 mice were sensitized and challenged with ovalbumin (OVA) to induce allergic asthma. Mice were treated orally with 8-oxo-dG or exposed to whole body irradiation (using (137)Cs gamma ray at a dose of 0.5 Gy) for three consecutive days 24 h after OVA challenge. Expression of extracellular matrix (ECM) proteins, TGF-β signaling molecules and NF-κB/AP-1 was determined in the BMMCs, bronchoalveolar lavage (BAL) cells or lung tissues using Western blot, polymerase chain reaction (PCR) and electrophoretic mobility shift assay (EMSA), respectively. Act-BMMCs increased expression of ECM proteins, TGF-β/TGF-β receptor I, TGF-β signaling molecules and cytokines; and increased both NF-κB and AP-1 activity. In addition, the population of mast cells; expression of mast cell markers, TGF-β signaling molecules, ECM proteins/amounts; OVA-specific serum IgE level; numbers of goblet cells; airway hyperresponsiveness; cytokines/chemokines were increased in BAL cells and lung tissues of OVA-challenged mice. All of the above end points were reduced by irradiation or 8-oxo-dG in vitro and in vivo, respectively. The data suggest that mast cells induce expression of ECM proteins through TGF-β produced in inflammatory cells of OVA mice and that post treatment of irradiation or 8-oxo-dG after OVA-challenge may reduce airway remodeling through down-regulating mast cell re-activation by

  6. Dark proteins: effect of inclusion body formation on quantification of protein expression.

    PubMed

    Iafolla, Marco A J; Mazumder, Mostafizur; Sardana, Vandit; Velauthapillai, Tharsan; Pannu, Karanbir; McMillen, David R

    2008-09-01

    Plasmid-borne gene expression systems have found wide application in the emerging fields of systems biology and synthetic biology, where plasmids are used to implement simple network architectures, either to test systems biology hypotheses about issues such as gene expression noise or as a means of exerting artificial control over a cell's dynamics. In both these cases, fluorescent proteins are commonly applied as a means of monitoring the expression of genes in the living cell, and efforts have been made to quantify protein expression levels through fluorescence intensity calibration and by monitoring the partitioning of proteins among the two daughter cells after division; such quantification is important in formulating the predictive models desired in systems and synthetic biology research. A potential pitfall of using plasmid-based gene expression systems is that the high protein levels associated with expression from plasmids can lead to the formation of inclusion bodies, insoluble aggregates of misfolded, nonfunctional proteins that will not generate fluorescence output; proteins caught in these inclusion bodies are thus "dark" to fluorescence-based detection methods. If significant numbers of proteins are incorporated into inclusion bodies rather than becoming biologically active, quantitative results obtained by fluorescent measurements will be skewed; we investigate this phenomenon here. We have created two plasmid constructs with differing average copy numbers, both incorporating an unregulated promoter (P(LtetO-1) in the absence of TetR) expressing the GFP derivative enhanced green fluorescent protein (EGFP), and inserted them into Escherichia coli bacterial cells (a common model organism for work on the dynamics of prokaryotic gene expression). We extracted the inclusion bodies, denatured them, and refolded them to render them active, obtaining a measurement of the average number of EGFP per cell locked into these aggregates; at the same time, we used

  7. β₂ adrenergic receptor activation suppresses bone morphogenetic protein (BMP)-induced alkaline phosphatase expression in osteoblast-like MC3T3E1 cells.

    PubMed

    Yamada, Takayuki; Ezura, Yoichi; Hayata, Tadayoshi; Moriya, Shuichi; Shirakawa, Jumpei; Notomi, Takuya; Arayal, Smriti; Kawasaki, Makiri; Izu, Yayoi; Harada, Kiyoshi; Noda, Masaki

    2015-06-01

    β adrenergic stimulation suppresses bone formation in vivo while its actions in osteoblastic differentiation are still incompletely understood. We therefore examined the effects of β2 adrenergic stimulation on osteoblast-like MC3T3-E1 cells focusing on BMP-induced alkaline phosphatase expression. Morphologically, isoproterenol treatment suppresses BMP-induced increase in the numbers of alkaline phosphatase-positive small foci in the cultures of MC3T3-E1 cells. Biochemically, isoproterenol treatment suppresses BMP-induced enzymatic activity of alkaline phosphatase in a dose-dependent manner. Isoproterenol suppression of alkaline phosphatase activity is observed even when the cells are treated with high concentrations of BMP. With respect to cell density, isoproterenol treatment tends to suppress BMP-induced increase in alkaline phosphatase expression more in osteoblasts cultured at higher cell density. In terms of treatment protocol, continuous isoproterenol treatment is compared to cyclic treatment. Continuous isoproterenol treatment is more suppressive against BMP-induced increase in alkaline phosphatase expression than cyclic regimen. At molecular level, isoproterenol treatment suppresses BMP-induced enhancement of alkaline phosphatase mRNA expression. Regarding the mode of isoproterenol action, isoproterenol suppresses BMP-induced BRE-luciferase activity. These data indicate that isoproterenol regulates BMP-induced alkaline phosphatase expression in osteoblast-like MC3T3E1 cells.

  8. Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts.

    PubMed

    Carmona-Rodríguez, Bruno; Alvarez-Pérez, Marco Antonio; Narayanan, A Sampath; Zeichner-David, Margarita; Reyes-Gasga, José; Molina-Guarneros, Juan; García-Hernández, Ana Lilia; Suárez-Franco, José Luis; Chavarría, Ivet Gil; Villarreal-Ramírez, Eduardo; Arzate, Higinio

    2007-07-06

    We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation.

  9. Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts

    SciTech Connect

    Carmona-Rodriguez, Bruno; Alvarez-Perez, Marco Antonio; Narayanan, A. Sampath; Zeichner-David, Margarita; Reyes-Gasga, Jose; Molina-Guarneros, Juan; Garcia-Hernandez, Ana Lilia; Suarez-Franco, Jose Luis; Chavarria, Ivet Gil; Villarreal-Ramirez, Eduardo; Arzate, Higinio . E-mail: harzate@servidor.unam.mx

    2007-07-06

    We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation.

  10. Examination of calcium-binding protein expression in the inner ear of wild-type, heterozygous and homozygous pituitary adenylate cyclase-activating polypeptide (PACAP)-knockout mice in kanamycin-induced ototoxicity.

