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  1. Identification of GBF1 as a Cellular Factor Required for Hepatitis C Virus RNA Replication▿

    PubMed Central

    Goueslain, Lucie; Alsaleh, Khaled; Horellou, Pauline; Roingeard, Philippe; Descamps, Véronique; Duverlie, Gilles; Ciczora, Yann; Wychowski, Czeslaw; Dubuisson, Jean; Rouillé, Yves

    2010-01-01

    In infected cells, hepatitis C virus (HCV) induces the formation of membrane alterations referred to as membranous webs, which are sites of RNA replication. In addition, HCV RNA replication also occurs in smaller membrane structures that are associated with the endoplasmic reticulum. However, cellular mechanisms involved in the formation of HCV replication complexes remain largely unknown. Here, we used brefeldin A (BFA) to investigate cellular mechanisms involved in HCV infection. BFA acts on cell membranes by interfering with the activation of several members of the family of ADP-ribosylation factors (ARF), which can lead to a wide range of inhibitory actions on membrane-associated mechanisms of the secretory and endocytic pathways. Our data show that HCV RNA replication is highly sensitive to BFA. Individual knockdown of the cellular targets of BFA using RNA interference and the use of a specific pharmacological inhibitor identified GBF1, a guanine nucleotide exchange factor for small GTPases of the ARF family, as a host factor critically involved in HCV replication. Furthermore, overexpression of a BFA-resistant GBF1 mutant rescued HCV replication in BFA-treated cells, indicating that GBF1 is the BFA-sensitive factor required for HCV replication. Finally, immunofluorescence and electron microscopy analyses indicated that BFA does not block the formation of membranous web-like structures induced by expression of HCV proteins in a nonreplicative context, suggesting that GBF1 is probably involved not in the formation of HCV replication complexes but, rather, in their activity. Altogether, our results highlight a functional connection between the early secretory pathway and HCV RNA replication. PMID:19906930

  2. Cellular factors required for multiple stages of SV40 DNA replication in vitro.

    PubMed Central

    Fairman, M P; Stillman, B

    1988-01-01

    Plasmids containing the SV40 origin replicate in the presence of SV40 T antigen and a cell free extract derived from human 293 cells. Upon fractionation of this extract, two essential replication factors have been identified. One of these is a multi-subunit DNA binding protein containing polypeptides of 70,000, 34,000 and 11,000 daltons which may function as a eukaryotic single strand DNA binding protein (SSB). The other partially purified fraction is required with T antigen for the first stage of DNA replication, the formation of a pre-synthesis complex at the replication origin. These results, and others, define multiple stages of SV40 DNA replication in vitro which are analogous to multiple stages of Escherichia coli and phage lambda replication, and may reflect similar events in the replication of cellular chromosomes. Images PMID:2841119

  3. Identification of cellular factors required for the budding of koala retrovirus.

    PubMed

    Shimode, Sayumi; Nakaoka, Rie; Hoshino, Shigeki; Abe, Masumi; Shogen, Hiroko; Yasuda, Jiro; Miyazawa, Takayuki

    2013-07-01

    Koala retrovirus (KoRV) is a unique gammaretrovirus that is currently endogenizing into its host and considered to be associated with leukemia, lymphoma and immunosuppression in koalas (Phascolactos cinereus). In this study, it was demonstrated that WWP2 or WWP2-like E3 ubiquitin ligases possessing the WW domain closely related to WWP2 and Vps4A/B are involved in KoRV budding. These data suggest that KoRV Gag recruits the cellular endosomal sorting complex required for transport machinery through interaction of the PPPY L-domain with the WW domain(s) of WWP2 and that progeny virions are released from cells by utilizing the multivesicular body sorting pathway.

  4. The cellular transcription factor SP1 and an unknown cellular protein are required to mediate Rep protein activation of the adeno-associated virus p19 promoter.

    PubMed Central

    Pereira, D J; Muzyczka, N

    1997-01-01

    Control of adeno-associated virus (AAV) transcription from the three AAV promoters (p5, p19, and p40) requires the adenovirus E1a protein and the AAV nonstructural (Rep) proteins. The Rep proteins have been shown to repress the AAV p5 promoter yet facilitate activation of the p19 and p40 promoters during a productive infection. To elucidate the mechanism of promoter regulation by the AAV Rep proteins, the cellular factors involved in mediating Rep activation of the p19 promoter were characterized. A series of protein-DNA binding experiments using extracts derived from uninfected HeLa cells was performed to identify cellular factors that bind to the p19 promoter. Electrophoretic mobility shift assays, DNase I protection analyses, and UV cross-linking experiments demonstrated specific interactions with the cellular factor SP1 (or an SP1-like protein) at positions -50 and -130 relative to the start of p19 transcription. Additionally, an unknown cellular protein (cellular AAV activating protein [cAAP]) with an approximate molecular mass of 34 kDa was found to interact with a CArG-like element at position -140. Mutational analysis of the p19 promoter suggested that the SP1 site at -50 and the cAAP site at -140 were necessary to mediate Rep activation of p19. Antibody precipitation experiments demonstrated that Rep-SP1 protein complexes can exist in vivo. Although Rep was demonstrated to interact with p19 DNA directly, the affinity of Rep binding was much lower than that seen for the Rep binding elements within the terminal repeat and the p5 promoter. Furthermore, the interaction of purified Rep68 with the p19 promoter in vitro was negligible unless purified SP1 was also added to the reaction. Thus, the ability of Rep to transactivate the p19 promoter is likely to involve SP1-Rep protein contacts that facilitate Rep interaction with p19 DNA. PMID:9032303

  5. Chromatin assembly factor CAF-1 is required for cellular differentiation during plant development.

    PubMed

    Exner, Vivien; Taranto, Patti; Schönrock, Nicole; Gruissem, Wilhelm; Hennig, Lars

    2006-11-01

    Chromatin assembly factor CAF-1 facilitates the formation of nucleosomes on newly replicated DNA in vitro. However, the role of CAF-1 in development is poorly understood because mutants are not available in most multicellular model organisms. Biochemical evidence suggests that FASCIATA1, FASCIATA2 and MSI1 form CAF-1 in Arabidopsis thaliana. Because fasciata mutants are viable, CAF-1 is not essential for cell division in plants. Arabidopsis CAF-1 mutants have defects in shoot apical meristems; in addition, CAF-1 is required to establish seedling architecture, leaf size and trichome differentiation. CAF-1 is needed to restrict branching of trichomes on rosette leaves. Increased trichome branching in CAF-1 mutants is not strictly correlated with increased nuclear DNA content. In addition, fas2 glabra3 double mutants show an additive genetic interaction, demonstrating that CAF-1 acts genetically parallel to the GLABRA3-containing, endoreduplication-coupled trichome branching pathway. However, CAF-1 is often needed to restrict endoreduplication, because seedlings of most CAF-1 mutants have increased ploidy. Notably, in the Landsberg erecta background, loss of CAF-1 does not affect ploidy, demonstrating that loss of CAF-1 can be compensated in some Arabidopsis accessions. These results reveal that the functions of FAS1, FAS2 and MSI1 are not restricted to meristems, but are also needed to control genome replication at multiple steps of development.

  6. Platelet-derived growth factor-dependent cellular transformation requires either phospholipase Cgamma or phosphatidylinositol 3 kinase.

    PubMed

    DeMali, K A; Whiteford, C C; Ulug, E T; Kazlauskas, A

    1997-04-04

    Although it has been well established that constitutive activation of receptor tyrosine kinases leads to cellular transformation, the signal relay pathways involved have not been systematically investigated. In this study we used a panel of platelet-derived growth factor (PDGF) beta receptor mutants (beta-PDGFR), which selectively activate various signal relay enzymes to define which signaling pathways are required for PDGF-dependent growth of cells in soft agar. The host cell line for these studies was Ph cells, a 3T3-like cell that expresses normal levels of the beta-PDGFR but no PDGF-alpha receptor (alpha-PDGFR). Hence, this cell system can be used to study signaling of mutant alphaPDGFRs or alpha/beta chimeras. We constructed chimeric receptors containing the alphaPDGFR extracellular domain and the betaPDGFR cytoplasmic domain harboring various phosphorylation site mutations. The mutants were expressed in Ph cells, and their ability to drive PDGF-dependent cellular transformation (growth in soft agar) was assayed. Cells infected with an empty expression vector failed to grow in soft agar, whereas introduction of the chimera with a wild-type beta-PDGFR cytoplasmic domain gave rise to a large number of colonies. In contrast, the N2F5 chimera, in which the binding sites for phospholipase Cgamma (PLC-gamma), RasGTPase-activating protein, phosphatidylinositol 3 kinase (PI3K), and SHP-2 were eliminated, failed to trigger proliferation. Restoring the binding sites for RasGTPase-activating protein or SHP-2 did not rescue the PDGF-dependent response. In contrast, receptors capable of associating with either PLC-gamma or PI3K relayed a growth signal that was comparable to wild-type receptors in the soft agar growth assay. These findings indicate that the PDGF receptor activates multiple signaling pathways that lead to cellular transformation, and that either PI3K or PLC-gamma are key initiators of such signal relay cascades.

  7. Identification and molecular characterization of cellular factors required for glucocorticoid receptor-mediated mRNA decay

    PubMed Central

    Park, Ok Hyun; Park, Joori; Yu, Mira; An, Hyoung-Tae; Ko, Jesang; Kim, Yoon Ki

    2016-01-01

    Glucocorticoid (GC) receptor (GR) has been shown recently to bind a subset of mRNAs and elicit rapid mRNA degradation. However, the molecular details of GR-mediated mRNA decay (GMD) remain unclear. Here, we demonstrate that GMD triggers rapid degradation of target mRNAs in a translation-independent and exon junction complex-independent manner, confirming that GMD is mechanistically distinct from nonsense-mediated mRNA decay (NMD). Efficient GMD requires PNRC2 (proline-rich nuclear receptor coregulatory protein 2) binding, helicase ability, and ATM-mediated phosphorylation of UPF1 (upstream frameshift 1). We also identify two GMD-specific factors: an RNA-binding protein, YBX1 (Y-box-binding protein 1), and an endoribonuclease, HRSP12 (heat-responsive protein 12). In particular, using HRSP12 variants, which are known to disrupt trimerization of HRSP12, we show that HRSP12 plays an essential role in the formation of a functionally active GMD complex. Moreover, we determine the hierarchical recruitment of GMD factors to target mRNAs. Finally, our genome-wide analysis shows that GMD targets a variety of transcripts, implicating roles in a wide range of cellular processes, including immune responses. PMID:27798850

  8. The cellular response to vascular endothelial growth factors requires co-ordinated signal transduction, trafficking and proteolysis.

    PubMed

    Smith, Gina A; Fearnley, Gareth W; Tomlinson, Darren C; Harrison, Michael A; Ponnambalam, Sreenivasan

    2015-08-18

    VEGFs (vascular endothelial growth factors) are a family of conserved disulfide-linked soluble secretory glycoproteins found in higher eukaryotes. VEGFs mediate a wide range of responses in different tissues including metabolic homoeostasis, cell proliferation, migration and tubulogenesis. Such responses are initiated by VEGF binding to soluble and membrane-bound VEGFRs (VEGF receptor tyrosine kinases) and co-receptors. VEGF and receptor splice isoform diversity further enhances complexity of membrane protein assembly and function in signal transduction pathways that control multiple cellular responses. Different signal transduction pathways are simultaneously activated by VEGFR-VEGF complexes with membrane trafficking along the endosome-lysosome network further modulating signal output from multiple enzymatic events associated with such pathways. Balancing VEGFR-VEGF signal transduction with trafficking and proteolysis is essential in controlling the intensity and duration of different intracellular signalling events. Dysfunction in VEGF-regulated signal transduction is important in chronic disease states including cancer, atherosclerosis and blindness. This family of growth factors and receptors is an important model system for understanding human disease pathology and developing new therapeutics for treating such ailments. © 2015 Authors.

  9. The cellular response to vascular endothelial growth factors requires co-ordinated signal transduction, trafficking and proteolysis

    PubMed Central

    Smith, Gina A.; Fearnley, Gareth W.; Tomlinson, Darren C.; Harrison, Michael A.; Ponnambalam, Sreenivasan

    2015-01-01

    VEGFs (vascular endothelial growth factors) are a family of conserved disulfide-linked soluble secretory glycoproteins found in higher eukaryotes. VEGFs mediate a wide range of responses in different tissues including metabolic homoeostasis, cell proliferation, migration and tubulogenesis. Such responses are initiated by VEGF binding to soluble and membrane-bound VEGFRs (VEGF receptor tyrosine kinases) and co-receptors. VEGF and receptor splice isoform diversity further enhances complexity of membrane protein assembly and function in signal transduction pathways that control multiple cellular responses. Different signal transduction pathways are simultaneously activated by VEGFR–VEGF complexes with membrane trafficking along the endosome–lysosome network further modulating signal output from multiple enzymatic events associated with such pathways. Balancing VEGFR–VEGF signal transduction with trafficking and proteolysis is essential in controlling the intensity and duration of different intracellular signalling events. Dysfunction in VEGF-regulated signal transduction is important in chronic disease states including cancer, atherosclerosis and blindness. This family of growth factors and receptors is an important model system for understanding human disease pathology and developing new therapeutics for treating such ailments. PMID:26285805

  10. Cellular Nuclear Export Factors TAP and Aly Are Required for HDAg-L-mediated Assembly of Hepatitis Delta Virus.

    PubMed

    Huang, Hsiu-Chen; Lee, Chung-Pei; Liu, Hui-Kang; Chang, Ming-Fu; Lai, Yu-Heng; Lee, Yu-Ching; Huang, Cheng

    2016-12-09

    Hepatitis delta virus (HDV) is a satellite virus of hepatitis B virus (HBV). HDV genome encodes two forms of hepatitis delta antigen (HDAg), small HDAg (HDAg-S), which is required for viral replication, and large HDAg (HDAg-L), which is essential for viral assembly. HDAg-L is identical to HDAg-S except that it bears a 19-amino acid extension at the C terminus. Both HDAgs contain a nuclear localization signal (NLS), but only HDAg-L contains a CRM1-independent nuclear export signal at its C terminus. The nuclear export activity of HDAg-L is important for HDV particle formation. However, the mechanisms of HDAg-L-mediated nuclear export of HDV ribonucleoprotein are not clear. In this study, the host cellular RNA export complex TAP-Aly was found to form a complex with HDAg-L, but not with an export-defective HDAg-L mutant, in which Pro(205) was replaced by Ala. HDAg-L was found to colocalize with TAP and Aly in the nucleus. The C-terminal domain of HDAg-L was shown to directly interact with the N terminus of TAP, whereas an HDAg-L mutant lacking the NLS failed to interact with full-length TAP. In addition, small hairpin RNA-mediated down-regulation of TAP or Aly reduced nuclear export of HDAg-L and assembly of HDV virions. Furthermore, a peptide, TAT-HDAg-L(198-210), containing the 10-amino acid TAT peptide and HDAg-L(198-210), inhibited the interaction between HDAg-L and TAP and blocked HDV virion assembly and secretion. These data demonstrate that formation and release of HDV particles are mediated by TAP and Aly.

  11. SWI/SNF factors required for cellular resistance to DNA damage include ARID1A and ARID1B and show interdependent protein stability.

    PubMed

    Watanabe, Reiko; Ui, Ayako; Kanno, Shin-Ichiro; Ogiwara, Hideaki; Nagase, Takahiro; Kohno, Takashi; Yasui, Akira

    2014-05-01

    The SWI/SNF chromatin-remodeling family contains various protein complexes, which regulate gene expression during cellular development and influence DNA damage response in an ATP- and complex-dependent manner, of which details remain elusive. Recent human genome sequencing of various cancer cells revealed frequent mutations in SWI/SNF factors, especially ARID1A, a variant subunit in the BRG1-associated factor (BAF) complex of the SWI/SNF family. We combined live-cell analysis and gene-suppression experiments to show that suppression of either ARID1A or its paralog ARID1B led to reduced nonhomologous end joining activity of DNA double-strand breaks (DSB), decreased accumulation of KU70/KU80 proteins at DSB, and sensitivity to ionizing radiation, as well as to cisplatin and UV. Thus, in contrast to transcriptional regulation, both ARID1 proteins are required for cellular resistance to various types of DNA damage, including DSB. The suppression of other SWI/SNF factors, namely SNF5, BAF60a, BAF60c, BAF155, or BAF170, exhibits a similar phenotype. Of these factors, ARID1A, ARID1B, SNF5, and BAF60c are necessary for the immediate recruitment of the ATPase subunit of the SWI/SNF complex to DSB, arguing that both ARID1 proteins facilitate the damage response of the complex. Finally, we found interdependent protein stability among the SWI/SNF factors, suggesting their direct interaction within the complex and the reason why multiple factors are frequently lost in parallel in cancer cells. Taken together, we show that cancer cells lacking in the expression of certain SWI/SNF factors, including ARID1A, are deficient in DNA repair and potentially vulnerable to DNA damage. ©2014 AACR.

  12. Epidermal growth factor-induced cellular invasion requires sphingosine-1-phosphate/sphingosine-1-phosphate 2 receptor-mediated ezrin activation

    PubMed Central

    Orr Gandy, K. Alexa; Adada, Mohamad; Canals, Daniel; Carroll, Brittany; Roddy, Patrick; Hannun, Yusuf A.; Obeid, Lina M.

    2013-01-01

    Ezrin, radixin, and moesin (ERM) proteins link cortical actin to the plasma membrane and coordinate cellular events that require cytoskeletal rearrangement, including cell division, migration, and invasion. While ERM proteins are involved in many important cellular events, the mechanisms regulating their function are not completely understood. Our laboratory previously identified reciprocal roles for the sphingolipids ceramide and sphingosine-1-phosphate (S1P) in the regulation of ERM proteins. We recently showed that ceramide-induced activation of PP1α leads to dephosphorylation and inactivation of ERM proteins, while S1P results in phosphorylation and activation of ERM proteins. Following these findings, we aimed to examine known inducers of the SK/S1P pathway and evaluate their ability to regulate ERM proteins. We examined EGF, a known inducer of the SK/S1P pathway, for its ability to regulate the ERM family of proteins. We found that EGF induces ERM c-terminal threonine phosphorylation via activation of the SK/S1P pathway, as this was prevented by siRNA knockdown or pharmacological inhibition of SK. Using pharmacological, as well as genetic, knockdown approaches, we determined that EGF induces ERM phosphorylation via activation of S1PR2. In addition, EGF led to cell polarization in the form of lamellipodia, and this occurred through a mechanism involving S1PR2-mediated phosphorylation of ezrin T567. EGF-induced cellular invasion was also found to be dependent on S1PR2-induced T567 ezrin phosphorylation, such that S1PR2 antagonist, JTE-013, and expression of a dominant-negative ezrin mutant prevented cellular invasion toward EGF. In this work, a novel mechanism of EGF-stimulated invasion is unveiled, whereby S1P-mediated activation of S1PR2 and phosphorylation of ezrin T567 is required.—Orr Gandy, K. A., Adada, M., Canals, D., Carroll, B., Roddy, P., Hannun, Y. A., Obeid, L. M. Epidermal growth factor-induced cellular invasion requires sphingosine-1-phosphate

  13. Attenuation of the suppressive activity of cellular splicing factor SRSF3 by Kaposi sarcoma–associated herpesvirus ORF57 protein is required for RNA splicing

    PubMed Central

    Majerciak, Vladimir; Lu, Mathew; Li, Xiaofan

    2014-01-01

    Kaposi sarcoma–associated herpesvirus (KSHV) ORF57 is a multifunctional post-transcriptional regulator essential for viral gene expression during KSHV lytic infection. ORF57 requires interactions with various cellular proteins for its function. Here, we identified serine/arginine-rich splicing factor 3 (SRSF3, formerly known as SRp20) as a cellular cofactor involved in ORF57-mediated splicing of KSHV K8β RNA. In the absence of ORF57, SRSF3 binds to a suboptimal K8β intron and inhibits K8β splicing. Knockdown of SRSF3 promotes K8β splicing, mimicking the effect of ORF57. The N-terminal half of ORF57 binds to the RNA recognition motif of SRSF3, which prevents SRSF3 from associating with the K8β intron RNA and therefore attenuates the suppressive effect of SRSF3 on K8β splicing. ORF57 also promotes splicing of heterologous non-KSHV transcripts that are negatively regulated by SRSF3, indicating that the effect of ORF57 on SRSF3 activity is independent of RNA target. SPEN proteins, previously identified as ORF57-interacting partners, suppress ORF57 splicing activity by displacing ORF57 from SRSF3–RNA complexes. In summary, we have identified modulation of SRSF3 activity as the molecular mechanism by which ORF57 promotes RNA splicing. PMID:25234929

  14. Attenuation of the suppressive activity of cellular splicing factor SRSF3 by Kaposi sarcoma-associated herpesvirus ORF57 protein is required for RNA splicing.

    PubMed

    Majerciak, Vladimir; Lu, Mathew; Li, Xiaofan; Zheng, Zhi-Ming

    2014-11-01

    Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 is a multifunctional post-transcriptional regulator essential for viral gene expression during KSHV lytic infection. ORF57 requires interactions with various cellular proteins for its function. Here, we identified serine/arginine-rich splicing factor 3 (SRSF3, formerly known as SRp20) as a cellular cofactor involved in ORF57-mediated splicing of KSHV K8β RNA. In the absence of ORF57, SRSF3 binds to a suboptimal K8β intron and inhibits K8β splicing. Knockdown of SRSF3 promotes K8β splicing, mimicking the effect of ORF57. The N-terminal half of ORF57 binds to the RNA recognition motif of SRSF3, which prevents SRSF3 from associating with the K8β intron RNA and therefore attenuates the suppressive effect of SRSF3 on K8β splicing. ORF57 also promotes splicing of heterologous non-KSHV transcripts that are negatively regulated by SRSF3, indicating that the effect of ORF57 on SRSF3 activity is independent of RNA target. SPEN proteins, previously identified as ORF57-interacting partners, suppress ORF57 splicing activity by displacing ORF57 from SRSF3-RNA complexes. In summary, we have identified modulation of SRSF3 activity as the molecular mechanism by which ORF57 promotes RNA splicing.

  15. Cellular Requirements for Building a Retinal Neuropil

    PubMed Central

    Randlett, Owen; MacDonald, Ryan B.; Yoshimatsu, Takeshi; Almeida, Alexandra D.; Suzuki, Sachihiro C.; Wong, Rachel O.; Harris, William A.

    2013-01-01

    Summary How synaptic neuropil is formed within the CNS is poorly understood. The retinal inner plexiform layer (IPL) is positioned between the cell bodies of amacrine cells (ACs) and retinal ganglion cells (RGCs). It consists of bipolar cell (BC) axon terminals that synapse on the dendrites of ACs and RGCs intermingled with projections from Müller glia (MG). We examined whether any of these cellular processes are specifically required for the formation of the IPL. Using genetic and pharmacological strategies, we eliminated RGCs, ACs, and MG individually or in combination. Even in the absence of all of these partner cells, an IPL-like neuropil consisting of only BC axon terminals still forms, complete with presynaptic specializations and sublaminar organization. Previous studies have shown that an IPL can form in the complete absence of BCs; therefore, we conclude that neither presynaptic nor postsynaptic processes are individually essential for the formation of this synaptic neuropil. PMID:23416047

  16. The simian retrovirus-1 constitutive transport element, unlike the HIV-1 RRE, uses factors required for cellular mRNA export.

    PubMed

    Saavedra, C; Felber, B; Izaurralde, E

    1997-09-01

    A hallmark of retroviral gene expression is that unspliced retroviral genomic RNA is exported to the cytoplasm, whereas endogenous intron-containing cellular RNAs are usually retained in the nucleus. In complex retroviruses, such as human immunodeficiency virus-1 (HIV-1), nuclear export is accomplished by the interaction of a virally encoded protein, Rev, with a cis-acting RNA element, the Rev-responsive element (RRE). In type D retroviruses, such as the simian retrovirus type 1 (SRV-1), however, genomic RNA is exported by cellular factor(s) that interact with a conserved cis-acting RNA element, the constitutive transport element (CTE). We found that the CTE was exported in a specific and saturable fashion from Xenopus oocyte nuclei. When inserted into the intron of an adenovirus-derived pre-mRNA, the CTE did not affect splicing efficiency but promoted the nuclear export of the excised intron lariat that is normally retained within the nucleus. Export of CTE-containing RNAs to the cytoplasm was not affected by the heterogeneous nuclear ribonucleoprotein A1 or an excess of peptides corresponding to the Rev nuclear export signal. Microinjection of saturating amounts of CTE RNA did not affect tRNA export or Rev-mediated export but did inhibit mRNA export. CTE-mediated export was found to be dependent on Ran-mediated GTP hydrolysis. The Rev-RRE system and the CTE direct intron-containing RNAs to distinct export pathways. Although previous data have suggested that Rev uses the same export pathway as uracil-rich small nuclear RNAs and 5S ribosomal RNA, the CTE seems to interact with evolutionarily conserved factors that are essential for cellular mRNA export.

  17. 47 CFR 22.901 - Cellular service requirements and limitations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... SERVICES PUBLIC MOBILE SERVICES Cellular Radiotelephone Service § 22.901 Cellular service requirements and... operates in compliance with this section. (a) Each cellular system must provide either mobile service, fixed service, or a combination of mobile and fixed service, subject to the requirements,...

  18. Cellular Restriction Factors of Feline Immunodeficiency Virus

    PubMed Central

    Zielonka, Jörg; Münk, Carsten

    2011-01-01

    Lentiviruses are known for their narrow cell- and species-tropisms, which are determined by cellular proteins whose absence or presence either support viral replication (dependency factors, cofactors) or inhibit viral replication (restriction factors). Similar to Human immunodeficiency virus type 1 (HIV-1), the cat lentivirus Feline immunodeficiency virus (FIV) is sensitive to recently discovered cellular restriction factors from non-host species that are able to stop viruses from replicating. Of particular importance are the cellular proteins APOBEC3, TRIM5α and tetherin/BST-2. In general, lentiviruses counteract or escape their species’ own variant of the restriction factor, but are targeted by the orthologous proteins of distantly related species. Most of the knowledge regarding lentiviral restriction factors has been obtained in the HIV-1 system; however, much less is known about their effects on other lentiviruses. We describe here the molecular mechanisms that explain how FIV maintains its replication in feline cells, but is largely prevented from cross-species infections by cellular restriction factors. PMID:22069525

  19. Cellular requirements for cutaneous sensitivity elicitation.

    PubMed

    Aoki, I

    1985-01-01

    The role of glass-adherent cells in cutaneous sensitivity (CS) elicitation has been analyzed in this study. CS responses have been revealed to be mediated by at least two distinct subsets of genetically restricted T cells: I-restricted 'DTH-like' T cells and K/D-restricted 'CTL-like' T cells. Both T-cell responses require I-A-positive glass-adherent cell populations, which lack T-cell markers, to manifest their activities. The role of the adherent cells is different in the 'DTH-like' responses and the 'CTL-like' responses. The disparities between the present results and previous contentions are discussed in this paper.

  20. Creating cellular diversity through transcription factor competition

    PubMed Central

    Göttgens, Berthold

    2015-01-01

    The development of blood cells has long served as a model system to study the generation of diverse mature cells from multipotent progenitors. The article by Org et al (2015) reveals how transcription factor competition on primed DNA templates may contribute to embryonic blood cell specification during the early stages of mesoderm development. The study not only provides new insights into the functionality of the key haematopoietic transcription factor Scl/Tal1, but also provides a potentially widely applicable framework for transcription factor-mediated cell fate specification. PMID:25680687

  1. Human cellular restriction factors that target HIV-1 replication

    PubMed Central

    Strebel, Klaus; Luban, Jeremy; Jeang, Kuan-Teh

    2009-01-01

    Recent findings have highlighted roles played by innate cellular factors in restricting intracellular viral replication. In this review, we discuss in brief the activities of apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G), bone marrow stromal cell antigen 2 (BST-2), cyclophilin A, tripartite motif protein 5 alpha (Trim5α), and cellular microRNAs as examples of host restriction factors that target HIV-1. We point to countermeasures encoded by HIV-1 for moderating the potency of these cellular restriction functions. PMID:19758442

  2. 47 CFR 22.939 - Site availability requirements for applications competing with cellular renewal applications.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... competing with cellular renewal applications. 22.939 Section 22.939 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES PUBLIC MOBILE SERVICES Cellular Radiotelephone Service § 22.939 Site availability requirements for applications competing with cellular renewal applications. In...

  3. [Regulation of uterine cellular proliferation with estrogens and growth factors].

    PubMed

    Alvarez-Rodríguez, C; Baiza-Guzmán, L A

    1996-09-01

    In this paper the role of estrogen and growth factors in the uterine cellular proliferation is analyzed. The evidences indicate that the estradiol-stimulate cell division is associated with the induction of expression of a variety of growth factors from the all major uterine cell types (epithelia, stroma and myometrium). These growth factors amplify the estrogen proliferation signal in autocrine and/or paracrin fashion. The best-studied growth factors in the uterine response to estradiol are epidermal growth factor (EGF) and insulin-like growth factor (IGF-1). Uterine cell proliferation is a complex process that involves interactions of several growth factors, ovarian steroids hormones action and cell to cell signaling.

  4. The major cellular sterol regulatory pathway is required for Andes virus infection.

    PubMed

    Petersen, Josiah; Drake, Mary Jane; Bruce, Emily A; Riblett, Amber M; Didigu, Chukwuka A; Wilen, Craig B; Malani, Nirav; Male, Frances; Lee, Fang-Hua; Bushman, Frederic D; Cherry, Sara; Doms, Robert W; Bates, Paul; Briley, Kenneth

    2014-02-01

    The Bunyaviridae comprise a large family of RNA viruses with worldwide distribution and includes the pathogenic New World hantavirus, Andes virus (ANDV). Host factors needed for hantavirus entry remain largely enigmatic and therapeutics are unavailable. To identify cellular requirements for ANDV infection, we performed two parallel genetic screens. Analysis of a large library of insertionally mutagenized human haploid cells and a siRNA genomic screen converged on components (SREBP-2, SCAP, S1P and S2P) of the sterol regulatory pathway as critically important for infection by ANDV. The significance of this pathway was confirmed using functionally deficient cells, TALEN-mediated gene disruption, RNA interference and pharmacologic inhibition. Disruption of sterol regulatory complex function impaired ANDV internalization without affecting virus binding. Pharmacologic manipulation of cholesterol levels demonstrated that ANDV entry is sensitive to changes in cellular cholesterol and raises the possibility that clinically approved regulators of sterol synthesis may prove useful for combating ANDV infection.

  5. Cellular monitoring systems for the assessment of space environmental factors

    NASA Astrophysics Data System (ADS)

    Hellweg, C. E.; Arenz, A.; Meier, M. M.; Baumstark-Khan, C.

    Harmful environmental factors - namely ionizing radiation - will continue to influence future manned space missions. The Cellular Biodiagnostic group at the German Aerospace Center (DLR) develops cellular monitoring systems, which include bacterial and mammalian cell systems capable of recognizing DNA damage as a consequence of the presence of genotoxic conditions. Such bioassay or biosensor systems will complement the physical detector systems used in space, insofar as they yield intrinsically biologically weighted measures of cellular responses. Furthermore, synergistic mutagenic and cancerogenic impacts of the radiation environment together with other potentially genotoxic constituents of the space habitat can be quantified using such systems, whose signals are especially relevant for the molecular damage to the DNA or the chromosomes. The experiment Cellular Responses to Radiation in Space (CERASP) has been selected by NASA to be performed on the International Space Station. It will supply basic information on the cellular response to radiation applied in microgravity. One of the biological end-points under investigation will be survival reflected by radiation-dependent reduction of constitutive expression of the enhanced variant of green fluorescent protein (EGFP), originally isolated from the bioluminescent jellyfish Aequorea victoria. A second end-point will be gene activation by space flight conditions in mammalian cells, based on fluorescent promoter reporter systems using the destabilized EGFP variant (d2EGFP). The promoter element to be investigated will reflect the activity of the NF-kB stress response pathway as an anti-apoptotic radiation response. DNA damage will be measured by fluorescent analysis of DNA unwinding (FADU). The systems have worked properly for terrestrial applications during the first experiments. Experiments using accelerated particles produced at the French heavy ion accelerator GANIL have given insights into cellular mechanisms

  6. Cellular Reprogramming Using Defined Factors and MicroRNAs

    PubMed Central

    Eguchi, Takanori; Kuboki, Takuo

    2016-01-01

    Development of human bodies, organs, and tissues contains numerous steps of cellular differentiation including an initial zygote, embryonic stem (ES) cells, three germ layers, and multiple expertized lineages of cells. Induced pluripotent stem (iPS) cells have been recently developed using defined reprogramming factors such as Nanog, Klf5, Oct3/4 (Pou5f1), Sox2, and Myc. This outstanding innovation is largely changing life science and medicine. Methods of direct reprogramming of cells into myocytes, neurons, chondrocytes, and osteoblasts have been further developed using modified combination of factors such as N-myc, L-myc, Sox9, and microRNAs in defined cell/tissue culture conditions. Mesenchymal stem cells (MSCs) and dental pulp stem cells (DPSCs) are also emerging multipotent stem cells with particular microRNA expression signatures. It was shown that miRNA-720 had a role in cellular reprogramming through targeting the pluripotency factor Nanog and induction of DNA methyltransferases (DNMTs). This review reports histories, topics, and idea of cellular reprogramming. PMID:27382371

  7. Telomeres and lifestyle factors: roles in cellular aging.

    PubMed

    Lin, Jue; Epel, Elissa; Blackburn, Elizabeth

    2012-02-01

    Recent research has demonstrated that telomere maintenance might be a key integrating point for the cumulative effects of genetic, environmental and lifestyle factors on aging and aging-related diseases. It is timely to 'take stock' of where this work has led the field. This review summarizes studies that have examined associations between lifestyle factors and telomere length and telomerase activity. In most of the studies described in this chapter, telomere length was measured in leukocytes (LTL) or peripheral blood mononuclear cells (PBMCs), taken from blood draws from the study subjects. Much of this chapter focuses on psychological stress, a widespread factor often intimately tied in with lifestyle or behavioral factors that in turn are related to risks of clinical diseases. Together, these findings suggest that cellular aging is linked to a range of influences, with an individual's life events and lifestyle parameters playing significant roles. Lastly, we propose possible biochemical mechanisms that mediate these associations and discuss future directions.

  8. Sirtuin 2 regulates cellular iron homeostasis via deacetylation of transcription factor NRF2.

    PubMed

    Yang, Xiaoyan; Park, Seong-Hoon; Chang, Hsiang-Chun; Shapiro, Jason S; Vassilopoulos, Athanassios; Sawicki, Konrad T; Chen, Chunlei; Shang, Meng; Burridge, Paul W; Epting, Conrad L; Wilsbacher, Lisa D; Jenkitkasemwong, Supak; Knutson, Mitchell; Gius, David; Ardehali, Hossein

    2017-03-13

    SIRT2 is a cytoplasmic sirtuin that plays a role in various cellular processes, including tumorigenesis, metabolism, and inflammation. Since these processes require iron, we hypothesized that SIRT2 directly regulates cellular iron homeostasis. Here, we have demonstrated that SIRT2 depletion results in a decrease in cellular iron levels both in vitro and in vivo. Mechanistically, we determined that SIRT2 maintains cellular iron levels by binding to and deacetylating nuclear factor erythroid-derived 2-related factor 2 (NRF2) on lysines 506 and 508, leading to a reduction in total and nuclear NRF2 levels. The reduction in nuclear NRF2 leads to reduced ferroportin 1 (FPN1) expression, which in turn results in decreased cellular iron export. Finally, we observed that Sirt2 deletion reduced cell viability in response to iron deficiency. Moreover, livers from Sirt2-/- mice had decreased iron levels, while this effect was reversed in Sirt2-/- Nrf2-/- double-KO mice. Taken together, our results uncover a link between sirtuin proteins and direct control over cellular iron homeostasis via regulation of NRF2 deacetylation and stability.

  9. Cellular factors targeting APCs to modulate adaptive T cell immunity.

    PubMed

    Visperas, Anabelle; Do, Jeongsu; Min, Booki

    2014-01-01

    The fate of adaptive T cell immunity is determined by multiple cellular and molecular factors, among which the cytokine milieu plays the most important role in this process. Depending on the cytokines present during the initial T cell activation, T cells become effector cells that produce different effector molecules and execute adaptive immune functions. Studies thus far have primarily focused on defining how these factors control T cell differentiation by targeting T cells themselves. However, other non-T cells, particularly APCs, also express receptors for the factors and are capable of responding to them. In this review, we will discuss how APCs, by responding to those cytokines, influence T cell differentiation and adaptive immunity.

  10. Cellular factors modulating the mechanism of tau protein aggregation

    PubMed Central

    Fontaine, Sarah N.; Sabbagh, Jonathan J.; Baker, Jeremy; Martinez-Licha, Carlos R.; Darling, April

    2015-01-01

    Pathological accumulation of the microtubule-associated protein tau, in the form of neurofibrillary tangles, is a major hallmark of Alzheimer’s disease, the most prevalent neurodegenerative condition worldwide. In addition to Alzheimer’s disease, a number of neurodegenerative diseases, called tauopathies, are characterized by the accumulation of aggregated tau in a variety of brain regions. While tau normally plays an important role in stabilizing the microtubule network of the cytoskeleton, its dissociation from microtubules and eventual aggregation into pathological deposits is an area of intense focus for therapeutic development. Here we discuss the known cellular factors that affect tau aggregation, from post-translational modifications to molecular chaperones. PMID:25666877

  11. 47 CFR 22.939 - Site availability requirements for applications competing with cellular renewal applications.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 2 2011-10-01 2011-10-01 false Site availability requirements for applications competing with cellular renewal applications. 22.939 Section 22.939 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES PUBLIC MOBILE SERVICES Cellular Radiotelephone Service §...

  12. Cellular monitoring systems for the assessment of space environmental factors

    NASA Astrophysics Data System (ADS)

    Hellweg, Christine E.; Arenz, Andrea; Meier, Matthias M.; Baumstark-Khan, Christa

    Detrimental environmental factors - namely ionizing radiation - will continue to affect future manned space missions. The Cellular Biodiagnostics group at the German Aerospace Center (DLR) develops cellular monitoring systems, which include bacterial and mammalian cell systems capable of responding to DNA damage as a consequence of the presence of genotoxic conditions. Such bioassays will complement the physical detector systems used in space, insofar as they yield intrinsically biologically weighted measures of cellular responses. Furthermore, synergistic toxic impacts of the radiation environment together with other potentially genotoxic constituents of the space habitat can be quantified using such systems. The biological end-point under investigation in this work is the gene activation by radiation in mammalian cells, based on fluorescent promoter reporter systems using the destabilized enhanced green fluorescent protein variant (d2EGFP). The promoter element to be investigated reflects the activity of the nuclear factor κB (NF-κB) pathway. The NF-κB family of proteins plays a major role in the inflammatory and immune response, cell proliferation and differentiation, apoptosis and tumorigenesis. After exposure to X-rays, an increase in NF-κB activation was seen only with high doses. Experiments using accelerated argon ions (95 MeV/u, LET ˜230 keV/μm) produced at the French heavy ion accelerator GANIL have shown activation of the NF-κB pathway with doses greater than 1 × 10 6 particles cm -2 (P cm -2), reaching its maximal activation at 2 × 10 7 P cm -2. These results suggest that the exceptional radiation field in space may activate the NF-κB pathway in human cells.

  13. The oxidative modification of cellular fibrin-stabilizing factor.

    PubMed

    Rosenfeld, M A; Shchegolikhin, A N; Leonova, V B; Kostanova, E A; Biryukova, M I; Bychkova, A V; Konstantinova, M L; Vasilyeva, A D

    2016-03-01

    For the first time, the induced oxidative modification of cellular fibrin-stabilizing factor (cFXIII) has been studied. According to the electrophoresis analysis, the conversion of oxidized cFXIII into FXIIIa resulted in producing the enzyme that significantly lost the initial enzymatic activity. At the same time, FXIIIa subjected to induced oxidation was completely devoid of enzymatic activity. The results of FTIR spectroscopy showed that the oxidation of cFXIII or FXIIIa was accompanied by profound changes both in chemical and spatial structures of the protein. The results of this study are in good agreement with our earlier assumption regarding the antioxidant role of the regulatory subunits B of plasma fibrin-stabilizing factor.

  14. Feline Leukemia Virus Infection Requires a Post-Receptor Binding Envelope-Dependent Cellular Component▿

    PubMed Central

    Hussain, Naveen; Thickett, Kelly R.; Na, Hong; Leung, Cherry; Tailor, Chetankumar S.

    2011-01-01

    Gammaretrovirus receptors have been suggested to contain the necessary determinants to mediate virus binding and entry. Here, we show that murine NIH 3T3 and baby hamster kidney (BHK) cells overexpressing receptors for subgroup A, B, and C feline leukemia viruses (FeLVs) are weakly susceptible (101 to 102 CFU/ml) to FeLV pseudotype viruses containing murine leukemia virus (MLV) core (Gag-Pol) proteins, whereas FeLV receptor-expressing murine Mus dunni tail fibroblast (MDTF) cells are highly susceptible (104 to 106 CFU/ml). However, NIH 3T3 cells expressing the FeLV subgroup B receptor PiT1 are highly susceptible to gibbon ape leukemia virus pseudotype virus, which differs from the FeLV pseudotype viruses only in the envelope protein. FeLV resistance is not caused by a defect in envelope binding, low receptor expression levels, or N-linked glycosylation. Resistance is not alleviated by substitution of the MLV core in the FeLV pseudotype virus with FeLV core proteins. Interestingly, FeLV resistance is alleviated by fusion of receptor-expressing NIH 3T3 and BHK cells with MDTF or human TE671 cells, suggesting the absence of an additional cellular component in NIH 3T3 and BHK cells that is required for FeLV infection. The putative FeLV-specific cellular component is not a secreted factor, as MDTF conditioned medium does not alleviate the block to FeLV infection. Together, our findings suggest that FeLV infection requires an additional envelope-dependent cellular component that is absent in NIH 3T3 and BHK cells but that is present in MDTF and TE671 cells. PMID:21917946

  15. Feline leukemia virus infection requires a post-receptor binding envelope-dependent cellular component.

    PubMed

    Hussain, Naveen; Thickett, Kelly R; Na, Hong; Leung, Cherry; Tailor, Chetankumar S

    2011-12-01

    Gammaretrovirus receptors have been suggested to contain the necessary determinants to mediate virus binding and entry. Here, we show that murine NIH 3T3 and baby hamster kidney (BHK) cells overexpressing receptors for subgroup A, B, and C feline leukemia viruses (FeLVs) are weakly susceptible (10(1) to 10(2) CFU/ml) to FeLV pseudotype viruses containing murine leukemia virus (MLV) core (Gag-Pol) proteins, whereas FeLV receptor-expressing murine Mus dunni tail fibroblast (MDTF) cells are highly susceptible (10(4) to 10(6) CFU/ml). However, NIH 3T3 cells expressing the FeLV subgroup B receptor PiT1 are highly susceptible to gibbon ape leukemia virus pseudotype virus, which differs from the FeLV pseudotype viruses only in the envelope protein. FeLV resistance is not caused by a defect in envelope binding, low receptor expression levels, or N-linked glycosylation. Resistance is not alleviated by substitution of the MLV core in the FeLV pseudotype virus with FeLV core proteins. Interestingly, FeLV resistance is alleviated by fusion of receptor-expressing NIH 3T3 and BHK cells with MDTF or human TE671 cells, suggesting the absence of an additional cellular component in NIH 3T3 and BHK cells that is required for FeLV infection. The putative FeLV-specific cellular component is not a secreted factor, as MDTF conditioned medium does not alleviate the block to FeLV infection. Together, our findings suggest that FeLV infection requires an additional envelope-dependent cellular component that is absent in NIH 3T3 and BHK cells but that is present in MDTF and TE671 cells.

  16. Cellular growth in plants requires regulation of cell wall biochemistry.

    PubMed

    Chebli, Youssef; Geitmann, Anja

    2017-02-01

    Cell and organ morphogenesis in plants are regulated by the chemical structure and mechanical properties of the extracellular matrix, the cell wall. The two primary load bearing components in the plant cell wall, the pectin matrix and the cellulose/xyloglucan network, are constantly remodelled to generate the morphological changes required during plant development. This remodelling is regulated by a plethora of loosening and stiffening agents such as pectin methyl-esterases, calcium ions, expansins, and glucanases. The tight spatio-temporal regulation of the activities of these agents is a sine qua non condition for proper morphogenesis at cell and tissue levels. The pectin matrix and the cellulose-xyloglucan network operate in concert and their behaviour is mutually dependent on their chemical, structural and mechanical modifications. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Transcription Factors in the Cellular Response to Charged Particle Exposure

    PubMed Central

    Hellweg, Christine E.; Spitta, Luis F.; Henschenmacher, Bernd; Diegeler, Sebastian; Baumstark-Khan, Christa

    2016-01-01

    Charged particles, such as carbon ions, bear the promise of a more effective cancer therapy. In human spaceflight, exposure to charged particles represents an important risk factor for chronic and late effects such as cancer. Biological effects elicited by charged particle exposure depend on their characteristics, e.g., on linear energy transfer (LET). For diverse outcomes (cell death, mutation, transformation, and cell-cycle arrest), an LET dependency of the effect size was observed. These outcomes result from activation of a complex network of signaling pathways in the DNA damage response, which result in cell-protective (DNA repair and cell-cycle arrest) or cell-destructive (cell death) reactions. Triggering of these pathways converges among others in the activation of transcription factors, such as p53, nuclear factor κB (NF-κB), activated protein 1 (AP-1), nuclear erythroid-derived 2-related factor 2 (Nrf2), and cAMP responsive element binding protein (CREB). Depending on dose, radiation quality, and tissue, p53 induces apoptosis or cell-cycle arrest. In low LET radiation therapy, p53 mutations are often associated with therapy resistance, while the outcome of carbon ion therapy seems to be independent of the tumor’s p53 status. NF-κB is a central transcription factor in the immune system and exhibits pro-survival effects. Both p53 and NF-κB are activated after ionizing radiation exposure in an ataxia telangiectasia mutated (ATM)-dependent manner. The NF-κB activation was shown to strongly depend on charged particles’ LET, with a maximal activation in the LET range of 90–300 keV/μm. AP-1 controls proliferation, senescence, differentiation, and apoptosis. Nrf2 can induce cellular antioxidant defense systems, CREB might also be involved in survival responses. The extent of activation of these transcription factors by charged particles and their interaction in the cellular radiation response greatly influences the destiny of the irradiated and also

  18. Lava lamp, a novel peripheral golgi protein, is required for Drosophila melanogaster cellularization.

    PubMed

    Sisson, J C; Field, C; Ventura, R; Royou, A; Sullivan, W

    2000-11-13

    Drosophila cellularization and animal cell cytokinesis rely on the coordinated functions of the microfilament and microtubule cytoskeletal systems. To identify new proteins involved in cellularization and cytokinesis, we have conducted a biochemical screen for microfilament/microtubule-associated proteins (MMAPs). 17 MMAPs were identified; seven have been previously implicated in cellularization and/or cytokinesis, including KLP3A, Anillin, Septins, and Dynamin. We now show that a novel MMAP, Lava Lamp (Lva), is also required for cellularization. Lva is a coiled-coil protein and, unlike other proteins previously implicated in cellularization or cytokinesis, it is Golgi associated. Our functional analysis shows that cellularization is dramatically inhibited upon injecting anti-Lva antibodies (IgG and Fab) into embryos. In addition, we show that brefeldin A, a potent inhibitor of membrane trafficking, also inhibits cellularization. Biochemical analysis demonstrates that Lva physically interacts with the MMAPs Spectrin and CLIP190. We suggest that Lva and Spectrin may form a Golgi-based scaffold that mediates the interaction of Golgi bodies with microtubules and facilitates Golgi-derived membrane secretion required for the formation of furrows during cellularization. Our results are consistent with the idea that animal cell cytokinesis depends on both actomyosin-based contraction and Golgi-derived membrane secretion.

