A Novel Regulatory Mechanism of Smooth Muscle α-Actin Expression by NRG-1/circACTA2/miR-548f-5p Axis.

PubMed

Sun, Yan; Yang, Zhan; Zheng, Bin; Zhang, Xin-Hua; Zhang, Man-Li; Zhao, Xue-Shan; Zhao, Hong-Ye; Suzuki, Toru; Wen, Jin-Kun

2017-09-01

Neuregulin-1 (NRG-1) includes an extracellular epidermal growth factor-like domain and an intracellular domain (NRG-1-ICD). In response to transforming growth factor-β1, its cleavage by proteolytic enzymes releases a bioactive fragment, which suppresses the vascular smooth muscle cell (VSMC) proliferation by activating ErbB (erythroblastic leukemia viral oncogene homolog) receptor. However, NRG-1-ICD function in VSMCs remains unknown. Here, we characterize the function of NRG-1-ICD and underlying mechanisms in VSMCs. Immunofluorescence staining, Western blotting, and quantitative real-time polymerase chain reaction showed that NRG-1 was expressed in rat, mouse, and human VSMCs and was upregulated and cleaved in response to transforming growth factor-β1. In the cytoplasm of HASMCs (human aortic smooth muscle cells), the NRG-1-ICD participated in filamentous actin formation by interacting with α-SMA (smooth muscle α-actin). In the nucleus, the Nrg-1-ICD induced circular ACTA2 (alpha-actin-2; circACTA2) formation by recruitment of the zinc-finger transcription factor IKZF1 (IKAROS family zinc finger 1) to the first intron of α-SMA gene. We further confirmed that circACTA2, acting as a sponge binding microRNA (miR)-548f-5p, interacted with miR-548f-5p targeting 3' untranslated region of α-SMA mRNA, which in turn relieves miR-548f-5p repression of the α-SMA expression and thus upregulates α-SMA expression, thereby facilitating stress fiber formation and cell contraction in HASMCs. Accordingly, in vivo studies demonstrated that the localization of the interaction of circACTA2 with miR-548f-5p is significantly decreased in human intimal hyperplastic arteries compared with normal arteries, implicating that dysregulation of circACTA2 and miR-548f-5p expression is involved in intimal hyperplasia. These results suggest that circACTA2 mediates NRG-1-ICD regulation of α-SMA expression in HASMCs via the NRG-1-ICD/circACTA2/miR-548f-5p axis. Our data provide a molecular

Repression of miR-17-5p with elevated expression of E2F-1 and c-MYC in non-metastatic hepatocellular carcinoma and enhancement of cell growth upon reversing this expression pattern

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

El Tayebi, H.M.; Omar, K.; Hegy, S.

2013-05-10

Highlights: •The oncogenic miR-17-5p is downregulated in non-metastatic hepatocellular carcinoma patients. •E2F-1 and c-MYC transcripts are upregulated in non-metastatic HCC patients. •miR-17-5p forced overexpression inhibited E2F-1 and c-MYC expression in HuH-7 cells. •miR-17-5p mimicking increased HuH-7 cell growth, proliferation, migration and colony formation. •miR-17-5p is responsible for HCC progression among the c-MYC/E2F-1/miR-17-5p triad members. -- Abstract: E2F-1, c-MYC, and miR-17-5p is a triad of two regulatory loops: a negative and a positive loop, where c-MYC induces the expression of E2F-1 that induces the expression of miR-17-5p which in turn reverses the expression of E2F-1 to close the loop. In thismore » study, we investigated this triad for the first time in hepatocellular carcinoma (HCC), where miR-17-5p showed a significant down-regulation in 23 non-metastatic HCC biopsies compared to 10 healthy tissues; however, E2F-1 and c-MYC transcripts were markedly elevated. Forced over-expression of miR-17-5p in HuH-7 cells resulted in enhanced cell proliferation, growth, migration and clonogenicity with concomitant inhibition of E2F-1 and c-MYC transcripts expressions, while antagomirs of miR-17-5p reversed these events. In conclusion, this study revealed a unique pattern of expression for miR-17-5p in non-metastatic HCC patients in contrast to metastatic HCC patients. In addition we show that miR-17-5p is the key player among the triad that tumor growth and spread.« less

The antitumor effect of tanshinone IIA on anti-proliferation and decreasing VEGF/VEGFR2 expression on the human non-small cell lung cancer A549 cell line.

PubMed

Xie, Jun; Liu, Jiahui; Liu, Heng; Liang, Shihui; Lin, Meigui; Gu, Yueyu; Liu, Taoli; Wang, Dongmei; Ge, Hui; Mo, Sui-Lin

2015-11-01

The effects of tanshinone IIA on the proliferation of the human non-small cell lung cancer cell line A549 and its possible mechanism on the VEGF/VEGFR signal pathway were investigated. The exploration of the interaction between tanshinone IIA and its target proteins provides a feasible platform for studying the anticancer mechanism of active components of herbs. The CCK-8 assay was used to evaluate the proliferative activity of A549 cells treated with tanshinone IIA (2.5-80 μmol/L) for 24, 48 and 72 h, respectively. Flow cytometry was used for the detection of cell apoptosis and cell cycle perturbation. VEGF and VEGFR2 expression were studied by Western blotting. The binding mode of tanshinone IIA within the crystal structure of the VEGFR2 protein was evaluated with molecular docking analysis by use of the CDOCKER algorithm in Discovery Studio 2.1. The CCK-8 results showed that tanshinone IIA can significantly inhibit A549 cell proliferation in a dose- and time-dependent manner. Flow cytometry results showed that the apoptosis rate of tested group was higher than the vehicle control, and tanshinone IIA-treated cells accumulated at the S phase, which was higher than the vehicle control. Furthermore, the expression of VEGF and VEGFR2 was decreased in Western blot. Finally, molecular docking analysis revealed that tanshinone IIA could be stably docked into the kinase domain of VEGFR2 protein with its unique modes to form H-bonds with Cys917 and π-π stacking interactions with Val848. In conclusion, tanshinone IIA may suppress A549 proliferation, induce apoptosis and cell cycle arrest at the S phase. This drug may suppress angiogenesis by targeting the protein kinase domains of VEGF/VEGFR2.

The antitumor effect of tanshinone IIA on anti-proliferation and decreasing VEGF/VEGFR2 expression on the human non-small cell lung cancer A549 cell line

PubMed Central

Xie, Jun; Liu, Jiahui; Liu, Heng; Liang, Shihui; Lin, Meigui; Gu, Yueyu; Liu, Taoli; Wang, Dongmei; Ge, Hui; Mo, Sui-lin

2015-01-01

The effects of tanshinone IIA on the proliferation of the human non-small cell lung cancer cell line A549 and its possible mechanism on the VEGF/VEGFR signal pathway were investigated. The exploration of the interaction between tanshinone IIA and its target proteins provides a feasible platform for studying the anticancer mechanism of active components of herbs. The CCK-8 assay was used to evaluate the proliferative activity of A549 cells treated with tanshinone IIA (2.5−80 μmol/L) for 24, 48 and 72 h, respectively. Flow cytometry was used for the detection of cell apoptosis and cell cycle perturbation. VEGF and VEGFR2 expression were studied by Western blotting. The binding mode of tanshinone IIA within the crystal structure of the VEGFR2 protein was evaluated with molecular docking analysis by use of the CDOCKER algorithm in Discovery Studio 2.1. The CCK-8 results showed that tanshinone IIA can significantly inhibit A549 cell proliferation in a dose- and time-dependent manner. Flow cytometry results showed that the apoptosis rate of tested group was higher than the vehicle control, and tanshinone IIA-treated cells accumulated at the S phase, which was higher than the vehicle control. Furthermore, the expression of VEGF and VEGFR2 was decreased in Western blot. Finally, molecular docking analysis revealed that tanshinone IIA could be stably docked into the kinase domain of VEGFR2 protein with its unique modes to form H-bonds with Cys917 and π–π stacking interactions with Val848. In conclusion, tanshinone IIA may suppress A549 proliferation, induce apoptosis and cell cycle arrest at the S phase. This drug may suppress angiogenesis by targeting the protein kinase domains of VEGF/VEGFR2. PMID:26713270

Promoter analysis of the rabbit POU5F1 gene and its expression in preimplantation stage embryos.

PubMed

Kobolak, Julianna; Kiss, Katalin; Polgar, Zsuzsanna; Mamo, Solomon; Rogel-Gaillard, Claire; Tancos, Zsuzsanna; Bock, Istvan; Baji, Arpad G; Tar, Krisztina; Pirity, Melinda K; Dinnyes, Andras

2009-09-04

The POU5F1 gene encodes the octamer-binding transcription factor-4 (Oct4). It is crucial in the regulation of pluripotency during embryonic development and widely used as molecular marker of embryonic stem cells (ESCs). The objective of this study was to identify and to analyse the promoter region of rabbit POU5F1 gene; furthermore to examine its expression pattern in preimplantation stage rabbit embryos. The upstream region of rabbit POU5F1 was subcloned sequenced and four highly conserved promoter regions (CR1-4) were identified. The highest degree of similarity on sequence level was found among the conserved domains between rabbit and human. Among the enhancers the proximal enhancer region (PE-1A) exhibited the highest degree of homology (96.4%). Furthermore, the CR4 regulator domain containing the distal enhancer (DE-2A) was responsible for stem cell-specific expression. Also, BAC library screen revealed the existence of a processed pseudogene of rabbit POU5F1. The results of quantitative real-time PCR experiments showed that POU5F1 mRNA was abundantly present in oocytes and zygotes, but it was gradually reduced until the activation of the embryonic genome, thereafter a continuous increase in POU5F1 mRNA level was observed until blastocyst stage. By using the XYClone laser system the inner cell mass (ICM) and trophoblast portions of embryos were microdissected and examined separately and POU5F1 mRNA was detected in both cell types. In this study we provide a comparative sequence analysis of the regulatory region of rabbit POU5F1 gene. Our data suggest that the POU5F1 gene is strictly regulated during early mammalian development. We proposed that the well conserved CR4 region containing the DE-2A enhancer is responsible for the highly conserved ESC specific gene expression. Notably, we are the first to report that the rabbit POU5F1 is not restricted to ICM cells only, but it is expressed in trophoblast cells as well. This information may be well applicable to

Comparative expression analysis of POU4F1, POU4F2 and ISL1 in developing mouse cochleovestibular ganglion neurons

PubMed Central

Deng, Min; Yang, Hua; Xie, Xiaoling; Liang, Guoqing; Gan, Lin

2014-01-01

POU-homeodomain and LIM-homeodomain transcription factors are expressed in developing projection neurons within retina, inner ear, dorsal root ganglion, and trigeminal ganglion, and play synergistic roles in their differentiation and survival. Here, using immunohistochemistry, we present a comparative analysis of the spatiotemporal expression pattern of POU4F1, POU4F2, and ISL1 during the development of cochleovestibular ganglion (CVG) neurons in mouse inner ear. At early stages, when otic neurons are first detected in the otic epithelium (OE) and migrate into periotic mesenchyme to form the CVG, POU4F1 and ISL1 are co-expressed in a majority of the delaminated CVG neurons, which are marked by NEUROD1 expression, but POU4F1 is absent in the otic epithelium. The onset of POU4F2 expression starts after that of POU4F1 and ISL1, and is observed in the NEUROD1-negative, post-mitotic CVG neurons. When the CVG neurons innervate the vestibular and cochlear sensory organs, the expression of POU4F1, POU4F2, and ISL1 continues in both vestibular and spiral ganglion cells. Later in development, POU4F1 expression becomes down-regulated in a majority of spiral ganglion (SG) neurons and more neurons express POU4F2 expression while ISL1 expression is maintained. The differential as well as overlapping expression of POU4F1, POU4F2, and ISL1 combined with previous studies suggests possible functional interaction and regulatory relationship of these transcription factors in the development of inner ear neurons. PMID:24709358

The anti-inflammatory activity of standard aqueous stem bark extract of Mangifera indica L. as evident in inhibition of Group IA sPLA2.

PubMed

Dhananjaya, Bhadrapura Lakkappa; Shivalingaiah, Sudharshan

2016-03-01

The standard aqueous stem bark extract is consumed as herbal drink and used in the pharmaceutical formulations to treat patients suffering from various disease conditions in Cuba. This study was carried out to evaluate the modulatory effect of standard aqueous bark extract of M. indica on Group IA sPLA2. M. indica extract, dose dependently inhibited the GIA sPLA2 (NN-XIa-PLA2) activity with an IC50 value 8.1 µg/ml. M. indica extract effectively inhibited the indirect hemolytic activity up to 98% at ~40 µg/ml concentration and at various concentrations (0-50 µg/ml), it dose dependently inhibited the edema formation. When examined as a function of increased substrate and calcium concentration, there was no relieve of inhibitory effect on the GIA sPLA2. Furthermore, the inhibition was irreversible as evidenced from binding studies. It is observed that the aqueous extract ofM. indica effectively inhibits sPLA2 and it is associated inflammatory activities, which substantiate their anti-inflammatory properties. The mode of inhibition could be due to direct interaction of components present in the extract, with sPLA2 enzyme. Further studies on understanding the principal constituents, responsible for the anti-inflammatory activity would be interesting to develop this into potent anti-inflammatory agent.

TFDP3 was expressed in coordination with E2F1 to inhibit E2F1-mediated apoptosis in prostate cancer.

PubMed

Ma, Yueyun; Xin, Yijuan; Li, Rui; Wang, Zhe; Yue, Qiaohong; Xiao, Fengjing; Hao, Xiaoke

2014-03-10

TFDP3 has been previously identified as an inhibitor of E2F molecules. It has been shown to suppress E2F1-induced apoptosis dependent P53 and to play a potential role in carcinogenesis. However, whether it indeed helps cancer cells tolerate apoptosis stress in cancer tissues remains unknown. TFDP3 expression was assessed by RT-PCR, in situ hybridization and immunohistochemistry in normal human tissues, cancer tissues and prostate cancer tissues. The association between TFDP3 and E2F1 in prostate cancer development was analyzed in various stages. Apoptosis was evaluated with annexin-V and propidium iodide staining and flow-cytometry. The results show that, in 96 samples of normal human tissues, TFDP3 could be detected in the cerebrum, esophagus, stomach, small intestine, bronchus, breast, ovary, uterus, and skin, but seldom in the lung, muscles, prostate, and liver. In addition, TFDP3 was highly expressed in numerous cancer tissues, such as brain-keratinous, lung squamous cell carcinoma, testicular seminoma, cervical carcinoma, skin squamous cell carcinoma, gastric adenocarcinoma, liver cancer, and prostate cancer. Moreover, TFDP3 was positive in 23 (62.2%) of 37 prostate cancer samples regardless of stage. Furthermore, immunohistochemistry results show that TFDP3 was always expressed in coordination with E2F1 at equivalent expression levels in prostate cancer tissues, and was highly expressed particularly in samples of high stage. When E2F1 was extrogenously expressed in LNCap cells, TFDP3 could be induced, and the apoptosis induced by E2F1 was significantly decreased. It was demonstrated that TFDP3 was a broadly expressed protein corresponding to E2F1 in human tissues, and suggested that TFDP3 is involved in prostate cancer cell survival by suppressing apoptosis induced by E2F1. Copyright © 2013 Elsevier B.V. All rights reserved.

Sexually Dimorphic Expression of Foxl2 and Ftz-F1 in Chinese Giant Salamander Andrias Davidianus.

PubMed

Hu, Qiaomu; Meng, Yan; Tian, Haifeng; Zhang, Y U; Xiao, Hanbing

2016-09-01

Foxl2 and FTZ-F1 play a crucial role in the regulation of gonad development in fish and mammals, but studies of their function in amphibians are scarce. We isolated the full length of Foxl2 (adFoxl2) and Ftz-F1 (adFtz-f1) cDNA from the Chinese giant salamander Andrias davidianus and quantified its expression in various tissues and developing gonads. The adFoxl2 gene encodes 301aa including a conserved forkhead box, and the adFtz-f1 gene encodes 467aa containing an Ftz-F1 box. The amino acid sequences showed high homology with other amphibians. adFoxl2 expression was high in ovary, whereas adFtz-f1 was higher in testis, moderate in pituitary, ovary, and kidney; and low in the remaining tested tissues. Expression of adFoxl2 gradually increased from 1Y to 5Y in ovary, whereas adFtz-f1 expression gradually decreased in testis. In addition, adFoxl2 and adFtz-f1 were detected in granulosa cell in ovary and in spermatocytes in testis. The adFoxl2 transcription was inhibited in brain and ovary after treatment with methyltestosterone and with letrozole, whereas adFtz-f1 expression was upregulated. High-temperature suppressed the expression of adFxl2 in ovary and enhanced the transcription of adFtz-f1. These results suggest that adFoxl2 functioned in ovary differentiation, whereas adFtz-f1 played a role in testis development, which lays a foundation for study of the sex differentiation mechanism in A. davidianus. © 2016 Wiley Periodicals, Inc.

Noble-Gas Difluoride Complexes of Mercury(II): The Syntheses and Structures of Hg(OTeF 5) 2·1.5NgF 2 (Ng = Xe, Kr) and Hg(OTeF 5) 2

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

DeBackere, John R.; Mercier, Helene P. A; Schrobilgen, Gary J.

2014-02-03

The synthesis of high-purity Hg(OTeF 5) 2 has resulted in its structural characterization in the solid state by Raman spectroscopy and single-crystal X-ray diffraction (XRD) and in solution by 19F NMR spectroscopy. The crystal structure of Hg(OTeF 5) 2 (-173 °C) consists of discrete Hg(OTeF 5) 2 units having gauche-conformations that interact through long Hg---O and Hg---F intramolecular contacts to give a chain structure. Furthermore, the Lewis acidity of Hg(OTeF 5) 2 toward NgF 2 (Ng = Xe, Kr) was investigated in SO 2ClF solvent and shown to form stable coordination complexes with NgF 2 at -78 °C. Both complexesmore » were characterized by low-temperature Raman spectroscopy (-155 °C) and single-crystal XRD. The complexes are isostructural and are formulated as Hg(OTeF 5) 2·1.5NgF 2. The Hg(OTeF 5) 2 units of Hg(OTeF 5) 2·1.5NgF 2 also have gauche-conformations and are linked through bridging NgF 2 molecules, also resulting in chain structures. The complexes represent the only examples of coordination compounds where NgF 2 coordinates to mercury in a neutral covalent compound and the only example of mercury coordinated to KrF 2. Moreover, the Hg(OTeF 5) 2·1.5KrF 2 complex is the only KrF 2 complex known to contain a bridging KrF 2 ligand. Energy-minimized gas-phase geometries and vibrational frequencies for the model compounds, [Hg(OTeF5) 2] 3 and [Hg(OTeF 5) 2] 3·2NgF 2, were obtained and provide good approximations of the local environments of Hg(OTeF 5) 2 and NgF 2 in the crystal structures of Hg(OTeF5)2 and Hg(OTeF 5) 2·1.5NgF 2. Assignments of the Raman spectra of Hg(OTeF 5) 2 and Hg(OTeF 5) 2·1.5NgF 2 are based on the calculated vibrational frequencies of the model compounds. Natural bond orbital analyses provided the associated bond orders, valencies, and natural population analysis charges.« less

Nonmuscle myosin IIA is required for lamellipodia formation through binding to WAVE2 and phosphatidylinositol 3,4,5-triphosphate.

PubMed

Morimura, Shigeru; Suzuki, Katsuo; Takahashi, Kazuhide

2011-01-21

Investigation of the mechanism underlying cell membrane-targeted WAVE2 capture by phosphatidylinositol 3,4,5-triphosphate (PIP(3)) through IRSp53 revealed an unidentified 250-kDa protein (p250) bound to PIP(3). We identified p250 as nonmuscle myosin IIA heavy chain (MYH9) by mass spectrometry and immunoblot analysis using anti-MYH9 antibody. After stimulation with insulin-like growth factor I (IGF-I), MYH9 colocalized with PIP(3) in lamellipodia at the leading edge of cells. Depletion of MYH9 expression by small interfering RNA (siRNA) and inhibition of myosin II activity by blebbistatin abrogated the formation of actin filament (F-actin) arcs and lamellipodia induced by IGF-I. MYH9 was constitutively associated with WAVE2, which was dependent on myosin II activity, and the MYH9-WAVE2 complex colocalized to PIP(3) at the leading edge after IGF-I stimulation. These results indicate that MYH9 is required for lamellipodia formation since it provides contractile forces and tension for the F-actin network to form convex arcs at the leading edge through constitutive binding to WAVE2 and colocalization with PIP(3) in response to IGF-I. Copyright © 2010 Elsevier Inc. All rights reserved.

Frequency measurement of the 5 S{1}/{2}(F = 3)-5 D{5}/{2}(F = 5) two-photon transition in rubidium

NASA Astrophysics Data System (ADS)

Touahri, D.; Acef, O.; Clairon, A.; Zondy, J.-J.; Felder, R.; Hilico, L.; de Beauvoir, B.; Biraben, F.; Nez, F.

1997-02-01

We have measured the frequencies of three diode lasers stabilized on the 5 S{1}/{2}(F = 3)-5 D{5}/{2}(F = 5) two-photon transition in rubidium at λ = 778.1 nm, with an uncertainty of 1 kHz, using BNM-LPTF frequency synthesis chain starting from a {CO 2}/{OsO 4} reference laser at 10.3 μm. We show that this frequency chain is able to reach the 10 -13 resolution level. After a discussion of the systematic effects that may shift the resonance, the transition frequency is found to be ν = 385 285 142 378.280 ± 2 kHz.

Detection and quantification of microparticles from different cellular lineages using flow cytometry. Evaluation of the impact of secreted phospholipase A2 on microparticle assessment.

PubMed

Rousseau, Matthieu; Belleannee, Clemence; Duchez, Anne-Claire; Cloutier, Nathalie; Levesque, Tania; Jacques, Frederic; Perron, Jean; Nigrovic, Peter A; Dieude, Melanie; Hebert, Marie-Josee; Gelb, Michael H; Boilard, Eric

2015-01-01

Microparticles, also called microvesicles, are submicron extracellular vesicles produced by plasma membrane budding and shedding recognized as key actors in numerous physio(patho)logical processes. Since they can be released by virtually any cell lineages and are retrieved in biological fluids, microparticles appear as potent biomarkers. However, the small dimensions of microparticles and soluble factors present in body fluids can considerably impede their quantification. Here, flow cytometry with improved methodology for microparticle resolution was used to detect microparticles of human and mouse species generated from platelets, red blood cells, endothelial cells, apoptotic thymocytes and cells from the male reproductive tract. A family of soluble proteins, the secreted phospholipases A2 (sPLA2), comprises enzymes concomitantly expressed with microparticles in biological fluids and that catalyze the hydrolysis of membrane phospholipids. As sPLA2 can hydrolyze phosphatidylserine, a phospholipid frequently used to assess microparticles, and might even clear microparticles, we further considered the impact of relevant sPLA2 enzymes, sPLA2 group IIA, V and X, on microparticle quantification. We observed that if enriched in fluids, certain sPLA2 enzymes impair the quantification of microparticles depending on the species studied, the source of microparticles and the means of detection employed (surface phosphatidylserine or protein antigen detection). This study provides analytical considerations for appropriate interpretation of microparticle cytofluorometric measurements in biological samples containing sPLA2 enzymes.

Emergence of Highly Pathogenic Avian Influenza A(H5N1) Virus PB1-F2 Variants and Their Virulence in BALB/c Mice

PubMed Central

Kamal, Ram P.; Kumar, Amrita; Davis, Charles T.; Tzeng, Wen-Pin; Nguyen, Tung; Donis, Ruben O.; Katz, Jacqueline M.

2015-01-01

ABSTRACT Influenza A viruses (IAVs) express the PB1-F2 protein from an alternate reading frame within the PB1 gene segment. The roles of PB1-F2 are not well understood but appear to involve modulation of host cell responses. As shown in previous studies, we find that PB1-F2 proteins of mammalian IAVs frequently have premature stop codons that are expected to cause truncations of the protein, whereas avian IAVs usually express a full-length 90-amino-acid PB1-F2. However, in contrast to other avian IAVs, recent isolates of highly pathogenic H5N1 influenza viruses had a high proportion of PB1-F2 truncations (15% since 2010; 61% of isolates in 2013) due to several independent mutations that have persisted and expanded in circulating viruses. One natural H5N1 IAV containing a mutated PB1-F2 start codon (i.e., lacking ATG) was 1,000-fold more virulent for BALB/c mice than a closely related H5N1 containing intact PB1-F2. In vitro, we detected expression of an in-frame protein (C-terminal PB1-F2) from downstream ATGs in PB1-F2 plasmids lacking the well-conserved ATG start codon. Transient expression of full-length PB1-F2, truncated (24-amino-acid) PB1-F2, and PB1-F2 lacking the initiating ATG in mammalian and avian cells had no effect on cell apoptosis or interferon expression in human lung epithelial cells. Full-length and C-terminal PB1-F2 mutants colocalized with mitochondria in A549 cells. Close monitoring of alterations of PB1-F2 and their frequency in contemporary avian H5N1 viruses should continue, as such changes may be markers for mammalian virulence. IMPORTANCE Although most avian influenza viruses are harmless for humans, some (such as highly pathogenic H5N1 avian influenza viruses) are capable of infecting humans and causing severe disease with a high mortality rate. A number of risk factors potentially associated with adaptation to mammalian infection have been noted. Here we demonstrate that the protein PB1-F2 is frequently truncated in recent isolates of

Hair Follicular Expression and Function of Group X Secreted Phospholipase A2 in Mouse Skin*

PubMed Central

Yamamoto, Kei; Taketomi, Yoshitaka; Isogai, Yuki; Miki, Yoshimi; Sato, Hiroyasu; Masuda, Seiko; Nishito, Yasumasa; Morioka, Kiyokazu; Ishimoto, Yoshikazu; Suzuki, Noriko; Yokota, Yasunori; Hanasaki, Kohji; Ishikawa, Yukio; Ishii, Toshiharu; Kobayashi, Tetsuyuki; Fukami, Kiyoko; Ikeda, Kazutaka; Nakanishi, Hiroki; Taguchi, Ryo; Murakami, Makoto

2011-01-01

Although perturbed lipid metabolism can often lead to skin abnormality, the role of phospholipase A2 (PLA2) in skin homeostasis is poorly understood. In the present study we found that group X-secreted PLA2 (sPLA2-X) was expressed in the outermost epithelium of hair follicles in synchrony with the anagen phase of hair cycling. Transgenic mice overexpressing sPLA2-X (PLA2G10-Tg) displayed alopecia, which was accompanied by hair follicle distortion with reduced expression of genes related to hair development, during a postnatal hair cycle. Additionally, the epidermis and sebaceous glands of PLA2G10-Tg skin were hyperplasic. Proteolytic activation of sPLA2-X in PLA2G10-Tg skin was accompanied by preferential hydrolysis of phosphatidylethanolamine species with polyunsaturated fatty acids as well as elevated production of some if not all eicosanoids. Importantly, the skin of Pla2g10-deficient mice had abnormal hair follicles with noticeable reduction in a subset of hair genes, a hypoplasic outer root sheath, a reduced number of melanin granules, and unexpected up-regulation of prostanoid synthesis. Collectively, our study highlights the spatiotemporal expression of sPLA2-X in hair follicles, the presence of skin-specific machinery leading to sPLA2-X activation, a functional link of sPLA2-X with hair follicle homeostasis, and compartmentalization of the prostanoid pathway in hair follicles and epidermis. PMID:21266583

E2F1/TS Immunophenotype and Survival of Patients with Colorectal Cancer Treated with 5FU-Based Adjuvant Therapy.

PubMed

Sulzyc-Bielicka, Violetta; Domagala, Pawel; Bielicki, Dariusz; Safranow, Krzysztof; Rogowski, Wojciech; Domagala, Wenancjusz

2016-07-01

The predictive value of thymidylate synthase (TS) expression alone for 5FU-based treatment of colorectal cancer (CRC) has not been clinically confirmed. Little is known on the association of expression of E2F1, which controls the transcription of genes encoding proteins engaged in DNA synthesis including TS, and survival of patients with CRC. The purpose of this study is to assess the correlation between expression of both E2F1 and TS in CRCs and survival of patients administered adjuvant 5FU-based chemotherapy, in order to find a better predictor of treatment outcome than expression of TS or E2F1 alone. Nuclear TS and E2F1 were detected by immunohistochemistry in tissue microarrays from 190 CRCs (Astler-Coller stage B2 or C). Multivariate analysis identified significant association of the combined E2F1+TS+ immunophenotype with worse OS (HR = 3,78, P = 0,009) and DFS (HR = 2,30, P = 0,03) of patients with colon cancer. There were significant differences between E2F1+TS+ and E2F1-TS- Kaplan-Meier survival curves in relation to DFS (P = 0.008) and OS (P = 0.01). About 37 and 31 % difference in 3-year DFS and OS respectively were seen between patients with E2F1+TS+ vs. E2F1-TS- colon cancer immunophenotype. The E2F1+TS+ immunophenotype may be a marker of poor prognosis (the worst DFS and OS) of patients with colon cancer treated with 5FU-based adjuvant therapy. A subgroup of patients with this immunophenotype may require different and perhaps more aggressive treatment than 5FU-based chemotherapy. Thus, the combined E2F1/TS immunophenotype could be a potential indicator of colon cancer sensitivity to 5FU.

Audiological findings in Usher syndrome types IIa and II (non-IIa).

PubMed

Sadeghi, Mehdi; Cohn, Edward S; Kelly, William J; Kimberling, William J; Tranebjoerg, Lisbeth; Möller, Claes

2004-03-01

The aim was to define the natural history of hearing loss in Usher syndrome type IIa compared to non-IIa. People with Usher syndrome type II show moderate-to-severe hearing loss, normal balance and retinitis pigmentosa. Several genes cause Usher syndrome type II. Our subjects formed two genetic groups: (1) subjects with Usher syndrome type IIa with a mutation and/or linkage to the Usher IIa gene; (2) subjects with the Usher II phenotype with no mutation and/or linkage to the Usher IIa gene. Four hundred and two audiograms of 80 Usher IIa subjects were compared with 435 audiograms of 87 non-IIa subjects. Serial audiograms with intervals of > or = 5 years were examined for progression in 109 individuals Those with Usher syndrome type IIa had significantly worse hearing thresholds than those with non-IIa Usher syndrome after the second decade. The hearing loss in Usher syndrome type IIa was found to be more progressive, and the progression started earlier than in non-IIa Usher syndrome. This suggests an auditory phenotype for Usher syndrome type IIa that is different from that of other types of Usher syndrome II. Thus, this is to our knowledge one of the first studies showing a genotype-phenotype auditory correlation.

PB1-F2 attenuates virulence of highly pathogenic avian H5N1 influenza virus in chickens.

PubMed

Leymarie, Olivier; Embury-Hyatt, Carissa; Chevalier, Christophe; Jouneau, Luc; Moroldo, Marco; Da Costa, Bruno; Berhane, Yohannes; Delmas, Bernard; Weingartl, Hana M; Le Goffic, Ronan

2014-01-01

Highly pathogenic avian influenza virus (HPAIV) is a permanent threat due to its capacity to cross species barriers and generate severe infections and high mortality in humans. Recent findings have highlighted the potential role of PB1-F2, a small accessory influenza protein, in the pathogenesis process mediated by HPAIV in mammals. In this study, using a recombinant H5N1 HPAIV (wt) and its PB1-F2-deleted mutant (ΔF2), we studied the effects of PB1-F2 in a chicken model. Unexpectedly, when using low inoculation dose we observed that the wt-infected chickens had a higher survival rate than the ΔF2-infected chickens, a feature that contrasts with what is usually observed in mammals. High inoculation dose had similar mortality rate for both viruses, and comparison of the bio-distribution of the two viruses indicated that the expression of PB1-F2 allows a better spreading of the virus within chicken embryos. Transcriptomic profiles of lungs and blood cells were characterized at two days post-infection in chickens inoculated with the wild type (wt) or the ΔF2 mutant viruses. In lungs, the expression of PB1-F2 during the infection induced pathways related to calcium signaling and repressed a large panel of immunological functions. In blood cells, PB1-F2 was associated with a gene signature specific for mitochondrial dysfunction and down-modulated leucocytes activation. Finally we compared the effect of PB1-F2 in lungs of chickens and mice. We identified that gene signature associated to tissue damages is a PB1-F2 feature shared by the two species; by contrast, the early inhibition of immune response mediated by PB1-F2 observed in chickens is not seen in mice. In summary, our data suggest that PB1-F2 expression deeply affect the immune response in chickens in a way that may attenuate pathogenicity at low infection dose, a feature differing from what was previously observed in mammal species.

PB1-F2 Attenuates Virulence of Highly Pathogenic Avian H5N1 Influenza Virus in Chickens

PubMed Central

Leymarie, Olivier; Embury-Hyatt, Carissa; Chevalier, Christophe; Jouneau, Luc; Moroldo, Marco; Da Costa, Bruno; Berhane, Yohannes; Delmas, Bernard

2014-01-01

Highly pathogenic avian influenza virus (HPAIV) is a permanent threat due to its capacity to cross species barriers and generate severe infections and high mortality in humans. Recent findings have highlighted the potential role of PB1-F2, a small accessory influenza protein, in the pathogenesis process mediated by HPAIV in mammals. In this study, using a recombinant H5N1 HPAIV (wt) and its PB1-F2-deleted mutant (ΔF2), we studied the effects of PB1-F2 in a chicken model. Unexpectedly, when using low inoculation dose we observed that the wt-infected chickens had a higher survival rate than the ΔF2-infected chickens, a feature that contrasts with what is usually observed in mammals. High inoculation dose had similar mortality rate for both viruses, and comparison of the bio-distribution of the two viruses indicated that the expression of PB1-F2 allows a better spreading of the virus within chicken embryos. Transcriptomic profiles of lungs and blood cells were characterized at two days post-infection in chickens inoculated with the wild type (wt) or the ΔF2 mutant viruses. In lungs, the expression of PB1-F2 during the infection induced pathways related to calcium signaling and repressed a large panel of immunological functions. In blood cells, PB1-F2 was associated with a gene signature specific for mitochondrial dysfunction and down-modulated leucocytes activation. Finally we compared the effect of PB1-F2 in lungs of chickens and mice. We identified that gene signature associated to tissue damages is a PB1-F2 feature shared by the two species; by contrast, the early inhibition of immune response mediated by PB1-F2 observed in chickens is not seen in mice. In summary, our data suggest that PB1-F2 expression deeply affect the immune response in chickens in a way that may attenuate pathogenicity at low infection dose, a feature differing from what was previously observed in mammal species. PMID:24959667

Regulation of myosin IIA and filamentous actin during insulin-stimulated glucose uptake in 3T3-L1 adipocytes

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

Stall, Richard; Ramos, Joseph; Kent Fulcher, F.

Insulin stimulated glucose uptake requires the colocalization of myosin IIA (MyoIIA) and the insulin-responsive glucose transporter 4 (GLUT4) at the plasma membrane for proper GLUT4 fusion. MyoIIA facilitates filamentous actin (F-actin) reorganization in various cell types. In adipocytes F-actin reorganization is required for insulin-stimulated glucose uptake. What is not known is whether MyoIIA interacts with F-actin to regulate insulin-induced GLUT4 fusion at the plasma membrane. To elucidate the relationship between MyoIIA and F-actin, we examined the colocalization of MyoIIA and F-actin at the plasma membrane upon insulin stimulation as well as the regulation of this interaction. Our findings demonstrated thatmore » MyoIIA and F-actin colocalized at the site of GLUT4 fusion with the plasma membrane upon insulin stimulation. Furthermore, inhibition of MyoII with blebbistatin impaired F-actin localization at the plasma membrane. Next we examined the regulatory role of calcium in MyoIIA-F-actin colocalization. Reduced calcium or calmodulin levels decreased colocalization of MyoIIA and F-actin at the plasma membrane. While calcium alone can translocate MyoIIA it did not stimulate F-actin accumulation at the plasma membrane. Taken together, we established that while MyoIIA activity is required for F-actin localization at the plasma membrane, it alone is insufficient to localize F-actin to the plasma membrane. - Highlights: • Insulin induces colocalization of MyoIIA and F-actin at the cortex in adipocytes. • MyoIIA is necessary but not sufficient to localize F-actin at the cell cortex. • MyoIIA-F-actin colocalization is regulated by calcium and calmodulin.« less

Cooption of secretory phospholipase (SPLA2) for different aspects of gravity receptor-associated mineralization in vertebrate phylogeny

NASA Astrophysics Data System (ADS)

Thalmann, R.; Lu, W.

2009-04-01

not induce formation of aragonite, but of calcite as default option, analogous to other aragonitic matrix proteins, e.g. AP8 alpha and beta of the avalone nacre. It has been shown that aragonitic proteins are able to express aragonite only in association with the appropriate insoluble matrix. The effects of the protein upon calcite modification are qualitatively identical to the effects of OC90 (nucleation density, crystal size and morphologic change), but are quantitatively much less. The most unexpected SPLA2 domain-related aspect is the recent discovery of an OC90 ortholog (Otoc1) in otoliths even though OMP, a derivative of melanotransferin is the principal soluble matrix protein of otoliths. We, heretofore, assumed that OC22, as a single SPLAL domain, constituted the earliest manifestation of this cooption in vertebrate phylogeny. Significantly, the presence of Otoc1 is not incidental. Knock-down of the gene results in agenesis or malformation of otolith with a change from aragonite to calcite. In summary, this presentation serves to illustrate the wide distribution and varied function of a coopted lipolytic enzyme in a wide range of mineralization-related contexts.

