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Sample records for teratocarcinoma f9 cells

  1. Radiation-induced apoptosis in F9 teratocarcinoma cells

    International Nuclear Information System (INIS)

    Langley, R.E.; Palayoor, S.T.; Coleman, C.N.; Bump, E.A.

    1994-01-01

    We have found that F9 murine teratocarcinoma cells undergo morphological changes and internucleosomal DNA fragmentation characteristic of apoptosis after exposure to ionizing radiation. We studied the time course, radiation dose-response, and the effects of protein and RNA synthesis inhibitors on this process. The response is dose dependent in the range 2-12 Gy. Internucleosomal DNA fragmentation can be detected as early as 6 h postirradiation and is maximal by 48 h. Cycloheximide, a protein synthesis inhibitor, and 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole, an RNA synthesis inhibitor, both induced internucleosomal DNA fragmentation in the unirradiated cells and enhanced radiation-induced DNA fragmentation. F9 cells can be induced to differentiate into cells resembling endoderm with retinoic acid. After irradiation, differentiated F9 cells exhibit less DNA fragmentation than stem cells. This indicates that ionizing radiation can induce apoptosis in non-lymphoid tumours. We suggest that embryonic tumour cells may be particularly susceptible to agents that induce apoptosis. (Author)

  2. Radiation-induced apoptosis in F9 teratocarcinoma cells

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    Langley, R E; Palayoor, S T; Coleman, C N; Bump, E A [Joint Center for Radiation Therapy and Dana Farber Cancer Inst., Boston (United States)

    1994-05-01

    We have found that F9 murine teratocarcinoma cells undergo morphological changes and internucleosomal DNA fragmentation characteristic of apoptosis after exposure to ionizing radiation. We studied the time course, radiation dose-response, and the effects of protein and RNA synthesis inhibitors on this process. The response is dose dependent in the range 2-12 Gy. Internucleosomal DNA fragmentation can be detected as early as 6 h postirradiation and is maximal by 48 h. Cycloheximide, a protein synthesis inhibitor, and 5,6-dichloro-1-[beta]-D-ribofuranosylbenzimidazole, an RNA synthesis inhibitor, both induced internucleosomal DNA fragmentation in the unirradiated cells and enhanced radiation-induced DNA fragmentation. F9 cells can be induced to differentiate into cells resembling endoderm with retinoic acid. After irradiation, differentiated F9 cells exhibit less DNA fragmentation than stem cells. This indicates that ionizing radiation can induce apoptosis in non-lymphoid tumours. We suggest that embryonic tumour cells may be particularly susceptible to agents that induce apoptosis. (Author).

  3. Characterization of protein kinase CK2 protein subunits and p53 in F9 teratocarcinoma cells in the absence and presence of cisplatin

    DEFF Research Database (Denmark)

    Küpper, M; Köster, M; Schmidt-Spaniol, I

    1994-01-01

    cell extracts treated with and without cisplatin were analyzed by ion exchange chromatography for protein kinase CK2 alpha/beta subunits and p53 distribution. The following results were obtained: (a) in crude extracts of cisplatin-treated cells, CK2 activity was sometimes reduced by as much as 50%; (b......The effect of cis-diaminedichloroplatinum(II) (cisplatin) on the induction of p53 and protein kinase CK2 activity was studied in the mouse teratocarcinoma cell line F9. Treatment of the cells with the chemotherapeutic agent cisplatin led to the detection of p53 3 h after addition of the drug. F9...... by immunostaining, we have detected, at a concentration of approximately 200 mM NaCl, a protein of approximately 46 kDa which reacted with the CK2 alpha-specific antibody. This fraction was devoid of CK2 activity; and (d) cisplatin-treated cells exhibited p53 protein, which was mostly eluting ahead but also partly...

  4. Dual functions of silver nanoparticles in F9 teratocarcinoma stem cells, a suitable model for evaluating cytotoxicity- and differentiation-mediated cancer therapy

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    Han JW

    2017-10-01

    Full Text Available Jae Woong Han, Sangiliyandi Gurunathan, Yun-Jung Choi, Jin-Hoi Kim Department of Stem Cell and Regenerative Biotechnology, Humanized Pig Research Center (SRC, Konkuk University, Seoul, Republic of Korea Background: Silver nanoparticles (AgNPs exhibit strong antibacterial and anticancer activity owing to their large surface-to-volume ratios and crystallographic surface structure. Owing to their various applications, understanding the mechanisms of action, biological interactions, potential toxicity, and beneficial effects of AgNPs is important. Here, we investigated the toxicity and differentiation-inducing effects of AgNPs in teratocarcinoma stem cells. Materials and methods: AgNPs were synthesized and characterized using various analytical techniques such as UV–visible spectroscopy, X-ray diffraction, energy-dispersive X-ray spectroscopy, and transmission electron microscopy. The cellular responses of AgNPs were analyzed by a series of cellular and biochemical assays. Gene and protein expressions were analyzed by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. Results: The AgNPs showed typical crystalline structures and spherical shapes (average size =20 nm. High concentration of AgNPs induced cytotoxicity in a dose-dependent manner by increasing lactate dehydrogenase leakage and reactive oxygen species. Furthermore, AgNPs caused mitochondrial dysfunction, DNA fragmentation, increased expression of apoptotic genes, and decreased expression of antiapoptotic genes. Lower concentrations of AgNPs induced neuronal differentiation by increasing the expression of differentiation markers and decreasing the expression of stem cell markers. Cisplatin reduced the viability of F9 cells that underwent AgNPs-induced differentiation. Conclusion: The results showed that AgNPs caused differentially regulated cytotoxicity and induced neuronal differentiation of F9 cells in a concentration-dependent manner

  5. A retinoic acid-inducible mRNA from F9 teratocarcinoma cells encodes a novel protease inhibitor homologue.

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    Wang, S Y; Gudas, L J

    1990-09-15

    We have previously isolated several cDNA clones specific for mRNA species that increase in abundance during the retinoic acid-associated differentiation of F9 teratocarcinoma stem cells. One of these mRNAs, J6, encodes a approximately 40 kDa protein as assayed by hybrid selection and in vitro translation (Wang, S.-Y., LaRosa, G., and Gudas, L. J. (1985) Dev. Biol. 107, 75-86). The time course of J6 mRNA expression is similar to those of both laminin B1 and collagen IV (alpha 1) messages following retinoic acid addition. To address the functional role of this protein, we have isolated a full-length cDNA clone complementary to this approximately 40-kDa protein mRNA. Sequence analysis reveals an open reading frame of 406 amino acids (Mr 45,652). The carboxyl-terminal portion of this predicted protein contains a region that is homologous to the reactive sites found among members of the serpin (serine protease inhibitor) family. The predicted reactive site (P1-P1') of this J6 protein is Arg-Ser, which is the same as that of antithrombin III. Like ovalbumin and human monocyte-derived plasminogen activator inhibitor (mPAI-2), which are members of the serpin gene family, the J6 protein appears to have no typical amino-terminal signal sequence.

  6. [Regulation of in vitro and in vivo differentiation of mouse embryonic stem cells, embryonic germ cells, and teratocarcinoma cells by TGFb family signaling factors].

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    Gordeeva, O F; Nikonova, T M; Lifantseva, N V

    2009-01-01

    The activity of specific signaling and transcription factors determines the cell fate in normal development and in tumor transformation. The transcriptional profiles of gene-components of different branches of TGFbeta family signaling pathways were studied in experimental models of initial stages of three-dimensional in vitro differentiation of embryonic stem cells, embryonic germ cells and teratocarcinoma cells and in teratomas and teratocarcinomas developed after their transplantation into immunodeficient Nude mice. Gene profile analysis of studied cell systems have revealed that expression patterns of ActivinA, Nodal, Lefty1, Lefty2, TGF TGFbeta1, BMP4, and GDF were identical in pluripotent stem cells whereas the mRNAs of all examined genes with the exception of Inhibin betaA/ActivinA were detected in the teratocarcinoma cells. These results indicate that differential activity of signaling pathways of the TGFbeta family factors regulates pluripotent state maintenance and pluripotent stem cell differentiation into the progenitors of three germ layers and extraembryonic structures and that normal expression pattern of TGFbeta family factors is rearranged in embryonic teratocarcinoma cells during tumor growth in vitro and in vivo.

  7. Transfected parvalbumin alters calcium homeostasis in teratocarcinoma PCC7 cells

    DEFF Research Database (Denmark)

    Müller, B K; Kabos, P; Belhage, B

    1996-01-01

    Indirect evidence supports a protective role of some EF-hand calcium-binding proteins against calcium-induced neurotoxicity. Little is known about how these proteins influence cytosolic calcium levels. After cloning the parvalbumin cDNA into an expression vector, teratocarcinoma cells (PCC7) were...

  8. Effect of cell cycle stage, dose rate and repair of sublethal damage of radiation-induced apoptosis in F9 teratocarcinoma cells

    International Nuclear Information System (INIS)

    Langley, R.E.; Quartuccio, S.G.; Kennealey, P.T.

    1995-01-01

    There are at least two different models of cell death after treatment with ionizing radiation. The first is a failure to undergo sustained cell division despite metabolic survival, and we refer to this end point as open-quotes classical reproductive cell death.close quotes The second is a process that results in loss of cell integrity. This second category includes cellular necrosis as well as apoptosis. Earlier studies in our laboratory showed that the predominant mechanism of cell death for irradiated F9 cell is apoptosis, and there is no indication that these cells die by necrosis. We have therefore used cells of this cell line to reassess basic radiobiological principles with respect to apoptosis. Classical reproductive cell death was determined by staining colonies derived from irradiated cells and scoring colonies of less than 50 cells as reproductively dead and colonies of more than 50 cells as survivors. Cells that failed to produce either type of colony (detached from the plate or disintegrated) were scored as having undergone apoptosis. Using these criteria we found that the fraction of the radiation-killed F9 cells that died by apoptosis did not vary when cells were irradiated at different stages of the cell cycle despite large variations in overall survival. This suggests that the factors that influence radiation sensitivity throughout the cell cycle have an equal impact on apoptosis and classical reproductive cell death. There was no difference in cell survival between split doses and single doses of X rays, suggesting that sublethal damage repair is not a factor in radiation-induced apoptosis of F9 cells. Apoptosis was not affected by changes in dose rate in the range of 0.038-4.96 Gy/min. 48 refs., 6 figs., 1 tab

  9. Regulation of TFIIIB during F9 cell differentiation.

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    Athineos, Dimitris; Marshall, Lynne; White, Robert J

    2010-03-12

    Differentiation of F9 embryonal carcinoma (EC) cells into parietal endoderm (PE) provides a tractable model system for studying molecular events during early and inaccessible stages of murine development. PE formation is accompanied by extensive changes in gene expression both in vivo and in culture. One of the most dramatic is the ~10-fold decrease in transcriptional output by RNA polymerase (pol) III. This has been attributed to changes in activity of TFIIIB, a factor that is necessary and sufficient to recruit pol III to promoters. The goal of this study was to identify molecular changes that can account for the low activity of TFIIIB following F9 cell differentiation. Three essential subunits of TFIIIB decrease in abundance as F9 cells differentiate; these are Brf1 and Bdp1, which are pol III-specific, and TBP, which is also used by pols I and II. The decreased levels of Brf1 and Bdp1 proteins can be explained by reduced expression of the corresponding mRNAs. However, this is not the case for TBP, which is regulated post-transcriptionally. In proliferating cells, pol III transcription is stimulated by the proto-oncogene product c-Myc and the mitogen-activated protein kinase Erk, both of which bind to TFIIIB. However, c-Myc levels fall during differentiation and Erk becomes inactive through dephosphorylation. The diminished abundance of TFIIIB is therefore likely to be compounded by changes to these positive regulators that are required for its full activity. In addition, PE cells have elevated levels of the retinoblastoma protein RB, which is known to bind and repress TFIIIB. The low activity of TFIIIB in PE can be attributed to a combination of changes, any one of which could be sufficient to inhibit pol III transcription. Declining levels of essential TFIIIB subunits and of activators that are required for maximal TFIIIB activity are accompanied by an increase in a potent repressor of TFIIIB. These events provide fail-safe guarantees to ensure that pol III

  10. Cervical mature teratoma 17 years after initial treatment of testicular teratocarcinoma: report of a late relapse

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    Alavion Mina

    2007-01-01

    Full Text Available Abstract Background Late relapses of testicular germ cell tumor are uncommon. We report a case of cervical mature teratoma appeared 17 years after treatment of testicular teratocarcinoma. Case presentation A 20- year- old patient underwent left sided orchiectomy followed by systemic therapy and retroperitoneal residual mass resection in 1989. He remained in complete remission for 200 months. In 2005 a huge left supraclavicular neck mass with extension to anterior mediastinum appeared. Radical surgical resection of the mass was performed and pathologic examination revealed mature teratoma. Conclusion This is one of the longest long-term reported intervals of a mature teratoma after treatment of a testicular nonseminoma germ cell tumor. This case emphasizes the necessity for follow up of testicular cancer throughout the patient's life.

  11. Establishment of 9L/F344 rat intracerebral glioma model of brain tumor stem cells

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    Zong-yu XIAO

    2015-04-01

    Full Text Available Objective To establish the 9L/F344 rat intracerebral glioma model of brain tumor stem cells.  Methods Rat 9L gliosarcoma stem-like cells were cultured in serum-free suspension. The expression of CD133 and nestin were tested by immunohistochemistry. A total of 48 inbredline male F344 rats were randomly divided into 2 groups, and 9L tumor sphere cells and 9L monolayer cells were respectively implanted into the right caudate nucleus of F344 rats in 2 groups. Survival time was observed and determined using the method of Kaplan-Meier survival analysis. Fourteen days after implantation or when the rats were dying, their brains were perfused and sectioned for HE staining, and CD133 and nestin were detected by immunohistochemistry.  Results Rat 9L tumor spheres were formed with suspension culture in serum-free medium. The gliomas formed in both groups were invasive without obvious capsule. More new vessels, bleeding and necrosis could be detected in 9L tumor spheres group. The tumor cells in both groups were positive for CD133 and nestin. There was no significant difference in the expression of CD133 and nestin between 2 groups (P > 0.05, for all. According to the expression of nestin, the tumors formed by 9L tumor sphere cells were more invasive. The median survival time of the rats bearing 9L tumor sphere cells was 15 d (95%CI: 15.219-15.781, and the median survival time of the rats bearing 9L monolayer cells was 21 d (95%CI: 20.395-21.605. There was significant difference between 2 groups (χ2 = 12.800, P = 0.000.  Conclusions 9L/F344 rat intracerebral glioma model of brain tumor stem cells is successfully established, which provides a glioma model for the future research. DOI: 10.3969/j.issn.1672-6731.2015.04.012

  12. Functional cooperativity between two TPA responsive elements in undifferentiated F9 embryonic stem cells.

    Science.gov (United States)

    Okuda, A; Imagawa, M; Sakai, M; Muramatsu, M

    1990-01-01

    We have recently identified an enhancer, termed GPEI, in the 5'-flanking region of the rat glutathione transferase P gene, that is composed of two imperfect TPA (phorbol 12-O-tetradecanoate 13-acetate) responsive elements (TREs). Unlike other TRE-containing enhancers, GPEI exhibits a strong transcriptional enhancing activity in F9 embryonic stem cells. Mutational analyses have revealed that the high activity of GPEI is mediated by two imperfect TREs. Each TRE-like sequence has no activity by itself but acts synergistically to form a strong enhancer which is active even in the very low level of AP-1 activity in F9 cells. Furthermore, we show that synthetic DNAs containing two perfect TREs in certain arrangements have strong transcriptional enhancing activities in F9 cells and the activity is greatly influenced by the relative orientation and the distance of two TREs. Images Fig. 1. Fig. 2. Fig. 3. PMID:2323334

  13. Advanced cell-based modeling of the royal disease: characterization of the mutated F9 mRNA.

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    Martorell, L; Luce, E; Vazquez, J L; Richaud-Patin, Y; Jimenez-Delgado, S; Corrales, I; Borras, N; Casacuberta-Serra, S; Weber, A; Parra, R; Altisent, C; Follenzi, A; Dubart-Kupperschmitt, A; Raya, A; Vidal, F; Barquinero, J

    2017-11-01

    Essentials The Royal disease (RD) is a form of hemophilia B predicted to be caused by a splicing mutation. We generated an iPSC-based model of the disease allowing mechanistic studies at the RNA level. F9 mRNA analysis in iPSC-derived hepatocyte-like cells showed the predicted abnormal splicing. Mutated F9 mRNA level was very low but we also found traces of wild type transcripts. Background The royal disease is a form of hemophilia B (HB) that affected many descendants of Queen Victoria in the 19th and 20th centuries. It was found to be caused by the mutation F9 c.278-3A>G. Objective To generate a physiological cell model of the disease and to study F9 expression at the RNA level. Methods Using fibroblasts from skin biopsies of a previously identified hemophilic patient bearing the F9 c.278-3A>G mutation and his mother, we generated induced pluripotent stem cells (iPSCs). Both the patient's and mother's iPSCs were differentiated into hepatocyte-like cells (HLCs) and their F9 mRNA was analyzed using next-generation sequencing (NGS). Results and Conclusion We demonstrated the previously predicted aberrant splicing of the F9 transcript as a result of an intronic nucleotide substitution leading to a frameshift and the generation of a premature termination codon (PTC). The F9 mRNA level in the patient's HLCs was significantly reduced compared with that of his mother, suggesting that mutated transcripts undergo nonsense-mediated decay (NMD), a cellular mechanism that degrades PTC-containing mRNAs. We also detected small proportions of correctly spliced transcripts in the patient's HLCs, which, combined with genetic variability in splicing and NMD machineries, could partially explain some clinical variability among affected members of the European royal families who had lifespans above the average. This work allowed the demonstration of the pathologic consequences of an intronic mutation in the F9 gene and represents the first bona fide cellular model of HB allowing the

  14. Wnt and Hedgehog Signaling Regulate the Differentiation of F9 Cells into Extraembryonic Endoderm

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    Gurjoth S. J. Deol

    2017-10-01

    Full Text Available Mouse F9 cells differentiate into primitive extraembryonic endoderm (PrE when treated with retinoic acid (RA, and this is accompanied by an up-regulation of Gata6. The role of the GATA6 network in PrE differentiation is known, and we have shown it directly activates Wnt6. Canonical Wnt/β-catenin signaling is required by F9 cells to differentiate to PrE, and this, like most developmental processes, requires input from one or more additional pathways. We found both RA and Gata6 overexpression, can induce the expression of Indian Hedgehog (Ihh and a subset of its target genes through Gli activation during PrE induction. Chemical activation of the Hh pathway using a Smoothened agonist (SAG also increased Gli reporter activity, and as expected, when Hh signaling was blocked with a Smoothened antagonist, cyclopamine, this RA-induced reporter activity was reduced. Interestingly, SAG alone failed to induce markers of PrE differentiation, and had no effect on Wnt/β-catenin-dependent TCF-LEF reporter activity. The expected increase in Wnt/β-catenin-dependent TCF-LEF reporter activity and PrE markers induced by RA was, however, blocked by cyclopamine. Finally, inhibiting GSK3 activity with BIO increased both TCF-LEF and Gli reporter activities. Together, we demonstrate the involvement of Hh signaling in the RA-induced differentiation of F9 cells into PrE, and while the activation of the Hh pathway itself is not sufficient, it as well as active Wnt/β-catenin are necessary for F9 cell differentiation.

  15. Evaluation of 18F-labeled exendin(9-39) derivatives targeting glucagon-like peptide-1 receptor for pancreatic β-cell imaging.

    Science.gov (United States)

    Kimura, Hiroyuki; Ogawa, Yu; Fujimoto, Hiroyuki; Mukai, Eri; Kawashima, Hidekazu; Arimitsu, Kenji; Toyoda, Kentaro; Fujita, Naotaka; Yagi, Yusuke; Hamamatsu, Keita; Murakami, Takaaki; Murakami, Atsushi; Ono, Masahiro; Nakamoto, Yuji; Togashi, Kaori; Inagaki, Nobuya; Saji, Hideo

    2018-01-15

    β-cell mass (BCM) is known to be decreased in subjects with type-2 diabetes (T2D). Quantitative analysis for BCM would be useful for understanding how T2D progresses and how BCM affects treatment efficacy and for earlier diagnosis of T2D and development of new therapeutic strategies. However, a noninvasive method to measure BCM has not yet been developed. We developed four 18 F-labeled exendin(9-39) derivatives for β-cell imaging by PET: [ 18 F]FB9-Ex(9-39), [ 18 F]FB12-Ex(9-39), [ 18 F]FB27-Ex(9-39), and [ 18 F]FB40-Ex(9-39). Affinity to the glucagon-like peptide-1 receptor (GLP-1R) was evaluated with dispersed islet cells of ddY mice. Uptake of exendin(9-39) derivatives in the pancreas as well as in other organs was evaluated by a biodistribution study. Small-animal PET study was performed after injecting [ 18 F]FB40-Ex(9-39). FB40-Ex(9-39) showed moderate affinity to the GLP-1R. Among all of the derivatives, [ 18 F]FB40-Ex(9-39) resulted in the highest uptake of radioactivity in the pancreas 30 min after injection. Moreover, it showed significantly less radioactivity accumulated in the liver and kidney, resulting in an overall increase in the pancreas-to-organ ratio. In the PET imaging study, pancreas was visualized at 30 min after injection of [ 18 F]FB40-Ex(9-39). [ 18 F]FB40-Ex(9-39) met the basic requirements for an imaging probe for GLP-1R in pancreatic β-cells. Further enhancement of pancreatic uptake and specific binding to GLP-1R will lead to a clear visualization of pancreatic β-cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Marked change in microRNA expression during neuronal differentiation of human teratocarcinoma NTera2D1 and mouse embryonal carcinoma P19 cells

    International Nuclear Information System (INIS)

    Hohjoh, Hirohiko; Fukushima, Tatsunobu

    2007-01-01

    MicroRNAs (miRNAs) are small noncoding RNAs, with a length of 19-23 nucleotides, which appear to be involved in the regulation of gene expression by inhibiting the translation of messenger RNAs carrying partially or nearly complementary sequences to the miRNAs in their 3' untranslated regions. Expression analysis of miRNAs is necessary to understand their complex role in the regulation of gene expression during the development, differentiation and proliferation of cells. Here we report on the expression profile analysis of miRNAs in human teratocarcinoma NTere2D1, mouse embryonic carcinoma P19, mouse neuroblastoma Neuro2a and rat pheochromocytoma PC12D cells, which can be induced into differentiated cells with long neuritic processes, i.e., after cell differentiation, such that the resultant cells look similar to neuronal cells. The data presented here indicate marked changes in the expression of miRNAs, as well as genes related to neuronal development, occurred in the differentiation of NTera2D1 and P19 cells. Significant changes in miRNA expression were not observed in Neuro2a and PC12D cells, although they showed apparent morphologic change between undifferentiated and differentiated cells. Of the miRNAs investigated, the expression of miRNAs belonging to the miR-302 cluster, which is known to be specifically expressed in embryonic stem cells, and of miR-124a specific to the brain, appeared to be markedly changed. The miR-302 cluster was potently expressed in undifferentiated NTera2D1 and P19 cells, but hardly in differentiated cells, such that miR-124a showed an opposite expression pattern to the miR-302 cluster. Based on these observations, it is suggested that the miR-302 cluster and miR-124a may be useful molecular indicators in the assessment of degree of undifferentiation and/or differentiation in the course of neuronal differentiation

  17. Synthesis and preliminary evaluation of 9-(4-[{sup 18}F]fluoro-3-hydroxymethylbutyl) guanine ([{sup 18}F]FHBG) in HSV1-tk gene transduced hepatoma cell

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    Moon, Byung Seok; Lee, Tae Sup [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Lee, Myoung Keun [Yonsei University, Wonju (Korea, Republic of)] (and others)

    2006-08-15

    The HSV1-tk reporter gene system is the most widely used system because of its advantage that direct monitoring is possible without the introduction of a separate reporter gene in case of HSV1-tk suicide gene therapy. In this study, we investigate the usefulness of the reporter probe (substrate), 9-(4-[{sup 18}F]fluoro-3-hydroxymethylbutyl) guanine ([{sup 18}F]FHBG) for non-invasive reporter gene imaging using PET in HSV1-tk expressing hepatoma model. Radiolabeled FHBG was prepared in 8 steps from a commercially available triester. The labeling reaction was carried out by NCA nucleophilic substitution with K[{sup 18}F]/K2.2.2. in acetonitrile using N2-monomethoxytrityl-9-[4-(tosly)-3-monomethoxytritylmethylbutl] guanine as a precursor, followed by deprotection with 1 N HCI. Preliminary biological properties of the probe were evaluated with MCA cells and MCA-tk cells transduced with HSV1-tk reporter gene. In vitro uptake and release-out studies of [{sup 18}F]FHBG were performed, and was analyzed correlation between [{sup 18}F]FHBG uptake ratio according to increasing numeric count of MCA-tk cells and degree of gene expression. MicroPET scan image was obtained with MCA and MCA-tk tumor beating Balb/c-nude mouse model. [{sup 18}F]FHBG was purified by reverse phase semi-HPLC system and collected at around 16-18 min. Radiochemical yield was about 20-25% (corrected for decay), radiochemical purity was > 95% and specific activity was around > 55.5 GBq/ {mu} mol. Specific accumulation of [{sup 18}F]FHBG was observed in HSV1-tk gene transduced MCA-tk cells but not MCA cells, and consecutive 1 hour release-out results showed more than 86% of uptaked [{sup 18}F]FHBG was retained inside of cells. The uptake of [{sup 18}F]FHBG was showed a highly significant linear correlation (R{sup 2} = 0.995) with increasing percentage of MCA-tk numeric cell count. In microPET scan images, remarkable difference of accumulation was observed for the two type of tumors. [{sup 18}F]FHBG appears

  18. The different shades of mammalian pluripotent stem cells

    NARCIS (Netherlands)

    Kuijk, E.W.; Lopes, S.M.; Geijsen, N.; Macklon, N.; Roelen, B.A.

    2011-01-01

    BACKGROUND: Pluripotent stem cells have been derived from a variety of sources such as from the inner cell mass of preimplantation embryos, from primordial germ cells, from teratocarcinomas and from male germ cells. The recent development of induced pluripotent stem cells demonstrates that somatic

  19. Functional cooperativity between two TPA responsive elements in undifferentiated F9 embryonic stem cells.

    OpenAIRE

    Okuda, A; Imagawa, M; Sakai, M; Muramatsu, M

    1990-01-01

    We have recently identified an enhancer, termed GPEI, in the 5'-flanking region of the rat glutathione transferase P gene, that is composed of two imperfect TPA (phorbol 12-O-tetradecanoate 13-acetate) responsive elements (TREs). Unlike other TRE-containing enhancers, GPEI exhibits a strong transcriptional enhancing activity in F9 embryonic stem cells. Mutational analyses have revealed that the high activity of GPEI is mediated by two imperfect TREs. Each TRE-like sequence has no activity by ...

  20. Intratumoral macrophages contribute to epithelial-mesenchymal transition in solid tumors

    International Nuclear Information System (INIS)

    Bonde, Anne-Katrine; Tischler, Verena; Kumar, Sushil; Soltermann, Alex; Schwendener, Reto A

    2012-01-01

    Several stromal cell subtypes including macrophages contribute to tumor progression by inducing epithelial-mesenchymal transition (EMT) at the invasive front, a mechanism also linked to metastasis. Tumor associated macrophages (TAM) reside mainly at the invasive front but they also infiltrate tumors and in this process they mainly assume a tumor promoting phenotype. In this study, we asked if TAMs also regulate EMT intratumorally. We found that TAMs through TGF-β signaling and activation of the β-catenin pathway can induce EMT in intratumoral cancer cells. We depleted macrophages in F9-teratocarcinoma bearing mice using clodronate-liposomes and analyzed the tumors for correlations between gene and protein expression of EMT-associated and macrophage markers. The functional relationship between TAMs and EMT was characterized in vitro in the murine F9 and mammary gland NMuMG cells, using a conditioned medium culture approach. The clinical relevance of our findings was evaluated on a tissue microarray cohort representing 491 patients with non-small cell lung cancer (NSCLC). Gene expression analysis of F9-teratocarcinomas revealed a positive correlation between TAM-densities and mesenchymal marker expression. Moreover, immunohistochemistry showed that TAMs cluster with EMT phenotype cells in the tumors. In vitro, long term exposure of F9-and NMuMG-cells to macrophage-conditioned medium led to decreased expression of the epithelial adhesion protein E-cadherin, activation of the EMT-mediating β-catenin pathway, increased expression of mesenchymal markers and an invasive phenotype. In a candidate based screen, macrophage-derived TGF-β was identified as the main inducer of this EMT-associated phenotype. Lastly, immunohistochemical analysis of NSCLC patient samples identified a positive correlation between intratumoral macrophage densities, EMT markers, intraepithelial TGF-β levels and tumor grade. Data presented here identify a novel role for macrophages in EMT

  1. Dual functions of silver nanoparticles in F9 teratocarcinoma stem cells, a suitable model for evaluating cytotoxicity- and differentiation-mediated cancer therapy

    OpenAIRE

    Han, Jae Woong; Gurunathan, Sangiliyandi; Choi, Yun-Jung; Kim, Jin-Hoi

    2017-01-01

    Jae Woong Han, Sangiliyandi Gurunathan, Yun-Jung Choi, Jin-Hoi Kim Department of Stem Cell and Regenerative Biotechnology, Humanized Pig Research Center (SRC), Konkuk University, Seoul, Republic of Korea Background: Silver nanoparticles (AgNPs) exhibit strong antibacterial and anticancer activity owing to their large surface-to-volume ratios and crystallographic surface structure. Owing to their various applications, understanding the mechanisms of action, biological interactions, potential...

  2. Synthesis and evaluation of [18F]exendin (9–39) as a potential biomarker to measure pancreatic β-cell mass

    International Nuclear Information System (INIS)

    Wang Yi; Lim, Keunpoong; Normandin, Marc; Zhao Xiaojian; Cline, Gary W.; Ding Yushin

    2012-01-01

    Introduction: Glucagon-like peptide 1 (GLP-1) is released in response to food intake and plays an important role in maintaining blood glucose homeostasis. Exendin (9–39), a potent glucagon-like peptide 1 receptor antagonist, has been labeled with In-111 for SPECT imaging. We report here the first radiosynthesis of [ 18 F]exendin (9–39) ([ 18 F]Ex(9–39)) and an evaluation of its potential as a biomarker for in vivo positron emission tomography (PET) imaging of pancreatic β-cell mass (BCM) in rats. Methods: F-18 label was introduced by conjugation of [ 18 F]4-fluorobenzaldehyde with an Ex(9–39) derivative containing a 6-hydrazinonicotinyl group on the ε-amine of Lys27. Positron emission tomography imaging was carried out in Sprague–Dawley rats (five control and five streptozotocin-induced diabetic) and BioBreeding diabetes-prone rats (three at 7 weeks and three at 12 weeks) using the high-resolution research tomograph (HRRT) after 0.187±0.084 mCi [ 18 F]Ex(9–39) administration. Time–activity curves were obtained from pancreas, liver and kidney. Pancreases were assayed for insulin content after the imaging study. Results: Site-specifically labeled [ 18 F]Ex(9–39) was purified on a G15 open column with radiochemical and chemical purities >98%. Positron emission tomography imaging showed pancreatic standardized uptake value (SUV) peaked at 10 min and plateaued by 50 min to the end of scan (240 min). No correlations of pancreatic SUV with postmortem measures of insulin content were seen. Conclusions: [ 18 F]Ex(9–39) was successfully prepared and used for PET imaging for the first time to measure pancreatic BCM. The results suggest that derivatization of the Lys27 residue might reduce binding affinity, as evidenced by the absence of specific binding. Exendin analogues radiolabeled at other sites may elucidate the active site required for binding.

  3. File list: Oth.EmF.20.Smarca5.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.EmF.20.Smarca5.AllCell mm9 TFs and others Smarca5 Embryonic fibroblast SRX10220...84 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.EmF.20.Smarca5.AllCell.bed ...

  4. File list: Oth.EmF.10.Ddit3.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.EmF.10.Ddit3.AllCell mm9 TFs and others Ddit3 Embryonic fibroblast SRX119350,SR...X119351 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.EmF.10.Ddit3.AllCell.bed ...

  5. File list: Oth.EmF.50.Ddit3.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.EmF.50.Ddit3.AllCell mm9 TFs and others Ddit3 Embryonic fibroblast SRX119350,SR...X119351 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.EmF.50.Ddit3.AllCell.bed ...

  6. File list: Oth.EmF.10.Rbfox2.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.EmF.10.Rbfox2.AllCell mm9 TFs and others Rbfox2 Embryonic fibroblast SRX551545,...SRX551553,SRX551551,SRX551546 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.EmF.10.Rbfox2.AllCell.bed ...

  7. File list: Oth.EmF.20.Rbfox2.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.EmF.20.Rbfox2.AllCell mm9 TFs and others Rbfox2 Embryonic fibroblast SRX551545,...SRX551553,SRX551551,SRX551546 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.EmF.20.Rbfox2.AllCell.bed ...

  8. File list: Oth.EmF.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.EmF.05.Epitope_tags.AllCell mm9 TFs and others Epitope tags Embryonic fibroblas...RX542102,SRX204644,SRX204643,SRX255462,SRX255460 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.EmF.05.Epitope_tags.AllCell.bed ...

  9. File list: Oth.EmF.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.EmF.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Embryonic fibroblas...RX255460,SRX204644,SRX542102,SRX204643,SRX204642 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.EmF.50.Epitope_tags.AllCell.bed ...

  10. File list: Pol.EmF.05.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.EmF.05.RNA_Polymerase_II.AllCell mm9 RNA polymerase RNA Polymerase II Embryonic...RX143288 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.EmF.05.RNA_Polymerase_II.AllCell.bed ...

  11. Basement membrane components secreted by mouse yolk sac carcinoma cell lines

    DEFF Research Database (Denmark)

    Damjanov, A; Wewer, U M; Tuma, B

    1990-01-01

    Three new cell lines (NE, ME, LRD) were cloned from mouse-embryo-derived teratocarcinomas and characterized on the basis of developmental, ultrastructural, and cytochemical criteria as nullipotent embryonal carcinoma (EC), pure parietal yolk sac (PYS) carcinoma and mixed parieto-visceral yolk sac...

  12. File list: InP.EmF.50.Input_control.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.EmF.50.Input_control.AllCell mm9 Input control Input control Embryonic fibrobla...363,SRX115361 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.EmF.50.Input_control.AllCell.bed ...

  13. File list: InP.EmF.05.Input_control.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.EmF.05.Input_control.AllCell mm9 Input control Input control Embryonic fibrobla...367,SRX115361 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.EmF.05.Input_control.AllCell.bed ...

  14. File list: InP.EmF.20.Input_control.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.EmF.20.Input_control.AllCell mm9 Input control Input control Embryonic fibrobla...367,SRX115361 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.EmF.20.Input_control.AllCell.bed ...

  15. E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation

    International Nuclear Information System (INIS)

    Hao, Hongying; Dong, Yanbin; Bowling, Maria T; Gomez-Gutierrez, Jorge G; Zhou, H Sam; McMasters, Kelly M

    2007-01-01

    PUMA is a pro-apoptotic Bcl-2 family member that has been shown to be involved in apoptosis in many cell types. We sought to ascertain whether induction of PUMA plays a crucial role in E2F-1-induced apoptosis in melanoma cells. PUMA gene and protein expression levels were detected by real-time PCR and Western blot in SK-MEL-2 and HCT116 cell lines after Ad-E2F-1 infection. Activation of the PUMA promoter by E2F-1 overexpression was detected by dual luciferase reporter assay. E2F-1-induced Bax translocation was shown by immunocytochemistry. The induction of caspase-9 activity was measured by caspase-9 colorimetric assay kit. Up-regulation of the PUMA gene and protein by E2F-1 overexpression was detected by real-time PCR and Western blot analysis in the SK-MEL-2 melanoma cell line. In support of this finding, we found six putative E2F-1 binding sites within the PUMA promoter. Subsequent dual luciferase reporter assay showed that E2F-1 expression could increase the PUMA gene promoter activity 9.3 fold in SK-MEL-2 cells. The role of PUMA in E2F-1-induced apoptosis was further investigated in a PUMA knockout cell line. Cell viability assay showed that the HCT116 PUMA-/- cell line was more resistant to Ad-E2F-1-mediated cell death than the HCT116 PUMA+/+ cell line. Moreover, a 2.2-fold induction of the PUMA promoter was also noted in the HCT116 PUMA+/+ colon cancer cell line after Ad-E2F-1 infection. Overexpression of a truncated E2F-1 protein that lacks the transactivation domain failed to up-regulate PUMA promoter, suggesting that PUMA may be a transcriptional target of E2F-1. E2F-1-induced cancer cell apoptosis was accompanied by Bax translocation from the cytosol to mitochondria and the induction of caspase-9 activity, suggesting that E2F-1-induced apoptosis is mediated by PUMA through the cytochrome C/Apaf-1-dependent pathway. Our studies strongly demonstrated that E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation. The signaling

  16. File list: Oth.EmF.05.Taf7l.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.EmF.05.Taf7l.AllCell mm9 TFs and others Taf7l Embryonic fibroblast SRX202798,SR...X202799 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.EmF.05.Taf7l.AllCell.bed ...

  17. Enterocin F4-9, a Novel O-Linked Glycosylated Bacteriocin.

    Science.gov (United States)

    Maky, Mohamed Abdelfattah; Ishibashi, Naoki; Zendo, Takeshi; Perez, Rodney Honrada; Doud, Jehan Ragab; Karmi, Mohamed; Sonomoto, Kenji

    2015-07-01

    Enterococcus faecalis F4-9 isolated from Egyptian salted-fermented fish produces a novel bacteriocin, termed enterocin F4-9. Enterocin F4-9 was purified from the culture supernatant by three steps, and its molecular mass was determined to be 5,516.6 Da by mass spectrometry. Amino acid and DNA sequencing showed that the propeptide consists of 67 amino acid residues, with a leader peptide containing a double glycine cleavage site to produce a 47-amino-acid mature peptide. Enterocin F4-9 is modified by two molecules of N-acetylglucosamine β-O-linked to Ser37 and Thr46. The O-linked N-acetylglucosamine moieties are essential for the antimicrobial activity of enterocin F4-9. Further analysis of the enterocin F4-9 gene cluster identified enfC, which has high sequence similarity to a glycosyltransferase. The antimicrobial activity of enterocin F4-9 covered a limited range of bacteria, including, interestingly, a Gram-negative strain, Escherichia coli JM109. Enterocin F4-9 is sensitive to protease, active at a wide pH range, and moderately resistant to heat. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  18. A simplified one-pot synthesis of 9-[(3-[18F]Fluoro-1-hydroxy-2-propoxy)methyl]guanine([18F]FHPG) and 9-(4-[18F]Fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) for gene therapy

    International Nuclear Information System (INIS)

    Shiue, Grace G.; Shiue, Chyng-Yann; Lee, Roland L.; MacDonald, Douglas; Hustinx, Roland; Eck, Stephen L.; Alavi, Abass A.

    2001-01-01

    9-[(3-[ 18 F]Fluoro-1-hydroxy-2-propoxy)methyl]guanine ([ 18 F]FHPG, 2) has been synthesized by nucleophilic substitution of N 2 -(p-anisyldiphenylmethyl)-9-{[1-(p-anisyldiphenylmethoxy)-3 -toluenesulfonyloxy-2-propoxy]methyl}guanine (1) with potassium [ 18 F]fluoride/Kryptofix 2.2.2 followed by deprotection with 1 N HCl and purification with different methods in variable yields. When both the nucleophilic substitution and deprotection were carried out at 90 deg. C and the product was purified by HPLC (method A), the yield of compound 2 was 5-10% and the synthesis time was 90 min from EOB. However, if both the nucleophilic substitution and deprotection were carried out at 120 deg. C and the product was purified by HPLC, the yield of compound 2 decreased to 2%. When compound 2 was synthesized at 90 deg. C and purified by Silica Sep-Pak (method B), the yield increased to 10-15% and the synthesis time was 60 min from EOB. Similarly, 9-(4-[ 18 F]fluoro-3-hydroxymethylbutyl)guanine ([ 18 F]FHBG, 4) was synthesized with method A and method B in 9% and 10-15% yield, respectively, in a synthesis time of 90 and 60 min, respectively, from EOB. Compound 2 was relatively unstable in acidic medium at 120 deg. C while compound 4 was stable under the same condition. Both compound 2 and compound 4 had low lipid/water partition coefficient (0.126±0.022, n=5 and 0.165±0.023, n=5, respectively). Although it contains non-radioactive ganciclovir (∼5-30 μg) as a chemical by-product, compound 2 synthesized by method B has a similar uptake in 9L glioma cells as that synthesized by method A, and is a potential tracer for imaging herpes simplex virus thymidine kinase gene expression in tumors using PET. Similarly, compound 4 synthesized by method B contains ∼10-25 μg of penciclovir as a chemical by-product. Thus, the simplified one pot synthesis (method B) is a useful method for synthesizing both compound 2 and compound 4 in good yield for routine clinical use, and the method is

  19. Flow sorting in the study of teratocarcinoma cell differentiation

    NARCIS (Netherlands)

    G.H. Schaap (Gerard Hendrik)

    1984-01-01

    textabstractFlow cytometry is a technique by which particles (cells, subcellular fragments, bacteria) in aqueous suspension are passed one by one through a sensing region where optical (or electrical) signals are generated. These signals for each individual cell are collected and processed, and may

  20. Enterocin F4-9, a Novel O-Linked Glycosylated Bacteriocin

    OpenAIRE

    Maky, Mohamed Abdelfattah; Ishibashi, Naoki; Zendo, Takeshi; Perez, Rodney Honrada; Doud, Jehan Ragab; Karmi, Mohamed; Sonomoto, Kenji

    2015-01-01

    Enterococcus faecalis F4-9 isolated from Egyptian salted-fermented fish produces a novel bacteriocin, termed enterocin F4-9. Enterocin F4-9 was purified from the culture supernatant by three steps, and its molecular mass was determined to be 5,516.6 Da by mass spectrometry. Amino acid and DNA sequencing showed that the propeptide consists of 67 amino acid residues, with a leader peptide containing a double glycine cleavage site to produce a 47-amino-acid mature peptide. Enterocin F4-9 is modi...

  1. The JAK2V617F and CALR exon 9 mutations are shared immunogenic neoantigens in hematological malignancy

    DEFF Research Database (Denmark)

    Holmstrom, Morten Orebo; Hasselbalch, Hans Carl; Andersen, Mads Hald

    2017-01-01

    Approximately 90% of patients with the hematological malignancies termed the chronic myeloproliferative neoplasms harbor either the JAK2V617F-mutation or CALR exon 9 mutation. Both of these are recognized by T-cells, which make the mutations ideal targets for cancer immune therapy as they are sha......Approximately 90% of patients with the hematological malignancies termed the chronic myeloproliferative neoplasms harbor either the JAK2V617F-mutation or CALR exon 9 mutation. Both of these are recognized by T-cells, which make the mutations ideal targets for cancer immune therapy...

  2. Stimulation of epithelial cell matrix metalloproteinase (MMP-2, -9, -13) and interleukin-8 secretion by fusobacteria.

    Science.gov (United States)

    Gursoy, U K; Könönen, E; Uitto, V-J

    2008-10-01

    Bacterial pathogens involved in periodontal diseases exert their destructive effects primarily by stimulating the host cells to increase their secretion of proinflammatory cytokines and matrix metalloproteinases (MMPs). This study aimed to determine the epithelial cell matrix metalloproteinase and interleukin-8 (IL-8) secretion upon exposure to fusobacteria. Eight different oral and non-oral Fusobacterium strains were incubated with HaCaT epithelial cells. Gelatin zymography and Western blot analysis were performed to detect collagenase 3 (MMP-13), gelatinase A (MMP-2), gelatinase B (MMP-9), and IL-8 secretion by epithelial cells. All Fusobacterium strains, especially Fusobacterium necrophorum ATCC 25286, Fusobacterium nucleatum ATCC 25586, and Fusobacterium varium ATCC 51644, increased MMP-9 and MMP-13 secretion. Fusobacterium simiae ATCC 33568, and to a lesser extent F. nucleatum and F. necrophorum, increased epithelial MMP-2 secretion. F. nucleatum and F. necrophorum also increased IL-8 secretion. F. varium ATCC 27725, a strain that only weakly stimulated MMP production, strongly increased the IL-8 production, suggesting that their expression is differently regulated. We conclude that the pathogenic potential of fusobacteria may partly result from their ability to stimulate secretion of MMP-9, MMP-13, and IL-8 from epithelial cells.

  3. Comparison of 68Ga-DOTA-Siglec-9 and 18F-Fluorodeoxyribose-Siglec-9: Inflammation Imaging and Radiation Dosimetry.

    Science.gov (United States)

    Virtanen, Helena; Silvola, Johanna M U; Autio, Anu; Li, Xiang-Guo; Liljenbäck, Heidi; Hellberg, Sanna; Siitonen, Riikka; Ståhle, Mia; Käkelä, Meeri; Airaksinen, Anu J; Helariutta, Kerttuli; Tolvanen, Tuula; Veres, Tibor Z; Saraste, Antti; Knuuti, Juhani; Jalkanen, Sirpa; Roivainen, Anne

    2017-01-01

    Sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) is a ligand of inflammation-inducible vascular adhesion protein-1 (VAP-1). We compared 68 Ga-DOTA- and 18 F-fluorodeoxyribose- (FDR-) labeled Siglec-9 motif peptides for PET imaging of inflammation. Methods . Firstly, we examined 68 Ga-DOTA-Siglec-9 and 18 F-FDR-Siglec-9 in rats with skin/muscle inflammation. We then studied 18 F-FDR-Siglec-9 for the detection of inflamed atherosclerotic plaques in mice and compared it with previous 68 Ga-DOTA-Siglec-9 results. Lastly, we estimated human radiation dosimetry from the rat data. Results . In rats, 68 Ga-DOTA-Siglec-9 (SUV, 0.88 ± 0.087) and 18 F-FDR-Siglec-9 (SUV, 0.77 ± 0.22) showed comparable ( P = 0.29) imaging of inflammation. In atherosclerotic mice, 18 F-FDR-Siglec-9 detected inflamed plaques with a target-to-background ratio (1.6 ± 0.078) similar to previously tested 68 Ga-DOTA-Siglec-9 ( P = 0.35). Human effective dose estimates for 68 Ga-DOTA-Siglec-9 and 18 F-FDR-Siglec-9 were 0.024 and 0.022 mSv/MBq, respectively. Conclusion . Both tracers are suitable for PET imaging of inflammation. The easier production and lower cost of 68 Ga-DOTA-Siglec-9 present advantages over 18 F-FDR-Siglec-9, indicating it as a primary choice for clinical studies.

  4. A simplified one-pot synthesis of 9-[(3-[{sup 18}F]Fluoro-1-hydroxy-2-propoxy)methyl]guanine([{sup 18}F]FHPG) and 9-(4-[{sup 18}F]Fluoro-3-hydroxymethylbutyl)guanine ([{sup 18}F]FHBG) for gene therapy

    Energy Technology Data Exchange (ETDEWEB)

    Shiue, Grace G.; Shiue, Chyng-Yann E-mail: Shiue@rad.upenn.edu; Lee, Roland L.; MacDonald, Douglas; Hustinx, Roland; Eck, Stephen L.; Alavi, Abass A

    2001-10-01

    9-[(3-[{sup 18}F]Fluoro-1-hydroxy-2-propoxy)methyl]guanine ([{sup 18}F]FHPG, 2) has been synthesized by nucleophilic substitution of N{sup 2}-(p-anisyldiphenylmethyl)-9-{l_brace}[1-(p-anisyldiphenylmethoxy)-3 -toluenesulfonyloxy-2-propoxy]methyl{r_brace}guanine (1) with potassium [{sup 18}F]fluoride/Kryptofix 2.2.2 followed by deprotection with 1 N HCl and purification with different methods in variable yields. When both the nucleophilic substitution and deprotection were carried out at 90 deg. C and the product was purified by HPLC (method A), the yield of compound 2 was 5-10% and the synthesis time was 90 min from EOB. However, if both the nucleophilic substitution and deprotection were carried out at 120 deg. C and the product was purified by HPLC, the yield of compound 2 decreased to 2%. When compound 2 was synthesized at 90 deg. C and purified by Silica Sep-Pak (method B), the yield increased to 10-15% and the synthesis time was 60 min from EOB. Similarly, 9-(4-[{sup 18}F]fluoro-3-hydroxymethylbutyl)guanine ([{sup 18}F]FHBG, 4) was synthesized with method A and method B in 9% and 10-15% yield, respectively, in a synthesis time of 90 and 60 min, respectively, from EOB. Compound 2 was relatively unstable in acidic medium at 120 deg. C while compound 4 was stable under the same condition. Both compound 2 and compound 4 had low lipid/water partition coefficient (0.126{+-}0.022, n=5 and 0.165{+-}0.023, n=5, respectively). Although it contains non-radioactive ganciclovir ({approx}5-30 {mu}g) as a chemical by-product, compound 2 synthesized by method B has a similar uptake in 9L glioma cells as that synthesized by method A, and is a potential tracer for imaging herpes simplex virus thymidine kinase gene expression in tumors using PET. Similarly, compound 4 synthesized by method B contains {approx}10-25 {mu}g of penciclovir as a chemical by-product. Thus, the simplified one pot synthesis (method B) is a useful method for synthesizing both compound 2 and compound 4 in

  5. Radiative electron capture for F8+ and F9+ ions in collisions with a He gas target

    International Nuclear Information System (INIS)

    Kawatsura, K.; Richard, P.; Tawara, H.

    1981-01-01

    The x rays from the radiative electron capture (REC) to the projectile K-shell were investigated for F 8+ and F 9+ ions incident on the He target atoms in the projectile energy range from 15 to 40 MeV. The peak energies of the K-REC x-ray spectra were found to decrease linearly with a decrease of the projectile energies as expected and extrapolated to the correct ls binding energies at zero velocity. According to theory the distribution width of the REC energies should be independent of the binding energy of electrons in the projectile ions. However, it is found that this width for F 8+ ions is systematically smaller by 20% than that for F 9+ ions. The measured REC cross sections for F 9+ ions are slightly larger than twice those for F 8+ ions and the total REC cross sections for F 8+ and F 9+ ions were found to be more than three orders of magnitude smaller than the total electron capture cross sections and the capture cross sections into excited states

  6. Phase resolved and coherence gated en face reflection imaging of multilayered embryonal carcinoma cells

    Science.gov (United States)

    Yamauchi, Toyohiko; Fukami, Tadashi; Iwai, Hidenao; Yamashita, Yutaka

    2012-03-01

    Embryonal carcinoma (EC) cells, which are cell lines derived from teratocarcinomas, have characteristics in common with stem cells and differentiate into many kinds of functional cells. Similar to embryonic stem (ES) cells, undifferentiated EC cells form multi-layered spheroids. In order to visualize the three-dimensional structure of multilayered EC cells without labeling, we employed full-field interference microscopy with the aid of a low-coherence quantitative phase microscope, which is a reflection-type interference microscope employing the digital holographic technique with a low-coherent light source. Owing to the low-coherency of the light-source (halogen lamp), only the light reflected from reflective surface at a specific sectioning height generates an interference image on the CCD camera. P19CL6 EC cells, derived from mouse teratocarcinomas, formed spheroids that are about 50 to 200 micrometers in diameter. Since the height of each cell is around 10 micrometers, it is assumed that each spheroid has 5 to 20 cell layers. The P19CL6 spheroids were imaged in an upright configuration and the horizontally sectioned reflection images of the sample were obtained by sequentially and vertically scanning the zero-path-length height. Our results show the threedimensional structure of the spheroids, in which plasma and nuclear membranes were distinguishably imaged. The results imply that our technique is further capable of imaging induced pluripotent stem (iPS) cells for the assessment of cell properties including their pluripotency.

  7. Cytotoxicity of the coagulant Moringa oleifera lectin (cMoL) to B16-F10 melanoma cells.

    Science.gov (United States)

    de Andrade Luz, Luciana; Rossato, Franco Aparecido; Costa, Rute Alves Pereira E; Napoleão, Thiago Henrique; Paiva, Patrícia Maria Guedes; Coelho, Luana Cassandra Breitenbach Barroso

    2017-10-01

    Moringa oleifera seeds are used in alternative medicine to treat inflammation, tumors and bacterial and protozoan infections, for example. The seeds contain lectins, which are carbohydrate-binding proteins with several biological properties including cytotoxicity to cancer cells. In this work, we examined the cytotoxicity of the coagulant M. oleifera lectin (cMoL) on B16-F10 murine melanoma cells. cMoL cytotoxic effects were evaluated through trypan blue assay and flow cytometry analysis. Mitochondrial superoxide levels and activation of caspases 3, 8 and 9 were measured. cMoL (1.5-16μM) reduced viability and caused cell death of B16-F10 cells with an IC 50 of 9.72μM. Flow cytometry analysis indicated induction of necrosis and suggested the presence of cells in late apoptosis. Specificity for tumor cells was observed since death of normal human fibroblasts (GN) was not higher than 20% in treatments with cMoL from 1.5 to 16μM. Microscopy images revealed rounded shape and reduction of volume in B16-F10 cells treated with cMoL. cMoL increased mitochondrial ROS production and promoted caspases 3, 8 and 9 activation in B16-F10 cells, indicating the activation of apoptosis-related pathway. In conclusion, this study demonstrates that cMoL is cytotoxic to B16-F10 cells, which stimulates more investigation on the anticancer potential of this lectin. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. The translocator protein radioligand 18F-DPA-714 monitors antitumor effect of erufosine in a rat 9L intracranial glioma model

    International Nuclear Information System (INIS)

    Awde, Ali R.; Boisgard, Raphael; Theze, Benoit; Dubois, Albertine; Zheng, Jinzi; Winkeler, Alexandra; Dolle, Frederic; Jacobs, Andreas H.; Tavitian, Bertrand

    2013-01-01

    On the one hand, the translocator protein (TSPO) radioligand N,N-diethyl-2-(2-(4-(2- 18 F-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a] pyrimidin-3-yl)acetamide ( 18 F-DPA-714) has been suggested to serve as an alternative radiotracer to image human glioma, and on the other hand the alkyl-phosphocholine erufosine (ErPC3) has been reported to induce apoptosis in otherwise highly apoptosis resistant glioma cell lines. The induction of apoptosis by ErPC3 requires TSPO, a mitochondrial membrane protein highly expressed in malignant gliomas. In this preclinical study, we monitored the effect of ErPC3 treatment in vivo using 18 F-DPA-714 PET. Methods: In vitro studies investigated the antitumor effect of ErPC3 in 9L rat gliosarcoma cells. In vivo, glioma-bearing rats were imaged with 18 F-DPA-714 for the time of treatment. Results: A significant decrease in 9L cell proliferation and viability and a significant increase in apoptosis and caspase-3 activation were demonstrated on ErPC3 treatment in cell culture. In the rat model, ErPC3 administration resulted in significant changes in 18 F-DPA-714 tumor uptake over the course of the treatment. Immunohistochemistry revealed reduced tumor volume and increased cell death in ErPC3-treated animals accompanied by infiltration of the tumor core by CD11b-positive micro-glia/macrophages and glial fibrillary acidic protein-positive astrocytes. Conclusion: Our findings demonstrate a potent antitumor effect of ErPC3 in vitro, in vivo, and ex vivo. PET imaging of TSPO expression using 18 F-DPA-714 allows effective monitoring and quantification of disease progression and response to ErPC3 therapy in intracranial 9L gliomas. (authors)

  9. NiF2/NaF:CaF2/Ca Solid-State High-Temperature Battery Cells

    Science.gov (United States)

    West, William; Whitacre, Jay; DelCastillo, Linda

    2009-01-01

    Experiments and theoretical study have demonstrated the promise of all-solid-state, high-temperature electrochemical battery cells based on NiF2 as the active cathode material, CaF2 doped with NaF as the electrolyte material, and Ca as the active anode material. These and other all-solid-state cells have been investigated in a continuing effort to develop batteries for instruments that must operate in environments much hotter than can be withstood by ordinary commercially available batteries. Batteries of this type are needed for exploration of Venus (where the mean surface temperature is about 450 C), and could be used on Earth for such applications as measuring physical and chemical conditions in geothermal wells and oil wells. All-solid-state high-temperature power cells are sought as alternatives to other high-temperature power cells based, variously, on molten anodes and cathodes or molten eutectic salt electrolytes. Among the all-solid-state predecessors of the present NiF2/NaF:CaF2/Ca cells are those described in "Solid-State High-Temperature Power Cells" (NPO-44396), NASA Tech Briefs, Vol. 32, No. 5 (May 2008), page 40. In those cells, the active cathode material is FeS2, the electrolyte material is a crystalline solid solution of equimolar amounts of Li3PO4 and LiSiO4, and the active anode material is Li contained within an alloy that remains solid in the intended high operational temperature range.

  10. Binding of 18F by cell membranes and cell walls of Streptococcus mutans

    International Nuclear Information System (INIS)

    Yotis, W.W.; Zeb, M.; McNulty, J.; Kirchner, F.; Reilly, C.; Glendenin, L.

    1983-01-01

    The binding of 18 F to isolated cell membranes and cell walls of Streptococcus mutans GS-5 or other bacteria was assayed. The attachment of 18 F to these cell envelopes proceeded slowly and reached equilibrium within 60 min. 18 F binding was stimulated by Ca 2+ (1 mM). The binding of 18 F to cellular components was dependent upon the pH, as well as the amount of 18 F and dose of the binder employed. The binding of 18 F by cell walls prepared from fluoride-sensitive and fluoride-resistant cells of S. salivarius and S. mutans did not differ significantly. The pretreatment of cell walls or cell membranes for 60 min at 30 degrees C with 1 mg of RNase, DNase, or trypsin per ml did not influence the binding of 18 F by the walls and membranes of S. mutans GS-5. However, prior exposure of cell membranes to sodium dodecyl sulfate caused a significant reduction in the number of 18 F atoms bound by the membranes. In saturated assay systems, cell membranes of S. mutans GS-5 bound 10(15) to 10(16) atoms of 18 F per mg (dry weight), whereas cell walls from S. mutans GS-5, FA-1, and HS-6 or Actinomyces viscosus T14V and T14AV bound 10(12) to 10(13) atoms of 18 F per mg (dry weight). 18 F in this quantity (10(12) to 10(13) atoms) cannot be detected with the fluoride electrode. The data provide, for the first time, a demonstration of 18 F binding by cell membranes and walls of oral flora

  11. Expression pattern of matrix metalloproteinases in human gynecological cancer cell lines

    International Nuclear Information System (INIS)

    Schröpfer, Andrea; Kammerer, Ulrike; Kapp, Michaela; Dietl, Johannes; Feix, Sonja; Anacker, Jelena

    2010-01-01

    Matrix metalloproteinases (MMPs) are involved in the degradation of protein components of the extracellular matrix and thus play an important role in tumor invasion and metastasis. Their expression is related to the progression of gynecological cancers (e.g. endometrial, cervical or ovarian carcinoma). In this study we investigated the expression pattern of the 23 MMPs, currently known in humans, in different gynecological cancer cell lines. In total, cell lines from three endometrium carcinomas (Ishikawa, HEC-1-A, AN3 CA), three cervical carcinomas (HeLa, Caski, SiHa), three chorioncarcinomas (JEG, JAR, BeWo), two ovarian cancers (BG-1, OAW-42) and one teratocarcinoma (PA-1) were examined. The expression of MMPs was analyzed by RT-PCR, Western blot and gelatin zymography. We demonstrated that the cell lines examined can constitutively express a wide variety of MMPs on mRNA and protein level. While MMP-2, -11, -14 and -24 were widely expressed, no expression was seen for MMP-12, -16, -20, -25, -26, -27 in any of the cell lines. A broad range of 16 MMPs could be found in the PA1 cells and thus this cell line could be used as a positive control for general MMP experiments. While the three cervical cancer cell lines expressed 10-14 different MMPs, the median expression in endometrial and choriocarcinoma cells was 7 different enzymes. The two investigated ovarian cancer cell lines showed a distinctive difference in the number of expressed MMPs (2 vs. 10). Ishikawa, Caski, OAW-42 and BeWo cell lines could be the best choice for all future experiments on MMP regulation and their role in endometrial, cervical, ovarian or choriocarcinoma development, whereas the teratocarcinoma cell line PA1 could be used as a positive control for general MMP experiments

  12. Polarized emission from light-emitting electrochemical cells using uniaxially oriented polymer thin films of poly(9,9-dioctylfluorene-co-bithiophene)

    Science.gov (United States)

    Miyazaki, Masumi; Sakanoue, Tomo; Takenobu, Taishi

    2018-03-01

    Uniaxially oriented poly(9,9-dioctylfluorene-co-bithiophene) (F8T2) films were prepared on rubbed polyimide substrates and applied to emitting layers of light-emitting electrochemical cells (LECs). The layered structure of the uniaxially oriented F8T2 film and ionic liquid electrolytes enabled us to demonstrate LEC operations with high anisotropic characteristics both in emission and charge transport. Polarized electroluminescence (EL) from electrochemically induced p-n junctions in the uniaxially oriented F8T2 was obtained. The dichroic ratios of EL were the same as those of photoluminescence, suggesting that the doping process into the oriented F8T2 did not interrupt the polymer ordering. This indicates the usefulness of the layered structure of the polymer/electrolyte for the fabrication of LECs based on highly oriented polymer films. In addition, uniaxially oriented F8T2 was found to show reduced threshold energy in optically pumped amplified spontaneous emission. These demonstrations suggest the advantage of uniaxially oriented polymer-based LECs for potential application in future electrically pumped lasers.

  13. iTRAQ-based proteomic analysis of LI-F type peptides produced by Paenibacillus polymyxa JSa-9 mode of action against Bacillus cereus.

    Science.gov (United States)

    Han, Jinzhi; Gao, Peng; Zhao, Shengming; Bie, Xiaomei; Lu, Zhaoxin; Zhang, Chong; Lv, Fengxia

    2017-01-06

    LI-F type peptides (AMP-jsa9) produced by Paenibacillus polymyxa JSa-9 are a group of cyclic lipodepsipeptide antibiotics that exhibit a broad antimicrobial spectrum against Gram-positive bacteria and filamentous fungi, especially Bacillus cereus and Fusarium moniliforme. In this study, to better understand the antibacterial mechanism of AMP-jsa9 against B. cereus, the ultrastructure of AMP-jsa9-treated B. cereus cells was observed by both atomic force microscopy and transmission electron microscopy, and quantitative proteomic analysis was performed on proteins extracted from treated and untreated bacterial cells by using isobaric tag for relative and absolute quantitation (iTRAQ) labeling and LC-MS/MS analysis to access differentially expressed proteins. Furthermore, multiple experiments were conducted to validate the results of the proteomic analysis, including determinations of ATP, NAD (+) H, NADP (+) H, reactive oxygen species (ROS), the activities of catalase (CAT) and superoxide dismutase (SOD), and the relative expression of target genes by quantitative real-time PCR. Bacterial cells exposed to AMP-jsa9 showed irregular surfaces with bleb projections and concaves; we hypothesize that AMP-jsa9 penetrated the cell wall and was anchored on the cytoplasmic membrane and that ROS accumulated in the cell membrane after treatment with AMP-jsa9, modulating the bacterial membrane properties and increasing membrane permeability. Consequently, the blebs were formed on the cell wall by the impulsive force of the leakage of intercellular contents. iTRAQ-based proteomic analysis detected a total of 1317 proteins, including 176 differentially expressed proteins (75 upregulated (fold >2) and 101 downregulated (fold AMP-jsa9 action against B. cereus can be summarized as: (i) inhibition of bacterial sporulation, thiamine biosynthesis, energy metabolism, DNA transcription and translation, and cell wall biosynthesis, through direct regulation of protein levels; and (ii

  14. Immunomodulatory and antitumor effects in vivo by the cytoplasmic fraction of Lactobacillus casei and Bifidobacterium longum.

    Science.gov (United States)

    Lee, Jung-Woo; Shin, Jung-Gul; Kim, Eun Hee; Kang, Hae Eun; Yim, In Been; Kim, Ji Yeon; Joo, Hong-Gu; Woo, Hee Jong

    2004-03-01

    The immunomodulatory and antitumor effects of lactic acid bacteria (LABs) were investigated. Cytoplasmic fraction of Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium longum were tested for the antiproliferative activity in vitro to SNUC2A, SNU1, NIH/3T3 and Jurkat cell lines by crystal violet assay. All cytoplasmic fraction suppressed proliferation of tumor cells, though L. casei and B. longum were more effective. From these results, cytoplasmic fraction of L. casei and B. longum with Y400 as a control were administered as dietary supplements to Balb/c mice for 2, and 4 consecutive wks. Administration for 4 wks enhanced the number of total T cells, NK cells and MHC class II+ cells, and CD4-CD8+ T cells in flow cytometry analysis. To determine of antitumor activity of LABs preparation in vivo, F9 teratocarcinoma cells were inoculated on mice at 14th day. Body weight was decreased with increased survival rate in all groups with the cytoplasm of LABs. Our results showed that cytoplasmic fraction of LABs had direct antiproliferative effects on tumor cell lines in vitro, effects on immune cells in vivo, and antitumor effects on tumor-bearing mice with prolonged survival periods.

  15. The clinical impact of {sup 18}F-FDG PET/CT in extracranial pediatric germ cell tumors

    Energy Technology Data Exchange (ETDEWEB)

    Hart, Adam; Vali, Reza; Marie, Eman; Shammas, Amer [The Hospital for Sick Children and University of Toronto, Department of Medical Imaging, Nuclear Medicine, Toronto, ON (Canada); Shaikh, Furqan [The Hospital for Sick Children and University of Toronto, Division of Haematology and oncology, Toronto, ON (Canada)

    2017-10-15

    Extracranial germ cell tumors are an uncommon pediatric malignancy with limited information on the clinical impact of {sup 18}F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) in the literature. The purpose of this study was to evaluate and compare the clinical impact on management of {sup 18}F-FDG PET/CT with diagnostic computed tomography (CT) in pediatric extracranial germ cell tumor. The list of {sup 18}F-FDG PET/CT performed for extracranial germ cell tumor between May 2007 and November 2015 was obtained from the nuclear medicine database. {sup 18}F-FDG PET/CT and concurrent diagnostic CT were obtained and independently reviewed. Additionally, the patients' charts were reviewed for duration of follow-up and biopsy when available. The impact of {sup 18}F-FDG PET/CT compared with diagnostic CT on staging and patient management was demonstrated by chart review, imaging findings and follow-up studies. During the study period, 9 children (5 males and 4 females; age range: 1.6-17 years, mode age: 14 years) had 11 {sup 18}F-FDG PET/CT studies for the evaluation of germ cell tumor. Diagnostic CTs were available for comparison in 8 patients (10 {sup 18}F-FDG PET/CT studies). The average interval between diagnostic CT and PET/CT was 7.2 days (range: 0-37 days). In total, five lesions concerning for active malignancy were identified on diagnostic CT while seven were identified on PET/CT. Overall, {sup 18}F-FDG PET/CT resulted in a change in management in 3 of the 9 patients (33%). {sup 18}F-FDG PET/CT had a significant impact on the management of pediatric germ cell tumors in this retrospective study. Continued multicenter studies are required secondary to the rarity of this tumor to demonstrate the benefit of {sup 18}F-FDG PET/CT in particular clinical scenarios. (orig.)

  16. Exploring Alternative Radiolabeling Strategies for Sialic Acid-Binding Immunoglobulin-Like Lectin 9 Peptide: [68Ga]Ga- and [18F]AlF-NOTA-Siglec-9

    Directory of Open Access Journals (Sweden)

    Olli Moisio

    2018-01-01

    Full Text Available Amino acid residues 283–297 from sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9 form a cyclic peptide ligand targeting vascular adhesion protein-1 (VAP-1. VAP-1 is associated with the transfer of leukocytes from blood to tissues upon inflammation. Therefore, analogs of Siglec-9 peptide are good candidates for visualizing inflammation non-invasively using positron emission tomography (PET. Gallium-68-labeled 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA-conjugated Siglec-9 has been evaluated extensively for this purpose. Here, we explored two alternative strategies for radiolabeling Siglec-9 peptide using a 1,4,7-triazacyclononane-triacetic acid (NOTA-chelator to bind [68Ga]Ga or [18F]AlF. The radioligands were evaluated by in vivo PET imaging and ex vivo γ-counting of turpentine-induced sterile skin/muscle inflammation in Sprague-Dawley rats. Both tracers showed clear accumulation in the inflamed tissues. The whole-body biodistribution patterns of the tracers were similar.

  17. Nesprin-2 epsilon: A novel nesprin isoform expressed in human ovary and Ntera-2 cells

    International Nuclear Information System (INIS)

    Lam, Le Thanh; Boehm, Sabrina V.; Roberts, Roland G.; Morris, Glenn E.

    2011-01-01

    Highlights: → A novel epsilon isoform of nesprin-2 has been discovered. → This 120 kDa protein was predicted by bioinformatic analysis, but has not previously been observed. → It is the main isoform expressed in a teratocarcinoma cell line and is also found in ovary. → Like other nesprins, it is located at the nuclear envelope. → We suggest it may have a role in very early development or in some ovary-specific function. -- Abstract: The nuclear envelope-associated cytoskeletal protein, nesprin-2, is encoded by a large gene containing several internal promoters that produce shorter isoforms. In a study of Ntera-2 teratocarcinoma cells, a novel isoform, nesprin-2-epsilon, was found to be the major mRNA and protein product of the nesprin-2 gene. Its existence was predicted by bioinformatic analysis, but this is the first direct demonstration of both the mRNA and the 120 kDa protein which is located at the nuclear envelope. In a panel of 21 adult and foetal human tissues, the nesprin-2-epsilon mRNA was strongly expressed in ovary but was a minor isoform elsewhere. The expression pattern suggests a possible link with very early development and a likely physiological role in ovary.

  18. Functional expression of BMP7 receptors in oral epithelial cells. Interleukin-17F production in response to BMP7.

    Science.gov (United States)

    Nishio, Kensuke; Ozawa, Yasumasa; Ito, Hisanori; Kifune, Takashi; Narita, Tatsuya; Iinuma, Toshimitsu; Gionhaku, Nobuhito; Asano, Masatake

    2017-10-01

    Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) superfamily. Recently, BMP7 has been demonstrated to be produced by salivary glands and contribute to embryonic branching in mice. The BMP7 in saliva is thought to be delivered to the oral cavity and is expected to contact with stratified squamous epithelial cells which line the surface of oral mucosa. In this study, we attempted to investigate the effects of BMP7 on oral epithelial cells. The expression of BMP receptors was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). OSCCs were stimulated with human recombinant BMP7 (hrBMP7) and the phosphorylation status of Smad1/5/8 was examined by western blotting. For microarray analysis, Ca9-22 cells were stimulated with 100 ng/mL of hrBMP7 and total RNA was extracted and subjected to real-time PCR. The 5'-untranslated region (5'-UTR) of IL-17 F gene was cloned to pGL4-basic vector and used for luciferase assay. Ca9-22 cells were pre-incubated with DM3189, a specific inhibitor of Smad1/5/8, for inhibition assay. All isoforms of type I and type II BMP receptors were expressed in both Ca9-22 and HSC3 cells and BMP7 stimulation resulted in the phosphorylation of Smad1/5/8 in both cell lines. The microarray analysis revealed the induction of interleukin-17 F (IL-17 F), netrin G2 (NTNG2) and hyaluronan synthase 1 (HAS1). Luciferase assay using the 5'-UTR of the IL-17 F gene revealed transcriptional regulation. Induced IL-17 F production was further confirmed at the protein level by ELISA. Smad1/5/8 inhibitor pretreatment decreased IL-17 F expression levels in the cells.

  19. Toxicity of the mycotoxin fumonisin B1 on the insect Sf9 cell line.

    Science.gov (United States)

    Zhang, He; Zhang, Liyang; Diao, Xue; Li, Na; Liu, Chenglan

    2017-04-01

    Fumonisins are a type of mycotoxin produced by Fusarium spp., mainly F. proliferatum and F. vertieilliodes, and represent a potential hazard to the health of animals and human beings. The toxicity and mechanism of action of fumonisins is ambiguous, and it is unclear whether fumonisins are toxic to insect cells. This study examines the toxicity of fumonisin B 1 (FB 1 ) and its mechanism of action in the Spodoptera frugiperda Sf9 cell line. We found that FB 1 inhibited Sf9 cellular proliferation and arrested cell growth at the G 2 /M phase. Morphological observation showed that FB 1 induced swelling, vacuole formation, and loss of adhesion in Sf9 cells. Flow cytometry analysis showed that FB 1 caused depolarization of the cell membrane potential and hyperpolarization of the mitochondrial membrane potential. To uncover potential genes associated with the molecular mechanisms of FB 1 , 41 differentially expressed genes were identified by transcriptome analyses after FB 1 treatment. These genes are putatively involved in detoxification metabolism, insect hormone regulation, cell apoptosis, and other related processes. Finally, six differentially expressed genes were chosen and validated by quantitative real-time PCR (QRT-PCR). Our test could provide a reference for other kinds of insect cells studies on FB 1 stress. At the same time, our studies try to provide a possible for FB 1 as a precursor compounds of biological insecticide. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Synthesis, in-vitro cytotoxicity of 1H-benzo[f]chromene derivatives and structure-activity relationships of the 1-aryl group and 9-position.

    Science.gov (United States)

    Mohamed, Hany M; Fouda, Ahmed M; Khattab, Essam S A E H; Agrody, Ahmed M El-; Afifi, Tarek H

    2017-05-01

    A series of 1H-benzo[f]chromene-2-carbonitriles was synthesized and evaluated for their cytotoxic activities against MCF-7, HCT-116, and HepG-2 cancer cells. The SAR studies reported that the substitution in the phenyl ring at 1-position of 1H-benzo[f]chromene nucleus with the specific group, H atom, or methoxy group at 9-position increases the ability of the molecule against the different cell lines.

  1. Generation of murine tumor cell lines deficient in MHC molecule surface expression using the CRISPR/Cas9 system.

    Directory of Open Access Journals (Sweden)

    Krishna Das

    Full Text Available In this study, the CRISPR/Cas9 technology was used to establish murine tumor cell lines, devoid of MHC I or MHC II surface expression, respectively. The melanoma cell line B16F10 and the murine breast cancer cell line EO-771, the latter stably expressing the tumor antigen NY-BR-1 (EO-NY, were transfected with an expression plasmid encoding a β2m-specific single guide (sgRNA and Cas9. The resulting MHC I negative cells were sorted by flow cytometry to obtain single cell clones, and loss of susceptibility of peptide pulsed MHC I negative clones to peptide-specific CTL recognition was determined by IFNγ ELISpot assay. The β2m knockout (KO clones did not give rise to tumors in syngeneic mice (C57BL/6N, unless NK cells were depleted, suggesting that outgrowth of the β2m KO cell lines was controlled by NK cells. Using sgRNAs targeting the β-chain encoding locus of the IAb molecule we also generated several B16F10 MHC II KO clones. Peptide loaded B16F10 MHC II KO cells were insusceptible to recognition by OT-II cells and tumor growth was unaltered compared to parental B16F10 cells. Thus, in our hands the CRISPR/Cas9 system has proven to be an efficient straight forward strategy for the generation of MHC knockout cell lines. Such cell lines could serve as parental cells for co-transfection of compatible HLA alleles together with human tumor antigens of interest, thereby facilitating the generation of HLA matched transplantable tumor models, e.g. in HLAtg mouse strains of the newer generation, lacking cell surface expression of endogenous H2 molecules. In addition, our tumor cell lines established might offer a useful tool to investigate tumor reactive T cell responses that function independently from MHC molecule surface expression by the tumor.

  2. Inhibition of VEGF-dependent angiogenesis by the anti-CD82 monoclonal antibody 4F9 through regulation of lipid raft microdomains

    International Nuclear Information System (INIS)

    Nomura, Sayaka; Iwata, Satoshi; Hatano, Ryo; Komiya, Eriko; Dang, Nam H.; Iwao, Noriaki; Ohnuma, Kei; Morimoto, Chikao

    2016-01-01

    CD82 (also known as KAI1) belongs to the tetraspanin superfamily of type III transmembrane proteins, and is involved in regulating cell adhesion, migration and proliferation. In contrast to these well-established roles of CD82 in tumor biology, its function in endothelial cell (EC) activity and tumor angiogenesis is yet to be determined. In this study, we show that suppression of CD82 negatively regulates vascular endothelial growth factor (VEGF)-induced angiogenesis. Moreover, we demonstrate that the anti-CD82 mAb 4F9 effectively inhibits phosphorylation of VEGF receptor 2 (VEGFR2), which is the principal mediator of the VEGF-induced angiogenic signaling process in tumor angiogenesis, by regulating the organization of the lipid raft microdomain signaling platform in human EC. Our present work therefore suggests that CD82 on EC is a potential target for anti-angiogenic therapy in VEGFR2-dependent tumor angiogenesis. -- Highlights: •Knockdown of CD82 decreases EC migration, proliferation and angiogenesis. •Anti-CD82 mAb 4F9 inhibits EC migration, proliferation and angiogenesis. •4F9 inhibits VEGFR2 phosphorylation via control of CD82 distribution in lipid rafts.

  3. Inhibition of VEGF-dependent angiogenesis by the anti-CD82 monoclonal antibody 4F9 through regulation of lipid raft microdomains

    Energy Technology Data Exchange (ETDEWEB)

    Nomura, Sayaka; Iwata, Satoshi; Hatano, Ryo [Division of Clinical Immunology, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639 (Japan); Komiya, Eriko [Department of Therapy Development and Innovation for Immune Disorders and Cancers, Graduate School of Medicine, Juntendo University, 2-1-1, Hongo, Bunkyo-ku, Tokyo, 113-8421 (Japan); Dang, Nam H. [Division of Hematology/Oncology, University of Florida, 1600 SW Archer Road- Box 100278, Room MSB M410A, Gainesville, FL, 32610 (United States); Iwao, Noriaki [Department of Hematology, School of Medicine, Juntendo University, 2-1-1, Hongo, Bunkyo-ku, Tokyo, 113-8421 (Japan); Ohnuma, Kei, E-mail: kohnuma@juntendo.ac.jp [Department of Rheumatology and Allergy, IMSUT Hospital, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639 (Japan); Morimoto, Chikao [Division of Clinical Immunology, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639 (Japan); Department of Rheumatology and Allergy, IMSUT Hospital, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639 (Japan)

    2016-05-20

    CD82 (also known as KAI1) belongs to the tetraspanin superfamily of type III transmembrane proteins, and is involved in regulating cell adhesion, migration and proliferation. In contrast to these well-established roles of CD82 in tumor biology, its function in endothelial cell (EC) activity and tumor angiogenesis is yet to be determined. In this study, we show that suppression of CD82 negatively regulates vascular endothelial growth factor (VEGF)-induced angiogenesis. Moreover, we demonstrate that the anti-CD82 mAb 4F9 effectively inhibits phosphorylation of VEGF receptor 2 (VEGFR2), which is the principal mediator of the VEGF-induced angiogenic signaling process in tumor angiogenesis, by regulating the organization of the lipid raft microdomain signaling platform in human EC. Our present work therefore suggests that CD82 on EC is a potential target for anti-angiogenic therapy in VEGFR2-dependent tumor angiogenesis. -- Highlights: •Knockdown of CD82 decreases EC migration, proliferation and angiogenesis. •Anti-CD82 mAb 4F9 inhibits EC migration, proliferation and angiogenesis. •4F9 inhibits VEGFR2 phosphorylation via control of CD82 distribution in lipid rafts.

  4. Long non-coding RNA TUG1 contributes to tumorigenesis of human osteosarcoma by sponging miR-9-5p and regulating POU2F1 expression.

    Science.gov (United States)

    Xie, Chu-Hai; Cao, Yan-Ming; Huang, Yan; Shi, Qun-Wei; Guo, Jian-Hong; Fan, Zi-Wen; Li, Ju-Gen; Chen, Bin-Wei; Wu, Bo-Yi

    2016-11-01

    Recent studies have shown that long non-coding RNAs (lncRNAs) have critical roles in tumorigenesis, including osteosarcoma. The lncRNA taurine-upregulated gene 1 (TUG1) was reported to be involved in the progression of osteosarcoma. Here, we investigated the role of TUG1 in osteosarcoma cells and the underlying mechanism. TUG1 expression was measured in osteosarcoma cell lines and human normal osteoblast cells by quantitative real-time PCR (qRT-PCR). The effects of TUG1 on osteosarcoma cells were studied by RNA interference in vitro and in vivo. The mechanism of competing endogenous RNA (ceRNA) was determined using bioinformatic analysis and luciferase assays. Our data showed that TUG1 knockdown inhibited cell proliferation and colony formation, and induced G0/G1 cell cycle arrest and apoptosis in vitro, and suppressed tumor growth in vivo. Besides, we found that TUG1 acted as an endogenous sponge to directly bind to miR-9-5p and downregulated miR-9-5p expression. Moreover, TUG1 overturned the effect of miR-9-5p on the proliferation, colony formation, cell cycle arrest, and apoptosis in osteosarcoma cells, which involved the derepression of POU class 2 homeobox 1 (POU2F1) expression. In conclusion, our study elucidated a novel TUG1/miR-9-5p/POU2F1 pathway, in which TUG1 acted as a ceRNA by sponging miR-9-5p, leading to downregulation of POU2F1 and facilitating the tumorigenesis of osteosarcoma. These findings may contribute to the lncRNA-targeted therapy for human osteosarcoma.

  5. Comparison of [18F]FLT and [18F]FDG in in vitro cancer cell uptake and glucose effect

    International Nuclear Information System (INIS)

    Soo Jung Lim; Jin-Sook Ryu; Heuiran Lee; Seok Young Kim; Seung Jun Oh; Dae Hyuk Moon

    2004-01-01

    [18F]FLT is a new radiopharmaceutical for cell proliferation. We compared [18F]FLT and [18F]FDG in in vitro cancer cell uptake and glucose effect. Method: In vitro cancer cell uptake of [18F]FLT was evaluated using SCC7(mouse squamous cell carcinoma). At 24 hours after seeding 1 x 106 cells/well in 6 well plates with RPMI 1640 medium, culture media were changed to medium with glucose free or glucose concentration of 100 mg/dl. Then, [18F]FLT 5 μCi/50 ml was added to each well. After incubation for 30, 60, 90, 120 minutes, cells were washed twice by PBS, and harvested using 0.25% trypsin-EDTA. After centrifugation and counting at gamma counter, cell uptake was calculated by % activity of cellular uptake to total activity of cell and supernatant. For comparison, same tumor cell uptake experiment was performed with [18F]FDG. Results: After incubation with SCC7 cell line for 30, 60, 90, 120 minutes, [18F]FLT showed 1.95%, 2.17%, 2.10% and 2.80% of cell uptake in glucose free media, respectively. The results [18F]FLT uptake in glucose 100 mg/dl media were 1.82%, 1.87%, 1.97%, and 2.94%, respectively. The results of [18F]FDG in glucose free media were 2.50%, 3.47%, 5.04%, and 10.4%, whereas those in glucose 100 mg/dl media were 1.60%, 1.79%, 1.53%, and 1.82%, respectively. Conclusion: In contrast to [18F]FDG, [18F]FLT uptake in cancer cell was not affected by glucose concentration. In physiologic glucose concentration, [18F]FLT uptake in SCC7 cell line was significantly higher than [18F]FDG uptake after 120 minutes incubation. In [18F]FLT PET imaging may not need fasting for preparation before imaging study. (authors)

  6. 18F-FET and 18F-FCH uptake in human glioblastoma T98G cell lines

    International Nuclear Information System (INIS)

    Persico, Marco Giovanni; Buroni, Federica Eleonora; Pasi, Francesca; Lodola, Lorenzo; Aprile, Carlo; Nano, Rosanna; Hodolic, Marina

    2016-01-01

    Despite complex treatment of surgery, radiotherapy and chemotherapy, high grade gliomas often recur. Differentiation between post-treatment changes and recurrence is difficult. 18 F-methyl-choline ( 18 F-FCH) is frequently used in staging and detection of recurrent prostate cancer disease as well as some brain tumours; however accumulation in inflammatory tissue limits its specificity. The 18 F-ethyl-tyrosine ( 18 F-FET) shows a specific uptake in malignant cells, resulting from increased expression of amino acid transporters or diffusing through the disrupted blood-brain barrier. 18 F-FET exhibits lower uptake in machrophages and other inflammatory cells. Aim of this study was to evaluate 18 F-FCH and 18 F-FET uptake by human glioblastoma T98G cells. Human glioblastoma T98G or human dermal fibroblasts cells, seeded at a density to obtain 2 × 10 5 cells per flask when radioactive tracers were administered, grew adherent to the plastic surface at 37°C in 5% CO 2 in complete medium. Equimolar amounts of radiopharmaceuticals were added to cells for different incubation times (20 to 120 minutes) for 18 F-FCH and 18 F-FET respectively. The cellular radiotracer uptake was determined with a gamma counter. All experiments were carried out in duplicate and repeated three times. The uptake measurements are expressed as the percentage of the administered dose of tracer per 2 × 10 5 cells. Data (expressed as mean values of % uptake of radiopharmaceuticals) were compared using parametric or non-parametric tests as appropriate. Differences were regarded as statistically significant when p<0.05. A significant uptake of 18 F-FCH was seen in T98G cells at 60, 90 and 120 minutes. The percentage uptake of 18 F-FET in comparison to 18 F-FCH was lower by a factor of more than 3, with different kinetic curves. 18 F-FET showed a more rapid initial uptake up to 40 minutes and 18 F-FCH showed a progressive rise reaching a maximum after 90 minutes. 18 F-FCH and 18 F-FET are candidates

  7. Curcumin inhibits oral squamous cell carcinoma SCC-9 cells proliferation by regulating miR-9 expression

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Can [Department of Occupational Medicine and Environmental Health, School of Public Health, Soochow University, Suzhou 215123 (China); Department of Stomatology, The First Affiliated Hospital of Soochow University, Suzhou 215006 (China); Wang, Lili; Zhu, Lifang [Department of Stomatology, The First Affiliated Hospital of Soochow University, Suzhou 215006 (China); Zhang, Chenping, E-mail: zhang_cping@163.com [Department of Head and Neck Tumors, Shanghai Ninth People’s Hospital Affiliated Shanghai JiaoTong University School of Medicine, Shanghai 200011 (China); Zhou, Jianhua [Department of Occupational Medicine and Environmental Health, School of Public Health, Soochow University, Suzhou 215123 (China)

    2014-11-28

    Highlights: • miR-9 expression level was significantly decreased in OSCC tissues. • Curcumin significantly inhibited SCC-9 cells proliferation. • miR-9 mediates the inhibition of SCC-9 proliferation by curcumin. • Curcumin suppresses Wnt/β-catenin signaling in SCC-9 cells. • miR-9 mediates the suppression of Wnt/β-catenin signaling by curcumin. - Abstract: Curcumin, a phytochemical derived from the rhizome of Curcuma longa, has shown anticancer effects against a variety of tumors. In the present study, we investigated the effects of curcumin on the miR-9 expression in oral squamous cell carcinoma (OSCC) and explored the potential relationships between miR-9 and Wnt/β-catenin pathway in curcumin-mediated OSCC inhibition in vitro. As the results shown, the expression levels of miR-9 were significantly lower in clinical OSCC specimens than those in the adjacent non-tumor tissues. Furthermore, our results indicated that curcumin inhibited OSCC cells (SCC-9 cells) proliferation through up-regulating miR-9 expression, and suppressing Wnt/β-catenin signaling by increasing the expression levels of the GSK-3β, phosphorylated GSK-3β and β-catenin, and decreasing the cyclin D1 level. Additionally, the up-regulation of miR-9 by curcumin in SCC-9 cells was significantly inhibited by delivering anti-miR-9 but not control oligonucleotides. Downregulation of miR-9 by anti-miR-9 not only attenuated the growth-suppressive effects of curcumin on SCC-9 cells, but also re-activated Wnt/β-catenin signaling that was inhibited by curcumin. Therefore, our findings would provide a new insight into the use of curcumin against OSCC in future.

  8. Curcumin inhibits oral squamous cell carcinoma SCC-9 cells proliferation by regulating miR-9 expression

    International Nuclear Information System (INIS)

    Xiao, Can; Wang, Lili; Zhu, Lifang; Zhang, Chenping; Zhou, Jianhua

    2014-01-01

    Highlights: • miR-9 expression level was significantly decreased in OSCC tissues. • Curcumin significantly inhibited SCC-9 cells proliferation. • miR-9 mediates the inhibition of SCC-9 proliferation by curcumin. • Curcumin suppresses Wnt/β-catenin signaling in SCC-9 cells. • miR-9 mediates the suppression of Wnt/β-catenin signaling by curcumin. - Abstract: Curcumin, a phytochemical derived from the rhizome of Curcuma longa, has shown anticancer effects against a variety of tumors. In the present study, we investigated the effects of curcumin on the miR-9 expression in oral squamous cell carcinoma (OSCC) and explored the potential relationships between miR-9 and Wnt/β-catenin pathway in curcumin-mediated OSCC inhibition in vitro. As the results shown, the expression levels of miR-9 were significantly lower in clinical OSCC specimens than those in the adjacent non-tumor tissues. Furthermore, our results indicated that curcumin inhibited OSCC cells (SCC-9 cells) proliferation through up-regulating miR-9 expression, and suppressing Wnt/β-catenin signaling by increasing the expression levels of the GSK-3β, phosphorylated GSK-3β and β-catenin, and decreasing the cyclin D1 level. Additionally, the up-regulation of miR-9 by curcumin in SCC-9 cells was significantly inhibited by delivering anti-miR-9 but not control oligonucleotides. Downregulation of miR-9 by anti-miR-9 not only attenuated the growth-suppressive effects of curcumin on SCC-9 cells, but also re-activated Wnt/β-catenin signaling that was inhibited by curcumin. Therefore, our findings would provide a new insight into the use of curcumin against OSCC in future

  9. Analysis list: E2f4 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available E2f4 Blood,Embryonic fibroblast,Liver,Muscle,Pluripotent stem cell + mm9 http://dba...rchive.biosciencedbc.jp/kyushu-u/mm9/target/E2f4.1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/E2f4....5.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/E2f4.10.tsv http://dbarchive.biosciencedbc....jp/kyushu-u/mm9/colo/E2f4.Blood.tsv,http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/E2f4....Embryonic_fibroblast.tsv,http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/E2f4.Liver.tsv,htt

  10. Sulforaphane induces cell cycle arrest by protecting RB-E2F-1 complex in epithelial ovarian cancer cells

    Directory of Open Access Journals (Sweden)

    Morris Robert

    2010-03-01

    Full Text Available Abstract Background Sulforaphane (SFN, an isothiocyanate phytochemical present predominantly in cruciferous vegetables such as brussels sprout and broccoli, is considered a promising chemo-preventive agent against cancer. In-vitro exposure to SFN appears to result in the induction of apoptosis and cell-cycle arrest in a variety of tumor types. However, the molecular mechanisms leading to the inhibition of cell cycle progression by SFN are poorly understood in epithelial ovarian cancer cells (EOC. The aim of this study is to understand the signaling mechanisms through which SFN influences the cell growth and proliferation in EOC. Results SFN at concentrations of 5 - 20 μM induced a dose-dependent suppression of growth in cell lines MDAH 2774 and SkOV-3 with an IC50 of ~8 μM after a 3 day exposure. Combination treatment with chemotherapeutic agent, paclitaxel, resulted in additive growth suppression. SFN at ~8 μM decreased growth by 40% and 20% on day 1 in MDAH 2774 and SkOV-3, respectively. Cells treated with cytotoxic concentrations of SFN have reduced cell migration and increased apoptotic cell death via an increase in Bak/Bcl-2 ratio and cleavage of procaspase-9 and poly (ADP-ribose-polymerase (PARP. Gene expression profile analysis of cell cycle regulated proteins demonstrated increased levels of tumor suppressor retinoblastoma protein (RB and decreased levels of E2F-1 transcription factor. SFN treatment resulted in G1 cell cycle arrest through down modulation of RB phosphorylation and by protecting the RB-E2F-1 complex. Conclusions SFN induces growth arrest and apoptosis in EOC cells. Inhibition of retinoblastoma (RB phosphorylation and reduction in levels of free E2F-1 appear to play an important role in EOC growth arrest.

  11. Cystatin F as a regulator of immune cell cytotoxicity.

    Science.gov (United States)

    Kos, Janko; Nanut, Milica Perišić; Prunk, Mateja; Sabotič, Jerica; Dautović, Esmeralda; Jewett, Anahid

    2018-05-10

    Cysteine cathepsins are lysosomal peptidases involved in the regulation of innate and adaptive immune responses. Among the diverse processes, regulation of granule-dependent cytotoxicity of cytotoxic T-lymphocytes (CTLs) and natural killer (NK) cells during cancer progression has recently gained significant attention. The function of cysteine cathepsins is regulated by endogenous cysteine protease inhibitors-cystatins. Whereas other cystatins are generally cytosolic or extracellular proteins, cystatin F is present in endosomes and lysosomes and is thus able to regulate the activity of its target directly. It is delivered to endosomal/lysosomal vesicles as an inactive, disulphide-linked dimer. Proteolytic cleavage of its N-terminal part leads to the monomer, the only form that is a potent inhibitor of cathepsins C, H and L, involved in the activation of granzymes and perforin. In NK cells and CTLs the levels of active cathepsin C and of granzyme B are dependent on the concentration of monomeric, active cystatin F. In tumour microenvironment, inactive dimeric cystatin F can be secreted from tumour cells or immune cells and further taken up by the cytotoxic cells. Subsequent monomerization and inhibition of cysteine cathepsins within the endosomal/lysosomal vesicles impairs granzyme and perforin activation, and provokes cell anergy. Further, the glycosylation pattern has been shown to be important in controlling secretion of cystatin F from target cells, as well as internalization by cytotoxic cells and trafficking to endosomal/lysosomal vesicles. Cystatin F is therefore an important mediator used by bystander cells to reduce NK and T-cell cytotoxicity.