  19. Lava Lamp, a Novel Peripheral Golgi Protein, Is Required for Drosophila melanogaster Cellularization

    PubMed Central

    Sisson, John C.; Field, Christine; Ventura, Richard; Royou, Anne; Sullivan, William

    2000-01-01

    Drosophila cellularization and animal cell cytokinesis rely on the coordinated functions of the microfilament and microtubule cytoskeletal systems. To identify new proteins involved in cellularization and cytokinesis, we have conducted a biochemical screen for microfilament/microtubule-associated proteins (MMAPs). 17 MMAPs were identified; seven have been previously implicated in cellularization and/or cytokinesis, including KLP3A, Anillin, Septins, and Dynamin. We now show that a novel MMAP, Lava Lamp (Lva), is also required for cellularization. Lva is a coiled-coil protein and, unlike other proteins previously implicated in cellularization or cytokinesis, it is Golgi associated. Our functional analysis shows that cellularization is dramatically inhibited upon injecting anti–Lva antibodies (IgG and Fab) into embryos. In addition, we show that brefeldin A, a potent inhibitor of membrane trafficking, also inhibits cellularization. Biochemical analysis demonstrates that Lva physically interacts with the MMAPs Spectrin and CLIP190. We suggest that Lva and Spectrin may form a Golgi-based scaffold that mediates the interaction of Golgi bodies with microtubules and facilitates Golgi-derived membrane secretion required for the formation of furrows during cellularization. Our results are consistent with the idea that animal cell cytokinesis depends on both actomyosin-based contraction and Golgi-derived membrane secretion. PMID:11076973

  20. Identification of Cellular Proteins Required for Replication of Human Immunodeficiency Virus Type 1

    PubMed Central

    Dziuba, Natallia; Ferguson, Monique R.; O'Brien, William A.; Sanchez, Anthony; Prussia, Andrew J.; McDonald, Natalie J.; Friedrich, Brian M.; Li, Guangyu; Shaw, Michael W.; Sheng, Jinsong; Hodge, Thomas W.; Rubin, Donald H.

    2012-01-01

    Abstract Cellular proteins are essential for human immunodeficiency virus type 1 (HIV-1) replication and may serve as viable new targets for treating infection. Using gene trap insertional mutagenesis, a high-throughput approach based on random inactivation of cellular genes, candidate genes were found that limit virus replication when mutated. Disrupted genes (N=87) conferring resistance to lytic infection with several viruses were queried for an affect on HIV-1 replication by utilizing small interfering RNA (siRNA) screens in TZM-bl cells. Several genes regulating diverse pathways were found to be required for HIV-1 replication, including DHX8, DNAJA1, GTF2E1, GTF2E2, HAP1, KALRN, UBA3, UBE2E3, and VMP1. Candidate genes were independently tested in primary human macrophages, toxicity assays, and/or Tat-dependent β-galactosidase reporter assays. Bioinformatics analyses indicated that several host factors present in this study participate in canonical pathways and functional processes implicated in prior genome-wide studies. However, the genes presented in this study did not share identity with those found previously. Novel antiviral targets identified in this study should open new avenues for mechanistic investigation. PMID:22404213

  1. Mechanotransduction and Growth Factor Signalling to Engineer Cellular Microenvironments.

    PubMed

    Cipitria, Amaia; Salmeron-Sanchez, Manuel

    2017-08-01

    Engineering cellular microenvironments involves biochemical factors, the extracellular matrix (ECM) and the interaction with neighbouring cells. This progress report provides a critical overview of key studies that incorporate growth factor (GF) signalling and mechanotransduction into the design of advanced microenvironments. Materials systems have been developed for surface-bound presentation of GFs, either covalently tethered or sequestered through physico-chemical affinity to the matrix, as an alternative to soluble GFs. Furthermore, some materials contain both GF and integrin binding regions and thereby enable synergistic signalling between the two. Mechanotransduction refers to the ability of the cells to sense physical properties of the ECM and to transduce them into biochemical signals. Various aspects of the physics of the ECM, i.e. stiffness, geometry and ligand spacing, as well as time-dependent properties, such as matrix stiffening, degradability, viscoelasticity, surface mobility as well as spatial patterns and gradients of physical cues are discussed. To conclude, various examples illustrate the potential for cooperative signalling of growth factors and the physical properties of the microenvironment for potential applications in regenerative medicine, cancer research and drug testing. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Regulation of Vif mRNA splicing by human immunodeficiency virus type 1 requires 5' splice site D2 and an exonic splicing enhancer to counteract cellular restriction factor APOBEC3G.

    PubMed

    Mandal, Dibyakanti; Exline, Colin M; Feng, Zehua; Stoltzfus, C Martin

    2009-06-01

    The human immunodeficiency virus type 1 (HIV-1) accessory protein Vif is encoded by an incompletely spliced mRNA resulting from splicing of the major splice donor in the HIV-1 genome, 5' splice site (5'ss) D1, to the first splice acceptor, 3'ss A1. We have shown previously that splicing of HIV-1 vif mRNA is tightly regulated by suboptimal 5'ss D2, which is 50 nucleotides downstream of 3'ss A1; a GGGG silencer motif proximal to 5'ss D2; and an SRp75-dependent exonic splicing enhancer (ESEVif). In agreement with the exon definition hypothesis, mutations within 5'ss D2 that are predicted to increase or decrease U1 snRNP binding affinity increase or decrease the usage of 3'ss A1 (D2-up and D2-down mutants, respectively). In this report, the importance of 5'ss D2 and ESEVif for avoiding restriction of HIV-1 by APOBEC3G (A3G) was determined by testing the infectivities of a panel of mutant viruses expressing different levels of Vif. The replication of D2-down and ESEVif mutants in permissive CEM-SS cells was not significantly different from that of wild-type HIV-1. Mutants that expressed Vif in 293T cells at levels greater than 10% of that of the wild type replicated similarly to the wild type in H9 cells, and Vif levels as low as 4% were affected only modestly in H9 cells. This is in contrast to Vif-deleted HIV-1, whose replication in H9 cells was completely inhibited. To test whether elevated levels of A3G inhibit replication of D2-down and ESEVif mutants relative to wild-type virus replication, a Tet-off Jurkat T-cell line that expressed approximately 15-fold-higher levels of A3G than control Tet-off cells was generated. Under these conditions, the fitness of all D2-down mutant viruses was reduced relative to that of wild-type HIV-1, and the extent of inhibition was correlated with the level of Vif expression. The replication of an ESEVif mutant was also inhibited only at higher levels of A3G. Thus, wild-type 5'ss D2 and ESEVif are required for production of

  3. Tumour-expressed tissue factor inhibits cellular cytotoxicity.

    PubMed

    Li, Chao; Colman, Lucy M; Collier, Mary E W; Dyer, Charlotte E; Greenman, John; Ettelaie, Camille

    2006-11-01

    The association between tissue factor (TF) expression and increased rate of tumour metastasis is well established. In this study, we have examined the hypothesis that the expression of TF by disseminated tumour cells confers protection against immune recognition and cytotoxicity. A hybrid EGFP-TF protein was expressed in HT29 colon carcinoma and K562 lymphoblast cell lines. To assess the cytotoxic activity against tumour cells over-expressing TF, a novel method was used, based on the direct measurement of fluorescently labelled HT29 or K562 target cells. Upon challenge with peripheral blood mononuclear cells (PBMC), tumour cells expressing TF partially evaded cellular cytotoxicity (Delta=15-40% reduction in cytotoxicity). Moreover, the influence of TF was not primarily dependent on its procoagulant function, although the inclusion of 20% (v/v) plasma did lower the rate of cytotoxicity against untransfected cells. However, expression of a truncated form of TF, devoid of the cytoplasmic domain, did not mediate any degree of inhibition of cytotoxicity, suggesting that the protective function of TF is principally due to this domain. We conclude that TF can promote immune evasion in tumour cells expressing this protein leading to increased survival and therefore metastatic rate in such cells.

  4. Mechanically Induced Chromatin Condensation Requires Cellular Contractility in Mesenchymal Stem Cells.

    PubMed

    Heo, Su-Jin; Han, Woojin M; Szczesny, Spencer E; Cosgrove, Brian D; Elliott, Dawn M; Lee, David A; Duncan, Randall L; Mauck, Robert L

    2016-08-23

    Mechanical cues play important roles in directing the lineage commitment of mesenchymal stem cells (MSCs). In this study, we explored the molecular mechanisms by which dynamic tensile loading (DL) regulates chromatin organization in this cell type. Our previous findings indicated that the application of DL elicited a rapid increase in chromatin condensation through purinergic signaling mediated by ATP. Here, we show that the rate and degree of condensation depends on the frequency and duration of mechanical loading, and that ATP release requires actomyosin-based cellular contractility. Increases in baseline cellular contractility via the addition of an activator of G-protein coupled receptors (lysophosphatidic acid) induced rapid ATP release, resulting in chromatin condensation independent of loading. Conversely, inhibition of contractility through pretreatment with either a RhoA/Rock inhibitor (Y27632) or MLCK inhibitor (ML7) abrogated ATP release in response to DL, blocking load-induced chromatin condensation. With loading, ATP release occurred very rapidly (within the first 10-20 s), whereas changes in chromatin occurred at a later time point (∼10 min), suggesting a downstream biochemical pathway mediating this process. When cells were pretreated with blockers of the transforming growth factor (TGF) superfamily, purinergic signaling in response to DL was also eliminated. Further analysis showed that this pretreatment decreased contractility, implicating activity in the TGF pathway in the establishment of the baseline contractile state of MSCs (in the absence of exogenous ligands). These data indicate that chromatin condensation in response to DL is regulated through the interplay between purinergic and RhoA/Rock signaling, and that ligandless activity in the TGF/bone morphogenetic proteins signaling pathway contributes to the establishment of baseline contractility in MSCs.

  5. 47 CFR 1.20007 - Additional assistance capability requirements for wireline, cellular, and PCS telecommunications...

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 1 2010-10-01 2010-10-01 false Additional assistance capability requirements for wireline, cellular, and PCS telecommunications carriers. 1.20007 Section 1.20007 Telecommunication... telecommunications carriers. (a) Definition—(1) Call-identifying information. Call identifying information...

  6. Differential requirements for clathrin endocytic pathway components in cellular entry by Ebola and Marburg glycoprotein pseudovirions.

    PubMed

    Bhattacharyya, Suchita; Hope, Thomas J; Young, John A T

    2011-10-10

    Clathrin-mediated endocytosis was previously implicated as one of the cellular pathways involved in filoviral glycoprotein mediated viral entry into target cells. Here we have further dissected the requirements for different components of this pathway in Ebola versus Marburg virus glycoprotein (GP) mediated viral infection. Although a number of these components were involved in both cases; Ebola GP-dependent viral entry specifically required the cargo recognition proteins Eps15 and DAB2 as well as the clathrin adaptor protein AP-2. In contrast, Marburg GP-mediated infection was independent of these three proteins and instead required beta-arrestin 1 (ARRB1). These findings have revealed an unexpected difference between the clathrin pathway requirements for Ebola GP versus Marburg GP pseudovirion infection. Anthrax toxin also uses a clathrin-, and ARRB1-dependent pathway for cellular entry, indicating that the mechanism used by Marburg GP pseudovirions may be more generally important for pathogen entry.

  7. Cellular Metabolism in Genetic Transformation of Pneumococci: Requirement for Protein Synthesis During Induction of Competence

    PubMed Central

    Tomasz, Alexander

    1970-01-01

    Metabolic inhibitors have differential effects on various phases of genetic transformation in pneumococci. Evidence is presented suggesting that, in addition to the competence factor, another specific protein or class of proteins is essential for the development of cellular “competence.” The precise role of this protein(s) in genetic transformation is not known, but it seems essential for some function subsequent to the interaction of competence factor and cells. PMID:4392399

  8. E2F transcription factor 1 regulates cellular and organismal senescence by inhibiting Forkhead box O transcription factors.

    PubMed

    Xie, Qi; Peng, Shengyi; Tao, Li; Ruan, Haihe; Yang, Yanglu; Li, Tie-Mei; Adams, Ursula; Meng, Songshu; Bi, Xiaolin; Dong, Meng-Qiu; Yuan, Zengqiang

    2014-12-05

    E2F1 and FOXO3 are two transcription factors that have been shown to participate in cellular senescence. Previous report reveals that E2F1 enhanced cellular senescence in human fibroblast cells, while FOXO transcription factors play against senescence by regulation reactive oxygen species scavenging proteins. However, their functional interplay has been unclear. Here we use E2F1 knock-out murine Embryonic fibroblasts (MEFs), knockdown RNAi constructs, and ectopic expression of E2F1 to show that it functions by negatively regulating FOXO3. E2F1 attenuates FOXO3-mediated expression of MnSOD and Catalase without affecting FOXO3 protein stability, subcellular localization, or phosphorylation by Akt. We mapped the interaction between E2F1 and FOXO3 to a region including the DNA binding domain of E2F1 and the C-terminal transcription-activation domain of FOXO3. We propose that E2F1 inhibits FOXO3-dependent transcription by directly binding FOXO3 in the nucleus and preventing activation of its target genes. Moreover, knockdown of the Caenorhabditis elegans E2F1 ortholog efl-1 significantly extends lifespan in a manner that requires the activity of the C. elegans FOXO gene daf-16. We conclude that there is an evolutionarily conserved signaling connection between E2F1 and FOXO3, which regulates cellular senescence and aging by regulating the activity of FOXO3. We speculate that drugs and/or therapies that inhibit this physical interaction might be good candidates for reducing cellular senescence and increasing longevity. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Selective transcription and cellular proliferation induced by PDGF require histone deacetylase activity

    SciTech Connect

    Catania, Annunziata; Iavarone, Carlo; Carlomagno, Stella M.; Chiariello, Mario . E-mail: chiariel@unina.it

    2006-05-05

    Histone deacetylases (HDACs) are key regulatory enzymes involved in the control of gene expression and their inhibition by specific drugs has been widely correlated to cell cycle arrest, terminal differentiation, and apoptosis. Here, we investigated whether HDAC activity was required for PDGF-dependent signal transduction and cellular proliferation. Exposure of PDGF-stimulated NIH3T3 fibroblasts to the HDAC inhibitor trichostatin A (TSA) potently repressed the expression of a group of genes correlated to PDGF-dependent cellular growth and pro-survival activity. Moreover, we show that TSA interfered with STAT3-dependent transcriptional activity induced by PDGF. Still, neither phosphorylation nor nuclear translocation and DNA-binding in vitro and in vivo of STAT3 were affected by using TSA to interfere with PDGF stimulation. Finally, TSA treatment resulted in the suppression of PDGF-dependent cellular proliferation without affecting cellular survival of NIH3T3 cells. Our data indicate that inhibition of HDAC activity antagonizes the mitogenic effect of PDGF, suggesting that these drugs may specifically act on the expression of STAT-dependent, PDGF-responsive genes.

  10. The epidermal growth factor receptor family as a central element for cellular signal transduction and diversification.

    PubMed

    Prenzel, N; Fischer, O M; Streit, S; Hart, S; Ullrich, A

    2001-03-01

    Homeostasis of multicellular organisms is critically dependent on the correct interpretation of the plethora of signals which cells are exposed to during their lifespan. Various soluble factors regulate the activation state of cellular receptors which are coupled to a complex signal transduction network that ultimately generates signals defining the required biological response. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases represents both key regulators of normal cellular development as well as critical players in a variety of pathophysiological phenomena. The aim of this review is to give a broad overview of signal transduction networks that are controlled by the EGFR superfamily of receptors in health and disease and its application for target-selective therapeutic intervention. Since the EGFR and HER2 were recently identified as critical players in the transduction of signals by a variety of cell surface receptors, such as G-protein-coupled receptors and integrins, our special focus is the mechanisms and significance of the interconnectivity between heterologous signalling systems.

  11. Cellular Factors Involved in HTLV-1 Entry and Pathogenicit

    PubMed Central

    Hoshino, Hiroo

    2012-01-01

    Human T cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T cell leukemia (ATL) and HTLV-1 – associated myelopathy and tropical spastic paraparesis (HAM/TSP). HTLV-1 has a preferential tropism for CD4 T cells in healthy carriers and ATL patients, while both CD4 and CD8 T cells serve as viral reservoirs in HAM/TSP patients. HTLV-1 has also been detected other cell types, including monocytes, endothelial cells, and dendritic cells. In contrast to the limited cell tropism of HTLV-1 in vivo, the HTLV receptor appears to be expressed in almost all human or animal cell lines. It remains to be examined whether this cell tropism is determined by host factors or by HTLV-1 heterogeneity. Unlike most retroviruses, cell-free virions of HTLV-1 are very poorly infectious. The lack of completely HTLV-1-resistant cells and the low infectivity of HTLV-1 have hampered research on the HTLV entry receptor. Entry of HTLV-1 into target cells is thought to involve interactions between the env (Env) glycoproteins, a surface glycoprotein (surface unit), and a transmembrane glycoprotein. Recent studies have shown that glucose transporter GLUT1, heparan sulfate proteoglycans (HSPGs), and neuropilin-1 (NRP-1) are the three proteins important for the entry of HTLV-1. Studies using adherent cell lines have shown that GLUT1 can function as a receptor for HTLV. HSPGs are required for efficient entry of HTLV-1 into primary CD4 T cells. NRP-1 is expressed in most established cell lines. Further studies have shown that these three molecules work together to promote HTLV-1 binding to cells and fusion of viral and cell membranes. The virus could first contact with HSPGs and then form complexes with NRP-1, followed by association with GLUT1. It remains to be determined whether these three molecules can explain HTLV-1 cell tropism. It also remains to be more definitively proven that these molecules are sufficient to permit HTLV-1 entry into completely HTLV-1-resistant cells. PMID

  12. Cellular Levels of Signaling Factors Are Sensed by β-actin Alleles to Modulate Transcriptional Pulse Intensity.

    PubMed

    Kalo, Alon; Kanter, Itamar; Shraga, Amit; Sheinberger, Jonathan; Tzemach, Hadar; Kinor, Noa; Singer, Robert H; Lionnet, Timothée; Shav-Tal, Yaron

    2015-04-21

    The transcriptional response of β-actin to extra-cellular stimuli is a paradigm for transcription factor complex assembly and regulation. Serum induction leads to a precisely timed pulse of β-actin transcription in the cell population. Actin protein is proposed to be involved in this response, but it is not known whether cellular actin levels affect nuclear β-actin transcription. We perturbed the levels of key signaling factors and examined the effect on the induced transcriptional pulse by following endogenous β-actin alleles in single living cells. Lowering serum response factor (SRF) protein levels leads to loss of pulse integrity, whereas reducing actin protein levels reveals positive feedback regulation, resulting in elevated gene activation and a prolonged transcriptional response. Thus, transcriptional pulse fidelity requires regulated amounts of signaling proteins, and perturbations in factor levels eliminate the physiological response, resulting in either tuning down or exaggeration of the transcriptional pulse.

  13. Resources required for topological quantum factoring

    SciTech Connect

    Baraban, M.; Bonesteel, N. E.; Simon, S. H.

    2010-06-15

    We consider a hypothetical topological quantum computer composed of either Ising or Fibonacci anyons. For each case, we calculate the time and number of qubits (space) necessary to execute the most computationally expensive step of Shor's algorithm, modular exponentiation. For Ising anyons, we apply Bravyi's distillation method [S. Bravyi, Phys. Rev. A 73, 042313 (2006)] which combines topological and nontopological operations to allow for universal quantum computation. With reasonable restrictions on the physical parameters we find that factoring a 128-bit number requires approximately 10{sup 3} Fibonacci anyons versus at least 3x10{sup 9} Ising anyons. Other distillation algorithms could reduce the resources for Ising anyons substantially.

  14. Regulation of Cellular Differentiation by Tissue Specific Transcription Factor

    DTIC Science & Technology

    2007-11-02

    EGFP (4). And it is shown that PKC and Ca2+ enhanced heat shock factor 1 ( HSF ) thorough phosphorylation 7. When the HSF is phosphorylated, it is...by PKC or calcium dependent pathway via HSF . As shown in Fig.6, electrically stimulated cells expressed EGFP fluorescence, which indicates

  15. Cellular Oxygen Sensing: Crystal Structure of Hypoxia-Inducible Factor Prolyl Hydroxylase (PHD2)

    SciTech Connect

    McDonough,M.; Li, V.; Flashman, E.; Chowdhury, R.; Mohr, C.; Lienard, B.; Zondlo, J.; Oldham, N.; Clifton, I.; et al.

    2006-01-01

    Cellular and physiological responses to changes in dioxygen levels in metazoans are mediated via the posttranslational oxidation of hypoxia-inducible transcription factor (HIF). Hydroxylation of conserved prolyl residues in the HIF-{alpha} subunit, catalyzed by HIF prolyl-hydroxylases (PHDs), signals for its proteasomal degradation. The requirement of the PHDs for dioxygen links changes in dioxygen levels with the transcriptional regulation of the gene array that enables the cellular response to chronic hypoxia; the PHDs thus act as an oxygen-sensing component of the HIF system, and their inhibition mimics the hypoxic response. We describe crystal structures of the catalytic domain of human PHD2, an important prolyl-4-hydroxylase in the human hypoxic response in normal cells, in complex with Fe(II) and an inhibitor to 1.7 Angstroms resolution. PHD2 crystallizes as a homotrimer and contains a double-stranded {beta}-helix core fold common to the Fe(II) and 2-oxoglutarate-dependant dioxygenase family, the residues of which are well conserved in the three human PHD enzymes (PHD 1-3). The structure provides insights into the hypoxic response, helps to rationalize a clinically observed mutation leading to familial erythrocytosis, and will aid in the design of PHD selective inhibitors for the treatment of anemia and ischemic disease.

  16. Hepatic to pancreatic switch defines a role for hemostatic factors in cellular plasticity in mice

    PubMed Central

    Shanmukhappa, Kumar; Mourya, Reena; Sabla, Gregg E.; Degen, Jay L.; Bezerra, Jorge A.

    2005-01-01

    In multiple systems, impaired proteolysis associated with the loss of the hemostatic factor plasminogen (Plg) results in fibrin-dependent defects in tissue repair. However, repair within the liver is known to be defective in Plg-deficient (Plgo) mice independent of fibrin clearance and appears to be compromised in part by the poor clearance of necrotic cells. Based on these findings, we examined the hepatic transcriptome after injury in search of transcriptional programs that are sensitive to the Plg/fibrinogen system. To this end, we generated biotinylated cRNA pools from livers of Plgo mice and controls before and after a single dose of the hepatotoxin carbon tetrachloride and hybridized them against high-density oligonucleotide arrays. Analysis of the gene expression platform identified an unexpected transcriptional signature within challenged livers of Plgo mice for pancreatic gene products, including trypsinogen-2, amylase-2, elastase-1, elastase-2, and cholesteryl-ester lipase. Validation studies found that this transcriptional program also contained products of the endocrine pancreas (Reg-1 and insulin genes) and the expression of the pancreatic transcription factors p48 and PDX-1. By using a LacZ transgene to trace the cellular source of pancreatic gene expression, we found that PDX-1 was expressed in albumin-positive cells that were morphologically indistinguishable from hepatocytes, and in albumin-negative epithelioid cells within zones of pericentral injury. More detailed studies revealed that the mechanisms of heterotopic gene expression in Plgo mice required fibrin(ogen). Collectively, these data reveal a regulatory role for the hemostatic factors plasmin(ogen) and fibrin(ogen) in cellular plasticity within adult tissues of the digestive system. PMID:16006527

  17. In Rhizobium etli symbiotic plasmid transfer, nodulation competitivity and cellular growth require interaction among different replicons.

    PubMed

    Brom, S; García-de los Santos, A; Cervantes, L; Palacios, R; Romero, D

    2000-07-01

    Bacteria belonging to the genus Rhizobium are able to develop two different lifestyles, in symbiotic association with plant roots or through saprophytic growth. The genome of Rhizobium strains is constituted by a chromosome and several large plasmids, one of them containing most of the genes involved in symbiosis (symbiotic plasmid or pSym). Our model strain Rhizobium etli CFN42 contains six plasmids. We have constructed multiple plasmid-cured derivatives of this strain and used them to analyze the contribution of these plasmids to free-living cellular viability, competitivity for nodulation, plasmid transfer, and utilization of diverse carbon sources. Our results show that the transfer of the pSym is strictly dependent on the presence of another plasmid; consequently under conditions where pSym transfer is required, nodulation relies on the presence of a plasmid devoid of nodulation genes. We also found a drastic decrease in competitivity for nodulation in multiple plasmid-cured derivatives when compared with single plasmid-cured strains. Cellular growth and viability were greatly diminished in some multiple plasmid-cured strains. The utilization of a number of carbon sources depends on the presence of specific plasmids. The results presented in this work indicate that functional interactions among sequences scattered in the different plasmids are required for successful completion of both lifestyles. Copyright 2000 Academic Press.

  18. TRIENNIAL GROWTH AND DEVELOPMENT SYMPOSIUM: Factors influencing bovine intramuscular adipose tissue development and cellularity.

    PubMed

    Albrecht, E; Schering, L; Liu, Y; Komolka, K; Kühn, C; Wimmers, K; Gotoh, T; Maak, S

    2017-05-01

    Appearance, distribution, and amount of intramuscular fat (IMF), often referred to as marbling, are highly variable and depend on environmental and genetic factors. On the molecular level, the concerted action of several drivers, including hormones, receptors, transcription factors, etc., determines where clusters of adipocytes arise. Therefore, the aim of future studies remains to identify such factors as biological markers of IMF to increase the ability to identify animals that deposit IMF early in age to increase efficiency of high-quality meat production. In an attempt to unravel the cellular development of marbling, we investigated the abundance of markers for adipogenic differentiation during fattening of cattle and the transcriptome of muscle and dissected IMF. Markers of different stages of adipogenic differentiation are well known from cell culture experiments. They are usually transiently expressed, such as delta-like homolog 1 (DLK1) that is abundant in preadipocytes and absent during differentiation to mature adipocytes. It is even a greater challenge to detect those markers in live animals. Within skeletal muscles, hyperplasia and hypertrophy of adipocytes can be observed throughout life. Therefore, development of marbling requires, on the cellular level, recruitment, proliferation, and differentiation of adipogenic cells to store excess energy in the form of lipids in new cells. In a recent study, we investigated the localization and abundance of early markers of adipogenic differentiation, such as DLK1, in bovine muscle tissue. An inverse relationship between IMF content and number of DLK1-positive cells in bovine muscle was demonstrated. Considering the cellular environment of differentiating adipocytes in muscle and the secretory action of adipocytes and myocytes, it becomes obvious that cross talk between cells via adipokines and myokines may be important for IMF development. Secreted proteins can act on other cells, inhibiting or stimulating

  19. Molecular and cellular biology of von Willebrand factor.

    PubMed

    Denis, Cécile V

    2002-01-01

    von Willebrand factor (VWF) is a plasma protein that performs 2 main functions in hemostasis: it mediates platelet adhesion to the injured vessel wall, and it carries and protects coagulation factor VIII. VWF is synthesized through a multistep process in endothelial cells and megakaryocytes as a very large polymer composed of identical disulfide-linked 250-kd subunits. In endothelial cells, VWF not only directs the formation of its own storage granules, the Weibel-Palade bodies, but it also acts as a chaperone molecule to direct other proteins, such as P-selectin, into these granules. Upon stimulation of the endothelium, the Weibel-Palade bodies will be translocated to the plasma membrane, and their contents will be secreted into the plasma milieu. The expression of VWF can be regulated at different levels by a number of genetic and environmental factors, resulting in control of its activity. New roles for VWF, especially in inflammatory processes, have recently been suggested, indicating that some aspects of this well-studied protein remain to be investigated.

  20. Cellular dynamics of the negative transcription elongation factor NELF

    SciTech Connect

    Yung, Tetsu M.C.; Narita, Takashi; Komori, Toshiharu; Yamaguchi, Yuki; Handa, Hiroshi

    2009-06-10

    Negative Elongation Factor (NELF) is a transcription factor discovered based on its biochemical activity to suppress transcription elongation, and has since been implicated in various diseases ranging from neurological disorders to cancer. Besides its role in promoter-proximal pausing of RNA polymerase II during early stages of transcription, recently we found that it also plays important roles in the 3'-end processing of histone mRNA. Furthermore, NELF has been found to form a distinct subnuclear structure, which we named NELF bodies. These recent developments point to a wide range of potential functions for NELF, and, as most studies on NELF thus far had been carried out in vitro, here, we prepared a complete set of fusion protein constructs of NELF subunits and carried out a general cell biological study of the intracellular dynamics of NELF. Our data show that NELF subunits exhibit highly specific subcellular localizations, such as in NELF bodies or in midbodies, and some shuttle actively between the nucleus and cytoplasm. We further show that loss of NELF from cells can lead to enlarged and/or multiple nuclei. This work serves as a foundation and starting point for further cell biological investigations of NELF in the future.

  1. Extreme cellular adaptations and cell differentiation required by a cyanobacterium for carbonate excavation.

    PubMed

    Guida, Brandon Scott; Garcia-Pichel, Ferran

    2016-05-17

    Some cyanobacteria, known as euendoliths, excavate and grow into calcium carbonates, with their activity leading to significant marine and terrestrial carbonate erosion and to deleterious effects on coral reef and bivalve ecology. Despite their environmental relevance, the mechanisms by which they can bore have remained elusive and paradoxical, in that, as oxygenic phototrophs, cyanobacteria tend to alkalinize their surroundings, which will encourage carbonate precipitation, not dissolution. Therefore, cyanobacteria must rely on unique adaptations to bore. Studies with the filamentous euendolith, Mastigocoleus testarum, indicated that excavation requires both cellular energy and transcellular calcium transport, mediated by P-type ATPases, but the cellular basis for this phenomenon remains obscure. We present evidence that excavation in M. testarum involves two unique cellular adaptations. Long-range calcium transport is based on active pumping at multiple cells along boring filaments, orchestrated by the preferential localization of calcium ATPases at one cell pole, in a ring pattern, facing the cross-walls, and by repeating this placement and polarity, a pattern that breaks at branching and apical cells. In addition, M. testarum differentiates specialized cells we call calcicytes, that which accumulate calcium at concentrations more than 500-fold those found in other cyanobacteria, concomitantly and drastically lowering photosynthetic pigments and enduring severe cytoplasmatic alkalinization. Calcicytes occur commonly, but not exclusively, in apical parts of the filaments distal to the excavation front. We suggest that calcicytes allow for fast calcium flow at low, nontoxic concentrations through undifferentiated cells by providing buffering storage for excess calcium before final excretion to the outside medium.

  2. Extreme cellular adaptations and cell differentiation required by a cyanobacterium for carbonate excavation

    PubMed Central

    Guida, Brandon Scott; Garcia-Pichel, Ferran

    2016-01-01

    Some cyanobacteria, known as euendoliths, excavate and grow into calcium carbonates, with their activity leading to significant marine and terrestrial carbonate erosion and to deleterious effects on coral reef and bivalve ecology. Despite their environmental relevance, the mechanisms by which they can bore have remained elusive and paradoxical, in that, as oxygenic phototrophs, cyanobacteria tend to alkalinize their surroundings, which will encourage carbonate precipitation, not dissolution. Therefore, cyanobacteria must rely on unique adaptations to bore. Studies with the filamentous euendolith, Mastigocoleus testarum, indicated that excavation requires both cellular energy and transcellular calcium transport, mediated by P-type ATPases, but the cellular basis for this phenomenon remains obscure. We present evidence that excavation in M. testarum involves two unique cellular adaptations. Long-range calcium transport is based on active pumping at multiple cells along boring filaments, orchestrated by the preferential localization of calcium ATPases at one cell pole, in a ring pattern, facing the cross-walls, and by repeating this placement and polarity, a pattern that breaks at branching and apical cells. In addition, M. testarum differentiates specialized cells we call calcicytes, that which accumulate calcium at concentrations more than 500-fold those found in other cyanobacteria, concomitantly and drastically lowering photosynthetic pigments and enduring severe cytoplasmatic alkalinization. Calcicytes occur commonly, but not exclusively, in apical parts of the filaments distal to the excavation front. We suggest that calcicytes allow for fast calcium flow at low, nontoxic concentrations through undifferentiated cells by providing buffering storage for excess calcium before final excretion to the outside medium. PMID:27140633

  3. THE TISSUE FACTOR REQUIREMENT IN BLOOD COAGULATION

    PubMed Central

    Orfeo, Thomas; Butenas, Saulius; Brummel-Ziedins, Kathleen E.; Mann, Kenneth G.

    2005-01-01

    Formation of thrombin is triggered when membrane-localized tissue factor (TF) is exposed to blood. In closed models of this process, thrombin formation displays an initiation phase (low rates of thrombin production cause platelet activation and fibrinogen clotting), a propagation phase (>95% of thrombin production occurs) and a termination phase (prothrombin activation ceases and free thrombin is inactivated). A current controversy centers on whether the TF stimulus requires supplementation from a circulating pool of blood TF in order to sustain an adequate procoagulant response. We have evaluated the requirement for TF during the progress of the blood coagulation reaction and have extended these analyses to assess the requirement for TF during resupply (“flow replacement”). Elimination of TF activity at various times during the initiation phase indicated: a period of absolute dependence (<10s); a transitional period in which the dependence on TF is partial and decreases as the reaction proceeds (10–240s); and a period in which the progress of the reaction is TF independent (>240s). Resupply of reactions late during the termination phase with fresh reactants, but no TF, yielded immediate bursts of thrombin formation similar in magnitude to the original propagation phases. Our data show that independence from the initial TF stimulus is achieved by the onset of the propagation phase and that the ensemble of coagulation products and intermediates which yield this TF independence maintain their prothrombin-activating potential for considerable time. These observations support the hypothesis that the transient, localized expression of TF is sufficient to sustain a TF-independent procoagulant response as long as flow persists. PMID:16215234

  4. The tissue factor requirement in blood coagulation.

    PubMed

    Orfeo, Thomas; Butenas, Saulius; Brummel-Ziedins, Kathleen E; Mann, Kenneth G

    2005-12-30

    Formation of thrombin is triggered when membrane-localized tissue factor (TF) is exposed to blood. In closed models of this process, thrombin formation displays an initiation phase (low rates of thrombin production cause platelet activation and fibrinogen clotting), a propagation phase (>95% of thrombin production occurs), and a termination phase (prothrombin activation ceases and free thrombin is inactivated). A current controversy centers on whether the TF stimulus requires supplementation from a circulating pool of blood TF to sustain an adequate procoagulant response. We have evaluated the requirement for TF during the progress of the blood coagulation reaction and have extended these analyses to assess the requirement for TF during resupply ("flow replacement"). Elimination of TF activity at various times during the initiation phase indicated: a period of absolute dependence (<10 s); a transitional period in which the dependence on TF is partial and decreases as the reaction proceeds (10-240 s); and a period in which the progress of the reaction is TF independent (>240 s). Resupply of reactions late during the termination phase with fresh reactants, but no TF, yielded immediate bursts of thrombin formation similar in magnitude to the original propagation phases. Our data show that independence from the initial TF stimulus is achieved by the onset of the propagation phase and that the ensemble of coagulation products and intermediates that yield this TF independence maintain their prothrombin activating potential for considerable time. These observations support the hypothesis that the transient, localized expression of TF is sufficient to sustain a TF-independent procoagulant response as long as flow persists.

  5. Cellular and Viral Requirements for Rapid Endocytic Entry of Herpes Simplex Virus

    PubMed Central

    Nicola, Anthony V.; Straus, Stephen E.

    2004-01-01

    It was recently demonstrated that herpes simplex virus (HSV) successfully infects Chinese hamster ovary (CHO) cells expressing glycoprotein D (gD) receptors and HeLa cells by an endocytic mechanism (A. V. Nicola, A. M. McEvoy, and S. E. Straus, J. Virol. 77:5324-5332, 2003). Here we define cellular and viral requirements of this pathway. Uptake of intact, enveloped HSV from the cell surface into endocytic vesicles was rapid (t1/2 of 8 to 9 min) and independent of the known cell surface gD receptors. Following uptake from the surface, recovery of intracellular, infectious virions increased steadily up to 20 min postinfection (p.i.), which corresponds to accumulation of enveloped virus in intracellular compartments. There was a sharp decline in recovery by 30 min p.i., suggesting loss of the virus envelope as a result of capsid penetration from endocytic organelles into the cytosol. In the absence of gD receptors, endocytosed virions did not successfully penetrate into the cytosol but were instead transported to lysosomes for degradation. Inhibitors of phosphatidylinositol (PI) 3-kinase, such as wortmannin, blocked transport of incoming HSV to the nuclear periphery and virus-induced gene expression but had no effect on virus binding or uptake. This suggests a role for PI 3-kinase activity in trafficking of HSV through the cytosol. Viruses that lack viral glycoproteins gB, gD, or gH-gL were defective in transport to the nucleus and had reduced infectivity. Thus, similar to entry via direct penetration at the cell surface, HSV entry into cells by wortmannin-sensitive endocytosis is efficient, involves rapid cellular uptake of viral particles, and requires gB, gD, and gH-gL. PMID:15220424

  6. The ATP requirements of adenovirus type 5 DNA replication and cellular DNA replication.

    PubMed

    De Jong, P J; Kwant, M M; van Driel, W; Jansz, H S; van der Vliet, P C

    1983-01-15

    Several in vitro DNA replication systems were employed to characterize the ATP dependency of adenovirus type 5 (Ad5) DNA replication. Ad5 DNA synthesis in isolated nuclei, representing the elongation of nascent DNA chains, was slightly ATP dependent. Reduction of the ATP concentration from the optimum (8 mM) to the endogenous value (0.16 microM) reduced Ad5 DNA replication only to 70%. No change in the pattern of replication was observed as indicated by the analysis of replicative intermediates using agarose gel electrophoresis. ATP could be replaced by dATP, but not by GTP or other nucleoside triphosphates. By contrast, cellular DNA replication in isolated nuclei from HeLa cells was reduced to 12% by the omission of ATP. These differences could not be explained by different ATP pools or by effects of ATP on dNTP pools. Cellular DNA replication in contrast to viral DNA replication was sensitive to low concentrations of adenosine 5'-O-(3-thiotriphosphate). Inhibition by this ATP analog was competitive with ATP (Ki = 0.4 mM). Adenovirus DNA replication by DNA-free nuclear extracts, representing initiation plus elongation (Challberg and Kelly, Proc. Nat. Acad. Sci. USA 76, 655-659, 1979), exhibited a nearly absolute requirement for ATP. ATP could be substituted not only by dATP, but also by GTP and dGTP and to a lesser extent by pyrimidine triphosphates. Similar results were found when the formation of a covalent complex between dCTP and the precursor terminal protein was studied. This reaction is essential for the initiation of Ad5 DNA replication. The results indicate that different ATP-requiring functions are employed during the initiation and elongation stages of adenovirus DNA replication.

  7. Pathogen virulence factors as molecular probes of basic plant cellular functions

    PubMed Central

    Speth, Elena Bray; Lee, Young Nam; He, Sheng Yang

    2007-01-01

    Summary To successfully colonize plants, pathogens have evolved a myriad of virulence factors that allow them to manipulate host cellular pathways in order to gain entry into, multiply and move within, and eventually exit the host for a new infection cycle. In the past few years, substantial progress has been made in characterizing the host targets of viral and bacterial virulence factors, providing unique insights into basic plant cellular processes such as gene silencing, vesicle trafficking, hormone signaling, and innate immunity. Identification of the host targets of additional pathogen virulence factors promises to continue shedding light on fundamental cellular mechanisms in plants, thus enhancing our understanding of plant signaling, metabolism and cell biology. PMID:17884715

  8. Insulin-like growth factor-1 regulates the SIRT1-p53 pathway in cellular senescence

    PubMed Central

    Tran, Duc; Bergholz, Johann; Zhang, Haibo; He, Hanbing; Wang, Yang; Zhang, Yujun; Li, Qintong; Kirkland, James L; Xiao, Zhi-Xiong

    2014-01-01

    Cellular senescence, which is known to halt proliferation of aged and stressed cells, plays a key role against cancer development and is also closely associated with organismal aging. While increased insulin-like growth factor (IGF) signaling induces cell proliferation, survival and cancer progression, disrupted IGF signaling is known to enhance longevity concomitantly with delay in aging processes. The molecular mechanisms involved in the regulation of aging by IGF signaling and whether IGF regulates cellular senescence are still poorly understood. In this study, we demonstrate that IGF-1 exerts a dual function in promoting cell proliferation as well as cellular senescence. While acute IGF-1 exposure promotes cell proliferation and is opposed by p53, prolonged IGF-1 treatment induces premature cellular senescence in a p53-dependent manner. We show that prolonged IGF-1 treatment inhibits SIRT1 deacetylase activity, resulting in increased p53 acetylation as well as p53 stabilization and activation, thus leading to premature cellular senescence. In addition, either expression of SIRT1 or inhibition of p53 prevented IGF-1-induced premature cellular senescence. Together, these findings suggest that p53 acts as a molecular switch in monitoring IGF-1-induced proliferation and premature senescence, and suggest a possible molecular connection involving IGF-1-SIRT1-p53 signaling in cellular senescence and aging. PMID:25070626

  9. Cellular factors in plant virus movement: at the leading edge of macromolecular trafficking in plants.

    PubMed

    Harries, Phillip; Ding, Biao

    2011-03-15

    To establish systemic infection, plant viruses must be localized to the correct subcellular sites to accomplish replication and then traffic from initially infected cells into neighboring cells and even distant organs. Viruses have evolved various strategies to interact with pre-existing cellular factors to achieve these functions. In this review we discuss plant virus intracellular, intercellular and long-distance movement, focusing on the host cellular factors involved. We emphasize that elucidating viral movement mechanisms will not only shed light on the molecular mechanisms of infection, but will also contribute valuable insights into the regulation of endogenous macromolecular trafficking. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Long-term inhibition of HIV-1 replication with RNA interference against cellular co-factors.

    PubMed

    Eekels, Julia J M; Geerts, Dirk; Jeeninga, Rienk E; Berkhout, Ben

    2011-01-01

    In this study we tested whether HIV-1 replication could be inhibited by stable RNAi-mediated knockdown of cellular co-factors. Cell lines capable of expressing shRNAs against 30 candidate co-factors implicated at different steps of the viral replication cycle were generated and analyzed for effects on cell viability and inhibition of HIV-1 replication. For half of these candidate co-factors we obtained knockdown cell lines that are less susceptible to virus replication. For three co-factors (ALIX, ATG16 and TRBP) the cell lines were resistant to HIV-1 replication for up to 2 months. With these cells we could test the hypothesis that HIV-1 is not able to escape from RNAi-mediated suppression of cellular co-factors, which was indeed not detected.

  11. Correlation of cellular factors and differential scrapie prion permissiveness in ovine microglia

    USDA-ARS?s Scientific Manuscript database

    Prion diseases are fatal neurodegenerative disorders by which the native cellular prion protein (PrP-C) is misfolded into an accumulating, disease-associated isoform (PrP-D). To improve the understanding of prion pathogenesis and develop effective treatments, it is essential to elucidate factors con...

  12. Herpesvirus saimiri STP-A oncoprotein utilizes Src family protein tyrosine kinase and tumor necrosis factor receptor-associated factors to elicit cellular signal transduction.

    PubMed

    Garcia, Maria I; Kaserman, Joseph; Chung, Young-Hwa; Jung, Jae U; Lee, Sun-Hwa

    2007-03-01

    The saimiri transforming protein oncogene, called STP-A, of herpesvirus saimiri (HVS) subgroup A is not required for viral replication but is required for lymphoid cell immortalization in culture and lymphoma induction in primates. Here we report that STP-A interacts with cellular tumor necrosis factor receptor-associated factors (TRAF2 and TRAF6) and Src family protein tyrosine kinases (SF-PTKs) in a genetically and functionally separable manner and that each interaction constitutively elicits independent cellular signal transduction. The amino-terminal and central proline-rich motifs of STP-A were responsible for TRAF6 and TRAF2 interactions, respectively, and STP-A and TRAF6 interaction contributed to the majority of NF-kappaB activation, whereas STP-A and TRAF2 interaction played a minor role in NF-kappaB activation. On the other hand, interaction of STP-A with SF-PTKs through its SH2 binding motif effectively elicited AP-1 and NF-AT transcription factor activity. One cellular gene targeted by STP-A is intercellular adhesion molecule 1 (ICAM-1), which participates in a wide range of inflammatory and immune responses. Both TRAF and SF-PTK signal transductions induced by STP-A were required for the marked increase of ICAM-1 expression. These results demonstrate that the viral oncogene STP-A independently targets two vital cellular signaling molecules and that these activities likely contribute to HVS-mediated lymphoid cell immortalization in culture and lymphoma induction in primates.

  13. Flow-dependent myosin recruitment during Drosophila cellularization requires zygotic dunk activity

    PubMed Central

    Martin, Adam; Wieschaus, Eric

    2016-01-01

    Actomyosin contractility underlies force generation in morphogenesis ranging from cytokinesis to epithelial extension or invagination. In Drosophila, the cleavage of the syncytial blastoderm is initiated by an actomyosin network at the base of membrane furrows that invaginate from the surface of the embryo. It remains unclear how this network forms and how it affects tissue mechanics. Here, we show that during Drosophila cleavage, myosin recruitment to the cleavage furrows proceeds in temporally distinct phases of tension-driven cortical flow and direct recruitment, regulated by different zygotic genes. We identify the gene dunk, which we show is transiently transcribed when cellularization starts and functions to maintain cortical myosin during the flow phase. The subsequent direct myosin recruitment, however, is Dunk-independent but requires Slam. The Slam-dependent direct recruitment of myosin is sufficient to drive cleavage in the dunk mutant, and the subsequent development of the mutant is normal. In the dunk mutant, cortical myosin loss triggers misdirected flow and disrupts the hexagonal packing of the ingressing furrows. Computer simulation coupled with laser ablation suggests that Dunk-dependent maintenance of cortical myosin enables mechanical tension build-up, thereby providing a mechanism to guide myosin flow and define the hexagonal symmetry of the furrows. PMID:27226317

  14. Protective cellular retroviral immunity requires both CD4+ and CD8+ immune T cells.

    PubMed Central

    Hom, R C; Finberg, R W; Mullaney, S; Ruprecht, R M

    1991-01-01

    We have found previously that postexposure chemoprophylaxis with 3'-azido-3'-deoxythymidine (also known as zidovudine or AZT) in combination with recombinant human alpha A/D interferon fully protected mice exposed to a lethal dose of Rauscher murine leukemia virus (RLV) against viremia and disease. After cessation of therapy, over 90% of these mice were able to resist rechallenge with live RLV, thus demonstrating an acquired immunity. Adoptive cell transfer of 4 x 10(7) cells from immunized mice fully protected naive recipients from viremia and splenomegaly after RLV challenge. However, when these immune T cells were fractionated into CD4+ and CD8+ subpopulations, only partial protection was found when 4 x 10(7) T cells of either subset were given. Full protection against RLV challenge was seen again when the T-cell subsets from immunized mice were recombined and transferred at the same number into naive mice. We conclude that cellular immunity alone is protective and that both CD4+ and CD8+ cell types are required for conferring full protection against live virus challenge. Images PMID:1898666

  15. Coordinated destruction of cellular messages in translation complexes by the gammaherpesvirus host shutoff factor and the mammalian exonuclease Xrn1.

    PubMed

    Covarrubias, Sergio; Gaglia, Marta M; Kumar, G Renuka; Wong, Wesley; Jackson, Andrew O; Glaunsinger, Britt A

    2011-10-01

    Several viruses encode factors that promote host mRNA degradation to silence gene expression. It is unclear, however, whether cellular mRNA turnover pathways are engaged to assist in this process. In Kaposi's sarcoma-associated herpesvirus this phenotype is enacted by the host shutoff factor SOX. Here we show that SOX-induced mRNA turnover is a two-step process, in which mRNAs are first cleaved internally by SOX itself then degraded by the cellular exonuclease Xrn1. SOX therefore bypasses the regulatory steps of deadenylation and decapping normally required for Xrn1 activation. SOX is likely recruited to translating mRNAs, as it cosediments with translation initiation complexes and depletes polysomes. Cleaved mRNA intermediates accumulate in the 40S fraction, indicating that recognition occurs at an early stage of translation. This is the first example of a viral protein commandeering cellular mRNA turnover pathways to destroy host mRNAs, and suggests that Xrn1 is poised to deplete messages undergoing translation in mammalian cells.