    PubMed

    Nemeth, A; Szabadfi, K; Fulop, B; Reglodi, D; Kiss, P; Farkas, J; Szalontai, B; Gabriel, R; Hashimoto, H; Tamas, A

    2014-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with diverse biological effects. It also occurs and exerts protective effects in sensory organs; however, little is known about its effects in the auditory system. Recently, we have shown that PACAP protects cochlear cells against oxidative-stress-induced apoptosis and homozygous PACAP-deficient animals show stronger expression of Ca(2+)-binding proteins in the hair cells of the inner ear, but there are no data about the consequences of the lack of endogenous PACAP in different ototoxic insults such as aminoglycoside-induced toxicity. In this study, we examined the effect of kanamycin treatment on Ca(2+)-binding protein expression in hair cells of wild-type, heterozygous and homozygous PACAP-deficient mice. We treated 5-day-old mice with kanamycin, and 2 days later, we examined the Ca(2+)-binding protein expression of the hair cells with immunohistochemistry. We found stronger expression of Ca(2+)-binding proteins in the hair cells of control heterozygous and homozygous PACAP-deficient mice compared with wild-type animals. Kanamycin induced a significant increase in Ca(2+)-binding protein expression in wild-type and heterozygous PACAP-deficient mice, but the baseline higher expression in homozygous PACAP-deficient mice did not show further changes after the treatment. Elevated endolymphatic Ca(2+) is deleterious for the cochlear function, against which the high concentration of Ca(2+)-buffers in hair cells may protect. Meanwhile, the increased immunoreactivity of Ca(2+)-binding proteins in the absence of PACAP provide further evidence for the important protective role of PACAP in ototoxicity, but further investigations are necessary to examine the exact role of endogenous PACAP in ototoxic insults.

  11. Follistatin‐like protein 1 contributes to dendritic cell and T‐lymphocyte activation in nasopharyngeal carcinoma patients by altering nuclear factor κb and Jun N‐terminal kinase expression

    PubMed Central

    Wang, Hong; Wu, Senyong; Huang, Shiping; Yin, Shaolin; Zou, Guilong; Huang, Kuan'en; Zhang, Zhe

    2016-01-01

    Follistatin‐like protein 1 (FSTL1) is a newly characterized protein that can regulate the immune response in various ways. Dendritic cells (DCs) are central to immune regulation. In this study, we explored the impact of FSTL1 on DC activity in nasopharyngeal carcinoma (NPC) patients. The surface expression of CD40, CD86, and HLA‐DR on DCs was analyzed and showed significantly elevated expression levels, indicating DC maturity. After FSTL1 was added to DCs collected from NPC patients (n = 50), controls (n = 47), and healthy donors (n = 10), interferon γ secretion and T‐cell receptor expression in cytotoxic T lymphocytes were also investigated. In the experimental groups, the expression of the critical immune protein nuclear factor (NF)‐κb was upregulated, whereas Jun N‐terminal kinase (JNK) was downregulated. Our findings demonstrate that FSTL1 plays a critical role in immune regulation, enhancing the antigen presentation ability of DCs by up‐regulating NF‐κb expression and down‐regulating JNK expression. PMID:27859422

  12. Polyphenol Oxidase Activity Expression in Ralstonia solanacearum

    PubMed Central

    Hernández-Romero, Diana; Solano, Francisco; Sanchez-Amat, Antonio

    2005-01-01

    Sequencing of the genome of Ralstonia solanacearum revealed several genes that putatively code for polyphenol oxidases (PPOs). To study the actual expression of these genes, we looked for and detected all kinds of PPO activities, including laccase, cresolase, and catechol oxidase activities, in cellular extracts of this microorganism. The conditions for the PPO assays were optimized for the phenolic substrate, pH, and sodium dodecyl sulfate concentration used. It was demonstrated that three different PPOs are expressed. The genes coding for the enzymes were unambiguously correlated with the enzymatic activities detected by generation of null mutations in the genes by using insertional mutagenesis with a suicide plasmid and estimating the changes in the levels of enzymatic activities compared to the levels in the wild-type strain. The protein encoded by the RSp1530 locus is a multicopper protein with laccase activity. Two other genes, RSc0337 and RSc1501, code for nonblue copper proteins exhibiting homology to tyrosinases. The product of RSc0337 has strong tyrosine hydroxylase activity, and it has been shown that this enzyme is involved in melanin synthesis by R. solanacearum. The product of the RSc1501 gene is an enzyme that shows a clear preference for oxidation of o-diphenols. Preliminary characterization of the mutants obtained indicated that PPOs expressed by R. solanacearum may participate in resistance to phenolic compounds since the mutants exhibited higher sensitivity to l-tyrosine than the wild-type strain. These results suggest a possible role in the pathogenic process to avoid plant resistance mechanisms involving the participation of phenolic compounds. PMID:16269713

  13. Palmitate induces tumor necrosis factor-alpha expression in C2C12 skeletal muscle cells by a mechanism involving protein kinase C and nuclear factor-kappaB activation.

    PubMed

    Jové, Mireia; Planavila, Anna; Sánchez, Rosa M; Merlos, Manuel; Laguna, Juan Carlos; Vázquez-Carrera, Manuel

    2006-01-01

    The mechanisms responsible for increased expression of TNF-alpha in skeletal muscle cells in diabetic states are not well understood. We examined the effects of the saturated acid palmitate on TNF-alpha expression. Exposure of C2C12 skeletal muscle cells to 0.75 mm palmitate enhanced mRNA (25-fold induction, P < 0.001) and protein (2.5-fold induction) expression of the proinflammatory cytokine TNF-alpha. This induction was inversely correlated with a fall in GLUT4 mRNA levels (57% reduction, P < 0.001) and glucose uptake (34% reduction, P < 0.001). PD98059 and U0126, inhibitors of the ERK-MAPK cascade, partially prevented the palmitate-induced TNF-alpha expression. Palmitate increased nuclear factor (NF)-kappaB activation and incubation of the cells with the NF-kappaB inhibitors pyrrolidine dithiocarbamate and parthenolide partially prevented TNF-alpha expression. Incubation of palmitate-treated cells with calphostin C, a strong and specific inhibitor of protein kinase C (PKC), abolished palmitate-induced TNF-alpha expression, and restored GLUT4 mRNA levels. Palmitate treatment enhanced the expression of phospho-PKCtheta, suggesting that this PKC isoform was involved in the changes reported, and coincubation of palmitate-treated cells with the PKC inhibitor chelerythrine prevented the palmitate-induced reduction in the expression of IkappaBalpha and insulin-stimulated Akt activation. These findings suggest that enhanced TNF-alpha expression and GLUT4 down-regulation caused by palmitate are mediated through the PKC activation, confirming that this enzyme may be a target for either the prevention or the treatment of fatty acid-induced insulin resistance.

  14. Maternal protein restriction during lactation modulated the expression and activity of rat offspring hepatic CYP1A1, CYP1A2, CYP2B1, CYP2B2, and CYP2E1 during development.

    PubMed

    Da Costa, N Meireles; Visoni, S B C; Dos Santos, I L; Barja-Fidalgo, T C; Ribeiro-Pinto, L F

    2016-01-01

    Early nutrition plays a long-term role in the predisposition to chronic diseases and influences the metabolism of several drugs. This may happen through cytochromes P450 (CYPs) regulation, which are the main enzymes responsible for the metabolism of xenobiotics. Here, we analyzed the effects of maternal protein restriction (MPR) on the expression and activity of hepatic offspring's CYPs during 90 days after birth, using Wistar rats as a mammal model. Hepatic CYP1A1, CYP1A2, CYP2B1, CYP2B2 and CYP2E1 mRNA and protein expression, and associated catalytic activities (ECOD, EROD, MROD, BROD, PROD and PNPH) were evaluated in 15-, 30-, 60-, and 90-day-old offspring from dams fed with either a 0% protein (MPR groups) or a standard diet (C groups) during the 10 first days of lactation. Results showed that most CYP genes were induced in 60- and 90-day-old MPR offspring. The inductions detected in MPR60 and MPR90 were of 5.0- and 2.0-fold (CYP1A2), 3.7- and 2.0-fold (CYP2B2) and 9.8- and 5.8- fold (CYP2E1), respectively, and a 3.8-fold increase of CYP2B1 in MPR90. No major alterations were detected in CYP protein expression. The most relevant CYP catalytic activities' alterations were observed in EROD, BROD and PNPH. Nevertheless, they did not follow the same pattern observed for mRNA expression, except for an induction of EROD in MPR90 (3.5-fold) and of PNPH in MPR60 (2.2-fold). Together, these results suggest that MPR during lactation was capable of altering the expression and activity of the hepatic CYP enzymes evaluated in the offspring along development.