E2F1-mediated human POMC expression in ectopic Cushing's syndrome.

PubMed

Araki, Takako; Liu, Ning-Ai; Tone, Yukiko; Cuevas-Ramos, Daniel; Heltsley, Roy; Tone, Masahide; Melmed, Shlomo

2016-11-01

Cushing's syndrome is caused by excessive adrenocorticotropic hormone (ACTH) secretion derived from pituitary corticotroph tumors (Cushing disease) or from non-pituitary tumors (ectopic Cushing's syndrome). Hypercortisolemic features of ectopic Cushing's syndrome are severe, and no definitive treatment for paraneoplastic ACTH excess is available. We aimed to identify subcellular therapeutic targets by elucidating transcriptional regulation of the human ACTH precursor POMC (proopiomelanocortin) and ACTH production in non-pituitary tumor cells and in cell lines derived from patients with ectopic Cushing's syndrome. We show that ectopic hPOMC transcription proceeds independently of pituitary-specific Tpit/Pitx1 and demonstrate a novel E2F1-mediated transcriptional mechanism regulating hPOMC We identify an E2F1 cluster binding to the proximal hPOMC promoter region (-42 to +68), with DNA-binding activity determined by the phosphorylation at Ser-337. hPOMC mRNA expression in cancer cells was upregulated (up to 40-fold) by the co-expression of E2F1 and its heterodimer partner DP1. Direct and indirect inhibitors of E2F1 activity suppressed hPOMC gene expression and ACTH by modifying E2F1 DNA-binding activity in ectopic Cushing's cell lines and primary tumor cells, and also suppressed paraneoplastic ACTH and cortisol levels in xenografted mice. E2F1-mediated hPOMC transcription is a potential target for suppressing ACTH production in ectopic Cushing's syndrome. © 2016 Society for Endocrinology.

Development of a potent 2-oxoamide inhibitor of secreted phospholipase A2 guided by molecular docking calculations and molecular dynamics simulations

PubMed Central

Vasilakaki, Sofia; Barbayianni, Efrosini; Leonis, Georgios; Papadopoulos, Manthos G.; Mavromoustakos, Thomas; Gelb, Michael H.; Kokotos, George

2016-01-01

Inhibition of group IIA secreted phospholipase A2 (GIIA sPLA2) has been an important objective for medicinal chemists. We have previously shown that inhibitors incorporating the 2-oxoamide functionality may inhibit human and mouse GIIA sPLA2s. Herein, the development of new potent inhibitors by molecular docking calculations using the structure of the known inhibitor 7 as scaffold, are described. Synthesis and biological evaluation of the new compounds revealed that the long chain 2-oxoamide based on (S)-valine GK241 led to improved activity (IC50 = 143 nM and 68 nM against human and mouse GIIA sPLA2, respectively). In addition, molecular dynamics simulations were employed to shed light on GK241 potent and selective inhibitory activity. PMID:26970660

Discovery of AZD2716: A Novel Secreted Phospholipase A2 (sPLA2) Inhibitor for the Treatment of Coronary Artery Disease

PubMed Central

2016-01-01

Expedited structure-based optimization of the initial fragment hit 1 led to the design of (R)-7 (AZD2716) a novel, potent secreted phospholipase A2 (sPLA2) inhibitor with excellent preclinical pharmacokinetic properties across species, clear in vivo efficacy, and minimized safety risk. Based on accumulated profiling data, (R)-7 was selected as a clinical candidate for the treatment of coronary artery disease. PMID:27774123

AMPK activation by Tanshinone IIA protects neuronal cells from oxygen-glucose deprivation

PubMed Central

Weng, Yingfeng; Lin, Jixian; Liu, Hui; Wu, Hui; Yan, Zhimin; Zhao, Jing

2018-01-01

The current study tested the potential neuroprotective function of Tanshinone IIA (ThIIA) in neuronal cells with oxygen-glucose deprivation (ODG) and re-oxygenation (OGDR). In SH-SY5Y neuronal cells and primary murine cortical neurons, ThIIA pre-treatment attenuated OGDR-induced viability reduction and apoptosis. Further, OGDR-induced mitochondrial depolarization, reactive oxygen species production, lipid peroxidation and DNA damages in neuronal cells were significantly attenuated by ThIIA. ThIIA activated AMP-activated protein kinase (AMPK) signaling, which was essential for neuroprotection against OGDR. AMPKα1 knockdown or complete knockout in SH-SY5Y cells abolished ThIIA-induced AMPK activation and neuroprotection against OGDR. Further studies found that ThIIA up-regulated microRNA-135b to downregulate the AMPK phosphatase Ppm1e. Notably, knockdown of Ppm1e by targeted shRNA or forced microRNA-135b expression also activated AMPK and protected SH-SY5Y cells from OGDR. Together, AMPK activation by ThIIA protects neuronal cells from OGDR. microRNA-135b-mediated silence of Ppm1e could be the key mechanism of AMPK activation by ThIIA. PMID:29435120

Estrogen directly and specifically downregulates NaPi-IIa through the activation of both estrogen receptor isoforms (ERα and ERβ) in rat kidney proximal tubule.

PubMed

Burris, Dara; Webster, Rose; Sheriff, Sulaiman; Faroqui, Rashma; Levi, Moshe; Hawse, John R; Amlal, Hassane

2015-03-15

We have previously demonstrated that estrogen (E2) downregulates phosphate transporter NaPi-IIa and causes phosphaturia and hypophosphatemia in ovariectomized rats. In the present study, we examined whether E2 directly targets NaPi-IIa in the proximal tubule (PT) and studied the respective roles of estrogen receptor isoforms (ERα and ERβ) in the downregulation of NaPi-IIa using both in vivo and an in vitro expression systems. We found that estrogen specifically downregulates NaPi-IIa but not NaPi-IIc or Pit2 in the kidney cortex. Proximal tubules incubated in a "shake" suspension with E2 for 24 h exhibited a dose-dependent decrease in NaPi-IIa protein abundance. Results from OVX rats treated with specific agonists for either ERα [4,4',4″;-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol, PPT] or ERβ [4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol, DPN] or both (PPT + DPN), indicated that only the latter caused a sharp downregulation of NaPi-IIa, along with significant phosphaturia and hypophosphatemia. Lastly, heterologous expression studies demonstrated that estrogen downregulated NaPi-IIa only in U20S cells expressing both ERα and ERβ, but not in cells expressing either receptor alone. In conclusion, these studies demonstrate that rat PT cells express both ERα and ERβ and that E2 induces phosphaturia by directly and specifically targeting NaPi-IIa in the PT cells. This effect is mediated via a mechanism involving coactivation of both ERα and ERβ, which likely form a functional heterodimer complex in the rat kidney proximal tubule. Copyright © 2015 the American Physiological Society.

Estrogen directly and specifically downregulates NaPi-IIa through the activation of both estrogen receptor isoforms (ERα and ERβ) in rat kidney proximal tubule

PubMed Central

Burris, Dara; Webster, Rose; Sheriff, Sulaiman; Faroqui, Rashma; Levi, Moshe; Hawse, John R.

2015-01-01

We have previously demonstrated that estrogen (E2) downregulates phosphate transporter NaPi-IIa and causes phosphaturia and hypophosphatemia in ovariectomized rats. In the present study, we examined whether E2 directly targets NaPi-IIa in the proximal tubule (PT) and studied the respective roles of estrogen receptor isoforms (ERα and ERβ) in the downregulation of NaPi-IIa using both in vivo and an in vitro expression systems. We found that estrogen specifically downregulates NaPi-IIa but not NaPi-IIc or Pit2 in the kidney cortex. Proximal tubules incubated in a “shake” suspension with E2 for 24 h exhibited a dose-dependent decrease in NaPi-IIa protein abundance. Results from OVX rats treated with specific agonists for either ERα [4,4′,4″;-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol, PPT] or ERβ [4,4′,4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol, DPN] or both (PPT + DPN), indicated that only the latter caused a sharp downregulation of NaPi-IIa, along with significant phosphaturia and hypophosphatemia. Lastly, heterologous expression studies demonstrated that estrogen downregulated NaPi-IIa only in U20S cells expressing both ERα and ERβ, but not in cells expressing either receptor alone. In conclusion, these studies demonstrate that rat PT cells express both ERα and ERβ and that E2 induces phosphaturia by directly and specifically targeting NaPi-IIa in the PT cells. This effect is mediated via a mechanism involving coactivation of both ERα and ERβ, which likely form a functional heterodimer complex in the rat kidney proximal tubule. PMID:25608964

Synthesis and In Vitro Evaluation of 5-[18F]Fluoroalkyl Pyrimidine Nucleosides for Molecular Imaging of Herpes Simplex Virus Type-1 Thymidine Kinase Reporter Gene Expression

PubMed Central

Chacko, Ann-Marie; Qu, Wenchao; Kung, Hank F.

2014-01-01

Two novel series of 5-fluoroalkyl-2′-deoxyuridines (FPrDU, FBuDU, FPeDU) and 2′-fluoro-2′-deoxy-5-fluoroalkylarabinouridines (FFPrAU, FFBuAU, FFPeAU), having three, four or five methylene units (propyl, butyl, or pentyl) at C-5, were prepared and tested as reporter probes for imaging HSV1-tk gene expression. The Negishi coupling methodology was employed to efficiently synthesize the radiolabeling precursors. All six 5-[18F]fluoroalkyl pyrimidines were prepared readily from 3-N-benzoyl-3′,5′-di-O-benzoyl-protected 5-O-mesylate precursors in 17–35% radiochemical yield (decay-corrected). In vitro studies highlighted that all six [18F]labeled nucleosides selectively accumulated in cells expressing the HSV1-TK protein, with negligible uptake in control cells. [18F]FPrDU, [18F]FBuDU, [18F]FPeDU, and [18F]FFBuAU had the best uptake profiles. Despite selective accumulation in HSV1-tk expressing cells, all 5-fluoroalkyl pyrimidine nucleosides had low to negligible cytotoxic activity (CC50>1000–209 μM). Ultimately, results demonstrated that 5-[18F]fluoropropyl, [18F]fluorobutyl, and [18F]fluoropentyl pyrimidine nucleosides have potential as in vivo HSV1-TK PET reporter probes over a dynamic range of reporter gene expression levels. PMID:18800764

Influenza A virus protein PB1-F2 exacerbates IFN-beta expression of human respiratory epithelial cells.

PubMed

Le Goffic, Ronan; Bouguyon, Edwige; Chevalier, Christophe; Vidic, Jasmina; Da Costa, Bruno; Leymarie, Olivier; Bourdieu, Christiane; Decamps, Laure; Dhorne-Pollet, Sophie; Delmas, Bernard

2010-10-15

The PB1-F2 protein of the influenza A virus (IAV) contributes to viral pathogenesis by a mechanism that is not well understood. PB1-F2 was shown to modulate apoptosis and to be targeted by the CD8(+) T cell response. In this study, we examined the downstream effects of PB1-F2 protein during IAV infection by measuring expression of the cellular genes in response to infection with wild-type WSN/33 and PB1-F2 knockout viruses in human lung epithelial cells. Wild-type virus infection resulted in a significant induction of genes involved in innate immunity. Knocking out the PB1-F2 gene strongly decreased the magnitude of expression of cellular genes implicated in antiviral response and MHC class I Ag presentation, suggesting that PB1-F2 exacerbates innate immune response. Biological network analysis revealed the IFN pathway as a link between PB1-F2 and deregulated genes. Using quantitative RT-PCR and IFN-β gene reporter assay, we determined that PB1-F2 mediates an upregulation of IFN-β expression that is dependent on NF-κB but not on AP-1 and IFN regulatory factor-3 transcription factors. Recombinant viruses knocked out for the PB1-F2 and/or the nonstructural viral protein 1 (the viral antagonist of the IFN response) genes provide further evidence that PB1-F2 increases IFN-β expression and that nonstructural viral protein 1 strongly antagonizes the effect of PB1-F2 on the innate response. Finally, we compared the effect of PB1-F2 variants taken from several IAV strains on IFN-β expression and found that PB1-F2-mediated IFN-β induction is significantly influenced by its amino acid sequence, demonstrating its importance in the host cell response triggered by IAV infection.

In vitro anti-Plasmodium falciparum properties of the full set of human secreted phospholipases A2.

PubMed

Guillaume, Carole; Payré, Christine; Jemel, Ikram; Jeammet, Louise; Bezzine, Sofiane; Naika, Gajendra S; Bollinger, James; Grellier, Philippe; Gelb, Michael H; Schrével, Joseph; Lambeau, Gérard; Deregnaucourt, Christiane

2015-06-01

We have previously shown that secreted phospholipases A2 (sPLA2s) from animal venoms inhibit the in vitro development of Plasmodium falciparum, the agent of malaria. In addition, the inflammatory-type human group IIA (hGIIA) sPLA2 circulates at high levels in the serum of malaria patients. However, the role of the different human sPLA2s in host defense against P. falciparum has not been investigated. We show here that 4 out of 10 human sPLA2s, namely, hGX, hGIIF, hGIII, and hGV, exhibit potent in vitro anti-Plasmodium properties with half-maximal inhibitory concentrations (IC50s) of 2.9 ± 2.4, 10.7 ± 2.1, 16.5 ± 9.7, and 94.2 ± 41.9 nM, respectively. Other human sPLA2s, including hGIIA, are inactive. The inhibition is dependent on sPLA2 catalytic activity and primarily due to hydrolysis of plasma lipoproteins from the parasite culture. Accordingly, purified lipoproteins that have been prehydrolyzed by hGX, hGIIF, hGIII, and hGV are more toxic to P. falciparum than native lipoproteins. However, the total enzymatic activities of human sPLA2s on purified lipoproteins or plasma did not reflect their inhibitory activities on P. falciparum. For instance, hGIIF is 9-fold more toxic than hGV but releases a lower quantity of nonesterified fatty acids (NEFAs). Lipidomic analyses of released NEFAs from lipoproteins demonstrate that sPLA2s with anti-Plasmodium properties are those that release polyunsaturated fatty acids (PUFAs), with hGIIF being the most selective enzyme. NEFAs purified from lipoproteins hydrolyzed by hGIIF were more potent at inhibiting P. falciparum than those from hGV, and PUFA-enriched liposomes hydrolyzed by sPLA2s were highly toxic, demonstrating the critical role of PUFAs. The selectivity of sPLA2s toward low- and high-density (LDL and HDL, respectively) lipoproteins and their ability to directly attack parasitized erythrocytes further explain their anti-Plasmodium activity. Together, our findings indicate that 4 human sPLA2s are active against P

In Vitro Anti-Plasmodium falciparum Properties of the Full Set of Human Secreted Phospholipases A2

PubMed Central

Guillaume, Carole; Payré, Christine; Jemel, Ikram; Jeammet, Louise; Bezzine, Sofiane; Naika, Gajendra S.; Bollinger, James; Grellier, Philippe; Gelb, Michael H.; Schrével, Joseph

2015-01-01

We have previously shown that secreted phospholipases A2 (sPLA2s) from animal venoms inhibit the in vitro development of Plasmodium falciparum, the agent of malaria. In addition, the inflammatory-type human group IIA (hGIIA) sPLA2 circulates at high levels in the serum of malaria patients. However, the role of the different human sPLA2s in host defense against P. falciparum has not been investigated. We show here that 4 out of 10 human sPLA2s, namely, hGX, hGIIF, hGIII, and hGV, exhibit potent in vitro anti-Plasmodium properties with half-maximal inhibitory concentrations (IC50s) of 2.9 ± 2.4, 10.7 ± 2.1, 16.5 ± 9.7, and 94.2 ± 41.9 nM, respectively. Other human sPLA2s, including hGIIA, are inactive. The inhibition is dependent on sPLA2 catalytic activity and primarily due to hydrolysis of plasma lipoproteins from the parasite culture. Accordingly, purified lipoproteins that have been prehydrolyzed by hGX, hGIIF, hGIII, and hGV are more toxic to P. falciparum than native lipoproteins. However, the total enzymatic activities of human sPLA2s on purified lipoproteins or plasma did not reflect their inhibitory activities on P. falciparum. For instance, hGIIF is 9-fold more toxic than hGV but releases a lower quantity of nonesterified fatty acids (NEFAs). Lipidomic analyses of released NEFAs from lipoproteins demonstrate that sPLA2s with anti-Plasmodium properties are those that release polyunsaturated fatty acids (PUFAs), with hGIIF being the most selective enzyme. NEFAs purified from lipoproteins hydrolyzed by hGIIF were more potent at inhibiting P. falciparum than those from hGV, and PUFA-enriched liposomes hydrolyzed by sPLA2s were highly toxic, demonstrating the critical role of PUFAs. The selectivity of sPLA2s toward low- and high-density (LDL and HDL, respectively) lipoproteins and their ability to directly attack parasitized erythrocytes further explain their anti-Plasmodium activity. Together, our findings indicate that 4 human sPLA2s are active against P

The Aryl Hydrocarbon Receptor Binds to E2F1 and Inhibits E2F1-induced Apoptosis

PubMed Central

Marlowe, Jennifer L.; Fan, Yunxia; Chang, Xiaoqing; Peng, Li; Knudsen, Erik S.; Xia, Ying

2008-01-01

Cellular stress by DNA damage induces checkpoint kinase-2 (CHK2)-mediated phosphorylation and stabilization of the E2F1 transcription factor, leading to induction of apoptosis by activation of a subset of proapoptotic E2F1 target genes, including Apaf1 and p73. This report characterizes an interaction between the aryl hydrocarbon (Ah) receptor (AHR), a ligand-activated transcription factor, and E2F1 that results in the attenuation of E2F1-mediated apoptosis. In Ahr−/− fibroblasts stably transfected with a doxycycline-regulated AHR expression vector, inhibition of AHR expression causes a significant elevation of oxidative stress, γH2A.X histone phosphorylation, and E2F1-dependent apoptosis, which can be blocked by small interfering RNA-mediated knockdown of E2F1 expression. In contrast, ligand-dependent AHR activation protects these cells from etoposide-induced cell death. In cells expressing both proteins, AHR and E2F1 interact independently of the retinoblastoma protein (RB), because AHR and E2F1 coimmunoprecipitate from extracts of RB-negative cells. Additionally, chromatin immunoprecipitation assays indicate that AHR and E2F1 bind to the Apaf1 promoter at a region containing a consensus E2F1 binding site but no AHR binding sites. AHR activation represses Apaf1 and TAp73 mRNA induction by a constitutively active CHK2 expression vector. Furthermore, AHR overexpression blocks the transcriptional induction of Apaf1 and p73 and the accumulation of sub-G0/G1 cells resulting from ectopic overexpression of E2F1. These results point to a proproliferative, antiapoptotic function of the Ah receptor that likely plays a role in tumor progression. PMID:18524851

Elevated autophagy gene expression in adipose tissue of obese humans: A potential non-cell-cycle-dependent function of E2F1

PubMed Central

Haim, Yulia; Blüher, Matthias; Slutsky, Noa; Goldstein, Nir; Klöting, Nora; Harman-Boehm, Ilana; Kirshtein, Boris; Ginsberg, Doron; Gericke, Martin; Guiu Jurado, Esther; Kovsan, Julia; Tarnovscki, Tanya; Kachko, Leonid; Bashan, Nava; Gepner, Yiftach; Shai, Iris; Rudich, Assaf

2015-01-01

Autophagy genes' expression is upregulated in visceral fat in human obesity, associating with obesity-related cardio-metabolic risk. E2F1 (E2F transcription factor 1) was shown in cancer cells to transcriptionally regulate autophagy. We hypothesize that E2F1 regulates adipocyte autophagy in obesity, associating with endocrine/metabolic dysfunction, thereby, representing non-cell-cycle function of this transcription factor. E2F1 protein (N=69) and mRNA (N=437) were elevated in visceral fat of obese humans, correlating with increased expression of ATG5 (autophagy-related 5), MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β), but not with proliferation/cell-cycle markers. Elevated E2F1 mainly characterized the adipocyte fraction, whereas MKI67 (marker of proliferation Ki-67) was elevated in the stromal-vascular fraction of adipose tissue. In human visceral fat explants, chromatin-immunoprecipitation revealed body mass index (BMI)-correlated increase in E2F1 binding to the promoter of MAP1LC3B, but not to the classical cell cycle E2F1 target, CCND1 (cyclin D1). Clinically, omental fat E2F1 expression correlated with insulin resistance, circulating free-fatty-acids (FFA), and with decreased circulating ADIPOQ/adiponectin, associations attenuated by adjustment for autophagy genes. Overexpression of E2F1 in HEK293 cells enhanced promoter activity of several autophagy genes and autophagic flux, and sensitized to further activation of autophagy by TNF. Conversely, mouse embryonic fibroblast (MEF)-derived adipocytes from e2f1 knockout mice (e2f1−/−) exhibited lower autophagy gene expression and flux, were more insulin sensitive, and secreted more ADIPOQ. Furthermore, e2f1−/− MEF-derived adipocytes, and autophagy-deficient (by Atg7 siRNA) adipocytes were resistant to cytokines-induced decrease in ADIPOQ secretion. Jointly, upregulated E2F1 sensitizes adipose tissue autophagy to inflammatory stimuli, linking visceral obesity to adipose and systemic

Infrared absorption spectra of N(CxF2x+1)3, x = 2-5 perfluoroamines

NASA Astrophysics Data System (ADS)

Bernard, François; Papanastasiou, Dimitrios K.; Papadimitriou, Vassileios C.; Burkholder, James B.

2018-05-01

Infrared absorption spectra of the perfluoroamines (N(C2F5)3, N(C3F7)3, N(C4F9)3, and N(C5F11)3) were measured over the 500-4000 cm-1 spectral region at 294 K using Fourier transform infrared (FTIR) spectroscopy at 1 cm-1 resolution. Spectral measurements were performed using static measurements of dilute perfluoroamines mixtures and by infusion of the pure compound into a calibrated gas flow. The perfluoroamines absorb strongly in the "atmospheric window" with integrated band strengths (10-17 cm2 molecule-1 cm-1) between 570 and 1500 cm-1 of 59.9, 74.9, 88.9, and 98.7 for N(C2F5)3, N(C3F7)3, N(C4F9)3, and N(C5F11)3, respectively. Radiative efficiencies (RE) for the perfluoroamines were estimated to be 0.61, 0.75, 0.87, and 0.95 W m-2 ppb-1 for atmospherically well-mixed conditions and including a +10% stratospheric temperature correction for N(C2F5)3, N(C3F7)3, N(C4F9)3, and N(C5F11)3, respectively. Theoretical calculations of the perfluoroamines were performed at the B97-1/6-311++G(2df,2p) level of theory and optimized perfluoroamine geometries, vibrational band positions, and band strengths are reported. The theoretically calculated infrared spectra are in good agreement with the experimental spectra, while comparison of individual bands was not attempted due to the significant overlap of vibrational bands in the experimental spectra.

Simultaneous induction of apoptosis and necroptosis by Tanshinone IIA in human hepatocellular carcinoma HepG2 cells.

PubMed

Lin, C-Y; Chang, T-W; Hsieh, W-H; Hung, M-C; Lin, I-H; Lai, S-C; Tzeng, Y-J

Tanshinone IIA (Tan IIA), a constituent of the traditional medicinal plant Salvia miltiorrhiza BUNGE, has been reported to possess anticancer activity through induction of apoptosis in many cancer cells. Surprisingly, the present study finds that Tan IIA simultaneously causes apoptosis and necroptosis in human hepatocellular carcinoma HepG2 cells. We further find that apoptosis can be converted to necroptosis by pan-caspase inhibitor Z-VAD-fmk, and the two death modes can be blocked by necroptotic inhibitor necrostatin-1. The underlying mechanisms are revealed by analysis of the signaling molecules using western blotting. In control cells, FLICE inhibitory protein in short form (FLIP S ) is expressed in relatively high levels and binds to caspase 8 in ripoptosome, which supposedly sustains cell survival. However, in Tan IIA-treated cells, FLIP S is down-regulated and may thus cause homodimer formation of cleaved caspase 8, cleavage of receptor-interacting serine/threonine-protein kinases 1, 3 (RIP1, RIP3), and mixed-lineage kinase domain-like (MLKL), in turn leads to cell apoptosis. In parallel, Tan IIA causes necroptosis by forming a suggested necrosomal complex composed of RIP1/RIP3. Regarding the inhibitors, z-VAD-fmk diminishes the cleaved caspase 8, RIP1, RIP3, and MLKL induced by Tan IIA, and reconstructs the ripoptosome complex, which marks cells moving from apoptosis to necroptosis. Nec-1 recovers the Tan IIA down-regulated FLIP S , consequently causes FLIP S to form heterodimer with caspase 8 and thus block apoptosis. Meanwhile, cleaved forms of RIP1 and RIP3 were observed preventing necroptosis. Intriguingly, the cytotoxicity of tumor necrosis factor-related apoptosis-inducing ligand to HepG2 cells is enhanced by Tan IIA in a pilot study, which may be attributed to low FLIP S levels induced by Tan IIA. In short, Tan IIA simultaneously induces both Nec-1 inhibition and FLIP S regulation-mediated apoptosis/necroptosis, which has not been previously

Group X secreted phospholipase A₂ specifically decreases sperm motility in mice.

PubMed

Escoffier, Jessica; Pierre, Virginie J; Jemel, Ikram; Munch, Léa; Boudhraa, Zied; Ray, Pierre F; De Waard, Michel; Lambeau, Gérard; Arnoult, Christophe

2011-10-01

Different mammalian secreted phospholipases A(2) (sPLA(2) s) are expressed in male reproductive organs and/or in sperm cells but their cellular functions are still not fully characterized. Because several reports indicate a link between cellular lipids and sperm motility, we have investigated the effect of mouse group IIA, IID, IIE, V, and X sPLA(2) s on sperm motility. Among these enzymes, only mouse group X sPLA(2) (mGX sPLA(2) ) acts as a potent inhibitor of sperm motility that decreases track speed (VCL) and lateral displacement of the head (ALH) of both noncapacitated and capacitated sperm. The inhibitory effect of mGX sPLA(2) is dependent on its enzymatic activity because (i) both the proenzyme form of mGX sPLA(2) (pro-mGX) and the H48Q mutant of mGX sPLA(2) have very weak enzymatic activity and are unable to modulate sperm motility and (ii) LY329722, a specific inhibitor of sPLA(2) s, blocks the inhibitory effect of mGX sPLA(2) . Moreover, mGX sPLA(2) exerts a gradual potency on sperm subpopulations with different velocities, an effect which may be linked to the heterogeneity of lipid composition in these sperm subpopulations. Finally, we found that endogenous mGX sPLA(2) released during spontaneous acrosome reaction modulates sperm motility of capacitated sperm. Together, our results suggest a new role of sPLA(2) in sperm physiology where the sPLA2 selects a sperm subpopulation for fertilization based on its effect on sperm motility. Copyright © 2010 Wiley-Liss, Inc.

Diabetes and exocrine pancreatic insufficiency in E2F1/E2F2 double-mutant mice.

PubMed

Iglesias, Ainhoa; Murga, Matilde; Laresgoiti, Usua; Skoudy, Anouchka; Bernales, Irantzu; Fullaondo, Asier; Moreno, Bernardino; Lloreta, José; Field, Seth J; Real, Francisco X; Zubiaga, Ana M

2004-05-01

E2F transcription factors are thought to be key regulators of cell growth control. Here we use mutant mouse strains to investigate the function of E2F1 and E2F2 in vivo. E2F1/E2F2 compound-mutant mice develop nonautoimmune insulin-deficient diabetes and exocrine pancreatic dysfunction characterized by endocrine and exocrine cell dysplasia, a reduction in the number and size of acini and islets, and their replacement by ductal structures and adipose tissue. Mutant pancreatic cells exhibit increased rates of DNA replication but also of apoptosis, resulting in severe pancreatic atrophy. The expression of genes involved in DNA replication and cell cycle control was upregulated in the E2F1/E2F2 compound-mutant pancreas, suggesting that their expression is repressed by E2F1/E2F2 activities and that the inappropriate cell cycle found in the mutant pancreas is likely the result of the deregulated expression of these genes. Interestingly, the expression of ductal cell and adipocyte differentiation marker genes was also upregulated, whereas expression of pancreatic cell marker genes were downregulated. These results suggest that E2F1/E2F2 activity negatively controls growth of mature pancreatic cells and is necessary for the maintenance of differentiated pancreatic phenotypes in the adult.

Activation of the 2-5OAS/RNase L pathway in CVB1 or HAV/18f infected FRhK-4 cells does not require induction of OAS1 or OAS2 expression

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

Kulka, Michael, E-mail: michael.kulka@fda.hhs.go; Calvo, Mona S., E-mail: mona.calvo@fda.hhs.go; Ngo, Diana T., E-mail: diana.ngo@fda.hhs.go

2009-05-25

The latent, constitutively expressed protein RNase L is activated in coxsackievirus and HAV strain 18f infected FRhK-4 cells. Endogenous oligoadenylate synthetase (OAS) from uninfected and virus infected cell extracts synthesizes active forms of the triphosphorylated 2-5A oligomer (the only known activator of RNase L) in vitro and endogenous 2-5A is detected in infected cell extracts. However, only the largest OAS isoform, OAS3, is readily detected throughout the time course of infection. While IFNbeta treatment results in an increase in the level of all three OAS isoforms in FRhK-4 cells, IFNbeta pretreatment does not affect the temporal onset or enhancement ofmore » RNase L activity nor inhibit virus replication. Our results indicate that CVB1 and HAV/18f activate the 2-5OAS/RNase L pathway in FRhK-4 cells during permissive infection through endogenous levels of OAS, but contrary to that reported for some picornaviruses, CVB1 and HAV/18f replication is insensitive to this activated antiviral pathway.« less

Effects of salvianolic acid B and tanshinone IIA on the pharmacokinetics of losartan in rats by regulating the activities and expression of CYP3A4 and CYP2C9.

PubMed

Wang, Rong; Zhang, Hai; Wang, Yujie; Yu, Xiaoyan; Yuan, Yongfang

2016-03-02

Losartan (LST) is a common chemical drug used to treat high blood pressure and reduce the risk of stroke in certain people with heart disease. Danshen, prepared from the dried root and rhizome of Salvia miltiorrhiza Bunge, has been widely used for prevention and treatment of various cardiovascular and cerebrovascular diseases. There are more than 35 formulations containing Danshen indexed in the 2010 Chinese Pharmacopoeia, which are often combined with LST to treat cardiovascular and cerebrovascular diseases in the clinic. The effects of the two major components of Danshen, salvianolic acid B (SA-B) and tanshinone IIA (Tan IIA), on the pharmacokinetics of losartan and its metabolite, EXP3174, in rats were investigated by liquid chromatography coupled with mass spectrometry (LC-MS). Male Sprague-Dawley rats were randomly assigned to 3 groups: LST, LST+SA-B and LST+Tan IIA, and the main pharmacokinetic parameters were estimated after oral administration of LST, LST+SA-B and LST+Tan IIA. It was found that there are significant differences in the pharmacokinetic parameters among the three groups: Cmax, t1/2, AUC, AUMC in the LST+SA-B group was smaller than those in group LST, while larger in group LST+Tan IIA. Further, the effects of SA-B and Tan IIA on the metabolism of losartan was also investigated using rat liver microsomes in vitro. The results indicated that SA-B can induce the metabolism of LST, while Tan IIA can inhibit the metabolism of LST in rat liver microsomes in vitro by regulating activities of CYP450 enzymes. In addition, the effect of SA-B and Tan IIA on CYP3A4 and CYP2C9 expression was studied in Chang liver cells by western-blotting and Real-time PCR. It was concluded that the two components of Danshen, SA-B and Tan IIA have different influences on the metabolism of LST: SA-B can obviously speed up the metabolism of LST by inducing CYP3A4/CYP2C9 activities and expression, however, Tan IIA can slow down the metabolism of LST by inhibiting CYP3A4/CYP2C

Effect of Chlorogenic Acid (5-Caffeoylquinic Acid) Isolated from Baccharis oxyodonta on the Structure and Pharmacological Activities of Secretory Phospholipase A2 from Crotalus durissus terrificus

PubMed Central

Toyama, Daniela O.; Ferreira, Marcelo J. P.; Romoff, Paulete; Fávero, Oriana A.; Gaeta, Henrique H.; Toyama, Marcos H.

2014-01-01

The aim of this paper was to investigate the effect of chlorogenic acid (5-caffeoylquinic acid, 5CQA), isolated from Baccharis oxyodonta, on the structure and pharmacological effect of secretory phospholipase A2 (sPLA2) from Crotalus durissus terrificus. All in vitro and in vivo experiments were conducted using a purified sPLA2 compared under the same experimental conditions with sPLA2 : 5CQA. 5CQA induced several discrete modifications in the secondary structure and the hydrophobic characteristics of native sPLA2 that induced slight changes in the α-helical content, increase in the random coil structure, and decrease of fluorescence of native sPLA2. Moreover, 5CQA significantly decreased the enzymatic activity and the oedema and myonecrosis induced by native sPLA2. As the catalytic activity of sPLA2 plays an important role in several of its biological and pharmacological properties, antibacterial activity was used to confirm the decrease in its enzymatic activity by 5CQA, which induced massive bacterial cell destruction. We found that 5CQA specifically abolished the enzymatic activity of sPLA2 and induced discrete protein unfolding that mainly involved the pharmacological site of sPLA2. These results showed the potential application of 5CQA in the snake poisoning treatment and modulation of the pathological effect of inflammation induced by secretory PLA2. PMID:25258715

Tanshinone IIA inhibits AGEs-induced proliferation and migration of cultured vascular smooth muscle cells by suppressing ERK1/2 MAPK signaling.

PubMed

Lu, Ming; Luo, Ying; Hu, Pengfei; Dou, Liping; Huang, Shuwei

2018-01-01

Vascular smooth muscle cells (VSMCs) play a key role in the pathogenesis of diabetic vascular disease. Our current study sought to explore the effects of tanshinone IIA on the proliferation and migration of VSMCs induced by advanced glycation end products (AGEs). In this study, we examined the effects of tanshinone IIA by cell proliferation assay and cell migration assay. And we explored the underlying mechanism by Western blotting. AGEs significantly induced the proliferation and migration of VSMCs, but treatment with tanshinone IIA attenuated these effects. AGEs could increase the activity of the ERK1/2 and p38 pathways but not the JNK pathway. Treatment with tanshinone IIA inhibited the AGEs-induced activation of the ERK1/2 pathway but not the p38 pathway. Tanshinone IIA inhibits AGEs-induced proliferation and migration of VSMCs by suppressing the ERK1/2 MAPK signaling pathway.

Self-limited growth of the CaF nanowire on the Si(5 5 12)-2 × 1 template

NASA Astrophysics Data System (ADS)

Kim, Hidong; Duvjir, Ganbat; Dugerjav, Otgonbayar; Li, Huiting; Motlak, Moaaed; Arvisbaatar, Amarmunkh; Seo, Jae M.

2012-10-01

The atomic structure and interfacial bonding of the ordered-and-isolated CaF nanowires on Si(5 5 12)-2 × 1 have been disclosed by scanning tunneling microscopy and synchrotron photoemission spectroscopy. Initially, CaF molecules dissociated from thermally deposited CaF2 molecules are adsorbed preferentially on the chain structures of Si(5 5 12)-2 × 1 held at 500 °C. With increasing CaF2 deposition amount, one-dimensional (1D) CaF nanowires composed of (113) and (111) facets are formed. The line density of these CaF nanowires increases as a function of deposition amount. Finally, at a submonolayer coverage, the surface is saturated with these 1D nanowires except for the (225) subunit, while the original period of Si(5 5 12)-2 × 1, 5.35 nm, is preserved. It has been deduced by the present studies that, owing to these preferential adsorption of CaF and facet-dependent growth of a CaF layer within a unit periodic length of Si(5 5 12)-2 × 1, such a self-limited growth of the CaF nanowire with a high aspect ratio becomes possible.

Identification of Epithelial Phospholipase A2 Receptor 1 as a Potential Target in Asthma

PubMed Central

Nolin, James D.; Ogden, H. Luke; Lai, Ying; Altemeier, William A.; Frevert, Charles W.; Bollinger, James G.; Naika, Gajendra S.; Kicic, Anthony; Stick, Stephen M.; Lambeau, Gerard; Henderson, William R.; Gelb, Michael H.

2016-01-01

Secreted phospholipase A2s (sPLA2s) regulate eicosanoid formation and have been implicated in asthma. Although sPLA2s function as enzymes, some of the sPLA2s bind with high affinity to a C-type lectin receptor, called PLA2R1, which has functions in both cellular signaling and clearance of sPLA2s. We sought to examine the expression of PLA2R1 in the airway epithelium of human subjects with asthma and the function of the murine Pla2r1 gene in a model of asthma. Expression of PLA2R1 in epithelial brushings was assessed in two distinct cohorts of children with asthma by microarray and quantitative PCR, and immunostaining for PLA2R1 was conducted on endobronchial tissue and epithelial brushings from adults with asthma. C57BL/129 mice deficient in Pla2r1 (Pla2r1−/−) were characterized in an ovalbumin (OVA) model of allergic asthma. PLA2R1 was differentially overexpressed in epithelial brushings of children with atopic asthma in both cohorts. Immunostaining for PLA2R1 in endobronchial tissue localized to submucosal glandular epithelium and columnar epithelial cells. After OVA sensitization and challenge, Pla2r1−/− mice had increased airway hyperresponsiveness, as well as an increase in cellular trafficking of eosinophils to the peribronchial space and bronchoalveolar lavage fluid, and an increase in airway permeability. In addition, Pla2r1−/− mice had more dendritic cells in the lung, higher levels of OVA-specific IgG, and increased production of both type-1 and type-2 cytokines by lung leukocytes. PLA2R1 is increased in the airway epithelium in asthma, and serves as a regulator of airway hyperresponsiveness, airway permeability, antigen sensitization, and airway inflammation. PMID:27448109

Identification of Epithelial Phospholipase A2 Receptor 1 as a Potential Target in Asthma.