  16. Effects of copper on CHO cells: cellular requirements and product quality considerations.

    PubMed

    Yuk, Inn H; Russell, Stephen; Tang, Yun; Hsu, Wei-Ting; Mauger, Jacob B; Aulakh, Rigzen P S; Luo, Jun; Gawlitzek, Martin; Joly, John C

    2015-01-01

    Recent reports highlight the impact of copper on lactate metabolism: CHO cell cultures with higher initial copper levels shift to net lactate consumption and yield lower final lactate and higher titers. These studies investigated the effects of copper on metabolite and transcript profiles, but did not measure in detail the dependences of cell culture performance and product quality on copper concentrations. To more thoroughly map these dependences, we explored the effects of various copper treatments on four recombinant CHO cell lines. In the first cell line, when extracellular copper remained above the limit of detection (LOD), cultures shifted to net lactate consumption and yielded comparable performances irrespective of the differences in copper levels; when extracellular copper dropped below LOD (∼13 nM), cultures failed to shift to net lactate consumption, and yielded significantly lower product titers. Across the four cell lines, the ability to grow and consume lactate seemed to depend on the presence of a minimum level of copper, beyond which there were no further gains in culture performance. Although this minimum cellular copper requirement could not be directly quantified, we estimated its probable range for the first cell line by applying several assumptions. Even when different copper concentrations did not affect cell culture performance, they affected product quality profiles: higher initial copper concentrations increased the basic variants in the recombinant IgG1 products. Therefore, in optimizing chemically defined media, it is important to select a copper concentration that is adequate and achieves desired product quality attributes. © 2014 American Institute of Chemical Engineers.

  17. N-Terminal signal sequence is required for cellular trafficking and hyaluronan-depolymerization of KIAA1199.

    PubMed

    Yoshida, Hiroyuki; Nagaoka, Aya; Nakamura, Sachiko; Tobiishi, Megumi; Sugiyama, Yoshinori; Inoue, Shintaro

    2014-01-03

    Recently, we disclosed that KIAA1199-mediated hyaluronan (HA) depolymerization requires an acidic cellular microenvironment (e.g. clathrin-coated vesicles or early endosomes), but no information about the structural basis underlying the cellular targeting and functional modification of KIAA1199 was available. Here, we show that the cleavage of N-terminal 30 amino acids occurs in functionally matured KIAA1199, and the deletion of the N-terminal portion results in altered intracellular trafficking of the molecule and loss of cellular HA depolymerization. These results suggest that the N-terminal portion of KIAA1199 functions as a cleavable signal sequence required for proper KIAA1199 translocation and KIAA1199-mediated HA depolymerization.

  18. A Genome-Wide Screen in Yeast Identifies Specific Oxidative Stress Genes Required for the Maintenance of Sub-Cellular Redox Homeostasis

    PubMed Central

    Ayer, Anita; Fellermeier, Sina; Fife, Christopher; Li, Simone S.; Smits, Gertien; Meyer, Andreas J.; Dawes, Ian W.; Perrone, Gabriel G.

    2012-01-01

    Maintenance of an optimal redox environment is critical for appropriate functioning of cellular processes and cell survival. Despite the importance of maintaining redox homeostasis, it is not clear how the optimal redox potential is sensed and set, and the processes that impact redox on a cellular/organellar level are poorly understood. The genetic bases of cellular redox homeostasis were investigated using a green fluorescent protein (GFP) based redox probe, roGFP2 and a pH sensitive GFP-based probe, pHluorin. The use of roGFP2, in conjunction with pHluorin, enabled determination of pH-adjusted sub-cellular redox potential in a non-invasive and real-time manner. A genome-wide screen using both the non-essential and essential gene collections was carried out in Saccharomyces cerevisiae using cytosolic-roGFP2 to identify factors essential for maintenance of cytosolic redox state under steady-state conditions. 102 genes of diverse function were identified that are required for maintenance of cytosolic redox state. Mutations in these genes led to shifts in the half-cell glutathione redox potential by 75-10 mV. Interestingly, some specific oxidative stress-response processes were identified as over-represented in the data set. Further investigation of the role of oxidative stress-responsive systems in sub-cellular redox homeostasis was conducted using roGFP2 constructs targeted to the mitochondrial matrix and peroxisome and EGSH was measured in cells in exponential and stationary phase. Analyses allowed for the identification of key redox systems on a sub-cellular level and the identification of novel genes involved in the regulation of cellular redox homeostasis. PMID:22970195

  19. A genome-wide screen in yeast identifies specific oxidative stress genes required for the maintenance of sub-cellular redox homeostasis.

    PubMed

    Ayer, Anita; Fellermeier, Sina; Fife, Christopher; Li, Simone S; Smits, Gertien; Meyer, Andreas J; Dawes, Ian W; Perrone, Gabriel G

    2012-01-01

    Maintenance of an optimal redox environment is critical for appropriate functioning of cellular processes and cell survival. Despite the importance of maintaining redox homeostasis, it is not clear how the optimal redox potential is sensed and set, and the processes that impact redox on a cellular/organellar level are poorly understood. The genetic bases of cellular redox homeostasis were investigated using a green fluorescent protein (GFP) based redox probe, roGFP2 and a pH sensitive GFP-based probe, pHluorin. The use of roGFP2, in conjunction with pHluorin, enabled determination of pH-adjusted sub-cellular redox potential in a non-invasive and real-time manner. A genome-wide screen using both the non-essential and essential gene collections was carried out in Saccharomyces cerevisiae using cytosolic-roGFP2 to identify factors essential for maintenance of cytosolic redox state under steady-state conditions. 102 genes of diverse function were identified that are required for maintenance of cytosolic redox state. Mutations in these genes led to shifts in the half-cell glutathione redox potential by 75-10 mV. Interestingly, some specific oxidative stress-response processes were identified as over-represented in the data set. Further investigation of the role of oxidative stress-responsive systems in sub-cellular redox homeostasis was conducted using roGFP2 constructs targeted to the mitochondrial matrix and peroxisome and E(GSH) was measured in cells in exponential and stationary phase. Analyses allowed for the identification of key redox systems on a sub-cellular level and the identification of novel genes involved in the regulation of cellular redox homeostasis.

  20. Transcriptome analyses identify key cellular factors associated with HIV-1 associated neuropathogenesis in infected men

    PubMed Central

    Venkatachari, Narasimhan J.; Jain, Siddhartha; Walker, Leah; Bivalkar-Mehla, Shalmali; Chattopadhyay, Ansuman; Bar-Joseph, Ziv; Rinaldo, Charles; Ragin, Ann; Seaberg, Eric; Levine, Andrew; Becker, James; Martin, Eileen; Sacktor, Ned; Ayyavoo, Velpandi

    2017-01-01

    Objective HIV-1 viral proteins and host inflammatory factors have a direct role in neuronal toxicity in in vitro, however, the contribution of these factors in vivo in HIV-1 associated neurocognitive disorder (HAND) is not fully understood. We applied novel Systems Biology approaches to identify specific cellular and viral factors and their related pathways that are associated with different stages of HAND. Design A cross-sectional study of individuals enrolled in the Multicenter AIDS Cohort Study (MACS) including HIV-1 seronegative (N=36) and HIV-1 seropositive individuals without neurocognitive symptoms (N=16), or with mild neurocognitive disorder (MND) (N=8) or HIV-associated dementia (HAD) (N=16). Methods A systematic evaluation of global transcriptome of peripheral blood mononuclear cells (PBMCs) obtained from HIV-1 seronegative individuals and from HIV-1 positive men without neurocognitive symptoms, or MND or HAD was performed. Results MND and HAD were associated with specific changes in mRNA transcripts and miRNAs in PBMCs. Comparison of upstream regulators and TimePath analyses identified specific cellular factors associated with MND and HAD, while HIV-1 viral proteins played a greater role in HAD. Additionally, expression of specific microRNAs – miR-let-7a, miR-124, miR-15a and others were found to correlate with mRNA gene expression and may have a potential protective role in asymptomatic HIV-1 seropositive individuals by regulating cellular signal transduction pathways downstream of chemokines and cytokines. Conclusions These results identify signature transcriptome changes in PBMCs associated with stages of HAND and shed light on the potential contribution of host cellular factors and viral proteins in HAND development. PMID:28005686

  1. Only signaling modules that discriminate sharply between stimulatory and nonstimulatory inputs require basal signaling for fast cellular responses

    NASA Astrophysics Data System (ADS)

    Artomov, Mykyta; Kardar, Mehran; Chakraborty, Arup K.

    2010-09-01

    In many types of cells, binding of molecules to their receptors enables cascades of intracellular chemical reactions to take place (signaling). However, a low level of signaling also occurs in most unstimulated cells. Such basal signaling in resting cells can have many functions, one of which is that it is thought to be required for fast cellular responses to external stimuli. A mechanistic understanding of why this is true and which features of cellular signaling networks make basal signaling necessary for fast responses is unknown. We address this issue by obtaining the time required for activation of common types of cell signaling modules with and without basal signaling. Our results show that the absence of basal signaling does not have any dramatic effects on the response time for signaling modules that exhibit a graded response to increasing stimulus levels. In sharp contrast, signaling modules that exhibit sharp dose-response curves which discriminate sensitively between stimuli to which the cell needs to respond and low-grade inputs (or stochastic noise) require basal signaling for fast cellular responses. In such cases, we find that an optimal level of basal signaling balances the requirements for fast cellular responses while minimizing spurious activation without appropriate stimulation.

  2. Discovery of Cellular Proteins Required for the Early Steps of HCV Infection Using Integrative Genomics

    PubMed Central

    Yang, Jae-Seong; Kwon, Oh Sung; Kim, Sanguk; Jang, Sung Key

    2013-01-01

    Successful viral infection requires intimate communication between virus and host cell, a process that absolutely requires various host proteins. However, current efforts to discover novel host proteins as therapeutic targets for viral infection are difficult. Here, we developed an integrative-genomics approach to predict human genes involved in the early steps of hepatitis C virus (HCV) infection. By integrating HCV and human protein associations, co-expression data, and tight junction-tetraspanin web specific networks, we identified host proteins required for the early steps in HCV infection. Moreover, we validated the roles of newly identified proteins in HCV infection by knocking down their expression using small interfering RNAs. Specifically, a novel host factor CD63 was shown to directly interact with HCV E2 protein. We further demonstrated that an antibody against CD63 blocked HCV infection, indicating that CD63 may serve as a new therapeutic target for HCV-related diseases. The candidate gene list provides a source for identification of new therapeutic targets. PMID:23593195

  3. Membrane Association via an Amino-terminal Amphipathic Helix Is Required for the Cellular Organization and Function of RNase II*

    PubMed Central

    Lu, Feng; Taghbalout, Aziz

    2013-01-01

    The subcellular localization of the exoribonuclease RNase II is not known despite the advanced biochemical characterization of the enzyme. Here we report that RNase II is organized into cellular structures that appear to coil around the Escherichia coli cell periphery and that RNase II is associated with the cytoplasmic membrane by its amino-terminal amphipathic helix. The helix also acts as an autonomous transplantable membrane binding domain capable of directing normally cytoplasmic proteins to the membrane. Assembly of the organized cellular structures of RNase II required the RNase II amphipathic membrane binding domain. Co-immunoprecipitation of the protein from cell extracts indicated that RNase II interacts with itself. The RNase II self-interaction and the ability of the protein to assemble into organized cellular structures required the membrane binding domain. The ability of RNase II to maintain cell viability in the absence of the exoribonuclease polynucleotide phosphorylase was markedly diminished when the RNase II cellular structures were lost due to changes in the amphipathicity of the amino-terminal helix, suggesting that membrane association and assembly of RNase II into organized cellular structures play an important role in the normal function of the protein within the bacterial cell. PMID:23344958

  4. Membrane association via an amino-terminal amphipathic helix is required for the cellular organization and function of RNase II.

    PubMed

    Lu, Feng; Taghbalout, Aziz

    2013-03-08

    The subcellular localization of the exoribonuclease RNase II is not known despite the advanced biochemical characterization of the enzyme. Here we report that RNase II is organized into cellular structures that appear to coil around the Escherichia coli cell periphery and that RNase II is associated with the cytoplasmic membrane by its amino-terminal amphipathic helix. The helix also acts as an autonomous transplantable membrane binding domain capable of directing normally cytoplasmic proteins to the membrane. Assembly of the organized cellular structures of RNase II required the RNase II amphipathic membrane binding domain. Co-immunoprecipitation of the protein from cell extracts indicated that RNase II interacts with itself. The RNase II self-interaction and the ability of the protein to assemble into organized cellular structures required the membrane binding domain. The ability of RNase II to maintain cell viability in the absence of the exoribonuclease polynucleotide phosphorylase was markedly diminished when the RNase II cellular structures were lost due to changes in the amphipathicity of the amino-terminal helix, suggesting that membrane association and assembly of RNase II into organized cellular structures play an important role in the normal function of the protein within the bacterial cell.

  5. Clinical, Cellular, and Molecular Factors That Contribute to Antifungal Drug Resistance

    PubMed Central

    White, Theodore C.; Marr, Kieren A.; Bowden, Raleigh A.

    1998-01-01

    In the past decade, the frequency of diagnosed fungal infections has risen sharply due to several factors, including the increase in the number of immunosuppressed patients resulting from the AIDS epidemic and treatments during and after organ and bone marrow transplants. Linked with the increase in fungal infections is a recent increase in the frequency with which these infections are recalcitrant to standard antifungal therapy. This review summarizes the factors that contribute to antifungal drug resistance on three levels: (i) clinical factors that result in the inability to successfully treat refractory disease; (ii) cellular factors associated with a resistant fungal strain; and (iii) molecular factors that are ultimately responsible for the resistance phenotype in the cell. Many of the clinical factors that contribute to resistance are associated with the immune status of the patient, with the pharmacology of the drugs, or with the degree or type of fungal infection present. At a cellular level, antifungal drug resistance can be the result of replacement of a susceptible strain with a more resistant strain or species or the alteration of an endogenous strain (by mutation or gene expression) to a resistant phenotype. The molecular mechanisms of resistance that have been identified to date in Candida albicans include overexpression of two types of efflux pumps, overexpression or mutation of the target enzyme, and alteration of other enzymes in the same biosynthetic pathway as the target enzyme. Since the study of antifungal drug resistance is relatively new, other factors that may also contribute to resistance are discussed. PMID:9564569

  6. 14 CFR 121.647 - Factors for computing fuel required.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 3 2010-01-01 2010-01-01 false Factors for computing fuel required. 121.647 Section 121.647 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF... § 121.647 Factors for computing fuel required. Each person computing fuel required for the purposes...

  7. Global analysis of bacterial transcription factors to predict cellular target processes.

    PubMed

    Doerks, Tobias; Andrade, Miguel A; Lathe, Warren; von Mering, Christian; Bork, Peer

    2004-03-01

    Whole-genome sequences are now available for >100 bacterial species, giving unprecedented power to comparative genomics approaches. We have applied genome-context methods to predict target processes that are regulated by transcription factors (TFs). Of 128 orthologous groups of proteins annotated as TFs, to date, 36 are functionally uncharacterized; in our analysis we predict a probable cellular target process or biochemical pathway for half of these functionally uncharacterized TFs.

  8. Using exploratory regression to identify optimal driving factors for cellular automaton modeling of land use change.

    PubMed

    Feng, Yongjiu; Tong, Xiaohua

    2017-09-22

    Defining transition rules is an important issue in cellular automaton (CA)-based land use modeling because these models incorporate highly correlated driving factors. Multicollinearity among correlated driving factors may produce negative effects that must be eliminated from the modeling. Using exploratory regression under pre-defined criteria, we identified all possible combinations of factors from the candidate factors affecting land use change. Three combinations that incorporate five driving factors meeting pre-defined criteria were assessed. With the selected combinations of factors, three logistic regression-based CA models were built to simulate dynamic land use change in Shanghai, China, from 2000 to 2015. For comparative purposes, a CA model with all candidate factors was also applied to simulate the land use change. Simulations using three CA models with multicollinearity eliminated performed better (with accuracy improvements about 3.6%) than the model incorporating all candidate factors. Our results showed that not all candidate factors are necessary for accurate CA modeling and the simulations were not sensitive to changes in statistically non-significant driving factors. We conclude that exploratory regression is an effective method to search for the optimal combinations of driving factors, leading to better land use change models that are devoid of multicollinearity. We suggest identification of dominant factors and elimination of multicollinearity before building land change models, making it possible to simulate more realistic outcomes.

  9. Cellular transcription factor YY1 mediates the varicella-zoster virus (VZV) IE62 transcriptional activation.

    PubMed

    Khalil, Mohamed I; Sommer, Marvin; Arvin, Ann; Hay, John; Ruyechan, William T

    2014-01-20

    Several cellular transcription factors have been shown to be involved in IE62-mediated activation. The YY1 cellular transcription factor has activating and repressive effects on gene transcription. Analysis of the VZV genome revealed 19 postulated YY1 binding sites located within putative promoters of 16 VZV genes. Electrophoretic mobility shift assays (EMSA) confirmed the binding of YY1 to ORF10, ORF28/29 and gI promoters and the mutation of these binding sites inhibited YY1 binding and the promoter activation by IE62 alone or following VZV infection. Mutation of the ORF28/29 YY1 site in the VZV genome displayed insignificant influence on virus growth in melanoma cells; but it inhibited the virus replication significantly at day 5 and 6 post infection in HELF cells. This work suggests a novel role for the cellular factor YY1 in VZV replication through the mediation of IE62 activation of viral gene expression. © 2013 Elsevier Inc. All rights reserved.

  10. Role of Fatty Acid Kinase in Cellular Lipid Homeostasis and SaeRS-Dependent Virulence Factor Expression in Staphylococcus aureus

    PubMed Central

    Ericson, Megan E.; Subramanian, Chitra; Frank, Matthew W.

    2017-01-01

    ABSTRACT The SaeRS two-component system is a master activator of virulence factor transcription in Staphylococcus aureus, but the cellular factors that control its activity are unknown. Fatty acid (FA) kinase is a two-component enzyme system required for extracellular FA uptake and SaeRS activity. Here, we demonstrate the existence of an intracellular nonesterified FA pool in S. aureus that is elevated in strains lacking FA kinase activity. SaeRS-mediated transcription is restored in FA kinase-negative strains when the intracellular FA pool is reduced either by growth with FA-depleted bovine serum albumin to extract the FA into the medium or by the heterologous expression of Neisseria gonorrhoeae acyl-acyl carrier protein synthetase to activate FA for phospholipid synthesis. These data show that FAs act as negative regulators of SaeRS signaling, and FA kinase activates SaeRS-dependent virulence factor production by lowering inhibitory FA levels. Thus, FA kinase plays a role in cellular lipid homeostasis by activating FA for incorporation into phospholipid, and it indirectly regulates SaeRS signaling by maintaining a low intracellular FA pool. PMID:28765222

  11. Ubiquitylation-dependent regulation of NEIL1 by Mule and TRIM26 is required for the cellular DNA damage response

    PubMed Central

    Edmonds, Matthew J.; Carter, Rachel J.; Nickson, Catherine M.; Williams, Sarah C.; Parsons, Jason L.

    2017-01-01

    Endonuclease VIII-like protein 1 (NEIL1) is a DNA glycosylase involved in initiating the base excision repair pathway, the major cellular mechanism for repairing DNA base damage. Here, we have purified the major E3 ubiquitin ligases from human cells responsible for regulation of NEIL1 by ubiquitylation. Interestingly, we have identified two enzymes that catalyse NEIL1 polyubiquitylation, Mcl-1 ubiquitin ligase E3 (Mule) and tripartite motif 26 (TRIM26). We demonstrate that these enzymes are capable of polyubiquitylating NEIL1 in vitro, and that both catalyse ubiquitylation of NEIL1 within the same C-terminal lysine residues. An siRNA-mediated knockdown of Mule or TRIM26 leads to stabilisation of NEIL1, demonstrating that these enzymes are important in regulating cellular NEIL1 steady state protein levels. Similarly, a mutant NEIL1 protein lacking residues for ubiquitylation is more stable than the wild type protein in vivo. We also demonstrate that cellular NEIL1 protein is induced in response to ionising radiation (IR), although this occurs specifically in a Mule-dependent manner. Finally we show that stabilisation of NEIL1, particularly following TRIM26 siRNA, contributes to cellular resistance to IR. This highlights the importance of Mule and TRIM26 in maintaining steady state levels of NEIL1, but also those required for the cellular DNA damage response. PMID:27924031

  12. Nuclear localization signal sequence is required for VACM-1/CUL5-dependent regulation of cellular growth.

    PubMed

    Willis, Angelica N; Dean, Shirley E Bradley; Habbouche, Joe A; Kempers, Brian T; Ludwig, Megan L; Sayfie, Aaron D; Lewis, Steven P; Harrier, Stephanie; DeBruine, Zachary J; Garrett, Richard; Burnatowska-Hledin, Maria A

    2017-04-01

    VACM-1/CUL5 is a member of the cullin family of proteins involved in the E3 ligase-dependent degradation of diverse proteins that regulate cellular proliferation. The ability of VACM-1/CUL5 to inhibit cellular growth is affected by its posttranslational modifications and its localization to the nucleus. Since the mechanism of VACM-1/CUL5 translocation to the nucleus is not clear, the goal of this project was to determine the role that the putative nuclear localization signal (NLS) we identified in the VACM-1/CUL5 ((640)PKLKRQ(646)) plays in the cellular localization of VACM-1/CUL5 and its effect on cellular growth. We used site-directed mutagenesis to change Lys642 and Lys644 to Gly and the mutated cDNA constructs were transfected into COS-1 cells. Mutation of the NLS in VACM-1/CUL5 significantly reduced its localization to the nucleus and compromised its effect on cellular growth. We have shown previously that the antiproliferative effect of VACM-1/CUL5 could be reversed by mutation of PKA-specific phosphorylation sequence ((S730A)VACM-1/CUL5), which was associated with its increased nuclear localization and modification by NEDD8. Thus, we examined whether these properties can be controlled by the NLS. The mutation of NLS in (S730A)VACM-1/CUL5 cDNA compromised its proliferative effect and reduced its localization to the nucleus. The immunocytochemistry results showed that, in cells transfected with the mutant cDNAs, the nuclear NEDD8 signal was decreased. Western blot analysis of total cell lysates, however, showed that VACM-1/CUL5 neddylation was not affected. Together, these results suggest that the presence of the NLS, both in VACM-1/CUL5 and in (S730A)VACM-1/CUL5 sequences, is critical for their control of cell proliferation.

  13. The cellular factor TRP-185 regulates RNA polymerase II binding to HIV-1 TAR RNA.

    PubMed Central

    Wu-Baer, F; Lane, W S; Gaynor, R B

    1995-01-01

    Activation of HIV-1 gene expression by the transactivator Tat is dependent on an RNA regulatory element located downstream of the transcription initiation site known as TAR. To characterize cellular factors that bind to TAR RNA and are involved in the regulation of HIV-1 transcription, HeLa nuclear extract was fractionated and RNA gel-retardation analysis was performed. This analysis indicated that only two cellular factors, RNA polymerase II and the previously characterized TAR RNA loop binding protein TRP-185, were capable of binding specifically to TAR RNA. To elucidate the function of TRP-185, it was purified from HeLa nuclear extract, amino acid microsequence analysis was performed and a cDNA encoding TRP-185 was isolated. TRP-185 is a novel protein of 1621 amino acids which contains a leucine zipper and potentially a novel RNA binding motif. In gel-retardation assays, the binding of both recombinant TRP-185 and RNA polymerase II was dependent on the presence of an additional group of proteins designated cellular cofactors. Both the TAR RNA loop and bulge sequences were critical for RNA polymerase II binding, while TRP-185 binding was dependent only on TAR RNA loop sequences. Since binding of TRP-185 and RNA polymerase II to TAR RNA was found to be mutually exclusive, our results suggest that TRP-185 may function either alone or in conjunction with Tat to disengage RNA polymerase II which is stalled upon binding to nascently synthesized TAR RNA during transcriptional elongation. Images PMID:8846792

  14. The transcription factor FOXM1 is a cellular target of the natural product thiostrepton

    NASA Astrophysics Data System (ADS)

    Hegde, Nagaratna S.; Sanders, Deborah A.; Rodriguez, Raphaël; Balasubramanian, Shankar

    2011-09-01

    Transcription factors are proteins that bind specifically to defined DNA sequences to promote gene expression. Targeting transcription factors with small molecules to modulate the expression of certain genes has been notoriously difficult to achieve. The natural product thiostrepton is known to reduce the transcriptional activity of FOXM1, a transcription factor involved in tumorigenesis and cancer progression. Herein we demonstrate that thiostrepton interacts directly with FOXM1 protein in the human breast cancer cells MCF-7. Biophysical analyses of the thiostrepton-FOXM1 interaction provide additional insights on the molecular mode of action of thiostrepton. In cellular experiments, we show that thiostrepton can inhibit the binding of FOXM1 to genomic target sites. These findings illustrate the potential druggability of transcription factors and provide a molecular basis for targeting the FOXM1 family with small molecules.

  15. Transforming growth factor-beta1 mediates cellular response to DNA damage in situ

    NASA Technical Reports Server (NTRS)

    Ewan, Kenneth B.; Henshall-Powell, Rhonda L.; Ravani, Shraddha A.; Pajares, Maria Jose; Arteaga, Carlos; Warters, Ray; Akhurst, Rosemary J.; Barcellos-Hoff, Mary Helen

    2002-01-01

    Transforming growth factor (TGF)-beta1 is rapidly activated after ionizing radiation, but its specific role in cellular responses to DNA damage is not known. Here we use Tgfbeta1 knockout mice to show that radiation-induced apoptotic response is TGF-beta1 dependent in the mammary epithelium, and that both apoptosis and inhibition of proliferation in response to DNA damage decrease as a function of TGF-beta1 gene dose in embryonic epithelial tissues. Because apoptosis in these tissues has been shown previously to be p53 dependent, we then examined p53 protein activation. TGF-beta1 depletion, by either gene knockout or by using TGF-beta neutralizing antibodies, resulted in decreased p53 Ser-18 phosphorylation in irradiated mammary gland. These data indicate that TGF-beta1 is essential for rapid p53-mediated cellular responses that mediate cell fate decisions in situ.

  16. Transforming growth factor-beta1 mediates cellular response to DNA damage in situ

    NASA Technical Reports Server (NTRS)

    Ewan, Kenneth B.; Henshall-Powell, Rhonda L.; Ravani, Shraddha A.; Pajares, Maria Jose; Arteaga, Carlos; Warters, Ray; Akhurst, Rosemary J.; Barcellos-Hoff, Mary Helen

    2002-01-01

    Transforming growth factor (TGF)-beta1 is rapidly activated after ionizing radiation, but its specific role in cellular responses to DNA damage is not known. Here we use Tgfbeta1 knockout mice to show that radiation-induced apoptotic response is TGF-beta1 dependent in the mammary epithelium, and that both apoptosis and inhibition of proliferation in response to DNA damage decrease as a function of TGF-beta1 gene dose in embryonic epithelial tissues. Because apoptosis in these tissues has been shown previously to be p53 dependent, we then examined p53 protein activation. TGF-beta1 depletion, by either gene knockout or by using TGF-beta neutralizing antibodies, resulted in decreased p53 Ser-18 phosphorylation in irradiated mammary gland. These data indicate that TGF-beta1 is essential for rapid p53-mediated cellular responses that mediate cell fate decisions in situ.

  17. Interacting factors and cellular localization of SR protein-specific kinase Dsk1

    SciTech Connect

    Tang, Zhaohua; Luca, Maria; Taggart-Murphy, Laura; Portillio, Jessica; Chang, Cathey; Guven, Ayse; Lin, Ren-Jang; Murray, Johanne; Carr, Antony

    2012-10-01

    Schizosaccharomyces pombe Dsk1 is an SR protein-specific kinase (SRPK), whose homologs have been identified in every eukaryotic organism examined. Although discovered as a mitotic regulator with protein kinase activity toward SR splicing factors, it remains largely unknown about what and how Dsk1 contributes to cell cycle and pre-mRNA splicing. In this study, we investigated the Dsk1 function by determining interacting factors and cellular localization of the kinase. Consistent with its reported functions, we found that pre-mRNA processing and cell cycle factors are prominent among the proteins co-purified with Dsk1. The identification of these factors led us to find Rsd1 as a novel Dsk1 substrate, as well as the involvement of Dsk1 in cellular distribution of poly(A){sup +} RNA. In agreement with its role in nuclear events, we also found that Dsk1 is mainly localized in the nucleus during G{sub 2} phase and at mitosis. Furthermore, we revealed the oscillation of Dsk1 protein in a cell cycle-dependent manner. This paper marks the first comprehensive analysis of in vivo Dsk1-associated proteins in fission yeast. Our results reflect the conserved role of SRPK family in eukaryotic organisms, and provide information about how Dsk1 functions in pre-mRNA processing and cell-division cycle.

  18. Cognitive effects of cellular phones: a possible role of non-radiofrequency radiation factors.

    PubMed

    Hareuveny, Ronen; Eliyahu, Ilan; Luria, Roy; Meiran, Nachshon; Margaliot, Menachem

    2011-10-01

    Some studies found that cognitive functions of human beings may be altered while exposed to radiofrequency radiation (RFR) emitted by cellular phones. In two recent studies, we have found that experiment duration and exposure side (i.e., phone's location--right or left) may have a major influence on the detection of such effects. In this brief follow-up experiment, 29 right-handed male subjects were divided into two groups. Each subject had two standard cellular phones attached to both sides of his head. The subjects performed a spatial working memory task that required either a left-hand or a right-hand response under one of the two exposure conditions: left side of the head or right side. Contrary to our previous studies, in this work external antennas located far away from the subjects were connected to the cellular phones. This setup prevents any emission of RFR from the internal antenna, thus drastically reducing RFR exposure. Despite that, the results remain similar to those obtained in our previous work. These results indicate that some of the effects previously attributed to RFR can be the result of some confounders. Copyright © 2011 Wiley-Liss, Inc.

  19. Cellular kinetics of murine lung: model system to determine basis for radioprotection with keratinocyte growth factor.

    PubMed

    Terry, Nicholas H A; Brinkley, John; Doig, Andrew J; Ma, Jun; Patel, Nalini; White, R Allen; Mahajan, Nitin; Kang, Yun

    2004-02-01

    Normal tissue toxicity remains a dose limitation for cancer radiotherapy and chemoradiotherapy. Growth factors offer a novel means of mitigating normal tissue radiotoxicity. In particular, keratinocyte growth factor (rHuKGF), whose proliferative activity is restricted to epithelial cells, holds promise on the basis of the findings of preclinical models of epithelial cytoprotection and the clinical developments to date. We report the radioprotection of murine lung by an increase in tissue cellularity after rHuKGF-induced proliferation. Flow cytometric and image analysis techniques after bromodeoxyuridine labeling were used to estimate proliferative parameters. Our specialized analytical methods measure not only labeling indexes, but also the durations of S and G(2)+M phases, potential doubling times, and the net cell production rate. Image analysis techniques were used to identify the specific cell types that were proliferating (type II pneumocytes). Lung labeling index control values (0.5%) rose to a maximum (5.5%) at 3 days after intratracheal rHuKGF, returning to normal by Day 7. The potential doubling time fell from 66 days to 4.4 days. The net cell production rate rose from a control value of 1%/d to >15%/d by Day 3. This resulted in a nearly twofold increase in alveolar epithelial cellularity, which remained significantly elevated on Day 7. Saline-treated control animals exhibited no significant changes in the proliferative parameter values or cellularity. On the basis of these data, mice were irradiated, solely to the thorax, with ranges of single doses of 250 kVp X-rays 7 days after either intratracheal administration of 5 mg/kg rHuKGF or phospate-buffered saline. This interval was chosen because the proliferative response of the type II cells was finished but the cellularity of the lung remained increased. Pretreatment with rHuKGF extended the latent period before onset of pneumonitis after all radiation doses. rHuKGF treatment 7 days before thoracic

  20. Modeling Cellular Noise Underlying Heterogeneous Cell Responses in the Epidermal Growth Factor Signaling Pathway

    PubMed Central

    Iwamoto, Kazunari; Shindo, Yuki; Takahashi, Koichi

    2016-01-01

    Cellular heterogeneity, which plays an essential role in biological phenomena, such as drug resistance and migration, is considered to arise from intrinsic (i.e., reaction kinetics) and extrinsic (i.e., protein variability) noise in the cell. However, the mechanistic effects of these types of noise to determine the heterogeneity of signal responses have not been elucidated. Here, we report that the output of epidermal growth factor (EGF) signaling activity is modulated by cellular noise, particularly by extrinsic noise of particular signaling components in the pathway. We developed a mathematical model of the EGF signaling pathway incorporating regulation between extracellular signal-regulated kinase (ERK) and nuclear pore complex (NPC), which is necessary for switch-like activation of the nuclear ERK response. As the threshold of switch-like behavior is more sensitive to perturbations than the graded response, the effect of biological noise is potentially critical for cell fate decision. Our simulation analysis indicated that extrinsic noise, but not intrinsic noise, contributes to cell-to-cell heterogeneity of nuclear ERK. In addition, we accurately estimated variations in abundance of the signal proteins between individual cells by direct comparison of experimental data with simulation results using Apparent Measurement Error (AME). AME was constant regardless of whether the protein levels varied in a correlated manner, while covariation among proteins influenced cell-to-cell heterogeneity of nuclear ERK, suppressing the variation. Simulations using the estimated protein abundances showed that each protein species has different effects on cell-to-cell variation in the nuclear ERK response. In particular, variability of EGF receptor, Ras, Raf, and MEK strongly influenced cellular heterogeneity, while others did not. Overall, our results indicated that cellular heterogeneity in response to EGF is strongly driven by extrinsic noise, and that such heterogeneity

  1. Structural requirements for the assembly of LINC complexes and their function in cellular mechanical stiffness

    SciTech Connect

    Stewart-Hutchinson, P.J.; Hale, Christopher M.; Wirtz, Denis; Hodzic, Didier

    2008-05-01

    The evolutionary-conserved interactions between KASH and SUN domain-containing proteins within the perinuclear space establish physical connections, called LINC complexes, between the nucleus and the cytoskeleton. Here, we show that the KASH domains of Nesprins 1, 2 and 3 interact promiscuously with luminal domains of Sun1 and Sun2. These constructs disrupt endogenous LINC complexes as indicated by the displacement of endogenous Nesprins from the nuclear envelope. We also provide evidence that KASH domains most probably fit a pocket provided by SUN domains and that post-translational modifications are dispensable for that interaction. We demonstrate that the disruption of endogenous LINC complexes affect cellular mechanical stiffness to an extent that compares to the loss of mechanical stiffness previously reported in embryonic fibroblasts derived from mouse lacking A-type lamins, a mouse model of muscular dystrophies and cardiomyopathies. These findings support a model whereby physical connections between the nucleus and the cytoskeleton are mediated by interactions between diverse combinations of Sun proteins and Nesprins through their respective evolutionary-conserved domains. Furthermore, they emphasize, for the first time, the relevance of LINC complexes in cellular mechanical stiffness suggesting a possible involvement of their disruption in various laminopathies, a group of human diseases linked to mutations of A-type lamins.

  2. Visual Genome-Wide RNAi Screening to Identify Human Host Factors Required for Trypanosoma cruzi Infection

    PubMed Central

    de Macedo Dossin, Fernando; Choi, Seo Yeon; Kim, Nam Youl; Kim, Hi Chul; Jung, Sung Yong; Schenkman, Sergio; Almeida, Igor C.; Emans, Neil; Freitas-Junior, Lucio H.

    2011-01-01

    The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical infection that affects millions of people in the Americas. Current chemotherapy relies on only two drugs that have limited efficacy and considerable side effects. Therefore, the development of new and more effective drugs is of paramount importance. Although some host cellular factors that play a role in T. cruzi infection have been uncovered, the molecular requirements for intracellular parasite growth and persistence are still not well understood. To further study these host-parasite interactions and identify human host factors required for T. cruzi infection, we performed a genome-wide RNAi screen using cellular microarrays of a printed siRNA library that spanned the whole human genome. The screening was reproduced 6 times and a customized algorithm was used to select as hits those genes whose silencing visually impaired parasite infection. The 162 strongest hits were subjected to a secondary screening and subsequently validated in two different cell lines. Among the fourteen hits confirmed, we recognized some cellular membrane proteins that might function as cell receptors for parasite entry and others that may be related to calcium release triggered by parasites during cell invasion. In addition, two of the hits are related to the TGF-beta signaling pathway, whose inhibition is already known to diminish levels of T. cruzi infection. This study represents a significant step toward unveiling the key molecular requirements for host cell invasion and revealing new potential targets for antiparasitic therapy. PMID:21625474

  3. Newborn human skin fibroblasts senesce in vitro without acquiring adult growth factor requirements

    SciTech Connect

    Wharton, W.

    1984-01-01

    Cultures of human fibroblasts were prepared from chest skin obtained either from newborns (less than 3 months old) or adults (more than 35 years old) and maintained in vitro until they senesced. Adult cells grew logarithmically in medium supplemented with whole blood serum but not with platelet-poor plasma. Early passage cells obtained from newborns grew equally well in either plasma- or serum-supplemented medium. The difference in growth factor requirements between adult and newborn cells persisted through the lifespan of the cells; i.e., newborn cells did not develop adult hormonal requirements when maintained in culture. Thus, in vitro cellular aging can be distinguished from some types of differentiation.

  4. Viral and Cellular Factors Involved in Phloem Transport of Plant Viruses

    PubMed Central

    Hipper, Clémence; Brault, Véronique; Ziegler-Graff, Véronique; Revers, Frédéric

    2013-01-01

    Phloem transport of plant viruses is an essential step in the setting-up of a complete infection of a host plant. After an initial replication step in the first cells, viruses spread from cell-to-cell through mesophyll cells, until they reach the vasculature where they rapidly move to distant sites in order to establish the infection of the whole plant. This last step is referred to as systemic transport, or long-distance movement, and involves virus crossings through several cellular barriers: bundle sheath, vascular parenchyma, and companion cells for virus loading into sieve elements (SE). Viruses are then passively transported within the source-to-sink flow of photoassimilates and are unloaded from SE into sink tissues. However, the molecular mechanisms governing virus long-distance movement are far from being understood. While most viruses seem to move systemically as virus particles, some viruses are transported in SE as viral ribonucleoprotein complexes (RNP). The nature of the cellular and viral factors constituting these RNPs is still poorly known. The topic of this review will mainly focus on the host and viral factors that facilitate or restrict virus long-distance movement. PMID:23745125

  5. Cellular factors controlling neuronal vulnerability in the brain: a lesson from the striatum.

    PubMed

    Calabresi, P; Centonze, D; Bernardi, G

    2000-11-14

    In many acute and chronic neurologic disorders, both deficits in energy metabolism and defects in glutamate-mediated excitatory synaptic transmission have been proposed as important pathogenic factors. Brain cells, however, express variable vulnerability to these insults, as indicated by the fact that certain brain areas and even different cell types in the same area are preferentially spared until the very late stages of various diseases. This can be clearly seen in the striatum, where GABAergic projection cells but not cholinergic interneurons are precociously damaged in the course of both acute metabolic insults (such as hypoxia, hypoglycemia, and ischemia) and chronic neurodegenerative disorders (such as Huntington's disease). A well-mapped pattern of cell loss, in fact, is a common finding in the striatum of patients suffering from these pathologic conditions. Physiologic and molecular studies have been directed in recent years to the identification of the cellular mechanisms underlying the cell-type specific vulnerability of striatal cells. These studied recognized that, in striatal spiny and aspiny cells, specific membrane ion channels, glutamate receptor subtypes and subunits, and intracellular enzymatic activities are involved in the cascade of events responsible for opposite responses and vulnerabilities to oxygen or glucose deprivation and to glutamate receptor-mediated toxicity. Specific molecules able to target these cellular factors might be employed as therapeutic agents during acute and chronic neurologic disorders affecting this brain area.

  6. Mitochondrial transcription factor A (TFAM): roles in maintenance of mtDNA and cellular functions.

    PubMed

    Kang, Dongchon; Kim, Sang Ho; Hamasaki, Naotaka

    2007-01-01

    A growing body of evidence suggests that mammalian mitochondrial DNA takes on higher structure called nucleoid or mitochromosome corresponding to that of nuclear DNA. Mitochondrial transcription factor A (TFAM), which was cloned as a transcription factor for mitochondrial DNA, has known to be essential for the maintenance of mitochondrial DNA. Human TFAM has an ability to bind to DNA in a sequence-independent manner and is abundant enough to cover whole region of mitochondrial DNA, owing to which TFAM stabilizes mitochondrial DNA through formation of nucleoid and regulates (or titrates) the amount of mitochondrial DNA. Overexpression of human TFAM in mice increases the amount of mitochondrial DNA and dramatically ameliorates the cardiac dysfunctions caused by myocardial infarction. The maintenance of integrity of mitochondrial DNA is important for keeping proper cellular functions both under physiological and pathological conditions. TFAM may play a crucial role in maintaining mitochondrial DNA as a main component of the nucleoid.

  7. CNS autoimmune disease after Streptococcus pyogenes infections: animal models, cellular mechanisms and genetic factors

    PubMed Central

    Cutforth, Tyler; DeMille, Mellissa MC; Agalliu, Ilir; Agalliu, Dritan

    2016-01-01

    Streptococcus pyogenes infections have been associated with two autoimmune diseases of the CNS: Sydenham’s chorea (SC) and Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus infections (PANDAS). Despite the high frequency of pharyngeal streptococcus infections among children, only a small fraction develops SC or PANDAS. This suggests that several factors in combination are necessary to trigger autoimmune complications: specific S. pyogenes strains that induce a strong immune response toward the host nervous system; genetic susceptibility that predispose children toward an autoimmune response involving movement or tic symptoms; and multiple infections of the throat or tonsils that lead to a robust Th17 cellular and humoral immune response when untreated. In this review, we summarize the evidence for each factor and propose that all must be met for the requisite neurovascular pathology and behavioral deficits found in SC/PANDAS. PMID:27110222

  8. Interactions between tobamovirus replication proteins and cellular factors: their impacts on virus multiplication.

    PubMed

    Ishibashi, Kazuhiro; Nishikiori, Masaki; Ishikawa, Masayuki

    2010-11-01

    Most viral gene products function inside cells in the presence of various host proteins, nucleic acids, and lipids. Thus, viral gene products come into direct contact with these molecules. The replication proteins of tobamovirus participate not only in viral genome replication but also in counterdefense mechanisms against RNA silencing and other plant defense systems. Accumulating evidence indicates that these functions are carried out through interactions with specific host components. Interactions with some cellular factors, however, are inhibitory to virus multiplication and contribute to host range restriction of tobamovirus. The interactions that have positive and negative impacts on virus multiplication should have been maintained and lost, respectively, during adaptation of the viruses to their respective natural hosts. This review lists the host factors that interact with the replication proteins of tobamovirus and discusses how they influence multiplication of the virus.

  9. The guanine nucleotide exchange factor Tiam1: a Janus-faced molecule in cellular signaling.

    PubMed

    Boissier, P; Huynh-Do, U

    2014-03-01

    The Rho family of GTPases consists of several small proteins that have been described as molecular switches, playing important roles in a wide variety of fundamental cellular processes and in human diseases such as cancer. These proteins, active in the GTP conformation and inactive in the GDP form, are in turn regulated by guanine nucleotide exchange factors (GEFs), guanine nucleotide activating proteins (GAPs) and guanine dissociation inhibitors (GDIs). Two decades ago, Tiam1 (T-lymphoma invasion and metastasis) was identified as a GEF specific for Rac1 activation, but also for Cdc42 and in a lesser extent RhoA. Acting principally upstream of Rac1, Tiam1 is mainly involved in the regulation of Rac1 mediated signaling pathways including cytoskeletal activities, cell polarity, endocytosis and membrane trafficking, cell migration, adhesion and invasion, cell growth and survival, metastasis and carcinogenesis. However, given the large number of protein interaction domains found in its structure, it is possible that Tiam1 affects cellular processes in another way than through its GEF activity by interactions with other signaling proteins. Due to its functional diversity, Tiam1 is involved in multiple steps of tumorigenesis. As its name suggests, Tiam1 has been shown to increase T-cell lymphoma invasion and metastasis. It also promotes migration of fibroblasts, neuronal and cancer cells. On the contrary, Tiam1-induced cell adhesion has also been described, as opposed to cell migration. Moreover, studies indicate that Tiam1 is involved in both anti-apoptotic and pro-apoptotic mechanisms. While increasing evidence has demonstrated Tiam1's contribution to tumorigenesis and metastasis, others suggest that Tiam1 could have anti-cancer properties. In the present review, we discuss the current knowledge about the controversial roles of Tiam1 in cellular signaling. In particular, we will focus on Tiam1's regulation, its biological functions and implication in cancer. Copyright

  10. Quantifying colocalization of a conditionally active transcription factor FOXP3 in three-dimensional cellular space

    NASA Astrophysics Data System (ADS)

    Abraham, Thomas; Allan, Sarah E.; Levings, Megan K.

    2009-02-01

    Biological macromolecular interactions between proteins, transcription factors, DNA and other types of biomolecules, are fundamentally important to several cellular and biological processes. 3D Multi-channel confocal microscopy and colocalization analysis of fluorescent signals have proven to be invaluable tools for detecting such molecular interactions. The aim of this work was to quantify colocalization of the FOXP3 transcription factor in 3D cellular space generated from the confocal 3D image sets. 293T cells transfected with a conditionally active form of FOXP3 were stained for nuclei with Hoechst, for FOXP3 with anti-FOXP3 conjugated to PE, and 4-hydroxytamoxifen used as protein translocation and activation agent. Since the protein signal was weak and nonspecific intensity contributions were strong, it was difficult to perform colocalization analysis and estimate colocalization quantities. We performed 3D restoration by deconvolution method on the confocal images using experimentally measured point spread functions (PSFs) and subsequently a color shift correction. The deconvolution method eliminated nonspecific intensity contributions originating from PSF imposed by optical microscopy diffraction resolution limits and noise since these factors significantly affected colocalization analysis and quantification. Visual inspection of the deconvolved 3D image suggested that the FOXP3 molecules are predominantly colocalized within the nuclei although the fluorescent signals from FOXP3 molecules were also present in the cytoplasm. A close inspection of the scatter plot (colocalization map) and correlation quantities such as the Pearsons and colocalization coefficients showed that the fluorescent signals from the FOXP3 molecules and DNA are strongly correlated. In conclusion, our colocalization quantification approach confirms the preferential association of the FOXP3 molecules with the DNA despite the presence of fluorescent signals from the former one both in the

  11. Robust mechanisms of ventral furrow invagination require the combination of cellular shape changes

    NASA Astrophysics Data System (ADS)

    Conte, Vito; Muñoz, José J.; Baum, Buzz; Miodownik, Mark

    2009-03-01

    Ventral furrow formation in Drosophila is the first large-scale morphogenetic movement during the life of the embryo, and is driven by co-ordinated changes in the shape of individual epithelial cells within the cellular blastoderm. Although many of the genes involved have been identified, the details of the mechanical processes that convert local changes in gene expression into whole-scale changes in embryonic form remain to be fully understood. Biologists have identified two main cell deformation modes responsible for ventral furrow invagination: constriction of the apical ends of the cells (apical wedging) and deformation along their apical-basal axes (radial lengthening/shortening). In this work, we used a computer 2D finite element model of ventral furrow formation to investigate the ability of different combinations of three plausible elementary active cell shape changes to bring about epithelial invagination: ectodermal apical-basal shortening, mesodermal apical-basal lengthening/shortening and mesodermal apical constriction. We undertook a systems analysis of the biomechanical system, which revealed many different combinations of active forces (invagination mechanisms) were able to generate a ventral furrow. Two important general features were revealed. First that combinations of shape changes are the most robust to environmental and mutational perturbation, in particular those combining ectodermal pushing and mesodermal wedging. Second, that ectodermal pushing plays a big part in all of the robust mechanisms (mesodermal forces alone do not close the furrow), and this provides evidence that it may be an important element in the mechanics of invagination in Drosophila.