  15. Protein glycosylation in pmt mutants of Saccharomyces cerevisiae. Influence of heterologously expressed cellobiohydrolase II of Trichoderma reesei and elevated levels of GDP-mannose and cis-prenyltransferase activity.

    PubMed

    Górka-Nieć, Wioletta; Bańkowska, Renata; Palamarczyk, Grazyna; Krotkiewski, Hubert; Kruszewska, Joanna S

    2007-05-01

    Protein O-mannosylation has been postulated to be critical for production and secretion of glycoproteins in fungi. Therefore, understanding the regulation of this process and the influence of heterologous expression of glycoproteins on the activity of enzymes engaged in O-glycosylation are of considerable interest. In this study we expressed cellobiohydrolase II (CBHII) of T. reesei, which is normally highly O-mannosylated, in Saccharomyces cerevisiae pmt mutants partially blocked in O-mannosylation. We found that the lack of Pmt1 or Pmt2 protein O-mannosyltransferase activity limited the glycosylation of CBHII, but it did not affect its secretion. The S. cerevisiae pmt1Delta and pmt2Delta mutants expressing T. reesei cbh2 gene showed a decrease of GDP-mannose level and a very high activity of cis-prenyltransferase compared to untransformed strains. On the other hand, elevation of cis-prenyltransferase activity by overexpression of the S. cerevisiae RER2 gene in these mutants led to an increase of dolichyl phosphate mannose synthase activity, but it did not influence the activity of O-mannosyltransferases. Overexpression of the MPG1 gene increased the level of GDP-mannose and stimulated the activity of mannosyltransferases elongating O-linked sugar chains, leading to partial restoration of CBHII glycosylation.

  16. Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish.

    PubMed

    Horstick, Eric J; Jordan, Diana C; Bergeron, Sadie A; Tabor, Kathryn M; Serpe, Mihaela; Feldman, Benjamin; Burgess, Harold A

    2015-04-20

    Many genetic manipulations are limited by difficulty in obtaining adequate levels of protein expression. Bioinformatic and experimental studies have identified nucleotide sequence features that may increase expression, however it is difficult to assess the relative influence of these features. Zebrafish embryos are rapidly injected with calibrated doses of mRNA, enabling the effects of multiple sequence changes to be compared in vivo. Using RNAseq and microarray data, we identified a set of genes that are highly expressed in zebrafish embryos and systematically analyzed for enrichment of sequence features correlated with levels of protein expression. We then tested enriched features by embryo microinjection and functional tests of multiple protein reporters. Codon selection, releasing factor recognition sequence and specific introns and 3' untranslated regions each increased protein expression between 1.5- and 3-fold. These results suggested principles for increasing protein yield in zebrafish through biomolecular engineering. We implemented these principles for rational gene design in software for codon selection (CodonZ) and plasmid vectors incorporating the most active non-coding elements. Rational gene design thus significantly boosts expression in zebrafish, and a similar approach will likely elevate expression in other animal models.

  17. The role of mitogen-activated protein kinases and sterol receptor coactivator-1 in TGF-β-regulated expression of genes implicated in macrophage cholesterol uptake

    PubMed Central

    Salter, Rebecca C.; Foka, Pelagia; Davies, Thomas S.; Gallagher, Hayley; Michael, Daryn R.; Ashlin, Tim G.; Ramji, Dipak P.

    2016-01-01

    The anti-atherogenic cytokine TGF-β inhibits macrophage foam cell formation by suppressing the expression of key genes implicated in the uptake of modified lipoproteins. We have previously shown a critical role for p38 MAPK and JNK in the TGF-β-mediated regulation of apolipoprotein E expression in human monocytes. However, the roles of these two MAPK pathways in the control of expression of key genes involved in the uptake of modified lipoproteins in human macrophages is poorly understood and formed the focus of this study. TGF-β activated both p38 MAPK and JNK, and knockdown of p38 MAPK or c-Jun, a key downstream target of JNK action, demonstrated their requirement in the TGF-β-inhibited expression of several key genes implicated in macrophage lipoprotein uptake. The potential role of c-Jun and specific co-activators in the action of TGF-β was investigated further by studies on the lipoprotein lipase gene. c-Jun did not directly interact with the minimal promoter region containing the TGF-β response elements and a combination of transient transfection and knock down assays revealed an important role for SRC-1. These studies provide novel insights into the mechanisms underlying the TGF-β-mediated inhibition of macrophage gene expression associated with the control of cholesterol homeostasis. PMID:27687241

  18. The role of mitogen-activated protein kinases and sterol receptor coactivator-1 in TGF-β-regulated expression of genes implicated in macrophage cholesterol uptake.

    PubMed

    Salter, Rebecca C; Foka, Pelagia; Davies, Thomas S; Gallagher, Hayley; Michael, Daryn R; Ashlin, Tim G; Ramji, Dipak P

    2016-09-30

    The anti-atherogenic cytokine TGF-β inhibits macrophage foam cell formation by suppressing the expression of key genes implicated in the uptake of modified lipoproteins. We have previously shown a critical role for p38 MAPK and JNK in the TGF-β-mediated regulation of apolipoprotein E expression in human monocytes. However, the roles of these two MAPK pathways in the control of expression of key genes involved in the uptake of modified lipoproteins in human macrophages is poorly understood and formed the focus of this study. TGF-β activated both p38 MAPK and JNK, and knockdown of p38 MAPK or c-Jun, a key downstream target of JNK action, demonstrated their requirement in the TGF-β-inhibited expression of several key genes implicated in macrophage lipoprotein uptake. The potential role of c-Jun and specific co-activators in the action of TGF-β was investigated further by studies on the lipoprotein lipase gene. c-Jun did not directly interact with the minimal promoter region containing the TGF-β response elements and a combination of transient transfection and knock down assays revealed an important role for SRC-1. These studies provide novel insights into the mechanisms underlying the TGF-β-mediated inhibition of macrophage gene expression associated with the control of cholesterol homeostasis.

  19. A pro-apoptotic 15-kDa protein from Bacopa monnieri activates caspase-3 and downregulates Bcl-2 gene expression in mouse mammary carcinoma cells.