PubMed

Nolin, James D; Ogden, H Luke; Lai, Ying; Altemeier, William A; Frevert, Charles W; Bollinger, James G; Naika, Gajendra S; Kicic, Anthony; Stick, Stephen M; Lambeau, Gerard; Henderson, William R; Gelb, Michael H; Hallstrand, Teal S

2016-12-01

Secreted phospholipase A 2 s (sPLA 2 s) regulate eicosanoid formation and have been implicated in asthma. Although sPLA 2 s function as enzymes, some of the sPLA 2 s bind with high affinity to a C-type lectin receptor, called PLA2R1, which has functions in both cellular signaling and clearance of sPLA 2 s. We sought to examine the expression of PLA2R1 in the airway epithelium of human subjects with asthma and the function of the murine Pla2r1 gene in a model of asthma. Expression of PLA2R1 in epithelial brushings was assessed in two distinct cohorts of children with asthma by microarray and quantitative PCR, and immunostaining for PLA2R1 was conducted on endobronchial tissue and epithelial brushings from adults with asthma. C57BL/129 mice deficient in Pla2r1 (Pla2r1 -/- ) were characterized in an ovalbumin (OVA) model of allergic asthma. PLA2R1 was differentially overexpressed in epithelial brushings of children with atopic asthma in both cohorts. Immunostaining for PLA2R1 in endobronchial tissue localized to submucosal glandular epithelium and columnar epithelial cells. After OVA sensitization and challenge, Pla2r1 -/- mice had increased airway hyperresponsiveness, as well as an increase in cellular trafficking of eosinophils to the peribronchial space and bronchoalveolar lavage fluid, and an increase in airway permeability. In addition, Pla2r1 -/- mice had more dendritic cells in the lung, higher levels of OVA-specific IgG, and increased production of both type-1 and type-2 cytokines by lung leukocytes. PLA2R1 is increased in the airway epithelium in asthma, and serves as a regulator of airway hyperresponsiveness, airway permeability, antigen sensitization, and airway inflammation.

Tanshinone IIA inhibits AGEs-induced proliferation and migration of cultured vascular smooth muscle cells by suppressing ERK1/2 MAPK signaling

PubMed Central

Lu, Ming; Luo, Ying; Hu, Pengfei; Dou, Liping; Huang, Shuwei

2018-01-01

Objective(s): Vascular smooth muscle cells (VSMCs) play a key role in the pathogenesis of diabetic vascular disease. Our current study sought to explore the effects of tanshinone IIA on the proliferation and migration of VSMCs induced by advanced glycation end products (AGEs). Materials and Methods: In this study, we examined the effects of tanshinone IIA by cell proliferation assay and cell migration assay. And we explored the underlying mechanism by Western blotting. Results: AGEs significantly induced the proliferation and migration of VSMCs, but treatment with tanshinone IIA attenuated these effects. AGEs could increase the activity of the ERK1/2 and p38 pathways but not the JNK pathway. Treatment with tanshinone IIA inhibited the AGEs-induced activation of the ERK1/2 pathway but not the p38 pathway. Conclusion: Tanshinone IIA inhibits AGEs-induced proliferation and migration of VSMCs by suppressing the ERK1/2 MAPK signaling pathway. PMID:29372041

Novel compounds TAD-1822-7-F2 and F5 inhibited HeLa cells growth through the JAK/Stat signaling pathway.

PubMed

Yang, Tianfeng; Shi, Xianpeng; Kang, Yuan; Zhu, Man; Fan, Mengying; Zhang, Dongdong; Zhang, Yanmin

2018-07-01

Cervical carcinoma remains the second most common malignancy with a high mortality rate among women worldwide. TAD-1822-7-F2 (F2) and TAD-1822-7-F5 (F5) are novel compounds synthesized on the chemical structure of taspine derivatives, and show an effective suppression for HeLa cells. Our study aims to confirm the potential targets of F2 and F5, and investigate the underlying mechanism of the inhibitory effect on HeLa cells. In this study, Real Time Cell Analysis and crystal violet staining assay were conducted to investigate the effect of F2 and F5 on HeLa cells proliferation. And the analytical methods of surface plasmon resonance and quartz crystal microbalance were established and employed to study the interaction between F2 and F5 and potential target protein JAK2, suggesting that both compounds have strong interaction with the JAK2 protein. Western blot analysis, immunofluorescence staining study and PCR was conducted to investigate the molecules of JAK/Stat signaling pathway. Interestingly, F2 and F5 showed diverse regulation for signaling molecules because of their different chemical structure. F2 increased the expression of JAK2 and downregulated the level of P-JAK1 and P-JAK2, and decreased P-Stat3 (Ser727). While F5 could increase the expression of JAK2 and naturally decrease the phosphorylation of JAK1 and Tyk2, and decreased the expression of P-Stat6. Moreover, F2 and F5 showed the same downregulation on the P-Stat3 (Tyr705). Therefore, F2 and F5 could target the JAK2 protein and prevent the phosphorylation of JAKs to suppress the phosphorylation of the downstream effector Stats, which suggested that F2 and F5 have great potential to be the inhibitors of the JAK/Stat signaling pathway. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Word Frequency Analysis. MOS: 95B. Skill Levels 1 & 2.

DTIC Science & Technology

1981-05-01

uU N D 1 A C P A C K1 ? G .I A 2 1 A’J Kt I S , 1 K L S 1 BaRp IF S I ~t2iIAED 5. 1.T’ 6 BATTERY 9!j 2 BECAUSE 17 67EI 2 BEHIND 13 IL 2 RE l1󈧱 3 b1...T S :t cTt lIION 5 U It :,4t 5 F L 1%T5 &:, JC.L ME NT 5 ESTIMS4EO 5 F -4 U0 . F- "CL5 UJ 7 "S 5 F-.[377 7i (, ID󈧏CAlN5.C1DEINTS 5 I’%SER71NkG .0

Signal transduction and functional selectivity of F15599, a preferential post-synaptic 5-HT1A receptor agonist

PubMed Central

Newman-Tancredi, A; Martel, J-C; Assié, M-B; Buritova, J; Lauressergues, E; Cosi, C; Heusler, P; Slot, L Bruins; Colpaert, FC; Vacher, B; Cussac, D

2009-01-01

Background and purpose: Activation of post-synaptic 5-HT1A receptors may provide enhanced therapy against depression. We describe the signal transduction profile of F15599, a novel 5-HT1A receptor agonist. Experimental approach: F15599 was compared with a chemical congener, F13714, and with (+)8-OH-DPAT in models of signal transduction in vitro and ex vivo. Key results: F15599 was highly selective for 5-HT1A receptors in binding experiments and in [35S]-GTPγS autoradiography of rat brain, where F15599 increased labelling in regions expressing 5-HT1A receptors. In cell lines expressing h5-HT1A receptors, F15599 more potently stimulated extracellular signal-regulated kinase (ERK1/2) phosphorylation, compared with G-protein activation, internalization of h5-HT1A receptors or inhibition of cAMP accumulation. F13714, (+)8-OH-DPAT and 5-HT displayed a different rank order of potency for these responses. F15599 stimulated [35S]-GTPγS binding more potently in frontal cortex than raphe. F15599, unlike 5-HT, more potently and efficaciously stimulated Gαi than Gαo activation. In rat prefrontal cortex (a region expressing post-synaptic 5-HT1A receptors), F15599 potently activated ERK1/2 phosphorylation and strongly induced c-fos mRNA expression. In contrast, in raphe regions (expressing pre-synaptic 5-HT1A receptors) F15599 only weakly or did not induce c-fos mRNA expression. Finally, despite its more modest affinity in vitro, F15599 bound to 5-HT1A receptors in vivo almost as potently as F13714. Conclusions and implications: F15599 showed a distinctive activation profiles for 5-HT1A receptor-mediated signalling pathways, unlike those of reference agonists and consistent with functional selectivity at 5-HT1A receptors. In rat, F15599 potently activated signalling in prefrontal cortex, a feature likely to underlie its beneficial effects in models of depression and cognition. PMID:19154445

Characterization of antigenic determinants in ApxIIA exotoxin capable of inducing protective immunity to Actinobacillus pleuropneumoniae challenge.

PubMed

Seo, Ki-Weon; Kim, Dong-Heon; Kim, Ah Hyun; Yoo, Han-Sang; Lee, Kyung-Yeol; Jang, Yong-Suk

2011-01-01

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia. Among the virulence factors of the pathogen, ApxIIA, a bacterial exotoxin, is expressed by many serotypes and presents a plausible target for vaccine development. We characterized the region within ApxIIA that induces a protective immune response against bacterial infection using mouse challenge model. Recombinant proteins spanning the length of ApxIIA were produced and antiserum to the full-length ApxIIA was induced in mice. This antiserum recognized fragments #2, #3 and #5 with high binding specificity, but showed poor recognition for fragments #1 and #4. Of the antisera induced in mice by injection of each fragments, only the antiserum to fragment #4 failed to efficiently recognize the full-length antigen, although the individual antisera recognized their cognate antigens with almost equal efficiency. The protective potency of the immunogenic proteins against a challenge injection of bacteria in vivo correlated well with the antibody titer. Fragment #5 induced the highest level of protective activity, comparable to that by the full-length protein. These results support the use of fragment #5 to produce a vaccine against A. pleuropneumoniae challenge, since the small antigen peptide is easier to handle than is the full-length protein and can be expressed efficiently in heterologous expression systems.

Ventana immunohistochemistry ALK (D5F3) detection of ALK expression in pleural effusion samples of lung adenocarcinoma.

PubMed

Wang, Zheng; Wu, Xiaonan; Shi, Yuankai; Han, Xiaohong; Cheng, Gang; Cui, Di; Li, Lin; Zhang, Yuhui; Mu, Xinlin; Zhang, Li; Yang, Li; Di, Jing; Yu, Qi; Liu, Dongge

2015-08-01

To evaluate the Ventana IHC ALK (D5F3) assay for detecting anaplastic lymphoma kinase (ALK) protein expression in pleural effusion samples. Historical, selected (wild-type EGFR, K-RAS) pleural effusion cytologic blocks of lung adenocarcinoma samples (Study 1) and unselected lung adenocarcinoma pleural effusion cytologic blocks (Study 2) were tested by Ventana IHC ALK (D5F3) assay. Quantitative real-time-PCR was used to verify immunohistochemistry results. A total of 17 out of 100 (Study 1) and ten out of 104 (Study 2) pleural effusion samples were ALK expression positive by the Ventana IHC ALK (D5F3) assay. The ALK fusion results with immunohistochemistry and quantitative real-time-PCR had a concordance rate of 87.5% (κ = 0.886; p < 0.001). The Ventana IHC ALK (D5F3) assay is a reliable tool for detecting ALK protein expression in pleural effusion samples.

Tanshinone IIA promotes the proliferation of WB-F344 hepatic oval cells via Wnt/β-catenin signaling

PubMed Central

ZE, XINGYU; JIA, JIDONG; LI, XINMIN; YOU, HONG; ZHAO, XINYAN; ZHANG, DONG; WANG, BAOEN

2016-01-01

Tanshinone IIA (TSA) is a widely used traditional Chinese medicine, which has been demonstrated to protect damaged liver cells and is currently administered in the treatment of liver fibrosis. Liver precursor cells, also termed oval cells, are key in the repair of liver tissues following injury. However, whether TSA improves the function of liver cells and protects the liver from injury by enhancing the growth and proliferation of hepatic oval cells remains to be elucidated. In the present study, low to moderate concentrations of TSA were observed to stimulate proliferation, did not induce apoptosis in WB-F344 rat hepatic oval cells and the increased expression levels of β-catenin. WB-F344 cells were treated with various concentrations of TSA (0–80 µg/ml) for 24, 48, 72 and 96 h. Cell proliferation was measured using a Cell Counting kit-8 (CCK-8) assay, a 5-ethynyl-2′-deoxyuridine assay and a carboxyfluorescein diacetate succinimidyl ester (CFSE) assay. The CCK-8 assay demonstrated that treatment of WB-F344 cells with 20–40 µg/ml TSA for up to 72 h significantly increased proliferation. Similar results were observed in the subsequent EdU and CFSE assays. Furthermore, a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay demonstrated that 20–40 µg/ml TSA treatment for up to 96 h did not induce apoptosis of the WB-F344 cells. Notably, the results of western blot, immunofluorescence and reverse transcription-quantitative polymerase chain reaction analyses demonstrated that treatment of the WB-F344 cells with 20–40 µg/ml TSA for up to 72 h significantly increased the expression levels of β-catenin. These data indicated that TSA at concentrations between 20 and 40 µg/ml may induce WB-F344 cell proliferation by activating the canonical Wnt signaling pathway. The results of the present study suggest that TSA may be a useful natural agent to enhance repair and regeneration of the injured liver, and improve liver regeneration

MYBPH inhibits NM IIA assembly via direct interaction with NMHC IIA and reduces cell motility

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

Hosono, Yasuyuki; Usukura, Jiro; Yamaguchi, Tomoya

2012-11-09

Highlights: Black-Right-Pointing-Pointer MYBPH inhibits NMHC IIA assembly and cell motility. Black-Right-Pointing-Pointer MYBPH interacts to assembly-competent NM IIA. Black-Right-Pointing-Pointer MYBPH inhibits RLC and NMHC IIA, independent components of NM IIA. -- Abstract: Actomyosin filament assembly is a critical step in tumor cell migration. We previously found that myosin binding protein H (MYBPH) is directly transactivated by the TTF-1 lineage-survival oncogene in lung adenocarcinomas and inhibits phosphorylation of the myosin regulatory light chain (RLC) of non-muscle myosin IIA (NM IIA) via direct interaction with Rho kinase 1 (ROCK1). Here, we report that MYBPH also directly interacts with an additional molecule, non-muscle myosinmore » heavy chain IIA (NMHC IIA), which was found to occur between MYBPH and the rod portion of NMHC IIA. MYBPH inhibited NMHC IIA assembly and reduced cell motility. Conversely, siMYBPH-induced increased motility was partially, yet significantly, suppressed by blebbistatin, a non-muscle myosin II inhibitor, while more profound effects were attained by combined treatment with siROCK1 and blebbistatin. Electron microscopy observations showed well-ordered paracrystals of NMHC IIA reflecting an assembled state, which were significantly less frequently observed in the presence of MYBPH. Furthermore, an in vitro sedimentation assay showed that a greater amount of NMHC IIA was in an unassembled state in the presence of MYBPH. Interestingly, treatment with a ROCK inhibitor that impairs transition of NM IIA from an assembly-incompetent to assembly-competent state reduced the interaction between MYBPH and NMHC IIA, suggesting that MYBPH has higher affinity to assembly-competent NM IIA. These results suggest that MYBPH inhibits RLC and NMHC IIA, independent components of NM IIA, and negatively regulates actomyosin organization at 2 distinct steps, resulting in firm inhibition of NM IIA assembly.« less

Krüppel-like factor 1 mutations and expression of hemoglobins F and A2 in homozygous hemoglobin E syndrome.

PubMed

Tepakhan, Wanicha; Yamsri, Supawadee; Fucharoen, Goonnapa; Sanchaisuriya, Kanokwan; Fucharoen, Supan

2015-07-01

The basis for variability of hemoglobin (Hb) F in homozygous Hb E disease is not well understood. We have examined multiple mutations of the Krüppel-like factor 1 (KLF1) gene; an erythroid specific transcription factor and determined their associations with Hbs F and A2 expression in homozygous Hb E. Four KLF1 mutations including G176AfsX179, T334R, R238H, and -154 (C-T) were screened using specific PCR assays on 461 subjects with homozygous Hb E and 100 normal controls. None of these four mutations were observed in 100 normal controls. Among 461 subjects with homozygous Hb E, 306 had high (≥5 %) and 155 had low (<5 %) Hb F. DNA analysis identified the KLF1 mutations in 35 cases of the former group with high Hb F, including the G176AfsX179 mutation (17/306 = 5.6 %), T334R mutation (9/306 = 2.9 %), -154 (C-T) mutation (7/306 = 2.3 %), and R328H mutation (2/306 = 0.7 %). Only two subjects in the latter group with low Hb F carried the G176AfsX179 and -154 (C-T) mutations. Significant higher Hb A2 level was observed in those of homozygous Hb E with the G176AfsX179 mutation as compared to those without KLF1 mutations. These results indicate that KLF1 is among the genetic factors associated with increased Hbs F and A2, and in combination with other factors could explain the variabilities of these Hb expression in Hb E syndrome.

Cloning and characterization of two duplicated interleukin-17A/F2 genes in common carp (Cyprinus carpio L.): Transcripts expression and bioactivity of recombinant IL-17A/F2.

PubMed

Li, Hongxia; Yu, Juhua; Li, Jianlin; Tang, Yongkai; Yu, Fan; Zhou, Jie; Yu, Wenjuan

2016-04-01

Interleukin-17 (IL-17) plays an important role in inflammation and host defense in mammals. In this study, we identified two duplicated IL-17A/F2 genes in the common carp (Cyprinus carpio) (ccIL-17A/F2a and ccIL-17A/F2b), putative encoded proteins contain 140 amino acids (aa) with conserved IL-17 family motifs. Expression analysis revealed high constitutive expression of ccIL-17A/F2s in mucosal tissues, including gill, skin and intestine, their expression could be induced by Aeromonas hydrophila, suggesting a potential role in mucosal immunity. Recombinant ccIL-17A/F2a protein (rccIL-17A/F2a) produced in Escherichia coli could induce the expression of proinflammatory cytokines (IL-1β) and the antimicrobial peptides S100A1, S100A10a and S100A10b in the primary kidney in a dose- and time-dependent manner. Above findings suggest that ccIL-17A/F2 plays an important role in both proinflammatory and innate immunity. Two duplicated ccIL-17A/F2s showed different expression level with ccIL-17A/F2a higher than b, comparison of two 5' regulatory regions indicated the length from anticipated promoter to transcriptional start site (TSS) and putative transcription factor binding site (TFBS) were different. Promoter activity of ccIL-17A/F2a was 2.5 times of ccIL-17A/F2b which consistent with expression results of two genes. These suggest mutations in 5'regulatory region contributed to the differentiation of duplicated genes. To our knowledge, this is the first report to analyze 5'regulatory region of piscine IL-17 family genes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Tanshinone IIA protects H9c2 cells from oxidative stress-induced cell death via microRNA-133 upregulation and Akt activation.

PubMed

Gu, Yunfei; Liang, Zhuo; Wang, Haijun; Jin, Jun; Zhang, Shouyan; Xue, Shufeng; Chen, Jianfeng; He, Huijuan; Duan, Kadan; Wang, Jing; Chang, Xuewei; Qiu, Chunguang

2016-08-01

The aim of the present study was to investigate the cardioprotective effect of tanshinone IIA and the underlying molecular mechanisms. An in vitro model of oxidative stress injury was established in cardiac H9c2 cells, and the effects of tanshinone IIa were investigated using cell viability, reverse transcription-quantitative polymerase chain reaction and western blotting assays. The results demonstrated that tanshinone IIA protects H9c2 cells from H 2 O 2 -induced cell death in a concentration-dependent manner, via a mechanism involving microRNA-133 (miR-133), and that treatment with TIIA alone exerted no cytotoxic effects on H9c2. In order to further elucidate the mechanisms underlying the actions of TIIA, reverse transcription-quantitative polymease chain reaction and western blot analysis were performed. Reductions in miR-133 expression levels induced by increasing concentrations of H 2 O 2 were reversed by treatment with tanshinone IIA. In addition, the inhibition of miR-133 by transfection with an miR-133 inhibitor abolished the cardioprotective effects of tanshinone IIA against H 2 O 2 -induced cell death. Furthermore, western blot analysis demonstrated that tanshinone IIA activated Akt kinase via the phosphorylation of serine 473. Inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway by pretreatment with the PI3K specific inhibitors wortmannin and LY294002 also eliminated the cardioprotective effects of tanshinone IIA against H 2 O 2 -induced cell death. Western blot analysis demonstrated that H 2 O 2 -induced reductions in B cell lymphoma 2 (Bcl-2) expression levels were reversed by tanshinone IIA. In addition, the effect of tanshinone IIA on Bcl-2 protein expression level in an oxidative environment was suppressed by a PI3K inhibitor, wortmannin, indicating that tanshinone IIA exerts cardioprotective effects against H 2 O 2 -induced cell death via the activation of the PI3K/Akt signal transduction pathway and the consequent

Deposition of lipid, protein, and secretory phospholipase A2 on hydrophilic contact lenses.

PubMed

Mochizuki, Hiroshi; Yamada, Masakazu; Hatou, Shin; Kawashima, Motoko; Hata, Seiichiro

2008-01-01

Recent studies have shown that low tear phospholipid levels are associated with tear film instability in hydrophilic contact lens wearers. The concentration of secretory phospholipase A2 (sPLA2), the enzyme that hydrolyzes phospholipids, in tears is known to exceed the levels found in serum by four orders of magnitude. This study was performed to determine the levels of sPLA2 from the deposition on two different frequent-replacement contact lens materials. Polymacon and etafilcon A contact lenses worn for 2 weeks by 16 experienced contact lens wearers were used for the analysis. Total lipids were determined by the sulfo-phospho-vanillin reaction. Phospholipids in lipid extracts were estimated by phosphorus determination with ammonium molybdate through enzymatic digestion. Total protein was measured by bicinchoninic acid analysis. Double-antibody sandwich enzyme-linked immunosorbent assay was used to determine sPLA2 concentrations. Total lipid deposition was found to be greater in the polymacon group (66.3+/-16.3 microg/lens) than in the etafilcon A group, although phospholipids were not detected in either group. The etafilcon A group had greater deposition of protein (3.7+/-0.7 mg/lens) than the polymacon group had. The etafilcon A group deposited statistically significantly more group IIa sPLA2 (1.1+/-0.3 microg/lens) than the polymacon group (0.07+/-0.04 microg/lens) did (P<0.001). There was a significant difference in the lipid and protein deposition profiles in the two lenses tested. A significant amount of sPLA2 in the deposition on contact lenses may play a role in tear film instability in hydrophilic contact lens wearers.

Ba2F2Fe(1.5)Se3: An Intergrowth Compound Containing Iron Selenide Layers.

PubMed

Driss, Dalel; Janod, Etienne; Corraze, Benoit; Guillot-Deudon, Catherine; Cario, Laurent

2016-03-21

The iron selenide compound Ba2F2Fe(1.5)Se3 was synthesized by a high-temperature ceramic method. The single-crystal X-ray structure determination revealed a layered-like structure built on [Ba2F2](2+) layers of the fluorite type and iron selenide layers [Fe(1.5)Se3](2-). These [Fe1.5Se3](2-) layers contain iron in two valence states, namely, Fe(II+) and Fe(III+) located in octahedral and tetrahedral sites, respectively. Magnetic measurements are consistent with a high-spin state for Fe(II+) and an intermediate-spin state for Fe(III+). Moreover, susceptibility and resistivity measurements demonstrate that Ba2F2Fe(1.5)Se3 is an antiferromagnetic insulator.

Genetic and antigenic characteristics of ApxIIA and ApxIIIA from Actinobacillus pleuropneumoniae serovars 2, 3, 4, 6, 8 and 15.

PubMed

To, Ho; Nagai, Shinya; Iwata, Akira; Koyama, Tomohiro; Oshima, Atsushi; Tsutsumi, Nobuyuki

2016-07-01

Apx toxins produced by Actinobacillus pleuropneumoniae are essential components of new generation vaccines. In this study, apxIIA and apxIIIA genes of serovars 2, 3, 4, 6, 8 and 15 were cloned and sequenced. Amino acid sequences of ApxIIA proteins of serovars 2, 3, 4, 6, 8 and 15 were almost identical to those of serovars 1, 5, 7, 9 and 11-13. Immunoblot analysis showed that rApxIIA from serovars 2 and 15 reacts strongly with sera from animals infected with various serovars. Sequence analysis revealed that ApxIIIA proteins has two variants, one in strains of serovar 2 and the other in strains of serovars 3, 4, 6, 8 and 15. A mouse cross-protection study showed that mice actively immunized with rApxIIIA/2 or rApxIIIA/15 are protected against challenge with A. pleuropneumoniae strains of serovars 3, 4, 6, 8, 15, and 2 expressing ApxIII/15 and ApxIII/2, respectively. Similarly, mice passively immunized with rabbit anti-rApxIIIA/2 or anti-rApxIIIA/15 sera were found to be protected against challenge with strains of serovars 2 and 15. Our study revealed antigenic and sequence similarities within ApxIIA and ApxIIIA proteins, which may help in the development of effective vaccines against disease caused by A. pleuropneumoniae. © 2016 The Societies and John Wiley & Sons Australia, Ltd.

Secreted Phospholipases A2 from Animal Venoms in Pain and Analgesia

PubMed Central

Zambelli, Vanessa O.; Picolo, Gisele; Fernandes, Carlos A. H.

2017-01-01

Animal venoms comprise a complex mixture of components that affect several biological systems. Based on the high selectivity for their molecular targets, these components are also a rich source of potential therapeutic agents. Among the main components of animal venoms are the secreted phospholipases A2 (sPLA2s). These PLA2 belong to distinct PLA2s groups. For example, snake venom sPLA2s from Elapidae and Viperidae families, the most important families when considering envenomation, belong, respectively, to the IA and IIA/IIB groups, whereas bee venom PLA2 belongs to group III of sPLA2s. It is well known that PLA2, due to its hydrolytic activity on phospholipids, takes part in many pathophysiological processes, including inflammation and pain. Therefore, secreted PLA2s obtained from animal venoms have been widely used as tools to (a) modulate inflammation and pain, uncovering molecular targets that are implicated in the control of inflammatory (including painful) and neurodegenerative diseases; (b) shed light on the pathophysiology of inflammation and pain observed in human envenomation by poisonous animals; and, (c) characterize molecular mechanisms involved in inflammatory diseases. The present review summarizes the knowledge on the nociceptive and antinociceptive actions of sPLA2s from animal venoms, particularly snake venoms. PMID:29311537

Synovial joint formation requires local Ext1 expression and heparan sulfate production in developing mouse embryo limbs and spine.

PubMed

Mundy, Christina; Yasuda, Tadashi; Kinumatsu, Takashi; Yamaguchi, Yu; Iwamoto, Masahiro; Enomoto-Iwamoto, Motomi; Koyama, Eiki; Pacifici, Maurizio

2011-03-01

Heparan sulfate proteoglycans (HSPGs) regulate a number of major developmental processes, but their roles in synovial joint formation remain unknown. Here we created conditional mouse embryo mutants lacking Ext1 in developing joints by mating Ext1(f/f) and Gdf5-Cre mice. Ext1 encodes a subunit of the Ext1/Ext2 Golgi-associated protein complex responsible for heparan sulfate (HS) synthesis. The proximal limb joints did form in the Gdf5-Cre;Ext1(f/f) mutants, but contained an uneven articulating superficial zone that expressed very low lubricin levels. The underlying cartilaginous epiphysis was deranged as well and displayed random patterns of cell proliferation and matrillin-1 and collagen IIA expression, indicative of an aberrant phenotypic definition of the epiphysis itself. Digit joints were even more affected, lacked a distinct mesenchymal interzone and were often fused likely as a result of local abnormal BMP and hedgehog activity and signaling. Interestingly, overall growth and lengthening of long bones were also delayed in the mutants. To test whether Ext1 function is needed for joint formation at other sites, we examined the spine. Indeed, entire intervertebral discs, normally composed by nucleus pulposus surrounded by the annulus fibrosus, were often missing in Gdf5-Cre;Ext1(f/f) mice. When disc remnants were present, they displayed aberrant organization and defective joint marker expression. Similar intervertebral joint defects and fusions occurred in Col2-Cre;β-catenin(f/f) mutants. The study provides novel evidence that local Ext1 expression and HS production are needed to maintain the phenotype and function of joint-forming cells and coordinate local signaling by BMP, hedgehog and Wnt/β-catenin pathways. The data indicate also that defects in joint formation reverberate on, and delay, overall long bone growth. Copyright © 2011 Elsevier Inc. All rights reserved.

Myosin IIA participates in docking of Glut4 storage vesicles with the plasma membrane in 3T3-L1 adipocytes

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

Chung, Le Thi Kim, E-mail: ngocanh@nutr.med.tokushima-u.ac.jp; Hosaka, Toshio; Harada, Nagakatsu

2010-01-01

In adipocytes and myocytes, insulin stimulation translocates glucose transporter 4 (Glut4) storage vesicles (GSVs) from their intracellular storage sites to the plasma membrane (PM) where they dock with the PM. Then, Glut4 is inserted into the PM and initiates glucose uptake into these cells. Previous studies using chemical inhibitors demonstrated that myosin II participates in fusion of GSVs and the PM and increase in the intrinsic activity of Glut4. In this study, the effect of myosin IIA on GSV trafficking was examined by knocking down myosin IIA expression. Myosin IIA knockdown decreased both glucose uptake and exposures of myc-tagged Glut4more » to the cell surface in insulin-stimulated cells, but did not affect insulin signal transduction. Interestingly, myosin IIA knockdown failed to decrease insulin-dependent trafficking of Glut4 to the PM. Moreover, in myosin IIA knockdown cells, insulin-stimulated binding of GSV SNARE protein, vesicle-associated membrane protein 2 (VAMP2) to PM SNARE protein, syntaxin 4 was inhibited. These data suggest that myosin IIA plays a role in insulin-stimulated docking of GSVs to the PM in 3T3-L1 adipocytes through SNARE complex formation.« less

On the origin of POU5F1

PubMed Central

2013-01-01

Background Pluripotency is a fundamental property of early mammalian development but it is currently unclear to what extent its cellular mechanisms are conserved in vertebrates or metazoans. POU5F1 and POU2 are the two principle members constituting the class V POU domain family of transcription factors, thought to have a conserved role in the regulation of pluripotency in vertebrates as well as germ cell maintenance and neural patterning. They have undergone a complex pattern of evolution which is poorly understood and controversial. Results By analyzing the sequences of POU5F1, POU2 and their flanking genes, we provide strong indirect evidence that POU5F1 originated at least as early as a common ancestor of gnathostomes but became extinct in a common ancestor of teleost fishes, while both POU5F1 and POU2 survived in the sarcopterygian lineage leading to tetrapods. Less divergent forms of POU5F1 and POU2 appear to have persisted among cartilaginous fishes. Conclusions Our study resolves the controversial evolutionary relationship between teleost pou2 and tetrapod POU2 and POU5F1, and shows that class V POU transcription factors have existed at least since the common ancestor of gnathostome vertebrates. It provides a framework for elucidating the basis for the lineage-specific extinctions of POU2 and POU5F1. PMID:23659605

Trifluorothymidine resistance is associated with decreased thymidine kinase and equilibrative nucleoside transporter expression or increased secretory phospholipase A2.

PubMed

Temmink, Olaf H; Bijnsdorp, Irene V; Prins, Henk-Jan; Losekoot, Nienke; Adema, Auke D; Smid, Kees; Honeywell, Richard J; Ylstra, Bauke; Eijk, Paul P; Fukushima, Masakazu; Peters, Godefridus J

2010-04-01

Trifluorothymidine (TFT) is part of the novel oral formulation TAS-102, which is currently evaluated in phase II studies. Drug resistance is an important limitation of cancer therapy. The aim of the present study was to induce resistance to TFT in H630 colon cancer cells using two different schedules and to analyze the resistance mechanism. Cells were exposed either continuously or intermittently to TFT, resulting in H630-cTFT and H630-4TFT, respectively. Cells were analyzed for cross-resistance, cell cycle, protein expression, and activity of thymidine phosphorylase (TP), thymidine kinase (TK), thymidylate synthase (TS), equilibrative nucleoside transporter (hENT), gene expression (microarray), and genomic alterations. Both cell lines were cross-resistant to 2'-deoxy-5-fluorouridine (>170-fold). Exposure to IC(75)-TFT increased the S/G(2)-M phase of H630 cells, whereas in the resistant variants, no change was observed. The two main target enzymes TS and TP remained unchanged in both TFT-resistant variants. In H630-4TFT cells, TK protein expression and activity were decreased, resulting in less activated TFT and was most likely the mechanism of TFT resistance. In H630-cTFT cells, hENT mRNA expression was decreased 2- to 3-fold, resulting in a 5- to 10-fold decreased TFT-nucleotide accumulation. Surprisingly, microarray-mRNA analysis revealed a strong increase of secretory phospholipase-A2 (sPLA2; 47-fold), which was also found by reverse transcription-PCR (RT-PCR; 211-fold). sPLA2 inhibition reversed TFT resistance partially. H630-cTFT had many chromosomal aberrations, but the exact role of sPLA2 in TFT resistance remains unclear. Altogether, resistance induction to TFT can lead to different mechanisms of resistance, including decreased TK protein expression and enzyme activity, decreased hENT expression, as well as (phospho)lipid metabolism. Mol Cancer Ther; 9(4); 1047-57. (c)2010 AACR.

Tanshinone IIA suppresses FcεRI-mediated mast cell signaling and anaphylaxis by activation of the Sirt1/LKB1/AMPK pathway.

PubMed

Li, Xian; Park, Soon Jin; Jin, Fansi; Deng, Yifeng; Yang, Ju Hye; Chang, Jae-Hoon; Kim, Dong-Young; Kim, Jung-Ae; Lee, Youn Ju; Murakami, Makoto; Son, Kun Ho; Chang, Hyeun Wook

2018-06-01

AMP-activated protein kinase (AMPK) and its upstream mediators liver kinase B1 (LKB1) and sirtuin 1 (Sirt1) are generally known as key regulators of metabolism. We have recently reported that the AMPK pathway negatively regulates mast cell activation and anaphylaxis. Tanshinone IIA (Tan IIA), an active component of Salvia miltiorrhiza extract that is currently used for the treatment of cardiovascular and cerebrovascular diseases, shows anti-diabetic activity and improves insulin resistance in db/db mice through activation of AMPK. The aim of this study was to evaluate the anti-allergic activity of Tan IIA in vivo and to investigate the underlying mechanism in vitro in the context of AMPK signaling. The anti-allergic effect of Tan IIA was evaluated using mouse bone marrow-derived mast cells (BMMCs) from AMPKα2 -/- or Sirt1 -/- mice, or BMMCs transfected with siRNAs specific for AMPKα2, LKB1, or Sirt1. AMPKα2 -/- and Sirt1 -/- mice were used to confirm the anti-allergic effect of Tan IIA in anaphylaxis in vivo. Tan IIA dose-dependently inhibited FcεRI-mediated degranulation and production of eicosanoids and cytokines in BMMCs. These inhibitory effects were diminished by siRNA-mediated knockdown or genetic deletion of AMPKα2 or Sirt1. Moreover, Tan IIA inhibited a mast cell-mediated local passive anaphylactic reaction in wild-type mice, but not in AMPKα2 -/- or Sirt1 -/- mice. In conclusion, Tan IIA suppresses FcεRI-mediated mast cell activation and anaphylaxis through activation of the inhibitory Sirt1-LKB1-AMPK pathway. Thus, Tan IIA may be useful as a new therapeutic agent for mast cell-mediated allergic diseases. Copyright © 2018 Elsevier Inc. All rights reserved.

Phospholipase A2 superfamily members play divergent roles after spinal cord injury

PubMed Central

López-Vales, Rubèn; Ghasemlou, Nader; Redensek, Adriana; Kerr, Bradley J.; Barbayianni, Efrosini; Antonopoulou, Georgia; Baskakis, Constantinos; Rathore, Khizr I.; Constantinou-Kokotou, Violetta; Stephens, Daren; Shimizu, Takao; Dennis, Edward A.; Kokotos, George; David, Samuel

2011-01-01

Spinal cord injury (SCI) results in permanent loss of motor functions. A significant aspect of the tissue damage and functional loss may be preventable as it occurs, secondary to the trauma. We show that the phospholipase A2 (PLA2) superfamily plays important roles in SCI. PLA2 enzymes hydrolyze membrane glycerophospholipids to yield a free fatty acid and lysophospholipid. Some free fatty acids (arachidonic acid) give rise to eicosanoids that promote inflammation, while some lysophospholipids (lysophosphatidylcholine) cause demyelination. We show in a mouse model of SCI that two cytosolic forms [calcium-dependent PLA2 group IVA (cPLA2 GIVA) and calcium-independent PLA2 group VIA (iPLA2 GVIA)], and a secreted form [secreted PLA2 group IIA (sPLA2 GIIA)] are up-regulated. Using selective inhibitors and null mice, we show that these PLA2s play differing roles. cPLA2 GIVA mediates protection, whereas sPLA2 GIIA and, to a lesser extent, iPLA2 GVIA are detrimental. Furthermore, completely blocking all three PLA2s worsens outcome, while the most beneficial effects are seen by partial inhibition of all three. The partial inhibitor enhances expression of cPLA2 and mediates its beneficial effects via the prostaglandin EP1 receptor. These findings indicate that drugs that inhibit detrimental forms of PLA2 (sPLA2 and iPLA2) and up-regulate the protective form (cPLA2) may be useful for the treatment of SCI.—López-Vales, R., Ghasemlou, N., Redensek, A., Kerr, B. J., Barbayianni, E., Antonopoulou, G., Baskakis, C., Rathore, K. I., Constantinou-Kokotou, V., Stephens, D., Shimizu, T., Dennis, E. A., Kokotos, G., David, S. Phospholipase A2 superfamily members play divergent roles after spinal cord injury. PMID:21868473

Syntheses, Raman spectra, and X-ray crystal structures of [XeF(5)][mu-F(OsO(3)F(2))(2)] and [M][OsO(3)F(3)] (M = XeF(5)(+), Xe(2)F(11)(+)).