  12. Increased cardiogenesis in P19-GFP teratocarcinoma cells expressing the propeptide IGF-1Ea

    Energy Technology Data Exchange (ETDEWEB)

    Poudel, Bhawana [Heart Science Centre, National Heart and Lung Institute, Imperial College, London (United Kingdom); Bilbao, Daniel [EMBL, Mouse Biology Unit, Monterotondo (Italy); Sarathchandra, Padmini; Germack, Renee [Heart Science Centre, National Heart and Lung Institute, Imperial College, London (United Kingdom); Rosenthal, Nadia [Heart Science Centre, National Heart and Lung Institute, Imperial College, London (United Kingdom); Australian Regenerative Medicine Institute, Monash University, Melbourne (Australia); Santini, Maria Paola, E-mail: m.santini@imperial.ac.uk [Heart Science Centre, National Heart and Lung Institute, Imperial College, London (United Kingdom)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer In this study, we explored the function of IGF-1Ea propeptide in inducing cardiogenesis of stem cells. Black-Right-Pointing-Pointer IGF-1Ea promoted cardiac mesodermal induction in uncommitted cells. Black-Right-Pointing-Pointer Under differentiation condition, IGF-1Ea increased expression of cardiac differentiation markers. Black-Right-Pointing-Pointer Furthermore, it promoted formation of finely organized sarcomeric structure. Black-Right-Pointing-Pointer IGF-1Ea propeptide may be a good candidate to improve production of cardiomyocytes from pluripotent cells. -- Abstract: The mechanism implicated in differentiation of endogenous cardiac stem cells into cardiomyocytes to regenerate the heart tissue upon an insult remains elusive, limiting the therapeutical goals to exogenous cell injection and/or gene therapy. We have shown previously that cardiac specific overexpression of the insulin-like growth factor 1 propeptide IGF-1Ea induces beneficial myocardial repair after infarct. Although the mechanism is still under investigation, the possibility that this propeptide may be involved in promoting stem cell differentiation into the cardiac lineage has yet to be explored. To investigate whether IGF-1Ea promote cardiogenesis, we initially modified P19 embryonal carcinoma cells to express IGF-1Ea. Taking advantage of their cardiomyogenic nature, we analyzed whether overexpression of this propeptide affected cardiac differentiation program. The data herein presented showed for the first time that constitutively overexpressed IGF-1Ea increased cardiogenic differentiation program in both undifferentiated and DMSO-differentiated cells. In details, IGF-1Ea overexpression promoted localization of alpha-actinin in finely organized sarcomeric structure compared to control cells and upregulated the cardiac mesodermal marker NKX-2.5 and the ventricular structural protein MLC2v. Furthermore, activated IGF-1 signaling promoted cardiac

  13. Increased cardiogenesis in P19-GFP teratocarcinoma cells expressing the propeptide IGF-1Ea

    International Nuclear Information System (INIS)

    Poudel, Bhawana; Bilbao, Daniel; Sarathchandra, Padmini; Germack, Renee; Rosenthal, Nadia; Santini, Maria Paola

    2011-01-01

    Highlights: ► In this study, we explored the function of IGF-1Ea propeptide in inducing cardiogenesis of stem cells. ► IGF-1Ea promoted cardiac mesodermal induction in uncommitted cells. ► Under differentiation condition, IGF-1Ea increased expression of cardiac differentiation markers. ► Furthermore, it promoted formation of finely organized sarcomeric structure. ► IGF-1Ea propeptide may be a good candidate to improve production of cardiomyocytes from pluripotent cells. -- Abstract: The mechanism implicated in differentiation of endogenous cardiac stem cells into cardiomyocytes to regenerate the heart tissue upon an insult remains elusive, limiting the therapeutical goals to exogenous cell injection and/or gene therapy. We have shown previously that cardiac specific overexpression of the insulin-like growth factor 1 propeptide IGF-1Ea induces beneficial myocardial repair after infarct. Although the mechanism is still under investigation, the possibility that this propeptide may be involved in promoting stem cell differentiation into the cardiac lineage has yet to be explored. To investigate whether IGF-1Ea promote cardiogenesis, we initially modified P19 embryonal carcinoma cells to express IGF-1Ea. Taking advantage of their cardiomyogenic nature, we analyzed whether overexpression of this propeptide affected cardiac differentiation program. The data herein presented showed for the first time that constitutively overexpressed IGF-1Ea increased cardiogenic differentiation program in both undifferentiated and DMSO-differentiated cells. In details, IGF-1Ea overexpression promoted localization of alpha-actinin in finely organized sarcomeric structure compared to control cells and upregulated the cardiac mesodermal marker NKX-2.5 and the ventricular structural protein MLC2v. Furthermore, activated IGF-1 signaling promoted cardiac mesodermal induction in undifferentiated cells independently of cell proliferation. This analysis suggests that IGF-1Ea may be a

  14. Tribulus terrestris Alters the Expression of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 in Rabbit Ovaries of Mothers and F1 Female Offspring

    Science.gov (United States)

    2016-01-01

    Although previous research has demonstrated the key role of the oocyte-derived factors, bone morphogenetic protein (BMP) 15 and growth differentiation factor (GDF) 9, in follicular development and ovulation, there is a lack of knowledge on the impact of external factors, which females are exposed to during folliculogenesis, on their expression. The present study investigated the effect of the aphrodisiac Tribulus terrestris on the GDF9 and BMP15 expression in the oocytes and cumulus cells at mRNA and protein levels during folliculogenesis in two generations of female rabbits. The experiment was conducted with 28 New Zealand rabbits. Only the diet of the experimental mothers group was supplemented with a dry extract of T. terrestris for the 45 days prior to insemination. The expression of BMP15 and GDF9 genes in the oocytes and cumulus cells of mothers and F1 female offspring was analyzed using real-time polymerase chain reaction (RT-PCR). The localization of the GDF9 and BMP15 proteins in the ovary tissues was determined by immunohistochemical analysis. The BMP15 and GDF9 transcripts were detected in the oocytes and cumulus cells of rabbits from all groups. T. terrestris caused a decrease in the BMP15 mRNA level in the oocytes and an increase in the cumulus cells. The GDF9 mRNA level increased significantly in both oocytes and cumulus cells. The downregulated expression of BMP15 in the treated mothers’ oocytes was inherited in the F1 female offspring born to treated mothers. BMP15 and GDF9 show a clearly expressed sensitivity to the bioactive compounds of T. terrestris. PMID:26928288

  15. Tribulus terrestris Alters the Expression of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 in Rabbit Ovaries of Mothers and F1 Female Offspring.

    Directory of Open Access Journals (Sweden)

    Desislava Abadjieva

    Full Text Available Although previous research has demonstrated the key role of the oocyte-derived factors, bone morphogenetic protein (BMP 15 and growth differentiation factor (GDF 9, in follicular development and ovulation, there is a lack of knowledge on the impact of external factors, which females are exposed to during folliculogenesis, on their expression. The present study investigated the effect of the aphrodisiac Tribulus terrestris on the GDF9 and BMP15 expression in the oocytes and cumulus cells at mRNA and protein levels during folliculogenesis in two generations of female rabbits. The experiment was conducted with 28 New Zealand rabbits. Only the diet of the experimental mothers group was supplemented with a dry extract of T. terrestris for the 45 days prior to insemination. The expression of BMP15 and GDF9 genes in the oocytes and cumulus cells of mothers and F1 female offspring was analyzed using real-time polymerase chain reaction (RT-PCR. The localization of the GDF9 and BMP15 proteins in the ovary tissues was determined by immunohistochemical analysis. The BMP15 and GDF9 transcripts were detected in the oocytes and cumulus cells of rabbits from all groups. T. terrestris caused a decrease in the BMP15 mRNA level in the oocytes and an increase in the cumulus cells. The GDF9 mRNA level increased significantly in both oocytes and cumulus cells. The downregulated expression of BMP15 in the treated mothers' oocytes was inherited in the F1 female offspring born to treated mothers. BMP15 and GDF9 show a clearly expressed sensitivity to the bioactive compounds of T. terrestris.

  16. Tribulus terrestris Alters the Expression of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 in Rabbit Ovaries of Mothers and F1 Female Offspring.

    Science.gov (United States)

    Abadjieva, Desislava; Kistanova, Elena

    2016-01-01

    Although previous research has demonstrated the key role of the oocyte-derived factors, bone morphogenetic protein (BMP) 15 and growth differentiation factor (GDF) 9, in follicular development and ovulation, there is a lack of knowledge on the impact of external factors, which females are exposed to during folliculogenesis, on their expression. The present study investigated the effect of the aphrodisiac Tribulus terrestris on the GDF9 and BMP15 expression in the oocytes and cumulus cells at mRNA and protein levels during folliculogenesis in two generations of female rabbits. The experiment was conducted with 28 New Zealand rabbits. Only the diet of the experimental mothers group was supplemented with a dry extract of T. terrestris for the 45 days prior to insemination. The expression of BMP15 and GDF9 genes in the oocytes and cumulus cells of mothers and F1 female offspring was analyzed using real-time polymerase chain reaction (RT-PCR). The localization of the GDF9 and BMP15 proteins in the ovary tissues was determined by immunohistochemical analysis. The BMP15 and GDF9 transcripts were detected in the oocytes and cumulus cells of rabbits from all groups. T. terrestris caused a decrease in the BMP15 mRNA level in the oocytes and an increase in the cumulus cells. The GDF9 mRNA level increased significantly in both oocytes and cumulus cells. The downregulated expression of BMP15 in the treated mothers' oocytes was inherited in the F1 female offspring born to treated mothers. BMP15 and GDF9 show a clearly expressed sensitivity to the bioactive compounds of T. terrestris.

  17. The promoter for intestinal cell kinase is head-to-head with F-Box 9 and contains functional sites for TCF7L2 and FOXA factors

    Directory of Open Access Journals (Sweden)

    Cohn Steven M

    2010-05-01

    Full Text Available Abstract Background Intestinal cell kinase (ICK; GeneID 22858 is a conserved MAPK and CDK-like kinase that is widely expressed in human tissues. Data from the Cancer Genome Anatomy Project indicated ICK mRNA is increased in cancer, and that its expression correlated with expression of mRNA for an uncharacterized F-box protein, FBX9 (GeneID: 26268. ICK and FBX9 genes are arranged head-to-head on opposite strands, with start sites for transcription separated by ~3.3 kb. We hypothesized ICK and FBX9 are potentially important genes in cancer controlled by a bidirectional promoter. Results We assessed promoter activity of the intergenic region in both orientations in cancer cell lines derived from breast (AU565, SKBR3, colon (HCT-15, KM12, and stomach (AGS cancers, as well as in embryonic human kidney (HEK293T cells. The intergenic segment was active in both orientations in all of these lines, and ICK promoter activity was greater than FBX9 promoter activity. Results from deletions and truncations defined a minimal promoter for ICK, and revealed that repressors and enhancers differentially regulate ICK versus FBX9 promoter activity. The ICK promoter contains consensus motifs for several FOX-family transcription factors that align when mouse and human are compared using EMBOSS. FOXA1 and FOXA2 increase luciferase activity of a minimal promoter 10-20 fold in HEK293T cells. Consensus sites for TCF7L2 (TCF4 (Gene Id: 6934 are also present in both mouse and human. The expression of β-catenin increased activity of the minimal promoter ~10 fold. ICK reference mRNAs (NM_014920.3, NM_016513 are expressed in low copy number and increased in some breast cancers, using a ten base tag 5'-TCAACCTTAT-3' specific for both ICK transcripts. Conclusion ICK and FBX9 are divergently transcribed from a bidirectional promoter that is GC-rich and contains a CpG island. A minimal promoter for ICK contains functional sites for β-cateinin/TCF7L2 and FOXA. These data are

  18. Pseudomonas orientalis F9: A Potent Antagonist against Phytopathogens with Phytotoxic Effect in the Apple Flower

    Directory of Open Access Journals (Sweden)

    Veronika Zengerer

    2018-02-01

    Full Text Available In light of public concerns over the use of pesticides and antibiotics in plant protection and the subsequent selection for spread of resistant bacteria in the environment, it is inevitable to broaden our knowledge about viable alternatives, such as natural antagonists and their mode of action. The genus Pseudomonas is known for its metabolic versatility and genetic plasticity, encompassing pathogens as well as antagonists. We characterized strain Pseudomonas orientalis F9, an isolate from apple flowers in a Swiss orchard, and determined its antagonistic activity against several phytopathogenic bacteria, in particular Erwinia amylovora, the causal agent of fire blight. P. orientalis F9 displayed antagonistic activity against a broad suite of phytopathogenic bacteria in the in vitro tests. The promising results from this analysis led to an ex vivo assay with E. amylovora CFBP1430Rif and P. orientalis F9 infected detached apple flowers. F9 diminished the fire blight pathogen in the flowers but also revealed phytotoxic traits. The experimental results were discussed in light of the complete genome sequence of F9, which revealed the strain to carry phenazine genes. Phenazines are known to contribute to antagonistic activity of bacterial strains against soil pathogens. When tested in the cress assay with Pythium ultimum as pathogen, F9 showed results comparable to the known antagonist P. protegens CHA0.

  19. Pseudomonas orientalis F9: A Potent Antagonist against Phytopathogens with Phytotoxic Effect in the Apple Flower.

    Science.gov (United States)

    Zengerer, Veronika; Schmid, Michael; Bieri, Marco; Müller, Denise C; Remus-Emsermann, Mitja N P; Ahrens, Christian H; Pelludat, Cosima

    2018-01-01

    In light of public concerns over the use of pesticides and antibiotics in plant protection and the subsequent selection for spread of resistant bacteria in the environment, it is inevitable to broaden our knowledge about viable alternatives, such as natural antagonists and their mode of action. The genus Pseudomonas is known for its metabolic versatility and genetic plasticity, encompassing pathogens as well as antagonists. We characterized strain Pseudomonas orientalis F9, an isolate from apple flowers in a Swiss orchard, and determined its antagonistic activity against several phytopathogenic bacteria, in particular Erwinia amylovora , the causal agent of fire blight. P. orientalis F9 displayed antagonistic activity against a broad suite of phytopathogenic bacteria in the in vitro tests. The promising results from this analysis led to an ex vivo assay with E. amylovora CFBP1430 Rif and P. orientalis F9 infected detached apple flowers. F9 diminished the fire blight pathogen in the flowers but also revealed phytotoxic traits. The experimental results were discussed in light of the complete genome sequence of F9, which revealed the strain to carry phenazine genes. Phenazines are known to contribute to antagonistic activity of bacterial strains against soil pathogens. When tested in the cress assay with Pythium ultimum as pathogen, F9 showed results comparable to the known antagonist P. protegens CHA0.

  20. Ab Initio f values for Fe II J=9/2 ->9/2^o transitions

    Science.gov (United States)

    Beck, Donald

    2005-05-01

    Relativistic configuration interaction f values have been obtained for 264 transitions between the lowest 12 J=9/2 and the 22 J=9/2^o levels. Length and velocity gauges agree to 3.8% for in-shell transitions and 10.0% for shell jump transitions. Two J=9/2^o levels are so nearly degenerate that it was necessary to introduce a semi-empirical correction to produce the correct level ordering. The results are in overall good agreement with the semi-empirical results of Kurucz ootnotetextR. L. Kurucz, http://kurucz.harvard.edu/atoms/2601/ and Raassen ootnotetextA. J. J. Raasen, ftp://ftp.wins.uva.nl/pub/orth/iron/FeII.E1 (1999). An efficient method of including magnetic Breit effects in the energy matrix is presented.

  1. Preparation and biological evaluation of 2-amino-6-[{sup 18}F]fluoro-9-(4-hydroxy-3-hydroxy-methylbutyl) purine (6-[{sup 18}F]FPCV) as a novel PET probe for imaging HSV1-tk reporter gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Cai Hancheng [Research Center of Radiopharmaceuticals, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800 (China); Graduate School of the Chinese Academy of Sciences, Beijing 100049 (China); Yin Duanzhi [Research Center of Radiopharmaceuticals, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800 (China)], E-mail: chcbati@yahoo.com.cn; Zhang Lan [Research Center of Radiopharmaceuticals, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800 (China); Zhejiang California International NanoSystems Institute, Hangzhou 310029 (China); Yang, Xiaofeng; Xu Xiaoyan; Liu Weiguo; Zheng Xuesheng [Institute of Brain Medical Science, Second affiliated Hospital, Medicine School of Zhejiang University, Hangzhou 310009 (China); Zhang Hong [Department of Nuclear Medicine, Second Affiliated Hospital, Zhejiang University Medical PET Center, Medicine School of Zhejiang University, Hangzhou 310009 (China); Wang Jing [Department of Nuclear Medicine, Second Affiliated Hospital, Zhejiang University Medical PET Center, Medicine School of Zhejiang University, Hangzhou 310009 (China); Zhejiang California International NanoSystems Institute, Hangzhou 310029 (China); Xu Yuhong [Zhejiang California International NanoSystems Institute, Hangzhou 310029 (China); Cheng Dengfeng; Zheng Mingqiang; Han Yanjiang; Wu Mingxing; Wang Yongxian [Research Center of Radiopharmaceuticals, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800 (China)

    2007-08-15

    Introduction: 2-Amino-6-[{sup 18}F]fluoro-9-(4-hydroxy-3-hydroxy-methylbutyl) purine (6-[{sup 18}F]FPCV) was prepared via a one-step nucleophilic substitution and evaluated as a novel probe for imaging the expression of herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene. Methods: Log P of 6-[{sup 18}F]FPCV was calculated in octanol/phosphate-buffered saline (PBS). Stability studies were performed in PBS and bovine serum albumin (BSA). Cell uptake was performed at various time points in wild-type cells and transduced cells. For in vivo studies, tumors were grown in nude mice by inoculation with C6 cells, wild type and tk positive. The radiotracer was intravenously injected to animals, and micro-PET imaging was performed. Biodistribution of 6-[{sup 18}F]FPCV was performed on another group of animals at different time points. Results: Log P of 6-[{sup 18}F]FPCV was -0.517. 6-[{sup 18}F]FPCV was fairly stable in PBS and BSA at 6 h. The tracer uptake in C6-tk cells was 5.5-18.8 times higher than that in wild-type cells. The plasma half-life of 6-[{sup 18}F]FPCV was as follows: {alpha} t{sub 1/2}=1.2 min and {beta} t{sub 1/2}=73.7 min. The average ratio of tumor uptake between the transduced tumor and the wild-type tumor was 1.69 at 15 min. Conclusion: Biological evaluation showed that 6-[{sup 18}F]FPCV is a potential probe for imaging HSV1-tk gene expression. However, its in vivo defluorination may limit its application in PET imaging of gene expression.

  2. Inhibition of cell proliferation, migration and invasion of B16-F10 melanoma cells by α-mangostin

    Energy Technology Data Exchange (ETDEWEB)

    Beninati, Simone, E-mail: beninati@bio.uniroma2.it [Department of Biology, University “Tor Vergata”, Rome (Italy); Oliverio, Serafina [Department of Biology, University “Tor Vergata”, Rome (Italy); Cordella, Martina [Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome (Italy); Rossi, Stefania; Senatore, Cinzia [Regina Elena National Cancer Institute, Rome (Italy); Liguori, Immacolata; Lentini, Alessandro; Piredda, Lucia [Department of Biology, University “Tor Vergata”, Rome (Italy); Tabolacci, Claudio [Department of Biology, University “Tor Vergata”, Rome (Italy); Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome (Italy)

    2014-08-08

    Highlights: • We studied the anticancer potential of a new emerging molecule, α-mangostin (α-M). • We provide first evidences on the effects of α-M on transglutaminase activity. • We deeply examined the antimetastatic effects of α-M through many in vitro assays. • Proteomic analysis revealed that α-M promotes a reorganization at cellular level. - Abstract: In this study, we have evaluated the potential antineoplastic effects of α-mangostin (α-M), the most representative xanthone in Garcinia mangostana pericarp, on melanoma cell lines. This xanthone markedly inhibits the proliferation of high-metastatic B16-F10 melanoma cells. Furthermore, by deeply analyzing which steps in the metastatic process are influenced by xanthone it was observed that α-M strongly interferes with homotypic aggregation, adhesion, plasticity and invasion ability of B16-F10 cells, probably by the observed reduction of metalloproteinase-9 activity. The antiproliferative and antimetastatic properties of α-M have been established in human SK-MEL-28 and A375 melanoma cells. In order to identify pathways potentially involved in the antineoplastic properties of α-M, a comparative mass spectrometry proteomic approach was employed. These findings may improve our understanding of the molecular mechanisms underlying the anti-cancer effects of α-M on melanoma.

  3. Cathepsins are required for Toll-like receptor 9 responses

    International Nuclear Information System (INIS)

    Matsumoto, Fumi; Saitoh, Shin-ichiroh; Fukui, Ryutaroh; Kobayashi, Toshihiko; Tanimura, Natsuko; Konno, Kazunori; Kusumoto, Yutaka; Akashi-Takamura, Sachiko; Miyake, Kensuke

    2008-01-01

    Toll-like receptors (TLR) recognize a variety of microbial products and activate defense responses. Pathogen sensing by TLR2/4 requires accessory molecules, whereas little is known about a molecule required for DNA recognition by TLR9. After endocytosis of microbes, microbial DNA is exposed and recognized by TLR9 in lysosomes. We here show that cathepsins, lysosomal cysteine proteases, are required for TLR9 responses. A cell line Ba/F3 was found to be defective in TLR9 responses despite enforced TLR9 expression. Functional cloning with Ba/F3 identified cathepsin B/L as a molecule required for TLR9 responses. The protease activity was essential for the complementing effect. TLR9 responses were also conferred by cathepsin S or F, but not by cathepsin H. TLR9-dependent B cell proliferation and CD86 upregulation were apparently downregulated by cathepsin B/L inhibitors. Cathepsin B inhibitor downregulated interaction of CpG-B with TLR9 in 293T cells. These results suggest roles for cathepsins in DNA recognition by TLR9

  4. Aqp 9 and Brain Tumour Stem Cells

    Directory of Open Access Journals (Sweden)

    Guri Fossdal

    2012-01-01

    Full Text Available Several studies have implicated the aquaporins (aqp 1, 4, and 9 in the pathogenesis of malignant brain tumours, suggesting that they contribute to motility, invasiveness, and oedema formation and facilitate metabolism in tumour cells under hypoxic conditions. We have studied the expression of aqp1, 4, and 9 in biopsies from glioblastomas, isolated tumour stem cells grown in a tumoursphere assay and analyzed the progenitor and differentiated cells from these cultures. We have compared these to the situation in normal rat brain, its stem cells, and differentiated cells derived thereof. In short, qPCR in tumour tissue showed presence of aqp1, 4, and 9. In the tumour progenitor population, aqp9 was markedly more highly expressed, whilst in tumour-derived differentiated cells, aqp4 was downregulated. However, immunostaining did not reveal increased protein expression of aqp9 in the tumourspheres containing progenitor cells; in contrast, its expression (both mRNA and protein was high in differentiated cultures. We, therefore, propose that aquaporin 9 may have a central role in the tumorigenesis of glioblastoma.

  5. LSD1 demethylase and the methyl-binding protein PHF20L1 prevent SET7 methyltransferase-dependent proteolysis of the stem-cell protein SOX2.

    Science.gov (United States)

    Zhang, Chunxiao; Hoang, Nam; Leng, Feng; Saxena, Lovely; Lee, Logan; Alejo, Salvador; Qi, Dandan; Khal, Anthony; Sun, Hong; Lu, Fei; Zhang, Hui

    2018-03-09

    The pluripotency-controlling stem-cell protein SRY-box 2 (SOX2) plays a pivotal role in maintaining the self-renewal and pluripotency of embryonic stem cells and also of teratocarcinoma or embryonic carcinoma cells. SOX2 is monomethylated at lysine 119 (Lys-119) in mouse embryonic stem cells by the SET7 methyltransferase, and this methylation triggers ubiquitin-dependent SOX2 proteolysis. However, the molecular regulators and mechanisms controlling SET7-induced SOX2 proteolysis are unknown. Here, we report that in human ovarian teratocarcinoma PA-1 cells, methylation-dependent SOX2 proteolysis is dynamically regulated by the LSD1 lysine demethylase and a methyl-binding protein, PHD finger protein 20-like 1 (PHF20L1). We found that LSD1 not only removes the methyl group from monomethylated Lys-117 (equivalent to Lys-119 in mouse SOX2), but it also demethylates monomethylated Lys-42 in SOX2, a reaction that SET7 also regulated and that also triggered SOX2 proteolysis. Our studies further revealed that PHF20L1 binds both monomethylated Lys-42 and Lys-117 in SOX2 and thereby prevents SOX2 proteolysis. Down-regulation of either LSD1 or PHF20L1 promoted SOX2 proteolysis, which was prevented by SET7 inactivation in both PA-1 and mouse embryonic stem cells. Our studies also disclosed that LSD1 and PHF20L1 normally regulate the growth of pluripotent mouse embryonic stem cells and PA-1 cells by preventing methylation-dependent SOX2 proteolysis. In conclusion, our findings reveal an important mechanism by which the stability of the pluripotency-controlling stem-cell protein SOX2 is dynamically regulated by the activities of SET7, LSD1, and PHF20L1 in pluripotent stem cells. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system.

    Science.gov (United States)

    Zhu, Xiaoxiao; Xu, Yajie; Yu, Shanshan; Lu, Lu; Ding, Mingqin; Cheng, Jing; Song, Guoxu; Gao, Xing; Yao, Liangming; Fan, Dongdong; Meng, Shu; Zhang, Xuewen; Hu, Shengdi; Tian, Yong

    2014-09-19

    The rapid generation of various species and strains of laboratory animals using CRISPR/Cas9 technology has dramatically accelerated the interrogation of gene function in vivo. So far, the dominant approach for genotyping of genome-modified animals has been the T7E1 endonuclease cleavage assay. Here, we present a polyacrylamide gel electrophoresis-based (PAGE) method to genotype mice harboring different types of indel mutations. We developed 6 strains of genome-modified mice using CRISPR/Cas9 system, and utilized this approach to genotype mice from F0 to F2 generation, which included single and multiplexed genome-modified mice. We also determined the maximal detection sensitivity for detecting mosaic DNA using PAGE-based assay as 0.5%. We further applied PAGE-based genotyping approach to detect CRISPR/Cas9-mediated on- and off-target effect in human 293T and induced pluripotent stem cells (iPSCs). Thus, PAGE-based genotyping approach meets the rapidly increasing demand for genotyping of the fast-growing number of genome-modified animals and human cell lines created using CRISPR/Cas9 system or other nuclease systems such as TALEN or ZFN.

  7. Targeted genome engineering in human induced pluripotent stem cells from patients with hemophilia B using the CRISPR-Cas9 system.

    Science.gov (United States)

    Lyu, Cuicui; Shen, Jun; Wang, Rui; Gu, Haihui; Zhang, Jianping; Xue, Feng; Liu, Xiaofan; Liu, Wei; Fu, Rongfeng; Zhang, Liyan; Li, Huiyuan; Zhang, Xiaobing; Cheng, Tao; Yang, Renchi; Zhang, Lei

    2018-04-06

    Replacement therapy for hemophilia remains a lifelong treatment. Only gene therapy can cure hemophilia at a fundamental level. The clustered regularly interspaced short palindromic repeats-CRISPR associated nuclease 9 (CRISPR-Cas9) system is a versatile and convenient genome editing tool which can be applied to gene therapy for hemophilia. A patient's induced pluripotent stem cells (iPSCs) were generated from their peripheral blood mononuclear cells (PBMNCs) using episomal vectors. The AAVS1-Cas9-sgRNA plasmid which targets the AAVS1 locus and the AAVS1-EF1α-F9 cDNA-puromycin donor plasmid were constructed, and they were electroporated into the iPSCs. When insertion of F9 cDNA into the AAVS1 locus was confirmed, whole genome sequencing (WGS) was carried out to detect the off-target issue. The iPSCs were then differentiated into hepatocytes, and human factor IX (hFIX) antigen and activity were measured in the culture supernatant. Finally, the hepatocytes were transplanted into non-obese diabetic/severe combined immunodeficiency disease (NOD/SCID) mice through splenic injection. The patient's iPSCs were generated from PBMNCs. Human full-length F9 cDNA was inserted into the AAVS1 locus of iPSCs of a hemophilia B patient using the CRISPR-Cas9 system. No off-target mutations were detected by WGS. The hepatocytes differentiated from the inserted iPSCs could secrete hFIX stably and had the ability to be transplanted into the NOD/SCID mice in the short term. PBMNCs are good somatic cell choices for generating iPSCs from hemophilia patients. The iPSC technique is a good tool for genetic therapy for human hereditary diseases. CRISPR-Cas9 is versatile, convenient, and safe to be used in iPSCs with low off-target effects. Our research offers new approaches for clinical gene therapy for hemophilia.

  8. In vitro metabolism studies of 18F-labeled 1-phenylpiperazine using mouse liver S9 fraction

    International Nuclear Information System (INIS)

    Ryu, Eun Kyoung; Choe, Yearn Seong; Kim, Dong Hyun; Ko, Bong-Ho; Choi, Yong; Lee, Kyung-Han; Kim, Byung-Tae

    2006-01-01

    The in vitro metabolism of 1-(4-[ 18 F]fluoromethylbenzyl)-4-phenylpiperazine ([ 18 F]1) and 1-(4-[ 18 F]fluorobenzyl)-4-phenylpiperazine ([ 18 F]2) was investigated using mouse liver S9 fraction. Results were compared to those of in vivo metabolism using mouse blood and bone and to in vitro metabolism using mouse liver microsomes. Defluorination was the main metabolic pathway for [ 18 F]1 in vitro and in vivo. Based on TLC, HPLC and LC-MS data, [ 18 F]fluoride ion and less polar radioactive metabolites derived from aromatic ring oxidation were detected in vitro, and the latter metabolites were rapidly converted into the former with time, whereas only the [ 18 F]fluoride ion was detected in vivo. Similarly, the in vitro metabolism of [ 18 F]2 using either S9 fraction or microsomes showed the same pattern as the in vivo method using blood; however, the radioactive metabolites derived from aromatic ring oxidation were not detected in vivo. These results demonstrate that liver S9 fraction can be widely used to investigate the intermediate radioactive metabolites and to predict the in vivo metabolism of radiotracers

  9. Adherence of B16-F10 melanoma cells to elastin

    International Nuclear Information System (INIS)

    Zetter, B.R.; Netland, P.A.

    1986-01-01

    B16-F10 melanoma cells selectivity colonize lung tissue in vivo. The authors have previously shown that these cells adhere preferentially to lung tissue in vitro. To quantify the binding of B16-F10 cells to isolated components of lung tissue, the authors devised a dot-blot cell adhesion assay. Samples were absorbed to 4 mm dots of nylon based paper under non-denaturing conditions, blocked with albumin or hemoglobin, and incubated with radiolabelled cells for 30 min. at 4 0 C. 125 -I labelled B16-F10 cells demonstrated a dose dependent binding to mouse lung elastin. Autoradiography and scanning electron microscopy demonstrated that cells localized preferentially to the elastin dots. The melanoma cells bound more strongly to elastin relative to laminin, fibronectin, collagen types I and IV or heparan sulfate. Neither elastin-associated microfibrillar protein nor fragments of elastin produced by alkali or acid treatment demonstrated significant binding activity for these cells. The findings demonstrate that in addition to its unique mechanical properties that confer elasticity to tissues, elastin can also function as a cell adhesion molecule. The localization of elastin in the lung and its adhesive properties reported here suggest that elastin may facilitate the arrest and eventual colonization of circulating B16-F10 melanoma cells in the mouse lung

  10. Tumor cell-released TLR4 ligands stimulate Gr-1+CD11b+F4/80+ cells to induce apoptosis of activated T cells.

    Science.gov (United States)

    Liu, Yan-Yan; Sun, Ling-Cong; Wei, Jing-Jing; Li, Dong; Yuan, Ye; Yan, Bin; Liang, Zhi-Hui; Zhu, Hui-Fen; Xu, Yong; Li, Bo; Song, Chuan-Wang; Liao, Sheng-Jun; Lei, Zhang; Zhang, Gui-Mei; Feng, Zuo-Hua

    2010-09-01

    Gr-1(+)CD11b(+)F4/80(+) cells play important roles in tumor development and have a negative effect on tumor immunotherapy. So far, the mechanisms underlying the regulation of their immunosuppressive phenotype by classical and alternative macrophage activation stimuli are not well elucidated. In this study, we found that molecules from necrotic tumor cells (NTC-Ms) stimulated Gr-1(+)CD11b(+)F4/80(+) cells to induce apoptosis of activated T cells but not nonstimulated T cells. The apoptosis-inducing capacity was determined by higher expression levels of arginase I and IL-10 relative to those of NO synthase 2 and IL-12 in Gr-1(+)CD11b(+)F4/80(+) cells, which were induced by NTC-Ms through TLR4 signaling. The apoptosis-inducing capacity of NTC-Ms-stimulated Gr-1(+)CD11b(+)F4/80(+) cells could be enhanced by IL-10. IFN-gamma may reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells only if their response to IFN-gamma was not attenuated. However, the potential of Gr-1(+)CD11b(+)F4/80(+) cells to express IL-12 in response to IFN-gamma could be attenuated by tumor, partially due to the existence of active STAT3 in Gr-1(+)CD11b(+)F4/80(+) cells and NTC-Ms from tumor. In this situation, IFN-gamma could not effectively reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells. Tumor immunotherapy with 4-1BBL/soluble programmed death-1 may significantly reduce, but not abolish the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells in local microenvironment. Blockade of TLR4 signaling could further reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells and enhance the suppressive effect of 4-1BBL/soluble form of programmed death-1 on tumor growth. These findings indicate the relationship of distinct signaling pathways with apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells and emphasize the importance of blocking TLR4 signaling to prevent the induction of T cell apoptosis by Gr-1(+)CD11b(+)F4/80(+) cells.

  11. F8 and F9 mutations in US haemophilia patients: correlation with history of inhibitor and race/ethnicity.

    Science.gov (United States)

    Miller, C H; Benson, J; Ellingsen, D; Driggers, J; Payne, A; Kelly, F M; Soucie, J M; Craig Hooper, W

    2012-05-01

    Both genetic and treatment-related risk factors contribute to the development of inhibitors in haemophilia. An inhibitor surveillance system piloted at 12 US sites has the goal of assessing risk factors through prospective data collection. This report examines the relationship of genotype and race/ethnicity to history of inhibitor in a large cohort of US haemophilia patients. Mutation analysis was performed on 676 haemophilia A (HA) and 153 haemophilia B (HB) patients by sequencing, Multiplex Ligation-dependent Probe Amplification, and PCR for inversions in F8 introns 22 (inv22) and 1 (inv1). Two HB patients with deletions had history of inhibitor. In severe HA, frequency of history of inhibitor was: large deletion 57.1%, splice site 35.7%, inv22 26.8%, nonsense 24.5%, frameshift 12.9%, inv1 11.1% and missense 9.5%. In HA, 19.6% of 321 White non-Hispanics (Whites), 37.1% of 35 Black non-Hispanics (Blacks) and 46.9% of 32 Hispanics had history of inhibitor (P = 0.0003). Mutation types and novel mutation rates were similar across ethnicities. When F8 haplotypes were constructed, Whites and Hispanics showed only H1 and H2. Within H1, history of inhibitor was 12.4% in Whites, 40.0% in Blacks (P = 0.009) and 32.4% in Hispanics (P = 0.002). Inhibitor frequency is confirmed to vary by mutation type and race in a large US population. White patients with history of inhibitor did not exhibit rare F8 haplotypes. F8 gene analysis did not reveal a cause for the higher inhibitor frequencies in Black and Hispanic patients. © 2011 Blackwell Publishing Ltd.

  12. The toxicity of NaF on BmN cells and a comparative proteomics approach to identify protein expression changes in cells under NaF-stress

    International Nuclear Information System (INIS)

    Chen, Liang; Chen, Huiqing; Yao, Chun; Chang, Cheng; Xia, Hengchuan; Zhang, Chunxia; Zhou, Yang; Yao, Qin; Chen, Keping

    2015-01-01

    Highlights: • On the cellular level, we identified IC 50 of NaF on BmN cell by flow cytometry. • High concentration of NaF gives effect on BmN cell morphology. • Five significantly differential proteins were identified by two-dimensional electrophoresis and mass spectrometry. • ALDH2 and WPH were up-regulated, while CRT and SCF were down-regulated, providing new information for metabolic pathway of fluoride. - Abstract: Fluorides negatively affect the development of organisms and are a threat to human health and environmental safety. In this study, Bombyx mori N cell line (BmN) were used to explore effects of NaF on insect cells. We found that 8 h (hrs) culture with high concentration of NaF (≥1 mM) induced significantly morphological changes. Dose-response curves of 72 h continuously cultured BmN treated with NaF showed that the half inhibitory concentration (IC 50 ) value was 56.60 μM. Treatment of BmN with 100 and 300 μM of NaF induced apoptosis and necrosis. 2-D electrophoresis of whole cell extracted from BmN showed that treatment with 300 μM NaF up-regulated 32 proteins and down-regulated 11 proteins when compared with controls. We identified 5 different proteins by MALDI-TOF MS, and 4 of them were identified for the first time, including 2 up-regulated proteins (mitochondrial aldehyde dehydrogenase ALDH2 and prohibitin protein WPH) and 2 down-regulated proteins (calreticulin precursor CRT and DNA supercoiling factor SCF). These observations were further confirmed by fluorescence quantitative PCR. Together, our data suggest that these target proteins could be regarded as targets influenced by NaF and also provide clues for studies on the response metabolism pathway under NaF stress

  13. The toxicity of NaF on BmN cells and a comparative proteomics approach to identify protein expression changes in cells under NaF-stress

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Liang; Chen, Huiqing [Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu 212013 (China); Yao, Chun [Department of Stomatology, Zhenjiang First People’s Hospital, Zhenjiang, Jiangsu 212013 (China); Chang, Cheng; Xia, Hengchuan; Zhang, Chunxia; Zhou, Yang; Yao, Qin [Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu 212013 (China); Chen, Keping, E-mail: kpchen@ujs.edu.cn [Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu 212013 (China)

    2015-04-09

    Highlights: • On the cellular level, we identified IC{sub 50} of NaF on BmN cell by flow cytometry. • High concentration of NaF gives effect on BmN cell morphology. • Five significantly differential proteins were identified by two-dimensional electrophoresis and mass spectrometry. • ALDH2 and WPH were up-regulated, while CRT and SCF were down-regulated, providing new information for metabolic pathway of fluoride. - Abstract: Fluorides negatively affect the development of organisms and are a threat to human health and environmental safety. In this study, Bombyx mori N cell line (BmN) were used to explore effects of NaF on insect cells. We found that 8 h (hrs) culture with high concentration of NaF (≥1 mM) induced significantly morphological changes. Dose-response curves of 72 h continuously cultured BmN treated with NaF showed that the half inhibitory concentration (IC{sub 50}) value was 56.60 μM. Treatment of BmN with 100 and 300 μM of NaF induced apoptosis and necrosis. 2-D electrophoresis of whole cell extracted from BmN showed that treatment with 300 μM NaF up-regulated 32 proteins and down-regulated 11 proteins when compared with controls. We identified 5 different proteins by MALDI-TOF MS, and 4 of them were identified for the first time, including 2 up-regulated proteins (mitochondrial aldehyde dehydrogenase ALDH2 and prohibitin protein WPH) and 2 down-regulated proteins (calreticulin precursor CRT and DNA supercoiling factor SCF). These observations were further confirmed by fluorescence quantitative PCR. Together, our data suggest that these target proteins could be regarded as targets influenced by NaF and also provide clues for studies on the response metabolism pathway under NaF stress.

  14. Fluoride induces apoptosis in H9c2 cardiomyocytes via the mitochondrial pathway.

    Science.gov (United States)

    Yan, Xiaoyan; Wang, Lu; Yang, Xia; Qiu, Yulan; Tian, Xiaolin; Lv, Yi; Tian, Fengjie; Song, Guohua; Wang, Tong

    2017-09-01

    Numerous studies have shown that chronic excessive fluoride intake can adversely affect different organ systems. In particular, the cardiovascular system is susceptible to disruption by a high concentration of fluoride. The objectives of this study were to explore the mechanism of apoptosis by detecting the toxic effects of different concentrations of sodium fluoride (NaF) in H9c2 cells exposed for up to 96 h. NaF not only inhibited H9c2 cell proliferation but also induced apoptosis and morphological damage. With increasing NaF concentrations, early apoptosis of H9c2 cells was increased while the mitochondrial membrane potential was decreased. Compared with the control group, the mRNA levels of caspase-3, caspase-9, and cytochrome c all increased with increasing concentrations of NaF. In summary, these data suggest that apoptosis is involved in NaF-induced H9c2 cell toxicity and that activation of the mitochondrial pathway may occur. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Hydrogen peroxide stimulates cell motile activity through LPA receptor-3 in liver epithelial WB-F344 cells

    Energy Technology Data Exchange (ETDEWEB)

    Shibata, Ayano; Tanabe, Eriko; Inoue, Serina; Kitayoshi, Misaho; Okimoto, Souta; Hirane, Miku; Araki, Mutsumi [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Fukushima, Nobuyuki [Division of Molecular Neurobiology, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Tsujiuchi, Toshifumi, E-mail: ttujiuch@life.kindai.ac.jp [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan)

    2013-04-12

    Highlights: •Hydrogen peroxide stimulates cell motility of WB-F344 cells. •LPA{sub 3} is induced by hydrogen peroxide in WB-F344 cells. •Cell motility by hydrogen peroxide is inhibited in LPA{sub 3} knockdown cells. •LPA signaling is involved in cell migration by hydrogen peroxide. -- Abstract: Hydrogen peroxide which is one of reactive oxygen species (ROS) mediates a variety of biological responses, including cell proliferation and migration. In the present study, we investigated whether lysophosphatidic acid (LPA) signaling is involved in cell motile activity stimulated by hydrogen peroxide. The rat liver epithelial WB-F344 cells were treated with hydrogen peroxide at 0.1 or 1 μM for 48 h. In cell motility assays, hydrogen peroxide treated cells showed significantly high cell motile activity, compared with untreated cells. To measure the expression levels of LPA receptor genes, quantitative real time RT-PCR analysis was performed. The expressions of LPA receptor-3 (Lpar3) in hydrogen peroxide treated cells were significantly higher than those in control cells, but not Lpar1 and Lpar2 genes. Next, to assess the effect of LPA{sub 3} on cell motile activity, the Lpar3 knockdown cells from WB-F344 cells were also treated with hydrogen peroxide. The cell motile activity of the knockdown cells was not stimulated by hydrogen peroxide. Moreover, in liver cancer cells, hydrogen peroxide significantly activated cell motility of Lpar3-expressing cells, but not Lpar3-unexpressing cells. These results suggest that LPA signaling via LPA{sub 3} may be mainly involved in cell motile activity of WB-F344 cells stimulated by hydrogen peroxide.

  16. A noncognate interaction with anti-receptor antibody-activated helper T cells induces small resting murine B cells to proliferate and to secrete antibody

    DEFF Research Database (Denmark)

    Owens, T

    1988-01-01

    on resting B cells (even in the presence of intact F23.1 antibody), but could induce antibody secretion by anti-Ig-preactivated B cells. Both F23.1+ clones (E9.D4 and 4.35F2) and one F23.1- clone (D2.2) could synergize with supernatants from activated E9.D4 T cells to induce B cell activation. F(ab')2......Culture of small resting allogeneic B cells (of an irrelevant haplotype) with two clones of T helper (Th) cells that were activated by the F23.1 anti-T cell receptor antibody led to the activation of B cells to proliferate and to secrete antibody. Th cell supernatants by themselves had no effect...... fragments of F23.1 induced E9.D4 to activate B cells as efficiently as intact F23.1 and B cell populations that had been incubated with F23.1 were not activated when cultured with E9.D4, although T cells recognized cell-presented F23.1 and were weakly activated. Reduction of the density of F23.1 adsorbed...

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

    Science.gov (United States)

    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.

  18. Wind-Tunnel Investigation at Low Speed of the Rolling Stability Derivatives of a 1/10-Scale Model of the Grumman F9F-9 Airplane, TED No. NACA AD 3109

    Science.gov (United States)

    Wolhart, Walter D.; Thomas, David F., Jr.

    1955-01-01

    An experimental investigation has been made in the Langley stability tunnel to determine the low-speed yawing, pitching, and static stability characteristics of a 1/10-scale model of the Grumman F9F-9 airplane. Tests were made to determine the effects of duct-entrance-fairing plugs on the static lateral and longitudinal stability characteristics of the complete model in the clean condition. The remaining tests were concerned with determining tail contributions as well as the effect of duct-entrance-fairing plugs, slats, flaps, and landing gear on the yawing and pitching stability derivatives. These data are presented without analysis in order to expedite distribution.

  19. Efficient CsF interlayer for high and low bandgap polymer solar cell

    Science.gov (United States)

    Mitul, Abu Farzan; Sarker, Jith; Adhikari, Nirmal; Mohammad, Lal; Wang, Qi; Khatiwada, Devendra; Qiao, Qiquan

    2018-02-01

    Low bandgap polymer solar cells have a great deal of importance in flexible photovoltaic market to absorb sun light more efficiently. Efficient wide bandgap solar cells are always available in nature to absorb visible photons. The development and incorporation of infrared photovoltaics (IR PV) with wide bandgap solar cells can improve overall solar device performance. Here, we have developed an efficient low bandgap polymer solar cell with CsF as interfacial layer in regular structure. Polymer solar cell devices with CsF shows enhanced performance than Ca as interfacial layer. The power conversion efficiency of 4.5% has been obtained for PDPP3T based polymer solar cell with CsF as interlayer. Finally, an optimal thickness with CsF as interfacial layer has been found to improve the efficiency in low bandgap polymer solar cells.

  20. Tensile Strength of the Al-9%Si Alloy Modified with Na, F and Cl Compounds

    Directory of Open Access Journals (Sweden)

    T. Lipiński

    2010-01-01

    Full Text Available The modification of the Al-9%Si alloy with the use of a complex modifier containing Na, F and Cl was investigated in the study. The modifier was composed of NaCl, Na3AlF6 and NaF compounds. The modifier and the liquid Al-Si alloy were kept in the crucible for 15 minutes. The modifier's effect relative to the weight of the processed alloy on its tensile strength was presented in graphic form. The results of the study indicate that the complex modifier altered the investigated properties of the eutectic Al-9%Si alloy.

  1. Isoginkgetin inhibits tumor cell invasion by regulating phosphatidylinositol 3-kinase/Akt-dependent matrix metalloproteinase-9 expression.

    Science.gov (United States)

    Yoon, Sang-Oh; Shin, Sejeong; Lee, Ho-Jae; Chun, Hyo-Kon; Chung, An-Sik

    2006-11-01

    Matrix metalloproteinase (MMP)-9 plays a key role in tumor invasion. Inhibitors of MMP-9 were screened from Metasequoia glyptostroboides (Dawn redwood) and one potent inhibitor, isoginkgetin, a biflavonoid, was identified. Noncytotoxic levels of isoginkgetin decreased MMP-9 production profoundly, but up-regulated the level of tissue inhibitor of metalloproteinase (TIMP)-1, an inhibitor of MMP-9, in HT1080 human fibrosarcoma cells. The major mechanism of Ras-dependent MMP-9 production in HT1080 cells was phosphatidylinositol 3-kinase (PI3K)/Akt/nuclear factor-kappaB (NF-kappaB) activation. Expression of dominant-active H-Ras and p85 (a subunit of PI3K) increased MMP-9 activity, whereas dominant-negative forms of these molecules decreased the level of MMP-9. H-Ras did not increase MMP-9 in the presence of a PI3K inhibitor, LY294002, and a NF-kappaB inhibitor, SN50. Further studies showed that isoginkgetin regulated MMP-9 production via PI3K/Akt/NF-kappaB pathway, as evidenced by the findings that isoginkgetin inhibited activities of both Akt and NF-kappaB. PI3K/Akt is a well-known key pathway for cell invasion, and isoginkgetin inhibited HT1080 tumor cell invasion substantially. Isoginkgetin was also quite effective in inhibiting the activities of Akt and MMP-9 in MDA-MB-231 breast carcinomas and B16F10 melanoma. Moreover, isoginkgetin treatment resulted in marked decrease in invasion of these cells. In summary, PI3K/Akt is a major pathway for MMP-9 expression and isoginkgetin markedly decreased MMP-9 expression and invasion through inhibition of this pathway. This suggests that isoginkgetin could be a potential candidate as a therapeutic agent against tumor invasion.

  2. Evaluation of 18F-FDG and 18F-FLT for monitoring therapeutic responses of colorectal cancer cells to radiotherapy

    International Nuclear Information System (INIS)

    Wang, Hui; Liu, Bo; Tian, Jiahe; Xu, Baixuan; Zhang, Jinming; Qu, Baolin; Chen, Yingmao

    2013-01-01

    In order to compare the efficacy of 18 F-fluorothymidine (FLT) and 18 F-fluorodeoxyglucose (FDG) for monitoring early responses to irradiation, two human colorectal cancer (CRC) cell lines SW480 and SW620, which were derived from the primary lesions and the metastatic lymph node, underwent X-ray irradiation of 0, 10, or 20 Gy and were examined at 0, 24 and 72 h After irradiation, reduced proliferation of both SW480 and SW620 cells was observed in a dose-dependent manner (P < 0.001), G0-G1 arrest was also noted in both cell types after 72 h in the 20 Gy group (P < 0.001). Although increased apoptosis was observed in both cell lines after irradiation (P < 0.001), a greater percentage of SW480 cells underwent apoptosis in response to irradiation than SW620 cells. Increased Hsp27 and decreased integrin β 3 , Ki67 and VEGFR2 expression was observed over time via immunocytochemistry and Western blot analysis (P < 0.001), however, no significant changes were noted in response to irradiation. Finally, reduced uptake of 18 F-FLT by SW480 or SW620 cells was observed at 24-h post-irradiation, however, reduced 18 F-FDG uptake was only observed after 72 h. Therefore, we conclude that 18 F-FLT is a more suitable positron emission tomography (PET) tracer for monitoring early responses to irradiation in primary and metastatic lymph node CRC cells

  3. Murine Th9 cells promote the survival of myeloid dendritic cells in cancer immunotherapy.

    Science.gov (United States)

    Park, Jungsun; Li, Haiyan; Zhang, Mingjun; Lu, Yong; Hong, Bangxing; Zheng, Yuhuan; He, Jin; Yang, Jing; Qian, Jianfei; Yi, Qing

    2014-08-01

    Dendritic cells (DCs) are professional antigen-presenting cells to initiate immune responses, and DC survival time is important for affecting the strength of T-cell responses. Interleukin (IL)-9-producing T-helper (Th)-9 cells play an important role in anti-tumor immunity. However, it is unclear how Th9 cells communicate with DCs. In this study, we investigated whether murine Th9 cells affected the survival of myeloid DCs. DCs derived from bone marrow of C57BL/6 mice were cocultured with Th9 cells from OT-II mice using transwell, and the survival of DCs was examined. DCs cocultured with Th9 cells had longer survival and fewer apoptotic cells than DCs cultured alone in vitro. In melanoma B16-OVA tumor-bearing mice, DCs conditioned by Th9 cells lived longer and induced stronger anti-tumor response than control DCs did in vivo. Mechanistic studies revealed that IL-3 but not IL-9 secreted by Th9 cells was responsible for the prolonged survival of DCs. IL-3 upregulated the expression of anti-apoptotic protein Bcl-xL and activated p38, ERK and STAT5 signaling pathways in DCs. Taken together, our data provide the first evidence that Th9 cells can promote the survival of DCs through IL-3, and will be helpful for designing Th9 cell immunotherapy and more effective DC vaccine for human cancers.

  4. CLEC4F is an inducible C-type lectin in F4/80-positive cells and is involved in alpha-galactosylceramide presentation in liver.

    Directory of Open Access Journals (Sweden)

    Chih-Ya Yang

    Full Text Available CLEC4F, a member of C-type lectin, was first purified from rat liver extract with high binding affinity to fucose, galactose (Gal, N-acetylgalactosamine (GalNAc, and un-sialylated glucosphingolipids with GalNAc or Gal terminus. However, the biological functions of CLEC4F have not been elucidated. To address this question, we examined the expression and distribution of murine CLEC4F, determined its binding specificity by glycan array, and investigated its function using CLEC4F knockout (Clec4f-/- mice. We found that CLEC4F is a heavily glycosylated membrane protein co-expressed with F4/80 on Kupffer cells. In contrast to F4/80, CLEC4F is detectable in fetal livers at embryonic day 11.5 (E11.5 but not in yolk sac, suggesting the expression of CLEC4F is induced as cells migrate from yolk cells to the liver. Even though CLEC4F is not detectable in tissues outside liver, both residential Kupffer cells and infiltrating mononuclear cells surrounding liver abscesses are CLEC4F-positive upon Listeria monocytogenes (L. monocytogenes infection. While CLEC4F has strong binding to Gal and GalNAc, terminal fucosylation inhibits CLEC4F recognition to several glycans such as Fucosyl GM1, Globo H, Bb3∼4 and other fucosyl-glycans. Moreover, CLEC4F interacts with alpha-galactosylceramide (α-GalCer in a calcium-dependent manner and participates in the presentation of α-GalCer to natural killer T (NKT cells. This suggests that CLEC4F is a C-type lectin with diverse binding specificity expressed on residential Kupffer cells and infiltrating monocytes in the liver, and may play an important role to modulate glycolipids presentation on Kupffer cells.

  5. Generation of GHR-modified pigs as Laron syndrome models via a dual-sgRNAs/Cas9 system and somatic cell nuclear transfer.

    Science.gov (United States)

    Yu, Honghao; Long, Weihu; Zhang, Xuezeng; Xu, Kaixiang; Guo, Jianxiong; Zhao, Heng; Li, Honghui; Qing, Yubo; Pan, Weirong; Jia, Baoyu; Zhao, Hong-Ye; Huang, Xingxu; Wei, Hong-Jiang

    2018-02-27

    Laron syndrome is an autosomal disease resulting from mutations in the growth hormone receptor (GHR) gene. The only therapeutic treatment for Laron syndrome is recombinant insulin-like growth factor I (IGF-I), which has been shown to have various side effects. The improved Laron syndrome models are important for better understanding the pathogenesis of the disease and developing corresponding therapeutics. Pigs have become attractive biomedical models for human condition due to similarities in anatomy, physiology, and metabolism relative to humans, which could serve as an appropriate model for Laron syndrome. To further improve the GHR knockout (GHRKO) efficiency and explore the feasibility of precise DNA deletion at targeted sites, the dual-sgRNAs/Cas9 system was designed to target GHR exon 3 in pig fetal fibroblasts (PFFs). The vectors encoding sgRNAs and Cas9 were co-transfected into PFFs by electroporation and GHRKO cell lines were established by single cell cloning culture. Two biallelic knockout cell lines were selected as the donor cell line for somatic cell nuclear transfer for the generation of GHRKO pigs. The genotype of colonies, cloned fetuses and piglets were identified by T7 endonuclease I (T7ENI) assay and sequencing. The GHR expression in the fibroblasts and piglets was analyzed by confocal microscopy, quantitative polymerase chain reaction (q-PCR), western blotting (WB) and immunohistochemical (IHC) staining. The phenotype of GHRKO pigs was recapitulated through level detection of IGF-I and glucose, and measurement of body weight and body size. GHRKO F1 generation were generated by crossing with wild-type pigs, and their genotype was detected by T7ENI assay and sequencing. GHRKO F2 generation was obtained via self-cross of GHRKO F1 pigs. Their genotypes of GHRKO F2 generation was also detected by Sanger sequencing. In total, 19 of 20 single-cell colonies exhibited biallelic modified GHR (95%), and the efficiency of DNA deletion mediated by dual

  6. Elastase production by B16-F10 melanoma cells

    International Nuclear Information System (INIS)

    Shrager, J.B.; Yusa, T.; Netland, P.A.; Zetter, B.R.

    1986-01-01

    Elastolytic activity was found in sonicates of mouse B16-F10 melanoma cells and in medium conditioned by these cells. Degradation of elastin was determined by measuring the release of soluble 3 H-peptides from labelled insoluble elastin. The activity secreted from B16-F10 cells was soluble and was not associated with membrane vesicles. The secreted activity was partially inhibited by incubation with phenymethylsulfonylfluoride (PMSF) and was abolished by incubation with the alpha-1-protease inhibitor, with pepstatin A or with L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK). In contrast, the activity was unaffected by incubation with elastatinal, with the plasmin inhibitor Σ-aminocaproic acid (EACA), the metalloproteinase inhibitor ethylenediamine-tetra-acetic acid (EDTA), the soybean trypsin inhibitor or the trypsin inhibitor N proportional to-p-tosyl-L-lysine chloromethyl ketone (TLCK). These results suggest that the majority of the tumor cell-derived elastolytic activity is attributable to a serine protease that differs in specificity from the well characterized elastases previously isolated from neutrophils, macrophages or from mammalian pancreas. The authors postulate that the release of elastase from lung-colonizing B16-F10 cells may facilitate their invasion of elastin-rich lung tissue

  7. Characterization of E2F8, a novel E2F-like cell-cycle regulated repressor of E2F-activated transcription

    DEFF Research Database (Denmark)

    Christensen, Jesper; Cloos, Paul; Toftegaard, Ulla

    2005-01-01

    The E2F family of transcription factors are downstream effectors of the retinoblastoma protein, pRB, pathway and are essential for the timely regulation of genes necessary for cell-cycle progression. Here we describe the characterization of human and murine E2F8, a new member of the E2F family...

  8. First human hNT neurons patterned on parylene-C/silicon dioxide substrates: Combining an accessible cell line and robust patterning technology for the study of the pathological adult human brain.

    Science.gov (United States)

    Unsworth, C P; Graham, E S; Delivopoulos, E; Dragunow, M; Murray, A F

    2010-12-15

    In this communication, we describe a new method which has enabled the first patterning of human neurons (derived from the human teratocarcinoma cell line (hNT)) on parylene-C/silicon dioxide substrates. We reveal the details of the nanofabrication processes, cell differentiation and culturing protocols necessary to successfully pattern hNT neurons which are each key aspects of this new method. The benefits in patterning human neurons on silicon chip using an accessible cell line and robust patterning technology are of widespread value. Thus, using a combined technology such as this will facilitate the detailed study of the pathological human brain at both the single cell and network level. Copyright © 2010 Elsevier B.V. All rights reserved.

  9. Preliminary 19F-MRS Study of Tumor Cell Proliferation with 3′-deoxy-3′-fluorothymidine and Its Metabolite (FLT-MP

    Directory of Open Access Journals (Sweden)

    In Ok Ko

    2017-01-01

    Full Text Available The thymidine analogue 3′-deoxy-3′-[18F]fluorothymidine, or [18F]fluorothymidine ([18F]FLT, is used to measure tumor cell proliferation with positron emission tomography (PET imaging technology in nuclear medicine. FLT is phosphorylated by thymidine kinase 1 (TK1 and then trapped inside cells; it is not incorporated into DNA. Imaging with 18F-radiolabeled FLT is a noninvasive technique to visualize cellular proliferation in tumors. However, it is difficult to distinguish between [18F]FLT and its metabolites by PET imaging, and quantification has not been attempted using current imaging methods. In this study, we successfully acquired in vivo F19 spectra of natural or nonradioactive 3′-deoxy-3′-fluorothymidine ([19F]FLT and its monophosphate metabolite (FLT-MP in a tumor xenograft mouse model using 9.4T magnetic resonance imaging (MRI. This preliminary result demonstrates that 19F magnetic resonance spectroscopy (MRS with FLT is suitable for the in vivo assessment of tumor aggressiveness and for early prediction of treatment response.

  10. The EspF N-Terminal of Enterohemorrhagic Escherichia coli O157:H7 EDL933w Imparts Stronger Toxicity Effects on HT-29 Cells than the C-Terminal.

    Science.gov (United States)

    Wang, Xiangyu; Du, Yanli; Hua, Ying; Fu, Muqing; Niu, Cong; Zhang, Bao; Zhao, Wei; Zhang, Qiwei; Wan, Chengsong

    2017-01-01

    Enterohemorrhagic Escherichia coli (EHEC) O157:H7 EspF is an important multifunctional protein that destroys the tight junctions of intestinal epithelial cells and promotes host cell apoptosis. However, its molecular mechanism remains elusive. We knocked out the espF sequence (747 bp, Δ espF ), N-terminal sequence (219 bp, Δ espF N ), and C-terminal sequence (528 bp, Δ espF C ) separately using the pKD46-mediated λ Red homologous recombination system. Then, we built the corresponding complementation strains, namely, Δ espF/pespF , Δ espF N /pespF N , and Δ espF C /pespF C by overlap PCR, which were used in infecting HT-29 cells and BALB/C mice. The level of reactive oxygen species, cell apoptosis, mitochondrial trans-membrane potential, inflammatory factors, transepithelial electrical resistance (TER), and animal mortality were evaluated by DCFH-DA, double staining of Annexin V-FITC/PI, JC-1 staining, ELISA kit, and a mouse assay. The wild-type (WT), Δ espF , Δ espF/pespF , Δ espF C , Δ espF C /pespF C , Δ espF N , and Δ espF N /pespF N groups exhibited apoptotic rates of 68.3, 27.9, 64.9, 65.7, 73.4, 41.3, and 35.3% respectively, and mean TNF-α expression levels of 428 pg/mL, 342, 466, 446, 381, 383, and 374 pg/mL, respectively. In addition, the apoptotic rates and TNF-α levels of the WT, Δ espF/pespF , and Δ espF C were significantly higher than that of Δ espF , Δ espF N , Δ espF C /pespF C , and Δ espF N /pespF N group ( p < 0.05). The N-terminal of EspF resulted in an increase in the number of apoptotic cells, TNF-α secretion, ROS generation, mitochondria apoptosis, and pathogenicity in BalB/c mice. In conclusion, the N-terminal domain of the Enterohemorrhagic E. coli O157:H7 EspF more strongly promotes apoptosis and inflammation than the C-terminal domain.

  11. Remaining Sites Verification Package for the 100-F-26:9, 1607-F2 Sanitary Sewer Pipelines. Attachment to Waste Site Reclassification Form 2008-029

    International Nuclear Information System (INIS)

    Capron, J.M.

    2008-01-01

    The 100-F-26:9 underground pipeline subsite consists of the sanitary sewers servicing the 105-F, 108-F, 184-F, 185-F, and 190-F buildings, and the 1700-F administration and service buildings (1704-F, 1707-F, 1707-FA, 1713-F, 1717-F, 1719-F, and 1722-F). In accordance with this evaluation, the confirmatory and verification sampling results support a reclassification of this site to Interim Closed Out. The current site conditions achieve the remedial action objectives and the corresponding remedial action goals established in the Remaining Sites ROD. The results of verification sampling show that residual contaminant concentrations do not preclude any future uses and allow for unrestricted use of shallow zone soils. The results also demonstrate that residual contaminant concentrations are protective of groundwater and the Columbia River

  12. Cell cycle-regulated expression of mammalian CDC6 is dependent on E2F

    DEFF Research Database (Denmark)

    Hateboer, G; Wobst, A; Petersen, B O

    1998-01-01

    The E2F transcription factors are essential regulators of cell growth in multicellular organisms, controlling the expression of a number of genes whose products are involved in DNA replication and cell proliferation. In Saccharomyces cerevisiae, the MBF and SBF transcription complexes have...... of this gene is dependent on E2F. In vivo footprinting data demonstrate that the identified E2F sites are occupied in resting cells and in exponentially growing cells, suggesting that E2F is responsible for downregulating the promoter in early phases of the cell cycle and the subsequent upregulation when cells...

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

    Science.gov (United States)

    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.

  14. Islet-specific T cell clones transfer diabetes to nonobese diabetic (NOD) F1 mice.

    Science.gov (United States)

    Peterson, J D; Pike, B; McDuffie, M; Haskins, K

    1994-09-15

    To investigate diabetes resistance to T cell-mediated disease transfer, we administered islet-specific T cell clones to the F1 progeny of nonobese diabetic (NOD) mice that were crossed with various nondiabetes-prone inbred mouse strains. We investigated four diabetogenic CD4+ T cell clones and all induced insulitis and full development of diabetes in (SWR x NOD)F1, (SJL x NOD)F1, and (C57BL/6 x NOD)F1 mice. In contrast, (BALB/c x NOD)F1 and (CBA x NOD)F1 mice were susceptible to disease transfer by some T cell clones but not others, and (C57/L x NOD)F1 mice seemed to be resistant to both insulitis and disease transfer by all of the clones tested. Disease induced by the T cell clones in susceptible F1 strains was age dependent and could only be observed in recipients younger than 13 days old. Full or partial disease resistance did not correlate with the presence or absence of I-E, different levels of Ag expression in islet cells, or differences in APC function. The results from this study suggest that there may be multiple factors contributing to susceptibility of F1 mice to T cell clone-mediated induction of diabetes, including non-MHC-related genetic background, the immunologic maturity of the recipient, and individual characteristics of the T cell clones.

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

    Science.gov (United States)

    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.

  16. Galectin-9 activates and expands human T-helper 1 cells.

    Directory of Open Access Journals (Sweden)

    Marloes J M Gooden

    Full Text Available Galectin-9 (Gal-9 is known for induction of apoptosis in IFN-γ and IL-17 producing T-cells and amelioration of autoimmunity in murine models. On the other hand, Gal-9 induced IFN-γ positive T-cells in a sarcoma mouse model and in food allergy, suggesting that Gal-9 can have diametric effects on T-cell immunity. Here, we aimed to delineate the immunomodulatory effect of Gal-9 on human resting and ex vivo activated peripheral blood lymphocytes. Treatment of resting lymphocytes with low concentrations of Gal-9 (5-30 nM induced apoptosis in ∼60% of T-cells after 1 day, but activated the surviving T-cells. These viable T-cells started to expand after 4 days with up to 6 cell divisions by day 7 and an associated shift from naïve towards central memory and IFN-γ producing phenotype. In the presence of T-cell activation signals (anti-CD3/IL-2 Gal-9 did not induce T-cell expansion, but shifted the CD4/CD8 balance towards a CD4-dominated T-cell response. Thus, Gal-9 activates resting T-cells in the absence of typical T-cell activating signals and promotes their transition to a TH1/C1 phenotype. In the presence of T-cell activating signals T-cell immunity is directed towards a CD4-driven response by Gal-9. Thus, Gal-9 may specifically enhance reactive immunological memory.

  17. Characterization of 18F-dipicolylamine (DPA) derivatives in cells infected with influenza virus

    International Nuclear Information System (INIS)

    Li, Junling; Gerlach, Rachael L.; Jonsson, Colleen B.; Gray, Brian D.; Pak, Koon Y.; Ng, Chin K.

    2015-01-01

    Objective: Bis(Zn-dipicolylamine (Zn-DPA)) coordination complexes represent a new class of synthetic small molecules that can target anionic phosphatidylserine (PS) in the apoptotic cells with high affinity and specificity. In this study, we labeled Zn-DPA and Cy7-Zn-DPA with different 18 F-prosthetic groups and characterized their uptake in A549 cells infected with influenza A virus from the 2009 pandemic (H1N1pdm). Methods: DPA was labeled with N-succinimidyl 4- 18 F-fluorobenzoate ( 18 F-SFB), 4-nitrophenyl 2- 18 F-fluoropropionate ( 18 F-NFP), 2- 18 F-Fluoroethyl toslyate ( 18 F-FET), and 18 F-aluminum (Al 18 F), respectively. Cy7-DPA was labeled with 18 F-SFB and 18 F-NFP only. The tracers were reconstituted with zinc nitrate before use. Apoptosis in A549 cells was induced by infection with the H1N1pdm virus for 48 h. Three μCi of each tracer was added to each well and incubated at 37 °C. The effect of different prosthetic groups, different MOI, and incubation time on percent cellular uptake was studied. Cell internalization and efflux was evaluated within 2 h of incubation. The competitive binding assay was performed with increasing concentration (10 −12 -10 −5 M) of Zn-DPA or Cy7-Zn-DPA prior to the addition of either 18 F-FB-Zn-DPA or 18 F-FB-Cy7-Zn-DPA into each well. IC 50 values for the two Zn-DPA analogues were estimated by GraphPad Prism 6.0. Results: Among all the four prosthetic groups, the 18 F-SFB method provided the highest conjugation yield for DPA and the highest uptake ratio between the infection cells and the control when both Zn-DPA and Cy7-Zn-DPA were present in the complex. The uptake ratio was similar for 18 F-FB-Zn-DPA and 18 F-FB-Cy7-Zn-DPA. Uptake of 18 F-FB-Zn-DPA and 18 F-FB-Cy7-Zn-DPA was proportional to the degree of apoptosis with a plateau at MOI 3. Uptake of 18 F-FB-Cy7-Zn-DPA also increased over incubation time and reached a plateau at 1 h, whereas uptake of 18 F-FB-Zn-DPA did not show any significant change over time

  18. Bone metastasis in patients with non-small cell lung cancer: The diagnostic role of F-18 FDG PET/CT

    International Nuclear Information System (INIS)

    Liu Ningbo; Ma Li; Zhou Wei; Pang Qingsong; Hu Man; Shi Fang; Fu Zheng; Li Minghuan; Yang Guoren; Yu Jinming

    2010-01-01

    Purpose: To evaluate the performance of F-18 FDG PET/CT in the detection of bone metastasis in non-small cell lung cancer (NSCLC) patients. Materials and methods: Three hundred and sixty-two consecutive NSCLC patients who underwent F-18 FDG PET/CT scanning were retrospectively analyzed. Each image of PET/CT, combined CT, and PET was performed at 10 separate areas and interpreted blindly and separately. The sensitivity, specificity and accuracy of F-18 FDG PET/CT, combined CT and F-18 FDG PET were calculated and the results were statistically analyzed. Results: Bone metastasis was confirmed in 82 patients with 331 positive segments based on the image findings and clinical follow-up. On patient-based analysis, the sensitivity of F-18 FDG PET/CT (93.9%) was significantly higher than those of combined CT (74.4%) and F-18 FDG PET (84.1%), respectively (p < 0.05). The overall specificity and accuracy of combined CT, F-18 FDG PET, and F-18 FDG PET/CT were 90.7%, 93.2%, 98.9% and 87.0%, 91.2%, and 97.8%, respectively (compared with PET/CT, p < 0.05). On segment-based analysis, the sensitivity of the three modalities were 79.5%, 94.3%, and 98.8%, respectively (compared with PET/CT, p < 0.05). The overall specificity and accuracy of the three modalities were 87.9%, 89.2%, 98.6% and 84.5%, 91.2%, 98.7%, respectively (compared with PET/CT, p < 0.05). Conclusion: F-18 FDG PET/CT is superior to F-18 FDG PET or combined CT in detecting bone metastasis of NSCLC patients because of the complementation of CT and PET. It is worth noting that the added value of F-18 FDG PET/CT may beneficially impact the clinical management of NSCLC.

  19. Regulation of cell proliferation by the E2F transcription factors

    DEFF Research Database (Denmark)

    Helin, K

    1998-01-01

    Experimental data generated in the past year have further emphasized the essential role for the E2F transcription factors in the regulation of cell proliferation. Genetic studies have shown that E2F activity is required for normal development in fruitflies, and the generation of E2F-1(-/-) mice h......Fs in the proteasomes. Novel target genes for the E2F transcription factors have been identified that link the E2Fs directly to the initiation of DNA replication.......Experimental data generated in the past year have further emphasized the essential role for the E2F transcription factors in the regulation of cell proliferation. Genetic studies have shown that E2F activity is required for normal development in fruitflies, and the generation of E2F-1(-/-) mice has...... demonstrated that individual members of the E2F transcription factor family are likely to have distinct roles in mammalian development and homeostasis. Additional mechanisms regulating the activity of the E2F transcription factors have been reported, including subcellular localization and proteolysis of the E2...

  20. F9 fimbriae of uropathogenic Escherichia coli are expressed at low temperature and recognise Galβ1-3GlcNAc-containing glycans.

    Directory of Open Access Journals (Sweden)

    Daniël J Wurpel

    Full Text Available Uropathogenic Escherichia coli (UPEC is the leading causative agent of urinary tract infections (UTI in the developed world. Among the major virulence factors of UPEC, surface expressed adhesins mediate attachment and tissue tropism. UPEC strains typically possess a range of adhesins, with type 1 fimbriae and P fimbriae of the chaperone-usher class the best characterised. We previously identified and characterised F9 as a new chaperone-usher fimbrial type that mediates biofilm formation. However, the regulation and specific role of F9 fimbriae remained to be determined in the context of wild-type clinical UPEC strains. In this study we have assessed the distribution and genetic context of the f9 operon among diverse E. coli lineages and pathotypes and demonstrated that f9 genes are significantly more conserved in a UPEC strain collection in comparison to the well-defined E. coli reference (ECOR collection. In the prototypic UPEC strain CFT073, the global regulator protein H-NS was identified as a transcriptional repressor of f9 gene expression at 37°C through its ability to bind directly to the f9 promoter region. F9 fimbriae expression was demonstrated at 20°C, representing the first evidence of functional F9 fimbriae expression by wild-type E. coli. Finally, glycan array analysis demonstrated that F9 fimbriae recognise and bind to terminal Galβ1-3GlcNAc structures.

  1. Male germ cell apoptosis and epigenetic histone modification induced by Tripterygium wilfordii Hook F.

    Directory of Open Access Journals (Sweden)

    Ji Xiong

    Full Text Available Multiglycosides of Tripterygium wilfordii Hook f (GTW, a Chinese herb-derived medicine used as a remedy for rheumatoid arthritis, are considered to be a reversible anti-fertility drug affecting the mammalian spermatids. However, the mechanism behind this effect is still unknown. To study the possible mechanism behind the impact of GTW on spermatogenesis, we administered 4 groups of 4-week-old male mice with different doses of GTW. We found a dose-dependent decrease in the number of germ cells after 40 days of GTW treatment, and an increase in apoptotic cells from the low-dose to the high-dose group. During this same period the dimethylated level of histone H3 lysine 9 (H3K9me2 in GTW-treated testes germ cells declined. Additionally, spermatogonial stem cells (SSCs from 6-day-old mice were isolated to evaluate the possible effect of GTW or triptolide on development of SSCs. We found a significantly higher incidence of apoptosis and lower dimethylation level of H3K9me2 in the SSCs of GTW or triptolide treatment than in controls. Thus, these data suggest that the GTW-induced apoptosis might be responsible for the fertility impairment in mice. This damage could be traced back to the early stages of spermatogenesis. GTW also affected the epigenetic modification of H3K9 in spermatogenesis. Molecular dynamics simulation suggested that triptolide and dimethylated or trimethylated H3K9 might have similar interaction mechanisms with EED (embryonic ectoderm development. These candidate activation mechanisms provide the first glimpse into the pathway of GTW-induced gonad toxicity, which is crucial for further research and clinical application.

  2. Semaphorin SEMA3F and VEGF Have Opposing Effects on Cell Attachment and Spreading

    Directory of Open Access Journals (Sweden)

    Patrick Nasarre

    2003-01-01

    Full Text Available SEMA3F, isolated from a 3p21.3 deletion, has antitumor activity in transfected cells, and protein expression correlates with tumor stage and histology. In primary tumors, SEMA3F and VEGF surface staining is inversely correlated. Coupled with SEMA3F at the leading edge of motile cells, we previously suggested that both proteins competitively regulate cell motility and adhesion. We have investigated this using the breast cancer cell line, MCF7. SEMA3F inhibited cell attachment and spreading as evidenced by loss of lamellipodia extensions, membrane ruffling, and cell-cell contacts, with cells eventually rounding-up and detaching. In contrast, VEGF had opposite effects. Although SEMA3F binds NRP2 with 10-fold greater affinity than NRP1, the effects in MCF7 were mediated by NRP1. This was determined by receptor expression and blocking of anti-NRP1 antibodies. Similar effects, but through NRP2, were observed in the C100 breast cancer cell line. Although we were unable to demonstrate changes in total GTPbound Rac1 or RhoA, we did observe changes in the localization of Rac1-GFP using time lapse microscopy. Following SEMA3F, Rac1 moved to the base of lamellipodia and — with their collapse — to the membrane. These results support the concept that SEMA3F and VEGF have antagonistic actions affecting motility in primary tumor cell.

  3. Glibenclamide Mimics Metabolic Effects of Metformin in H9c2 Cells.

    Science.gov (United States)

    Salani, Barbara; Ravera, Silvia; Fabbi, Patrizia; Garibaldi, Silvano; Passalacqua, Mario; Brunelli, Claudio; Maggi, Davide; Cordera, Renzo; Ameri, Pietro

    2017-01-01

    Sulfonylureas, such as glibenclamide, are antidiabetic drugs that stimulate beta-cell insulin secretion by binding to the sulfonylureas receptors (SURs) of adenosine triphosphate-sensitive potassium channels (KATP). Glibenclamide may be also cardiotoxic, this effect being ascribed to interference with the protective function of cardiac KATP channels for which glibenclamide has high affinity. Prompted by recent evidence that glibenclamide impairs energy metabolism of renal cells, we investigated whether this drug also affects the metabolism of cardiac cells. The cardiomyoblast cell line H9c2 was treated for 24 h with glibenclamide or metformin, a known inhibitor of the mitochondrial respiratory chain. Cell viability was evaluated by sulforodhamine B assay. ATP and AMP were measured according to the enzyme coupling method and oxygen consumption by using an amperometric electrode, while Fo-F1 ATP synthase activity assay was evaluated by chemiluminescent method. Protein expression was measured by western blot. Glibenclamide deregulated energy balance of H9c2 cardiomyoblasts in a way similar to that of metformin. It inhibited mitochondrial complexes I, II and III with ensuing impairment of oxygen consumption and ATP synthase activity, ATP depletion and increased AMPK phosphorylation. Furthermore, glibenclamide disrupted mitochondrial subcellular organization. The perturbation of mitochondrial energy balance was associated with enhanced anaerobic glycolysis, with increased activity of phosphofructo kinase, pyruvate kinase and lactic dehydrogenase. Interestingly, some additive effects of glibenclamide and metformin were observed. Glibenclamide deeply alters cell metabolism in cardiac cells by impairing mitochondrial organization and function. This may further explain the risk of cardiovascular events associated with the use of this drug, alone or in combination with metformin. © 2017 The Author(s). Published by S. Karger AG, Basel.

  4. Wind-Tunnel Investigation at Low Speed of the Yawing, Pitching, and Static Stability Characteristics of a 1/10-Scale Model of the Grumman F9F-9 Airplane, TED No. NACA AD 3109

    Science.gov (United States)

    Wolhart, Walter D.; Thomas, David F., Jr.

    1955-01-01

    An experimental investigation has been made in the Langley stability tunnel to determine the low-speed yawing, pitching, and static stability characteristics of a 1/10-scale model of the Grumman F9F-9 airplane. Tests were made to determine the effects of duct-entrance-fairing plugs on the static lateral and longitudinal stability characteristics of the complete model in the clean condition. The remaining tests were concerned with determining tail contributions as well as the effect of duct-entrance-fairing plugs, slats, flaps, and landing gear on the yawing and pitching stability derivatives. These data are presented without analysis in order to expedite distribution.

  5. Microwave mediated radiosynthesis of [F-18] FLT and its in-vitro study with androgen independent human prostate cancer cell line (PC-3)

    International Nuclear Information System (INIS)

    Ponde, D.E.; Dence, C.S.; Oyama, N.; Welch, M.J.

    2003-01-01

    The aim of this work was to improve the radiosynthesis of [F-18] FLT and to study its usefulness in monitoring change of proliferative activity of prostate cancer cells in the early phase of therapy. Method: Starting with anhydrothymidine, [F-18] FLT was synthesized by microwave mediated nucleophilic displacement by fluoride ion followed by acid hydrolysis in a synthesis time of just 55 minutes, which included Oasis solid phase and HPLC purification. The total radiochemical yield was 10-15% (at EOS), and the radiochemical purity was >99%. An in vitro study was carried out with androgen-independent human prostate cancer cell line PC-3. Two X 10e5 cells were seeded in 6 well plates with Ham's F-12K medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate supplemented with 10% heat activated FBS. One day later, PC-3 cells were at 50% confluent, the media was removed and the cells divided into two groups. In one group, cells were suspended in fresh media as above with 10% FBS, whereas in the other group cells were suspended in fresh media as above but without serum. Twenty-four hours later, [F-18] FLT was added to each flask (n=3). The cell-associated uptake of [F-18] FLT at 37 deg C was determined at 0, 1, 3, and 6 h after incubation. [F-18] FLT uptake in PC-3 cells decreased by 55% (from 9% to 4%) after 24h incubation with serum free media, indicating its potential usefulness to monitor cell proliferation in androgen-independent human prostate cancer. Studies to ascertain the uptake-mechanism are in the way. NIH grant HL13851

  6. TLR9 ligation in pancreatic stellate cells promotes tumorigenesis.

    Science.gov (United States)

    Zambirinis, Constantinos P; Levie, Elliot; Nguy, Susanna; Avanzi, Antonina; Barilla, Rocky; Xu, Yijie; Seifert, Lena; Daley, Donnele; Greco, Stephanie H; Deutsch, Michael; Jonnadula, Saikiran; Torres-Hernandez, Alejandro; Tippens, Daniel; Pushalkar, Smruti; Eisenthal, Andrew; Saxena, Deepak; Ahn, Jiyoung; Hajdu, Cristina; Engle, Dannielle D; Tuveson, David; Miller, George

    2015-11-16

    Modulation of Toll-like receptor (TLR) signaling can have protective or protumorigenic effects on oncogenesis depending on the cancer subtype and on specific inflammatory elements within the tumor milieu. We found that TLR9 is widely expressed early during the course of pancreatic transformation and that TLR9 ligands are ubiquitous within the tumor microenvironment. TLR9 ligation markedly accelerates oncogenesis, whereas TLR9 deletion is protective. We show that TLR9 activation has distinct effects on the epithelial, inflammatory, and fibrogenic cellular subsets in pancreatic carcinoma and plays a central role in cross talk between these compartments. Specifically, TLR9 activation can induce proinflammatory signaling in transformed epithelial cells, but does not elicit oncogene expression or cancer cell proliferation. Conversely, TLR9 ligation induces pancreatic stellate cells (PSCs) to become fibrogenic and secrete chemokines that promote epithelial cell proliferation. TLR9-activated PSCs mediate their protumorigenic effects on the epithelial compartment via CCL11. Additionally, TLR9 has immune-suppressive effects in the tumor microenvironment (TME) via induction of regulatory T cell recruitment and myeloid-derived suppressor cell proliferation. Collectively, our work shows that TLR9 has protumorigenic effects in pancreatic carcinoma which are distinct from its influence in extrapancreatic malignancies and from the mechanistic effects of other TLRs on pancreatic oncogenesis. © 2015 Zambirinis et al.

  7. 9 CFR 101.6 - Cell cultures.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Cell cultures. 101.6 Section 101.6..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to as tissue cultures...

  8. The EspF N-Terminal of Enterohemorrhagic Escherichia coli O157:H7 EDL933w Imparts Stronger Toxicity Effects on HT-29 Cells than the C-Terminal

    Directory of Open Access Journals (Sweden)

    Xiangyu Wang

    2017-09-01

    Full Text Available Enterohemorrhagic Escherichia coli (EHEC O157:H7 EspF is an important multifunctional protein that destroys the tight junctions of intestinal epithelial cells and promotes host cell apoptosis. However, its molecular mechanism remains elusive. We knocked out the espF sequence (747 bp, ΔespF, N-terminal sequence (219 bp, ΔespFN, and C-terminal sequence (528 bp, ΔespFC separately using the pKD46-mediated λ Red homologous recombination system. Then, we built the corresponding complementation strains, namely, ΔespF/pespF, ΔespFN/pespFN, and ΔespFC/pespFC by overlap PCR, which were used in infecting HT-29 cells and BALB/C mice. The level of reactive oxygen species, cell apoptosis, mitochondrial trans-membrane potential, inflammatory factors, transepithelial electrical resistance (TER, and animal mortality were evaluated by DCFH-DA, double staining of Annexin V-FITC/PI, JC-1 staining, ELISA kit, and a mouse assay. The wild-type (WT, ΔespF, ΔespF/pespF, ΔespFC, ΔespFC/pespFC, ΔespFN, and ΔespFN/pespFN groups exhibited apoptotic rates of 68.3, 27.9, 64.9, 65.7, 73.4, 41.3, and 35.3% respectively, and mean TNF-α expression levels of 428 pg/mL, 342, 466, 446, 381, 383, and 374 pg/mL, respectively. In addition, the apoptotic rates and TNF-α levels of the WT, ΔespF/pespF, and ΔespFC were significantly higher than that of ΔespF, ΔespFN, ΔespFC/pespFC, and ΔespFN/pespFN group (p < 0.05. The N-terminal of EspF resulted in an increase in the number of apoptotic cells, TNF-α secretion, ROS generation, mitochondria apoptosis, and pathogenicity in BalB/c mice. In conclusion, the N-terminal domain of the Enterohemorrhagic E. coli O157:H7 EspF more strongly promotes apoptosis and inflammation than the C-terminal domain.

  9. Oct3/4 directly regulates expression of E2F3a in mouse embryonic stem cells

    International Nuclear Information System (INIS)

    Kanai, Dai; Ueda, Atsushi; Akagi, Tadayuki; Yokota, Takashi; Koide, Hiroshi

    2015-01-01

    Embryonic stem (ES) cells, derived from the inner cell mass of blastocysts, have a characteristic cell cycle with truncated G1 and G2 phases. Recent findings that suppression of Oct3/4 expression results in a reduced proliferation rate of ES cells suggest the involvement of Oct3/4 in the regulation of ES cell growth, although the underlying molecular mechanism remains unclear. In the present study, we identified E2F3a as a direct target gene of Oct3/4 in ES cells. Oct3/4 directly bound to the promoter region of the E2F3a gene and positively regulated expression of E2F3a in mouse ES cells. Suppression of E2F3a activity by E2F6 overexpression led to the reduced proliferation in ES cells, which was relieved by co-expression of E2F3a. Furthermore, cell growth retardation caused by loss of Oct3/4 was rescued by E2F3a expression. These results suggest that Oct3/4 upregulates E2F3a expression to promote ES cell growth. - Highlights: • Oct3/4 positively regulates E2F3a expression in ES cells. • Oct3/4 binds to the promoter region of the E2F3a gene. • Overexpression of E2F6, an inhibitor of E2F3a, reduces ES cell growth. • E2F3a recovers growth retardation of ES cells caused by Oct3/4 reduction

  10. Oct3/4 directly regulates expression of E2F3a in mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Kanai, Dai; Ueda, Atsushi; Akagi, Tadayuki; Yokota, Takashi; Koide, Hiroshi, E-mail: hkoide@med.kanazawa-u.ac.jp

    2015-04-10

    Embryonic stem (ES) cells, derived from the inner cell mass of blastocysts, have a characteristic cell cycle with truncated G1 and G2 phases. Recent findings that suppression of Oct3/4 expression results in a reduced proliferation rate of ES cells suggest the involvement of Oct3/4 in the regulation of ES cell growth, although the underlying molecular mechanism remains unclear. In the present study, we identified E2F3a as a direct target gene of Oct3/4 in ES cells. Oct3/4 directly bound to the promoter region of the E2F3a gene and positively regulated expression of E2F3a in mouse ES cells. Suppression of E2F3a activity by E2F6 overexpression led to the reduced proliferation in ES cells, which was relieved by co-expression of E2F3a. Furthermore, cell growth retardation caused by loss of Oct3/4 was rescued by E2F3a expression. These results suggest that Oct3/4 upregulates E2F3a expression to promote ES cell growth. - Highlights: • Oct3/4 positively regulates E2F3a expression in ES cells. • Oct3/4 binds to the promoter region of the E2F3a gene. • Overexpression of E2F6, an inhibitor of E2F3a, reduces ES cell growth. • E2F3a recovers growth retardation of ES cells caused by Oct3/4 reduction.

  11. IL-9-Producing Mast Cell Precursors and Food Allergy

    Science.gov (United States)

    2017-10-01

    AWARD NUMBER: W81XWH-15-1-0517 TITLE: IL-9-Producing Mast Cell Precursors and Food Allergy PRINCIPAL INVESTIGATOR: Dr. Simon P. Hogan PhD...IL-9-Producing Mast Cell Precursors and Food Allergy 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Yui Hsi Wang, Sunil...threatening anaphylaxis. We have identified a novel multi-functional IL-9-producing mucosal mast cells (MMC9s) that produce large amounts of IL-9, IL

  12. Evaluation of {sup 18}F-FDG and {sup 18}F-FLT for monitoring therapeutic responses of colorectal cancer cells to radiotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hui [Nuclear Medicine Department, The General Hospital of the Chinese People' s Liberation Army and Military Medical Postgraduate College, Beijing 100853 (China); Liu, Bo [Department of Hepatobiliary Surgery, The General Hospital of the Chinese People' s Liberation Army and Military Medical Postgraduate College, Beijing 100853 (China); Tian, Jiahe, E-mail: tianjh@vip.sina.com [Nuclear Medicine Department, The General Hospital of the Chinese People' s Liberation Army and Military Medical Postgraduate College, Beijing 100853 (China); Xu, Baixuan; Zhang, Jinming [Nuclear Medicine Department, The General Hospital of the Chinese People' s Liberation Army and Military Medical Postgraduate College, Beijing 100853 (China); Qu, Baolin [Department of Radiation Oncology, The General Hospital of the Chinese People' s Liberation Army and Military Medical Postgraduate College, Beijing 100853 (China); Chen, Yingmao [Nuclear Medicine Department, The General Hospital of the Chinese People' s Liberation Army and Military Medical Postgraduate College, Beijing 100853 (China)

    2013-09-15

    In order to compare the efficacy of {sup 18}F-fluorothymidine (FLT) and {sup 18}F-fluorodeoxyglucose (FDG) for monitoring early responses to irradiation, two human colorectal cancer (CRC) cell lines SW480 and SW620, which were derived from the primary lesions and the metastatic lymph node, underwent X-ray irradiation of 0, 10, or 20 Gy and were examined at 0, 24 and 72 h After irradiation, reduced proliferation of both SW480 and SW620 cells was observed in a dose-dependent manner (P < 0.001), G0-G1 arrest was also noted in both cell types after 72 h in the 20 Gy group (P < 0.001). Although increased apoptosis was observed in both cell lines after irradiation (P < 0.001), a greater percentage of SW480 cells underwent apoptosis in response to irradiation than SW620 cells. Increased Hsp27 and decreased integrin β{sub 3}, Ki67 and VEGFR2 expression was observed over time via immunocytochemistry and Western blot analysis (P < 0.001), however, no significant changes were noted in response to irradiation. Finally, reduced uptake of {sup 18}F-FLT by SW480 or SW620 cells was observed at 24-h post-irradiation, however, reduced {sup 18}F-FDG uptake was only observed after 72 h. Therefore, we conclude that {sup 18}F-FLT is a more suitable positron emission tomography (PET) tracer for monitoring early responses to irradiation in primary and metastatic lymph node CRC cells.

  13. Streptococcal cell wall-induced arthritis and adjuvant arthritis in F344----Lewis and in Lewis----F344 bone marrow chimeras

    International Nuclear Information System (INIS)

    van Bruggen, M.C.; van den Broek, M.F.; van den Berg, W.B.

    1991-01-01

    Streptococcal cell wall (SCW)-induced arthritis and adjuvant arthritis (AA) are rat models for chronic, erosive polyarthritis. Both models can be induced in susceptible Lewis rats, whereas F344 rats are resistant. In AA as well as in SCW arthritis, antigen-specific T lymphocytes have been demonstrated to be crucial for chronic disease. In this communication the authors describe their studies to probe the cellular mechanism responsible for the difference in susceptibility of Lewis and F344, using bone marrow chimeras. By transplanting bone marrow cells from F344 into lethally irradiated Lewis recipients, Lewis rats were rendered resistant to SCW arthritis induction. F344 rats reconstituted with Lewis bone marrow, i.e., Lewis----F344 chimeras, develop an arthritis upon SCW injection. For AA comparable results were obtained. These data suggest that both resistance and susceptibility to bacterium-induced chronic arthritis are mediated by hemopoietic/immune cells and that the recipiental environment does not influence the susceptibility to chronic joint inflammation

  14. F.F.F.F. armastab mõtestada ennast / Reet Varblane

    Index Scriptorium Estoniae

    Varblane, Reet, 1952-

    2002-01-01

    Rühmituse F.F.F.F. (Kristi Paap, Kaire Rannik, Berit Teeäär, Ketli Tiitsar, Maria Valdma) näitus "Meie igapäevane leib" 9. novembrini Hansapanga galeriis ja näitus "Pentagramm" 26. X-1. XII Tarbekunstimuuseumis

  15. Knockdown of E2f1 by RNA interference impairs proliferation of rat cells in vitro

    Directory of Open Access Journals (Sweden)

    Luciana dos Reis Vasques

    2010-01-01

    Full Text Available E2F1 plays a key role in cell-cycle regulation in mammals, since its transcription factor activity controls genes required for DNA synthesis and apoptosis. E2F1 deregulation is a common feature among different tumor types and can be a major cause of cell proliferation. Thus, blocking E2F1 expression by RNA interference represents a promising therapeutic approach. In this study, the introduction of specific short hairpin RNAs (shRNAs reduced E2f1 expression by up to 77%, and impaired rat glioma cell proliferation by approximately 70%, as compared to control cells. Furthermore, we investigated the expression of E2f1 target genes, Cyclin A and Cyclin E. Cyclin A was found to be down-regulated, whereas Cyclin E had similar expression to control cells, indicating that gene(s other than E2f1 control its transcription. Other E2f family members, E2f2 and E2f3, which have been classified in the same subgroup of transcriptional activators, were also analyzed. Expression of both E2f2 and E2f3 was similar to control cells, showing no cross-inactivation or up-regulation to compensate for the absence of E2f1. Nevertheless, their expression was insufficient to maintain the initial proliferation potential. Taken together, our results suggest that shE2f1 is a promising therapy to control tumor cell proliferation.