    PubMed

    Kalyani, Manjula Ishwara; Lingaraju, Sheela Mysore; Salimath, Bharathi P

    2013-01-01

    In diseases such as cancer, induction of apoptosis has been a new target for mechanism-based drug discovery. The central component of the process of apoptosis is a proteolytic system involving a family of proteases called caspases. Apoptosis involves characteristic morphological and biochemical events ultimately leading to cell demise. Apoptotic induction is evidently central to the mechanism of action of plant-derived anticancer drugs. Extract of the medicinal plant, Bacopa monnieri, inhibits tumor cell proliferation and accumulation of malignant ascites fluid. The crude sample when subjected to Soxhlet extraction yielded different solvent extracts of which the aqueous extract showed biological activity of apoptosis in Ehrlich ascites tumor cell lines (EAT). Bacopa monnieri water extract (BMWE) treatment of EAT cells produced apoptotic morphological characteristics and in-vivo DNA fragmentation, which is due to the activity of an endogenous endonuclease. The endonuclease responsible for DNA fragmentation acts downstream of caspase-3 activity and is also referred to as caspase-activated DNase (CAD). The CAD constitutively expressed in the cell cytoplasm is translocated into the nucleus upon BMWE treatment, as verified by Western blotting, leading to DNA fragmentation and to programmed cell death. The expression of the pro-apoptotic gene Bax was increased and the expression of the anti-apoptotic gene Bcl-2 was decreased by BMWE treatment. Considering the above results, BMWE was able induce apoptosis in EAT cells via Bax-related caspase-3 activation. This may provide experimental data for the further clinical use of BMWE in cancer.

  20. Molecular cloning, expression of a big defensin gene from bay scallop Argopecten irradians and the antimicrobial activity of its recombinant protein.

    PubMed

    Zhao, Jianmin; Song, Linsheng; Li, Chenghua; Ni, Duojiao; Wu, Longtao; Zhu, Ling; Wang, Hao; Xu, Wei

    2007-01-01

    Antimicrobial peptides are important components of the host innate immune responses by exerting broad-spectrum microbicidal activity against pathogenic microbes. The first mollusk big defensin (designated AiBD) cDNA was cloned from bay scallop Argopecten irradians by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The scallop AiBD consisted of 531 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, encoding a polypeptide of 122 amino acids. The high similarity of AiBD deduced amino acid sequence with big defensin from Tachypleus tridentatus and Branchiostoma belcheri tsingtaunese indicated that AiBD should be a member of big defensin family. The expression of AiBD in various tissues was measured by using Northern blotting analysis. mRNA transcripts of AiBD could be detected in haemocytes of unchallenged scallops. The temporal expression of AiBD in haemolymph after Vibrio anguilarum challenge was recorded by quantitative real time PCR. The relative expression level of AiBD in haemolymph was up-regulated evenly in the first 8 h, followed by a drastic increase, and increased 131.1-fold at 32 h post-injection. These results indicated that AiBD could be induced by bacterial challenge, and it should participate in the immune responses of A. irradians. Biological activity assay revealed that recombinant AiBD could inhibit the growth of both Gram-positive and Gram-negative bacteria, and also showed strong fungicidal activity towards the expression host. Recombinant expression of AiBD made it possible to further characterize its functions involved in immune responses, and also provided a potential therapeutic agent for disease control in aquaculture.

  1. DNA-based control of protein activity

    PubMed Central

    Engelen, W.; Janssen, B. M. G.

    2016-01-01

    DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control. PMID:26812623

  2. Expression profile of mitrogen-activated protein kinase (MAPK) signaling genes in the skeletal muscle & liver of rat with type 2 diabetes: Role in disease pathology

    PubMed Central

    Tang, Xiaoli; Deng, Libin; Xiong, Huangui; Li, Guilin; Lin, Jiari; Liu, Shuangmei; Xie, Jinyan; Liu, Jun; Kong, Fanjun; Tu, Guihua; Peng, Haiying; Liang, Shangdong

    2014-01-01

    Background & objectives: Type 2 diabetes (T2D) is characterized as hyperglycaemia caused by defects in insulin secretion, and it affects target tissues, such as skeletal muscle, liver and adipose tissue. Therefore, analyzing the changes of gene expression profiles in these tissues is important to elucidate the pathogenesis of T2D. We, therefore, measured the gene transcript alterations in liver and skeletal muscle of rat with induced T2D, to detect differentially expressed genes in liver and skeletal muscle and perform gene-annotation enrichment analysis. Methods: In the present study, skeletal muscle and liver tissue from 10 streptozotocin-induced diabetic rats and 10 control rats were analyzed using gene expression microarrays. KEGG pathways enriched by differentially expressed genes (DEGs) were identified by WebGestalt Expander and GATHER software. DEGs were validated by the method of real-time PCR and western blot. Results: From the 9,929 expressed genes across the genome, 1,305 and 997 differentially expressed genes (DEGs, P<0.01) were identified in comparisons of skeletal muscle and liver, respectively. Large numbers of DEGs (200) were common in both comparisons, which was clearly more than the predicted number (131 genes, P<0.001). For further interpretation of the gene expression data, three over-representation analysis softwares (WebGestalt, Expander and GATHER) were used. All the tools detected one KEGG pathway (MAPK signaling) and two GO (gene ontology) biological processes (response to stress and cell death), with enrichment of DEGs in both tissues. In addition, PPI (protein-protein interaction) networks constructed using human homologues not only revealed the tendency of DEGs to form a highly connected module, but also suggested a “hub” role of p38-MAPK-related genes (such as MAPK14) in the pathogenesis of T2D. Interpretation & conclusions: Our results indicated the considerably aberrant MAPK signaling in both insulin-sensitive tissues of T2D rat

  3. Peroxisome proliferator-activated receptor-γ activation enhances insulin-stimulated glucose disposal by reducing ped/pea-15 gene expression in skeletal muscle cells: evidence for involvement of activator protein-1.

    PubMed

    Ungaro, Paola; Mirra, Paola; Oriente, Francesco; Nigro, Cecilia; Ciccarelli, Marco; Vastolo, Viviana; Longo, Michele; Perruolo, Giuseppe; Spinelli, Rosa; Formisano, Pietro; Miele, Claudia; Beguinot, Francesco

    2012-12-14

    The gene network responsible for inflammation-induced insulin resistance remains enigmatic. In this study, we show that, in L6 cells, rosiglitazone- as well as pioglitazone-dependent activation of peroxisome proliferator-activated receptor-γ (PPARγ) represses transcription of the ped/pea-15 gene, whose increased activity impairs glucose tolerance in mice and humans. Rosiglitazone enhanced insulin-induced glucose uptake in L6 cells expressing the endogenous ped/pea-15 gene but not in cells expressing ped/pea-15 under the control of an exogenous promoter. The ability of PPARγ to affect ped/pea-15 expression was also lost in cells and in C57BL/6J transgenic mice expressing ped/pea-15 under the control of an exogenous promoter, suggesting that ped/pea-15 repression may contribute to rosiglitazone action on glucose disposal. Indeed, high fat diet mice showed insulin resistance and increased ped/pea-15 levels, although these effects were reduced by rosiglitazone treatment. Both supershift and ChIP assays revealed the presence of the AP-1 component c-JUN at the PED/PEA-15 promoter upon 12-O-tetradecanoylphorbol-13-acetate stimulation of the cells. In these experiments, rosiglitazone treatment reduced c-JUN presence at the PED/PEA-15 promoter. This effect was not associated with a decrease in c-JUN expression. In addition, c-jun silencing in L6 cells lowered ped/pea-15 expression and caused nonresponsiveness to rosiglitazone, although c-jun overexpression enhanced the binding to the ped/pea-15 promoter and blocked the rosiglitazone effect. These results indicate that PPARγ regulates ped/pea-15 transcription by inhibiting c-JUN binding at the ped/pea-15 promoter. Thus, ped/pea-15 is downstream of a major PPARγ-regulated inflammatory network. Repression of ped/pea-15 transcription might contribute to the PPARγ regulation of muscle sensitivity to insulin.