  12. Administrative and research policies required to bring cellular therapies from the research laboratory to the patient's bedside.

    PubMed

    Yim, Robyn

    2005-10-01

    presidential administrations on cellular therapy, variations in individual state laws, and states becoming involved in research funding, such as California's Proposition 71. Legal concerns include expanding private litigation with diversity of lawsuits, expanding lists of defendants, and the use of class-action lawsuits in research cases. Ownership issues also arise in terms of intellectual property, patents, and ownership of stem cells collected from minors, as in umbilical cord blood donations. Situations that challenge the regulatory processes established to ensure participant safety include differences in reporting requirements for private- and public-funded research and the lack of adequate funding and resources to implement and support the institutional review board (IRB) process. Financial considerations influence the development of clinical protocols, because funding is often limited. Financial incentives, personal investment in companies funding research activities, and fundraising pressures may present potential conflicts. In addition, the increasing role of emerging biotechnology start-up companies and pharmaceutical companies in clinical research introduces additional financial considerations. Administrative policies are needed to address these possible conflicts and ensure research participant safety as cellular therapies progress from the research laboratories to the patient's bedside. Administrative policies to ensure minimum standards of quality for emerging products before human clinical trials, policies to enforce consistent reporting requirements for private and public cellular research, policies to minimize financial conflicts of interest, policies to strengthen implementation of the existing IRB process and to structure into the process a consistent, systematic review of these identified conflicts, and policies to limit private litigation will help to preserve the objectivity of the review process and ultimately increase participant safety.

  13. 47 CFR 1.20007 - Additional assistance capability requirements for wireline, cellular, and PCS telecommunications...

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... Assistance for Law Enforcement Act § 1.20007 Additional assistance capability requirements for wireline... lawfully authorized intercepted communications and call-identifying information is collected by a law... monitor the content of conversations by all parties connected via a conference call when the...

  14. 47 CFR 1.20007 - Additional assistance capability requirements for wireline, cellular, and PCS telecommunications...

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... Assistance for Law Enforcement Act § 1.20007 Additional assistance capability requirements for wireline... lawfully authorized intercepted communications and call-identifying information is collected by a law... monitor the content of conversations by all parties connected via a conference call when the...

  15. MYB64 and MYB119 Are Required for Cellularization and Differentiation during Female Gametogenesis in Arabidopsis thaliana

    PubMed Central

    Rabiger, David S.; Drews, Gary N.

    2013-01-01

    In angiosperms, the egg cell forms within the multicellular, haploid female gametophyte. Female gametophyte and egg cell development occurs through a unique process in which a haploid spore initially undergoes several rounds of synchronous nuclear divisions without cytokinesis, resulting in a single cell containing multiple nuclei. The developing gametophyte then forms cell walls (cellularization) and the resulting cells differentiate to generate the egg cell and several accessory cells. The switch between free nuclear divisions and cellularization-differentiation occurs during developmental stage FG5 in Arabidopsis, and we refer to it as the FG5 transition. The molecular regulators that initiate the FG5 transition during female gametophyte development are unknown. In this study, we show using mutant analysis that two closely related MYB transcription factors, MYB64 and MYB119, act redundantly to promote this transition. MYB64 and MYB119 are expressed during the FG5 transition, and most myb64 myb119 double mutant gametophytes fail to initiate the FG5 transition, resulting in uncellularized gametophytes with supernumerary nuclei. Analysis of cell-specific markers in myb64 myb119 gametophytes that do cellularize suggests that gametophytic polarity and differentiation are also affected. We also show using multiple-mutant analysis that MYB119 expression is regulated by the histidine kinase CKI1, the primary activator of two-component signaling (TCS) during female gametophyte development. Our data establish a molecular pathway regulating the FG5 transition and implicates CKI1-dependent TCS in the promotion of cellularization, differentiation, and gamete specification during female gametogenesis. PMID:24068955

  16. MYB64 and MYB119 are required for cellularization and differentiation during female gametogenesis in Arabidopsis thaliana.

    PubMed

    Rabiger, David S; Drews, Gary N

    2013-01-01

    In angiosperms, the egg cell forms within the multicellular, haploid female gametophyte. Female gametophyte and egg cell development occurs through a unique process in which a haploid spore initially undergoes several rounds of synchronous nuclear divisions without cytokinesis, resulting in a single cell containing multiple nuclei. The developing gametophyte then forms cell walls (cellularization) and the resulting cells differentiate to generate the egg cell and several accessory cells. The switch between free nuclear divisions and cellularization-differentiation occurs during developmental stage FG5 in Arabidopsis, and we refer to it as the FG5 transition. The molecular regulators that initiate the FG5 transition during female gametophyte development are unknown. In this study, we show using mutant analysis that two closely related MYB transcription factors, MYB64 and MYB119, act redundantly to promote this transition. MYB64 and MYB119 are expressed during the FG5 transition, and most myb64 myb119 double mutant gametophytes fail to initiate the FG5 transition, resulting in uncellularized gametophytes with supernumerary nuclei. Analysis of cell-specific markers in myb64 myb119 gametophytes that do cellularize suggests that gametophytic polarity and differentiation are also affected. We also show using multiple-mutant analysis that MYB119 expression is regulated by the histidine kinase CKI1, the primary activator of two-component signaling (TCS) during female gametophyte development. Our data establish a molecular pathway regulating the FG5 transition and implicates CKI1-dependent TCS in the promotion of cellularization, differentiation, and gamete specification during female gametogenesis.

  17. Viral Replication Protein Inhibits Cellular Cofilin Actin Depolymerization Factor to Regulate the Actin Network and Promote Viral Replicase Assembly

    PubMed Central

    Kovalev, Nikolay; de Castro Martín, Isabel Fernández; Barajas, Daniel; Risco, Cristina; Nagy, Peter D.

    2016-01-01

    RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions. PMID:26863541

  18. Protein synthesis during cellular quiescence is inhibited by phosphorylation of a translational elongation factor.

    PubMed

    Pereira, Sandro F F; Gonzalez, Ruben L; Dworkin, Jonathan

    2015-06-23

    In nature, most organisms experience conditions that are suboptimal for growth. To survive, cells must fine-tune energy-demanding metabolic processes in response to nutrient availability. Here, we describe a novel mechanism by which protein synthesis in starved cells is down-regulated by phosphorylation of the universally conserved elongation factor Tu (EF-Tu). Phosphorylation impairs the essential GTPase activity of EF-Tu, thereby preventing its release from the ribosome. As a consequence, phosphorylated EF-Tu has a dominant-negative effect in elongation, resulting in the overall inhibition of protein synthesis. Importantly, this mechanism allows a quick and robust regulation of one of the most abundant cellular proteins. Given that the threonine that serves as the primary site of phosphorylation is conserved in all translational GTPases from bacteria to humans, this mechanism may have important implications for growth-rate control in phylogenetically diverse organisms.

  19. Identification of pivotal cellular factors involved in HPV-induced dysplastic and neoplastic cervical pathologies.

    PubMed

    Mattarocci, Stefano; Abbruzzese, Claudia; Mileo, Anna M; Carosi, Mariantonia; Pescarmona, Edoardo; Vico, Carmen; Federico, Antonio; Vizza, Enrico; Corrado, Giacomo; Arisi, Ivan; Felsani, Armando; Paggi, Marco G

    2014-04-01

    Cervical carcinoma represents the paradigm of virus-induced cancers, where virtually all cervical cancers come from previous "high-risk" HPV infection. The persistent expression of the HPV viral oncoproteins E6 and E7 is responsible for the reprogramming of fundamental cellular functions in the host cell, thus generating a noticeable, yet only partially explored, imbalance in protein molecular networks and cell signaling pathways. Eighty-eight cellular factors, identified as HPV direct or surrogate targets, were chosen and monitored in a retrospective analysis for their mRNA expression in HPV-induced cervical lesions, from dysplasia to cancer. Real-time quantitative PCR (qPCR) was performed by using formalin-fixed, paraffin embedded archival samples. Gene expression analysis identified 40 genes significantly modulated in LSIL, HSIL, and squamous cervical carcinoma. Interestingly, among these, the expression level of a panel of four genes, TOP2A, CTNNB1, PFKM, and GSN, was able to distinguish between normal tissues and cervical carcinomas. Immunohistochemistry was also done to assess protein expression of two genes among those up-regulated during the transition between dysplasia and carcinoma, namely E2F1 and CDC25A, and their correlation with clinical parameters. Besides the possibility of significantly enhancing the use of some of these factors in diagnostic or prognostic procedures, these data clearly outline specific pathways, and thus key biological processes, altered in cervical dysplasia and carcinoma. Deeper insight on how these molecular mechanisms work may help widen the spectrum of novel innovative approaches to these virus-induced cell pathologies. © 2013 Wiley Periodicals, Inc.

  20. Molecular and cellular factors control signal transduction via switchable allosteric modulator proteins (SAMPs).

    PubMed

    Babel, Heiko; Bischofs, Ilka B

    2016-04-27

    Rap proteins from Bacilli directly target response regulators of bacterial two-component systems and modulate their activity. Their effects are controlled by binding of signaling peptides to an allosteric site. Hence Raps exemplify a class of monomeric signaling receptors, which we call switchable allosteric modulator proteins (SAMPs). These proteins have potential applications in diverse biomedical and biotechnical settings, but a quantitative understanding of the impact of molecular and cellular factors on signal transduction is lacking. Here we introduce mathematical models that elucidate how signals are propagated though the network upon receptor stimulation and control the level of active response regulator. Based on a systematic parameter analysis of the models, we show that key features of the dose-response behavior at steady state are controlled either by the molecular properties of the modulator or the signaling context. In particular, we find that the biochemical activity (i.e. non-enzymatic vs. enzymatic) and allosteric properties of the modulator control the response amplitude. The Hill coefficient and the EC50 are controlled in addition by the relative ligand affinities. By tuning receptor properties, either graded or more switch-like (memory-less) response functions can be fashioned. Furthermore, we show that other contextual factors (e.g. relative concentrations of network components and kinase activity) have a substantial impact on the response, and we predict that there exists a modulator concentration which is optimal for response amplitude. We discuss data on Rap-Phr systems in B. subtilis to show how our models can contribute to an integrated view of SAMP signaling by combining biochemical, structural and physiological insights. Our results also suggest that SAMPs could be evolved or engineered to implement diverse response behaviors. However-without additional regulatory controls-they can generate rather variable cellular outputs.

  1. Clearance of Bordetella parapertussis from the Lower Respiratory Tract Requires Humoral and Cellular Immunity

    PubMed Central

    Wolfe, Daniel N.; Kirimanjeswara, Girish S.; Harvill, Eric T.

    2005-01-01

    Bordetella parapertussis and Bordetella pertussis are closely related species that cause whooping cough, an acute, immunizing disease. Their coexistence in the same host populations at the same time and vaccine studies showing that B. pertussis vaccines have little effect on B. parapertussis infection or disease suggest that the protective immunity induced by each does not efficiently cross protect against the other. Although the mechanisms of protective immunity to B. pertussis have been well studied, those of B. parapertussis have not. The present study explores the mechanism by which B. parapertussis is cleared from the lower respiratory tract by anamnestic immunity. Serum antibodies are necessary and sufficient for elimination of this bacterium, and CD4+ T cells, complement, and neutrophils are required for serum antibody-mediated clearance. Mice lacking immunoglobulin A had no defect in their ability to control or clear infection. Interestingly, serum antibody-mediated clearance of B. parapertussis did not require Fc receptors that are required for antibody-mediated clearance of B. pertussis. Together these data support a model for the mechanism of protective immunity to B. parapertussis that is similar but distinct from that of B. pertussis. PMID:16177324

  2. Nerve growth factor stimulates cellular proliferation of human epithelial ovarian cancer.

    PubMed

    Urzua, U; Tapia, V; Geraldo, M P; Selman, A; Vega, M; Romero, C

    2012-09-01

    Due to its ability to induce vascular endothelial growth factor expression and proliferation, migration, and vasculogenesis of endothelial cells, nerve growth factor (NGF) has been considered as an angiogenic factor in epithelial ovarian cancer (EOC). In this work, we evaluated the angiogenic and proliferative mRNA expression profiles of EOC and addressed the responsiveness of EOC explants to NGF stimulation. Twenty EOC samples were obtained from Obstetrics and Gynecology Department, University of Chile's Clinical Hospital. Global gene expression profiles of selected poorly differentiated serous EOC samples were obtained with DNA oligonucleotide microarrays. In addition, EOC explants were subjected to NGF stimulation and levels of p-AKT, BAX, BCL2, Ki-67, c-MYC, and FOXL2 proteins were determined by immunohistochemistry. Results showed that mRNAs coding for specific transcriptional regulators and antiapoptotic components of the NGF signaling pathway were upregulated in EOC cells. At the protein level, key members of the NGF pathway including p-AKT, BCL2/BAX, Ki-67, and c-MYC were found increased, while FOXL2 was decreased in response to NGF stimulation. These findings strongly suggest that NGF stimulates cellular proliferation of human EOC. © Georg Thieme Verlag KG Stuttgart · New York.

  3. Regulation of Cellular Diacylglycerol through Lipid Phosphate Phosphatases Is Required for Pathogenesis of the Rice Blast Fungus, Magnaporthe oryzae

    PubMed Central

    Mir, Albely Afifa; Choi, Jaeyoung; Choi, Jaehyuk; Lee, Yong-Hwan

    2014-01-01

    Considering implication of diacylglycerol in both metabolism and signaling pathways, maintaining proper levels of diacylglycerol (DAG) is critical to cellular homeostasis and development. Except the PIP2-PLC mediated pathway, metabolic pathways leading to generation of DAG converge on dephosphorylation of phosphatidic acid catalyzed by lipid phosphate phosphatases. Here we report the role of such enzymes in a model plant pathogenic fungus, Magnaporthe oryzae. We identified five genes encoding putative lipid phosphate phosphatases (MoLPP1 to MoLPP5). Targeted disruption of four genes (except MoLPP4) showed that MoLPP3 and MoLPP5 are required for normal progression of infection-specific development and proliferation within host plants, whereas MoLPP1 and MoLPP2 are indispensable for fungal pathogenicity. Reintroduction of MoLPP3 and MoLPP5 into individual deletion mutants restored all the defects. Furthermore, exogenous addition of saturated DAG not only restored defect in appressorium formation but also complemented reduced virulence in both mutants. Taken together, our data indicate differential roles of lipid phosphate phosphatase genes and requirement of proper regulation of cellular DAGs for fungal development and pathogenesis. PMID:24959955

  4. Cellular localization of interleukin-8 and its inducer, tumor necrosis factor-alpha in psoriasis.

    PubMed Central

    Nickoloff, B. J.; Karabin, G. D.; Barker, J. N.; Griffiths, C. E.; Sarma, V.; Mitra, R. S.; Elder, J. T.; Kunkel, S. L.; Dixit, V. M.

    1991-01-01

    transforming growth factor-alpha mRNAs. Further elucidation of the cellular and molecular basis for the genesis and evolution of psoriasis will provide the framework for a better evaluation of the cause and treatment of this skin disease. Images Figure 1 Figure 1 Figure 2 Figure 14 Figure 15 Figure 16 PMID:1702929

  5. Cellular Membrane Composition Requirement by Antimicrobial and Anticancer Peptide GA-K4.

    PubMed

    Mishig-Ochir, Tsogbadrakh; Gombosuren, Davaadulam; Jigjid, Altanchimeg; Tuguldur, Badamkhatan; Chuluunbaatar, Galbadrakh; Urnukhsaikhan, Enerelt; Pathak, Chinar; Lee, Bong-Jin

    2017-01-01

    Naturally occurring antimicrobial peptides important for innate immunity are widely studied for their antimicrobial and anticancer activity. The primary target of these AMPs is believed to be the bacterial cytoplasmic membrane. However, the interaction between cytoplasmic membrane and the antimicrobial peptides remains poorly understood. Therefore to focus on the target membrane composition that is required by AMPs to interact with membranes, we have examined the interaction of the antimicrobial and anticancer active 11-residue GA-K4 (FLKWLFKWAKK) peptide with model and intact cell membranes. Effect on the structural conformational properties of GA-K4 peptide was investigated by means of far-UV CD and fluorescence spectroscopic methods. The different conformation of GA-K4 peptide in large unilamellar vesicles (LUV) bilayer and micelle environment suggest that the curvature has an influence on the secondary structure acquired by the peptide. Furthermore, the leakage experiment result confirmed that GA-K4 induced the leakage of cytoplasmic membrane in Staphylococcus аureus bacterial cells. Fluorescence data revealed the interfacial location of GA-K4 peptide in the model membranes. The blue-shift in emission wavelength by tryptophan residues in fluorescence data indicated the penetration of GA-K4 peptide in micelles and phospholipid bilayers. These results showed that the GA-K4 peptide is a membrane-active peptide and its activity depends on membrane curvature and lipid composition. Although further studies are required to confirm the mechanism of action, the data suggest mechanism of toroidal pore formation for the interaction of GA-K4 peptide with membranes. Our studies will be helpful in better understanding of the membrane requirment of peptides to express their therapeutic effects. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  6. The Arabidopsis homeobox gene SHOOT MERISTEMLESS has cellular and meristem-organisational roles with differential requirements for cytokinin and CYCD3 activity.

    PubMed

    Scofield, Simon; Dewitte, Walter; Nieuwland, Jeroen; Murray, James A H

    2013-07-01

    The Arabidopsis class-1 KNOX gene SHOOT MERISTEMLESS (STM) encodes a homeodomain transcription factor essential for shoot apical meristem (SAM) formation and sustained activity. STM activates cytokinin (CK) biosynthesis in the SAM, but the extent to which STM function is mediated through CK is unclear. Here we show that STM inhibits cellular differentiation and endoreduplication, acting through CK and the CK-inducible CYCD3 cell cycle regulators, establishing a mechanistic link to cell cycle control which provides sustained mitotic activity to maintain a pool of undifferentiated cells in the SAM. Equivalent functions are revealed for the related KNOX genes KNAT1/BP and KNAT2 through ectopic expression. STM is also required for proper meristem organisation and can induce de novo meristem formation when expressed ectopically, even when CK levels are reduced or CK signaling is impaired. This function in meristem establishment and organisation can be replaced by KNAT1/BP, but not KNAT2, despite its activation of CK responses, suggesting that promotion of CK responses alone is insufficient for SAM organisation. We propose that STM has dual cellular and meristem-organisational functions that are differentially represented in the class-1 KNOX gene family and have differing requirements for CK and CYCD3. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  7. Cyclin A2 Mutagenesis Analysis: A New Insight into CDK Activation and Cellular Localization Requirements

    PubMed Central

    Bendris, Nawal; Lemmers, Bénédicte; Blanchard, Jean-Marie; Arsic, Nikola

    2011-01-01

    Cyclin A2 is essential at two critical points in the somatic cell cycle: during S phase, when it activates CDK2, and during the G2 to M transition when it activates CDK1. Based on the crystal structure of Cyclin A2 in association with CDKs, we generated a panel of mutants to characterize the specific amino acids required for partner binding, CDK activation and subcellular localization. We find that CDK1, CDK2, p21, p27 and p107 have overlapping but distinct requirements for association with this protein. Our data highlight the crucial importance of the N-terminal α helix, in conjunction with the α3 helix within the cyclin box, in activating CDK. Several Cyclin A2 mutants selectively bind to either CDK1 or CDK2. We demonstrate that association of Cyclin A2 to proteins such as CDK2 that was previously suggested as crucial is not a prerequisite for its nuclear localization, and we propose that the whole protein structure is involved. PMID:21829545

  8. Cellular requirements for the formation of EA rosettes by human monocytes.

    PubMed Central

    Passwell, J H; Schneeberger, E; Merler, E

    1978-01-01

    The binding of sensitized red cells to Fc receptors in human monocytes was studied by evaluating the effects of various pharmacological reagents and other treatments on EA rosette formation. Cytochalasin B and 2-deoxyglucose inhibited rosette formation in a dose-dependent manner. Sodium azide and incubation at 4 degrees also inhibited rosette formation, while at 37 degrees increased numbers of RBCs bound to the monocytes. The microtubular poisons, vinblastine and colchicine at high concentrations resulted in decreased adherence of monocytes and inhibition of rosette formation, while at low concentrations of colchicine, enhanced rosette formation was sometimes observed. Contrary to the effects on rosette formation, binding of [125I] IgG to monocyte monolayers was not altered by treatment of the monocytes with drugs. Magnesium ions were required to promote monocyte adherence, but both magnesium and calcium were needed for the best rosette formation. We conclude that the formation of EA rosettes is dependent not merely on binding of IgG to the Fc receptor but requires metabolically active monocytes, an intact cytostructure and suitable environmental conditions (temperature and cation concentration). Images Figure 3 PMID:738764

  9. Cyclin A2 mutagenesis analysis: a new insight into CDK activation and cellular localization requirements.

    PubMed

    Bendris, Nawal; Lemmers, Bénédicte; Blanchard, Jean-Marie; Arsic, Nikola

    2011-01-01

    Cyclin A2 is essential at two critical points in the somatic cell cycle: during S phase, when it activates CDK2, and during the G2 to M transition when it activates CDK1. Based on the crystal structure of Cyclin A2 in association with CDKs, we generated a panel of mutants to characterize the specific amino acids required for partner binding, CDK activation and subcellular localization. We find that CDK1, CDK2, p21, p27 and p107 have overlapping but distinct requirements for association with this protein. Our data highlight the crucial importance of the N-terminal α helix, in conjunction with the α3 helix within the cyclin box, in activating CDK. Several Cyclin A2 mutants selectively bind to either CDK1 or CDK2. We demonstrate that association of Cyclin A2 to proteins such as CDK2 that was previously suggested as crucial is not a prerequisite for its nuclear localization, and we propose that the whole protein structure is involved.

  10. Voltage-gated Nav channel targeting in the heart requires an ankyrin-G dependent cellular pathway.

    PubMed

    Lowe, John S; Palygin, Oleg; Bhasin, Naina; Hund, Thomas J; Boyden, Penelope A; Shibata, Erwin; Anderson, Mark E; Mohler, Peter J

    2008-01-14

    Voltage-gated Na(v) channels are required for normal electrical activity in neurons, skeletal muscle, and cardiomyocytes. In the heart, Na(v)1.5 is the predominant Na(v) channel, and Na(v)1.5-dependent activity regulates rapid upstroke of the cardiac action potential. Na(v)1.5 activity requires precise localization at specialized cardiomyocyte membrane domains. However, the molecular mechanisms underlying Na(v) channel trafficking in the heart are unknown. In this paper, we demonstrate that ankyrin-G is required for Na(v)1.5 targeting in the heart. Cardiomyocytes with reduced ankyrin-G display reduced Na(v)1.5 expression, abnormal Na(v)1.5 membrane targeting, and reduced Na(+) channel current density. We define the structural requirements on ankyrin-G for Na(v)1.5 interactions and demonstrate that loss of Na(v)1.5 targeting is caused by the loss of direct Na(v)1.5-ankyrin-G interaction. These data are the first report of a cellular pathway required for Na(v) channel trafficking in the heart and suggest that ankyrin-G is critical for cardiac depolarization and Na(v) channel organization in multiple excitable tissues.

  11. Robotic technology applied to meet the requirements of tools to support microsurgery and cellular surgery.

    PubMed

    Brett, Peter; Ma, Xianghong; Tritto, Giuseppe

    2004-01-01

    Microsurgery and processes involving cell manipulation or cell surgery are clinical practices where the operator works at or beyond the threshold of human dexterity. Current tools available are conventional in their design, and this limits consistency and the level of reliability and achievement. Surgical robotic devices have been explored to improve precision in minimal access surgical procedures to augment control of tool points in tissues, and have enabled feedback of sensory data from which the operator is able to deduce information on the tool at the working site. In this paper, relevant technologies are described that can be harnessed to improve perception of tool point interaction with tissues at the working site and to improve tool control at the small scale required in clinical practice for microsurgery and for cell surgery.

  12. Aged blood factors decrease cellular responses associated with delayed gingival wound repair

    PubMed Central

    Morgan, Alejandra; Díaz, Catalina; Morales, Diego; Jaña, Fabián; Gómez, Alvaro; Silva, Alonso; Briceño, Fernanda; Oyarzún, Alejandro; Maldonado, Felipe; Cerda, Oscar; Smith, Patricio C.

    2017-01-01

    Aging is a gradual biological process characterized by a decrease in cell and organism functions. Gingival wound healing is one of the impaired processes found in old rats. Here, we studied the in vivo wound healing process using a gingival repair rat model and an in vitro model using human gingival fibroblast for cellular responses associated to wound healing. To do that, we evaluated cell proliferation of both epithelial and connective tissue cells in gingival wounds and found decreased of Ki67 nuclear staining in old rats when compared to their young counterparts. We next evaluated cellular responses of primary gingival fibroblast obtained from young subjects in the presence human blood serum of individuals of different ages. Eighteen to sixty five years old masculine donors were classified into 3 groups: “young” from 18 to 22 years old, “middle-aged” from 30 to 48 years old and “aged” over 50 years old. Cell proliferation, measured through immunofluorescence for Ki67 and flow cytometry for DNA content, was decreased when middle-aged and aged serum was added to gingival fibroblast compared to young serum. Myofibroblastic differentiation, measured through alpha-smooth muscle actin (α-SMA), was stimulated with young but not middle-aged or aged serum both the protein levels and incorporation of α-SMA into actin stress fibers. High levels of PDGF, VEGF, IL-6R were detected in blood serum from young subjects when compared to middle-aged and aged donors. In addition, the pro-inflammatory cytokines MCP-1 and TNF were increased in the serum of aged donors. In old rat wound there is an increased of staining for TNF compared to young wound. Moreover, healthy gingiva (non injury) shows less staining compared to a wound site, suggesting a role in wound healing. Moreover, serum from middle-aged and aged donors was able to stimulate cellular senescence in young cells as determined by the expression of senescence associated beta-galactosidase and histone H2A

  13. Downregulation of Cellular Protective Factors of Rumen Epithelium in Goats Fed High Energy Diet

    PubMed Central

    Hollmann, Manfred; Miller, Ingrid; Hummel, Karin; Sabitzer, Sonja; Metzler-Zebeli, Barbara U.; Razzazi-Fazeli, Ebrahim; Zebeli, Qendrim

    2013-01-01

    Energy-rich diets can challenge metabolic and protective functions of the rumen epithelial cells, but the underlying factors are unclear. This study sought to evaluate proteomic changes of the rumen epithelium in goats fed a low, medium, or high energy diet. Expression of protein changes were compared by two-dimensional differential gel electrophoresis followed by protein identification with matrix assisted laser desorption ionisation tandem time-of-flight mass spectrometry. Of about 2,000 spots commonly detected in all gels, 64 spots were significantly regulated, which were traced back to 24 unique proteins. Interestingly, the expression profiles of several chaperone proteins with important cellular protective functions such as heat shock cognate 71 kDa protein, peroxiredoxin-6, serpin H1, protein disulfide-isomerase, and selenium-binding protein were collectively downregulated in response to high dietary energy supply. Similar regulation patterns were obtained for some other proteins involved in transport or metabolic functions. In contrast, metabolic enzymes like retinal dehydrogenase 1 and ATP synthase subunit beta, mitochondrial precursor were upregulated in response to high energy diet. Lower expressions of chaperone proteins in the rumen epithelial cells in response to high energy supply may suggest that these cells were less protected against the potentially harmful rumen toxic compounds, which might have consequences for rumen and systemic health. Our findings also suggest that energy-rich diets and the resulting acidotic insult may render rumen epithelial cells more vulnerable to cellular damage by attenuating their cell defense system, hence facilitating the impairment of rumen barrier function, typically observed in energy-rich fed ruminants. PMID:24349094

  14. Graphene Enhances Cellular Proliferation through Activating the Epidermal Growth Factor Receptor.

    PubMed

    Liu, Wei; Sun, Cheng; Liao, Chunyang; Cui, Lin; Li, Haishan; Qu, Guangbo; Yu, Wenlian; Song, Naining; Cui, Yuan; Wang, Zheng; Xie, Wenping; Chen, Huiming; Zhou, Qunfang

    2016-07-27

    Graphene has promising applications in food packaging, water purification, and detective sensors for contamination monitoring. However, the biological effects of graphene are not fully understood. It is necessary to clarify the potential risks of graphene exposure to humans through diverse routes, such as foods. In the present study, graphene, as the model nanomaterial, was used to test its potential effects on the cell proliferation based on multiple representative cell lines, including HepG2, A549, MCF-7, and HeLa cells. Graphene was characterized by Raman spectroscopy, particle size analysis, atomic force microscopy, and transmission electron microscopy. The cellular responses to graphene exposure were evaluated using flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and alamarBlue assays. Rat cerebral astrocyte cultures, as the non-cancer cells, were used to assess the potential cytotoxicity of graphene as well. The results showed that graphene stimulation enhanced cell proliferation in all tested cell cultures and the highest elevation in cell growth was up to 60%. A western blot assay showed that the expression of epidermal growth factor (EGF) was upregulated upon graphene treatment. The phosphorylation of EGF receptor (EGFR) and the downstream proteins, ShC and extracellular regulating kinase (ERK), were remarkably induced, indicating that the activation of the mitogen-activated protein kinase (MAPK)/ERK signaling pathway was triggered. The activation of PI3 kinase p85 and AKT showed that the PI3K/AKT signaling pathway was also involved in graphene-induced cell proliferation, causing the increase of cell ratios in the G2/M phase. No influences on cell apoptosis were observed in graphene-treated cells when compared to the negative controls, proving the low cytotoxicity of this emerging nanomaterial. The findings in this study revealed the potential cellular biological effect of graphene, which may give useful hints on its biosafety

  15. Cellular Uptake of Clostridium botulinum C2 Toxin Requires Acid Sphingomyelinase Activity.

    PubMed

    Nagahama, Masahiro; Takehara, Masaya; Takagishi, Teruhisa; Seike, Soshi; Miyamoto, Kazuaki; Kobayashi, Keiko

    2017-04-01

    Clostridium botulinum C2 toxin consists of an enzyme component (C2I) and a binding component (C2II). Activated C2II (C2IIa) binds to a cell receptor, giving rise to lipid raft-dependent oligomerization, and it then assembles with C2I. The whole toxin complex is then endocytosed into the cytosol, resulting in the destruction of the actin cytoskeleton and cell rounding. Here, we showed that C2 toxin requires acid sphingomyelinase (ASMase) activity during internalization. In this study, inhibitors of ASMase and lysosomal exocytosis blocked C2 toxin-induced cell rounding. C2IIa induced Ca(2+) influx from the extracellular medium to cells. C2 toxin-induced cell rounding was enhanced in the presence of Ca(2+) ASMase was released extracellularly when cells were incubated with C2IIa in the presence of Ca(2+) Small interfering RNA (siRNA) knockdown of ASMase reduced C2 toxin-induced cell rounding. ASMase hydrolyzes sphingomyelin to ceramide on the outer leaflet of the membrane at acidic pH. Ceramide was detected in cytoplasmic vesicles containing C2IIa. These results indicated that ASMase activity is necessary for the efficient internalization of C2 toxin into cells. Inhibitors of ASMase may confer protection against infection. Copyright © 2017 American Society for Microbiology.

  16. Viral and Cellular Requirements for the Nuclear Entry of Retroviral Preintegration Nucleoprotein Complexes

    PubMed Central

    Matreyek, Kenneth A.; Engelman, Alan

    2013-01-01

    Retroviruses integrate their reverse transcribed genomes into host cell chromosomes as an obligate step in virus replication. The nuclear envelope separates the chromosomes from the cell cytoplasm during interphase, and different retroviral groups deal with this physical barrier in different ways. Gammaretroviruses are dependent on the passage of target cells through mitosis, where they are believed to access chromosomes when the nuclear envelope dissolves for cell division. Contrastingly, lentiviruses such as HIV-1 infect non-dividing cells, and are believed to enter the nucleus by passing through the nuclear pore complex. While numerous virally encoded elements have been proposed to be involved in HIV-1 nuclear import, recent evidence has highlighted the importance of HIV-1 capsid. Furthermore, capsid was found to be responsible for the viral requirement of various nuclear transport proteins, including transportin 3 and nucleoporins NUP153 and NUP358, during infection. In this review, we describe our current understanding of retroviral nuclear import, with emphasis on recent developments on the role of the HIV-1 capsid protein. PMID:24103892

  17. siRNA and shRNA screens advance key understanding of host factors required for HIV-1 replication.

    PubMed

    Kok, Kin-Hang; Lei, Ting; Jin, Dong-Yan

    2009-08-27

    A recent RNAi screen used a genome-wide shRNA library to search for cellular factors required for HIV-1 replication. This work complements three other siRNA-based screening studies and potentially opens the door to the discovery of factors that are important for HIV-1 replication in physiological host cells such as T lymphocytes. shRNA screens can be further improved, and they could promise to unravel new pathways and new facets of virus-cell interactions.

  18. Genome-wide siRNA screen identifies the retromer as a cellular entry factor for human papillomavirus

    PubMed Central

    Lipovsky, Alex; Popa, Andreea; Pimienta, Genaro; Wyler, Michael; Bhan, Ashima; Kuruvilla, Leena; Guie, Marie-Aude; Poffenberger, Adrian C.; Nelson, Christian D. S.; Atwood, Walter J.; DiMaio, Daniel

    2013-01-01

    Despite major advances in our understanding of many aspects of human papillomavirus (HPV) biology, HPV entry is poorly understood. To identify cellular genes required for HPV entry, we conducted a genome-wide screen for siRNAs that inhibited infection of HeLa cells by HPV16 pseudovirus. Many retrograde transport factors were required for efficient infection, including multiple subunits of the retromer, which initiates retrograde transport from the endosome to the trans-Golgi network (TGN). The retromer has not been previously implicated in virus entry. Furthermore, HPV16 capsid proteins arrive in the TGN/Golgi in a retromer-dependent fashion during entry, and incoming HPV proteins form a stable complex with retromer subunits. We propose that HPV16 directly engages the retromer at the early or late endosome and traffics to the TGN/Golgi via the retrograde pathway during cell entry. These results provide important insights into HPV entry, identify numerous potential antiviral targets, and suggest that the role of the retromer in infection by other viruses should be assessed. PMID:23569269

  19. Genome-wide siRNA screen identifies the retromer as a cellular entry factor for human papillomavirus.

    PubMed

    Lipovsky, Alex; Popa, Andreea; Pimienta, Genaro; Wyler, Michael; Bhan, Ashima; Kuruvilla, Leena; Guie, Marie-Aude; Poffenberger, Adrian C; Nelson, Christian D S; Atwood, Walter J; DiMaio, Daniel

    2013-04-30

    Despite major advances in our understanding of many aspects of human papillomavirus (HPV) biology, HPV entry is poorly understood. To identify cellular genes required for HPV entry, we conducted a genome-wide screen for siRNAs that inhibited infection of HeLa cells by HPV16 pseudovirus. Many retrograde transport factors were required for efficient infection, including multiple subunits of the retromer, which initiates retrograde transport from the endosome to the trans-Golgi network (TGN). The retromer has not been previously implicated in virus entry. Furthermore, HPV16 capsid proteins arrive in the TGN/Golgi in a retromer-dependent fashion during entry, and incoming HPV proteins form a stable complex with retromer subunits. We propose that HPV16 directly engages the retromer at the early or late endosome and traffics to the TGN/Golgi via the retrograde pathway during cell entry. These results provide important insights into HPV entry, identify numerous potential antiviral targets, and suggest that the role of the retromer in infection by other viruses should be assessed.

  20. Essential requirement of cytochrome c release for caspase activation by procaspase-activating compound defined by cellular models

    PubMed Central

    Seervi, M; Joseph, J; Sobhan, P K; Bhavya, B C; Santhoshkumar, T R

    2011-01-01

    Mitochondrial cytochrome c (cyt. c) release and caspase activation are often impaired in tumors with Bcl-2 overexpression or Bax and Bak-defective status. Direct triggering of cell death downstream of Bax and Bak is an attractive strategy to kill such cancers. Small molecule compounds capable of direct caspase activation appear to be the best mode for killing such tumors. However, there is no precise model to screen such compounds. The currently employed cell-free systems possess the inherent drawback of lacking cellular contents and organelles that operate in integrating cell death signaling. We have developed highly refined cell-based approaches to validate direct caspase activation in cancer cells. Using this approach, we show that PAC-1 (first procaspase-activating compound), the first direct activator of procaspases identified in a cell-free system, in fact requires mitochondrial cyt. c release for triggering caspase activation similar to other antitumor agents. It can induce significant caspase activation and cell death in the absence of Bax and Bak, and in cells overexpressing Bcl-2 and Bcl-xL. This study for the first time defines precise criteria for the validation of direct caspase-activating compounds using specialized cellular models that is expected to accelerate the discovery of potential direct caspase activators. PMID:21900958

  1. Tissue factor expression in human arterial smooth muscle cells. TF is present in three cellular pools after growth factor stimulation.

    PubMed Central

    Schecter, A D; Giesen, P L; Taby, O; Rosenfield, C L; Rossikhina, M; Fyfe, B S; Kohtz, D S; Fallon, J T; Nemerson, Y; Taubman, M B

    1997-01-01

    Tissue factor (TF) is a transmembrane glycoprotein that initiates the coagulation cascade. Because of the potential role of TF in mediating arterial thrombosis, we have examined its expression in human aortic and coronary artery smooth muscle cells (SMC). TF mRNA and protein were induced in SMC by a variety of growth agonists. Exposure to PDGF AA or BB for 30 min provided all of the necessary signals for induction of TF mRNA and protein. This result was consistent with nuclear runoff analyses, demonstrating that PDGF-induced TF transcription occurred within 30 min. A newly developed assay involving binding of digoxigenin-labeled FVIIa (DigVIIa) and digoxigenin-labeled Factor X (DigX) was used to localize cellular TF. By light and confocal microscopy, prominent TF staining was seen in the perinuclear cytoplasm beginning 2 h after agonist treatment and persisting for 10-12 h. Surface TF activity, measured on SMC monolayers under flow conditions, increased transiently, peaking 4-6 h after agonist stimulation and returning to baseline within 16 h. Peak surface TF activity was only approximately 20% of total TF activity measured in cell lysates. Surface TF-blocking experiments demonstrated that the remaining TF was found as encrypted surface TF, and also in an intracellular pool. The relatively short-lived surface expression of TF may be critical for limiting the thrombotic potential of intact SMC exposed to growth factor stimulation. In contrast, the encrypted surface and intracellular pools may provide a rich source of TF under conditions associated with SMC damage, such as during atherosclerotic plaque rupture or balloon arterial injury. PMID:9410905

  2. Microscopic analysis of the cellular events during scatter factor/hepatocyte growth factor-induced epithelial tubulogenesis.

    PubMed

    Williams, M J; Clark, P

    2003-11-01

    Scatter factor/hepatocyte growth factor (SF/HGF), a large multifunctional polypeptide growth and motility factor, is known to play important roles during embryonic development, adult tissue growth and repair. In an established three-dimensional type I collagen model, SF/HGF induces Madin-Darby canine kidney (MDCK) epithelial cysts to form long, branching tubules (tubulogenesis). In addition, the composition of the surrounding extracellular matrix (ECM) has been shown to modulate SF/HGF-induced morphogenesis, where tubulogenesis was completely abrogated in Matrigel basement membrane. Many cellular events that occur during SF/HGF-mediated remodelling, and its modulation by the ECM, remain unclear. We have investigated these mechanisms through microscopic examination of the time-course of SF/HGF-induced responses in MDCK cysts cultured in type I collagen or Matrigel. We found that early responses to SF/HGF were matrix-independent. Changes included increased paracellular spacing between normally closely apposed lateral membranes, and the formation of filopodial processes, indicating a partial motile response. Cell-cell contact was maintained, with the persistence of cell junctions. Therefore, while one or a number of ECM components are preventing SF/HGF-primed cells from undergoing an invasive and/or migratory programme, non-permissive matrices are not preventing SF/HGF signalling to the cell. Later matrix-dependent responses, which occurred in type I collagen but not Matrigel, included the formation of basal protrusions that comprise two or more neighbouring cells, which extend to form nascent tubules. Modified polarity of cells comprising the basal protrusions was evident, with a marker for the apical membrane being found in the same region as adherens junctions and desmosomes, typically localized at lateral membranes. We propose a model for SF/HGF-induced tubulogenesis in which tubules form from basal protrusions of adjacent cells. This mechanism of in vitro tubule

  3. DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus

    PubMed Central

    Qi, Yonghe; Gao, Zhenchao; Peng, Bo; Yan, Huan; Tang, Dingbin; Song, Zilin; He, Wenhui; Sun, Yinyan; Guo, Ju-Tao; Li, Wenhui

    2016-01-01

    Hepatitis B virus (HBV) infection of hepatocytes begins by binding to its cellular receptor sodium taurocholate cotransporting polypeptide (NTCP), followed by the internalization of viral nucleocapsid into the cytoplasm. The viral relaxed circular (rc) DNA genome in nucleocapsid is transported into the nucleus and converted into covalently closed circular (ccc) DNA to serve as a viral persistence reservoir that is refractory to current antiviral therapies. Host DNA repair enzymes have been speculated to catalyze the conversion of rcDNA to cccDNA, however, the DNA polymerase(s) that fills the gap in the plus strand of rcDNA remains to be determined. Here we conducted targeted genetic screening in combination with chemical inhibition to identify the cellular DNA polymerase(s) responsible for cccDNA formation, and exploited recombinant HBV with capsid coding deficiency which infects HepG2-NTCP cells with similar efficiency of wild-type HBV to assure cccDNA synthesis is exclusively from de novo HBV infection. We found that DNA polymerase κ (POLK), a Y-family DNA polymerase with maximum activity in non-dividing cells, substantially contributes to cccDNA formation during de novo HBV infection. Depleting gene expression of POLK in HepG2-NTCP cells by either siRNA knockdown or CRISPR/Cas9 knockout inhibited the conversion of rcDNA into cccDNA, while the diminished cccDNA formation in, and hence the viral infection of, the knockout cells could be effectively rescued by ectopic expression of POLK. These studies revealed that POLK is a crucial host factor required for cccDNA formation during a de novo HBV infection and suggest that POLK may be a potential target for developing antivirals against HBV. PMID:27783675

  4. Homogenizing cellular tension by hepatocyte growth factor in expanding epithelial monolayer

    PubMed Central

    Jang, Hwanseok; Notbohm, Jacob; Gweon, Bomi; Cho, Youngbin; Park, Chan Young; Kee, Sun-Ho; Fredberg, Jeffrey J.; Shin, Jennifer H.; Park, Yongdoo

    2017-01-01

    Hepatocyte growth factor (HGF) induces cell migration and scattering by mechanisms that are thought to tip a local balance of competing physical forces; cell-to-cell and cell-to-substrate forces. In this local process, HGF is known to attenuate local cadherin-dependent adhesion forces for cell-cell junction development and enhance local integrin-dependent contractile forces for pulling neighboring cells apart. Here we use an expanding island of confluent Madin-Darby canine kidney (MDCK) cells as a model system to quantify the collective cell migration. In the absence of HGF, cell trajectories are highly tortuous whereas in the presence of HGF, they become far less so, resembling free expansion of a gas. At the level of cell-to-cell junctions, HGF attenuates the linkage of stress fibers to cell-to-cell junctions with concomitant decrease in intercellular stress. At the level of cell-to-substrate junctions, HGF augments the linkage of stress fibers to cell-to-substrate junctions with no apparent effect on traction. Together, HGF induces both structural changes in the actin-bound junctional protein complex and physical forces spanning multicellular clusters, which further promotes the expansion of confluent cellular layer. PMID:28374776

  5. Homogenizing cellular tension by hepatocyte growth factor in expanding epithelial monolayer.

    PubMed

    Jang, Hwanseok; Notbohm, Jacob; Gweon, Bomi; Cho, Youngbin; Park, Chan Young; Kee, Sun-Ho; Fredberg, Jeffrey J; Shin, Jennifer H; Park, Yongdoo

    2017-04-04

    Hepatocyte growth factor (HGF) induces cell migration and scattering by mechanisms that are thought to tip a local balance of competing physical forces; cell-to-cell and cell-to-substrate forces. In this local process, HGF is known to attenuate local cadherin-dependent adhesion forces for cell-cell junction development and enhance local integrin-dependent contractile forces for pulling neighboring cells apart. Here we use an expanding island of confluent Madin-Darby canine kidney (MDCK) cells as a model system to quantify the collective cell migration. In the absence of HGF, cell trajectories are highly tortuous whereas in the presence of HGF, they become far less so, resembling free expansion of a gas. At the level of cell-to-cell junctions, HGF attenuates the linkage of stress fibers to cell-to-cell junctions with concomitant decrease in intercellular stress. At the level of cell-to-substrate junctions, HGF augments the linkage of stress fibers to cell-to-substrate junctions with no apparent effect on traction. Together, HGF induces both structural changes in the actin-bound junctional protein complex and physical forces spanning multicellular clusters, which further promotes the expansion of confluent cellular layer.

  6. Intracellular localization and cellular factors interaction of HTLV-1 and HTLV-2 Tax proteins: similarities and functional differences.

    PubMed

    Bertazzoni, Umberto; Turci, Marco; Avesani, Francesca; Di Gennaro, Gianfranco; Bidoia, Carlo; Romanelli, Maria Grazia

    2011-05-01

    Human T-lymphotropic viruses type 1 (HTLV-1) and type 2 (HTLV-2) present very similar genomic structures but HTLV-1 is more pathogenic than HTLV-2. Is this difference due to their transactivating Tax proteins, Tax-1 and Tax-2, which are responsible for viral and cellular gene activation? Do Tax-1 and Tax-2 differ in their cellular localization and in their interaction pattern with cellular factors? In this review, we summarize Tax-1 and Tax-2 structural and phenotypic properties, their interaction with factors involved in signal transduction and their localization-related behavior within the cell. Special attention will be given to the distinctions between Tax-1 and Tax-2 that likely play an important role in their transactivation activity.

  7. Intracellular Localization and Cellular Factors Interaction of HTLV-1 and HTLV-2 Tax Proteins: Similarities and Functional Differences

    PubMed Central

    Bertazzoni, Umberto; Turci, Marco; Avesani, Francesca; Di Gennaro, Gianfranco; Bidoia, Carlo; Romanelli, Maria Grazia

    2011-01-01

    Human T-lymphotropic viruses type 1 (HTLV-1) and type 2 (HTLV-2) present very similar genomic structures but HTLV-1 is more pathogenic than HTLV-2. Is this difference due to their transactivating Tax proteins, Tax-1 and Tax-2, which are responsible for viral and cellular gene activation? Do Tax-1 and Tax-2 differ in their cellular localization and in their interaction pattern with cellular factors? In this review, we summarize Tax-1 and Tax-2 structural and phenotypic properties, their interaction with factors involved in signal transduction and their localization-related behavior within the cell. Special attention will be given to the distinctions between Tax-1 and Tax-2 that likely play an important role in their transactivation activity. PMID:21994745

  8. Communication requirements of sparse Cholesky factorization with nested dissection ordering

    NASA Technical Reports Server (NTRS)

    Naik, Vijay K.; Patrick, Merrell L.

    1989-01-01

    Load distribution schemes for minimizing the communication requirements of the Cholesky factorization of dense and sparse, symmetric, positive definite matrices on multiprocessor systems are presented. The total data traffic in factoring an n x n sparse symmetric positive definite matrix representing an n-vertex regular two-dimensional grid graph using n exp alpha, alpha not greater than 1, processors are shown to be O(n exp 1 + alpha/2). It is O(n), when n exp alpha, alpha not smaller than 1, processors are used. Under the conditions of uniform load distribution, these results are shown to be asymptotically optimal.