  16. Isolated thymic Langerhans cell histiocytosis discovered on F-18 fluorodeoxyglucose positron emission tomography/computed tomography (F-18 FDG PET/CT).

    Science.gov (United States)

    Turpin, Sophie; Carret, Anne-Sophie; Dubois, Josée; Buteau, Chantal; Patey, Natalie

    2015-11-01

    The thymic infiltration in young patients with multisystemic Langerhans cell histiocytosis and its radiologic features are well known. However, isolated thymic disease has seldom been reported in the literature. We report the case of a 10-month-old child admitted for fever of unknown origin. Whole-body F-18 fluorodeoxyglucose positron emission tomography/computed tomography (F-18 FDG PET/CT) was performed to identify a focus of infection. It demonstrated an unusual aspect of the thymus, which led to further investigation and revealed isolated infiltration of the thymus by Langerhans cell histiocytosis. The patient was treated accordingly and is now disease free. As evaluation of Langerhans cell histiocytosis patients with F-18 FDG PET/CT is becoming more frequent, it is important to be aware of the scintigraphical characteristics of thymic Langerhans cell histiocytosis.

  17. Isolated thymic Langerhans cell histiocytosis discovered on F-18 fluorodeoxyglucose positron emission tomography/computed tomography (F-18 FDG PET/CT)

    Energy Technology Data Exchange (ETDEWEB)

    Turpin, Sophie [CHU Sainte-Justine, Nuclear Medicine, Montreal (Canada); Carret, Anne-Sophie [CHU Sainte-Justine, Hemato-Oncology, Montreal (Canada); Dubois, Josee [CHU Sainte-Justine, Radiology, Montreal (Canada); Buteau, Chantal [CHU Sainte-Justine, Infectious Diseases, Montreal (Canada); Patey, Natalie [CHU Sainte-Justine, Pathology, Montreal (Canada)

    2015-11-15

    The thymic infiltration in young patients with multisystemic Langerhans cell histiocytosis and its radiologic features are well known. However, isolated thymic disease has seldom been reported in the literature. We report the case of a 10-month-old child admitted for fever of unknown origin. Whole-body F-18 fluorodeoxyglucose positron emission tomography/computed tomography (F-18 FDG PET/CT) was performed to identify a focus of infection. It demonstrated an unusual aspect of the thymus, which led to further investigation and revealed isolated infiltration of the thymus by Langerhans cell histiocytosis. The patient was treated accordingly and is now disease free. As evaluation of Langerhans cell histiocytosis patients with F-18 FDG PET/CT is becoming more frequent, it is important to be aware of the scintigraphical characteristics of thymic Langerhans cell histiocytosis. (orig.)

  18. Regulation of immune responses in SJL and F1 hybrid mice by gamma-irradiated syngeneic lymphoma cells

    International Nuclear Information System (INIS)

    Katz, I.R.; Nagase, F.; Bell, M.K.; Ponzio, N.M.; Thorbecke, G.J.

    1984-01-01

    Syngeneic mixed lymphocyte-stimulating la+ lymphomas of SJL mice [reticulum cell sarcoma(s) (RCS)] were found to modulate immune responses in vivo. Simultaneous injection of 2 X 10(7) gamma-irradiated or glutaraldehyde-fixed RCS cells with the antigen sheep red blood cells (SRBC) or 2,4,6-trinitrophenol (TNP)-Ficoll markedly suppressed the subsequent plaque-forming cell response in the spleen. The suppression of the anti-SRBC response was prevented by pretreatment of the mice with cyclophosphamide, whereas the suppression of the anti-TNP-Ficoll response was not affected. RCS injection induced high interferon serum titers within 24 hours after injection, which were not prevented by pretreatment with cyclophosphamide. Injection of gamma-irradiated RCS cells (gamma-RCS) or RCS cell extract 2 days prior to antigen enhanced the anti-SRBC but markedly suppressed the anti- TNP-Ficoll response. Injection of RCS both on day -2 and day 0 enhanced the anti-SRBC response. SJL mice 8-9 months of age showed much less or no suppression when gamma-RCS cells were injected on day 0. Certain F1 hybrids of SJL also showed the gamma-RCS-induced suppression of the anti-SRBC response. Suppression was seen in SJL X BALB.B but not in SJL X BALB/c mice and in SJL X A.TH but not in SJL X A.TL mice, suggesting an I-region effect. F1 hybrids of SJL by B10 background mice showed no significant suppression. Enhancement of the anti-SRBC response by prior injection of gamma-RCS was seen in all F1 hybrid mice examined

  19. Contrasting feature in the repopulation of host-type T cells in the spleens of F1----P and P----F1 radiation bone marrow chimeras

    International Nuclear Information System (INIS)

    Hirokawa, K.; Sado, T.; Kubo, S.; Kamisaku, H.; Utsuyama, M.

    1986-01-01

    The regeneration and persistence of host- and donor-derived T cells were examined in the thymus as well as the spleen of mouse radiation bone marrow chimeras of two semiallogeneic combinations (F1----P, P----F1) with different Thy-1 markers on T cells of donor and host origins. An unexpectedly large number of host-type T cells were recovered from the spleens of F1----P chimeras, amounting to as high as 45 and 25% of total T cells at 6 and 14 weeks after bone marrow transplantation (BMT), respectively. To the contrary, the residual host-type T cells in the spleens of P----F1 chimeras disappeared quickly, resulting in less than 0.1% of total T cells at 6 weeks after BMT. It was also revealed that the number of host-type T cells in the spleens of F1----P chimeras decreased in proportion to increase of radiation dose given to the recipients

  20. Expression of the fructose receptor BmGr9 and its involvement in the promotion of feeding, suggested by its co-expression with neuropeptide F1 in Bombyx mori.

    Science.gov (United States)

    Mang, Dingze; Shu, Min; Tanaka, Shiho; Nagata, Shinji; Takada, Tomoyuki; Endo, Haruka; Kikuta, Shingo; Tabunoki, Hiroko; Iwabuchi, Kikuo; Sato, Ryoichi

    2016-08-01

    Insect gustatory receptors (Grs) are members of a large family of proteins with seven transmembrane domains that provide insects with the ability to detect chemical signals critical for feeding, mating, and oviposition. To date, 69 Bombyx mori Grs (BmGrs) genes have been identified via genome studies. BmGr9 has been shown to respond specifically to fructose and to function as a ligand-gated ion channel selectively activated by fructose. However, the sites where this Gr are expressed remain unclear. We demonstrated using reverse transcription (RT)-PCR that BmGr9 is widely expressed in the central nervous system (CNS), as well as oral sensory organs. Additionally, immunohistochemistry was performed using anti-BmGr9 antiserum to show that BmGr9 is expressed in cells of the oral sensory organs, including the maxillary galea, maxillary palps, labrum, and labium, as well as in putative neurosecretory cells of the CNS. Furthermore, double immunohistochemical analysis showed that most BmGr9-expressing cells co-localized with putative neuropeptide F1-expressing cells in the brain, suggesting that BmGr9 is involved in the promotion of feeding behaviors. In addition, a portion of BmGr9-expressing cells in the brain co-localized with cells expressing BmGr6, a molecule of the sugar receptor clade, suggesting that sugars other than fructose are involved in the regulation of feeding behaviors in B. mori larvae. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Human umbilical cord mesenchymal stem cells increase interleukin-9 production of CD4+ T cells

    Science.gov (United States)

    Yang, Zhou Xin; Chi, Ying; Ji, Yue Ru; Wang, You Wei; Zhang, Jing; Luo, Wei Feng; Li, Li Na; Hu, Cai Dong; Zhuo, Guang Sheng; Wang, Li Fang; Han, Zhi-Bo; Han, Zhong Chao

    2017-01-01

    Mesenchymal stem cells (MSC) are able to differentiate into cells of multiple lineage, and additionally act to modulate the immune response. Interleukin (IL)-9 is primarily produced by cluster of differentiation (CD)4+ T cells to regulate the immune response. The present study aimed to investigate the effect of human umbilical cord derived-MSC (UC-MSC) on IL-9 production of human CD4+ T cells. It was demonstrated that the addition of UC-MSC to the culture of CD4+ T cells significantly enhanced IL-9 production by CD4+ T cells. Transwell experiments suggested that UC-MSC promotion of IL-9 production by CD4+ T cells was dependent on cell-cell contact. Upregulated expression of CD106 was observed in UC-MSC co-cultured with CD4+ T cells, and the addition of a blocking antibody of CD106 significantly impaired the ability of UC-MSC to promote IL-9 production by CD4+ T cells. Therefore, the results of the present study demonstrated that UC-MSC promoted the generation of IL-9 producing cells, which may be mediated, in part by CD106. The findings may act to expand understanding and knowledge of the immune modulatory role of UC-MSC. PMID:29042945

  2. Inhibition of H3K9 methyltransferase G9a induces autophagy and apoptosis in oral squamous cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Aishu; Qiu, Yu [Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences, Chongqing Medical University, Chongqing, 401147 (China); Affiliated Hospital of Stomatology, Chongqing Medical University, Chongqing, 401147 (China); Cui, Hongjuan [State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400716 (China); Fu, Gang, E-mail: fg.ras@hotmail.com [Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences, Chongqing Medical University, Chongqing, 401147 (China); Affiliated Hospital of Stomatology, Chongqing Medical University, Chongqing, 401147 (China)

    2015-03-27

    Objective: To explore whether inhibition of H3K9 Methyltransferase G9a could exert an antitumoral effect in oral squamous cell carcinoma (OSCC). Materials and methods: First we checked G9a expression in two OSCC cell lines Tca8113 and KB. Next we used a special G9a inhibitor BIX01294 (BIX) to explore the effect of inhibition of G9a on OSCC in vitro. Cell growth was tested by typlan blue staining, MTT assay and Brdu immunofluorescence staining. Cell autophagy was examined by monodansylcadaverine (MDC) staining, LC3-II immunofluorescence staining and LC3-II western blot assay. Cell apoptosis was checked by FITC Annexin-V and PI labeling, tunnel staining and caspase 3 western blot assay. Finally, the effect of inhibition of G9a on clonogenesis and tumorigenesis capacity of OSCC was analyzed by soft agar growth and xenograft model. Results: Here we showed that G9a was expressed in both Tca8113 and KB cells. Inhibition of G9a using BIX significantly reduced cell growth and proliferation in Tca8113 and KB. Inhibition of G9a induced cell autophagy with conversion of LC3-I to LC3-II and cell apoptosis with the expression of cleaved caspase 3. We also found that inhibition of G9a reduced colony formation in soft agar and repressed tumor growth in mouse xenograph model. Conclusion: Our results suggested that G9a might be a potential epigenetic target for OSCC treatment. - Highlights: • Inhibition of G9a reduced cell growth and proliferation in OSCC cells. • Inhibition of G9a induces autophagy and apoptosis in OSCC cells. • Inhibition of G9a repressed tumor growth in mouse xenograph model.

  3. Evaluation of two new STR loci 9q2h2 and wg3f12 in a Japanese population.

    Science.gov (United States)

    Mizutani, M; Huang, X L; Tamaki, K; Yoshimoto, T; Uchihi, R; Yamamoto, T; Katsumata, Y; Armour, J A

    1999-09-01

    Two short tandem repeat (STR) loci (9q2h2 and wg3f12) have been evaluated in a Japanese population. Ten and seven different alleles were observed in 9q2h2 and wg3f12 respectively. 9q2h2 displayed simple polymorphism in tetrameric repeat structure; by contrast, wg3f12 contained variable numbers of tetrameric repeats and a 30-bp deletion/insertion polymorphism. No "interalleles" were found. The expected heterozygosities of 9q2h2 and wg3fl2 were 0.749 and 0.574, respectively. No deviation from Hardy-Weinberg equilibrium was found.

  4. The translocator protein ligand [{sup 18}F]DPA-714 images glioma and activated microglia in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Winkeler, Alexandra; Boisgard, Raphael; Awde, Ali R.; Dubois, Albertine; Theze, Benoit; Zheng, Jinzi [Universite Paris Sud, Inserm, U1023, Laboratoire d' Imagerie Moleculaire Experimentale, Orsay (France); CEA, I2BM, SHFJ, Orsay (France); Ciobanu, Luisa [CEA, DSV, I2BM, NeuroSpin, LRMN, Gif sur Yvette (France); Dolle, Frederic [CEA, I2BM, SHFJ, Orsay (France); Viel, Thomas; Jacobs, Andreas H. [Westfaelische Wilhelm-Universitaet Muenster (WWU), European Institute for Molecular Imaging (EIMI), Muenster (Germany); Tavitian, Bertrand [Universite Paris Sud, Inserm, U1023, Laboratoire d' Imagerie Moleculaire Experimentale, Orsay (France)

    2012-05-15

    In recent years there has been an increase in the development of radioligands targeting the 18-kDa translocator protein (TSPO). TSPO expression is well documented in activated microglia and serves as a biomarker for imaging neuroinflammation. In addition, TSPO has also been reported to be overexpressed in a number of cancer cell lines and human tumours including glioma. Here we investigated the use of [{sup 18}F]DPA-714, a new TSPO positron emission tomography (PET) radioligand to image glioma in vivo. We studied the uptake of [{sup 18}F]DPA-714 in three different rat strains implanted with 9L rat glioma cells: Fischer (F), Wistar (W) and Sprague Dawley (SD) rats. Dynamic [{sup 18}F]DPA-714 PET imaging, kinetic modelling of PET data and in vivo displacement studies using unlabelled DPA-714 and PK11195 were performed. Validation of TSPO expression in 9L glioma cell lines and intracranial 9L gliomas were investigated using Western blotting and immunohistochemistry of brain tissue sections. All rats showed significant [{sup 18}F]DPA-714 PET accumulation at the site of 9L tumour implantation compared to the contralateral brain hemisphere with a difference in uptake among the three strains (F > W > SD). The radiotracer showed high specificity for TSPO as demonstrated by the significant reduction of [{sup 18}F]DPA-714 binding in the tumour after administration of unlabelled DPA-714 or PK11195. TSPO expression was confirmed by Western blotting in 9L cells in vitro and by immunohistochemistry ex vivo. The TSPO radioligand [{sup 18}F]DPA-714 can be used for PET imaging of intracranial 9L glioma in different rat strains. This preclinical study demonstrates the feasibility of employing [{sup 18}F]DPA-714 as an alternative radiotracer to image human glioma. (orig.)

  5. Effects of LiF/Al back electrode on the amorphous/crystalline silicon heterojunction solar cells

    International Nuclear Information System (INIS)

    Kim, Sunbo; Lee, Jaehyeong; Dao, Vinh Ai; Lee, Seungho; Balaji, Nagarajan; Ahn, Shihyun; Hussain, Shahzada Qamar; Han, Sangmyeong; Jung, Junhee; Jang, Juyeon; Lee, Yunjung; Yi, Junsin

    2013-01-01

    Highlights: ► We have employed a LiF dielectric layer as a new back-contact electrode. ► Increasing LiF thickness will decrease barrier for electrons transport, thus yield J sc . ► Increasing LiF thickness will reduced shunt leakage and enhanced internal field, thus yield V oc . ► Employing LiF layer, improvement of performance of HIT solar cells up to 17.13%. -- Abstract: To improve the quantum efficiency (QE) and hence the efficiency of the amorphous/crystalline silicon heterojunction solar cell, we have employed a LiF dielectric layer on the rear side. The high dipole moment of the LiF reduces the aluminum electrode's work–function and then lowers the energy barrier at back contact. This lower energy barrier height helps to enhance both the operating voltage and the QE at longer wavelength region, in turn improves the open-circuit voltage (V oc ), short-circuit current density (J sc ), and then overall cell efficiency. With optimized LiF layer thickness of 20 nm, 1 cm 2 heterojunction with intrinsic thin layer (HIT) solar cells were produced with industry-compatible process, yielding V oc of 690 mV, J sc of 33.62 mA/cm 2 , and cell efficiencies of 17.13%. Therefore LiF/Al electrode on rear side is proposed as an alternate back electrode for high efficiency HIT solar cells

  6. Effect of F-spondin on cementoblastic differentiation of human periodontal ligament cells

    International Nuclear Information System (INIS)

    Kitagawa, Masae; Kudo, Yasusei; Iizuka, Shinji; Ogawa, Ikuko; Abiko, Yoshimitsu; Miyauchi, Mutsumi; Takata, Takashi

    2006-01-01

    Cementum is a mineralized tissue produced by cementoblasts covering the roots of teeth that provides for the attachment of periodontal ligament to roots and surrounding alveolar bone. To study the mechanism of proliferation and differentiation of cementoblasts is important for understanding periodontal physiology and pathology including periodontal tissue regeneration. However, the detailed mechanism of the proliferation and differentiation of human cementoblasts is still unclear. We previously established human cementoblast-like (HCEM) cell lines. We thought that comparing the transcriptional profiles of HCEM cells and human periodontal ligament (HPL) cells derived from the same teeth could be a good approach to identify genes that influence the nature of cementoblasts. We identified F-spondin as the gene demonstrating the high fold change expression in HCEM cells. Interestingly, F-spondin highly expressing HPL cells showed similar phenotype of cementoblasts, such as up-regulation of mineralized-related genes. Overall, we identified F-spondin as a promoting factor for cementoblastic differentiation

  7. Stochastic E2F activation and reconciliation of phenomenological cell-cycle models.

    Science.gov (United States)

    Lee, Tae J; Yao, Guang; Bennett, Dorothy C; Nevins, Joseph R; You, Lingchong

    2010-09-21

    The transition of the mammalian cell from quiescence to proliferation is a highly variable process. Over the last four decades, two lines of apparently contradictory, phenomenological models have been proposed to account for such temporal variability. These include various forms of the transition probability (TP) model and the growth control (GC) model, which lack mechanistic details. The GC model was further proposed as an alternative explanation for the concept of the restriction point, which we recently demonstrated as being controlled by a bistable Rb-E2F switch. Here, through a combination of modeling and experiments, we show that these different lines of models in essence reflect different aspects of stochastic dynamics in cell cycle entry. In particular, we show that the variable activation of E2F can be described by stochastic activation of the bistable Rb-E2F switch, which in turn may account for the temporal variability in cell cycle entry. Moreover, we show that temporal dynamics of E2F activation can be recast into the frameworks of both the TP model and the GC model via parameter mapping. This mapping suggests that the two lines of phenomenological models can be reconciled through the stochastic dynamics of the Rb-E2F switch. It also suggests a potential utility of the TP or GC models in defining concise, quantitative phenotypes of cell physiology. This may have implications in classifying cell types or states.

  8. Embryonic hybrid cells: a powerful tool for studying pluripotency and reprogramming of the differentiated cell chromosomes

    Directory of Open Access Journals (Sweden)

    SEROV OLEG

    2001-01-01

    Full Text Available The properties of embryonic hybrid cells obtained by fusion of embryonic stem (ES or teratocarcinoma (TC cells with differentiated cells are reviewed. Usually, ES-somatic or TC-somatic hybrids retain pluripotent capacity at high levels quite comparable or nearly identical with those of the pluripotent partner. When cultured in vitro, ES-somatic- and TC-somatic hybrid cell clones, as a rule, lose the chromosomes derived from the somatic partner; however, in some clones the autosomes from the ES cell partner were also eliminated, i.e. the parental chromosomes segregated bilaterally in the ES-somatic cell hybrids. This opens up ways for searching correlation between the pluripotent status of the hybrid cells and chromosome segregation patterns and therefore for identifying the particular chromosomes involved in the maintenance of pluripotency. Use of selective medium allows to isolate in vitro the clones of ES-somatic hybrid cells in which "the pluripotent" chromosome can be replaced by "the somatic" counterpart carrying the selectable gene. Unlike the TC-somatic cell hybrids, the ES-somatic hybrids with a near-diploid complement of chromosomes are able to contribute to various tissues of chimeric animals after injection into the blastocoel cavity. Analysis of the chimeric animals showed that the "somatic" chromosome undergoes reprogramming during development. The prospects for the identification of the chromosomes that are involved in the maintenance of pluripotency and its cis- and trans-regulation in the hybrid cell genome are discussed.

  9. OP9 bone marrow stroma cells differentiate into megakaryocytes and platelets.

    Directory of Open Access Journals (Sweden)

    Yumiko Matsubara

    Full Text Available Platelets are essential for hemostatic plug formation and thrombosis. The mechanisms of megakaryocyte (MK differentiation and subsequent platelet production from stem cells remain only partially understood. The manufacture of megakaryocytes (MKs and platelets from cell sources including hematopoietic stem cells and pluripotent stem cells have been highlighted for studying the platelet production mechanisms as well as for the development of new strategies for platelet transfusion. The mouse bone marrow stroma cell line OP9 has been widely used as feeder cells for the differentiation of stem cells into MK lineages. OP9 cells are reported to be pre-adipocytes. We previously reported that 3T3-L1 pre-adipocytes differentiated into MKs and platelets. In the present study, we examined whether OP9 cells differentiate into MKs and platelets using MK lineage induction (MKLI medium previously established to generate MKs and platelets from hematopoietic stem cells, embryonic stem cells, and pre-adipocytes. OP9 cells cultured in MKLI medium had megakaryocytic features, i.e., positivity for surface markers CD41 and CD42b, polyploidy, and distinct morphology. The OP9-derived platelets had functional characteristics, providing the first evidence for the differentiation of OP9 cells into MKs and platelets. We then analyzed gene expressions of critical factors that regulate megakaryopoiesis and thrombopoiesis. The gene expressions of p45NF-E2, FOG, Fli1, GATA2, RUNX1, thrombopoietin, and c-mpl were observed during the MK differentiation. Among the observed transcription factors of MK lineages, p45NF-E2 expression was increased during differentiation. We further studied MK and platelet generation using p45NF-E2-overexpressing OP9 cells. OP9 cells transfected with p45NF-E2 had enhanced production of MKs and platelets. Our findings revealed that OP9 cells differentiated into MKs and platelets in vitro. OP9 cells have critical factors for megakaryopoiesis and

  10. The F309S mutation increases factor VIII secretion in human cell line

    Directory of Open Access Journals (Sweden)

    Daianne Maciely Carvalho Fantacini

    2016-06-01

    Full Text Available ABSTRACT OBJECTIVES: The capacity of a human cell line to secrete recombinant factor VIII with a F309S point mutation was investigated, as was the effect of the addition of chemical chaperones (betaine and sodium-4-phenylbutyrate on the secretion of factor VIII. METHODS: This work used a vector with a F309S mutation in the A1 domain to investigate FVIII production in the HEK 293 human cell line. Factor VIII activity was measured by chromogenic assay. Furthermore, the effects of chemical drugs on the culture were evaluated. RESULTS: The addition of the F309S mutation to a previously described FVIII variant increased FVIII secretion by 4.5 fold. Moreover, the addition of betaine or sodium-4-phenylbutyrate increased the secretion rate of FVIIIΔB proteins in HEK 293 cells, but the same effect was not seen for FVIIIΔB-F309S indicating that all the recombinant protein produced had been efficiently secreted. CONCLUSION: Bioengineering factor VIII expressed in human cells may lead to an efficient production of recombinant factor VIII and contribute toward low-cost coagulation factor replacement therapy for hemophilia A. FVIII-F309S produced in human cells can be effective in vivo.

  11. The anti-(+-methamphetamine monoclonal antibody mAb7F9 attenuates acute (+-methamphetamine effects on intracranial self-stimulation in rats.

    Directory of Open Access Journals (Sweden)

    Andrew C Harris

    Full Text Available Passive immunization with monoclonal antibodies (mAbs against (+-methamphetamine (METH is being evaluated for the treatment of METH addiction. A human/mouse chimeric form of the murine anti-METH mAb7F9 has entered clinical trials. This study examined the effects of murine mAb7F9 on certain addiction-related behavioral effects of METH in rats as measured using intracranial self-stimulation (ICSS. Initial studies indicated that acute METH (0.1-0.56 mg/kg, s.c. lowered the minimal (threshold stimulation intensity that maintained ICSS. METH (0.3 mg/kg, s.c. also blocked elevations in ICSS thresholds (anhedonia-like behavior during spontaneous withdrawal from a chronic METH infusion (10 mg/kg/day x 7 days. In studies examining effects of i.v. pretreatment with mAb7F9 (at 30, 100, or 200 mg/kg, 200 mg/kg blocked the ability of an initial injection of METH (0.3 mg/kg, s.c. to reduce baseline ICSS thresholds, but was less capable of attenuating the effect of subsequent daily injections of METH. MAb7F9 (200 mg/kg also produced a small but significant reduction in the ability of METH (0.3 mg/kg, s.c. to reverse METH withdrawal-induced elevations in ICSS thresholds. These studies demonstrate that mAb7F9 can partially attenuate some addiction-related effects of acute METH in an ICSS model, and provide some support for the therapeutic potential of mAb7F9 for the treatment of METH addiction.

  12. El fútbol 9 como alternativa al fútbol 11, a partir del estudio de la utilización del espacio de juego

    OpenAIRE

    Daniel Lapresa Ajamil; Javier Arana Idiakez; Belén Garzón Echevarría

    2006-01-01

    El presente trabajo de investigación pretende demostrar que la actual configuración de la competición existente en la Comunidad Autónoma de La Rioja –entre otras–, en el primer año de la categoría infantil, en relación con el tránsito de la modalidad de fútbol 7 a fútbol 11, presenta diferencias manifiestas que implican un desajuste entre las características del juego y las posibilidades reales del niño. Consecuencia de ello se propone una modalidad intermedia, el fútbol 9 –una alternativa co...

  13. C5 Extract Induces Apoptosis in B16F10 Murine Melanoma Cells ...

    African Journals Online (AJOL)

    Purpose: To investigate the anti-cancer activities of C5 extract (C5E), a new herbal preparation from Korea, on B16F10 cells. Methods: The anti-proliferative effects of C5E were assessed by culturing B16F10 cells in the presence or absence of C5E. Cell cycle progression was analyzed by PI staining using flow cytometry.

  14. Downregulation of HDAC9 inhibits cell proliferation and tumor formation by inducing cell cycle arrest in retinoblastoma

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yiting; Wu, Dan; Xia, Fengjie; Xian, Hongyu; Zhu, Xinyue [Medical School of Nanjing University, Department of Ophthalmology, Jinling Hospital, Nanjing, 210002 (China); Cui, Hongjuan, E-mail: hcui@swu.edu.cn [State Key Laboratory of Silkworm Genome Biology, Institute of Sericulture and Systems Biology, Southwest University, Chongqing, 400716 (China); Huang, Zhenping, E-mail: huangzhenping19633@163.com [Medical School of Nanjing University, Department of Ophthalmology, Jinling Hospital, Nanjing, 210002 (China)

    2016-04-29

    Histone deacetylase 9 (HDAC9) is a member of class II HDACs, which regulates a wide variety of normal and abnormal physiological functions. Recently, HDAC9 has been found to be overexpressed in some types of human cancers. However, the role of HDAC9 in retinoblastoma remains unclear. In this study, we found that HDAC9 was commonly expressed in retinoblastoma tissues and HDAC9 was overexpressed in prognostically poor retinoblastoma patients. Through knocking down HDAC9 in Y79 and WERI-Rb-1 cells, the expression level of HDAC9 was found to be positively related to cell proliferation in vitro. Further investigation indicated that knockdown HDAC9 could significantly induce cell cycle arrest at G1 phase in retinoblastoma cells. Western blot assay showed downregulation of HDAC9 could significantly decrease cyclin E2 and CDK2 expression. Lastly, xenograft study in nude mice showed that downregulation of HDAC9 inhibited tumor growth and development in vivo. Therefore, our results suggest that HDAC9 could serve as a novel potential therapeutic target in the treatment of retinoblastoma. - Highlights: • High expression of HDAC9 correlates with poor patient prognosis. • Downregulation of HDAC9 inhibits cell proliferation in retinoblastoma cells. • Downregulation of HDAC9 induces cell cycle arrest at G1 phase in retinoblastoma cells. • Downregulation of HDAC9 suppresses tumor growth in nude mice.

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

    International Nuclear Information System (INIS)

    Hong, Wei; Li, Jinru; Wang, Bo; Chen, Linfeng; Niu, Wenyan; Yao, Zhi; Baniahmad, Aria

    2011-01-01

    Highlights: ► Corepressor Alien interacts with histone methyltransferase ESET in vivo. ► Alien/ESET complex is recruited to nTRE of T3-responsive gene by liganded TRβ1. ► ESET-mediated H3K9 methylation is required for liganded TRβ1-repressed transcription. ► 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 which 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 TRβ1 within the E2F1 gene promoter. We show that, overexpression of ESET promotes, whereas knockdown of ESET releases, the inhibition of TRβ1-regulated gene transcription upon T3 stimulation; and H3K9 methylation is required for TRβ1-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 TRβ1-mediated transcription repression and provide a molecular basis of thyroid hormone-induced repression of proliferation.

  16. F2α-isoprostane, Na+-K+ ATPase and membrane fluidity of placental syncytiotrophoblast cell in preeclamptic women with vitamin E supplementation

    Directory of Open Access Journals (Sweden)

    Franciscus D. Suyatna

    2012-11-01

    Full Text Available Background: The aim of our study was to analyze F2α-isoprostane level, Na+-K+ ATPase activity and placental syncytiotrophoblast cell membrane fluidity in preeclamptic women who received vitamin E supplementation.Methods: The study was conducted between September 2003 and February 2005 at Budi Kemuliaan Maternity Hospital, Central Jakarta. Samples were 6 preeclamptic women with vitamin E supplementation, 6 preeclamptic women without vitamin E supplementation and 6 normal pregnant women. The dose of vitamin E was 200 mg daily. F2α-isoprostane was measured with ELISA reader at λ of 450 nm. Cell membrane fluidity was measured by comparing the molar ratio of total cholesterol and cell membrane phospholipid concentration. The cholesterol was measured by Modular C800 using Roche reagent. Phospholipid was measured by Shimadzu RF5301PC spectrofluorometer (excitation 267 nm, emission 307 nm. Na+-K+ ATPase activity was inhibited by ouabain. Pi production was measured with Fiske and Subbarow method using spectrophotometer at λ of 660 nm. Data was analyzed using F test with one-way ANOVA.Results: Vitamin E supplementation in preeclamptic women decreased the oxidative stress, indicated by significantly lower level of F2α-isoprostane compared to those without vitamin E (26.72 ± 11.21 vs 41.85 ± 7.09 ng/mL, respectively, p = 0.017. Membrane fluidity in syncytiotrophoblast cell of preeclampsia with vitamin E group was maintained at 0.39 ± 0.08 while in those without vitamin E was 0.53 ± 0.14 (p = 0.04. Na+-K+ ATPase activity in syncytiotrophoblast cell membrane was not affected by vitamin E (p = 0.915.Conclusion: Vitamin E supplementation in preeclamptic women decreases F2α-isoprostane level and maintains cell membrane fluidity of syncytiotrophoblast cells; however, it does not increase Na+-K+ ATPase enzyme activity. (Med J Indones. 2012;21:225-9Keywords: F2α-isoprostane, membrane fluidity, Na+-K+ ATPase, preeclampsia, vitamin E

  17. Effects of LiF/Al back electrode on the amorphous/crystalline silicon heterojunction solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sunbo; Lee, Jaehyeong; Dao, Vinh Ai; Lee, Seungho [School of Information and Communication Engineering, Sungkyunkwan University, Suwon, 440-746 (Korea, Republic of); Balaji, Nagarajan [Department of Energy Science, Sungkyunkwan University, Suwon, 440-746 (Korea, Republic of); Ahn, Shihyun [School of Information and Communication Engineering, Sungkyunkwan University, Suwon, 440-746 (Korea, Republic of); Hussain, Shahzada Qamar [Department of Energy Science, Sungkyunkwan University, Suwon, 440-746 (Korea, Republic of); Han, Sangmyeong [School of Information and Communication Engineering, Sungkyunkwan University, Suwon, 440-746 (Korea, Republic of); Jung, Junhee [Department of Energy Science, Sungkyunkwan University, Suwon, 440-746 (Korea, Republic of); Jang, Juyeon; Lee, Yunjung [School of Information and Communication Engineering, Sungkyunkwan University, Suwon, 440-746 (Korea, Republic of); Yi, Junsin, E-mail: yi@yurim.skku.ac.kr [School of Information and Communication Engineering, Sungkyunkwan University, Suwon, 440-746 (Korea, Republic of); Department of Energy Science, Sungkyunkwan University, Suwon, 440-746 (Korea, Republic of)

    2013-05-15

    Highlights: ► We have employed a LiF dielectric layer as a new back-contact electrode. ► Increasing LiF thickness will decrease barrier for electrons transport, thus yield J{sub sc}. ► Increasing LiF thickness will reduced shunt leakage and enhanced internal field, thus yield V{sub oc}. ► Employing LiF layer, improvement of performance of HIT solar cells up to 17.13%. -- Abstract: To improve the quantum efficiency (QE) and hence the efficiency of the amorphous/crystalline silicon heterojunction solar cell, we have employed a LiF dielectric layer on the rear side. The high dipole moment of the LiF reduces the aluminum electrode's work–function and then lowers the energy barrier at back contact. This lower energy barrier height helps to enhance both the operating voltage and the QE at longer wavelength region, in turn improves the open-circuit voltage (V{sub oc}), short-circuit current density (J{sub sc}), and then overall cell efficiency. With optimized LiF layer thickness of 20 nm, 1 cm{sup 2} heterojunction with intrinsic thin layer (HIT) solar cells were produced with industry-compatible process, yielding V{sub oc} of 690 mV, J{sub sc} of 33.62 mA/cm{sup 2}, and cell efficiencies of 17.13%. Therefore LiF/Al electrode on rear side is proposed as an alternate back electrode for high efficiency HIT solar cells.

  18. The human Vδ2+ T-cell compartment comprises distinct innate-like Vγ9+ and adaptive Vγ9- subsets.

    Science.gov (United States)

    Davey, Martin S; Willcox, Carrie R; Hunter, Stuart; Kasatskaya, Sofya A; Remmerswaal, Ester B M; Salim, Mahboob; Mohammed, Fiyaz; Bemelman, Frederike J; Chudakov, Dmitriy M; Oo, Ye H; Willcox, Benjamin E

    2018-05-02

    Vδ2 + T cells form the predominant human γδ T-cell population in peripheral blood and mediate T-cell receptor (TCR)-dependent anti-microbial and anti-tumour immunity. Here we show that the Vδ2 + compartment comprises both innate-like and adaptive subsets. Vγ9 + Vδ2 + T cells display semi-invariant TCR repertoires, featuring public Vγ9 TCR sequences equivalent in cord and adult blood. By contrast, we also identify a separate, Vγ9 - Vδ2 + T-cell subset that typically has a CD27 hi CCR7 + CD28 + IL-7Rα + naive-like phenotype and a diverse TCR repertoire, however in response to viral infection, undergoes clonal expansion and differentiation to a CD27 lo CD45RA + CX 3 CR1 + granzymeA/B + effector phenotype. Consistent with a function in solid tissue immunosurveillance, we detect human intrahepatic Vγ9 - Vδ2 + T cells featuring dominant clonal expansions and an effector phenotype. These findings redefine human γδ T-cell subsets by delineating the Vδ2 + T-cell compartment into innate-like (Vγ9 + ) and adaptive (Vγ9 - ) subsets, which have distinct functions in microbial immunosurveillance.

  19. Identification of a novel centrosomal protein CrpF46 involved in cell cycle progression and mitosis

    International Nuclear Information System (INIS)

    Wei Yi; Shen Enzhi; Zhao Na; Liu Qian; Fan Jinling; Marc, Jan; Wang Yongchao; Sun Le; Liang Qianjin

    2008-01-01

    A novel centrosome-related protein Crp F46 was detected using a serum F46 from a patient suffering from progressive systemic sclerosis. We identified the protein by immunoprecipitation and Western blotting followed by tandem mass spectrometry sequencing. The protein Crp F46 has an apparent molecular mass of ∼ 60 kDa, is highly homologous to a 527 amino acid sequence of the C-terminal portion of the protein Golgin-245, and appears to be a splice variant of Golgin-245. Immunofluorescence microscopy of synchronized HeLa cells labeled with an anti-Crp F46 monoclonal antibody revealed that Crp F46 localized exclusively to the centrosome during interphase, although it dispersed throughout the cytoplasm at the onset of mitosis. Domain analysis using Crp F46 fragments in GFP-expression vectors transformed into HeLa cells revealed that centrosomal targeting is conferred by a C-terminal coiled-coil domain. Antisense Crp F46 knockdown inhibited cell growth and proliferation and the cell cycle typically stalled at S phase. The knockdown also resulted in the formation of poly-centrosomal and multinucleate cells, which finally became apoptotic. These results suggest that Crp F46 is a novel centrosome-related protein that associates with the centrosome in a cell cycle-dependent manner and is involved in the progression of the cell cycle and M phase mechanism

  20. In vivo and in vitro characterization of [18F]-FE-(+)-DTBZ as a tracer for beta-cell mass

    International Nuclear Information System (INIS)

    Eriksson, Olof; Jahan, Mahabuba; Johnstroem, Peter; Korsgren, Olle; Sundin, Anders; Halldin, Christer; Johansson, Lars

    2010-01-01

    Introduction: The positron emission tomography (PET) tracer 9-[ 18 F]fluoroethyl-(+)-dihydrotetrabenazine ([ 18 F]-FE-(+)-DTBZ) is a potential candidate for quantifying beta-cell mass in vivo. The purpose was to investigate in vitro and in vivo utility of this tracer for the assessment of beta-cell mass. Methods: Three pigs were intravenously administered [ 18 F]-FE-(+)-DTBZ and examined by PET/computed tomography. Binding parameters were estimated by kinetic modeling. In vitro k D and B max were determined by saturation binding studies of endocrine and exocrine human tissue homogenates. In vitro pancreatic uptake was determined by tissue autoradiography with pancreases from patients with types 1 (T1DM) and 2 diabetes mellitus (T2DM) and healthy controls. Results: [ 18 F]-FE-(+)-DTBZ had a k D of 3.5±1.0 nM, a B max of 382±108 fmol/mg protein and a specificity of 89±1.8% in islet homogenates. The total exocrine uptake was lower and 65% was nondisplaceable. No uptake difference was observed in pancreatic tissue slices from patients with T1DM, T2DM or healthy controls. The in vivo porcine pancreatic uptake reached a peak of standardized uptake value (SUV) of 2.8 with a low distribution volume ratio in all animals. Moderate to high tracer uptake was identified in the bile system and in bone. Conclusions: [ 18 F]-FE-(+)-DTBZ binds to vesicular monoamine transporter 2 (VMAT2) with high specificity in pure islet tissue in vitro. However, there is high nondisplaceable binding to exocrine tissue. In addition, in vivo tracer metabolism and dehalogenation result in severe underestimation of porcine pancreatic VMAT2 expression and BCM. The results do not support [ 18 F]-FE-(+)-DTBZ as a suitable tracer for in vivo beta-cell imaging.

  1. Atmospheric chemistry of C4F9O(CH2)3OC4F9 and CF3CFHCF2O (CH2)3OCF3CFHCF2

    DEFF Research Database (Denmark)

    Toft, A. M.; Hurley, M. D.; Wallington, T. J.

    2006-01-01

    FTIR smog chamber techniques were used to measure k(Cl + CF3CFHCF2O(CH2)(3)OCF2CFHCF3) = (2.97 +/- 0.17) x 10(-12) k(OH + CF3CFHCF2O(CH2)(3)OCF2CFHCF3) = (2.45 +/- 0.14) x 10(-13), k(Cl + C4F9O(CH2)(3)OC4F9) = (1.45 +/- 0.16) x 10(-12), and k(OH + C4F9O(CH2)(3)OC4F9) = (1.44 +/- 0.10) x 10(-13) c...

  2. Lineshape test on overlapped transitions (R9F1, R9F2) of the 2v3 band of 12CH4 by frequency-stabilized cavity ring-down spectroscopy

    Science.gov (United States)

    Yang, L.; Lin, H.; Plimmer, M. D.; Feng, X. J.; Zhang, J. T.

    2018-05-01

    The performances of a multi-spectral fit for the spectra of pressure-broadened overlapping lines (R9F1, R9F2) of 12CH4 in binary mixtures with N2 were studied by applying different lineshape models, from the simplest Voigt profile (VP) to the Harmann-Tran profile (HTP). Line-mixing was approximated in the first order in the spectral fits. Data were acquired using a high-resolution cavity ring-down spectrometer of minimum detectable absorption coefficient of 2.8 × 10-12 cm-1. The lines were observed with a signal-to-noise ratio of 19 365 for pressures from 5 to 40 kPa. The study reveals that the multi-spectral fits using the HTP and the speed-dependent Nelkin-Ghatak profile (SDNGP) yield the best among all tested. The two models gave the maximum relative residuals of less than 0.065 %. All things considered, the HTP and the SDNGP appear to be the most reliable models for treating the present case of multi-spectral fitting of unresolved dual-component spectra.

  3. Nuclear donor cell lines considerably influence cloning efficiency and the incidence of large offspring syndrome in bovine somatic cell nuclear transfer.

    Science.gov (United States)

    Liu, J; Wang, Y; Su, J; Luo, Y; Quan, F; Zhang, Y

    2013-08-01

    Total five ear skin fibroblast lines (named F1, F2, F3, F4 and F5) from different newborn Holstein cows have been used as nuclear donor cells for producing cloned cows by somatic cell nuclear transfer (SCNT). The effects of these cell lines on both in vitro and in vivo developmental rates of cloned embryos, post-natal survivability and incidence of large offspring syndrome (LOS) were examined in this study. We found that the different cell lines possessed the same capacity to support pre-implantation development of cloned embryos, the cleavage and blastocyst formation rates ranged from 80.2 ± 0.9 to 84.5 ± 2.5% and 28.5 ± 0.9 to 33.3 ± 1.4%, respectively. However, their capacities to support the in vivo development of SCNT embryos showed significant differences (p cloning efficiency was significantly higher in group F5 than those in group F1, F2, F3 and F4 (9.3% vs 4.1%, 1.2%, 2.0% and 5.0%, respectively, p cloned offspring from cell line F1, F2, F3 and F4 showed LOS and gestation length delay, while all cloned offspring from F5 showed normal birthweight and gestation length. We concluded that the nuclear donor cell lines have significant impact on the in vivo development of cloned embryos and the incidence of LOS in cloned calves. © 2013 Blackwell Verlag GmbH.

  4. In vivo imaging of cellular proliferation in renal cell carcinoma using 18F-fluorothymidine PET

    International Nuclear Information System (INIS)

    Wong, Peter K.; Lee, Sze Ting; Murone, Carmel; Eng, John; Lawrentschuk, Nathan; Berlangieri, Salvatore University; Pathmaraj, Kunthi; O’Keefe, Graeme J.; Sachinidis, John; Byrne, Amanda J.; Bolton, Damien M.; Davis, Ian D.; Scott, Andrew M.

    2014-01-01

    The ability to measure cellular proliferation non-invasively in renal cell carcinoma may allow prediction of tumour aggressiveness and response to therapy. The aim of this study was to evaluate the uptake of 18F-fluorothymidine (FLT) PET in renal cell carcinoma (RCC), and to compare this to 18F-fluorodeoxyglucose (FDG), and to an immunohistochemical measure of cellular proliferation (Ki-67). Twenty seven patients (16 male, 11 females; age 42-77) with newly diagnosed renal cell carcinoma suitable for resection were prospectively enrolled. All patients had preoperative FLT and FDG PET scans. Visual identification of tumour using FLT PET compared to normal kidney was facilitated by the use of a pre-operative contrast enhanced CT scan. After surgery tumour was taken for histologic analysis and immunohistochemical staining by Ki-67. The SUVmax (maximum standardized uptake value) mean±SD for FLT in tumour was 2.59±1.27, compared to normal kidney (2.47±0.34). The mean SUVmax for FDG in tumour was similar to FLT (2.60±1.08). There was a significant correlation between FLT uptake and the immunohistochemical marker Ki-67 (r=0.72, P<0.0001) in RCC. Ki-67 proliferative index was mean ± SD of 13.3%±9.2 (range 2.2% - 36.3%). There is detectable uptake of FLT in primary renal cell carcinoma, which correlates with cellular proliferation as assessed by Ki-67 labelling index. This finding has relevance to the use of FLT PET in molecular imaging studies of renal cell carcinoma biology

  5. Melanogenesis inhibits respiration in B16-F10 melanoma cells whereas enhances mitochondrial cell content

    International Nuclear Information System (INIS)

    Meira, Willian Vanderlei; Heinrich, Tassiele Andréa; Cadena, Silvia Maria Suter Correia; Martinez, Glaucia Regina

    2017-01-01

    Melanoma is a rare and aggressive skin tumor; the survival of patients diagnosed late is fairly low. This high mortality rate is due to the characteristics of the cells that allow them to be resistant to radiotherapy and conventional chemotherapy, besides of being able to evade the immune system. Melanin, the pigment responsible for skin, hair and eye color, seems to be involved in this resistance. The main function of melanin is to protect the cells against ultraviolet (UV) light by absorbing this radiation and reactive oxygen species (ROS) scavenging. But this pigment may have also a role as photosensitizer, because when it is irradiated with UVA light (320-400 nm), the generation of ROS was detected. Besides, the melanogenesis stimulation on B16-F10 cells resulted in cell cycle arrest, induction of a quiescent state, change in the expression of several proteins and alterations on ADP/ATP ratio. The present study aimed to investigate the influence of melanogenesis stimulation in mitochondrial function of B16-F10 melanoma cells. Therefore, we analyzed cells respiration, mitochondrial membrane potential (Δψ_m) and mitochondria mass in B16-F10 melanoma cells stimulated with 0.4 mM L-tyrosine and 10 mM NH_4Cl. Our results showed that the induction of melanin synthesis was able to reduce significantly the oxygen consumption after 48 h of stimulation, without changes of mitochondrial membrane potential when compared to non-stimulated cells. Despite of respiration inhibition, the mitochondria mass was higher in cells with melanogenesis stimulation. We suggest that the stimulation in the melanin synthesis might be promoting the inhibition of electrons transport chain by some intermediate compound from the synthesis of the pigment and this effect could contribute to explain the entry in the quiescent state. - Highlights: • Melanoma pigmentation alters mitochondrial respiration. • Induction of melanin synthesis by 48 h do not change mitochondrial membrane potential

  6. Melanogenesis inhibits respiration in B16-F10 melanoma cells whereas enhances mitochondrial cell content

    Energy Technology Data Exchange (ETDEWEB)

    Meira, Willian Vanderlei; Heinrich, Tassiele Andréa; Cadena, Silvia Maria Suter Correia; Martinez, Glaucia Regina, E-mail: grmartinez@ufpr.br

    2017-01-01

    Melanoma is a rare and aggressive skin tumor; the survival of patients diagnosed late is fairly low. This high mortality rate is due to the characteristics of the cells that allow them to be resistant to radiotherapy and conventional chemotherapy, besides of being able to evade the immune system. Melanin, the pigment responsible for skin, hair and eye color, seems to be involved in this resistance. The main function of melanin is to protect the cells against ultraviolet (UV) light by absorbing this radiation and reactive oxygen species (ROS) scavenging. But this pigment may have also a role as photosensitizer, because when it is irradiated with UVA light (320-400 nm), the generation of ROS was detected. Besides, the melanogenesis stimulation on B16-F10 cells resulted in cell cycle arrest, induction of a quiescent state, change in the expression of several proteins and alterations on ADP/ATP ratio. The present study aimed to investigate the influence of melanogenesis stimulation in mitochondrial function of B16-F10 melanoma cells. Therefore, we analyzed cells respiration, mitochondrial membrane potential (Δψ{sub m}) and mitochondria mass in B16-F10 melanoma cells stimulated with 0.4 mM L-tyrosine and 10 mM NH{sub 4}Cl. Our results showed that the induction of melanin synthesis was able to reduce significantly the oxygen consumption after 48 h of stimulation, without changes of mitochondrial membrane potential when compared to non-stimulated cells. Despite of respiration inhibition, the mitochondria mass was higher in cells with melanogenesis stimulation. We suggest that the stimulation in the melanin synthesis might be promoting the inhibition of electrons transport chain by some intermediate compound from the synthesis of the pigment and this effect could contribute to explain the entry in the quiescent state. - Highlights: • Melanoma pigmentation alters mitochondrial respiration. • Induction of melanin synthesis by 48 h do not change mitochondrial membrane

  7. Automated Synthesis of 18F-Fluoropropoxytryptophan for Amino Acid Transporter System Imaging

    Directory of Open Access Journals (Sweden)

    I-Hong Shih

    2014-01-01

    Full Text Available Objective. This study was to develop a cGMP grade of [18F]fluoropropoxytryptophan (18F-FTP to assess tryptophan transporters using an automated synthesizer. Methods. Tosylpropoxytryptophan (Ts-TP was reacted with K18F/kryptofix complex. After column purification, solvent evaporation, and hydrolysis, the identity and purity of the product were validated by radio-TLC (1M-ammonium acetate : methanol = 4 : 1 and HPLC (C-18 column, methanol : water = 7 : 3 analyses. In vitro cellular uptake of 18F-FTP and 18F-FDG was performed in human prostate cancer cells. PET imaging studies were performed with 18F-FTP and 18F-FDG in prostate and small cell lung tumor-bearing mice (3.7 MBq/mouse, iv. Results. Radio-TLC and HPLC analyses of 18F-FTP showed that the Rf and Rt values were 0.9 and 9 min, respectively. Radiochemical purity was >99%. The radiochemical yield was 37.7% (EOS 90 min, decay corrected. Cellular uptake of 18F-FTP and 18F-FDG showed enhanced uptake as a function of incubation time. PET imaging studies showed that 18F-FTP had less tumor uptake than 18F-FDG in prostate cancer model. However, 18F-FTP had more uptake than 18F-FDG in small cell lung cancer model. Conclusion. 18F-FTP could be synthesized with high radiochemical yield. Assessment of upregulated transporters activity by 18F-FTP may provide potential applications in differential diagnosis and prediction of early treatment response.

  8. Matrix metalloproteinase-9 expression in folliculostellate cells of rat anterior pituitary gland.

    Science.gov (United States)

    Ilmiawati, Cimi; Horiguchi, Kotaro; Fujiwara, Ken; Yashiro, Takashi

    2012-03-01

    Folliculostellate (FS) cells of the anterior pituitary gland express a variety of regulatory molecules. Using transgenic rats that express green fluorescent protein specifically in FS cells, we recently demonstrated that FS cells in vitro showed marked changes in motility, proliferation, and that formation of cellular interconnections in the presence of laminin, a component of the extracellular matrix, closely resembled those observed in vivo. These findings suggested that FS cells express matrix metalloproteinase-9 (MMP-9), which assists their function on laminin. In the present study, we investigate MMP-9 expression in rat anterior pituitary gland and examine its role in motility and proliferation of FS cells on laminin. Immunohistochemistry, RT-PCR, immunoblotting, and gelatin zymography were performed to assess MMP-9 expression in the anterior pituitary gland and cultured FS cells. Real-time RT-PCR was used to quantify MMP-9 expression in cultured FS cells under different conditions and treatments. MMP-9 expression was inhibited by pharmacological inhibitor or downregulated by siRNA and time-lapse images were acquired. A 5-bromo-2'-deoxyuridine assay was performed to analyze the proliferation of FS cells. Our results showed that MMP-9 was expressed in FS cells, that this expression was upregulated by laminin, and that laminin induced MMP-9 secretion by FS cells. MMP-9 inhibition and downregulation did not impair FS motility; however, it did impair the capacity of FS cells to form interconnections and it significantly inhibited proliferation of FS cells on laminin. We conclude that MMP-9 is necessary in FS cell interconnection and proliferation in the presence of laminin.

  9. The effect of Cytochalasin D on F-Actin behavior of single-cell electroendocytosis using multi-chamber micro cell chip

    KAUST Repository

    Lin, Ran

    2012-03-01

    Electroendocytosis (EED) is a pulsed-electric-field (PEF) induced endocytosis, facilitating cells uptake molecules through nanometer-sized EED vesicles. We herein investigate the effect of a chemical inhibitor, Cytochalasin D (CD) on the actin-filaments (F-Actin) behavior of single-cell EED. The CD concentration (C CD) can control the depolymerization of F-actin. A multi-chamber micro cell chip was fabricated to study the EED under different conditions. Large-scale single-cell data demonstrated EED highly depends on both electric field and C CD. © 2012 IEEE.

  10. The effect of Cytochalasin D on F-Actin behavior of single-cell electroendocytosis using multi-chamber micro cell chip

    KAUST Repository

    Lin, Ran; Chang, Donald C.; Lee, Yi Kuen

    2012-01-01

    Electroendocytosis (EED) is a pulsed-electric-field (PEF) induced endocytosis, facilitating cells uptake molecules through nanometer-sized EED vesicles. We herein investigate the effect of a chemical inhibitor, Cytochalasin D (CD) on the actin-filaments (F-Actin) behavior of single-cell EED. The CD concentration (C CD) can control the depolymerization of F-actin. A multi-chamber micro cell chip was fabricated to study the EED under different conditions. Large-scale single-cell data demonstrated EED highly depends on both electric field and C CD. © 2012 IEEE.

  11. Tetraspanin CD9 regulates cell contraction and actin arrangement via RhoA in human vascular smooth muscle cells.

    Directory of Open Access Journals (Sweden)

    Michael J Herr

    Full Text Available The most prevalent cardiovascular diseases arise from alterations in vascular smooth muscle cell (VSMC morphology and function. Tetraspanin CD9 has been previously implicated in regulating vascular pathologies; however, insight into how CD9 may regulate adverse VSMC phenotypes has not been provided. We utilized a human model of aortic smooth muscle cells to understand the consequences of CD9 deficiency on VSMC phenotypes. Upon knocking down CD9, the cells developed an abnormally small and rounded morphology. We determined that this morphological change was due to a lack of typical parallel actin arrangement. We also found similar total RhoA but decreased GTP-bound (active RhoA levels in CD9 deficient cells. As a result, cells lacking a full complement of CD9 were less contractile than their control treated counterparts. Upon restoration of RhoA activity in the CD9 deficient cells, the phenotype was reversed and cell contraction was restored. Conversely, inhibition of RhoA activity in the control cells mimicked the CD9-deficient cell phenotype. Thus, alteration in CD9 expression was sufficient to profoundly disrupt cellular actin arrangement and endogenous cell contraction by interfering with RhoA signaling. This study provides insight into how CD9 may regulate previously described vascular smooth muscle cell pathophysiology.

  12. A single tyrosine of the interleukin-9 (IL-9) receptor is required for STAT activation, antiapoptotic activity, and growth regulation by IL-9.

    Science.gov (United States)

    Demoulin, J B; Uyttenhove, C; Van Roost, E; DeLestré, B; Donckers, D; Van Snick, J; Renauld, J C

    1996-09-01

    Interleukin-9 (IL-9), a T-cell-derived cytokine, interacts with a specific receptor associated with the IL-2 receptor gamma chain. In this report, we analyze the functional domains of the human IL-9 receptor transfected into mouse lymphoid cell lines. Three different functions were examined: growth stimulation in factor-dependent pro-B Ba/F3 cells, protection against dexamethasone-induced apoptosis, and Ly-6A2 induction in BW5147 lymphoma cells. The results indicated that a single tyrosine, at position 116 in the cytoplasmic domain, was required for all three activities. In addition, we observed that human IL-9 reduced the proliferation rate of transfected BW5147 cells, an effect also dependent on the same tyrosine. This amino acid was necessary for IL-9-mediated tyrosine phosphorylation of the receptor and for STAT activation but not for IRS-2/4PS activation or for JAK1 phosphorylation, which depended on a domain closer to the plasma membrane. We also showed that JAK1 was constitutively associated with the IL-9 receptor. Activated STAT complexes induced by IL-9 were found to contain STAT1, STAT3, and STAT5 transcription factors. Moreover, sequence homologies between human IL-9 receptor tyrosine 116 and tyrosines (of other receptors activating STAT3 and STAT5 were observed. Taken together, these data indicate that a single tyrosine of the IL-9 receptor, required for activation of three different STAT proteins, is necessary for distinct activities of this cytokine, including proliferative responses.

  13. Cell cycle regulator E2F4 is essential for the development of the ventral telencephalon.

    Science.gov (United States)

    Ruzhynsky, Vladimir A; McClellan, Kelly A; Vanderluit, Jacqueline L; Jeong, Yongsu; Furimsky, Marosh; Park, David S; Epstein, Douglas J; Wallace, Valerie A; Slack, Ruth S

    2007-05-30

    Early forebrain development is characterized by extensive proliferation of neural precursors coupled with complex structural transformations; however, little is known regarding the mechanisms by which these processes are integrated. Here, we show that deficiency of the cell cycle regulatory protein, E2F4, results in the loss of ventral telencephalic structures and impaired self-renewal of neural precursor cells. The mechanism underlying aberrant ventral patterning lies in a dramatic loss of Sonic hedgehog (Shh) expression specifically in this region. The E2F4-deficient phenotype can be recapitulated by interbreeding mice heterozygous for E2F4 with those lacking one allele of Shh, suggesting a genetic interaction between these pathways. Treatment of E2F4-deficient cells with a Hh agonist rescues stem cell self-renewal and cells expressing the homeodomain proteins that specify the ventral telencephalic structures. Finally, we show that E2F4 deficiency results in impaired activity of Shh forebrain-specific enhancers. In conclusion, these studies establish a novel requirement for the cell cycle regulatory protein, E2F4, in the development of the ventral telencephalon.

  14. Isotope Shifts and Hyperfine Structure in the[Xe]4f(7)5d 6s(2) D-2(J)->[Xe]4f(7)5d 6s 6p F-9(J+1) Transitions of Gadolinium

    International Nuclear Information System (INIS)

    Blaum, K.; Bushaw, Bruce A.; Diel, S; Geppert, Ch; Kuschnick, A; Muller, P.; Nortershauser, W.; Schmitt, A.; Wendt, K.

    1999-01-01

    High-resolution resonance ionization mass spectrometry has been used to measure isotope shifts and hyperfine structure in all[Xe] 4f 7 5d 6s2 9DJ ---[Xe] 4f 7 5d 6s 6p 9FJ+1 (J= 2-6) and the[Xe] 4f 7 5d 6s2 9D6---[Xe] 4f 7 5d 6s 6p 9D5 transitions of gadolinium (Gd I). Gadolinium atoms in an atomic beam were excited with a tunable single-frequency laser in the wavelength range of 422 - 429 nm. Resonant excitation was followed by photoionization with the 363.8 nm line of an argon ion laser and resulting ions were mass separated and detected with a quadrupole mass spectrometer. Isotope shifts for all stable gadolinium isotopes in these transitions have been measured for the first time. Additionally, the hyperfine structure constants of the upper states have been derived for the isotopes 155, 157Gd and are compared with previous work. Using prior experimental values for the mean nuclear charge radii, derived from the combination of muonic atoms and electron scattering data, field shift a nd specific mass shift coefficients for the investigated transitions have been determined and nuclear charge parameters l for the minor isotopes 152, 154Gd have been calculated

  15. A new F-actin structure in fungi: actin ring formation around the cell nucleus of Cryptococcus neoformans.

    Science.gov (United States)

    Kopecká, Marie; Kawamoto, Susumu; Yamaguchi, Masashi

    2013-04-01

    The F-actin cytoskeleton of Cryptococcus neoformans is known to comprise actin cables, cortical patches and cytokinetic ring. Here, we describe a new F-actin structure in fungi, a perinuclear F-actin collar ring around the cell nucleus, by fluorescent microscopic imaging of rhodamine phalloidin-stained F-actin. Perinuclear F-actin rings form in Cryptococcus neoformans treated with the microtubule inhibitor Nocodazole or with the drug solvent dimethyl sulfoxide (DMSO) or grown in yeast extract peptone dextrose (YEPD) medium, but they are absent in cells treated with Latrunculin A. Perinuclear F-actin rings may function as 'funicular cabin' for the cell nucleus, and actin cables as intracellular 'funicular' suspending nucleus in the central position in the cell and moving nucleus along the polarity axis along actin cables.

  16. In vivo and in vitro characterization of [{sup 18}F]-FE-(+)-DTBZ as a tracer for beta-cell mass

    Energy Technology Data Exchange (ETDEWEB)

    Eriksson, Olof [Division of Radiology, Department of Oncology, Radiology and Clinical Immunology, University Hospital, 751 87 Uppsala (Sweden)], E-mail: olof.eriksson@radiol.uu.se; Jahan, Mahabuba [Department of Clinical Neuroscience, Karolinska Institute, Section of Psychiatry, Karolinska Hospital, 171 76, Stockholm (Sweden); Johnstroem, Peter [Department of Clinical Neuroscience, Karolinska Institute, Section of Psychiatry, Karolinska Hospital, 171 76, Stockholm (Sweden); CNS and Pain Control, AstraZeneca R and D, 151 36, Soedertaelje (Sweden); Korsgren, Olle [Department of Oncology, Radiology and Clinical Immunology, Division of Clinical Immunology, University Hospital, 751 87 Uppsala (Sweden); Sundin, Anders [Division of Radiology, Department of Oncology, Radiology and Clinical Immunology, University Hospital, 751 87 Uppsala (Sweden); Department of Radiology, Karolinska University Hospital, 171 76, Stockholm (Sweden); Halldin, Christer [Department of Clinical Neuroscience, Karolinska Institute, Section of Psychiatry, Karolinska Hospital, 171 76, Stockholm (Sweden); Johansson, Lars [Division of Radiology, Department of Oncology, Radiology and Clinical Immunology, University Hospital, 751 87 Uppsala (Sweden); AstraZeneca R and D, 431 50, Moelndal (Sweden)

    2010-04-15

    Introduction: The positron emission tomography (PET) tracer 9-[{sup 18}F]fluoroethyl-(+)-dihydrotetrabenazine ([{sup 18}F]-FE-(+)-DTBZ) is a potential candidate for quantifying beta-cell mass in vivo. The purpose was to investigate in vitro and in vivo utility of this tracer for the assessment of beta-cell mass. Methods: Three pigs were intravenously administered [{sup 18}F]-FE-(+)-DTBZ and examined by PET/computed tomography. Binding parameters were estimated by kinetic modeling. In vitro k{sub D} and B{sub max} were determined by saturation binding studies of endocrine and exocrine human tissue homogenates. In vitro pancreatic uptake was determined by tissue autoradiography with pancreases from patients with types 1 (T1DM) and 2 diabetes mellitus (T2DM) and healthy controls. Results: [{sup 18}F]-FE-(+)-DTBZ had a k{sub D} of 3.5{+-}1.0 nM, a B{sub max} of 382{+-}108 fmol/mg protein and a specificity of 89{+-}1.8% in islet homogenates. The total exocrine uptake was lower and 65% was nondisplaceable. No uptake difference was observed in pancreatic tissue slices from patients with T1DM, T2DM or healthy controls. The in vivo porcine pancreatic uptake reached a peak of standardized uptake value (SUV) of 2.8 with a low distribution volume ratio in all animals. Moderate to high tracer uptake was identified in the bile system and in bone. Conclusions: [{sup 18}F]-FE-(+)-DTBZ binds to vesicular monoamine transporter 2 (VMAT2) with high specificity in pure islet tissue in vitro. However, there is high nondisplaceable binding to exocrine tissue. In addition, in vivo tracer metabolism and dehalogenation result in severe underestimation of porcine pancreatic VMAT2 expression and BCM. The results do not support [{sup 18}F]-FE-(+)-DTBZ as a suitable tracer for in vivo beta-cell imaging.

  17. Tracking of [18F]FDG-labeled natural killer cells to HER2/neu-positive tumors

    International Nuclear Information System (INIS)

    Meier, Reinhard; Piert, Morand; Piontek, Guido; Rudelius, Martina; Oostendorp, Robert A.; Senekowitsch-Schmidtke, Reingard; Henning, Tobias D.; Wels, Winfried S.; Uherek, Christoph; Rummeny, Ernst J.; Daldrup-Link, Heike E.

    2008-01-01

    Introduction: The objective of this study was to label the human natural killer (NK) cell line NK-92 with [ 18 F]fluoro-deoxy-glucose (FDG) for subsequent in vivo tracking to HER2/neu-positive tumors. Methods: NK-92 cells were genetically modified to NK-92-scFv(FRP5)-zeta cells, which express a chimeric antigen receptor that is specific to the tumor-associated ErbB2 (HER2/neu) antigen. NK-92 and NK-92-scFv(FRP5)-zeta cells were labeled with [ 18 F]FDG by simple incubation at different settings. Labeling efficiency was evaluated by a gamma counter. Subsequently, [ 18 F]FDG-labeled parental NK-92 or NK-92-scFv(FRP5)-zeta cells were intravenously injected into mice with implanted HER2/neu-positive NIH/3T3 tumors. Radioactivity in tumors was quantified by digital autoradiography and correlated with histopathology. Results: The NK-92 and NK-92-scFv(FRP5)-zeta cells could be efficiently labeled with [ 18 F]FDG by simple incubation. Optimal labeling efficiencies (80%) were achieved using an incubation period of 60 min and additional insulin (10 IU/ml). After injection of 5x10 6 [ 18 F]FDG-labeled NK-92-scFv(FRP5)-zeta cells into tumor-bearing mice, digital autoradiography showed an increased uptake of radioactivity in HER2/neu-positive tumors at 60 min postinjection. Conversely, injection of 5x10 6 NK-92 cells not directed against HER2/neu receptors did not result in increased uptake of radioactivity in the tumors. Histopathology confirmed an accumulation of the NK-92-scFv(FRP5)-zeta cells, but not the parental NK cells, in tumor tissues. Conclusion: The human NK cell line NK-92 can be directed against HER2/neu antigens by genetic modification. The genetically modified NK cells can be efficiently labeled with [ 18 F]FDG, and the accumulation of these labeled NK cells in HER2/neu-positive tumors can be monitored with autoradiography

  18. Cholesterol negatively regulates IL-9-producing CD8+ T cell differentiation and antitumor activity.

    Science.gov (United States)

    Ma, Xingzhe; Bi, Enguang; Huang, Chunjian; Lu, Yong; Xue, Gang; Guo, Xing; Wang, Aibo; Yang, Maojie; Qian, Jianfei; Dong, Chen; Yi, Qing

    2018-05-09

    CD8 + T cells can be polarized into IL-9-secreting (Tc9) cells. We previously showed that adoptive therapy using tumor-specific Tc9 cells generated stronger antitumor responses in mouse melanoma than classical Tc1 cells. To understand why Tc9 cells exert stronger antitumor responses, we used gene profiling to compare Tc9 and Tc1 cells. Tc9 cells expressed different levels of cholesterol synthesis and efflux genes and possessed significantly lower cholesterol content than Tc1 cells. Unique to Tc9, but not other CD8 + or CD4 + T cell subsets, manipulating cholesterol content in polarizing Tc9 cells significantly affected IL-9 expression and Tc9 differentiation and antitumor response in vivo. Mechanistic studies showed that IL-9 was indispensable for Tc9 cell persistence and antitumor effects, and cholesterol or its derivatives inhibited IL-9 expression by activating liver X receptors (LXRs), leading to LXR Sumoylation and reduced p65 binding to Il9 promoter. Our study identifies cholesterol as a critical regulator of Tc9 cell differentiation and function. © 2018 Ma et al.

  19. F4/80+ Host Macrophages Are a Barrier to Murine Embryonic Stem Cell-Derived Hematopoietic Progenitor Engraftment In Vivo.

    Science.gov (United States)

    Thompson, Heather L; van Rooijen, Nico; McLelland, Bryce T; Manilay, Jennifer O

    2016-01-01

    Understanding how embryonic stem cells and their derivatives interact with the adult host immune system is critical to developing their therapeutic potential. Murine embryonic stem cell-derived hematopoietic progenitors (ESHPs) were generated via coculture with the bone marrow stromal cell line, OP9, and then transplanted into NOD.SCID.Common Gamma Chain (NSG) knockout mice, which lack B, T, and natural killer cells. Compared to control mice transplanted with adult lineage-negative bone marrow (Lin - BM) progenitors, ESHP-transplanted mice attained a low but significant level of donor hematopoietic chimerism. Based on our previous studies, we hypothesized that macrophages might contribute to the low engraftment of ESHPs in vivo . Enlarged spleens were observed in ESHP-transplanted mice and found to contain higher numbers of host F4/80 + macrophages compared to BM-transplanted controls. In vivo depletion of host macrophages using clodronate-loaded liposomes improved the ESHP-derived hematopoietic chimerism in the spleen but not in the BM. F4/80 + macrophages demonstrated a striking propensity to phagocytose ESHP targets in vitro . Taken together, these results suggest that macrophages are a barrier to both syngeneic and allogeneic ESHP engraftment in vivo .

  20. Possible interrelationship between changes in F-actin and myosin II, protein phosphorylation, and cell volume regulation in Ehrlich ascites tumor cells

    DEFF Research Database (Denmark)

    Pedersen, S F; Hoffmann, E K

    2002-01-01

    effects on F-actin. The subsequent F-actin depolymerization, however, appeared MLCK- and PKC-dependent, and the initial swelling-induced F-actin depolymerization was MLCK-dependent; both effects were apparently secondary to kinase-mediated effects on cell volume changes. NHE1 in EATC is activated both....... Moreover, Rho kinase inhibition did not significantly affect NHE1 activation, neither by shrinkage nor by CL-A. Implications for the possible interrelationship between changes in F-actin and myosin II, protein phosphorylation, and cell volume regulation are discussed....

  1. 18F-PEG-biotin: Precursor (boroaryl-PEG-biotin) synthesis, 18F-labelling and an in-vitro assessment of its binding with NeutravidinTM-trastuzumab pre-treated cells

    International Nuclear Information System (INIS)

    Smith, Tim A.D.; Simpson, Michael; Cheyne, Richard; Trembleau, Laurent

    2011-01-01

    In terms of nuclear decay 18 F is the most ideal PET nuclide but its short t 1/2 precludes its use for directly labelling whole antibodies due to their long blood residence times. Pre-targeted imaging using affinity systems such as Neutravidin TM -biotin facilitates the application of short-lived nuclides by their attachment to biotin for imaging cell surface proteins targeted with Neutravidin TM -conjugated antibodies. Methods: Boroaryl functionalised biotin was prepared with a PEG linker and radiolabelled by incubation with 18 F in acidified aqueous solution. Cells expressing high (SKBr3), medium (MDA-MB-453) and low (MDA-MB-468) levels of HER-2 were pre-incubated with Neutravidin TM -conjugated trastuzumab, washed, and then incubated with 18 F-PEG-biotin. Results: The 18 F-fluorination of boroaryl-PEG-biotin was much more efficient than reported for other versions of boroaryl-biotin. The novel 18 F-PEG-biotin was demonstrated to bind to HER-2-expressing cells in-vitro pre-incubated with Neutravidin TM -conjugated trastuzumab. Conclusion: Biotin can be functionalised with boroaryl and readily 18 F-radiolabelled in aqueous solution and will bind to cells pre-incubated with Neutravidin TM -antibody conjugates. - Highlights: → Boroaryl-biotin precursor is prepared. → Rapid 18 F-fluorination is demonstrated. → HER-2 expressing breast cancer cells pre-treated with trastuzumab-Neutravidin TM . → 18 F-PEG-biotin binding to pre-treated cells corresponds with HER-2 expression.

  2. Imaging cellular pharmacokinetics of 18F-FDG and 6-NBDG uptake by inflammatory and stem cells.

    Directory of Open Access Journals (Sweden)

    Raiyan T Zaman

    Full Text Available Myocardial infarction (MI causes significant loss of cardiomyocytes, myocardial tissue damage, and impairment of myocardial function. The inability of cardiomyocytes to proliferate prevents the heart from self-regeneration. The treatment for advanced heart failure following an MI is heart transplantation despite the limited availability of the organs. Thus, stem-cell-based cardiac therapies could ultimately prevent heart failure by repairing injured myocardium that reverses cardiomyocyte loss. However, stem-cell-based therapies lack understanding of the mechanisms behind a successful therapy, including difficulty tracking stem cells to provide information on cell migration, proliferation and differentiation. In this study, we have investigated the interaction between different types of stem and inflammatory cells and cell-targeted imaging molecules, 18F-FDG and 6-NBDG, to identify uptake patterns and pharmacokinetics in vitro.Macrophages (both M1 and M2, human induced pluripotent stem cells (hiPSCs, and human amniotic mesenchymal stem cells (hAMSCs were incubated with either 18F-FDG or 6-NBDG. Excess radiotracer and fluorescence were removed and a 100 μm-thin CdWO4 scintillator plate was placed on top of the cells for radioluminescence microscopy imaging of 18F-FDG uptake, while no scintillator was needed for fluorescence imaging of 6-NBDG uptake. Light produced following beta decay was imaged with a highly sensitive inverted microscope (LV200, Olympus and an Electron Multiplying Charge-Couple Device (EM-CCD camera. Custom-written software was developed in MATLAB for image processing.The average cellular activity of 18F-FDG in a single cell of hAMSCs (0.670±0.028 fCi/μm2, P = 0.001 was 20% and 36% higher compared to uptake in hiPSCs (0.540±0.026 fCi/μm2, P = 0.003 and macrophages (0.430±0.023 fCi/μm2, P = 0.002, respectively. hAMSCs exhibited the slowest influx (0.210 min-1 but the fastest efflux (0.327 min-1 rate compared to the other

  3. Imaging cellular pharmacokinetics of 18F-FDG and 6-NBDG uptake by inflammatory and stem cells.

    Science.gov (United States)

    Zaman, Raiyan T; Tuerkcan, Silvan; Mahmoudi, Morteza; Saito, Toshinobu; Yang, Phillip C; Chin, Frederick T; McConnell, Michael V; Xing, Lei

    2018-01-01

    Myocardial infarction (MI) causes significant loss of cardiomyocytes, myocardial tissue damage, and impairment of myocardial function. The inability of cardiomyocytes to proliferate prevents the heart from self-regeneration. The treatment for advanced heart failure following an MI is heart transplantation despite the limited availability of the organs. Thus, stem-cell-based cardiac therapies could ultimately prevent heart failure by repairing injured myocardium that reverses cardiomyocyte loss. However, stem-cell-based therapies lack understanding of the mechanisms behind a successful therapy, including difficulty tracking stem cells to provide information on cell migration, proliferation and differentiation. In this study, we have investigated the interaction between different types of stem and inflammatory cells and cell-targeted imaging molecules, 18F-FDG and 6-NBDG, to identify uptake patterns and pharmacokinetics in vitro. Macrophages (both M1 and M2), human induced pluripotent stem cells (hiPSCs), and human amniotic mesenchymal stem cells (hAMSCs) were incubated with either 18F-FDG or 6-NBDG. Excess radiotracer and fluorescence were removed and a 100 μm-thin CdWO4 scintillator plate was placed on top of the cells for radioluminescence microscopy imaging of 18F-FDG uptake, while no scintillator was needed for fluorescence imaging of 6-NBDG uptake. Light produced following beta decay was imaged with a highly sensitive inverted microscope (LV200, Olympus) and an Electron Multiplying Charge-Couple Device (EM-CCD) camera. Custom-written software was developed in MATLAB for image processing. The average cellular activity of 18F-FDG in a single cell of hAMSCs (0.670±0.028 fCi/μm2, P = 0.001) was 20% and 36% higher compared to uptake in hiPSCs (0.540±0.026 fCi/μm2, P = 0.003) and macrophages (0.430±0.023 fCi/μm2, P = 0.002), respectively. hAMSCs exhibited the slowest influx (0.210 min-1) but the fastest efflux (0.327 min-1) rate compared to the other tested

  4. All-in-One CRISPR-Cas9/FokI-dCas9 Vector-Mediated Multiplex Genome Engineering in Cultured Cells.

    Science.gov (United States)

    Sakuma, Tetsushi; Sakamoto, Takuya; Yamamoto, Takashi

    2017-01-01

    CRISPR-Cas9 enables highly convenient multiplex genome engineering in cultured cells, because it utilizes generic Cas9 nuclease and an easily customizable single-guide RNA (sgRNA) for site-specific DNA double-strand break induction. We previously established a multiplex CRISPR-Cas9 assembly system for constructing an all-in-one vector simultaneously expressing multiple sgRNAs and Cas9 nuclease or other Cas9 variants including FokI-dCas9, which supersedes the wild-type Cas9 with regard to high specificity. In this chapter, we describe a streamlined protocol to design and construct multiplex CRISPR-Cas9 or FokI-dCas9 vectors, to introduce them into cultured cells by lipofection or electroporation, to enrich the genomically edited cells with a transient puromycin selection, to validate the mutation efficiency by Surveyor nuclease assay, and to perform off-target analyses. We show that our protocol enables highly efficient multiplex genome engineering even in hard-to-transfect HepG2 cells.

  5. E2F-dependent induction of p14ARF during cell cycle re-entry in human T cells

    DEFF Research Database (Denmark)

    del Arroyo, Ana Gutierrez; El Messaoudi, Selma; Clark, Paula A

    2007-01-01

    The ARF protein, encoded by alternate exon usage within the CDKN2A locus, provides a link between the retinoblastoma (pRb) and p53 tumor suppressor pathways. Agents that disable pRb or otherwise impinge on the E2F family of transcription factors induce expression of ARF, resulting in stabilization...... of p53 and activation of p53-regulated genes. However, in some cell types ARF is not induced upon cell cycle re-entry, as expected of a conventional E2F target gene, leading to the suggestion that the ARF promoter only responds to supra-physiological or aberrant levels of E2F. These properties have...

  6. The effect of P-glycoprotein on 18F-FDG uptake in vitro

    International Nuclear Information System (INIS)

    Yu Chunjing; Zhang Bin; Deng Shengming; Wan Weixing; Wu Yiwei

    2013-01-01

    Objective: To evaluate the effect of P-gp inhibitors of verapamil (VER) and GF120918 on 18 F-FDG uptake in Bcap37 and Bcap37/multidrug resistance (MDR)1 cell lines in vitro, and to explore the relationship between 18 F-FDG uptake and P-gp expression at cellular level. Methods: Bcap37 and Bcap37/MDR1 cells were seeded into 6-well plates at a density of 1 × 10 6 per well. Three days later,37 kBq/ml 18 F-FDG, or 37 kBq/ml 18 F-FDG + 100 μmol/L VER, or 37 kBq/ml 18 F-FDG + 50 μmol/L GF120918 were added into each well. After incubated for 10, 30, 60 and 120 min at 37 ℃ and in 5% CO 2 , the medium was removed and the cells were washed three times with 1 ml ice-cold PBS immediately. The radioactivity of 18 F-FDG was measured using a gamma counter. The uptake of 18 F-FDG was expressed as the ratio of 18 F-FDG radioactivity in Bcap37 or Bcap37/MDR1 cells and the overall radioactivity added to the cells in each well.The t test was used for statistical analysis. Results: 18 F-FDG uptake was higher in Bcap37/MDR1 cells than that in Bcap37 cells after incubated for 10 min. The uptake rate was (1.88 ±0.19) % in Bcap37/MDR1 cells and (1.37 ± 0.18) % in Bcap37 cells (t=7.832, P<0.05). On the contrary, 18 F-FDG uptake was significantly higher in Bcap37 cells than that in Bcap37/MDR1 cells after incubated for 60 and 120 min. The uptake rates were (2.29 ±0.23)% and (2.34 ±0.15)% in Bcap37 cells, (1.47 ±0.14)% and (1.53 ±0.22)% in Bcap37/MDR1 cells (t=8.437, 8.283, both P<0.05). 18 F-FDG uptake was significantly higher with VER or GF120918 in Bcap37/MDR1 cells than that without VER or GF120918 after the incubation of 60 and 120 min (t=9.032, 9.243 and 8.765, 8.803, all P<0.05). The uptake rates with VER or GF120918 were (2.45 ±0.21)% and (2.46 ±0.25)%, (2.50 ±0.24)% and (2.48 ±0.27)%. There was no significant difference of 18 F-FDG uptake in Bcap37 cells with or without VER or GF120918. Conclusions: 18 F-FDG is a substrate of P-gp at cellular level. P-gp may act as an

  7. Cell Labeling for 19F MRI: New and Improved Approach to Perfluorocarbon Nanoemulsion Design

    Directory of Open Access Journals (Sweden)

    Jonathan Williams

    2013-09-01

    Full Text Available This report describes novel perfluorocarbon (PFC nanoemulsions designed to improve ex vivo cell labeling for 19F magnetic resonance imaging (MRI. 19F MRI is a powerful non-invasive technique for monitoring cells of the immune system in vivo, where cells are labeled ex vivo with PFC nanoemulsions in cell culture. The quality of 19F MRI is directly affected by the quality of ex vivo PFC cell labeling. When co-cultured with cells for longer periods of time, nanoemulsions tend to settle due to high specific weight of PFC oils (1.5–2.0 g/mL. This in turn can decrease efficacy of excess nanoemulsion removal and reliability of the cell labeling in vitro. To solve this problem, novel PFC nanoemulsions are reported which demonstrate lack of sedimentation and high stability under cell labeling conditions. They are monodisperse, have small droplet size (~130 nm and low polydispersity (<0.15, show a single peak in the 19F nuclear magnetic resonance spectrum at −71.4 ppm and possess high fluorine content. The droplet size and polydispersity remained unchanged after 160 days of follow up at three temperatures (4, 25 and 37 °C. Further, stressors such as elevated temperature in the presence of cells, and centrifugation, did not affect the nanoemulsion droplet size and polydispersity. Detailed synthetic methodology and in vitro testing for these new PFC nanoemulsions is presented.

  8. F-BAR family proteins, emerging regulators for cell membrane dynamic changes-from structure to human diseases.

    Science.gov (United States)

    Liu, Suxuan; Xiong, Xinyu; Zhao, Xianxian; Yang, Xiaofeng; Wang, Hong

    2015-05-09

    Eukaryotic cell membrane dynamics change in curvature during physiological and pathological processes. In the past ten years, a novel protein family, Fes/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain proteins, has been identified to be the most important coordinators in membrane curvature regulation. The F-BAR domain family is a member of the Bin/Amphiphysin/Rvs (BAR) domain superfamily that is associated with dynamic changes in cell membrane. However, the molecular basis in membrane structure regulation and the biological functions of F-BAR protein are unclear. The pathophysiological role of F-BAR protein is unknown. This review summarizes the current understanding of structure and function in the BAR domain superfamily, classifies F-BAR family proteins into nine subfamilies based on domain structure, and characterizes F-BAR protein structure, domain interaction, and functional relevance. In general, F-BAR protein binds to cell membrane via F-BAR domain association with membrane phospholipids and initiates membrane curvature and scission via Src homology-3 (SH3) domain interaction with its partner proteins. This process causes membrane dynamic changes and leads to seven important cellular biological functions, which include endocytosis, phagocytosis, filopodium, lamellipodium, cytokinesis, adhesion, and podosome formation, via distinct signaling pathways determined by specific domain-binding partners. These cellular functions play important roles in many physiological and pathophysiological processes. We further summarize F-BAR protein expression and mutation changes observed in various diseases and developmental disorders. Considering the structure feature and functional implication of F-BAR proteins, we anticipate that F-BAR proteins modulate physiological and pathophysiological processes via transferring extracellular materials, regulating cell trafficking and mobility, presenting antigens, mediating extracellular matrix degradation, and transmitting

  9. Isolation and characterization of multiple F-box genes linked to the S9- and S10-RNase in apple (Malus × domestica Borkh.).

    Science.gov (United States)

    Okada, Kazuma; Moriya, Shigeki; Haji, Takashi; Abe, Kazuyuki

    2013-06-01

    Using 11 consensus primer pairs designed from S-linked F-box genes of apple and Japanese pear, 10 new F-box genes (MdFBX21 to 30) were isolated from the apple cultivar 'Spartan' (S(9)S(10)). MdFBX21 to 23 and MdFBX24 to 30 were completely linked to the S(9) -RNase and S(10-)RNase, respectively, and showed pollen-specific expression and S-haplotype-specific polymorphisms. Therefore, these 10 F-box genes are good candidates for the pollen determinant of self-incompatibility in apple. Phylogenetic analysis and comparison of deduced amino acid sequences of MdFBX21 to 30 with those of 25 S-linked F-box genes previously isolated from apple showed that a deduced amino acid identity of greater than 88.0 % can be used as the tentative criterion to classify F-box genes into one type. Using this criterion, 31 of 35 F-box genes of apple were classified into 11 types (SFBB1-11). All types included F-box genes derived from S(3-) and S(9-)haplotypes, and seven types included F-box genes derived from S(3-), S(9-), and S(10-)haplotypes. Moreover, comparison of nucleotide sequences of S-RNases and multiple F-box genes among S(3-), S(9-), and S(10-)haplotypes suggested that F-box genes within each type showed high nucleotide identity regardless of the identity of the S-RNase. The large number of F-box genes as candidates for the pollen determinant and the high degree of conservation within each type are consistent with the collaborative non-self-recognition model reported for Petunia. These findings support that the collaborative non-self-recognition system also exists in apple.

  10. Lipofection of insulin-producing RINm5F cells: methodological improvements.

    Science.gov (United States)

    Barbu, Andreea; Welsh, Nils

    2007-01-01

    Cationic lipid/DNA-complexes have been widely used as gene transfer vectors because they are less toxic and immunogenic than viral vectors. The aim of the present study was to improve and characterize lipofection of an insulin-producing cell line. We compared the transfection efficiency of seven commercially available lipid formulations (Lipotaxi, SuperFect, Fugene, TransFast, Dosper, GenePORTER and LipofectAMINE) by flow cytometry analysis of GFP-expression. In addition, we have determined the influences of centrifugation, serum and a nuclear localization signal peptide on the lipofection efficiency. We observed that two lipid formulations, GenePORTER and LipofectAMINE, were able to promote efficient gene transfer in RINm5F cells. However, GenePORTER exhibited the important advantage of being able to transfect cells in the presence of serum and with less cytotoxicity than LipofectAMINE. LipofectAMINE-induced RINm5F cell death could partially be counteracted by TPA, forskolin or fumonisin beta(1). Finally, both centrifugation and a nuclear localization signal peptide increased transfection efficiency.

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

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Wei, E-mail: hongwei@tijmu.edu.cn [Department of Immunology, Tianjin Medical University, 300070 Tianjin (China); College of Basic Medicine, Tianjin Medical University, 300070 Tianjin (China); Li, Jinru; Wang, Bo [College of Basic Medicine, Tianjin Medical University, 300070 Tianjin (China); Chen, Linfeng [Department of Medical Oncology, Harvard Medical School, Dana Farber Cancer Institute, Boston, 02115 MA (United States); Niu, Wenyan; Yao, Zhi [Department of Immunology, Tianjin Medical University, 300070 Tianjin (China); Baniahmad, Aria, E-mail: aban@mti.uni-jena.de [Institute for Human Genetics, Jena University Hospital, 07740 Jena (Germany)

    2011-12-02

    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 which 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.

  12. Differentiation of Odontoblast-Like Cells From Mouse Induced Pluripotent Stem Cells by Pax9 and Bmp4 Transfection.

    Science.gov (United States)

    Seki, Daisuke; Takeshita, Nobuo; Oyanagi, Toshihito; Sasaki, Shutaro; Takano, Ikuko; Hasegawa, Masakazu; Takano-Yamamoto, Teruko

    2015-09-01

    The field of tooth regeneration has progressed in recent years, and human tooth regeneration could become viable in the future. Because induced pluripotent stem (iPS) cells can differentiate into odontogenic cells given appropriate conditions, iPS cells are a potential cell source for tooth regeneration. However, a definitive method to induce iPS cell-derived odontogenic cells has not been established. We describe a novel method of odontoblast differentiation from iPS cells using gene transfection. We generated mouse iPS cell-derived neural crest-like cells (iNCLCs), which exhibited neural crest markers. Next, we differentiated iNCLCs into odontoblast-like cells by transfection of Pax9 and Bmp4 expression plasmids. Exogenous Pax9 upregulated expression of Msx1 and dentin matrix protein 1 (Dmp1) in iNCLCs but not bone morphogenetic protein 4 (Bmp4) or dentin sialophosphoprotein (Dspp). Exogenous Bmp4 upregulated expression of Msx1, Dmp1, and Dspp in iNCLCs, but not Pax9. Moreover, cotransfection of Pax9 and Bmp4 plasmids in iNCLCs revealed a higher expression of Pax9 than when Pax9 plasmid was used alone. In contrast, exogenous Pax9 downregulated Bmp4 overexpression. Cotransfection of Pax9 and Bmp4 synergistically upregulated Dmp1 expression; however, Pax9 overexpression downregulated exogenous Bmp4-induced Dspp expression. Together, these findings suggest that an interaction between exogenous Pax9- and Bmp4-induced signaling modulated Dmp1 and Dspp expression. In conclusion, transfection of Pax9 and Bmp4 expression plasmids in iNCLCs induced gene expression associated with odontoblast differentiation, suggesting that iNCLCs differentiated into odontoblast-like cells. The iPS cell-derived odontoblast-like cells could be a useful cell source for tooth regeneration. It has been reported that induced pluripotent stem (iPS) cells differentiate into odontogenic cells by administration of recombinant growth factors and coculture with odontogenic cells. Therefore, they can

  13. METACASPASE9 modulates autophagy to confine cell death to the target cells during Arabidopsis vascular xylem differentiation

    Directory of Open Access Journals (Sweden)

    Sacha Escamez

    2016-02-01

    Full Text Available We uncovered that the level of autophagy in plant cells undergoing programmed cell death determines the fate of the surrounding cells. Our approach consisted of using Arabidopsis thaliana cell cultures capable of differentiating into two different cell types: vascular tracheary elements (TEs that undergo programmed cell death (PCD and protoplast autolysis, and parenchymatic non-TEs that remain alive. The TE cell type displayed higher levels of autophagy when expression of the TE-specific METACASPASE9 (MC9 was reduced using RNAi (MC9-RNAi. Misregulation of autophagy in the MC9-RNAi TEs coincided with ectopic death of the non-TEs, implying the existence of an autophagy-dependent intercellular signalling from within the TEs towards the non-TEs. Viability of the non-TEs was restored when AUTOPHAGY2 (ATG2 was downregulated specifically in MC9-RNAi TEs, demonstrating the importance of autophagy in the spatial confinement of cell death. Our results suggest that other eukaryotic cells undergoing PCD might also need to tightly regulate their level of autophagy to avoid detrimental consequences for the surrounding cells.

  14. Responses of proenkephalin Peptide F to aerobic exercise stress in the plasma and white blood cell biocompartments.

    Science.gov (United States)

    Kraemer, William J; Fragala, Maren S; van Henegouwen, Wendy R H Beijersbergen; Gordon, Scott E; Bush, Jill A; Volek, Jeff S; Triplett, N Travis; Dunn-Lewis, Courtenay; Comstock, Brett A; Szivak, Tunde K; Flanagan, Shawn D; Hooper, David R; Luk, Hui-Ying; Mastro, Andrea M

    2013-04-01

    Proenkephalin Peptide F [107-140] is an enkephalin-containing peptide found predominantly within the adrenal medulla, co-packaged with epinephrine within the chromaffin granules. In vivo studies indicate that Peptide F has classic opioid analgesia effects; in vitro studies suggest potential immune cell interactions. In this investigation we examined patterns of Peptide F concentrations in different bio-compartments of the blood at rest and following sub-maximal cycle exercise to determine if Peptide F interacts with the white blood cell (WBC) bio-compartment during aerobic exercise. Eight physically active men (n=8) performed sub-maximal (80-85% V˙O2peak) cycle ergometer exercise for 30 min. Plasma Peptide F and WBC Peptide F immunoreactivity were examined pre-exercise, mid-exercise and immediately post-, 5-min post-, 15-min post-, 30-min post- and 60-min post-exercise and at similar time-points during a control condition (30 min rest). Peptide F concentrations significantly (pexercise, compared to pre-exercise concentrations. No significant increases in Peptide F concentrations in the WBC fraction were observed during or after exercise. However, a significant decrease was observed at 30 min post-exercise. An ultradian pattern of Peptide F distribution was apparent during rest. Furthermore, concentrations of T cells, B cells, NK cells, and total WBCs demonstrated significant changes in response to aerobic exercise. Data indicated that Peptide F was bound in significant molar concentrations in the WBC fraction and that this biocompartment may be one of the tissue targets for binding interactions. These data indicate that Peptide F is involved with immune cell modulation in the white blood circulatory biocompartment of blood. Copyright © 2013. Published by Elsevier Inc.

  15. Advancing chimeric antigen receptor T cell therapy with CRISPR/Cas9.

    Science.gov (United States)

    Ren, Jiangtao; Zhao, Yangbing

    2017-09-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (CRISPR/Cas9) system, an RNA-guided DNA targeting technology, is triggering a revolution in the field of biology. CRISPR/Cas9 has demonstrated great potential for genetic manipulation. In this review, we discuss the current development of CRISPR/Cas9 technologies for therapeutic applications, especially chimeric antigen receptor (CAR) T cell-based adoptive immunotherapy. Different methods used to facilitate efficient CRISPR delivery and gene editing in T cells are compared. The potential of genetic manipulation using CRISPR/Cas9 system to generate universal CAR T cells and potent T cells that are resistant to exhaustion and inhibition is explored. We also address the safety concerns associated with the use of CRISPR/Cas9 gene editing and provide potential solutions and future directions of CRISPR application in the field of CAR T cell immunotherapy. As an integration-free gene insertion method, CRISPR/Cas9 holds great promise as an efficient gene knock-in platform. Given the tremendous progress that has been made in the past few years, we believe that the CRISPR/Cas9 technology holds immense promise for advancing immunotherapy.