PubMed

Hughes, Michael J; Mercier, Hélène P A; Schrobilgen, Gary J

2010-04-05

Stoichiometric amounts of XeF(6) and (OsO(3)F(2))(infinity) react at 25-50 degrees C to form salts of the known XeF(5)(+) and Xe(2)F(11)(+) cations, namely, [XeF(5)][mu-F(OsO(3)F(2))(2)], [XeF(5)][OsO(3)F(3)], and [Xe(2)F(11)][OsO(3)F(3)]. Although XeF(6) is oxophilic toward a number of transition metal and main-group oxides and oxide fluorides, fluoride/oxide metathesis was not observed. The series provides the first examples of noble-gas cations that are stabilized by metal oxide fluoride anions and the first example of a mu-F(OsO(3)F(2))(2)(-) salt. Both [XeF(5)][mu-F(OsO(3)F(2))(2)] and [Xe(2)F(11)][OsO(3)F(3)] are orange solids at room temperature. The [XeF(5)][OsO(3)F(3)] salt is an orange liquid at room temperature that solidifies at 5-0 degrees C. When the salts are heated at 50 degrees C under 1 atm of N(2) for more than 2 h, significant XeF(6) loss occurs. The X-ray crystal structures (-173 degrees C) show that the salts exist as discrete ion pairs and that the osmium coordination spheres in OsO(3)F(3)(-) and mu-F(OsO(3)F(2))(2)(-) are pseudo-octahedral OsO(3)F(3)-units having facial arrangements of oxygen and fluorine atoms. The mu-F(OsO(3)F(2))(2)(-) anion is comprised of two symmetry-related OsO(3)F(2)-groups that are fluorine-bridged to one another. Ion pairing results from secondary bonding interactions between the fluorine/oxygen atoms of the anions and the xenon atom of the cation, with the Xe...F/O contacts occurring opposite the axial fluorine and from beneath the equatorial XeF(4)-planes of the XeF(5)(+) and Xe(2)F(11)(+) cations so as to avoid the free valence electron lone pairs of the xenon atoms. The xenon atoms of [XeF(5)][mu-F(OsO(3)F(2))(2)] and [Xe(2)F(11)][OsO(3)F(3)] are nine-coordinate and the xenon atom of [XeF(5)][OsO(3)F(3)] is eight-coordinate. Quantum-chemical calculations at SVWN and B3LYP levels of theory were used to obtain the gas-phase geometries, vibrational frequencies, and NBO bond orders, valencies, and NPA charges of

Atmospheric chemistry of C 2F 5CHO: reaction with Cl atoms and OH radicals, IR spectrum of C 2F 5C(O)O 2NO 2

NASA Astrophysics Data System (ADS)

Sulbaek Andersen, M. P.; Hurley, M. D.; Wallington, T. J.; Ball, J. C.; Martin, J. W.; Ellis, D. A.; Mabury, S. A.; Nielsen, O. J.

2003-09-01

Smog chamber/FTIR techniques were used to measure k(Cl + C 2F 5CHO)=(1.96 ± 0.28) × 10 -12 and k(OH + C 2F 5CHO)=(5.26 ± 0.80) × 10 -13 cm 3 molecule -1 s -1 in 700 Torr of N 2 or air at 296 ± 2 K. The Cl initiated oxidation of C 2F 5CHO in the presence of NO in air diluent gave COF 2, CF 3ONO 2, and C 2F 5C(O)O 2NO 2 in molar yields of 180 ± 19%, 6.1 ± 0.6%, and 1.0 ± 0.3%, respectively. The IR spectrum for C 2F 5C(O)O 2NO 2 is reported. Results are discussed with respect to the potential for the atmospheric degradation of fluorinated aldehydes, C nF 2 n+1 CHO, to contribute to the observed environmental burden of fluorinated carboxylic acids, C nF 2 n+1 C(O)OH.

Expression, purification, refolding, and enzymatic characterization of two secretory phospholipases A₂ from Neurospora crassa.

PubMed

Takayanagi, Ayumi; Miyakawa, Takuya; Asano, Atsuko; Ohtsuka, Jun; Tanokura, Masaru; Arioka, Manabu

2015-11-01

Secretory phospholipase A2 (sPLA2) catalyzes the hydrolysis of sn-2 linkage in the glycerophospholipid, thereby releasing fatty acid and 1-acyl lysophospholipid. Among sPLA2s from various organisms and tissues, group XIV fungal/bacterial sPLA2s are relatively less characterized compared to their mammalian counterparts. Here we report cloning, recombinant expression, refolding, and enzymatic characterization of two sPLA2s, NCU06650 and NCU09423, from the filamentous fungus Neurospora crassa. The hexahistidine-tagged putative mature region of both proteins was expressed in Escherichia coli. Inclusion bodies were solubilized using a high hydrostatic pressure refolding technique. NCU06650 was solubilized without any additives at alkaline pH, and the addition of arginine or non-detergent sulfobetain (NDSB) significantly improved the process at acidic pH. In contrast, NCU09423 was solubilized only when NDSB was added at alkaline pH. Both enzymes displayed a Ca(2+)-dependent lipolytic activity toward E. coli membrane. Mass spectrometry analysis using the synthetic phospholipids as substrates demonstrated that both enzymes preferentially cleaved the sn-2 ester linkage of substrates and generated 1-acyl lysophospholipids, demonstrating that they are bona fide PLA2. Copyright © 2015 Elsevier Inc. All rights reserved.

Laser-induced fluorescence studies of excited Sr reactions: II. Sr(3P1)+CH3F, C2H5F, C2H4F2

NASA Astrophysics Data System (ADS)

Teule, J. M.; Janssen, M. H. M.; Bulthuis, J.; Stolte, S.

1999-06-01

The vibrational and rotational energy distributions of ground state SrF(X 2Σ) formed in the reactions of electronically excited Sr(3P1) with methylfluoride, ethylfluoride, and 1,1-difluoroethane have been studied by laser-induced fluorescence. Although the reactions of ground state Sr with these reactants are exothermic, no SrF products are observed for those reactions in this study. The fraction of available energy disposed into the sum of rotational and vibrational energy of the SrF(X 2Σ) product is approximately the same for all three reactions, i.e., 40%. The reaction of Sr(3P1) with CH3F results in very low vibrational excitation in the SrF reaction product. The product vibration increases in going to C2H5F and C2H4F2. It is concluded that the alkyl group influences the energy disposal mechanism in these reactions, and some suggestions are given for a partial explanation of the observations.

Molecular and functional characterization of polymorphisms in the secreted phospholipase A2 group X gene: relevance to coronary artery disease.

PubMed

Gora, Sarah; Perret, Claire; Jemel, Ikram; Nicaud, Viviane; Lambeau, Gérard; Cambien, François; Ninio, Ewa; Blankenberg, Stefan; Tiret, Laurence; Karabina, Sonia-Athina

2009-07-01

Among secreted phospholipases A2 (sPLA2s), human group X sPLA2 (hGX sPLA2) is emerging as a novel attractive therapeutic target due to its implication in inflammatory diseases. To elucidate whether hGX sPLA2 plays a causative role in coronary artery disease (CAD), we screened the human PLA2G10 gene to identify polymorphisms and possible associations with CAD end-points in a prospective study, AtheroGene. We identified eight polymorphisms, among which, one non-synonymous polymorphism R38C in the propeptide region of the sPLA2. The T-512C polymorphism located in the 5' untranslated region was associated with a decreased risk of recurrent cardiovascular events during follow-up. The functional analysis of the R38C polymorphism showed that it leads to a profound change in expression and activity of hGX sPLA2, although there was no detectable impact on CAD risk. Due to the potential role of hGX sPLA2 in inflammatory processes, these polymorphisms should be investigated in other inflammatory diseases.

A lentiviral vector with expression controlled by E2F-1: A potential tool for the study and treatment of proliferative diseases

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

Strauss, Bryan E.; Patricio, Juliana Rotelli; Program in Biotechnology, University of Sao Paulo

2006-10-06

We have constructed a lentiviral vector with expression limited to cells presenting active E2F-1 protein, a potential advantage for gene therapy of proliferative diseases. For the FE2FLW vector, the promoter region of the human E2F-1 gene was utilized to drive expression of luciferase cDNA, included as a reporter of viral expression. Primary, immortalized, and transformed cells were transduced with the FE2FLW vector and cell cycle alterations were induced with serum starvation/replacement, contact inhibition or drug treatment, revealing cell cycle-dependent changes in reporter activity. Forced E2F-1 expression, but not E2F-2 or E2F-3, increased reporter activity, indicating a major role for thismore » factor in controlling expression from the FE2FLW virus. We show the utility of this vector as a reporter of E2F-1 and proliferation-dependent cellular alterations upon cytotoxic/cytostatic treatment, such as the introduction of tumor suppressor genes. We propose that the FE2FLW vector may be a starting point for the development of gene therapy strategies for proliferative diseases, such as cancer or restinosis.« less

E2F1 and E2F2 induction in response to DNA damage preserves genomic stability in neuronal cells.

PubMed

Castillo, Daniela S; Campalans, Anna; Belluscio, Laura M; Carcagno, Abel L; Radicella, J Pablo; Cánepa, Eduardo T; Pregi, Nicolás

2015-01-01

E2F transcription factors regulate a wide range of biological processes, including the cellular response to DNA damage. In the present study, we examined whether E2F family members are transcriptionally induced following treatment with several genotoxic agents, and have a role on the cell DNA damage response. We show a novel mechanism, conserved among diverse species, in which E2F1 and E2F2, the latter specifically in neuronal cells, are transcriptionally induced after DNA damage. This upregulation leads to increased E2F1 and E2F2 protein levels as a consequence of de novo protein synthesis. Ectopic expression of these E2Fs in neuronal cells reduces the level of DNA damage following genotoxic treatment, while ablation of E2F1 and E2F2 leads to the accumulation of DNA lesions and increased apoptotic response. Cell viability and DNA repair capability in response to DNA damage induction are also reduced by the E2F1 and E2F2 deficiencies. Finally, E2F1 and E2F2 accumulate at sites of oxidative and UV-induced DNA damage, and interact with γH2AX DNA repair factor. As previously reported for E2F1, E2F2 promotes Rad51 foci formation, interacts with GCN5 acetyltransferase and induces histone acetylation following genotoxic insult. The results presented here unveil a new mechanism involving E2F1 and E2F2 in the maintenance of genomic stability in response to DNA damage in neuronal cells.

E2F1 and E2F2 induction in response to DNA damage preserves genomic stability in neuronal cells

PubMed Central

Castillo, Daniela S; Campalans, Anna; Belluscio, Laura M; Carcagno, Abel L; Radicella, J Pablo; Cánepa, Eduardo T; Pregi, Nicolás

2015-01-01

E2F transcription factors regulate a wide range of biological processes, including the cellular response to DNA damage. In the present study, we examined whether E2F family members are transcriptionally induced following treatment with several genotoxic agents, and have a role on the cell DNA damage response. We show a novel mechanism, conserved among diverse species, in which E2F1 and E2F2, the latter specifically in neuronal cells, are transcriptionally induced after DNA damage. This upregulation leads to increased E2F1 and E2F2 protein levels as a consequence of de novo protein synthesis. Ectopic expression of these E2Fs in neuronal cells reduces the level of DNA damage following genotoxic treatment, while ablation of E2F1 and E2F2 leads to the accumulation of DNA lesions and increased apoptotic response. Cell viability and DNA repair capability in response to DNA damage induction are also reduced by the E2F1 and E2F2 deficiencies. Finally, E2F1 and E2F2 accumulate at sites of oxidative and UV-induced DNA damage, and interact with γH2AX DNA repair factor. As previously reported for E2F1, E2F2 promotes Rad51 foci formation, interacts with GCN5 acetyltransferase and induces histone acetylation following genotoxic insult. The results presented here unveil a new mechanism involving E2F1 and E2F2 in the maintenance of genomic stability in response to DNA damage in neuronal cells. PMID:25892555

Class IIa Histone Deacetylases Are Conserved Regulators of Circadian Function*

PubMed Central

Fogg, Paul C. M.; O'Neill, John S.; Dobrzycki, Tomasz; Calvert, Shaun; Lord, Emma C.; McIntosh, Rebecca L. L.; Elliott, Christopher J. H.; Sweeney, Sean T.; Hastings, Michael H.; Chawla, Sangeeta

2014-01-01

Class IIa histone deacetylases (HDACs) regulate the activity of many transcription factors to influence liver gluconeogenesis and the development of specialized cells, including muscle, neurons, and lymphocytes. Here, we describe a conserved role for class IIa HDACs in sustaining robust circadian behavioral rhythms in Drosophila and cellular rhythms in mammalian cells. In mouse fibroblasts, overexpression of HDAC5 severely disrupts transcriptional rhythms of core clock genes. HDAC5 overexpression decreases BMAL1 acetylation on Lys-537 and pharmacological inhibition of class IIa HDACs increases BMAL1 acetylation. Furthermore, we observe cyclical nucleocytoplasmic shuttling of HDAC5 in mouse fibroblasts that is characteristically circadian. Mutation of the Drosophila homolog HDAC4 impairs locomotor activity rhythms of flies and decreases period mRNA levels. RNAi-mediated knockdown of HDAC4 in Drosophila clock cells also dampens circadian function. Given that the localization of class IIa HDACs is signal-regulated and influenced by Ca2+ and cAMP signals, our findings offer a mechanism by which extracellular stimuli that generate these signals can feed into the molecular clock machinery. PMID:25271152

Mitochondrial ribosomal protein S18-2 is highly expressed in endometrial cancers along with free E2F1

PubMed Central

Iurchenko, Natalia; Kovalevska, Larysa; Stip, Maria C; Budnikova, Daria; Andersson, Sonia; Polischuk, Ludmila; Buchynska, Lubov; Kashuba, Elena

2016-01-01

Endometrial cancer (EC) is one of the most frequent causes of cancer death among women in developed countries. Histopathological diagnosis and imaging techniques for EC are limited, thus new prognostic markers are needed to offer patients the best treatment and follow-up. In the present paper we showed that the level of mitochondrial ribosomal protein MRPS18-2 (S18-2) increased in EC compared with the normal endometrium and hyperplasia, based on a study of 42 patient biopsies. Importantly, high expression of free E2F1 in EC correlates well with high S18-2 expression. The EC cell line HEC-1-A, which overexpresses S18-2 constitutively, showed an increased proliferation capacity in vitro and in vivo (in SCID mice). Moreover, pan-keratin, beta-catenin and E-cadherin signals are diminished in these cells, compared to the parental HEC-1-A line, in contrast to vimentin signal that is increased. This may be associated with epithelial-mesenchymal cell transition (EMT). We conclude that high expression of S18-2 and free E2F1, and low pan-keratin, beta-catenin, and E-cadherin signals might be a good set of prognostic markers for EC. PMID:26959119

Effects of ethanol and anesthetics on type 1 and 5 metabotropic glutamate receptors expressed in Xenopus laevis oocytes.

PubMed

Minami, K; Gereau, R W; Minami, M; Heinemann, S F; Harris, R A

1998-01-01

Previous studies have demonstrated that ethanol and volatile anesthetics inhibit the function of some metabotropic (G protein-coupled) receptors, including the 5-hydroxytryptamine2 and muscarinic cholinergic receptors. The metabotropic glutamate receptors (mGluRs) show little sequence homology with most other metabotropic receptors and are important modulators of synaptic transmission in the mammalian central nervous system. It was of interest to determine drug actions on these receptors, and we investigated the effects of ethanol, halothane, the anesthetic compound F3 (1-chloro-1,2,2-trifluorocyclobutane), and the nonanesthetics F6 (1,2-dichlorohexafluorocyclobutane) and F8 (2,3-chlorooctafluorobutane) on the function of mGluR1 and mGluR5 expressed in Xenopus laevis oocytes. Halothane, F3, and ethanol inhibited mGluR5-induced Ca(2+)-dependent Cl- currents, yet pharmacologically relevant concentrations of these compounds had little effect on the glutamate-induced currents in the oocytes expressing mGluR1. F6 had inhibitory effects on both receptors, and F8 did not affect either mGluR1 or mGluR5 function. The protein kinase C (PKC) inhibitor GF109203X enhanced the glutamate-induced current, and the PKC activator phorbol-12-myristate-13-acetate inhibited this current in the oocytes expressing mGluR5, but these compounds had little effect on mGluR1 function. GF109203X abolished the inhibitory effects of halothane, F3, and ethanol on mGluR5s. Conversely, the phosphatase inhibitor calyculin A prolonged the action of halothane and ethanol. Furthermore, mutation of a PKC consensus site (Ser890) of mGluR5 abolished the inhibitory effects of halothane, F3, and ethanol. These results suggest that ethanol and volatile anesthetics inhibit mGluR5 because they promote PKC-mediated phosphorylation.

TiF(4) and NaF at pH 1.2 but not at pH 3.5 are able to reduce dentin erosion.

PubMed

Wiegand, Annette; Magalhães, Ana Carolina; Sener, Beatrice; Waldheim, Elena; Attin, Thomas

2009-08-01

This study aimed to analyse and compare the protective effect of buffered (pH 3.5) and native (pH 1.2) TiF(4) in comparison to NaF solutions of same pH on dentin erosion. Bovine samples were pretreated with 1.50% TiF(4) or 2.02% NaF (both 0.48M F) solutions, each with a pH of 1.2 and 3.5. The control group received no fluoride pretreatment. Ten samples in each group were eroded with HCl (pH 2.6) for 10x60s. Erosion was analysed by determination of calcium release into the acid. Additionally, the surface and the elemental surface composition were examined by scanning electron microscopy (two samples in each group) and X-ray energy-dispersive spectroscopy in fluoridated but not eroded samples (six samples in each group). Cumulative calcium release (nmol/mm(2)) was statistically analysed by repeated measures ANOVA and one-way ANOVA at t=10min. TiF(4) and NaF at pH 1.2 decreased calcium release significantly, while TiF(4) and NaF at pH 3.5 were not effective. Samples treated with TiF(4) at pH 1.2 showed a significant increase of Ti, while NaF pretreatment increased F concentration significantly. TiF(4) at pH 1.2 led to the formation of globular precipitates occluding dentinal tubules, which could not be observed on samples treated with TiF(4) at pH 3.5. NaF at pH 1.2 but not at pH 3.5 induced the formation of surface precipitates covering dentinal tubules. Dentin erosion can be significantly reduced by TiF(4) and NaF at pH 1.2, but not at pH 3.5.

Cloning and differential expression of steroid 5 alpha-reductase type 1 (Srd5a1) and type 2 (Srd5a2) from the Harderian glands of hamsters.

PubMed

Ramos, Luis; Chávez, Bertha; Vilchis, Felipe

2010-04-01

In hamsters, the Harderian glands (HGs) exhibit a marked sexual dimorphism which is thought to depend on dihydrotestosterone (DHT); however, it is unclear whether hamster HGs contain one or more 5 alpha-reductases and whether these enzymes are differentially expressed in males and females. In this study, we isolated specific cDNAs for 5 alpha-reductase 1 (Srd5a1) and 5 alpha-reductase 2 (Srd5a2), determined their sequences and investigated their expression in the HG of both sexes. Isozyme 1, cloned from liver mRNA, encodes a protein of 255 amino acids (aa); isozyme 2 cDNA, isolated from the epididymis encodes a 254-aa protein. When assayed in transfected HEK-293 cells, the type 1 isozyme displayed activity over a broad pH range (6.5-8), while isozyme 2 had a pH optimum of 5.5. Both isoenzymes efficiently catalyzed the in vitro transformation of T into DHT, with apparent K(m) values of 7.1 and 1.9 micromol/L for Srd5a1 and Srd5a2, respectively. Real-time PCR analysis revealed higher mRNA levels for Srd5a1 than for Srd5a2. Expression of both isoenzymes increased slightly in HGs of castrated males and showed variations during the estrous cycle in females. Hormonal replacement with 17beta-estradiol administered to spayed females induced the up-regulation of Srd5a2 mRNA levels. Altogether, our results demonstrated that both Srd5a1 and Srd5a2 are expressed in HGs without clear differences between males and females. The biochemical characteristics and relative expression of these 5 alpha-reductases support the view that both isozymes may play a relevant role in modulating androgen signaling in HG. (c) 2009 Elsevier Inc. All rights reserved.

Expression of progesterone metabolizing enzyme genes (AKR1C1, AKR1C2, AKR1C3, SRD5A1, SRD5A2) is altered in human breast carcinoma

PubMed Central

Lewis, Michael J; Wiebe, John P; Heathcote, J Godfrey

2004-01-01

Background Recent evidence suggests that progesterone metabolites play important roles in regulating breast cancer. Previous studies have shown that tumorous tissues have higher 5α-reductase (5αR) and lower 3α-hydroxysteroid oxidoreductase (3α-HSO) and 20α-HSO activities. The resulting higher levels of 5α-reduced progesterone metabolites such as 5α-pregnane-3,20-dione (5αP) in tumorous tissue promote cell proliferation and detachment, whereas the 4-pregnene metabolites, 4-pregnen-3α-ol-20-one (3αHP) and 4-pregnen-20α-ol-3-one (20αDHP), more prominent in normal tissue, have the opposite (anti-cancer-like) effects. The aim of this study was to determine if the differences in enzyme activities between tumorous and nontumorous breast tissues are associated with differences in progesterone metabolizing enzyme gene expression. Methods Semi-quantitative RT-PCR was used to compare relative expression (as a ratio of 18S rRNA) of 5αR type 1 (SRD5A1), 5αR type 2 (SRD5A2), 3α-HSO type 2 (AKR1C3), 3α-HSO type 3 (AKR1C2) and 20α-HSO (AKR1C1) mRNAs in paired (tumorous and nontumorous) breast tissues from 11 patients, and unpaired tumor tissues from 17 patients and normal tissues from 10 reduction mammoplasty samples. Results Expression of 5αR1 and 5αR2 in 11/11 patients was higher (mean of 4.9- and 3.5-fold, respectively; p < 0.001) in the tumor as compared to the paired normal tissues. Conversely, expression of 3α-HSO2, 3α-HSO3 and 20α-HSO was higher (2.8-, 3.9- and 4.4-fold, respectively; p < 0.001) in normal than in tumor sample. The mean tumor:normal expression ratios for 5αR1 and 5αR2 were about 35–85-fold higher than the tumor:normal expression ratios for the HSOs. Similarly, in the unmatched samples, the tumor:normal ratios for 5αR were significantly higher than the ratios for the HSOs. Conclusions The study shows changes in progesterone metabolizing enzyme gene expression in human breast carcinoma. Expression of SRD5A1 (5αR1) and SRD5A2 (5αR2

Expression of progesterone metabolizing enzyme genes (AKR1C1, AKR1C2, AKR1C3, SRD5A1, SRD5A2) is altered in human breast carcinoma.

PubMed

Lewis, Michael J; Wiebe, John P; Heathcote, J Godfrey

2004-06-22

Recent evidence suggests that progesterone metabolites play important roles in regulating breast cancer. Previous studies have shown that tumorous tissues have higher 5alpha-reductase (5alphaR) and lower 3alpha-hydroxysteroid oxidoreductase (3alpha-HSO) and 20alpha-HSO activities. The resulting higher levels of 5alpha-reduced progesterone metabolites such as 5alpha-pregnane-3,20-dione (5alphaP) in tumorous tissue promote cell proliferation and detachment, whereas the 4-pregnene metabolites, 4-pregnen-3alpha-ol-20-one (3alphaHP) and 4-pregnen-20alpha-ol-3-one (20alphaDHP), more prominent in normal tissue, have the opposite (anti-cancer-like) effects. The aim of this study was to determine if the differences in enzyme activities between tumorous and nontumorous breast tissues are associated with differences in progesterone metabolizing enzyme gene expression. Semi-quantitative RT-PCR was used to compare relative expression (as a ratio of 18S rRNA) of 5alphaR type 1 (SRD5A1), 5alphaR type 2 (SRD5A2), 3alpha-HSO type 2 (AKR1C3), 3alpha-HSO type 3 (AKR1C2) and 20alpha-HSO (AKR1C1) mRNAs in paired (tumorous and nontumorous) breast tissues from 11 patients, and unpaired tumor tissues from 17 patients and normal tissues from 10 reduction mammoplasty samples. Expression of 5alphaR1 and 5alphaR2 in 11/11 patients was higher (mean of 4.9- and 3.5-fold, respectively; p < 0.001) in the tumor as compared to the paired normal tissues. Conversely, expression of 3alpha-HSO2, 3alpha-HSO3 and 20alpha-HSO was higher (2.8-, 3.9- and 4.4-fold, respectively; p < 0.001) in normal than in tumor sample. The mean tumor:normal expression ratios for 5alphaR1 and 5alphaR2 were about 35-85-fold higher than the tumor:normal expression ratios for the HSOs. Similarly, in the unmatched samples, the tumor:normal ratios for 5alphaR were significantly higher than the ratios for the HSOs. The study shows changes in progesterone metabolizing enzyme gene expression in human breast carcinoma. Expression of

Nucleoprotein of influenza A virus negatively impacts antiapoptotic protein API5 to enhance E2F1-dependent apoptosis and virus replication.

PubMed

Mayank, A K; Sharma, S; Nailwal, H; Lal, S K

2015-12-17

Apoptosis of host cells profoundly influences virus propagation and dissemination, events that are integral to influenza A virus (IAV) pathogenesis. The trigger for activation of apoptosis is regulated by an intricate interplay between cellular and viral proteins, with a strong bearing on IAV replication. Though the knowledge of viral proteins and mechanisms employed by IAV to induce apoptosis has advanced considerably of late, we know relatively little about the repertoire of host factors targeted by viral proteins. Thus, identification of cellular proteins that are hijacked by the virus will help us not only to understand the molecular underpinnings of IAV-induced apoptosis, but also to design future antiviral therapies. Here we show that the nucleoprotein (NP) of IAV directly interacts with and suppresses the expression of API5, a host antiapoptotic protein that antagonizes E2F1-dependent apoptosis. siRNA-mediated depletion of API5, in NP-overexpressed as well as IAV-infected cells, leads to upregulation of apoptotic protease activating factor 1 (APAF1), a downstream modulator of E2F1-mediated apoptosis, and cleavage of caspases 9 and 3, although a reciprocal pattern of these events was observed on ectopic overexpression of API5. In concordance with these observations, annexin V and 7AAD staining assays exhibit downregulation of early and late apoptosis in IAV-infected or NP-transfected cells on overexpression of API5. Most significantly, while overexpression of API5 decreases viral titers, cellular NP protein as well as mRNA levels in IAV-infected A549 cells, silencing of API5 expression causes a steep rise in the same parameters. From the data reported in this manuscript, we propose a proapoptotic role for NP in IAV pathogenesis, whereby it suppresses expression of antiapoptotic factor API5, thus potentiating the E2F1-dependent apoptotic pathway and ensuring viral replication.

Genomic structure, expression pattern, and functional characterization of transcription factor E2F-2 from black tiger shrimp (Penaeus monodon)

PubMed Central

Zhao, Chao; Qiu, Lihua

2017-01-01

Transcription factor E2F-2 is a regulator of cell cycle. Researchers identified E2F-2 genes from yeasts to humans, but few reports investigated E2F-2 gene from black tiger shrimp. In the present study, we cloned E2F-2 gene from black tiger shrimp (Penaeus monodon). Full-length PmE2F-2 complementary DNA sequence measures 3,189 bp with an open reading frame of 1,371 bp. Complete PmE2F-2 genomic sequence (17,305 bp) of P. monodon contains nine exons, which are separated by eight introns. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that PmE2F-2 is highly expressed in hepatopancreas and ovaries of P. monodon. Highest PmE2F-2 expression levels were observed in stage III ovarian development of P. monodon. PmE2F-2 expression levels were significantly augmented in ovaries of P. monodon after 5-hydroxytryptamine injection and eyestalk ablation. RNA interference experiments were conducted to examine PmE2F-2, PmCDK2, and PmCyclin E expression profiles. PmE2F-2 was successfully knocked down in ovaries and hepatopancreas via double-stranded RNA (dsRNA)–E2F-2 injection. In the same organs, PmE2F-2 expression localization and level were investigated through in situ hybridization, which revealed consistent results with those of qRT-PCR. After dsRNA—E2F-2 injection, gonadosomatic index of shrimp was significantly lower than those following dsRNA—GFP and phosphate-buffered solution injections. Therefore, PmE2F-2 may be involved in ovarian maturation in P. monodon. PMID:28558060

Effect of POU5F1 Expression Level in Clonal Subpopulations of Bovine Fibroblasts Used as Nuclear Donors for Somatic Cell Nuclear Transfer.

PubMed

Sá, André Luiz; Sampaio, Rafael V; da Costa Almeida, Nathália Nogueira; Sangalli, Juliano Rodrigues; Brito, Karynne Nazaré Lins; Bressan, Fabiana Fernandes; Rissino, Joirge Dores; do Socorro Damasceno Santos, Simone; Meirelles, Flavio Vieira; Ohashi, Otávio Mitio; Dos Santos Miranda, Moysés

2017-10-01

Somatic cell nuclear transfer (SCNT) success is partially hindered by the low epigenetic reprogramming efficiency of the donor cell. Previous studies suggest cellular heterogeneity among donor nuclei in regard to reprogramming potential, which precludes comparison among different strategies to increase cloning success. In this context, we evaluated the effect of using clonal cell populations (CPs) of bovine adult fibroblasts established by single-cell plating in SCNT. Different CPs were evaluated in regard to proliferation rate, senescence level, and chromosome stability, as well as for POU5F1 (POU class 5 homeobox 1) mRNA expression levels. In total, 9 of 24 CPs (37.5%) were successfully expanded in vitro up to the fourth passage and shown to proliferate following cryopreservation, at which time cell analyses were performed. The use of a CP with low senescence level, normal karyotype, and highest POU5F1 expression levels did not improve embryo development rates or quality following SCNT. As previously suggested, this study supports the notion that levels of POU5F1 expression in the donor nucleus do not impact the SCNT results. Notably, the single-cell seeding approach used herein to isolate CPs may be extended to the evaluation of additional predictor markers of reprogrammability success for SCNT in future experiments.

Phospholipase A2 in experimental allergic bronchitis: a lesson from mouse and rat models.

PubMed

Mruwat, Rufayda; Yedgar, Saul; Lavon, Iris; Ariel, Amiram; Krimsky, Miron; Shoseyov, David

2013-01-01

Phospholipases A2 (PLA2) hydrolyzes phospholipids, initiating the production of inflammatory lipid mediators. We have previously shown that in rats, sPLA2 and cPLA2 play opposing roles in the pathophysiology of ovalbumin (OVA)-induced experimental allergic bronchitis (OVA-EAB), an asthma model: Upon disease induction sPLA2 expression and production of the broncho-constricting CysLTs are elevated, whereas cPLA2 expression and the broncho-dilating PGE2 production are suppressed. These were reversed upon disease amelioration by treatment with an sPLA2 inhibitor. However, studies in mice reported the involvement of both sPLA2 and cPLA2 in EAB induction. To examine the relevance of mouse and rat models to understanding asthma pathophysiology. OVA-EAB was induced in mice using the same methodology applied in rats. Disease and biochemical markers in mice were compared with those in rats. As in rats, EAB in mice was associated with increased mRNA of sPLA2, specifically sPLA2gX, in the lungs, and production of the broncho-constricting eicosanoids CysLTs, PGD2 and TBX2 in bronchoalveolar lavage (BAL). In contrast, EAB in mice was associated also with elevated cPLA2 mRNA and PGE2 production. Yet, treatment with an sPLA2 inhibitor ameliorated the EAB concomitantly with reverting the expression of both cPLA2 and sPLA2, and eicosanoid production. In both mice and rats sPLA2 is pivotal in OVA-induced EAB. Yet, amelioration of asthma markers in mouse models, and human tissues, was observed also upon cPLA2 inhibition. It is plausible that airway conditions, involving multiple cell types and organs, require the combined action of more than one, essential, PLA2s.

Class IIa histone deacetylases are conserved regulators of circadian function.

PubMed

Fogg, Paul C M; O'Neill, John S; Dobrzycki, Tomasz; Calvert, Shaun; Lord, Emma C; McIntosh, Rebecca L L; Elliott, Christopher J H; Sweeney, Sean T; Hastings, Michael H; Chawla, Sangeeta

2014-12-05

Class IIa histone deacetylases (HDACs) regulate the activity of many transcription factors to influence liver gluconeogenesis and the development of specialized cells, including muscle, neurons, and lymphocytes. Here, we describe a conserved role for class IIa HDACs in sustaining robust circadian behavioral rhythms in Drosophila and cellular rhythms in mammalian cells. In mouse fibroblasts, overexpression of HDAC5 severely disrupts transcriptional rhythms of core clock genes. HDAC5 overexpression decreases BMAL1 acetylation on Lys-537 and pharmacological inhibition of class IIa HDACs increases BMAL1 acetylation. Furthermore, we observe cyclical nucleocytoplasmic shuttling of HDAC5 in mouse fibroblasts that is characteristically circadian. Mutation of the Drosophila homolog HDAC4 impairs locomotor activity rhythms of flies and decreases period mRNA levels. RNAi-mediated knockdown of HDAC4 in Drosophila clock cells also dampens circadian function. Given that the localization of class IIa HDACs is signal-regulated and influenced by Ca(2+) and cAMP signals, our findings offer a mechanism by which extracellular stimuli that generate these signals can feed into the molecular clock machinery. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

High resolution laser spectroscopy of the [13.8]0.5 - X2Δ3/2 and [14.2]1.5 - X2Δ3/2 transitions of hafnium monofluoride (HfF)

NASA Astrophysics Data System (ADS)

Adam, A. G.; Esson, L. M.; Linton, C.; Smith, A. M.; Tokaryk, D. W.

2018-07-01

Laser-induced fluorescence (LIF) spectra of the (0, 0) and (1, 0) bands of the [13.8]0.5 - X2Δ3/2 and [14.2]1.5 - X2Δ3/2 transitions of HfF have been obtained at high resolution (∼120 MHz) using a laser ablation source. Spectra of all five isotopologues, from 180HfF to 176HfF have been clearly resolved and the two most abundant, 180HfF and 178HfF, were chosen for analysis. The rotational levels of the [13.8]0.5 state showed clear Ω - doubling closely resembling that of a 2Σ state in the v = 0 level. Irregularity in the doubling in the v = 1 level and in the isotope shift indicated a strong perturbation affecting this level. The final fit included all the previously analysed bands in the visible region and the doubling of all the Ω = 0.5 states and the isotope effect are examined and discussed.

The prostaglandin E2 receptor PTGER2 and prostaglandin F2α receptor PTGFR mediate oviductal glycoprotein 1 expression in bovine oviductal epithelial cells.

PubMed

Zhang, Nan; Mao, Wei; Zhang, Ying; Huang, Na; Liu, Bo; Gao, Long; Zhang, Shuangyi; Cao, Jinshan

2018-04-13

Oviductal glycoprotein 1 (OVGP1), an oviductin, is involved in the maintenance of sperm viability and motility and contributes to sperm capacitation in the oviduct. In this study, the regulatory effects exerted by prostaglandin E 2 (PGE 2 ) and F 2α (PGF 2α ) on OVGP1 expression via their corresponding receptors in bovine oviductal epithelial cells (BOECs) were investigated. BOECs were cultured in vitro, and their expression of receptors of PGE 2 (PTGER1, PTGER2, PTGER3, and PTGER4) and PGF 2α (PTGFR) was measured using RT-qPCR. Ca 2+ concentration was determined with a fluorescence-based method and cAMP was quantified by enzyme-linked immunosorbent assays to verify activation of PTGER2 and PTGFR by their corresponding agonists in these cells. OVGP1 mRNA and protein expression was measured using RT-qPCR and western blotting, respectively, following PTGER2 and PTGFR agonist-induced activation. PTGER1, PTGER2, PTGER4, and PTGFR were found to be present in BOECs; however, PTGER3 expression was not detected. OVGP1 expression was significantly promoted by 10 -6 M butaprost (a PTGER2 agonist) and decreased by 10 -6 M fluprostenol (a PTGFR agonist). In addition, 3 μM H-89 (a PKA inhibitor) and 3 μM U0126 (an ERK inhibitor) effectively inhibited PGE 2 -induced upregulation of OVGP1, and 5 μM chelerythrine chloride (a PKC inhibitor) and 3 μM U0126 negated OVGP1 downregulation by PGF 2α . In conclusion, this study demonstrates that OVGP1 expression in BOECs is enhanced by PGE 2 via PTGER2-cAMP-PKA signaling, and reduced by PGF 2α through the PTGFR-Ca 2+ -PKC pathway.