  9. Communication requirements of sparse Cholesky factorization with nested dissection ordering

    NASA Technical Reports Server (NTRS)

    Naik, Vijay K.; Patrick, Merrell L.

    1989-01-01

    Load distribution schemes for minimizing the communication requirements of the Cholesky factorization of dense and sparse, symmetric, positive definite matrices on multiprocessor systems are presented. The total data traffic in factoring an n x n sparse symmetric positive definite matrix representing an n-vertex regular two-dimensional grid graph using n exp alpha, alpha not greater than 1, processors are shown to be O(n exp 1 + alpha/2). It is O(n), when n exp alpha, alpha not smaller than 1, processors are used. Under the conditions of uniform load distribution, these results are shown to be asymptotically optimal.

  10. Evaluation of microbiological, cellular and risk factors associated with subclinical mastitis in female buffaloes

    PubMed Central

    de Oliveira Moura, Emmanuella; do Nascimento Rangel, Adriano Henrique; de Melo, Maria Celeste Nunes; Borba, Luiz Henrique Fernandes; de Lima Júnior, Dorgival Morais; Novaes, Luciano Patto; Urbano, Stela Antas; de Andrade Neto, Júlio César

    2017-01-01

    Objective This study aimed to evaluate the microbiological and cellular milk profile for the diagnosis of subclinical mastitis in female buffaloes and to assess risk factors for predisposition of the disease. Methods Analyses were carried out by standard plate count (SPC), identification of species and antibiotic resistance, somatic cell count (SCC), electrical electrical conductivity of milk (ECM), and lactoferrin content in milk. Teat cups were swabbed to evaluate risk factors, observing hyperkeratosis, milking vacuum pressure and cleanliness of the site. Hence, 30 female buffaloes were randomly selected (15 from a group in early lactation and 15 in late lactation). Results The most common bacteria in the microbiological examination were Staphylococcus spp., Streptococcus spp. and Corynebacterium sp. In the antibiotic sensitivity test, 10 (58.82%) of the 17 antibiotics tested were sensitive to all isolates, and resistant bacteria were Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus haemolyticus, and Escherichia coli. It was observed that positive samples in the microbiological examination showed total bacterial count between 9.10×103 to 6.94×106 colony forming units/mL, SCC between 42,000 to 4,320,000 cells/mL and ECM ranging from 1.85 to 7.40 mS/cm. It was also found that the teat cups had high microbial counts indicating poor hygiene, and even faults in the cleanliness of the animals’ waiting room were observed. It is concluded that values of SCC above 537,000 cells/mL and ECM above 3.0 mS/mL are indications of mammary gland infection for this herd; however, the association of these values with a microbiological analysis is necessary to more accurately evaluate the health status of mammary glands with subclinical mastitis. Conclusion Through phenotypic characterization of bacteria involved in the samples, the genera Staphylococcus spp., Streptococcus spp., and Corynebacterimum bovis were the most prevalent in this study. Faults in

  11. Evaluation of microbiological, cellular and risk factors associated with subclinical mastitis in female buffaloes.

    PubMed

    de Oliveira Moura, Emmanuella; do Nascimento Rangel, Adriano Henrique; de Melo, Maria Celeste Nunes; Borba, Luiz Henrique Fernandes; de Lima Júnior, Dorgival Morais; Novaes, Luciano Patto; Urbano, Stela Antas; de Andrade Neto, Júlio César

    2017-09-01

    This study aimed to evaluate the microbiological and cellular milk profile for the diagnosis of subclinical mastitis in female buffaloes and to assess risk factors for predisposition of the disease. Analyses were carried out by standard plate count (SPC), identification of species and antibiotic resistance, somatic cell count (SCC), electrical electrical conductivity of milk (ECM), and lactoferrin content in milk. Teat cups were swabbed to evaluate risk factors, observing hyperkeratosis, milking vacuum pressure and cleanliness of the site. Hence, 30 female buffaloes were randomly selected (15 from a group in early lactation and 15 in late lactation). The most common bacteria in the microbiological examination were Staphylococcus spp., Streptococcus spp. and Corynebacterium sp. In the antibiotic sensitivity test, 10 (58.82%) of the 17 antibiotics tested were sensitive to all isolates, and resistant bacteria were Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus haemolyticus, and Escherichia coli. It was observed that positive samples in the microbiological examination showed total bacterial count between 9.10×10(3) to 6.94×10(6) colony forming units/mL, SCC between 42,000 to 4,320,000 cells/mL and ECM ranging from 1.85 to 7.40 mS/cm. It was also found that the teat cups had high microbial counts indicating poor hygiene, and even faults in the cleanliness of the animals' waiting room were observed. It is concluded that values of SCC above 537,000 cells/mL and ECM above 3.0 mS/mL are indications of mammary gland infection for this herd; however, the association of these values with a microbiological analysis is necessary to more accurately evaluate the health status of mammary glands with subclinical mastitis. Through phenotypic characterization of bacteria involved in the samples, the genera Staphylococcus spp., Streptococcus spp., and Corynebacterimum bovis were the most prevalent in this study. Faults in environment and equipment hygienization are

  12. Transcriptional control of fungal cell cycle and cellular events by Fkh2, a forkhead transcription factor in an insect pathogen

    PubMed Central

    Wang, Juan-Juan; Qiu, Lei; Cai, Qing; Ying, Sheng-Hua; Feng, Ming-Guang

    2015-01-01

    Transcriptional control of the cell cycle by forkhead (Fkh) transcription factors is likely associated with fungal adaptation to host and environment. Here we show that Fkh2, an ortholog of yeast Fkh1/2, orchestrates cell cycle and many cellular events of Beauveria bassiana, a filamentous fungal insect pathogen. Deletion of Fkh2 in B. bassiana resulted in dramatic down-regulation of the cyclin-B gene cluster and hence altered cell cycle (longer G2/M and S, but shorter G0/G1, phases) in unicellular blastospores. Consequently, ΔFkh2 produced twice as many, but smaller, blastospores than wild-type under submerged conditions, and formed denser septa and shorter/broader cells in aberrantly branched hyphae. In these hyphae, clustered genes required for septation and conidiation were remarkedly up-regulated, followed by higher yield and slower germination of aerial conidia. Moreover, ΔFkh2 displayed attenuated virulence and decreased tolerance to chemical and environmental stresses, accompanied with altered transcripts and activities of phenotype-influencing proteins or enzymes. All the changes in ΔFkh2 were restored by Fkh2 complementation. All together, Fkh2-dependent transcriptional control is vital for the adaptation of B. bassiana to diverse habitats of host insects and hence contributes to its biological control potential against arthropod pests. PMID:25955538

  13. Genomic and proteomic analysis of transcription factor TFII-I reveals insight into the response to cellular stress

    PubMed Central

    Fan, Alex Xiucheng; Papadopoulos, Giorgio L.; Hossain, Mir A.; Lin, I.-Ju; Hu, Jianhong; Tang, Tommy Ming; Kilberg, Michael S.; Renne, Rolf; Strouboulis, John; Bungert, Jörg

    2014-01-01

    The ubiquitously expressed transcription factor TFII-I exerts both positive and negative effects on transcription. Using biotinylation tagging technology and high-throughput sequencing, we determined sites of chromatin interactions for TFII-I in the human erythroleukemia cell line K562. This analysis revealed that TFII-I binds upstream of the transcription start site of expressed genes, both upstream and downstream of the transcription start site of repressed genes, and downstream of RNA polymerase II peaks at the ATF3 and other stress responsive genes. At the ATF3 gene, TFII-I binds immediately downstream of a Pol II peak located 5 kb upstream of exon 1. Induction of ATF3 expression increases transcription throughout the ATF3 gene locus which requires TFII-I and correlates with increased association of Pol II and Elongin A. Pull-down assays demonstrated that TFII-I interacts with Elongin A. Partial depletion of TFII-I expression caused a reduction in the association of Elongin A with and transcription of the DNMT1 and EFR3A genes without a decrease in Pol II recruitment. The data reveal different interaction patterns of TFII-I at active, repressed, or inducible genes, identify novel TFII-I interacting proteins, implicate TFII-I in the regulation of transcription elongation and provide insight into the role of TFII-I during the response to cellular stress. PMID:24875474

  14. Functional genomics identifies a requirement of pre-mRNA splicing factors for sister chromatid cohesion.

    PubMed

    Sundaramoorthy, Sriramkumar; Vázquez-Novelle, María Dolores; Lekomtsev, Sergey; Howell, Michael; Petronczki, Mark

    2014-11-18

    Sister chromatid cohesion mediated by the cohesin complex is essential for chromosome segregation during cell division. Using functional genomic screening, we identify a set of 26 pre-mRNA splicing factors that are required for sister chromatid cohesion in human cells. Loss of spliceosome subunits increases the dissociation rate of cohesin from chromatin and abrogates cohesion after DNA replication, ultimately causing mitotic catastrophe. Depletion of splicing factors causes defective processing of the pre-mRNA encoding sororin, a factor required for the stable association of cohesin with chromatin, and an associated reduction of sororin protein level. Expression of an intronless version of sororin and depletion of the cohesin release protein WAPL suppress the cohesion defect in cells lacking splicing factors. We propose that spliceosome components contribute to sister chromatid cohesion and mitotic chromosome segregation through splicing of sororin pre-mRNA. Our results highlight the loss of cohesion as an early cellular consequence of compromised splicing. This may have clinical implications because SF3B1, a splicing factor that we identify to be essential for cohesion, is recurrently mutated in chronic lymphocytic leukaemia.

  15. Adrenal Gland Infection by Serotype 5 Adenovirus Requires Coagulation Factors

    PubMed Central

    Franken, Philippe R.; Darcourt, Jacques; Cornilleau, Gaétan; Benihoud, Karim; Vassaux, Georges

    2013-01-01

    Recombinant, replication-deficient serotype 5 adenovirus infects the liver upon in vivo, systemic injection in rodents. This infection requires the binding of factor X to the capsid of this adenovirus. Another organ, the adrenal gland is also infected upon systemic administration of Ad, however, whether this infection is dependent on the cocksackie adenovirus receptor (CAR) or depends on the binding of factor X to the viral capsid remained to be determined. In the present work, we have used a pharmacological agent (warfarin) as well as recombinant adenoviruses lacking the binding site of Factor X to elucidate this mechanism in mice. We demonstrate that, as observed in the liver, adenovirus infection of the adrenal glands in vivo requires Factor X. Considering that the level of transduction of the adrenal glands is well-below that of the liver and that capsid-modified adenoviruses are unlikely to selectively infect the adrenal glands, we have used single-photon emission computed tomography (SPECT) imaging of gene expression to determine whether local virus administration (direct injection in the kidney) could increase gene transfer to the adrenal glands. We demonstrate that direct injection of the virus in the kidney increases gene transfer in the adrenal gland but liver transduction remains important. These observations strongly suggest that serotype 5 adenovirus uses a similar mechanism to infect liver and adrenal gland and that selective transgene expression in the latter is more likely to be achieved through transcriptional targeting. PMID:23638001

  16. Cellular localization of transforming growth factor-beta expression in bleomycin-induced pulmonary fibrosis.

    PubMed Central

    Zhang, K.; Flanders, K. C.; Phan, S. H.

    1995-01-01

    Bleomycin-induced pulmonary fibrosis is associated with increased lung transforming growth factor-beta (TGF-beta) gene expression, but cellular localization of the source of this expression has not been unequivocally established. In this study, lung fibrosis was induced in rats by endotracheal bleomycin injection on day 0 and, on selected days afterwards, lungs were harvested for in situ hybridization, immunohistochemical and histochemical analyses for TGF-beta 1 mRNA and protein expression, and cell identification. The results show that control lungs express essentially no detectable TGF-beta 1 mRNA or protein in the parenchyma. Before day 3 after bleomycin treatment, scattered bronchiolar epithelial cells, mononuclear cells, and eosinophils expressed elevated levels of TGF-beta 1. Between days 3 and 14, there was a major increase in the number of eosinophils, myofibroblasts, and fibroblasts strongly expressing TGF-beta 1 mRNA and protein. TGF-beta 1-producing cells were predominantly localized within areas of injury and active fibrosis. After day 14, the intensity and number of TGF-beta 1-expressing cells significantly declined and were predominantly found in fibroblasts in fibrotic areas. The expression of TGF-beta 1 protein was generally coincident with that for mRNA with the exception of bronchiolar epithelial cells in which strong protein expression was unaccompanied by a commensurate increase in mRNA. The study demonstrates that myofibroblasts, fibroblasts, and eosinophils represent the major sources of increased lung TGF-beta 1 expression in this model of pulmonary fibrosis. Images Figure 2 Figure 3 Figure 4 PMID:7543734

  17. Pancreatic cellular injury after cardiac surgery with cardiopulmonary bypass: frequency, time course and risk factors.

    PubMed

    Nys, Monique; Venneman, Ingrid; Deby-Dupont, Ginette; Preiser, Jean-Charles; Vanbelle, Sophie; Albert, Adelin; Camus, Gérard; Damas, Pierre; Larbuisson, Robert; Lamy, Maurice

    2007-05-01

    Although often clinically silent, pancreatic cellular injury (PCI) is relatively frequent after cardiac surgery with cardiopulmonary bypass; and its etiology and time course are largely unknown. We defined PCI as the simultaneous presence of abnormal values of pancreatic isoamylase and immunoreactive trypsin (IRT). The frequency and time evolution of PCI were assessed in this condition using assays for specific exocrine pancreatic enzymes. Correlations with inflammatory markers were searched for preoperative risk factors. One hundred ninety-three patients submitted to cardiac surgery were enrolled prospectively. Blood IRT, amylase, pancreatic isoamylase, lipase, and markers of inflammation (alpha1-protease inhibitor, alpha2-macroglobulin, myeloperoxidase) were measured preoperatively and postoperatively until day 8. The postoperative increase in plasma levels of pancreatic enzymes and urinary IRT was biphasic in all patients: early after surgery and later (from day 4 to 8 after surgery). One hundred thirty-three patients (69%) experienced PCI, with mean IRT, isoamylase, and alpha1-protease inhibitor values higher for each sample than that in patients without PCI. By multiple regression analysis, we found preoperative values of plasma IRT >or=40 ng/mL, amylase >or=42 IU/mL, and pancreatic isoamylase >or=20 IU/L associated with a higher incidence of postsurgery PCI (P < 0.005). In the PCI patients, a significant correlation was found between the 4 pancreatic enzymes and urinary IRT, total calcium, myeloperoxidase, alpha1-protease inhibitor, and alpha2-macroglobulin. These data support a high prevalence of postoperative PCI after cardiac surgery with cardiopulmonary bypass, typically biphasic and clinically silent, especially when pancreatic enzymes were elevated preoperatively.

  18. Role of cellular bioenergetics in smooth muscle cell proliferation induced by platelet-derived growth factor.

    PubMed

    Perez, Jessica; Hill, Bradford G; Benavides, Gloria A; Dranka, Brian P; Darley-Usmar, Victor M

    2010-05-13

    Abnormal smooth muscle cell proliferation is a hallmark of vascular disease. Although growth factors are known to contribute to cell hyperplasia, the changes in metabolism associated with this response, particularly mitochondrial respiration, remain unclear. Given the increased energy requirements for proliferation, we hypothesized that PDGF (platelet-derived growth factor) would stimulate glycolysis and mitochondrial respiration and that this elevated bioenergetic capacity is required for smooth muscle cell hyperplasia. To test this hypothesis, cell proliferation, glycolytic flux and mitochondrial oxygen consumption were measured after treatment of primary rat aortic VSMCs (vascular smooth muscle cells) with PDGF. PDGF increased basal and maximal rates of glycolytic flux and mitochondrial oxygen consumption; enhancement of these bioenergetic pathways led to a substantial increase in the mitochondrial reserve capacity. Interventions with the PI3K (phosphoinositide 3-kinase) inhibitor LY-294002 or the glycolysis inhibitor 2-deoxy-D-glucose abrogated PDGF-stimulated proliferation and prevented augmentation of glycolysis and mitochondrial reserve capacity. Similarly, when L-glucose was substituted for D-glucose, PDGF-dependent proliferation was abolished, as were changes in glycolysis and mitochondrial respiration. Interestingly, LDH (lactate dehydrogenase) protein levels and activity were significantly increased after PDGF treatment. Moreover, substitution of L-lactate for D-glucose was sufficient to increase mitochondrial reserve capacity and cell proliferation after treatment with PDGF; these effects were inhibited by the LDH inhibitor oxamate. These results suggest that glycolysis, by providing substrates that enhance the mitochondrial reserve capacity, plays an essential role in PDGF-induced cell proliferation, underscoring the integrated metabolic response required for proliferation of VSMCs in the diseased vasculature.

  19. Melanin is required for the formation of the multi-cellular conidia in the endophytic fungus Pestalotiopsis microspora.

    PubMed

    Yu, Xi; Huo, Liang; Liu, Heng; Chen, Longfei; Wang, Yu; Zhu, Xudong

    2015-10-01

    Melanin plays an important role in regulating various biological processes in many fungi. However, its biological role in conidiation remains largely elusive. We report here that conidia production, morphogenesis, integrity, germination and their viability in Pestalotiopsis microspora require the polyketide-derived melanin. A polyketide synthase gene, pks1, was identified and demonstrated responsible for melanin biosynthesis in this fungus. A targeted deletion mutant strain Δpks1 displayed a defect in pigmentation of conidia and had an albino colonial phenotype. Interestingly, Δpks1 produced approximately 6-fold as many conidia as the wild type did, suggesting a negative modulation of melanin on conidia production in this fungus. Moreover, the conidia failed to develop into the normal five-cell morphology, rather the three main-body cells separated via constriction at the original septum position to generate three independent mutant conidia. This result suggests a novel role of melanin in the formation of the multi-cellular conidia. Germ tubes could develop from the three different types of mutant conidia and kept elongating, despite a significantly lower germination rate was observed for them. Still more, the unpigmented conidia became permeable to Calcofluor White and DAPI, suggesting the integrity of the conidia was impaired. Deliberate inhibition of melanin biosynthesis by a specific inhibitor, tricyclazole, led to a similar phenotypes. This work demonstrates a new function of fungal melanin in conidial development.

  20. The Adaptor Protein and Arf GTPase-activating Protein Cat-1/Git-1 Is Required for Cellular Transformation

    PubMed Central

    Yoo, Sungsoo M.; Antonyak, Marc A.; Cerione, Richard A.

    2012-01-01

    Cat-1/Git-1 is a multifunctional protein that acts as a GTPase-activating protein (GAP) for Arf GTPases, as well as serves as a scaffold for a number of different signaling proteins. Cat-1 is best known for its role in regulating cell shape and promoting cell migration. However, whether Cat-1 might also contribute to cellular transformation is currently unknown. Here we show that ∼95% of cervical tumor samples examined overexpress Cat-1, suggesting that the up-regulation of Cat-1 expression is a frequent occurrence in this type of cancer. We demonstrate further that knocking down Cat-1 from NIH3T3 fibroblasts expressing an activated form of Cdc42 (Cdc42 F28L), or from the human cervical carcinoma (HeLa) cell line, inhibits the ability of these cells to form colonies in soft agar, an in vitro measure of tumorgenicity. The requirement for Cat-1 when assaying the anchorage-independent growth of transformed fibroblasts and HeLa cells is dependent on its ability to bind paxillin, while being negatively impacted by its Arf-GAP activity. Moreover, the co-expression of Cat-1 and an activated form of Arf6 in fibroblasts was sufficient to induce their transformation. These findings highlight novel roles for Cat-1 and its interactions with the Arf GTPases and paxillin in oncogenic transformation. PMID:22807447

  1. The maize pentatricopeptide repeat gene empty pericarp4 (emp4) is required for proper cellular development in vegetative tissues.

    PubMed

    Gabotti, Damiano; Caporali, Elisabetta; Manzotti, Priscilla; Persico, Martina; Vigani, Gianpiero; Consonni, Gabriella

    2014-06-01

    The empty pericarp4 (emp4) gene encodes a mitochondrion-targeted pentatricopeptide repeat (ppr) protein that is involved in the regulation of mitochondrial gene expression and is required for seed development. In homozygous mutant emp4-1 kernels the endosperm is drastically reduced and the embryo is retarded in its development and unable to germinate. With the aim of investigating the role of emp4 during post-germinative development, homozygous mutant seedlings were obtained by cultivation of excised immature embryos on a synthetic medium. In the mutants both germination frequency as well as the proportion of seedlings reaching the first and second leaf stages were reduced. The anatomy of the leaf blades and the root cortex was not affected by the mutation, however severe alterations such as the presence of empty cells or cells containing poorly organized organelles, were observed. Moreover both mitochondria and chloroplast functionality was impaired in the mutants. Our hypothesis is that mitochondrial impairment, the primary effect of the mutation, causes secondary effects on the development of other cellular organelles. Ultra-structural features of mutant leaf blade mesophyll cells are reminiscent of cells undergoing senescence. Interestingly, both structural and functional damage was less severe in seedlings grown in total darkness compared with those exposed to light, thus suggesting that the effects of the mutation are enhanced by the presence of light. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  2. Chemical structure requirements and cellular targeting of microRNA-122 by peptide nucleic acids anti-miRs

    PubMed Central

    Torres, Adrian G.; Fabani, Martin M.; Vigorito, Elena; Williams, Donna; Al-Obaidi, Naowras; Wojciechowski, Filip; Hudson, Robert H. E.; Seitz, Oliver; Gait, Michael J.

    2012-01-01

    Anti-miRs are oligonucleotide inhibitors complementary to miRNAs that have been used extensively as tools to gain understanding of specific miRNA functions and as potential therapeutics. We showed previously that peptide nucleic acid (PNA) anti-miRs containing a few attached Lys residues were potent miRNA inhibitors. Using miR-122 as an example, we report here the PNA sequence and attached amino acid requirements for efficient miRNA targeting and show that anti-miR activity is enhanced substantially by the presence of a terminal-free thiol group, such as a Cys residue, primarily due to better cellular uptake. We show that anti-miR activity of a Cys-containing PNA is achieved by cell uptake through both clathrin-dependent and independent routes. With the aid of two PNA analogues having intrinsic fluorescence, thiazole orange (TO)-PNA and [bis-o-(aminoethoxy)phenyl]pyrrolocytosine (BoPhpC)-PNA, we explored the subcellular localization of PNA anti-miRs and our data suggest that anti-miR targeting of miR-122 may take place in or associated with endosomal compartments. Our findings are valuable for further design of PNAs and other oligonucleotides as potent anti-miR agents. PMID:22070883

  3. Foxd1-dependent signals control cellularity in the renal capsule, a structure required for normal renal development.

    PubMed

    Levinson, Randy S; Batourina, Ekatherina; Choi, Christopher; Vorontchikhina, Marina; Kitajewski, Jan; Mendelsohn, Cathy L

    2005-02-01

    Development of the metanephric kidney involves the establishment of discrete zones of induction and differentiation that are crucial to the future radial patterning of the organ. Genetic deletion of the forkhead transcription factor, Foxd1, results in striking renal abnormalities, including the loss of these discrete zones and pelvic fused kidneys. We have investigated the molecular and cellular basis of the kidney phenotypes displayed by Foxd1-null embryos and report here that they are likely to be caused by a failure in the correct formation of the renal capsule. Unlike the single layer of Foxd1-positive stroma that comprises the normal renal capsule, the mutant capsule contains heterogeneous layers of cells, including Bmp4-expressing cells, which induce ectopic phospho-Smad1 signaling in nephron progenitors. This missignaling disrupts their early patterning, which, in turn, causes mispatterning of the ureteric tree, while delaying and disorganizing nephrogenesis. In addition, the defects in capsule formation prevent the kidneys from detaching from the body wall, thus explaining their fusion and pelvic location. For the first time, functions have been ascribed to the renal capsule that include delineation of the organ and acting as a barrier to inappropriate exogenous signals, while providing a source of endogenous signals that are crucial to the establishment of the correct zones of induction and differentiation.

  4. An in vivo RNAi assay identifies major genetic and cellular requirements for primary piRNA biogenesis in Drosophila

    PubMed Central

    Olivieri, Daniel; Sykora, Martina M; Sachidanandam, Ravi; Mechtler, Karl; Brennecke, Julius

    2010-01-01

    In Drosophila, PIWI proteins and bound PIWI-interacting RNAs (piRNAs) form the core of a small RNA-mediated defense system against selfish genetic elements. Within germline cells, piRNAs are processed from piRNA clusters and transposons to be loaded into Piwi/Aubergine/AGO3 and a subset of piRNAs undergoes target-dependent amplification. In contrast, gonadal somatic support cells express only Piwi, lack signs of piRNA amplification and exhibit primary piRNA biogenesis from piRNA clusters. Neither piRNA processing/loading nor Piwi-mediated target silencing is understood at the genetic, cellular or molecular level. We developed an in vivo RNAi assay for the somatic piRNA pathway and identified the RNA helicase Armitage, the Tudor domain containing RNA helicase Yb and the putative nuclease Zucchini as essential factors for primary piRNA biogenesis. Lack of any of these proteins leads to transposon de-silencing, to a collapse in piRNA levels and to a failure in Piwi-nuclear accumulation. We show that Armitage and Yb interact physically and co-localize in cytoplasmic Yb bodies, which flank P bodies. Loss of Zucchini leads to an accumulation of Piwi and Armitage in Yb bodies, indicating that Yb bodies are sites of primary piRNA biogenesis. PMID:20818334

  5. Wnt inhibitory factor 1 suppresses cancer stemness and induces cellular senescence

    PubMed Central

    Ramachandran, I; Ganapathy, V; Gillies, E; Fonseca, I; Sureban, S M; Houchen, C W; Reis, A; Queimado, L

    2014-01-01

    Hyperactivation of the Wingless-type (Wnt)/β-catenin pathway promotes tumor initiation, tumor growth and metastasis in various tissues. Although there is evidence for the involvement of Wnt/β-catenin pathway activation in salivary gland tumors, the precise mechanisms are unknown. Here we report for the first time that downregulation of the Wnt inhibitory factor 1 (WIF1) is a widespread event in salivary gland carcinoma ex-pleomorphic adenoma (CaExPA). We also show that WIF1 downregulation occurs in the CaExPA precursor lesion pleomorphic adenoma (PA) and indicates a higher risk of progression from benign to malignant tumor. Our results demonstrate that diverse mechanisms including WIF1 promoter hypermethylation and loss of heterozygosity contribute to WIF1 downregulation in human salivary gland tumors. In accordance with a crucial role in suppressing salivary gland tumor progression, WIF1 re-expression in salivary gland tumor cells inhibited cell proliferation, induced more differentiated phenotype and promoted cellular senescence, possibly through upregulation of tumor-suppressor genes, such as p53 and p21. Most importantly, WIF1 significantly diminished the number of salivary gland cancer stem cells and the anchorage-independent cell growth. Consistent with this observation, WIF1 caused a reduction in the expression of pluripotency and stemness markers (OCT4 and c-MYC), as well as adult stem cell self-renewal and multi-lineage differentiation markers, such as WNT3A, TCF4, c-KIT and MYB. Furthermore, WIF1 significantly increased the expression of microRNAs pri-let-7a and pri-miR-200c, negative regulators of stemness and cancer progression. In addition, we show that WIF1 functions as a positive regulator of miR-200c, leading to downregulation of BMI1, ZEB1 and ZEB2, with a consequent increase in downstream targets such as E-cadherin. Our study emphasizes the prognostic and therapeutic potential of WIF1 in human salivary gland CaExPA. Moreover, our findings

  6. Cellular Transcription Factor Sp1 Recruits Simian Virus 40 Capsid Proteins to the Viral Packaging Signal, ses

    PubMed Central

    Gordon-Shaag, Ariela; Ben-Nun-Shaul, Orly; Roitman, Vered; Yosef, Yael; Oppenheim, Ariella

    2002-01-01

    Simian virus 40 (SV40) capsid assembly occurs in the nucleus. All three capsid proteins bind DNA nonspecifically, raising the dilemma of how they attain specificity to the SV40 minichromosome in the presence of a large excess of genomic DNA. The SV40 packaging signal, ses, which is required for assembly, is composed of multiple DNA elements that bind transcription factor Sp1. Our previous studies showed that Sp1 participates in SV40 assembly and that it cooperates in DNA binding with VP2/3. We hypothesized that Sp1 recruits the capsid proteins to the viral minichromosome, conferring upon them specific DNA recognition. Here, we have tested the hypothesis. Computer analysis showed that the combination of six tandem GC boxes at ses is not found at cellular promoters and therefore is unique to SV40. Cooperativity in DNA binding between Sp1 and VP2/3 was not abolished at even a 1,000-fold excess of cellular DNA, providing strong support for the recruitment hypothesis. Sp1 also binds VP1 and cooperates with VP1 in DNA binding. VP1 pentamers (VP15) avidly interact with VP2/3, utilizing the same VP2/3 domain as described for polyomavirus. We conclude that VP15-VP2/3 building blocks are recruited by Sp1 to ses, where they form the nucleation center for capsid assembly. By this mechanism the virus ensures that capsid formation is initiated at a single site around its minichromosome. Sp1 enhances the formation of SV40 pseudovirions in vitro, providing additional support for the model. Analyses of Sp1 and VP3 deletion mutants showed that Sp1 and VP2/3 bind one another and cooperate in DNA binding through their DNA-binding domains, with additional contacts outside these domains. VP1 contacts Sp1 at residues outside the Sp1 DNA-binding domain. These and additional data allowed us to propose a molecular model for the VP15-VP2/3-DNA-Sp1 complex. PMID:12021324

  7. Role of Eukaryotic Initiation Factors during Cellular Stress and Cancer Progression

    PubMed Central

    Sharma, Divya Khandige; Bressler, Kamiko; Patel, Harshil; Balasingam, Nirujah

    2016-01-01

    Protein synthesis can be segmented into distinct phases comprising mRNA translation initiation, elongation, and termination. Translation initiation is a highly regulated and rate-limiting step of protein synthesis that requires more than 12 eukaryotic initiation factors (eIFs). Extensive evidence shows that the transcriptome and corresponding proteome do not invariably correlate with each other in a variety of contexts. In particular, translation of mRNAs specific to angiogenesis, tumor development, and apoptosis is altered during physiological and pathophysiological stress conditions. In cancer cells, the expression and functions of eIFs are hampered, resulting in the inhibition of global translation and enhancement of translation of subsets of mRNAs by alternative mechanisms. A precise understanding of mechanisms involving eukaryotic initiation factors leading to differential protein expression can help us to design better strategies to diagnose and treat cancer. The high spatial and temporal resolution of translation control can have an immediate effect on the microenvironment of the cell in comparison with changes in transcription. The dysregulation of mRNA translation mechanisms is increasingly being exploited as a target to treat cancer. In this review, we will focus on this context by describing both canonical and noncanonical roles of eIFs, which alter mRNA translation. PMID:28083147

  8. Identification of RNA Binding Proteins Associated with Dengue Virus RNA in Infected Cells Reveals Temporally Distinct Host Factor Requirements

    PubMed Central

    Viktorovskaya, Olga V.; Greco, Todd M.; Cristea, Ileana M.; Thompson, Sunnie R.

    2016-01-01

    Background There are currently no vaccines or antivirals available for dengue virus infection, which can cause dengue hemorrhagic fever and death. A better understanding of the host pathogen interaction is required to develop effective therapies to treat DENV. In particular, very little is known about how cellular RNA binding proteins interact with viral RNAs. RNAs within cells are not naked; rather they are coated with proteins that affect localization, stability, translation and (for viruses) replication. Methodology/Principal Findings Seventy-nine novel RNA binding proteins for dengue virus (DENV) were identified by cross-linking proteins to dengue viral RNA during a live infection in human cells. These cellular proteins were specific and distinct from those previously identified for poliovirus, suggesting a specialized role for these factors in DENV amplification. Knockdown of these proteins demonstrated their function as viral host factors, with evidence for some factors acting early, while others late in infection. Their requirement by DENV for efficient amplification is likely specific, since protein knockdown did not impair the cell fitness for viral amplification of an unrelated virus. The protein abundances of these host factors were not significantly altered during DENV infection, suggesting their interaction with DENV RNA was due to specific recruitment mechanisms. However, at the global proteome level, DENV altered the abundances of proteins in particular classes, including transporter proteins, which were down regulated, and proteins in the ubiquitin proteasome pathway, which were up regulated. Conclusions/Significance The method for identification of host factors described here is robust and broadly applicable to all RNA viruses, providing an avenue to determine the conserved or distinct mechanisms through which diverse viruses manage the viral RNA within cells. This study significantly increases the number of cellular factors known to interact with

  9. The Effects of Imatinib Mesylate on Cellular Viability, Platelet Derived Growth Factor and Stem Cell Factor in Mouse Testicular Normal Leydig Cells.

    PubMed

    Kheradmand, Fatemeh; Hashemnia, Seyyed Mohammad Reza; Valizadeh, Nasim; Roshan-Milani, Shiva

    2016-01-01

    Growth factors play an essential role in the development of tumor and normal cells like testicular leydig cells. Treatment of cancer with anti-cancer agents like imatinib mesylate may interfere with normal leydig cell activity, growth and fertility through failure in growth factors' production or their signaling pathways. The purpose of the study was to determine cellular viability and the levels of, platelet derived growth factor (PDGF) and stem cell factor (SCF) in normal mouse leydig cells exposed to imatinib, and addressing the effect of imatinib on fertility potential. The mouse TM3 leydig cells were treated with 0 (control), 2.5, 5, 10 and 20 μM imatinib for 2, 4 and 6 days. Each experiment was repeated three times (15 experiments in each day).The cellular viability and growth factors levels were assessed by MTT and ELISA methods, respectively. For statistical analysis, one-way ANOVA with Tukey's post hoc and Kruskal-Wallis test were performed. A p-value less than 0.05 was considered statistically significant. With increasing drug concentration, cellular viability decreased significantly (p<0.05) and in contrast, PDGF levels increased (p<0.05). Different imatinib concentrations had no significant effect on SCF level. Increasing the duration of treatment from 2 to 6 days had no obvious effect on cellular viability, PDGF and SCF levels. Imatinib may reduce fertility potential especially at higher concentrations in patients treated with this drug by decreasing cellular viability. The effect of imatinib on leydig cells is associated with PDGF stimulation. Of course future studies can be helpful in exploring the long term effects of this drug.

  10. Epidermal Growth Factor Receptor Cell Survival Signaling Requires Phosphatidylcholine Biosynthesis

    PubMed Central

    Crook, Matt; Upadhyay, Awani; Ido, Liyana J.; Hanna-Rose, Wendy

    2016-01-01

    Identification of pro-cell survival signaling pathways has implications for cancer, cardiovascular, and neurodegenerative disease. We show that the Caenorhabditis elegans epidermal growth factor receptor LET-23 (LET-23 EGFR) has a prosurvival function in counteracting excitotoxicity, and we identify novel molecular players required for this prosurvival signaling. uv1 sensory cells in the C. elegans uterus undergo excitotoxic death in response to activation of the OSM-9/OCR-4 TRPV channel by the endogenous agonist nicotinamide. Activation of LET-23 EGFR can effectively prevent this excitotoxic death. We investigate the roles of signaling pathways known to act downstream of LET-23 EGFR in C. elegans and find that the LET-60 Ras/MAPK pathway, but not the IP3 receptor pathway, is required for efficient LET-23 EGFR activity in its prosurvival function. However, activation of LET-60 Ras/MAPK pathway does not appear to be sufficient to fully mimic LET-23 EGFR activity. We screen for genes that are required for EGFR prosurvival function and uncover a role for phosphatidylcholine biosynthetic enzymes in EGFR prosurvival function. Finally, we show that exogenous application of phosphatidylcholine is sufficient to prevent some deaths in this excitotoxicity model. Our work implicates regulation of lipid synthesis downstream of EGFR in cell survival and death decisions. PMID:27605519

  11. Risk factors for endophthalmitis requiring evisceration or enucleation

    PubMed Central

    Lu, Xuehui; Ng, Danny Siu-Chun; Zheng, Kangkeng; Peng, Kun; Jin, Chuang; Xia, Honghe; Chen, Weiqi; Chen, Haoyu

    2016-01-01

    Endophthalmitis has devastating sequelae resulting in blindness and even loss of eyeball. Although the prognosis of endophthalmitis has much improved with the advances of antibiotics and vitreoretinal surgery, of the number of patients that required evisceration or enucleation is still significant. We retrospectively reviewed the charts of 210 eyes of 210 patients with endophthalmitis andcompared the group that required evisceration or enucleation with those that received salvaging therapies. Regression analysis was used to identify the risk factors for evisceration or enucleation. Thirty eyes (14.3%) underwent enucleation or evisceration. The group of eviscerated or enucleated eyes were older (58.7 vs. 42.2 years, p < 0.001), had more women (56.7% vs. 22.2%, p = 0.003), had poorer initial visual acuity (2.79 vs. 2.10 LogMAR, p < 0.001), and had longer duration before intervention (18.03 vs. 5.74 days, p = 0.031). The most common primary indications for endophthalmitis were infections from corneal ulcer (50.0% vs. 4.4%, p < 0.001) andfrom endogenous source (23.3% vs. 5.6%, p < 0.001). Less common indications were trauma (26.7% vs. 67.8%, p < 0.001) and postoperative (6.7% vs. 22.2%, p = 0.049) endophthalmitis. After adjusting for confounding factors, corneal ulcer-related endophthalmitis, endogenous endophthalmitis and initial visual acuity were the independent risk factors for evisceration or enucleation. PMID:27302573

  12. Factors Associated with Higher Oxytocin Requirements in Labor

    PubMed Central

    Frey, Heather A.; Tuuli, Methodius G.; England, Sarah K.; Roehl, Kimberly A.; Odibo, Anthony O.; Macones, George A.; Cahill, Alison G.

    2014-01-01

    Objective To identify clinical characteristics associated with high maximum oxytocin doses in women who achieve complete cervical dilation. Methods A retrospective nested case-control study was performed within a cohort of all term women at a single center between 2004-2008 who reached the second stage of labor. Cases were defined as women who had a maximum oxytocin dose during labor >20 mu/min, while women in the control group had a maximum oxytocin dose during labor of ≤20 mu/min. Exclusion criteria included no oxytocin administration during labor, multiple gestations, major fetal anomalies, nonvertex presentation, and prior cesarean delivery. Multiple maternal, fetal, and labor factors were evaluated with univariable analysis and multivariable logistic regression. Results Maximum oxytocin doses >20 mu/min were administered to 108 women (3.6%), while 2864 women received doses ≤20 mu/min. Factors associated with higher maximum oxytocin dose after adjusting for relevant confounders included maternal diabetes, birth weight >4000 gm, intrapartum fever, administration of magnesium, and induction of labor. Conclusions Few women who achieve complete cervical dilation require high doses of oxytocin. We identified maternal, fetal and labor factors that characterize this group of parturients. PMID:25204333

  13. Temperature, template topology, and factor requirements of archaeal transcription

    PubMed Central

    Bell, Stephen D.; Jaxel, Christine; Nadal, Marc; Kosa, Peter F.; Jackson, Stephen P.

    1998-01-01

    Although Archaea are prokaryotic and resemble Bacteria morphologically, their transcription apparatus is remarkably similar to those of eukaryotic cell nuclei. Because some Archaea exist in environments with temperatures of around 100°C, they are likely to have evolved unique strategies for transcriptional control. Here, we investigate the effects of temperature and DNA template topology in a thermophilic archaeal transcription system. Significantly, and in marked contrast with characterized eucaryal systems, archaeal DNA template topology has negligible effect on transcription levels at physiological temperatures using highly purified polymerase and recombinant transcription factors. Furthermore, archaeal transcription does not require hydrolysis of the β-γ phosphoanhydride bond of ATP. However, at lower temperatures, negatively supercoiled templates are transcribed more highly than those that are positively supercoiled. Notably, the block to transcription on positively supercoiled templates at lowered temperatures is at the level of polymerase binding and promoter opening. These data imply that Archaea do not possess a functional homologue of transcription factor TFIIH, and that for the promoters studied, transcription is mediated by TATA box-binding protein, transcription factor TFB, and RNA polymerase alone. Furthermore, they suggest that the reduction of plasmid linking number by hyperthermophilic Archaea in vivo in response to cold shock is a mechanism to maintain gene expression under these adverse circumstances. PMID:9860949

  14. Factors associated with higher oxytocin requirements in labor.

    PubMed

    Frey, Heather A; Tuuli, Methodius G; England, Sarah K; Roehl, Kimberly A; Odibo, Anthony O; Macones, George A; Cahill, Alison G

    2015-09-01

    To identify clinical characteristics associated with high maximum oxytocin doses in women who achieve complete cervical dilation. A retrospective nested case-control study was performed within a cohort of all term women at a single center between 2004 and 2008 who reached the second stage of labor. Cases were defined as women who had a maximum oxytocin dose during labor >20 mu/min, while women in the control group had a maximum oxytocin dose during labor of ≤20 mu/min. Exclusion criteria included no oxytocin administration during labor, multiple gestations, major fetal anomalies, nonvertex presentation, and prior cesarean delivery. Multiple maternal, fetal, and labor factors were evaluated with univariable analysis and multivariable logistic regression. Maximum oxytocin doses >20 mu/min were administered to 108 women (3.6%), while 2864 women received doses ≤20 mu/min. Factors associated with higher maximum oxytocin dose after adjusting for relevant confounders included maternal diabetes, birthweight >4000 g, intrapartum fever, administration of magnesium, and induction of labor. Few women who achieve complete cervical dilation require high doses of oxytocin. We identified maternal, fetal and labor factors that characterize this group of parturients.

  15. Nuclear Factor E2-related Factor-2 (Nrf2) Is Required for NLRP3 and AIM2 Inflammasome Activation*

    PubMed Central

    Zhao, Changcheng; Gillette, Devyn D.; Li, Xinghui; Zhang, Zhibin; Wen, Haitao

    2014-01-01

    Despite the number of extensive studies on the immune function and signaling of inflammasomes in various diseases, the activating mechanism of inflammasome, especially the NLRP3 inflammasome, is not fully understood. Nuclear factor E2-related Factor-2 (Nrf2), a key transcription factor that regulates cellular redox homeostasis, has been reported to play both protective and pathogenic roles depending on the disease conditions through undefined mechanism. This study reveals an essential role of Nrf2 in inflammasome activation. LPS stimulation increased Nrf2 protein levels in a Myd88-dependent manner. When compared with wild-type controls, Nrf2-deficient (Nrf2−/−) macrophages showed decreased maturation and secretion of caspase-1 and IL-1β and reduced apoptosis-associated speck-like protein containing CARD (ASC) speck formation in response to various NLRP3 and AIM2 inflammasome stimuli. In contrast, NLRC4 inflammasome activation was not controlled by Nrf2. Biochemical analysis revealed that Nrf2 appeared in the ASC-enriched cytosolic compartment after NLRP3 inflammasome activation. Furthermore, mitochondrial reactive oxygen species-induced NLRP3 activation also required Nrf2. Nrf2−/− mice showed a dramatic decrease in immune cell recruitment and IL-1β generation in alum-induced peritonitis, which is a typical IL-1 signaling-dependent inflammation animal model. This work discovered a critical proinflammatory effect of Nrf2 by mediating inflammasome activation. PMID:24798340

  16. Nuclear factor E2-related factor-2 (Nrf2) is required for NLRP3 and AIM2 inflammasome activation.

    PubMed

    Zhao, Changcheng; Gillette, Devyn D; Li, Xinghui; Zhang, Zhibin; Wen, Haitao

    2014-06-13

    Despite the number of extensive studies on the immune function and signaling of inflammasomes in various diseases, the activating mechanism of inflammasome, especially the NLRP3 inflammasome, is not fully understood. Nuclear factor E2-related Factor-2 (Nrf2), a key transcription factor that regulates cellular redox homeostasis, has been reported to play both protective and pathogenic roles depending on the disease conditions through undefined mechanism. This study reveals an essential role of Nrf2 in inflammasome activation. LPS stimulation increased Nrf2 protein levels in a Myd88-dependent manner. When compared with wild-type controls, Nrf2-deficient (Nrf2(-/-)) macrophages showed decreased maturation and secretion of caspase-1 and IL-1β and reduced apoptosis-associated speck-like protein containing CARD (ASC) speck formation in response to various NLRP3 and AIM2 inflammasome stimuli. In contrast, NLRC4 inflammasome activation was not controlled by Nrf2. Biochemical analysis revealed that Nrf2 appeared in the ASC-enriched cytosolic compartment after NLRP3 inflammasome activation. Furthermore, mitochondrial reactive oxygen species-induced NLRP3 activation also required Nrf2. Nrf2(-/-) mice showed a dramatic decrease in immune cell recruitment and IL-1β generation in alum-induced peritonitis, which is a typical IL-1 signaling-dependent inflammation animal model. This work discovered a critical proinflammatory effect of Nrf2 by mediating inflammasome activation.

  17. Resuscitation of Pseudomonas aeruginosa from dormancy requires hibernation promoting factor (PA4463) for ribosome preservation.

    PubMed

    Akiyama, Tatsuya; Williamson, Kerry S; Schaefer, Robert; Pratt, Shawna; Chang, Connie B; Franklin, Michael J

    2017-03-21

    Pseudomonas aeruginosa biofilm infections are difficult to treat with antibiotic therapy in part because the biofilms contain subpopulations of dormant antibiotic-tolerant cells. The dormant cells can repopulate the biofilms following alleviation of antibiotic treatments. While dormant, the bacteria must maintain cellular integrity, including ribosome abundance, to reinitiate the de novo protein synthesis required for resuscitation. Here, we demonstrate that the P. aeruginosa gene PA4463 [hibernation promoting factor (HPF)], but not the ribosome modulation factor (PA3049), is required for ribosomal RNA preservation during prolonged nutrient starvation conditions. Single-cell-level studies using fluorescence in situ hybridization (FISH) and growth in microfluidic drops demonstrate that, in the absence of hpf, the rRNA abundances of starved cells decrease to levels that cause them to lose their ability to resuscitate from starvation, leaving intact nondividing cells. P. aeruginosa defective in the stringent response also had reduced ability to resuscitate from dormancy. However, FISH analysis of the starved stringent response mutant showed a bimodal response where the individual cells contained either abundant or low ribosome content, compared with the wild-type strain. The results indicate that ribosome maintenance is key for maintaining the ability of P. aeruginosa to resuscitate from starvation-induced dormancy and that HPF is the major factor associated with P. aeruginosa ribosome preservation.

  18. Resuscitation of Pseudomonas aeruginosa from dormancy requires hibernation promoting factor (PA4463) for ribosome preservation

    PubMed Central

    Akiyama, Tatsuya; Williamson, Kerry S.; Schaefer, Robert; Pratt, Shawna; Chang, Connie B.; Franklin, Michael J.

    2017-01-01

    Pseudomonas aeruginosa biofilm infections are difficult to treat with antibiotic therapy in part because the biofilms contain subpopulations of dormant antibiotic-tolerant cells. The dormant cells can repopulate the biofilms following alleviation of antibiotic treatments. While dormant, the bacteria must maintain cellular integrity, including ribosome abundance, to reinitiate the de novo protein synthesis required for resuscitation. Here, we demonstrate that the P. aeruginosa gene PA4463 [hibernation promoting factor (HPF)], but not the ribosome modulation factor (PA3049), is required for ribosomal RNA preservation during prolonged nutrient starvation conditions. Single-cell–level studies using fluorescence in situ hybridization (FISH) and growth in microfluidic drops demonstrate that, in the absence of hpf, the rRNA abundances of starved cells decrease to levels that cause them to lose their ability to resuscitate from starvation, leaving intact nondividing cells. P. aeruginosa defective in the stringent response also had reduced ability to resuscitate from dormancy. However, FISH analysis of the starved stringent response mutant showed a bimodal response where the individual cells contained either abundant or low ribosome content, compared with the wild-type strain. The results indicate that ribosome maintenance is key for maintaining the ability of P. aeruginosa to resuscitate from starvation-induced dormancy and that HPF is the major factor associated with P. aeruginosa ribosome preservation. PMID:28270601

  19. GADS is Required for TCR-Mediated Calcium Influx and Cytokine Release, but not Cellular Adhesion, in Human T Cells

    PubMed Central

    Bilal, Mahmood Y.; Zhang, Elizabeth Y.; Dinkel, Brittney; Hardy, Daimon; Yankee, Thomas M.; Houtman, Jon C.D.