  4. Expression and purification of membrane proteins.

    PubMed

    Kubicek, Jan; Block, Helena; Maertens, Barbara; Spriestersbach, Anne; Labahn, Jörg

    2014-01-01

    Approximately 30% of a genome encodes for membrane proteins. They are one of the most important classes of proteins in that they can receive, differentiate, and transmit intra- and intercellular signals. Some examples of classes of membrane proteins include cell-adhesion molecules, translocases, and receptors in signaling pathways. Defects in membrane proteins may be involved in a number of serious disorders such as neurodegenerative diseases (e.g., Alzheimer's) and diabetes. Furthermore, membrane proteins provide natural entry and anchoring points for the molecular agents of infectious diseases. Thus, membrane proteins constitute ~50% of known and novel drug targets. Progress in this area is slowed by the requirement to develop methods and procedures for expression and isolation that are tailored to characteristic properties of membrane proteins. A set of standard protocols for the isolation of the targets in quantities that allow for the characterization of their individual properties for further optimization is required. The standard protocols given below represent a workable starting point. If optimization of yields is desired, a variation of conditions as outlined in the theory section is recommended.

  5. Engineering Genes for Predictable Protein Expression

    PubMed Central

    Gustafsson, Claes; Minshull, Jeremy; Govindarajan, Sridhar; Ness, Jon; Villalobos, Alan; Welch, Mark

    2013-01-01

    The DNA sequence used to encode a polypeptide can have dramatic effects on its expression. Lack of readily available tools has until recently inhibited meaningful experimental investigation of this phenomenon. Advances in synthetic biology and the application of modern engineering approaches now provide the tools for systematic analysis of the sequence variables affecting heterologous expression of recombinant proteins. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches, highlighting some of the surprising and promising results emerging from the developing field of gene engineering. PMID:22425659

  6. Engineering genes for predictable protein expression.

    PubMed

    Gustafsson, Claes; Minshull, Jeremy; Govindarajan, Sridhar; Ness, Jon; Villalobos, Alan; Welch, Mark

    2012-05-01

    The DNA sequence used to encode a polypeptide can have dramatic effects on its expression. Lack of readily available tools has until recently inhibited meaningful experimental investigation of this phenomenon. Advances in synthetic biology and the application of modern engineering approaches now provide the tools for systematic analysis of the sequence variables affecting heterologous expression of recombinant proteins. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches, highlighting some of the surprising and promising results emerging from the developing field of gene engineering.

  7. Maternal protein restriction during lactation modulated the expression and activity of rat offspring hepatic CYP1A1, CYP1A2, CYP2B1, CYP2B2, and CYP2E1 during development

    PubMed Central

    Da Costa, N. Meireles; Visoni, S.B.C.; Dos Santos, I.L.; Barja-Fidalgo, T.C.; Ribeiro-Pinto, L.F.

    2016-01-01

    Early nutrition plays a long-term role in the predisposition to chronic diseases and influences the metabolism of several drugs. This may happen through cytochromes P450 (CYPs) regulation, which are the main enzymes responsible for the metabolism of xenobiotics. Here, we analyzed the effects of maternal protein restriction (MPR) on the expression and activity of hepatic offspring’s CYPs during 90 days after birth, using Wistar rats as a mammal model. Hepatic CYP1A1, CYP1A2, CYP2B1, CYP2B2 and CYP2E1 mRNA and protein expression, and associated catalytic activities (ECOD, EROD, MROD, BROD, PROD and PNPH) were evaluated in 15-, 30-, 60-, and 90-day-old offspring from dams fed with either a 0% protein (MPR groups) or a standard diet (C groups) during the 10 first days of lactation. Results showed that most CYP genes were induced in 60- and 90-day-old MPR offspring. The inductions detected in MPR60 and MPR90 were of 5.0- and 2.0-fold (CYP1A2), 3.7- and 2.0-fold (CYP2B2) and 9.8- and 5.8– fold (CYP2E1), respectively, and a 3.8-fold increase of CYP2B1 in MPR90. No major alterations were detected in CYP protein expression. The most relevant CYP catalytic activities’ alterations were observed in EROD, BROD and PNPH. Nevertheless, they did not follow the same pattern observed for mRNA expression, except for an induction of EROD in MPR90 (3.5-fold) and of PNPH in MPR60 (2.2-fold). Together, these results suggest that MPR during lactation was capable of altering the expression and activity of the hepatic CYP enzymes evaluated in the offspring along development. PMID:27828666

  8. Mitogen-activated protein kinases regulate expression of neuronal nitric oxide synthase and neurite outgrowth via non-classical retinoic acid receptor signaling in human neuroblastoma SH-SY5Y cells.

    PubMed

    Fujibayashi, Tatsuya; Kurauchi, Yuki; Hisatsune, Akinori; Seki, Takahiro; Shudo, Koichi; Katsuki, Hiroshi

    2015-10-01

    We have previously shown that retinoic acid receptor (RAR) stimulation by an agonist Am80 recruits nitric oxide-dependent signaling via increased expression of neuronal nitric oxide synthase (nNOS) in rat midbrain slice cultures. Using neuroblastoma SH-SY5Y cells, here we investigated the mechanisms of RAR-induced nNOS expression, together with relationship between nNOS expression and neurite outgrowth. Am80 promoted neurite outgrowth, which was attenuated by inhibitors of phosphoinositide 3-kinase (PI3K; LY294002), c-Jun N-terminal kinase (JNK; SP600125) and p38 mitogen-activated protein kinase (p38 MAPK; SB203580). A selective nNOS inhibitor 3-bromo-nitroindazole also suppressed Am80-induced neurite outgrowth. Am80-induced increase in nNOS protein expression was attenuated by LY294002, SP600125 and SB203580, whereas increase in nNOS mRNA expression was attenuated only by LY294002. Am80-induced activation of JNK and p38 MAPK was blocked by LY294002, suggesting that these kinases acted downstream of PI3K. We also confirmed that DAX1, a nuclear receptor reported to regulate nNOS expression, was up-regulated in response to Am80. siRNA-mediated knockdown of DAX1 abrogated Am80-induced nNOS expression and neurite outgrowth. These results reveal for the first time that nNOS expression is crucial for RAR-mediated neurite outgrowth, and that non-genomic signaling such as JNK and p38 MAPK is involved in RAR-mediated nNOS expression.