Expression and Functional Pathway Analysis of Nuclear Receptor NR2F2 in Ovarian Cancer

PubMed Central

Hawkins, Shannon M.; Loomans, Holli A.; Wan, Ying-Wooi; Ghosh-Choudhury, Triparna; Coffey, Donna; Xiao, Weimin; Liu, Zhandong; Sangi-Haghpeykar, Haleh

2013-01-01

Context: Recent evidence implicates the orphan nuclear receptor, nuclear receptor subfamily 2, group F, member 2 (NR2F2; chicken ovalbumin upstream promoter-transcription factor II) as both a master regulator of angiogenesis and an oncogene in prostate and other human cancers. Objective: The objective of the study was to determine whether NR2F2 plays a role in ovarian cancer and dissect its potential mechanisms of action. Design, Setting, and Patients: We examined NR2F2 expression in healthy ovary and ovarian cancers using quantitative PCR and immunohistochemistry. NR2F2 expression was targeted in established ovarian cancer cell lines to assess the impact of dysregulated NR2F2 expression in the epithelial compartment of ovarian cancers. Results: Our results indicate that NR2F2 is robustly expressed in the stroma of healthy ovary with little or no expression in epithelia lining the ovarian surface, clefts, or crypts. This pattern of NR2F2 expression was markedly disrupted in ovarian cancers, in which decreased levels of stromal expression and ectopic epithelial expression were frequently observed. Ovarian cancers with the most disrupted patterns of NR2F2 were associated with significantly shorter disease-free interval by Kaplan-Meier analysis. Targeting NR2F2 expression in established ovarian cancer cell lines enhanced apoptosis and increased proliferation. In addition, we found that NR2F2 regulates the expression of NEK2, RAI14, and multiple other genes involved in the cell cycle, suggesting potential pathways by which dysregulated expression of NR2F2 impacts ovarian cancer. Conclusions: These results uncover novel roles for NR2F2 in ovarian cancer and point to a unique scenario in which a single nuclear receptor plays potentially distinct roles in the stromal and epithelial compartments of the same tissue. PMID:23690307

Insulin Inhibits Nrf2 Gene Expression via Heterogeneous Nuclear Ribonucleoprotein F/K in Diabetic Mice

PubMed Central

Ghosh, Anindya; Abdo, Shaaban; Zhao, Shuiling; Wu, Chin-Han; Shi, Yixuan; Lo, Chao-Sheng; Chenier, Isabelle; Alquier, Thierry; Filep, Janos G.; Ingelfinger, Julie R.; Zhang, Shao-Ling

2017-01-01

Oxidative stress induces endogenous antioxidants via nuclear factor erythroid 2–related factor 2 (Nrf2), potentially preventing tissue injury. We investigated whether insulin affects renal Nrf2 expression in type 1 diabetes (T1D) and studied its underlying mechanism. Insulin normalized hyperglycemia, hypertension, oxidative stress, and renal injury; inhibited renal Nrf2 and angiotensinogen (Agt) gene expression; and upregulated heterogeneous nuclear ribonucleoprotein F and K (hnRNP F and hnRNP K) expression in Akita mice with T1D. In immortalized rat renal proximal tubular cells, insulin suppressed Nrf2 and Agt but stimulated hnRNP F and hnRNP K gene transcription in high glucose via p44/42 mitogen-activated protein kinase signaling. Transfection with small interfering RNAs of p44/42 MAPK, hnRNP F, or hnRNP K blocked insulin inhibition of Nrf2 gene transcription. Insulin curbed Nrf2 promoter activity via a specific DNA-responsive element that binds hnRNP F/K, and hnRNP F/K overexpression curtailed Nrf2 promoter activity. In hyperinsulinemic-euglycemic mice, renal Nrf2 and Agt expression was downregulated, whereas hnRNP F/K expression was upregulated. Thus, the beneficial actions of insulin in diabetic nephropathy appear to be mediated, in part, by suppressing renal Nrf2 and Agt gene transcription and preventing Nrf2 stimulation of Agt expression via hnRNP F/K. These findings identify hnRNP F/K and Nrf2 as potential therapeutic targets in diabetes. PMID:28324005

Induction of Heme Oxygenase-1 by Sodium 9-Hydroxyltanshinone IIA Sulfonate Derivative Contributes to Inhibit LPS-Mediated Inflammatory Response in Macrophages.

PubMed

Liu, Xin-Hua; Wang, Xi-Ling; Xin, Hong; Wu, Dan; Xin, Xiao-Ming; Miao, Lei; Zhang, Qiu-Yan; Zhou, Yang; Liu, Qian; Zhang, Qian; Zhu, Yi-Zhun

2015-01-01

Sodium 9-acetoxyltanshinone IIA sulfonate (ZY-1A4), a novel compound derived from sodium 9-hydroxyltanshinone IIA sulfonate, was synthesized with potential biological activities. This study aimed to explore the effects of ZY-1A4 on lipopolysaccharide (LPS)-triggered inflammatory response and the underlying mechanisms. Activation of RAW264.7 macrophages was induced by LPS. The effects of ZY-1A4 on inducible nitric oxide synthase (iNOS) expression, nitric oxide (NO) generation, nuclear factor-κB (NF-κB) activation, heme oxygenase-1 (HO-1) expression, and nuclear factor-erythroid 2 related factor 2 (Nrf2) pathway were evaluated to elucidate its underlying mechanisms on inflammatory responses. ZY-1A4 concentration-dependently reduced iNOS expression and NO production, and inhibited c-Jun-N-terminal kinase 1/2 (JNK1/2) phosphorylation and NF-κB activation in LPS-stimulated macrophages. In addition, ZY-1A4 concentration- and time-dependently induced HO-1 expression associated with degradation of Kelch-like ECH-associated protein 1 (Keap1) and nuclear translocation of Nrf2, while the effect of ZY-1A4 was abolished by a phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Intriguingly, pharmacological inactivation of HO-1 with zinc protoporphyrin IX reversed anti-inflammatory effect of ZY- 1A4, but the anti-inflammatory effect of ZY-1A4 was largely mimicked by HO-1 by-products carbon monoxide and bilirubin. Furthermore, the inhibitory effect of ZY-1A4 on LPS-induced iNOS expression and NO release was abolished by HO-1 siRNA or LY294002. Our results demonstrated that ZY-1A4 suppressed LPS-induced iNOS expression and NO generation via modulation of NF-κB activation and HO-1 expression. This new finding might shed light to the prevention and therapy of cardiovascular diseases. © 2015 S. Karger AG, Basel.

Pou5f1-dependent EGF expression controls E-cad endocytosis, cell adhesion, and zebrafish epiboly movements

PubMed Central

Song, Sungmin; Eckerle, Stephanie; Onichtchouk, Daria; Marrs, James A.; Nitschke, Roland; Driever, Wolfgang

2013-01-01

Summary Initiation of motile cell behavior in embryonic development occurs during late blastula stages when gastrulation begins. At this stage, the strong adhesion of blastomeres has to be modulated to enable dynamic behavior, similar to epithelial-to-mesenchymal transitions. We show that in zebrafish MZspg embryos mutant for the stem cell transcription factor Pou5f1/Oct4, which are severely delayed in the epiboly gastrulation movement, all blastomeres are defective in E-cad endosomal trafficking and E-cad accumulates at the plasma membrane. We find that Pou5f1-dependent control of EGF expression regulates endosomal E-cad trafficking. EGFR may act via modulation of p120 activity. Loss of E-cad dynamics reduces cohesion of cells in reaggregation assays. Quantitative analysis of cell behavior indicates that dynamic E-cad endosomal trafficking is required for epiboly cell movements. We hypothesize that dynamic control of E-cad trafficking is essential to effectively generate new adhesion sites when cells move relative to each other. PMID:23484854

ROS Production Is Essential for the Apoptotic Function of E2F1 in Pheochromocytoma and Neuroblastoma Cell Lines

PubMed Central

Espada, Lilia; Meo-Evoli, Nathalie; Sancho, Patricia; Real, Sebastian; Fabregat, Isabel; Ambrosio, Santiago; Tauler, Albert

2012-01-01

In this study we demonstrate that accumulation of reactive oxygen species (ROS) is essential for E2F1 mediated apoptosis in ER-E2F1 PC12 pheochromocytoma, and SH-SY5Y and SK-N-JD neuroblastoma stable cell lines. In these cells, the ER-E2F1 fusion protein is expressed in the cytosol; the addition of 4-hydroxytamoxifen (OHT) induces its translocation to the nucleus and activation of E2F1target genes. Previously we demonstrated that, in ER-E2F1 PC12 cells, OHT treatment induced apoptosis through activation of caspase-3. Here we show that caspase-8 activity did not change upon treatment with OHT. Moreover, over-expression of Bcl-xL arrested OHT-induced apoptosis; by contrast, over-expression of c-FLIP, did not have any effect on OHT-induced apoptosis. OHT addition induces BimL expression, its translocation to mitochondria and activation of Bax, which is paralleled by diminished mitochondrial enrichment of Bcl-xL. Treatment with a Bax-inhibitory peptide reduced OHT-induced apoptosis. These results point out the essential role of mitochondria on the apoptotic process driven by E2F1. ROS accumulation followed E2F1 induction and treatment with the antioxidant N-acetylcysteine, inhibited E2F1-induced Bax translocation to mitochondria and subsequent apoptosis. The role of ROS in mediating OHT-induced apoptosis was also studied in two neuroblastoma cell lines, SH-SY5Y and SK-N-JD. In SH-SY5Y cells, activation of E2F1 by the addition of OHT induced ROS production and apoptosis, whereas over-expression of E2F1 in SK-N-JD cells failed to induce either response. Transcriptional profiling revealed that many of the genes responsible for scavenging ROS were down-regulated following E2F1-induction in SH-SY5Y, but not in SK-N-JD cells. Finally, inhibition of GSK3β blocked ROS production, Bax activation and the down regulation of ROS scavenging genes. These findings provide an explanation for the apparent contradictory role of E2F1 as an apoptotic agent versus a cell cycle activator

Tanshinone IIA inhibits cervix carcinoma stem cells migration and invasion via inhibiting YAP transcriptional activity.

PubMed

Qin, Jinghao; Shi, Hongbing; Xu, Yanjie; Zhao, Fang; Wang, Qing

2018-06-14

This study aims to explore the effects and related mechanisms of Tanshinone IIA in cervix carcinoma (CC) stemness-like cells migration, invasion, stemness and chemotherapeutical sensitivity. Here, we found that Tanshinone IIA suppressed CC stemness-like cells migration and invasion in a concentration- and time-dependent manner. And consistent results were obtained in CC cells stemness characterized as the decrease of CC stemness markers expression and cells spheroid formation ability. Mechanistically, we found that Tanshinone IIA suppressed RNA binding protein HuR translocation from nuclear to cytoplasm, and thus reduced YAP mRNAs stability and transcriptional activity. Importantly, overexpression YAP-5SA rescued the inhibition of Tanshinone IIA on CC cells stemness. Furthermore, Tanshinone IIA enhanced adriamycin sensitivity in CC stemness-like cells, this effect was attenuated by YAP-5SA overexpression too. Therefore, Tanshinone IIA could suppress CC stemness-like cells migration and invasion by inhibiting YAP transcriptional activity. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Generation of transgenic corn-derived Actinobacillus pleuropneumoniae ApxIIA fused with the cholera toxin B subunit as a vaccine candidate

PubMed Central

Shin, Min-Kyoung; Jung, Myung Hwan; Lee, Won-Jung; Choi, Pil Son; Jang, Yong-Suk

2011-01-01

Corn, one of the most important forage crops worldwide, has proven to be a useful expression vehicle due to the availability of established transformation procedures for this well-studied plant. The exotoxin Apx, a major virulence factor, is recognized as a common antigen of Actinobacillus (A.) pleuropneumoniae, the causative agent of porcine pleuropneumonia. In this study, a cholera toxin B (CTB)-ApxIIA#5 fusion protein and full-size ApxIIA expressed in corn seed, as a subunit vaccine candidate, were observed to induce Apx-specific immune responses in mice. These results suggest that transgenic corn-derived ApxIIA and CTB-ApxIIA#5 proteins are potential vaccine candidates against A. pleuropneumoniae infection. PMID:22122907

30 CFR 57.22232 - Actions at 0.5 percent methane (I-B, II-A, II-B, IV, V-B, and VI mines).

Code of Federal Regulations, 2011 CFR

2011-07-01

... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Actions at 0.5 percent methane (I-B, II-A, II-B, IV, V-B, and VI mines). 57.22232 Section 57.22232 Mineral Resources MINE SAFETY AND HEALTH....22232 Actions at 0.5 percent methane (I-B, II-A, II-B, IV, V-B, and VI mines). If methane reaches 0.5...

30 CFR 57.22232 - Actions at 0.5 percent methane (I-B, II-A, II-B, IV, V-B, and VI mines).

Code of Federal Regulations, 2010 CFR

2010-07-01

... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Actions at 0.5 percent methane (I-B, II-A, II-B...-UNDERGROUND METAL AND NONMETAL MINES Safety Standards for Methane in Metal and Nonmetal Mines Ventilation § 57.22232 Actions at 0.5 percent methane (I-B, II-A, II-B, IV, V-B, and VI mines). If methane reaches 0.5...

Crystal structure of MboIIA methyltransferase.

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

Osipiuk, J.; Walsh, M. A.; Joachimiak, A.

2003-09-15

DNA methyltransferases (MTases) are sequence-specific enzymes which transfer a methyl group from S-adenosyl-L-methionine (AdoMet) to the amino group of either cytosine or adenine within a recognized DNA sequence. Methylation of a base in a specific DNA sequence protects DNA from nucleolytic cleavage by restriction enzymes recognizing the same DNA sequence. We have determined at 1.74 {angstrom} resolution the crystal structure of a {beta}-class DNA MTase MboIIA (M {center_dot} MboIIA) from the bacterium Moraxella bovis, the smallest DNA MTase determined to date. M {center_dot} MboIIA methylates the 3' adenine of the pentanucleotide sequence 5'-GAAGA-3'. The protein crystallizes with two molecules inmore » the asymmetric unit which we propose to resemble the dimer when M {center_dot} MboIIA is not bound to DNA. The overall structure of the enzyme closely resembles that of M {center_dot} RsrI. However, the cofactor-binding pocket in M {center_dot} MboIIA forms a closed structure which is in contrast to the open-form structures of other known MTases.« less

Crystal structure of MboIIA methyltransferase.

PubMed

Osipiuk, Jerzy; Walsh, Martin A; Joachimiak, Andrzej

2003-09-15

DNA methyltransferases (MTases) are sequence-specific enzymes which transfer a methyl group from S-adenosyl-L-methionine (AdoMet) to the amino group of either cytosine or adenine within a recognized DNA sequence. Methylation of a base in a specific DNA sequence protects DNA from nucleolytic cleavage by restriction enzymes recognizing the same DNA sequence. We have determined at 1.74 A resolution the crystal structure of a beta-class DNA MTase MboIIA (M.MboIIA) from the bacterium Moraxella bovis, the smallest DNA MTase determined to date. M.MboIIA methylates the 3' adenine of the pentanucleotide sequence 5'-GAAGA-3'. The protein crystallizes with two molecules in the asymmetric unit which we propose to resemble the dimer when M.MboIIA is not bound to DNA. The overall structure of the enzyme closely resembles that of M.RsrI. However, the cofactor-binding pocket in M.MboIIA forms a closed structure which is in contrast to the open-form structures of other known MTases.

Detection of Hearing Loss Using 2f2-f1 and 2f1-f2 Distortion-Product Otoacoustic Emissions

ERIC Educational Resources Information Center

Fitzgerald, Tracy S.; Prieve, Beth A.

2005-01-01

Although many distortion-product otoacoustic emissions (DPOAEs) may be measured in the ear canal in response to 2 pure tone stimuli, the majority of clinical studies have focused exclusively on the DPOAE at the frequency 2f1-f2. This study investigated another DPOAE, 2f2-f1, in an attempt to determine the following: (a) the optimal stimulus…

Overexpression of heterogeneous nuclear ribonucleoprotein F stimulates renal Ace-2 gene expression and prevents TGF-β1-induced kidney injury in a mouse model of diabetes.

PubMed

Lo, Chao-Sheng; Shi, Yixuan; Chang, Shiao-Ying; Abdo, Shaaban; Chenier, Isabelle; Filep, Janos G; Ingelfinger, Julie R; Zhang, Shao-Ling; Chan, John S D

2015-10-01

We investigated whether heterogeneous nuclear ribonucleoprotein F (hnRNP F) stimulates renal ACE-2 expression and prevents TGF-β1 signalling, TGF-β1 inhibition of Ace-2 gene expression and induction of tubulo-fibrosis in an Akita mouse model of type 1 diabetes. Adult male Akita transgenic (Tg) mice overexpressing specifically hnRNP F in their renal proximal tubular cells (RPTCs) were studied. Non-Akita littermates and Akita mice served as controls. Immortalised rat RPTCs stably transfected with plasmid containing either rat Hnrnpf cDNA or rat Ace-2 gene promoter were also studied. Overexpression of hnRNP F attenuated systemic hypertension, glomerular filtration rate, albumin/creatinine ratio, urinary angiotensinogen (AGT) and angiotensin (Ang) II levels, renal fibrosis and profibrotic gene (Agt, Tgf-β1, TGF-β receptor II [Tgf-βrII]) expression, stimulated anti-profibrotic gene (Ace-2 and Ang 1-7 receptor [MasR]) expression, and normalised urinary Ang 1-7 level in Akita Hnrnpf-Tg mice as compared with Akita mice. In vitro, hnRNP F overexpression stimulated Ace-2 gene promoter activity, mRNA and protein expression, and attenuated Agt, Tgf-β1 and Tgf-βrII gene expression. Furthermore, hnRNP F overexpression prevented TGF-β1 signalling and TGF-β1 inhibition of Ace-2 gene expression. These data demonstrate that hnRNP F stimulates Ace-2 gene transcription, prevents TGF-β1 inhibition of Ace-2 gene transcription and induction of kidney injury in diabetes. HnRNP F may be a potential target for treating hypertension and renal fibrosis in diabetes.

Isolation and expression analysis of FTZ-F1 encoding gene of black rock fish ( Sebastes schlegelii)

NASA Astrophysics Data System (ADS)

Shafi, Muhammad; Wang, Yanan; Zhou, Xiaosu; Ma, Liman; Muhammad, Faiz; Qi, Jie; Zhang, Quanqi

2013-03-01

Sex related FTZ-F1 is a transcriptional factor regulating the expression of fushi tarazu (a member of the orphan nuclear receptors) gene. In this study, FTZ-F1 gene ( FTZ-F1) was isolated from the testis of black rockfish ( Sebastes schlegeli) by homology cloning. The full-length cDNA of S. schlegeli FTZ-F1 ( ssFTZ-F1) contained a 232bp 5' UTR, a 1449bp ORF encoding FTZ-F1 (482 amino acid residules in length) with an estimated molecular weight of 5.4kD and a 105bp 3' UTR. Sequence, tissue distribution and phylogenic analysis showed that ssFTZ-F1 belonged to FTZ group, holding highly conserved regions including I, II and III FTZ-F1 boxes and an AF-2 hexamer. Relatively high expression was observed at different larva stages. In juveniles (105 days old), the transcript of ssFTZ-F1 can be detected in all tissues and the abuncance of the gene transcript in testis, ovary, spleen and brain was higher than that in other tissues. In mature fish, the abundance of gene transcript was higher in testis, ovary, spleen and brain than that in liver (trace amount), and the gene was not transcribed in other tissues. The highest abundance of gene transcript was always observed in gonads of both juvenile and mature fish. In addition, the abundance of gene transcript in male tissues were higher than that in female tissue counterparts ( P<0.05).

The Finding of a Group IIE Phospholipase A2 Gene in a Specified Segment of Protobothrops flavoviridis Genome and Its Possible Evolutionary Relationship to Group IIA Phospholipase A2 Genes

PubMed Central

Yamaguchi, Kazuaki; Chijiwa, Takahito; Ikeda, Naoki; Shibata, Hiroki; Fukumaki, Yasuyuki; Oda-Ueda, Naoko; Hattori, Shosaku; Ohno, Motonori

2014-01-01

The genes encoding group IIE phospholipase A2, abbreviated as IIE PLA2, and its 5' and 3' flanking regions of Crotalinae snakes such as Protobothrops flavoviridis, P. tokarensis, P. elegans, and Ovophis okinavensis, were found and sequenced. The genes consisted of four exons and three introns and coded for 22 or 24 amino acid residues of the signal peptides and 134 amino acid residues of the mature proteins. These IIE PLA2s show high similarity to those from mammals and Colubridae snakes. The high expression level of IIE PLA2s in Crotalinae venom glands suggests that they should work as venomous proteins. The blast analysis indicated that the gene encoding OTUD3, which is ovarian tumor domain-containing protein 3, is located in the 3' downstream of IIE PLA2 gene. Moreover, a group IIA PLA2 gene was found in the 5' upstream of IIE PLA2 gene linked to the OTUD3 gene (OTUD3) in the P. flavoviridis genome. It became evident that the specified arrangement of IIA PLA2 gene, IIE PLA2 gene, and OTUD3 in this order is common in the genomes of humans to snakes. The present finding that the genes encoding various secretory PLA2s form a cluster in the genomes of humans to birds is closely related to the previous finding that six venom PLA2 isozyme genes are densely clustered in the so-called NIS-1 fragment of the P. flavoviridis genome. It is also suggested that venom IIA PLA2 genes may be evolutionarily derived from the IIE PLA2 gene. PMID:25529307

Potential proteins targeted by let-7f-5p in HeLa cells.

PubMed

Wang, Yu; Chen, Xiujuan; Zhang, Yi; Song, Jiandong

2017-07-24

MicroRNAs are a class of small, endogenous, non-coding RNAs mediating posttranscriptional gene silencing. The current authors hypothesized that let-7f-5p is likely involved in cell invasion and proliferation by regulating the expression of target genes. The current study combined let-7f-5p with iTRAQ to assess its effect on gene expression in HeLa cells. Results indicated that 164 proteins were expressed at different levels in HeLa cells overexpressing let-7f-5p and negative controls and that 172 proteins were expressed at different levels in let-7f-5p-silenced HeLa cells and negative controls. Results indicated that let-7f-5p may suppress insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) in HeLa cells.

Distinct enzymatic and cellular characteristics of two secretory phospholipases A2 in the filamentous fungus Aspergillus oryzae.

PubMed

Nakahama, Tomoyuki; Nakanishi, Yoshito; Viscomi, Arturo R; Takaya, Kohei; Kitamoto, Katsuhiko; Ottonello, Simone; Arioka, Manabu

2010-04-01

Microbial secretory phospholipases A(2) (sPLA(2)s) are among the last discovered and least known members of this functionally diverse family of enzymes. We analyzed here two sPLA(2)s, named sPlaA and sPlaB, of the filamentous ascomycete Aspergillus oryzae. sPlaA and sPlaB consist of 222 and 160 amino acids, respectively, and share the conserved Cys and catalytic His-Asp residues typical of microbial sPLA(2)s. Two sPLA(2)s differ in pH optimum, Ca(2+) requirement and expression profile. The splaA mRNA was strongly upregulated in response to carbon starvation, oxidative stress and during conidiation, while splaB was constitutively expressed at low levels and was weakly upregulated by heat shock. Experiments with sPLA(2) overexpressing strains demonstrated that two enzymes produce subtly different phospholipid composition variations and also differ in their subcellular localization: sPlaA is most abundant in hyphal tips and secreted to the medium, whereas sPlaB predominantly localizes to the ER-like intracellular compartment. Both sPLA(2) overexpressing strains were defective in conidiation, which was more pronounced for sPlaB overexpressors. Although no major morphological abnormality was detected in either DeltasplaA or DeltasplaB mutants, hyphal growth of DeltasplaB, but not that of DeltasplaA, displayed increased sensitivity to H(2)O(2) treatment. These data indicate that two A. oryzae sPLA(2) enzymes display distinct, presumably non-redundant, physiological functions.

Tanshinone IIA combined with adriamycin inhibited malignant biological behaviors of NSCLC A549 cell line in a synergistic way.

PubMed

Xie, Jun; Liu, Jia-Hui; Liu, Heng; Liao, Xiao-Zhong; Chen, Yuling; Lin, Mei-Gui; Gu, Yue-Yu; Liu, Tao-Li; Wang, Dong-Mei; Ge, Hui; Mo, Sui-Lin

2016-11-18

The study was designed to develop a platform to verify whether the extract of herbs combined with chemotherapy drugs play a synergistic role in anti-tumor effects, and to provide experimental evidence and theoretical reference for finding new effective sensitizers. Inhibition of tanshinone IIA and adriamycin on the proliferation of A549, PC9 and HLF cells were assessed by CCK8 assays. The combination index (CI) was calculated with the Chou-Talalay method, based on the median-effect principle. Migration and invasion ability of A549 cells were determined by wound healing assay and transwell assay. Flow cytometry was used to detect the cell apoptosis and the distribution of cell cycles. TUNEL staining was used to detect the apoptotic cells. Immunofluorescence staining was used to detect the expression of Cleaved Caspase-3. Western blotting was used to detect the proteins expression of relative apoptotic signal pathways. CDOCKER module in DS 2.5 was used to detect the binding modes of the drugs and the proteins. Both tanshinone IIA and adriamycin could inhibit the growth of A549, PC9, and HLF cells in a dose- and time-dependent manner, while the proliferative inhibition effect of tanshinone IIA on cells was much weaker than that of adriamycin. Different from the cancer cells, HLF cells displayed a stronger sensitivity to adriamycin, and a weaker sensitivity to tanshinone IIA. When tanshinone IIA combined with adriamycin at a ratio of 20:1, they exhibited a synergistic anti-proliferation effect on A549 and PC9 cells, but not in HLF cells. Tanshinone IIA combined with adriamycin could synergistically inhibit migration, induce apoptosis and arrest cell cycle at the S and G2 phases in A549 cells. Both groups of the single drug treatment and the drug combination up-regulated the expressions of Cleaved Caspase-3 and Bax, but down-regulated the expressions of VEGF, VEGFR2, p-PI3K, p-Akt, Bcl-2, and Caspase-3 protein. Compared with the single drug treatment groups, the drug

Copy number variations of E2F1: a new genetic risk factor for testicular cancer.

PubMed

Rocca, Maria Santa; Di Nisio, Andrea; Marchiori, Arianna; Ghezzi, Marco; Opocher, Giuseppe; Foresta, Carlo; Ferlin, Alberto

2017-03-01

Testicular germ cell tumor (TGCT) is one of the most heritable forms of cancer. In last years, many evidence suggested that constitutional genetic factors, mainly single nucleotide polymorphisms, can increase its risk. However, the possible contribution of copy number variations (CNVs) in TGCT susceptibility has not been substantially addressed. Indeed, an increasing number of studies have focused on the effect of CNVs on gene expression and on the role of these structural genetic variations as risk factors for different forms of cancer. E2F1 is a transcription factor that plays an important role in regulating cell growth, differentiation, apoptosis and response to DNA damage. Therefore, deficiency or overexpression of this protein might significantly influence fundamental biological processes involved in cancer development and progression, including TGCT. We analyzed E2F1 CNVs in 261 cases with TGCT and 165 controls. We found no CNVs in controls, but 17/261 (6.5%) cases showed duplications in E2F1 Blot analysis demonstrated higher E2F1 expression in testicular samples of TGCT cases with three copies of the gene. Furthermore, we observed higher phosphorylation of Akt and mTOR in samples with E2F1 duplication. Interestingly, normal, non-tumoral testicular tissue in patient with E2F1 duplication showed lower expression of E2F1 and lower AKT/mTOR phosphorylation with respect to adjacent tumor tissue. Furthermore, increased expression of E2F1 obtained in vitro in NTERA-2 testicular cell line induced increased AKT/mTOR phosphorylation. This study suggests for the first time an involvement of E2F1 CNVs in TGCT susceptibility and supports previous preliminary data on the importance of AKT/mTOR signaling pathway in this cancer. © 2017 Society for Endocrinology.

Ifosfamide metabolites CAA, 4-OH-Ifo and Ifo-mustard reduce apical phosphate transport by changing NaPi-IIa in OK cells.

PubMed

Patzer, L; Hernando, N; Ziegler, U; Beck-Schimmer, B; Biber, J; Murer, H

2006-11-01

Renal Fanconi syndrome occurs in about 1-5% of all children treated with Ifosfamide (Ifo) and impairment of renal phosphate reabsorption in about 20-30% of them. Pathophysiological mechanisms of Ifo-induced nephropathy are ill defined. The aim has been to investigate whether Ifo metabolites affect the type IIa sodium-dependent phosphate transporter (NaPi-IIa) in viable opossum kidney cells. Ifo did not influence viability of cells or NaPi-IIa-mediated transport up to 1 mM/24 h. Incubation of confluent cells with chloroacetaldehyde (CAA) and 4-hydroperoxyIfosfamide (4-OH-Ifo) led to cell death by necrosis in a concentration-dependent manner. At low concentrations (50-100 microM/24 h), cell viability was normal but apical phosphate transport, NaPi-IIa protein, and -mRNA expression were significantly reduced. Coincubation with sodium-2-mercaptoethanesulfonate (MESNA) prevented the inhibitory action of CAA but not of 4-OH-Ifo; DiMESNA had no effect. Incubation with Ifosfamide-mustard (Ifo-mustard) did alter cell viability at concentrations above 500 microM/24 h. At lower concentrations (50-100 microM/24 h), it led to significant reduction in phosphate transport, NaPi-IIa protein, and mRNA expression. MESNA did not block these effects. The effect of Ifo-mustard was due to internalization of NaPi-IIa. Cyclophosphamide-mustard (CyP-mustard) did not have any influence on cell survival up to 1000 microM, but the inhibitory effect on phosphate transport and on NaPi-IIa protein was the same as found after Ifo-mustard. In conclusion, CAA, 4-OH-Ifo, and Ifo- and CyP-mustard are able to inhibit sodium-dependent phosphate cotransport in viable opossum kidney cells. The Ifo-mustard effect took place via internalization and reduction of de novo synthesis of NaPi-IIa. Therefore, it is possible that Ifo-mustard plays an important role in pathogenesis of Ifo-induced nephropathy.

YB-1, the E2F Pathway, and Regulation of Tumor Cell Growth

PubMed Central

Samuel, Weini; Cao, Helen; Patel, Rachna; Mehta, Reena; Stern, J. Lewis; Reid, Glen; Woolley, Adele G.; Miller, Lance D.; Black, Michael A; Shelling, Andrew N.; Print, Cristin G.; Braithwaite, Antony W.

2012-01-01

Background Y-box binding factor 1 (YB-1) has been associated with prognosis in many tumor types. Reduced YB-1 expression inhibits tumor cell growth, but the mechanism is unclear. Methods YB-1 mRNA levels were compared with tumor grade and histology using microarray data from 771 breast cancer patients and with disease-free survival and distant metastasis–free survival using data from 375 of those patients who did not receive adjuvant therapy. Microarrays were further searched for genes that had correlated expression with YB-1 mRNA. Small interfering RNA (siRNA) was used to study the effects of reduced YB-1 expression on growth of three tumor cell lines (MCF-7 breast, HCT116 colon, and A549 lung cancer cells), on tumorigenesis by A549 cells in nude mice, and on global transcription in the three cancer cell lines. Reporter gene assays were used to determine whether YB-1 siRNAs affected the expression of E2F1, and chromatin immunoprecipitation was used to determine whether YB-1 bound to various E2F promoters as well as E2F1-regulated promoters. All P values were from two-sided tests. Results YB-1 levels were elevated in more aggressive tumors and were strongly associated with poor disease-free survival and distant metastasis–free survival. YB-1 expression was often associated with the expression of genes with E2F sites in their promoters. Cells expressing YB-1 siRNA grew substantially more slowly than control cells and formed tumors less readily in nude mice. Transcripts that were altered in cancer cell lines with YB-1 siRNA included 32 genes that are components of prognostic gene expression signatures. YB-1 regulated expression of an E2F1 promoter–reporter construct in A549 cells (eg, relative E2F1 promoter activity with control siRNA = 4.04; with YB-1 siRNA = 1.40, difference= −2.64, 95% confidence interval = −3.57 to −1.71, P < .001) and bound to the promoters of several well-defined E2F1 target genes. Conclusion YB-1 expression is associated with the

H5N1 Influenza A Virus PB1-F2 Relieves HAX-1-Mediated Restriction of Avian Virus Polymerase PA in Human Lung Cells.

PubMed

Mazel-Sanchez, B; Boal-Carvalho, I; Silva, F; Dijkman, R; Schmolke, M

2018-06-01

Highly pathogenic influenza A viruses (IAV) from avian hosts were first reported to directly infect humans 20 years ago. However, such infections are rare events, and our understanding of factors promoting or restricting zoonotic transmission is still limited. One accessory protein of IAV, PB1-F2, was associated with pathogenicity of pandemic and zoonotic IAV. This short (90-amino-acid) peptide does not harbor an enzymatic function. We thus identified host factors interacting with H5N1 PB1-F2, which could explain its importance for virulence. PB1-F2 binds to HCLS1-associated protein X1 (HAX-1), a recently identified host restriction factor of the PA subunit of IAV polymerase complexes. We demonstrate that the PA of a mammal-adapted H1N1 IAV is resistant to HAX-1 imposed restriction, while the PA of an avian-origin H5N1 IAV remains sensitive. We also showed HAX-1 sensitivity for PAs of A/Brevig Mission/1/1918 (H1N1) and A/Shanghai/1/2013 (H7N9), two avian-origin zoonotic IAV. Inhibition of H5N1 polymerase by HAX-1 can be alleviated by its PB1-F2 through direct competition. Accordingly, replication of PB1-F2-deficient H5N1 IAV is attenuated in the presence of large amounts of HAX-1. Mammal-adapted H1N1 and H3N2 viruses do not display this dependence on PB1-F2 for efficient replication in the presence of HAX-1. We propose that PB1-F2 plays a key role in zoonotic transmission of avian H5N1 IAV into humans. IMPORTANCE Aquatic and shore birds are the natural reservoir of influenza A viruses from which the virus can jump into a variety of bird and mammal host species, including humans. H5N1 influenza viruses are a good model for this process. They pose an ongoing threat to human and animal health due to their high mortality rates. However, it is currently unclear what restricts these interspecies jumps on the host side or what promotes them on the virus side. Here we show that a short viral peptide, PB1-F2, helps H5N1 bird influenza viruses to overcome a human restriction

Biodegradation of 5-chloro-2-picolinic acid by novel identified co-metabolizing degrader Achromobacter sp. f1.

PubMed

Wu, Zhi-Guo; Wang, Fang; Ning, Li-Qun; Stedtfeld, Robert D; Yang, Zong-Zheng; Cao, Jing-Guo; Sheng, Hong-Jie; Jiang, Xin

2017-06-01

Several bacteria have been isolated to degrade 4-chloronitrobenzene. Degradation of 4-chloronitrobenzene by Cupriavidus sp. D4 produces 5-chloro-2-picolinic acid as a dead-end by-product, a potential pollutant. To date, no bacterium that degrades 5-chloro-2-picolinic acid has been reported. Strain f1, isolated from a soil polluted by 4-chloronitrobenzene, was able to co-metabolize 5-chloro-2-picolinic acid in the presence of ethanol or other appropriate carbon sources. The strain was identified as Achromobacter sp. based on its physiological, biochemical characteristics, and 16S rRNA gene sequence analysis. The organism completely degraded 50, 100 and 200 mg L -1 of 5-chloro-2-picolinic acid within 48, 60, and 72 h, respectively. During the degradation of 5-chloro-2-picolinic acid, Cl - was released. The initial metabolic product of 5-chloro-2-picolinic acid was identified as 6-hydroxy-5-chloro-2-picolinic acid by LC-MS and NMR. Using a mixed culture of Achromobacter sp. f1 and Cupriavidus sp. D4 for degradation of 4-chloronitrobenzen, 5-chloro-2-picolinic acid did not accumulate. Results infer that Achromobacter sp. f1 can be used for complete biodegradation of 4-chloronitrobenzene in remedial applications.

The expression of NFATc1 in adult rat skeletal muscle fibres.

PubMed

Mutungi, Gabriel

2008-03-01

Although numerous studies have recently implicated the calcineurin-nuclear factor of activated T-cells (Cn-NFAT) signalling pathway in the regulation of activity-dependent fibre type switching in adult mammalian skeletal muscles, little is known about the endogenous expression of NFAT proteins in the various fibre types present in these muscles. In this study, the immunolocalization of NFATc1 (also known as NFATc or NFAT2) in the extensor digitorum longus (EDL; a mainly fast-twitch muscle) and the soleus (a predominantly slow-twitch muscle) muscles of adult ( approximately 90-day-old) Wistar rats was investigated. The results show that NFATc1 is expressed only in oxidative fibres (i.e. type I and type IIA fibres) that stain intensely for succinate dehydrogenase activity irrespective of whether they are from the fast- or slow-twitch muscle. Thus, 99 +/- 4% (n = 7 rats) of the muscle fibres in the soleus and 42 +/- 2% (n = 7 rats) of those in the EDL expressed NFATc1. In the soleus muscle fibres, NFATc1 was localized mainly in the fibre nuclei, whereas in the EDL fibres it was localized in both the cytoplasm and the nuclei. However, no difference in its localization was observed between type I and type IIA fibres in both muscles. Western blot experiments showed that the soleus expressed more NFATc1 proteins than the EDL. From these results, we suggest that NFATc1 controls the number and distribution of both type I and type IIA fibres, as well as the oxidative capacity of adult mammalian skeletal muscles.