    2015-01-01

    GRB2 related adaptor protein downstream of Shc (GADS) is a member of the GRB2 family of adaptors and is critical for TCR-induced signaling. The current model is that GADS recruits SLP-76 to the LAT complex, which facilitates the phosphorylation of SLP-76, the activation of PLC-γ1, T cell adhesion and cytokine production. However, this model is largely based on studies of disruption of the GADS/SLP-76 interaction and murine T cell differentiation in GADS deficient mice. The role of GADS in mediating TCR-induced signals in human CD4+ T cells has not been thoroughly investigated. In this study, we have suppressed the expression of GADS in human CD4+ HuT78 T cells. GADS deficient HuT78 T cells displayed similar levels of TCR-induced SLP-76 and PLC-γ1 phosphorylation but exhibited substantial decrease in TCR-induced IL-2 and IFN-γ release. The defect in cytokine production occurred because of impaired calcium mobilization due to reduced recruitment of SLP-76 and PLC-γ1 to the LAT complex. Surprisingly, both GADS deficient HuT78 and GADS deficient primary murine CD8+ T cells had similar TCR-induced adhesion when compared to control T cells. Overall, our results show that GADS is required for calcium influx and cytokine production, but not cellular adhesion, in human CD4+ T cells, suggesting that the current model for T cell regulation by GADS is incomplete. PMID:25636200

  20. Decoding Cellular Dynamics in Epidermal Growth Factor Signaling Using a New Pathway-Based Integration Approach for Proteomics and Transcriptomics Data

    PubMed Central

    Wachter, Astrid; Beißbarth, Tim

    2016-01-01

    Identification of dynamic signaling mechanisms on different cellular layers is now facilitated as the increased usage of various high-throughput techniques goes along with decreasing costs for individual experiments. A lot of these signaling mechanisms are known to be coordinated by their dynamics, turning time-course data sets into valuable information sources for inference of regulatory mechanisms. However, the combined analysis of parallel time-course measurements from different high-throughput platforms still constitutes a major challenge requiring sophisticated bioinformatic tools in order to ease biological interpretation. We developed a new pathway-based integration approach for the analysis of coupled omics time-series data, which we implemented in the R package pwOmics. Unlike many other approaches, our approach acknowledges the role of the different cellular layers of measurement and infers consensus profiles and time profile clusters for further biological interpretation. We investigated a time-course data set on epidermal growth factor stimulation of human mammary epithelial cells generated on the two layers of RNA and proteins. The data was analyzed using our new approach with a focus on feedback signaling and pathway crosstalk. We could confirm known regulatory patterns relevant in the physiological cellular response to epidermal growth factor stimulation as well as identify interesting new interactions in this signaling context, such as the regulatory influence of the connective tissue growth factor on transferrin receptor or the influence of growth arrest and DNA-damage-inducible alpha on the connective tissue growth factor. Thus, we show that integrated cross-platform analysis provides a deeper understanding of regulatory signaling mechanisms. Combined with time-course information it enables the characterization of dynamic signaling processes and leads to the identification of important regulatory interactions which might be dysregulated in disease

  1. The Effects of Imatinib Mesylate on Cellular Viability, Platelet Derived Growth Factor and Stem Cell Factor in Mouse Testicular Normal Leydig Cells

    PubMed Central

    Kheradmand, Fatemeh; Hashemnia, Seyyed Mohammad Reza; Valizadeh, Nasim; Roshan-Milani, Shiva

    2016-01-01

    Background: Growth factors play an essential role in the development of tumor and normal cells like testicular leydig cells. Treatment of cancer with anti-cancer agents like imatinib mesylate may interfere with normal leydig cell activity, growth and fertility through failure in growth factors’ production or their signaling pathways. The purpose of the study was to determine cellular viability and the levels of, platelet derived growth factor (PDGF) and stem cell factor (SCF) in normal mouse leydig cells exposed to imatinib, and addressing the effect of imatinib on fertility potential. Methods: The mouse TM3 leydig cells were treated with 0 (control), 2.5, 5, 10 and 20 μM imatinib for 2, 4 and 6 days. Each experiment was repeated three times (15 experiments in each day).The cellular viability and growth factors levels were assessed by MTT and ELISA methods, respectively. For statistical analysis, one-way ANOVA with Tukey’s post hoc and Kruskal-Wallis test were performed. A p-value less than 0.05 was considered statistically significant. Results: With increasing drug concentration, cellular viability decreased significantly (p<0.05) and in contrast, PDGF levels increased (p<0.05). Different imatinib concentrations had no significant effect on SCF level. Increasing the duration of treatment from 2 to 6 days had no obvious effect on cellular viability, PDGF and SCF levels. Conclusion: Imatinib may reduce fertility potential especially at higher concentrations in patients treated with this drug by decreasing cellular viability. The effect of imatinib on leydig cells is associated with PDGF stimulation. Of course future studies can be helpful in exploring the long term effects of this drug. PMID:27141462

  2. Human Papillomavirus 16 Oncoprotein Expression Is Controlled by the Cellular Splicing Factor SRSF2 (SC35)

    PubMed Central

    McFarlane, Melanie; MacDonald, Alasdair I.; Stevenson, Andrew

    2015-01-01

    ABSTRACT High-risk human papillomaviruses (HR-HPV) cause anogenital cancers, including cervical cancer, and head and neck cancers. Human papillomavirus 16 (HPV16) is the most prevalent HR-HPV. HPV oncogenesis is driven by two viral oncoproteins, E6 and E7, which are expressed through alternative splicing of a polycistronic RNA to yield four major splice isoforms (E6 full length, E6*I, E6*II, E6*X). The production of multiple mRNA isoforms from a single gene is controlled by serine/arginine-rich splicing factors (SRSFs), and HPV16 infection induces overexpression of a subset of these, SRSFs 1, 2, and 3. In this study, we examined whether these proteins could control HPV16 oncoprotein expression. Small interfering RNA (siRNA) depletion experiments revealed that SRSF1 did not affect oncoprotein RNA levels. While SRSF3 knockdown caused some reduction in E6E7 expression, depletion of SRSF2 resulted in a significant loss of E6E7 RNAs, resulting in reduced levels of the E6-regulated p53 proteins and E7 oncoprotein itself. SRSF2 contributed to the tumor phenotype of HPV16-positive cervical cancer cells, as its depletion resulted in decreased cell proliferation, reduced colony formation, and increased apoptosis. SRSF2 did not affect transcription from the P97 promoter that controls viral oncoprotein expression. Rather, RNA decay experiments showed that SRSF2 is required to maintain stability of E6E7 mRNAs. These data show that SRSF2 is a key regulator of HPV16 oncoprotein expression and cervical tumor maintenance. IMPORTANCE Expression of the HPV16 oncoproteins E7 and E6 drives HPV-associated tumor formation. Although increased transcription may yield increased levels of E6E7 mRNAs, it is known that the RNAs can have increased stability upon integration into the host genome. SR splicing factors (SRSFs) control splicing but can also control other events in the RNA life cycle, including RNA stability. Previously, we demonstrated increased levels of SRSFs 1, 2, and 3 during

  3. Epstein–Barr virus transcription factor Zta acts through distal regulatory elements to directly control cellular gene expression

    PubMed Central

    Ramasubramanyan, Sharada; Osborn, Kay; Al-Mohammad, Rajaei; Naranjo Perez-Fernandez, Ijiel B.; Zuo, Jianmin; Balan, Nicolae; Godfrey, Anja; Patel, Harshil; Peters, Gordon; Rowe, Martin; Jenner, Richard G.; Sinclair, Alison J.

    2015-01-01

    Lytic replication of the human gamma herpes virus Epstein-Barr virus (EBV) is an essential prerequisite for the spread of the virus. Differential regulation of a limited number of cellular genes has been reported in B-cells during the viral lytic replication cycle. We asked whether a viral bZIP transcription factor, Zta (BZLF1, ZEBRA, EB1), drives some of these changes. Using genome-wide chromatin immunoprecipitation coupled to next-generation DNA sequencing (ChIP-seq) we established a map of Zta interactions across the human genome. Using sensitive transcriptome analyses we identified 2263 cellular genes whose expression is significantly changed during the EBV lytic replication cycle. Zta binds 278 of the regulated genes and the distribution of binding sites shows that Zta binds mostly to sites that are distal to transcription start sites. This differs from the prevailing view that Zta activates viral genes by binding exclusively at promoter elements. We show that a synthetic Zta binding element confers Zta regulation at a distance and that distal Zta binding sites from cellular genes can confer Zta-mediated regulation on a heterologous promoter. This leads us to propose that Zta directly reprograms the expression of cellular genes through distal elements. PMID:25779048

  4. Effect of extraction protocols and epidermal growth factor on the cellular repopulation of decellularized anterior cruciate ligament allografts.

    PubMed

    Harrison, Robert D; Gratzer, Paul F

    2005-12-15

    We are developing a decellularized bone-anterior cruciate ligament (ACL)-bone allograft for treatment of ACL disruption in young or active patients. This study demonstrates the feasibility of seeding decellularized ACL tissue with primary ligament fibroblasts. Porcine ACLs were decellularized by one of three protocols, each differing only by the detergent/solvent used during the second wash (SDS, Triton-X, or TnBP). Porcine ACL fibroblasts were obtained by explant and seeded onto tissue samples of decellularized ACL. Culture conditions were varied to compare the relative effect of three different decellularization protocols on cellular repopulation. Culture condition variables included (1) the number of cells used for seeding, (2) the addition of epidermal growth factor (EGF), and (3) culture duration. Cellular ingrowth was assessed by metabolic activity (MTT assay), DNA quantification (Hoescht dye), and histology (H&E staining). Cell counting on histological sections demonstrated that Triton-X-and TnBP-treated ligaments were more receptive to cellular ingrowth than SDS-treated samples. The addition of EGF to culture medium did not significantly increase cellular ingrowth. Both the Triton-X and TnBP decellularization treatments provide suitable, naturally derived scaffolds for the ingrowth of primary ACL fibroblasts, and should be further investigated in the development of an allograft-derived bone-ACL-bone graft.

  5. Cellular Human CLE/C14orf166 Protein Interacts with Influenza Virus Polymerase and Is Required for Viral Replication ▿

    PubMed Central

    Rodriguez, Ariel; Pérez-González, Alicia; Nieto, Amelia

    2011-01-01

    The influenza A virus polymerase associates with a number of cellular transcription-related factors, including RNA polymerase II. We previously described the interaction of influenza virus polymerase subunit PA with human CLE/C14orf166 protein (hCLE), a positive modulator of this cellular RNA polymerase. Here, we show that hCLE also interacts with the influenza virus polymerase complex and colocalizes with viral ribonucleoproteins. Silencing of hCLE causes reduction of viral polymerase activity, viral RNA transcription and replication, virus titer, and viral particle production. Altogether, these findings indicate that the cellular transcription factor hCLE is an important protein for influenza virus replication. PMID:21900157

  6. Carp thrombocyte phagocytosis requires activation factors secreted from other leukocytes.

    PubMed

    Nagasawa, Takahiro; Somamoto, Tomonori; Nakao, Miki

    2015-10-01

    Thrombocytes are nucleated blood cells in non-mammalian vertebrates, which were recently focused on not only as hemostatic cells but also as immune cells with potent phagocytic activities. We have analyzed the phagocytic activation mechanisms in common carp (Cyprinus carpio) thrombocytes. MACS-sorted mAb(+) thrombocytes showed no phagocytic activity even in the presence of several stimulants. However, remixing these thrombocytes with other anti-thrombocyte mAb(-) leukocyte populations restored their phagocytic activities, indicating that carp thrombocyte phagocytosis requires an appropriate exogenous stimulation. Culture supernatant from anti-thrombocyte mAb(-) leukocytes harvested after PMA or LPS stimulation, but not culture supernatant from unstimulated leukocytes, could activate thrombocyte phagocytosis. This proposed mechanism of thrombocyte phagocytosis activation involving soluble factors produced by activated leukocytes suggests that thrombocyte activation is restricted to areas proximal to injured tissues, ensuring suppression of excessive thrombocyte activation and a balance between inflammation and tissue repair.

  7. Biochemical, Biophysical and Cellular Techniques to Study the Guanine Nucleotide Exchange Factor, GIV/Girdin.

    PubMed

    Ghosh, Pradipta; Aznar, Nicolas; Swanson, Lee; Lo, I-Chung; Lopez-Sanchez, Inmaculada; Ear, Jason; Rohena, Cristina; Kalogriopoulos, Nicholas; Joosen, Linda; Dunkel, Ying; Sun, Nina; Nguyen, Peter; Bhandari, Deepali

    2016-12-07

    Canonical signal transduction via heterotrimeric G proteins is spatiotemporally restricted, i.e., triggered exclusively at the plasma membrane, only by agonist activation of G protein-coupled receptors via a finite process that is terminated within a few hundred milliseconds. Recently, a rapidly emerging paradigm has revealed a noncanonical pathway for activation of heterotrimeric G proteins via the nonreceptor guanidine-nucleotide exchange factor, GIV/Girdin. Biochemical, biophysical, and functional studies evaluating this pathway have unraveled its unique properties and distinctive spatiotemporal features. As in the case of any new pathway/paradigm, these studies first required an in-depth optimization of tools/techniques and protocols, governed by rationale and fundamentals unique to the pathway, and more specifically to the large multimodular GIV protein. Here we provide the most up-to-date overview of protocols that have generated most of what we know today about noncanonical G protein activation by GIV and its relevance in health and disease. © 2016 by John Wiley & Sons, Inc.

  8. Induction of Specific Cellular and Humoral Responses against Renal Cell Carcinoma after Combination Therapy with Cryoablation and Granulocyte-Macrophage Colony Stimulating Factor: A Pilot Study

    PubMed Central

    Thakur, Archana; Littrup, Peter; Paul, Elyse N.; Adam, Barbara; Heilbrun, Lance K.; Lum, Lawrence G.

    2013-01-01

    Cryotherapy offers a minimally invasive treatment option for the management of both irresectable and localized prostate, liver, pulmonary and renal tumors. The anti-neoplastic effects of cryotherapy are mediated by direct tumor lysis and by indirect effects such as intracellular dehydration, pH changes, and microvascular damage resulting in ischemic necrosis. In this study, we investigated whether percutaneous cryoablation of lung metastasis from renal cell carcinoma (RCC) in combination with aerosolized granulocyte-macrophage colony stimulating factor (GM-CSF) can induce systemic cellular and humoral immune responses in 6 RCC patients. Peripheral blood mononuclear cells (PBMC) were sequentially studied up to 63 days post cryoimmunotherapy (CI). PBMC from pre and post CI were phenotyped for lymphocyte subsets and tested for cytotoxicity and IFNγ Elispots directed at RCC cells. Humoral responses were measured by in vitro antibody synthesis assay directed at RCC cells. The immune monitoring data showed that CI induced tumor specific CTL, specific in vitro anti-tumor antibody responses, and enhanced Th1 cytokine production in 4 out of 6 patients. More importantly, the magnitude of cellular and humoral anti-tumor response appears to be associated with clinical responses. These pilot data show that CI can induce robust and brisk cellular and humoral immune responses in metastatic RCC patients, but requires further evaluation in optimized protocols. PMID:21577139

  9. Inactivation of Heat Shock Factor Hsf4 Induces Cellular Senescence and Suppresses Tumorigenesis In Vivo

    PubMed Central

    Jin, Xiongjie; Eroglu, Binnur; Cho, Wonkyoung; Yamaguchi, Yukihiro; Moskophidis, Demetrius; Mivechi, Nahid F.

    2013-01-01

    Studies suggest that Hsf4 expression correlates with its role in cell growth and differentiation. However, the role of Hsf4 in tumorigenesis in vivo remains unexplored. In this article, we provide evidence that absence of the Hsf4 gene suppresses evolution of spontaneous tumors arising in p53- or Arf-deficient mice. Furthermore, deletion of hsf4 alters the tumor spectrum by significantly inhibiting development of lymphomas that are normally observed in the majority of mice lacking p53 or Arf tumor suppressor genes. Using mouse embryo fibroblasts deficient in the hsf4 gene, we have found that these cells exhibit reduced proliferation that is associated with induction of senescence and senescence-associated β-galactosidase (SA-β-gal). Cellular senescence in hsf4-deficient cells is associated with the increased expression of the cyclin-dependent kinase inhibitors, p21 and p27 proteins. Consistent with the cellular senescence observed in vitro, specific normal tissues of hsf4−/− mice and tumors that arose in mice deficient in both hsf4 and p53 genes exhibit increased SA-β-gal activity and elevated levels of p27 compared with wild-type mice. These results suggest that hsf4 deletion-induced senescence is also present in vivo. Our results therefore indicate that Hsf4 is involved in modulation of cellular senescence, which can be exploited during cancer therapy. PMID:22355043

  10. CRISPR Immunological Memory Requires a Host Factor for Specificity.

    PubMed

    Nuñez, James K; Bai, Lawrence; Harrington, Lucas B; Hinder, Tracey L; Doudna, Jennifer A

    2016-06-16

    Bacteria and archaea employ adaptive immunity against foreign genetic elements using CRISPR-Cas systems. To generate immunological memory, the Cas1-Cas2 protein complex captures 30-40 base pair segments of foreign DNA and catalyzes their integration into the host genome as unique spacer sequences. Although spacers are inserted strictly at the A-T-rich leader end of CRISPR loci in vivo, the molecular mechanism of leader-specific spacer integration remains poorly understood. Here we show that the E. coli integration host factor (IHF) protein is required for spacer acquisition in vivo and for integration into linear DNA in vitro. IHF binds to the leader sequence and induces a sharp DNA bend, allowing the Cas1-Cas2 integrase to catalyze the first integration reaction at the leader-repeat border. Together, these results reveal that Cas1-Cas2-mediated spacer integration requires IHF-induced target DNA bending and explain the elusive role of CRISPR leader sequences during spacer acquisition.

  11. Predictive factors of open globe injury in patients requiring vitrectomy.

    PubMed

    Pimolrat, Weeraya; Choovuthayakorn, Janejit; Watanachai, Nawat; Patikulsila, Direk; Kunavisarut, Paradee; Chaikitmongkol, Voraporn; Ittipunkul, Nimitr

    2014-01-01

    To determine the outcomes and predictive factors of patients with open globe injury requiring pars plana vitrectomy (PPV). The medical records of 114 patients age 10 years or older who had undergone PPV due to ocular trauma, with at least 6 months follow up, were retrospectively reviewed. The mean age of the patients was 42 (SD14) years, with males accounting for 89% of the cases. Penetrating eye injury was the most common injury mechanism (43%) with most injuries occurring secondary to work related incidents (54%). After surgical interventions, 78% of the patients had visual improvement of one or more Snellen lines, while no light perception occurred in 10%. Anatomical attachment was achieved in 87% of eyes at the final follow up. Logistic regression analysis showed that the presence of a relative afferent pupillary defect (RAPD) was a significant predictive factor of visual outcome, while initial retinal detachment was a significant predictor of anatomical outcome. Pupillary reaction is an important presenting ocular sign in estimating the post-vitrectomy poor visual outcome for open globe injury. Vision was restored and improved in more than half of the patients in this study; however, long-term sequelae should be monitored. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Receptor binding and cellular uptake studies of macrophage migration inhibitory factor (MIF): use of biologically active labeled MIF derivatives.

    PubMed

    Kleemann, Robert; Grell, Matthias; Mischke, Ralf; Zimmermann, Gudrun; Bernhagen, Jürgen

    2002-03-01

    Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine for which a receptor has not been identified. That MIF has intracellular functions has been suggested by its enzymatic activity and constitutive expression profile. The discovery of functional MIF-c-Jun activation domain binding protein 1 (JAB1) binding has confirmed this notion and indicated that nonreceptor-based signaling mechanisms are important for MIF function. Here, we have generated and tested several biologically active labeled MIF derivatives to further define target protein binding by MIF and its cellular uptake characteristics. (35)S-MIF, biotinylated MIF, and fluoresceinated MIF were demonstrated to exhibit full biologic activity. Neither by applying a standard iodinated MIF preparation nor by using the biologically active (35)S-MIF derivative in receptor-binding studies were we able to measure any receptor-binding activity on numerous cells, confirming that uptake of MIF into target cells and MIF signaling can occur by receptor-independent pathways. When MIF derivatives were applied in cellular uptake studies, MIF was found to be endocytosed into both immune and nonimmune cells and targeted to the cytosol and lysosomes. The entry of MIF was temperature and energy dependent and was inhibited by monodansylcadaverine but not by ouabain. Endocytosed biotin-MIF bound JAB1 not only in macrophages, as shown previously, but also in nonimmune cells. A tagged MIF construct, MIF-enhanced green fluorescent protein (EGFP), was shown to be a valuable tool, as EGFP constructs of critical MIF cysteine mutants exhibited identical cellular localization properties to those of wild-type MIF (wtMIF). Our results indicate that MIF membrane receptors are not widely expressed, if at all, and suggest that the cellular uptake of MIF occurs by nonreceptor-mediated endocytosis rather than penetration. All the derivatives investigated, except for iodinated MIF, represent valuable tools for further MIF target

  13. Cloning of a cellular factor, interleukin binding factor, that binds to NFAT-like motifs in the human immunodeficiency virus long terminal repeat.

    PubMed Central

    Li, C; Lai, C F; Sigman, D S; Gaynor, R B

    1991-01-01

    Human immunodeficiency virus (HIV) gene expression is regulated by both general transcription factors and factors induced by activation of T lymphocytes such as NF-kappa B and the nuclear factor of activated T cells (NFAT). Within the HIV long terminal repeat (LTR), two purine-rich domains between nucleotides -283 and -195 have homology to a regulatory region found in the interleukin 2 promoter, which binds NFAT and other cellular factors. In the HIV LTR, this region has been demonstrated to have both positive and negative regulatory effects on HIV gene expression. In an attempt to clone genes encoding cellular factors that bind to these NFAT-like elements in the HIV LTR, we used lambda gt11 expression cloning with oligonucleotides corresponding to these binding motifs. A ubiquitously expressed cDNA encoding a 60-kDa protein, which we termed interleukin binding factor (ILF), binds specifically to these purine-rich motifs in the HIV LTR. This factor also binds to similar purine-rich motifs in the interleukin 2 promoter, through with lower affinity than to HIV LTR sequences. Sequence analysis reveals that the DNA binding domain of ILF has strong homology to the recently described fork head DNA binding domain found in the Drosophila homeotic protein fork head and a family of hepatocyte nuclear factors, HNF-3. Other domains found in ILF include a nucleotide binding site, an N-glycosylation motif, a signal for ubiquitin-mediated degradation, and a potential nuclear localization signal. These results describe a DNA binding protein that may be involved in both positive and negative regulation of important viral and cellular promoter elements. Images PMID:1909027

  14. Requirement of rRNA Methylation for 80S Ribosome Assembly on a Cohort of Cellular Internal Ribosome Entry Sites▿

    PubMed Central

    Basu, Abhijit; Das, Priyanka; Chaudhuri, Sujan; Bevilacqua, Elena; Andrews, Joel; Barik, Sailen; Hatzoglou, Maria; Komar, Anton A.; Mazumder, Barsanjit

    2011-01-01

    Protein syntheses mediated by cellular and viral internal ribosome entry sites (IRESs) are believed to have many features in common. Distinct mechanisms for ribosome recruitment and preinitiation complex assembly between the two processes have not been identified thus far. Here we show that the methylation status of rRNA differentially influenced the mechanism of 80S complex formation on IRES elements from the cellular sodium-coupled neutral amino acid transporter 2 (SNAT2) versus the hepatitis C virus mRNA. Translation initiation involves the assembly of the 48S preinitiation complex, followed by joining of the 60S ribosomal subunit and formation of the 80S complex. Abrogation of rRNA methylation did not affect the 48S complex but resulted in impairment of 80S complex assembly on the cellular, but not the viral, IRESs tested. Impairment of 80S complex assembly on the amino acid transporter SNAT2 IRES was rescued by purified 60S subunits containing fully methylated rRNA. We found that rRNA methylation did not affect the activity of any of the viral IRESs tested but affected the activity of numerous cellular IRESs. This work reveals a novel mechanism operating on a cohort of cellular IRESs that involves rRNA methylation for proper 80S complex assembly and efficient translation initiation. PMID:21930789

  15. Efficient Hepatitis Delta Virus RNA Replication in Avian Cells Requires a Permissive Factor(s) from Mammalian Cells

    PubMed Central

    Liu, Yu-Tsueng; Brazas, Rob; Ganem, Don

    2001-01-01

    Hepatitis delta virus (HDV) is a highly pathogenic human RNA virus whose genome is structurally related to those of plant viroids. Although its spread from cell to cell requires helper functions supplied by hepatitis B virus (HBV), intracellular HDV RNA replication can proceed in the absence of HBV proteins. As HDV encodes no RNA-dependent RNA polymerase, the identity of the (presumably cellular) enzyme responsible for this reaction remains unknown. Here we show that, in contrast to mammalian cells, avian cells do not support efficient HDV RNA replication and that this defect cannot be rescued by provision of HDV gene products in trans. Contrary to earlier assertions, this defect is not due to enhanced apoptosis triggered in avian cells by HDV. Fusion of avian cells to mammalian cells rescues HDV replication in avian nuclei, indicating that the nonpermissive phenotype of avian cells is not due to the presence of dominantly acting inhibitors of replication. Rather, avian cells lack one or more essential permissive factors present in mammalian cells. These results set the stage for the identification of such factors and also explain the failure of earlier efforts to transmit HDV infection to avian hosts harboring indigenous hepadnaviruses. PMID:11462021

  16. Mammary tumorigenesis induced by fibroblast growth factor receptor 1 requires activation of the epidermal growth factor receptor.

    PubMed

    Bade, Lindsey K; Goldberg, Jodi E; Dehut, Hazel A; Hall, Majken K; Schwertfeger, Kathryn L

    2011-09-15

    Fibroblast growth factor receptor 1 (FGFR1) is an oncoprotein with known involvement in mammary tumorigenesis. To understand how FGFR1 signaling promotes mammary tumorigenesis, an inducible FGFR1 (iFGFR1) system was created previously. Previous studies have demonstrated that upon iFGFR1 activation in vivo, the epidermal growth factor (EGF) ligands amphiregulin (AREG) and epiregulin (EREG) are upregulated. Both AREG and EREG interact with the EGF receptor (EGFR). Here, we investigated whether the FGFR1-induced increase in AREG and EREG expression might coordinately increase EGFR signaling to promote mammary tumorigenesis. Treatment of mouse mammary epithelial cells with either AREG or EREG conferred a greater migratory potential, increased cellular proliferation and increased extracellular regulated kinase 1/2 (ERK1/2) activation. These effects could be blocked with the EGFR-specific inhibitor erlotinib, suggesting that they are EGFR-dependent. In transgenic mice with iFGFR1 under the control of the mouse mammary tumor virus (MMTV) promoter, iFGFR1 activation also led to increased mammary epithelial cell proliferation that was inhibited with erlotinib. Taken together, these data suggest that AREG and EREG mediate tumorigenic phenotypes by activating EGFR signaling, and that the oncogenic potential of FGFR1 requires EGFR activation to promote mammary tumorigenesis.

  17. Human Cortical Neural Stem Cells Expressing Insulin-Like Growth Factor-I: A Novel Cellular Therapy for Alzheimer's Disease.

    PubMed

    McGinley, Lisa M; Sims, Erika; Lunn, J Simon; Kashlan, Osama N; Chen, Kevin S; Bruno, Elizabeth S; Pacut, Crystal M; Hazel, Tom; Johe, Karl; Sakowski, Stacey A; Feldman, Eva L

    2016-03-01

    Alzheimer's disease (AD) is the most prevalent age-related neurodegenerative disorder and a leading cause of dementia. Current treatment fails to modify underlying disease pathologies and very little progress has been made to develop effective drug treatments. Cellular therapies impact disease by multiple mechanisms, providing increased efficacy compared with traditional single-target approaches. In amyotrophic lateral sclerosis, we have shown that transplanted spinal neural stem cells (NSCs) integrate into the spinal cord, form synapses with the host, improve inflammation, and reduce disease-associated pathologies. Our current goal is to develop a similar "best in class" cellular therapy for AD. Here, we characterize a novel human cortex-derived NSC line modified to express insulin-like growth factor-I (IGF-I), HK532-IGF-I. Because IGF-I promotes neurogenesis and synaptogenesis in vivo, this enhanced NSC line offers additional environmental enrichment, enhanced neuroprotection, and a multifaceted approach to treating complex AD pathologies. We show that autocrine IGF-I production does not impact the cell secretome or normal cellular functions, including proliferation, migration, or maintenance of progenitor status. However, HK532-IGF-I cells preferentially differentiate into gamma-aminobutyric acid-ergic neurons, a subtype dysregulated in AD; produce increased vascular endothelial growth factor levels; and display an increased neuroprotective capacity in vitro. We also demonstrate that HK532-IGF-I cells survive peri-hippocampal transplantation in a murine AD model and exhibit long-term persistence in targeted brain areas. In conclusion, we believe that harnessing the benefits of cellular and IGF-I therapies together will provide the optimal therapeutic benefit to patients, and our findings support further preclinical development of HK532-IGF-I cells into a disease-modifying intervention for AD. ©AlphaMed Press.

  18. The nociception genes painless and Piezo are required for the cellular immune response of Drosophila larvae to wasp parasitization.

    PubMed

    Tokusumi, Yumiko; Tokusumi, Tsuyoshi; Schulz, Robert A

    2017-05-13

    In vertebrates, interaction between the nervous system and immune system is important to protect a challenged host from stress inputs from external sources. In this study, we demonstrate that sensory neurons are involved in the cellular immune response elicited by wasp infestation of Drosophila larvae. Multidendritic class IV neurons sense contacts from external stimuli and induce avoidance behaviors for host defense. Our findings show that inactivation of these sensory neurons impairs the cellular response against wasp parasitization. We also demonstrate that the nociception genes encoding the mechanosensory receptors Painless and Piezo, both expressed in class IV neurons, are essential for the normal cellular immune response to parasite challenge. Copyright © 2017. Published by Elsevier Inc.

  19. Lipase maturation factor 1 is required for endothelial lipase activity[S

    PubMed Central

    Ben-Zeev, Osnat; Hosseini, Maryam; Lai, Ching-Mei; Ehrhardt, Nicole; Wong, Howard; Cefalù, Angelo B.; Noto, Davide; Averna, Maurizio R.; Doolittle, Mark H.; Péterfy, Miklós

    2011-01-01

    Lipase maturation factor 1 (Lmf1) is an endoplasmic reticulum (ER) membrane protein involved in the posttranslational folding and/or assembly of lipoprotein lipase (LPL) and hepatic lipase (HL) into active enzymes. Mutations in Lmf1 are associated with diminished LPL and HL activities (“combined lipase deficiency”) and result in severe hypertriglyceridemia in mice as well as in human subjects. Here, we investigate whether endothelial lipase (EL) also requires Lmf1 to attain enzymatic activity. We demonstrate that cells harboring a (cld) loss-of-function mutation in the Lmf1 gene are unable to generate active EL, but they regain this capacity after reconstitution with the Lmf1 wild type. Furthermore, we show that cellular EL copurifies with Lmf1, indicating their physical interaction in the ER. Finally, we determined that post-heparin phospholipase activity in a patient with the LMF1W464X mutation is reduced by more than 95% compared with that in controls. Thus, our study indicates that EL is critically dependent on Lmf1 for its maturation in the ER and demonstrates that Lmf1 is a required factor for all three vascular lipases, LPL, HL, and EL. PMID:21447484

  20. Cytosolic Iron-Sulfur Cluster Assembly (CIA) System: Factors, Mechanism, and Relevance to Cellular Iron Regulation*

    PubMed Central

    Sharma, Anil K.; Pallesen, Leif J.; Spang, Robert J.; Walden, William E.

    2010-01-01

    FeS cluster biogenesis is an essential process in virtually all forms of life. Complex protein machineries that are conserved from bacteria through higher eukaryotes facilitate assembly of the FeS cofactor in proteins. In the last several years, significant strides have been made in our understanding of FeS cluster assembly and the functional overlap of this process with cellular iron homeostasis. This minireview summarizes the present understanding of the cytosolic iron-sulfur cluster assembly (CIA) system in eukaryotes, with a focus on information gained from studies in budding yeast and mammalian systems. PMID:20522543

  1. Cellular aggregate size as the critical factor for flavonoid production by suspension cultures of Saussurea medusa.

    PubMed

    Fu, Chun-xiang; Zhao, De-xiu; Huang, Yan; Ma, Feng-shan

    2005-01-01

    Three previously established cell lines (yellow, red and white) of Saussurea medusa were investigated for jaceosidin and hispidulin production. Maximum yields of the jaceosidin and hispidulin were obtained in the red cell line at 75+/-0.41 and 6.4+/-0.31 mg l-1. Production of jaceosidin and hispidulin correlated with the sizes of compact callus aggregates (CCA) and cellular viability. In the red cell line, the sizes of CCA were predominantly of 2-4 mm diameter and accounted for 64% biomass. This line had a sustained cell viability over 10 successive sub-cultures.

  2. Cytosolic iron-sulfur cluster assembly (CIA) system: factors, mechanism, and relevance to cellular iron regulation.

    PubMed

    Sharma, Anil K; Pallesen, Leif J; Spang, Robert J; Walden, William E

    2010-08-27

    FeS cluster biogenesis is an essential process in virtually all forms of life. Complex protein machineries that are conserved from bacteria through higher eukaryotes facilitate assembly of the FeS cofactor in proteins. In the last several years, significant strides have been made in our understanding of FeS cluster assembly and the functional overlap of this process with cellular iron homeostasis. This minireview summarizes the present understanding of the cytosolic iron-sulfur cluster assembly (CIA) system in eukaryotes, with a focus on information gained from studies in budding yeast and mammalian systems.

  3. Cellular and humoral factors in host susceptibility to Lewis lung carcinoma.

    PubMed

    Gundersen, S; Wibe, E; Gidlund, M; Godal, T

    1981-04-01

    Cellular and humoral anti-tumour reactivity in strains of mice highly susceptible (C57Bl/6) or less susceptible (C57Cl/6 x DBA/2 = B6D2F1) to Lewis lung carcinoma (LLC) was investigated. Natural killer cell activity in a 51Cr release assay against this tumour could be demonstrated with a good correlation to in vivo susceptibility. This has not been demonstrated earlier for solid, spontaneous tumours. T-cell deficiency (congenital athymic (nude mice)) did not affect the cumulative incidence of tumour take. However, the number of lung metastases was significantly reduced in nude mice. Treatment with antilymphocyte serum (ALS) increased the susceptibility to LLC in both strains. In a soft agar colony assay a marked reduction in the number of colonies was observed when tumour cells were incubated with serum from B6D2F1 mice as compared to serum from C57Bl/6 mice, prior to seeding. Apparently naturally occurring cellular, as well as humoral effector mechanisms are involved in host resistance to Lewis lung carcinoma in the mouse.

  4. Shear stress is required for the endocytic uptake of the factor VIII-von Willebrand factor complex by macrophages.

    PubMed

    Castro-Núñez, L; Dienava-Verdoold, I; Herczenik, E; Mertens, K; Meijer, A B

    2012-09-01

    Low-density lipoprotein (LDL) receptor family members contribute to the cellular uptake of factor VIII. How von Willebrand factor fits into this endocytic pathway has remained poorly understood. It has been suggested that macrophages contribute to the clearance of the factor VIII (FVIII)-von Willebrand factor (VWF) complex. We now assessed the mechanisms of uptake employing human monocyte-derived macrophages. A confocal microscopy study was employed to study the uptake by monocyte-derived macrophages of a functional green fluorescent FVIII-GFP derivative in the presence and absence of VWF. The results revealed that FVIII-GFP is internalized by macrophages. We found that FVIII-GFP co-localizes with LDL receptor-related protein (LRP), and that the LRP antagonist Receptor Associated Protein (RAP) blocks the uptake of FVIII-GFP. However, FVIII-GFP was not detected in the macrophages in the presence of VWF, suggesting that the FVIII-VWF complex is not internalized by these cells at all. Apart from static conditions, we also investigated the effect of shear stress on the uptake of FVIII-GFP in presence of VWF. Immunofluorescence studies demonstrated that VWF does not block endocytosis of FVIII-GFP under flow conditions. Moreover, VWF itself was also internalized by the macrophages. Strikingly, in the presence of RAP, endocytosis of FVIII-GFP and VWF was inhibited. The results show that shear stress is required for macrophages to internalize both constituents of the FVIII-VWF complex. © 2012 International Society on Thrombosis and Haemostasis.

  5. Matrix Metalloproteinase 3 Promotes Cellular Anti-Dengue Virus Response via Interaction with Transcription Factor NFκB in Cell Nucleus

    PubMed Central

    Zuo, Xiangyang; Pan, Wen; Feng, Tingting; Shi, Xiaohong; Dai, Jianfeng

    2014-01-01

    Dengue virus (DENV), the causative agent of human Dengue hemorrhagic fever, is a mosquito-borne virus of immense global health importance. Characterization of cellular factors promoting or inhibiting DENV infection is important for understanding the mechanism of DENV infection. In this report, MMP3 (stromelysin-1), a secretory endopeptidase that degrades extracellular matrices, has been shown promoting cellular antiviral response against DENV infection. Quantitative RT-PCR and Western Blot showed that the expression of MMP3 was upregulated in DENV-infected RAW264.7 cells. The intracellular viral loads were significantly higher in MMP3 silenced cells compared with controls. The expression level of selective anti-viral cytokines were decreased in MMP3 siRNA treated cells, and the transcription factor activity of NFκB was significantly impaired upon MMP3 silencing during DENV infection. Further, we found that MMP3 moved to cell nucleus upon DENV infection and colocalized with NFκB P65 in nucleus. Co-immunoprecipitation analysis suggested that MMP3 directly interacted with NFκB in nucleus during DENV infection and the C-terminal hemopexin-like domain of MMP3 was required for the interaction. This study suggested a novel role of MMP3 in nucleus during viral infection and provided new evidence for MMPs in immunomodulation. PMID:24416274

  6. Matrix metalloproteinase 3 promotes cellular anti-dengue virus response via interaction with transcription factor NFκB in cell nucleus.

    PubMed

    Zuo, Xiangyang; Pan, Wen; Feng, Tingting; Shi, Xiaohong; Dai, Jianfeng

    2014-01-01

    Dengue virus (DENV), the causative agent of human Dengue hemorrhagic fever, is a mosquito-borne virus of immense global health importance. Characterization of cellular factors promoting or inhibiting DENV infection is important for understanding the mechanism of DENV infection. In this report, MMP3 (stromelysin-1), a secretory endopeptidase that degrades extracellular matrices, has been shown promoting cellular antiviral response against DENV infection. Quantitative RT-PCR and Western Blot showed that the expression of MMP3 was upregulated in DENV-infected RAW264.7 cells. The intracellular viral loads were significantly higher in MMP3 silenced cells compared with controls. The expression level of selective anti-viral cytokines were decreased in MMP3 siRNA treated cells, and the transcription factor activity of NFκB was significantly impaired upon MMP3 silencing during DENV infection. Further, we found that MMP3 moved to cell nucleus upon DENV infection and colocalized with NFκB P65 in nucleus. Co-immunoprecipitation analysis suggested that MMP3 directly interacted with NFκB in nucleus during DENV infection and the C-terminal hemopexin-like domain of MMP3 was required for the interaction. This study suggested a novel role of MMP3 in nucleus during viral infection and provided new evidence for MMPs in immunomodulation.

  7. Requirements for Receptor Engagement during Infection by Adenovirus Complexed with Blood Coagulation Factor X

    PubMed Central

    Bradshaw, Angela C.; Parker, Alan L.; Duffy, Margaret R.; Coughlan, Lynda; van Rooijen, Nico; Kähäri, Veli-Matti; Nicklin, Stuart A.; Baker, Andrew H.

    2010-01-01

    Human adenoviruses from multiple species bind to coagulation factor X (FX), yet the importance of this interaction in adenovirus dissemination is unknown. Upon contact with blood, vectors based on adenovirus serotype 5 (Ad5) binds to FX via the hexon protein with nanomolar affinity, leading to selective uptake of the complex into the liver and spleen. The Ad5:FX complex putatively targets heparan sulfate proteoglycans (HSPGs). The aim of this study was to elucidate the specific requirements for Ad5:FX-mediated cellular uptake in this high-affinity pathway, specifically the HSPG receptor requirements as well as the role of penton base-mediated integrin engagement in subsequent internalisation. Removal of HS sidechains by enzymatic digestion or competition with highly-sulfated heparins/heparan sulfates significantly decreased FX-mediated Ad5 cell binding in vitro and ex vivo. Removal of N-linked and, in particular, O-linked sulfate groups significantly attenuated the inhibitory capabilities of heparin, while the chemical inhibition of endogenous HSPG sulfation dose-dependently reduced FX-mediated Ad5 cellular uptake. Unlike native heparin, modified heparins lacking O- or N-linked sulfate groups were unable to inhibit Ad5 accumulation in the liver 1h after intravascular administration of adenovirus. Similar results were observed in vitro using Ad5 vectors possessing mutations ablating CAR- and/or αv integrin binding, demonstrating that attachment of the Ad5:FX complex to the cell surface involves HSPG sulfation. Interestingly, Ad5 vectors ablated for αv integrin binding showed markedly delayed cell entry, highlighting the need for an efficient post-attachment internalisation signal for optimal Ad5 uptake and transport following surface binding mediated through FX. This study therefore integrates the established model of αv integrin-dependent adenoviral infection with the high-affinity FX-mediated pathway. This has important implications for mechanisms that define

  8. The expression level of the transcription factor Aryl hydrocarbon receptor nuclear translocator (ARNT) determines cellular survival after radiation treatment.

    PubMed

    Mandl, Markus; Lieberum, Maria- Katharina; Dunst, Juergen; Depping, Reinhard

    2015-11-16

    Tumour hypoxia promotes radioresistance and is associated with poor prognosis. The transcription factor Aryl hydrocarbon receptor nuclear translocator (ARNT), also designated as Hypoxia-inducible factor (HIF)-1β, is part of the HIF pathway which mediates cellular adaptations to oxygen deprivation and facilitates tumour progression. The subunits HIF-1α and ARNT are key players within this pathway. HIF-1α is regulated in an oxygen-dependent manner whereas ARNT is considered to be constitutively expressed. However, there is mounting evidence that certain tumour cells are capable to elevate ARNT in hypoxia which suggests a survival benefit. Therefore the objective of this study was to elucidate effects of an altered ARNT expression level on the cellular response to radiation. Different human cell lines (Hep3B, MCF-7, 786-Owt, 786-Ovhl, RCC4wt and RCC4vhl) originating from various tumour entities (Hepatocellular carcinoma, breast cancer and renal cell carcinoma respectively) were X-irradiated using a conventional linear accelerator. Knockdown of ARNT expression was achieved by transient siRNA transfection. Complementary experiments were performed by forced ARNT overexpression using appropriate plasmids. Presence/absence of ARNT protein was confirmed by Western blot analysis. Clonogenic survival assays were performed in order to determine cellular survival post irradiation. Statistical comparison of two groups was achieved by the unpaired t-test. The results of this study indicate that ARNT depletion renders tumour cells susceptible to radiation whereas overexpression of this transcription factor confers radioresistance. These findings provide evidence to consider ARNT as a drug target and as a predictive marker in clinical applications concerning the response to radiation.

  9. [Molecular-cellular and hormonal mechanisms of induced brain tolerance of extreme factors].

    PubMed

    Samoĭlov, M O; Rybnikova, E A

    2012-01-01

    This review includes results of own studies and literature data on the topical problem of neurobiology and medicine: discovery of the mechanisms of increased brain resistance to extreme exposures. The emphasis is made on the molecular-cellular and hormonal mechanisms of hypoxic preconditioning-induced brain tolerance to injurious hypoxia, psychoemotional and traumatic stress. A role of basic hormonal and intracellular cascade pro-adaptive processes mediating the neuroprotective action of hypoxic preconditioning is reviewed. A dynamics of the mechanisms of development of induced susceptible brain areas (hippocampus, neocortex) tolerance which includes phases of induction, transformation and expression, is presented. New data on preconditioning-induced cross-tolerance providing increased brain resistance not only to hypoxia but also to other stresses are reported. For the first time neuroprotective effects of hypoxic postconditioning are described.

  10. Total cellular protein presence of the transcription factor IRF8 does not necessarily correlate with its nuclear presence.

    PubMed

    Minderman, Hans; Maguire, Orla; O'Loughlin, Kieran L; Muhitch, Jason; Wallace, Paul K; Abrams, Scott I

    2017-01-01

    The transcription factor interferon regulatory factor-8 (IRF8) plays an essential role in myeloid differentiation and lineage commitment, based largely on molecular and genetic studies. The detection of IRF8 in specific cell populations by flow cytometry (FCM) has the potential to provide new insights into normal and pathologic myelopoiesis, but critical validation of this protein-based approach, particularly in human samples, is lacking. In this study, the assessment of total cellular IRF8 presence was compared to its specific nuclear presence as assessed by imaging flow cytometry (IFC) analysis. Peptide neutralization of the IRF8-specific antibody that has been predominantly used to date in the literature served as a negative control for the immunofluorescent labeling. Expression of total IRF8 was analyzed by total cellular fluorescence analogous to the mean fluorescence intensity readout of conventional FCM. Additionally, specific nuclear fluorescence and the similarity score between the nuclear image (DAPI) and the corresponding IRF8 image for each cell were analyzed as parameters for nuclear localization of IRF8. IFC showed that peptide blocking eliminated binding of the IRF8 antibody in the nucleus. It also reduced cytoplasmic binding of the antibody but not to the extent observed in the nucleus. In agreement with the similarity score data, the total cellular IRF8 as well as nuclear IRF8 intensities decreased with peptide blocking. In healthy donor peripheral blood subpopulations and a positive control cell line (THP-1), the assessment of IRF8 by total cellular presence correlated well with its specific nuclear presence and correlated with the known distribution of IRF8 in these cells. In clinical samples of myeloid-derived suppressors cells derived from patients with renal carcinoma, however, total cellular IRF8 did not necessarily correlate with its nuclear presence. Discordance was primarily associated with peptide blocking having a proportionally greater

  11. Fibrillarin, a nucleolar protein, is required for normal nuclear morphology and cellular growth in HeLa cells

    SciTech Connect

    Amin, Mohammed Abdullahel; Matsunaga, Sachihiro; Ma, Nan; Takata, Hideaki; Yokoyama, Masami; Uchiyama, Susumu; Fukui, Kiichi . E-mail: kfukui@bio.eng.osaka-u.ac.jp

    2007-08-24

    Fibrillarin is a key small nucleolar protein in eukaryotes, which has an important role in pre-rRNA processing during ribosomal biogenesis. Though several functions of fibrillarin are known, its function during the cell cycle is still unknown. In this study, we confirmed the dynamic localization of fibrillarin during the cell cycle of HeLa cells and also performed functional studies by using a combination of immunofluorescence microscopy and RNAi technique. We observed that depletion of fibrillarin has almost no effect on the nucleolar structure. However, fibrillarin-depleted cells showed abnormal nuclear morphology. Moreover, fibrillarin depletion resulted in the reduction of the cellular growth and modest accumulation of cells with 4n DNA content. Our data suggest that fibrillarin would play a critical role in the maintenance of nuclear shape and cellular growth.

  12. von Willebrand factor storage requires intact prosequence cleavage site.