  16. Advancing chimeric antigen receptor T cell therapy with CRISPR/Cas9

    Directory of Open Access Journals (Sweden)

    Jiangtao Ren

    2017-04-01

    Full Text Available ABSTRACT The clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR-associated 9 (CRISPR/Cas9 system, an RNA-guided DNA targeting technology, is triggering a revolution in the field of biology. CRISPR/Cas9 has demonstrated great potential for genetic manipulation. In this review, we discuss the current development of CRISPR/Cas9 technologies for therapeutic applications, especially chimeric antigen receptor (CAR T cell-based adoptive immunotherapy. Different methods used to facilitate efficient CRISPR delivery and gene editing in T cells are compared. The potential of genetic manipulation using CRISPR/Cas9 system to generate universal CAR T cells and potent T cells that are resistant to exhaustion and inhibition is explored. We also address the safety concerns associated with the use of CRISPR/Cas9 gene editing and provide potential solutions and future directions of CRISPR application in the field of CAR T cell immunotherapy. As an integration-free gene insertion method, CRISPR/Cas9 holds great promise as an efficient gene knock-in platform. Given the tremendous progress that has been made in the past few years, we believe that the CRISPR/Cas9 technology holds immense promise for advancing immunotherapy.

  17. Estradiol Receptors Regulate Differential Connexin 43 Expression in F98 and C6 Glioma Cell Lines.

    Directory of Open Access Journals (Sweden)

    Zahra Moinfar

    Full Text Available Glioma is the most common malignant primary brain tumour with male preponderance and poor prognosis. Glioma cells express variable amounts of connexin 43 (Cx43 and estrogen receptors (ERs. Both, Cx43 and ERs, play important roles in cell proliferation and migration. Therefore, we investigated the effects of 17-ß estradiol (E2 on Cx43 expression in two glioma cell lines with variable native expression of Cx43.F98 and C6 rat glioma cells were cultured for 24 h in the presence of 10 nM or 100 nM E2, and the E2-antagonist, Fulvestrant. An MTT assay was performed to evaluate cell viability. ERα, ERβ and Cx43 protein expressions were analysed by western blotting and Cx43 mRNA expression was analysed by real-time polymerase chain reaction. To quantify cell migration, an exclusive zone migration assay was used. Functional coupling of cells via gap junctions was examined using whole-cell patch-clamp technique.E2 reduced Cx43 expression in C6 cells, but increased Cx43 expression in F98 cultures. These effects were mediated via ERs. Moreover, E2 promoted C6 cell migration, but it did not affect F98 cell migration. The expression level of ERα was found to be high in C6, but low in F98 cells. ERβ was exclusively expressed in C6 cells. In addition, E2 treatment induced a significant decrease of ERβ in C6 cultures, while it decreased ERα expression in F98 glioma cells.These findings show that E2 differentially modulates Cx43 expression in F98 and C6 glioma cells, likely due to the differential expression of ERs in each of these cell lines. Our findings point to the molecular mechanisms that might contribute to the gender-specific differences in the malignancy of glioma and could have implications for therapeutic strategies against glioma.

  18. Characterization and expression of the human T cell receptor-T3 complex by monoclonal antibody F101.01

    DEFF Research Database (Denmark)

    Geisler, C; Plesner, T; Pallesen, G

    1988-01-01

    A murine monoclonal antibody (MoAb) F101.01 reacting with the T cell receptor (TCR)-T3 complex is presented. Immunohistological studies showed that F101.01 specifically stains T-zone lymphocytes in lymph nodes, tonsils, and splenic tissue. Two-colour immunofluorescence and flow cytometry...... demonstrated co-expression of the antigen defined by F101.01 and the pan-T cell antigens defined by CD2, CD3, CD5, and CD7 antibodies. Cells stained with CD4 and CD8 antibodies were both included in the F101.01-positive population, whereas CD16-positive natural killer cells (NK), B cells (CD19 and CD20......), and myeloid cells (CD13 and CD33) were excluded. The target antigen of F101.01 co-modulated with the CD3-defined antigen (T3) and the TCR recognized by the MoAb WT-31. CD3 antibody and WT-31 both blocked binding of F101.01. F101.01 precipitated the TCR-T3 complex from lysates of 125I-labelled peripheral blood...

  19. CCR5 Gene Disruption via Lentiviral Vectors Expressing Cas9 and Single Guided RNA Renders Cells Resistant to HIV-1 Infection

    Science.gov (United States)

    Liu, Jingjing; Zhang, Di; Kimata, Jason T.; Zhou, Paul

    2014-01-01

    CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN) or Transcription activator-like effector nuclease (TALEN). In the present study, we silenced CCR5 via CRISPR associated protein 9 (Cas9) and single guided RNAs (sgRNAs). We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates, but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of CCR5 via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1. PMID:25541967

  20. Combined use of (18)F-FDG and (18)F-FMISO in unresectable non-small cell lung cancer patients planned for radiotherapy: a dynamic PET/CT study.

    Science.gov (United States)

    Sachpekidis, Christos; Thieke, Christian; Askoxylakis, Vasileios; Nicolay, Nils H; Huber, Peter E; Thomas, Michael; Dimitrakopoulou, Georgia; Debus, Juergen; Haberkorn, Uwe; Dimitrakopoulou-Strauss, Antonia

    2015-01-01

    Aim of this study was to evaluate and compare, by means of dynamic and static PET/CT, the distribution patterns and pharmacokinetics of fluorine-18 fluorodeoxyglucose ((18)F-FDG) and of fluorine-18-fluoromisonidazole ((18)F-FMISO) in non-small cell lung cancer (NSCLC) patients scheduled for intensity modulated radiation therapy (IMRT). Thirteen patients suffering from inoperable stage III NSCLC underwent PET/CTs with (18)F-FDG and (18)F-FMISO for tumor metabolism and hypoxia assessment accordingly. Evaluation of PET/CT studies was based on visual analysis, semi-quantitative (SUV) calculations and absolute quantitative estimations, after application of a two-tissue compartment model and a non-compartmental approach. (18)F-FDG PET/CT revealed all thirteen primary lung tumors as sites of increased (18)F-FDG uptake. Six patients demonstrated also in total 43 (18)F-FDG avid metastases; these patients were excluded from radiotherapy. (18)F-MISO PET/CT demonstrated 12/13 primary lung tumors with faint tracer uptake. Only one tumor was clearly (18)F-FMISO avid, (SUVaverage = 3.4, SUVmax = 5.0). Mean values for (18)F-FDG, as derived from dPET/CT data, were SUVaverage = 8.9, SUVmax = 15.1, K1 = 0.23, k2 = 0.53, k3 = 0.17, k4 = 0.02, influx = 0.05 and fractal dimension (FD) = 1.25 for the primary tumors. The respective values for (18)F-FMISO were SUVaverage = 1.4, SUVmax = 2.2, K1 = 0.26, k2 = 0.56, k3 = 0.06, k4 = 0.06, influx = 0.02 and FD = 1.14. No statistically significant correlation was observed between the two tracers. (18)F-FDG PET/CT changed therapy management in six patients, by excluding them from planned IMRT. (18)F-FMISO PET/CT revealed absence of significant tracer uptake in the majority of the (18)F-FDG avid NSCLCs. Lack of correlation between the two tracers' kinetics indicates that they reflect different molecular mechanisms and implies the discordance between increased glycolysis and hypoxia in the malignancy.

  1. In-vivo imaging of cellular proliferation in renal cell carcinoma using 18F-fluorothymidine (FLT) PET

    International Nuclear Information System (INIS)

    Wong, P.; Lee, S. T.; Eng, J.; Berlangieri, S. U.; Pathmaraj, K.; O'Keefe, G. J.; Lawrentschuk, N.

    2009-01-01

    Full text:Background: The ability to measure cellular proliferation non-invasively in renal cell carcinoma may allow prediction of tumour aggressiveness and response to therapy. The aim of this study was to evaluate the uptake of 18F-fluorothymidine (FLT) in renal cell carcinoma, and to compare this to 18F-fluorodeoxyglucose (FDG), and to an immunohistochemical measure of cellular proliferation (Ki-67). Methods: Twenty seven patients (16 men, 11 women; age 42-77) with newly diagnosed renal cell carcinoma suitable for resection were prospectively enrolled. All patients had preoperative FLT and FDG PET scans. After surgery tumour was taken for histologic analysis and immunohistochemical staining by Ki-67. Results: The mean SUVmax (maximum standardized uptake value) ± SD for FLT in tumour was 2.53 ± 1.26, compared to normal kidney (2.47 ± 0.34). The mean SUVmax for FDG in tumour was similar to FLT (2.60 ± 1.08). Visual identification of tumour using FLT PET compared to normal kidney was facilitated by the use of a pre-operative contrast enhanced CT scan. There was a significant correlation between FLT uptake and the immunohistochemical marker Ki-67 (r=0.624, p=0.0008) in RCC. Ki-67 labelling index was mean ± SD of 13.3% ± 9.2 (range 2.2% to 36.3%). Conclusion: There is detectable uptake of FLT in primary renal cell carcinoma, which correlates with cellular proliferation as assessed by Ki-67 labelling index. This finding has relevance to the use of FLT PET in molecular imaging studies of renal cell carcinoma biology.

  2. In vivo evaluation of 2'-deoxy-2'-[{sup 18}F]fluoro-5-iodo-1-{beta}-D-arabinofuranosyluracil ([{sup 18}F]FIAU) and 2'-deoxy-2'-[{sup 18}F]fluoro-5-ethyl-1-{beta}-D-arabinofuranosyluracil ([{sup 18}F]FEAU) as markers for suicide gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Alauddin, Mian M. [University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd. T8.3895, P.O. Box 059, Houston, TX (United States); Shahinian, Antranic; Park, Ryan; Fissekis, John D.; Conti, Peter S. [University of Southern California, PET Imaging Science Center, Keck School of Medicine, Los Angeles, CA (United States); Tohme, Michel [University of Southern California, PET Imaging Science Center, Keck School of Medicine, Los Angeles, CA (United States); Department of Biomedical Engineering, UC Davis, Davis, CA (United States)

    2007-06-15

    FIAU and FEAU were evaluated in vitro and in vivo as markers for HSV1-tk gene expression. In vitro and biodistribution studies were performed in wild type and transduced HT-29 cells using [{sup 14}C]FIAU and [{sup 3}H]FEAU. PET imaging was performed using [{sup 18}F]FIAU and [{sup 18}F]FEAU. In vitro uptake of [{sup 14}C]FIAU in tk-positive cells was 39-fold, 49-fold, and 43-fold higher (p < 0.001) than in wild type cells at 30, 60, and 120 min, respectively. Uptake of [{sup 3}H]FEAU in transduced cells was 46-fold, 62-fold, and 121-fold higher (p < 0.001) than in wild type cells at the same time points. In vivo uptake of [{sup 14}C]FIAU at 2 h in HSV1-tk positive tumors was 15.48 {+-} 3.94, 6.7-fold higher (p < 0.001) than in wild type tumors. Uptake of [{sup 3}H]FEAU in transduced tumors was 9.98 {+-} 1.99, 5.0-fold higher (p < 0.001) than in wild type tumors. Micro-PET images using [{sup 18}F]FIAU and [{sup 18}F]FEAU also showed very high uptake in HSV-tk tumors. [{sup 18}F]FIAU and [{sup 18}F]FEAU appear to be potential PET imaging agents for gene expression. (orig.)

  3. Self-renewal of single mouse hematopoietic stem cells is reduced by JAK2V617F without compromising progenitor cell expansion.

    Science.gov (United States)

    Kent, David G; Li, Juan; Tanna, Hinal; Fink, Juergen; Kirschner, Kristina; Pask, Dean C; Silber, Yvonne; Hamilton, Tina L; Sneade, Rachel; Simons, Benjamin D; Green, Anthony R

    2013-01-01

    Recent descriptions of significant heterogeneity in normal stem cells and cancers have altered our understanding of tumorigenesis, emphasizing the need to understand how single stem cells are subverted to cause tumors. Human myeloproliferative neoplasms (MPNs) are thought to reflect transformation of a hematopoietic stem cell (HSC) and the majority harbor an acquired V617F mutation in the JAK2 tyrosine kinase, making them a paradigm for studying the early stages of tumor establishment and progression. The consequences of activating tyrosine kinase mutations for stem and progenitor cell behavior are unclear. In this article, we identify a distinct cellular mechanism operative in stem cells. By using conditional knock-in mice, we show that the HSC defect resulting from expression of heterozygous human JAK2V617F is both quantitative (reduced HSC numbers) and qualitative (lineage biases and reduced self-renewal per HSC). The defect is intrinsic to individual HSCs and their progeny are skewed toward proliferation and differentiation as evidenced by single cell and transplantation assays. Aged JAK2V617F show a more pronounced defect as assessed by transplantation, but mice that transform reacquire competitive self-renewal ability. Quantitative analysis of HSC-derived clones was used to model the fate choices of normal and JAK2-mutant HSCs and indicates that JAK2V617F reduces self-renewal of individual HSCs but leaves progenitor expansion intact. This conclusion is supported by paired daughter cell analyses, which indicate that JAK2-mutant HSCs more often give rise to two differentiated daughter cells. Together these data suggest that acquisition of JAK2V617F alone is insufficient for clonal expansion and disease progression and causes eventual HSC exhaustion. Moreover, our results show that clonal expansion of progenitor cells provides a window in which collaborating mutations can accumulate to drive disease progression. Characterizing the mechanism(s) of JAK2V617F

  4. Treatment response evaluation with 18F-FDG PET/CT and 18F-NaF PET/CT in multiple myeloma patients undergoing high-dose chemotherapy and autologous stem cell transplantation.

    Science.gov (United States)

    Sachpekidis, Christos; Hillengass, J; Goldschmidt, H; Wagner, B; Haberkorn, U; Kopka, K; Dimitrakopoulou-Strauss, A

    2017-01-01

    The aim of this study was to assess the combined use of the radiotracers 18 F-FDG and 18 F-NaF in treatment response evaluation of a group of multiple myeloma (MM) patients undergoing high-dose chemotherapy (HDT) followed by autologous stem cell transplantation (ASCT) by means of static (whole-body) and dynamic PET/CT (dPET/CT). Thirty-four patients with primary, previously untreated MM scheduled for treatment with HDT followed by ASCT were enrolled in the study. All patients underwent PET/CT scanning with 18 F-FDG and 18 F-NaF before and after therapy. Treatment response by means of PET/CT was assessed according to the European Organization for Research and Treatment of Cancer (EORTC) 1999 criteria. The evaluation of dPET/CT studies was based on qualitative evaluation, semi-quantitative (SUV) calculation, and quantitative analysis based on two-tissue compartment modelling and a non-compartmental approach leading to the extraction of fractal dimension (FD). An analysis was possible in 29 patients: three with clinical complete response (CR) and 26 with non-CR (13 patients near complete response-nCR, four patients very good partial response-VGPR, nine patients partial response-PR). After treatment, 18 F-FDG PET/CT was negative in 14/29 patients and positive in 15/29 patients, showing a sensitivity of 57.5 % and a specificity of 100 %. According to the EORTC 1999 criteria, 18 F-FDG PET/CT-based treatment response revealed CR in 14 patients ( 18 F-FDG PET/CT CR), PR in 11 patients ( 18 F-FDG PET/CT PR) and progressive disease in four patients ( 18 F-FDG PET/CT PD). In terms of 18 F-NaF PET/CT, 4/29 patients (13.8 %) had a negative baseline scan, thus failed to depict MM. Regarding the patients for which a direct lesion-to-lesion comparison was feasible, 18 F-NaF PET/CT depicted 56 of the 129 18 F-FDG positive lesions (43 %). Follow-up 18 F-NaF PET/CT showed persistence of 81.5 % of the baseline 18 F-NaF positive MM lesions after treatment, despite the fact that 64

  5. Cytotoxicity and cell death mechanisms induced by the polyamine-vectorized anti-cancer drug F14512 targeting topoisomerase II.

    Science.gov (United States)

    Brel, Viviane; Annereau, Jean-Philippe; Vispé, Stéphane; Kruczynski, Anna; Bailly, Christian; Guilbaud, Nicolas

    2011-12-15

    The polyamines transport system (PTS) is usually enhanced in cancer cells and can be exploited to deliver anticancer drugs. The spermine-conjugated epipodophyllotoxin derivative F14512 is a topoisomerase II poison that exploits the PTS to target preferentially tumor cells. F14512 has been characterized as a potent anticancer drug candidate and is currently in phase 1 clinical trials. Here we have analyzed the mechanisms of cell death induced by F14512, compared to the parent drug etoposide lacking the polyamine tail. F14512 proved to be >30-fold more cytotoxic than etoposide against A549 non-small cell lung cancer cells and triggers less but unrecoverable DNA damages. The cytotoxic action of F14512 is extremely rapid (within 3 h) and does not lead to a marked accumulation in the S-phase of the cell cycle, unlike etoposide. Interestingly, A549 cells treated with F14512 were less prone to undergo apoptosis (neither caspases-dependent nor caspases-independent pathways) or autophagy but preferentially entered into senescence. Drug-induced senescence was characterized qualitatively and quantitatively by an increased β-galactosidase activity, both by cytochemical staining and by flow cytometry. A morphological analysis by electron microscopy revealed the presence of numerous multi-lamellar and vesicular bodies and large electron-lucent (methuosis-like) vacuoles in F14512-treated cell samples. The mechanism of drug-induced cell death is thus distinct for F14512 compared to etoposide, and this difference may account for their distinct pharmacological profiles and the markedly superior activity of F14512 in vivo. This study suggests that senescence markers should be considered as potential pharmacodynamic biomarkers of F14512 antitumor activity. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. MicroRNAs regulate T-cell production of interleukin-9 and identify hypoxia-inducible factor-2α as an important regulator of T helper 9 and regulatory T-cell differentiation.

    Science.gov (United States)

    Singh, Yogesh; Garden, Oliver A; Lang, Florian; Cobb, Bradley S

    2016-09-01

    MicroRNAs (miRNAs) regulate many aspects of helper T cell (Th) development and function. Here we found that they are required for the suppression of interleukin-9 (IL-9) expression in Th9 cells and other Th subsets. Two highly related miRNAs (miR-15b and miR-16) that we previously found to play an important role in regulatory T (Treg) cell differentiation were capable of suppressing IL-9 expression when they were over-expressed in Th9 cells. We used these miRNAs as tools to identify novel regulators of IL-9 expression and found that they could regulate the expression of Epas1, which encodes hypoxia-inducible factor (HIF)-2α. HIF proteins regulate metabolic pathway usage that is important in determining appropriate Th differentiation. The related protein, HIF-1α enhances Th17 differentiation and inhibits Treg cell differentiation. Here we found that HIF-2α was required for IL-9 expression in Th9 cells, but its expression was not sufficient in other Th subsets. Furthermore, HIF-2α suppressed Treg cell differentiation like HIF-1α, demonstrating both similar and distinct roles of the HIF proteins in Th differentiation and adding a further dimension to their function. Ironically, even though miR-15b and miR-16 suppressed HIF-2α expression in Treg cells, inhibiting their function in Treg cells did not lead to an increase in IL-9 expression. Therefore, the physiologically relevant miRNAs that regulate IL-9 expression in Treg cells and other subsets remain unknown. Nevertheless, the analysis of miR-15b and miR-16 function led to the discovery of the importance of HIF-2α so this work demonstrated the utility of studying miRNA function to identify novel regulatory pathways in helper T-cell development. © 2016 John Wiley & Sons Ltd.

  7. Electric dipole f values for Fe II J=9/2->11/2^o transitions

    Science.gov (United States)

    Beck, Donald R.

    2007-06-01

    Relativistic configuration-interaction f values have been obtained for all transitions between the lowest 17 J=9/2 and the lowest 27 J=11/2^o levels. The 35 f values >0.01 have an average gauge agreement of 3.6% and generally agree well with the semi-empirical results of Raassen ootnotetextA. J. J. Raassen, ftp://ftp.wins.uva.nl/pub/orth/iron/FeII.E1 (1999).. The near degeneracy of many of the odd levels requires the introduction of small semi-empirical energy shifts ootnotetextD. R. Beck, Phys. Scr. 71, 447 (2005).. A systematic way of estimating radial convergence is discussed.

  8. Ultraviolet light induces double-strand breaks in DNA of cultured human P3 cells as measured by neutral filter elution

    International Nuclear Information System (INIS)

    Peak, J.G.; Peak, M.J.

    1990-01-01

    Neutral filter elution at pH 7.2 and 9.6 was used to measure the induction of DNA lesions in human P3 teratocarcinoma cells by monochromatic 254-, 270-, 313-, 334-, 334-,365-, and 405-nm radiation and by 60 gamma rays. In this assay DNA double-strand breaks (dsb) increase the rate of elution of DNA from cell lysates on a filter. Yields of dsb as measured by this procedure were determined by using a calibration of the assay that correlates elution parameters with number of dsb caused by disintegration of 125 I incorporated into the DNA. Analysis of fluence responses obtained by using the calibrated assay indicated that the number of dsb induced per dalton of DNA as measured by this assay is proportional to the square of the fluence at all the energies of radiation studied, implying that the induction of these lesions may be a two-hit event. Analysis of the relative efficiencies for the induction of dsb by ultraviolet radiation, corrected for quantum efficiency, revealed a spectrum that coincided closely with that for the induction of single-strand breaks (ssb) in the same cells, having a close fit with the spectrum of nucleic acid in the UVC and UVB region below 313 nm, and a shoulder in the UVA region. It was calculated, however, that there may be too few ssb for dsb to result from randomly distributed closely opposed ssb. (author)

  9. The E2F transcription factors: key regulators of cell proliferation

    DEFF Research Database (Denmark)

    Müller, H; Helin, K

    2000-01-01

    Ever since its discovery, the RB-1 gene and the corresponding protein, pRB, have been a focal point of cancer research. The isolation of E2F transcription factors provided the key to our current understanding of RB-1 function in the regulation of the cell cycle and in tumor suppression....... It is becoming more and more evident that the regulatory circuits governing the cell cycle are very complex and highly interlinked. Certain aspects of RB-1 function, for instance its role in differentiation, cannot be easily explained by the current models of pRB-E2F interaction. One reason is that pRB has...

  10. Human umbilical cord mesenchymal stem cells increase interleukin-9 production of CD4+ T cells

    OpenAIRE

    Yang, Zhou Xin; Chi, Ying; Ji, Yue Ru; Wang, You Wei; Zhang, Jing; Luo, Wei Feng; Li, Li Na; Hu, Cai Dong; Zhuo, Guang Sheng; Wang, Li Fang; Han, Zhi-Bo; Han, Zhong Chao

    2017-01-01

    Mesenchymal stem cells (MSC) are able to differentiate into cells of multiple lineage, and additionally act to modulate the immune response. Interleukin (IL)-9 is primarily produced by cluster of differentiation (CD)4+ T cells to regulate the immune response. The present study aimed to investigate the effect of human umbilical cord derived-MSC (UC-MSC) on IL-9 production of human CD4+ T cells. It was demonstrated that the addition of UC-MSC to the culture of CD4+ T cells significantly enhance...

  11. Long-Term Expandable SOX9+ Chondrogenic Ectomesenchymal Cells from Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Katsutsugu Umeda

    2015-04-01

    Full Text Available Here we report the successful generation and long-term expansion of SOX9-expressing CD271+PDGFRα+CD73+ chondrogenic ectomesenchymal cells from the PAX3/SOX10/FOXD3-expressing MIXL1−CD271hiPDGFRαloCD73− neural crest-like progeny of human pluripotent stem cells in a chemically defined medium supplemented with Nodal/Activin/transforming growth factorβ (TGFβ inhibitor and fibroblast growth factor (FGF. When “primed” with TGFβ, such cells efficiently formed translucent cartilage particles, which were completely mineralized in 12 weeks in immunocompromized mice. The ectomesenchymal cells were expandable without loss of chondrogenic potential for at least 16 passages. They maintained normal karyotype for at least 10 passages and expressed genes representing embryonic progenitors (SOX4/12, LIN28A/B, cranial mesenchyme (ALX1/3/4, and chondroprogenitors (SOX9, COL2A1 of neural crest origin (SOX8/9, NGFR, NES. Ectomesenchyme is a source of many craniofacial bone and cartilage structures. The method we describe for obtaining a large quantity of human ectomesenchymal cells will help to model craniofacial disorders in vitro and potentially provide cells for the repair of craniofacial damage.

  12. New triarylamine organic dyes containing the 9-hexyl-2-(hexyloxy)-9H-carbazole for dye-sensitized solar cells

    International Nuclear Information System (INIS)

    Su, Jianyang; Chen, Yu; Wu, Yungen; Ghimire, Raju Prasad; Xu, Yingjun; Liu, Xiujie; Wang, Zhihui; Liang, Mao

    2017-01-01

    Highlights: •9-hexyl-2-(hexyloxy)-9H-carbazole (HHCBZ) was synthesized for organic dyes. •Three new triarylamine sensitizers based on the HHCBZ unit were synthesized. •The HHCBZ unit outperforms the HCBZ when used as an electron donor. •An efficiency of 8.67% was achieved by M92 with the HHCBZ donor. -- Abstract: Developing carbazole derivatives as the electron donor for organic dyes have attracted extensive interest recently. Three organic dyes M92-94 based on the 9-hexyl-2-(hexyloxy)-9H-carbazole (HHCBZ) electron donor have been successfully designed and synthesized for dye-sensitized solar cells. M95 with the 9-hexyl-9H-carbazole (HCBZ) unit has also been synthesized for comparison. An introduction of the HHCBZ unit in triarylamine brings several advantages: (i) red shifting the absorption peak and increasing the maximum molar absorption coefficient of absorption bands; (ii) decreasing the charge recombination in cobalt cells as well as iodine cells; (iii) enhancing photocurrent/photovoltage and thus the power conversion efficiencies of cobalt cells as well as iodine cells. Devices prepared with M92 show consistently higher light-to-electric energy conversion efficiencies, with the champion device reaching 8.67%, surpassing M93-95.

  13. Cyclin F suppresses B-Myb activity to promote cell cycle checkpoint control

    DEFF Research Database (Denmark)

    Klein, Ditte Kjærsgaard; Hoffmann, Saskia; Ahlskog, Johanna K

    2015-01-01

    an important role in checkpoint control following ionizing radiation. Cyclin F-depleted cells initiate checkpoint signalling after ionizing radiation, but fail to maintain G2 phase arrest and progress into mitosis prematurely. Importantly, cyclin F suppresses the B-Myb-driven transcriptional programme...... that promotes accumulation of crucial mitosis-promoting proteins. Cyclin F interacts with B-Myb via the cyclin box domain. This interaction is important to suppress cyclin A-mediated phosphorylation of B-Myb, a key step in B-Myb activation. In summary, we uncover a regulatory mechanism linking the F-box protein...

  14. Tracking of [{sup 18}F]FDG-labeled natural killer cells to HER2/neu-positive tumors

    Energy Technology Data Exchange (ETDEWEB)

    Meier, Reinhard [Department of Radiology, University of California San Francisco (United States)], E-mail: reinhardt.meier@gmail.com; Piert, Morand [Department of Radiology, Division of Nuclear Medicine, University of Michigan (United States); Piontek, Guido; Rudelius, Martina [Institute of Pathology, Klinikum rechts der Isar, Technische Universitaet Muenchen (Germany); Oostendorp, Robert A. [3rd Department of Internal Medicine, Klinikum rechts der Isar, Technische Universitaet Muenchen (Germany); Senekowitsch-Schmidtke, Reingard [Department of Nuclear Medicine, Klinikum rechts der Isar, Technische Universitaet Muenchen (Germany); Henning, Tobias D. [Department of Radiology, University of California San Francisco (United States); Wels, Winfried S.; Uherek, Christoph [Chemotherapeutisches Forschungsinstitut, Georg-Speyer-Haus, Frankfurt am Main (Germany); Rummeny, Ernst J. [Department of Radiology, Klinikum rechts der Isar, Technische Universitaet Muenchen (Germany); Daldrup-Link, Heike E. [Department of Radiology, University of California San Francisco (United States)

    2008-07-15

    Introduction: The objective of this study was to label the human natural killer (NK) cell line NK-92 with [{sup 18}F]fluoro-deoxy-glucose (FDG) for subsequent in vivo tracking to HER2/neu-positive tumors. Methods: NK-92 cells were genetically modified to NK-92-scFv(FRP5)-zeta cells, which express a chimeric antigen receptor that is specific to the tumor-associated ErbB2 (HER2/neu) antigen. NK-92 and NK-92-scFv(FRP5)-zeta cells were labeled with [{sup 18}F]FDG by simple incubation at different settings. Labeling efficiency was evaluated by a gamma counter. Subsequently, [{sup 18}F]FDG-labeled parental NK-92 or NK-92-scFv(FRP5)-zeta cells were intravenously injected into mice with implanted HER2/neu-positive NIH/3T3 tumors. Radioactivity in tumors was quantified by digital autoradiography and correlated with histopathology. Results: The NK-92 and NK-92-scFv(FRP5)-zeta cells could be efficiently labeled with [{sup 18}F]FDG by simple incubation. Optimal labeling efficiencies (80%) were achieved using an incubation period of 60 min and additional insulin (10 IU/ml). After injection of 5x10{sup 6} [{sup 18}F]FDG-labeled NK-92-scFv(FRP5)-zeta cells into tumor-bearing mice, digital autoradiography showed an increased uptake of radioactivity in HER2/neu-positive tumors at 60 min postinjection. Conversely, injection of 5x10{sup 6} NK-92 cells not directed against HER2/neu receptors did not result in increased uptake of radioactivity in the tumors. Histopathology confirmed an accumulation of the NK-92-scFv(FRP5)-zeta cells, but not the parental NK cells, in tumor tissues. Conclusion: The human NK cell line NK-92 can be directed against HER2/neu antigens by genetic modification. The genetically modified NK cells can be efficiently labeled with [{sup 18}F]FDG, and the accumulation of these labeled NK cells in HER2/neu-positive tumors can be monitored with autoradiography.

  15. Survival Analysis of F98 Glioma Rat Cells Following Minibeam or Broad-Beam Synchrotron Radiation Therapy

    International Nuclear Information System (INIS)

    Gil, Silvia; Sarun, Sukhéna; Biete, Albert; Prezado, Yolanda; Sabés, Manel

    2011-01-01

    In the quest of a curative radiotherapy treatment for gliomas new delivery modes are being explored. At the Biomedical Beamline of the European Synchrotron Radiation Facility (ESRF), a new spatially-fractionated technique, called Minibeam Radiation Therapy (MBRT) is under development. The aim of this work is to compare the effectiveness of MBRT and broad-beam (BB) synchrotron radiation to treat F98 glioma rat cells. A dose escalation study was performed in order to delimit the range of doses where a therapeutic effect could be expected. These results will help in the design and optimization of the forthcoming in vivo studies at the ESRF. Two hundred thousand F98 cells were seeded per well in 24-well plates, and incubated for 48 hours before being irradiated with spatially fractionated and seamless synchrotron x-rays at several doses. The percentage of each cell population (alive, early apoptotic and dead cells, where either late apoptotic as necrotic cells are included) was assessed by flow cytometry 48 hours after irradiation, whereas the metabolic activity of surviving cells was analyzed on days 3, 4, and 9 post-irradiation by using QBlue test. The endpoint (or threshold dose from which an important enhancement in the effectiveness of both radiation treatments is achieved) obtained by flow cytometry could be established just before 12 Gy in the two irradiation schemes, whilst the endpoints assessed by the QBlue reagent, taking into account the cell recovery, were set around 18 Gy in both cases. In addition, flow cytometric analysis pointed at a larger effectiveness for minibeams, due to the higher proportion of early apoptotic cells. When the valley doses in MBRT equal the dose deposited in the BB scheme, similar cell survival ratio and cell recovery were observed. However, a significant increase in the number of early apoptotic cells were found 48 hours after the minibeam radiation in comparison with the seamless mode

  16. Different (direct and indirect) mechanisms for the induction of DNA-protein crosslinks in human cells by far- and near-ultraviolet radiations (290 and 405 nm)

    International Nuclear Information System (INIS)

    Peak, M.J.; Peak, J.G.; Jones, C.A.

    1985-01-01

    Apparent DNA-protein crosslinking induced by monochromatic 290 and 405 nm radiations was measured in cultured human P3 teratocarcinoma cells with DNA alkaline elution techniques. The rates of the induction of crosslinks by 290 nm radiation were the same when the cells were irradiated either aerobically or anaerobically or when the cells were in an H 2 O or D 2 O aqueous environment. With 405 nm radiation, anaerobic irradiation reduced the induction of the crosslinks (dose modifying factor is about 0.2), and about twice as many crosslinks were observed when the cells were irradiated in an environment of D 2 O rather than H 2 O. The results are consistent with the hypothesis that far-UV radiation induces DNA-protein crosslinks by a direct mechanism, whereas near-UV radiation induces crosslinks via indirect photodynamic photosensitizations in which unidentified cellular endogenous photosensitizers and reactive species of oxygen are used. (author)

  17. Selective cell culture on UV transparent polymer by F2 laser surface modification

    International Nuclear Information System (INIS)

    Hanada, Yasutaka; Sugioka, Koji; Kawano, Hiroyuki; Tsuchimoto, Takayoshi; Miyamoto, Iwao; Miyawaki, Atsushi; Midorikawa, Katsumi

    2009-01-01

    A microchip made of UV transparent polymer (CYTOP) that can perform selective cell culture has been fabricated by F 2 laser surface modification. The refractive index of CYTOP is almost the same as that of culture medium, which is essential for three-dimensional (3D) observation of cells. The F 2 laser modification of CYTOP achieves hydrophilicity only on the laser irradiated area with little deterioration of the optical properties and surface smoothness. After the laser modification, HeLa cells were successfully cultured and strongly adhered only on the modified area of CYTOP. The cells patterned on CYTOP were applied for clear 3D observation using an optical microscope in phase contrast mode.

  18. Oral administration of a select mixture of Bacillus probiotics generates Tr1 cells in weaned F4ab/acR- pigs challenged with an F4+ ETEC/VTEC/EPEC strain.

    Science.gov (United States)

    Zhou, Dong; Zhu, Yao-Hong; Zhang, Wei; Wang, Meng-Ling; Fan, Wen-Yi; Song, Dan; Yang, Gui-Yan; Jensen, Bent Borg; Wang, Jiu-Feng

    2015-09-17

    Although breeding of F4 receptor - negative (F4R(-)) pigs may prevent post-weaning diarrhea, the underlying immunity is poorly understood. Here, various doses of a Bacillus licheniformis and Bacillus subtilis mixture (BLS-mix) were orally administered to F4ab/acR(-) pigs for 1 week before F4 (K88) - positive ETEC/VTEC/EPEC challenge. Administration of BLS-mix increased the percentage of Foxp3(-)IL-10(+) T cells but not of Foxp3(+)IL-10(+) regulatory T (Treg) cells among peripheral blood CD4(+) T cells. A low dose of BLS-mix feeding resulted in increased the expression of IL-6, TNF-α, IL-10, and the transcription factors Foxp3 and T-bet mRNAs in the jejunum. Administration of either a low or high dose BLS-mix also led to an increase in the percentage of CD4(+)Foxp3(+) Treg cells among intraepithelial lymphocytes and CD4(+)IL-10(+) T cells in the small intestinal Peyer's patches and the lamina propria of F4ab/acR(-) pigs following F4(+) ETEC/VTEC/EPEC challenge. The increased number of IL-10-producing CD4(+) T cells was attributed to an increase in the proportion of Foxp3(-)IL-10(+) Treg cells rather than Foxp3(+)IL-10(+) Treg cells. Our data indicate that oral administration of BLS-mix to newly weaned F4ab/acR(-) pigs ameliorates enteritis in an F4(+) ETEC/VTEC/EPEC model; however, induction of IL-10-producing Foxp3(-) Treg cells by BLS-mix administration cannot account for the protection of newly weaned F4ab/acR(-) pigs from F4(+) ETEC/VTEC/EPEC infection, and that excessive generation of CD4(+)IL-10(+) T cells following consumption of BLS-mix during episodes of intestinal inflammation that is caused by enteric pathogens might prohibit clearance of the pathogen. Select probiotic mixtures may allow for tailoring strategies to prevent infectious diseases.

  19. Association of Increased F4/80high Macrophages With Suppression of Serum-Transfer Arthritis in Mice With Reduced FLIP in Myeloid Cells.

    Science.gov (United States)

    Huang, Qi-Quan; Birkett, Robert; Doyle, Renee E; Haines, G Kenneth; Perlman, Harris; Shi, Bo; Homan, Philip; Xing, Lianping; Pope, Richard M

    2017-09-01

    Macrophages are critical in the pathogenesis of rheumatoid arthritis (RA). We recently demonstrated that FLIP is necessary for the differentiation and/or survival of macrophages. We also showed that FLIP is highly expressed in RA synovial macrophages. This study was undertaken to determine if a reduction in FLIP in mouse macrophages reduces synovial tissue macrophages and ameliorates serum-transfer arthritis. Mice with Flip deleted in myeloid cells (Flip f/f LysM c/+ mice) and littermate controls were used. Arthritis was induced by intraperitoneal injection of K/BxN serum. Disease severity was evaluated by clinical score and change in ankle thickness, and joints were examined by histology and immunohistochemistry. Cells were isolated from the ankles and bone marrow of the mice and examined by flow cytometry, real-time quantitative reverse transcriptase-polymerase chain reaction, or Western blotting. In contrast to expectations, Flip f/f LysM c/+ mice developed more severe arthritis early in the clinical course, but peak arthritis was attenuated and the resolution phase more complete than in control mice. Prior to the induction of serum-transfer arthritis, the number of tissue-resident macrophages was reduced. On day 9 after arthritis induction, the number of F4/80 high macrophages in the joints of the Flip f/f LysM c/+ mice was not decreased, but increased. FLIP was reduced in the F4/80 high macrophages in the ankles of the Flip f/f LysM c/+ mice, while F4/80 high macrophages expressed an antiinflammatory phenotype in both the Flip f/f LysM c/+ and control mice. Our observations suggest that reducing FLIP in macrophages by increasing the number of antiinflammatory macrophages may be an effective therapeutic approach to suppress inflammation, depending on the disease stage. © 2017, American College of Rheumatology.

  20. Sodium butyrate stimulates cellular recovery from UV damage in xeroderma pigmentosum cells belonging to complementation group F

    International Nuclear Information System (INIS)

    Nishigori, Chikako; Takebe, Hiraku

    1987-01-01

    Possible stimulation of the DNA repair capacity by sodium butyrate in normal and xeroderma pigmentosum (XP) cells was investigated. XP cells belonging to the complementation group F showed considerable stimulation of DNA repair by sodium butyrate in terms of both the amount of unscheduled DNA synthesis (UDS) and the colony-forming ability after UV irradiation. UDS in XP cells belonging to the complementation group A was not enhanced, while normal cells showed slight enhancement, but less than that of XP F cells. In XP A, XP C, and normal cells, sodium butyrate treatment enhanced the killing effect of UV irradiation. The residual repair capacity in XP F cells appeared to be stimulated by sodium butyrate. (author)

  1. The prevalence of maternal F cells in a pregnant population and potential overestimation of foeto-maternal haemorrhage as a consequence.

    LENUS (Irish Health Repository)

    Corcoran, Deirdre

    2014-06-12

    Acid elution (AE) is used to estimate foeto-maternal haemorrhage (FMH). However AE cannot differentiate between cells containing foetal or adult haemoglobin F (F cells), potentially leading to false positive results or an overestimate of the amount of FMH. The prevalence of F cells in pregnant populations remains poorly characterised. The purpose of this study was to ascertain the incidence of HbF-containing red cells in our pregnant population using anti-HbF-fluorescein isothiocyanate flow cytometry (anti-HbF FC) and to assess whether its presence leads to a significant overestimate of FMH.

  2. Higher cell stiffness indicating lower metastatic potential in B16 melanoma cell variants and in (-)-epigallocatechin gallate-treated cells.

    Science.gov (United States)

    Watanabe, Tatsuro; Kuramochi, Hiromi; Takahashi, Atsushi; Imai, Kazue; Katsuta, Naoko; Nakayama, Tomonobu; Fujiki, Hirota; Suganuma, Masami

    2012-05-01

    To understand how nanomechanical stiffness affects metastatic potential, we studied the relationship between cell migration, a characteristic of metastasis, and cell stiffness using atomic force microscopy (AFM), which can measure stiffness (elasticity) of individual living cells. Migration and cell stiffness of three metastatic B16 melanoma variants (B16-F10, B16-BL6, and B16-F1 cells), and also effects of (-)-epigallocatechin gallate (EGCG), were studied using Transwell assay and AFM. Migration of B16-F10 and B16-BL6 cells was 3 and 2 times higher than that of B16-F1 cells in Transwell assay, and cell stiffness determined by AFM was also different among the three variants, although they have similar morphologies and the same growth rates: Means of Young's modulus were 350.8 ± 4.8 Pa for B16-F10 cells, 661.9 ± 16.5 Pa for B16-BL6 cells, and 727.2 ± 13.0 Pa for B16-F1 cells. AFM measurements revealed that highly motile B16-F10 cells have low cell stiffness, and low motile and metastatic B16-F1 cells have high cell stiffness: Nanomechanical stiffness is inversely correlated with migration potential. Treatment of highly motile B16-F10 cells with EGCG increased cell stiffness 2-fold and inhibited migration of the cells. Our study with AFM clearly demonstrates that cell stiffness is a reliable quantitative indicator of migration potential, and very likely metastatic potential, even in morphologically similar cells. And increased cell stiffness may be a key nanomechanical feature in inhibition of metastasis.

  3. Evaluation of granulated BGO, GSO:Ce, YAG:Ce, CaF2:Eu and ZnS:Ag for alpha/beta pulse shape discrimination in a flow-cell radiation detector

    International Nuclear Information System (INIS)

    DeVol, T.A.; Chotoo, S.B.; Fjeld, R.A.

    1999-01-01

    Granulated BGO, GSO:Ce, YAG:Ce, and CaF 2 :Eu; CaF 2 :Eu coated with a fluorescent polymer, and combinations of coated and uncoated CaF 2 :Eu with ZnS:Ag were evaluated for their ability to discriminate between alpha and beta particles in a flow-cell radiation detector. The evaluations were based on the analysis of pulse shape spectra. Various granulated scintillators were packed into flow cell detectors that were coils of 3.0 mm ODx1.5 mm ID fluorinated ethylene propylene Teflon[reg] tubing positioned between dual photomultiplier tubes for analysis. The best pulse shape discrimination was obtained for a combination of equal masses of uncoated CaF 2 :Eu (63-90 μm) and ZnS:Ag (10 μm), which had a 9% spillover. Additional research is needed to reduce the spillover

  4. Application of a molecular diagnostic algorithm for haemophilia A and B using next-generation sequencing of entire F8, F9 and VWF genes.

    Science.gov (United States)

    Bastida, Jose Maria; González-Porras, Jose Ramon; Jiménez, Cristina; Benito, Rocio; Ordoñez, Gonzalo R; Álvarez-Román, Maria Teresa; Fontecha, M Elena; Janusz, Kamila; Castillo, David; Fisac, Rosa María; García-Frade, Luis Javier; Aguilar, Carlos; Martínez, María Paz; Bermejo, Nuria; Herrero, Sonia; Balanzategui, Ana; Martin-Antorán, Jose Manuel; Ramos, Rafael; Cebeiro, Maria Jose; Pardal, Emilia; Aguilera, Carmen; Pérez-Gutierrez, Belen; Prieto, Manuel; Riesco, Susana; Mendoza, Maria Carmen; Benito, Ana; Hortal Benito-Sendin, Ana; Jiménez-Yuste, Víctor; Hernández-Rivas, Jesus Maria; García-Sanz, Ramon; González-Díaz, Marcos; Sarasquete, Maria Eugenia

    2017-01-05

    Currently, molecular diagnosis of haemophilia A and B (HA and HB) highlights the excess risk-inhibitor development associated with specific mutations, and enables carrier testing of female relatives and prenatal or preimplantation genetic diagnosis. Molecular testing for HA also helps distinguish it from von Willebrand disease (VWD). Next-generation sequencing (NGS) allows simultaneous investigation of several complete genes, even though they may span very extensive regions. This study aimed to evaluate the usefulness of a molecular algorithm employing an NGS approach for sequencing the complete F8, F9 and VWF genes. The proposed algorithm includes the detection of inversions of introns 1 and 22, an NGS custom panel (the entire F8, F9 and VWF genes), and multiplex ligation-dependent probe amplification (MLPA) analysis. A total of 102 samples (97 FVIII- and FIX-deficient patients, and five female carriers) were studied. IVS-22 screening identified 11 out of 20 severe HA patients and one female carrier. IVS-1 analysis did not reveal any alterations. The NGS approach gave positive results in 88 cases, allowing the differential diagnosis of mild/moderate HA and VWD in eight cases. MLPA confirmed one large exon deletion. Only one case did have no pathogenic variants. The proposed algorithm had an overall success rate of 99 %. In conclusion, our evaluation demonstrates that this algorithm can reliably identify pathogenic variants and diagnose patients with HA, HB or VWD.

  5. Importancia de la práctica físico-deportiva y del género en el autoconcepto físico de los 9 a los 23 años

    Directory of Open Access Journals (Sweden)

    Juan Antonio Moreno

    2008-01-01

    Full Text Available El propósito de este estudio descriptivo mediante encuesta es comprobar los efectos de la edad, el género y la práctica físico-deportiva en el autoconcepto físico en estudiantes de 9 a 23 años. La muestra está compuesta por 2.332 alumnos a los que se les pasó el Physical Self-concept Questionary (PSQ. Tras distintos análisis de correlación, univariantes, multivariantes y de regresión, los resultados revelan que tanto la autoestima como el autoconcepto físico están influenciados por la edad, el género y la práctica físico-deportiva, destacando como principal aportación que la práctica de alguna actividad física o deportiva es la que más predice la autoestima en particular y el autoconcepto físico, en general. Por otro lado, son los varones los que presentan una mayor autoestima, imagen corporal, competencia y condición física. En cuanto a la evolución de la autoestima y el autoconcepto físico de los 9 a los 23 años, solo se observan diferencias significativas en la competencia percibida y la condición física. Como conclusión, este estudio muestra la relevancia que la edad, el género y la práctica físico-deportiva extraescolar parecen tener sobre la elaboración del autoconcepto físico.

  6. Functional interrelationship between TFII-I and E2F transcription factors at specific cell cycle gene loci.

    Science.gov (United States)

    Shen, Yong; Nar, Rukiye; Fan, Alex X; Aryan, Mahmoud; Hossain, Mir A; Gurumurthy, Aishwarya; Wassel, Paul C; Tang, Ming; Lu, Jianrong; Strouboulis, John; Bungert, Jörg

    2018-01-01

    Transcription factor TFII-I is a multifunctional protein implicated in the regulation of cell cycle and stress-response genes. Previous studies have shown that a subset of TFII-I associated genomic sites contained DNA-binding motifs for E2F family transcription factors. We analyzed the co-association of TFII-I and E2Fs in more detail using bioinformatics, chromatin immunoprecipitation, and co-immunoprecipitation experiments. The data show that TFII-I interacts with E2F transcription factors. Furthermore, TFII-I, E2F4, and E2F6 interact with DNA-regulatory elements of several genes implicated in the regulation of the cell cycle, including DNMT1, HDAC1, CDKN1C, and CDC27. Inhibition of TFII-I expression led to a decrease in gene expression and in the association of E2F4 and E2F6 with these gene loci in human erythroleukemia K562 cells. Finally, TFII-I deficiency reduced the proliferation of K562 cells and increased the sensitivity toward doxorubicin toxicity. The results uncover novel interactions between TFII-I and E2Fs and suggest that TFII-I mediates E2F function at specific cell cycle genes. © 2017 Wiley Periodicals, Inc.

  7. Non-steroidal anti-inflammatory drugs decrease E2F1 expression and inhibit cell growth in ovarian cancer cells.

    Directory of Open Access Journals (Sweden)

    Blanca L Valle

    Full Text Available Epidemiological studies have shown that the regular use of non-steroidal anti-inflammatory (NSAIDs drugs is associated with a reduced risk of various cancers. In addition, in vitro and experiments in mouse models have demonstrated that NSAIDs decrease tumor initiation and/or progression of several cancers. However, there are limited preclinical studies investigating the effects of NSAIDs in ovarian cancer. Here, we have studied the effects of two NSAIDs, diclofenac and indomethacin, in ovarian cancer cell lines and in a xenograft mouse model. Diclofenac and indomethacin treatment decreased cell growth by inducing cell cycle arrest and apoptosis. In addition, diclofenac and indomethacin reduced tumor volume in a xenograft model of ovarian cancer. To identify possible molecular pathways mediating the effects of NSAID treatment in ovarian cancer, we performed microarray analysis of ovarian cancer cells treated with indomethacin or diclofenac. Interestingly, several of the genes found downregulated following diclofenac or indomethacin treatment are transcriptional target genes of E2F1. E2F1 was downregulated at the mRNA and protein level upon treatment with diclofenac and indomethacin, and overexpression of E2F1 rescued cells from the growth inhibitory effects of diclofenac and indomethacin. In conclusion, NSAIDs diclofenac and indomethacin exert an anti-proliferative effect in ovarian cancer in vitro and in vivo and the effects of NSAIDs may be mediated, in part, by downregulation of E2F1.

  8. Clinical values for abnormal 18F-FDG uptake in the head and neck region of patients with head and neck squamous cell carcinoma

    International Nuclear Information System (INIS)

    Lee, Hwan Seo; Kim, Jae Seung; Roh, Jong-Lyel; Choi, Seung-Ho; Nam, Soon Yuhl; Kim, Sang Yoon

    2014-01-01

    Highlights: • Abnormal 18 F-FDG uptakes in the head and neck (HN) region can be carefully interpreted as being index primary, second primary cancer (SP) or benign. • 18 F-FDG PET/CT identified 91.9% primary HN squamous cell carcinomas (HNSCC). • The specificity and negative predictive value of 18 F-FDG PET/CT for identification of SP were as high as 98.7% and 99.3%, respectively. • Proper detection of primary tumors and SP in the HN region may promote appropriate therapeutic planning of HNSCC patients. - Abstract: Purpose: Fluorine 18-fluorodeoxyglucose ( 18 F-FDG) positron emission tomography (PET)/computed tomography (CT) is used to identify index or second primary cancer (SP) of the head and neck (HN) through changes in 18 F-FDG uptake. However, both physiologic and abnormal lesions increase 18 F-FDG uptake. Therefore, we evaluated 18 F-FDG uptake in the HN region to determine clinical values of abnormal tracer uptake. Methods: A prospective study approved by the institutional review board was conducted in 314 patients with newly diagnosed HN squamous cell carcinoma (HNSCC) and informed consent was obtained from all enrolled patients. The patients received initial staging workups including 18 F-FDG PET/CT and biopsies. All lesions with abnormal HN 18 F-FDG uptake were recorded and most of those were confirmed by biopsies. Diagnostic values for abnormal 18 F-FDG uptake were calculated. Results: Abnormal 18 F-FDG uptake was identified in primary tumors from 285 (91.9%) patients. False-negative results were obtained for 22.3% (23/103) T1 tumors and 2.2% (2/93) T2 tumors (P < 0.001). Thirty-eight regions of abnormal 18 F-FDG uptake were identified in 36 (11.5%) patients: the thyroid (n = 13), maxillary sinus (n = 7), palatine tonsil (n = 6), nasopharynx (n = 5), parotid gland (n = 2) and others (n = 5). Synchronous SP of the HN was identified in eight (2.5%) patients: the thyroid (n = 5), palatine tonsil (n = 2), and epiglottis (n = 1). The sensitivity and

  9. S-phase checkpoint elements of the E2F-1 family increase radiosensitivity in fibrosarcoma cells lacking p53

    International Nuclear Information System (INIS)

    Bodis, Stephan; Pruschy, Martin; Wirbelauer, Christiane; Glanzmann, Christoph; Krek, Wilhelm

    1997-01-01

    Purpose: Correct advance of cells through the S-phase of the mammalian cell cycle depends on the timely controlled activity of the E2F-1 transcription factor by cyclin A-cdk2. We are studying the reproductive integrity and radiosensitation of isogenic mouse fibrosarcoma cells, differing only in their p53 status, after expression of E2F-1 wildtype (wt) and specific E2F-1 mutants (mt) lacking the cyclin-A-binding domain. In this tumor model system only p53 wild-type expressing tumor cells are sensitive to ionizing radiation in vitro and in vivo. Material and Methods: Either wild-type p53 or genetically engineered p53 'null' mouse embryo fibroblasts were transfected with the oncogenes E1A and ras. These otherwise isogenic fibrosarcoma cells, with a malignant phenotype and tumorigenic in nude mice, were transfected with retroviruses containing either E2F-1 wild-type or specific E2F-1 mutants lacking the cyclin-A binding domain. Reproductive integrity after E2F-1 transfection with or without ionizing radiation (RT) was tested using the clonogenic assay. Tumor cell morphology of treated cells is analyzed for cell death mechanism. Results: E2F-1 wild-type expression in fibrosarcoma cells induced a clear p53 dependent cell death. While clonogenic survival of p53 'null' tumor cells was only slightly reduced with the expression of E2F-1 wild type (survival fraction of 0.5), the clonogenic survival of p53 wild-type fibrosarcoma tumor cells was reduced by at least one logarithm (survival fraction of 0.05). However, expression of the specific E2F-1 mutant lacking the cyclin-A binding domain reduced clonogenic survival in both the p53 'null' and the p53 wild-type fibrosarcoma cells by at least 2 logarithms (survival fraction 0.01 for p53 'null' and 0.002 for p53 wild-type). The mean values of the survival fractions after 2 and 5 Gy radiation alone in p53 'null' fibrosarcoma cells (SF 2 and SF 5) were SF 2 0.7, SF 5 = 0.15, respectively. The combination of ionizing RT in the p53

  10. Synthesis, uptake mechanism characterization and biological evaluation of {sup 18}F labeled fluoroalkyl phenylalanine analogs as potential PET imaging agents

    Energy Technology Data Exchange (ETDEWEB)

    Wang Limin [Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104 (United States); Qu Wenchao; Lieberman, Brian P.; Ploessl, Karl [Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104 (United States); Kung, Hank F., E-mail: kunghf@gmail.co [Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104 (United States); Department of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104 (United States)

    2011-01-15

    Introduction: Amino acids based tracers represent a promising class of tumor metabolic imaging agents with successful clinical applications. Two new phenylalanine derivatives, p-(2-[{sup 18}F]fluoroethyl)-L-phenylalanine (FEP, [{sup 18}F]2) and p-(3-[{sup 18}F]fluoropropyl)-L-phenylalanine (FPP, [{sup 18}F]3) were synthesized and evaluated in comparison to clinically utilized O-(2-[{sup 18}F]fluoroethyl)-L-tyrosine (FET, [{sup 18}F]1). Methods: FEP ([{sup 18}F]2) and FPP ([{sup 18}F]3) were successfully synthesized by a rapid and efficient two-step nucleophilic fluorination of tosylate precursors and deprotection reaction. In vitro cell uptake studies were carried out in 9L glioma cells. In vivo studies, 9L tumor xenografts were implanted in Fisher 344 rats. Results: FEP ([{sup 18}F]2) and FPP ([{sup 18}F]3) could be efficiently labeled within 90 min with good enantiomeric purity (>95%), good yield (11-37%) and high specific activity (21-69 GBq/{mu}mol). Cell uptake studies showed FEP had higher uptake than FPP as well as reference ligand FET ([{sup 18}F]1). Uptake mechanism studies suggested that FEP is a selective substrate for system L and prefers its subtype LAT1. In vivo biodistribution studies demonstrated FEP had specific accumulation in tumor cells and tumor to background ratio reached 1.45 at 60 min. Small animal positron emission tomography (PET) imaging studies showed FEP was comparable to FET for imaging rats bearing 9L tumor model. FEP had high uptake in 9L tumor compared to surrounding tissue and was quickly excreted through urinary tract. Conclusion: Biological evaluations indicate that FEP ([{sup 18}F]2) is a potential useful tracer for tumor imaging with PET.

  11. F NMR measurement of intracellular free calcium in human red blood cells

    International Nuclear Information System (INIS)

    Gupta, R.K.; Schanne, F.A.X.

    1986-01-01

    Optical techniques for the measurement of intracellular Ca are not readily applicable to the human red cell because of the intense absorption of hemoglobin. The authors have therefore examined the use of 19 F NMR of 5,5'-difluoro-1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetra acetic acid (5FBAPTA) introduced non-disruptively by intracellular hydrolysis of the membrane-permeant acetoxymethyl ester derivative. 19 F NMR spectra of 5FBAPTA-containing erythrocytes at 188 MHz displayed two well resolved resonances corresponding to the free and Ca-bound forms of the chelator, the resonance of the free form being ten-fold larger than that of the Ca-bound form. Addition of the ionophore A23187 resulted in the disappearance of the resonance of the free anion and a quantitative increase in the intensity of the resonance of the Ca-complex. From these data, and a K/sub D/ of 708 nM for the Ca-5FBAPTA complex, the authors estimate red cell free Ca to be 70 nM, which is in the range of values obtained for other cells, despite the fact that the human red cell, which lacks intracellular organelles for storing Ca, possesses only 1 μmol total Ca/1. cells in comparison to mmols of total Ca found in other cells. The authors ability to use 19 F NMR to measure free Ca in the red blood cell paves the way for future NMR studies of red cell free Ca concentrations in human essential hypertension as well as in other diseases states in which alterations in cellular Ca homeostasis may be involved

  12. Inhibitory effects of kaempferol on the invasion of human breast carcinoma cells by downregulating the expression and activity of matrix metalloproteinase-9.

    Science.gov (United States)

    Li, Chenglin; Zhao, Yuanwei; Yang, Dan; Yu, Yanyan; Guo, Hao; Zhao, Ziming; Zhang, Bei; Yin, Xiaoxing

    2015-02-01

    Matrix metalloproteinases (MMPs) have been regarded as major critical molecules assisting tumor cells during metastasis, for excessive ECM (ECM) degradation, and cancer cell invasion. In the present study, in vitro and in vivo assays were employed to examine the inhibitory effects of kaempferol, a natural polyphenol of flavonoid family, on tumor metastasis. Data showed that kaempferol could inhibit adhesion, migration, and invasion of MDA-MB-231 human breast carcinoma cells. Moreover, kaempferol led to the reduced activity and expression of MMP-2 and MMP-9, which were detected by gelatin zymography, real-time PCR, and western blot analysis, respectively. Further elucidation of the mechanism revealed that kaempferol treatment inhibited the activation of transcription factor activator protein-1 (AP-1) and MAPK signaling pathway. Moreover, kaempferol repressed phorbol-12-myristate-13-acetate (PMA)-induced MMP-9 expression and activity through suppressing the translocation of protein kinase Cδ (PKCδ) and MAPK signaling pathway. Our results also indicated that kaempferol could block the lung metastasis of B16F10 murine melanoma cells as well as the expression of MMP-9 in vivo. Taken together, these results demonstrated that kaempferol could inhibit cancer cell invasion through blocking the PKCδ/MAPK/AP-1 cascade and subsequent MMP-9 expression and its activity. Therefore, kaempferol might act as a therapeutic potential candidate for cancer metastasis.

  13. Sperm associated antigen 9 plays an important role in bladder transitional cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Deepika Kanojia

    Full Text Available BACKGROUND: Majority of bladder cancer deaths are caused due to transitional cell carcinoma (TCC which is the most prevalent and chemoresistant malignancy of urinary bladder. Therefore, we analyzed the role of Sperm associated antigen 9 (SPAG9 in bladder TCC. METHODOLOGY AND FINDINGS: We examined SPAG9 expression and humoral response in 125 bladder TCC patients. Four bladder cancer cell lines were assessed for SPAG9 expression. In addition, we investigated the effect of SPAG9 ablation on cellular proliferation, cell cycle, migration and invasion in UM-UC-3 bladder cancer cells by employing gene silencing approach. Our SPAG9 gene and protein expression analysis revealed SPAG9 expression in 81% of bladder TCC tissue specimens. High SPAG9 expression (>60% SPAG9 positive cells was found to be significantly associated with superficial non-muscle invasive stage (P = 0.042 and low grade tumors (P = 0.002 suggesting SPAG9 putative role in early spread and tumorigenesis. Humoral response against SPAG9 was observed in 95% of patients found positive for SPAG9 expression. All four bladder cancer cell lines revealed SPAG9 expression. In addition, SPAG9 gene silencing in UM-UC-3 cells resulted in induction of G0-G1 arrest characterized by up-regulation of p16 and p21 and consequent down-regulation of cyclin E, cyclin D and cyclin B, CDK4 and CDK1. Further, SPAG9 gene silencing also resulted in reduction in cellular growth, and migration and invasion ability of cancer cells in vitro. CONCLUSIONS: Collectively, our data in clinical specimens indicated that SPAG9 is potential biomarker and therapeutic target for bladder TCC.

  14. Combining Vγ9Vδ2 T Cells with a Lipophilic Bisphosphonate Efficiently Kills Activated Hepatic Stellate Cells

    Directory of Open Access Journals (Sweden)

    Xiaoying Zhou

    2017-10-01

    Full Text Available Activated hepatic stellate cells (aHSCs are now established as a central driver of fibrosis in human liver injury. In the presence of chronic or repeated injury, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC can occur, so there is interest in down-regulating aHSCs activity in order to treat these diseases. Here, we report that Vγ9Vδ2 T cells are reduced in patients with liver cirrhosis, stimulating us to investigate possible interactions between Vγ9Vδ2 T cells and aHSCs. We find that Vγ9Vδ2 T cells kill aHSCs and killing is enhanced when aHSCs are pretreated with BPH-1236, a lipophilic analog of the bone resorption drug zoledronate. Cytotoxicity is mediated by direct cell-to-cell contact as shown by Transwell experiments and atomic force microscopy, with BPH-1236 increasing the adhesion between aHSCs and Vγ9Vδ2 T cells. Mechanistically, BPH-1236 functions by inhibiting farnesyl diphosphate synthase, leading to accumulation of the phosphoantigen isopentenyl diphosphate and recognition by Vγ9Vδ2 T cells. The cytolytic process is largely dependent on the perforin/granzyme B pathway. In a Rag2−/−γc−/− immune-deficient mouse model, we find that Vγ9Vδ2 T cells home-in to the liver, and when accompanied by BPH-1236, kill not only orthotopic aHSCs but also orthotopic HCC tumors. Collectively, our results provide the first proof-of-concept of a novel immunotherapeutic strategy for the treatment of fibrosis–cirrhosis–HCC diseases using adoptively transferred Vγ9Vδ2 T cells, combined with a lipophilic bisphosphonate.

  15. Cell stress promotes the association of phosphorylated HspB1 with F-actin.

    Directory of Open Access Journals (Sweden)

    Joseph P Clarke

    Full Text Available Previous studies have suggested that the small heat shock protein, HspB1, has a direct influence on the dynamics of cytoskeletal elements, in particular, filamentous actin (F-actin polymerization. In this study we have assessed the influence of HspB1 phosphorylation on its interaction(s with F-actin. We first determined the distribution of endogenous non-phosphorylated HspB1, phosphorylated HspB1 and F-actin in neuroendocrine PC12 cells by immunocytochemistry and confocal microscopy. We then investigated a potential direct interaction between HspB1 with F-actin by precipitating F-actin directly with biotinylated phalloidin followed by Western analyses; the reverse immunoprecipitation of HspB1 was also carried out. The phosphorylation influence of HspB1 in this interaction was investigated by using pharmacologic inhibition of p38 MAPK. In control cells, HspB1 interacts with F-actin as a predominantly non-phosphorylated protein, but subsequent to stress there is a redistribution of HspB1 to the cytoskeletal fraction and a significantly increased association of pHspB1 with F-actin. Our data demonstrate HspB1 is found in a complex with F-actin both in phosphorylated and non-phosphorylated forms, with an increased association of pHspB1 with F-actin after heat stress. Overall, our study combines both cellular and biochemical approaches to show cellular localization and direct demonstration of an interaction between endogenous HspB1 and F-actin using methodolgy that specifically isolates F-actin.

  16. CRISPR/Cas9 system and its applications in human hematopoietic cells.

    Science.gov (United States)

    Hu, Xiaotang

    2016-11-01

    Since 2012, the CRISPR-Cas9 system has been quickly and successfully tested in a broad range of organisms and cells including hematopoietic cells. The application of CRISPR-Cas9 in human hematopoietic cells mainly involves the genes responsible for HIV infection, β-thalassemia and sickle cell disease (SCD). The successful disruption of CCR5 and CXCR4 genes in T cells by CRISPR-Cas9 promotes the prospect of the technology in the functional cure of HIV. More recently, eliminating CCR5 and CXCR4 in induced pluripotent stem cells (iPSCs) derived from patients and targeting the HIV genome have been successfully carried out in several laboratories. The outcome from these approaches bring us closer to the goal of eradicating HIV infection. For hemoglobinopathies the ability to produce iPSC-derived from patients with the correction of hemoglobin (HBB) mutations by CRISPR-Cas9 has been tested in a number of laboratories. These corrected iPSCs also show the potential to differentiate into mature erythrocytes expressing high-level and normal HBB. In light of the initial success of CRESPR-Cas9 in target mutated gene(s) in the iPSCs, a combination of genomic editing and autogenetic stem cell transplantation would be the best strategy for root treatment of the diseases, which could replace traditional allogeneic stem cell transplantation. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Linc-POU3F3 is overexpressed in hepatocellular carcinoma and regulates cell proliferation, migration and invasion.

    Science.gov (United States)

    Li, Yichun; Li, Yannan; Wang, Dan; Meng, Qingdong

    2018-06-12

    Linc-POU3F3 showed an up-regulated tendency and functioned as tumor promoter in glioma, esophageal cancer and colorectal cancer. There was no report about the expression pattern and clinical value of linc-POU3F3 in hepatocellular carcinoma. Thus, the purpose of our study is to explore the clinical significance and biological role of linc-POU3F3 in hepatocellular carcinoma. Our results suggested that levels of linc-POU3F3 were dramatically increased in hepatocellular carcinoma tissues and cell lines compared with paired normal hepatic tissues and normal hepatic cell line, respectively. Levels of linc-POU3F3 were positively correlated with clinical stage, tumor size, vascular invasion and metastasis. Moreover, high-expression of linc-POU3F3 was an independent prognostic factor for hepatocellular carcinoma patients. The gain- and loss-of-function experiments showed that linc-POU3F3 expression significantly promoted tumor cell proliferation, migration and invasion. In addition, linc-POU3F3 expression was negatively correlated with POU3F3 mRNA and protein expressions in hepatocellular carcinoma tissues, and negatively regulated POU3F3 mRNA and protein expressions in hepatocellular carcinoma cells. In conclusion, our study supports the first evidence that linc-POU3F3 plays an oncogenic role in hepatocellular carcinoma, and represents a potential therapeutic strategy for hepatocellular carcinoma patients. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  18. ASC-J9 Suppresses Castration-Resistant Prostate Cancer Growth through Degradation of Full-length and Splice Variant Androgen Receptors

    Directory of Open Access Journals (Sweden)

    Shinichi Yamashita

    2012-01-01

    Full Text Available Early studies suggested androgen receptor (AR splice variants might contribute to the progression of prostate cancer (PCa into castration resistance. However, the therapeutic strategy to target these AR splice variants still remains unresolved. Through tissue survey of tumors from the same patients before and after castration resistance, we found that the expression of AR3, a major AR splice variant that lacks the AR ligand-binding domain, was substantially increased after castration resistance development. The currently used antiandrogen, Casodex, showed little growth suppression in CWR22Rv1 cells. Importantly, we found that AR degradation enhancer ASC-J9 could degrade both full-length (fAR and AR3 in CWR22Rv1 cells as well as in C4-2 and C81 cells with addition of AR3. The consequences of such degradation of both fAR and AR3 might then result in the inhibition of AR transcriptional activity and cell growth in vitro. More importantly, suppression of AR3 specifically by short-hairpin AR3 or degradation of AR3 by ASC-J9 resulted in suppression of AR transcriptional activity and cell growth in CWR22Rv1-fARKD (fAR knockdown cells in which DHT failed to induce, suggesting the importance of targeting AR3. Finally, we demonstrated the in vivo therapeutic effects of ASC-J9 by showing the inhibition of PCa growth using the xenografted model of CWR22Rv1 cells orthotopically implanted into castrated nude mice with undetectable serum testosterone. These results suggested that targeting both fAR- and AR3-mediated PCa growth by ASC-J9 may represent the novel therapeutic approach to suppress castration-resistant PCa. Successful clinical trials targeting both fAR and AR3 may help us to battle castration-resistant PCa in the future.

  19. Live imaging of H3K9 acetylation in plant cells

    Science.gov (United States)

    Kurita, Kazuki; Sakamoto, Takuya; Yagi, Noriyoshi; Sakamoto, Yuki; Ito, Akihiro; Nishino, Norikazu; Sako, Kaori; Yoshida, Minoru; Kimura, Hiroshi; Seki, Motoaki; Matsunaga, Sachihiro

    2017-01-01

    Proper regulation of histone acetylation is important in development and cellular responses to environmental stimuli. However, the dynamics of histone acetylation at the single-cell level remains poorly understood. Here we established a transgenic plant cell line to track histone H3 lysine 9 acetylation (H3K9ac) with a modification-specific intracellular antibody (mintbody). The H3K9ac-specific mintbody fused to the enhanced green fluorescent protein (H3K9ac-mintbody-GFP) was introduced into tobacco BY-2 cells. We successfully demonstrated that H3K9ac-mintbody-GFP interacted with H3K9ac in vivo. The ratio of nuclear/cytoplasmic H3K9ac-mintbody-GFP detected in quantitative analysis reflected the endogenous H3K9ac levels. Under chemically induced hyperacetylation conditions with histone deacetylase inhibitors including trichostatin A, Ky-2 and Ky-14, significant enhancement of H3K9ac was detected by H3K9ac-mintbody-GFP dependent on the strength of inhibitors. Conversely, treatment with a histone acetyltransferase inhibitor, C646 caused a reduction in the nuclear to cytoplasmic ratio of H3K9ac-mintbody-GFP. Using this system, we assessed the environmental responses of H3K9ac and found that cold and salt stresses enhanced H3K9ac in tobacco BY-2 cells. In addition, a combination of H3K9ac-mintbody-GFP with 5-ethynyl-2′-deoxyuridine labelling confirmed that H3K9ac level is constant during interphase. PMID:28418019

  20. Preliminary study on the evaluation of Langerhans cell histiocytosis using F-18-fluoro-deoxy-glucose PET/CT

    Institute of Scientific and Technical Information of China (English)

    Zhou Wenlan; Wu Hubing; Han Yanjiang; Wang Shaobo; Dong Ye; Wang Quanshi

    2014-01-01

    Background Limited number of studies have been reported regarding the utilization of F-18-fluoro-deoxy-glucose (F-18-FDG) positron emission tomography/computed tomography (F-18-FDG PET/CT) in Langerhans cell histiocytosis (LCH).The aim of this study was to assess the role of F-18-FDG PET/CT in the diagnosis and treatment of LCH.Methods Eight newly diagnosed and seven recurrent patients with LCH received F-18-FDG PET/CT scans.The diagnosis of LCH was established by pathology,multi-modality imaging,and clinical follow-up.Results F-18-FDG PET/CT was positive in 14 patients with 13 true positives and one false positive.All 45 LCH lesions were F-18-FDG avid including six small bone lesions <1.0 cm in diameter.The mean maximal standardized uptake value (SUVmax) was 7.13±4.91.F-18-FDG uptake showed no significant difference between newly diagnosed lesions vs recurrent lesions (SUVmax:6.50±2.97 vs.7.93±6.60,t=-0.901,P=0.376).Among 45 LCH lesions,68.9% (31/45) were found in bones and 31.1% (14/45) in soft tissue.The most commonly involved bones were the pelvis and vertebrae.There was no significant difference in F-18-FDG uptake between bone lesions vs.non-bone lesions (SUVmax:6.30±2.87 vs.8.97±7.58,t=1.277,P=0.221).In two patients,changes in F-18-FDG uptake on serial PET/CT scans reflected response of lesions to treatment.Conclusions The present study suggests that F-18-FDG PET/CT may be useful for diagnosis and assessing the treatment response of LCH.Because of the small sample size,further research is warranted to confirm our findings.

  1. High CD49f expression is associated with osteosarcoma tumor progression: a study using patient-derived primary cell cultures.

    Science.gov (United States)

    Penfornis, Patrice; Cai, David Z; Harris, Michael R; Walker, Ryan; Licini, David; Fernandes, Joseph D A; Orr, Griffin; Koganti, Tejaswi; Hicks, Chindo; Induru, Spandana; Meyer, Mark S; Khokha, Rama; Barr, Jennifer; Pochampally, Radhika R

    2014-08-01

    Overall prognosis for osteosarcoma (OS) is poor despite aggressive treatment options. Limited access to primary tumors, technical challenges in processing OS tissues, and the lack of well-characterized primary cell cultures has hindered our ability to fully understand the properties of OS tumor initiation and progression. In this study, we have isolated and characterized cell cultures derived from four central high-grade human OS samples. Furthermore, we used the cell cultures to study the role of CD49f in OS progression. Recent studies have implicated CD49f in stemness and multipotency of both cancer stem cells and mesenchymal stem cells. Therefore, we investigated the role of CD49f in osteosarcomagenesis. First, single cell suspensions of tumor biopsies were subcultured and characterized for cell surface marker expression. Next, we characterized the growth and differentiation properties, sensitivity to chemotherapy drugs, and anchorage-independent growth. Xenograft assays showed that cell populations expressing CD49f(hi) /CD90(lo) cell phenotype produced an aggressive tumor. Multiple lines of evidence demonstrated that inhibiting CD49f decreased the tumor-forming ability. Furthermore, the CD49f(hi) /CD90(lo) cell population is generating more aggressive OS tumor growth and indicating this cell surface marker could be a potential candidate for the isolation of an aggressive cell type in OSs. © 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  2. Chronic lymphocytic leukemia cells acquire regulatory B-cell properties in response to TLR9 and CD40 activation.

    Science.gov (United States)

    Ringelstein-Harlev, Shimrit; Avivi, Irit; Fanadka, Mona; Horowitz, Netanel A; Katz, Tami

    2018-02-15

    Circulating chronic lymphocytic leukemia (CLL) cells share phenotypic features with certain subsets of regulatory B-cells (Bregs). The latter cells have been reported to negatively regulate immune cell responses, mostly by provision of IL-10. The purpose of the current study was to identify and delineate Breg properties of CLL cells. B-cells and T-cells were obtained from the peripheral blood of untreated CLL patients diagnosed according to the 2008 Guidelines of the International Workshop on Chronic Lymphocytic Leukemia. Co-culture assays were used to examine the ability of CLL cells to suppress autologous T-cell immune responses. IL-10 potency of CLL cells was assessed following stimulation with activators of the toll-like receptor 9 (TLR9) or CD40 and was correlated with the inhibitory activity of the cells. TLR9-activated CLL cells were found to increase the frequency of CD4 + CD25 hi FOXp3 + regulatory T-cells (Tregs) and to inhibit autologous CD4 + T-cell proliferation. This signaling cascade proved to control IL-10 generation in CLL cells, which in turn promoted the inhibition of T-cell proliferation by CLL cells. However, CD40 activation of CLL cells, while exhibiting a similar ability to augment Treg frequency, did not either affect IL-10 generation or T-cell proliferation. In conclusion, CLL cells demonstrate a unique clonal quality of adopting Breg properties which promote modulation of T-cell characteristics. TLR9 appears to be a potent activator of regulatory abilities in CLL cells, possibly contributing to preferential immune escape of TLR9-responsive cells.

  3. Treatment response evaluation with {sup 18}F-FDG PET/CT and {sup 18}F-NaF PET/CT in multiple myeloma patients undergoing high-dose chemotherapy and autologous stem cell transplantation

    Energy Technology Data Exchange (ETDEWEB)

    Sachpekidis, Christos [German Cancer Research Center (DKFZ), Medical PET Group-Biological Imaging, Clinical Cooperation Unit Nuclear Medicine, Heidelberg (Germany); University of Bern, Department of Nuclear Medicine, Inselspital, Bern University Hospital, Bern (Switzerland); Hillengass, J.; Wagner, B. [University Hospital Heidelberg, Department of Internal Medicine V, Heidelberg (Germany); Goldschmidt, H. [University Hospital Heidelberg, Department of Internal Medicine V, Heidelberg (Germany); National Center for Tumor Diseases (NCT) Heidelberg, Heidelberg (Germany); Haberkorn, U. [German Cancer Research Center (DKFZ), Medical PET Group-Biological Imaging, Clinical Cooperation Unit Nuclear Medicine, Heidelberg (Germany); University of Heidelberg, Department of Nuclear Medicine, Heidelberg (Germany); Kopka, K. [German Cancer Research Center (DKFZ), Department of Radiopharmaceutical Chemistry, Heidelberg (Germany); Dimitrakopoulou-Strauss, A. [German Cancer Research Center (DKFZ), Medical PET Group-Biological Imaging, Clinical Cooperation Unit Nuclear Medicine, Heidelberg (Germany)

    2017-01-15

    The aim of this study was to assess the combined use of the radiotracers {sup 18}F-FDG and {sup 18}F-NaF in treatment response evaluation of a group of multiple myeloma (MM) patients undergoing high-dose chemotherapy (HDT) followed by autologous stem cell transplantation (ASCT) by means of static (whole-body) and dynamic PET/CT (dPET/CT). Thirty-four patients with primary, previously untreated MM scheduled for treatment with HDT followed by ASCT were enrolled in the study. All patients underwent PET/CT scanning with {sup 18}F-FDG and {sup 18}F-NaF before and after therapy. Treatment response by means of PET/CT was assessed according to the European Organization for Research and Treatment of Cancer (EORTC) 1999 criteria. The evaluation of dPET/CT studies was based on qualitative evaluation, semi-quantitative (SUV) calculation, and quantitative analysis based on two-tissue compartment modelling and a non-compartmental approach leading to the extraction of fractal dimension (FD). An analysis was possible in 29 patients: three with clinical complete response (CR) and 26 with non-CR (13 patients near complete response-nCR, four patients very good partial response-VGPR, nine patients partial response-PR). After treatment, {sup 18}F-FDG PET/CT was negative in 14/29 patients and positive in 15/29 patients, showing a sensitivity of 57.5 % and a specificity of 100 %. According to the EORTC 1999 criteria, {sup 18}F-FDG PET/CT-based treatment response revealed CR in 14 patients ({sup 18}F-FDG PET/CT CR), PR in 11 patients ({sup 18}F-FDG PET/CT PR) and progressive disease in four patients ({sup 18}F-FDG PET/CT PD). In terms of {sup 18}F-NaF PET/CT, 4/29 patients (13.8 %) had a negative baseline scan, thus failed to depict MM. Regarding the patients for which a direct lesion-to-lesion comparison was feasible, {sup 18}F-NaF PET/CT depicted 56 of the 129 {sup 18}F-FDG positive lesions (43 %). Follow-up {sup 18}F-NaF PET/CT showed persistence of 81.5 % of the baseline {sup 18}F-NaF

  4. LncRNA Taurine-Upregulated Gene 1 Promotes Cell Proliferation by Inhibiting MicroRNA-9 in MCF-7 Cells.

    Science.gov (United States)

    Zhao, Xiao-Bo; Ren, Guo-Sheng

    2016-12-01

    This study was designed to investigate the role of taurine-upregulated gene 1 ( TUG1 ) in MCF-7 breast cancer cells and the molecular mechanism involved in the regulation of microRNA-9 (miR-9). The expression of TUG1 in breast cancer tissues and cells was evaluated using quantitative reverse transcription polymerase chain reaction. Cell viability was examined using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay; cell cycle progression and apoptosis were analyzed using flow cytometry. A dual luciferase reporter assay was used to detect the relationship between TUG1 and miR-9. The expression of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) was measured by western blot. Higher expression of TUG1 was observed in breast cancer tissues and cell lines than in the corresponding controls. TUG1 knockdown reduced proliferation, suppressed cell cycle progression, and promoted apoptosis of MCF-7 cells. The dual luciferase reporter assay showed that TUG1 could negatively regulate the expression of miR-9. MiR-9 inhibition abrogated the effect of TUG1 knockdown on the proliferation, cell cycle progression, and apoptosis of MCF-7 cells. TUG1 positively regulated the expression of MTHFD2 in breast cancer cells. TUG1 knockdown was significantly associated with decreased cell proliferation and it promoted apoptosis of breast cancer cells through the regulation of miR-9.

  5. Application of 18F-FDG PET/CT combined with carbohydrate antigen 19-9 for differentiating pancreatic carcinoma from chronic mass-forming pancreatitis in Chinese elderly

    Directory of Open Access Journals (Sweden)

    Gu X

    2016-09-01

    Full Text Available Xinjin Gu,1 Rong Liu2 1Department of Hepatobiliary Surgery, Chinese People’s Liberation Army General Hospital and Hainan Branch, Sanya, 2Department of Hepatobiliary and Pancreatic Surgical Oncology, Chinese People’s Liberation Army General Hospital, Beijing, People’s Republic of China Objective: The current study was designed to analyze the value of 18F-FDG positron emission tomography/computed tomography (PET/CT combined with carbohydrate antigen 19-9 (CA19-9 in differentiating pancreatic carcinoma (PC from chronic mass-forming pancreatitis (CMFP in Chinese elderly. Methods: As it is impossible to differentially diagnose PC from CMFP, 60 participants older than 65 years with focal pancreatic lesions were scanned by 18F-FDG PET/CT and their CA19-9 levels were tested. Diagnoses of all participants were confirmed by comprehensive methods including aspiration biopsy, surgical pathology, and clinical follow-up of 12 months. Twenty participants with CMFP were included in CMFP group and 40 participants with PC in PC group.Results: In CMFP and PC groups, 46 participants showed increased 18F-FDG uptake, 43 had elevated CA19-9 levels, and 38 participants had both increased 18F-FDG uptake and elevated CA19-9 levels. Standardized uptake value maximum of PC group (5.98±2.27 was significantly different from CMFP group (2.58±1.81, P<0.05. Sensitivity, specificity, and accuracy of 18F-FDG PET/CT in differentiating PC from CMFP were 95%, 60%, and 83.3%, respectively. CA19-9 levels of PC group (917.44±1,088.24 were significantly different from CMFP group (19.09±19.54, P<0.05. Sensitivity, specificity, and accuracy of CA19-9 levels in differentiating PC from CMFP were 87.5%, 60%, and 78.3%, respectively. Sensitivity, specificity, and accuracy of 18F-FDG PET/CT combined with CA19-9 levels in differentiating PC from CMFP were 90%, 90%, and 90%, respectively.Conclusion: 18F-FDG PET/CT had reliable sensitivity, specificity, and accuracy in differentiating

  6. DPL-1 DP, LIN-35 Rb and EFL-1 E2F act with the MCD-1 zinc-finger protein to promote programmed cell death in Caenorhabditis elegans.

    Science.gov (United States)

    Reddien, Peter W; Andersen, Erik C; Huang, Michael C; Horvitz, H Robert

    2007-04-01

    The genes egl-1, ced-9, ced-4, and ced-3 play major roles in programmed cell death in Caenorhabditis elegans. To identify genes that have more subtle activities, we sought mutations that confer strong cell-death defects in a genetically sensitized mutant background. Specifically, we screened for mutations that enhance the cell-death defects caused by a partial loss-of-function allele of the ced-3 caspase gene. We identified mutations in two genes not previously known to affect cell death, dpl-1 and mcd-1 (modifier of cell death). dpl-1 encodes the C. elegans homolog of DP, the human E2F-heterodimerization partner. By testing genes known to interact with dpl-1, we identified roles in cell death for four additional genes: efl-1 E2F, lin-35 Rb, lin-37 Mip40, and lin-52 dLin52. mcd-1 encodes a novel protein that contains one zinc finger and that is synthetically required with lin-35 Rb for animal viability. dpl-1 and mcd-1 act with efl-1 E2F and lin-35 Rb to promote programmed cell death and do so by regulating the killing process rather than by affecting the decision between survival and death. We propose that the DPL-1 DP, MCD-1 zinc finger, EFL-1 E2F, LIN-35 Rb, LIN-37 Mip40, and LIN-52 dLin52 proteins act together in transcriptional regulation to promote programmed cell death.

  7. Antimelanogenic, Antioxidant and Antiproliferative Effects of Antrodia camphorata Fruiting Bodies on B16-F0 Melanoma Cells.

    Directory of Open Access Journals (Sweden)

    Jyh-Jye Wang

    Full Text Available Antrodia camphorata is a fungus that is endemic to Taiwan, and its fruiting body has been used as a folk medicine for the prevention or treatment of diverse diseases. The present study is aimed at investigating the antimelanogenesis and antioxidation effect of the ethanolic extract of Antrodia camphorata fruiting body (EE-AC, as well as its antiproliferation effects in B16-F0 melanoma cells. Regarding antimelanogenic effects, EE-AC had effective cupric ions reducing capacity and expressed more potent inhibitory effect than kojic acid on mushroom tyrosinase activity. Moreover, EE-AC significantly inhibited cellular tyrosinase activity and the melanin content in B16-F0 cells at 12.5 μg/mL concentration without cell toxicities. Regarding antioxidant effects, EE-AC exhibited potent DPPH radical- and SOD-like-scavenging activities. Regarding antiproliferative effects, EE-AC exhibited a selective cytotoxic effect and markedly inhibited the migration ability of B16-F0 cells. EE-AC increased the population of B16-F0 cells at sub-G1 phase of the cell cycle. EE-AC also caused the increase of early apoptotic cells and chromatin condensation, which indicated the apoptotic effects in B16-F0 cells. We demonstrated that EE-AC possessed antimelanogenic, antioxidant and anti-skin cancer actions. The results would contribute to the development and application of cosmetics, healthy food and pharmaceuticals.

  8. In-Vivo Detection and Tracking of T Cells in Various Organs in a Melanoma Tumor Model by 19F-Fluorine MRS/MRI.

    Directory of Open Access Journals (Sweden)

    Christine Gonzales

    Full Text Available 19F-MRI and 19F-MRS can identify specific cell types after in-vitro or in-vivo 19F-labeling. Knowledge on the potential to track in-vitro 19F-labeled immune cells in tumor models by 19F-MRI/MRS is scarce.To study 19F-based MR techniques for in-vivo tracking of adoptively transferred immune cells after in-vitro 19F-labeling, i.e. to detect and monitor their migration non-invasively in melanoma-bearing mice.Splenocytes (SP were labeled in-vitro with a perfluorocarbon (PFC and IV-injected into non-tumor bearing mice. In-vitro PFC-labeled ovalbumin (OVA-specific T cells from the T cell receptor-transgenic line OT-1, activated with anti-CD3 and anti-CD28 antibodies (Tact or OVA-peptide pulsed antigen presenting cells (TOVA-act, were injected into B16 OVA melanoma-bearing mice. The distribution of the 19F-labelled donor cells was determined in-vivo by 19F-MRI/MRS. In-vivo 19F-MRI/MRS results were confirmed by ex-vivo 19F-NMR and flow cytometry.SP, Tact, and TOVA-act were successfully PFC-labeled in-vitro yielding 3x1011-1.4x1012 19F-atoms/cell in the 3 groups. Adoptively transferred 19F-labeled SP, TOVA-act, and Tact were detected by coil-localized 19F-MRS in the chest, abdomen, and left flank in most animals (corresponding to lungs, livers, and spleens, respectively, with highest signal-to-noise for SP vs TOVA-act and Tact, p<0.009 for both. SP and Tact were successfully imaged by 19F-MRI (n = 3; liver. These in-vivo data were confirmed by ex-vivo high-resolution 19F-NMR-spectroscopy. By flow cytometric analysis, however, TOVA-act tended to be more abundant versus SP and Tact (liver: p = 0.1313; lungs: p = 0.1073; spleen: p = 0.109. Unlike 19F-MRI/MRS, flow cytometry also identified transferred immune cells (SP, Tact, and TOVA-act in the tumors.SP, Tact, and TOVA-act were successfully PFC-labeled in-vitro and detected in-vivo by non-invasive 19F-MRS/MRI in liver, lung, and spleen. The portion of 19F-labeled T cells in the adoptively transferred cell

  9. E2F8 is essential for polyploidization in mammalian cells.

    Science.gov (United States)

    Pandit, Shusil K; Westendorp, Bart; Nantasanti, Sathidpak; van Liere, Elsbeth; Tooten, Peter C J; Cornelissen, Peter W A; Toussaint, Mathilda J M; Lamers, Wouter H; de Bruin, Alain

    2012-11-01

    Polyploidization is observed in all mammalian species and is a characteristic feature of hepatocytes, but its molecular mechanism and biological significance are unknown. Hepatocyte polyploidization in rodents occurs through incomplete cytokinesis, starts after weaning and increases with age. Here, we show in mice that atypical E2F8 is induced after weaning and required for hepatocyte binucleation and polyploidization. A deficiency in E2f8 led to an increase in the expression level of E2F target genes promoting cytokinesis and thereby preventing polyploidization. In contrast, loss of E2f1 enhanced polyploidization and suppressed the polyploidization defect of hepatocytes deficient for atypical E2Fs. In addition, E2F8 and E2F1 were found on the same subset of target promoters. Contrary to the long-standing hypothesis that polyploidization indicates terminal differentiation and senescence, we show that prevention of polyploidization through inactivation of atypical E2Fs has, surprisingly, no impact on liver differentiation, zonation, metabolism and regeneration. Together, these results identify E2F8 as a repressor and E2F1 as an activator of a transcriptional network controlling polyploidization in mammalian cells.

  10. Regulation of Trib2 by an E2F1-C/EBPα feedback loop in AML cell proliferation.

    LENUS (Irish Health Repository)

    Rishi, Loveena

    2014-04-10

    The loss of regulation of cell proliferation is a key event in leukemic transformation, and the oncogene tribbles (Trib)2 is emerging as a pivotal target of transcription factors in acute leukemias. Deregulation of the transcription factor E2F1, normally repressed by CCAAT enhancer-binding protein α (C\\/EBPα)-p42, occurs in acute myeloid leukemia (AML), resulting in the perturbation of cell cycle and apoptosis, emphasizing its importance in the molecular pathogenesis of AML. Here we show that E2F family members directly regulate Trib2 in leukemic cells and identify a feedback regulatory loop for E2F1, C\\/EBPα, and Trib2 in AML cell proliferation and survival. Further analyses revealed that E2F1-mediated Trib2 expression was repressed by C\\/EBPα-p42, and in normal granulocyte\\/macrophage progenitor cells, we detect C\\/EBPα bound to the Trib2 promoter. Pharmacological inhibition of the cell cycle or Trib2 knockdown resulted in a block in AML cell proliferation. Our work proposes a novel paradigm whereby E2F1 plays a key role in the regulation of Trib2 expression important for AML cell proliferation control. Importantly, we identify the contribution of dysregulated C\\/EBPα and E2F1 to elevated Trib2 expression and leukemic cell survival, which likely contributes to the initiation and maintenance of AML and may have significant implications for normal and malignant hematopoiesis.

  11. p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells.

    Science.gov (United States)

    Ihry, Robert J; Worringer, Kathleen A; Salick, Max R; Frias, Elizabeth; Ho, Daniel; Theriault, Kraig; Kommineni, Sravya; Chen, Julie; Sondey, Marie; Ye, Chaoyang; Randhawa, Ranjit; Kulkarni, Tripti; Yang, Zinger; McAllister, Gregory; Russ, Carsten; Reece-Hoyes, John; Forrester, William; Hoffman, Gregory R; Dolmetsch, Ricardo; Kaykas, Ajamete

    2018-06-11

    CRISPR/Cas9 has revolutionized our ability to engineer genomes and conduct genome-wide screens in human cells 1-3 . Whereas some cell types are amenable to genome engineering, genomes of human pluripotent stem cells (hPSCs) have been difficult to engineer, with reduced efficiencies relative to tumour cell lines or mouse embryonic stem cells 3-13 . Here, using hPSC lines with stable integration of Cas9 or transient delivery of Cas9-ribonucleoproteins (RNPs), we achieved an average insertion or deletion (indel) efficiency greater than 80%. This high efficiency of indel generation revealed that double-strand breaks (DSBs) induced by Cas9 are toxic and kill most hPSCs. In previous studies, the toxicity of Cas9 in hPSCs was less apparent because of low transfection efficiency and subsequently low DSB induction 3 . The toxic response to DSBs was P53/TP53-dependent, such that the efficiency of precise genome engineering in hPSCs with a wild-type P53 gene was severely reduced. Our results indicate that Cas9 toxicity creates an obstacle to the high-throughput use of CRISPR/Cas9 for genome engineering and screening in hPSCs. Moreover, as hPSCs can acquire P53 mutations 14 , cell replacement therapies using CRISPR/Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function.

  12. Effect of selected insecticides on SF9 insect cell line

    International Nuclear Information System (INIS)

    Saleh, M.; Rahmo, A.; Hajjar, J.