CYP79F1 and CYP79F2 have distinct functions in the biosynthesis of aliphatic glucosinolates in Arabidopsis.

PubMed

Chen, Sixue; Glawischnig, Erich; Jørgensen, Kirsten; Naur, Peter; Jørgensen, Bodil; Olsen, Carl-Erik; Hansen, Carsten H; Rasmussen, Hasse; Pickett, John A; Halkier, Barbara A

2003-03-01

Cytochromes P450 of the CYP79 family catalyze the conversion of amino acids to oximes in the biosynthesis of glucosinolates, a group of natural plant products known to be involved in plant defense and as a source of flavor compounds, cancer-preventing agents and bioherbicides. We report a detailed biochemical analysis of the substrate specificity and kinetics of CYP79F1 and CYP79F2, two cytochromes P450 involved in the biosynthesis of aliphatic glucosinolates in Arabidopsis thaliana. Using recombinant CYP79F1 and CYP79F2 expressed in Escherichia coli and Saccharomyces cerevisiae, respectively, we show that CYP79F1 metabolizes mono- to hexahomomethionine, resulting in both short- and long-chain aliphatic glucosinolates. In contrast, CYP79F2 exclusively metabolizes long-chain elongated penta- and hexahomomethionines. CYP79F1 and CYP79F2 are spatially and developmentally regulated, with different gene expression patterns. CYP79F2 is highly expressed in hypocotyl and roots, whereas CYP79F1 is strongly expressed in cotyledons, rosette leaves, stems, and siliques. A transposon-tagged CYP79F1 knockout mutant completely lacks short-chain aliphatic glucosinolates, but has an increased level of long-chain aliphatic glucosinolates, especially in leaves and seeds. The level of long-chain aliphatic glucosinolates in a transposon-tagged CYP79F2 knockout mutant is substantially reduced, whereas the level of short-chain aliphatic glucosinolates is not affected. Biochemical characterization of CYP79F1 and CYP79F2, and gene expression analysis, combined with glucosinolate profiling of knockout mutants demonstrate the functional role of these enzymes. This provides valuable insights into the metabolic network leading to the biosynthesis of aliphatic glucosinolates, and into metabolic engineering of altered aliphatic glucosinolate profiles to improve nutritional value and pest resistance.

MiR-205-5p and miR-342-3p cooperate in the repression of the E2F1 transcription factor in the context of anticancer chemotherapy resistance

PubMed Central

Lai, Xin; Gupta, Shailendra K; Schmitz, Ulf; Marquardt, Stephan; Knoll, Susanne; Spitschak, Alf; Wolkenhauer, Olaf; Pützer, Brigitte M; Vera, Julio

2018-01-01

High rates of lethal outcome in tumour metastasis are associated with the acquisition of invasiveness and chemoresistance. Several clinical studies indicate that E2F1 overexpression across high-grade tumours culminates in unfavourable prognosis and chemoresistance in patients. Thus, fine-tuning the expression of E2F1 could be a promising approach for treating patients showing chemoresistance. Methods: We integrated bioinformatics, structural and kinetic modelling, and experiments to study cooperative regulation of E2F1 by microRNA (miRNA) pairs in the context of anticancer chemotherapy resistance. Results: We showed that an enhanced E2F1 repression efficiency can be achieved in chemoresistant tumour cells through two cooperating miRNAs. Sequence and structural information were used to identify potential miRNA pairs that can form tertiary structures with E2F1 mRNA. We then employed molecular dynamics simulations to show that among the identified triplexes, miR-205-5p and miR-342-3p can form the most stable triplex with E2F1 mRNA. A mathematical model simulating the E2F1 regulation by the cooperative miRNAs predicted enhanced E2F1 repression, a feature that was verified by in vitro experiments. Finally, we integrated this cooperative miRNA regulation into a more comprehensive network to account for E2F1-related chemoresistance in tumour cells. The network model simulations and experimental data indicate the ability of enhanced expression of both miR-205-5p and miR-342-3p to decrease tumour chemoresistance by cooperatively repressing E2F1. Conclusions: Our results suggest that pairs of cooperating miRNAs could be used as potential RNA therapeutics to reduce E2F1-related chemoresistance. PMID:29464002

Word Frequency Analysis. MOS: 57H. Skill Levels 1 & 2.

DTIC Science & Technology

1981-05-01

8217 . -4 1a’ _________ Palms 1t. FLL& T - VI;:.P - -- S LtrTZE5 I Lu" I rprz l * 4 2lE p:Zn.uAwn PLCU4LLE 14 Piz;* I P RTICULAR 4 PZR1S 7 J _________ 2...I’ I VIn 1 fIV 7 IaIIA!’I TIP 2 1-111 L LAlT P i;10! S II*,s , 11- 1 L Ii1! p UIIIc tInlG Z :𔃻 S -W (INSf4 2 IZ 1111tE~ ;l ,’I I z a5T p Oil ’(1$I1...p --G 6 RIGGING- I~ V~. 1 5EL~ .1 50:L I SF AFY~~3!.rS_7I; 7 SAFETY I----c- 7 -- ss1 ’_F T_-------- 1 HPPI 7A! I1CEUE SCP .~LaG 3 SHUTWI ~ SC’~J’!LI

Improved electrochemical performance of spinel LiMn(1.5)Ni(0.5)O4 through MgF2 nano-coating.

PubMed

Wu, Qing; Zhang, Xiaoping; Sun, Shuwei; Wan, Ning; Pan, Du; Bai, Ying; Zhu, Huiyuan; Hu, Yong-Sheng; Dai, Sheng

2015-10-14

A spinel LiMn1.5Ni0.5O4 (LMNO) cathode material synthesized by a sol-gel method is modified by MgF2 nano-coating via a wet coating strategy. The results of X-ray diffraction (XRD), Raman spectroscopy, field emission scanning electron microscopy (FESEM) and high resolution transmission electron microscopy (HRTEM) showed that the MgF2 nano-coating layers do not physically change the bulk structure of the pristine material. Compared with the pristine compound, the MgF2-coated LMNO electrodes display enhanced cycling stabilities. Particularly, the 5 wt% MgF2-coated LMNO demonstrates the best reversibility, with a capacity retention of 89.9% after 100 cycles, much higher than that of the pristine material, 69.3%. The dQ/dV analysis and apparent Li(+) diffusion coefficient calculation prove that the kinetic properties are enhanced after MgF2 surface modification, which partly explains the improved electrochemical performances. Electrochemical impedance spectroscopy (EIS) and Fourier transform infrared spectroscopy (FTIR) data confirm that the MgF2 coating layer helps in suppressing the fast growth of the solid electrolyte interface (SEI) film in repeated cycling, which effectively stabilizes the spinel structure. Additionally, differential scanning calorimetry (DSC) tests show that the MgF2 nano-coating layer also helps in enhancing the thermal stability of the LMNO cathode.

A WAVE2-Abi1 complex mediates CSF-1-induced F-actin-rich membrane protrusions and migration in macrophages.

PubMed

Kheir, Wassim Abou; Gevrey, Jean-Claude; Yamaguchi, Hideki; Isaac, Beth; Cox, Dianne

2005-11-15

Colony-stimulating factor 1 (CSF-1) is an important physiological chemoattractant for macrophages. The mechanisms by which CSF-1 elicits the formation of filamentous actin (F-actin)-rich membrane protrusions and induces macrophage migration are not fully understood. In particular, very little is known regarding the contribution of the different members of the Wiskott-Aldrich Syndrome protein (WASP) family of actin regulators in response to CSF-1. Although a role for WASP itself in macrophage chemotaxis has been previously identified, no data was available regarding the function of WASP family verprolin-homologous (WAVE) proteins in this cell type. We found that WAVE2 was the predominant isoform to be expressed in primary macrophages and in cells derived from the murine monocyte/macrophage RAW264.7 cell line (RAW/LR5). CSF-1 treatment of macrophages resulted in WAVE2 accumulation in F-actin-rich protrusions induced by CSF-1. Inhibition of WAVE2 function by expressing a dominant-negative mutant or introducing anti-WAVE2 antibodies in RAW/LR5 cells, as well as reduction of endogenous WAVE2 expression by RNA-mediated interference (RNAi), resulted in a significant reduction of CSF-1-elicited F-actin protrusions. WAVE2 was found in a protein complex together with Abelson kinase interactor 1 (Abi1) in resting or stimulated cells. Both WAVE2 and Abi1 were recruited to and necessary for the formation of F-actin protrusions in response to CSF-1. Reducing the levels of WAVE2, directly or by targeting Abi1, resulted in an impaired cell migration to CSF-1. Altogether these data identify a WAVE2-Abi1 complex crucial for the normal actin cytoskeleton reorganization and migration of macrophages in response to CSF-1.

New organic superconductors beta-(BDA-TTP)2X [BDA-TTP + 2,5-bis(1,3-dithian-2ylidene)-1,3,4,6-tetrathiapentalene; X(-) = SbF6(-), AsF6(-), and PF6(-)].

PubMed

Yamada, J; Watanabe, M; Akutsu, H; Nakatsuji, S; Nishikawa, H; Ikemoto, I; Kikuchi, K

2001-05-09

The synthesis, electrochemical properties, and molecular structure of a new pi-electron donor, 2,5-bis(1,3-dithian-2-ylidene)-1,3,4,6-tetrathiapentalene (BDA-TTP), is described. In contrast to the hitherto-known tetrachalcogenafulvalene pi-donors providing organic superconductors, this donor contains only the bis-fused 1,3-dithiole-2-ylidene unit as a pi-electron system, yet produces a series of ambient-pressure superconductors beta-(BDA-TTP)2X [X = SbF6 (magnetic T(c) = 6.9 K, resistive T(c) = 7.5 K), AsF6 (magnetic T(c) = 5.9 K, resistive T(c) = 5.8 K), and PF6 (magnetic T(c) = 5.9 K)], which are isostructural. The values of the intermolecular overlap integrals calculated on the donor layers of these superconductors suggest a two-dimensional (2D) electronic structure with loose donor packing. Tight-binding band calculations also indicate that these superconductors have the 2D band dispersion relations and closed Fermi surfaces.

[miR-503-5p inhibits the proliferation of T24 and EJ bladder cancer cells by interfering with the Rb/E2F signaling pathway].

PubMed

Li, Xiaohui; Han, Xingtao; Yang, Jinhui; Sun, Jiantao; Wei, Pengtao

2017-10-01

Objective To observe the effect of microRNA-503-5p (miR-503-5p) on the growth of T24 and EJ bladder cancer cells, and explore the possible molecular mechanism. Methods The miR-504-5p mimics or miR-NC was transfected into T24 and EJ cells. The target gene of miR-503-5p was predicted by bioinformatics. The expressions of E2F transcription factor 3 (E2F3) mRNA and Rb/E2F signaling pathway mRNA were detected by the real-time quantitative PCR (qPCR). The expressions of Rb/E2F signal pathway proteins E2F3, cyclin E, CDK2, Rb and p-Rb were detected by Western blotting. The cell cycle of bladder cancer cell lines was determined by flow cytometry. MTT assay and plate cloning assay were performed to observe the proliferation ability of bladder cancer cells. Results After miR-503-5p mimics transfection, the expression of miR-503-5p in bladder cancer cells significantly increased. The increased expression of miR-503-5p significantly reduced the expressions of E2F3 mRNA and Rb/E2F signaling pathway mRNA in bladder cancer cells. What's more, the expressions of Rb/E2F signal pathway proteins were down-regulated. The bladder cancer cells were arrested in G0/G1 phase, and their growth was significantly inhibited by miR-503-5p. Conclusion The miR-503-5p over-expression can inhibit the growth of bladder cancer cell lines T24 and EJ by down-regulating the expression of the Rb/E2F signaling pathway.

DNA Methyl Transferase 1 Reduces Expression of SRD5A2 in the Aging Adult Prostate

PubMed Central

Ge, Rongbin; Wang, Zongwei; Bechis, Seth K.; Otsetov, Alexander G.; Hua, Shengyu; Wu, Shulin; Wu, Chin-Lee; Tabatabaei, Shahin; Olumi, Aria F.

2016-01-01

5-α Reductase type 2 (SRD5A2) is a critical enzyme for prostatic development and growth. Inhibition of SRD5A2 by finasteride is used commonly for the management of urinary obstruction caused by benign prostatic hyperplasia. Contrary to common belief, we have found that expression of SRD5A2 is variable and absent in one third of benign adult prostates. In human samples, absent SRD5A2 expression is associated with hypermethylation of the SRD5A2 promoter, and in vitro SRD5A2 promoter activity is suppressed by methylation. We show that methylation of SRD5A2 is regulated by DNA methyltransferase 1, and inflammatory mediators such as tumor necrosis factor α, NF-κB, and IL-6 regulate DNA methyltransferase 1 expression and thereby affect SRD5A2 promoter methylation and gene expression. Furthermore, we show that increasing age in mice and humans is associated with increased methylation of the SRD5A2 promoter and concomitantly decreased protein expression. Artificial induction of inflammation in prostate primary epithelial cells leads to hypermethylation of the SRD5A2 promoter and silencing of SRD5A2, whereas inhibition with tumor necrosis factor α inhibitor reactivates SRD5A2 expression. Therefore, expression of SRD5A2 is not static and ubiquitous in benign adult prostate tissues. Methylation and expression of SRD5A2 may be used as a gene signature to tailor therapies for more effective treatment of prostatic diseases. PMID:25700986

Investigation of the 2f1-f2 and 2f2-f1 distortion product otoacoustic emissions using a computational model of the gerbil ear.

PubMed

Wen, Haiqi; Bowling, Thomas; Meaud, Julien

2018-05-19

In this work, a three-dimensional computational model of the gerbil ear is used to investigate the generation of the 2f 1 -f 2 and 2f 2 -f 1 distortion product otoacoustic emissions (DPOAEs). In order to predict both the distortion and reflection sources, cochlear roughness is modeled by introducing random inhomogeneities in the outer hair cell properties. The model was used to simulate the generation of DPOAEs in response to a two-tone stimulus for various primary stimulus levels and frequency ratios. As in published experiments, the 2f 1 -f 2 DPOAEs are mostly dominated by the distortion component while the 2f 2 -f 1 DPOAEs are dominated by the reflection component; furthermore, the influence of the levels and frequency ratio of the primaries are consistent with measurements. Analysis of the intracochlear response shows that the distortion component has the highest magnitude at all longitudinal locations for the 2f 1 -f 2 distortion product (DP) while the distortion component only dominates close to the DP best place in the case of the 2f 2 -f 1 DP. Decomposition of the intracochlear DPs into forward and reverse waves demonstrates that the 2f 1 -f 2 DP generates reverse waves for both the distortion and reflection components; however, a reverse wave is only generated for the reflection component in the case of the 2f 2 -f 1 DP. As in experiments in the gerbil, the group delay of the reflection component of the DPOAE is between 1× and 2× the forward group delay, which is consistent with the propagation of DP towards the stapes as slow reverse waves. Copyright © 2018 Elsevier B.V. All rights reserved.

CDH1 regulates E2F1 degradation in response to differentiation signals in keratinocytes

PubMed Central

Singh, Randeep K.; Dagnino, Lina

2017-01-01

The E2F1 transcription factor plays key roles in skin homeostasis. In the epidermis, E2F1 expression is essential for normal proliferation of undifferentiated keratinocytes, regeneration after injury and DNA repair following UV radiation-induced photodamage. Abnormal E2F1 expression promotes nonmelanoma skin carcinoma. In addition, E2F1 must be downregulated for proper keratinocyte differentiation, but the relevant mechanisms involved remain poorly understood. We show that differentiation signals induce a series of post-translational modifications in E2F1 that are jointly required for its downregulation. Analysis of the structural determinants that govern these processes revealed a central role for S403 and T433. In particular, substitution of these two amino acid residues with non-phosphorylatable alanine (E2F1 ST/A) interferes with E2F1 nuclear export, K11- and K48-linked polyubiquitylation and degradation in differentiated keratinocytes. In contrast, replacement of S403 and T433 with phosphomimetic aspartic acid to generate a pseudophosphorylated E2F1 mutant protein (E2F1 ST/D) generates a protein that is regulated in a manner indistinguishable from that of wild type E2F1. Cdh1 is an activating cofactor that interacts with the anaphase-promoting complex/cyclosome (APC/C) ubiquitin E3 ligase, promoting proteasomal degradation of various substrates. We found that Cdh1 associates with E2F1 in keratinocytes. Inhibition or RNAi-mediated silencing of Cdh1 prevents E2F1 degradation in response to differentiation signals. Our results reveal novel regulatory mechanisms that jointly modulate post-translational modifications and downregulation of E2F1, which are necessary for proper epidermal keratinocyte differentiation. PMID:27903963

CDH1 regulates E2F1 degradation in response to differentiation signals in keratinocytes.

PubMed

Singh, Randeep K; Dagnino, Lina

2017-01-17

The E2F1 transcription factor plays key roles in skin homeostasis. In the epidermis, E2F1 expression is essential for normal proliferation of undifferentiated keratinocytes, regeneration after injury and DNA repair following UV radiation-induced photodamage. Abnormal E2F1 expression promotes nonmelanoma skin carcinoma. In addition, E2F1 must be downregulated for proper keratinocyte differentiation, but the relevant mechanisms involved remain poorly understood. We show that differentiation signals induce a series of post-translational modifications in E2F1 that are jointly required for its downregulation. Analysis of the structural determinants that govern these processes revealed a central role for S403 and T433. In particular, substitution of these two amino acid residues with non-phosphorylatable alanine (E2F1 ST/A) interferes with E2F1 nuclear export, K11- and K48-linked polyubiquitylation and degradation in differentiated keratinocytes. In contrast, replacement of S403 and T433 with phosphomimetic aspartic acid to generate a pseudophosphorylated E2F1 mutant protein (E2F1 ST/D) generates a protein that is regulated in a manner indistinguishable from that of wild type E2F1. Cdh1 is an activating cofactor that interacts with the anaphase-promoting complex/cyclosome (APC/C) ubiquitin E3 ligase, promoting proteasomal degradation of various substrates. We found that Cdh1 associates with E2F1 in keratinocytes. Inhibition or RNAi-mediated silencing of Cdh1 prevents E2F1 degradation in response to differentiation signals. Our results reveal novel regulatory mechanisms that jointly modulate post-translational modifications and downregulation of E2F1, which are necessary for proper epidermal keratinocyte differentiation.

E2F1 and E2F2 prevent replicative stress and subsequent p53-dependent organ involution.

PubMed

Iglesias-Ara, A; Zenarruzabeitia, O; Buelta, L; Merino, J; Zubiaga, A M

2015-10-01

Tissue homeostasis requires tight regulation of cellular proliferation, differentiation and apoptosis. E2F1 and E2F2 transcription factors share a critical role in tissue homeostasis, since their combined inactivation results in overall organ involution, specially affecting the pancreatic gland, which subsequently triggers diabetes. We have examined the mechanism by which these E2Fs regulate tissue homeostasis. We show that pancreas atrophy in E2F1/E2F2 double-knockout (DKO) mice is associated with mitochondrial apoptosis and activation of the p53 pathway in young animals, before the development of diabetes. A deregulated expression of E2F target genes was detected in pancreatic cells of young DKO animals, along with unscheduled DNA replication and activation of a DNA damage response. Importantly, suppression of DNA replication in vivo with aphidicolin led to a significant inhibition of the p53 pathway in DKO pancreas, implying a causal link between DNA replication stress and p53 activation in this model. We further show that activation of the p53 pathway has a key role in the aberrant phenotype of DKO mice, since targeted inactivation of p53 gene abrogated cellular apoptosis and prevented organ involution and insulin-dependent diabetes in mice lacking E2F1/E2F2. Unexpectedly, p53 inactivation unmasked oncogenic features of E2F1/E2F2-depleted cells, as evidenced by an accelerated tumor development in triple-knockout mice compared with p53(-/-) mice. Collectively, our data reveal a role for E2F1 and E2F2 as suppressors of replicative stress in differentiating cells, and uncover the existence of a robust E2F-p53 regulatory axis to enable tissue homeostasis and prevent tumorigenesis. These findings have implications in the design of approaches targeting E2F for cancer therapy.

miR-132 targeting E2F5 suppresses cell proliferation, invasion, migration in ovarian cancer cells

PubMed Central

Tian, Hang; Hou, Lei; Xiong, Yu-Mei; Huang, Jun-Xiang; Zhang, Wen-Hua; Pan, Yong-Ying; Song, Xing-Rong

2016-01-01

Accumulating evidence showed that microRNA-132 (miR-132) are involved in development and progression of several types of cancers, however, the function and underlying molecular mechanism of miR-132 in ovarian cancer remains unclear. In this study we investigated the biological roles and molecular mechanism of miR-132 in ovarian cancer. Here, we found that that the expression levels of miR-132 were dramatically decreased in ovarian cancer cell lines and clinical ovarian cancer tissue samples. Then, we found that introduction of miR-132 significantly suppressed the proliferation, colony formation, migration and invasion of ovarian cancer cells. Mechanism investigation revealed that miR-132 inhibited the expression of transcription factor E2F5 by specifically targeting its mRNA 3’UTR. Moreover, the expression level of E2F5 was significantly increased in ovarian cancer tissues than in the adjacent normal tissues, and its expression was inversely correlated with miR-132 expression in clinical ovarian cancer tissues. Additionally, silencing E2F5 was able to inhibit the proliferation, colony formation, migration and invasion of ovarian cancer cells, parallel to the effect of miR-132 overexpression on the ovarian cancer cells. Meanwhile, overexpression of E2F5 reversed the inhibition effect mediated by miR-132 overexpression. These results indicate that miR-132 suppresses the cell proliferation, invasion, migration in ovarian cancer cells by targeting E2F5. PMID:27186275

miR-132 targeting E2F5 suppresses cell proliferation, invasion, migration in ovarian cancer cells.

PubMed

Tian, Hang; Hou, Lei; Xiong, Yu-Mei; Huang, Jun-Xiang; Zhang, Wen-Hua; Pan, Yong-Ying; Song, Xing-Rong

2016-01-01

Accumulating evidence showed that microRNA-132 (miR-132) are involved in development and progression of several types of cancers, however, the function and underlying molecular mechanism of miR-132 in ovarian cancer remains unclear. In this study we investigated the biological roles and molecular mechanism of miR-132 in ovarian cancer. Here, we found that that the expression levels of miR-132 were dramatically decreased in ovarian cancer cell lines and clinical ovarian cancer tissue samples. Then, we found that introduction of miR-132 significantly suppressed the proliferation, colony formation, migration and invasion of ovarian cancer cells. Mechanism investigation revealed that miR-132 inhibited the expression of transcription factor E2F5 by specifically targeting its mRNA 3'UTR. Moreover, the expression level of E2F5 was significantly increased in ovarian cancer tissues than in the adjacent normal tissues, and its expression was inversely correlated with miR-132 expression in clinical ovarian cancer tissues. Additionally, silencing E2F5 was able to inhibit the proliferation, colony formation, migration and invasion of ovarian cancer cells, parallel to the effect of miR-132 overexpression on the ovarian cancer cells. Meanwhile, overexpression of E2F5 reversed the inhibition effect mediated by miR-132 overexpression. These results indicate that miR-132 suppresses the cell proliferation, invasion, migration in ovarian cancer cells by targeting E2F5.

Protein expression and genetic variability of canine Can f 1 in golden and Labrador retriever service dogs.

PubMed

Breitenbuecher, Christina; Belanger, Janelle M; Levy, Kerinne; Mundell, Paul; Fates, Valerie; Gershony, Liza; Famula, Thomas R; Oberbauer, Anita M

2016-01-01

Valued for trainability in diverse tasks, dogs are the primary service animal used to assist individuals with disabilities. Despite their utility, many people in need of service dogs are sensitive to the primary dog allergen, Can f 1, encoded by the Lipocalin 1 gene (LCN1). Several organizations specifically breed service dogs to meet special needs and would like to reduce allergenic potential if possible. In this study, we evaluated the expression of Can f 1 protein and the inherent variability of LCN1 in two breeds used extensively as service dogs. Saliva samples from equal numbers of male and female Labrador retrievers (n = 12), golden retrievers (n = 12), and Labrador-golden crosses (n = 12) were collected 1 h after the morning meal. Can f 1 protein concentrations in the saliva were measured by ELISA, and the LCN1 5' and 3' UTRs and exons sequenced. There was no sex effect (p > 0.2) nor time-of-day effect; however, Can f 1 protein levels varied by breed with Labrador retrievers being lower than golden retrievers (3.18 ± 0.51 and 5.35 ± 0.52 μg/ml, respectively, p < 0.0075), and the Labrador-golden crosses having intermediate levels (3.77 ± 0.48 μg/ml). Although several novel SNPs were identified in LCN1, there were no significant breed-specific sequence differences in the gene and no association of LCN1 genotypes with Can f 1 expression. As service dogs, Labrador retrievers likely have lower allergenic potential and, though there were no DNA sequence differences identified, classical genetic selection on the estimated breeding values associated with salivary Can f 1 expression may further reduce that potential.

Improved electrochemical performance of spinel LiMn 1.5Ni 0.5O 4 through MgF 2 nano-coating

DOE PAGES

Wu, Qing; Zhang, Xiaoping; Sun, Shuwei; ...

2015-07-08

In this paper, a spinel LiMn 1.5Ni 0.5O 4 (LMNO) cathode material synthesized by a sol–gel method is modified by MgF 2 nano-coating via a wet coating strategy. The results of X-ray diffraction (XRD), Raman spectroscopy, field emission scanning electron microscopy (FESEM) and high resolution transmission electron microscopy (HRTEM) showed that the MgF 2 nano-coating layers do not physically change the bulk structure of the pristine material. Compared with the pristine compound, the MgF 2-coated LMNO electrodes display enhanced cycling stabilities. Particularly, the 5 wt% MgF 2-coated LMNO demonstrates the best reversibility, with a capacity retention of 89.9% after 100more » cycles, much higher than that of the pristine material, 69.3%. The dQ/dV analysis and apparent Li + diffusion coefficient calculation prove that the kinetic properties are enhanced after MgF 2 surface modification, which partly explains the improved electrochemical performances. Electrochemical impedance spectroscopy (EIS) and Fourier transform infrared spectroscopy (FTIR) data confirm that the MgF 2 coating layer helps in suppressing the fast growth of the solid electrolyte interface (SEI) film in repeated cycling, which effectively stabilizes the spinel structure. Finally and additionally, differential scanning calorimetry (DSC) tests show that the MgF 2 nano-coating layer also helps in enhancing the thermal stability of the LMNO cathode.« less

Transient expression of the influenza A virus PB1-F2 protein using a plum pox virus-based vector in Nicotiana benthamiana.

PubMed

Kamencayová, M; Košík, I; Hunková, J; Subr, Z W

2014-01-01

PB1-F2 protein of influenza A virus (IAV) was cloned in a plum pox virus (PPV) genome-based vector and attempts to express it in biolistically transfected Nicotiana benthamiana plants were performed. The vector-insert construct replicated in infected plants properly and was stable during repeated passage by mechanical inoculation, as demonstrated by disease symptoms and immunoblot detection of PPV capsid protein, while PB1-F2-specific band was more faint. We showed that it was due its low solubility. Modification of sample preparation (denaturation/solubilization preceding the centrifugation of cell debris) led to substantial signal enhancement. Maximal level of PB1-F2 expression in plants was observed 12 days post inoculation (dpi). Only 1% SDS properly solubilized the protein, other detergents were much less efficient. Solubilization with 8M urea released approximately 50% of PB1-F2 from the plant tissues, thus the treatment with this removable chaotropic agent may be a good starting point for the purification of the protein for eventual functional studies in the future.

FGF-23 transgenic mice demonstrate hypophosphatemic rickets with reduced expression of sodium phosphate cotransporter type IIa.

PubMed

Shimada, Takashi; Urakawa, Itaru; Yamazaki, Yuji; Hasegawa, Hisashi; Hino, Rieko; Yoneya, Takashi; Takeuchi, Yasuhiro; Fujita, Toshiro; Fukumoto, Seiji; Yamashita, Takeyoshi

2004-02-06

Fibroblast growth factor (FGF)-23 was identified as a causative factor of tumor-induced osteomalacia and also as a responsible gene for autosomal dominant hypophosphatemic rickets. To clarify the pathophysiological roles of FGF-23 in these diseases, we generated its transgenic mice. The transgenic mice expressing human FGF-23 reproduced the common clinical features of these diseases such as hypophosphatemia probably due to increased renal phosphate wasting, inappropriately low serum 1,25-dihydroxyvitamin D level, and rachitic bone. The renal phosphate wasting in the transgenic mice was accompanied by the reduced expression of sodium phosphate cotransporter type IIa in renal proximal tubules. These results reinforce the notion that the excessive action of FGF-23 plays a causative role in the development of several hypophosphatemic rickets/osteomalacia.

Comparative Studies of Class IIa Bacteriocins of Lactic Acid Bacteria

PubMed Central

Eijsink, Vincent G. H.; Skeie, Marianne; Middelhoven, P. Hans; Brurberg, May Bente; Nes, Ingolf F.

1998-01-01

Four class IIa bacteriocins (pediocin PA-1, enterocin A, sakacin P, and curvacin A) were purified to homogeneity and tested for activity toward a variety of indicator strains. Pediocin PA-1 and enterocin A inhibited more strains and had generally lower MICs than sakacin P and curvacin A. The antagonistic activity of pediocin-PA1 and enterocin A was much more sensitive to reduction of disulfide bonds than the antagonistic activity of sakacin P and curvacin A, suggesting that an extra disulfide bond that is present in the former two may contribute to their high levels of activity. The food pathogen Listeria monocytogenes was among the most sensitive indicator strains for all four bacteriocins. Enterocin A was most effective in inhibiting Listeria, having MICs in the range of 0.1 to 1 ng/ml. Sakacin P had the interesting property of being very active toward Listeria but not having concomitant high levels of activity toward lactic acid bacteria. Strains producing class IIa bacteriocins displayed various degrees of resistance toward noncognate class IIa bacteriocins; for the sakacin P producer, it was shown that this resistance is correlated with the expression of immunity genes. It is hypothesized that variation in the presence and/or expression of such immunity genes accounts in part for the remarkably large variation in bacteriocin sensitivity displayed by lactic acid bacteria. PMID:9726871

Anti-inflammatory effects of tanshinone IIA on atherosclerostic vessels of ovariectomized ApoE mice are mediated by estrogen receptor activation and through the ERK signaling pathway.

PubMed

Liu, Xin; Guo, Chun-Yu; Ma, Xiao-Juan; Wu, Cai-Feng; Zhang, Ying; Sun, Ming-Yue; Pan, Yu-Ting; Yin, Hui-Jun

2015-01-01

Estrogen plays a protective role in atherosclerosis. Our preliminary work demonstrated that the active conformation of Tanshinone IIA(TanIIA) is similar to the 17β-estradiol and it can bind to the estrogen receptor. Here, we hypothesized that Tanshinone IIA might have anti-inflammatory and anti-oxidative effects in atherosclerosis, mediated through estrogen receptor activation. Subjects for this study were 120 apoE(-/-) female mice and 20 C57/BL female mice. The apoE(-/-) mice were ovariectomized (OVX) and the C57/BL mice were sham ovariectomized. The sham OVX mice were maintained on a normal diet (NOR) group. The OVX apoE(-/-) mice were fed a high fat diet and randomly divided into 6 groups: Model (MOD) group which was fed a high fat diet only, E2 group were given estrogen (E2) 0.13 mg/kg/d; E2+ICI group were given E2:0.13 mg/kg/d and ICI182780:65 mg/kg/m; TLD group (TanIIA low dose) were given TanIIA: 30 mg/kg/d; THD group (TanIIA high dose) were given TanIIA:60 mg/kg/d; and TLD+ICI group were given TanIIA 30 mg/kg/d and ICI182780 65 mg/kg/m. After three months of treatment, the aorta and the blood of the mice from each group was collected. The aorta were used for testing the lipid deposition by using hematoxylin and eosin(HE) and oil red O staining and for testing the expression of p-ERK1/2 by Western blot. The blood was used for testing the serum cholesterol, superoxide dismutase (SOD), methane dicarboxylic aldehyde (MDA), nuclear factor kappa (NF-κB), soluble intercellular cell adhesion molecule-1 (sICAM-1), activating protein-1 (AP-1), E-selectin and 17β-estradiol in serum. Tanshinone IIA significantly reduced the lipid deposition in aorta, decreased the levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), very low density lipoprotein (VLDL), MDA, NF-κB, sICAM-1, AP-1, and E-selectin in serum but increased the levels of high density lipoprotein (HDL) and SOD in serum. Tanshinone IIA also suppressed the expression of p-ERK1/2

Extended analysis of the 5g. -->. 4f emissions in H/sub 2/

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

Chang, E.S.; Pulchtopek, S.; Eyler, E.E.

1984-01-15

An analysis starting from Hund's case d has been used to extend the work of Herzberg and Jungen on the 5g..-->..4f emissions in H/sub 2/. A simple analytical expression for the line intensities is presented that agrees with their calculations is about 1%. All of the experimentally observed lines have been accounted for by including higher vibrational levels in our calculations.

Essential role for Stat5a/b in myeloproliferative neoplasms induced by BCR-ABL1 and JAK2V617F in mice

PubMed Central

Walz, Christoph; Ahmed, Wesam; Lazarides, Katherine; Betancur, Monica; Patel, Nihal; Hennighausen, Lothar; Zaleskas, Virginia M.

2012-01-01

STAT5 proteins are constitutively activated in malignant cells from many patients with leukemia, including the myeloproliferative neoplasms (MPNs) chronic myeloid leukemia (CML) and polycythemia vera (PV), but whether STAT5 is essential for the pathogenesis of these diseases is not known. In the present study, we used mice with a conditional null mutation in the Stat5a/b gene locus to determine the requirement for STAT5 in MPNs induced by BCR-ABL1 and JAK2V617F in retroviral transplantation models of CML and PV. Loss of one Stat5a/b allele resulted in a decrease in BCR-ABL1–induced CML-like MPN and the appearance of B-cell acute lymphoblastic leukemia, whereas complete deletion of Stat5a/b prevented the development of leukemia in primary recipients. However, BCR-ABL1 was expressed and active in Stat5-null leukemic stem cells, and Stat5 deletion did not prevent progression to lymphoid blast crisis or abolish established B-cell acute lymphoblastic leukemia. JAK2V617F failed to induce polycythemia in recipients after deletion of Stat5a/b, although the loss of STAT5 did not prevent the development of myelofibrosis. These results demonstrate that STAT5a/b is essential for the induction of CML-like leukemia by BCR-ABL1 and of polycythemia by JAK2V617F, and validate STAT5a/b and the genes they regulate as targets for therapy in these MPNs. PMID:22234689

Idiopathic hirsutism: local and peripheral expression of aromatase (CYP19A) and 5α-reductase genes (SRD5A1 and SRD5A2).

PubMed

Caglayan, A Okay; Dundar, Munis; Tanriverdi, Fatih; Baysal, Nuran A; Unluhizarci, Kursad; Ozkul, Yusuf; Borlu, Murat; Batukan, Cem; Kelestimur, Fahrettin

2011-08-01

To evaluate idiopathic hirsutism etiology via molecular studies testing peripheral and local aromatase and 5α-reductase expression. Assessment of the expression of messenger RNA (mRNA) for type 1 and 2,5α-reductase isoenzyme gene (SDR5A1, SDR5A2) and aromatase (CYP19A) in dermal papillae cells and peripheral blood mononuclear cells. University hospital. 28 untreated idiopathic hirsute patients and 20 healthy women (controls). Human skin biopsies and peripheral venous blood. SDR5A1, SDR5A2, CYP19A gene expression in skin biopsies and peripheral blood. A statistically significant reduction of SRD5A1, SRD5A2, and CYP19A gene expression was found in the dermal papillae cells and peripheral blood mononuclear cell between the study and control group. Further study, including protein expression and enzyme activity assays, are warranted to characterize the paradoxically low gene expression levels of local 5α-reductase and aromatase in women with idiopathic hirsutism. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Epigenetic involvement of Alien/ESET complex in thyroid hormone-mediated repression of E2F1 gene expression and cell proliferation

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

Hong, Wei, E-mail: hongwei@tijmu.edu.cn; College of Basic Medicine, Tianjin Medical University, 300070 Tianjin; Li, Jinru

Highlights: Black-Right-Pointing-Pointer Corepressor Alien interacts with histone methyltransferase ESET in vivo. Black-Right-Pointing-Pointer Alien/ESET complex is recruited to nTRE of T3-responsive gene by liganded TR{beta}1. Black-Right-Pointing-Pointer ESET-mediated H3K9 methylation is required for liganded TR{beta}1-repressed transcription. Black-Right-Pointing-Pointer ESET is involved in T3-repressed G1/S phase transition and proliferation. -- Abstract: The ligand-bound thyroid hormone receptor (TR) is known to repress via a negative TRE (nTRE) the expression of E2F1, a key transcription factor that controls the G1/S phase transition. Alien has been identified as a novel interacting factor of E2F1 and acts as a corepressor of E2F1. The detailed molecular mechanism by whichmore » Alien inhibits E2F1 gene expression remains unclear. Here, we report that the histone H3 lysine 9 (H3K9) methyltransferase (HMT) ESET is an integral component of the corepressor Alien complex and the Alien/ESET complex is recruited to both sites, the E2F1 and the nTRE site of the E2F1 gene while the recruitment to the negative thyroid hormone response element (nTRE) is induced by the ligand-bound TR{beta}1 within the E2F1 gene promoter. We show that, overexpression of ESET promotes, whereas knockdown of ESET releases, the inhibition of TR{beta}1-regulated gene transcription upon T3 stimulation; and H3K9 methylation is required for TR{beta}1-repressed transcription. Furthermore, depletion of ESET impairs thyroid hormone-repressed proliferation as well as the G1/S transition of the cell cycle. Taken together, our data indicate that ESET is involved in TR{beta}1-mediated transcription repression and provide a molecular basis of thyroid hormone-induced repression of proliferation.« less

Downregulation of Forkhead box F1 gene expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia.