  9. Expression of the p66Shc protein adaptor is regulated by the activator of transcription STAT4 in normal and chronic lymphocytic leukemia B cells

    PubMed Central

    Cattaneo, Francesca; Patrussi, Laura; Capitani, Nagaja; Frezzato, Federica; D'Elios, Mario Milco; Trentin, Livio; Semenzato, Gianpietro; Baldari, Cosima T.

    2016-01-01

    p66Shc attenuates mitogenic, prosurvival and chemotactic signaling and promotes apoptosis in lymphocytes. Consistently, p66Shc deficiency contributes to the survival and trafficking abnormalities of chronic lymphocytic leukemia (CLL) B cells. The mechanism of p66shc silencing in CLL B cells is methylation-independent, at variance with other cancer cell types. Here we identify STAT4 as a novel transcriptional regulator of p66Shc in B cells. Chromatin immunoprecipitation and reporter gene assays showed that STAT4 binds to and activates the p66shc promoter. Silencing or overexpression of STAT4 resulted in a co-modulation of p66Shc. IL-12-dependent STAT4 activation caused a coordinate increase in STAT4 and p66Shc expression, which correlated with enhanced B cell apoptosis. Treatment with the STAT4 inhibitor lisofylline reverted partly this effect, suggesting that STAT4 phosphorylation is not essential for but enhances p66shc transcription. Additionally, we demonstrate that CLL B lymphocytes have a STAT4 expression defect which partly accounts for their p66Shc deficiency, as supported by reconstitution experiments. Finally, we show that p66Shc participates in a positive feedback loop to promote STAT4 expression. These results provide new insights into the mechanism of p66Shc expression in B cells and its defect in CLL, identifying the STAT4/IL-12 pathway as a potential therapeutic target in this neoplasia. PMID:27494881

  10. Inhibition of tumor necrosis factor-alpha-induced interleukin-6 expression by telmisartan through cross-talk of peroxisome proliferator-activated receptor-gamma with nuclear factor kappaB and CCAAT/enhancer-binding protein-beta.

    PubMed

    Tian, Qingping; Miyazaki, Ryohei; Ichiki, Toshihiro; Imayama, Ikuyo; Inanaga, Keita; Ohtsubo, Hideki; Yano, Kotaro; Takeda, Kotaro; Sunagawa, Kenji

    2009-05-01

    Telmisartan, an angiotensin II type 1 receptor antagonist, was reported to be a partial agonist of peroxisome proliferator-activated receptor-gamma. Although peroxisome proliferator-activated receptor-gamma activators have been shown to have an anti-inflammatory effect, such as inhibition of cytokine production, it has not been determined whether telmisartan has such effects. We examined whether telmisartan inhibits expression of interleukin-6 (IL-6), a proinflammatory cytokine, in vascular smooth muscle cells. Telmisartan, but not valsartan, attenuated IL-6 mRNA expression induced by tumor necrosis factor-alpha (TNF-alpha). Telmisartan decreased TNF-alpha-induced IL-6 mRNA and protein expression in a dose-dependent manner. Because suppression of IL-6 mRNA expression was prevented by pretreatment with GW9662, a specific peroxisome proliferator-activated receptor-gamma antagonist, peroxisome proliferator-activated receptor-gamma may be involved in the process. Telmisartan suppressed IL-6 gene promoter activity induced by TNF-alpha. Deletion analysis suggested that the DNA segment between -150 bp and -27 bp of the IL-6 gene promoter that contains nuclear factor kappaB and CCAAT/enhancer-binding protein-beta sites was responsible for telmisartan suppression. Telmisartan attenuated TNF-alpha-induced nuclear factor kappaB- and CCAAT/enhancer-binding protein-beta-dependent gene transcription and DNA binding. Telmisartan also attenuated serum IL-6 level in TNF-alpha-infused mice and IL-6 production from rat aorta stimulated with TNF-alpha ex vivo. These data suggest that telmisartan may attenuate inflammatory process induced by TNF-alpha in addition to the blockade of angiotensin II type 1 receptor. Because both TNF-alpha and angiotensin II play important roles in atherogenesis through enhancement of vascular inflammation, telmisartan may be beneficial for treatment of not only hypertension but also vascular inflammatory change.

  11. Expression Differentiation Is Constrained to Low-Expression Proteins over Ecological Timescales.

    PubMed

    Margres, Mark J; Wray, Kenneth P; Seavy, Margaret; McGivern, James J; Herrera, Nathanael D; Rokyta, Darin R

    2016-01-01

    Protein expression level is one of the strongest predictors of protein sequence evolutionary rate, with high-expression protein sequences evolving at slower rates than low-expression protein sequences largely because of constraints on protein folding and function. Expression evolutionary rates also have been shown to be negatively correlated with expression level across human and mouse orthologs over relatively long divergence times (i.e., ∼100 million years). Long-term evolutionary patterns, however, often cannot be extrapolated to microevolutionary processes (and vice versa), and whether this relationship holds for traits evolving under directional selection within a single species over ecological timescales (i.e., <5000 years) is unknown and not necessarily expected. Expression is a metabolically costly process, and the expression level of a particular protein is predicted to be a tradeoff between the benefit of its function and the costs of its expression. Selection should drive the expression level of all proteins close to values that maximize fitness, particularly for high-expression proteins because of the increased energetic cost of production. Therefore, stabilizing selection may reduce the amount of standing expression variation for high-expression proteins, and in combination with physiological constraints that may place an upper bound on the range of beneficial expression variation, these constraints could severely limit the availability of beneficial expression variants. To determine whether rapid-expression evolution was restricted to low-expression proteins owing to these constraints on highly expressed proteins over ecological timescales, we compared venom protein expression levels across mainland and island populations for three species of pit vipers. We detected significant differentiation in protein expression levels in two of the three species and found that rapid-expression differentiation was restricted to low-expression proteins. Our

  12. Expression and Activity of Metalloproteinases in Depression

    PubMed Central

    Bobińska, Kinga; Szemraj, Janusz; Czarny, Piotr; Gałecki, Piotr

    2016-01-01

    Background Depression is one of the most common mental disorders and often co-exists with somatic diseases. The most probable cause of comorbidity is a generalized inflammatory process that occurs in both depression and somatic diseases. Matrix metalloproteinases MMPs play a role in modulating inflammation and their impact in many inflammatory diseases has been investigated. The purpose of this study was to evaluate gene expression for selected polymorphisms of MMP-2 (C-735T), MMP-7 (A-181G), and MMP-9 (T-1702A, C1562T), which have been confirmed to participate in development of depression, and TIMP-2 (G-418C, tissue inhibitor of MMP). Activity variability of pro-MMP-2 and pro-MMP-9 was measured in a group of people with depression and a group of healthy individuals. Material/Methods The examined population comprised 142 individuals suffering from depression and 100 individuals who formed a control group (CG). Designations were carried out for MMP-2 (C-735T), MMP-7 (A-181G), MMP-9 (T-1702A, C1562T), and TIMP-2 (G-418C). Results For all examined and tested MMPs and for TIMP-2, gene expression at the mRNA level was higher in patients with depression than in the CG. Similar results were recorded for gene expression at the protein level, while expression on the protein level for TIMP-2 was higher in the CG. Change in activity of MMP-2 and pro-MMP-2 was statistically more significant in the group with depression. The opposite result was recorded for MMP-9 and pro-MMP-9, in which the change in activity was statistically more significant in the CG. Conclusions Changes in MMPs and TIMP expression may be a common element in, or perhaps even a marker for, recurrent depressive disorders and somatic diseases. PMID:27098106

  13. PARP-1 protein expression in glioblastoma multiforme

    PubMed Central

    Galia, A.; Calogero, A.E.; Condorelli, R.A.; Fraggetta, F.; La Corte, C.; Ridolfo, F.; Bosco, P.; Castiglione, R.; Salemi, M.