    2013-01-01

    The toxic effect of three insecticides: dimethoate (organophosphate insecticide), acetamiprid (neonicotinoid insecticide) and deltamethrin (pyrethroid insecticide) were evaluated in vitro on cultured Sf9 cell line. Cell growth inhibition was measured by the 3- (4,5- dimethylthiazol - 2-yl) - 2,5 - diphenyl tetrazolium bromide (MTT) assay. Regression Analysis was used to estimate the 20% inhibition of cells growth (IC 20). The IC 20 values obtained for deltamethrin, acetamipridand dimethoate were: 46.8, 61.6 and 68.9 μM, respectively. The proportion of phagocytic cells was positively correlated with the applied concentrations of the insecticides. (author)

  13. Clinical values for abnormal {sup 18}F-FDG uptake in the head and neck region of patients with head and neck squamous cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Hwan Seo [Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Kim, Jae Seung [Department of Nuclear Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Roh, Jong-Lyel, E-mail: rohjl@amc.seoul.kr [Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Choi, Seung-Ho; Nam, Soon Yuhl [Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Kim, Sang Yoon [Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Biomedical Research Institute, Korea Institute of Science and Technology, Seoul (Korea, Republic of)

    2014-08-15

    Highlights: • Abnormal {sup 18}F-FDG uptakes in the head and neck (HN) region can be carefully interpreted as being index primary, second primary cancer (SP) or benign. • {sup 18}F-FDG PET/CT identified 91.9% primary HN squamous cell carcinomas (HNSCC). • The specificity and negative predictive value of {sup 18}F-FDG PET/CT for identification of SP were as high as 98.7% and 99.3%, respectively. • Proper detection of primary tumors and SP in the HN region may promote appropriate therapeutic planning of HNSCC patients. - Abstract: Purpose: Fluorine 18-fluorodeoxyglucose ({sup 18}F-FDG) positron emission tomography (PET)/computed tomography (CT) is used to identify index or second primary cancer (SP) of the head and neck (HN) through changes in {sup 18}F-FDG uptake. However, both physiologic and abnormal lesions increase {sup 18}F-FDG uptake. Therefore, we evaluated {sup 18}F-FDG uptake in the HN region to determine clinical values of abnormal tracer uptake. Methods: A prospective study approved by the institutional review board was conducted in 314 patients with newly diagnosed HN squamous cell carcinoma (HNSCC) and informed consent was obtained from all enrolled patients. The patients received initial staging workups including {sup 18}F-FDG PET/CT and biopsies. All lesions with abnormal HN {sup 18}F-FDG uptake were recorded and most of those were confirmed by biopsies. Diagnostic values for abnormal {sup 18}F-FDG uptake were calculated. Results: Abnormal {sup 18}F-FDG uptake was identified in primary tumors from 285 (91.9%) patients. False-negative results were obtained for 22.3% (23/103) T1 tumors and 2.2% (2/93) T2 tumors (P < 0.001). Thirty-eight regions of abnormal {sup 18}F-FDG uptake were identified in 36 (11.5%) patients: the thyroid (n = 13), maxillary sinus (n = 7), palatine tonsil (n = 6), nasopharynx (n = 5), parotid gland (n = 2) and others (n = 5). Synchronous SP of the HN was identified in eight (2.5%) patients: the thyroid (n = 5), palatine

  14. Calbindin-D9k (CaBP9k) localization and levels of expression in trophoblast cells from human term placenta.

    Science.gov (United States)

    Belkacemi, Louiza; Gariépy, Gilles; Mounier, Catherine; Simoneau, Lucie; Lafond, Julie

    2004-01-01

    During pregnancy, the calcium (Ca(2+)) transport machinery of the placenta is solely responsible for the nutrient supply to the developing fetus, where active Ca(2+) transport occurs from the mother to the fetus. As part of a larger study to determine the role of Ca(2+) in placental transport in vivo, we questioned whether calbindin-D9k (CaBP9k), which is mainly expressed in duodenum, uterus, and placenta of several mammals, is present in cytotrophoblast cells and syncytiotrophoblasts of human term placenta. We were interested in this protein because of its potential importance in serving as an indicator of Ca(2+) availability and utilization in the placenta. Here, we demonstrated that CaBP9k transcript is present in both cell types, with a lower expression in cytotrophoblast cells as compared to syncytiotrophoblasts. Moreover, we showed by immunochemistry that CaBP9k protein was present in cytotrophoblast and syncytiotrophoblast placental tissue sections as well as in cultured cells. The occurrence of CaBP9k protein in trophoblast cells was further confirmed by Western blot analysis. Thus, these results indicate for the first time that CaBP9k is unequivocally expressed by trophoblast cells from human term placenta.

  15. Δ9-Tetrahydrocannabinol enhances MCF-7 cell proliferation via cannabinoid receptor-independent signaling

    International Nuclear Information System (INIS)

    Takeda, Shuso; Yamaori, Satoshi; Motoya, Erina; Matsunaga, Tamihide; Kimura, Toshiyuki; Yamamoto, Ikuo; Watanabe, Kazuhito

    2008-01-01

    We recently reported that Δ 9 -tetrahydrocannabinol (Δ 9 -THC) has the ability to stimulate the proliferation of human breast carcinoma MCF-7 cells. However, the mechanism of action remains to be clarified. The present study focused on the relationship between receptor expression and the effects of Δ 9 -THC on cell proliferation. RT-PCR analysis demonstrated that there was no detectable expression of CB receptors in MCF-7 cells. In accordance with this, no effects of cannabinoid 1/2 (CB1/2) receptor antagonists and pertussis toxin on cell proliferation were observed. Although MCF-7 cell proliferation is suggested to be suppressed by Δ 9 -THC in the presence of CB receptors, it was revealed that Δ 9 -THC could exert upregulation of living cells in the absence of the receptors. Interestingly, Δ 9 -THC upregulated human epithelial growth factor receptor type 2 (HER2) expression, which is known to be a predictive factor of human breast cancer and is able to stimulate cancer cells as well as MCF-7 cells. Actinomycin D-treatment interfered with the upregulation of HER2 and cell proliferation by cannabinoid. Taken together, these studies suggest that, in the absence of CB receptors, Δ 9 -THC can stimulate the proliferation of MCF-7 cells by modulating, at least in part, HER2 transcription

  16. Regulated production and anti-HIV type 1 activities of cytidine deaminases APOBEC3B, 3F, and 3G.

    Science.gov (United States)

    Rose, Kristine M; Marin, Mariana; Kozak, Susan L; Kabat, David

    2005-07-01

    APOBEC3G and 3F (A3G and A3F) cytidine deaminases incorporate into retroviral cores where they lethally hypermutate nascent DNA reverse transcripts. As substantiated here, the viral infectivity factor (Vif) encoded by human immunodeficiency virus type-1 (HIV-1) binds A3G and A3F and induces their degradation, thereby precluding their incorporation into viral progeny. Previous evidence suggested that A3G is expressed in H9 and other nonpermissive cells that contain this antiviral defense but not in several permissive cells, and that overexpression of A3G or A3F makes permissive cells nonpermissive. Using a broader panel of cell lines, we confirmed a correlation between A3G and cellular abilities to inactivate HIV-1(Deltavif). However, there was a quantitative discrepancy because several cells with weak antiviral activities had similar amounts of wild-type A3G mRNA and protein compared to H9 cells. Antiviral activity of H9 cells was also attenuated in some conditions. These quantitative discrepancies could not be explained by the presence of A3F or other A3G paralogs in some of the cell lines. Thus, A3A, A3B, and A3C had weak but significant anti-HIV-1 activities and did not dominantly interfere with A3G or A3F antiviral functions. Control of A3G synthesis by the protein kinase C/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway was also similar in permissive and nonpermissive cells. A3G in highly permissive cells is degraded by Vif, suggesting that it is not in a sequestered site, and is specifically incorporated in low amounts into HIV-1(Deltavif). Although A3G and/or A3F inactivate HIV-1(Deltavif) and are neutralized by Vif, the antiviral properties of cell lines are also influenced by other cellular and viral factors.

  17. Synthesis of Multicolor Core/Shell NaLuF4:Yb3+/Ln3+@CaF2 Upconversion Nanocrystals

    Directory of Open Access Journals (Sweden)

    Hui Li

    2017-02-01

    Full Text Available The ability to synthesize high-quality hierarchical core/shell nanocrystals from an efficient host lattice is important to realize efficacious photon upconversion for applications ranging from bioimaging to solar cells. Here, we describe a strategy to fabricate multicolor core @ shell α-NaLuF4:Yb3+/Ln3+@CaF2 (Ln = Er, Ho, Tm upconversion nanocrystals (UCNCs based on the newly established host lattice of sodium lutetium fluoride (NaLuF4. We exploited the liquid-solid-solution method to synthesize the NaLuF4 core of pure cubic phase and the thermal decomposition approach to expitaxially grow the calcium fluoride (CaF2 shell onto the core UCNCs, yielding cubic core/shell nanocrystals with a size of 15.6 ± 1.2 nm (the core ~9 ± 0.9 nm, the shell ~3.3 ± 0.3 nm. We showed that those core/shell UCNCs could emit activator-defined multicolor emissions up to about 772 times more efficient than the core nanocrystals due to effective suppression of surface-related quenching effects. Our results provide a new paradigm on heterogeneous core/shell structure for enhanced multicolor upconversion photoluminescence from colloidal nanocrystals.

  18. The nature of tolerance in adult recipient mice made tolerant of alloantigens with supralethal irradiation followed by syngeneic bone marrow cell transplantation plus injection of F1 spleen cells

    International Nuclear Information System (INIS)

    Tomita, Y.; Himeno, K.; Mayumi, H.; Tokuda, N.; Nomoto, K.

    1989-01-01

    The length of time after syngeneic bone marrow reconstitution when tolerance to alloantigens can be induced in adult mice during T cell differentiation from bone marrow cells was studied by exposing those T cells to (recipient x donor)F1 spleen cells. Supralethally irradiated C3H/He Slc(C3H; H-2k) mice were reconstituted with 1 x 10(7) syngeneic T cell-depleted bone marrow cells and then injected intravenously with 5 x 10(7) (C3H x C57BL/6[B6])F1 (B6C3F1; H-2bxk) or (C3H x AKR/J[AKR])F1 (AKC3F1; H-2kxk) spleen cells at various intervals. In the fully allogeneic combination of B6C3F1----C3H, EL-4 tumor originating from B6 was accepted, and survival of grafted B6 skin was significantly prolonged in the tolerant C3H mice treated with irradiation on day -1 followed by injection of syngeneic bone marrow cells on day 0 plus B6C3F1 spleen cells on days 0, 5, or 10, in a tolerogen-specific manner. In the multiminor histocompatibility antigen-disparate combination of AKC3F1----C3H, AKR skin grafts were permanently accepted in the tolerant C3H mice treated with AKC3F1 spleen cells on days 0, 5, 10, or 15. Immunological parameters, including cytotoxic T lymphocyte activity and delayed foot-pad reaction (DFR), were almost completely suppressed in C3H mice made tolerant of B6 or AKR antigens. A chimeric assay using a direct immunofluorescence method revealed that the tolerant C3H mice given B6C3F1 spleen cells on day 0 were mixed-chimeric for at least 8 weeks after syngeneic bone marrow reconstitution, but not definitely chimeric thereafter. The C3H mice given AKC3F1 spleen cells on day 0 were chimeric even 43 weeks after syngeneic bone marrow reconstitution, but the C3H mice given AKC3F1 spleen cells on day 15 showed temporal chimerism that disappeared within 43 weeks. The untolerant mice were never detectably chimeric

  19. Isolation and structures of sulfonium salts derived from thioethers: [{o-C(6)H(4)(CH(2)SMe)(2)}H][NbF(6)] and [{[9]aneS(3)}H][NbF(6)].

    Science.gov (United States)

    Jura, Marek; Levason, William; Reid, Gillian; Webster, Michael

    2009-10-07

    Two very unusual sulfonium salts, [{o-C(6)H(4)(CH(2)SMe)(2)}H][NbF(6)] and [{[9]aneS(3)}H][NbF(6)], obtained from reaction of the thioethers with NbF(5) in CH(2)Cl(2) solution, are reported and their structures described; the eight-coordinate tetrafluoro Nb(v) cation of the dithioether is obtained from the same reaction.

  20. Selective cell culture on UV transparent polymer by F{sub 2} laser surface modification

    Energy Technology Data Exchange (ETDEWEB)

    Hanada, Yasutaka, E-mail: y-hanada@riken.jp [RIKEN-Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Sugioka, Koji [RIKEN-Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Kawano, Hiroyuki [RIKEN-Brain Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Tsuchimoto, Takayoshi [RIKEN-Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Department of Applied Electronics, Faculty of Industrial Science and Technology Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510 (Japan); Miyamoto, Iwao [Department of Applied Electronics, Faculty of Industrial Science and Technology Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510 (Japan); Miyawaki, Atsushi [RIKEN-Brain Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Midorikawa, Katsumi [RIKEN-Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan)

    2009-09-30

    A microchip made of UV transparent polymer (CYTOP) that can perform selective cell culture has been fabricated by F{sub 2} laser surface modification. The refractive index of CYTOP is almost the same as that of culture medium, which is essential for three-dimensional (3D) observation of cells. The F{sub 2} laser modification of CYTOP achieves hydrophilicity only on the laser irradiated area with little deterioration of the optical properties and surface smoothness. After the laser modification, HeLa cells were successfully cultured and strongly adhered only on the modified area of CYTOP. The cells patterned on CYTOP were applied for clear 3D observation using an optical microscope in phase contrast mode.

  1. Bim and Mcl-1 exert key roles in regulating JAK2V617F cell survival

    International Nuclear Information System (INIS)

    Rubert, Joëlle; Qian, Zhiyan; Andraos, Rita; Guthy, Daniel A; Radimerski, Thomas

    2011-01-01

    The JAK2 V617F mutation plays a major role in the pathogenesis of myeloproliferative neoplasms and is found in the vast majority of patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia or from primary myelofibrosis. The V617F mutation is thought to provide hematopoietic stem cells and myeloid progenitors with a survival and proliferation advantage. It has previously been shown that activated JAK2 promotes cell survival by upregulating the anti-apoptotic STAT5 target gene Bcl-xL. In this study, we have investigated the role of additional apoptotic players, the pro-apoptotic protein Bim as well as the anti-apoptotic protein Mcl-1. Pharmacological inhibition of JAK2/STAT5 signaling in JAK2 V617F mutant SET-2 and MB-02 cells was used to study effects on signaling, cell proliferation and apoptosis by Western blot analysis, WST-1 proliferation assays and flow cytometry. Cells were transfected with siRNA oligos to deplete candidate pro- and anti-apoptotic proteins. Co-immunoprecipitation assays were performed to assess the impact of JAK2 inhibition on complexes of pro- and anti-apoptotic proteins. Treatment of JAK2 V617F mutant cell lines with a JAK2 inhibitor was found to trigger Bim activation. Furthermore, Bim depletion by RNAi suppressed JAK2 inhibitor-induced cell death. Bim activation following JAK2 inhibition led to enhanced sequestration of Mcl-1, besides Bcl-xL. Importantly, Mcl-1 depletion by RNAi was sufficient to compromise JAK2 V617F mutant cell viability and sensitized the cells to JAK2 inhibition. We conclude that Bim and Mcl-1 have key opposing roles in regulating JAK2 V617F cell survival and propose that inactivation of aberrant JAK2 signaling leads to changes in Bim complexes that trigger cell death. Thus, further preclinical evaluation of combinations of JAK2 inhibitors with Bcl-2 family antagonists that also tackle Mcl-1, besides Bcl-xL, is warranted to assess the therapeutic potential

  2. Cell-type-specific genome editing with a microRNA-responsive CRISPR–Cas9 switch

    Science.gov (United States)

    Hirosawa, Moe; Fujita, Yoshihiko; Parr, Callum J. C.; Hayashi, Karin; Kashida, Shunnichi; Hotta, Akitsu; Woltjen, Knut

    2017-01-01

    Abstract The CRISPR–Cas9 system is a powerful genome-editing tool useful in a variety of biotechnology and biomedical applications. Here we developed a synthetic RNA-based, microRNA (miRNA)-responsive CRISPR–Cas9 system (miR-Cas9 switch) in which the genome editing activity of Cas9 can be modulated through endogenous miRNA signatures in mammalian cells. We created miR-Cas9 switches by using a miRNA-complementary sequence in the 5΄-UTR of mRNA encoding Streptococcus pyogenes Cas9. The miR-21-Cas9 or miR-302-Cas9 switches selectively and efficiently responded to miR-21-5p in HeLa cells or miR-302a-5p in human induced pluripotent stem cells, and post-transcriptionally attenuated the Cas9 activity only in the target cells. Moreover, the miR-Cas9 switches could differentially control the genome editing by sensing endogenous miRNA activities within a heterogeneous cell population. Our miR-Cas9 switch system provides a promising framework for cell-type selective genome editing and cell engineering based on intracellular miRNA information. PMID:28525578

  3. Role for chondroitin sulfate glycosaminoglycan in NEDD9-mediated breast cancer cell growth.

    Science.gov (United States)

    Iida, Joji; Dorchak, Jesse; Clancy, Rebecca; Slavik, Juliana; Ellsworth, Rachel; Katagiri, Yasuhiro; Pugacheva, Elena N; van Kuppevelt, Toin H; Mural, Richard J; Cutler, Mary Lou; Shriver, Craig D

    2015-01-15

    There are lines of evidence demonstrating that NEDD9 (Cas-L, HEF-1) plays a key role in the development, progression, and metastasis of breast cancer cells. We previously reported that NEDD9 plays a critical role for promoting migration and growth of MDA-MB-231. In order to further characterize the mechanisms of NEDD9-mediated cancer migration and growth, stable cells overexpressing NEDD9 were generated using HCC38 as a parental cell line which expresses low level of endogenous NEDD9. Microarray studies demonstrated that core proteins of CD44 and Serglycin were markedly upregulated in HCC38(NEDD9) cells compared to HCC38(Vector) cells, while those of Syndecan-1, Syndecan-2, and Versican were downregulated in HCC38(NEDD9). Importantly, enzymes generating chondroitin sulfate glycosaminoglycans (CS) such as CHST11, CHST15, and CSGALNACT1 were upregulated in HCC38(NEDD9) compared to HCC38(Vector). Immunofluorescence studies using specific antibody, GD3G7, confirmed the enhanced expression of CS-E subunit in HCC38(NEDD9). Immunoprecipitation and western blotting analysis demonstrated that CS-E was attached to CD44 core protein. We demonstrated that removing CS by chondroitinase ABC significantly inhibited anchorage-independent colony formation of HCC38(NEDD9) in methylcellulose. Importantly, the fact that GD3G7 significantly inhibited colony formation of HCC38(NEDD9) cells suggests that CS-E subunit plays a key role in this process. Furthermore, treatment of HCC38(NEDD9) cells with chondroitinase ABC or GD3G7 significantly inhibited mammosphere formation. Exogenous addition of CS-E enhanced colony formation and mammosphere formation of HCC38 parental and HCC38(Vector) cells. These results suggest that NEDD9 regulates the synthesis and expression of tumor associated glycocalyx structures including CS-E, which plays a key role in promoting and regulating breast cancer progression and metastasis and possibly stem cell phenotypes. Copyright © 2014 Elsevier Inc. All rights

  4. Structure-activity relationship of 9-methylstreptimidone, a compound that induces apoptosis selectively in adult T-cell leukemia cells.

    Science.gov (United States)

    Takeiri, Masatoshi; Ota, Eisuke; Nishiyama, Shigeru; Kiyota, Hiromasa; Umezawa, Kazuo

    2012-01-01

    We previously reported that 9-methylstreptimidone, a piperidine compound isolated from a culture filtrate of Streptomyces, induces apoptosis selectively in adult T-cell leukemia cells. It was screened for a compound that inhibits LPS-induced NF-kappaB and NO production in mouse macrophages. However, 9-methystreptimidone is poorly obtained from the producing microorganism and difficult to synthesize. Therefore, in the present research, we studied the structure-activity relationship to look for new selective inhibitors. We found that the structure of the unsaturated hydrophobic portion of 9-methylstreptimidone was essential for the inhibition of LPS-induced NO production. Among the 9-methylstreptimidone-related compounds tested, (+/-)-4,alpha-diepi-streptovitacin A inhibited NO production in macrophage-like cells as potently as 9-methylstreptimidone and without cellular toxicity. Moreover, this compound selectively induced apoptosis in adult T-cell leukemia MT-1 cells.

  5. Beneficial Effect of Jojoba Seed Extracts on Hyperglycemia-Induced Oxidative Stress in RINm5f Beta Cells.

    Science.gov (United States)

    Belhadj, Sahla; Hentati, Olfa; Hamdaoui, Ghaith; Fakhreddine, Khaskhoussi; Maillard, Elisa; Dal, Stéphanie; Sigrist, Séverine

    2018-03-20

    Hyperglycemia occurs during diabetes and insulin resistance. It causes oxidative stress by increasing reactive oxygen species (ROS) levels, leading to cellular damage. Polyphenols play a central role in defense against oxidative stress. In our study, we investigated the antioxidant properties of simmondsin, a pure molecule present in jojoba seeds, and of the aqueous extract of jojoba seeds on fructose-induced oxidative stress in RINm5f beta cells. The exposure of RINm5f beta cells to fructose triggered the loss of cell viability (-48%, p jojoba seed extract makes jojoba a powerful agent to prevent the destruction of RINm5f beta cells induced by hyperglycemia.

  6. {sup 18}F-PEG-biotin: Precursor (boroaryl-PEG-biotin) synthesis, {sup 18}F-labelling and an in-vitro assessment of its binding with Neutravidin{sup TM}-trastuzumab pre-treated cells

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Tim A.D., E-mail: t.smith@abdn.ac.uk [Biomedical Physics Building, John Mallard PET Unit, Aberdeen Biomedical Imaging Centre, School of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD (United Kingdom); Simpson, Michael; Cheyne, Richard [Biomedical Physics Building, John Mallard PET Unit, Aberdeen Biomedical Imaging Centre, School of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD (United Kingdom); School of Natural and Computing Sciences, University of Aberdeen, Aberdeen AB24 3UE (United Kingdom); Trembleau, Laurent [School of Natural and Computing Sciences, University of Aberdeen, Aberdeen AB24 3UE (United Kingdom)

    2011-10-15

    In terms of nuclear decay {sup 18}F is the most ideal PET nuclide but its short t{sub 1/2} precludes its use for directly labelling whole antibodies due to their long blood residence times. Pre-targeted imaging using affinity systems such as Neutravidin{sup TM}-biotin facilitates the application of short-lived nuclides by their attachment to biotin for imaging cell surface proteins targeted with Neutravidin{sup TM}-conjugated antibodies. Methods: Boroaryl functionalised biotin was prepared with a PEG linker and radiolabelled by incubation with {sup 18}F in acidified aqueous solution. Cells expressing high (SKBr3), medium (MDA-MB-453) and low (MDA-MB-468) levels of HER-2 were pre-incubated with Neutravidin{sup TM}-conjugated trastuzumab, washed, and then incubated with {sup 18}F-PEG-biotin. Results: The {sup 18}F-fluorination of boroaryl-PEG-biotin was much more efficient than reported for other versions of boroaryl-biotin. The novel {sup 18}F-PEG-biotin was demonstrated to bind to HER-2-expressing cells in-vitro pre-incubated with Neutravidin{sup TM}-conjugated trastuzumab. Conclusion: Biotin can be functionalised with boroaryl and readily {sup 18}F-radiolabelled in aqueous solution and will bind to cells pre-incubated with Neutravidin{sup TM}-antibody conjugates. - Highlights: > Boroaryl-biotin precursor is prepared. > Rapid {sup 18}F-fluorination is demonstrated. > HER-2 expressing breast cancer cells pre-treated with trastuzumab-Neutravidin{sup TM}. > {sup 18}F-PEG-biotin binding to pre-treated cells corresponds with HER-2 expression.

  7. Brachytherapy model with sodium pertechnetate-"9"9"mTc balloon (Na"9"9"mTcO_4"-) for breast cancer: evaluation of dosimetry and cell response

    International Nuclear Information System (INIS)

    Lima, Carla Flavia de

    2016-01-01

    Breast cancer is the most common type of cancer that affects more women worldwide. Among various treatment options, radiotherapy which is often used as a treatment for locoregional recurrences control or to decrease tumor size. In patients with breast cancer at an early stage, a booster dose (boost) in the primary tumor area can be applied after conventional radiation therapy. There are several drawbacks to applying this technique. In this work we aimed to perform a dosimetric analysis in a breast model, where it put a balloon filled with sodium pertechnetate-"9"9"mTc (Na"9"9"mTcO_4"-) which in future could be used in preference to other possible therapies. The methodology involved the development of dosimetry in water based on radiochromic films and in a computational voxel thorax model. Calibration protocol achieved a mathematical relation between absorbed dose versus optical density (OD) measured at a set of radiochromic sample films placed at the surface of the balloon plus 1 cm up to 10 cm far, in which theoretical dose values were provided by MCNP modeling, reproducing the water equivalent physical simulator. A voxel model of a female thorax, developed at the SISCODES/MCNP codes, received a filled balloon inside. Spatial dose distribution was generated, illustrating the dose received in the chest wall, glandular tissue, breast skin and lung. The dosimetric findings contribute to present the Na"9"9"mTcO_4"- balloon modality which provides a suitable spatial dose distribution in the tumor bed preserving adjacent health tissues. We also studied the radiobiological response radio resistant mammary adenocarcinoma cells (MDAMB231) by exposure of these cells to Na"9"9"mTcO_4"- balloon. The findings include the presence of apoptotic cells in the balloon around point out a favorable response. In conclusion, the balloon may represent a viable option in the supplementary therapy of breast cancer in patients who have appropriate indication. Irradiation with Na"9"9"mTcO_4

  8. Inflammatory cytokines and hypoxia contribute to 18F-FDG uptake by cells involved in pannus formation in rheumatoid arthritis.

    Science.gov (United States)

    Matsui, Tamiko; Nakata, Norihito; Nagai, Shigenori; Nakatani, Akira; Takahashi, Miwako; Momose, Toshimitsu; Ohtomo, Kuni; Koyasu, Shigeo

    2009-06-01

    Assessment of the activity of rheumatoid arthritis (RA) is important for the prediction of future articular destruction. (18)F-FDG PET is known to represent the metabolic activity of inflammatory disease, which correlates with the pannus volume measured by MRI or ultrasonography. To evaluate the correlation between (18)F-FDG accumulation and RA pathology, we assessed (18)F-FDG accumulation in vivo using collagen-induced arthritis (CIA) animal models and (3)H-FDG uptake in vitro using various cells involved in arthritis. (18)F-FDG PET images of rats with CIA were acquired on days 10, 14, and 17 after arthritis induction. The specimens were subsequently subjected to macroautoradiography, and the (18)F-FDG accumulation was compared with the histologic findings. (3)H-FDG uptake in vitro in inflammatory cells (neutrophils, macrophages, T cells, and fibroblasts) was measured to evaluate the contributions of these cells to (18)F-FDG accumulation. In addition, the influence on (3)H-FDG uptake of inflammatory factors, such as cytokines (tumor necrosis factor alpha [TNFalpha], interleukin 1 [IL-1], and IL-6), and hypoxia was examined. (18)F-FDG PET depicted swollen joints, and (18)F-FDG accumulation increased with the progression of arthritis. Histologically, a higher level of (18)F-FDG accumulation correlated with the pannus rather than the infiltration of inflammatory cells around the joints. In the in vitro (3)H-FDG uptake assay, fibroblasts showed the highest (3)H-FDG uptake, followed by neutrophils. Although only a small amount of (3)H-FDG was incorporated by resting macrophages, a dramatic increase in (3)H-FDG uptake in both fibroblasts and macrophages was observed when these cells were exposed to inflammatory cytokines, such as TNFalpha and IL-1, and hypoxia. Although neutrophils showed relatively high (3)H-FDG uptake without activation, no increase in (3)H-FDG uptake was observed in response to inflammatory cytokines. (3)H-FDG uptake by T cells was much lower than

  9. Regulation of cancer stem cell properties by CD9 in human B-acute lymphoblastic leukemia

    Energy Technology Data Exchange (ETDEWEB)

    Yamazaki, Hiroto [Division of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Wilson Xu, C. [Drug Development Program, Nevada Cancer Institute, Las Vegas, NV (United States); Naito, Motohiko [Division of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Nishida, Hiroko [Division of Hematology, Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan); Okamoto, Toshihiro; Ghani, Farhana Ishrat; Iwata, Satoshi [Division of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Inukai, Takeshi; Sugita, Kanji [Department of Pediatrics, School of Medicine, University of Yamanashi, Yamanashi (Japan); Morimoto, Chikao, E-mail: morimoto@ims.u-tokyo.ac.jp [Division of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Drug Development Program, Nevada Cancer Institute, Las Vegas, NV (United States)

    2011-05-27

    Highlights: {yields} We performed more detailed analysis of CD9 function for CSC properties in B-ALL. {yields} Leukemogenic fusion/Src family proteins were markedly regulated in the CD9{sup +} cells. {yields} Proliferation of B-ALL cells was inhibited by anti-CD9 monoclonal antibody. {yields} Knockdown of CD9 by RNAi remarkably reduced the leukemogenic potential. {yields} CD9-knockdown affected the expression and phosphorylation of Src family and USP22. -- Abstract: Although the prognosis of acute lymphoblastic leukemia (ALL) has improved considerably in recent years, some of the cases still exhibit therapy-resistant. We have previously reported that CD9 was expressed heterogeneously in B-ALL cell lines and CD9{sup +} cells exhibited an asymmetric cell division with greater tumorigenic potential than CD9{sup -} cells. CD9{sup +} cells were also serially transplantable in immunodeficient mice, indicating that CD9{sup +} cell possess self-renewal capacity. In the current study, we performed more detailed analysis of CD9 function for the cancer stem cell (CSC) properties. In patient sample, CD9 was expressed in the most cases of B-ALL cells with significant correlation of CD34-expression. Gene expression analysis revealed that leukemogenic fusion proteins and Src family proteins were significantly regulated in the CD9{sup +} population. Moreover, CD9{sup +} cells exhibited drug-resistance, but proliferation of bulk cells was inhibited by anti-CD9 monoclonal antibody. Knockdown of CD9 remarkably reduced the leukemogenic potential. Furthermore, gene ablation of CD9 affected the expression and tyrosine-phosphorylation of Src family proteins and reduced the expression of histone-deubiquitinase USP22. Taken together, our results suggest that CD9 links to several signaling pathways and epigenetic modification for regulating the CSC properties of B-ALL.

  10. Evaluation of granulated BGO, GSO:Ce, YAG:Ce, CaF sub 2 :Eu and ZnS:Ag for alpha/beta pulse shape discrimination in a flow-cell radiation detector

    CERN Document Server

    Devol, T A; Fjeld, R A

    1999-01-01

    Granulated BGO, GSO:Ce, YAG:Ce, and CaF sub 2 :Eu; CaF sub 2 :Eu coated with a fluorescent polymer, and combinations of coated and uncoated CaF sub 2 :Eu with ZnS:Ag were evaluated for their ability to discriminate between alpha and beta particles in a flow-cell radiation detector. The evaluations were based on the analysis of pulse shape spectra. Various granulated scintillators were packed into flow cell detectors that were coils of 3.0 mm ODx1.5 mm ID fluorinated ethylene propylene Teflon[reg] tubing positioned between dual photomultiplier tubes for analysis. The best pulse shape discrimination was obtained for a combination of equal masses of uncoated CaF sub 2 :Eu (63-90 mu m) and ZnS:Ag (10 mu m), which had a 9% spillover. Additional research is needed to reduce the spillover.

  11. Vacuole formation in mast cells responding to osmotic stress and to F-actin disassembly

    DEFF Research Database (Denmark)

    Koffer, Anna; Williams, Mark; Johansen, Torben

    2002-01-01

    Fluorescent probes were used to visualize the morphology of membranes and of F-actin in rat peritoneal mast cells, exposed to hyperosmotic medium and consequently reversed to isotonicity. Hypertonicity induced cell shrinkage followed by a regulatory volume increase, and cell alkalinization...

  12. F4/80+ Macrophages Contribute to Clearance of Senescent Cells in the Mouse Postpartum Uterus.

    Science.gov (United States)

    Egashira, Mahiro; Hirota, Yasushi; Shimizu-Hirota, Ryoko; Saito-Fujita, Tomoko; Haraguchi, Hirofumi; Matsumoto, Leona; Matsuo, Mitsunori; Hiraoka, Takehiro; Tanaka, Tomoki; Akaeda, Shun; Takehisa, Chiaki; Saito-Kanatani, Mayuko; Maeda, Kei-Ichiro; Fujii, Tomoyuki; Osuga, Yutaka

    2017-07-01

    Cellular senescence, defined as an irreversible cell cycle arrest, exacerbates the tissue microenvironment. Our previous study demonstrated that mouse uterine senescent cells were physiologically increased according to gestational days and that their abnormal accumulation was linked to the onset of preterm delivery. We hypothesized that there is a mechanism for removal of senescent cells after parturition to maintain uterine function. In the current study, we noted abundant uterine senescent cells and their gradual disappearance in wild-type postpartum mice. F4/80+ macrophages were present specifically around the area rich in senescent cells. Depletion of macrophages in the postpartum mice using anti-F4/80 antibody enlarged the area of senescent cells in the uterus. We also found excessive uterine senescent cells and decreased second pregnancy success rate in a preterm birth model using uterine p53-deleted mice. Furthermore, a decrease in F4/80+ cells and an increase in CD11b+ cells with a senescence-associated inflammatory microenvironment were observed in the p53-deleted uterus, suggesting that uterine p53 deficiency affects distribution of the macrophage subpopulation, interferes with senescence clearance, and promotes senescence-induced inflammation. These findings indicate that the macrophage is a key player in the clearance of uterine senescent cells to maintain postpartum uterine function. Copyright © 2017 Endocrine Society.

  13. Knockdown of HSPA9 induces TP53-dependent apoptosis in human hematopoietic progenitor cells.

    Directory of Open Access Journals (Sweden)

    Tuoen Liu

    Full Text Available Myelodysplastic syndromes (MDS are the most common adult myeloid blood cancers in the US. Patients have increased apoptosis in their bone marrow cells leading to low peripheral blood counts. The full complement of gene mutations that contribute to increased apoptosis in MDS remains unknown. Up to 25% of MDS patients harbor and acquired interstitial deletion on the long arm of chromosome 5 [del(5q], creating haploinsufficiency for a large set of genes including HSPA9. Knockdown of HSPA9 in primary human CD34+ hematopoietic progenitor cells significantly inhibits growth and increases apoptosis. We show here that HSPA9 knockdown is associated with increased TP53 expression and activity, resulting in increased expression of target genes BAX and p21. HSPA9 protein interacts with TP53 in CD34+ cells and knockdown of HSPA9 increases nuclear TP53 levels, providing a possible mechanism for regulation of TP53 by HSPA9 haploinsufficiency in hematopoietic cells. Concurrent knockdown of TP53 and HSPA9 rescued the increased apoptosis observed in CD34+ cells following knockdown of HSPA9. Reduction of HSPA9 below 50% results in severe inhibition of cell growth, suggesting that del(5q cells may be preferentially sensitive to further reductions of HSPA9 below 50%, thus providing a genetic vulnerability to del(5q cells. Treatment of bone marrow cells with MKT-077, an HSPA9 inhibitor, induced apoptosis in a higher percentage of cells from MDS patients with del(5q compared to non-del(5q MDS patients and normal donor cells. Collectively, these findings indicate that reduced levels of HSPA9 may contribute to TP53 activation and increased apoptosis observed in del(5q-associated MDS.

  14. Decreased Siglec-9 Expression on Natural Killer Cell Subset Associated With Persistent HBV Replication

    Directory of Open Access Journals (Sweden)

    Di Zhao

    2018-05-01

    Full Text Available Siglec-9 is an MHC-independent inhibitory receptor selectively expressed on CD56dim NK cells. Its role in infection diseases has not been investigated yet. Here, we studied the potential regulatory roles of NK Siglec-9 in the pathogenesis of chronic hepatitis B (CHB infection. Flow cytometry evaluated the expression of Siglec-9 and other receptors on peripheral NK cells. Immunofluorescence staining was used to detect Siglec-9 ligands on liver biopsy tissues and cultured hepatocyte cell lines. Siglec-9 blocking assay was carried out and cytokine synthesis and CD107a degranulation was detected by flow cytometry. Compared to healthy donors, CHB patients had decreased Siglec-9+ NK cells, which reversely correlated with serum hepatitis B e antigen and HBV DNA titer. Siglec-9 expression on NK cells from patients achieving sustained virological response recovered to the level of normal donors. Neutralization of Siglec-9 restored cytokine synthesis and degranulation of NK cells from CHB patients. Immunofluorescence staining showed increased expression of Siglec-9 ligands in liver biopsy tissues from CHB patients and in hepatocyte cell lines infected with HBV or stimulated with inflammatory cytokines (IL-6 or TGF-β. These findings identify Siglec-9 as a negative regulator for NK cells contributing to HBV persistence and the intervention of Siglec-9 signaling might be of potentially translational significance.

  15. Interaction of Prevotella intermedia strain 17 leucine-rich repeat domain protein AdpF with eukaryotic cells promotes bacterial internalization.

    Science.gov (United States)

    Sengupta, Dipanwita; Kang, Dae-Joong; Anaya-Bergman, Cecilia; Wyant, Tiana; Ghosh, Arnab K; Miyazaki, Hiroshi; Lewis, Janina P

    2014-06-01

    Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells.

  16. In Silico Prediction of T and B Cell Epitopes of Der f 25 in Dermatophagoides farinae

    Directory of Open Access Journals (Sweden)

    Xiaohong Li

    2014-01-01

    Full Text Available The house dust mites are major sources of indoor allergens for humans, which induce asthma, rhinitis, dermatitis, and other allergic diseases. Der f 25 is a triosephosphate isomerase, representing the major allergen identified in Dermatophagoides farinae. The objective of this study was to predict the B and T cell epitopes of Der f 25. In the present study, we analyzed the physiochemical properties, function motifs and domains, and structural-based detailed features of Der f 25 and predicted the B cell linear epitopes of Der f 25 by DNAStar protean system, BPAP, and BepiPred 1.0 server and the T cell epitopes by NetMHCIIpan-3.0 and NetMHCII-2.2. As a result, the sequence and structure analysis identified that Der f 25 belongs to the triosephosphate isomerase family and exhibited a triosephosphate isomerase pattern (PS001371. Eight B cell epitopes (11–18, 30–35, 71–77, 99–107, 132–138, 173–187, 193–197, and 211–224 and five T cell epitopes including 26–34, 38–54, 66–74, 142–151, and 239–247 were predicted in this study. These results can be used to benefit allergen immunotherapies and reduce the frequency of mite allergic reactions.

  17. Toxicology and carcinogenesis studies of nitrofurantoin (CAS No. 67-20-9) in F344/n rats and B6C3F1 mice (feed studies). Technical report

    Energy Technology Data Exchange (ETDEWEB)

    French, J.E.

    1989-09-01

    Two-year toxicology and carcinogenesis studies were conducted by administering diets containing 0, 600, or 1,300 ppm nitrofurantoin to groups of 50 female rats for 103 weeks. Groups of 50 male rats and 50 mice of each sex were fed diets containing 0, 1,300 or 2,500 ppm for 103 weeks. Under the conditions of these 2-year feed studies, there was some evidence of carcinogenic activity of nitrofurantoin for male F344/N rats as shown by increased incidences of uncommon kidney tubular cell neoplasms. Uncommon osteosarcomas of the bone and neoplasms of the subcutaneous tissue were observed in dosed male rats. Incidences of interstitial cell adenomas of the testis and neoplasms of the preputial gland were decreased in the 2,500-ppm group of male rats. There was no evidence of carcinogenic activity of nitrofurantoin for female F344/N rats fed diets containing 600 ppm or 1,300 ppm for 2 years. Female rats may have been able to tolerate higher doses. There was no evidence of carcinogenic activity of nitrofurantoin for male B6C3F(1) mice fed diets containing 1,300 ppm or 2,500 ppm for 2 years. There was clear evidence of carcinogenic activity of nitrofurantoin for female B6C3F(1) mice as shown by increased incidences of tubular adenomas, benign mixed tumors, and granulosa cell tumors of the ovary.

  18. Overexpression of SAMD9 suppresses tumorigenesis and progression during non small cell lung cancer

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Qing; Yu, Tao; Ren, Yao-Yao; Gong, Ting; Zhong, Dian-Sheng, E-mail: zhongdsyx@126.com

    2014-11-07

    Highlights: • SAMD9 is down-regulated in human non-small cell lung cancer (NSCLC). • Knockdown of SAMD9 expression is increased the invasion, migration and proliferation in H1299 cells in vitro. • Overexpression of SAMD9 suppressed proliferation and invasion in A549 cells in vitro. • Depletion of SAMD9 increases tumor formation in vivo. - Abstract: The Sterile Alpha Motif Domain-containing 9 (SAMD9) gene has been recently emphasized during the discovery that it is expressed at a lower level in aggressive fibromatosis and some cases of breast and colon cancer, however, the underlying mechanisms are poorly understood. Here, we found that SAMD9 is down-regulated in human non-small cell lung cancer (NSCLC). Furthermore, knockdown of SAMD9 expression is increased the invasion, migration and proliferation in H1299 cells in vitro and overexpression of SAMD9 suppressed proliferation and invasion in A549 cells. Finally, depletion of SAMD9 increases tumor formation in vivo. Our results may provide a strategy for blocking NSCLC tumorigenesis and progression.

  19. Cell wall metabolism and hexose allocation contribute to biomass accumulation in high yielding extreme segregants of a Saccharum interspecific F2 population.

    Science.gov (United States)

    Wai, Ching Man; Zhang, Jisen; Jones, Tyler C; Nagai, Chifumi; Ming, Ray

    2017-10-11

    Sugarcane is an emerging dual-purpose biofuel crop for energy and sugar production, owing to its rapid growth rate, high sucrose storage in the stems, and high lignocellulosic yield. It has the highest biomass production reaching 1.9 billion tonnes in 2014 worldwide. To improve sugarcane biomass accumulation, we developed an interspecific cross between Saccharum officinarum 'LA Purple' and Saccharum robustum 'MOL5829'. Selected F1 individuals were self-pollinated to generate a transgressive F2 population with a wide range of biomass yield. Leaf and stem internodes of fourteen high biomass and eight low biomass F2 extreme segregants were used for RNA-seq to decipher the molecular mechanism of rapid plant growth and dry weight accumulation. Gene Ontology terms involved in cell wall metabolism and carbohydrate catabolism were enriched among 3274 differentially expressed genes between high and low biomass groups. Up-regulation of cellulose metabolism, pectin degradation and lignin biosynthesis genes were observed in the high biomass group, in conjunction with higher transcript levels of callose metabolic genes and the cell wall loosening enzyme expansin. Furthermore, UDP-glucose biosynthesis and sucrose conversion genes were differentially expressed between the two groups. A positive correlation between stem glucose, but not sucrose, levels and dry weight was detected. We thus postulated that the high biomass sugarcane plants rapidly convert sucrose to UDP-glucose, which is the building block of cell wall polymers and callose, in order to maintain the rapid plant growth. The gene interaction of cell wall metabolism, hexose allocation and cell division contributes to biomass yield.

  20. Cell-type-specific genome editing with a microRNA-responsive CRISPR-Cas9 switch.

    Science.gov (United States)

    Hirosawa, Moe; Fujita, Yoshihiko; Parr, Callum J C; Hayashi, Karin; Kashida, Shunnichi; Hotta, Akitsu; Woltjen, Knut; Saito, Hirohide

    2017-07-27

    The CRISPR-Cas9 system is a powerful genome-editing tool useful in a variety of biotechnology and biomedical applications. Here we developed a synthetic RNA-based, microRNA (miRNA)-responsive CRISPR-Cas9 system (miR-Cas9 switch) in which the genome editing activity of Cas9 can be modulated through endogenous miRNA signatures in mammalian cells. We created miR-Cas9 switches by using a miRNA-complementary sequence in the 5΄-UTR of mRNA encoding Streptococcus pyogenes Cas9. The miR-21-Cas9 or miR-302-Cas9 switches selectively and efficiently responded to miR-21-5p in HeLa cells or miR-302a-5p in human induced pluripotent stem cells, and post-transcriptionally attenuated the Cas9 activity only in the target cells. Moreover, the miR-Cas9 switches could differentially control the genome editing by sensing endogenous miRNA activities within a heterogeneous cell population. Our miR-Cas9 switch system provides a promising framework for cell-type selective genome editing and cell engineering based on intracellular miRNA information. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Inhibition of ERK1/2 or AKT Activity Equally Enhances Radiation Sensitization in B16F10 Cells

    Science.gov (United States)

    Kalal, Bhuvanesh Sukhlal; Fathima, Faraz; Pai, Vinitha Ramanath; Sanjeev, Ganesh; Krishna, Chilakapati Murali; Upadhya, Dinesh

    2018-01-01

    Background The aim of the study was to evaluate the radiation sensitizing ability of ERK1/2, PI3K-AKT and JNK inhibitors in highly radiation resistant and metastatic B16F10 cells which carry wild-type Ras and Braf. Methods Mouse melanoma cell line B16F10 was exposed to 1.0, 2.0 and 3.0 Gy of electron beam radiation. Phosphorylated ERK1/2, AKT and JNK levels were estimated by ELISA. Cells were exposed to 2.0 and 3.0 Gy of radiation with or without prior pharmacological inhibition of ERK1/2, AKT as well as JNK pathways. Cell death induced by radiation as well as upon inhibition of these pathways was measured by TUNEL assay using flow cytometry. Results Exposure of B16F10 cells to 1.0, 2.0 and 3.0 Gy of electron beam irradiation triggered an increase in all the three phosphorylated proteins compared to sham-treated and control groups. B16F10 cells pre-treated with either ERK1/2 or AKT inhibitors equally enhanced radiation-induced cell death at 2.0 as well as 3.0 Gy (P < 0.001), while inhibition of JNK pathway increased radiation-induced cell death to a lesser extent. Interestingly combined inhibition of ERK1/2 or AKT pathways did not show additional cell death compared to individual ERK1/2 or AKT inhibition. This indicates that ERK1/2 or AKT mediates radiation resistance through common downstream molecules in B16F10 cells. Conclusions Even without activating mutations in Ras or Braf genes, ERK1/2 and AKT play a critical role in B16F10 cell survival upon radiation exposure and possibly act through common downstream effector/s. PMID:29581812

  2. Gene complementation. Neither Ir-GLphi gene need be present in the proliferative I cell to generate an immune response to Poly(Glu55Lys36Phe9)n

    International Nuclear Information System (INIS)

    Longo, D.L.; Schwartz, R.H.

    1980-01-01

    The cellular requirements for immune response (Ir) gene expression in a T cell proliferative response under dual Ir gene control were examined with radiation-induced bone marrow chimeras. The response to poly(Glu55Lys36Phe9)n (GLphi) requires two responder alleles that in the [B10.A x B10.A(18R)]F1 map in I-Ab and I-Ek/Cd. Chimeras in which a mixture of the nonresponder B10.A parental cells and the nonresponder B10.A(18R) parental cells were allowed to mature in a responder F1 environment did not respond to GLphi. When T cells from such A + 18R leads to F1 chimeras were primed in the presence of responder antigen-presenting cells (APC), the chimeric T cells responded to GLphi. When bone marrow cells from (B10.A X B10)F1 responder animals were allowed to mature in a low-responder B10 of B10.A parental environment, neither chimera could respond to GLphi. This demonstrated that the presence of high-responder APC, which derive from the donar bone marrow, was not sufficient to generate a GLphi response. Finally, B10.A(4R) T cells, which possess neither Ir-GLphi responder allele, could be educated to mount a GLphi-proliferative response provided that they matured in a responder environment and were primed with APC expressing both responder alleles. Therefore, the gene products of the complementing Ir-GLphi responder alleles appear to function as a single restriction element at the level of the APC

  3. Regulation of Trib2 by an E2F1-C/EBPα feedback loop in AML cell proliferation

    DEFF Research Database (Denmark)

    Rishi, Loveena; Hannon, Maura; Salomè, Mara

    2014-01-01

    α (C/EBPα)-p42, occurs in acute myeloid leukemia (AML), resulting in the perturbation of cell cycle and apoptosis, emphasizing its importance in the molecular pathogenesis of AML. Here we show that E2F family members directly regulate Trib2 in leukemic cells and identify a feedback regulatory loop......The loss of regulation of cell proliferation is a key event in leukemic transformation, and the oncogene tribbles (Trib)2 is emerging as a pivotal target of transcription factors in acute leukemias. Deregulation of the transcription factor E2F1, normally repressed by CCAAT enhancer-binding protein...... for E2F1, C/EBPα, and Trib2 in AML cell proliferation and survival. Further analyses revealed that E2F1-mediated Trib2 expression was repressed by C/EBPα-p42, and in normal granulocyte/macrophage progenitor cells, we detect C/EBPα bound to the Trib2 promoter. Pharmacological inhibition of the cell cycle...

  4. Involvement of F-Actin in Chaperonin-Containing t-Complex 1 Beta Regulating Mouse Mesangial Cell Functions in a Glucose-Induction Cell Model

    Directory of Open Access Journals (Sweden)

    Jin-Shuen Chen

    2011-01-01

    Full Text Available The aim of this study is to investigate the role of chaperonin-containing t-complex polypeptide 1 beta (CCT2 in the regulation of mouse mesangial cell (mMC contraction, proliferation, and migration with filamentous/globular-(F/G- actin ratio under high glucose induction. A low CCT2 mMC model induced by treatment of small interference RNA was established. Groups with and without low CCT2 induction examined in normal and high (H glucose conditions revealed the following major results: (1 low CCT2 or H glucose showed the ability to attenuate F/G-actin ratio; (2 groups with low F/G-actin ratio all showed less cell contraction; (3 suppression of CCT2 may reduce the proliferation and migration which were originally induced by H glucose. In conclusion, CCT2 can be used as a specific regulator for mMC contraction, proliferation, and migration affected by glucose, which mechanism may involve the alteration of F-actin, particularly for cell contraction.

  5. Lanthanide-activated Na5Gd9F32 nanocrystals precipitated from a borosilicate glass: Phase-separation-controlled crystallization and optical property

    International Nuclear Information System (INIS)

    Chen, Daqin; Wan, Zhongyi; Zhou, Yang; Chen, Yan; Yu, Hua; Lu, Hongwei; Ji, Zhenguo; Huang, Ping

    2015-01-01

    Highlights: • Na 5 Gd 9 F 32 nanocrystals embedded glass ceramics were fabricated for the first time. • Such glass ceramics were achieved by phase-separation-controlled crystallization. • Elemental mapping evidenced the segregation of activators into the Na 5 Gd 9 F 32 lattice. • Luminescent color could be tuned by controlling glass crystallization temperature. - Abstract: Lanthanide-activated cubic Na 5 Gd 9 F 32 nanocrystals were precipitated from a borosilicate glass with a specifically designed composition. The precursor glass is already phase-separated after melt-quenching, which is beneficial to the realization of the controllable glass crystallization for affording desirable size, morphology and activator partition. Elemental mapping in the scanning transmission electron microscopy evidenced that the segregation of lanthanide ions into the Na 5 Gd 9 F 32 lattice was in situ formed without the requirement of long-range ionic diffusion. Impressively, such fabricated glass ceramic co-doped with Yb 3+ /Er 3+ ions exhibited intense upconversion luminescence, which was about 500 times higher than that of the precursor glass, and its luminescent color could be easily tuned from red to green by controlling glass crystallization temperature. It is anticipated that such phase-separation synthesis strategy with precise control over nanostructure of glass ceramics offer a great opportunity to design other highly transparent nanocomposites with a wide range of tunable optical properties

  6. Evaluation of the antitumor effects of c-Myc-Max heterodimerization inhibitor 100258-F4 in ovarian cancer cells.

    Science.gov (United States)

    Wang, Jiandong; Ma, Xiaoli; Jones, Hannah M; Chan, Leo Li-Ying; Song, Fang; Zhang, Weiyuan; Bae-Jump, Victoria L; Zhou, Chunxiao

    2014-08-21

    Epithelial ovarian carcinoma is the most lethal gynecological cancer due to its silent onset and recurrence with resistance to chemotherapy. Overexpression of oncogene c-Myc is one of the most frequently encountered events present in ovarian carcinoma. Disrupting the function of c-Myc and its downstream target genes is a promising strategy for cancer therapy. Our objective was to evaluate the potential effects of small-molecule c-Myc inhibitor, 10058-F4, on ovarian carcinoma cells and the underlying mechanisms by which 10058-F4 exerts its actions. Using MTT assay, colony formation, flow cytometry and Annexin V FITC assays, we found that 10058-F4 significantly inhibited cell proliferation of both SKOV3 and Hey ovarian cancer cells in a dose dependent manner through induction of apoptosis and cell cycle G1 arrest. Treatment with 10058-F4 reduced cellular ATP production and ROS levels in SKOV3 and Hey cells. Consistently, primary cultures of ovarian cancer treated with 10058-F4 showed induction of caspase-3 activity and inhibition of cell proliferation in 15 of 18 cases. The response to 10058-F4 was independent the level of c-Myc protein over-expression in primary cultures of ovarian carcinoma. These novel findings suggest that the growth of ovarian cancer cells is dependent upon c-MYC activity and that targeting c-Myc-Max heterodimerization could be a potential therapeutic strategy for ovarian cancer.

  7. SVCT2 vitamin C transporter expression in progenitor cells of the postnatal neurogenic niche

    Science.gov (United States)

    Pastor, Patricia; Cisternas, Pedro; Salazar, Katterine; Silva-Alvarez, Carmen; Oyarce, Karina; Jara, Nery; Espinoza, Francisca; Martínez, Agustín D.; Nualart, Francisco

    2013-01-01

    Known as a critical antioxidant, recent studies suggest that vitamin C plays an important role in stem cell generation, proliferation and differentiation. Vitamin C also enhances neural differentiation during cerebral development, a function that has not been studied in brain precursor cells. We observed that the rat neurogenic niche is structurally organized at day 15 of postnatal development, and proliferation and neural differentiation increase at day 21. In the human brain, a similar subventricular niche was observed at 1-month of postnatal development. Using immunohistochemistry, sodium-vitamin C cotransporter 2 (SVCT2) expression was detected in the subventricular zone (SVZ) and rostral migratory stream (RMS). Low co-distribution of SVCT2 and βIII-tubulin in neuroblasts or type-A cells was detected, and minimal co-localization of SVCT2 and GFAP in type-B or precursor cells was observed. Similar results were obtained in the human neurogenic niche. However, BrdU-positive cells also expressed SVCT2, suggesting a role of vitamin C in neural progenitor proliferation. Primary neurospheres prepared from rat brain and the P19 teratocarcinoma cell line, which forms neurospheres in vitro, were used to analyze the effect of vitamin C in neural stem cells. Both cell types expressed functional SVCT2 in vitro, and ascorbic acid (AA) induced their neural differentiation, increased βIII-tubulin and SVCT2 expression, and amplified vitamin C uptake. PMID:23964197

  8. Supplementary Investigation in the Free-Spinning Tunnel of a 1/24-Scale Model of the Grumman F9F-6 Airplane Incorporating only Flaperons for Lateral Control, TED No. NACA DE 364

    Science.gov (United States)

    Klinar, Walter J.; Lee, Henry A.

    1954-01-01

    A supplementary investigation was conducted in the Langley 20-foot free-spinning tunnel on a 1/24-scale model of the Grumman F9F-6 airplane. The primary purpose of the investigation was to reevaluate the spin-recovery characteristics of the airplane in view of the fact that the ailerons had been eliminated from the flaperon-aileron lateral control system of the airplane. A spin-tunnel investigation on a model of the earlier version of the F9F-6 airplane had indicated that use of ailerons with the spin (stick right in a right spin) was essential to insure recovery. The results indicate that with.ailerons eliminated, it may be difficult to obtain an erect developed spin but if a fully developed spin is obtained on the airplane, recovery therefrom may be difficult or impossible. Flaperon deflection should have little effect on spins or recoveries.

  9. Characterization and expression of the human T cell receptor-T3 complex by monoclonal antibody F101.01

    DEFF Research Database (Denmark)

    Geisler, C; Plesner, T; Pallesen, G

    1988-01-01

    demonstrated co-expression of the antigen defined by F101.01 and the pan-T cell antigens defined by CD2, CD3, CD5, and CD7 antibodies. Cells stained with CD4 and CD8 antibodies were both included in the F101.01-positive population, whereas CD16-positive natural killer cells (NK), B cells (CD19 and CD20...

  10. Crystal structure of difluorochloronium hexafluoroniobate and hexafluorotantalate, ClF2NbF6 and ClF2TaF6

    International Nuclear Information System (INIS)

    Ehllern, A.M.; Antipin, M.Yu.; Sharabarin, A.V.; Struchkov, Yu.T.

    1991-01-01

    Crystal structure of ClF 2 NbF 6 (1) and ClF 2 TaF 6 (2) were investigated by the method of X-ray diffraction analysis. Salts 1 and 2 are isostructural, crystals are rhombic: a = 9.981(2) and 10.049(2), b = 5.781(1) and 5.775(1), c = 10.552(2) and 10.670(2) A, V = 608.9(3) and 619.2(3) A 3 , Z = 4, d calcd 3.058 and 3.952 g/cm 3 , sp. gr. Pcca. Both salts are characterized by ionic structure. Bond lengths and valent angles, general view of 1 crystal structure are presented

  11. Galectin-9 enhances cytokine secretion, but suppresses survival and degranulation, in human mast cell line.

    Directory of Open Access Journals (Sweden)

    Reiji Kojima

    Full Text Available Galectin-9 (Gal-9, a lectin having a β-galactoside-binding domain, can induce apoptosis of Th1 cells by binding to TIM-3. In addition, Gal-9 inhibits IgE/Ag-mediated degranulation of mast cell/basophilic cell lines by binding to IgE, thus blocking IgE/Ag complex formation. However, the role of Gal-9 in mast cell function in the absence of IgE is not fully understood. Here, we found that recombinant Gal-9 directly induced phosphorylation of Erk1/2 but not p38 MAPK in a human mast cell line, HMC-1, which does not express FcεRI. Gal-9 induced apoptosis and inhibited PMA/ionomycin-mediated degranulation of HMC-1 cells. On the other hand, Gal-9 induced cytokine and/or chemokine production by HMC-1 cells, dependent on activation of ERK1/2 but not p38 MAPK. In addition, the lectin activity of Gal-9 was required for Gal-9-mediated cytokine secretion by HMC-1 cells. These observations suggest that Gal-9 has dual properties as both a regulator and an activator of mast cells.

  12. Autophagy plays an important role in Sunitinib-mediated cell death in H9c2 cardiac muscle cells

    International Nuclear Information System (INIS)

    Zhao Yuqin; Xue Tao; Yang Xiaochun; Zhu Hong; Ding Xiaofei; Lou Liming; Lu Wei; Yang Bo; He Qiaojun

    2010-01-01

    Sunitinib, which is a multitargeted tyrosine-kinase inhibitor, exhibits antiangiogenic and antitumor activity, and extends survival of patients with metastatic renal-cell carcinoma (mRCC) and gastrointestinal stromal tumors (GIST). This molecule has also been reported to be associated with cardiotoxicity at a high frequency, but the mechanism is still unknown. In the present study, we observed that Sunitinib showed high anti-proliferative effect on H9c2 cardiac muscle cells measured by PI staining and the MTT assay. But apoptotic markers (PARP cleavage, caspase 3 cleavage and chromatin condensation) were uniformly negative in H9c2 cells after Sunitinib treatment for 48 h, indicating that another cell death pathway may be involved in Sunitinib-induced cardiotoxicity. Here we found Sunitinib dramatically increased autophagic flux in H9c2 cells. Acidic vesicle fluorescence and high expression of LC3-II in H9c2 cells identified autophagy as a Sunitinib-induced process that might be associated with cytotoxicity. Furthermore, knocking down Beclin 1 by RNA-interference to block autophagy in H9c2 cells revealed that the death rate was decreased when treated with Sunitinib in comparison to control cells. These results confirmed that autophagy plays an important role in Sunitinib-mediated H9c2 cells cytotoxicity. Taken together, the data presented here strongly suggest that autophagy is associated with Sunitinib-induced cardiotoxicity, and that inhibition of autophagy constitutes a viable strategy for reducing Sunitinib-induced cardiomyocyte death thereby alleviating Sunitinib cardiotoxicity.

  13. F4/80 inhibits osteoclast differentiation via downregulation of nuclear factor of activated T cells, cytoplasmic 1.

    Science.gov (United States)

    Kang, Ju-Hee; Sim, Jung-Sun; Zheng, Ting; Yim, Mijung

    2017-04-01

    Osteoclastogenesis is an essential process in bone metabolism, which can be induced by RANKL stimulation. The F4/80 glycoprotein is a member of the EGF-transmembrane 7 (TM7) family and has been established as a specific cell-surface marker for murine macrophages. This study aimed to identify the role of F4/80 in osteoclastogenesis. Using mouse bone marrow-derived macrophages (BMMs), we observed that the mRNA level of F4/80 was dramatically reduced as these cells differentiated into osteoclasts. Furthermore, osteoclastogenesis was decreased in F4/80 high BMMs compared to F4/80 -/low BMMs. The inhibitory effect of F4/80 was associated with decreased expression of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1). Ectopic overexpression of a constitutively active form of NFATc1 rescued the anti-osteoclastogenic effect of F4/80 completely, suggesting that the anti-osteoclastogenic effect of F4/80 was mainly due to reduction in NFATc1 expression. As an underlying mechanism, we demonstrated that the presence of F4/80 abrogated the effect of RANKL on the phosphorylation of CREB and activated the expression of IFN-β, which are restored by cyclic AMP. Collectively, our results demonstrate that the presence of F4/80 suppresses RANKL-induced osteoclastogenesis by impairing the expression of NFATc1 via CREB and IFN-β. Therefore, F4/80 may hold therapeutic potential for bone destructive diseases.

  14. Downregulation of NEDD9 by apigenin suppresses migration, invasion, and metastasis of colorectal cancer cells

    International Nuclear Information System (INIS)

    Dai, Jin; Van Wie, Peter G.; Fai, Leonard Yenwong; Kim, Donghern; Wang, Lei; Poyil, Pratheeshkumar; Luo, Jia; Zhang, Zhuo

    2016-01-01

    Apigenin is a natural flavonoid which possesses multiple anti-cancer properties such as anti-proliferation, anti-inflammation, and anti-metastasis in many types of cancers including colorectal cancer. Neural precursor cell expressed developmentally downregulated 9 (NEDD9) is a multi-domain scaffolding protein of the Cas family which has been shown to correlate with cancer metastasis and progression. The present study investigates the role of NEDD9 in apigenin-inhibited cell migration, invasion, and metastasis of colorectal adenocarcinoma DLD1 and SW480 cells. The results show that knockdown of NEDD9 inhibited cell migration, invasion, and metastasis and that overexpression of NEDD9 promoted cell migration and invasion of DLD1 cells and SW4890 cells. Apigenin treatment attenuated NEDD9 expression at protein level, resulting in reduced phosphorylations of FAK, Src, and Akt, leading to inhibition on cell migration, invasion, and metastasis of both DLD1 and SW480 cells. The present study has demonstrated that apigenin inhibits cell migration, invasion, and metastasis through NEDD9/Src/Akt cascade in colorectal cancer cells. NEDD9 may function as a biomarker for evaluation of cancer aggressiveness and for selection of therapeutic drugs against cancer progression. - Highlights: • Apigenin inhibits migration, invasion, and metastasis of colorectal cancer cells. • Apigenin downregulates NEDD9. • Apigenin decreases phosphorylations of FAK, Src, and Akt. • Apigenin inhibits cell migration, invasion, and metastasis through NEDD9/Src/Akt.

  15. Downregulation of NEDD9 by apigenin suppresses migration, invasion, and metastasis of colorectal cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Dai, Jin; Van Wie, Peter G.; Fai, Leonard Yenwong; Kim, Donghern [Department of Toxicology and Cancer Biology, University of Kentucky, Lexington, KY 40536 (United States); Wang, Lei; Poyil, Pratheeshkumar [Center for Research on Environmental Disease, University of Kentucky, Lexington, KY 40536 (United States); Luo, Jia [Department of Pharmacology and Nutritional Sciences, University of Kentucky, Lexington, KY 40536 (United States); Zhang, Zhuo, E-mail: Zhuo.Zhang@uky.edu [Department of Toxicology and Cancer Biology, University of Kentucky, Lexington, KY 40536 (United States)

    2016-11-15

    Apigenin is a natural flavonoid which possesses multiple anti-cancer properties such as anti-proliferation, anti-inflammation, and anti-metastasis in many types of cancers including colorectal cancer. Neural precursor cell expressed developmentally downregulated 9 (NEDD9) is a multi-domain scaffolding protein of the Cas family which has been shown to correlate with cancer metastasis and progression. The present study investigates the role of NEDD9 in apigenin-inhibited cell migration, invasion, and metastasis of colorectal adenocarcinoma DLD1 and SW480 cells. The results show that knockdown of NEDD9 inhibited cell migration, invasion, and metastasis and that overexpression of NEDD9 promoted cell migration and invasion of DLD1 cells and SW4890 cells. Apigenin treatment attenuated NEDD9 expression at protein level, resulting in reduced phosphorylations of FAK, Src, and Akt, leading to inhibition on cell migration, invasion, and metastasis of both DLD1 and SW480 cells. The present study has demonstrated that apigenin inhibits cell migration, invasion, and metastasis through NEDD9/Src/Akt cascade in colorectal cancer cells. NEDD9 may function as a biomarker for evaluation of cancer aggressiveness and for selection of therapeutic drugs against cancer progression. - Highlights: • Apigenin inhibits migration, invasion, and metastasis of colorectal cancer cells. • Apigenin downregulates NEDD9. • Apigenin decreases phosphorylations of FAK, Src, and Akt. • Apigenin inhibits cell migration, invasion, and metastasis through NEDD9/Src/Akt.

  16. Intracellular Drug Uptake-A Comparison of Single Cell Measurements Using ToF-SIMS Imaging and Quantification from Cell Populations with LC/MS/MS.

    Science.gov (United States)

    Newman, Carla F; Havelund, Rasmus; Passarelli, Melissa K; Marshall, Peter S; Francis, Ian; West, Andy; Alexander, Morgan R; Gilmore, Ian S; Dollery, Colin T

    2017-11-21

    ToF-SIMS is a label-free imaging method that has been shown to enable imaging of amiodarone in single rat macrophage (NR8383) cells. In this study, we show that the method extends to three other cell lines relevant to drug discovery: human embryonic kidney (HEK293), cervical cancer (HeLa), and liver cancer (HepG2). There is significant interest in the variation of drug uptake at the single cell level, and we use ToF-SIMS to show that there is great diversity between individual cells and when comparing each of the cell types. These single cell measurements are compared to quantitative measurements of cell-associated amiodarone for the population using LC/MS/MS and cell counting with flow cytometry. NR8383 and HepG2 cells uptake the greatest amount of amiodarone with an average of 2.38 and 2.60 pg per cell, respectively, and HeLa and Hek 293 have a significantly lower amount of amiodarone at 0.43 and 0.36 pg per cell, respectively. The amount of cell-associated drug for the ensemble population measurement (LC/MS/MS) is compared with the ToF-SIMS single cell data: a similar amount of drug was detected per cell for the NR8383, and HepG2 cells at a greater level than that for the HEK293 cells. However, the two techniques did not agree for the HeLa cells, and we postulate potential reasons for this.

  17. Clathrin-mediated endocytosis and transcytosis of enterotoxigenic Escherichia coli F4 fimbriae in porcine intestinal epithelial cells.

    Science.gov (United States)

    Rasschaert, Kristien; Devriendt, Bert; Favoreel, Herman; Goddeeris, Bruno M; Cox, Eric

    2010-10-15

    Enterotoxigenic Escherichia coli (ETEC) cause severe diarrhea in neonatal and recently weaned piglets. Previously, we demonstrated that oral immunization of F4 receptor positive piglets with purified F4 fimbriae induces a protective F4-specific intestinal immune response. However, in F4 receptor negative animals no F4-specific immune response can be elicited, indicating that the induction of an F4-specific mucosal immune response upon oral immunisation is receptor-dependent. Although F4 fimbriae undergo transcytosis across the intestinal epithelium in vivo, the endocytosis pathways used remain unknown. In the present study, we characterized the internalization of F4 fimbriae in the porcine intestinal epithelial cell line IPEC-J2. The results in the present study demonstrate that F4 fimbriae are internalized through a clathrin-dependent pathway. Furthermore, our results suggest that F4 fimbriae are transcytosed across differentiated IPEC-J2 cells. This receptor-dependent transcytosis of F4 fimbriae may explain the immunogenicity of these fimbriae upon oral administration in vivo. (c) 2010 Elsevier B.V. All rights reserved.

  18. CRISPR/Cas9-induced transgene insertion and telomere-associated truncation of a single human chromosome for chromosome engineering in CHO and A9 cells.

    Science.gov (United States)

    Uno, Narumi; Hiramatsu, Kei; Uno, Katsuhiro; Komoto, Shinya; Kazuki, Yasuhiro; Oshimura, Mitsuo

    2017-10-06

    Chromosome engineering techniques including gene insertion, telomere-associated truncation and microcell-mediated chromosome transfer (MMCT) are powerful tools for generation of humanised model animal, containing megabase-sized genomic fragments. However, these techniques require two cell lines: homologous recombination (HR)-proficient DT40 cells for chromosome modification, and CHO cells for transfer to recipient cells. Here we show an improved technique using a combination of CRISPR/Cas9-induced HR in CHO and mouse A9 cells without DT40 cells following MMCT to recipient cells. Transgene insertion was performed in CHO cells with the insertion of enhanced green fluorescence protein (EGFP) using CRISPR/Cas9 and a circular targeting vector containing two 3 kb HR arms. Telomere-associated truncation was performed in CHO cells using CRISPR/Cas9 and a linearised truncation vector containing a single 7 kb HR arm at the 5' end, a 1 kb artificial telomere at the 3' end. At least 11% and 6% of the targeting efficiency were achieved for transgene insertion and telomere-associated truncation, respectively. The transgene insertion was also confirmed in A9 cells (29%). The modified chromosomes were transferrable to other cells. Thus, this CHO and A9 cell-mediated chromosome engineering using the CRISPR/Cas9 for direct transfer of the modified chromosome is a rapid technique that will facilitate chromosome manipulation.

  19. Characterization of oligosaccharide structures on a chimeric respiratory syncytial virus protein expressed in insect cell line Sf9

    International Nuclear Information System (INIS)

    Wathen, M.W.; Aeed, P.A.; Elhammer, A.P.

    1991-01-01

    The oligosaccharide structures added to a chimeric protein (FG) composed of the extracellular domains of respiratory syncytial virus F and G proteins, expressed in the insect cell line Sf9, were investigated. Cells were labeled in vivo with [ 3 H]glucosamine and infected wit a recombinant baculovirus containing the FG gene. The secreted chimeric protein was isolated by immunoprecipitation and subjected to oligosaccharide analysis. The FG protein contains two types of O-linked oligosaccharides: GalNAc and Galβ1-3GalNAc constituting 17 and 66% of the total number of structures respectively. Only one type of N-linked oligosaccharide, constituting the remaining 17% of the structures on FG, was detected: a trimannosyl core structure with a fucose residue linked α1-6 to the asparagine-linked N-acetylglucosamine

  20. The F-actin modifier villin regulates insulin granule dynamics and exocytosis downstream of islet cell autoantigen 512

    Directory of Open Access Journals (Sweden)

    Hassan Mziaut

    2016-08-01

    Full Text Available Objective: Insulin release from pancreatic islet β cells should be tightly controlled to avoid hypoglycemia and insulin resistance. The cortical actin cytoskeleton is a gate for regulated exocytosis of insulin secretory granules (SGs by restricting their mobility and access to the plasma membrane. Prior studies suggest that SGs interact with F-actin through their transmembrane cargo islet cell autoantigen 512 (Ica512 (also known as islet antigen 2/Ptprn. Here we investigated how Ica512 modulates SG trafficking and exocytosis. Methods: Transcriptomic changes in Ica512−/− mouse islets were analyzed. Imaging as well as biophysical and biochemical methods were used to validate if and how the Ica512-regulated gene villin modulates insulin secretion in mouse islets and insulinoma cells. Results: The F-actin modifier villin was consistently downregulated in Ica512−/− mouse islets and in Ica512-depleted insulinoma cells. Villin was enriched at the cell cortex of β cells and dispersed villin−/− islet cells were less round and less deformable. Basal mobility of SGs in villin-depleted cells was enhanced. Moreover, in cells depleted either of villin or Ica512 F-actin cages restraining cortical SGs were enlarged, basal secretion was increased while glucose-stimulated insulin release was blunted. The latter changes were reverted by overexpressing villin in Ica512-depleted cells, but not vice versa. Conclusion: Our findings show that villin controls the size of the F-actin cages restricting SGs and, thus, regulates their dynamics and availability for exocytosis. Evidence that villin acts downstream of Ica512 also indicates that SGs directly influence the remodeling properties of the cortical actin cytoskeleton for tight control of insulin secretion. Keywords: F-actin, Granules, Ica512, Insulin, Secretion, Villin

  1. TLR4 induces CREB-mediated IL-6 production via upregulation of F-spondin to promote vascular smooth muscle cell migration

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Guan-Lin [Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan (China); Graduate Institutes of Life Sciences, National Defense Medical Center, Taipei, Taiwan (China); Wu, Jing-Yiing [Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan (China); Yeh, Chang-Ching [Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan (China); Graduate Institutes of Life Sciences, National Defense Medical Center, Taipei, Taiwan (China); Kuo, Cheng-Chin, E-mail: kuocc@nhri.org.tw [Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan (China); Graduate Institutes of Life Sciences, National Defense Medical Center, Taipei, Taiwan (China); Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China)

    2016-05-13

    Toll-like receptor 4 (TLR4) is important in promoting inflammation and vascular smooth muscle cell (VSMC) migration, both of which contribute to atherosclerosis development and progression. But the mechanism underlying the regulation of TLR4 in VSMC migration remains unclear. Stimulation of VSMCs with LPS increased the cellular level of F-spondin which is associated with the regulation of proinflammatory cytokine production. The LPS-induced F-spondin expression depended on TLR4-mediated PI3K/Akt pathway. Suppression of F-spondin level by siRNA inhibited not only F-spondin expression but also LPS-induced phosphorylation of cAMP response element binding protein (CREB) and IL-6 expression, VSMC migration and proliferation as well as MMP9 expression. Moreover, suppression of CREB level by siRNA inhibited TLR4-induced IL-6 production and VSMC migration. Inhibition of F-spondin siRNA on LPS-induced migration was restored by addition of exogenous recombinant mouse IL-6. We conclude that upon ligand binding, TLR4 activates PI3K/Akt signaling to induce F-spondin expression, subsequently control CREB-mediated IL-6 production to promote VSMC migration. These findings provide vital insights into the essential role of F-spondin in VSMC function and will be valuable for developing new therapeutic strategies against atherosclerosis. -- Highlights: •LPS-induced F-spondin expression of VSMCs is via a TLR4/PI3K/Akt signaling. •F-spondin is pivotal for LPS-induced CREB-mediated IL-6 production. •F-spondin is required for LPS-induced VSMC migration and proliferation.

  2. TLR4 induces CREB-mediated IL-6 production via upregulation of F-spondin to promote vascular smooth muscle cell migration

    International Nuclear Information System (INIS)

    Lee, Guan-Lin; Wu, Jing-Yiing; Yeh, Chang-Ching; Kuo, Cheng-Chin

    2016-01-01

    Toll-like receptor 4 (TLR4) is important in promoting inflammation and vascular smooth muscle cell (VSMC) migration, both of which contribute to atherosclerosis development and progression. But the mechanism underlying the regulation of TLR4 in VSMC migration remains unclear. Stimulation of VSMCs with LPS increased the cellular level of F-spondin which is associated with the regulation of proinflammatory cytokine production. The LPS-induced F-spondin expression depended on TLR4-mediated PI3K/Akt pathway. Suppression of F-spondin level by siRNA inhibited not only F-spondin expression but also LPS-induced phosphorylation of cAMP response element binding protein (CREB) and IL-6 expression, VSMC migration and proliferation as well as MMP9 expression. Moreover, suppression of CREB level by siRNA inhibited TLR4-induced IL-6 production and VSMC migration. Inhibition of F-spondin siRNA on LPS-induced migration was restored by addition of exogenous recombinant mouse IL-6. We conclude that upon ligand binding, TLR4 activates PI3K/Akt signaling to induce F-spondin expression, subsequently control CREB-mediated IL-6 production to promote VSMC migration. These findings provide vital insights into the essential role of F-spondin in VSMC function and will be valuable for developing new therapeutic strategies against atherosclerosis. -- Highlights: •LPS-induced F-spondin expression of VSMCs is via a TLR4/PI3K/Akt signaling. •F-spondin is pivotal for LPS-induced CREB-mediated IL-6 production. •F-spondin is required for LPS-induced VSMC migration and proliferation.

  3. Structural, spectroscopic and cytotoxicity studies of TbF3@CeF3 and TbF3@CeF3@SiO2 nanocrystals

    International Nuclear Information System (INIS)

    Grzyb, Tomasz; Runowski, Marcin; Dąbrowska, Krystyna; Giersig, Michael; Lis, Stefan

    2013-01-01

    Terbium fluoride nanocrystals, covered by a shell, composed of cerium fluoride were synthesized by a co-precipitation method. Their complex structure was formed spontaneously during the synthesis. The surface of these core/shell nanocrystals was additionally modified by silica. The properties of TbF 3 @CeF 3 and TbF 3 @CeF 3 @SiO 2 nanocrystals, formed in this way, were investigated. Spectroscopic studies showed that the differences between these two groups of products resulted from the presence of the SiO 2 shell. X-ray diffraction patterns confirmed the trigonal crystal structure of TbF 3 @CeF 3 nanocrystals. High resolution transmission electron microscopy in connection with energy-dispersive X-ray spectroscopy showed a complex structure of the formed nanocrystals. Crystallized as small discs, ‘the products’, with an average diameter around 10 nm, showed an increase in the concentration of Tb 3+ ions from surface to the core of nanocrystals. In addition to photo-physical analyses, cytotoxicity studies were performed on HSkMEC (Human Skin Microvascular Endothelial Cells) and B16F0 mouse melanoma cancer cells. The cytotoxicity of the nanomaterials was neutral for the investigated cells with no toxic or antiproliferative effect in the cell cultures, either for normal or for cancer cells. This fact makes the obtained nanocrystals good candidates for biological applications and further modifications of the SiO 2 shell.Graphical Abstract

  4. Growth factors and medium hyperglycemia induce Sox9+ ductal cell differentiation into β cells in mice with reversal of diabetes

    Science.gov (United States)

    Zhang, Mingfeng; Lin, Qing; Qi, Tong; Wang, Tiankun; Chen, Ching-Cheng; Riggs, Arthur D.; Zeng, Defu

    2016-01-01

    We previously reported that long-term administration of a low dose of gastrin and epidermal growth factor (GE) augments β-cell neogenesis in late-stage diabetic autoimmune mice after eliminating insulitis by induction of mixed chimerism. However, the source of β-cell neogenesis is still unknown. SRY (sex-determining region Y)-box 9+ (Sox9+) ductal cells in the adult pancreas are clonogenic and can give rise to insulin-producing β cells in an in vitro culture. Whether Sox9+ ductal cells in the adult pancreas can give rise to β cells in vivo remains controversial. Here, using lineage-tracing with genetic labeling of Insulin- or Sox9-expressing cells, we show that hyperglycemia (>300 mg/dL) is required for inducing Sox9+ ductal cell differentiation into insulin-producing β cells, and medium hyperglycemia (300–450 mg/dL) in combination with long-term administration of low-dose GE synergistically augments differentiation and is associated with normalization of blood glucose in nonautoimmune diabetic C57BL/6 mice. Short-term administration of high-dose GE cannot augment differentiation, although it can augment preexisting β-cell replication. These results indicate that medium hyperglycemia combined with long-term administration of low-dose GE represents one way to induce Sox9+ ductal cell differentiation into β cells in adult mice. PMID:26733677

  5. Diagnostic usefulness of 18F-FAMT PET and L-type amino acid transporter 1 (LAT1) expression in oral squamous cell carcinoma

    International Nuclear Information System (INIS)

    Nobusawa, Aiko; Kim, Mai; Kaira, Kyoichi; Miyashita, Go; Negishi, Akihide; Yokoo, Satoshi; Oriuchi, Noboru; Higuchi, Tetsuya; Tsushima, Yoshito; Kanai, Yoshikatsu; Oyama, Tetsunari

    2013-01-01

    l-[3- 18 F]-α-Methyltyrosine ( 18 F-FAMT) was developed as an amino acid tracer for PET imaging to provide better specificity than 2-[ 18 F]fluoro-2-deoxy-d-glucose ( 18 F-FDG) PET for cancer diagnosis. We investigated the diagnostic usefulness of 18 F-FAMT in oral squamous cell carcinoma (OSCC). The correlation between tumour uptake of 18 F-FAMT and L-type amino acid transporter 1 (LAT1) expression was determined. The study group comprised 68 OSCC patients who underwent both 18 F-FAMT and 18 F-FDG PET. Resected tumour sections were stained by immunohistochemistry for LAT1, CD98 and Ki-67, and microvessel density was determined in terms of CD34 and p53 expression. The sensitivity of primary tumour detection by 18 F-FAMT and 18 F-FDG PET was 98 % and 100 %, respectively. The sensitivity, specificity and accuracy of 18 F-FAMT PET for detecting malignant lymph nodes were 68 %, 99 % and 97 %, respectively, and equivalent values for 18 F-FDG PET were 84 %, 94 % and 94 %, respectively. The specificity and accuracy of 18 F-FAMT were significantly higher than those of 18 F-FDG. The uptake of 18 F-FAMT was significantly correlated with LAT1 expression, cell proliferation and advanced stage. The expression of LAT1 in OSCC cells was closely correlated with CD98 levels, cell proliferation and angiogenesis. 18 F-FAMT PET showed higher specificity for detecting malignant lesions than 18 F-FDG PET. The uptake of 18 F-FAMT by OSCC cells can be determined by the presence of LAT1 expression and tumour cell proliferation. (orig.)

  6. Epigenetic mechanisms regulate MHC and antigen processing molecules in human embryonic and induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Beatriz Suárez-Alvarez

    2010-04-01

    Full Text Available Human embryonic stem cells (hESCs are an attractive resource for new therapeutic approaches that involve tissue regeneration. hESCs have exhibited low immunogenicity due to low levels of Mayor Histocompatibility Complex (MHC class-I and absence of MHC class-II expression. Nevertheless, the mechanisms regulating MHC expression in hESCs had not been explored.We analyzed the expression levels of classical and non-classical MHC class-I, MHC class-II molecules, antigen-processing machinery (APM components and NKG2D ligands (NKG2D-L in hESCs, induced pluripotent stem cells (iPSCs and NTera2 (NT2 teratocarcinoma cell line. Epigenetic mechanisms involved in the regulation of these genes were investigated by bisulfite sequencing and chromatin immunoprecipitation (ChIP assays. We showed that low levels of MHC class-I molecules were associated with absent or reduced expression of the transporter associated with antigen processing 1 (TAP-1 and tapasin (TPN components in hESCs and iPSCs, which are involved in the transport and load of peptides. Furthermore, lack of beta2-microglobulin (beta2m light chain in these cells limited the expression of MHC class I trimeric molecule on the cell surface. NKG2D ligands (MICA, MICB were observed in all pluripotent stem cells lines. Epigenetic analysis showed that H3K9me3 repressed the TPN gene in undifferentiated cells whilst HLA-B and beta2m acquired the H3K4me3 modification during the differentiation to embryoid bodies (EBs. Absence of HLA-DR and HLA-G expression was regulated by DNA methylation.Our data provide fundamental evidence for the epigenetic control of MHC in hESCs and iPSCs. Reduced MHC class I and class II expression in hESCs and iPSCs can limit their recognition by the immune response against these cells. The knowledge of these mechanisms will further allow the development of strategies to induce tolerance and improve stem cell allograft acceptance.

  7. Epigenetic Mechanisms Regulate MHC and Antigen Processing Molecules in Human Embryonic and Induced Pluripotent Stem Cells

    Science.gov (United States)

    Suárez-Álvarez, Beatriz; Rodriguez, Ramón M.; Calvanese, Vincenzo; Blanco-Gelaz, Miguel A.; Suhr, Steve T.; Ortega, Francisco; Otero, Jesus; Cibelli, Jose B.; Moore, Harry; Fraga, Mario F.; López-Larrea, Carlos

    2010-01-01

    Background Human embryonic stem cells (hESCs) are an attractive resource for new therapeutic approaches that involve tissue regeneration. hESCs have exhibited low immunogenicity due to low levels of Mayor Histocompatibility Complex (MHC) class-I and absence of MHC class-II expression. Nevertheless, the mechanisms regulating MHC expression in hESCs had not been explored. Methodology/Principal Findings We analyzed the expression levels of classical and non-classical MHC class-I, MHC class-II molecules, antigen-processing machinery (APM) components and NKG2D ligands (NKG2D-L) in hESCs, induced pluripotent stem cells (iPSCs) and NTera2 (NT2) teratocarcinoma cell line. Epigenetic mechanisms involved in the regulation of these genes were investigated by bisulfite sequencing and chromatin immunoprecipitation (ChIP) assays. We showed that low levels of MHC class-I molecules were associated with absent or reduced expression of the transporter associated with antigen processing 1 (TAP-1) and tapasin (TPN) components in hESCs and iPSCs, which are involved in the transport and load of peptides. Furthermore, lack of β2-microglobulin (β2m) light chain in these cells limited the expression of MHC class I trimeric molecule on the cell surface. NKG2D ligands (MICA, MICB) were observed in all pluripotent stem cells lines. Epigenetic analysis showed that H3K9me3 repressed the TPN gene in undifferentiated cells whilst HLA-B and β2m acquired the H3K4me3 modification during the differentiation to embryoid bodies (EBs). Absence of HLA-DR and HLA-G expression was regulated by DNA methylation. Conclusions/Significance Our data provide fundamental evidence for the epigenetic control of MHC in hESCs and iPSCs. Reduced MHC class I and class II expression in hESCs and iPSCs can limit their recognition by the immune response against these cells. The knowledge of these mechanisms will further allow the development of strategies to induce tolerance and improve stem cell allograft acceptance

  8. Clinical Application of 18F-FDG PET in Non-Small Cell Lung Cancer

    International Nuclear Information System (INIS)

    Choi, Joon Young

    2008-01-01

    This review focuses on the clinical use of 18 F-FDG PET to evaluate solitary pulmonary nodule (SPN) and non-small cell lung cancer (NSCLC). When SPN or mass without calcification is found on chest X-ray or CT, 18 F-FDG PET is an effective modality to differentiate benign from malignant lesions. For initial staging of NSCLC, 18 F-FDG PET is useful, and proved to be cost-effective in several countries. 18 F-FDG PET is useful for detecting recurrence, restaging and evaluating residual tumor after curative therapy in NSCLC. For therapy response assessment, 18 F-FDG PET may be effective after chemotherapy or radiation therapy. 18 F-FDG PET is useful to predict pathological response after neoadjuvant therapy in NSCLC. For radiation therapy planning, 18 F-FDG PET may be helpful, but requires further investigations. PET/CT is better for evaluating NSCLC than conventional PET

  9. Tetraspanin CD9: A Key Regulator of Cell Adhesion in the Immune System

    Directory of Open Access Journals (Sweden)

    Raquel Reyes

    2018-04-01

    Full Text Available The tetraspanin CD9 is expressed by all the major subsets of leukocytes (B cells, CD4+ T cells, CD8+ T cells, natural killer cells, granulocytes, monocytes and macrophages, and immature and mature dendritic cells and also at a high level by endothelial cells. As a typical member of the tetraspanin superfamily, a prominent feature of CD9 is its propensity to engage in a multitude of interactions with other tetraspanins as well as with different transmembrane and intracellular proteins within the context of defined membranal domains termed tetraspanin-enriched microdomains (TEMs. Through these associations, CD9 influences many cellular activities in the different subtypes of leukocytes and in endothelial cells, including intracellular signaling, proliferation, activation, survival, migration, invasion, adhesion, and diapedesis. Several excellent reviews have already covered the topic of how tetraspanins, including CD9, regulate these cellular processes in the different cells of the immune system. In this mini-review, however, we will focus particularly on describing and discussing the regulatory effects exerted by CD9 on different adhesion molecules that play pivotal roles in the physiology of leukocytes and endothelial cells, with a particular emphasis in the regulation of adhesion molecules of the integrin and immunoglobulin superfamilies.

  10. The function of BCL9 in Wnt/β-catenin signaling and colorectal cancer cells

    International Nuclear Information System (INIS)

    Roche, Marc de la; Worm, Jesper; Bienz, Mariann

    2008-01-01

    Most cases of colorectal cancer are initiated by hyperactivation of the Wnt/β-catenin pathway due to mutations in the APC tumour suppressor, or in β-catenin itself. A recently discovered component of this pathway is Legless, which is essential for Wnt-induced transcription during Drosophila development. Limited functional information is available for its two mammalian relatives, BCL9 and B9L/BCL9-2: like Legless, these proteins bind to β-catenin, and RNAi-mediated depletion of B9L/BCL9-2 has revealed that this protein is required for efficient β-catenin-mediated transcription in mammalian cell lines. No loss-of-function data are available for BCL9. We have used overexpression of dominant-negative forms of BCL9, and RNAi-mediated depletion, to study its function in human cell lines with elevated Wnt pathway activity, including colorectal cancer cells. We found that BCL9 is required for efficient β-catenin-mediated transcription in Wnt-stimulated HEK 293 cells, and in the SW480 colorectal cancer cell line whose Wnt pathway is active due to APC mutation. Dominant-negative mutants of BCL9 indicated that its function depends not only on its β-catenin ligand, but also on an unknown ligand of its C-terminus. Finally, we show that BCL9 and B9L are both Wnt-inducible genes, hyperexpressed in colorectal cancer cell lines, indicating that they are part of a positive feedback loop. BCL9 is required for efficient β-catenin-mediated transcription in human cell lines whose Wnt pathway is active, including colorectal cancer cells, indicating its potential as a drug target in colorectal cancer

  11. CRISPR/Cas9-mediated genome editing of Epstein-Barr virus in human cells.

    Science.gov (United States)

    Yuen, Kit-San; Chan, Chi-Ping; Wong, Nok-Hei Mickey; Ho, Chau-Ha; Ho, Ting-Hin; Lei, Ting; Deng, Wen; Tsao, Sai Wah; Chen, Honglin; Kok, Kin-Hang; Jin, Dong-Yan

    2015-03-01

    The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a highly efficient and powerful tool for RNA-guided editing of the cellular genome. Whether CRISPR/Cas9 can also cleave the genome of DNA viruses such as Epstein-Barr virus (EBV), which undergo episomal replication in human cells, remains to be established. Here, we reported on CRISPR/Cas9-mediated editing of the EBV genome in human cells. Two guide RNAs (gRNAs) were used to direct a targeted deletion of 558 bp in the promoter region of BART (BamHI A rightward transcript) which encodes viral microRNAs (miRNAs). Targeted editing was achieved in several human epithelial cell lines latently infected with EBV, including nasopharyngeal carcinoma C666-1 cells. CRISPR/Cas9-mediated editing of the EBV genome was efficient. A recombinant virus with the desired deletion was obtained after puromycin selection of cells expressing Cas9 and gRNAs. No off-target cleavage was found by deep sequencing. The loss of BART miRNA expression and activity was verified, supporting the BART promoter as the major promoter of BART RNA. Although CRISPR/Cas9-mediated editing of the multicopy episome of EBV in infected HEK293 cells was mostly incomplete, viruses could be recovered and introduced into other cells at low m.o.i. Recombinant viruses with an edited genome could be further isolated through single-cell sorting. Finally, a DsRed selectable marker was successfully introduced into the EBV genome during the course of CRISPR/Cas9-mediated editing. Taken together, our work provided not only the first genetic evidence that the BART promoter drives the expression of the BART transcript, but also a new and efficient method for targeted editing of EBV genome in human cells. © 2015 The Authors.

  12. Ca2+ signals, cell membrane disintegration, and activation of TMEM16F during necroptosis.

    Science.gov (United States)

    Ousingsawat, Jiraporn; Cabrita, Inês; Wanitchakool, Podchanart; Sirianant, Lalida; Krautwald, Stefan; Linkermann, Andreas; Schreiber, Rainer; Kunzelmann, Karl

    2017-01-01

    Activated receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain like (MLKL) are essential components of the necroptotic pathway. Phosphorylated MLKL (pMLKL) is thought to induce membrane leakage, leading to cell swelling and disintegration of the cell membrane. However, the molecular identity of the necroptotic membrane pore remains unclear, and the role of pMLKL for membrane permeabilization is currently disputed. We observed earlier that the phospholipid scramblase and ion channel TMEM16F/anoctamin 6 cause large membrane currents, cell swelling, and cell death when activated by a strong increase in intracellular Ca 2+ . We, therefore, asked whether TMEM16F is also central to necroptotic cell death and other cellular events during necroptosis. Necroptosis was induced by TNFα, smac mimetic, and Z-VAD (TSZ) in NIH3T3 fibroblasts and the four additional cell lines HT 29 , 16HBE, H441, and L929. Time-dependent changes in intracellular Ca 2+ , cell morphology, and membrane currents were recorded. TSZ induced a small and only transient oscillatory rise in intracellular Ca 2+ , which was paralleled by the activation of outwardly rectifying Cl - currents, which were typical for TMEM16F/ANO6. Ca 2+ oscillations were due to Ca 2+ release from endoplasmic reticulum, and were independent of extracellular Ca 2+ . The initial TSZ-induced cell swelling was followed by cell shrinkage. Using typical channel blockers and siRNA-knockdown, the Cl - currents were shown to be due to the activation of ANO6. However, the knockdown of ANO6 or inhibitors of ANO6 did not inhibit necroptotic cell death. The present data demonstrate the activation of ANO6 during necroptosis, which, however, is not essential for cell death.