PubMed

Zimmer, J; Takahashi, T; Hofmann, A D; Puri, Prem

2016-12-01

High mortality and morbidity in infants born with congenital diaphragmatic hernia (CDH) are attributed to pulmonary hypoplasia and pulmonary hypertension (PH). Forkhead box (Fox) transcription factors are known to be crucial for cell proliferation and homeostasis. FoxF1 is essential for lung morphogenesis, vascular development, and endothelial proliferation. Mutations in FoxF1 and also the Fox family member FoxC2 have been identified in neonates with PH. In human and experimental models of arterial PH, the Fox protein FoxO1 was found to be downregulated. We hypothesized that Fox expression is altered in the lungs of the nitrofen-induced CDH rat model and investigated the expression of FoxF1, FoxC2, and FoxO1. Following ethical approval (Rec 913b), time-pregnant Sprague-Dawley rats received nitrofen or vehicle on gestational day (D9). Fetuses were sacrificed on D21, inspected for CDH and divided into CDH (n = 11) and control group (n = 11). Gene expression of FoxF1, FoxC2, and FoxO1 was evaluated with qRT-PCR. Detected alterations of mRNA levels were subsequently assessed on the protein level by performing western blot analysis and laser scanning confocal microscopy. The relative mRNA level of FoxF1 was significantly downregulated in CDH lungs compared to controls (FoxF1 CDH 1.047 ± 0.108, FoxF1 Ctrl 1.419 ± 0.01, p = 0.014). Relative mRNA levels of FoxC2 and FoxO1 were not found to be altered between the experimental groups (FoxC2 CDH 30.74 ± 8.925, FoxC2 Ctrl 27.408 ± 7.487, p = 0.776; FoxO1 CDH 0.011 ± 0.002, FoxO1 Ctrl 0.011 ± 0.001, p = 0.809). On the protein level, western blotting demonstrated a reduced pulmonary protein expression of FoxF1 in CDH lungs. Confocal microscopy showed a markedly diminished expression of FoxF1 in the pulmonary vasculature of CDH lungs compared to controls. Our study demonstrates a strikingly reduced expression of FoxF1 in the pulmonary vasculature of nitrofen-induced CDH. Altered FoxF1 gene expression

Molecular details of secretory phospholipase A2 from flax (Linum usitatissimum L.) provide insight into its structure and function.

PubMed

Gupta, Payal; Dash, Prasanta K

2017-09-11

Secretory phospholipase A 2 (sPLA 2 ) are low molecular weight proteins (12-18 kDa) involved in a suite of plant cellular processes imparting growth and development. With myriad roles in physiological and biochemical processes in plants, detailed analysis of sPLA 2 in flax/linseed is meagre. The present work, first in flax, embodies cloning, expression, purification and molecular characterisation of two distinct sPLA 2 s (I and II) from flax. PLA 2 activity of the cloned sPLA 2 s were biochemically assayed authenticating them as bona fide phospholipase A 2 . Physiochemical properties of both the sPLA 2 s revealed they are thermostable proteins requiring di-valent cations for optimum activity.While, structural analysis of both the proteins revealed deviations in the amino acid sequence at C- & N-terminal regions; hydropathic study revealed LusPLA 2 I as a hydrophobic protein and LusPLA 2 II as a hydrophilic protein. Structural analysis of flax sPLA 2 s revealed that secondary structure of both the proteins are dominated by α-helix followed by random coils. Modular superimposition of LusPLA 2 isoforms with rice sPLA 2 confirmed monomeric structural preservation among plant phospholipase A 2 and provided insight into structure of folded flax sPLA 2 s.

Protective effect of tanshinone IIA against cardiac hypertrophy in spontaneously hypertensive rats through inhibiting the Cys-C/Wnt signaling pathway

PubMed Central

Feng, Jun; Chen, Hua-Wen; Pi, Li-Juan; Wang, Jin; Zhan, Da-Qian

2017-01-01

The study aimed to investigate the protective effect of tanshinone IIA against cardiac hypertrophy in spontaneously hypertensive rats (SHRs) through the Cys-C/Wnt signaling pathway. Thirty SHRs were randomly divided into cardiac hypertrophy, low- and high-dose tanshinone IIA groups. Ten Wistar-Kyoto rats were selected as control group. The systolic blood pressure (SBP), heart weight (HW), left ventricular weight (LVW) and body weight (BW) of all rats were recorded. HE staining and qRT-PCR were applied to observe the morphology of myocardial tissue and mRNA expressions of COL1A1 and COL3A1. ELISA and Western blotting were used to measure the serum asymmetric dimethylarginine (ADMA), nitric oxide (NO) and cardiac troponin I (cTnI) levels, and the expressions of the Cys-C/Wnt signaling pathway-related proteins, eNOS and Nox4. Compared with the cardiac hypertrophy group, the SBP, HW/BW, LVW/BW, swelling degree of myocardial cells, COL1A1 and COL3A1 mRNA expressions, serum cTnI and ADMA levels, and the Cys-C/Wnt signaling pathway-related proteins and Nox4 expressions in the low- and high-dose tanshinone IIA groups were decreased, but the endothelial NO synthase (eNOS), phosphorylated eNOS (Ser1177) and NO expressions were increased. No significant difference was found between the low- and high-dose tanshinone IIA groups. Our study indicated a protective effect of tanshinone IIA against cardiac hypertrophy in SHRs through inhibiting the Cys-C/Wnt signaling pathway. PMID:28053285

E2F1 and E2F2 prevent replicative stress and subsequent p53-dependent organ involution

PubMed Central

Iglesias-Ara, A; Zenarruzabeitia, O; Buelta, L; Merino, J; Zubiaga, A M

2015-01-01

Tissue homeostasis requires tight regulation of cellular proliferation, differentiation and apoptosis. E2F1 and E2F2 transcription factors share a critical role in tissue homeostasis, since their combined inactivation results in overall organ involution, specially affecting the pancreatic gland, which subsequently triggers diabetes. We have examined the mechanism by which these E2Fs regulate tissue homeostasis. We show that pancreas atrophy in E2F1/E2F2 double-knockout (DKO) mice is associated with mitochondrial apoptosis and activation of the p53 pathway in young animals, before the development of diabetes. A deregulated expression of E2F target genes was detected in pancreatic cells of young DKO animals, along with unscheduled DNA replication and activation of a DNA damage response. Importantly, suppression of DNA replication in vivo with aphidicolin led to a significant inhibition of the p53 pathway in DKO pancreas, implying a causal link between DNA replication stress and p53 activation in this model. We further show that activation of the p53 pathway has a key role in the aberrant phenotype of DKO mice, since targeted inactivation of p53 gene abrogated cellular apoptosis and prevented organ involution and insulin-dependent diabetes in mice lacking E2F1/E2F2. Unexpectedly, p53 inactivation unmasked oncogenic features of E2F1/E2F2-depleted cells, as evidenced by an accelerated tumor development in triple-knockout mice compared with p53−/− mice. Collectively, our data reveal a role for E2F1 and E2F2 as suppressors of replicative stress in differentiating cells, and uncover the existence of a robust E2F-p53 regulatory axis to enable tissue homeostasis and prevent tumorigenesis. These findings have implications in the design of approaches targeting E2F for cancer therapy. PMID:25656653

Five of 12 forms of vaccinia virus-expressed human hepatic cytochrome P450 metabolically activate aflatoxin B sub 1

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

Aoyama, Toshifumi; Yamano, Shigeru; Gelboin, H.V.

Twelve forms of human hepatic cytochrome P450 were expressed in hepatoma cells by means of recombinant vaccinia viruses. The expressed P450s were analyzed for their abilities to activate the potent hepatocarcinogen aflatoxin B{sub 1} to metabolites having mutagenic or DNA-binding properties. Five forms, P450s IA2, IIA3, IIB7, IIIA3, and IIIA4, activated aflatoxin B{sub 1} to mutagenic metabolites as assessed by the production of His revertants of Salmonella typhimurium in the Ames test. The same P450s catalyzed conversion of aflatoxin B{sub 1} to DNA-bound derivatives as judged by an in situ assay in which the radiolabeled carcinogen was incubated with cellsmore » expressing the individual P450 forms. Seven other human P450s, IIC8, IIC9, IID6, IIE1, IIF1, and IIIA5, and IVB1, did not significantly activate aflatoxin B{sub 1} as measured by both the Ames test and the DNA-binding assay. Moreover, polyclonal anti-rat liver P450 antibodies that crossreact with individual human P450s IA2, IIA3, IIIA3, and IIIA4 each inhibited aflatoxin B{sub 1} activation catalyzed by human liver S-9 extracts. Inhibition ranged from as low as 10% with antibody against IIA3 to as high as 65% with antibody against IIIA3 and IIIA4. These results establish that metabolic activation of aflatoxin B{sub 1} in human liver involves the contribution of multiple forms of P450.« less

Inhibition of class IIa histone deacetylase activity by gallic acid, sulforaphane, TMP269, and panobinostat.

PubMed

Choi, Sin Young; Kee, Hae Jin; Jin, Li; Ryu, Yuhee; Sun, Simei; Kim, Gwi Ran; Jeong, Myung Ho

2018-05-01

Histone deacetylase (HDAC) inhibitors are gaining increasing attention as potential therapeutics for cardiovascular diseases as well as cancer. We recently reported that the class II HDAC inhibitor, MC1568, and the phytochemical, gallic acid, lowered high blood pressure in mouse models of hypertension. We hypothesized that class II HDACs may be involved in the regulation of hypertension. The aim of this study was to determine and compare the effects of well-known HDAC inhibitors (TMP269, panobinostat, and MC1568), phytochemicals (gallic acid, sulforaphane, and piceatannol), and anti-hypertensive drugs (losartan, carvedilol, and furosemide) on activities of class IIa HDACs (HDAC4, 5, 7, and 9). The selective class IIa HDAC inhibitor, TMP269, and the pan-HDAC inhibitor, panobinostat, but not MC1568, clearly inhibited class IIa HDAC activities. Among the three phytochemicals, gallic acid showed remarkable inhibition, whereas sulforaphane presented mild inhibition of class IIa HDACs. Piceatannol inhibited only HDAC7 activity. As expected, the anti-hypertensive drugs losartan, carvedilol, and furosemide did not affect the activity of any class IIa HDAC. In addition, we evaluated the inhibitory effect of several compounds on the activity of class l HDACs (HDAC1, 2, 3, and 8) and class IIb HDAC (HDAC6). MC1568 did not affect the activities of HDAC1, HDAC2, and HDAC3, but it reduced the activity of HDAC8 at concentrations of 1 and 10 μM. Gallic acid weakly inhibited HDAC1 and HDAC6 activities, but strongly inhibited HDAC8 activity with effectiveness comparable to that of trichostatin A. Inhibition of HDAC2 activity by sulforaphane was stronger than that by piceatnnaol. These results indicated that gallic acid is a powerful dietary inhibitor of HDAC8 and class IIa/b HDAC activities. Sulforaphane may also be used as a dietary inhibitor of HDAC2 and class IIa HDAC. Our findings suggest that the class II HDAC inhibitor, MC1568, does not inhibit class IIa HDAC, but inhibits

[Study on solid dispersion of copovidone-based tanshinone II(A)].

PubMed

Jiang, Yan-Rong; Zhang, Zhen-Hai; Xia, Hai-Jiang; Jia, Xiao-Bin

2013-01-01

To apply PVP-S630 in the preparation of tanshinone II(A) (TS II(A)) solid dispersion, in order to improve its dissolution in vitro and reduce the moisture absorption of the solid dispersion. Tanshinone II(A) solid dispersion was prepared by spray drying method. Such analytical methods as SEM, DSC, XRD were used to characterize their phases and detect their dissolution, moisture absorption and stability. In the solid dispersion prepared with tanshinone II(A) and copovidone with proportion of 1:10, tanshinone II(A) was scattered on the surface of the carrier in the amorphous form, with a dissolution in vitro up to 100% at 0.5 h and a lower moisture absorption than PVP-K30 solid dispersion prepared with the same proportion. After a three-month accelerated stability test, it showed no significant change in drug dissolution and content. The solid dispersion prepared with copovidone as the carrier can significantly improve the dissolution of tanshinone II(A), with a relatively low moisture absorption and high stability, thereby having a good prospect of application.

Is chloroplastic class IIA aldolase a marine enzyme?

PubMed

Miyasaka, Hitoshi; Ogata, Takeru; Tanaka, Satoshi; Ohama, Takeshi; Kano, Sanae; Kazuhiro, Fujiwara; Hayashi, Shuhei; Yamamoto, Shinjiro; Takahashi, Hiro; Matsuura, Hideyuki; Hirata, Kazumasa

2016-11-01

Expressed sequence tag analyses revealed that two marine Chlorophyceae green algae, Chlamydomonas sp. W80 and Chlamydomonas sp. HS5, contain genes coding for chloroplastic class IIA aldolase (fructose-1, 6-bisphosphate aldolase: FBA). These genes show robust monophyly with those of the marine Prasinophyceae algae genera Micromonas, Ostreococcus and Bathycoccus, indicating that the acquisition of this gene through horizontal gene transfer by an ancestor of the green algal lineage occurred prior to the divergence of the core chlorophytes (Chlorophyceae and Trebouxiophyceae) and the prasinophytes. The absence of this gene in some freshwater chlorophytes, such as Chlamydomonas reinhardtii, Volvox carteri, Chlorella vulgaris, Chlorella variabilis and Coccomyxa subellipsoidea, can therefore be explained by the loss of this gene somewhere in the evolutionary process. Our survey on the distribution of this gene in genomic and transcriptome databases suggests that this gene occurs almost exclusively in marine algae, with a few exceptions, and as such, we propose that chloroplastic class IIA FBA is a marine environment-adapted enzyme. This hypothesis was also experimentally tested using Chlamydomonas W80, for which we found that the transcript levels of this gene to be significantly lower under low-salt (that is, simulated terrestrial) conditions. Expression analyses of transcriptome data for two algae, Prymnesium parvum and Emiliania huxleyi, taken from the Sequence Read Archive database also indicated that the expression of this gene under terrestrial conditions (low NaCl and low sulfate) is significantly downregulated. Thus, these experimental and transcriptome data provide support for our hypothesis.

Is chloroplastic class IIA aldolase a marine enzyme?

PubMed Central

Miyasaka, Hitoshi; Ogata, Takeru; Tanaka, Satoshi; Ohama, Takeshi; Kano, Sanae; Kazuhiro, Fujiwara; Hayashi, Shuhei; Yamamoto, Shinjiro; Takahashi, Hiro; Matsuura, Hideyuki; Hirata, Kazumasa

2016-01-01

Expressed sequence tag analyses revealed that two marine Chlorophyceae green algae, Chlamydomonas sp. W80 and Chlamydomonas sp. HS5, contain genes coding for chloroplastic class IIA aldolase (fructose-1, 6-bisphosphate aldolase: FBA). These genes show robust monophyly with those of the marine Prasinophyceae algae genera Micromonas, Ostreococcus and Bathycoccus, indicating that the acquisition of this gene through horizontal gene transfer by an ancestor of the green algal lineage occurred prior to the divergence of the core chlorophytes (Chlorophyceae and Trebouxiophyceae) and the prasinophytes. The absence of this gene in some freshwater chlorophytes, such as Chlamydomonas reinhardtii, Volvox carteri, Chlorella vulgaris, Chlorella variabilis and Coccomyxa subellipsoidea, can therefore be explained by the loss of this gene somewhere in the evolutionary process. Our survey on the distribution of this gene in genomic and transcriptome databases suggests that this gene occurs almost exclusively in marine algae, with a few exceptions, and as such, we propose that chloroplastic class IIA FBA is a marine environment-adapted enzyme. This hypothesis was also experimentally tested using Chlamydomonas W80, for which we found that the transcript levels of this gene to be significantly lower under low-salt (that is, simulated terrestrial) conditions. Expression analyses of transcriptome data for two algae, Prymnesium parvum and Emiliania huxleyi, taken from the Sequence Read Archive database also indicated that the expression of this gene under terrestrial conditions (low NaCl and low sulfate) is significantly downregulated. Thus, these experimental and transcriptome data provide support for our hypothesis. PMID:27058504

Studies of styrene, styrene oxide and 4-hydroxystyrene toxicity in CYP2F2 knockout and CYP2F1 humanized mice support lack of human relevance for mouse lung tumors.

PubMed

Cruzan, G; Bus, J; Hotchkiss, J; Sura, R; Moore, C; Yost, G; Banton, M; Sarang, S

2013-06-01

Styrene (S) is lung tumorigenic in mice but not in rats. S and its alkene-oxidized metabolite styrene oxide (SO) were not lung toxic in CYP2F2(-/-) [knockout] mice, indicating S-induced mouse lung tumors are mediated through mouse-specific CYP2F2-generated ring-oxidized metabolite(s) in lung bronchioles. The human relevance of the CYP2F MOA was assessed by insertion of a human CYP2F1, 2A13, 2B6 transgene into CYP2F2(-/-) mice; CYP2F1 expression and activity were confirmed in the transgenic (TG) mice. No evidence of cytotoxicity or increased cell proliferation (BrdU labeling) was seen in TG mice treated with either S or SO (200mg/kg/day ip for 5days). In contrast to S and SO, 4HS (105mg/kg/day ip for 5days) increased BrdU labeling 5-10-fold in WT mice, <3-fold increase in KO mice and 2-4-fold in TG mice. The limited response of 4HS in KO and TG mice may result from intrinsic toxicity or from further metabolism; regardless of the MOA, these findings indicate that the CYP2F-mediated tumorigenic MOA in WT mice is not operative for S, SO, or for 4HS putatively derived from metabolism of S by CYP2F1 in humans, and thus S-induced mouse lung tumors are unlikely to be relevant to human risk. Copyright © 2013. Published by Elsevier Inc.

Loss of GCN5 leads to increased neuronal apoptosis by upregulating E2F1- and Egr-1-dependent BH3-only protein Bim.

PubMed

Wu, Yanna; Ma, Shanshan; Xia, Yong; Lu, Yangpeng; Xiao, Shiyin; Cao, Yali; Zhuang, Sidian; Tan, Xiangpeng; Fu, Qiang; Xie, Longchang; Li, Zhiming; Yuan, Zhongmin

2017-01-26

Cellular acetylation homeostasis is a kinetic balance precisely controlled by histone acetyl-transferase (HAT) and histone deacetylase (HDAC) activities. The loss of the counterbalancing function of basal HAT activity alters the precious HAT:HDAC balance towards enhanced histone deacetylation, resulting in a loss of acetylation homeostasis, which is closely associated with neuronal apoptosis. However, the critical HAT member whose activity loss contributes to neuronal apoptosis remains to be identified. In this study, we found that inactivation of GCN5 by either pharmacological inhibitors, such as CPTH2 and MB-3, or by inactivation with siRNAs leads to a typical apoptosis in cultured cerebellar granule neurons. Mechanistically, the BH3-only protein Bim is transcriptionally upregulated by activated Egr-1 and E2F1 and mediates apoptosis following GCN5 inhibition. Furthermore, in the activity withdrawal- or glutamate-evoked neuronal apoptosis models, GCN5 loses its activity, in contrast to Bim induction. Adenovirus-mediated overexpression of GCN5 suppresses Bim induction and apoptosis. Interestingly, the loss of GCN5 activity and the induction of Egr-1, E2F1 and Bim are involved in the early brain injury (EBI) following subarachnoid haemorrhage (SAH) in rats. HDAC inhibition not only significantly rescues Bim expression and apoptosis induced by either potassium deprivation or GCN5 inactivation but also ameliorates these events and EBI in SAH rats. Taken together, our results highlight a new mechanism by which the loss of GCN5 activity promotes neuronal apoptosis through the transcriptional upregulation of Bim, which is probably a critical event in triggering neuronal death when cellular acetylation homeostasis is impaired.

XoxF Is Required for Expression of Methanol Dehydrogenase in Methylobacterium extorquens AM1 ▿

PubMed Central

Skovran, Elizabeth; Palmer, Alexander D.; Rountree, Austin M.; Good, Nathan M.; Lidstrom, Mary E.

2011-01-01

In Gram-negative methylotrophic bacteria, the first step in methylotrophic growth is the oxidation of methanol to formaldehyde in the periplasm by methanol dehydrogenase. In most organisms studied to date, this enzyme consists of the MxaF and MxaI proteins, which make up the large and small subunits of this heterotetrameric enzyme. The Methylobacterium extorquens AM1 genome contains two homologs of MxaF, XoxF1 and XoxF2, which are ∼50% identical to MxaF and ∼90% identical to each other. It was previously reported that xoxF is not required for methanol growth in M. extorquens AM1, but here we show that when both xoxF homologs are absent, strains are unable to grow in methanol medium and lack methanol dehydrogenase activity. We demonstrate that these defects result from the loss of gene expression from the mxa promoter and suggest that XoxF is part of a complex regulatory cascade involving the 2-component systems MxcQE and MxbDM, which are required for the expression of the methanol dehydrogenase genes. PMID:21873495

Pressure-induced phase transition in La 1 – x Sm x O 0.5 F 0.5 BiS 2

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

Fang, Y.; Yazici, D.; White, B. D.

Electrical resistivity measurements on La 1–xSm xO 0.5F 0.5BiS 2 (x = 0.1, 0.3, 0.6, 0.8) have been performed under applied pressures up to 2.6 GPa from 2 K to room temperature. The superconducting transition temperature T c of each sample significantly increases at a Sm-concentration dependent pressure P t, indicating a pressure-induced phase transition from a low-T c to a high-T c phase. At ambient pressure, T c increases dramatically from 2.8 K at x = 0.1 to 5.4 K at x = 0.8; however, the T c values at P > P t decrease slightly with x andmore » P t shifts to higher pressures with Sm substitution. In the normal state, semiconducting-like behavior is suppressed and metallic conduction is induced with increasing pressure in all of the samples. Furthermore, these results suggest that the pressure dependence of T c for the BiS 2-based superconductors is related to the lattice parameters at ambient pressure and enable us to estimate the evolution of T c for SmO 0.5F 0.5BiS 2 under pressure.« less

Pressure-induced phase transition in La 1 – x Sm x O 0.5 F 0.5 BiS 2

DOE PAGES

Fang, Y.; Yazici, D.; White, B. D.; ...

2015-09-15

Electrical resistivity measurements on La 1–xSm xO 0.5F 0.5BiS 2 (x = 0.1, 0.3, 0.6, 0.8) have been performed under applied pressures up to 2.6 GPa from 2 K to room temperature. The superconducting transition temperature T c of each sample significantly increases at a Sm-concentration dependent pressure P t, indicating a pressure-induced phase transition from a low-T c to a high-T c phase. At ambient pressure, T c increases dramatically from 2.8 K at x = 0.1 to 5.4 K at x = 0.8; however, the T c values at P > P t decrease slightly with x andmore » P t shifts to higher pressures with Sm substitution. In the normal state, semiconducting-like behavior is suppressed and metallic conduction is induced with increasing pressure in all of the samples. Furthermore, these results suggest that the pressure dependence of T c for the BiS 2-based superconductors is related to the lattice parameters at ambient pressure and enable us to estimate the evolution of T c for SmO 0.5F 0.5BiS 2 under pressure.« less

INHIBITION OF ERN1 SIGNALING ENZYME AFFECTS HYPOXIC REGULATION OF THE EXPRESSION OF E2F8, EPAS1, HOXC6, ATF3, TBX3 AND FOXF1 GENES IN U87 GLIOMA CELLS.

PubMed

Minchenko, O H; Tsymbal, D O; Minchenko, D O; Kovalevska, O V; Karbovskyi, L L; Bikfalvi, A

2015-01-01

Hypoxia as well as the endoplasmic reticulum stress are important factors of malignant tumor growth and control of the expression of genes, which regulate numerous metabolic processes and cell proliferation. Furthermore, blockade of ERN1 (endoplasmic reticulum to nucleus 1) suppresses cell proliferation and tumor growth. We studied the effect of hypoxia on the expression of genes encoding the transcription factors such as E2F8 (E2F transcription factor 8), EPAS1 (endothelial PAS domain protein 1), TBX3 (T-box 3), ATF3 (activating transcription factor 3), FOXF1 (forkhead box F), and HOXC6 (homeobox C6) in U87 glioma cells with and without ERN1 signaling enzyme function. We have established that hypoxia enhances the expression of HOXC6, E2F8, ATF3, and EPAS1 genes but does not change TBX3 and FOXF1 gene expression in glioma cells with ERNI function. At the same time, the expression level of all studied genes is strongly decreased, except for TBX3 gene, in glioma cells without ERN1 function. Moreover, the inhibition of ERN1 signaling enzyme function significantly modifies the effect of hypoxia on the expression of these transcription factor genes. removes or introduces this regulation as well as changes a direction or magnitude of hypoxic regulation. Present study demonstrates that fine-tuning of the expression of proliferation related genes depends upon hypoxia and ERN1-mediated endoplasmic reticulum stress signaling and correlates with slower proliferation rate of glioma cells without ERN1 function.

Short communication: expression and alternative splicing of POU1F1 pathway genes in preimplantation bovine embryos.

PubMed

Laporta, J; Driver, A; Khatib, H

2011-08-01

Early embryo loss is a major contributing factor to cow infertility and that 70 to 80% of this loss occurs between d 8 and 16 postfertilization. However, little is known about the molecular mechanisms and the nature of genes involved in normal and abnormal embryonic development. Moreover, information is limited on the contributions of the genomes of dams and of embryos to the development and survival of preimplantation embryos. We hypothesized that proper gene expression level in the developing embryo is essential for embryo survival and pregnancy success. As such, the characterization of expression profiles in early embryos could lead to a better understanding of the mechanisms involved in normal and abnormal embryo development. To test this hypothesis, 2 d-8 embryo populations (degenerate embryos and blastocysts) that differed in morphology and developmental status were investigated. Expression levels of POU1F1 pathway genes were estimated in 4 sets of biological replicate pools of degenerate embryos and blastocysts. The OPN and STAT5A genes were found to be upregulated in degenerate embryos compared with blastocysts, whereas STAT5B showed similar expression levels in both embryo groups. Analysis of splice variants of OPN and STAT5A revealed expression patterns different from the total expression values of these genes. As such, measuring expression of individual transcripts should be considered in gene expression studies. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Arachidonic acid and lipoxin A4 attenuate alloxan-induced cytotoxicity to RIN5F cells in vitro and type 1 diabetes mellitus in vivo.

PubMed

Gundala, Naveen K V; Naidu, Vegi G M; Das, Undurti N

2017-03-01

We studied whether polyunsaturated fatty acids (PUFAs) can protect rat insulinoma (RIN5F) cells against alloxan-induced apoptosis in vitro and type 1 diabetes mellitus (type 1 DM) in vivo and if so, mechanism of this beneficial action. In vitro study was conducted using RIN5F cells while in vivo study was performed in Wistar rats. The effect of PUFAs, cyclo-oxygenase and lipoxygenase inhibitors, various eicosanoids and PUFAs metabolites: lipoxin A4 (LXA4), resolvin D2 and protectin against alloxan-induced cytotoxicity to RIN5F cells and type 1 DM was studied. Expression of PDX1, P65 NF-kB and IKB in RIN5F cells and Nrf2, GLUT2, COX2, iNOS protein levels in the pancreatic tissue and plasma glucose, insulin and tumor necrosis factor-α and antioxidants, lipid peroxides and nitric oxide were measured. Of all, arachidonic acid (AA) was found to be the most effective against alloxan-induced cytotoxicity to RIN5F cells and preventing type 1 DM. Both cyclo-oxygenase and lipoxygenase inhibitors did not block the beneficial actions of AA in vitro and in vivo. Alloxan inhibited LXA4 production by RIN5F cells and in alloxan-induced type 1 DM Wistar rats. AA-treatment restored LXA4 levels to normal both in vitro and in vivo. LXA4 protected RIN5F cells against alloxan-induced cytotoxicity and prevented type 1 DM and restored expression of Nrf2, Glut2, COX2, and iNOS genes and abnormal antioxidants to near normal. AA seems to bring about its beneficial actions against alloxan-induced cytotoxicity and type 1 DM by enhancing the production of LXA4. © 2016 BioFactors, 43(2):251-271, 2017. © 2016 International Union of Biochemistry and Molecular Biology.

Regulation of p53 expression and apoptosis by vault RNA2-1-5p in cervical cancer cells.

PubMed

Kong, Lu; Hao, Qi; Wang, Ying; Zhou, Ping; Zou, Binbin; Zhang, Yu-xiang

2015-09-29

nc886 or VRNA2-1 has recently been identified as a noncoding RNA instead of a vault RNA or a pre-microRNA. Several studies have reported that pre-miR-886 plays a tumor-suppressive role in a wide range of cancer cells through its activity as a cellular protein kinase RNA-activated (PKR) ligand and repressor. However, by sequencing stem-PCR products, we found that a microRNA originating from this precursor, vault RNA2-1-5p (VTRNA2-1-5p), occurs in cervical cancer cells. The expression levels of the predicted targets of VTRNA2-1-5p are negatively correlated with VTRNA2-1-5p levels by quantitative reversion transcription PCR (qRT-PCR). Previous results have shown that VTRNA2-1-5p is overexpressed in human cervical squamous cell carcinomas (CSCCs) compared with adjacent healthy tissues. Inhibition of VTRNA2-1-5p increases Bax protein expression and apoptotic cell death in cervical cancer cells. Our findings suggest that VTRNA2-1-5p has oncogenic activity related to the progression of cervical cancer. Here, we report that VTRNA2-1-5p directly targeted p53 expression and functioned as an oncomir in cervical cancer. VTRNA2-1-5p inhibition decreased cervical cancer cell invasion, proliferation, and tumorigenicity while increasing apoptosis and p53 expression. Interestingly, VTRNA2-1-5p inhibition also increased cisplatin-induced apoptosis of HeLa and SiHa cells. In human clinical cervical cancer specimens, low p53 expression and high VTRNA2-1-5p expression were positively associated.In addition, VTRNA2-1-5p was found to directly target the 5' and 3' untranslated regions (UTRs) of p53. We propose that VTRNA2-1-5p is a direct regulator of p53 and suggest that it plays an essential role in the apoptosis and proliferation of cervical cancer cells.

Correlation of Glut-1 and Glut-3 expression with F-18 FDG uptake in pulmonary inflammatory lesions

PubMed Central

Wang, Zhen Guang; Yu, Ming Ming; Han, Yu; Wu, Feng Yu; Yang, Guang Jie; Li, Da Cheng; Liu, Si Min

2016-01-01

Abstract The aim of the study was to investigate the correlation of glucose transporter-1 (Glut-1) and glucose transporter-3 (Glut-3) expression with F-18 FDG uptake in pulmonary inflammatory lesions. Twenty-two patients with pulmonary inflammatory lesions underwent positron emission tomography/computed tomography (PET/CT) examination preoperatively, and Glut-1 and Glut-3 expression were detected by immunohistochemistry in these lesions. Correlations of Glut-1 and Glut-3 with 18F-FDG uptake were assessed using Spearman's rank correlation test. The maximum standardized uptake value (SUVmax) of pulmonary inflammatory lesions in 22 patients was 0.50 to 7.50, with a mean value of 3.66 ± 1.62. Immunohistochemical staining scores of Glut-1 and Glut-3 were 2.18 ± 0.96 and 2.82 ± 1.37, respectively. The expression of Glut-1 and Glut-3 was positively correlated with F-18 FDG uptake. Glut-3 expression was evidently higher than Glut-1 expression in 22 patients. Glut-1 and Glut-3 expressions are high in pulmonary inflammatory lesions, and Glut-3 plays a more important role in F-18 FDG uptake in pulmonary inflammatory lesions. PMID:27902598

Effect of 3,3',5-triiodothyronine and 3,5-diiodothyronine on progesterone production, cAMP synthesis, and mRNA expression of STAR, CYP11A1, and HSD3B genes in granulosa layer of chicken preovulatory follicles.

PubMed

Sechman, A; Pawlowska, K; Hrabia, A

2011-10-01

In vitro studies were performed to assess whether stimulatory effects of triiodothyronine (T3) on progesterone (P4) production in a granulosa layer (GL) of chicken preovulatory follicles are associated with 3',5'-cyclic adenosine monophosphate (cAMP) synthesis and mRNA expression of STAR protein, CYP11A1, and HSD3B. Effects of 3,5-diiodothyronine (3,5-T2) on steroidogenic function in these follicles were also investigated. The GL of F3 to F1 follicles was incubated in medium supplemented with T3 or 3,5-T2, LH, or forskolin (F), and a combination of each iodothyronine with LH or F. Levels of P4 and cAMP in culture media were determined by RIA. Expression of genes involved in P4 synthesis (ie, STAR protein, CYP11A1, and HSD3B) in the GL of F3 to F1 follicles incubated in medium with T3 or 3,5-T2 and their combination with LH was performed by real-time PCR. Triiodothyronine increased basal and LH- and F-stimulated P4 secretion by preovulatory follicles. The 3,5-T2 elevated P4 synthesis by F3, had no effect on F2 follicles, and diminished P4 production by the GL of F1 follicles. It had no effect on LH-stimulated P4 production; however, it augmented F-stimulated P4 production by F2 and F1 follicles. Although T3 did not affect basal and F-stimulated cAMP synthesis by the GL of preovulatory follicles, it increased LH-stimulated synthesis of this nucleotide. However, 3,5-T2 elevated F-stimulated cAMP synthesis in F3 and F2 follicles; it did not change basal and LH-stimulated cAMP production. Triiodothyronine decreased basal STAR and CYP11A1 mRNAs in F3 follicles, increased them in F1 follicles, and elevated HSD3B mRNA levels in F1 follicles. Triiodothyronine augmented LH-stimulated STAR, CYP11A1, and HSD3B mRNA levels in F2 and CYP11A1 in F1 follicles. However, T3 decreased LH-stimulated STAR and HSD3B mRNA levels in F1 follicles. The 3,5-T2 did not affect basal STAR and CYP11A1 mRNA expression in all investigated follicles; however, it decreased LH-stimulated STAR

Micro-RNA-128 (miRNA-128) down-regulation in glioblastoma targets ARP5 (ANGPTL6), Bmi-1 and E2F-3a, key regulators of brain cell proliferation.

PubMed

Cui, J G; Zhao, Y; Sethi, P; Li, Y Y; Mahta, A; Culicchia, F; Lukiw, W J

2010-07-01

High density micro-RNA (miRNA) arrays, fluorescent-reporter miRNA assay and Northern miRNA dot-blot analysis show that a brain-enriched miRNA-128 is significantly down-regulated in glioblastoma multiforme (GBM) and in GBM cell lines when compared to age-matched controls. The down-regulation of miRNA-128 was found to inversely correlate with WHO tumor grade. Three bioinformatics-verified miRNA-128 targets, angiopoietin-related growth factor protein 5 (ARP5; ANGPTL6), a transcription suppressor that promotes stem cell renewal and inhibits the expression of known tumor suppressor genes involved in senescence and differentiation, Bmi-1, and a transcription factor critical for the control of cell-cycle progression, E2F-3a, were found to be up-regulated. Addition of exogenous miRNA-128 to CRL-1690 and CRL-2610 GBM cell lines (a) restored 'homeostatic' ARP5 (ANGPTL6), Bmi-1 and E2F-3a expression, and (b) significantly decreased the proliferation of CRL-1690 and CRL-2610 cell lines. Our data suggests that down-regulation of miRNA-128 may contribute to glioma and GBM, in part, by coordinately up-regulating ARP5 (ANGPTL6), Bmi-1 and E2F-3a, resulting in the proliferation of undifferentiated GBM cells.

Chronic 5-HT2 receptor blockade unmasks the role of 5-HT1F receptors in the inhibition of rat cardioaccelerator sympathetic outflow.