    2012-01-01

    One of the most common type of primary brain tumors in adults is the glioblastoma multiforme (GBM) (World Health Organization grade IV astrocytoma). It is the most common malignant and aggressive form of glioma and it is among the most lethal ones. Poly (ADP-ribose) polymerase 1 (PARP-1) gene, located to 1q42, plays an important role for the efficient maintenance of genome integrity. PARP-1 protein is required for the apoptosis-inducing factor (AIF) translocation from the mitochondria to the nucleus. PARP-1 is proteolytically cleaved at the onset of apoptosis by caspase-3. Microarray analysis of PARP-1 gene expression in more than 8000 samples revealed that PARP-1 is more highly expressed in several types of cancer compared with the equivalent normal tissues. Overall, the most differences in PARP-1 gene expression have been observed in breast, ovarian, endometrial, lung, and skin cancers, and non-Hodgkin's lymphoma. We evaluated the expression of PARP-1 protein in normal brain tissues and primary GBM by immunohistochemistry. Positive nuclear PARP-1 staining was found in all samples with GBM, but not in normal neurons from controls (n=4) and GBM patients (n=27). No cytoplasmic staining was observed in any sample. In conclusion, PARP-1 gene is expressed in GBM. This finding may be envisioned as an attempt to trigger apoptosis in this tumor, as well as in many other malignancies. The presence of the protein exclusively at the nucleus further support the function played by this gene in genome integrity maintenance and apoptosis. Finally, PARP-1 staining may be used as GBM cell marker. PMID:22472897

  14. Effect of polysaccharides extract of rhizoma atractylodis macrocephalae on thymus, spleen and cardiac indexes, caspase-3 activity ratio, Smac/DIABLO and HtrA2/Omi protein and mRNA expression levels in aged rats.

    PubMed

    Guo, Ling; Sun, Yong Le; Wang, Ai Hong; Xu, Chong En; Zhang, Meng Yuan

    2012-10-01

    This study was designed to determine the possible protective effect of polysaccharides extract of rhizoma atractylodis macrocephalae on heart function in aged rats. Polysaccharides extract of rhizoma atractylodis macrocephalae was administered to aged rats. Results showed that thymus, spleen and cardiac indexs were significantly increased, whereas caspase-3 activity ratio, Smac/DIABLO and HtrA2/Omi protein expression, Smac/DIABLO and HtrA2/Omi mRNA expression levels were markedly reduced. It can be concluded that polysaccharides extract of rhizoma atractylodis macrocephalae may enhance immunity and improve heart function in aged rats.

  15. Structure of the β-Galactosidase Gene from Thermus sp. Strain T2: Expression in Escherichia coli and Purification in a Single Step of an Active Fusion Protein

    PubMed Central

    Vian, Alejandro; Carrascosa, Alfonso V.; García, José L.; Cortés, Estrella

    1998-01-01

    The nucleotide sequence of both the bgaA gene, coding for a thermostable β-galactosidase of Thermus sp. strain T2, and its flanking regions was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 645 amino acids (Mr, 73,595). Comparative analysis of the open reading frames located in the flanking regions of the bgaA gene revealed that they might encode proteins involved in the transport and hydrolysis of sugars. The observed homology between the deduced amino acid sequences of BgaA and the β-galactosidase of Bacillus stearothermophilus allows us to classify the new enzyme within family 42 of glycosyl hydrolases. BgaA was overexpressed in its active form in Escherichia coli, but more interestingly, an active chimeric β-galactosidase was constructed by fusing the BgaA protein to the choline-binding domain of the major pneumococcal autolysin. This chimera illustrates a novel approach for producing an active and thermostable hybrid enzyme that can be purified in a single step by affinity chromatography on DEAE-cellulose, retaining the catalytic properties of the native enzyme. The chimeric enzyme showed a specific activity of 191,000 U/mg at 70°C and a Km value of 1.6 mM with o-nitrophenyl-β-d-galactopyranoside as a substrate, and it retained 50% of its initial activity after 1 h of incubation at 70°C. PMID:9603833

  16. DNA methyltransferase 3a and mitogen-activated protein kinase signaling regulate the expression of fibroblast growth factor-inducible 14 (Fn14) during denervation-induced skeletal muscle atrophy.

    PubMed

    Tajrishi, Marjan M; Shin, Jonghyun; Hetman, Michal; Kumar, Ashok

    2014-07-18

    The TWEAK-fibroblast growth factor-inducible 14 (Fn14) system is a critical regulator of denervation-induced skeletal muscle atrophy. Although the expression of Fn14 is a rate-limiting step in muscle atrophy on denervation, mechanisms regulating gene expression of Fn14 remain unknown. Methylation of CpG sites within promoter region is an important epigenetic mechanism for gene silencing. Our study demonstrates that Fn14 promoter contains a CpG island close to transcription start site. Fn14 promoter also contains multiple consensus DNA sequence for transcription factors activator protein 1 (AP1) and specificity protein 1 (SP1). Denervation diminishes overall genomic DNA methylation and causes hypomethylation at specific CpG sites in Fn14 promoter leading to the increased gene expression of Fn14 in skeletal muscle. Abundance of DNA methyltransferase 3a (Dnmt3a) and its interaction with Fn14 promoter are repressed in denervated skeletal muscle of mice. Overexpression of Dnmt3a inhibits the gene expression of Fn14 and attenuates skeletal muscle atrophy upon denervation. Denervation also causes the activation of ERK1/2, JNK1/2, and ERK5 MAPKs and AP1 and SP1, which stimulate the expression of Fn14 in skeletal muscle. Collectively, our study provides novel evidence that Dnmt3a and MAPK signaling regulate the levels of Fn14 in skeletal muscle on denervation.