  13. Arsenate reduction and methylation in the cells of Trichoderma asperellum SM-12F1, Penicillium janthinellum SM-12F4, and Fusarium oxysporum CZ-8F1 investigated with X-ray absorption near edge structure

    Energy Technology Data Exchange (ETDEWEB)

    Su, S.M., E-mail: shimingsu@163.com [Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural Sciences/Key Laboratory of Agro-Environment, Ministry of Agriculture, Beijing (China); Zeng, X.B., E-mail: zengxb@ieda.org.cn [Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural Sciences/Key Laboratory of Agro-Environment, Ministry of Agriculture, Beijing (China); Li, L.F.; Duan, R.; Bai, L.Y. [Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural Sciences/Key Laboratory of Agro-Environment, Ministry of Agriculture, Beijing (China); Li, A.G.; Wang, J.; Jiang, S. [Shanghai Synchrotron Radiation Facility, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai (China)

    2012-12-15

    Highlights: Black-Right-Pointing-Pointer Three fungal strains are capable of As(V) reduction and methylation. Black-Right-Pointing-Pointer As(V) reduction might be more easily processed than the methylation in fungal cells. Black-Right-Pointing-Pointer As sequestration and speciation transformation might be the detoxification processes. - Abstract: Synchrotron radiation-based X-ray absorption near edge structure (XANES) was introduced to directly analysis chemical species of arsenic (As) in the cells of Trichoderma asperellum SM-12F1, Penicillium janthinellum SM-12F4, and Fusarium oxysporum CZ-8F1 capable of As accumulation and volatilisation. After exposure to As(V) of 500 mg L{sup -1} for 15 days, a total of 60.5% and 65.3% of the accumulated As in the cells of T. asperellum SM-12F1 and P. janthinellum SM-12F4, respectively, was As(III), followed by 31.3% and 32.4% DMA (dimethylarsinic acid), 8.3% and 2.3% MMA (monomethylarsonic acid), respectively. However, for F. oxysporum CZ-8F1, 54.5% of the accumulated As was As(III), followed by 37.8% MMA and 7.7% As(V). The reduction and methylation of As(V) formed As(III), MMA, and DMA as the primacy products, and the reduction of As(V) might be more easily processed than the methylation. These results will help to understanding the mechanisms of As detoxification and its future application in bioremediation.

  14. Arsenate reduction and methylation in the cells of Trichoderma asperellum SM-12F1, Penicillium janthinellum SM-12F4, and Fusarium oxysporum CZ-8F1 investigated with X-ray absorption near edge structure

    International Nuclear Information System (INIS)

    Su, S.M.; Zeng, X.B.; Li, L.F.; Duan, R.; Bai, L.Y.; Li, A.G.; Wang, J.; Jiang, S.

    2012-01-01

    Highlights: ► Three fungal strains are capable of As(V) reduction and methylation. ► As(V) reduction might be more easily processed than the methylation in fungal cells. ► As sequestration and speciation transformation might be the detoxification processes. - Abstract: Synchrotron radiation-based X-ray absorption near edge structure (XANES) was introduced to directly analysis chemical species of arsenic (As) in the cells of Trichoderma asperellum SM-12F1, Penicillium janthinellum SM-12F4, and Fusarium oxysporum CZ-8F1 capable of As accumulation and volatilisation. After exposure to As(V) of 500 mg L −1 for 15 days, a total of 60.5% and 65.3% of the accumulated As in the cells of T. asperellum SM-12F1 and P. janthinellum SM-12F4, respectively, was As(III), followed by 31.3% and 32.4% DMA (dimethylarsinic acid), 8.3% and 2.3% MMA (monomethylarsonic acid), respectively. However, for F. oxysporum CZ-8F1, 54.5% of the accumulated As was As(III), followed by 37.8% MMA and 7.7% As(V). The reduction and methylation of As(V) formed As(III), MMA, and DMA as the primacy products, and the reduction of As(V) might be more easily processed than the methylation. These results will help to understanding the mechanisms of As detoxification and its future application in bioremediation.

  15. (19)F-heptuloses as tools for the non-invasive imaging of GLUT2-expressing cells

    DEFF Research Database (Denmark)

    Malaisse, Willy J; Zhang, Ying; Louchami, Karim

    2012-01-01

    Suitable analogs of d-mannoheptulose are currently considered as possible tools for the non-invasive imaging of pancreatic islet insulin-producing cells. Here, we examined whether (19)F-heptuloses could be used for non-invasive imaging of GLUT2-expressing cells. After 20 min incubation, the uptake......-mannoheptulose in inhibiting insulin release. The 1-deoxy-1-fluoro-d-mannoheptulose and 3-deoxy-3-fluoro-d-mannoheptulose only marginally affected INS-1 cell viability. These findings are compatible with the view that selected (19)F-heptuloses may represent suitable tools for the non-invasive imaging of hepatocytes...

  16. SEPT9_v1 Functions in Breast Cancer Cell Division

    Science.gov (United States)

    2012-01-01

    in PBS containing 3% PFA and stained with antibodies to SEPT2, gp135/podocalyxin (3F2/D8 mouse hybridoma supernatant), and Na/K-ATPase ( chicken ...Science 317(5836):372–376. Rohatgi R, Snell WJ. 2010. The ciliary membrane. Curr Opin Cell Biol 22(4):541–546. Romio L, Fry AM, Winyard PJD, Malcolm...Making sense of cilia and flag- ella. J Cell Biol 179(4):575–582. Snell WJ, Pan J, Wang Q. 2004. Cilia and flagella revealed: From flagellar assembly in

  17. Cell proliferation kinetics and radiation response in 9L tumor spheroids

    Energy Technology Data Exchange (ETDEWEB)

    Sweigert, S.E.

    1984-05-01

    Cell kinetic parameters, including population doubling-time, cell cycle time, and growth fraction, were measured in 9L gliosarcoma spheroids. These parameters were studied as the spheroids grew from 50 ..mu..m to over 900 ..mu..m in diameter. Experiments relating the cell kinetic parameters to the radiation response of 9L spheroids were also carried out. The major findings were that the average cell cycle time (T/sub c/), is considerably longer in large spheroids than in exponentially-growing monolayers, the radiosensitivity of noncycling (but still viable) cells in spheroids is not significantly different from that of cycling spheroid cells, and the radiation-induced division delay is approximately twice as long in spheroid cells as in monolayer cells given equal radiation doses. The cell loss factor for spheroids of various sizes was calculated, by using the measured kinetic parameters in the basic equations for growth of a cell population. 157 references, 6 figures, 3 tables.

  18. Cell proliferation kinetics and radiation response in 9L tumor spheroids

    International Nuclear Information System (INIS)

    Sweigert, S.E.

    1984-05-01

    Cell kinetic parameters, including population doubling-time, cell cycle time, and growth fraction, were measured in 9L gliosarcoma spheroids. These parameters were studied as the spheroids grew from 50 μm to over 900 μm in diameter. Experiments relating the cell kinetic parameters to the radiation response of 9L spheroids were also carried out. The major findings were that the average cell cycle time (T/sub c/), is considerably longer in large spheroids than in exponentially-growing monolayers, the radiosensitivity of noncycling (but still viable) cells in spheroids is not significantly different from that of cycling spheroid cells, and the radiation-induced division delay is approximately twice as long in spheroid cells as in monolayer cells given equal radiation doses. The cell loss factor for spheroids of various sizes was calculated, by using the measured kinetic parameters in the basic equations for growth of a cell population. 157 references, 6 figures, 3 tables

  19. The compressibility mechanism of Li3Na3In2F12 garnet

    DEFF Research Database (Denmark)

    Grzechnik, Andrzej; Balic Zunic, Tonci; Makovicky, Emil

    2006-01-01

    The high pressure behaviour of Li3Na3In2F12 garnet (Ia¯3d, Z = 8) is studied up to 9.2 GPa at room temperature in diamond anvil cells using xray diffraction. Its equation of state to 9.2 GPa and the pressure dependences of the structural parameters to 4.07 GPa are determined from synchrotron angle......-dispersive powder and laboratory single-crystal data, respectively. No indication of any structural phase transition in this material has been found up to 9.2 GPa. The fitting of the Murnaghan equation of state yields B0 = 36.2(5) GPa, B0 = 5.38(18), and V0 = 2051.76(0.69) °A 3. The compressibility mechanism of Li3......Na3In2F12 is attributed to the substantial bending of the In-F-Li angles linking the InF6 octahedra and LiF4 tetrahedra. The most compressible polyhedral units are the NaF8 triangulated dodecahedra. These results are discussed in relation to previous high pressure photoluminescence measurements...

  20. Imprinting of CCR9 on CD4 T cells requires IL-4 signaling on mesenteric lymph node dendritic cells.

    Science.gov (United States)

    Elgueta, Raul; Sepulveda, Fernando E; Vilches, Felipe; Vargas, Leonardo; Mora, J Rodrigo; Bono, Maria Rosa; Rosemblatt, Mario

    2008-05-15

    It has recently been shown that IL-4 can educate dendritic cells (DC) to differentially affect T cell effector activity. In this study, we show that IL-4 can also act upon DC to instruct naive T cells to express the gut-associated homing receptor CCR9. Thus, effector T cells generated after coculture with mesenteric lymph node (MLN)-DC show a higher expression of CCR9 when activated in the presence of IL-4. In contrast, IL-4 had no effect on CCR9 expression when naive T cells were polyclonally activated in the absence of MLN-DC, suggesting that the effect of IL-4 on CCR9 expression passed through DC. Indeed, T cells activated by MLN-DC from IL-4Ralpha(-/-) mice showed a much lower CCR9 expression and a greatly reduced migration to the small intestine than T cells activated by wild-type MLN-DC even in the presence of IL-4. Consistent with the finding that the vitamin A metabolite retinoic acid (RA) induces gut-homing molecules on T cells, we further demonstrate that IL-4 up-regulated retinaldehyde dehydrogenase 2 mRNA on MLN-DC, a critical enzyme involved in the synthesis of RA. Moreover, LE135, a RA receptor antagonist, blocked the increased expression of CCR9 driven by IL-4-treated MLN-DC. Thus, besides the direct effect of RA on T cell gut tropism, our results show that the induction of a gut-homing phenotype on CD4(+) T cells is also influenced by the effect of IL-4 on gut-associated DC.

  1. In vitro production of haploid cells after coculture of CD49f+ with Sertoli cells from testicular sperm extraction in nonobstructive azoospermic patients.

    Science.gov (United States)

    Riboldi, Marcia; Rubio, Carmen; Pellicer, Antonio; Gil-Salom, Manuel; Simón, Carlos

    2012-09-01

    To isolate CD49f+ cells from testicular sperm extraction (TESE) samples of azoospermic patients and induce meiosis by coculturing these cells with Sertoli cells. Prospective analysis. Research center. Obstructive azoospermic (OA) and nonobstructive azoospermic (NOA) patients. TESE, with enzymatic dissociation of samples to obtain a cell suspension, which was cultured for 4 days with 4 ng/mL GDNF. The CD49f+ cells were sorted using fluorescence-activated cell sorting (FACS) as a marker to identify spermatogonial stem cells (SSCs), which were cocultured with Sertoli cells expressing red fluorescent protein (RFP) in knockout serum replacement (KSR) media with addition of 1,000 IU/mL of follicle-stimulating hormone (FSH), 1 μM testosterone, 40 ng/mL of GDNF, and 2 μM retinoic acid (RA) for 15 days in culture at 37°C and 5% CO(2) to induce meiotic progression. Cells were collected and analyzed by immunofluorescence for meiosis progression with specific markers SCP3 and CREST, and they were confirmed by fluorescence in situ hybridization (FISH). Isolation of CD49f+ cells and coculture with Sertoli cells, meiosis progression in vitro, assessment of SSCs and meiotic markers real-time polymerase chain reaction (RT-PCR), immunohistochemical analysis, and FISH. The CD49f+ isolated from the of total cell count in the TESE samples of azoospermic patients varied from 5.45% in OA to 2.36% in NOA. Sertoli cells were obtained from the same TESE samples, and established protocols were used to characterize them as positive for SCF, rGDNF, WT1, GATA-4, and vimentin, with the presence of tight junctions and lipid droplets shown by oil red staining. After isolation, the CD49f+ cells were cocultured with RFP Sertoli cells in a 15-day time-course experiment. Positive immunostaining for meiosis markers SCP3 and CREST on days 3 to 5 was noted in the samples obtained from one NOA patient. A FISH analysis for chromosomes 13, 18, 21, X, and Y confirmed the presence of haploid cells on day

  2. Combination of nitric oxide stimulation with high-dose 18F-FDG promotes apoptosis and enhances radiation therapy of endothelial cells

    International Nuclear Information System (INIS)

    Paik, Jin-Young; Park, Jin-Won; Jung, Kyung-Ho; Lee, Eun Jeong; Lee, Kyung-Han

    2012-01-01

    Introduction: High-dose 18 F-FDG can provide targeted nuclear therapy of cancer. Endothelial cell injury is a key determinant of tumor response to radiotherapy. Here, we tested the hypothesis that activation of endothelial cell glycolytic metabolism with nitric oxide can enhance the therapeutic effect of high-dose 18 F-FDG. Methods: Calf pulmonary artery endothelial (CPAE) cells were treated with graded doses of 18 F-FDG. Glycolysis was stimulated by 24 h of exposure to the nitric oxide donor, sodium nitroprusside (SNP). Cell viability was assessed by MTT and clonogenic assays. Apoptosis was evaluated by ELISA of cytosolic DNA fragments and Western blots of cleaved caspase-3. Results: SNP stimulation (0.1 and 1 mM) augmented CPAE cell 18 F-FDG uptake to 2.6- and 4.6-fold of controls without adverse effects. Treatment with 333 μCi/ml 18 F-FDG alone reduced viable cell number to 35.4% of controls by Day 3. Combining 0.1 mM SNP stimulation significantly enhanced the killing effect, reducing cell numbers to 19.2% and 39.2% of controls by 333 and 167 μCi/ml of 18 F-FDG, respectively. 18 F-FDG also suppressed clonogenic survival to 80.8% and 43.2% of controls by 83 and 167 μCi/ml, which was again intensified by SNP to 59.7% and 21.1% of controls. The cytotoxic effect of 18 F-FDG was attributed to induction of apoptosis as shown by increased cytosolic fragmented DNA and cleaved caspase-3 levels (26.4% and 30.7% increases by 167 μCi/ml). Combining SNP stimulation significantly increased both of these levels to 1.8-fold of control cells. Conclusion: High-dose 18 F-FDG combined with nitric oxide-stimulated glycolysis is an effective method to inhibit endothelial cell survival and promote apoptosis. These results suggest a potential role of this strategy for targeted radiotherapy of angiogenic vasculature.

  3. New Azalomycin F Analogs from Mangrove Streptomyces sp. 211726 with Activity against Microbes and Cancer Cells

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    Haipeng Lin

    2013-03-01

    Full Text Available Seven new azalomycin F analogs (1–7 were isolated from the broth of mangrove Streptomyces sp. 211726, and respectively identified as 25-malonyl demalonylazalomycin F5a monoester (1, 23-valine demalonylazalomycin F5a ester (2, 23-(6-methylheptanoic acid demalonylazalomycins F3a ester (3, F4a ester (4 and F5a ester (5, 23-(9-methyldecanoic acid demalonylazalomycin F4a ester (6 and 23-(10-methylundecanoic acid demalony lazalomycin F4a ester (7. Their structures were established by their spectroscopic data and by comparing with those of azalomycins F3a, F4a and F5a. Biological assays exhibited that 1–7 showed broad-spectrum antimicrobial and anti HCT-116 activities.

  4. Population heterogeneity in the surface expression of Ulex europaeus I-lectin (UEA I)-binding sites in cultured malignant and transformed cells

    Energy Technology Data Exchange (ETDEWEB)

    Virtanen, I.; Lehtonen, E.; Naervaenen, O.; Leivo, I.; Lehto, V.P.

    1985-11-01

    We studied the binding of fluorochrome-coupled Ulex europaeus I-lectin (UEA-I) to cultured malignant cells: all human malignant and transformed cells and also mouse teratocarcinoma cells examined gave a homogeneous cell membrane-type of surface staining only in some of the cells. Such a population heterogeneity appeared to be independent of the cell cycle. Instead, other lectin conjugates used bound homogeneously to all cell. In permeabilized cells, a juxtanuclear reticular staining of the Golgi apparatus was seen in the UEA-I-positive cells. No staining of the pericellular matrix components, produced by malignant cells grown in serum-free culture medium, could be obtained with TRITC-UEA-I. UEA-I-lectin recognized most polypeptides from A8387 fibrosarcoma cells and HeLa cells, metabolically labelled with (/sup 3/H)fucose. Furthermore, surface labelling of these cells with the neuraminidase-galactose oxidase/sodium borohydride method disclosed that both UEA-I and Ricinus communis agglutinin I revealed the same major surface glycoproteins. Results with metabolically labelled cells showed, in addition, that UEA-I-lectin did not bind to secreted glycoproteins produced by A8387 cells and recognized by other lectins. The results indicate that transformed and malignant cells show a distinct population heterogeneity in their expression of some cell surface-associated fucosyl glycoconjugates. The results also suggest that malignant cells can glycosylate their membrane and secreted glycoproteins in a different manner.

  5. Phage display-derived inhibitor of the essential cell wall biosynthesis enzyme MurF

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    Blewett Ann

    2008-12-01

    Full Text Available Abstract Background To develop antibacterial agents having novel modes of action against bacterial cell wall biosynthesis, we targeted the essential MurF enzyme of the antibiotic resistant pathogen Pseudomonas aeruginosa. MurF catalyzes the formation of a peptide bond between D-Alanyl-D-Alanine (D-Ala-D-Ala and the cell wall precursor uridine 5'-diphosphoryl N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid (UDP-MurNAc-Ala-Glu-meso-A2pm with the concomitant hydrolysis of ATP to ADP and inorganic phosphate, yielding UDP-N-acetylmuramyl-pentapeptide. As MurF acts on a dipeptide, we exploited a phage display approach to identify peptide ligands having high binding affinities for the enzyme. Results Screening of a phage display 12-mer library using purified P. aeruginosa MurF yielded to the identification of the MurFp1 peptide. The MurF substrate UDP-MurNAc-Ala-Glumeso-A2pm was synthesized and used to develop a sensitive spectrophotometric assay to quantify MurF kinetics and inhibition. MurFp1 acted as a weak, time-dependent inhibitor of MurF activity but was a potent inhibitor when MurF was pre-incubated with UDP-MurNAc-Ala-Glu-meso-A2pm or ATP. In contrast, adding the substrate D-Ala-D-Ala during the pre-incubation nullified the inhibition. The IC50 value of MurFp1 was evaluated at 250 μM, and the Ki was established at 420 μM with respect to the mixed type of inhibition against D-Ala-D-Ala. Conclusion MurFp1 exerts its inhibitory action by interfering with the utilization of D-Ala-D-Ala by the MurF amide ligase enzyme. We propose that MurFp1 exploits UDP-MurNAc-Ala-Glu-meso-A2pm-induced structural changes for better interaction with the enzyme. We present the first peptide inhibitor of MurF, an enzyme that should be exploited as a target for antimicrobial drug development.

  6. IL-9-Producing Mast Cell Precursors and Food Allergy

    Science.gov (United States)

    2016-10-01

    that Stat6-/- BM progenitors in sensitized wild type recipients that were competent in GFP- CD4+ST2+TH2 and ILC2s ( innate lymphoid cells ) generation, and...report demonstrated that type 2 innate lymphoid cells (ILC2s) lack cell lineage markers and have the potential to pro- duce IL-9 (Wilhelm et al., 2011...Fujii, H., and Koyasu, S. (2010). Innate production of T(H)2 cytokines by adipose tissue-associated c-Kit(+)Sca-1(+) lymphoid cells . Nature 463, 540–544

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

    Science.gov (United States)

    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.

  8. Delta-9-tetrahydrocannabinol-induced dopamine release as a function of psychosis risk: 18F-fallypride positron emission tomography study.

    Directory of Open Access Journals (Sweden)

    Rebecca Kuepper

    Full Text Available Cannabis use is associated with psychosis, particularly in those with expression of, or vulnerability for, psychotic illness. The biological underpinnings of these differential associations, however, remain largely unknown. We used Positron Emission Tomography and (18F-fallypride to test the hypothesis that genetic risk for psychosis is expressed by differential induction of dopamine release by Δ(9-THC (delta-9-tetrahydrocannabinol, the main psychoactive ingredient of cannabis. In a single dynamic PET scanning session, striatal dopamine release after pulmonary administration of Δ(9-THC was measured in 9 healthy cannabis users (average risk psychotic disorder, 8 patients with psychotic disorder (high risk psychotic disorder and 7 un-related first-degree relatives (intermediate risk psychotic disorder. PET data were analyzed applying the linear extension of the simplified reference region model (LSRRM, which accounts for time-dependent changes in (18F-fallypride displacement. Voxel-based statistical maps, representing specific D2/3 binding changes, were computed to localize areas with increased ligand displacement after Δ(9-THC administration, reflecting dopamine release. While Δ(9-THC was not associated with dopamine release in the control group, significant ligand displacement induced by Δ(9-THC in striatal subregions, indicative of dopamine release, was detected in both patients and relatives. This was most pronounced in caudate nucleus. This is the first study to demonstrate differential sensitivity to Δ(9-THC in terms of increased endogenous dopamine release in individuals at risk for psychosis.

  9. Unexpected heterogeneity derived from Cas9 ribonucleoprotein-introduced clonal cells at the HPRT1 locus.

    Science.gov (United States)

    Sakuma, Tetsushi; Mochida, Keiji; Nakade, Shota; Ezure, Toru; Minagawa, Sachi; Yamamoto, Takashi

    2018-04-01

    Single-cell cloning is an essential technique for establishing genome-edited cell clones mediated by programmable nucleases such as CRISPR-Cas9. However, residual genome-editing activity after single-cell cloning may cause heterogeneity in the clonal cells. Previous studies showed efficient mutagenesis and rapid degradation of CRISPR-Cas9 components in cultured cells by introducing Cas9 ribonucleoproteins (RNPs). In this study, we investigated how the timing for single-cell cloning of Cas9 RNP-transfected cells affected the heterogeneity of the resultant clones. We carried out transfection of Cas9 RNPs targeting several loci in the HPRT1 gene in HCT116 cells, followed by single-cell cloning at 24, 48, 72 hr and 1 week post-transfection. After approximately 3 weeks of incubation, the clonal cells were collected and genotyped by high-resolution microchip electrophoresis and Sanger sequencing. Unexpectedly, long-term incubation before single-cell cloning resulted in highly heterogeneous clones. We used a lipofection method for transfection, and the media containing transfectable RNPs were not removed before single-cell cloning. Therefore, the active Cas9 RNPs were considered to be continuously incorporated into cells during the precloning incubation. Our findings provide a warning that lipofection of Cas9 RNPs may cause continuous introduction of gene mutations depending on the experimental procedures. © 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  10. Is cell survival a determinant of the in situ response of 9L tumours

    International Nuclear Information System (INIS)

    Wheeler, K.T.; Wallen, C.A.

    1980-01-01

    The influence of growth rate, location, size and potential lethal damage (PLD) recovery on the cellular radiosensitivity and the tumour response was studied in 9L/Ro and 9L/SF rat tumours. The median day of death of rats bearing the intracerebral (i.c.) 9L/Ro tumours was 16-18 days; for i.c. 9L/SF tumours it was 23-25 days. The doubling time of 9L/Ro cells was slightly faster than for 9L/SF cells both in culture and in the brain. The cellular radiosensitivity of both i.c. tumour cell sublines was identical. However, subcutaneous (s.c.) 9L/Ro tumour cells were more resistant. There was no evidence of a substantial hypoxic fraction in either site. When i.c. 9L/Ro and 9L/SF tumours of similar size were treated with fractionated doses of BCNU, X-rays or combinations of the two, the responses of the two tumours were essentially identical. The rate of recovery from radiation-induced PLD was identical in the two sublines and the two sites. Increase in life-span of rats bearing i.c. 9L/Ro tumours appeared to be correlated with the tumour cell kill measured after completion of PLD recovery rather than with the tumour cell kill determined immediately after irradiation. (author)

  11. SU-D-201-03: Imaging Cellular Pharmacokinetics of 18F-FDG in Inflammatory/Stem Cells

    Energy Technology Data Exchange (ETDEWEB)

    Zaman, R; Tuerkcan, S; Mahmoudi, M; Toshinobu, T; Kosuge, H; Yang, P; Chin, F; McConnell, M; Xing, L [Stanford University School of Medicine, Stanford, CA (United States)

    2015-06-15

    Purpose: Atherosclerosis is a progressive inflammatory condition that underlies coronary artery disease (CAD)—the leading cause of death in the USA. Thus, understating the metabolism of inflammatory cells can be a valuable tool for investigating CAD. To the best of our knowledge, we are the first to successfully investigate the pharmacokinetics of [18F]fluoro-deoxyglucose (18F-FDG) uptake in a single macrophages and compared with induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs) with a novel imaging technique, radioluminescence microscopy, initially developed for cancer imaging. Methods: Live cells were cultured sparsely on Matrigel in a glass-bottom dish and starved for 1 hour before incubation with 250 microCi of 18F-FDG for 45 minutes. Excess radiotracer was removed using DMEM medium without glucose. Before imaging, DMEM (1 mL) was added to the cell culture and a 100 microm-thin CdWO4 scintillator plate was placed on top of the cells. Light produced following beta decay was imaged with a highly sensitive inverted microscope (LV200, Olympus) fitted with a 40x/1.3 high-NA oil objective, and an EM-CCD camera. The images were collected over 18,000 frames with 4×4 binning (1200 MHz EM Gain, 300ms exposure). Custom-written software was developed in MATLAB for image processing (Figure 1). For statistical analysis 10 different region-of-interests (ROIs) were selected for each cell type. Results: Figures 2A–2B show bright-field/fusion images for all three different cell types. The relationship between cell-to-cell comparisons was found to be linear for macrophages unlike iPSCs and MSCs, which were best fitted with moving or rolling average (Figure 2C). The average observed decay of 18F-FDG in a single cell of MSCs per second (0.067) was 20% and 36% higher compared to iPSCs (0.054) and macrophages (0.043), respectively (Figure 2D). Conclusion: MSCs was found to be 2–3x more sensitive to glucose molecule despite constant parameters for each

  12. SU-D-201-03: Imaging Cellular Pharmacokinetics of 18F-FDG in Inflammatory/Stem Cells

    International Nuclear Information System (INIS)

    Zaman, R; Tuerkcan, S; Mahmoudi, M; Toshinobu, T; Kosuge, H; Yang, P; Chin, F; McConnell, M; Xing, L

    2015-01-01

    Purpose: Atherosclerosis is a progressive inflammatory condition that underlies coronary artery disease (CAD)—the leading cause of death in the USA. Thus, understating the metabolism of inflammatory cells can be a valuable tool for investigating CAD. To the best of our knowledge, we are the first to successfully investigate the pharmacokinetics of [18F]fluoro-deoxyglucose (18F-FDG) uptake in a single macrophages and compared with induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs) with a novel imaging technique, radioluminescence microscopy, initially developed for cancer imaging. Methods: Live cells were cultured sparsely on Matrigel in a glass-bottom dish and starved for 1 hour before incubation with 250 microCi of 18F-FDG for 45 minutes. Excess radiotracer was removed using DMEM medium without glucose. Before imaging, DMEM (1 mL) was added to the cell culture and a 100 microm-thin CdWO4 scintillator plate was placed on top of the cells. Light produced following beta decay was imaged with a highly sensitive inverted microscope (LV200, Olympus) fitted with a 40x/1.3 high-NA oil objective, and an EM-CCD camera. The images were collected over 18,000 frames with 4×4 binning (1200 MHz EM Gain, 300ms exposure). Custom-written software was developed in MATLAB for image processing (Figure 1). For statistical analysis 10 different region-of-interests (ROIs) were selected for each cell type. Results: Figures 2A–2B show bright-field/fusion images for all three different cell types. The relationship between cell-to-cell comparisons was found to be linear for macrophages unlike iPSCs and MSCs, which were best fitted with moving or rolling average (Figure 2C). The average observed decay of 18F-FDG in a single cell of MSCs per second (0.067) was 20% and 36% higher compared to iPSCs (0.054) and macrophages (0.043), respectively (Figure 2D). Conclusion: MSCs was found to be 2–3x more sensitive to glucose molecule despite constant parameters for each

  13. Bimodal cell death induced by high radiation doses in the radioresistant sf9 insect cell line

    International Nuclear Information System (INIS)

    Chandna, S.

    2003-01-01

    Full text: This study was conducted to investigate the mode(s) of cell death induced by high radiation doses in the highly radioresistant Sf9 insect ovarian cell line. Methods: Cells were exposed to γ-radiation doses 200Gy and 500Gy, harvested at various time intervals (6h-72h) following irradiation, and subjected to cell morphology assay, DNA agarose gel electrophoresis, single cell gel electrophoresis (SCGE; comet assay) and Annexin-V labeling for the detection of membrane phosphatidylserine externalization. Cell morphology was assessed in cells entrapped and fixed in agarose gel directly from the cell suspension, thus preventing the possible loss of fragments/ apoptotic bodies. Surviving fraction of Sf9 cells was 0.01 at 200Gy and 98%) undergoing extensive DNA fragmentation at 500Gy, whereas the frequency of cells with DNA fragmentation was considerably less (∼12%) at 200Gy. Conclusions: While the mode of cell death at 200Gy seems to be different from typical apoptosis, a dose of 500Gy induced bimodal cell death, with typical apoptotic as well as the atypical cell death observed at 200Gy

  14. Simple Meets Single: The Application of CRISPR/Cas9 in Haploid Embryonic Stem Cells

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    Zixi Yin

    2017-01-01

    Full Text Available The CRISPR/Cas9 system provides a powerful method for the genetic manipulation of the mammalian genome, allowing knockout of individual genes as well as the generation of genome-wide knockout cell libraries for genetic screening. However, the diploid status of most mammalian cells restricts the application of CRISPR/Cas9 in genetic screening. Mammalian haploid embryonic stem cells (haESCs have only one set of chromosomes per cell, avoiding the issue of heterozygous recessive mutations in diploid cells. Thus, the combination of haESCs and CRISPR/Cas9 facilitates the generation of genome-wide knockout cell libraries for genetic screening. Here, we review recent progress in CRISPR/Cas9 and haPSCs and discuss their applications in genetic screening.

  15. Translation of LINE-1 DNA elements in vitro and in human cells

    International Nuclear Information System (INIS)

    Leibold, D.M.; Swergold, G.D.; Thayer, R.E.; Singer, M.F.; Fanning, T.G.; Dombroski, B.A.

    1990-01-01

    The LINE-1(L1) family of interspread DNA sequences found throughout the human genome (L1 Homo sapiens, L1Hs) includes active transposable elements. Current models for the mechanism of transposition involve reverse transcription of an RNA intermediate and utilization of element-encoded proteins. The authors report that an antiserum against the polypeptide encoded by the L1Hs 5' open reading frame (ORF1) detects, in human cells, an endogenous ORF1 protein as well as the ORG1 product of an appropriate transfecting recombinant vector. The endogenous polypeptide is most abundant in teratocarcinoma and choriocarcinoma cells, among those cell lines tested; it appears to be a single species of ∼38 kDa. In contrast, RNAs synthesized in vitro from cDNAs representing full-length, polyadenylylated cytoplasmic L1Hs RNA yield, upon in vitro translation, ORF1 products of slightly different sizes. This is consistent with the fact that the various cDNAs are different and represent transcription of different genomic L1Hs elements. In vitro studies additionally suggest that translation of ORF1 is initiated at the first AUG codon. Finally, in no case was an ORF1-ORF2 fusion protein detected

  16. File list: His.PSC.20.H3K9ac.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.PSC.20.H3K9ac.AllCell mm9 Histone H3K9ac Pluripotent stem cell SRX185844,SRX185...848,SRX873351,SRX873349,SRX873345,SRX097641,SRX130021,SRX873347,SRX187620 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.PSC.20.H3K9ac.AllCell.bed ...

  17. Human chromosome 9 can complement UV sensitivity of xeroderma pigmentosum group A cells

    International Nuclear Information System (INIS)

    Ishizaki, Kanji; Sasaki, Masao S.; Ikenaga, Mituo; Nakamura, Yusuke

    1990-01-01

    A single human chromosome derived from normal human fibroblasts and tagged with the G418 resistance gene was transferred into SV40-transformed xeroderma pigmentosum group A (XP-A) cells via microcell fusion. When chromosome 1 or 12 was transferred, UV sensitivity of microcell hybrid cells was not changed. By contrast, after transferring chromosome 9,7 of 11 reipient clones were as UV-resistant as normal human cells. Four other clones were still as UV-sensitive as the parental XP-A cells. Southern hybridization analysis using a polymorphic probe, pEKZ19.3, which is homologous to a sequence of the D9S17 locus on chromosome 9, has confirmed that at least a part of normal human chromosome 9 was transferred into the recipient clones. However, amounts iof UV-induced unscheduled DNA synthesis in the UV-resistant clones were only one-third of those in normal human cells. These results indicate that a gene on chromosome 9 can confer complementation of high UV sensitivity of XP-A cells although it is still possible that 2 or more genes might be involved in the defective-repair phenotypes of XP-A. (author). 20 refs.; 3 figs.; 1 tab

  18. Human TM9SF4 Is a New Gene Down-Regulated by Hypoxia and Involved in Cell Adhesion of Leukemic Cells.

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    Rosa Paolillo

    Full Text Available The transmembrane 9 superfamily protein member 4, TM9SF4, belongs to the TM9SF family of proteins highly conserved through evolution. TM9SF4 homologs, previously identified in many different species, were mainly involved in cellular adhesion, innate immunity and phagocytosis. In human, the function and biological significance of TM9SF4 are currently under investigation. However, TM9SF4 was found overexpressed in human metastatic melanoma and in a small subset of acute myeloid leukemia (AMLs and myelodysplastic syndromes, consistent with an oncogenic function of this gene.In this study, we first analyzed the expression and regulation of TM9SF4 in normal and leukemic cells and identified TM9SF4 as a gene highly expressed in human quiescent CD34+ hematopoietic progenitor cells (HPCs, regulated during monocytic and granulocytic differentiation of HPCs, both lineages giving rise to mature myeloid cells involved in adhesion, phagocytosis and immunity. Then, we found that TM9SF4 is markedly overexpressed in leukemic cells and in AMLs, particularly in M2, M3 and M4 AMLs (i.e., in AMLs characterized by the presence of a more or less differentiated granulocytic progeny, as compared to normal CD34+ HPCs. Proliferation and differentiation of HPCs occurs in hypoxia, a physiological condition in bone marrow, but also a crucial component of cancer microenvironment. Here, we investigated the impact of hypoxia on TM9SF4 expression in leukemic cells and identified TM9SF4 as a direct target of HIF-1α, downregulated in these cells by hypoxia. Then, we found that the hypoxia-mediated downregulation of TM9SF4 expression is associated with a decrease of cell adhesion of leukemic cells to fibronectin, thus demonstrating that human TM9SF4 is a new molecule involved in leukemic cell adhesion.Altogether, our study reports for the first time the expression of TM9SF4 at the level of normal and leukemic hematopoietic cells and its marked expression at the level of AMLs

  19. Impact of dual-time-point F-18 FDG PET/CT in the assessment of pleural effusion in patients with non-small-cell lung cancer.

    Science.gov (United States)

    Alkhawaldeh, Khaled; Biersack, Hans-J; Henke, Anna; Ezziddin, Samer

    2011-06-01

    The aim of this study was to assess the utility of dual-time-point F-18 fluorodeoxyglucose positron emission tomography (F-18 FDG PET) in differentiating benign from malignant pleural disease, in patients with non-small-cell lung cancer. A total of 61 patients with non-small-cell lung cancer and pleural effusion were included in this retrospective study. All patients had whole-body FDG PET/CT imaging at 60 ± 10 minutes post-FDG injection, whereas 31 patients had second-time delayed imaging repeated at 90 ± 10 minutes for the chest. Maximum standardized uptake values (SUV(max)) and the average percent change in SUV(max) (%SUV) between time point 1 and time point 2 were calculated. Malignancy was defined using the following criteria: (1) visual assessment using 3-points grading scale; (2) SUV(max) ≥2.4; (3) %SUV ≥ +9; and (4) SUV(max) ≥2.4 and/or %SUV ≥ +9. Analysis of variance test and receiver operating characteristic analysis were used in statistical analysis. P < 0.05 was considered significant. Follow-up revealed 29 patient with malignant pleural disease and 31 patients with benign pleural effusion. The average SUV(max) in malignant effusions was 6.5 ± 4 versus 2.2 ± 0.9 in benign effusions (P < 0.0001). The average %SUV in malignant effusions was +13 ± 10 versus -8 ± 11 in benign effusions (P < 0.0004). Sensitivity, specificity, and accuracy for the 5 criteria were as follows: (1) 86%, 72%, and 79%; (2) 93%, 72%, and 82%; (3) 67%, 94%, and 81%; (4) 100%, 94%, and 97%. Dual-time-point F-18 FDG PET can improve the diagnostic accuracy in differentiating benign from malignant pleural disease, with high sensitivity and good specificity.

  20. Matrix metalloproteinase-9 is involved in chronic lymphocytic leukemia cell response to fludarabine and arsenic trioxide.

    Directory of Open Access Journals (Sweden)

    Irene Amigo-Jiménez

    Full Text Available Matrix metalloproteinase-9 (MMP-9 contributes to chronic lymphocytic leukemia (CLL pathology by regulating cell migration and preventing spontaneous apoptosis. It is not known if MMP-9 is involved in CLL cell response to chemotherapy and we address this in the present study, using arsenic trioxide (ATO and fludarabine as examples of cytotoxic drugs.We used primary cells from the peripheral blood of CLL patients and MEC-1 cells stably transfected with an empty vector or a vector containing MMP-9. The effect of ATO and fludarabine was determined by flow cytometry and by the MTT assay. Expression of mRNA was measured by RT-PCR and qPCR. Secreted and cell-bound MMP-9 was analyzed by gelatin zymography and flow cytometry, respectively. Protein expression was analyzed by Western blotting and immunoprecipitation. Statistical analyses were performed using the two-tailed Student's t-test.In response to ATO or fludarabine, CLL cells transcriptionally upregulated MMP-9, preceding the onset of apoptosis. Upregulated MMP-9 primarily localized to the membrane of early apoptotic cells and blocking apoptosis with Z-VAD prevented MMP-9 upregulation, thus linking MMP-9 to the apoptotic process. Culturing CLL cells on MMP-9 or stromal cells induced drug resistance, which was overcome by anti-MMP-9 antibodies. Accordingly, MMP-9-MEC-1 transfectants showed higher viability upon drug treatment than Mock-MEC-1 cells, and this effect was blocked by silencing MMP-9 with specific siRNAs. Following drug exposure, expression of anti-apoptotic proteins (Mcl-1, Bcl-xL, Bcl-2 and the Mcl-1/Bim, Mcl-1/Noxa, Bcl-2/Bax ratios were higher in MMP-9-cells than in Mock-cells. Similar results were obtained upon culturing primary CLL cells on MMP-9.Our study describes for the first time that MMP-9 induces drug resistance by modulating proteins of the Bcl-2 family and upregulating the corresponding anti-apoptotic/pro-apoptotic ratios. This is a novel role for MMP-9 contributing to CLL

  1. Measurements of red cell deformability and hydration reflect HbF and HbA2 in blood from patients with sickle cell anemia.

    Science.gov (United States)

    Parrow, Nermi L; Tu, Hongbin; Nichols, James; Violet, Pierre-Christian; Pittman, Corinne A; Fitzhugh, Courtney; Fleming, Robert E; Mohandas, Narla; Tisdale, John F; Levine, Mark

    2017-06-01

    Decreased erythrocyte deformability, as measured by ektacytometry, may be associated with disease severity in sickle cell anemia (SCA). Heterogeneous populations of rigid and deformable cells in SCA blood result in distortions of diffraction pattern measurements that correlate with the concentration of hemoglobin S (HbS) and the percentage of irreversibly sickled cells. We hypothesize that red cell heterogeneity, as well as deformability, will also be influenced by the concentration of alternative hemoglobins such as fetal hemoglobin (HbF) and the adult variant, HbA 2 . To test this hypothesis, we investigate the relationship between diffraction pattern distortion, osmotic gradient ektacytometry parameters, and the hemoglobin composition of SCA blood. We observe a correlation between the extent of diffraction pattern distortions and percentage of HbF and HbA 2 . Osmotic gradient ektacytometry data indicate that minimum elongation in the hypotonic region is positively correlated with HbF, as is the osmolality at which it occurs. The osmolality at both minimum and maximum elongation is inversely correlated with HbS and HbA 2 . These data suggest that HbF may effectively improve surface-to-volume ratio and osmotic fragility in SCA erythrocytes. HbA 2 may be relatively ineffective in improving these characteristics or cellular hydration at the levels found in this patient cohort. Copyright © 2017. Published by Elsevier Inc.

  2. Combinatorial study of NaF addition in CIGSe films for high efficiency solar cells

    KAUST Repository

    Eid, Jessica; Liang, Haifan; Gereige, Issam; Lee, Sang; Van Duren, Jeroen K J

    2013-01-01

    We report on a sodium fluoride (NaF) thickness variation study for the H2Se batch furnace selenization of sputtered Cu(In,Ga) films in a wide range of Cu(In,Ga) film compositions to form Cu(In,Ga)Se2 (CIGSe) films and solar cells. Literature review indicates lack of consensus on the mechanisms involved in Na altering CIGSe film properties. In this work, for sputtered and batch-selenized CIGSe, NaF addition results in reduced gallium content and an increase in grain size for the top portion of the CIGSe film, as observed by scanning electron microscopy and secondary ion mass spectrometry. The addition of up to 20nm of NaF resulted in an improvement in all relevant device parameters: open-circuit voltage, short-circuit current, and fill factor. The best results were found for 15nm NaF addition, resulting in solar cells with 16.0% active-area efficiency (without anti-reflective coating) at open-circuit voltage (VOC) of 674mV. © 2013 John Wiley & Sons, Ltd.

  3. Combinatorial study of NaF addition in CIGSe films for high efficiency solar cells

    KAUST Repository

    Eid, Jessica

    2013-12-04

    We report on a sodium fluoride (NaF) thickness variation study for the H2Se batch furnace selenization of sputtered Cu(In,Ga) films in a wide range of Cu(In,Ga) film compositions to form Cu(In,Ga)Se2 (CIGSe) films and solar cells. Literature review indicates lack of consensus on the mechanisms involved in Na altering CIGSe film properties. In this work, for sputtered and batch-selenized CIGSe, NaF addition results in reduced gallium content and an increase in grain size for the top portion of the CIGSe film, as observed by scanning electron microscopy and secondary ion mass spectrometry. The addition of up to 20nm of NaF resulted in an improvement in all relevant device parameters: open-circuit voltage, short-circuit current, and fill factor. The best results were found for 15nm NaF addition, resulting in solar cells with 16.0% active-area efficiency (without anti-reflective coating) at open-circuit voltage (VOC) of 674mV. © 2013 John Wiley & Sons, Ltd.

  4. Expression of matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinases 1 (TIMP-1) by colorectal cancer cells and adjacent stroma cells - associations with histopathology and patients outcome

    DEFF Research Database (Denmark)

    Jensen, Søren Astrup; Vainer, Ben; Bartels, Annette

    2010-01-01

    AIM: To elucidate cellular features accountable for colorectal cancers' (CRC) capability to invade normal tissue and to metastasize, we investigated the level of the collagenase matrix metalloproteinase 9 (MMP-9) and its physiological inhibitor tissue inhibitor of metalloproteinases 1 (TIMP-1) in...... cells is associated with poor prognosis independent of its function as inhibitor of MMP-9. MMP-9 and TIMP-1 are important mediators of the host-cancer cell interaction in the tumour microenvironment with significant influence on the histopathology and on prognosis of CRC....

  5. Research of TGF-beta1 Inducing Lung Adencarcinoma PC9 Cells to Mesenchymal Cells Transition

    Directory of Open Access Journals (Sweden)

    Xiaofeng CHEN

    2010-01-01

    Full Text Available Background and objective It has been proven that epithelial-mesenchymal transition (EMT not only correlated with embryonic development but also could promote tumor invasion and metastasis. Transforming growth factor beta-1 (TGF-β1 has been identified as the main inducer of tumor EMT. The aim of this study was to investigate the effects of TGF-β1 on EMT and PI3K/AKT signaling pathway in lung adencarcinoma PC9 cells. Methods Cultured PC9 cells were treated with different concentrations of TGF-β1 for 48 h. The morphological changes were observed under phase-contrast microscopy; EMT relative marker protein changes were assessed by Western blot and immunoflurescence staining. In addition, the expression of AKT and P-AKT were also measured by Western blot. Results The data showed that TGF-β1 could induce PC9 morphological alteration from epithelial to mesenchymal and upregulate the expression of mesenchymal maker protein Fibronectin. Obviously, the expression of P-AKT was downregulated by TGF-β1 treatment for 48 h. Conclusion TGF-β1 might induce EMT of PC9 cells , accompanied by the changes of PI3K/AKT signaling pathway.

  6. RhCd{sub 9+δ} (-1.18 ≤δ≤0.29) a γ-brass related cubic giant cell structure

    Energy Technology Data Exchange (ETDEWEB)

    Jana, Partha Pratim [Indian Institute of Technology, Kharagpur (India). Dept. of Chemistry

    2017-09-01

    The compound RhCd{sub 9+δ} (-1.18 ≤δ≤0.29) has been synthesized and the average structure has been analyzed by single crystal X-ray diffraction. The average structure crystallizes in the face centered cubic space group F43m (216) and contains ∝405 atoms/unit cell. It represents a (2a{sub γ}){sup 3}-superstructure of cubic γ-brass and is isostructural to Rh{sub 7-x}Mg{sub 44+x}. The comparison between the structures of RhCd{sub 9+δ} and Rh{sub 7-x}Mg{sub 44+x} has been presented using a layer description. The structure of the title phase has also been described by a ''cluster'' concept. The electronic structure of RhCd{sub 9+δ} (-1.18 ≤δ≤0.29) shows that the phase is stabilized by a Hume-Rothery mechanism.

  7. 26 CFR 5c.168(f)(8)-9 - Pass-through leases-transfer of only the investment tax credit to a party other than the ultimate...

    Science.gov (United States)

    2010-04-01

    ... investment tax credit to a party other than the ultimate user of the property. [Reserved] 5c.168(f)(8)-9...) INCOME TAX (CONTINUED) TEMPORARY INCOME TAX REGULATIONS UNDER THE ECONOMIC RECOVERY TAX ACT OF 1981 § 5c.168(f)(8)-9 Pass-through leases—transfer of only the investment tax credit to a party other than the...

  8. Spermatogenic Cell-Specific Gene Mutation in Mice via CRISPR-Cas9.

    Science.gov (United States)

    Bai, Meizhu; Liang, Dan; Wang, Yinghua; Li, Qing; Wu, Yuxuan; Li, Jinsong

    2016-05-20

    Tissue-specific knockout technology enables the analysis of the gene function in specific tissues in adult mammals. However, conventional strategy for producing tissue-specific knockout mice is a time- and labor-consuming process, restricting rapid study of the gene function in vivo. CRISPR-Cas9 system from bacteria is a simple and efficient gene-editing technique, which has enabled rapid generation of gene knockout lines in mouse by direct injection of CRISPR-Cas9 into zygotes. Here, we demonstrate CRISPR-Cas9-mediated spermatogenic cell-specific disruption of Scp3 gene in testes in one step. We first generated transgenic mice by pronuclear injection of a plasmid containing Hspa2 promoter driving Cas9 expression and showed Cas9 specific expression in spermatogenic cells. We then produced transgenic mice carrying Hspa2 promoter driven Cas9 and constitutive expressed sgRNA targeting Scp3 gene. Male founders were infertile due to developmental arrest of spermatogenic cells while female founders could produce progeny normally. Consistently, male progeny from female founders were infertile and females could transmit the transgenes to the next generation. Our study establishes a CRISPR-Cas9-based one-step strategy to analyze the gene function in adult tissues by a temporal-spatial pattern. Copyright © 2016 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

  9. New cascade laser transitions in CH2F2 pumped with the 9R32 line of a cw CO2 laser

    International Nuclear Information System (INIS)

    Nieswand, C.

    1991-11-01

    New cascade laser transitions of 12 CH 2 F 2 at 172.50μm, 208.83μm, 220.44μm, 223.99μm,and 250.61μm are reported. A waveguide FIR laser was pumped with a quasi cw 12 C 16 O 2 laser operating on the 9R32 line. Together with the already known lines at 184.3μm, 196.1μm and 235.9μm, the laser lines can be assigned to rotational transitions in the ν 9 vibrational band of 12 CH 2 F 2 and to refill transitions of the vibrational ground state ν 0 . 1 fig., 2 tabs., 6 refs. (author)

  10. Diagnostic usefulness of {sup 18}F-FAMT PET and L-type amino acid transporter 1 (LAT1) expression in oral squamous cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Nobusawa, Aiko [Gunma University Graduate School of Medicine, Department of Stomatology and Maxillofacial Surgery, Maebashi, Gunma (Japan); Gunma University Graduate School of Medicine, Department of Diagnostic Pathology, Maebashi, Gunma (Japan); Kim, Mai [Gunma University Graduate School of Medicine, Department of Stomatology and Maxillofacial Surgery, Maebashi, Gunma (Japan); Gunma University Graduate School of Medicine, Department of Diagnostic Radiology and Nuclear Medicine, Maebashi, Gunma (Japan); Kaira, Kyoichi [Gunma University Graduate School of Medicine, Department of Diagnostic Pathology, Maebashi, Gunma (Japan); Gunma University Hospital, Oncology Center, Maebashi, Gunma (Japan); Miyashita, Go; Negishi, Akihide; Yokoo, Satoshi [Gunma University Graduate School of Medicine, Department of Stomatology and Maxillofacial Surgery, Maebashi, Gunma (Japan); Oriuchi, Noboru; Higuchi, Tetsuya; Tsushima, Yoshito [Gunma University Graduate School of Medicine, Department of Diagnostic Radiology and Nuclear Medicine, Maebashi, Gunma (Japan); Kanai, Yoshikatsu [Osaka University, Division of Bio-system Pharmacology, Graduate School of Medicine, Osaka (Japan); Oyama, Tetsunari [Gunma University Graduate School of Medicine, Department of Diagnostic Pathology, Maebashi, Gunma (Japan)

    2013-10-15

    l-[3-{sup 18}F]-{alpha}-Methyltyrosine ({sup 18}F-FAMT) was developed as an amino acid tracer for PET imaging to provide better specificity than 2-[{sup 18}F]fluoro-2-deoxy-d-glucose ({sup 18}F-FDG) PET for cancer diagnosis. We investigated the diagnostic usefulness of {sup 18}F-FAMT in oral squamous cell carcinoma (OSCC). The correlation between tumour uptake of {sup 18}F-FAMT and L-type amino acid transporter 1 (LAT1) expression was determined. The study group comprised 68 OSCC patients who underwent both {sup 18}F-FAMT and {sup 18}F-FDG PET. Resected tumour sections were stained by immunohistochemistry for LAT1, CD98 and Ki-67, and microvessel density was determined in terms of CD34 and p53 expression. The sensitivity of primary tumour detection by {sup 18}F-FAMT and {sup 18}F-FDG PET was 98 % and 100 %, respectively. The sensitivity, specificity and accuracy of {sup 18}F-FAMT PET for detecting malignant lymph nodes were 68 %, 99 % and 97 %, respectively, and equivalent values for {sup 18}F-FDG PET were 84 %, 94 % and 94 %, respectively. The specificity and accuracy of {sup 18}F-FAMT were significantly higher than those of {sup 18}F-FDG. The uptake of {sup 18}F-FAMT was significantly correlated with LAT1 expression, cell proliferation and advanced stage. The expression of LAT1 in OSCC cells was closely correlated with CD98 levels, cell proliferation and angiogenesis. {sup 18}F-FAMT PET showed higher specificity for detecting malignant lesions than {sup 18}F-FDG PET. The uptake of {sup 18}F-FAMT by OSCC cells can be determined by the presence of LAT1 expression and tumour cell proliferation. (orig.)

  11. Both flagella and F4 fimbriae from F4ac+ enterotoxigenic Escherichia coli contribute to attachment to IPEC-J2 cells in vitro.

    Science.gov (United States)

    Zhou, Mingxu; Duan, Qiangde; Zhu, Xiaofang; Guo, Zhiyan; Li, Yinchau; Hardwidge, Philip R; Zhu, Guoqiang

    2013-05-13

    The role of flagella in the pathogenesis of F4ac+ Enterotoxigenic Escherichia coli (ETEC) mediated neonatal and post-weaning diarrhea (PWD) is not currently understood. We targeted the reference C83902 ETEC strain (O8:H19:F4ac+ LT+ STa+ STb+), to construct isogenic mutants in the fliC (encoding the major flagellin protein), motA (encoding the flagella motor), and faeG (encoding the major subunit of F4 fimbriae) genes. Both the ΔfliC and ΔfaeG mutants had a reduced ability to adhere to porcine intestinal epithelial IPEC-J2 cells. F4 fimbriae expression was significantly down-regulated after deleting fliC, which revealed that co-regulation exists between flagella and F4 fimbriae. However, there was no difference in adhesion between the ΔmotA mutant and its parent strain. These data demonstrate that both flagella and F4 fimbriae are required for efficient F4ac+ ETEC adhesion in vitro.

  12. [Efficient genome editing in human pluripotent stem cells through CRISPR/Cas9].

    Science.gov (United States)

    Liu, Gai-gai; Li, Shuang; Wei, Yu-da; Zhang, Yong-xian; Ding, Qiu-rong

    2015-11-01

    The RNA-guided CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas9 nuclease has offered a new platform for genome editing with high efficiency. Here, we report the use of CRISPR/Cas9 technology to target a specific genomic region in human pluripotent stem cells. We show that CRISPR/Cas9 can be used to disrupt a gene by introducing frameshift mutations to gene coding region; to knock in specific sequences (e.g. FLAG tag DNA sequence) to targeted genomic locus via homology directed repair; to induce large genomic deletion through dual-guide multiplex. Our results demonstrate the versatile application of CRISPR/Cas9 in stem cell genome editing, which can be widely utilized for functional studies of genes or genome loci in human pluripotent stem cells.

  13. Li{sub 35}Ln{sub 9}Si{sub 30}N{sub 59}O{sub 2}F with Ln = Ce, Pr - highly condensed nitridosilicates

    Energy Technology Data Exchange (ETDEWEB)

    Lupart, Saskia; Durach, Dajana; Schnick, Wolfgang [Department Chemie, Lehrstuhl fuer Anorganische Festkoerperchemie, Ludwig-Maximilians-Universitaet Muenchen (Germany)

    2011-10-15

    The isotypic nitridosilicates Li{sub 35}Ln{sub 9}Si{sub 30}N{sub 59}O{sub 2}F (Ln = Ce, Pr) were synthesized by reaction of LnF{sub 3} and LiN{sub 3} with Si(NH){sub 2} in liquid lithium flux in weld shut tantalum ampoules. The crystal structures of the isotypic compounds were solved and refined on the basis of single-crystal X-ray diffraction (P anti 3c1 (no. 165), Z = 2; Li{sub 35}Ce{sub 9}Si{sub 30}N{sub 59}O{sub 2}F:, a = 1479.9(2), c = 1538.3(3) pm, R{sub 1} = 0.0526, 1671 data, 175 parameters; Li{sub 35}Pr{sub 9}Si{sub 30}N{sub 59}O{sub 2}F: a = 1477.3(2), c = 1533.9(3) pm, R{sub 1} = 0.0441, 1331 data, 175 parameters). The silicate substructure represents a 3D network of all side corner sharing SiN{sub 4} tetrahedra. At one discrete and not condensed mixed anion position an atomic ratio O:F = 2:1 is assumed in order to achieve charge neutrality. With an atomic ratio Si:N = 30:59, the degree of condensation of the silicate substructure is slightly above κ = 1/2. Accordingly, there are triply crosslinking N{sup [3]} atoms in the silicate substructure. The obtained structures prove that by employing the lithium flux technique not only nitridosilicates with a low degree of condensation can be obtained by using rather mild reaction conditions at low temperatures. Lattice energy calculations (MAPLE) and EDX measurements confirmed the electrostatic bonding interactions and the chemical composition. (Copyright copyright 2011 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

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

    Science.gov (United States)

    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.

  15. Modulation of the E2F1-driven cancer cell fate by the DNA damage response machinery and potential novel E2F1 targets in osteosarcomas

    DEFF Research Database (Denmark)

    Liontos, Michalis; Niforou, Katerina; Velimezi, Georgia

    2009-01-01

    Osteosarcoma is the most common primary bone cancer. Mutations of the RB gene represent the most frequent molecular defect in this malignancy. A major consequence of this alteration is that the activity of the key cell cycle regulator E2F1 is unleashed from the inhibitory effects of pRb. Studies...... in a clinical setting of human primary osteosarcomas and in E2F1-inducible osteosarcoma cell line models that are wild-type and deficient for p53. Collectively, our data demonstrated that high E2F1 levels exerted a growth-suppressing effect that relied on the integrity of the DNA damage response network...

  16. Leu-9 (CD 7) positivity in acute leukemias: a marker of T-cell lineage?

    Science.gov (United States)

    Ben-Ezra, J; Winberg, C D; Wu, A; Rappaport, H

    1987-01-01

    Monoclonal antibody Leu-9 (CD 7) has been reported to be a sensitive and specific marker for T-cell lineage in leukemic processes, since it is positive in patients whose leukemic cells fail to express other T-cell antigens. To test whether Leu-9 is indeed specific for T-cell leukemias, we examined in detail 10 cases of acute leukemia in which reactions were positive for Leu-9 and negative for other T-cell-associated markers including T-11, Leu-1, T-3, and E-rosettes. Morphologically and cytochemically, 2 of these 10 leukemias were classified as lymphoblastic, 4 as myeloblastic, 2 as monoblastic, 1 as megakaryoblastic, and 1 as undifferentiated. The case of acute megakaryoblastic leukemia is the first reported case to be Leu-9 positive. None of the 10 were TdT positive. Of six cases (two monoblastic, one lymphoblastic, one myeloblastic, one megakaryoblastic, and one undifferentiated) in which we evaluated for DNA gene rearrangements, only one, a peroxidase-positive leukemia, showed a novel band on study of the T-cell-receptor beta-chain gene. We therefore conclude that Leu-9 is not a specific marker to T-cell lineage and that, in the absence of other supporting data, Leu-9 positivity should not be used as the sole basis of classifying an acute leukemia as being T-cell derived.

  17. Type 1 Responses of Human Vγ9Vδ2 T Cells to Influenza A Viruses▿

    Science.gov (United States)

    Qin, Gang; Liu, Yinping; Zheng, Jian; Ng, Iris H. Y.; Xiang, Zheng; Lam, Kwok-Tai; Mao, Huawei; Li, Hong; Peiris, J. S. Malik; Lau, Yu-Lung; Tu, Wenwei

    2011-01-01

    γδ T cells are essential constituents of antimicrobial and antitumor defenses. We have recently reported that phosphoantigen isopentenyl pyrophosphate (IPP)-expanded human Vγ9Vδ2 T cells participated in anti-influenza virus immunity by efficiently killing both human and avian influenza virus-infected monocyte-derived macrophages (MDMs) in vitro. However, little is known about the noncytolytic responses and trafficking program of γδ T cells to influenza virus. In this study, we found that Vγ9Vδ2 T cells expressed both type 1 cytokines and chemokine receptors during influenza virus infection, and IPP-expanded cells had a higher capacity to produce gamma interferon (IFN-γ). Besides their potent cytolytic activity against pandemic H1N1 virus-infected cells, IPP-activated γδ T cells also had noncytolytic inhibitory effects on seasonal and pandemic H1N1 viruses via IFN-γ but had no such effects on avian H5N1 or H9N2 virus. Avian H5N1 and H9N2 viruses induced significantly higher CCL3, CCL4, and CCL5 production in Vγ9Vδ2 T cells than human seasonal H1N1 virus. CCR5 mediated the migration of Vγ9Vδ2 T cells toward influenza virus-infected cells. Our findings suggest a novel therapeutic strategy of using phosphoantigens to boost the antiviral activities of human Vγ9Vδ2 T cells against influenza virus infection. PMID:21752902

  18. Promising Noninvasive Cellular Phenotype in Prostate Cancer Cells Knockdown of Matrix Metalloproteinase 9

    Directory of Open Access Journals (Sweden)

    Aditi Gupta

    2013-01-01

    Full Text Available Cell surface interaction of CD44 and MMP9 increases migration and invasion of PC3 cells. We show here that stable knockdown of MMP9 in PC3 cells switches CD44 isoform expression from CD44s to CD44v6 which is more glycosylated. These cells showed highly adhesive morphology with extensive cell spreading which is due to the formation of focal adhesions and well organized actin-stress fibers. MMP9 knockdown blocks invadopodia formation and matrix degradation activity as well. However, CD44 knockdown PC3 cells failed to develop focal adhesions and stress fibers; hence these cells make unstable adhesions. A part of the reason for these changes could be caused by silencing of CD44v6 as well. Immunostaining of prostate tissue microarray sections illustrated significantly lower levels of CD44v6 in adenocarcinoma than normal tissue. Our results suggest that interaction between CD44 and MMP9 is a potential mechanism of invadopodia formation. CD44v6 expression may be essential for the protection of non-invasive cellular phenotype. CD44v6 decrease may be a potential marker for prognosis and therapeutics.

  19. Giant cell tumor of the rib: Two cases of F-18 FDG PET/CT findings

    Energy Technology Data Exchange (ETDEWEB)

    Park, Hye Lim; Yoo, Le Ryung; Lee, Yeong Joo; Jung, Chan Kwon [Seoul St. Mary' s Hospital, College of MedicineThe Catholic University of Korea, Seoul (Korea, Republic of); Park, Sonya Young Ju [Molecular Imaging Program, Dept. of Radiology, Stanford Hospital and Clinics, Stanford (Korea, Republic of)

    2017-06-15

    We report two cases of giant cell tumor arising from the rib and their F-18 FDG PET/CT findings. The two patients complained of chest wall pain, and large lobulated soft tissue masses with intense FDG uptake were seen on F-18 FDG PET/CT. A malignant tumor such as osteosarcoma or chondrosarcoma was suspected due to the large size of the mass, bony destruction, and intense FDG uptake. En bloc resection was performed and final pathologic results revealed giant cell tumor of the rib. Giant cell tumor of the rib is very rare, and larger lesions with high FDG uptake can be misdiagnosed as an intrathoracic malignancy arising from the rib, pleura, or chest wall.

  20. Coronin 3 involvement in F-actin-dependent processes at the cell cortex

    International Nuclear Information System (INIS)

    Rosentreter, Andre; Hofmann, Andreas; Xavier, Charles-Peter; Stumpf, Maria; Noegel, Angelika A.; Clemen, Christoph S.

    2007-01-01

    The actin interaction of coronin 3 has been mainly documented by in vitro experiments. Here, we discuss coronin 3 properties in the light of new structural information and focus on assays that reflect in vivo roles of coronin 3 and its impact on F-actin-associated functions. Using GFP-tagged coronin 3 fusion proteins and RNAi silencing we show that coronin 3 has roles in wound healing, protrusion formation, cell proliferation, cytokinesis, endocytosis, axonal growth, and secretion. During formation of cell protrusions actin accumulation precedes the focal enrichment of coronin 3 suggesting a role for coronin 3 in events that follow the initial F-actin assembly. Moreover, we show that coronin 3 similar to other coronins interacts with the Arp2/3-complex and cofilin indicating that this family in general is involved in regulating Arp2/3-mediated events

  1. Characteristics of Metastatic Mediastinal Lymph Nodes of Non-Small Cell Lung Cancer on Preoperative F-18 FDG PET/CT

    International Nuclear Information System (INIS)

    Lee, Ah Young; Choi, Su Jung; Jung, Kyung Pyo; Park, Ji Sun; Lee, Seok Mo; Bae, Sang Kyun

    2014-01-01

    The aim of this study was to evaluate the characteristics of PET and CT features of mediastinal metastatic lymph nodes on F-18 FDG PET/CT and to determine the diagnostic criteria in nodal staging of non-small cell lung cancer. One hundred four non-small cell lung cancer patients who had preoperative F-18 FDG PET/CT were included. For quantitative analysis, the maximum SUV of the primary tumor, maximum SUV of the lymph nodes (SUVmax), size of the lymph nodes, and average Hounsfield units (aHUs) and maximum Hounsfield units (mHUs) of the lymph nodes were measured. The SUVmax, SUV ratio of the lymph node to blood pool (LN SUV/blood pool SUV), SUV ratio of the lymph node to primary tumor (LN SUV/primary tumor SUV), size, aHU, and mHU were compared between the benign and malignant lymph nodes. Among 372 dissected lymph node stations that were pathologically diagnosed after surgery, 49 node stations were malignant and 323 node stations benign. SUVmax, LN SUV/blood pool SUV, and size were significantly different between the malignant and benign lymph node stations (P <0.0001). However, there was no significant difference in LN SUV/primary tumor SUV (P =0.18), mHU (P =0.42), and aHU (P =0.98). Using receiver-operating characteristic curve analyses, there was no significant difference among these three variables (SUVmax, LN SUV/blood pool SUV, and size). The optimal cutoff values were 2.9 for SUVmax, 1.4 for LN SUV/blood pool SUV, and 5 mm for size. When the cutoff value of SUVmax≥2.9 and size≥5 mm were used in combination, the positive predictive value was 44.2%, and the negative predictive value was 90.9 %. When we evaluated the results based on the histology of the primary tumor, the negative predictive value was 92.3 % in adenocarcinoma (cutoff values of SUVmax≥2.3 and size≥5 mm) and 97.2 % in squamous cell carcinoma (cutoff values of SUVmax≥3.6 and size≥8 mm), separately. In the lymph node staging of non-small cell lung cancer, SUVmax, LN SUV/blood pool SUV

  2. 9-β-arabinofuranosyladenine preferentially sensitizes radioresistant squamous cell carcinoma cell lines to x-rays

    International Nuclear Information System (INIS)

    Heaton, D.

    1992-06-01

    The effect of 9-β-arabinofuranosyladenine (ara-A) on sensitivity to the deleterious effects of x-rays was studied in six squamous cell carcinoma cell lines. Three lines were relatively radioresistant, having D 0 values of 2.31 to 2.89 Gy, and the other three lines were relatively radiosensitive, having D 0 values of between 1.07 and 1.45 Gy. Ara-A (50 or 500 μM) was added to cultures 30 min prior to irradiation and removed 30 min after irradiation, and sensitivity was measured in terms of cell survival. The radiosensitizing effect of ara-A was very dependent on the inherent radiosensitivity of the tumor cell line. Fifty micromolar concentrations of ara-A sensitized only the two most radioresistant lines, SCC-12B.2 and JSQ-3. Five hundred micromolar concentrations of ara-A sensitized the more sensitive cell lines, SQ-20B and SQ-9G, but failed to have any effect on the radiation response of the two most sensitive cell lines, SQ-38 and SCC-61. Concentrations of ara-A as low as 10 μM were equally efficient in inhibiting DNA synthesis in all six cell lines. These results suggest that the target for the radiosensitizing effect of ara-A is probably related to the factor controlling the inherent radiosensitivity of human tumor cells. Therefore, ara-A might be useful in overcoming radiation resistance in vivo

  3. 9-{beta}-arabinofuranosyladenine preferentially sensitizes radioresistant squamous cell carcinoma cell lines to x-rays

    Energy Technology Data Exchange (ETDEWEB)

    Heaton, D. [Rush Univ. Medical Center, Chicago, IL (United States). Therapeutic Radiology; Mustafi, R. [Chicago Univ., IL (United States). Dept. of Radiation and Cellular Oncology; Schwartz, J.L. [Chicago Univ., IL (United States). Dept. of Radiation and Cellular Oncology]|[Argonne National Lab., IL (United States)

    1992-06-01

    The effect of 9-{beta}-arabinofuranosyladenine (ara-A) on sensitivity to the deleterious effects of x-rays was studied in six squamous cell carcinoma cell lines. Three lines were relatively radioresistant, having D{sub 0} values of 2.31 to 2.89 Gy, and the other three lines were relatively radiosensitive, having D{sub 0} values of between 1.07 and 1.45 Gy. Ara-A (50 or 500 {mu}M) was added to cultures 30 min prior to irradiation and removed 30 min after irradiation, and sensitivity was measured in terms of cell survival. The radiosensitizing effect of ara-A was very dependent on the inherent radiosensitivity of the tumor cell line. Fifty micromolar concentrations of ara-A sensitized only the two most radioresistant lines, SCC-12B.2 and JSQ-3. Five hundred micromolar concentrations of ara-A sensitized the more sensitive cell lines, SQ-20B and SQ-9G, but failed to have any effect on the radiation response of the two most sensitive cell lines, SQ-38 and SCC-61. Concentrations of ara-A as low as 10 {mu}M were equally efficient in inhibiting DNA synthesis in all six cell lines. These results suggest that the target for the radiosensitizing effect of ara-A is probably related to the factor controlling the inherent radiosensitivity of human tumor cells. Therefore, ara-A might be useful in overcoming radiation resistance in vivo.

  4. HOXA9 inhibits migration of lung cancer cells and its hypermethylation is associated with recurrence in non-small cell lung cancer.

    Science.gov (United States)

    Hwang, Jung-Ah; Lee, Bo Bin; Kim, Yujin; Hong, Seung-Hyun; Kim, Young-Ho; Han, Joungho; Shim, Young Mog; Yoon, Chae-Yeong; Lee, Yeon-Su; Kim, Duk-Hwan

    2015-06-01

    This study was aimed at understanding the clinicopathological significance of HOXA9 hypermethylation in non-small cell lung cancer (NSCLC). HOXA9 hypermethylation was characterized in six lung cancer cell lines, and its clinicopathological significance was analyzed using methylation-specific PCR in 271 formalin-fixed paraffin-embedded tissues and 27 fresh-frozen tumor and matched normal tissues from 298 NSCLC patients, and Ki-67 expression was analyzed using immunohistochemistry. The promoter region of HOXA9 was highly methylated in six lung cancer cell lines, but not in normal bronchial epithelial cells. The loss of expression was restored by treatment of the cells with a demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC). Transient transfection of HOXA9 into H23 lung cancer cells resulted in the inhibition of cell migration but not proliferation. Conversely, sequence-specific siRNA-mediated knockdown of HOXA9 enhanced cell migration. The mRNA levels of HOXA9 in 27 fresh-frozen tumor tissues were significantly lower than in matched normal tissues (Precurrence-free survival (hazard ratio=3.98, 95% confidence interval = 1.07-17.09, P=0.01) in never-smokers, after adjusting for age, sex, tumor size, adjuvant therapy, pathologic stage, and histology. In conclusion, the present study suggests that HOXA9 inhibits migration of lung cancer cells and its hypermethylation is an independent prognostic factor for recurrence-free survival in never-smokers with NSCLC. © 2014 Wiley Periodicals, Inc.

  5. JAK2V617F expression in mice amplifies early hematopoietic cells and gives them a competitive advantage that is hampered by IFNα.

    Science.gov (United States)

    Hasan, Salma; Lacout, Catherine; Marty, Caroline; Cuingnet, Marie; Solary, Eric; Vainchenker, William; Villeval, Jean-Luc

    2013-08-22

    The acquired gain-of-function V617F mutation in the Janus Kinase 2 (JAK2(V617F)) is the main mutation involved in BCR/ABL-negative myeloproliferative neoplasms (MPNs), but its effect on hematopoietic stem cells as a driver of disease emergence has been questioned. Therefore, we reinvestigated the role of endogenous expression of JAK2(V617F) on early steps of hematopoiesis as well as the effect of interferon-α (IFNα), which may target the JAK2(V617F) clone in humans by using knock-in mice with conditional expression of JAK2(V617F) in hematopoietic cells. These mice develop a MPN mimicking polycythemia vera with large amplification of myeloid mature and precursor cells, displaying erythroid endogenous growth and progressing to myelofibrosis. Interestingly, early hematopoietic compartments [Lin-, LSK, and SLAM (LSK/CD48-/CD150+)] increased with the age. Competitive repopulation assays demonstrated disease appearance and progressive overgrowth of myeloid, Lin-, LSK, and SLAM cells, but not lymphocytes, from a low number of engrafted JAK2(V617F) SLAM cells. Finally, IFNα treatment prevented disease development by specifically inhibiting JAK2(V617F) cells at an early stage of differentiation and eradicating disease-initiating cells. This study shows that JAK2(V617F) in mice amplifies not only late but also early hematopoietic cells, giving them a proliferative advantage through high cell cycling and low apoptosis that may sustain MPN emergence but is lost upon IFNα treatment.

  6. Cell adhesion of F{sup +} ion implantation of intraocular lens

    Energy Technology Data Exchange (ETDEWEB)

    Li, D.J. E-mail: dejunli@hotmail.com; Cui, F.Z.; Gu, H.Q

    1999-04-01

    The cell adhesion of ion implanted polymethylmethacrylate (PMMA) intraocular lens was studied using cultured cells in vitro. F{sup +} ion implantation was performed at the energies of 40, 60, 80, 100 keV with the fluences ranging from 5x10{sup 13} to 1x10{sup 15} ions/cm{sup 2} at room temperature. The cell adhesion tests gave interesting results that the number of the neutral granulocytes and the macrophages adhering on surface were reduced significantly after ion implantation. The optimal fluence was about 4x10{sup 14} ions/cm{sup 2}. The hydrophobicity imparted to the lens surface was also enhanced. The results of X-ray photoelectron spectroscopy analysis indicated that ion implantation resulted in the cleavage of some pendant groups, the oxidation of the surface, and the formation of some new chemical bonds, which was probably the main reason for the cell adhesion change.

  7. Intravenous administration of bone marrow-derived multipotent mesenchymal stromal cells enhances the recruitment of CD11b{sup +} myeloid cells to the lungs and facilitates B16-F10 melanoma colonization

    Energy Technology Data Exchange (ETDEWEB)

    Souza, Lucas E.B., E-mail: lucasebsouza@usp.br [Department of Clinical Medicine, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil); Hemotherapy Center of Ribeirão Preto, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil); Almeida, Danilo C., E-mail: gudaalmeida@gmail.com [Department of Medicine – Nephrology, Laboratory of Clinical and Experimental Immunology, Federal University of São Paulo, São Paulo, SP (Brazil); Yaochite, Juliana N.U., E-mail: ueda.juliana@gmail.com [Department of Biochemistry and Immunology, Basic and Applied Immunology Program, School of Medicine of Ribeirão Preto, University of São Paulo (Brazil); Covas, Dimas T., E-mail: dimas@fmrp.usp.br [Department of Clinical Medicine, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil); Hemotherapy Center of Ribeirão Preto, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil); Fontes, Aparecida M., E-mail: aparecidamfontes@usp.br [Department of Genetics, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil)

    2016-07-15

    The discovery that the regenerative properties of bone marrow multipotent mesenchymal stromal cells (BM-MSCs) could collaterally favor neoplastic progression has led to a great interest in the function of these cells in tumors. However, the effect of BM-MSCs on colonization, a rate-limiting step of the metastatic cascade, is unknown. In this study, we investigated the effect of BM-MSCs on metastatic outgrowth of B16-F10 melanoma cells. In in vitro experiments, direct co-culture assays demonstrated that BM-MSCs stimulated the proliferation of B16-F10 cells in a dose-dependent manner. For in vivo experiments, luciferase-expressing B16-F10 cells were injected through tail vein and mice were subsequently treated with four systemic injections of BM-MSCs. In vivo bioluminescent imaging during 16 days demonstrated that BM-MSCs enhanced the colonization of lungs by B16-F10 cells, which correlated with a 2-fold increase in the number of metastatic foci. Flow cytometry analysis of lungs demonstrated that although mice harboring B16-F10 metastases displayed more endothelial cells, CD4 T and CD8 T lymphocytes in the lungs in comparison to metastases-free mice, BM-MSCs did not alter the number of these cells. Interestingly, BM-MSCs inoculation resulted in a 2-fold increase in the number of CD11b{sup +} myeloid cells in the lungs of melanoma-bearing animals, a cell population previously described to organize “premetastatic niches” in experimental models. These findings indicate that BM-MSCs provide support to B16-F10 cells to overcome the constraints that limit metastatic outgrowth and that these effects might involve the interplay between BM-MSCs, CD11b{sup +} myeloid cells and tumor cells. - Highlights: • BM-MSCs enhanced B16-F10 proliferation in a dose-dependent manner in vitro. • BM-MSCs facilitated lung colonization by B16-F10 melanoma cells. • BM-MSCs administration did not alter the number of endothelial cells and T lymphocytes in the lungs. • BM-MSCs enhanced

  8. Imaging grafted cells with [18F]FHBG using an optimized HSV1-TK mammalian expression vector in a brain injury rodent model.

    Directory of Open Access Journals (Sweden)

    Anne-Sophie Salabert

    Full Text Available Cell transplantation is an innovative therapeutic approach after brain injury to compensate for tissue damage. To have real-time longitudinal monitoring of intracerebrally grafted cells, we explored the feasibility of a molecular imaging approach using thymidine kinase HSV1-TK gene encoding and [18F]FHBG as a reporter probe to image enzyme expression.A stable neuronal cell line expressing HSV1-TK was developed with an optimised mammalian expression vector to ensure long-term transgene expression. After [18F]FHBG incubation under defined parameters, calibration ranges from 1 X 104 to 3 X 106 Neuro2A-TK cells were analysed by gamma counter or by PET-camera. In parallel, grafting with different quantities of [18F]FHBG prelabelled Neuro2A-TK cells was carried out in a rat brain injury model induced by stereotaxic injection of malonate toxin. Image acquisition of the rats was then performed with PET/CT camera to study the [18F]FHBG signal of transplanted cells in vivo.Under the optimised incubation conditions, [18F]FHBG cell uptake rate was around 2.52%. In-vitro calibration range analysis shows a clear linear correlation between the number of cells and the signal intensity. The PET signal emitted into rat brain correlated well with the number of cells injected and the number of surviving grafted cells was recorded via the in-vitro calibration range. PET/CT acquisitions also allowed validation of the stereotaxic injection procedure. Technique sensitivity was evaluated under 5 X 104 grafted cells in vivo. No [18F]FHBG or [18F]metabolite release was observed showing a stable cell uptake even 2 h post-graft.The development of this kind of approach will allow grafting to be controlled and ensure longitudinal follow-up of cell viability and biodistribution after intracerebral injection.

  9. Usefulness of (18)F-FDG PET/CT in recurrent basal cell carcinoma: Report of a case.

    Science.gov (United States)

    Ayala, S; Perlaza, P; Puig, S; Prats, E; Vidal-Sicart, S

    2016-01-01

    We analyze the case of a patient with left periorbital infiltrating basal cell carcinoma treated with surgical excision in October 2010. Surgery included orbital exenteration and reconstruction using skin graft and radiotherapy. In May 2013 a MR imaging showed a mass in the left orbital fossa, suggesting a recurrence in the graft. A basal cell carcinoma recurrence with perineural invasion was confirmed in the biopsy. On (18)F-FDG PET/CT performed, a hypermetabolic activity was observed in the left periorbital area with extension to surrounding sinus and bones. The use of (18)F-FDG PET/CT in patients with advanced basal cell carcinoma has not been fully explored due to the rarity of this entity. This case demonstrates the usefulness of this technique to determine the extent of non-melanocytic recurrent skin tumors, and its value in the staging and treatment control, supporting the incorporation of (18)F-FDG PET/CT in the management of advanced basal cell carcinoma. Copyright © 2015 Elsevier España, S.L.U. and SEMNIM. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-08-17

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

  12. [18F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase activity.

    Science.gov (United States)

    Kim, Woosuk; Le, Thuc M; Wei, Liu; Poddar, Soumya; Bazzy, Jimmy; Wang, Xuemeng; Uong, Nhu T; Abt, Evan R; Capri, Joseph R; Austin, Wayne R; Van Valkenburgh, Juno S; Steele, Dalton; Gipson, Raymond M; Slavik, Roger; Cabebe, Anthony E; Taechariyakul, Thotsophon; Yaghoubi, Shahriar S; Lee, Jason T; Sadeghi, Saman; Lavie, Arnon; Faull, Kym F; Witte, Owen N; Donahue, Timothy R; Phelps, Michael E; Herschman, Harvey R; Herrmann, Ken; Czernin, Johannes; Radu, Caius G

    2016-04-12

    Deoxycytidine kinase (dCK), a rate-limiting enzyme in the cytosolic deoxyribonucleoside (dN) salvage pathway, is an important therapeutic and positron emission tomography (PET) imaging target in cancer. PET probes for dCK have been developed and are effective in mice but have suboptimal specificity and sensitivity in humans. To identify a more suitable probe for clinical dCK PET imaging, we compared the selectivity of two candidate compounds-[(18)F]Clofarabine; 2-chloro-2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-adenine ([(18)F]CFA) and 2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-guanine ([(18)F]F-AraG)-for dCK and deoxyguanosine kinase (dGK), a dCK-related mitochondrial enzyme. We demonstrate that, in the tracer concentration range used for PET imaging, [(18)F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [(18)F]F-AraG is a better substrate for dGK than for dCK. [(18)F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [(18)F]CFA uptake was reduced by deoxycytidine (dC) competition, this inhibition required high dC concentrations present in murine, but not human, plasma. Expression of cytidine deaminase, a dC-catabolizing enzyme, in leukemia cells both in cell culture and in mice reduced the competition between dC and [(18)F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [(18)F]CFA PET/CT studies showed probe accumulation in tissues with high dCK expression: e.g., hematopoietic bone marrow and secondary lymphoid organs. The selectivity of [(18)F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [(18)F]CFA PET as a new cancer biomarker for treatment stratification and monitoring.

  13. Modulation of Δ9-tetrahydrocannabinol-induced MCF-7 breast cancer cell growth by cyclooxygenase and aromatase

    International Nuclear Information System (INIS)

    Takeda, Shuso; Yamamoto, Ikuo; Watanabe, Kazuhito

    2009-01-01

    Δ 9 -Tetrahydrocannabinol (Δ 9 -THC), a major constituent of marijuana, has been shown to stimulate the growth of MCF-7 breast cancer cells through cannabinoid receptor-independent signaling [Takeda, S., Yamaori, S., Motoya, E., Matsunaga, T., Kimura, T., Yamamoto, I., Watanabe, K., 2008. Δ 9 -Tetrahydrocannabinol enhances MCF-7 cell proliferation via cannabinoid receptor-independent signaling. Toxicology 245, 141-146]. Although the growth of MCF-7 cells is known to be stimulated by 17β-estradiol (E 2 ), the interaction of Δ 9 -THC and E 2 in MCF-7 cell growth is not fully clarified so far. In the present study, by using E 2 -sensitive MCF-7 cells that have expressed cyclooxygenase-2 (COX-2) and cytochrome P450 19 (aromatase), we studied whether or not COX-2 and aromatase are involved in Δ 9 -THC-mediated MCF-7 cell proliferation. It was shown that Δ 9 -THC-induced MCF-7 cell growth was inhibited by COX-2 inhibitors and was stimulated by arachidonic acid (a COX substrate). However, the growth of MCF-7 cells induced by Δ 9 -THC was not stimulated by PGE 2 , and the expression of aromatase was not affected by COX-2 inhibitors, arachidonic acid, and PGE 2 , suggesting that there is a disconnection between COX-2 (PGE 2 ) and aromatase in Δ 9 -THC-mediated MCF-7 cell proliferation. On the other hand, Δ 9 -THC-induced MCF-7 cell growth was elevated by two kinds of aromatase inhibitors. Taken together with the evidence that Δ 9 -THC-induced MCF-7 cell proliferation was interfered with testosterone (an aromatase substrate) and exogenously provided E 2 , it is suggested that (1) the growth stimulatory effects of Δ 9 -THC are mediated by the product(s) of COX-2 except for PGE 2 , (2) the action of Δ 9 -THC is modulated by E 2 , and (3) COX-2 and aromatase are individually engaged in the proliferation of MCF-7 cells induced by Δ 9 -THC.

  14. Toll like Receptor 2 engagement on CD4+ T cells promotes TH9 differentiation and function.

    Science.gov (United States)

    Karim, Ahmad Faisal; Reba, Scott M; Li, Qing; Boom, W Henry; Rojas, Roxana E

    2017-09-01

    We have recently demonstrated that mycobacterial ligands engage Toll like receptor 2 (TLR2) on CD4 + T cells and up-regulate T-cell receptor (TCR) triggered Th1 responses in vitro and in vivo. To better understand the role of T-cell expressed TLR2 on CD4 + T-cell differentiation and function, we conducted a gene expression analysis of murine naïve CD4 + T-cells stimulated in the presence or absence of TLR2 co-stimulation. Unexpectedly, naïve CD4 + T-cells co-stimulated via TLR2 showed a significant up-regulation of Il9 mRNA compared to cells co-stimulated via CD28. Under TH9 differentiation, we observed up-regulation of TH9 differentiation, evidenced by increases in both percent of IL-9 secreting cells and IL-9 in culture supernatants in the presence of TLR2 agonist both in polyclonal and Ag85B cognate peptide specific stimulations. Under non-polarizing conditions, TLR2 engagement on CD4 + T-cells had minimal effect on IL-9 secretion and TH9 differentiation, likely due to a prominent effect of TLR2 signaling on IFN-γ secretion and TH1 differentiation. We also report that, TLR2 signaling in CD4 + T cells increased expression of transcription factors BATF and PU.1, known to positively regulate TH9 differentiation. These results reveal a novel role of T-cell expressed TLR2 in enhancing the differentiation and function of TH9 T cells. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Influenza virus PB1-F2 protein induces cell death through mitochondrial ANT3 and VDAC1.

    Directory of Open Access Journals (Sweden)

    Dmitriy Zamarin

    2005-09-01

    Full Text Available The influenza virus PB1-F2 is an 87-amino acid mitochondrial protein that previously has been shown to induce cell death, although the mechanism of apoptosis induction has remained unclear. In the process of characterizing its mechanism of action we found that the viral PB1-F2 protein sensitizes cells to apoptotic stimuli such as tumor necrosis factor alpha, as demonstrated by increased cleavage of caspase 3 substrates in PB1-F2-expressing cells. Moreover, treatment of purified mouse liver mitochondria with recombinant PB1-F2 protein resulted in cytochrome c release, loss of the mitochondrial membrane potential, and enhancement of tBid-induced mitochondrial permeabilization, suggesting a possible mechanism for the observed cellular sensitization to apoptosis. Using glutathione-S-transferase pulldowns with subsequent mass spectrometric analysis, we identified the mitochondrial interactors of the PB1-F2 protein and showed that the viral protein uniquely interacts with the inner mitochondrial membrane adenine nucleotide translocator 3 and the outer mitochondrial membrane voltage-dependent anion channel 1, both of which are implicated in the mitochondrial permeability transition during apoptosis. Consistent with this interaction, blockers of the permeability transition pore complex (PTPC inhibited PB1-F2-induced mitochondrial permeabilization. Based on our findings, we propose a model whereby the proapoptotic PB1-F2 protein acts through the mitochondrial PTPC and may play a role in the down-regulation of the host immune response to infection.

  16. Variations among Japanese of the factor IX gene (F9) detected by PCR-denaturing gradient gel electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Satoh, Chiyoko; Takahashi, Norio; Asakawa, Junichi; Hiyama, Keiko; Kodaira, Meiko (Radiation Effects Research Foundation, Hiroshima (Japan))

    1993-01-01

    In the course of feasibility studies to examine the efficiencies and practicalities of various techniques for screening for genetic variations, the human coagulation factor IX (F9) genes of 63 Japanese families were examined by PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Four target sequences with lengths of 983-2,891 bp from the F9 genes of 126 unrelated individuals from Hiroshima and their 100 children were amplified by PCR, digested with restriction enzymes to approximately 500-bp fragments, and examined by DGGE - a total of 6,724 bp being examined per individual. GC-rich sequences (GC-clamps) of 40 bp were attached to both ends of the target sequences, as far as was feasible. Eleven types of new nucleotide substitutions were detected in the population, none of which produced RFLPs or caused hemophilia B. By examining two target sequences in a single lane, approximately 8,000 bp in a diploid individual could be examined. This approach is very effective for the detection of variations in DNA and is applicable to large-scale population studies. 46 refs., 3 figs., 1 tab.

  17. Analysis of the Arabidopsis IRX9/IRX9-L and IRX14/IRX14-L pairs of glycosyltransferase genes reveals critical contributions to biosynthesis of the hemicellulose glucuronoxylan.

    Science.gov (United States)

    Wu, Ai-Min; Hörnblad, Emma; Voxeur, Aline; Gerber, Lorenz; Rihouey, Christophe; Lerouge, Patrice; Marchant, Alan

    2010-06-01

    The hemicellulose glucuronoxylan (GX) is a major component of plant secondary cell walls. However, our understanding of GX synthesis remains limited. Here, we identify and analyze two new genes from Arabidopsis (Arabidopsis thaliana), IRREGULAR XYLEM9-LIKE (IRX9-L) and IRX14-LIKE (IRX14-L) that encode glycosyltransferase family 43 members proposed to function during xylan backbone elongation. We place IRX9-L and IRX14-L in a genetic framework with six previously described glycosyltransferase genes (IRX9, IRX10, IRX10-L, IRX14, FRAGILE FIBER8 [FRA8], and FRA8 HOMOLOG [F8H]) and investigate their function in GX synthesis. Double-mutant analysis identifies IRX9-L and IRX14-L as functional homologs of IRX9 and IRX14, respectively. Characterization of irx9 irx10 irx14 fra8 and irx9-L irx10-L irx14-L f8h quadruple mutants allows definition of a set of genes comprising IRX9, IRX10, IRX14, and FRA8 that perform the main role in GX synthesis during vegetative development. The IRX9-L, IRX10-L, IRX14-L, and F8H genes are able to partially substitute for their respective homologs and normally perform a minor function. The irx14 irx14-L double mutant virtually lacks xylan, whereas irx9 irx9-L and fra8 f8h double mutants form lowered amounts of GX displaying a greatly reduced degree of backbone polymerization. Our findings reveal two distinct sets of four genes each differentially contributing to GX biosynthesis.

  18. Apoptotic effects of antilymphocyte globulins on human pro-inflammatory CD4+CD28- T-cells.

    Directory of Open Access Journals (Sweden)

    Christina Duftner

    Full Text Available BACKGROUND: Pro-inflammatory, cytotoxic CD4(+CD28(- T-cells with known defects in apoptosis have been investigated as markers of premature immuno-senescence in various immune-mediated diseases. In this study we evaluated the influence of polyclonal antilymphocyte globulins (ATG-Fresenius, ATG-F on CD4(+CD28(- T-cells in vivo and in vitro. PRINCIPAL FINDINGS: Surface and intracellular three colour fluorescence activated cell sorting analyses of peripheral blood mononuclear cells from 16 consecutive transplant recipients and short-term cell lines were performed. In vivo, peripheral levels of CD3(+CD4(+CD28(- T-cells decreased from 3.7 ± 7.1% before to 0 ± 0% six hours after ATG-F application (P = 0.043 in 5 ATG-F treated but not in 11 control patients (2.9 ± 2.9% vs. 3.9 ± 3.0%. In vitro, ATG-F induced apoptosis even in CD4(+CD28(- T-cells, which was 4.3-times higher than in CD4(+CD28(+ T-cells. ATG-F evoked apoptosis was partially reversed by the broad-spectrum caspase inhibitor benzyloxycarbonyl (Cbz-Val-Ala-Asp(OMe-fluoromethylketone (zVAD-fmk and prednisolon-21-hydrogensuccinate. ATG-F triggered CD25 expression and production of pro-inflammatory cytokines, and induced down-regulation of the type 1 chemokine receptors CXCR-3, CCR-5, CX3CR-1 and the central memory adhesion molecule CD62L predominately in CD4(+CD28(- T-cells. CONCLUSION: In summary, in vivo depletion of peripheral CD3(+CD4(+CD28(- T-cells by ATG-F in transplant recipients was paralleled in vitro by ATG-F induced apoptosis. CD25 expression and chemokine receptor down-regulation in CD4(+CD28(- T-cells only partly explain the underlying mechanism.

  19. Differential protection by cell wall components of Lactobacillus amylovorus DSM 16698Tagainst alterations of membrane barrier and NF-kB activation induced by enterotoxigenic F4+ Escherichia coli on intestinal cells.

    Science.gov (United States)

    Roselli, Marianna; Finamore, Alberto; Hynönen, Ulla; Palva, Airi; Mengheri, Elena

    2016-09-29

    The role of Lactobacillus cell wall components in the protection against pathogen infection in the gut is still largely unexplored. We have previously shown that L. amylovorus DSM 16698 T is able to reduce the enterotoxigenic F4 + Escherichia coli (ETEC) adhesion and prevent the pathogen-induced membrane barrier disruption through the regulation of IL-10 and IL-8 expression in intestinal cells. We have also demonstrated that L. amylovorus DSM 16698 T protects host cells through the inhibition of NF-kB signaling. In the present study, we investigated the role of L. amylovorus DSM 16698 T cell wall components in the protection against F4 + ETEC infection using the intestinal Caco-2 cell line. Purified cell wall fragments (CWF) from L. amylovorus DSM 16698 T were used either as such (uncoated, U-CWF) or coated with S-layer proteins (S-CWF). Differentiated Caco-2/TC7 cells on Transwell filters were infected with F4 + ETEC, treated with S-CWF or U-CWF, co-treated with S-CWF or U-CWF and F4 + ETEC for 2.5 h, or pre-treated with S-CWF or U-CWF for 1 h before F4 + ETEC addition. Tight junction (TJ) and adherens junction (AJ) proteins were analyzed by immunofluorescence and Western blot. Membrane permeability was determined by phenol red passage. Phosphorylated p65-NF-kB was measured by Western blot. We showed that both the pre-treatment with S-CWF and the co- treatment of S-CWF with the pathogen protected the cells from F4 + ETEC induced TJ and AJ injury, increased membrane permeability and activation of NF-kB expression. Moreover, the U-CWF pre-treatment, but not the co-treatment with F4 + ETEC, inhibited membrane damage and prevented NF-kB activation. The results indicate that the various components of L. amylovorus DSM 16698 T cell wall may counteract the damage caused by F4 + ETEC through different mechanisms. S-layer proteins are essential for maintaining membrane barrier function and for mounting an anti-inflammatory response against F4 + ETEC infection. U-CWF are

  20. Depletion of kidney CD11c+ F4/80+ cells impairs the recovery process in ischaemia/reperfusion-induced acute kidney injury.