PubMed

García-Pedraza, José Ángel; Hernández-Abreu, Oswaldo; García, Mónica; Morán, Asunción; Villalón, Carlos M

2018-04-01

Serotonin (5-hydroxytryptamine; 5-HT) inhibits the rat cardioaccelerator sympathetic outflow by 5-HT 1B/1D/5 receptors. Because chronic blockade of sympatho-excitatory 5-HT 2 receptors is beneficial in several cardiovascular pathologies, this study investigated whether sarpogrelate (a 5-HT 2 receptor antagonist) alters the pharmacological profile of the above sympatho-inhibition. Rats were pretreated for 2 weeks with sarpogrelate in drinking water (30 mg/kg per day; sarpogrelate-treated group) or equivalent volumes of drinking water (control group). Animals were pithed and prepared for spinal stimulation (C 7 -T 1 ) of the cardioaccelerator sympathetic outflow or for intravenous (i.v.) bolus injections of noradrenaline. Both procedures produced tachycardic responses remaining unaltered after saline. Continuous i.v. infusions of 5-HT induced a cardiac sympatho-inhibition that was mimicked by the 5-HT receptor agonists 5-carboxamidotryptamine (5-CT; 5-HT 1/5A ), CP 93,129 (5-HT 1B ), or PNU 142633 (5-HT 1D ), but not by indorenate (5-HT 1A ) in both groups; whereas LY344864 (5-HT 1F ) mimicked 5-HT only in sarpogrelate-treated rats. In sarpogrelate-treated animals, i.v. GR 127935 (310 μg/kg; 5-HT 1B/1D/1F receptor antagonist) attenuated 5-CT-induced sympatho-inhibition and abolished LY344864-induced sympatho-inhibition; while GR 127935 plus SB 699551 (1 mg/kg; 5-HT 5A receptor antagonist) abolished 5-CT-induced inhibition. These results confirm the cardiac sympatho-inhibitory role of 5-HT 1B , 5-HT 1D , and 5-HT 5A receptors in both groups; nevertheless, sarpogrelate treatment specifically unmasked a cardiac sympatho-inhibition mediated by 5-HT 1F receptors.

Evidence for a non-canonical role of HDAC5 in regulation of the cardiac Ncx1 and Bnp genes.

PubMed

Harris, Lillianne G; Wang, Sabina H; Mani, Santhosh K; Kasiganesan, Harinath; Chou, C James; Menick, Donald R

2016-05-05

Class IIa histone deacetylases (HDACs) are very important for tissue specific gene regulation in development and pathology. Because class IIa HDAC catalytic activity is low, their exact molecular roles have not been fully elucidated. Studies have suggested that class IIa HDACs may serve as a scaffold to recruit the catalytically active class I HDAC complexes to their substrate. Here we directly address whether the class IIa HDAC, HDAC5 may function as a scaffold to recruit co-repressor complexes to promoters. We examined two well-characterized cardiac promoters, the sodium calcium exchanger (Ncx1) and the brain natriuretic peptide (Bnp) whose hypertrophic upregulation is mediated by both class I and IIa HDACs. Selective inhibition of class IIa HDACs did not prevent adrenergic stimulated Ncx1 upregulation, however HDAC5 knockout prevented pressure overload induced Ncx1 upregulation. Using the HDAC5((-/-)) mouse we show that HDAC5 is required for the interaction of the HDAC1/2/Sin3a co-repressor complexes with the Nkx2.5 and YY1 transcription factors and critical for recruitment of the HDAC1/Sin3a co-repressor complex to either the Ncx1 or Bnp promoter. Our novel findings support a non-canonical role of class IIa HDACs in the scaffolding of transcriptional regulatory complexes, which may be relevant for therapeutic intervention for pathologies. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Parameters to Maximize 2f2-f1 Distortion Product Otoacoustic Emission Levels

ERIC Educational Resources Information Center

Horn, Jennifer H.; Pratt, Shiela R.; Durrant, John D.

2008-01-01

Purpose: Past research has established parameters for the 2f1-f2 distortion product otoacoustic emissions (DPOAEs) that enhance response levels (e.g., L1 - L2 = 10 dB; f2/f1 = 1.22; L1, L2 = 65, 55 dB SPL). These same parameters do not optimize 2f2-f1 DPOAEs. Therefore, this study was conducted to evaluate more completely those parameters that…

Dissociative electron attachment to C{sub 2}F{sub 5} radicals

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

Haughey, Sean A.; Field, Thomas A.; Langer, Judith

Dissociative electron attachment to the reactive C{sub 2}F{sub 5} molecular radical has been investigated with two complimentary experimental methods; a single collision beam experiment and a new flowing afterglow Langmuir probe technique. The beam results show that F{sup -} is formed close to zero electron energy in dissociative electron attachment to C{sub 2}F{sub 5}. The afterglow measurements also show that F{sup -} is formed in collisions between electrons and C{sub 2}F{sub 5} molecules with rate constants of 3.7 Multiplication-Sign 10{sup -9} cm{sup 3} s{sup -1} to 4.7 Multiplication-Sign 10{sup -9} cm{sup 3} s{sup -1} at temperatures of 300-600 K. Themore » rate constant increases slowly with increasing temperature, but the rise observed is smaller than the experimental uncertainty of 35%.« less

Influenza A virus PB1-F2 protein expression is regulated in a strain-specific manner by sequences located downstream of the PB1-F2 initiation codon

USDA-ARS?s Scientific Manuscript database

Translation of influenza A virus PB1-F2 occurs in a second open reading frame (ORF) of the PB1 gene segment. PB1-F2 has been implicated in regulation of polymerase activity, immunopathology, susceptibility to secondary bacterial infection, and induction of apoptosis. Experimental evidence of PB1-F2 ...

Synthesis of fused 1,2,4-dithiazines and 1,2,3,5-trithiazepines.

PubMed

Koyioni, Maria; Manoli, Maria; Koutentis, Panayiotis A

2014-10-17

Reacting (Z)-N-(4-chloro-5H-1,2,3-dithiazol-5-ylidene)-1H-pyrazol-5-amines 5 with Et2NH and then with concd H2SO4 gives 5H-pyrazolo[3,4-e][1,2,4]dithiazine-3-carbonitriles 7 in good yields (74-85%) and 6H-pyrazolo[3,4-f][1,2,3,5]trithiazepine-4-carbonitriles 9 as minor products (0-6%). Furthermore, the 1,3-dimethylpyrazole analogue 5a was transformed into the dithiazine 7a in two discrete steps, allowing the isolation of a disulfide intermediate (Z)-2-[(diethylamino)disulfan-yl]-2-[(1H-pyrazol-5-yl)imino]acetonitrile (8a). The one-pot, two-step reaction also worked with electron-rich hydroxy- and methoxy-substituted anilines. Thermolysis of the pyrazolo[3,4-e][1,2,4]dithiazines 7 gave the ring-contracted 1H-pyrazolo[3,4-d]thiazole-5-carbonitriles 6 (94-100%). With active sulfur, 1,3-dimethyl-5H-pyrazolo[3,4-e][1,2,4]dithiazine-3-carbonitrile (7a) gave 1,3-dimethyl-6H-pyrazolo[3,4-f][1,2,3,5]trithiazepine-4-carbonitrile (9a), but on prolonged reaction times, it gave 5,7-dimethyl-5H-[1,2,3]dithiazolo[4,5-b]pyrazolo[3,4-e][1,4]thiazine (13). Finally, in the absence of acid, heating a solution of (Z)-2-[(diethylamino)disulfanyl]-2-[(1,3-dimethyl-1H-pyrazol-5-yl)imino]acetonitrile (8a) gave 4,6,10,12-tetramethyl-6H-pyrazolo[3,4-f]pyrazolo[3',4':4,5]pyrimido[6,1-d][1,2,3,5]trithiazepine-8,12b(10H)-dicarbonitrile (19) (67%).

E2F3a gene expression has prognostic significance in childhood acute lymphoblastic leukemia.

PubMed

Wang, Kai-Ling; Mei, Yan-Yan; Cui, Lei; Zhao, Xiao-Xi; Li, Wei-Jing; Gao, Chao; Liu, Shu-Guang; Jiao, Ying; Liu, Fei-Fei; Wu, Min-Yuan; Ding, Wei; Li, Zhi-Gang

2014-10-01

To study E2F3a expression and its clinical significance in children with acute lymphoblastic leukemia (ALL). We quantified E2F3a expression at diagnosis in 148 children with ALL by real-time PCR. In the test cohort (n = 48), receiver operating characteristic (ROC) curve was used to find the best cut-off point to divide the patients into E2F3a low- and high-expression groups. The prognostic significance of E2F3a expression was investigated in the test cohort and confirmed in the validation cohort (n = 100). The correlations of E2F3a expression with the clinical features and treatment outcome of these patients were analyzed. ROC curve analysis indicated that the best cut-off point of E2F3a expression was 0.3780. In the test cohort, leukemia-free survival (LFS) and event-free survival (EFS) of the low-expression group were lower than those of the high-expression group (log rank: P = 0.026 for both). This finding was verified in the validation cohort. LFS, EFS, and overall survival were also lower in the low-expression group than in the high-expression group (log rank, P = 0.015, 0.008, and 0.002 respectively). E2F3a low expression was correlated with the existence of BCR-ABL fusion. An algorithm composed of E2F3a expression and minimal residual disease (MRD) could predict relapse or induction failure more precisely than current risk stratification. These results were still significant in the ALL patients without BCR-ABL fusion. Low expression of E2F3a was associated with inferior prognosis in childhood ALL. An algorithm composed of E2F3a expression and MRD could predict relapse or induction failure more precisely than that of the current risk stratification. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Study of the 4p2, 5p2 and 5s5f excited configurations of the Zn and Cd isoelectronic sequences, using relativistic and non-relativistic semiempirical approaches

NASA Astrophysics Data System (ADS)

Di Rocco, Héctor O.; Raineri, Mónica; Reyna-Almandos, Jorge G.

2016-11-01

The consistency of the energy levels published for configurations 4p2, 5p2 and 5s5f belonging to Zn and Cd isoelectronic sequences is studied. Different semiempirical approaches considering the linearity of the Slater integrals for large Zc, the smoothness of the sF screening parameters, the energy values in terms of Z (or Zc), and the differences of the Ecalc - Eexp values are used, where Ecalc values are energies calculated with a Hartree-Hock method with relativistic corrections and superposition of configurations (HFR-SOC), and Eexp are the experimental values. For the np2 configurations both LS and relativistic jj expressions are considered. Configuration 5s5f is also analyzed taking into account the Landé's interval rule.

Efficiency, safety, and patient preference of switching from dorzolamide 1%/timolol 0.5% to brinzolamide 1%/timolol 0.5% while maintaining the prostaglandin F2α analog.

PubMed

Shimizu, Yoshie; Nakakura, Shunsuke; Nishiyama, Makiko; Tabuchi, Hitoshi; Kiuchi, Yoshiaki

2015-01-01

We investigated the efficiency, safety and patient preference of switching from dorzolamide 1%/timolol 0.5% to brinzolamide 1%/timolol 0.5% while maintaining the prostaglandin F2α analog. We initially enrolled 44 eyes from 44 primary open angle glaucoma patients, and a total of 42 patients completed the study. All patients were under treatment with various prostaglandin F2α analogs and dorzolamide 1%/timolol 0.5%. While maintaining the prostaglandin F2α analog, dorzolamide 1%/timolol 0.5% was switched to brinzolamide 1%/timolol 0.5%. Conjunctival hyperemia, superficial punctate keratopathy, and intraocular pressure (IOP) were evaluated at baseline and at 4, 12, and 24 weeks. Adverse events and patient preferences, measured using a questionnaire at study initiation and at 24 weeks, were also noted. The IOP was 17.7±1.7, 16.8±2.6, 16.7±2.2, and 16.7±2.4 mmHg at baseline and at 4, 12, and 24 weeks, respectively, with no significant differences in IOP values at any time point (P=0.117, one-way analysis of variance). In addition, no significant differences were found in the incidence of conjunctival hyperemia or SPK score at any time point (all P>0.5, by Kruskal-Wallis test). Based on the evaluation of side effects using the questionnaire, stinging/burning was less common (P=0.042), while blurred vision was more common (P=0.003), after switching to brinzolamide 1%/timolol 0.5%. Regarding patient preferences, 13 patients (31%) preferred dorzolamide 1%/timolol 0.5%, 12 patients (29%) preferred brinzolamide 1%/timolol 0.5%, and 17 patients (40%) preferred neither. When switching from dorzolamide 1%/timolol 0.5% to brinzolamide 1%/timolol 0.5%, the IOP values and incidence of superficial punctate keratopathy and conjunctival hyperemia were sustained throughout the 24-week observation period, and the patient preferences were similar for the two regimens. However, differences were observed in the ocular sensations of stinging/burning with dorzolamide 1%/timolol 0.5

Efficiency, safety, and patient preference of switching from dorzolamide 1%/timolol 0.5% to brinzolamide 1%/timolol 0.5% while maintaining the prostaglandin F2α analog

PubMed Central

Shimizu, Yoshie; Nakakura, Shunsuke; Nishiyama, Makiko; Tabuchi, Hitoshi; Kiuchi, Yoshiaki

2015-01-01

Background We investigated the efficiency, safety and patient preference of switching from dorzolamide 1%/timolol 0.5% to brinzolamide 1%/timolol 0.5% while maintaining the prostaglandin F2α analog. Methods We initially enrolled 44 eyes from 44 primary open angle glaucoma patients, and a total of 42 patients completed the study. All patients were under treatment with various prostaglandin F2α analogs and dorzolamide 1%/timolol 0.5%. While maintaining the prostaglandin F2α analog, dorzolamide 1%/timolol 0.5% was switched to brinzolamide 1%/timolol 0.5%. Conjunctival hyperemia, superficial punctate keratopathy, and intraocular pressure (IOP) were evaluated at baseline and at 4, 12, and 24 weeks. Adverse events and patient preferences, measured using a questionnaire at study initiation and at 24 weeks, were also noted. Results The IOP was 17.7±1.7, 16.8±2.6, 16.7±2.2, and 16.7±2.4 mmHg at baseline and at 4, 12, and 24 weeks, respectively, with no significant differences in IOP values at any time point (P=0.117, one-way analysis of variance). In addition, no significant differences were found in the incidence of conjunctival hyperemia or SPK score at any time point (all P>0.5, by Kruskal–Wallis test). Based on the evaluation of side effects using the questionnaire, stinging/burning was less common (P=0.042), while blurred vision was more common (P=0.003), after switching to brinzolamide 1%/timolol 0.5%. Regarding patient preferences, 13 patients (31%) preferred dorzolamide 1%/timolol 0.5%, 12 patients (29%) preferred brinzolamide 1%/timolol 0.5%, and 17 patients (40%) preferred neither. Conclusion When switching from dorzolamide 1%/timolol 0.5% to brinzolamide 1%/timolol 0.5%, the IOP values and incidence of superficial punctate keratopathy and conjunctival hyperemia were sustained throughout the 24-week observation period, and the patient preferences were similar for the two regimens. However, differences were observed in the ocular sensations of stinging

Key Role of Group V Secreted Phospholipase A2 in Th2 Cytokine and Dendritic Cell-Driven Airway Hyperresponsiveness and Remodeling

PubMed Central

Henderson Jr, William R.; Ye, Xin; Lai, Ying; Ni, Zhanglin; Bollinger, James G.; Tien, Ying-Tzang; Chi, Emil Y.; Gelb, Michael H.

2013-01-01

Background Previous work has shown that disruption of the gene for group X secreted phospholipase A2 (sPLA2-X) markedly diminishes airway hyperresponsiveness and remodeling in a mouse asthma model. With the large number of additional sPLA2s in the mammalian genome, the involvement of other sPLA2s in the asthma model is possible – in particular, the group V sPLA2 (sPLA2-V) that like sPLA2-X is highly active at hydrolyzing membranes of mammalian cells. Methodology and Principal Findings The allergen-driven asthma phenotype was significantly reduced in sPLA2-V-deficient mice but to a lesser extent than observed previously in sPLA2-X-deficient mice. The most striking difference observed between the sPLA2-V and sPLA2-X knockouts was the significant impairment of the primary immune response to the allergen ovalbumin (OVA) in the sPLA2-V−/− mice. The impairment in eicosanoid generation and dendritic cell activation in sPLA2-V−/− mice diminishes Th2 cytokine responses in the airways. Conclusions This paper illustrates the diverse roles of sPLA2s in the immunopathogenesis of the asthma phenotype and directs attention to developing specific inhibitors of sPLA2-V as a potential new therapy to treat asthma and other allergic disorders. PMID:23451035

E2F1 somatic mutation within miRNA target site impairs gene regulation in colorectal cancer.

PubMed

Lopes-Ramos, Camila M; Barros, Bruna P; Koyama, Fernanda C; Carpinetti, Paola A; Pezuk, Julia; Doimo, Nayara T S; Habr-Gama, Angelita; Perez, Rodrigo O; Parmigiani, Raphael B

2017-01-01

Genetic studies have largely concentrated on the impact of somatic mutations found in coding regions, and have neglected mutations outside of these. However, 3' untranslated regions (3' UTR) mutations can also disrupt or create miRNA target sites, and trigger oncogene activation or tumor suppressor inactivation. We used next-generation sequencing to widely screen for genetic alterations within predicted miRNA target sites of oncogenes associated with colorectal cancer, and evaluated the functional impact of a new somatic mutation. Target sequencing of 47 genes was performed for 29 primary colorectal tumor samples. For 71 independent samples, Sanger methodology was used to screen for E2F1 mutations in miRNA predicted target sites, and the functional impact of these mutations was evaluated by luciferase reporter assays. We identified germline and somatic alterations in E2F1. Of the 100 samples evaluated, 3 had germline alterations at the MIR205-5p target site, while one had a somatic mutation at MIR136-5p target site. E2F1 gene expression was similar between normal and tumor tissues bearing the germline alteration; however, expression was increased 4-fold in tumor tissue that harbored a somatic mutation compared to that in normal tissue. Luciferase reporter assays revealed both germline and somatic alterations increased E2F1 activity relative to wild-type E2F1. We demonstrated that somatic mutation within E2F1:MIR136-5p target site impairs miRNA-mediated regulation and leads to increased gene activity. We conclude that somatic mutations that disrupt miRNA target sites have the potential to impact gene regulation, highlighting an important mechanism of oncogene activation.

Rsp5-Bul1/2 complex is necessary for the HSE-mediated gene expression in budding yeast.

PubMed

Kaida, Daisuke; Toh-e, Akio; Kikuchi, Yoshiko

2003-07-11

Rsp5 is an essential ubiquitin ligase in Saccharomyces cerevisiae and is concerned with many functions such as endocytosis and transcription through ubiquitination of various substrates. Bul1 or its homologue Bul2 binds to Rsp5 through the PY-motif and the bul1 bul2 double mutant is sensitive to various stresses. We demonstrate here that heat shock element (HSE)-mediated gene expression was defective in both rsp5-101 and bul1 bul2 mutants under high temperature condition. The bul1 gene containing mutations in the PY motif region did not recover this defective gene expression of the bul1 bul2 mutant. The protein level and phosphorylation state of the HSE-binding transcription factor, Hsf1, was not affected by these mutations. Thus, the Rsp5-Bul1/2 complex has a new function for the HSE-mediated gene expression and may regulate it through other factors than Hsf1.

Properties and Crystallization Phenomena in Li2Si2O5-Ca5(PO4)3F and Li2Si2O5-Sr5(PO4)3F Glass-Ceramics Via Twofold Internal Crystallization.

PubMed

Rampf, Markus; Dittmer, Marc; Ritzberger, Christian; Schweiger, Marcel; Höland, Wolfram

2015-01-01

The combination of specific mechanical, esthetic, and chemical properties is decisive for the application of materials in prosthodontics. Controlled twofold crystallization provides a powerful tool to produce special property combinations for glass-ceramic materials. The present study outlines the potential of precipitating Ca5(PO4)3F as well as Sr5(PO4)3F as minor crystal phases in Li2Si2O5 glass-ceramics. Base glasses with different contents of CaO/SrO, P2O5, and F(-) were prepared within the glasses of the SiO2-Li2O-K2O-CaO/SrO-Al2O3-P2O5-F system. Preliminary studies of nucleation by means of XRD and scanning electron microscopy (SEM) of the nucleated base glasses revealed X-ray amorphous phase separation phenomena. Qualitative and quantitative crystal phase analyses after crystallization were conducted using XRD in combination with Rietveld refinement. As a main result, a direct proportional relationship between the content of apatite-forming components in the base glasses and the content of apatite in the glass-ceramics was established. The microstructures of the glass-ceramics were investigated using SEM. Microstructural and mechanical properties were found to be dominated by Li2Si2O5 crystals and quite independent of the content of the apatite present in the glass-ceramics. Biaxial strengths of up to 540 MPa were detected. Ca5(PO4)3F and Sr5(PO4)3F influence the translucency of the glass-ceramics and, hence, help to precisely tailor the properties of Li2Si2O5 glass-ceramics. The authors conclude that the twofold crystallization of Li2Si2O5-Ca5(PO4)3F or Li2Si2O5-Sr5(PO4)3F glass-ceramics involves independent solid-state reactions, which can be controlled via the chemical composition of the base glasses. The influence of the minor apatite phase on the optical properties helps to achieve new combinations of features of the glass-ceramics and, hence, displays new potential for dental applications.

EBP1 is a novel E2F target gene regulated by transforming growth factor-β.

PubMed

Judah, David; Chang, Wing Y; Dagnino, Lina

2010-11-10

Regulation of gene expression requires transcription factor binding to specific DNA elements, and a large body of work has focused on the identification of such sequences. However, it is becoming increasingly clear that eukaryotic transcription factors can exhibit widespread, nonfunctional binding to genomic DNA sites. Conversely, some of these proteins, such as E2F, can also modulate gene expression by binding to non-consensus elements. E2F comprises a family of transcription factors that play key roles in a wide variety of cellular functions, including survival, differentiation, activation during tissue regeneration, metabolism, and proliferation. E2F factors bind to the Erb3-binding protein 1 (EBP1) promoter in live cells. We now show that E2F binding to the EBP1 promoter occurs through two tandem DNA elements that do not conform to typical consensus E2F motifs. Exogenously expressed E2F1 activates EBP1 reporters lacking one, but not both sites, suggesting a degree of redundancy under certain conditions. E2F1 increases the levels of endogenous EBP1 mRNA in breast carcinoma and other transformed cell lines. In contrast, in non-transformed primary epidermal keratinocytes, E2F, together with the retinoblastoma family of proteins, appears to be involved in decreasing EBP1 mRNA abundance in response to growth inhibition by transforming growth factor-β1. Thus, E2F is likely a central coordinator of multiple responses that culminate in regulation of EBP1 gene expression, and which may vary depending on cell type and context.

ATP binding cassette G1-dependent cholesterol efflux during inflammation.

PubMed

de Beer, Maria C; Ji, Ailing; Jahangiri, Anisa; Vaughan, Ashley M; de Beer, Frederick C; van der Westhuyzen, Deneys R; Webb, Nancy R

2011-02-01

ATP binding cassette transporter G1 (ABCG1) mediates the transport of cellular cholesterol to HDL, and it plays a key role in maintaining macrophage cholesterol homeostasis. During inflammation, HDL undergoes substantial remodeling, acquiring lipid changes and serum amyloid A (SAA) as a major apolipoprotein. In the current study, we investigated whether remodeling of HDL that occurs during acute inflammation impacts ABCG1-dependent efflux. Our data indicate that lipid free SAA acts similarly to apolipoprotein A-I (apoA-I) in mediating sequential efflux from ABCA1 and ABCG1. Compared with normal mouse HDL, acute phase (AP) mouse HDL containing SAA exhibited a modest but significant 17% increase in ABCG1-dependent efflux. Interestingly, AP HDL isolated from mice lacking SAA (SAAKO mice) was even more effective in promoting ABCG1 efflux. Hydrolysis with Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) significantly reduced the ability of AP HDL from SAAKO mice to serve as a substrate for ABCG1-mediated cholesterol transfer, indicating that phospholipid (PL) enrichment, and not the presence of SAA, is responsible for alterations in efflux. AP human HDL, which is not PL-enriched, was somewhat less effective in mediating ABCG1-dependent efflux compared with normal human HDL. Our data indicate that inflammatory remodeling of HDL impacts ABCG1-dependent efflux independent of SAA.

The E2F3—Oncomir 1 axis is activated in Wilms Tumor

PubMed Central

Kort, Eric J.; Farber, Leslie; Tretiakova, Maria; Petillo, David; Furge, Kyle A.; Yang, Ximing J.; Cornelius, Albert; Teh, Bin T.

2008-01-01

Oncomir-1 is an oncogenic cluster of microRNAs located on chromosome 13. Previous in vitro studies demonstrated that it is transcriptionally regulated by the transcription factor E2F3. In this report we combine expression profiling of both messenger RNA (mRNA) and micro RNAs (miRNA) in Wilms tumor (WT) samples to provide the first evidence that the E2F3—Oncomir 1 axis, previously identified in cell culture, is deregulated in primary human tumors. Analysis of RNA expression signatures demonstrated that an E2F3 gene signature was activated in all Wilms tumor samples analyzed, in contrast to other kidney tumors. This finding was validated by immunohistochemistry (IHC) on the protein level. Expression of E2F3 was lowest in early stage tumors, and highest in metastatic tissue. Expression profiling of miRNAs in WT showed that expression of each measured member of the Oncomir-1 family was highest in WT relative to other kidney tumor subtypes. Quantitative polymerase chain reaction (PCR) confirmed that these microRNAs were overexpressed in Wilms tumor relative to normal kidney tissue. These results suggest that the E2F3—Oncomir-1 axis is activated in Wilms tumor. Our study also demonstrates the utility of integrated genomics combining gene signature analysis with miRNA expression profiling to identify protein-miRNA interactions that are perturbed in disease states. PMID:18519660

Die Interhalogenkationen [Br2F5]+ und [Br3F8].

PubMed

Ivlev, Sergei; Karttunen, Antti; Buchner, Magnus; Conrad, Matthias; Kraus, Florian

2018-05-02

Wir berichten über die Synthese und Charakterisierung der bislang einzigen Polyhalogenkationen, in denen verbrückende Fluoratome vorliegen. Das [Br2F5]+-Kation enthält eine symmetrische [F2Br-µ-F-BrF2]-Brücke, das [Br3F8]+-Kation enthält unsymmetrische µ-F-Brücken. Die Fluoronium-Ionen wurden in Form ihrer [SbF6]--Salze erhalten und Raman-, und 19F-NMR-spektroskopisch, sowie durch Röntgenbeugung am Einkristall untersucht. Quantenchemische Rechnungen, sowohl für die isolierten Kationen in der Gasphase, als auch für die Festkörper selbst, wurden durchgeführt. Populationsanalysen zeigen, dass die µ-F-Atome die am stärksten negativ partialgeladenen Atome der Kationen sind. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Limiting prothrombin activation to meizothrombin is compatible with survival but significantly alters hemostasis in mice

PubMed Central

Shaw, Maureen A.; Kombrinck, Keith W.; McElhinney, Kathryn E.; Sweet, David R.; Flick, Matthew J.; Palumbo, Joseph S.; Cheng, Mei; Esmon, Naomi L.; Esmon, Charles T.; Brill, Alexander; Wagner, Denisa D.; Degen, Jay L.

2016-01-01

Thrombin-mediated proteolysis is central to hemostatic function but also plays a prominent role in multiple disease processes. The proteolytic conversion of fII to α-thrombin (fIIa) by the prothrombinase complex occurs through 2 parallel pathways: (1) the inactive intermediate, prethrombin; or (2) the proteolytically active intermediate, meizothrombin (fIIaMZ). FIIaMZ has distinct catalytic properties relative to fIIa, including diminished fibrinogen cleavage and increased protein C activation. Thus, fII activation may differentially influence hemostasis and disease depending on the pathway of activation. To determine the in vivo physiologic and pathologic consequences of restricting thrombin generation to fIIaMZ, mutations were introduced into the endogenous fII gene, resulting in expression of prothrombin carrying 3 amino acid substitutions (R157A, R268A, and K281A) to limit activation events to yield only fIIaMZ. Homozygous fIIMZ mice are viable, express fII levels comparable with fIIWT mice, and have reproductive success. Although in vitro studies revealed delayed generation of fIIaMZ enzyme activity, platelet aggregation by fIIMZ is similar to fIIWT. Consistent with prior analyses of human fIIaMZ, significant prolongation of clotting times was observed for fIIMZ plasma. Adult fIIMZ animals displayed significantly compromised hemostasis in tail bleeding assays, but did not demonstrate overt bleeding. More notably, fIIMZ mice had 2 significant phenotypic advantages over fIIWT animals: protection from occlusive thrombosis after arterial injury and markedly diminished metastatic potential in a setting of experimental tumor metastasis to the lung. Thus, these novel animals will provide a valuable tool to assess the role of both fIIa and fIIaMZ in vivo. PMID:27252233

Tory II-A: a nuclear ramjet test reactor

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

Hadley, J.W.

Declassified 28 Nov 1973. The first test reactor in the Pluto program, leading to development of a nuclear ramjet engine, is called Tory II-A. While it is not an actual prototype engine, this reactor embodies a core design which is considered feasible for an engine, and operation of the reactor will provide a test of that core type as well as more generalized values in reactor design and testing. The design of Tory II-A and construction of the reactor and of its test facility are described. Operation of the Tory II-A core at a total power of 160 megawatts, withmore » 800 pounds of air per second passing through the core and emerging at a temperature of 2000 deg F, is the central objective of the test program. All other reactor and facility components exist to support operation of the core, and preliminary steps in the test program itself will be directed primarily toward ensuring attalnment of full-power operation and collection of meaningful data on core behavior during that operation. The core, 3 feet in diameter and 41/2 feet long, will be composed of bundled ceramic tubes whose central holes will provide continuous air passages from end to end of the reactor. These tubes are to be composed of a homogeneous mixture of UO/sub 2/ fuel and BeO moderator, compacted and sintered to achieve high strength and density. (30 references) (auth)« less

Differential Contribution of PB1-F2 to the Virulence of Highly Pathogenic H5N1 Influenza A Virus in Mammalian and Avian Species

PubMed Central

Schmolke, Mirco; Manicassamy, Balaji; Pena, Lindomar; Sutton, Troy; Hai, Rong; Varga, Zsuzsanna T.; Hale, Benjamin G.; Steel, John; Pérez, Daniel R.; García-Sastre, Adolfo

2011-01-01

Highly pathogenic avian influenza A viruses (HPAIV) of the H5N1 subtype occasionally transmit from birds to humans and can cause severe systemic infections in both hosts. PB1-F2 is an alternative translation product of the viral PB1 segment that was initially characterized as a pro-apoptotic mitochondrial viral pathogenicity factor. A full-length PB1-F2 has been present in all human influenza pandemic virus isolates of the 20th century, but appears to be lost evolutionarily over time as the new virus establishes itself and circulates in the human host. In contrast, the open reading frame (ORF) for PB1-F2 is exceptionally well-conserved in avian influenza virus isolates. Here we perform a comparative study to show for the first time that PB1-F2 is a pathogenicity determinant for HPAIV (A/Viet Nam/1203/2004, VN1203 (H5N1)) in both mammals and birds. In a mammalian host, the rare N66S polymorphism in PB1-F2 that was previously described to be associated with high lethality of the 1918 influenza A virus showed increased replication and virulence of a recombinant VN1203 H5N1 virus, while deletion of the entire PB1-F2 ORF had negligible effects. Interestingly, the N66S substituted virus efficiently invades the CNS and replicates in the brain of Mx+/+ mice. In ducks deletion of PB1-F2 clearly resulted in delayed onset of clinical symptoms and systemic spreading of virus, while variations at position 66 played only a minor role in pathogenesis. These data implicate PB1-F2 as an important pathogenicity factor in ducks independent of sequence variations at position 66. Our data could explain why PB1-F2 is conserved in avian influenza virus isolates and only impacts pathogenicity in mammals when containing certain amino acid motifs such as the rare N66S polymorphism. PMID:21852950

Differential contribution of PB1-F2 to the virulence of highly pathogenic H5N1 influenza A virus in mammalian and avian species.

PubMed

Schmolke, Mirco; Manicassamy, Balaji; Pena, Lindomar; Sutton, Troy; Hai, Rong; Varga, Zsuzsanna T; Hale, Benjamin G; Steel, John; Pérez, Daniel R; García-Sastre, Adolfo

2011-08-01

Highly pathogenic avian influenza A viruses (HPAIV) of the H5N1 subtype occasionally transmit from birds to humans and can cause severe systemic infections in both hosts. PB1-F2 is an alternative translation product of the viral PB1 segment that was initially characterized as a pro-apoptotic mitochondrial viral pathogenicity factor. A full-length PB1-F2 has been present in all human influenza pandemic virus isolates of the 20(th) century, but appears to be lost evolutionarily over time as the new virus establishes itself and circulates in the human host. In contrast, the open reading frame (ORF) for PB1-F2 is exceptionally well-conserved in avian influenza virus isolates. Here we perform a comparative study to show for the first time that PB1-F2 is a pathogenicity determinant for HPAIV (A/Viet Nam/1203/2004, VN1203 (H5N1)) in both mammals and birds. In a mammalian host, the rare N66S polymorphism in PB1-F2 that was previously described to be associated with high lethality of the 1918 influenza A virus showed increased replication and virulence of a recombinant VN1203 H5N1 virus, while deletion of the entire PB1-F2 ORF had negligible effects. Interestingly, the N66S substituted virus efficiently invades the CNS and replicates in the brain of Mx+/+ mice. In ducks deletion of PB1-F2 clearly resulted in delayed onset of clinical symptoms and systemic spreading of virus, while variations at position 66 played only a minor role in pathogenesis. These data implicate PB1-F2 as an important pathogenicity factor in ducks independent of sequence variations at position 66. Our data could explain why PB1-F2 is conserved in avian influenza virus isolates and only impacts pathogenicity in mammals when containing certain amino acid motifs such as the rare N66S polymorphism.

The over expression of long non-coding RNA ANRIL promotes epithelial-mesenchymal transition by activating the ATM-E2F1 signaling pathway in pancreatic cancer: An in vivo and in vitro study.

PubMed

Chen, Shi; Zhang, Jia-Qiang; Chen, Jiang-Zhi; Chen, Hui-Xing; Qiu, Fu-Nan; Yan, Mao-Lin; Chen, Yan-Ling; Peng, Cheng-Hong; Tian, Yi-Feng; Wang, Yao-Dong

2017-09-01

This study aims to investigate the roles of lncRNA ANRIL in epithelial-mesenchymal transition (EMT) by regulating the ATM-E2F1 signaling pathway in pancreatic cancer (PC). PC rat models were established and ANRIL overexpression and interference plasmids were transfected. The expression of ANRIL, EMT markers (E-cadherin, N-cadherin and Vimentin) and ATM-E2F1 signaling pathway-related proteins (ATM, E2F1, INK4A, INK4B and ARF) were detected. Small molecule drugs were applied to activate and inhibit the ATM-E2F1 signaling pathway. Transwell assay and the scratch test were adopted to detect cell invasion and migration abilities. ANRIL expression in the PC cells was higher than in normal pancreatic duct epithelial cells. In the PC rat models and PC cells, ANRIL interference promoted the expressions of INK4B, INK4A, ARF and E-cadherin, while reduced N-cadherin and Vimentin expression. Over-expressed ANRIL decreased the expression of INK4B, INK4A, ARF and E-cadherin, but raised N-cadherin and Vimentin expressions. By inhibiting the ATM-E2F1 signaling pathway in PC cells, E-cadherin expression increased but N-cadherin and Vimentin expressions decreased. After ANRIL was silenced or the ATM-E2F1 signaling pathway inhibited, PC cell migration and invasion abilities were decreased. In conclusion, over-expression of lncRNA ANRIL can promote EMT of PC cells by activating the ATM-E2F1 signaling pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

Myosin IIA-related Actomyosin Contractility Mediates Oxidative Stress-induced Neuronal Apoptosis

PubMed Central

Wang, Yan; Xu, Yingqiong; Liu, Qian; Zhang, Yuanyuan; Gao, Zhen; Yin, Mingzhu; Jiang, Nan; Cao, Guosheng; Yu, Boyang; Cao, Zhengyu; Kou, Junping

2017-01-01

Oxidative stress-induced neuronal apoptosis plays an important role in the progression of central nervous system (CNS) diseases. In our study, when neuronal cells were exposed to hydrogen peroxide (H2O2), an exogenous oxidant, cell apoptosis was observed with typical morphological changes including membrane blebbing, neurite retraction and cell contraction. The actomyosin system is considered to be responsible for the morphological changes, but how exactly it regulates oxidative stress-induced neuronal apoptosis and the distinctive functions of different myosin II isoforms remain unclear. We demonstrate that myosin IIA was required for neuronal contraction, while myosin IIB was required for neuronal outgrowth in normal conditions. During H2O2-induced neuronal apoptosis, myosin IIA, rather than IIB, interacted with actin filaments to generate contractile forces that lead to morphological changes. Moreover, myosin IIA knockout using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) reduced H2O2-induced neuronal apoptosis and the associated morphological changes. We further demonstrate that caspase-3/Rho-associated kinase 1 (ROCK1) dependent phosphorylation of myosin light chain (MLC) was required for the formation of the myosin IIA-actin complex. Meanwhile, either inhibition of myosin II ATPase with blebbistatin or knockdown of myosin IIA with siRNA reversely attenuated caspase-3 activation, suggesting a positive feedback loop during oxidative stress-induced apoptosis. Based on our observation, myosin IIA-actin complex contributes to actomyosin contractility and is associated with the positive feedback loop of caspase-3/ROCK1/MLC pathway. This study unravels the biochemical and mechanistic mechanisms during oxidative stress-induced neuronal apoptosis and may be applicable for the development of therapies for CNS diseases. PMID:28352215