  17. CREPT/RPRD1B, a Recently Identified Novel Protein Highly Expressed in Tumors, Enhances the β-Catenin·TCF4 Transcriptional Activity in Response to Wnt Signaling*

    PubMed Central

    Zhang, Yanquan; Liu, Chunxiao; Duan, Xiaolin; Ren, Fangli; Li, Shan; Jin, Zhe; Wang, Yinyin; Feng, Yarui; Liu, Zewen; Chang, Zhijie

    2014-01-01

    CREPT (cell cycle-related and expression elevated protein in tumor)/RPRD1B (regulation of nuclear pre-mRNA domain-containing protein 1B), highly expressed during tumorigenesis, was shown to enhance transcription of CCND1 and to promote cell proliferation by interacting with RNA polymerase II. However, which signaling pathway is involved in CREPT-mediated activation of gene transcription remains unclear. In this study, we reveal that CREPT participates in transcription of the Wnt/β-catenin signaling activated genes through the β-catenin and the TCF4 complex. Our results demonstrate that CREPT interacts with both β-catenin and TCF4, and enhances the association of β-catenin with TCF4, in response to Wnt stimulation. Furthermore, CREPT was shown to occupy at TCF4 binding sites (TBS) of the promoters of Wnt-targeted genes under Wnt stimulation. Interestingly, depletion of CREPT resulted in decreased occupancy of β-catenin on TBS, and over-expression of CREPT enhances the activity of the β-catenin·TCF4 complex to initiate transcription of Wnt target genes, which results in up-regulated cell proliferation and invasion. Our study suggests that CREPT acts as an activator to promote transcriptional activity of the β-catenin·TCF4 complex in response to Wnt signaling. PMID:24982424

  18. Dehydroeburicoic Acid from Antrodia camphorata Prevents the Diabetic and Dyslipidemic State via Modulation of Glucose Transporter 4, Peroxisome Proliferator-Activated Receptor α Expression and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice

    PubMed Central

    Kuo, Yueh-Hsiung; Lin, Cheng-Hsiu; Shih, Chun-Ching

    2016-01-01

    This study investigated the potential effects of dehydroeburicoic acid (TT), a triterpenoid compound from Antrodia camphorata, in vitro and examined the effects and mechanisms of TT on glucose and lipid homeostasis in high-fat-diet (HFD)-fed mice. The in vitro study examined the effects of a MeOH crude extract (CruE) of A. camphorata and Antcin K (AnK; the main constituent of fruiting body of this mushroom) on membrane glucose transporter 4 (GLUT4) and phospho-Akt in C2C12 myoblasts cells. The in vitro study demonstrated that treatment with CruE, AnK and TT increased the membrane levels of glucose transporter 4 (GLUT4) and phospho-Akt at different concentrations. The animal experiments were performed for 12 weeks. Diabetic mice were randomly divided into six groups after 8 weeks of HFD-induction and treated with daily oral gavage doses of TT (at three dose levels), fenofibrate (Feno) (at 0.25 g/kg body weight), metformin (Metf) (at 0.3 g/kg body weight) or vehicle for another 4 weeks while on an HFD diet. HFD-fed mice exhibited increased blood glucose levels. TT treatment dramatically lowered blood glucose levels by 34.2%~43.4%, which was comparable to the antidiabetic agent-Metf (36.5%). TT-treated mice reduced the HFD-induced hyperglycemia, hypertriglyceridemia, hyperinsulinemia, hyperleptinemia, and hypercholesterolemia. Membrane levels of GLUT4 were significantly higher in CruE-treated groups in vitro. Skeletal muscle membrane levels of GLUT4 were significantly higher in TT-treated mice. These groups of mice also displayed lower mRNA levels of glucose-6-phosphatase (G6 Pase), an inhibitor of hepatic glucose production. The combination of these agents produced a net hypoglycemic effect in TT-treated mice. TT treatment enhanced the expressions of hepatic and skeletal muscle AMP-activated protein kinase (AMPK) phosphorylation in mice. TT-treated mice exhibited enhanced expression of hepatic fatty acid oxidation enzymes, including peroxisome proliferator-activated

  19. Dehydroeburicoic Acid from Antrodia camphorata Prevents the Diabetic and Dyslipidemic State via Modulation of Glucose Transporter 4, Peroxisome Proliferator-Activated Receptor α Expression and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice.

    PubMed

    Kuo, Yueh-Hsiung; Lin, Cheng-Hsiu; Shih, Chun-Ching

    2016-06-03

    This study investigated the potential effects of dehydroeburicoic acid (TT), a triterpenoid compound from Antrodia camphorata, in vitro and examined the effects and mechanisms of TT on glucose and lipid homeostasis in high-fat-diet (HFD)-fed mice. The in vitro study examined the effects of a MeOH crude extract (CruE) of A. camphorata and Antcin K (AnK; the main constituent of fruiting body of this mushroom) on membrane glucose transporter 4 (GLUT4) and phospho-Akt in C2C12 myoblasts cells. The in vitro study demonstrated that treatment with CruE, AnK and TT increased the membrane levels of glucose transporter 4 (GLUT4) and phospho-Akt at different concentrations. The animal experiments were performed for 12 weeks. Diabetic mice were randomly divided into six groups after 8 weeks of HFD-induction and treated with daily oral gavage doses of TT (at three dose levels), fenofibrate (Feno) (at 0.25 g/kg body weight), metformin (Metf) (at 0.3 g/kg body weight) or vehicle for another 4 weeks while on an HFD diet. HFD-fed mice exhibited increased blood glucose levels. TT treatment dramatically lowered blood glucose levels by 34.2%~43.4%, which was comparable to the antidiabetic agent-Metf (36.5%). TT-treated mice reduced the HFD-induced hyperglycemia, hypertriglyceridemia, hyperinsulinemia, hyperleptinemia, and hypercholesterolemia. Membrane levels of GLUT4 were significantly higher in CruE-treated groups in vitro. Skeletal muscle membrane levels of GLUT4 were significantly higher in TT-treated mice. These groups of mice also displayed lower mRNA levels of glucose-6-phosphatase (G6 Pase), an inhibitor of hepatic glucose production. The combination of these agents produced a net hypoglycemic effect in TT-treated mice. TT treatment enhanced the expressions of hepatic and skeletal muscle AMP-activated protein kinase (AMPK) phosphorylation in mice. TT-treated mice exhibited enhanced expression of hepatic fatty acid oxidation enzymes, including peroxisome proliferator-activated

  20. The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells.

    PubMed

    Whisenant, Thomas C; Peralta, Eigen R; Aarreberg, Lauren D; Gao, Nina J; Head, Steven R; Ordoukhanian, Phillip; Williamson, Jamie R; Salomon, Daniel R

    2015-01-01

    Activation of CD4 T cells is a reaction to challenges such as microbial pathogens, cancer and toxins that defines adaptive immune responses. The roles of T cell receptor crosslinking, intracellular signaling, and transcription factor activation are well described, but the importance of post-transcriptional regulation by RNA-binding proteins (RBPs) has not been considered in depth. We describe a new model expanding and activating primary human CD4 T cells and applied this to characterizing activation-induced assembly of splicing factors centered on U2AF2. We immunoprecipitated U2AF2 to identify what mRNA transcripts were bound as a function of activation by TCR crosslinking and costimulation. In parallel, mass spectrometry revealed the proteins incorporated into the U2AF2-centered RNA/protein interactome. Molecules that retained interaction with the U2AF2 complex after RNAse treatment were designated as "central" interactome members (CIMs). Mass spectrometry also identified a second class of activation-induced proteins, "peripheral" interactome members (PIMs), that bound to the same transcripts but were not in physical association with U2AF2 or its partners. siRNA knockdown of two CIMs and two PIMs caused ch