    Science.gov (United States)

    Kim, Myung-Gyu; Boo, Chang Su; Ko, Yoon Sook; Lee, Hee Young; Cho, Won Yong; Kim, Hyoung Kyu; Jo, Sang-Kyung

    2010-09-01

    Recent studies provided evidence of the potential role of CD11c(+) F4/80(+) dendritic subset in mediating injury and repair. The purpose of this study was to examine the role of kidney CD11c(+) F4/80(+) dendritic subset in the recovery phase of ischaemia/reperfusion injury (IRI). Following ischaemia/reperfusion (I/R), liposome clodronate or phosphate buffered saline (PBS) was administered, and on day 7 biochemical and histologic kidney damage was assessed. Activation and depletion of CD11c(+) F4/80(+) dendritic subset were confirmed by flow cytometry. Isolation of kidney CD11c(+) cells on days 1 and 7 with in vitro culture for measuring cytokines was performed to define functional characteristics of these cells, and adoptive transfer of CD11c(+) cells was also done. Following kidney IRI, the percentage of CD11c(+) F4/80(+) kidney dendritic cell subset that co-expresses maturation marker increased. Liposome clodronate injection after I/R resulted in preferential depletion of CD11c(+) F4/80(+) kidney dendritic subset, and depletion of these cells was associated with persistent kidney injury, more apoptosis, inflammation and impaired tubular cell proliferation. CD11c(+) F4/80(+) cell depletion was also associated with higher tissue levels of pro-inflammatory cytokines and lower level of IL-10, indicating the persistence of inflammatory milieu. Isolated kidney CD11c(+) cells on day 7 showed different phenotype with increased production of IL-10 compared with those on day 1. Adoptive transfer of CD11c(+) cells partially reversed impaired tissue recovery. Our results suggest that kidney CD11c(+) F4/80(+) dendritic subset might contribute to the recovery process by dynamic phenotypic change from pro-inflammatory to anti-inflammatory with modulation of immune response.

  1. Rad9 contribution to radiosensitivity and the G2 checkpoint in a DT40 cell line

    Energy Technology Data Exchange (ETDEWEB)

    Kumano, Tomoyasu [Kanazawa Univ. (Japan). Graduate School of Medical Science

    2002-12-01

    In fission yeast, the rad9 (radiation sensitive) gene was cloned from a mutant that is sensitive to ionizing radiation, ultraviolet and hydroxyurea. This gene has also been shown to be required for a DNA damage checkpoint. Orthologues of the rad9 gene have recently been identified in higher eukaryote cells including human. Here we generated Rad9 knockout (Rad9-/-) cells from the chicken B lymphocyte line DT40 to examine the role of Rad9 in higher eukaryotes. First we isolated a part of the chicken Rad9 gene which was 54% identical with human Rad9 at the amino acid sequence level. Next we isolated genomic clones, determined exons and introns, and constructed targeting vectors designed to disrupt exon 1-3 of the chicken Rad9 gene by replacement with a drug-resistant gene. Successful targeted integration was verified by Southern blot analysis and the disruption of the Rad9 gene was confirmed by reverse transcription polymerase chain reaction (RT-PCR). To analyze the radiosensitivity of these Rad9-/- cells, we monitored the clonogenic survival after various degrees of X-ray irradiation. Rad9-/- cells were more sensitive to X-rays than wild type cells at all dosages. However, these cells were less sensitive than ATM knockout (ATM-/-) cells that are known to be X-ray sensitive and that showed a defective checkpoint control. In contrast, Rad9-/- cells were markedly more sensitive to ultraviolet and hydroxyruea. In addition, we assessed the G2 checkpoint by measurement of the mitotic index that is the fraction of the accumulating number of cells in mitosis at various times after X-ray irradiation. While the number of mitotic wild type cells did not increase until 2 hrs after X-ray irradiation, the number of mitotic Rad9-/- cells showed an increase similar to that of ATM-/- cells. These results suggest that just as in fission yeast, in higher eukaryotes Rad9 also contributes to X-ray, ultraviolet and hydroxyurea sensitivity, and plays an important role in the G2 checkpoint

  2. Rad9 contribution to radiosensitivity and the G2 checkpoint in a DT40 cell line

    International Nuclear Information System (INIS)

    Kumano, Tomoyasu

    2002-01-01

    In fission yeast, the rad9 (radiation sensitive) gene was cloned from a mutant that is sensitive to ionizing radiation, ultraviolet and hydroxyurea. This gene has also been shown to be required for a DNA damage checkpoint. Orthologues of the rad9 gene have recently been identified in higher eukaryote cells including human. Here we generated Rad9 knockout (Rad9-/-) cells from the chicken B lymphocyte line DT40 to examine the role of Rad9 in higher eukaryotes. First we isolated a part of the chicken Rad9 gene which was 54% identical with human Rad9 at the amino acid sequence level. Next we isolated genomic clones, determined exons and introns, and constructed targeting vectors designed to disrupt exon 1-3 of the chicken Rad9 gene by replacement with a drug-resistant gene. Successful targeted integration was verified by Southern blot analysis and the disruption of the Rad9 gene was confirmed by reverse transcription polymerase chain reaction (RT-PCR). To analyze the radiosensitivity of these Rad9-/- cells, we monitored the clonogenic survival after various degrees of X-ray irradiation. Rad9-/- cells were more sensitive to X-rays than wild type cells at all dosages. However, these cells were less sensitive than ATM knockout (ATM-/-) cells that are known to be X-ray sensitive and that showed a defective checkpoint control. In contrast, Rad9-/- cells were markedly more sensitive to ultraviolet and hydroxyruea. In addition, we assessed the G2 checkpoint by measurement of the mitotic index that is the fraction of the accumulating number of cells in mitosis at various times after X-ray irradiation. While the number of mitotic wild type cells did not increase until 2 hrs after X-ray irradiation, the number of mitotic Rad9-/- cells showed an increase similar to that of ATM-/- cells. These results suggest that just as in fission yeast, in higher eukaryotes Rad9 also contributes to X-ray, ultraviolet and hydroxyurea sensitivity, and plays an important role in the G2 checkpoint

  3. Structural, spectroscopic and cytotoxicity studies of TbF{sub 3}@CeF{sub 3} and TbF{sub 3}@CeF{sub 3}@SiO{sub 2} nanocrystals

    Energy Technology Data Exchange (ETDEWEB)

    Grzyb, Tomasz; Runowski, Marcin [Adam Mickiewicz University, Department of Rare Earths, Faculty of Chemistry (Poland); Dabrowska, Krystyna [Polish Academy of Sciences, Bacteriophage Laboratory, Institute of Immunology and Experimental Therapy (Poland); Giersig, Michael; Lis, Stefan, E-mail: blis@amu.edu.pl [Adam Mickiewicz University, Department of Rare Earths, Faculty of Chemistry (Poland)

    2013-10-15

    Terbium fluoride nanocrystals, covered by a shell, composed of cerium fluoride were synthesized by a co-precipitation method. Their complex structure was formed spontaneously during the synthesis. The surface of these core/shell nanocrystals was additionally modified by silica. The properties of TbF{sub 3}@CeF{sub 3} and TbF{sub 3}@CeF{sub 3}@SiO{sub 2} nanocrystals, formed in this way, were investigated. Spectroscopic studies showed that the differences between these two groups of products resulted from the presence of the SiO{sub 2} shell. X-ray diffraction patterns confirmed the trigonal crystal structure of TbF{sub 3}@CeF{sub 3} nanocrystals. High resolution transmission electron microscopy in connection with energy-dispersive X-ray spectroscopy showed a complex structure of the formed nanocrystals. Crystallized as small discs, 'the products', with an average diameter around 10 nm, showed an increase in the concentration of Tb{sup 3+} ions from surface to the core of nanocrystals. In addition to photo-physical analyses, cytotoxicity studies were performed on HSkMEC (Human Skin Microvascular Endothelial Cells) and B16F0 mouse melanoma cancer cells. The cytotoxicity of the nanomaterials was neutral for the investigated cells with no toxic or antiproliferative effect in the cell cultures, either for normal or for cancer cells. This fact makes the obtained nanocrystals good candidates for biological applications and further modifications of the SiO{sub 2} shell.Graphical Abstract.

  4. Difference in prognostic significance of maximum standardized uptake value on [18F]-fluoro-2-deoxyglucose positron emission tomography between adenocarcinoma and squamous cell carcinoma of the lung

    International Nuclear Information System (INIS)

    Tsutani, Yasuhiro; Miyata, Yoshihiro; Misumi, Keizo; Ikeda, Takuhiro; Mimura, Takeshi; Hihara, Jun; Okada, Morihito

    2011-01-01

    This study evaluates the prognostic significance of [18F]-fluoro-2-deoxyglucose positron emission tomography/computed tomography findings according to histological subtypes in patients with completely resected non-small cell lung cancer. We examined 176 consecutive patients who had undergone preoperative [18F]-fluoro-2-deoxyglucose-positron emission tomography/computed tomography imaging and curative surgical resection for adenocarcinoma (n=132) or squamous cell carcinoma (n=44). Maximum standardized uptake values for the primary lesions in all patients were calculated as the [18F]-fluoro-2-deoxyglucose uptake and the surgical results were analyzed. The median values of maximum standardized uptake value for the primary tumors were 2.60 in patients with adenocarcinoma and 6.95 in patients with squamous cell carcinoma (P 6.95 (P=0.83) among patients with squamous cell carcinoma, 2-year disease-free survival rates were 93.9% for maximum standardized uptake value ≤3.7 and 52.4% for maximum standardized uptake value >3.7 (P<0.0001) among those with adenocarcinoma, and notably, 100 and 57.2%, respectively, in patients with Stage I adenocarcinoma (P<0.0001). On the basis of the multivariate Cox analyses of patients with adenocarcinoma, maximum standardized uptake value (P=0.008) was a significantly independent factor for disease-free survival as well as nodal metastasis (P=0.001). Maximum standardized uptake value of the primary tumor was a powerful prognostic determinant for patients with adenocarcinoma, but not with squamous cell carcinoma of the lung. (author)

  5. Induction of DNA-protein crosslinks in human cells by ultraviolet and visible radiations: action spectrum

    International Nuclear Information System (INIS)

    Peak, J.G.; Peak, M.J.; Sikorski, R.S.; Jones, C.A.

    1985-01-01

    DNA-protein crosslinking was induced in cultured human P3 teratocarcinoma cells by irradiation with monochromatic radiation with wavelengths in the range 254-434 nm (far-UV, near-UV, and blue light). Wavelength 545 nm green light did not induce these crosslinks, using the method of alkaline elution of the DNA from membrane filters. The action spectrum for the formation of DNA-protein crosslinks revealed two maxima, one in the far-UV spectrum that closely coincided with the relative spectrum of DNA at 254 and 290 nm, and one in the visible light spectrum at 405 nm, which has no counterpart in the DNA spectrum. The primary events for the formation of DNA-protein crosslinks by such long-wavelength radiation probably involve photosensitizers. This dual mechanism for DNA-protein crosslink formation is in strong contrast to the single mechanism for pyrimidine dimer formation in DNA, which apparently has no component in the visible light spectrum

  6. Both core and F proteins of hepatitis C virus could enhance cell proliferation in transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Wen-Ta [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Li, Hui-Chun [Department of Biochemistry, Tzu Chi University, Hualien, Taiwan (China); Lee, Shen-Kao; Ma, Hsin-Chieh; Yang, Chee-Hing; Chen, Hung-Ling [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Lo, Shih-Yen, E-mail: losylo@mail.tcu.edu.tw [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Department of Laboratory Medicine, Buddhist Tzu Chi General Hospital, Hualien, Taiwan (China)

    2013-05-24

    Highlights: •HCV core and F proteins could induce hepatocyte proliferation in the transgenic mice. •β-Catenin signaling pathway was activated by core protein in the transgenic mice. •β-Catenin signaling pathway was activated by myc-F protein in the transgenic mice. •Expression of SMA protein was enhanced by core but not myc-F protein. -- Abstract: The role of the protein encoded by the alternative open reading frame (ARF/F/core+1) of the Hepatitis C virus (HCV) genome in viral pathogenesis remains unknown. The different forms of ARF/F/core+1 protein were labile in cultured cells, a myc-tag fused at the N-terminus of the F protein made it more stable. To determine the role of core and F proteins in HCV pathogenesis, transgenic mice with either protein expression under the control of Albumin promoter were generated. Expression of core protein and F protein with myc tag (myc-F) could be detected by Western blotting analysis in the livers of these mice. The ratio of liver to body weight is increased for both core and myc-F transgenic mice compared to that of wild type mice. Indeed, the proliferating cell nuclear antigen protein, a proliferation marker, was up-regulated in the transgenic mice with core or myc-F protein. Further analyses by microarray and Western blotting suggested that β-catenin signaling pathway was activated by either core or myc-F protein in the transgenic mice. These transgenic mice were further treated with either Diethynitrosamine (a tumor initiator) or Phenobarbital (a tumor promoter). Phenobarbital but not Diethynitrosamine treatment could increase the liver/body weight ratio of these mice. However, no tumor formation was observed in these mice. In conclusion, HCV core and myc-F proteins could induce hepatocyte proliferation in the transgenic mice possibly through β-catenin signaling pathway.

  7. Both core and F proteins of hepatitis C virus could enhance cell proliferation in transgenic mice

    International Nuclear Information System (INIS)

    Hu, Wen-Ta; Li, Hui-Chun; Lee, Shen-Kao; Ma, Hsin-Chieh; Yang, Chee-Hing; Chen, Hung-Ling; Lo, Shih-Yen

    2013-01-01

    Highlights: •HCV core and F proteins could induce hepatocyte proliferation in the transgenic mice. •β-Catenin signaling pathway was activated by core protein in the transgenic mice. •β-Catenin signaling pathway was activated by myc-F protein in the transgenic mice. •Expression of SMA protein was enhanced by core but not myc-F protein. -- Abstract: The role of the protein encoded by the alternative open reading frame (ARF/F/core+1) of the Hepatitis C virus (HCV) genome in viral pathogenesis remains unknown. The different forms of ARF/F/core+1 protein were labile in cultured cells, a myc-tag fused at the N-terminus of the F protein made it more stable. To determine the role of core and F proteins in HCV pathogenesis, transgenic mice with either protein expression under the control of Albumin promoter were generated. Expression of core protein and F protein with myc tag (myc-F) could be detected by Western blotting analysis in the livers of these mice. The ratio of liver to body weight is increased for both core and myc-F transgenic mice compared to that of wild type mice. Indeed, the proliferating cell nuclear antigen protein, a proliferation marker, was up-regulated in the transgenic mice with core or myc-F protein. Further analyses by microarray and Western blotting suggested that β-catenin signaling pathway was activated by either core or myc-F protein in the transgenic mice. These transgenic mice were further treated with either Diethynitrosamine (a tumor initiator) or Phenobarbital (a tumor promoter). Phenobarbital but not Diethynitrosamine treatment could increase the liver/body weight ratio of these mice. However, no tumor formation was observed in these mice. In conclusion, HCV core and myc-F proteins could induce hepatocyte proliferation in the transgenic mice possibly through β-catenin signaling pathway

  8. Prenatal exposure to chromium induces early reproductive senescence by increasing germ cell apoptosis and advancing germ cell cyst breakdown in the F1 offspring.

    Science.gov (United States)

    Sivakumar, Kirthiram K; Stanley, Jone A; Arosh, Joe A; Pepling, Melissa E; Burghardt, Robert C; Banu, Sakhila K

    2014-04-01

    Hexavalent chromium (CrVI), one of the more toxic heavy metals, is widely used in more than 50 industries such as chrome plating, welding, wood processing and tanneries. As one of the world's leading producers of chromium compounds, the U.S. is facing growing challenges in protecting human health against multiple adverse effects of CrVI. CrVI is rapidly converted to CrIII intracellularly, and can induce apoptosis through different mechanisms. Our previous studies demonstrated postnatal exposure to CrVI results in a delay or arrest in follicle development and puberty. Pregnant rats were treated with 25 ppm potassium dichromate (CrVI) from gestational day (GD) 9.5 to 14.5 through drinking water, placentae were removed on GD 20, and total Cr was estimated in the placentae; ovaries were removed from the F1 offspring on postnatal day (PND)-1 and various analyses were performed. Our results show that gestational exposure to CrVI resulted in (i) increased Cr concentration in the placenta, (ii) increased germ cell apoptosis by up-regulating p53/p27-Bax-caspase-3 proteins and by increasing p53-SOD-2 co-localization; (iii) accelerated germ cell cyst (GCC) breakdown; (iv) advanced primordial follicle assembly and primary follicle transition and (v) down regulation of p-AKT, p-ERK and XIAP. As a result of the above events, CrVI induced early reproductive senescence and decrease in litter size in F1 female progeny. Published by Elsevier Inc.

  9. BMP9-Induced Osteogenetic Differentiation and Bone Formation of Muscle-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Li Xiang

    2012-01-01

    Full Text Available Efficient osteogenetic differentiation and bone formation from muscle-derived stem cells (MDSCs should have potential clinical applications in treating nonunion fracture healing or bone defects. Here, we investigate osteogenetic differentiation ability of MDSCs induced by bone morphogenetic protein 9 (BMP9 in vitro and bone formation ability in rabbit radius defects repairing model. Rabbit's MDSCs were extracted by type I collagenase and trypsin methods, and BMP9 was introduced into MDSCs by infection with recombinant adenovirus. Effects of BMP9-induced osteogenetic differentiation of MDSCs were identified with alkaline phosphatase (ALP activity and expression of later marker. In stem-cell implantation assay, MDSCs have also shown valuable potential bone formation ability induced by BMP9 in rabbit radius defects repairing test. Taken together, our findings suggest that MDSCs are potentiated osteogenetic stem cells which can be induced by BMP9 to treat large segmental bone defects, nonunion fracture, and/or osteoporotic fracture.

  10. Shedding of CD9 antigen into cerebrospinal fluid by acute lymphoblastic leukemia cells.

    Science.gov (United States)

    Komada, Y; Ochiai, H; Shimizu, K; Azuma, E; Kamiya, H; Sakurai, M

    1990-07-01

    The accurate identification of small numbers of leukemic cells in the cerebrospinal fluid (CSF) presents a diagnostic problem in the treatment of acute lymphoblastic leukemia (ALL). We demonstrated that soluble CD9 antigen was shed into CSF obtained from children with ALL, using enzyme-linked immunosorbent assay (ELISA), which used the activity of CD9 antigen to bind the Ricinus communis agglutinin (RCA1) and a monoclonal antibody, SJ-9A4, simultaneously. Using RCA1/SJ-9A4 ELISA, CD9 antigen was detectable in CSF but not in plasma from 12 cases of CD9+ ALL in central nervous system (CNS) relapse. However, CD9 antigen was not released into CSF from 11 cases of CD9- ALL with CNS involvement, 136 cases of CD9+ ALL in complete remission (CR), 29 cases of CD9- ALL in CR, or 21 cases of aseptic meningitis. Interestingly, the levels of CD9 antigen were elevated in CSF from 7 of 10 CD9+ ALL patients without cytologically proven CNS involvement at diagnosis, with subsequent return to undetectable levels after initial induction chemotherapy was begun. In addition, sequential analysis of CSF from a 5-year-old boy with CD9+ ALL in CNS relapse showed that levels of CD9 antigen correlated well with the number of leukemic cells in CSF. Serial quantitative analysis of CD9 antigen in CSF could be useful to detect the proliferation of residual leukemic cells before the clinical manifestation.

  11. Matrix metalloproteinase-9 (MMP9) is involved in the TNF-α-induced fusion of human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells.

    Science.gov (United States)

    Weiler, Julian; Mohr, Marieke; Zänker, Kurt S; Dittmar, Thomas

    2018-04-10

    In addition to physiological events such as fertilisation, placentation, osteoclastogenesis, or tissue regeneration/wound healing, cell fusion is involved in pathophysiological conditions such as cancer. Cell fusion, which applies to both the proteins and conditions that induce the merging of two or more cells, is not a fully understood process. Inflammation/pro-inflammatory cytokines might be a positive trigger for cell fusion. Using a Cre-LoxP-based cell fusion assay we demonstrated that the fusion between human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells was induced by the pro-inflammatory cytokine tumour necrosis factor-α (TNF-α). The gene expression profile of the cells in the presence of TNF-α and under normoxic and hypoxic conditions was analysed by cDNA microarray analysis. cDNA microarray data were verified by qPCR, PCR, Western blot and zymography. Quantification of cell fusion events was determined by flow cytometry. Proteins of interest were either blocked or knocked-down using a specific inhibitor, siRNA or a blocking antibody. The data showed an up-regulation of various genes, including claudin-1 (CLDN1), ICAM1, CCL2 and MMP9 in M13SV1-Cre and/or MDA-MB-435-pFDR1 cells. Inhibition of these proteins using a blocking ICAM1 antibody, CLDN1 siRNA or an MMP9 inhibitor showed that only the blockage of MMP9 was correlated with a decreased fusion rate of the cells. Likewise, the tetracycline-based antibiotic minocycline, which exhibits anti-inflammatory properties, was also effective in both inhibiting the TNF-α-induced MMP9 expression in M13SV1-Cre cells and blocking the TNF-α-induced fusion frequency of human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells. The matrix metalloproteinase-9 (MMP9) is most likely involved in the TNF-α-mediated fusion of human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells. Likewise, our data indicate that the tetracycline

  12. Cyclic mechanical stretch enhances BMP9-induced osteogenic differentiation of mesenchymal stem cells.

    Science.gov (United States)

    Song, Yang; Tang, Yinhong; Song, Jinlin; Lei, Mingxing; Liang, Panpan; Fu, Tiwei; Su, Xudong; Zhou, Pengfei; Yang, Li; Huang, Enyi

    2018-04-01

    The purpose of this study was to investigate whether mechanical stretch can enhance the bone morphogenetic protein 9 (BMP9)-induced osteogenic differentiation in MSCs. Recombinant adenoviruses were used to overexpress the BMP9 in C3H10T1/2 MSCs. Cells were seeded onto six-well BioFlex collagen I-coated plates and subjected to cyclic mechanical stretch [6% elongation at 60 cycles/minute (1 Hz)] in a Flexercell FX-4000 strain unit for up to 12 hours. Immunostaining and confocal microscope were used to detect cytoskeleton organization. Cell cycle progression was checked by flow cytometry. Alkaline phosphatase activity was measured with a Chemiluminescence Assay Kit and was quantified with a histochemical staining assay. Matrix mineralization was examined by Alizarin Red S Staining. Mechanical stretch induces cytoskeleton reorganization and inhibits cell proliferation by preventing cells entry into S phase of the cell cycle. Although mechanical stretch alone does not induce the osteogenic differentiation of C3H10T1/2 MSCs, co-stimulation with mechanical stretch and BMP9 enhances alkaline phosphatase activity. The expression of key lineage-specific regulators (e.g., osteocalcin (OCN), SRY-related HMG-box 9, and runt-related transcription factor 2) is also increased after the co-stimulation, compared to the mechanical stretch stimulation along. Furthermore, mechanical stretch augments the BMP9-mediated bone matrix mineralization of C3H10T1/2 MSCs. Our results suggest that mechanical stretch enhances BMP9-induced osteoblastic lineage specification in C3H10T1/2 MSCs.

  13. Repair of ultraviolet radiation damage in xeroderma pigmentosum cells belonging to complementation group F

    International Nuclear Information System (INIS)

    Hayakawa, H.; Ishizaki, K.; Yagi, T.; Takebe, H.; Inoue, M.; Sekiguchi, M.; Kyoto Univ.

    1981-01-01

    DNA-repair characteristics of xeroderma pigmentosum belonging to complementation group F were investigated. The cells exhibited an intermediate level of repair as measured in terms of (1) disappearance of T4 endonuclease-V-susceptible sites from DNA, (2) formation of ultraviolet-induced strand breaks in DNA, and (3) ultraviolet-induced unscheduled DNA synthesis during post-irradiation incubation. The impaired ability of XP3YO to perform unscheduled DNA synthesis was restored, to half the normal level, by the concomitant treatment with T4 endonuclease V and ultraviolet-inactivated Sendai virus. It is suggested that xeroderma pigmentosum cells of group F may be defective, at least in part, in the incision step of excision repair. (orig.)

  14. [Knockdown of NEDD9 inhibits the proliferation, invasion and migration of esophageal carcinoma EC109 cells].

    Science.gov (United States)

    Zhang, Wen; Li, Shaojun; Zhao, Yunlong; Guo, Nannan; Li, Yingjie

    2016-12-01

    Objective To observe the expression of the neural precursor cell expressed, developmentally down-regulated 9 (NEDD9) in esophageal cancer, to investigate the impact of decreased expression of NEDD9 on invasion and migration, and to explicit the function of NEDD9 in EC109 human esophageal cancer cell line. Methods Immunohistochemical staining was used to detect the expression of NEDD9 in human esophageal cancer tissues and paracancerous normal tissues. RNA interfering (RNAi) was used to knockdown NEDD9 in EC109 cells. The interference efficiency was detected by reverse transcription PCR (RT-PCR) and Western blot analysis. Cell proliferation was determined by MTT assay and the invasion and migration abilities of EC109 cells were monitored by Transwell TM assay. The protein levels of proliferating cell nuclear antigen (PCNA), Bax and Bcl-2 were tested by Western blotting. Results The positive expression rate of NEDD9 in esophageal carcinoma tissues was significantly higher compared with that in the paracancerous tissues. After NEDD9 expression was successfully downregulated in EC109 cells by siRNA, the proliferation, invasion and migration rates in transfection group were significantly lower than those in control group; meanwhile, the expression of Bcl-2 was reduced and Bax expression was enhanced. Conclusion The protein expression level of NEDD9 is higher in esophageal carcinoma tissues than that in adjacent normal tissues. Knockdown of NEDD9 expression can restrain the proliferation, invasion and migration of EC109 cells.

  15. In vitro and in vivo studies with [18F]fluorocholine on digestive tumoral cell lines and in an animal model of metastasized endocrine tumor

    International Nuclear Information System (INIS)

    Nejjari, Mimoun; Kryza, David; Poncet, Gilles; Roche, Colette; Perek, Nathalie; Chayvialle, Jean-Alain; Le Bars, Didier; Scoazec, Jean-Yves; Janier, Marc; Borson-Chazot, Francoise

    2008-01-01

    Purpose: The aim of this study was to investigate (a) in vitro the relationship between [ 18 F]fluorocholine ([ 18 F]FCH) uptake and cell growth in endocrine cell lines and (b) in vivo the uptake of [ 18 F]FCH by tumoral sites in an animal model of metastasized endocrine tumor. Methods: In vitro studies were conducted on three endocrine and two nonendocrine digestive tumoral cell lines. The proliferative ratio was estimated using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The uptake of [ 18 F]FCH and that of [ 18 F]fluorodeoxyglucose ([ 18 F]FDG) were measured before and after cytotoxic therapy. [ 18 F]FCH biodistribution was studied in nude mice and in an endocrine xenografted mice model. Results: The [ 18 F]FCH uptake in tumoral cell lines was related to their proliferative capacities as measured by the MTT assay in basal conditions. After cytotoxic therapy, the IC 50 values calculated with the [ 18 F]FCH incorporation test were very close to those determined with the MTT assay. Biodistribution studies showed that [ 18 F]FCH was predominantly concentrated in the liver and kidney of nude mice. In the STC-1 xenografted animal model, the uptake of [ 18 F]FCH in the primary tumor was only 1.1%. On autoradiography and micro-positron emission tomography, there was no uptake of [ 18 F]FCH in liver metastases but there was a significant uptake of [ 18 F]FDG. Conclusions: In vitro studies suggested that the incorporation of [ 18 F]FCH in endocrine tumor cell lines was related to their growth capacities; however, in vivo studies conducted in an endocrine xenografted animal model showed an uptake of [ 18 F]FCH in hepatic metastases lower than that in normal liver cells. An influence of the microenvironment or a competition phenomenon for [ 18 F]FCH uptake between normal liver and endocrine tumor cells cannot be excluded

  16. Complete study demonstrating the absence of rhabdovirus in a distinct Sf9 cell line.

    Directory of Open Access Journals (Sweden)

    Yoshifumi Hashimoto

    Full Text Available A putative novel rhabdovirus (SfRV was previously identified in a Spodoptera frugiperda cell line (Sf9 cells [ATCC CRL-1711 lot 58078522] by next generation sequencing and extensive bioinformatic analysis. We performed an extensive analysis of our Sf9 cell bank (ATCC CRL-1711 lot 5814 [Sf9L5814] to determine whether this virus was already present in cells obtained from ATCC in 1987. Inverse PCR of DNA isolated from Sf9 L5814 cellular DNA revealed integration of SfRV sequences in the cellular genome. RT-PCR of total RNA showed a deletion of 320 nucleotides in the SfRV RNA that includes the transcriptional motifs for genes X and L. Concentrated cell culture supernatant was analyzed by sucrose density gradient centrifugation and revealed a single band at a density of 1.14 g/ml. This fraction was further analysed by electron microscopy and showed amorphous and particulate debris that did not resemble a rhabdovirus in morphology or size. SDS-PAGE analysis confirmed that the protein composition did not contain the typical five rhabdovirus structural proteins and LC-MS/MS analysis revealed primarily of exosomal marker proteins, the SfRV N protein, and truncated forms of SfRV N, P, and G proteins. The SfRV L gene fragment RNA sequence was recovered from the supernatant after ultracentrifugation of the 1.14 g/ml fraction treated with diethyl ether suggesting that the SfRV L gene fragment sequence is not associated with a diethyl ether resistant nucleocapsid. Interestingly, the 1.14 g/ml fraction was able to transfer baculovirus DNA into Sf9L5814 cells, consistent with the presence of functional exosomes. Our results demonstrate the absence of viral particles in ATCC CRL-1711 lot 5814 Sf9 cells in contrast to a previous study that suggested the presence of infectious rhabdoviral particles in Sf9 cells from a different lot. This study highlights how cell lines with different lineages may present different virosomes and therefore no general conclusions can

  17. Differentiation of Human Malignant Melanoma Cells that Escape Apoptosis After Treatment with 9-Nitrocamptothecin In Vitro

    Directory of Open Access Journals (Sweden)

    Panayotis Pantazis

    1999-08-01

    Full Text Available After in-vitro exposure to 0.05 μmol/L 9-nitrocamptothecin (9NC for periods of time longer than 5 days, 65% to 80% of the human malignant melanoma SB1 B cells die by apoptosis, whereas the remaining cells are arrested at the G2-phase of the cell cycle. Upon discontinuation of exposure to 9NC the G2-arrested cells resume cell cycling or remain arrested depending on the duration of 9NC exposure. In contrast to cycling malignant cells, the cells irreversibly arrested at G2 exhibit features of normal-like cells, the melanocytes, as assessed by the appearance of dendrite-like structures; loss of proliferative activity; synthesis of the characteristic pigment, melanin; and, particularly, loss of tumorigenic ability after xenografting in immunodeficient mice. Further, the expression of the cyclin-dependent kinase inhibitor p16 is upregulated in the 9NC-treated, G1-arrested, but downregulated in density G1-arrested cells, whereas the reverse is observed in the expression of another cyclin-dependent kinase inhibitor, p21. These results suggest that malignant melanoma SB1B cells that escape 9NC-induced death by apoptosis undergo differentiation toward nonmalignant, normal-like cells.

  18. The systematic study of the electroporation and electrofusion of B16-F1 and CHO cells in isotonic and hypotonic buffer.

    Science.gov (United States)

    Usaj, Marko; Kanduser, Masa

    2012-09-01

    The fusogenic state of the cell membrane can be induced by external electric field. When two fusogenic membranes are in close contact, cell fusion takes place. An appropriate hypotonic treatment of cells before the application of electric pulses significantly improves electrofusion efficiency. How hypotonic treatment improves electrofusion is still not known in detail. Our results indicate that at given induced transmembrane potential electroporation was not affected by buffer osmolarity. In contrast to electroporation, cells' response to hypotonic treatment significantly affects their electrofusion. High fusion yield was observed when B16-F1 cells were used; this cell line in hypotonic buffer resulted in 41 ± 9 % yield, while in isotonic buffer 32 ± 11 % yield was observed. Based on our knowledge, these fusion yields determined in situ by dual-color fluorescence microscopy are among the highest in electrofusion research field. The use of hypotonic buffer was more crucial for electrofusion of CHO cells; the fusion yield increased from below 1 % in isotonic buffer to 10 ± 4 % in hypotonic buffer. Since the same degree of cell permeabilization was achieved in both buffers, these results indicate that hypotonic treatment significantly improves fusion yield. The effect could be attributed to improved physical contact of cell membranes or to enhanced fusogenic state of the cell membrane itself.

  19. miR-335 negatively regulates osteosarcoma stem cell-like properties by targeting POU5F1.

    Science.gov (United States)

    Guo, Xiaodong; Yu, Ling; Zhang, Zhengpei; Dai, Guo; Gao, Tian; Guo, Weichun

    2017-01-01

    Evidence is accumulating to link cancer stem cells to the pathogenesis and progression of osteosarcoma. The aim of this study is to investigate the role of miR-335 in osteosarcoma stem cells. Tumor spheroid culture and flow cytometry were applied to screen out osteosarcoma stem cells. Real-time quantitative PCR was used to detect the expression level of miR-335 in MG63, U2OS and 143B osteosarcoma stem cells. The relationship of miR-335 expression with osteosarcoma stem cells was then analyzed. Transwell assay and transplantation assay were performed to elucidate biological effects of miR-335 on cell invasion and vivo tumor formation. Western Blot and luciferase assays were executed to investigate the regulation of POU5F1 by miR-335. The expression of miR-335 in osteosarcoma stem cells was lower than their differentiated counterparts. Cells expressing miR-335 possessed decreased stem cell-like properties. Gain or loss of function assays were applied to find that miR-335 antagonist promoted stem cell-like properties as well as invasion. Luciferase report and transfection assay showed that POU5F1 was downregulated by miR-335. Pre-miR-335 resulted in tumor enhanced sensitivity to traditional chemotherapy, whereas anti-miR-335 promoted chemoresistance. Finally, the inhibitory effect of miR-335 on in vivo tumor formation showed that combination of pre-miR-335 with cisplatin further reduced the tumor size, and miR-335 brought down the sphere formation capacity induced by cisplatin. The current study demonstrates that miR-335 negatively regulates osteosarcoma stem cell-like properties by targeting POU5F1, and miR-335 could target CSCs to synergize with traditional chemotherapeutic agents to overcome osteosarcoma.

  20. [Alterations in expression of F-actin and DNA of fluid shear stress treated-mesenchymal stem cells affected by titanium particles loading].

    Science.gov (United States)

    Wu, Jiang; Chen, Huiqing; Cao, Hui; Zhou, Jiang; Zhang, Li; Sung, K L

    2004-02-01

    Particulate wear debris within the bone-prosthesis microenvironment generated by normal wear and corrosion of orthopaedic implants is considered to be one of the main factors responsible for chronic aseptic inflammation and development of osteolysis in the long-term instability and failure of total joint arthroplasty. While the decrease in bone volume caused by wear debris-induced osteolysis could have been compensated by enough new bone matrix secreted by osteoblasts. Actually, the normal osteoblastic population depend on the regular differentiation and proliferation of their progenitor cells--bone marrow mesenchymal stem cells (MSCs). This study aims to investigate the potential mechanism for the rat MSCs cytotoxicity upon exposure to Titanium (Ti) particles. Rat mesenchymal stem cells (rMSCs) isolated from 3-month-old male Sprague-Dawley rats by Percoll intensity gradient method were cultured in DMEM medium (low glucose) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 micrograms/ml streptomycin in a humidified incubator with 5% CO2 at 37 degrees C. In order to gain the homogenous cell population, rMSCs were passaged to 3-4th subpassage which were used in all the experiment groups. Then rMSCs were seeded in the 6 well culture plates and exposed to three different circle diameters (mean size, TD1: 0.9 micron, TD2: 2.7 microns, TD3: 6.9 microns) with three different concentrations (0.1 wt%, 0.05 wt%, 0.01 wt%, W/V) at different durations (8 h, 16 h, 24 h,), respectively. Unexposed rMSCs were used as control. In the given periods of Ti loading, fluid shear stress (FSS) was applied to each group cells. The expression of F-actin and DNA of the rMSCs at the indicated time were determined with laser confocal scanning microscopy and image analysis software. The results showed that there was up-regulation expression of F-actin in the rMSCs without Ti particles loading but in the presence of FSS. Ti particles loading can suppress the expression of F

  1. Triage of Limited Versus Extensive Disease on 18F-FDG PET/CT Scan in Small Cell lung Cancer

    Directory of Open Access Journals (Sweden)

    Saima Riaz

    2017-06-01

    Full Text Available Objective(s: Small cell lung cancer (SCLC is an aggressive neuroendocrine carcinoma, which accounts for 10-15% of pulmonary cancers and exhibits early metastatic spread. This study aimed to determine the added value of 18F-FDG PET/CT imaging in tumor, node, and metastasis (TNM staging of SCLC, compared to the conventional computed tomography (CT scan and its potential role as a prognosticator.Methods: This retrospective review was conducted on 23 patients, who were histopathologically diagnosed to have SCLC and referred for undergoing 18F-FDG PET/CT scanning during October 2009-December 2015. The rate of agreement between the CT and 18F-FDG PET/CT findings for TNM staging was calculated using the Cohen’s kappa (κ. The median follow-up time was eight months, ranging 27-3 months. The overall and disease-free survival rates were calculated based on the extent of disease.Results: 19 cases were male and four female with the mean age of 58±9 years. The 18F-FDG PET/CT identified limited and extensive diseases in 2 (8.7% and 21 (91.3% patients, respectively. In addition, the results of the Cohen’s kappa demonstrated a strong (κ=0.82, fair (κ=0.24, and poor (κ=0.12 agreement between the PET/CT and CT findings for determining tumor, node, and metastasis stages, respectively. The 18F-FDG PET/CT scans upstaged disease in 47% of the cases with visceral and osseous metastasis. The disease-free survival rates for the limited and extensive diseases were 100% and 23% within the 12-month follow-up. In addition, 8 (35% patients expired during the follow-up period.Conclusion: Improved nodal and metastatic disease identification highlights the role of 18F-FDG PET/CT scanning in initial staging of SCLC with prognostic implications.

  2. Analysis list: E2f1 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available E2f1 Blood,Liver + mm9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/E2f1.1....tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/E2f1.5.tsv http://dbarchive.biosciencedbc.jp/kyus...hu-u/mm9/target/E2f1.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/E2f1.Blood.tsv,http://dbarchive.bioscience...dbc.jp/kyushu-u/mm9/colo/E2f1.Liver.tsv http://dbarchive.bioscience...dbc.jp/kyushu-u/mm9/colo/Blood.gml,http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Liver.gml ...

  3. Human CD4+ T cell responses to the dog major allergen Can f 1 and its human homologue tear lipocalin resemble each other.

    Directory of Open Access Journals (Sweden)

    Aino L K Liukko

    Full Text Available Lipocalin allergens form a notable group of proteins, as they contain most of the significant respiratory allergens from mammals. The basis for the allergenic capacity of allergens in the lipocalin family, that is, the development of T-helper type 2 immunity against them, is still unresolved. As immunogenicity has been proposed to be a decisive feature of allergens, the purpose of this work was to examine human CD4+ T cell responses to the major dog allergen Can f 1 and to compare them with those to its human homologue, tear lipocalin (TL. For this, specific T cell lines were induced in vitro from the peripheral blood mononuclear cells of Can f 1-allergic and healthy dog dust-exposed subjects with peptides containing the immunodominant T cell epitopes of Can f 1 and the corresponding TL peptides. We found that the frequency of Can f 1 and TL-specific T cells in both subject groups was low and close to each other, the difference being about two-fold. Importantly, we found that the proliferative responses of both Can f 1 and TL-specific T cell lines from allergic subjects were stronger than those from healthy subjects, but that the strength of the responses within the subject groups did not differ between these two antigens. Moreover, the phenotype of the Can f 1 and TL-specific T cell lines, determined by cytokine production and expression of cell surface markers, resembled each other. The HLA system appeared to have a minimal role in explaining the allergenicity of Can f 1, as the allergic and healthy subjects' HLA background did not differ, and HLA binding was very similar between Can f 1 and TL peptides. Along with existing data on lipocalin allergens, we conclude that strong antigenicity is not decisive for the allergenicity of Can f 1.

  4. Evaluation of recurrent disease in the re-staging of colorectal cancer by 18F-FDG PET/CT: Use of CEA and CA 19-9 in patient selection.

    Science.gov (United States)

    Chiaravalloti, Agostino; Fiorentini, Alessandro; Palombo, Erika; Rinino, Davide; Lacanfora, Annamaria; Danieli, Roberta; Di Russo, Carmen; Di Biagio, Daniele; Squillaci, Ettore; Schillaci, Orazio

    2016-11-01

    The aim of the present retrospective study was to evaluate the sensitivity and specificity of fluorodeoxyglucose ( 18 F-FDG) positron emission tomography/computed tomography (PET/CT) in assessing the recurrence of colorectal cancer (CRC) with regard to carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9). 18 F-FDG PET/CT was performed in 100 patients for the re-staging of CRC. Therapy was discontinued prior to the examination. The mean (± standard deviation) CEA value (measured ~30 days prior to PET/CT examination) was 23.71 (±107) ng/ml, whereas the CA 19-9 value was 72 (±190.3) U/ml. Differences in CEA and CA 19-9 values in patients with scans that were positive or negative for recurrence were analyzed by means of a receiver operating characteristic (ROC) curve. ROC curves were used for the calculation of the sensitivity and specificity of 18 F-FDG PET/CT for the CEA and CA 19-9 levels. The results of the 18 F-FDG PET/CT were found to be associated with the CEA level (P=0.001), but not with the CA 19-9 level (P=0.43). PET/CT was positive for recurrence in 60 patients (60.0%), whose mean CEA and CA 19-9 values were 33.07±136.7 ng/ml and 75.24±192.3 U/ml, respectively. PET/CT was negative for recurrence in 40 patients (40.0%), whose mean CEA and CA 19-9 values were 10.15±30 ng/ml and 67.76±190 U/ml, respectively. On the basis of ROC curve analysis, the best compromise between sensitivity and specificity was achieved for CEA levels of 3.5 ng/ml [sensitivity, 80%; 95% confidence interval (CI), 67-89%; and specificity, 60%; 95% CI, 45-78%]. The study concluded that the detection of recurrence by 18 F-FDG PET/CT in patients treated for CRC is associated with CEA, but not CA 19-9 serum levels. Moreover, 18 F-FDG PET/CT should be recommended in patients with suspected CRC recurrence even when they present with CEA levels below the normal cut-off.

  5. The OsO(3)F(+) and mu-F(OsO(3)F)(2)(+) cations: their syntheses and study by Raman and (19)F NMR spectroscopy and electron structure calculations and X-ray crystal structures of [OsO(3)F][PnF(6)] (Pn = As, Sb), [OsO(3)F][HF](2)[AsF(6)], [OsO(3)F][HF][SbF(6)], and [OsO(3)F][Sb(3)F(16)].

    Science.gov (United States)

    Gerken, Michael; Dixon, David A; Schrobilgen, Gary J

    2002-01-28

    The fluoride ion donor properties of OsO(3)F(2) have been investigated. The salts [OsO(3)F][AsF(6)], [OsO(3)F][HF](2)[AsF(6)], mu-F(OsO(3)F)(2)[AsF(6)], [OsO(3)F][HF](2)[SbF(6)], and [OsO(3)F][HF][SbF(6)] have been prepared by reaction of OsO(3)F(2) with AsF(5) and SbF(5) in HF solvent and have been characterized in the solid state by Raman spectroscopy. The single-crystal X-ray diffraction studies of [OsO(3)F][AsF(6)] (P2(1)/n, a = 7.0001(11) A, c = 8.8629(13) A, beta = 92.270(7) degrees, Z = 4, and R(1) = 0.0401 at -126 degrees C), [OsO(3)F][SbF(6)] (P2(1)/c, a = 5.4772(14) A, b = 10.115(3) A, c = 12.234(3) A, beta = 99.321(5) degrees, Z = 4, and R(1) = 0.0325 at -173 degrees C), [OsO(3)F][HF](2)[AsF(6)] (P2(1)/n, a = 5.1491(9) A, b = 8.129(2) A, c = 19.636(7) A, beta = 95.099(7) degrees, Z = 4, and R(1) = 0.0348 at -117 degrees C), and [OsO(3)F][HF][SbF(6)] (Pc, a = 5.244(4) A, b = 9.646(6) A, c = 15.269(10) A, beta = 97.154(13) degrees, Z = 4, and R(1) = 0.0558 at -133 degrees C) have shown that the OsO(3)F(+) cations exhibit strong contacts to the anions and HF solvent molecules giving rise to cyclic, dimeric structures in which the osmium atoms have coordination numbers of 6. The reaction of OsO(3)F(2) with neat SbF(5) yielded [OsO(3)F][Sb(3)F(16)], which has been characterized by (19)F NMR spectroscopy in SbF(5) and SO(2)ClF solvents and by Raman spectroscopy and single-crystal X-ray diffraction in the solid state (P4(1)m, a = 10.076(6) A, c = 7.585(8) A, Z = 2, and R(1) = 0.0858 at -113 degrees C). The weak fluoride ion basicity of the Sb(3)F(16)(-) anion resulted in an OsO(3)F(+) cation (C(3)(v) point symmetry) that is well isolated from the anion and in which the osmium is four-coordinate. The geometrical parameters and vibrational frequencies of OsO(3)F(+), ReO(3)F, mu-F(OsO(3)F)(2)(+), (FO(3)Os--FPnF(5))(2), and (FO(3)Os--(HF)(2)--FPnF(5))(2) (Pn = As, Sb) have been calculated using density functional theory methods.

  6. Factor VIII (F8) inversions in severe hemophilia A: Male germ cell origin and diagnosis with RT-PCR

    Energy Technology Data Exchange (ETDEWEB)

    Antonarakis, S.E. [Geneva Medical School (Switzerland)]|[Johns Hopkins School of Medicine, Baltimore, MD (United States); Rossiter, J.P. [Johns Hopkins School of Medicine, Baltimore, MD (United States); Young, M. [Geneva Medical School (Switzerland)] [and others

    1994-09-01

    The Factor VIII (F8) gene, which is defective in hemophilia A, is located in the most telomeric megabase of Xq. Inversions due to intrachromosomal homologous recombination between mispaired copies of gene A located within intron 22 of the gene and about 500 kb telomeric to it account for nearly half of the cases of severe hemophilia A. We hypothesized that pairing of Xq with its homolog inhibits the inversion process, and that therefore the event originates predominantly in male germ cells. In all 21 informative cases in which the inversion originated in a maternal grandparent, DNA polymorphism analysis using markers within or very closely linked to F8, determined that it occurred in the male germline. In addition, all but one of 56 mothers of sporadic cases due to inversions were carriers. The data indicate that the F8 gene inversions leading to severe hemophilia A occur almost exclusively in male germ cells. The mean age of maternal grandfathers at the birth of their carrier daughters was 29.9 years (13 cases), i.e. not different from the mean paternal age in the general population, supporting the hypothesis that the inversions occur in meiosis. The inversions can be diagnosed by Southern blot analysis. For more rapid diagnosis we have used RT-PCR of RNA ectopically expressed in blood. Oligonucleotides were used to PCR amplify, after the initial RT reaction of RNA samples using random hexamers, either the normal transcript (F8 exons 21 to 24;312 bp product) or the novel abnormal transcript that is generated after the inversion. Both type 1 and 2 inversions can be recognized in affecteds and carriers by the presence of the diagnostic PcR product of 248 bp. Correct diagnoses were made in samples from 6 patients and 2 carriers with type 1 inversions, 2 patients and 2 carriers with type 2 inversions and 5 normal controls.

  7. Pulsed-dosing with oral sodium phenylbutyrate increases hemoglobin F in a patient with sickle cell anemia.

    Science.gov (United States)

    Hines, Patrick; Dover, George J; Resar, Linda M S

    2008-02-01

    Increasing hemoglobin F (HbF) appears to be beneficial for patients with sickle cell anemia. We previously demonstrated that daily, oral sodium phenylbutyrate (OSPB) induces HbF synthesis in pediatric and adult patients with hemoglobin SS (HbSS). The high doses and need for daily therapy, however, have limited its use. Here, we report a patient treated with pulsed-dosing of OSPB for over 3 years. This patient developed a modest, but sustained elevation in HbF over the course of therapy without side effects. Although larger studies are needed, this case demonstrates that pulsed-dosing with OSPB enhances HbF synthesis. (c) 2007 Wiley-Liss, Inc.

  8. DEPDC1 promotes cell proliferation and tumor growth via activation of E2F signaling in prostate cancer.

    Science.gov (United States)

    Huang, Lin; Chen, Keng; Cai, Zhao-Peng; Chen, Fu-Chao; Shen, Hui-Yong; Zhao, Wei-Hua; Yang, Song-Jie; Chen, Xu-Biao; Tang, Guo-Xue; Lin, Xi

    2017-08-26

    DEP domain containing 1 (DEPDC1) is recently reported to be overexpressed in several types of human cancer; however the role of DEPDC1 in prostate cancer remains to be investigated. Herein, we identified that the DEPDC1 mRNA and protein expression levels were dramatically increased in prostate cancer tissues and cell lines. Overexpression of DEPDC1 promoted, but depletion of DEPDC1 inhibited cell proliferation by regulating the G1-S phase cell cycle transition. Importantly, we found that DEPDC1 was essential for the tumor growth and formation of bone metastases of prostate cancer cells in vivo. Finally, we demonstrated that DEPDC1 interacted with E2F1 and increased its transcriptional activity, leading to hyper-activation of E2F signaling in prostate cancer cells. Our findings reveal an oncogenic role of DEPDC1 in prostate cancer progression via activation of E2F signaling, and suggest DEPDC1 might be a potential therapeutic target against the disease. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Selective targeting of tumour neovasculature by a radiohalogenated human antibody fragment specific for the ED-B domain of fibronectin

    International Nuclear Information System (INIS)

    Demartis, S.; Tarli, L.; Neri, D.; Borsi, L.; Zardi, L.

    2001-01-01

    Angiogenesis is a characteristic feature of many aggressive tumours and other disorders. Antibodies capable of binding to new blood vessels, but not to mature vessels, could be used as selective targeting agents for immunoscintigraphic and radioimmunotherapeutic applications. Here we show that scFv(L19), a recombinant human antibody fragment with sub-nanomolar affinity for the ED-B domain of fibronectin, a marker of angiogenesis, can be stably labelled with iodine-125 and astatine-211 with full retention of immunoreactivity, using a trimethyl-stannyl benzoate bifunctional derivative. Biodistribution studies in mice bearing two different types of tumour grafted subcutaneously, followed by ex vivo micro-autoradiographic analysis, revealed that scFv(L19) rapidly localises around tumour blood vessels, but not around normal vessels. Four hours after intravenous injection of the stably radioiodinated scFv(L19), tumour to blood ratios were 6:1 in mice bearing the F9 murine teratocarcinoma and 9:1 in mice bearing an FE8 rat sarcoma. As expected, all other organs (including kidney) contained significantly less radioactivity than the tumour. Since the ED-B domain of fibronectin has an identical sequence in mouse and man, scFv(L19) is a pan-species antibody and the results presented here suggest clinical utility of radiolabelled scFv(L19) for the scintigraphic detection of angiogenesis in vivo. Furthermore, it should now be possible to investigate scFv(L19) for the selective delivery of 211 At to the tumour neovasculature, causing the selective death of tumour endothelial cells and tumour collapse. (orig.)

  10. In Vivo Interplay between p27Kip1, GATA3, ATOH1, and POU4F3 Converts Non-sensory Cells to Hair Cells in Adult Mice

    Directory of Open Access Journals (Sweden)

    Bradley J. Walters

    2017-04-01

    Full Text Available Summary: Hearing loss is widespread and persistent because mature mammalian auditory hair cells (HCs are nonregenerative. In mice, the ability to regenerate HCs from surrounding supporting cells (SCs declines abruptly after postnatal maturation. We find that combining p27Kip1 deletion with ectopic ATOH1 expression surmounts this age-related decline, leading to conversion of SCs to HCs in mature mouse cochleae and after noise damage. p27Kip1 deletion, independent of canonical effects on Rb-family proteins, upregulated GATA3, a co-factor for ATOH1 that is lost from SCs with age. Co-activation of GATA3 or POU4F3 and ATOH1 promoted conversion of SCs to HCs in adult mice. Activation of POU4F3 alone also converted mature SCs to HCs in vivo. These data illuminate a genetic pathway that initiates auditory HC regeneration and suggest p27Kip1, GATA3, and POU4F3 as additional therapeutic targets for ATOH1-mediated HC regeneration. : Auditory hair cells are nonregenerative, resulting in persistent hearing loss upon damage. Walters et al. find that manipulating two genes, p27Kip1 and Atoh1, induces the conversion of nonsensory cells to hair cells in adult mice. This effect is mediated by GATA3 and POU4F3, where POU4F3 alone was found to convert nonsensory cells. Keywords: regeneration, aging, differentiation, proliferation, development, cancer, sensory, cochlea, hearing

  11. 18F-FAC PET selectively images hepatic infiltrating CD4 and CD8 T cells in a mouse model of autoimmune hepatitis.

    Science.gov (United States)

    Salas, Jessica R; Chen, Bao Ying; Wong, Alicia; Cheng, Donghui; Van Arnam, John S; Witte, Owen N; Clark, Peter M

    2018-04-26

    Immune cell-mediated attack on the liver is a defining feature of autoimmune hepatitis and hepatic allograft rejection. Despite an assortment of diagnostic tools, invasive biopsies remain the only method for identifying immune cells in the liver. We evaluated whether PET imaging with radiotracers that quantify immune activation ( 18 F-FDG and 18 F-FAC) and hepatocyte biology ( 18 F-DFA) can visualize and quantify hepatic infiltrating immune cells and hepatocyte inflammation, respectively, in a preclinical model of autoimmune hepatitis. Methods: Mice treated with Concanavalin A (ConA) to induce a model of autoimmune hepatitis or vehicle were imaged with 18 F-FDG, 18 F-FAC, and 18 F-DFA PET. Immunohistochemistry, digital autoradiography, and ex vivo accumulation assays were used to localize areas of altered radiotracer accumulation in the liver. For comparison, mice treated with an adenovirus to induce a viral hepatitis or vehicle were imaged with 18 F-FDG, 18 F-FAC, and 18 F-DFA PET. 18 F-FAC PET was performed on mice treated with ConA, and vehicle or dexamethasone. Biopsy samples of patients suffering from autoimmune hepatitis were immunostained for deoxycytidine kinase (dCK). Results: Hepatic accumulation of 18 F-FDG and 18 F-FAC was 173% and 61% higher, respectively, and hepatic accumulation of 18 F-DFA was 41% lower in a mouse model of autoimmune hepatitis compared to control mice. Increased hepatic 18 F-FDG accumulation was localized to infiltrating leukocytes and inflamed sinusoidal endothelial cells, increased hepatic 18 F-FAC accumulation was concentrated in infiltrating CD4 and CD8 cells, and decreased hepatic 18 F-DFA accumulation was apparent in hepatocytes throughout the liver. In contrast, viral hepatitis increased hepatic 18 F-FDG accumulation by 109% and decreased hepatic 18 F-DFA accumulation by 20% but had no effect on hepatic 18 F-FAC accumulation (non-significant 2% decrease). 18 F-FAC PET provided a non-invasive biomarker of the efficacy of

  12. Melanogenesis stimulation in B16-F10 melanoma cells induces cell cycle alterations, increased ROS levels and a differential expression of proteins as revealed by proteomic analysis

    Energy Technology Data Exchange (ETDEWEB)

    Cunha, Elizabeth S.; Kawahara, Rebeca [Departamento de Bioquimica e Biologia Molecular, Setor de Ciencias Biologicas, Universidade Federal do Parana, P.O. Box 19046, CEP 81531-990, Curitiba, PR (Brazil); Kadowaki, Marina K. [Universidade Estadual do Oeste do Parana, Cascavel, PR (Brazil); Amstalden, Hudson G.; Noleto, Guilhermina R.; Cadena, Silvia Maria S.C.; Winnischofer, Sheila M.B. [Departamento de Bioquimica e Biologia Molecular, Setor de Ciencias Biologicas, Universidade Federal do Parana, P.O. Box 19046, CEP 81531-990, Curitiba, PR (Brazil); Martinez, Glaucia R., E-mail: grmartinez@ufpr.br [Departamento de Bioquimica e Biologia Molecular, Setor de Ciencias Biologicas, Universidade Federal do Parana, P.O. Box 19046, CEP 81531-990, Curitiba, PR (Brazil)

    2012-09-10

    Considering that stimulation of melanogenesis may lead to alterations of cellular responses, besides melanin production, our main goal was to study the cellular effects of melanogenesis stimulation of B16-F10 melanoma cells. Our results show increased levels of the reactive oxygen species after 15 h of melanogenesis stimulation. Following 48 h of melanogenesis stimulation, proliferation was inhibited (by induction of cell cycle arrest in the G1 phase) and the expression levels of p21 mRNA were increased. In addition, melanogenesis stimulation did not induce cellular senescence. Proteomic analysis demonstrated the involvement of proteins from other pathways besides those related to the cell cycle, including protein disulfide isomerase A3, heat-shock protein 70, and fructose biphosphate aldolase A (all up-regulated), and lactate dehydrogenase (down-regulated). In RT-qPCR experiments, the levels of pyruvate kinase M2 mRNA dropped, whereas the levels of ATP synthase (beta-F1) mRNA increased. These data indicate that melanogenesis stimulation of B16-F10 cells leads to alterations in metabolism and cell cycle progression that may contribute to an induction of cell quiescence, which may provide a mechanism of resistance against cellular injury promoted by melanin synthesis. -- Highlights: Black-Right-Pointing-Pointer Melanogenesis stimulation by L-tyrosine+NH{sub 4}Cl in B16-F10 melanoma cells increases ROS levels. Black-Right-Pointing-Pointer Melanogenesis inhibits cell proliferation, and induced cell cycle arrest in the G1 phase. Black-Right-Pointing-Pointer Proteomic analysis showed alterations in proteins of the cell cycle and glucose metabolism. Black-Right-Pointing-Pointer RT-qPCR analysis confirmed alterations of metabolic targets after melanogenesis stimulation.

  13. Nlrp9b inflammasome restricts rotavirus infection in intestinal epithelial cells.

    Science.gov (United States)

    Zhu, Shu; Ding, Siyuan; Wang, Penghua; Wei, Zheng; Pan, Wen; Palm, Noah W; Yang, Yi; Yu, Hua; Li, Hua-Bing; Wang, Geng; Lei, Xuqiu; de Zoete, Marcel R; Zhao, Jun; Zheng, Yunjiang; Chen, Haiwei; Zhao, Yujiao; Jurado, Kellie A; Feng, Ningguo; Shan, Liang; Kluger, Yuval; Lu, Jun; Abraham, Clara; Fikrig, Erol; Greenberg, Harry B; Flavell, Richard A

    2017-06-29

    Rotavirus, a leading cause of severe gastroenteritis and diarrhoea in young children, accounts for around 215,000 deaths annually worldwide. Rotavirus specifically infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-κB signalling, raising the question as to whether other host factors participate in antiviral responses in intestinal mucosa. The mechanism by which enteric viruses are sensed and restricted in vivo, especially by NOD-like receptor (NLR) inflammasomes, is largely unknown. Here we uncover and mechanistically characterize the NLR Nlrp9b that is specifically expressed in intestinal epithelial cells and restricts rotavirus infection. Our data show that, via RNA helicase Dhx9, Nlrp9b recognizes short double-stranded RNA stretches and forms inflammasome complexes with the adaptor proteins Asc and caspase-1 to promote the maturation of interleukin (Il)-18 and gasdermin D (Gsdmd)-induced pyroptosis. Conditional depletion of Nlrp9b or other inflammasome components in the intestine in vivo resulted in enhanced susceptibility of mice to rotavirus replication. Our study highlights an important innate immune signalling pathway that functions in intestinal epithelial cells and may present useful targets in the modulation of host defences against viral pathogens.

  14. Nlrp9b inflammasome restricts rotavirus infection in intestinal epithelial cells

    Science.gov (United States)

    Zhu, Shu; Ding, Siyuan; Wang, Penghua; Wei, Zheng; Pan, Wen; Palm, Noah W; Yang, Yi; Yu, Hua; Li, Hua-Bing; Wang, Geng; Lei, Xuqiu; de Zoete, Marcel R.; Zhao, Jun; Zheng, Yunjiang; Chen, Haiwei; Zhao, Yujiao; Jurado, Kellie A.; Feng, Ningguo; Shan, Liang; Kluger, Yuval; Lu, Jun; Abraham, Clara; Fikrig, Erol; Greenberg, Harry B.; Flavell, Richard A.

    2018-01-01

    Rotavirus, a leading cause of severe gastroenteritis and diarrhoea in young children, accounts for around 215,000 deaths annually worldwide1. Rotavirus specifically infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-κB signalling2–5, raising the question as to whether other host factors participate in antiviral responses in intestinal mucosa. The mechanism by which enteric viruses are sensed and restricted in vivo, especially by NOD-like receptor (NLR) inflammasomes, is largely unknown. Here we uncover and mechanistically characterize the NLR Nlrp9b that is specifically expressed in intestinal epithelial cells and restricts rotavirus infection. Our data show that, via RNA helicase Dhx9, Nlrp9b recognizes short double-stranded RNA stretches and forms inflammasome complexes with the adaptor proteins Asc and caspase-1 to promote the maturation of interleukin (Il)-18 and gasdermin D (Gsdmd)-induced pyroptosis. Conditional depletion of Nlrp9b or other inflammasome components in the intestine in vivo resulted in enhanced susceptibility of mice to rotavirus replication. Our study highlights an important innate immune signalling pathway that functions in intestinal epithelial cells and may present useful targets in the modulation of host defences against viral pathogens. PMID:28636595

  15. IL-9-producing cells in the development of IgE-mediated food allergy.

    Science.gov (United States)

    Shik, Dana; Tomar, Sunil; Lee, Jee-Boong; Chen, Chun-Yu; Smith, Andrew; Wang, Yui-Hsi

    2017-01-01

    Food allergy is a harmful immune reaction driven by uncontrolled type 2 immune responses. Considerable evidence demonstrates the key roles of mast cells, IgE, and TH2 cytokines in mediating food allergy. However, this evidence provides limited insight into why only some, rather than all, food allergic individuals are prone to develop life-threatening anaphylaxis. Clinical observations suggest that patients sensitized to food through the skin early in life may later develop severe food allergies. Aberrant epidermal thymic stromal lymphopoietin and interleukin (IL) 33 production and genetic predisposition can initiate an allergic immune response mediated by dendritic cells and CD4 + TH2 cells in inflamed skin. After allergic sensitization, intestinal IL-25 and food ingestion enhance concerted interactions between type 2 innate lymphoid cells (ILC2s) and CD4 + TH2 cells, which perpetuate allergic reactions from the skin to the gut. IL-4 and cross-linking of antigen/IgE/FcεR complexes induce emigrated mast cell progenitors to develop into the multi-functional IL-9-producing mucosal mast cells, which produce prodigious amounts of IL-9 and mast cell mediators to drive intestinal mastocytosis in an autocrine loop. ILC2s and TH9 cells may also serve as alternative cellular sources of IL-9 to augment the amplification of intestinal mastocytosis, which is the key cellular checkpoint in developing systemic anaphylaxis. These findings provide a plausible view of how food allergy develops and progresses in a stepwise manner and that atopic signals, dietary allergen ingestion, and inflammatory cues are fundamental in promoting life-threatening anaphylaxis. This information will aid in improving diagnosis and developing more effective therapies for food allergy-triggered anaphylaxis.

  16. A COMPREHENSIVE STUDY ON APOPTOSIS INDUCTION BY AZADIRACHTIN IN Spodoptera frugiperda CULTURED CELL LINE Sf9.

    Science.gov (United States)

    Shu, Benshui; Wang, Wenxiang; Hu, Qingbo; Huang, Jingfei; Hu, Meiying; Zhong, Guohua

    2015-07-01

    The induction of apoptosis by azadirachtin, a well-known botanical tetranortriterpenoid isolated from the neem tree (Azadirachta indica A. Juss) and other members of the Meliaceae, was investigated in Spodoptera frugiperda cultured cell line (Sf9). Morphological changes in Sf9 cells treated by various concentrations of azadirachtin were observed at different times under light microscopy. Morphological and biochemical analysis indicated that Sf9 cells treated by 1.5 μg/mL azadirachtin showed typical morphological changes, which were indicative of apoptosis and a clear DNA ladder. The flow cytometry analysis showed the apoptosis rate reached a maximum value of 32.66% at 24 h with 1.5 μg/mL azadirachtin in Sf9 cells. The inhibition of Sf9 cell proliferation suggested that the effect of azadirachtin was dose dependent and the EC50 at 48 and 72 h was 2.727 × 10(-6) and 6.348 × 10(-9) μg/mL, respectively. The treatment of azadirachtin in Sf9 cells could significantly increase the activity of Sf caspase-1, but showed no effect on the activity of Topo I, suggesting that the apoptosis induced by azadirachtinin Sf9 cells is through caspase-dependent pathway. These results provided not only a series of morphological, biochemical, and toxicological comprehensive evidences for induction of apoptosis by azadirachtin, but also a reference model for screening insect cell apoptosis inducers from natural compounds. © 2015 Wiley Periodicals, Inc.

  17. Lanthanide-activated Na{sub 5}Gd{sub 9}F{sub 32} nanocrystals precipitated from a borosilicate glass: Phase-separation-controlled crystallization and optical property

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Daqin, E-mail: dqchen@hdu.edu.cn [College of Materials and Environmental Engineering, Hangzhou Dianzi University, Hangzhou 310018 (China); Key Laboratory of Design and Assembly of Functional Nanostructures, Fujian Institute of Research on the Structure of Matter, CAS, Fuzhou 350002 (China); Wan, Zhongyi; Zhou, Yang [College of Materials and Environmental Engineering, Hangzhou Dianzi University, Hangzhou 310018 (China); Chen, Yan, E-mail: chenyan@hdu.edu.cn [College of Materials and Environmental Engineering, Hangzhou Dianzi University, Hangzhou 310018 (China); Yu, Hua; Lu, Hongwei; Ji, Zhenguo [College of Materials and Environmental Engineering, Hangzhou Dianzi University, Hangzhou 310018 (China); Huang, Ping, E-mail: phuang@fjirsm.ac.cn [Key Laboratory of Design and Assembly of Functional Nanostructures, Fujian Institute of Research on the Structure of Matter, CAS, Fuzhou 350002 (China)

    2015-03-15

    Highlights: • Na{sub 5}Gd{sub 9}F{sub 32} nanocrystals embedded glass ceramics were fabricated for the first time. • Such glass ceramics were achieved by phase-separation-controlled crystallization. • Elemental mapping evidenced the segregation of activators into the Na{sub 5}Gd{sub 9}F{sub 32} lattice. • Luminescent color could be tuned by controlling glass crystallization temperature. - Abstract: Lanthanide-activated cubic Na{sub 5}Gd{sub 9}F{sub 32} nanocrystals were precipitated from a borosilicate glass with a specifically designed composition. The precursor glass is already phase-separated after melt-quenching, which is beneficial to the realization of the controllable glass crystallization for affording desirable size, morphology and activator partition. Elemental mapping in the scanning transmission electron microscopy evidenced that the segregation of lanthanide ions into the Na{sub 5}Gd{sub 9}F{sub 32} lattice was in situ formed without the requirement of long-range ionic diffusion. Impressively, such fabricated glass ceramic co-doped with Yb{sup 3+}/Er{sup 3+} ions exhibited intense upconversion luminescence, which was about 500 times higher than that of the precursor glass, and its luminescent color could be easily tuned from red to green by controlling glass crystallization temperature. It is anticipated that such phase-separation synthesis strategy with precise control over nanostructure of glass ceramics offer a great opportunity to design other highly transparent nanocomposites with a wide range of tunable optical properties.

  18. OP9-Lhx2 stromal cells facilitate derivation of hematopoietic progenitors both in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Xiaoli Chen

    2015-09-01

    Full Text Available Generating engraftable hematopoietic stem cells (HSCs from pluripotent stem cells (PSCs is an ideal approach for obtaining induced HSCs for cell therapy. However, the path from PSCs to robustly induced HSCs (iHSCs in vitro remains elusive. We hypothesize that the modification of hematopoietic niche cells by transcription factors facilitates the derivation of induced HSCs from PSCs. The Lhx2 transcription factor is expressed in fetal liver stromal cells but not in fetal blood cells. Knocking out Lhx2 leads to a fetal hematopoietic defect in a cell non-autonomous role. In this study, we demonstrate that the ectopic expression of Lhx2 in OP9 cells (OP9-Lhx2 accelerates the hematopoietic differentiation of PSCs. OP9-Lhx2 significantly increased the yields of hematopoietic progenitor cells via co-culture with PSCs in vitro. Interestingly, the co-injection of OP9-Lhx2 and PSCs into immune deficient mice also increased the proportion of hematopoietic progenitors via the formation of teratomas. The transplantation of phenotypic HSCs from OP9-Lhx2 teratomas but not from the OP9 control supported a transient repopulating capability. The upregulation of Apln gene by Lhx2 is correlated to the hematopoietic commitment property of OP9-Lhx2. Furthermore, the enforced expression of Apln in OP9 cells significantly increased the hematopoietic differentiation of PSCs. These results indicate that OP9-Lhx2 is a good cell line for regeneration of hematopoietic progenitors both in vitro and in vivo.

  19. Nanotopography induced contact guidance of the F11 cell line during neuronal differentiation: a neuronal model cell line for tissue scaffold development

    International Nuclear Information System (INIS)

    Wieringa, Paul; Micera, Silvestro; Tonazzini, Ilaria; Cecchini, Marco

    2012-01-01

    The F11 hybridoma, a dorsal root ganglion-derived cell line, was used to investigate the response of nociceptive sensory neurons to nanotopographical guidance cues. This established this cell line as a model of peripheral sensory neuron growth for tissue scaffold design. Cells were seeded on substrates of cyclic olefin copolymer (COC) films imprinted via nanoimprint lithography (NIL) with a grating pattern of nano-scale grooves and ridges. Different ridge widths were employed to alter the focal adhesion formation, thereby changing the cell/substrate interaction. Differentiation was stimulated with forskolin in culture medium consisting of either 1 or 10% fetal bovine serum (FBS). Per medium condition, similar neurite alignment was achieved over the four day period, with the 1% serum condition exhibiting longer, more aligned neurites. Immunostaining for focal adhesions found the 1% FBS condition to also have fewer, less developed focal adhesions. The robust response of the F11 to guidance cues further builds on the utility of this cell line as a sensory neuron model, representing a useful tool to explore the design of regenerative guidance tissue scaffolds. (paper)

  20. Uptake of [18F]fluorodeoxyglucose in human monocyte-macrophages in vitro

    International Nuclear Information System (INIS)

    Deichen, Jan Thiess; Prante, Olaf; Gack, Michaela; Schmiedehausen, Kristin; Kuwert, Torsten

    2003-01-01

    The fact that fluorine-18 fluorodeoxyglucose ([ 18 F]FDG) accumulates in inflammatory lesions as well as in tumours reduces the diagnostic specificity of positron emission tomography (PET) in oncology. The aim of this study was to characterise the uptake of [ 18 F]FDG in isolated human monocyte-macrophages (HMMs) in vitro in comparison with that in human glioblastoma (GLI) and pancreatic carcinoma cells (PAN). The purity of HMM preparations was determined by immunohistochemical staining and their functional integrity was assessed by long-term incubation with iodine-131 acetylated bovine serum albumin. [ 18 F]FDG uptake in HMMs was quantified as percent of whole [ 18 F]FDG activity per well (% ID) or as % ID in relation to total protein mass. [ 18 F]FDG uptake in HMMs significantly increased with culture duration, yielding 7.5%±0.9% (% ID/100 μg) at day 14. Stimulation by lipopolysaccharide further enhanced [ 18 F]FDG uptake in HMMs by a factor of 2. [ 18 F]FDG uptake significantly decreased with increasing glucose concentration in the medium. Radio-thin layer chromatography of intracellular metabolites revealed that [ 18 F]FDG was trapped by HMMs mainly as [ 18 F]FDG-6-phosphate and [ 18 F]FDG-1,6-diphosphate. [ 18 F]FDG uptake was in the range of uptake values measured in GLI and PAN. By accumulating [ 18 F]FDG in a manner analogous to uptake by tumour cells, activated HMMs may contribute to the [ 18 F]FDG uptake values measured by PET in neoplasms. (orig.)

  1. TGFβ activated kinase 1 (TAK1 at the crossroad of B cell receptor and Toll-like receptor 9 signaling pathways in human B cells.

    Directory of Open Access Journals (Sweden)

    Dániel Szili

    Full Text Available B cell development and activation are regulated by combined signals mediated by the B cell receptor (BCR, receptors for the B-cell activating factor of the tumor necrosis factor family (BAFF-R and the innate receptor, Toll-like receptor 9 (TLR9. However, the underlying mechanisms by which these signals cooperate in human B cells remain unclear. Our aim was to elucidate the key signaling molecules at the crossroads of BCR, BAFF-R and TLR9 mediated pathways and to follow the functional consequences of costimulation.Therefore we stimulated purified human B cells by combinations of anti-Ig, B-cell activating factor of the tumor necrosis factor family (BAFF and the TLR9 agonist, CpG oligodeoxynucleotide. Phosphorylation status of various signaling molecules, B cell proliferation, cytokine secretion, plasma blast generation and the frequency of IgG producing cells were investigated. We have found that BCR induced signals cooperate with BAFF-R- and TLR9-mediated signals at different levels of cell activation. BCR and BAFF- as well as TLR9 and BAFF-mediated signals cooperate at NFκB activation, while BCR and TLR9 synergistically costimulate mitogen activated protein kinases (MAPKs, ERK, JNK and p38. We show here for the first time that the MAP3K7 (TGF beta activated kinase, TAK1 is responsible for the synergistic costimulation of B cells by BCR and TLR9, resulting in an enhanced cell proliferation, plasma blast generation, cytokine and antibody production. Specific inhibitor of TAK1 as well as knocking down TAK1 by siRNA abrogates the synergistic signals. We conclude that TAK1 is a key regulator of receptor crosstalk between BCR and TLR9, thus plays a critical role in B cell development and activation.

  2. Release of Liposomal Contents by Cell-Secreted Matrix Metalloproteinase-9

    Science.gov (United States)

    Banerjee, Jayati; Hanson, Andrea J.; Gadam, Bhushan; Elegbede, Adekunle I.; Tobwala, Shakila; Ganguly, Bratati; Wagh, Anil; Muhonen, Wallace W.; Law, Benedict; Shabb, John B.; Srivastava, D. K.; Mallik, Sanku

    2011-01-01

    Liposomes have been widely used as a drug delivery vehicle and currently, more than 10 liposomal formulations are approved by the Food and Drug Administration for clinical use. However, upon targeting, the release of the liposome-encapsulated contents is usually slow. We have recently demonstrated that contents from appropriately-formulated liposomes can be rapidly released by the cancer-associated enzyme matrix metalloproteinase-9 (MMP-9). Herein, we report our detailed studies to optimize the liposomal formulations. By properly selecting the lipopeptide, the major lipid component and their relative amounts, we demonstrate that the contents are rapidly released in the presence of cancer-associated levels of recombinant human MMP-9. We observed that the degree of lipid mismatch between the lipopepides and the major lipid component profoundly affects the release profiles from the liposomes. By utilizing the optimized liposomal formulations, we also demonstrate that cancer cells (HT-29) which secrete low levels of MMP-9 failed to release significant amount of the liposomal contents. Metastatic cancer cells (MCF7) secreting high levels of the enzyme rapidly release the encapsulated contents from the liposomes. PMID:19601658

  3. Targeting of Escherichia coli F4 fimbriae to Fcgamma receptors enhances the maturation of porcine dendritic cells.

    Science.gov (United States)

    Devriendt, Bert; Verdonck, Frank; Summerfield, Artur; Goddeeris, Bruno M; Cox, Eric

    2010-06-15

    F4(+) enterotoxigenic Escherichia coli (ETEC) infections are an important cause of postweaning diarrhoea in piglets and an oral immunization of piglets with purified F4 fimbriae protects them from a subsequent F4(+) ETEC infection. However, oral immunization of suckling piglets is hampered due to the immature status of their immune system. Targeting of antigens to Fcgamma receptors (FcgammaR) on human and murine dendritic cells (DC) has been shown to enhance DC maturation and both humoral and cellular immune responses. To investigate the effect of F4 fimbriae incorporated in immune complexes (F4-IC) on porcine DC, we used porcine monocytic-derived DC (MoDC) as a model system. The results in this study demonstrate that FcgammaRI, II and III mRNA is expressed by porcine MoDC. Furthermore, we show that FcgammaRII and III are expressed on the cell surface and that F4-IC are internalized by MoDC via FcgammaR. This FcgammaR ligation induced a significantly enhanced expression of Major Histocompatibility complex (MHCII) class II and the costimulatory molecules CD80/86 and CD40 by MoDC compared with immature MoDC. Furthermore, the phagocytic capacity of F4-IC stimulated MoDC was reduced as evidenced by a reduced uptake of DQ-ovalbumin and FITC-dextran. In an allogenic and autologous mixed lymphocyte reaction, these F4-IC-activated MoDC showed an improved T cell stimulatory capacity in comparison with immature MoDC. The F4-IC induced DC maturation correlated with significant higher expression levels of several pro-inflammatory cytokines such as interleukine (IL) 1beta, IL-6 and Tumor necrosis factor alpha, the chemokine IL-8 and IL-12p40 in comparison with immature MoDC. Altogether, these results clearly demonstrate that FcgammaR engagement enhances the maturation of porcine MoDC, which may suggest that antigen targeting to FcgammaR on DC could improve vaccine design against infections. Copyright 2009 Elsevier B.V. All rights reserved.

  4. The Cytotoxicity of Dacarbazine Potentiated by Sea Cucumber Saponin in Resistant B16F10 Melanoma Cells through Apoptosis Induction.

    Science.gov (United States)

    Baharara, Javad; Amini, Elaheh; Nikdel, Najme; Salek-Abdollahi, Farzaneh

    2016-01-01

    Malignant melanoma is a highly aggressive malignant melanocytic neoplasm which resists against the most conventional therapies. Sea cucumber as one of marine organisms contains bioactive compounds such as polysaccharide, terpenoid and other metabolites which have anti-cancer, anti-tumor, anti-inflammatory and antioxidant properties. The present study was designed to investigate the anticancer potential of saponin extracted from sea cucumber Holothuria leucospilata alone and in combination with dacarbazine on B16F10 melanoma cell line. The B16F10 cell line was treated with different concentrations of saponin (0, 4, 8, 12, 16, 20 μg/ml), dacarbazine (0, 1200, 1400, 1600, 18000, 1200, 1400, 1600, 2000 μg/ml) and co-administration of saponin-dacarbazine (1200 da+8 sp, 1200 da+4 sp) for 24 and 48 hr and the cytotoxic effect was examined by MTT, DAPI, acridine orange/propodium iodide, flow cytometry and caspase colorimetric assay. The results exhibited that sea cucumber saponin, dacarbazine, and co-administration of saponin-dacarbazine inhibited the proliferation of melanoma cells in a dose and time dependent manner with IC50 values of 10, 1400 and 4+1200 μg/ml, respectively. Morphological observation of DAPI and acridine orange/propodium iodide staining documented typical characteristics of apoptotic cell death. Flow cytometry assay indicated accumulation of IC50 treated cells in sub-G1 peak. Additionally, saponin extracted induced intrinsic apoptosis via up-regulation of caspase-3 and caspase-9. These results revealed that the saponin extracted from sea cucumber as a natural anti-cancer compound may be a new treatment modality for metastatic melanoma and the application of sea cucumber saponin in combination with dacarbazine demonstrated the strongest anti-cancer activity as compared with the drug alone.

  5. Immunomodulatory effects of intravenous bis-1 f(ab')(2) administration in renal-cell cancer-patients

    NARCIS (Netherlands)

    Janssen, R. A. J.; Kroesen, B. J.; Mesander, G.; Sleijfer, D. T.; The, T. Hauw; Mulder, N. H.; de Leij, L

    We report the immunomodulatory effects of an intravenous treatment with F(ab')(2) fragments of the bispecific monoclonal antibody BIS-1 during subcutaneous recombinant interleukin 2 (rIL-2) therapy of renal cell cancer (RCC) patients. BIS-1 is directed against both the CD3 antigen on T cells and the

  6. F508del-CFTR rescue: a matter of cell stress response.

    Science.gov (United States)

    Nieddu, Erika; Pollarolo, Benedetta; Merello, Luisa; Schenone, Silvia; Mazzei, Mauro

    2013-01-01

    Cystic fibrosis (CF) is a common inherited fatal disease affecting 70,000 people worldwide, with a median predicted age of survival of approximately 38 years. The deletion of Phenylalanine in position 508 of the Cystic Fibrosis Transmembrane conductance Regulator (F508del-CFTR) is the most common mutation in CF patients: the deleted protein, not properly folded, is degraded. To date no commercial drugs are available. Low temperature, some osmolytes and conditions able to induce heat shock protein 70 (Hsp70) expression and heat shock cognate 70 (Hsc70) inhibition result in F508del-CFTR rescue, hence restoring its physiological function: this review sheds light on the correlation between these several evidences. Interestingly, all these approaches have a role in the cell stress response (CSR), a set of cell reactions to stress. In addition, unpredictably, F508del-CFTR rescue has to be considered in the frame of CSR: entities that induce - or are induced during - the CSR are, in general, also able to correct trafficking defect of CFTR. Specifically, the low temperature induces, by definition, a CSR; osmolytes, such as glycerol and trimethylamine N-oxide (TMAO), are products of the CSR; pharmacological correctors, such as Matrine and 4-phenylbutirric acid (4PBA), down-regulate the constitutive Hsc70 in favor of an up-regulation of the inducible chaperone Hsp70, another component of the CSR. The identification of a common mechanism of action for different types of correctors could drive the discovery of new active molecules in CF, overcoming methods clinically inapplicable, such as the low temperature.

  7. Anti-metastasis effect of fucoidan from Undaria pinnatifida sporophylls in mouse hepatocarcinoma Hca-F cells.

    Directory of Open Access Journals (Sweden)

    Peisheng Wang

    Full Text Available Metastasis is one of the major causes of cancer-related death. It is a complex biological process involving multiple genes, steps, and phases. It is also closely connected to many biological activities of cancer cells, such as growth, invasion, adhesion, hematogenous metastasis, and lymphatic metastasis. Fucoidan derived from Undaria pinnatifida sporophylls (Ups-fucoidan is a sulfated polysaccharide with more biological activities than other fucoidans. However, there is no information on the effects of Ups-fucoidan on tumor invasion and metastasis. We used the mouse hepatocarcinoma Hca-F cell line, which has high invasive and lymphatic metastasis potential in vitro and in vivo, to examine the effect of Ups-fucoidan on cancer cell invasion and metastasis. Ups-fucoidan exerted a concentration- and time-dependent inhibitory effect on tumor metastasis in vivo and inhibited Hca-F cell growth, migration, invasion, and adhesion capabilities in vitro. Ups-fucoidan inhibited growth and metastasis by downregulating vascular endothelial growth factor (VEGF C/VEGF receptor 3, hepatocyte growth factor/c-MET, cyclin D1, cyclin-dependent kinase 4, phosphorylated (p phosphoinositide 3-kinase, p-Akt, p-extracellular signal regulated kinase (ERK 1/2, and nuclear transcription factor-κB (NF-κB, and suppressed adhesion and invasion by downregulating L-Selectin, and upregulating protein levels of tissue inhibitor of metalloproteinases (TIMPs. The results suggest that Ups-fucoidan suppresses Hca-F cell growth, adhesion, invasion, and metastasis capabilities and that these functions are mediated through the mechanism involving inactivation of the NF-κB pathway mediated by PI3K/Akt and ERK signaling pathways.

  8. Effect of gamma irradiation on proliferation and survival of Sf9 cells: radioresistance in a Lepidopteran insect cell line

    International Nuclear Information System (INIS)

    Seth, R.K.; Lovell, K.V.; Reynolds, S.E.

    2003-01-01

    Sf9 cells of Spodoptera frugiperda, when exposed to gamma-irradiation from a 60 Co source, were found markedly less sensitive to ionising radiation than mammalian cells in terms of both growth kinetics and survival. Following irradiation at 1.2 Gy S -1 there was a dose-dependent delay in Sf9 cell proliferation and plateau cell density was reduced. These effects were dependent on dose rate too. In the range 0.3 - 1.2 Gy s -1 , growth was delayed longer and reached a lower plateau with increasing dose rate. Exposure to radiation caused a decrease in adherence of cells to the substrate, and an increase in number of enlarged ('giant') cells. Analysis of colony formation after irradiation at 1.2 Gy s -1 gave a survival curve of conventional shape but with a very large D o value of 24 Gy. Extrapolation number (N) was 2.9, a value within the normal range for mammalian cells. At 0.12 Gy s -1 N had a similar value of 3.2, but D o was higher (30 Gy) than at the higher dose rate. This study indicates that the relative insensitivity of lepidoptera insects may be attributed to some extent to the intrinsic properties of their constituent cells. (author)

  9. FGF signalling controls the specification of hair placode-derived SOX9 positive progenitors to Merkel cells.

    Science.gov (United States)

    Nguyen, Minh Binh; Cohen, Idan; Kumar, Vinod; Xu, Zijian; Bar, Carmit; Dauber-Decker, Katherine L; Tsai, Pai-Chi; Marangoni, Pauline; Klein, Ophir D; Hsu, Ya-Chieh; Chen, Ting; Mikkola, Marja L; Ezhkova, Elena

    2018-06-13

    Merkel cells are innervated mechanosensory cells responsible for light-touch sensations. In murine dorsal skin, Merkel cells are located in touch domes and found in the epidermis around primary hairs. While it has been shown that Merkel cells are skin epithelial cells, the progenitor cell population that gives rise to these cells is unknown. Here, we show that during embryogenesis, SOX9-positive (+) cells inside hair follicles, which were previously known to give rise to hair follicle stem cells (HFSCs) and cells of the hair follicle lineage, can also give rise to Merkel Cells. Interestingly, while SOX9 is critical for HFSC specification, it is dispensable for Merkel cell formation. Conversely, FGFR2 is required for Merkel cell formation but is dispensable for HFSCs. Together, our studies uncover SOX9(+) cells as precursors of Merkel cells and show the requirement for FGFR2-mediated epithelial signalling in Merkel cell specification.

  10. Antiproliferative constituents in plants 9. Aerial parts of Lippia dulcis and Lippia canescens.

    Science.gov (United States)

    Abe, Fumiko; Nagao, Tsuneatsu; Okabe, Hikaru

    2002-07-01

    The antiproliferative constituents in the MeOH extracts of the aerial parts of Lippia dulcis Trev. and Lippia canescens Kunth (Verbenaceae) were investigated. Activity-guided chemical investigation of the MeOH extracts resulted in the isolation of the three bisabolane-type sesquiterpenes [(+)-hernandulcin (1), (-)-epihernandulcin (2), and (+)-anymol (3)] and four phenylethanoid glycosides [acteoside (4), isoacteoside (5), martynoside (6), and a new diacetylmartynoside (7)] from the former, and four phenylethanoid glycosides [acteoside (4), isoacteoside (5), arenarioside (8), and leucosceptoside A (9)] and three flavones [desmethoxycentaureidin (10), eupafolin (11), and 6-hydroxyluteolin (12)] from the latter. Antiproliferative activity of the isolated compounds against murine melanoma (B16F10), human gastric adenocarcinoma (MK-1), and human uterine carcinoma (HeLa) cells was estimated. (+)-Anymol (3), acteoside (4), isoacteoside (5), arenarioside (8), eupafolin (11), and 6-hydroxyluteolin (12) had GI50 values of 10-16 microM against B16F10 cell. Desmethoxycentaureidin (10) and eupafolin (11) showed high inhibitory activity against HeLa cell growth (GI50 9 microM, and 6 microM, respectively).

  11. BID-F1 and BID-F2 domains of Bartonella henselae effector protein BepF trigger together with BepC the formation of invasome structures.

    Science.gov (United States)

    Truttmann, Matthias C; Guye, Patrick; Dehio, Christoph

    2011-01-01

    The gram-negative, zoonotic pathogen Bartonella henselae (Bhe) translocates seven distinct Bartonella effector proteins (Beps) via the VirB/VirD4 type IV secretion system (T4SS) into human cells, thereby interfering with host cell signaling [1], [2]. In particular, the effector protein BepG alone or the combination of effector proteins BepC and BepF trigger massive F-actin rearrangements that lead to the establishment of invasome structures eventually resulting in the internalization of entire Bhe aggregates [2], [3]. In this report, we investigate the molecular function of the effector protein BepF in the eukaryotic host cell. We show that the N-terminal [E/T]PLYAT tyrosine phosphorylation motifs of BepF get phosphorylated upon translocation but do not contribute to invasome-mediated Bhe uptake. In contrast, we found that two of the three BID domains of BepF are capable to trigger invasome formation together with BepC, while a mutation of the WxxxE motif of the BID-F1 domain inhibited its ability to contribute to the formation of invasome structures. Next, we show that BepF function during invasome formation can be replaced by the over-expression of constitutive-active Rho GTPases Rac1 or Cdc42. Finally we demonstrate that BID-F1 and BID-F2 domains promote the formation of filopodia-like extensions in NIH 3T3 and HeLa cells as well as membrane protrusions in HeLa cells, suggesting a role for BepF in Rac1 and Cdc42 activation during the process of invasome formation.

  12. Hsc70 facilitates TGF-β-induced activation of Smad2/3 in fibroblastic NRK-49F cells

    Energy Technology Data Exchange (ETDEWEB)

    Ikezaki, Midori; Higashimoto, Natsuki; Matsumura, Ko; Ihara, Yoshito, E-mail: y-ihara@wakayama-med.ac.jp

    2016-08-26

    Heat-shock cognate protein 70 (Hsc70), a molecular chaperone constitutively expressed in the cell, is involved in the regulation of several cellular signaling pathways. In this study, we found that TGF-β-induced phosphorylation and nuclear translocation of Smad2/3 were suppressed in fibroblastic NRK-49F cells treated with small interfering RNA (siRNA) for Hsc70. In the cells underexpressing Hsc70, transcriptional induction of connective tissue growth factor (CTGF), a target gene of the TGF-β signaling, was also suppressed in the early phase of TGF-β stimulation. Upon stimulation with TGF-β, Hsc70 interacted with Smad2/3, suggesting functional interactions of Hsc70 and Smad2/3 for the activation of TGF-β-induced Smad signaling. Although the expression of heat-shock protein 70 (Hsp70) was upregulated in the cells treated with Hsc70 siRNA, TGF-β-induced Smad activation was not affected in the cells overexpressing Hsp70. Collectively, these results indicate that Hsc70, but not Hsp70, supportively regulates TGF-β-induced Smad signaling in NRK-49F cells. - Highlights: • Hsc70 siRNA treatment suppressed the expression of Hsc70 but induced the expression of Hsp70 in NRK-49F cells. • Hsc70 siRNA treatment suppressed the activation of Smad2/3 in the cells treated with TGF-β. • Hsc70 interacted with Smad2/3 on stimulation with TGF-β in the cells. • Hsp70 did not influence the TGF-β-induced activation of Smad2/3 in the cells overexpressing Hsp70.

  13. Bioactive spirans and other constituents from the leaves of Cannabis sativa f. sativa.

    Science.gov (United States)

    Guo, Tian-Tian; Zhang, Jian-Chun; Zhang, Hai; Liu, Qing-Chao; Zhao, Yong; Hou, Yu-Fei; Bai, Lu; Zhang, Li; Liu, Xue-Qiang; Liu, Xue-Ying; Zhang, Sheng-Yong; Bai, Nai-Sheng

    2017-08-01

    In this paper, 17 compounds (1-17) were isolated from the leaves of Hemp (Cannabis sativa f. sativa). Among the isolates, two were determined to be new spirans: cannabispirketal (1), and α-cannabispiranol 4'-O-β-D-glucopyranose (2) by 1D and 2D NMR spectroscopy, LC-MS, and HRESIMS. The known compounds 7, 8, 10, 13, 15, and 16 were isolated from Hemp (C. sativa f. sativa) for the first time. Furthermore, compounds 8 and 13 were isolated from the nature for the first time. All isolated compounds were evaluated for cytotoxicity on different tissue-derived passage cancer cell lines through cell viability and apoptosis assay. Among these compounds, compounds 5, 9 and 16 exhibited a broad-spectrum antitumor effect via inhibiting cell proliferation and promoting apoptosis. These results obtained have provided valuable clues to the understanding of the cytotoxic profile for these isolated compounds from Hemp (C. sativa f. sativa).