    PubMed

    Journet, A M; Saffaripour, S; Cramer, E M; Tenza, D; Wagner, D D

    1993-02-01

    Large multimers of the adhesive glycoprotein von Willebrand factor (vWf) are stored in endothelial cells in rod-shaped granules called Weibel-Palade bodies, while small multimers are secreted constitutively. Expression of pro-vWf in other cells with a regulated pathway of secretion, results in formation of vWf-containing storage granules that have a morphology similar to Weibel-Palade bodies. vWf expressed without its prosequence is not stored. To evaluate the importance of prosequence cleavage in vWf storage, the Arg at position -1, known to be necessary for cleavage, was mutated to Gly. Transfection of this cleavage mutant into two cell lines with a regulated pathway of secretion (RIN 5F and AtT-20 cells) led to the formation of large multimers. However, treatment of the cell lysates by the enzyme endoglycosidase H (Endo-H) did not reveal significant amounts of intracellular Endo-H-resistant vWf, which indicates the absence of a pool of stored processed vWf. In addition, no Weibel-Palade body-like structure was detected in these cells by immunofluorescence labeling with anti-vWf antiserum. Electron microscopy and immunocytochemistry of RIN 5F cells expressing the pro-vWf mutant confirmed the absence of Weibel-Palade body-like structures. In addition, anti-vWf-linked gold particles were found in the ER, occasionally in rounded granules and particularly in lysosomal structures which were abundant. We conclude that the formation of large aggregates is not sufficient to induce efficient vWf storage, and that the lack of cleavage of the prosequence may direct the mutant pro-vWf molecule to a degradative pathway. Therefore, the prosequence cleavage is a requirement for vWf storage.

  13. A Viral Protein Mediates Superinfection Exclusion at the Whole-Organism Level but Is Not Required for Exclusion at the Cellular Level

    PubMed Central

    Bergua, María; Zwart, Mark P.; El-Mohtar, Choaa; Shilts, Turksen; Elena, Santiago F.

    2014-01-01

    ABSTRACT Superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondary infection by the same or a closely related virus, has been described for different viruses, including important pathogens of humans, animals, and plants. Citrus tristeza virus (CTV), a positive-sense RNA virus, represents a valuable model system for studying SIE due to the existence of several phylogenetically distinct strains. Furthermore, CTV allows SIE to be examined at the whole-organism level. Previously, we demonstrated that SIE by CTV is a virus-controlled function that requires the viral protein p33. In this study, we show that p33 mediates SIE at the whole-organism level, while it is not required for exclusion at the cellular level. Primary infection of a host with a fluorescent protein-tagged CTV variant lacking p33 did not interfere with the establishment of a secondary infection by the same virus labeled with a different fluorescent protein. However, cellular coinfection by both viruses was rare. The obtained observations, along with estimates of the cellular multiplicity of infection (MOI) and MOI model selection, suggested that low levels of cellular coinfection appear to be best explained by exclusion at the cellular level. Based on these results, we propose that SIE by CTV is operated at two levels—the cellular and the whole-organism levels—by two distinct mechanisms that could function independently. This novel aspect of viral SIE highlights the intriguing complexity of this phenomenon, further understanding of which may open up new avenues to manage virus diseases. IMPORTANCE Many viruses exhibit superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondary infection by related viruses. SIE plays an important role in the pathogenesis and evolution of virus populations. The observations described here suggest that SIE could be controlled independently at different levels of the host

  14. Enabled (Xena) regulates neural plate morphogenesis, apical constriction, and cellular adhesion required for neural tube closure in Xenopus

    PubMed Central

    Roffers-Agarwal, Julaine; Xanthos, Jennifer B.; Kragtorp, Katherine A.; Miller, Jeffrey R.

    2008-01-01

    Regulation of cellular adhesion and cytoskeletal dynamics is essential for neurulation, though it remains unclear how these two processes are coordinated. Members of the Ena/VASP family of proteins are localized to sites of cellular adhesion and actin dynamics and lack of two family members, Mena and VASP, in mice results in failure of neural tube closure. The precise mechanism by which Ena/VASP proteins regulate this process, however, is not understood. In this report, we show that Xenopus Ena (Xena) is localized to apical adhesive junctions of neuroepithelial cells during neurulation and that Xena knockdown disrupts cell behaviors integral to neural tube closure. Changes in the shape of the neural plate as well as apical constriction within the neural plate are perturbed in Xena knockdown embryos. Additionally, we demonstrate that Xena is essential for cell-cell adhesion. These results demonstrate that Xena plays an integral role in coordinating the regulation of cytoskeletal dynamics and cellular adhesion during neurulation in Xenopus. PMID:18201691

  15. p53, cellular proliferation, and apoptosis-related factors in thymic neuroendocrine tumors.

    PubMed

    Gal, Anthony A; Sheppard, Mary N; Nolen, John D L; Cohen, Cynthia

    2004-01-01

    Thymic neuroendocrine tumors are biologically aggressive neoplasms with extensive local invasion and high mortality. Although various markers of cellular proliferation and apoptosis have correlated with degrees of tumor differentiation in pulmonary neuroendocrine neoplasms, they have not been systematically studied in thymic neuroendocrine tumors. We immunostained 21 cases of thymic neuroendocrine tumors for p53, MIB-1, and the apoptosis-related markers Bcl-2, Bcl-x, and Bax. By histological classification the cases were low-grade (nine cases), intermediate-grade (eight cases), and high-grade (four cases) thymic neuroendocrine tumors. p53 was expressed in five cases: 1/9 low grade, 3/8 intermediate grade, and 2/4 high grade. The mean cellular proliferation (MIB-1) was 7.1% (range 2-12%) in low-grade thymic neuroendocrine tumors, 6.1% (range 2-15%) in intermediate-grade thymic neuroendocrine tumors, and 34.2% (range 2-80%) in high-grade thymic neuroendocrine tumors. Bcl-2 was expressed in 16 cases: 7/9 low grade, 5/8 intermediate grade, and 4/4 high grade. Bcl-x was expressed in 16 cases: 7/9 low grade, 6/8 intermediate grade, and 3/4 high grade. Bax was expressed in 13 cases: 5/9 low grade, 4/8 intermediate grade, and 4/4 high grade. The presence of mutant p53 in the tumor was associated with a statistically significant decreased mean survival (P<0.05). In contrast, either by positive or negative staining or by the score technique (staining intensity x percentage of cells staining), the presence of Bcl-x was associated with an increased mean survival (P<0.05). Finally, a Bcl-x : Bax ratio >or=1 was also associated with an increased mean survival, as compared to a Bcl-x : Bax ratio >or=1 (P<0.05). Our study shows that p53 expression and certain apoptosis markers correlate with survival. The expression of these markers may account for differences in biological behavior.

  16. Off the shelf cellular therapeutics: Factors to consider during cryopreservation and storage of human cells for clinical use.

    PubMed

    Woods, Erik J; Thirumala, Sreedhar; Badhe-Buchanan, Sandhya S; Clarke, Dominic; Mathew, Aby J

    2016-06-01

    The field of cellular therapeutics has immense potential, affording an exciting array of applications in unmet medical needs. One of several key issues is an emphasis on getting these therapies from bench to bedside without compromising safety and efficacy. The successful commercialization of cellular therapeutics will require many to extend the shelf-life of these therapies beyond shipping "fresh" at ambient or chilled temperatures for "just in time" infusion. Cryopreservation is an attractive option and offers potential advantages, such as storing and retaining patient samples in case of a relapse, banking large quantities of allogeneic cells for broader distribution and use and retaining testing samples for leukocyte antigen typing and matching. However, cryopreservation is only useful if cells can be reanimated to physiological life with negligible loss of viability and functionality. Also critical is the logistics of storing, processing and transporting cells in clinically appropriate packaging systems and storage devices consistent with quality and regulatory standards. Rationalized approaches to develop commercial-scale cell therapies require an efficient cryopreservation system that provides the ability to inventory standardized products with maximized shelf life for later on-demand distribution and use, as well as a method that is scientifically sound and optimized for the cell of interest. The objective of this review is to bridge this gap between the basic science of cryobiology and its application in this context by identifying several key aspects of cryopreservation science in a format that may be easily integrated into mainstream cell therapy manufacture.

  17. The bacterial virulence factor NleA inhibits cellular protein secretion by disrupting mammalian COPII function.

    PubMed

    Kim, Jinoh; Thanabalasuriar, Ajitha; Chaworth-Musters, Tessa; Fromme, J Chris; Frey, Elizabeth A; Lario, Paula I; Metalnikov, Pavel; Rizg, Keyrillos; Thomas, Nikhil A; Lee, Sau Fung; Hartland, Elizabeth L; Hardwidge, Philip R; Pawson, Tony; Strynadka, Natalie C; Finlay, B Brett; Schekman, Randy; Gruenheid, Samantha

    2007-09-13

    Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) maintain an extracellular lifestyle and use a type III secretion system to translocate effector proteins into the host cytosol. These effectors manipulate host pathways to favor bacterial replication and survival. NleA is an EHEC/EPEC- and related species-specific translocated effector protein that is essential for bacterial virulence. However, the mechanism by which NleA impacts virulence remains undetermined. Here we demonstrate that NleA compromises the Sec23/24 complex, a component of the mammalian COPII protein coat that shapes intracellular protein transport vesicles, by directly binding Sec24. Expression of an NleA-GFP fusion protein reduces the efficiency of cellular secretion by 50%, and secretion is inhibited in EPEC-infected cells. Direct biochemical experiments show that NleA inhibits COPII-dependent protein export from the endoplasmic reticulum. Collectively, these findings indicate that disruption of COPII function in host cells contributes to the virulence of EPEC and EHEC.

  18. Cleavage of Interferon Regulatory Factor 7 by Enterovirus 71 3C Suppresses Cellular Responses

    PubMed Central

    Lei, Xiaobo; Xiao, Xia; Xue, Qinghua; Jin, Qi

    2013-01-01

    Enterovirus 71 (EV71) is a positive-stranded RNA virus which is capable of inhibiting innate immunity. Among virus-encoded proteins, the 3C protein compromises the type I interferon (IFN-I) response mediated by retinoid acid-inducible gene-I (RIG-I) or Toll-like receptor 3 that activates interferon regulatory 3 (IRF3) and IRF7. In the present study, we report that enterovirus 71 downregulates IRF7 through the 3C protein, which inhibits the function of IRF7. When expressed in mammalian cells, the 3C protein mediates cleavage of IRF7 rather than that of IRF3. This process is insensitive to inhibitors of caspase, proteasome, lysosome, and autophagy. H40D substitution in the 3C active site abolishes its activity, whereas R84Q or V154S substitution in the RNA binding motif has no effect. Furthermore, 3C-mediated cleavage occurs at the Q189-S190 junction within the constitutive activation domain of IRF7, resulting in two cleaved IRF7 fragments that are incapable of activating IFN expression. Ectopic expression of wild-type IRF7 limits EV71 replication. On the other hand, expression of the amino-terminal domain of IRF7 enhances EV71 infection, which correlates with its ability to interact with and inhibit IRF3. These results suggest that control of IRF7 by the 3C protein may represent a viral mechanism to escape cellular responses. PMID:23175366

  19. Cellular factors promoting resistance to effective treatment of glioma with oncolytic myxoma virus.

    PubMed

    Zemp, Franz J; McKenzie, Brienne A; Lun, Xueqing; Reilly, Karlyne M; McFadden, Grant; Yong, V Wee; Forsyth, Peter A

    2014-12-15

    Oncolytic virus therapy is being evaluated in clinical trials for human glioma. While it is widely assumed that the immune response of the patient to the virus infection limits the utility of the therapy, investigations into the specific cell type(s) involved in this response have been performed using nonspecific pharmacologic inhibitors or allogeneic models with compromised immunity. To identify the immune cells that participate in clearing an oncolytic infection in glioma, we used flow cytometry and immunohistochemistry to immunophenotype an orthotopic glioma model in immunocompetent mice after Myxoma virus (MYXV) administration. These studies revealed a large resident microglia and macrophage population in untreated tumors, and robust monocyte, T-, and NK cell infiltration 3 days after MYXV infection. To determine the role on the clinical utility of MYXV therapy for glioma, we used a combination of knockout mouse strains and specific immunocyte ablation techniques. Collectively, our experiments identify an important role for tumor-resident myeloid cells and overlapping roles for recruited NK and T cells in the clearance and efficacy of oncolytic MYXV from gliomas. Using a cyclophosphamide regimen to achieve lymphoablation prior and during MYXV treatment, we prevented treatment-induced peripheral immunocyte recruitment and, surprisingly, largely ablated the tumor-resident macrophage population. Virotherapy of cyclophosphamide-treated animals resulted in sustained viral infection within the glioma as well as a substantial survival advantage. This study demonstrates that resistance to MYXV virotherapy in syngeneic glioma models involves a multifaceted cellular immune response that can be overcome with cyclophosphamide-mediated lymphoablation.

  20. The catalytic subunit of DNA-dependent protein kinase is required for cellular resistance to oxidative stress independent of DNA double-strand break repair.

    PubMed

    Li, Mengxia; Lin, Yu-Fen; Palchik, Guillermo A; Matsunaga, Shinji; Wang, Dong; Chen, Benjamin P C

    2014-11-01

    DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) are the two major kinases involved in DNA double-strand break (DSB) repair, and are required for cellular resistance to ionizing radiation. Whereas ATM is the key upstream kinase for DSB signaling, DNA-PKcs is primarily involved in DSB repair through the nonhomologous end-joining (NHEJ) mechanism. In addition to DSB repair, ATM has been shown to be involved in the oxidative stress response and could be activated directly in vitro on hydrogen peroxide (H2O2) treatment. However, the role of DNA-PKcs in cellular response to oxidative stress is not clear. We hypothesize that DNA-PKcs may participate in the regulation of ATM activation in response to oxidative stress, and that this regulatory role is independent of its role in DNA double-strand break repair. Our findings reveal that H2O2 induces hyperactivation of ATM signaling in DNA-PKcs-deficient, but not Ligase 4-deficient cells, suggesting an NHEJ-independent role for DNA-PKcs. Furthermore, DNA-PKcs deficiency leads to the elevation of reactive oxygen species (ROS) production, and to a decrease in cellular survival against H2O2. For the first time, our results reveal that DNA-PKcs plays a noncanonical role in the cellular response to oxidative stress, which is independent from its role in NHEJ. In addition, DNA-PKcs is a critical regulator of the oxidative stress response and contributes to the maintenance of redox homeostasis. Our findings reveal that DNA-PKcs is required for cellular resistance to oxidative stress and suppression of ROS buildup independently of its function in DSB repair.

  1. Epigenetic and genetic factors in the cellular response to radiations and DNA-damaging chemicals

    SciTech Connect

    Williams, J.R.; D'Arpa, P.

    1981-07-01

    DNA-damaging agents are widely used as therapeutic tools for a variety of disease states. Many such agents are considered to produce detrimental side effects. Thus, it is important to evaluate both therapeutic efficacy and potential risk. DNA-damaging agents can be so evaluated by comparison to agents whose therapeutic benefit and potential hazards are better known. We propose a framework for such comparison, demonstrating that a simple transformation of cytotoxicity-dose response patterns permits a facile comparison of variation between cells exposed to a single DNA-damaging agent or to different cytotoxic agents. Further, by transforming data from experiments which compare responses of 2 cell populations to an effects ratio, different patterns for the changes in cytotoxicity produced by epigenetic and genetic factors were compared. Using these transformations, we found that there is a wide variation (a factor of 4) between laboratories for a single agent (UVC) and only a slightly larger variation (factor of 6) between normal cell response for different types of DNA-damaging agents (x-ray, UVC, alkylating agents, crosslinking agents). Epigenetic factors such as repair and recovery appear to be a factor only at higher dose levels. Comparison in the cytotoxic effect of a spectrum of DNA-damaging agents in xeroderma pigmentosum, ataxia telangiectasia, and Fanconi's anemia cells indicates significantly different patterns, implying that the effect, and perhaps the nature, of these genetic conditions are quite different.

  2. The GATA transcription factor gene gtaG is required for terminal differentiation in Dictyostelium.

    PubMed

    Katoh-Kurasawa, Mariko; Santhanam, Balaji; Shaulsky, Gad

    2016-03-09

    The GATA transcription factor GtaG is conserved in Dictyostelids and essential for terminal differentiation in Dictyostelium discoideum, but its function is not well understood. Here we show that gtaG is expressed in prestalk cells at the anterior region of fingers and in the extending stalk during culmination. The gtaG(-) phenotype is cell-autonomous in prestalk cells and non-cell-autonomous in prespore cells. Transcriptome analyses reveal that GtaG regulates prestalk gene expression during cell differentiation before culmination and is required for progression into culmination. GtaG-dependent genes include genetic suppressors of the Dd-STATa-defective phenotype as well as Dd-STATa target-genes, including extra cellular matrix genes. We show that GtaG may be involved in the production of two culmination-signaling molecules, cyclic di-GMP and the spore differentiation factor SDF-1 and that addition of c-di-GMP rescues the gtaG(-) culmination and spore formation deficiencies. We propose that GtaG is a regulator of terminal differentiation that functions in concert with Dd-STATa and controls culmination through regulating c-di-GMP and SDF-1 production in prestalk cells.

  3. SMARCA3, a Chromatin-Remodeling Factor, Is Required for p11-Dependent Antidepressant Action

    PubMed Central

    Oh, Yong-Seok; Gao, Pu; Lee, Ko-Woon; Ceglia, Ilaria; Seo, Ji-Seon; Zhang, Xiaozhu; Ahn, Jung-Hyuck; Chait, Brian T.; Patel, Dinshaw J.; Kim, Yong; Greengard, Paul

    2013-01-01

    SUMMARY p11, through unknown mechanisms, is required for behavioral and cellular responses to selective serotonin-reuptake inhibitors (SSRIs). Here we have identified SMARCA3, a chromatin-remodeling factor, as a novel target for the p11/annexin A2 heterotetrameric complex. Determination of the crystal structure indicates that SMARCA3 peptide binds to a hydrophobic pocket in the heterotetramer. Formation of this complex increases the DNA binding affinity of SMARCA3 and its localization to the nuclear matrix fraction. In the dentate gyrus, both p11 and SMARCA3 are highly enriched in hilar mossy cells and basket cells. In response to the SSRI, fluoxetine, the expression of p11 is induced in both cell types, and the amount of the ternary complex of p11/annexin A2/SMARCA3 is increased. SSRI-induced neurogenesis and behavioral responses are abolished by constitutive knockout of SMARCA3. Our studies indicate a central role for a chromatin-remodeling factor in the SSRI/p11 signaling pathway, and suggest a novel approach to the development of improved antidepressant therapies. PMID:23415230

  4. DNA binding site for a factor(s) required to initiate simian virus 40 DNA replication.

    PubMed Central

    Yamaguchi, M; DePamphilis, M L

    1986-01-01

    Efficient initiation of DNA replication in the absence of nonspecific DNA repair synthesis was obtained by using a modification of the system developed by J.J. Li and T.J. Kelly [(1984) Proc. Natl. Acad. Sci. USA 81, 6973-6977]. Circular double-stranded DNA plasmids replicated in extracts of CV-1 cells only when the plasmids contained the cis-acting origin sequence for simian virus 40 DNA replication (ori) and the extract contained simian virus 40 large tumor antigen. Competition between plasmids containing ori and plasmids carrying deletions in and about ori served to identify a sequence that binds the rate-limiting factor(s) required to initiate DNA replication. The minimum binding site (nucleotides 72-5243) encompassed one-half of the simian virus 40 ori sequence that is required for initiation of replication (ori-core) plus the contiguous sequence on the late gene side of ori-core containing G + C-rich repeats that facilitates initiation (ori-auxiliary). This initiation factor binding site was specific for the simian virus 40 ori region, even though it excluded the high-affinity large tumor antigen DNA binding sites. Images PMID:3006062

  5. Space environmental factors affecting responses to radiation at the cellular level.

    PubMed

    Planel, H; Gaubin, Y; Pianezzi, B; Gasset, G

    1989-01-01

    Previous space experiments suggest a high value for the RBE of cosmic radiation. A possible explanation could be a change in cell radiosensitivity due to a combined effect of radiation and other factors related to the space environment and to the space flight. Results of the EXOBLOC II experiment support this assumption. On earth, vibrations or accelerations applied before or after irradiation can change the responses to radiation. Microgravity could be the main factor affecting the radiosensitivity and DNA repair but this hypothesis must be confirmed by additional experiments.

  6. Cellular factors promoting resistance to effective treatment of glioma with oncolytic Myxoma virus

    PubMed Central

    Zemp, Franz J.; McKenzie, Brienne A.; Lun, Xueqing; Reilly, Karlyne M.; McFadden, Grant; Yong, V. Wee; Forsyth, Peter A.

    2014-01-01

    Oncolytic virus therapy is being evaluated in clinical trials for human glioma. While it is widely assumed that the patient's immune response to the virus infection limits the therapy's utility, investigations into the specific cell type(s) involved in this response have been performed using non-specific pharmacological inhibitors or allogeneic models with compromised immunity. To identify the immune cells that participate in clearing an oncolytic infection in glioma, we used flow cytometry and immunohistochemistry to immunophenotype an orthotopic glioma model in immunocompetent mice after Myxoma virus (MYXV) administration. These studies revealed a large resident microglia and macrophage population in untreated tumours, and robust monocyte, T and NK cell infiltration 3 days following MYXV infection. To determine the role on the clinical utility of MYXV therapy for glioma, we used a combination of knockout mouse strains and specific immunocyte ablation techniques. Collectively, our experiments identify an important role for tumour-resident myeloid cells and overlapping roles for recruited NK and T cells in the clearance and efficacy of oncolytic MYXV from gliomas. Using a cyclophosphamide regimen to achieve lymphoablation prior and during MYXV treatment, we prevented treatment-induced peripheral immunocyte recruitment and, surprisingly, largely ablated the tumour-resident macrophage population. Virotherapy of CPA-treated animals resulted in sustained viral infection within the glioma as well as a substantial survival advantage. This study demonstrates that resistance to MYXV virotherapy in syngeneic glioma models involves a multi-faceted cellular immune response that can be overcome with CPA-mediated lymphoablation. PMID:25336188

  7. Cellular Fibronectin Expression in Human Trabecular Meshwork and Induction by Transforming Growth Factor-β2

    PubMed Central

    Medina-Ortiz, Wanda E.; Belmares, Ricardo; Neubauer, Sandra; Wordinger, Robert J.; Clark, Abbot F.

    2013-01-01

    Purpose. Levels of TGF-β2 are higher in POAG aqueous humor, causing deposition of extracellular matrix (ECM) proteins, including fibronectin (FN), in the glaucomatous human trabecular meshwork (HTM) that may be responsible for elevated IOP. The purpose of this study was to identify the expression of cellular FN (cFN) isoforms (EDA and EDB) in HTM cells and tissues, and to determine whether TGF-β2 can induce cFN expression and fibril formation in cultured HTM cells. Methods. Expression of cFN mRNA isoforms and induction by recombinant TGF-β2 (5 ng/mL) were determined by quantitative RT-PCR. The TGF-β2 induction of EDA isoform protein expression and FN fibril formation were analyzed using Western immunoblots and immunocytochemistry (ICC), respectively. Immunohistochemistry (IHC) analysis was used to examine total FN and EDA isoform expression in normal (NTM) and glaucomatous (GTM) trabecular meshwork (TM) tissues. Results. Both cFN mRNA isoforms were expressed in cultured HTM cells and were induced by TGF-β2 after 2, 4, and 7 days (P < 0.05). Similarly, EDA isoform protein and fibril formation were increased after 4 and 7 days of TGF-β2 treatment. Finally, GTM tissues had significantly greater EDA isoform protein levels (1.7-fold, P < 0.05) compared to NTM tissues. Conclusions. This study demonstrated that cFN isoforms are expressed and induced in HTM cells by TGF-β2. Also, increased EDA isoform protein levels were seen in GTM tissues. Our findings suggest that induction of cFN isoform expression in the TM ECM may be a novel pathologic mechanism involved in the TM changes associated with glaucoma. PMID:24030464

  8. Information System Requirements Determination: Factors Impeding Stakeholders from Reaching Common Understandings and Agreements on Requirements

    ERIC Educational Resources Information Center

    Gissel, Richard L.

    2010-01-01

    Information system implementations require developers to first know what they must create and then determine how best to create it. The requirements determination phase of the system development life cycle typically determines what functions a system must perform and how well it must accomplish required functions. Implementation success depends on…

  9. Information System Requirements Determination: Factors Impeding Stakeholders from Reaching Common Understandings and Agreements on Requirements

    ERIC Educational Resources Information Center

    Gissel, Richard L.

    2010-01-01

    Information system implementations require developers to first know what they must create and then determine how best to create it. The requirements determination phase of the system development life cycle typically determines what functions a system must perform and how well it must accomplish required functions. Implementation success depends on…

  10. Transcriptional activation of the herpes simplex virus type 1 UL38 promoter conferred by the cis-acting downstream activation sequence is mediated by a cellular transcription factor.

    PubMed

    Guzowski, J F; Singh, J; Wagner, E K

    1994-12-01

    The herpes simplex virus (HSV) type 1 strict late (gamma) UL38 promoter contains three cis-acting transcriptional elements: a TATA box, a specific initiator element, and the downstream activation sequence (DAS). DAS is located between positions +20 and +33 within the 5' untranslated leader region and strongly influences transcript levels during productive infection. In this communication, we further characterize DAS and investigate its mechanism of action. DAS function has a strict spacing requirement, and DAS contains an essential 6-bp core element. A similarly positioned element from the gamma gC gene (UL44) has partial DAS function within the UL38 promoter context, and the promoter controlling expression of the gamma US11 transcript contains an identically located element with functional and sequence similarity to UL38 DAS. These data suggest that downstream elements are a common feature of many HSV gamma promoters. Results with recombinant viruses containing modifications of the TATA box or initiator element of the UL38 promoter suggest that DAS functions to increase transcription initiation and not the efficiency of transcription elongation. In vitro transcription assays using uninfected HeLa nuclear extracts show that, as in productive infection with recombinant viruses, the deletion of DAS from the UL38 promoter dramatically decreases RNA expression. Finally, electrophoretic mobility shift assays and UV cross-linking experiments show that DAS DNA forms a specific, stable complex with a cellular protein (the DAS-binding factor) of approximately 35 kDa. These data strongly suggest that the interaction of cellular DAS-binding factor with DAS is required for efficient expression of UL38 and other HSV late genes.

  11. Modulation of enhancer looping and differential gene targeting by Epstein-Barr virus transcription factors directs cellular reprogramming.

    PubMed

    McClellan, Michael J; Wood, C David; Ojeniyi, Opeoluwa; Cooper, Tim J; Kanhere, Aditi; Arvey, Aaron; Webb, Helen M; Palermo, Richard D; Harth-Hertle, Marie L; Kempkes, Bettina; Jenner, Richard G; West, Michelle J

    2013-09-01

    Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors, we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets (WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular basis for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of

  12. Deconvolution and chromatic aberration corrections in quantifying colocalization of a transcription factor in three-dimensional cellular space.

    PubMed

    Abraham, Thomas; Allan, Sarah E; Levings, Megan K

    2010-08-01

    In the realm of multi-dimensional confocal microscopy, colocalization analysis of fluorescent emission signals has proven to be an invaluable tool for detecting molecular interactions between biological macromolecules at the subcellular level. We show here that image processing operations such as the deconvolution and chromatic corrections play a crucial role in the accurate determination of colocalization between biological macromolecules particularly when the fluorescent signals are faint, and when the fluorescent signals are in the blue and red emission regions. The cellular system presented here describes quantification of an activated forkhead box P3 (FOXP3) transcription factor in three-dimensional (3D) cellular space. 293T cells transfected with a conditionally active form of FOXP3 were stained for anti-FOXP3 conjugated to a fluorescent red dye (Phycoerythrin), and counterstained for DNA (nucleus) with fluorescent blue dye (Hoechst). Due to the broad emission spectra of these dyes, the fluorescent signals were collected only from peak regions and were acquired sequentially. Since the PE signal was weak, a confocal pinhole size of two Airy size was used to collect the 3D image data sets. The raw images supplemented with the spectral data show the preferential association of activated FOXP3 molecules with the nucleus. However, the PE signals were found to be highly diffusive and colocalization quantification from these raw images was not possible. In order to deconvolve the 3D raw image data set, point spread functions (PSFs) of these emissions were measured. From the measured PSF, we found that chromatic shifts between the blue and red colors were quite considerable. Followed by the applications of both the axial and lateral chromatic corrections, colocalization analysis performed on the deconvolved-chromatic corrected-3D image data set showed that 98% of DNA molecules were associated with FOXP3 molecules, whereas only 66% of FOXP3 molecules were colocalized

  13. Cleavage of the HPV16 Minor Capsid Protein L2 during Virion Morphogenesis Ablates the Requirement for Cellular Furin during De Novo Infection

    PubMed Central

    Cruz, Linda; Biryukov, Jennifer; Conway, Michael J.; Meyers, Craig

    2015-01-01

    Infections by high-risk human papillomaviruses (HPV) are the causative agents for the development of cervical cancer. As with other non-enveloped viruses, HPVs are taken up by the cell through endocytosis following primary attachment to the host cell. Through studies using recombinant pseudovirus particles (PsV), many host cellular proteins have been implicated in the process. The proprotein convertase furin has been demonstrated to cleave the minor capsid protein, L2, post-attachment to host cells and is required for infectious entry by HPV16 PsV. In contrast, using biochemical inhibition by a furin inhibitor and furin-negative cells, we show that tissue-derived HPV16 native virus (NV) initiates infection independent of cellular furin. We show that HPV16 L2 is cleaved during virion morphogenesis in differentiated tissue. In addition, HPV45 is also not dependent on cellular furin, but two other alpha papillomaviruses, HPV18 and HPV31, are dependent on the activity of cellular furin for infection. PMID:26569287

  14. Mechanism of Diphtheria Toxin Catalytic Domain Delivery to the Eukaryotic Cell Cytosol and the Cellular Factors that Directly Participate in the Process

    PubMed Central

    Murphy, John R.

    2011-01-01

    Research on diphtheria and anthrax toxins over the past three decades has culminated in a detailed understanding of their structure function relationships (e.g., catalytic (C), transmembrane (T), and receptor binding (R) domains), as well as the identification of their eukaryotic cell surface receptor, an understanding of the molecular events leading to the receptor-mediated internalization of the toxin into an endosomal compartment, and the pH triggered conformational changes required for pore formation in the vesicle membrane. Recently, a major research effort has been focused on the development of a detailed understanding of the molecular interactions between each of these toxins and eukaryotic cell factors that play an essential role in the efficient translocation of their respective catalytic domains through the trans-endosomal vesicle membrane pore and delivery into the cell cytosol. In this review, I shall focus on recent findings that have led to a more detailed understanding of the mechanism by which the diphtheria toxin catalytic domain is delivered to the eukaryotic cell cytosol. While much work remains, it is becoming increasingly clear that the entry process is facilitated by specific interactions with a number of cellular factors in an ordered sequential fashion. In addition, since diphtheria, anthrax lethal factor and anthrax edema factor all carry multiple coatomer I complex binding motifs and COPI complex has been shown to play an essential role in entry process, it is likely that the initial steps in catalytic domain entry of these divergent toxins follow a common mechanism. PMID:22069710

  15. Factors affecting energy and macronutrient requirements in elderly people.

    PubMed

    Ritz, P

    2001-04-01

    (i) to describe energy and macronutrient requirements in healthy and diseased elderly patients from knowledge acquired about the age-related changes in energy balance (ii) to describe changes in body composition and the consequences of physical activity and exercise programs. Aging in individuals considered healthy is associated with a reduction in muscle mass and strength (with consequences on autonomy), and an increase in fat mass mainly in the central area, the latter might increase the risk of cardiovascular disease. Body composition changes can be seen as a positive energy (fat) balance. The reduced fat-free mass is responsible for a low resting metabolic rate. Therefore, energy requirements are reduced all the more since physical activity is decreased. A simple means for calculating individuals' energy requirements from estimated resting metabolic rate and physical activity is not yet available in a validated form and is much required. Protein requirements are still debated. Exercise programs can be implemented for increasing muscle mass and strength (resistance training) or for improving aerobic fitness and reducing fat mass (endurance exercise). It is not yet clear whether structured exercise programs or spontaneous physical activity have similar advantages. It is not known in which cases resistance, endurance, or a combination of both exercises should be recommended. The consequences of physical activity and exercise programs on energy and macronutrient requirements is not clear. Diseased elderly persons are prone to malnutrition which impairs clinical and functional outcome. Malnutrition is the result of an energy intake inadequate to match energy requirements. Literature is very short of data on energy requirements in diseased elderly persons, who are under the complex influences of stress (increasing resting energy requirements), reduced body mass and physical activity (reducing energy requirements), plus potential effects of drugs. Almost nothing is

  16. Poxvirus tumor necrosis factor receptor (TNFR)-like T2 proteins contain a conserved preligand assembly domain that inhibits cellular TNFR1-induced cell death.

    PubMed

    Sedger, Lisa M; Osvath, Sarah R; Xu, Xiao-Ming; Li, Grace; Chan, Francis K-M; Barrett, John W; McFadden, Grant

    2006-09-01

    The poxvirus tumor necrosis factor receptor (TNFR) homologue T2 has immunomodulatory properties; secreted myxoma virus T2 (M-T2) protein binds and inhibits rabbit TNF-alpha, while intracellular M-T2 blocks virus-induced lymphocyte apoptosis. Here, we define the antiapoptotic function as inhibition of TNFR-mediated death via a highly conserved viral preligand assembly domain (vPLAD). Jurkat cell lines constitutively expressing M-T2 were generated and shown to be resistant to UV irradiation-, etoposide-, and cycloheximide-induced death. These cells were also resistant to human TNF-alpha, but M-T2 expression did not alter surface expression levels of TNFRs. Previous studies indicated that T2's antiapoptotic function was conferred by the N-terminal region of the protein, and further examination of this region revealed a highly conserved N-terminal vPLAD, which is present in all poxvirus T2-like molecules. In cellular TNFRs and TNF-alpha-related apoptosis-inducing ligand (TRAIL) receptors (TRAILRs), PLAD controls receptor signaling competency prior to ligand binding. Here, we show that M-T2 potently inhibits TNFR1-induced death in a manner requiring the M-T2 vPLAD. Furthermore, we demonstrate that M-T2 physically associates with and colocalizes with human TNFRs but does not prevent human TNF-alpha binding to cellular receptors. Thus, M-T2 vPLAD is a species-nonspecific dominant-negative inhibitor of cellular TNFR1 function. Given that the PLAD is conserved in all known poxvirus T2-like molecules, we predict that it plays an important function in each of these proteins. Moreover, that the vPLAD confers an important antiapoptotic function confirms this domain as a potential target in the development of the next generation of TNF-alpha/TNFR therapeutics.

  17. SOX family transcription factors involved in diverse cellular events during development.

    PubMed

    She, Zhen-Yu; Yang, Wan-Xi

    2015-12-01

    In metazoa, SOX family transcription factors play many diverse roles. In vertebrate, they are well-known regulators of numerous developmental processes. Wide-ranging studies have demonstrated the co-expression of SOX proteins in various developing tissues and that they occur in an overlapping manner and show functional redundancy. In particular, studies focusing on the HMG box of SOX proteins have revealed that the HMG box regulates DNA-binding properties, and mediates both the nucleocytoplasmic shuttling of SOX proteins and their physical interactions with partner proteins. Posttranslational modifications are further implicated in the regulation of the transcriptional activities of SOX proteins. In this review, we discuss the underlying molecular mechanisms involved in the SOX-partner factor interactions and the functional modes of SOX-partner complexes during development. We particularly emphasize the representative roles of the SOX group proteins in major tissues during developmental and physiological processes.

  18. Activation of the transcription factor c-Jun in acute cellular and antibody-mediated rejection after kidney transplantation.

    PubMed

    Kobayashi, Akimitsu; Takahashi, Takamune; Horita, Shigeru; Yamamoto, Izumi; Yamamoto, Hiroyasu; Teraoka, Satoshi; Tanabe, Kazunari; Hosoya, Tatsuo; Yamaguchi, Yutaka

    2010-12-01

    c-Jun is a transcription factor that belongs to the activator protein-1 family of proteins. In human kidney disease, c-Jun is activated in glomerular and tubular cells and plays a major role in renal pathophysiology. However, the contribution of this pathway to renal allograft rejection has not been determined. We investigated whether c-Jun is activated in acute allograft rejection. c-Jun activation was assessed with immunohistochemistry using phospho-specific c-Jun antibodies in control human renal tissue and renal tissue from patients with acute cellular rejection, acute antibody-mediated rejection, and no rejection in the month after transplantation. In patients with acute cellular rejection, c-Jun activation was observed primarily in infiltrated T cells associated with tubulitis, interstitial cell infiltration, and endarteritis. The number of infiltrated phosphorylated c-Jun-positive cells in the tubules and interstitium was correlated with the Banff classification "t" and "i" scores. In patients with acute antibody-mediated rejection, c-Jun activation was observed in injured endothelial cells as well as in infiltrated cells, including macrophages, in the glomerular and peritubular capillaries. Furthermore, the serum creatinine levels and changes in serum creatinine from the previous year were significantly correlated with the total tubulointerstitial phosphorylated c-Jun-positive score (representing the number of positive nuclei in the tubules, interstitium, and peritubular capillaries). In conclusion, c-Jun was activated in acute antibody-mediated rejection and acute cellular rejection and was associated with reduced graft function. These findings suggest that c-Jun plays a key role in pathological events and may represent a novel therapeutic target in acute renal allograft rejection.

  19. Differential expression of growth factors at the cellular level in virus-infected brain

    PubMed Central

    Prosniak, Mikhail; Zborek, Anna; Scott, Gwen S.; Roy, Anirban; Phares, Timothy W.; Koprowski, Hilary; Hooper, D. Craig

    2003-01-01

    The contribution of host factors to rabies virus (RV) transcription/replication and axonal/transsynaptic spread is largely unknown. We previously identified several host genes that are up-regulated in the mouse brain during RV infection, including neuroleukin, which is involved in neuronal growth and survival, cell motility, and differentiation, and fibroblast growth factor homologous factor 4 (FHF4), which has been implicated in limb and nervous system development. In this study, we used real-time quantitative RT-PCR to assess the expression of mRNAs specific for neuroleukin, the two isoforms of FHF4 (FHF4-1a and -1b) encoded by the FHF4 gene, and N protein of RV in neurons and astrocytes isolated by laser capture microdissection from mouse brains infected with the laboratory-adapted RV strain CVS-N2c or with a street RV of silver-haired bat origin. Differences in the gene expression patterns suggest that the capacity of RV strains to infect nonneuronal cells and differentially modulate host gene expression may be important in virus replication and spread in the CNS. PMID:12736376

  20. Cellular characterization of epidermal growth factor-expanded free-floating neurospheres.

    PubMed

    Lobo, Maria V T; Alonso, F Javier M; Redondo, Carolina; López-Toledano, Miguel A; Caso, Enrique; Herranz, Antonio S; Paíno, Carlos Luis; Reimers, Diana; Bazán, Eulalia

    2003-01-01

    Neural stem cells proliferate in liquid culture as cell clusters (neurospheres). This study was undertaken to characterize the epidermal growth factor (EGF)-expanded free-floating neurospheres derived from rat fetal striatum. We examined the ultrastructural and antigenic characteristics of these spheres. They consisted of two cell types, electron-dense and electron-lucent cells. Lucent cells were immunopositive to actin, vimentin, and nestin, whereas dense cells were immunopositive to actin, weakly positive to vimentin, and nestin-negative. Neurospheres contained healthy, apoptotic, and necrotic cells. Healthy cells were attached to each other by adherens junctions. They showed many pseudopodia and occasionally a single cilium. Sphere cells showed phagocytic capability because healthy cells phagocytosed the cell debris derived from dead cells in a particular process that involves the engulfment of dying cells by cell processes from healthy cells. Sphere cells showed a cytoplasmic and a nuclear pool of fibroblast growth factor (FGF) receptors. They expressed E- and N-cadherin, alpha- and beta-catenin, EGF receptor, and a specific subset of FGF receptors. Because sphere cells expressed this factor in the absence of exogenous FGF-2, we propose that they are able to synthesize FGF-2.

  1. Factors affecting power requirements for chipping whole trees

    Treesearch

    Bryce J. Stokes; William F. Watson; Donald L. Sirois

    1987-01-01

    Large and small in-woods disk chippers were used in field tests to determine the power requirements for chipping whole trees. Hardwood and softwood species were evaluated over a range of diameter classes and moisture contents.

  2. Development of water requirement factors for biomass conversion pathway.

    PubMed

    Singh, Shikhar; Kumar, Amit

    2011-01-01

    Published data were used to develop an integrated spreadsheet-based model to estimate total water requirement for 12 biomass conversion pathways. The water requirement for crop production was attributed only to the grains in the estimates since agricultural residues are produced irrespective of their use for fuel or electricity. Corn stover- and wheat straw-based ethanol production pathways are water efficient, requiring only 0.3 l, whereas biopower production pathways (i.e. direct combustion and bio-oil production) require about 0.8-0.9 l of water per MJ. Wheat- and corn-based ethanol production pathways consume 77 and 108 l of water per MJ, respectively. Utilization of switchgrass for production of ethanol, biopower through the direct combustion, and pyrolysis consume 128, 187 and 229 l of water per MJ, respectively. Biodiesel production from canola seed consumes 124 l of water per MJ. Corn stover- and wheat straw-based conversion pathways are most water efficient.

  3. Acetylation mediated by the p300/CBP-associated factor determines cellular energy metabolic pathways in cancer.

    PubMed

    Rajendran, Ramkumar; Garva, Richa; Ashour, Hassan; Leung, Travis; Stratford, Ian; Krstic-Demonacos, Marija; Demonacos, Constantinos

    2013-06-01

    Normal cells produce energy either through OXPHOS in the presence of oxygen or glycolysis in its absence. Cancer cells produce energy preferably through glycolysis even in the presence of oxygen, thereby, acquiring survival and proliferative advantages. Oncogenes and tumour suppressors control these metabolic pathways by regulating the expression of their target genes involved in these processes. During hypoxia, HIF-1 favours high glycolytic flux by upregulating glycolytic enzymes. Conversely, p53 inhibits glycolysis and increases OXPHOS expression through TIGAR and SCO2 gene expression, respectively. We hypothesise that the p300/CBP-associated factor (PCAF) as a common co-factor shared between p53 and HIF-1 plays an important role in the regulation of energy production by modulating SCO2 and TIGAR gene expression mediated by these two transcription factors. The possible involvement of HIF-1 in the regulation of SCO2 and TIGAR gene expression was investigated in cells with different p53 status in normoxia- and hypoxia-mimicking conditions. Putative hypoxia response elements (HREs) were identified in the regulatory region of SCO2 and TIGAR gene promoters. Chromatin immunoprecipitation experiments suggested that HIF-1 was recruited to the putative HREs present in the SCO2 and TIGAR promoters in a cell type-dependent manner. Transcriptional assays endorsed the notion that PCAF may be involved in the determination of the SCO2 and TIGAR cellular levels, thereby, regulating cellular energy metabolism, a view supported by assays measuring lactic acid production and oxygen consumption in cells ectopically expressing PCAF. The present study identified HIF-1 as a potential regulator of SCO2 and TIGAR gene expression. Furthermore, evidence to suggest that PCAF is involved in the regulation of cellular energy production pathways in hypoxia-mimicking conditions is presented. This effect of PCAF is exerted by orchestrating differential recruitment of HIF-1α and p53 to the

  4. Nuclear transcription factors: a new approach to enhancing cellular responses to ALA-mediated photodynamic therapy

    NASA Astrophysics Data System (ADS)

    Maytin, Edward V.; Anand, Sanjay; Sato, Nobuyuki; Moore, Brian; Mack, Judith; Gasbarre, Christopher; Keevey, Samantha; Ortel, Bernhard; Sinha, Alok; Khachemoune, Amor

    2006-02-01

    Photodynamic therapy (PDT) using aminolevulinic acid (ALA) relies upon the uptake of ALA into cancer cells, where it is converted into a porphyrin intermediate, protoporphyrin IX (PpIX) that is highly photosensitizing. For large or resistant tumors, however, ALA/PDT is often not completely effective due to inadequate PpIX levels. Therefore, new approaches to enhance the intracellular production of PpIX are sought. Here, we describe a general approach to improve intracellular PpIX accumulation via manipulations that increase the expression of an enzyme, coproporphyrinogen oxidase (CPO), that is rate-determining for PpIX production. We show that nuclear hormones that promote terminal differentiation, e.g. vitamin D or androgens, can also increase the accumulation of PpIX and the amount of killing of the target cells upon exposure to light. These hormones bind to intracellular hormone receptors that translocate to the nucleus, where they act as transcription factors to increase the expression of target genes. We have found that several other transcription factors associated with terminal differentiation, including members of the CCAAT enhancer binding (C/EBP) family, and a homeobox protein named Hoxb13, are also capable of enhancing PpIX accumulation. These latter transcription factors appear to interact directly with the CPO gene promoter, resulting in enhanced CPO transcriptional activity. Our data in several different cell systems, including epithelial cells of the skin and prostate cancer cells, indicate that enhancement of CPO expression and PpIX accumulation represents a viable new approach toward improving the efficacy of ALA/PDT.

  5. Targeting against epidermal growth factor receptors. Cellular processing of astatinated EGF after binding to cultured carcinoma cells.

    PubMed

    Orlova, Anna; Sjöstrom, Anna; Lebeda, Ondrej; Lundqvist, Hans; Carlsson, Jorgen; Tolmachev, Vladimir

    2004-01-01

    The alpha-emitting nuclide 211At is of great interest for radionuclide therapy when coupled to a tumor-targeting biomolecule, e.g. epidermal growth factor (EGF) the receptors of which are overexpressed in many malignancies. However, almost no information concerning the cellular processing of astatinated targeting agents is available. We indirectly astatinated EGF ([211At]-benzoate-EGF) and studied its cellular processing in A-431 carcinoma cells in comparison with data concerning [125I]-benzoate-EGF. The biological half-life of astatine (3.5 h) was longer than the half-life of the iodine label (1.5 h). The increase of the half-life was due to longer retention of the internalised astatine radioactivity. The maximum accumulation for the astatine label occurred later (4-6h) than that for the iodine label (2-4h), indicating a slower excretion of astatine that was confirmed in experiment with 211At/1251-benzoate-EGF. The long retention of astatine might be advantageous for radionuclide therapy.

  6. Direct cellular effects of some mediators, hormones and growth factor-like agents on denervated (isolated) rat gastric mucosal cells.

    PubMed

    Bódis, B; Karádi, O; Nagy, L; Dohoczky, C; Kolega, M; Mózsik, G

    1997-01-01

    The brain-gut axis has an important role in the mechanism of gastric cytoprotection in vivo. The aim of this study was to evaluate the in vitro effect of protective agents without any central and peripheral innervation. A mixed population of rat gastric mucosal cells was isolated by the method of Nagy et al (Gastroenterology (1994) 77, 433-443). Cells were incubated for 60 min with cytoprotective drugs such as prostacyclin, histamine, pentagastrin and PL-10 substances (synthesized parts of BPC). At the end of this incubation cells were treated by 15% ethanol for 5 min. Cell viability was tested by trypan blue exclusion test and succinic dehydrogenase activity. The following results were obtained: 1) prostacyclin, histamine and pentagastrin had no direct cytoprotective effect on isolated cells; and 2) PL-10 substances significantly protected the cells against ethanol-induced cellular damage. This led to the following conclusions: 1) in the phenomenon of gastric cytoprotection only the growth factor-like agents have a direct cellular effect; and 2) the intact peripheral innervation is basically necessary for the development of mediators and hormone-induced gastric cytoprotection.

  7. Pigment epithelium-derived factor delays cellular senescence of human mesenchymal stem cells in vitro by reducing oxidative stress.