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

    Science.gov (United States)

    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

  15. Evaluation of recurrent disease in the re-staging of colorectal cancer by 18F-FDG PET/CT: Use of CEA and CA 19–9 in patient selection

    Science.gov (United States)

    Chiaravalloti, Agostino; Fiorentini, Alessandro; Palombo, Erika; Rinino, Davide; Lacanfora, Annamaria; Danieli, Roberta; Di Russo, Carmen; Di Biagio, Daniele; Squillaci, Ettore; Schillaci, Orazio

    2016-01-01

    The aim of the present retrospective study was to evaluate the sensitivity and specificity of fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) in assessing the recurrence of colorectal cancer (CRC) with regard to carcinoembryonic antigen (CEA) and carbohydrate antigen 19–9 (CA 19–9). 18F-FDG PET/CT was performed in 100 patients for the re-staging of CRC. Therapy was discontinued prior to the examination. The mean (± standard deviation) CEA value (measured ~30 days prior to PET/CT examination) was 23.71 (±107) ng/ml, whereas the CA 19–9 value was 72 (±190.3) U/ml. Differences in CEA and CA 19–9 values in patients with scans that were positive or negative for recurrence were analyzed by means of a receiver operating characteristic (ROC) curve. ROC curves were used for the calculation of the sensitivity and specificity of 18F-FDG PET/CT for the CEA and CA 19–9 levels. The results of the 18F-FDG PET/CT were found to be associated with the CEA level (P=0.001), but not with the CA 19–9 level (P=0.43). PET/CT was positive for recurrence in 60 patients (60.0%), whose mean CEA and CA 19–9 values were 33.07±136.7 ng/ml and 75.24±192.3 U/ml, respectively. PET/CT was negative for recurrence in 40 patients (40.0%), whose mean CEA and CA 19–9 values were 10.15±30 ng/ml and 67.76±190 U/ml, respectively. On the basis of ROC curve analysis, the best compromise between sensitivity and specificity was achieved for CEA levels of 3.5 ng/ml [sensitivity, 80%; 95% confidence interval (CI), 67–89%; and specificity, 60%; 95% CI, 45–78%]. The study concluded that the detection of recurrence by 18F-FDG PET/CT in patients treated for CRC is associated with CEA, but not CA 19–9 serum levels. Moreover, 18F-FDG PET/CT should be recommended in patients with suspected CRC recurrence even when they present with CEA levels below the normal cut-off. PMID:27895793

  16. Preferential Th1 cytokine profile of phosphoantigen-stimulated human Vγ9Vδ2 T cells.

    LENUS (Irish Health Repository)

    Dunne, Margaret R

    2010-01-01

    Human Vγ9Vδ2 T cells recognise pyrophosphate-based antigens (phosphoantigens) and have multiple functions in innate and adaptive immunity, including a unique ability to activate other cells of the immune system. We used flow cytometry and ELISA to define the early cytokine profiles of Vγ9Vδ2 T cells stimulated in vitro with isopentenyl pyrophosphate (IPP) and (E)-4-hydroxy-3-methyl-but-2 enyl pyrophosphate (HMB-PP) in the absence and presence of IL-2 and IL-15. We show that fresh Vγ9Vδ2 T cells produce interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) within 4 hours of stimulation with phosphoantigen, but neither IL-10, IL-13, nor IL-17 was detectable up to 72 hours under these conditions. Cytokine production was not influenced by expression or lack, thereof, of CD4 or CD8. Addition of IL-2 or IL-15 caused expansion of IFN-γ-producing Vγ9Vδ2 T cells, but did not enhance IFN-γ secretion after 24-72 hours. Thus, phosphoantigen-stimulated Vγ9Vδ2 T cells have potential as Th1-biasing adjuvants for immunotherapy.

  17. Preferential Th1 Cytokine Profile of Phosphoantigen-Stimulated Human Vγ9Vδ2 T Cells

    Directory of Open Access Journals (Sweden)

    Margaret R. Dunne

    2010-01-01

    Full Text Available Human Vγ9Vδ2 T cells recognise pyrophosphate-based antigens (phosphoantigens and have multiple functions in innate and adaptive immunity, including a unique ability to activate other cells of the immune system. We used flow cytometry and ELISA to define the early cytokine profiles of Vγ9Vδ2 T cells stimulated in vitro with isopentenyl pyrophosphate (IPP and (E-4-hydroxy-3-methyl-but-2 enyl pyrophosphate (HMB-PP in the absence and presence of IL-2 and IL-15. We show that fresh Vγ9Vδ2 T cells produce interferon-γ (IFN-γ and tumour necrosis factor-α (TNF-α within 4 hours of stimulation with phosphoantigen, but neither IL-10, IL-13, nor IL-17 was detectable up to 72 hours under these conditions. Cytokine production was not influenced by expression or lack, thereof, of CD4 or CD8. Addition of IL-2 or IL-15 caused expansion of IFN-γ-producing Vγ9Vδ2 T cells, but did not enhance IFN-γ secretion after 24–72 hours. Thus, phosphoantigen-stimulated Vγ9Vδ2 T cells have potential as Th1-biasing adjuvants for immunotherapy.

  18. Enhancing Effect of Hydroxyurea on Hb F in Sickle Cell Disease: Ten-Year Egyptian Experience.

    Science.gov (United States)

    Youssry, Ilham; Abdel-Salam, Amina; Ismail, Rania; Bou-Fakhredin, Rayan; Mohamed Samy, Rania; Ezz El-Deen, Fatma; Taher, Ali T

    Patients with sickle cell disease experience hemolytic anemia and vaso-occlusions that result in pain, organ injury, and premature mortality. Several prospective studies have verified the efficacy and tolerability of hydroxyurea (HU), and demonstrated its efficacy in reducing painful vaso-occlusive crises (VOCs) in addition to its ability to increase Hb F levels. We aimed to evaluate the long-term effects of HU therapy on Hb F and assess its long term efficacy and safety in sickle cell disease patients. A retrospective study on 60 sickle cell disease patients was conducted. We studied the laboratory changes, frequency of VOCs per year, frequency of hospital admisions per year and number of transfusions per year, both before and after HU therapy. The follow-up period was 4 to 120 months. Hb F levels after HU therapy positively correlated with the duration of HU therapy, baseline Hb F levels and baseline total hemoglobin (Hb) (r = 0.4, p = 0.04; r = 0.45, p = 0.001; r = 0.5, p = 0.019, respectively) and inversely correlated with baseline total leucocyte count (r = -0.33, p = 0.034). Hydroxyurea therapy was associated with an increase in the total Hb and mean corpuscular volume (MCV) (p = 0.009, p = 0.000; respectively) and with a decrease in total leucocyte count, platelet count and reticulocyte count (p = 0.00, p = 0.03, p = 0.02, respectively). Moreover, a significant reduction in the frequency of VOCs, transfusion frequency and hospital admissions per year after HU therapy was shown in the studied subjects. Hydroxyurea induced an increase in Hb F level, which was maintained over time and was associated with clinical efficacy and acceptable safety.

  19. Regulation of retinoschisin secretion in Weri-Rb1 cells by the F-actin and microtubule cytoskeleton.

    Directory of Open Access Journals (Sweden)

    Eiko Kitamura

    Full Text Available Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS, an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process.

  20. Regulation of Retinoschisin Secretion in Weri-Rb1 Cells by the F-Actin and Microtubule Cytoskeleton

    Science.gov (United States)

    Kitamura, Eiko; Gribanova, Yekaterina E.; Farber, Debora B.

    2011-01-01

    Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS), an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process. PMID:21738583

  1. Effects of miR-424 on Proliferation and Migration Abilities in Non-small Cell Lung Cancer A549 Cells and Its Molecular Mechanism

    Directory of Open Access Journals (Sweden)

    Hongmin LI

    2016-09-01

    Full Text Available Background and objective The inhibitory ability of miR-424 on the proliferation of renal carcinoma cell and the migration and invasion of cancer cells has been widely explored and demonstrated. However, the effects of miR-424 on non-small cell lung cancer (NSCLC have not been systematically examined. In this study, detected the growth and invasion effect of miR-424 in NSCLC A549 cell. The migration and molecular mechanism of this cell are also detected. Methods NSCLC A549 cell was transfected with miR-424 and its inhibitor. After transfection, the proliferation ability of A549 cell was detectedby CCK8 assay. Then, the migration ability in A549 cell was detected by migration assays. Furthermore, the expression level of MMP2 and MMP9 in A549 was detected by Western blot and immune fluorescence. The 3'UTR of E2F6 was cloned into luciferase reporter vector and its enzymatic activitywas detected to verify whether miR-424 can target E2F6. The expression level of E2F6 in a549 cell after transfecing with miR-424 was detected by Western blot. Results After transfection of miR-424, the proliferation and migration abilities were remarkably decreased and the expression level of MMP-2 and MMP-9 were down-regulated in A549. Moreover, MiR-424 inhibited the enzymatic activity of luviferase reporter vector of E2F6. Specifically, the expression level of E2F6 was down-regulated in A549. Conclusion miR-424 can inhibit the proliferation and migration abilities of A549 by negatively regulating the expression of E2F6.

  2. OP9 Feeder Cells Are Superior to M2-10B4 Cells for the Generation of Mature and Functional Natural Killer Cells from Umbilical Cord Hematopoietic Progenitors

    Directory of Open Access Journals (Sweden)

    Lara Herrera

    2017-06-01

    Full Text Available Adoptive natural killer (NK cell therapy relies on the acquisition of large numbers of mature and functional NK cells. An option for future immunotherapy treatments is to use large amounts of NK cells derived and differentiated from umbilical cord blood (UCB CD34+ hematopoietic stem cells (HSCs, mainly because UCB is one of the most accessible HSC sources. In our study, we compared the potential of two stromal cell lines, OP9 and M2-10B4, for in vitro generation of mature and functional CD56+ NK cells from UCB CD34+ HSC. We generated higher number of CD56+ NK cells in the presence of the OP9 cell line than when they were generated in the presence of M2-10B4 cells. Furthermore, higher frequency of CD56+ NK cells was achieved earlier when cultures were performed with the OP9 cells than with the M2-10B4 cells. Additionally, we studied in detail the maturation stages of CD56+ NK cells during the in vitro differentiation process. Our data show that by using both stromal cell lines, CD34+ HSC in vitro differentiated into the terminal stages 4–5 of maturation resembled the in vivo differentiation pattern of human NK cells. Higher frequencies of more mature NK cells were reached earlier by using OP9 cell line than M2-10B4 cells. Alternatively, we observed that our in vitro NK cells expressed similar levels of granzyme B and perforin, and there were no significant differences between cultures performed in the presence of OP9 cell line or M2-10B4 cell line. Likewise, degranulation and cytotoxic activity against K562 target cells were very similar in both culture conditions. The results presented here provide an optimal strategy to generate high numbers of mature and functional NK cells in vitro, and point toward the use of the OP9 stromal cell line to accelerate the culture procedure to obtain them. Furthermore, this method could establish the basis for the generation of mature NK cells ready for cancer immunotherapy.

  3. CRISPR/Cas9-mediated genome engineering of CHO cell factories: application and perspectives

    DEFF Research Database (Denmark)

    Lee, Jae Seong; Grav, Lise Marie; Lewis, Nathan E.

    2015-01-01

    repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system enables rapid,easy and efficient engineering of mammalian genomes. It has a wide range of applications frommodification of individual genes to genome-wide screening or regulation of genes. Facile genomeediting using CRISPR/Cas9 empowers...... researchers in the CHO community to elucidate the mechanisticbasis behind high level production of proteins and product quality attributes of interest. Inthis review, we describe the basis of CRISPR/Cas9-mediated genome editing and its applicationfor development of next generation CHO cell factories while...... highlighting both future perspectivesand challenges. As one of the main drivers for the CHO systems biology era, genome engineeringwith CRISPR/Cas9 will pave the way for rational design of CHO cell factories....

  4. A PET Imaging Strategy to Visualize Activated T Cells in Acute Graft-versus-Host Disease Elicited by Allogenic Hematopoietic Cell Transplant.

    Science.gov (United States)

    Ronald, John A; Kim, Byung-Su; Gowrishankar, Gayatri; Namavari, Mohammad; Alam, Israt S; D'Souza, Aloma; Nishikii, Hidekazu; Chuang, Hui-Yen; Ilovich, Ohad; Lin, Chih-Feng; Reeves, Robert; Shuhendler, Adam; Hoehne, Aileen; Chan, Carmel T; Baker, Jeanette; Yaghoubi, Shahriar S; VanBrocklin, Henry F; Hawkins, Randall; Franc, Benjamin L; Jivan, Salma; Slater, James B; Verdin, Emily F; Gao, Kenneth T; Benjamin, Jonathan; Negrin, Robert; Gambhir, Sanjiv Sam

    2017-06-01

    A major barrier to successful use of allogeneic hematopoietic cell transplantation is acute graft-versus-host disease (aGVHD), a devastating condition that arises when donor T cells attack host tissues. With current technologies, aGVHD diagnosis is typically made after end-organ injury and often requires invasive tests and tissue biopsies. This affects patient prognosis as treatments are dramatically less effective at late disease stages. Here, we show that a novel PET radiotracer, 2'-deoxy-2'-[18F]fluoro-9-β-D-arabinofuranosylguanine ([18F]F-AraG), targeted toward two salvage kinase pathways preferentially accumulates in activated primary T cells. [18F]F-AraG PET imaging of a murine aGVHD model enabled visualization of secondary lymphoid organs harboring activated donor T cells prior to clinical symptoms. Tracer biodistribution in healthy humans showed favorable kinetics. This new PET strategy has great potential for early aGVHD diagnosis, enabling timely treatments and improved patient outcomes. [18F]F-AraG may be useful for imaging activated T cells in various biomedical applications. Cancer Res; 77(11); 2893-902. ©2017 AACR . ©2017 American Association for Cancer Research.

  5. Secretion of Galectin-9 as a DAMP during Dengue Virus Infection in THP-1 Cells.

    Science.gov (United States)

    Dapat, Isolde C; Pascapurnama, Dyshelly Nurkartika; Iwasaki, Hiroko; Labayo, Hannah Karen; Chagan-Yasutan, Haorile; Egawa, Shinichi; Hattori, Toshio

    2017-07-28

    Damage-associated molecular patterns (DAMPs) are endogenous cellular molecules released to the extracellular environment in response to stress conditions such as virus infection. Galectins are β-galactoside-binding proteins that are widely expressed in cells and tissues of the immune system, are localized in the cell cytoplasm, and have roles in inflammatory responses and immune responses against infection. Elevated levels of galectin-9 (Gal-9) in natural human infections have been documented in numerous reports. To investigate the effect of dengue virus (DENV) infection on expression of endogenous Gal-9, monocytic THP-1 cells were infected with varying doses of DENV-3 (multiplicity of infection (MOI) 0.01, 0.03 and 0.1) and incubated at varying time points (Day 1, Day 2, Day 3). Results showed augmentation of Gal-9 levels in the supernatant, reduction of Gal-9 levels in the cells and decreased expression of LGALS9 mRNA, while DENV-3 mRNA copies for all three doses remained stable through time. Dengue virus induced the secretion of Gal-9 as a danger response; in turn, Gal-9 and other inflammatory factors, and stimulated effector responses may have limited further viral replication. The results in this pilot experiment add to the evidence of Gal-9 as a potential DAMP.

  6. On process model representation and AlF{sub 3} dynamics of aluminium electrolysis cells

    Energy Technology Data Exchange (ETDEWEB)

    Drengstig, Tormod

    1997-12-31

    This thesis develops a formal graphical based process representation scheme for modelling complex, non-standard unit processes. The scheme is based on topological and phenomenological decompositions. The topological decomposition is the modularization of processes into modules representing volumes and boundaries, whereas the phenomenological decomposition focuses on physical phenomena and characteristics inside these topological modules. This defines legal and illegal connections between components at all levels and facilitates a full implementation of the methodology into a computer aided modelling tool that can interpret graphical symbols and guide modelers towards a consistent mathematical model of the process. The thesis also presents new results on the excess AlF{sub 3} and bath temperature dynamics of an aluminium electrolysis cell. A dynamic model of such a cell is developed and validated against known behaviour and real process data. There are dynamics that the model does not capture and this is further discussed. It is hypothesized that long-term prediction of bath temperature and excess AlF{sub 3} is impossible with a current efficiency model considering only bath composition and temperature. A control strategy for excess AlF{sub 3} and bath temperature is proposed based on an almost constant AlF{sub 3} input close to average consumption and energy manipulations to compensate for the disturbances. 96 refs., 135 figs., 22 tabs.

  7. CRISPR/Cas9 nuclease-mediated gene knock-in in bovine-induced pluripotent cells.

    Science.gov (United States)

    Heo, Young Tae; Quan, Xiaoyuan; Xu, Yong Nan; Baek, Soonbong; Choi, Hwan; Kim, Nam-Hyung; Kim, Jongpil

    2015-02-01

    Efficient and precise genetic engineering in livestock such as cattle holds great promise in agriculture and biomedicine. However, techniques that generate pluripotent stem cells, as well as reliable tools for gene targeting in livestock, are still inefficient, and thus not routinely used. Here, we report highly efficient gene targeting in the bovine genome using bovine pluripotent cells and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease. First, we generate induced pluripotent stem cells (iPSCs) from bovine somatic fibroblasts by the ectopic expression of yamanaka factors and GSK3β and MEK inhibitor (2i) treatment. We observed that these bovine iPSCs are highly similar to naïve pluripotent stem cells with regard to gene expression and developmental potential in teratomas. Moreover, CRISPR/Cas9 nuclease, which was specific for the bovine NANOG locus, showed highly efficient editing of the bovine genome in bovine iPSCs and embryos. To conclude, CRISPR/Cas9 nuclease-mediated homologous recombination targeting in bovine pluripotent cells is an efficient gene editing method that can be used to generate transgenic livestock in the future.

  8. Hinokitiol Exerts Anticancer Activity through Downregulation of MMPs 9/2 and Enhancement of Catalase and SOD Enzymes: In Vivo Augmentation of Lung Histoarchitecture.

    Science.gov (United States)

    Huang, Chien-Hsun; Jayakumar, Thanasekaran; Chang, Chao-Chien; Fong, Tsorng-Harn; Lu, Shing-Hwa; Thomas, Philip Aloysius; Choy, Cheuk-Sing; Sheu, Joen-Rong

    2015-09-25

    Melanoma is extremely resistant to chemotherapy and the death rate is increasing hastily worldwide. Extracellular matrix promotes the migration and invasion of tumor cells through the production of matrix metalloproteinase (MMP)-2 and -9. Evidence has shown that natural dietary antioxidants are capable of inhibiting cancer cell growth. Our recent studies showed that hinokitiol, a natural bioactive compound, inhibited vascular smooth muscle cell proliferation and platelets aggregation. The present study is to investigate the anticancer efficacy of hinokitiol against B16-F10 melanoma cells via modulating tumor invasion factors MMPs, antioxidant enzymes in vitro. An in vivo mice model of histological investigation was performed to study the patterns of elastic and collagen fibers. Hinokitiol inhibited the expression and activity of MMPs-2 and -9 in B16-F10 melanoma cells, as measured by western blotting and gelatin zymography, respectively. An observed increase in protein expression of MMPs 2/9 in melanoma cells was significantly inhibited by hinokitiol. Notably, hinokitiol (1-5 μM) increased the activities of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) from the reduction in melanoma cells. Also, hinokitiol (2-10 µM) concentration dependently reduced in vitro Fenton reaction induced hydroxyl radical (OH·) formation. An in vivo study showed that hinokitiol treatment increased elastic fibers (EF), collagens dispersion, and improved alveolar alterations in the lungs of B16/F10 injected mice. Overall, our findings propose that hinokitiol may be a potent anticancer candidate through down regulation of MMPs 9/2, reduction of OH· production and enhancement of antioxidant enzymes SOD and CAT.

  9. Regulation of matrix metalloproteinase-9 expression between gingival fibroblast cells from old and young rats

    International Nuclear Information System (INIS)

    Kim, Su-Jung; Chung, Yong-Koo; Chung, Tae-Wook; Kim, Jeong-Ran; Moon, Sung-Kwon; Kim, Cheorl-Ho; Park, Young-Guk

    2009-01-01

    Gingival fibroblast cells (rGF) from aged rats have an age-related decline in proliferative capacity compared with young rats. We investigated G1 phase cell cycle regulation and MMP-9 expression in both young and aged rGF. G1 cell cycle protein levels and activity were significantly reduced in response to interleukin-1β (IL-1β) stimulation with increasing in vitro age. Tumor necrosis factor-α (TNF-α)-induced matrix metalloproteinase-9 (MMP-9) expression was also decreased in aged rGF in comparison with young rGF. Mutational analysis and gel shift assays demonstrated that the lower MMP-9 expression in aged rGF is associated with lower activities of transcription factors NF-κB and AP-1. These results suggest that cell cycle dysregulation and down-regulation of MMP-9 expression in rGF may play a role in gingival remodeling during in vitro aging.

  10. Establishing clonal cell lines with endothelial-like potential from CD9(hi, SSEA-1(- cells in embryonic stem cell-derived embryoid bodies.

    Directory of Open Access Journals (Sweden)

    Qizhou Lian

    Full Text Available BACKGROUND: Differentiation of embryonic stem cells (ESCs into specific cell types with minimal risk of teratoma formation could be efficiently directed by first reducing the differentiation potential of ESCs through the generation of clonal, self-renewing lineage-restricted stem cell lines. Efforts to isolate these stem cells are, however, mired in an impasse where the lack of purified lineage-restricted stem cells has hindered the identification of defining markers for these rare stem cells and, in turn, their isolation. METHODOLOGY/PRINCIPAL FINDINGS: We describe here a method for the isolation of clonal lineage-restricted cell lines with endothelial potential from ESCs through a combination of empirical and rational evidence-based methods. Using an empirical protocol that we have previously developed to generate embryo-derived RoSH lines with endothelial potential, we first generated E-RoSH lines from mouse ESC-derived embryoid bodies (EBs. Despite originating from different mouse strains, RoSH and E- RoSH lines have similar gene expression profiles (r(2 = 0.93 while that between E-RoSH and ESCs was 0.83. In silico gene expression analysis predicted that like RoSH cells, E-RoSH cells have an increased propensity to differentiate into vasculature. Unlike their parental ESCs, E-RoSH cells did not form teratomas and differentiate efficiently into endothelial-like cells in vivo and in vitro. Gene expression and FACS analysis revealed that RoSH and E-RoSH cells are CD9(hi, SSEA-1(- while ESCs are CD9(lo, SSEA-1(+. Isolation of CD9(hi, SSEA-1(- cells that constituted 1%-10% of EB-derived cultures generated an E-RoSH-like culture with an identical E-RoSH-like gene expression profile (r(2 = 0.95 and a propensity to differentiate into endothelial-like cells. CONCLUSIONS: By combining empirical and rational evidence-based methods, we identified definitive selectable surface antigens for the isolation and propagation of lineage-restricted stem cells

  11. miR-24 inhibits cell proliferation by suppressing expression of E2F2, MYC and other cell cycle regulatory genes by binding to “seedless” 3′UTR microRNA recognition elements

    Science.gov (United States)

    Lal, Ashish; Navarro, Francisco; Maher, Christopher; Maliszewski, Laura E.; Yan, Nan; O'Day, Elizabeth; Chowdhury, Dipanjan; Dykxhoorn, Derek M.; Tsai, Perry; Hofman, Oliver; Becker, Kevin G.; Gorospe, Myriam; Hide, Winston; Lieberman, Judy

    2009-01-01

    Summary miR-24, up-regulated during terminal differentiation of multiple lineages, inhibits cell cycle progression. Antagonizing miR-24 restores post-mitotic cell proliferation and enhances fibroblast proliferation, while over-expressing miR-24 increases the G1 compartment. The 248 mRNAs down-regulated upon miR-24 over-expression are highly enriched for DNA repair and cell cycle regulatory genes that form a direct interaction network with prominent nodes at genes that enhance (MYC, E2F2, CCNB1, CDC2) or inhibit (p27Kip1, VHL) cell cycle progression. miR-24 directly regulates MYC and E2F2 and some genes they transactivate. Enhanced proliferation from antagonizing miR-24 is abrogated by knocking down E2F2, but not MYC, and cell proliferation, inhibited by miR-24 over-expression, is rescued by miR-24-insensitive E2F2. Therefore, E2F2 is a critical miR-24 target. The E2F2 3′UTR lacks a predicted miR-24 recognition element. In fact, miR-24 regulates expression of E2F2, MYC, AURKB, CCNA2, CDC2, CDK4 and FEN1 by recognizing seedless, but highly complementary, sequences. PMID:19748357

  12. Parental resistance of irradiated mice to (CBAXM523)F/sub 1/ lymphocytes: the destiny of transplanted cells, duration of resistance and its specificity

    Energy Technology Data Exchange (ETDEWEB)

    Kondrat' eva, T K; Fontalin, L N; Nagurskaya, E V; Novikova, T K; Blandova, Z K; Chernousov, A D [Akademiya Meditsinskikh Nauk SSSR, Moscow. Inst. Ehpidemiologii i Mikrobiologii

    1979-05-01

    The immunologic reactivity of mouse (CBAxM523)F/sub 1/ lymphocytes to alien antigens (ram erythrocytes) in the organisms of lethally irradiated CBA mice was studied. If the irradiation, cell transfer and antigen test-injection were performed on the same day, the activity of the transplant was suppressed as compared to the syngenic system. If the intervals between these procedures increased to three days the activity of donor cells was recovered. The retransplantation of recipient spleen cells to the irradiated CBA and F/sub 1/ mice demonstrated the viability of the transplanted cells and the absence of their transadaptation to nonsyngenic microenvironment. The resistance of the recipients could be overcome specifically by preliminary injection of F/sub 1/ mouse cells in combination with cyclophosphamide or without it. The results obtained suggest to conclude, that the genetic parental resistance of CBA mice to F/sub 1/ mouse cells is caused by the immunologically competent recipient cells that are inactivated after three days following irradiation. They do not produce a cytotoxic effect on donor cells, but limit temporarily the activity of the latter.

  13. Identification of T-cell epitopes of Lol p 9, a major allergen of ryegrass (Lolium perenne) pollen.

    Science.gov (United States)

    Blaher, B; Suphioglu, C; Knox, R B; Singh, M B; McCluskey, J; Rolland, J M

    1996-07-01

    T-cell recognition of Lol p 9, a major allergen of ryegrass pollen, was investigated by using a T-cell line and T-cell clones generated from the peripheral blood of an atopic donor. The T-cell line reacted with purified Lol p 9, as well as with crude ryegrass pollen extract, but failed to cross-react with Bermuda grass pollen extract. All of six T-cell clones generated from this line proliferated in response to Lol p 9. Epitope mapping was carried out with a panel of 34 overlapping synthetic peptides, which spanned the entire sequence of the Lol p 9 12R isoform. The T-cell line responded to two of the peptides, Lol p 9 (105-116) and Lol p 9 (193-204), whereas reactivity with one or other of these peptides was shown by five T-cell clones. These two peptides contained sequences consistent with motifs previously reported for major histocompatibility complex class II-restricted peptides. HLA antibody blocking studies showed that presentation of peptide Lol p 9 (105-116) to one T-cell clone was HLA-DR-restricted; this clone expressed a T helper cell phenotype (CD3+, CD4+) and the T-cell receptor alpha beta. The identification of immunodominant T-cell epitope(s) on allergens is essential for devising safer and more effective immunotherapy strategies, which can interrupt the chain of events leading to allergic disease.

  14. PET imaging of EGF receptors using [{sup 18}F]FBEM-EGF in a head and neck squamous cell carcinoma model

    Energy Technology Data Exchange (ETDEWEB)

    Li, Weihua [Harbin Medical University, Department of Medical Imaging and Nuclear Medicine, Fourth Affiliated Hospital, Harbin (China); National Institutes of Health (NIH), Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), Bethesda, MD (United States); Niu, Gang; Lang, Lixin; Guo, Ning; Ma, Ying; Kiesewetter, Dale O.; Chen, Xiaoyuan [National Institutes of Health (NIH), Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), Bethesda, MD (United States); Backer, Joseph M. [SibTech Inc., Brookfield, CT (United States); Shen, Baozhong [Harbin Medical University, Department of Medical Imaging and Nuclear Medicine, Fourth Affiliated Hospital, Harbin (China)

    2012-02-15

    To prepare and evaluate a new radiotracer for molecular imaging of cell surface receptors for epidermal growth factor (EGF). Cys-tagged EGF (cEGF) was labeled with {sup 18}F by coupling the free thiol group of the Cys tag with N-[2-(4-[{sup 18}F]fluorobenzamido)ethyl]maleimide ([{sup 18}F]FBEM) to form [{sup 18}F]FBEM-cEGF. Cell uptake, internalization and efflux of [{sup 18}F]FBEM-cEGF were tested in human head and neck squamous carcinoma UM-SCC1 cells. In vivo tumor targeting and pharmacokinetics of the radiotracers were evaluated in UM-SCC1 tumor-bearing athymic nude mice by static and dynamic microPET imaging. Ex vivo biodistribution assays were performed to confirm the noninvasive imaging results. The radiolabeling yield for [{sup 18}F]FBEM-cEGF was over 60%, based on starting [{sup 18}F]FBEM. [{sup 18}F]FBEM-cEGF exhibited rapid blood clearance through both hepatobiliary and renal excretion. UM-SCC1 tumors were clearly visualized and showed modest tracer uptake of 2.60 {+-} 0.59 %ID/g at 30 min after injection. Significantly higher tumor uptake of [{sup 18}F]FBEM-cEGF (5.99 {+-} 1.61%ID/g at 30 min after injection, p < 0.01) and tumor/nontumor ratio were achieved by coinjection of 50 {mu}g of unlabeled EGF. Decreased liver uptake of [{sup 18}F]FBEM-cEGF was observed when unlabeled EGF was coadministered. With optimized liver blocking, [{sup 18}F]FBEM-cEGF has the potential to be used in a noninvasive and quantitative manner for detection of malignant lesions and evaluation of EGFR activity. (orig.)

  15. Atmospheric histories and growth trends of C4F10, C5F12, C6F14, C7F16 and C8F18

    Directory of Open Access Journals (Sweden)

    R. F. Weiss

    2012-05-01

    Full Text Available Atmospheric observations and trends are presented for the high molecular weight perfluorocarbons (PFCs: decafluorobutane (C4F10, dodecafluoropentane (C5F12, tetradecafluorohexane (C6F14, hexadecafluoroheptane (C7F16 and octadecafluorooctane (C8F18. Their atmospheric histories are based on measurements of 36 Northern Hemisphere and 46 Southern Hemisphere archived air samples collected between 1973 to 2011 using the Advanced Global Atmospheric Gases Experiment (AGAGE "Medusa" preconcentration gas chromatography-mass spectrometry systems. A new calibration scale was prepared for each PFC, with estimated accuracies of 6.8% for C4F10, 7.8% for C5F12, 4.0% for C6F14, 6.6% for C7F16 and 7.9% for C8F18. Based on our observations the 2011 globally averaged dry air mole fractions of these heavy PFCs are: 0.17 parts-per-trillion (ppt, i.e., parts per 1012 for C4F10, 0.12 ppt for C5F12, 0.27 ppt for C6F14, 0.12 ppt for C7F16 and 0.09 ppt for C8F18. These atmospheric mole fractions combine to contribute to a global average radiative forcing of 0.35 mW m−2, which is 6% of the total anthropogenic PFC radiative forcing (Montzka and Reimann, 2011; Oram et al., 2012. The growth rates of the heavy perfluorocarbons were largest in the late 1990s peaking at 6.2 parts per quadrillion (ppq, i.e., parts per 1015 per year (yr for C4F10, at 5.0 ppq yr−1 for C5F12 and 16.6 ppq yr−1 for C6F14 and in the early 1990s for C7F16 at 4.7 ppq yr−1 and in the mid 1990s for C8F18 at 4.8 ppq yr−1. The 2011 globally averaged mean atmospheric growth rates of these PFCs are subsequently lower at 2.2 ppq yr−1 for C4F10, 1.4 ppq yr−1 for C5F12, 5.0 ppq yr−1 for C6F14, 3.4 ppq yr−1 for C7F16 and 0.9 ppq yr−1 for C8F18. The more recent slowdown in the growth rates suggests that emissions are declining as compared to the 1980s and 1990s.

  16. Genetic disruption of the pHi-regulating proteins Na+/H+ exchanger 1 (SLC9A1) and carbonic anhydrase 9 severely reduces growth of colon cancer cells.

    Science.gov (United States)

    Parks, Scott K; Cormerais, Yann; Durivault, Jerome; Pouyssegur, Jacques

    2017-02-07

    Hypoxia and extracellular acidosis are pathophysiological hallmarks of aggressive solid tumors. Regulation of intracellular pH (pHi) is essential for the maintenance of tumor cell metabolism and proliferation in this microenvironment and key proteins involved in pHi regulation are of interest for therapeutic development. Carbonic anhydrase 9 (CA9) is one of the most robustly regulated proteins by the hypoxia inducible factor (HIF) and contributes to pHi regulation. Here, we have investigated for the first time, the role of CA9 via complete genomic knockout (ko) and compared its impact on tumor cell physiology with the essential pHi regulator Na+/H+ exchanger 1 (NHE1). Initially, we established NHE1-ko LS174 cells with inducible CA9 knockdown. While increased sensitivity to acidosis for cell survival in 2-dimensions was not observed, clonogenic proliferation and 3-dimensional spheroid growth in particular were greatly reduced. To avoid potential confounding variables with use of tetracycline-inducible CA9 knockdown, we established CA9-ko and NHE1/CA9-dko cells. NHE1-ko abolished recovery from NH4Cl pre-pulse cellular acid loading while both NHE1 and CA9 knockout reduced resting pHi. NHE1-ko significantly reduced tumor cell proliferation both in normoxia and hypoxia while CA9-ko dramatically reduced growth in hypoxic conditions. Tumor xenografts revealed substantial reductions in tumor growth for both NHE1-ko and CA9-ko. A notable induction of CA12 occurred in NHE1/CA9-dko tumors indicating a potential means to compensate for loss of pH regulating proteins to maintain growth. Overall, these genomic knockout results strengthen the pursuit of targeting tumor cell pH regulation as an effective anti-cancer strategy.

  17. Regional differentiation of retinoic acid-induced human pluripotent embryonic carcinoma stem cell neurons.

    Directory of Open Access Journals (Sweden)

    Dennis E Coyle

    Full Text Available The NTERA2 cl D1 (NT2 cell line, derived from human teratocarcinoma, exhibits similar properties as embryonic stem (ES cells or very early neuroepithelial progenitors. NT2 cells can be induced to become postmitotic central nervous system neurons (NT2N with retinoic acid. Although neurons derived from pluripotent cells, such as NT2N, have been characterized for their neurotransmitter phenotypes, their potential suitability as a donor source for neural transplantation also depends on their ability to respond to localized environmental cues from a specific region of the CNS. Therefore, our study aimed to characterize the regional transcription factors that define the rostocaudal and dorsoventral identity of NT2N derived from a monolayer differentiation paradigm using quantitative PCR (qPCR. Purified NT2N mainly expressed both GABAergic and glutamatergic phenotypes and were electrically active but did not form functional synapses. The presence of immature astrocytes and possible radial glial cells was noted. The NT2N expressed a regional transcription factor code consistent with forebrain, hindbrain and spinal cord neural progenitors but showed minimal expression of midbrain phenotypes. In the dorsoventral plane NT2N expressed both dorsal and ventral neural progenitors. Of major interest was that even under the influence of retinoic acid, a known caudalization factor, the NT2N population maintained a rostral phenotype subpopulation which expressed cortical regional transcription factors. It is proposed that understanding the regional differentiation bias of neurons derived from pluripotent stem cells will facilitate their successful integration into existing neuronal networks within the CNS.

  18. Fibroblast Growth Factor-9 Activates c-Kit Progenitor Cells and Enhances Angiogenesis in the Infarcted Diabetic Heart

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    Dinender Singla

    2016-01-01

    Full Text Available We hypothesized that fibroblast growth factor-9 (FGF-9 would enhance angiogenesis via activating c-kit positive stem cells in the infarcted nondiabetic and diabetic heart. In brief, animals were divided into three groups: Sham, MI, and MI+FGF-9. Two weeks following MI or sham surgery, our data suggest that treatment with FGF-9 significantly diminished vascular apoptosis compared to the MI group in both C57BL/6 and db/db mice (p<0.05. Additionally, the number of c-kit+ve/SM α-actin+ve cells and c-kit+ve/CD31+ve cells were greatly enhanced in the MI+FGF-9 groups relative to the MI suggesting FGF-9 enhances c-Kit cell activation and their differentiation into vascular smooth muscle cells and endothelial cells, respectively (p<0.05. Histology shows that the total number of vessels were quantified for all groups and our data suggest that the FGF-9 treated groups had significantly more vessels than their MI counterparts (p<0.05. Finally, echocardiographic data suggests a significant improvement in left ventricular output, as indicated by fractional shortening and ejection fraction in both nondiabetic and diabetic animals treated with FGF-9 (p<0.05. Overall, our data suggests FGF-9 has the potential to attenuate vascular cell apoptosis, activate c-Kit progenitor cells, and enhance angiogenesis and neovascularization in C57BL/6 and db/db mice leading to improved cardiac function.

  19. Impact of calcium ion on cytotoxic effect of the boroxine derivative, K2[B3O3F4OH].

    Science.gov (United States)

    Ivankovic, Sinisa; Stojkovic, Ranko; Maksimovic, Milka; Galic, Borivoj; Milos, Mladen

    2016-01-01

    The effect of Ca 2+ ions on the cytotoxic ability of boron heterocyclic compound dipotassium-trioxohydroxytetrafluorotriborate (K 2 [B 3 O 3 F 4 OH]), on in vitro tumor cells (mammary adenocarcinoma 4T1, melanoma B16F10 and squamous cell carcinoma SCCVII) and non-tumoral fibroblast cells (mouse dermal L929 and hamster lung V79) was examined. At small concentrations of Ca 2+ ions (0.42 mM), K 2 [B 3 O 3 F 4 OH] (3.85 mM) has a very strong cytotoxic effect on all cancer cells tested (89.1, 85.6 and 84.6%) and significantly less effect on normal cells (19.5 and 24.2%), respectively. Applying larger concentrations of Ca 2+ ions (9.42-72.42 mM), at the same concentration of K 2 [B 3 O 3 F 4 OH], no significant cytotoxic effect was detected on cancer cells and normal cells investigated. The selective ability of K 2 [B 3 O 3 F 4 OH], in the medium with a low concentration of Ca 2+ ions has a strong cytotoxic effect on cancer cells and very weak effect in normal cells, opens up the possibility of its application in antitumor therapy.

  20. NADPH oxidase/ROS-dependent PYK2 activation is involved in TNF-α-induced matrix metalloproteinase-9 expression in rat heart-derived H9c2 cells

    International Nuclear Information System (INIS)

    Yang, Chuen-Mao; Lee, I-Ta; Hsu, Ru-Chun; Chi, Pei-Ling; Hsiao, Li-Der

    2013-01-01

    TNF-α plays a mediator role in the pathogenesis of chronic heart failure contributing to cardiac remodeling and peripheral vascular disturbances. The implication of TNF-α in inflammatory responses has been shown to be mediated through up-regulation of matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of TNF-α-induced MMP-9 expression in rat embryonic-heart derived H9c2 cells are largely not defined. We demonstrated that in H9c2 cells, TNF-α induced MMP-9 mRNA and protein expression associated with an increase in the secretion of pro-MMP-9. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of ROS (N-acetyl-L-cysteine, NAC), NADPH oxidase [apocynin (APO) or diphenyleneiodonium chloride (DPI)], MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), NF-κB (Bay11-7082), or PYK2 (PF-431396) and transfection with siRNA of TNFR1, p47 phox , p42, p38, JNK1, p65, or PYK2. Moreover, TNF-α markedly induced NADPH oxidase-derived ROS generation in these cells. TNF-α-enhanced p42/p44 MAPK, p38 MAPK, JNK1/2, and NF-κB (p65) phosphorylation and in vivo binding of p65 to the MMP-9 promoter were inhibited by U0126, SB202190, SP600125, NAC, DPI, or APO. In addition, TNF-α-mediated PYK2 phosphorylation was inhibited by NAC, DPI, or APO. PYK2 inhibition could reduce TNF-α-stimulated MAPKs and NF-κB activation. Thus, in H9c2 cells, we are the first to show that TNF-α-induced MMP-9 expression is mediated through a TNFR1/NADPH oxidase/ROS/PYK2/MAPKs/NF-κB cascade. We demonstrated that NADPH oxidase-derived ROS generation is involved in TNF-α-induced PYK2 activation in these cells. Understanding the regulation of MMP-9 expression and NADPH oxidase activation by TNF-α on H9c2 cells may provide potential therapeutic targets of chronic heart failure. - Highlights: • TNF-α induces MMP-9 secretion and expression via a TNFR1-dependent pathway. • TNF-α induces ROS/PYK2-dependent MMP-9 expression in H9c2 cells. • TNF-α induces

  1. SPAG9 is involved in hepatocarcinoma cell migration and invasion via modulation of ELK1 expression

    Directory of Open Access Journals (Sweden)

    Yan QY

    2016-03-01

    Full Text Available Qiuyue Yan,1,2 Guohua Lou,3 Ying Qian,1 Bo Qin,1 Xiuping Xu,1,2 Yanan Wang,1,2 Yanning Liu,3 Xuejun Dong1 1Shaoxing People’s Hospital, Shaoxing Hospital Zhejiang University, Shaoxing, Zhejiang, 2The Key Laboratory of Laboratory Medicine, Ministry of Education of China, School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, 3State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, People’s Republic of China Background: Sperm-associated antigen 9 (SPAG9 is upregulated in several malignancies and its overexpression is positively correlated with cancer cell malignancies. However, the specific biological roles of SPAG9 in hepatocellular carcinoma (HCC are less understood. Methods: We analyzed SPAG9 and ETS-like gene 1, tyrosine kinase (ELK1 expression in 50 paired HCC specimens and adjacent noncancerous liver specimens using immunohistochemistry. SPAG9 small interfering RNA (siRNA was used to knockdown SPAG9 expression in HCCLM3 and HuH7 cell lines. We used plasmids to upregulate ELK1 expression and siRNA to downregulate ELK1 expression in HuH7 cells. Quantitative real-time polymerase chain reaction and Western blot were used to evaluate the expression of SPAG9 and ELK1 at the mRNA and protein level, respectively. Wound healing, matrigel migration, and invasion analyses were performed to determine the effect of SPAG9 and ELK1 on HCC metastasis. Results: SPAG9 and ELK1 were overexpressed in HCC tissue specimens and their expressions were higher in HCCLM3 and HuH7 cells compared to the low-metastatic HepG2 cells. Overexpression of SPAG9 was positively associated with tumor-node-metastasis staging (P=0.032, metastasis parameters (P=0.018 of HCC patients, and ELK1 expression (r=0.422, P<0.001 in HCC tissue specimens. In addition

  2. BID-F1 and BID-F2 domains of Bartonella henselae effector protein BepF trigger together with BepC the formation of invasome structures.

    Directory of Open Access Journals (Sweden)

    Matthias C Truttmann

    Full Text Available The gram-negative, zoonotic pathogen Bartonella henselae (Bhe translocates seven distinct Bartonella effector proteins (Beps via the VirB/VirD4 type IV secretion system (T4SS into human cells, thereby interfering with host cell signaling [1], [2]. In particular, the effector protein BepG alone or the combination of effector proteins BepC and BepF trigger massive F-actin rearrangements that lead to the establishment of invasome structures eventually resulting in the internalization of entire Bhe aggregates [2], [3]. In this report, we investigate the molecular function of the effector protein BepF in the eukaryotic host cell. We show that the N-terminal [E/T]PLYAT tyrosine phosphorylation motifs of BepF get phosphorylated upon translocation but do not contribute to invasome-mediated Bhe uptake. In contrast, we found that two of the three BID domains of BepF are capable to trigger invasome formation together with BepC, while a mutation of the WxxxE motif of the BID-F1 domain inhibited its ability to contribute to the formation of invasome structures. Next, we show that BepF function during invasome formation can be replaced by the over-expression of constitutive-active Rho GTPases Rac1 or Cdc42. Finally we demonstrate that BID-F1 and BID-F2 domains promote the formation of filopodia-like extensions in NIH 3T3 and HeLa cells as well as membrane protrusions in HeLa cells, suggesting a role for BepF in Rac1 and Cdc42 activation during the process of invasome formation.

  3. Electroporation of DC-3F cells is a dual process.

    Science.gov (United States)

    Wegner, Lars H; Frey, Wolfgang; Silve, Aude

    2015-04-07

    Treatment of biological material by pulsed electric fields is a versatile technique in biotechnology and biomedicine used, for example, in delivering DNA into cells (transfection), ablation of tumors, and food processing. Field exposure is associated with a membrane permeability increase usually ascribed to electroporation, i.e., formation of aqueous membrane pores. Knowledge of the underlying processes at the membrane level is predominantly built on theoretical considerations and molecular dynamics (MD) simulations. However, experimental data needed to monitor these processes with sufficient temporal resolution are scarce. The whole-cell patch-clamp technique was employed to investigate the effect of millisecond pulsed electric fields on DC-3F cells. Cellular membrane permeabilization was monitored by a conductance increase. For the first time, to our knowledge, it could be established experimentally that electroporation consists of two clearly separate processes: a rapid membrane poration (transient electroporation) that occurs while the membrane is depolarized or hyperpolarized to voltages beyond so-called threshold potentials (here, +201 mV and -231 mV, respectively) and is reversible within ∼100 ms after the pulse, and a long-term, or persistent, permeabilization covering the whole voltage range. The latter prevailed after the pulse for at least 40 min, the postpulse time span tested experimentally. With mildly depolarizing or hyperpolarizing pulses just above threshold potentials, the two processes could be separated, since persistent (but not transient) permeabilization required repetitive pulse exposure. Conductance increased stepwise and gradually with depolarizing and hyperpolarizing pulses, respectively. Persistent permeabilization could also be elicited by single depolarizing/hyperpolarizing pulses of very high field strength. Experimental measurements of propidium iodide uptake provided evidence of a real membrane phenomenon, rather than a mere

  4. Noninvasive monitoring of cancer therapy induced activated T cells using [18F]FB-IL-2 PET imaging

    NARCIS (Netherlands)

    Hartimath, S.V.; Draghiciu, O.; Wall, S. van de; Manuelli, V.; Dierckx, R.A.J.O.; Nijman, H.W.; Daemen, T.; Vries, E.F.J. de

    2017-01-01

    Cancer immunotherapy urgently calls for methods to monitor immune responses at the site of the cancer. Since activated T lymphocytes may serve as a hallmark for anticancer responses, we targeted these cells using the radiotracer N-(4-[18F]fluorobenzoyl)-interleukin-2 ([18F]FB-IL-2) for positron

  5. Direct detection of hemophilia B F9 gene mutation using multiplex PCR and conformation sensitive gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Ki Young Yoo

    2010-03-01

    Full Text Available Purpose : The F9 gene is known to be the causative gene for hemophilia B, but unfortunately the detection rate for restriction fragment length polymorphism-based linkage analysis is only 55.6%. Direct DNA sequencing can detect 98% of mutations, but this alternative procedure is very costly. Here, we conducted multiplex polymerase chain reactions (PCRs and conformation sensitive gel electrophoresis (CSGE to perform a screened DNA sequencing for the F9 gene, and we compared the results with direct sequencing in terms of accuracy, cost, simplicity, and time consumption. Methods : A total of 27 unrelated hemophilia B patients were enrolled. Direct DNA sequencing was performed for 27 patients by a separate institute, and multiplex PCR-CSGE screened sequencing was done in our laboratory. Results of the direct DNA sequencing were used as a reference, to which the results of the multiplex PCR-CSGE screened sequencing were compared. For the patients whose mutation was not detected by the 2 methods, multiplex ligation-dependent probe amplification (MLPA was conducted. Results : With direct sequencing, the mutations could be identified from 26 patients (96.3%, whereas for multiplex PCR- CSGE screened sequencing, the mutations could be detected in 23 (85.2%. One patient’s mutation was identified by MLPA. A total of 21 different mutations were found among the 27 patients. Conclusion : Multiplex PCR-CSGE screened DNA sequencing detected 88.9% of mutations and reduced costs by 55.7% compared with direct DNA sequencing. However, it was more labor-intensive and time-consuming.

  6. Genome Editing in Induced Pluripotent Stem Cells using CRISPR/Cas9.

    Science.gov (United States)

    Ben Jehuda, Ronen; Shemer, Yuval; Binah, Ofer

    2018-06-01

    The development of the reprogramming technology led to generation of induced Pluripotent Stem Cells (iPSC) from a variety of somatic cells. Ever since, fast growing knowledge of different efficient protocols enabled the differentiation of these iPSCs into different cells types utilized for disease modeling. Indeed, iPSC-derived cells have been increasingly used for investigating molecular and cellular pathophysiological mechanisms underlying inherited diseases. However, a major barrier in the field of iPSC-based disease modeling relies on discriminating between the effects of the causative mutation and the genetic background of these cells. In the past decade, researchers have made great improvement in genome editing techniques, with one of the latest being CRISPR/Cas9. Using a single non-sequence specific protein combined with a small guiding RNA molecule, this state-of-the-art approach enables modifications of genes with high efficiency and accuracy. By so doing, this technique enables the generation of isogenic controls or isogenic mutated cell lines in order to focus on the pathologies caused by a specific mutation. In this article, we review the latest studies combining iPSC and CRISPR/Cas9 technologies for the investigation of the molecular and cellular mechanisms underlying inherited diseases including immunological, metabolic, hematological, neurodegenerative and cardiac diseases.

  7. E2F6: a member of the E2F family that does not modulate squamous differentiation

    International Nuclear Information System (INIS)

    Wong, C.F.; Barnes, Liam M.; Smith, Louise; Popa, Claudia; Serewko-Auret, Magdalena M.; Saunders, Nicholas A.

    2004-01-01

    The inhibition of E2F has been demonstrated to be important in the initiation of squamous differentiation by two independent manners: promotion of growth arrest and the relief of the differentiation-suppressive properties of E2Fs. E2F6 is reported to behave as a transcriptional repressor of the E2F family. In this study, we examined the ability of E2F6 to act as the molecular switch required for E2F inhibition in order for keratinocytes to enter a terminal differentiation programme. Results demonstrated that whilst E2F6 was able to suppress E2F activity in proliferating keratinocytes, it did not modulate squamous differentiation in a differentiated keratinocyte. Furthermore, inhibition of E2F, by overexpressing E2F6, was not sufficient to sensitise either proliferating keratinocytes or the squamous cell carcinoma cell line, KJD-1/SV40, to differentiation-inducing agents. Significantly, although E2F6 could suppress E2F activity in proliferating cells, it could not inhibit proliferation of KJD-1/SV40 cells. These results demonstrate that E2F6 does not contain the domains required for modulation of squamous differentiation and imply isoform-specific functions for individual E2F family members

  8. Hinokitiol, a tropolone derivative, inhibits mouse melanoma (B16-F10) cell migration and in vivo tumor formation.

    Science.gov (United States)

    Huang, Chien-Hsun; Lu, Shing-Hwa; Chang, Chao-Chien; Thomas, Philip Aloysius; Jayakumar, Thanasekaran; Sheu, Joen-Rong

    2015-01-05

    Invasion and metastasis are the major causes of treatment failure in patients with cancer. Hinokitiol, a natural bioactive compound found in Chamacyparis taiwanensis, has been used in hair tonics, cosmetics, and food as an antimicrobial agent. In this study, we investigated the effects and possible mechanisms of action of hinokitiol on migration by the metastatic melanoma cell line, B16-F10, in which matrix metalloproteinase-1 (MMP-1) is found to be highly- expressed. Treatment with hinokitiol revealed a concentration-dependent inhibition of migration of B16-F10 melanoma cells. Hinokitiol appeared to achieve this effect by reducing the expression of MMP-1 and by suppressing the phosphorylation of mitogen- activated protein kinase (MAPK) signaling molecules such as extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK and c-Jun N-terminal kinases (JNK). On the other hand, hinokitiol treatment reversed IκB-α degradation and inhibited the phosphorylation of p65 nuclear factor kappa B (NF-κB) and cJun in B16-F10 cells. In addition, hinokitiol suppressed the translocation of p65 NF-κB from the cytosol to the nucleus, suggesting reduced NF-κB activation. Consistent with these in vitro findings, our in vivo study demonstrated that hinokitiol treatment significantly reduced the total number of mouse lung metastatic nodules and improved histological alterations in B16-F10 injected C57BL/6 mice. These findings suggest that treatment of B16-F10 cells with hinokitiol significantly inhibits metastasis, possibly by blocking MMP-1 activation, MAPK signaling pathways and inhibition of the transcription factors, NF-κB and c-Jun, involved in cancer cell migration. These results may accelerate the development of novel therapeutic agents for the treatment of malignant cancers. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Molecular composition of vapor in the NaF-ZrF4 system

    International Nuclear Information System (INIS)

    Korenev, Yu.M.; Sidorov, L.N.; Rykov, A.N.; Novoselova, A.V.

    1980-01-01

    The NaF-ZrF 4 system is studied. It is established that Na 2 ZrF 6 , NaZrF 5 , (NaZrF 5 ) 2 , NaZr 2 F 9 complex molecules are present in the saturated vapor alongside with pure components. Partial pressures of all vapor components are determined. The values of partial pressure and evaporation heat have been used to calculate the vapor composition above the system; T-x and P-T projections of the phase diagram of the NaF-ZrF 4 system are plotted

  10. Novel Biophysical Marker of Aggressive Prostate Cancer Cells

    Science.gov (United States)

    2013-06-01

    756 30.33 PrEC LHSR 8.60 687 19.71 PC-3 9.31 744 48.42 TEM 4–18 8.55 683 45.00 22Rv1 7.27 581 41.00 MD.MBA.231 8.16 652 32.43 B16.f0 9.11 728 37.37 Panc ...established human cancer cell lines evaluated, PANC -1 pancreatic cancer cells and Jurkat leukemia cells, were considerably more sensitive to the FSS

  11. Full restoration of Brucella-infected dendritic cell functionality through Vγ9Vδ2 T helper type 1 crosstalk.

    Directory of Open Access Journals (Sweden)

    Ming Ni

    Full Text Available Vγ9Vδ2 T cells play an important role in the immune response to infectious agents but the mechanisms contributing to this immune process remain to be better characterized. Following their activation, Vγ9Vδ2 T cells develop cytotoxic activity against infected cells, secrete large amounts of cytokines and influence the function of other effectors of immunity, notably cells playing a key role in the initiation of the adaptive immune response such as dendritic cells. Brucella infection dramatically impairs dendritic cell maturation and their capacity to present antigens to T cells. Herein, we investigated whether V T cells have the ability to restore the full functional capacities of Brucella-infected dendritic cells. Using an in vitro multicellular infection model, we showed that: 1/Brucella-infected dendritic cells activate Vγ9Vδ2 T cells through contact-dependent mechanisms, 2/activated Vγ9Vδ2 T cells induce full differentiation into IL-12 producing cells of Brucella-infected dendritic cells with functional antigen presentation activity. Furthermore, phosphoantigen-activated Vγ9Vδ2 T cells also play a role in triggering the maturation process of dendritic cells already infected for 24 h. This suggests that activated Vγ9Vδ2 T cells could be used to modulate the outcome of infectious diseases by promoting an adjuvant effect in dendritic cell-based cellular therapies.

  12. Substituting mouse transcription factor Pou4f2 with a sea urchin orthologue restores retinal ganglion cell development.

    Science.gov (United States)

    Mao, Chai-An; Agca, Cavit; Mocko-Strand, Julie A; Wang, Jing; Ullrich-Lüter, Esther; Pan, Ping; Wang, Steven W; Arnone, Maria Ina; Frishman, Laura J; Klein, William H

    2016-03-16

    Pou domain transcription factor Pou4f2 is essential for the development of retinal ganglion cells (RGCs) in the vertebrate retina. A distant orthologue of Pou4f2 exists in the genome of the sea urchin (class Echinoidea) Strongylocentrotus purpuratus (SpPou4f1/2), yet the photosensory structure of sea urchins is strikingly different from that of the mammalian retina. Sea urchins have no obvious eyes, but have photoreceptors clustered around their tube feet disc. The mechanisms that are associated with the development and function of photoreception in sea urchins are largely unexplored. As an initial approach to better understand the sea urchin photosensory structure and relate it to the mammalian retina, we asked whether SpPou4f1/2 could support RGC development in the absence of Pou4f2. To answer this question, we replaced genomic Pou4f2 with an SpPou4f1/2 cDNA. In Pou4f2-null mice, retinas expressing SpPou4f1/2 were outwardly identical to those of wild-type mice. SpPou4f1/2 retinas exhibited dark-adapted electroretinogram scotopic threshold responses, indicating functionally active RGCs. During retinal development, SpPou4f1/2 activated RGC-specific genes and in S. purpuratus, SpPou4f2 was expressed in photoreceptor cells of tube feet in a pattern distinct from Opsin4 and Pax6. Our results suggest that SpPou4f1/2 and Pou4f2 share conserved components of a gene network for photosensory development and they maintain their conserved intrinsic functions despite vast morphological differences in mouse and sea urchin photosensory structures. © 2016 The Authors.

  13. Beneficial Effect of Jojoba Seed Extracts on Hyperglycemia-Induced Oxidative Stress in RINm5f Beta Cells

    Directory of Open Access Journals (Sweden)

    Sahla Belhadj

    2018-03-01

    Full Text Available Hyperglycemia occurs during diabetes and insulin resistance. It causes oxidative stress by increasing reactive oxygen species (ROS levels, leading to cellular damage. Polyphenols play a central role in defense against oxidative stress. In our study, we investigated the antioxidant properties of simmondsin, a pure molecule present in jojoba seeds, and of the aqueous extract of jojoba seeds on fructose-induced oxidative stress in RINm5f beta cells. The exposure of RINm5f beta cells to fructose triggered the loss of cell viability (−48%, p < 0.001 and disruption of insulin secretion (p < 0.001 associated with of reactive oxygen species (ROS production and a modulation of pro-oxidant and antioxidant signaling pathway. Cell pre-treatments with extracts considerably increased cell viability (+86% p < 0.001 for simmondsin and +74% (p < 0.001 for aqueous extract and insulin secretion. The extracts also markedly decreased ROS (−69% (p < 0.001 for simmondsin and −59% (p < 0.001 for aqueous extract and caspase-3 activation and improved antioxidant defense, inhibiting p22phox and increasing nuclear factor (erythroid-derived 2-like 2 (Nrf2 levels (+70%, p < 0.001 for aqueous extract. Simmondsin had no impact on Nrf2 levels. The richness and diversity of molecules present in jojoba seed extract makes jojoba a powerful agent to prevent the destruction of RINm5f beta cells induced by hyperglycemia.

  14. Infra-red laser ablative micromachining of parylene-C on SiO2 substrates for rapid prototyping, high yield, human neuronal cell patterning

    International Nuclear Information System (INIS)

    Raos, B J; Unsworth, C P; Costa, J L; Rohde, C A; Simpson, M C; Doyle, C S; Dickinson, M E; Bunting, A S; Murray, A F; Delivopoulos, E; Graham, E S

    2013-01-01

    Cell patterning commonly employs photolithographic methods for the micro fabrication of structures on silicon chips. These require expensive photo-mask development and complex photolithographic processing. Laser based patterning of cells has been studied in vitro and laser ablation of polymers is an active area of research promising high aspect ratios. This paper disseminates how 800 nm femtosecond infrared (IR) laser radiation can be successfully used to perform laser ablative micromachining of parylene-C on SiO 2 substrates for the patterning of human hNT astrocytes (derived from the human teratocarcinoma cell line (hNT)) whilst 248 nm nanosecond ultra-violet laser radiation produces photo-oxidization of the parylene-C and destroys cell patterning. In this work, we report the laser ablation methods used and the ablation characteristics of parylene-C for IR pulse fluences. Results follow that support the validity of using IR laser ablative micromachining for patterning human hNT astrocytes cells. We disseminate the variation in yield of patterned hNT astrocytes on parylene-C with laser pulse spacing, pulse number, pulse fluence and parylene-C strip width. The findings demonstrate how laser ablative micromachining of parylene-C on SiO 2 substrates can offer an accessible alternative for rapid prototyping, high yield cell patterning with broad application to multi-electrode arrays, cellular micro-arrays and microfluidics. (paper)

  15. Dissection of the transformation of primary human hematopoietic cells by the oncogene NUP98-HOXA9.

    Directory of Open Access Journals (Sweden)

    Enas R Yassin

    2009-08-01

    Full Text Available NUP98-HOXA9 is the prototype of a group of oncoproteins associated with acute myeloid leukemia. It consists of an N-terminal portion of NUP98 fused to the homeodomain of HOXA9 and is believed to act as an aberrant transcription factor that binds DNA through the homeodomain. Here we show that NUP98-HOXA9 can regulate transcription without binding to DNA. In order to determine the relative contributions of the NUP98 and HOXA9 portions to the transforming ability of NUP98-HOXA9, the effects of NUP98-HOXA9 on primary human CD34+ cells were dissected and compared to those of wild-type HOXA9. In contrast to previous findings in mouse cells, HOXA9 had only mild effects on the differentiation and proliferation of primary human hematopoietic cells. The ability of NUP98-HOXA9 to disrupt the differentiation of primary human CD34+ cells was found to depend primarily on the NUP98 portion, whereas induction of long-term proliferation required both the NUP98 moiety and an intact homeodomain. Using oligonucleotide microarrays in primary human CD34+ cells, a group of genes was identified whose dysregulation by NUP98-HOXA9 is attributable primarily to the NUP98 portion. These include RAP1A, HEY1, and PTGS2 (COX-2. Their functions may reflect the contribution of the NUP98 moiety of NUP98-HOXA9 to leukemic transformation. Taken together, these results suggest that the effects of NUP98-HOXA9 on gene transcription and cell transformation are mediated by at least two distinct mechanisms: one that involves promoter binding through the homeodomain with direct transcriptional activation, and another that depends predominantly on the NUP98 moiety and does not involve direct DNA binding.

  16. A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cell fate determination.

    Science.gov (United States)

    Zhao, Chunnian; Sun, GuoQiang; Li, Shengxiu; Shi, Yanhong

    2009-04-01

    MicroRNAs have been implicated as having important roles in stem cell biology. MicroRNA-9 (miR-9) is expressed specifically in neurogenic areas of the brain and may be involved in neural stem cell self-renewal and differentiation. We showed previously that the nuclear receptor TLX is an essential regulator of neural stem cell self-renewal. Here we show that miR-9 suppresses TLX expression to negatively regulate neural stem cell proliferation and accelerate neural differentiation. Introducing a TLX expression vector that is not prone to miR-9 regulation rescued miR-9-induced proliferation deficiency and inhibited precocious differentiation. In utero electroporation of miR-9 in embryonic brains led to premature differentiation and outward migration of the transfected neural stem cells. Moreover, TLX represses expression of the miR-9 pri-miRNA. By forming a negative regulatory loop with TLX, miR-9 provides a model for controlling the balance between neural stem cell proliferation and differentiation.

  17. A receptor tyrosine kinase inhibitor, Tyrphostin A9 induces cancer cell death through Drp1 dependent mitochondria fragmentation

    International Nuclear Information System (INIS)

    Park, So Jung; Park, Young Jun; Shin, Ji Hyun; Kim, Eun Sung; Hwang, Jung Jin; Jin, Dong-Hoon; Kim, Jin Cheon; Cho, Dong-Hyung

    2011-01-01

    Highlights: → We screened and identified Tyrphostin A9, a receptor tyrosine kinase inhibitor as a strong mitochondria fission inducer. → Tyrphostin A9 treatment promotes mitochondria dysfunction and contributes to cytotoxicity in cancer cells. → Tyrphostin A9 induces apoptotic cell death through a Drp1-mediated pathway. → Our studies suggest that Tyrphostin A9 induces mitochondria fragmentation and apoptotic cell death via Drp1 dependently. -- Abstract: Mitochondria dynamics controls not only their morphology but also functions of mitochondria. Therefore, an imbalance of the dynamics eventually leads to mitochondria disruption and cell death. To identify specific regulators of mitochondria dynamics, we screened a bioactive chemical compound library and selected Tyrphostin A9, a tyrosine kinase inhibitor, as a potent inducer of mitochondrial fission. Tyrphostin A9 treatment resulted in the formation of fragmented mitochondria filament. In addition, cellular ATP level was decreased and the mitochondrial membrane potential was collapsed in Tyr A9-treated cells. Suppression of Drp1 activity by siRNA or over-expression of a dominant negative mutant of Drp1 inhibited both mitochondrial fragmentation and cell death induced by Tyrpohotin A9. Moreover, treatment of Tyrphostin A9 also evoked mitochondrial fragmentation in other cells including the neuroblastomas. Taken together, these results suggest that Tyrphostin A9 induces Drp1-mediated mitochondrial fission and apoptotic cell death.

  18. Immunohistochemical demonstration of a hitherto undescribed localization of hemoglobin A and F in endodermal cells of normal human yolk sac and endodermal sinus tumor

    DEFF Research Database (Denmark)

    Albrechtsen, R; Wewer, U; Wimberley, P D

    1980-01-01

    In this study of 4 human yolk sacs, the presence of hemoglobin A and F (HbA and HbF) is demonstrated for the first time in epithelial cells (type 1) and erythroid-like cells (type 2) in the endodermal layer by immunoperoxidase technique. Our findings strongly support the hypothesis previously...... proposed that the red blood cells formed in the yolk sac are of endodermal origin. Tumor with yolk sac differentiation (8 endodermal sinus tumors and 1 embryonal carcinoma with vitelline areas) similarly showed HbA and HbF localisation in endodermal cells. None of 59 germ cell tumors of other types...

  19. Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells.

    Science.gov (United States)

    Dong, Zhan-Qi; Chen, Ting-Ting; Zhang, Jun; Hu, Nan; Cao, Ming-Ya; Dong, Fei-Fan; Jiang, Ya-Ming; Chen, Peng; Lu, Cheng; Pan, Min-Hui

    2016-06-01

    Although current antiviral strategies can inhibit baculovirus infection and decrease viral DNA replication to a certain extent, novel tools are required for specific and accurate elimination of baculovirus genomes from infected insects. Using the newly developed clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology, we disrupted a viral genome in infected insect cells in vitro as a defense against viral infection. We optimized the CRISPR/Cas9 system to edit foreign and viral genome in insect cells. Using Bombyx mori nucleopolyhedrovirus (BmNPV) as a model, we found that the CRISPR/Cas9 system was capable of cleaving the replication key factor ie-1 in BmNPV thus effectively inhibiting virus proliferation. Furthermore, we constructed a virus-inducible CRISPR/Cas9 editing system, which minimized the probability of off-target effects and was rapidly activated after viral infection. This is the first report describing the application of the CRISPR/Cas9 system in insect antiviral research. Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells provides insights to produce virus-resistant transgenic strains for future. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. SOX9 as a Predictor for Neurogenesis Potentiality of Amniotic Fluid Stem Cells

    Science.gov (United States)

    Wei, Pei-Cih; Chao, Angel; Peng, Hsiu-Huei; Chao, An-Shine; Chang, Yao-Lung; Chang, Shuenn-Dyh; Wang, Hsin-Shih; Chang, Yu-Jen; Tsai, Ming-Song; Sieber, Martin; Chen, Hua-Chien; Chen, Shu-Jen; Lee, Yun-Shien

    2014-01-01

    Preclinical studies of amniotic fluid-derived cell therapy have been successful in the research of neurodegenerative diseases, peripheral nerve injury, spinal cord injury, and brain ischemia. Transplantation of human amniotic fluid stem cells (AFSCs) into rat brain ventricles has shown improvement in symptoms of Parkinson's disease and also highlighted the minimal immune rejection risk of AFSCs, even between species. Although AFSCs appeared to be a promising resource for cell-based regenerative therapy, AFSCs contain a heterogeneous pool of distinct cell types, rendering each preparation of AFSCs unique. Identification of predictive markers for neuron-prone AFSCs is necessary before such stem cell-based therapeutics can become a reality. In an attempt to identify markers of AFSCs to predict their ability for neurogenesis, we performed a two-phase study. In the discovery phase of 23 AFSCs, we tested ZNF521/Zfp521, OCT6, SOX1, SOX2, SOX3, and SOX9 as predictive markers of AFSCs for neural differentiation. In the validation phase, the efficacy of these predictive markers was tested in independent sets of 18 AFSCs and 14 dental pulp stem cells (DPSCs). We found that high expression of SOX9 in AFSCs is associated with good neurogenetic ability, and these positive correlations were confirmed in independent sets of AFSCs and DPSCs. Furthermore, knockdown of SOX9 in AFSCs inhibited their neuronal differentiation. In conclusion, the discovery of SOX9 as a predictive marker for neuron-prone AFSCs could expedite the selection of useful clones for regenerative medicine, in particular, in neurological diseases and injuries. PMID:25154783

  1. Effect of 0.4 mT power frequency magnetic field on F-actin assembly of CHL cells

    International Nuclear Information System (INIS)

    Chu Keping; Cai Zhiyin; Zhang Yukun; Xia Nuohong

    2007-01-01

    Objective: To investigate the effect of 0.4mT power frequency magnetic field on the microfilament (F- actin) assembly of Chinese hamster lung (CHL) cells. Methods: F-actin were marked with immunohistochemical method, then observed under a confocal microscope. The content of ECFRs in the preparation of the detergent-insoluble cytoskeleton was measured with Western-blotting. Results: The stress fiber's of CHL cells decreased after exposure to 0.4mT power frequency magnetic field for 30min, as well as after treatment with epidermal growth factor (ECF) of 50nM. Filopodias appeared at the periphery after exposure to magnetic field as well as treatment with EGF. The EGF receptor mass associated with the detergent-insoluble cytoskeleton increased after exposure to magnetic field as well as treatment with EGF. Conclusion: 0.4mT power frequency magnetic field induced assembly of F-actin in CHL cells. The change induced by magnetic field would be related to clustering of EGFR induced by magnetic field and passing the signal down. (authors)

  2. Radiosynthesis of a new PSMA targeting ligand ([18F]FPy-DUPA-Pep)

    International Nuclear Information System (INIS)

    Malik, Noeen; Machulla, Hans-Juergen; Solbach, Christoph; Winter, Gordon; Reske, Sven N.; Zlatopolskiy, Boris

    2011-01-01

    Due to the specificity of expression of PSMA (prostate specific membrane antigen) particularly in prostate cancer cells (e.g. LNCaP), numerous PSMA ligands have been synthesized until now. In the current study, we synthesized DUPA-Pep having 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA) linked via 8-aminooctanoic acid to two phenylalanine residues and chose 6-[ 18 F]fluoronicotinic acid 2,3,5,6-tetrafluorophenyl ester [ 18 F]FPy-TFP as a prosthetic group for coupling. [ 18 F]FPy-DUPA-Pep was obtained in a radiochemical yield of 48±0.9% (decay uncorrected) within 50 min with a chemical purity of >98%.

  3. Avaliação da resistência à corrosão de implantes em aços inoxidáveis ISO 5832-9 e F138 removidos de pacientes

    Directory of Open Access Journals (Sweden)

    Elison da Fonseca e Silva

    2012-01-01

    Full Text Available This work has compared the surfaces of two different steel samples used as orthopedical implants, classified as ASTM F138 and ISO5832-9, through optical emission spectroscopy, by means of SEM and EDS. The samples (implants were also submitted to potentiodynamic cyclic polarization in Ringer lactate and NaCl 0.9 M L-1 solutions; ISO5832-9 sample did not show any kind of localized corrosion, but in the case of F138 steel was observed a pit localized corrosion in both solutions. In Ringer lactate solution it was observed a loss of about 63% for nickel and 26% for iron for F138 stell, compared to the initial composition.

  4. Manufacturing of the L band 9-cell niobium cavity

    International Nuclear Information System (INIS)

    Matsuoka, Masanori; Ohkubo, Kohichi; Yamanaka, Toshiyuki; Kako, Eiji; Saito, Kenji; Shishido, Toshio; Ono, Masaaki; Noguchi, Shuichi.

    1993-01-01

    Since 1990, L-band niobium superconducting cavities have been developed with collaboration between our company and National Laboratory for High Energy Physics (KEK). The manufacturing procedure and the performance of 9-cell superconducting cavity are presented. The maximum accelerating gradient of 12 MV/m was attained in a cold test. (author)

  5. STAT3/5-dependent IL9 overexpression contributes to neoplastic cell survival in mycosis fungoides

    DEFF Research Database (Denmark)

    Vieyra-Garcia, Pablo A.; Wei, Tianling; Naym, David Gram

    2016-01-01

    preparations. To explore the mechanism of IL9 secretion, we knocked down STAT3/5 and IRF4 by siRNA transfection in CTCL cell lines receiving psoralen+UVA (PUVA) ± anti-IL9 antibody. To further examine the role of IL9 in tumor development, the EL-4 T-cell lymphoma model was used in C57BL/6 mice.  Results...

  6. Acute damage by naphthalene triggers expression of the neuroendocrine marker PGP9.5 in airway epithelial cells

    DEFF Research Database (Denmark)

    Poulsen, T.T.; Naizhen, X.; Linnoila, R.I.

    2008-01-01

    Protein Gene Product 9.5 (PGP9.5) is highly expressed in nervous tissue. Recently PGP9.5 expression has been found to be upregulated in the pulmonary epithelium of smokers and in non-small cell lung cancer, suggesting that it also plays a role in carcinogen-inflicted lung epithelial injury...... neuroendocrine markers was found in the non-neuroendocrine epithelial cells after naphthalene exposure. In contrast, immunostaining for the cell cycle regulator p27(Kip1), which has previously been associated with PGP9.5 in lung cancer cells, revealed transient downregulation of p27(Kip1) in naphthalene exposed...... and further strengthens the accumulating evidence of PGP9.5 as a central player in lung epithelial damage and early carcinogenesis Udgivelsesdato: 2008/9/26...

  7. Comparison of six methods of segmentation of tumor volume on the 18F-F.D.G. PET scan with reference histological volume in non small cell bronchopulmonary cancers

    International Nuclear Information System (INIS)

    Venel, Y.; Garhi, H.; Baulieu, J.L.; Prunier-Aesch, C.; Muret, A. de; Barillot, I.

    2008-01-01

    The 18 F-F.D.G. PET has demonstrated its importance in oncology, for initial extension and efficacy of anti tumoral therapeutics. Several studies have attempted to prove its utility to define tumoral volumes for conformational radiotherapy in non small cell lung cancers. Some authors have suggested the use of threshold of tumor intensity uptake with 40 or 50% of maximal intensity. Black et al. have determined contouring with linear regression formula of mean semi-quantitative index of tumor uptake (standard uptake value): SUV threshold = 0.307 Sub average + 0.588. Nestle et al. have taken into account the background noise intensity and mean intensity of the tumor: I threshold = β I average +I noise with β 0.15. Our study was done in collaboration with Inserm U618 team and has compared volumes defined on PET scan defined according to different methods based on intensity or S.U.V. to the tumour volume determined on CT scan by radio physicist. We have compared those volumes with histological volume that we considered for reference. Four patients have been included. They had 18 F-F.D.G. PET scan followed by complete tumoral removal surgery. Specific histological procedure allowed to define complete size of the tumor in re expanded lung. Comparatively to pathology, the volumes obtained using I max 40 and I max 50 are all underestimated. The volumes defined by Black's et al. method are under evaluated for the two largest tumours (15.8% to 22%) and overestimated for the two smallest ones (17.9 to 82.9%). Nestle's et al. method, using β = 0.15, correctly estimates two tumor volumes over 2 cm, but overestimates the two small tumors (79.6 to 124%). Finally, the corrected Nestle's et al. formula (using β = 0.264) overestimates three tumours. Volumes defined on CT scan by radio physicist are correct for one lesion, underestimated for one and overestimated for two other ones (44 and 179.5%). Nestle's et al. method seems to be the most accurate for tumours over 2 cm of

  8. File list: His.PSC.50.H3K9K14ac.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.PSC.50.H3K9K14ac.AllCell hg19 Histone H3K9K14ac Pluripotent stem cell SRX037086... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.PSC.50.H3K9K14ac.AllCell.bed ...

  9. Signalling in the epidermis: the E2F cell cycle regulatory pathway in epidermal morphogenesis, regeneration and transformation.

    Science.gov (United States)

    Ivanova, Iordanka A; D'Souza, Sudhir J A; Dagnino, Lina

    2005-01-01

    The epidermis is the outermost layer in the skin, and it is the first line of defence against the environment. The epidermis also provides a barrier against loss of fluids and electrolytes, which is crucial for life. Essential in the maintenance of this tissue is its ability to continually self-renew and regenerate after injury. These two characteristics are critically dependent on the ability of the principal epidermal cell type, the keratinocyte, to proliferate and to respond to differentiation cues. Indeed, the epidermis is a multilayered tissue composed of keratinocyte stem cells and their differentiated progeny. Central for the control of cell proliferation is the E2F transcription factor regulatory network. This signaling network also includes cyclins, cdk, cdk inhibitors and the retinoblastoma (pRb) family of proteins. The biological importance of the E2F/pRb pathway is emphasized by the fact that a majority of human tumours exhibit alterations that disrupt the ability of pRb proteins to inhibit E2F, leading to permanent activation of the latter. Further, E2F is essential for normal epidermal regeneration after injury. Other member of the E2F signaling pathway are also involved in epidermal development and pathophysiology. Thus, whereas the pRb family of proteins is essential for epidermal morphogenesis, abnormal regulation of cyclins and E2F proteins results in tumorgenesis in this tissue. In this review, we discuss the role of each member of this important growth regulatory network in epidermal formation, homeostasis and carcinogenesis.

  10. Feedback regulation of mitochondria by caspase-9 in the B cell receptor-mediated apoptosis.

    Science.gov (United States)

    Eeva, J; Nuutinen, U; Ropponen, A; Mättö, M; Eray, M; Pellinen, R; Wahlfors, J; Pelkonen, J

    2009-12-01

    During the germinal centre reaction (GC), B cells with non-functional or self-reactive antigen receptors are negatively selected by apoptosis to generate B cell repertoire with appropriate antigen specificities. We studied the molecular mechanism of Fas/CD95- and B cell receptor (BCR)-induced apoptosis to shed light on the signalling events involved in the negative selection of GC B cells. As an experimental model, we used human follicular lymphoma (FL) cell line HF1A3, which originates from a GC B cell, and transfected HF1A3 cell lines overexpressing Bcl-x(L), c-FLIP(long) or dominant negative (DN) caspase-9. Fas-induced apoptosis was dependent on the caspase-8 activation, since the overexpression of c-FLIP(long), a natural inhibitor of caspase-8 activation, blocked apoptosis induced by Fas. In contrast, caspase-9 activation was not involved in Fas-induced apoptosis. BCR-induced apoptosis showed the typical characteristics of mitochondria-dependent (intrinsic) apoptosis. Firstly, the activation of caspase-9 was involved in BCR-induced DNA fragmentation, while caspase-8 showed only marginal role. Secondly, overexpression of Bcl-x(L) could block all apoptotic changes induced by BCR. As a novel finding, we demonstrate that caspase-9 can enhance the cytochrome-c release and collapse of mitochondrial membrane potential (DeltaPsi(m)) during BCR-induced apoptosis. The requirement of different signalling pathways in apoptosis induced by BCR and Fas may be relevant, since Fas- and BCR-induced apoptosis can thus be regulated independently, and targeted to different subsets of GC B cells.

  11. No Evidence for JAK2(V617F) Mutation in Monoclonal B Cells in 2 Patients with Polycythaemia Vera and Concurrent Monoclonal B Cell Disorder

    NARCIS (Netherlands)

    Stijnis, C.; Kroes, W. G. M.; Balkassmi, S.; Marijt, E. W. A.; van Rossum, A. P.; Bakker, E.; Vlasveld, L. T.

    2012-01-01

    Occurrence of Philadelphia chromosome-negative myeloproliferative neoplasms (Ph- MPN) and lymphoproliferative disorders, like B cell chronic lymphocytic leukaemia (B-CLL), in the same patient is rare. JAK2(V617F) mutation was recently introduced as a powerful diagnostic tool for Ph- MPN. JAK2(V617F)

  12. F-18 FDG PET/CT Findings of Subcutaneous Panniculitis - Like T- Cell Lymphoma : A Case Report

    International Nuclear Information System (INIS)

    Kong, Eun Jung; Cho, Ihn Ho; Chun, Kyung Ah; Bae, Yeung Kyung; Choi, Joon Hyuk; Hyun, Myung Soo

    2009-01-01

    F-18 FDG PET is a metabolic imaging modality that is efficacious in staging and assessment of treatment response for variety of lymphomas. We report usefulness of F-18 FDG PET/CT in evaluating severity of the disease and response to therapy in a patient with subcutaneous panniculitis- like T-cell lymphoma (SPTCL). Here we describe a case of SPTCL in 24-year-old man who had wide spread firm and tender nodular lesions with increased F-18 FDG uptake. After chemotherapy follow up F-18 FDG PET/CT image shows disseminated malignancy and then the patient died with hemophagocytic syndrome. This report suggests that F-18 FDG PET/CT may be useful in determining disease activity at the time of initial diagnosis, after treatment, and evaluating a suspected outcome of SPTCL

  13. GLUT3 is present in Clone 9 liver cells and translocates to the plasma membrane in response to insulin.

    Science.gov (United States)

    Defries, Danielle M; Taylor, Carla G; Zahradka, Peter

    2016-08-26

    Clone 9 cells have been reported to express only the GLUT1 facilitative glucose transporter; however, previous studies have not examined Clone 9 cells for GLUT3 content. The current study sought to profile the presence of glucose transporters in Clone 9 cells, H4IIE hepatoma cells, and L6 myoblasts and myotubes. While the other cell types contained the expected complement of transporters, Clone 9 cells had GLUT3 which was previously not reported. Interestingly, both GLUT3 mRNA and protein were detected in Clone 9 cells, but only mRNA for GLUT1 was detected. Glucose transport in Clone 9 cells was insulin-sensitive in a concentration-dependent manner, concomitant with the presence of GLUT3 in the plasma membrane after insulin treatment. Although basal glucose uptake was unaffected, insulin-stimulated glucose uptake was abolished with siRNA-mediated GLUT3 knockdown. These results contradict previous reports that Clone 9 cells exclusively express GLUT1 and suggest GLUT3 is a key insulin-sensitive glucose transporter required for insulin-stimulated glucose uptake by Clone 9 cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. 9Vδ2 T cell activation by strongly agonistic nucleotidic phosphoantigens.

    Science.gov (United States)

    Moulin, Morgane; Alguacil, Javier; Gu, Siyi; Mehtougui, Asmaa; Adams, Erin J; Peyrottes, Suzanne; Champagne, Eric

    2017-12-01

    Human Vγ9Vδ2 T cells can sense through their TCR tumor cells producing the weak endogenous phosphorylated antigen isopentenyl pyrophosphate (IPP), or bacterially infected cells producing the strong agonist hydroxyl dimethylallyl pyrophosphate (HDMAPP). The recognition of the phosphoantigen is dependent on its binding to the intracellular B30.2 domain of butyrophilin BTN3A1. Most studies have focused on pyrophosphate phosphoantigens. As triphosphate nucleotide derivatives are naturally co-produced with IPP and HDMAPP, we analyzed their specific properties using synthetic nucleotides derived from HDMAPP. The adenylated, thymidylated and uridylated triphosphate derivatives were found to activate directly Vγ9Vδ2 cell lines as efficiently as HDMAPP in the absence of accessory cells. These antigens were inherently resistant to terminal phosphatases, but apyrase, when added during a direct stimulation of Vγ9Vδ2 cells, abrogated their stimulating activity, indicating that their activity required transformation into strong pyrophosphate agonists by a nucleotide pyrophosphatase activity which is present in serum. Tumor cells can be sensitized with nucleotide phosphoantigens in the presence of apyrase to become stimulatory, showing that this can occur before their hydrolysis into pyrophosphates. Whereas tumors sensitized with HDMAPP rapidly lost their stimulatory activity, sensitization with nucleotide derivatives, in particular with the thymidine derivative, induced long-lasting stimulating ability. Using isothermal titration calorimetry, binding of some nucleotide derivatives to BTN3A1 intracellular domain was found to occur with an affinity similar to that of IPP, but much lower than that of HDMAPP. Thus, nucleotide phosphoantigens are precursors of pyrophosphate antigens which can deliver strong agonists intracellularly resulting in prolonged and strengthened activity.

  15. Bone morphogenetic protein 9 as a key regulator of liver progenitor cells in DDC-induced cholestatic liver injury.

    Science.gov (United States)

    Addante, Annalisa; Roncero, Cesáreo; Almalé, Laura; Lazcanoiturburu, Nerea; García-Álvaro, María; Fernández, Margarita; Sanz, Julián; Hammad, Seddik; Nwosu, Zeribe C; Lee, Se-Jin; Fabregat, Isabel; Dooley, Steven; Ten Dijke, Peter; Herrera, Blanca; Sánchez, Aránzazu

    2018-05-11

    Bone morphogenetic protein 9 (BMP9) interferes with liver regeneration upon acute injury, while promoting fibrosis upon carbon tetrachloride-induced chronic injury. We have now addressed the role of BMP9 in 3,5 diethoxicarbonyl-1,4 dihydrocollidine (DDC)-induced cholestatic liver injury, a model of liver regeneration mediated by hepatic progenitor cell (known as oval cell), exemplified as ductular reaction and oval cell expansion. WT and BMP9KO mice were submitted to DDC diet. Livers were examined for liver injury, fibrosis, inflammation and oval cell expansion by serum biochemistry, histology, RT-qPCR and western blot. BMP9 signalling and effects in oval cells were studied in vitro using western blot and transcriptional assays, plus functional assays of DNA synthesis, cell viability and apoptosis. Crosslinking assays and short hairpin RNA approaches were used to identify the receptors mediating BMP9 effects. Deletion of BMP9 reduces liver damage and fibrosis, but enhances inflammation upon DDC feeding. Molecularly, absence of BMP9 results in overactivation of PI3K/AKT, ERK-MAPKs and c-Met signalling pathways, which together with an enhanced ductular reaction and oval cell expansion evidence an improved regenerative response and decreased damage in response to DDC feeding. Importantly, BMP9 directly targets oval cells, it activates SMAD1,5,8, decreases cell growth and promotes apoptosis, effects that are mediated by Activin Receptor-Like Kinase 2 (ALK2) type I receptor. We identify BMP9 as a negative regulator of oval cell expansion in cholestatic injury, its deletion enhancing liver regeneration. Likewise, our work further supports BMP9 as an attractive therapeutic target for chronic liver diseases. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Effects of matrix metalloproteinase inhibitor doxycycline and CD147 antagonist peptide-9 on gallbladder carcinoma cell lines.

    Science.gov (United States)

    Wang, Shihang; Liu, Chao; Liu, Xinjiang; He, Yanxin; Shen, Dongfang; Luo, Qiankun; Dong, Yuxi; Dong, Haifeng; Pang, Zhigang

    2017-10-01

    Gallbladder carcinoma is the most common and aggressive malignancy of the biliary tree and highly expresses CD147, which is closely related to disease prognosis in a variety of human cancers. Doxycycline exhibited anti-tumor properties in many cancer cells. CD147 antagonist peptide-9 is a polypeptide and can specifically bind to CD147. The effect of these two drugs on gallbladder cancer cells has not been studied. The aim of this study is to investigate the effect of doxycycline and antagonist peptide-9 on gallbladder carcinoma cells and the possible mechanism of inhibition on cancer cell of doxycycline. To investigate the effects of doxycycline and antagonist peptide-9 on gallbladder carcinoma cells (GBC-SD and SGC-996), cell proliferation, CD147 expression, and early-stage apoptosis rate were measured after treated with doxycycline. Matrix metalloproteinase-2 and matrix metalloproteinase-9 activities were measured after treated with different concentrations of doxycycline, antagonist peptide-9, and their combination. The results demonstrated that doxycycline inhibited cell proliferation, reduced CD147 expression level, and induced an early-stage apoptosis response in GBC-SD and SGC-996 cells. The matrix metalloproteinase-2 and matrix metalloproteinase-9 activities were inhibited by antagonist peptide-9 and doxycycline, and the inhibitory effects were enhanced by combined drugs in gallbladder carcinoma cell lines. Taken together, doxycycline showed inhibitory effects on gallbladder carcinoma cell lines and reduced the expression of CD147, and this may be the mechanism by which doxycycline inhibits cancer cells. This study provides new information and tries to implement the design of adjuvant therapy method for gallbladder carcinoma.

  17. Microfluidic system for monitoring of cardiac (H9C2) cell proliferation

    Science.gov (United States)

    Kobuszewska, A.; Cwik, P.; Jastrzebska, E.; Brzozka, Z.; Chudy, M.; Renaud, P.; Dybko, A.

    2017-05-01

    The paper presents the application of electrical impedance spectroscopy (EIS) analysis for investigation of cardiac cell (H9C2 - rat cardiomyoblast) proliferation after verapamil hydrochloride exposure. For this purpose, two different PDMS/glass microsystems with circular microchamber and longitudinal microchannel integrated with Pt/Al electrodes were used. The microchambers were fabricated in PDMS using photolithography and replica moulding techniques. Pt/Al electrodes were fabricated on a 4-inch glass substrate using Physical Vapor Deposition (PVD). Solution of verapamil hydrochloride was continuously introduced into the microsystems with H9C2 cell culture (a flow rate of 1 μl/min) for 72 h. The impedance spectra were recorded from 100 Hz to 1 MHz. We confirmed that impedance spectroscopy can be used for non-invasive, label-free and real-time analysis of cardiac cells proliferation based on cells dielectric properties and biological structure.

  18. Inverse relationship of tumors and mononuclear cell leukemia infiltration in the lungs of F344 rats

    Energy Technology Data Exchange (ETDEWEB)

    Lundgren, D.L.; Griffith, W.C.; Hahn, F.F.

    1995-12-01

    In 1970 and F344 rat, along with the B6C3F{sub 1} mouse, were selected as the standard rodents for the National Cancer Institute Carcinogenic Bioassay program for studies of potentially carcinogenic chemicals. The F344 rat has also been used in a variety of other carcinogenesis studies, including numerous studies at ITRI. A major concern to be considered in evaluating carcinogenic bioassay studies using the F344 rat is the relatively high background incidence of mononuclear cell leukemia (MCL) (also referred to as large granular lymphocytic leukemia, Fischer rat leukemia, or monocytic leukemia). Incidences of MCL ranging from 10 to 72% in male F344 rats to 6 to 31% in female F344 rats have been reported. Gaining the understanding of the mechanisms involved in the negative correlations noted should enhance our understanding of the mechanisms involved in the development of lung cancer.

  19. 8,9-Epoxyeicosatrienoic acid analog protects pulmonary artery smooth muscle cells from apoptosis via ROCK pathway

    International Nuclear Information System (INIS)

    Ma, Jun; Zhang, Lei; Li, Shanshan; Liu, Shulin; Ma, Cui; Li, Weiyang; Falck, J.R.; Manthati, Vijay L.; Reddy, D. Sudarshan; Medhora, Meetha; Jacobs, Elizabeth R.; Zhu, Daling

    2010-01-01

    Epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acid (AA) catalyzed by cytochrome P450 (CYP), have many essential biologic roles in the cardiovascular system including inhibition of apoptosis in cardiomyocytes. In the present study, we tested the potential of 8,9-EET and derivatives to protect pulmonary artery smooth muscle cells (PASMCs) from starvation induced apoptosis. We found 8,9-epoxy-eicos-11(Z)-enoic acid (8,9-EET analog (214)), but not 8,9-EET, increased cell viability, decreased activation of caspase-3 and caspase-9, and decreased TUNEL-positive cells or nuclear condensation induced by serum deprivation (SD) in PASMCs. These effects were reversed after blocking the Rho-kinase (ROCK) pathway with Y-27632 or HA-1077. Therefore, 8,9-EET analog (214) protects PASMC from serum deprivation-induced apoptosis, mediated at least in part via the ROCK pathway. Serum deprivation of PASMCs resulted in mitochondrial membrane depolarization, decreased expression of Bcl-2 and enhanced expression of Bax, all effects were reversed by 8,9-EET analog (214) in a ROCK dependent manner. Because 8,9-EET and not the 8,9-EET analog (214) protects pulmonary artery endothelial cells (PAECs), these observations suggest the potential to differentially promote apoptosis or survival with 8,9-EET or analogs in pulmonary arteries.

  20. 8,9-Epoxyeicosatrienoic acid analog protects pulmonary artery smooth muscle cells from apoptosis via ROCK pathway

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Jun; Zhang, Lei; Li, Shanshan [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin, Heilongjiang 150081 (China); Liu, Shulin [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin, Heilongjiang 150081 (China); Bio-pharmaceutical Key Laboratory of Heilongjiang Province, Harbin 150081 (China); Ma, Cui [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin, Heilongjiang 150081 (China); Li, Weiyang [Mudanjiang Medical College, Mudanjiang 157011 (China); Falck, J.R.; Manthati, Vijay L.; Reddy, D. Sudarshan [University of Texas Southwestern Medical Center, Dallas, TX 75390 (United States); Medhora, Meetha; Jacobs, Elizabeth R. [Division of Pulmonary and Critical Care, Department of Medicine, Cardiovascular Center, Medical College of Wisconsin, Milwaukee, WI 53226 (United States); Zhu, Daling, E-mail: dalingz@yahoo.com [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin, Heilongjiang 150081 (China); Bio-pharmaceutical Key Laboratory of Heilongjiang Province, Harbin 150081 (China)

    2010-08-15

    Epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acid (AA) catalyzed by cytochrome P450 (CYP), have many essential biologic roles in the cardiovascular system including inhibition of apoptosis in cardiomyocytes. In the present study, we tested the potential of 8,9-EET and derivatives to protect pulmonary artery smooth muscle cells (PASMCs) from starvation induced apoptosis. We found 8,9-epoxy-eicos-11(Z)-enoic acid (8,9-EET analog (214)), but not 8,9-EET, increased cell viability, decreased activation of caspase-3 and caspase-9, and decreased TUNEL-positive cells or nuclear condensation induced by serum deprivation (SD) in PASMCs. These effects were reversed after blocking the Rho-kinase (ROCK) pathway with Y-27632 or HA-1077. Therefore, 8,9-EET analog (214) protects PASMC from serum deprivation-induced apoptosis, mediated at least in part via the ROCK pathway. Serum deprivation of PASMCs resulted in mitochondrial membrane depolarization, decreased expression of Bcl-2 and enhanced expression of Bax, all effects were reversed by 8,9-EET analog (214) in a ROCK dependent manner. Because 8,9-EET and not the 8,9-EET analog (214) protects pulmonary artery endothelial cells (PAECs), these observations suggest the potential to differentially promote apoptosis or survival with 8,9-EET or analogs in pulmonary arteries.