    PubMed

    Cao, Yukun; Yang, Ting; Gu, Chunhu; Yi, Dinghua

    2013-04-01

    Mesenchymal stem cells (MSCs) are multipotent progenitor cells that represent a promising approach in the field of regenerative medicine; however, this potential diminishes with senescence. Pigment epithelium-derived factor (PEDF) gives some protection by reducing oxidative stress, which is known to accelerate cellular senescence. Thus we hypothesized that PEDF could delay senescence during MSC expansion by reducing oxidative stress. Proliferation and differentiation potentials, oxidative stress, senescence and p53/p16 expressions have been examined. In MSCs cultured under normoxic conditions treated with PEDF, proliferative lifespan in vitro was significantly increased compared with control group not given PEDF, with ∼10 additional population doublings (PD) occurring before terminal growth arrest. Most of the MSCs cultured under normoxic conditions ceased to proliferate after 20-28 PD, while few senescent cells were found in the hypoxic, PEDF-hypoxic and PEDF-normoxic cultures; this was associated with downregulation of p53 and p16 expression and decreased oxidative stress. PEDF also preserved differentiation potentials of MSCs compared with the control group. Thus PEDF suppression of oxidative stress delays cellular senescence and allows greater expansion of MSCs.

  8. Ionizing radiation induces upregulation of cellular procoagulability and tissue factor expression in human peripheral blood mononuclear cells.

    PubMed

    Goldin-Lang, Petra; Niebergall, Florian; Antoniak, Silvio; Szotowski, Bjoern; Rosenthal, Peter; Pels, Klaus; Schultheiss, Heinz-Peter; Rauch, Ursula

    2007-01-01

    The therapeutic application of ionizing radiation is associated with thrombotic events, but the exact underlying molecular mechanisms are still unclear. Tissue factor (TF), the primary initiator of blood coagulation, is essentially involved in the pathophysiology of thrombosis. Circulating monocytes have been identified to upregulate TF under inflammatory conditions and, thereby, enhance blood thrombogenicity. The study examines the effect of irradiation on the cellular procoagulability and TF protein expression of human peripheral blood mononuclear cells (PBMNCs) in a time period of 7 days. Human PBMNCs were irradiated with 20 Gy. Procoagulability of PBMNCs, released microparticles and microparticle-free cell supernatant was analyzed by a chromogenic assay and TF protein expression quantified by TF ELISA. To determine whether irradiated PBMNCs and shed microparticles initiate plasma clotting, a one stage clotting assay was performed. We found a significant increase of PBMNC-associated procoagulant activity over a time period of 7 days post irradiation. Moreover, 3 days post irradiation PBMNCs initiated the plasma clotting faster than non-irradiated cells. An enhanced cellular TF protein concentration was persistently observed throughout the investigated time up to 7 days post irradiation. Microparticle-associated TF activity significantly increased 3 days post irradiation compared with the non-irradiated controls. PBMNC-derived microparticles post irradiation also initiated the plasma clotting faster than microparticles derived from controls. The results show irradiation to induce TF expression and to increase procoagulability of PBMNCs and cell-derived microparticles. This could be a possible mechanism by which ionizing radiation enhances blood thrombogenicity.

  9. Molecular and Cellular Interactions between Mother and Fetus. Pregnancy as a Rejuvenating Factor.

    PubMed

    Popkov, V A; Silachev, D N; Jankauskas, S S; Zorova, L D; Pevzner, I B; Babenko, V A; Plotnikov, E Y; Zorov, D B

    2016-12-01

    Aging is associated with a decline of various body functions, including ability to regenerate. Over recent decades, it has been demonstrated that some of these changes could be reversed in response to factors originating from a young organism, for example, fetal stem cells or "young blood" in models of heterochronic parabiosis. Pregnancy might be considered as parabiotic model of the interaction between two organisms of different age. In this work, we analyzed and summarized data on the effects of pregnancy on the maternal organism that confirm the hypothesis that pregnancy rejuvenates the mother's organism or slows its aging.

  10. Increased tumor necrosis factor alpha mRNA after cellular exposure to ionizing radiation.

    PubMed Central

    Hallahan, D E; Spriggs, D R; Beckett, M A; Kufe, D W; Weichselbaum, R R

    1989-01-01

    We report that tumor necrosis factor alpha (TNF-alpha) mRNA is increased after treatment with x-rays in certain human sarcoma cells. An increase in TNF-alpha mRNA is accompanied by the increased production of TNF-alpha protein. TNF-alpha enhances radiation lethality in both TNF-alpha-producing and -nonproducing tumor cells. These data suggest that, in addition to the direct cytotoxic effects of x-rays, production of TNF-alpha may add to radiation lethality through autocrine and paracrine mechanisms. Combinations of TNF-alpha and therapeutic radiation may be useful in clinical cancer therapy. Images PMID:2602359

  11. Influenza virus polymerase confers independence of the cellular cap-binding factor eIF4E for viral mRNA translation

    PubMed Central

    Yángüez, Emilio; Rodriguez, Paloma; Goodfellow, Ian; Nieto, Amelia

    2012-01-01

    The influenza virus mRNAs are structurally similar to cellular mRNAs nevertheless; the virus promotes selective translation of viral mRNAs despite the inhibition of host cell protein synthesis. The infection proceeds normally upon functional impairment of eIF4E cap-binding protein, but requires functional eIF4A helicase and eIF4G factor. Here, we have studied whether the presence of cis elements in viral mRNAs or the action of viral proteins are responsible for this eIF4E-independence. The eIF4E protein is required for viral mRNAs translation in vitro, indicating that cis-acting RNA sequences are not involved in this process. We also show that PB2 viral polymerase subunit interacts with the eIF4G protein. In addition, a chimeric mRNA containing viral UTRs sequences transcribed by the viral polymerase out of the infection is successfully translated independently of an impaired eIF4E factor. These data support that the viral polymerase is responsible for the eIF4E independence of influenza virus mRNAs translation. PMID:22112850

  12. The Oxidative Stress Response in Caenorhabditis elegans Requires the GATA Transcription Factor ELT-3 and SKN-1/Nrf2.

    PubMed

    Hu, Queenie; D'Amora, Dayana R; MacNeil, Lesley T; Walhout, Albertha J M; Kubiseski, Terrance J

    2017-08-01

    Cellular damage caused by reactive oxygen species is believed to be a major contributor to age-associated diseases. Previously, we characterized the Caenorhabditis elegans Brap2 ortholog (BRAP-2) and found that it is required to prevent larval arrest in response to elevated levels of oxidative stress. Here, we report that C. elegans brap-2 mutants display increased expression of SKN-1-dependent, phase II detoxification enzymes that is dependent on PMK-1 (a p38 MAPK C. elegans ortholog). An RNA-interference screen was conducted using a transcription factor library to identify genes required for increased expression of the SKN-1 target gst-4 in brap-2 mutants. We identified ELT-3, a member of the GATA transcription factor family, as a positive regulator of gst-4p::gfp expression. We found that ELT-3 interacts with SKN-1 to activate gst-4 transcription in vitro and that elt-3 is required for enhanced gst-4 expression in the brap-2(ok1492) mutant in vivo Furthermore, nematodes overexpressing SKN-1 required ELT-3 for life-span extension. Taken together, these results suggest a model where BRAP-2 acts as negative regulator of SKN-1 through inhibition of p38 MAPK activity, and that the GATA transcription factor ELT-3 is required along with SKN-1 for the phase II detoxification response in C. elegans. Copyright © 2017 by the Genetics Society of America.

  13. Cellular glycosylation affects Herceptin binding and sensitivity of breast cancer cells to doxorubicin and growth factors

    PubMed Central

    Peiris, Diluka; Spector, Alexander F.; Lomax-Browne, Hannah; Azimi, Tayebeh; Ramesh, Bala; Loizidou, Marilena; Welch, Hazel; Dwek, Miriam V.

    2017-01-01

    Alterations in protein glycosylation are a key feature of oncogenesis and have been shown to affect cancer cell behaviour perturbing cell adhesion, favouring cell migration and metastasis. This study investigated the effect of N-linked glycosylation on the binding of Herceptin to HER2 protein in breast cancer and on the sensitivity of cancer cells to the chemotherapeutic agent doxorubicin (DXR) and growth factors (EGF and IGF-1). The interaction between Herceptin and recombinant HER2 protein and cancer cell surfaces (on-rate/off-rate) was assessed using a quartz crystal microbalance biosensor revealing an increase in the accessibility of HER2 to Herceptin following deglycosylation of cell membrane proteins (deglycosylated cells Bmax: 6.83 Hz; glycosylated cells Bmax: 7.35 Hz). The sensitivity of cells to DXR and to growth factors was evaluated using an MTT assay. Maintenance of SKBR-3 cells in tunicamycin (an inhibitor of N-linked glycosylation) resulted in an increase in sensitivity to DXR (0.1 μM DXR P < 0.001) and a decrease in sensitivity to IGF-1 alone and to IGF-1 supplemented with EGF (P < 0.001). This report illustrates the importance of N-linked glycosylation in modulating the response of cancer cells to chemotherapeutic and biological treatments and highlights the potential of glycosylation inhibitors as future combination treatments for breast cancer. PMID:28223691

  14. Potential for cellular stress response to hepatic factor VIII expression from AAV vector

    PubMed Central

    Zolotukhin, Irene; Markusic, David M; Palaschak, Brett; Hoffman, Brad E; Srikanthan, Meera A; Herzog, Roland W

    2016-01-01

    Hemophilia A and B are coagulation disorders resulting from the loss of functional coagulation factor VIII (FVIII) or factor IX proteins, respectively. Gene therapy for hemophilia with adeno-associated virus vectors has shown efficacy in hemophilia B patients. Although hemophilia A patients are more prevalent, the development of therapeutic adeno-associated virus vectors has been impeded by the size of the F8 cDNA and impaired secretion of FVIII protein. Further, it has been reported that over-expression of the FVIII protein induces endoplasmic reticulum stress and activates the unfolded protein response pathway both in vitro and in hepatocytes in vivo, presumably due to retention of misfolded FVIII protein within the endoplasmic reticulum. Engineering of the F8 transgene, including removal of the B domain (BDD-FVIII) and codon optimization, now allows for the generation of adeno-associated virus vectors capable of expressing therapeutic levels of FVIII. Here we sought to determine if the risks of inducing the unfolded protein response in murine hepatocytes extend to adeno-associated virus gene transfer. Although our data show a mild activation of unfolded protein response markers following F8 gene delivery at a certain vector dose in C57BL/6 mice, it was not augmented upon further elevated dosing, did not induce liver pathology or apoptosis, and did not impact FVIII immunogenicity. PMID:27738644

  15. The S2 Cu(i) site in CupA from Streptococcus pneumoniae is required for cellular copper resistance.

    PubMed

    Fu, Yue; Bruce, Kevin E; Wu, Hongwei; Giedroc, David P

    2016-01-01

    Pathogenic bacteria have evolved copper homeostasis and resistance systems for fighting copper toxicity imposed by the human immune system. Streptococcus pneumoniae is a respiratory pathogen that encodes an obligatorily membrane-anchored Cu(i) binding protein, CupA, and a P1B-type ATPase efflux transporter, CopA. The soluble, cytoplasmic domain of CupA (sCupA) contains a binuclear Cu(i) cluster consisting of S1 and S2 Cu(i) ions. The NMR solution structure of apo-sCupA reveals the same cupredoxin fold of Cu2-sCupA, except that the Cu(i) binding loop (residues 112-116, harboring S2 Cu ligands M113 and M115) is highly dynamic as documented by both backbone and side chain methionine methyl order parameters. In contrast to the more solvent exposed, lower affinity S2 Cu site, the high affinity S1 Cu-coordinating cysteines (C74, C111) are pre-organized in the apo-sCupA structure. Biological experiments reveal that the S1 site is largely dispensable for cellular Cu resistance and may be involved in buffering low cytoplasmic Cu(i). In contrast, the S2 site is essential for Cu resistance. Expression of a chimeric CopZ chaperone fused to the CupA transmembrane helix does not protect S. pneumoniae from copper toxicity and substitution of a predicted cytoplasm-facing Cu(i) entry metal-binding site (MBS) on CopA also gives rise to a Cu-sensitivity phenotype. These findings suggest that CupA and CopA may interact and filling of the CupA S2 site with Cu(i) results in stimulation of cellular copper efflux by CopA.

  16. Cellular Effects of Pyocyanin, a Secreted Virulence Factor of Pseudomonas aeruginosa

    PubMed Central

    Hall, Susan; McDermott, Catherine; Anoopkumar-Dukie, Shailendra; McFarland, Amelia J.; Forbes, Amanda; Perkins, Anthony V.; Davey, Andrew K.; Chess-Williams, Russ; Kiefel, Milton J.; Arora, Devinder; Grant, Gary D.

    2016-01-01

    Pyocyanin has recently emerged as an important virulence factor produced by Pseudomonas aeruginosa. The redox-active tricyclic zwitterion has been shown to have a number of potential effects on various organ systems in vitro, including the respiratory, cardiovascular, urological, and central nervous systems. It has been shown that a large number of the effects to these systems are via the formation of reactive oxygen species. The limitations of studies are, to date, focused on the localized effect of the release of pyocyanin (PCN). It has been postulated that, given its chemical properties, PCN is able to readily cross biological membranes, however studies have yet to be undertaken to evaluate this effect. This review highlights the possible manifestations of PCN exposure; however, most studies to date are in vitro. Further high quality in vivo studies are needed to fully assess the physiological manifestations of PCN exposure on the various body systems. PMID:27517959

  17. Identification of the homolog of cell-counting factor in the cellular slime mold Dictyostelium discoideum.

    PubMed

    Okuwa, Takako; Katayama, Takahiro; Takano, Akinori; Yasukawa, Hiroo

    2002-10-01

    Genes for the cell-counting factors in Dictyostelium discoideum, countin and countin2, are considered to control the size of the multicellular structure of this organism. A novel gene, countin3, that is homologous to countin and countin2 genes (49 and 39% identity in amino acid sequence, respectively) was identified in the D. discoideum genome. The expression of countin3 was observed in the vegetatively growing cells, decreased in the aggregating stage, increased in the mid-developmental stage and decreased again in subsequent stages. This expression pattern is different from that of countin and countin2. The distinct expression kinetics of three genes suggests that they would have unique roles in size control of D. discoideum.

  18. Two cell-counting factors regulate the aggregate size of the cellular slime mold Dictyostelium discoideum.

    PubMed

    Okuwa, T; Katayama, T; Takano, A; Kodaira, K; Yasukawa, H

    2001-12-01

    Countin, a cell-counting factor in Dictyostelium discoideum, is considered to limit the maximum size of the multicellular structure, because a countin null strain forms a huge fruiting body compared to that of the wild-type. A novel gene, countin2, that is highly homologous to countin (40% identity in amino acid sequence) was identified in the D. discoideum genome. The countin2 null strain formed a 1.7-fold higher number of the aggregates, resulting in smaller fruiting bodies compared with those of wild-type cells. Thus, the Countin2 protein is thought to limit the minimum size of the multicellular structure. The size and number of aggregates formed by a mixture of countin null and countin2 null strains were the same as those of the wild-type. These findings demonstrate that a combination of Countin and Countin2 proteins determines the appropriate size of the multicellular structure of D. discoideum.

  19. Binding and regulation of cellular functions by monoclonal antibodies against human tumor necrosis factor receptors

    PubMed Central

    1990-01-01

    The present study was undertaken to further characterize the interaction of monoclonal antibodies (mAbs) against tumor necrosis factor (TNF) receptors with different targets, and to assess their ability to influence TNF effects on U937 and human endothelial cell (HEC) functions. Actions of recombinant TNF-alpha on U937 and HEC were effectively inhibited by Htr-5 and Utr-1, and to a greater extent by a combination of both mAbs. These observations indicate that TNF interaction with antigenically different components of membrane receptors (p55 and p75) represents a crucial step in transduction of signals for TNF toxicity against U937 and TNF activation of HEC functions. PMID:2172437

  20. Cellular distribution, subcellular localization and possible functions of basic and acidic fibroblast growth factors.

    PubMed

    Eckenstein, F P; Kuzis, K; Nishi, R; Woodward, W R; Meshul, C; Sherman, L; Ciment, G

    1994-01-13

    The distribution in the rat nervous system of acidic and basic fibroblast growth factors (FGFs) was analysed by a combination of biochemical and anatomical methods. Acidic FGF (aFGF) was found to be present exclusively in specific neuronal populations, such as motor neurons and basal forebrain cholinergic neurons. Basic FGF (bFGF) was found in astrocytes and in neurons in hippocampal area CA2. Within labelled astrocytes and CA2-neurons, bFGF was detected in both the cytoplasm and the nucleus. The levels of intracellular bFGF were manipulated by antisense oligonucleotide treatment of cultures of developing neural crest cells. Results indicated that the amount of melanogenesis in the cultures is likely to be regulated by intracellular, possibly nuclear bFGF.

  1. Microwaves and cellular immunity. I. Effect of whole body microwave irradiation on tumor necrosis factor production in mouse cells.

    PubMed

    Fesenko, E E; Makar, V R; Novoselova, E G; Sadovnikov, V B

    1999-10-01

    Whole body microwave sinusoidal irradiation of male NMRI mice with 8.15-18 GHz (1 Hz within) at a power density of 1 microW/cm2 caused a significant enhancement of TNF production in peritoneal macrophages and splenic T lymphocytes. Microwave radiation affected T cells, facilitating their capacity to proliferate in response to mitogenic stimulation. The exposure duration necessary for the stimulation of cellular immunity ranged from 5 h to 3 days. Chronic irradiation of mice for 7 days produced the decreasing of TNF production in peritoneal macrophages. The exposure of mice for 24 h increased the TNF production and immune proliferative response, and these stimulatory effects persisted over 3 days after the termination of exposure. Microwave treatment increased the endogenously produced TNF more effectively than did lipopolysaccharide, one of the most potential stimuli of synthesis of this cytokine. The role of microwaves as a factor interfering with the process of cell immunity is discussed.

  2. Cytoplasmic domains determine signal specificity, cellular routing characteristics and influence ligand binding of epidermal growth factor and insulin receptors.

    PubMed Central

    Riedel, H; Dull, T J; Honegger, A M; Schlessinger, J; Ullrich, A

    1989-01-01

    The cell surface receptors for insulin and epidermal growth factor (EGF) both employ a tyrosine-specific protein kinase activity to fulfil their distinct biological roles. To identify the structural domains responsible for various receptor activities, we have generated chimeric receptor polypeptides consisting of major EGF and insulin receptor structural domains and examined their biochemical properties and cellular signalling activities. The EGF-insulin receptor hybrids are properly synthesized and transported to the cell surface, where they form binding competent structures that are defined by the origin of their extracellular domains. While their ligand binding affinities are altered, we find that these chimeric receptors are fully functional in transmitting signals across the plasma membrane and into the cell. Thus, EGF receptor and insulin receptor cytoplasmic domain signalling capabilities are independent of their new heterotetrameric or monomeric environments respectively. Furthermore, the cytoplasmic domains carry the structural determinants that define kinase specificity, mitogenic and transforming potential, and receptor routing. Images PMID:2583088

  3. Requirement of Tumor Necrosis Factor Receptor–Associated Factor (Traf)6 in Interleukin 17 Signal Transduction

    PubMed Central

    Schwandner, Ralf; Yamaguchi, Kyoko; Cao, Zhaodan

    2000-01-01

    Signaling through its widely distributed cell surface receptor, interleukin (IL)-17 enhances the transcription of genes encoding proinflammatory molecules. Although it has been well documented that IL-17 activates the transcription factor nuclear factor (NF)-κB and c-Jun NH2-terminal kinase (JNK), the upstream signaling events are largely unknown. Here we report the requirement of tumor necrosis factor receptor–associated factor (TRAF)6 in IL-17–induced NF-κB and JNK activation. In embryonic fibroblasts (EFs) derived from TRAF6 knockout mice, IL-17 failed to activate the IκB kinases (IKKs) and JNK. Consequently, IL-17–induced IL-6 and intercellular adhesion molecule 1 expression in the TRAF6-deficient cells was abolished. Lack of TRAF6 appeared to be the sole defect responsible for the observed failure to respond to IL-17, because transient transfection of TRAF6 expression plasmid into the TRAF6-deficient cells restored IL-17–induced NF-κB activation in a luciferase reporter assay. Furthermore, the levels of IL-17 receptor (IL-17R) on the TRAF6-deficient EFs were comparable to those on the wild-type control cells. Defect in IL-17 response was not observed in TRAF2-deficient EFs. Moreover, when TRAF6 and IL-17R were coexpressed in 293 cells, TRAF6 coimmunoprecipitated with IL-17R. Together, these results indicate that TRAF6, but not TRAF2, is a crucial component in the IL-17 signaling pathway leading to proinflammatory responses. PMID:10748240

  4. Down-regulation of phosphoglucose isomerase/autocrine motility factor expression sensitizes human fibrosarcoma cells to oxidative stress leading to cellular senescence.

    PubMed

    Funasaka, Tatsuyoshi; Hu, Huankai; Hogan, Victor; Raz, Avraham

    2007-12-14

    Phosphoglucose isomerase/autocrine motility factor (PGI/AMF) is a housekeeping gene product present in all cells, is an essential enzyme of catabolic glycolysis and anabolic gluconeogenesis, and regulates tumor cell growth and metastasis. Because glycolytic enzyme up-regulation of expression contributes to glycolytic flux, leading to increased of cell growth and a resistance to cellular stress of normal fibroblasts whereas down-regulation of PGI/AMF leads to mesenchymal-to-epithelial transition in tumor cells, we examined the involvement of PGI/AMF in overcoming cellular senescence in cancer cells. PGI/AMF cellular expression in HT1080 human fibrosarcoma was down-regulated by small interfering RNA methodology, which resulted in an increased sensitivity to oxidative stress and oxidative stress-induced cellular senescence. Signaling analysis revealed that the senescence pathway involving p21 cyclin-dependent kinase inhibitor was up-regulated in PGI/AMF knockdown cells and that superoxide dismutase is the upstream regulator protein of p21-mediated cellular senescence. A specific inhibitor of PGI/AMF induced cellular senescence and p21 expression in tumor cells exposed to an oxidative stress environment. Taken together, the results presented here suggest that PGI/AMF is involved in oxidative stress-induced cellular senescence and should bring novel insights into the control of cellular growth leading to a new methodology for cancer treatment.

  5. Sleep Loss as a Factor to Induce Cellular and Molecular Inflammatory Variations

    PubMed Central

    Hurtado-Alvarado, Gabriela; Castillo-García, Stephanie Ariadne; Hernández, María Eugenia; Domínguez-Salazar, Emilio; Velázquez-Moctezuma, Javier; Gómez-González, Beatriz

    2013-01-01

    A reduction in the amount of time spent sleeping occurs chronically in modern society. Clinical and experimental studies in humans and animal models have shown that immune function is impaired when sleep loss is experienced. Sleep loss exerts a strong regulatory influence on peripheral levels of inflammatory mediators of the immune response. An increasing number of research projects support the existence of reciprocal regulation between sleep and low-intensity inflammatory response. Recent studies show that sleep deficient humans and rodents exhibit a proinflammatory component; therefore, sleep loss is considered as a risk factor for developing cardiovascular, metabolic, and neurodegenerative diseases (e.g., diabetes, Alzheimer's disease, and multiple sclerosis). Circulating levels of proinflammatory mediators depend on the intensity and duration of the method employed to induce sleep loss. Recognizing the fact that the concentration of proinflammatory mediators is different between acute and chronic sleep-loss may expand the understanding of the relationship between sleep and the immune response. The aim of this review is to integrate data from recent published reports (2002–2013) on the effects of sleep loss on the immune response. This review may allow readers to have an integrated view of the mechanisms involved in central and peripheral deficits induced by sleep loss. PMID:24367384

  6. Cellular myeloperoxidase activity in human monocytes stimulated by hyposialylated immunoglobulins and rheumatoid factors.

    PubMed Central

    Dodon, M D; Gazzolo, L; Quash, G A

    1984-01-01

    When hyposialylated , immunoglobulins become immunogenic and tend to form aggregates. In pursuit of the possibility that hyposialylated immunoglobulins (hs-Ig) can trigger human mononuclear phagocytic cells, we have investigated the effects of such hs-Ig on the myeloperoxidase (MPO) activity of these cells. The incubation of human monocytes with aggregated hs-Ig leads to the decrease of intracellular MPO activity. This decrease is dependent on the incubation time, on the amount of hs-Ig added, and on the degree of aggregation. Incubation with unaggregated hs-Ig has a similar effect, thus providing evidence that the loss of sialic acid residues per se is enough to render these molecules capable of decreasing the MPO content of phagocytic cells. Furthermore, human rheumatoid factors, isolated from the sera of rheumatoid arthritis patients, and previously characterized as hyposailylated Ig, interact in the same way with monocytes in triggering the MPO decrease. These observations imply that hs-Ig may be considered as active stimuli in the induction of inflammatory processes, through the initiation of oxidative reactions. PMID:6329948

  7. Cellular Behavior on Epidermal Growth Factor (EGF)-Immobilized PCL/Gelatin Nanofibrous Scaffolds.

    PubMed

    Tığlı, R Seda; Kazaroğlu, N Merve; Mavış, Bora; Gümüşderelioğlu, Menemşe

    2011-01-01

    Nano-scaled poly(ε-caprolactone) (PCL) and PCL/gelatin fibrous scaffolds with immobilized epidermal growth factor (EGF) were prepared for the purpose of wound-healing treatments. The tissue scaffolds were fabricated by electrospinning and the parameters that affect the electrospinning process were optimized. While the fiber diameters were 488 ± 114 nm and 663 ± 107 nm for PCL and PCL/gelatin scaffolds, respectively, the porosities were calculated as 79% for PCL and 68% for PCL/gelatin scaffolds. Electrospun PCL and PCL/gelatin scaffolds were first modified with 1,6-diaminohexane to introduce amino groups on their surfaces, then EGF was chemically conjugated to the surface of nanofibers. The results obtained from Attenuated Total Reflectance Fourier Transform Infrared (ATR-FT-IR) spectroscopy and quantitative measurements showed that EGF was successfully immobilized on nanofibrous scaffolds. L929 mouse fibroblastic cells were cultivated on both neat and EGF-immobilized PCL and PCL/gelatin scaffolds in order to investigate the effect of EGF on cell spreading and proliferation. According to the results, especially EGF-immobilized PCL/gelatin scaffolds exerted early cell spreading and superior and rapid proliferation compared to EGF-immobilized PCL scaffolds and neat PCL, PCL/gelatin scaffolds. Consequently, EGF-immobilized PCL/gelatin scaffolds could potentially be employed as novel scaffolds for skin tissueengineering applications.

  8. Allopurinol ameliorates thioacetamide-induced acute liver failure by regulating cellular redox-sensitive transcription factors in rats.

    PubMed

    Demirel, Ulvi; Yalniz, Mehmet; Aygün, Cem; Orhan, Cemal; Tuzcu, Mehmet; Sahin, Kazim; Ozercan, Ibrahim Hanifi; Bahçecioğlu, Ibrahim Halil

    2012-08-01

    Oxidative stress plays important role in the development of acute liver failure. In this study, we investigated effects of allopurinol (AP) upon thioacetamide (TAA)-induced liver injury and the potential mechanisms leading to amelioration in inflammation with AP treatment. Acute liver failure was induced by intraperitoneal administration of TAA (300 mg/kg/day for 2 days). Thirty-five rats were divided into five groups as control (group 1), TAA (group 2), TAA + 25AP (group 3), TAA + 50 AP (group 4), and TAA + 100AP (group 5). The number of animals in each group was seven. At the end of the study, histopathological, biochemical, and western blot analysis were done. TAA treatment significantly increased serum levels of aminotransferases, liver malondialdehyde (MDA), nuclear factor-kappa B (NF-қB ), activator protein-1 (AP-1), tumor necrosis factor-alpha (TNF-α), cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6) levels, and the necro-inflammation scores. Nevertheless, nuclear factor E2-related factor-2 and heme oxygenase-1 (HO-1) expressions in the liver were decreased by TAA. AP treatment significantly lowered the serum levels of aminotransferases (P < 0.01) and liver MDA, NF-κB, AP-1, TNF-α, COX-2, and IL-6 expressions (P < 0.05). Moreover, AP restored the liver Nrf2 and HO-1 expressions and improved the necro-inflammation scores significantly. AP improves oxidative stress-induced liver damage by regulating cellular redox-sensitive transcriptor factors and expression of pro-inflammatory and antioxidant defense mechanisms. AP probably exerts these beneficiary features by its free radical scavenging ability in a dose-dependent manner.

  9. Immortalization by c-myc, H-ras, and Ela oncogenes induces differential cellular gene expression and growth factor responses

    SciTech Connect

    Kelekar, A.; Cole, M.D.

    1987-11-01

    Early-passage rat kidney cells were immortalized or rescued from senescence with three different oncogenes: viral promoter-driven c-myc, H-ras (Val-12), and adenovirus type 5 E1a. The normal c-myc and H-ras (Gly-12) were unable to immortalize cells under similar conditions. Quantitation of RNA in the ras-immortalized lines demonstrated that the H-ras oncogene was expressed at a level equivalent to that of the normal H-ras gene in established human or rat cell lines. Cell lines immortalized by different oncogenes were found to have distinct growth responses to individual growth factors in a short-term assay. E1a-immortalized cells were largely independent of serum growth factors, whereas c-myc-immortalized cells responded to serum better than to epidermal growth factor and insulin. H-ras-immortalized cells responded significantly to insulin alone and gave a maximal response to epidermal growth factor and insulin. Several cellular genes associated with platelet-derived growth factor stimulation, including c-myc, were expressed at high levels in the H-ras-immortalized cells, and c-myc expression was deregulated, suggesting that the H-ras oncogene has provided a ''competence'' function. H-ras-immortalized cells could not be morphologically transformed by secondary transfection with a long terminal repeat-c-myc oncogene, but secondary transfection of the same cells with H-ras (Val-12) produced morphologically transformed colonies that had 20- to 40-fold higher levels of H-ras oncogene expression. Thus transformation in this system is dependent on high levels of H-ras oncogene expression rather than on the presence of activated H-ras and c-myc oncogenes in the same cell.

  10. Measurement of immunotargeted plasmonic nanoparticles' cellular binding: a key factor in optimizing diagnostic efficacy

    NASA Astrophysics Data System (ADS)

    Fu, Kun; Sun, Jiantang; Bickford, Lissett R.; Lin, Alex W. H.; Halas, Naomi J.; Yu, Tse-Kuan; Drezek, Rebekah A.

    2008-01-01

    In this study, we use polarized light scattering to study immunotargeted plasmonic nanoparticles which bind to live SK-BR-3 human breast carcinoma cells. Gold nanoparticles can be conjugated to various biomolecules in order to target specific molecular signatures of disease. This specific targeting provides enhanced contrast in scattering-based optical imaging techniques. While there are papers which report the number of antibodies that bind per nanoparticle, there are almost no reports of the key factor which influences diagnostic or therapeutic efficacy using nanoparticles: the number of targeted nanoparticles that bind per cell. To achieve this goal, we have developed a 'negative' method of determining the binding concentration of those antibody/nanoparticle bioconjugates which are targeted specifically to breast cancer cells. Unlike previously reported methods, we collected unbound nanoparticle bioconjugates and measured the light scattering from dilute solutions of these particles so that quantitative binding information can be obtained. By following this process, the interaction effects of adjacent bound nanoparticles on the cell membrane can be avoided simply by measuring the light scattering from the unbound nanoparticles. Specifically, using nanoshells of two different sizes, we compared the binding concentrations of anti-HER2/nanoshell and anti-IgG/nanoshell bioconjugates targeted to HER2-positive SK-BR-3 breast cancer cells. The results indicate that, for anti-HER2/nanoshell bioconjugates, there are approximately 800-1600 nanoshells bound per cell; for anti-IgG/nanoshell bioconjugates, the binding concentration is significantly lower at nearly 100 nanoshells bound per cell. These results are also supported by dark-field microscopy images of the cells labeled with anti-HER2/nanoshell and anti-IgG/nanoshell bioconjugates.

  11. Mosquito Cellular Factors and Functions in Mediating the Infectious entry of Chikungunya Virus

    PubMed Central

    Lee, Regina Ching Hua; Hapuarachchi, Hapuarachchige Chanditha; Chen, Karen Caiyun; Hussain, Khairunnisa' Mohamed; Chen, Huixin; Low, Swee Ling; Ng, Lee Ching; Lin, Raymond; Ng, Mary Mah-Lee; Chu, Justin Jang Hann

    2013-01-01

    Chikungunya virus (CHIKV) is an arthropod-borne virus responsible for recent epidemics in the Asia Pacific regions. A customized gene expression microarray of 18,760 transcripts known to target Aedes mosquito genome was used to identify host genes that are differentially regulated during the infectious entry process of CHIKV infection on C6/36 mosquito cells. Several genes such as epsin I (EPN1), epidermal growth factor receptor pathway substrate 15 (EPS15) and Huntingtin interacting protein I (HIP1) were identified to be differentially expressed during CHIKV infection and known to be involved in clathrin-mediated endocytosis (CME). Transmission electron microscopy analyses further revealed the presence of CHIKV particles within invaginations of the plasma membrane, resembling clathrin-coated pits. Characterization of vesicles involved in the endocytic trafficking processes of CHIKV revealed the translocation of the virus particles to the early endosomes and subsequently to the late endosomes and lysosomes. Treatment with receptor-mediated endocytosis inhibitor, monodansylcadaverine and clathrin-associated drug inhibitors, chlorpromazine and dynasore inhibited CHIKV entry, whereas no inhibition was observed with caveolin-related drug inhibitors. Inhibition of CHIKV entry upon treatment with low-endosomal pH inhibitors indicated that low pH is essential for viral entry processes. CHIKV entry by clathrin-mediated endocytosis was validated via overexpression of a dominant-negative mutant of Eps15, in which infectious entry was reduced, while siRNA-based knockdown of genes associated with CME, low endosomal pH and RAB trafficking proteins exhibited significant levels of CHIKV inhibition. This study revealed, for the first time, that the infectious entry of CHIKV into mosquito cells is mediated by the clathrin-dependent endocytic pathway. PMID:23409203

  12. Human coronaviruses: viral and cellular factors involved in neuroinvasiveness and neuropathogenesis.

    PubMed

    Desforges, Marc; Le Coupanec, Alain; Stodola, Jenny K; Meessen-Pinard, Mathieu; Talbot, Pierre J

    2014-12-19

    Among the various respiratory viruses infecting human beings, coronaviruses are important pathogens, which usually infect the upper respiratory tract, where they are mainly associated with common colds. However, in more vulnerable populations, such as newborns, infants, the elderly and immune-compromised individuals, these opportunistic pathogens can also affect the lower respiratory tract, leading to pneumonia, exacerbations of asthma, and various types of respiratory distress syndrome. The respiratory involvement of human coronaviruses has been clearly established since the 1960s. Nevertheless, for almost three decades now, data reported in the scientific literature has also demonstrated that, like it was described for other human viruses, coronaviruses have neuroinvasive capacities since they can spread from the respiratory tract to the central nervous system (CNS). Once there, infection of CNS cells (neurotropism) could lead to human health problems, such as encephalitis and long-term neurological diseases. Neuroinvasive coronaviruses could damage the CNS as a result of misdirected host immune responses that could be associated with autoimmunity in susceptible individuals (virus-induced neuroimmunopathology) and/or viral replication, which directly induces damage to CNS cells (virus-induced neuropathology). Given all these properties, it has been suggested that these opportunistic human respiratory pathogens could be associated with the triggering or the exacerbation of neurologic diseases for which the etiology remains poorly understood. Herein, we present host and viral factors that participate in the regulation of the possible pathogenic processes associated with CNS infection by human coronaviruses and we try to decipher the intricate interplay between virus and host target cells in order to characterize their role in the virus life cycle as well as in the capacity of the cell to respond to viral invasion.

  13. Cellular turnover and expression of hypoxic-inducible factor in acute acalculous and calculous cholecystitis

    PubMed Central

    Vakkala, Merja; Laurila, Jouko J; Saarnio, Juha; Koivukangas, Vesa; Syrjälä, Hannu; Karttunen, Tuomo; Soini, Ylermi; Ala-Kokko, Tero I

    2007-01-01

    Introduction Epithelial corrective and destructive mechanisms have not been studied in inflammatory gallbladder disease. Methods Epithelial apoptosis, cell proliferation and expression of hypoxia-inducible factor (HIF)-1α were compared in gallbladders from patients with acute acalculous cholecystitis (AAC; n = 30) and acute calculous cholecystitis (ACC; n = 21), and from patients undergoing surgery for other reasons (normal gallbladders; n = 9), which were removed during open cholecystectomy. The immunohistochemical stains included antibodies to Ki-67 (proliferation), M30 (apoptosis) and HIF-1α. Proliferation and apoptosis were expressed as percentages of positive cells. HIF-1α expression was expressed as absent, weak, or strong. Results Apoptosis (median [25th to 75th percentile]) was significantly increased in AAC (1.31% [0.75% to 1.8%], P < 0.001) and ACC (1.10% [0.63% to 1.64%], P = 0.001), compared with control samples (0.20% [0.07% to 0.45%]. The proliferation rate was significantly increased in AAC (8.0% [4.0% to 17.0%], P < 0.001) and ACC (14% [7.5% to 26.5%], P = 0.001) compared with control samples (1.0% [1.0% to 3.0%]). Strong HIF-1α staining was observed in 57% of AAC, in 100% of ACC and in 44% of control specimens (P < 0.001). Intense HIF-1α expression was associated with increased cell proliferation (P = 0.002). Conclusion Cell proliferation and apoptosis were increased in AAC and ACC, as compared with normal gallbladders. Expression of HIF-1α was lower in AAC than in ACC. PMID:17974017

  14. Structural changes and cellular localization of resuscitation-promoting factor in environmental isolates of Micrococcus luteus.

    PubMed

    Koltunov, Viktoria; Greenblatt, Charles L; Goncharenko, Anna V; Demina, Galya R; Klein, Benjamin Y; Young, Michael; Kaprelyants, Arseny S

    2010-02-01

    Dormancy among nonsporulating actinobacteria is now a widely accepted phenomenon. In Micrococcus luteus, the resuscitation of dormant cells is caused by a small secreted protein (resuscitation-promoting factor, or Rpf) that is found in "spent culture medium." Rpf is encoded by a single essential gene in M. luteus. Homologs of Rpf are widespread among the high G + C Gram-positive bacteria, including mycobacteria and streptomycetes, and most organisms make several functionally redundant proteins. M. luteus Rpf comprises a lysozyme-like domain that is necessary and sufficient for activity connected through a short linker region to a LysM motif, which is present in a number of cell-wall-associated enzymes. Muralytic activity is responsible for resuscitation. In this report, we characterized a number of environmental isolates of M. luteus, including several recovered from amber. There was substantial variation in the predicted rpf gene product. While the lysozyme-like and LysM domains showed little variation, the linker region was elongated from ten amino acid residues in the laboratory strains to as many as 120 residues in one isolate. The genes encoding these Rpf proteins have been characterized, and a possible role for the Rpf linker in environmental adaptation is proposed. The environmental isolates show enhanced resistance to lysozyme as compared with the laboratory strains and this correlates with increased peptidoglycan acetylation. In strains that make a protein with an elongated linker, Rpf was bound to the cell wall, rather than being released to the growth medium, as occurs in reference strains. This rpf gene was introduced into a lysozyme-sensitive reference strain. Both rpf genes were expressed in transformants which showed a slight but statistically significant increase in lysozyme resistance.

  15. The Modeling of Factors That Influence Coast Guard Manpower Requirements

    DTIC Science & Technology

    2014-12-01

    with accurate manpower requirements determination. There is benefit to the USCG and the Navy remaining appraised of each service’s best practices ...Regardless of similarities and differences, there is benefit to the USCG and the Navy remaining appraised of the other service’s best practices and...process to generate alternatives to support the best business case for each alternative (Commandant (CG-1B1), 2008b, p. 17). To date, the contractor

  16. Osteocytes exposed to far field of therapeutic ultrasound promotes osteogenic cellular activities in pre-osteoblasts through soluble factors.

    PubMed

    Fung, Chak-Hei; Cheung, Wing-Hoi; Pounder, Neill M; Harrison, Andrew; Leung, Kwok-Sui

    2014-07-01

    Low intensity pulsed ultrasound (LIPUS) was reported to accelerate the rate of fracture healing. When LIPUS is applied to fractures transcutaneously, bone tissues at different depths are exposed to different ultrasound fields. Measurement of LIPUS shows pressure variations in near field (nearby transducer); uniform profile was found beyond it (far field). Moreover, we have reported that the therapeutic effect of LIPUS is dependent on the axial distance of ultrasound beam in rat fracture model. However, the mechanisms of how different axial distances of LIPUS influence the mechanotransduction of bone cells are not understood. To understand the cellular mechanisms underlying far field LIPUS on enhanced fracture healing in rat model, the present study investigated the effect of ultrasound axial distances on (1) osteocyte, the mechanosensor, and (2) mechanotransduction between osteocyte and pre-osteoblast (bone-forming cell) through paracrine signaling. We hypothesized that far field LIPUS could enhance the osteogenic activities of osteoblasts via paracrine factors secreted from osteocytes. The objective of this study was to investigate the effect of axial distances of LIPUS on osteocytes and osteocyte-osteoblast mechanotransduction. In this study, LIPUS (plane; 2.2 cm in diameter, 1.5MHz sine wave, ISATA=30 mW/cm(2)) was applied to osteocytes (mechanosensor) at three axial distances: 0mm (near field), 60mm (mid-near field) and 130 mm (far field). The conditioned medium of osteocytes (OCM) collected from these three groups were used to culture pre-osteoblasts (effector cell). In this study, (1) the direct effect of ultrasound fields on the mechanosensitivity of osteocytes; and (2) the osteogenic effect of different OCM treatments on pre-osteoblasts were assessed. The immunostaining results indicated the ultrasound beam at far field resulted in more β-catenin nuclear translocation in osteocytes than all other groups. This indicated that osteocytes could detect the

  17. Development of the Cellular Immune System of Drosophila Requires the Membrane Attack Complex/Perforin-Like Protein Torso-Like.

    PubMed

    Forbes-Beadle, Lauren; Crossman, Tova; Johnson, Travis K; Burke, Richard; Warr, Coral G; Whisstock, James C

    2016-10-01

    Pore-forming members of the membrane attack complex/perforin-like (MACPF) protein superfamily perform well-characterized roles as mammalian immune effectors. For example, complement component 9 and perforin function to directly form pores in the membrane of Gram-negative pathogens or virally infected/transformed cells, respectively. In contrast, the only known MACPF protein in Drosophila melanogaster, Torso-like, plays crucial roles during development in embryo patterning and larval growth. Here, we report that in addition to these functions, Torso-like plays an important role in Drosophila immunity. However, in contrast to a hypothesized effector function in, for example, elimination of Gram-negative pathogens, we find that torso-like null mutants instead show increased susceptibility to certain Gram-positive pathogens such as Staphylococcus aureus and Enterococcus faecalis We further show that this deficit is due to a severely reduced number of circulating immune cells and, as a consequence, an impaired ability to phagocytose bacterial particles. Together these data suggest that Torso-like plays an important role in controlling the development of the Drosophila cellular immune system. Copyright © 2016 by the Genetics Society of America.

  18. A heteromeric transcription factor required for mammalian RNA polymerase II.

    PubMed Central

    Kitajima, S; Tanaka, Y; Kawaguchi, T; Nagaoka, T; Weissman, S M; Yasukochi, Y

    1990-01-01

    A general transcription factor, FC, essential for specific initiation of in vitro transcription by mammalian RNA polymerase II was identified and a procedure developed to purify it to near homogeneity from HeLa cell nuclei. Purified FC is composed of two polypeptides of apparent molecular masses 80 kDa and 30 kDa, on SDS-PAGE, and has a native size of 280 kDa estimated by gel filtration column. Both polypeptides were shown to be essential for reconstituting in vitro transcription activity. Biochemical analysis showed that the 80 kDa and 30 kDa components were present in a 1:1 molar ratio. FC was also demonstrated to interact directly or indirectly with purified RNA polymerase II. Similarities between FC and transcription factors reported by others from human, rat or Drosophila cells are discussed. Images PMID:2395645

  19. A human factors analysis of EVA time requirements

    NASA Technical Reports Server (NTRS)

    Pate, D. W.

    1996-01-01

    Human Factors Engineering (HFE), also known as Ergonomics, is a discipline whose goal is to engineer a safer, more efficient interface between humans and machines. HFE makes use of a wide range of tools and techniques to fulfill this goal. One of these tools is known as motion and time study, a technique used to develop time standards for given tasks. A human factors motion and time study was initiated with the goal of developing a database of EVA task times and a method of utilizing the database to predict how long an ExtraVehicular Activity (EVA) should take. Initial development relied on the EVA activities performed during the STS-61 mission (Hubble repair). The first step of the analysis was to become familiar with EVAs and with the previous studies and documents produced on EVAs. After reviewing these documents, an initial set of task primitives and task time modifiers was developed. Videotaped footage of STS-61 EVAs were analyzed using these primitives and task time modifiers. Data for two entire EVA missions and portions of several others, each with two EVA astronauts, was collected for analysis. Feedback from the analysis of the data will be used to further refine the primitives and task time modifiers used. Analysis of variance techniques for categorical data will be used to determine which factors may, individually or by interactions, effect the primitive times and how much of an effect they have.

  20. Cellular processing of the nerve growth factor precursor by the mammalian pro-protein convertases.

    PubMed Central

    Seidah, N G; Benjannet, S; Pareek, S; Savaria, D; Hamelin, J; Goulet, B; Laliberte, J; Lazure, C; Chrétien, M; Murphy, R A

    1996-01-01

    In order to define the enzymes responsible for the maturation of the precursor of nerve growth factor (proNGF), its biosynthesis and intracellular processing by the pro-protein convertases furin, PC1, PC2, PACE4, PC5 and the PC5 isoform PC5/6-B were analysed using the vaccinia virus expression system in cells containing a regulated and/or a constitutive secretory pathway. Results demonstrate that in both cell types furin, and to a lesser extent PACE4 and PC5/6-B, are the best candidate proNGF convertases. Furthermore, two processed NGF forms of 16.5 and 13.5 kDa were evident in constitutively secreting cell lines such as LoVo and BSC40 cells, whereas only the 13.5 kDa form was observed in AtT20 cells, which contain secretory granules. Both forms display the same N-terminal sequence as mature NGF, and were also produced following site-directed mutagenesis of the C-terminal Arg-Arg sequence of NGF into Ala-Ala, suggesting that the difference between them is not at the C-terminus. Co-expression of proNGF with furin and either chromogranin B or secretogranin II (but not chromogranin A) in BSC40 cells eliminated the 16.5 kDa form. Data also show that N-glycosylation of the pro-segment of proNGF and trimming of the oligosaccharide chains are necessary for the exit of this precursor from the endoplasmic reticulum and its eventual processing and secretion. Sulphate labelling experiments demonstrated that proNGF is processed into mature NGF following the arrival of the precursor in the trans-Golgi network. This comparative study shows that the three candidate mammalian subtilisin/kexin-like convertases identified process proNGF into NGF and that the nature of the final processed products is dependent on the intracellular environment. PMID:8615794

  1. A Human Factors Analysis of EVA Time Requirements

    NASA Technical Reports Server (NTRS)

    Pate, Dennis W.

    1997-01-01

    Human Factors Engineering (HFE) is a discipline whose goal is to engineer a safer, more efficient interface between humans and machines. HFE makes use of a wide range of tools and techniques to fulfill this goal. One of these tools is known as motion and time study, a technique used to develop time standards for given tasks. During the summer of 1995, a human factors motion and time study was initiated with the goals of developing a database of EVA task times and developing a method of utilizing the database to predict how long an EVA should take. Initial development relied on the EVA activities performed during the STS-61 (Hubble) mission. The first step of the study was to become familiar with EVA's, the previous task-time studies, and documents produced on EVA's. After reviewing these documents, an initial set of task primitives and task-time modifiers was developed. Data was collected from videotaped footage of two entire STS-61 EVA missions and portions of several others, each with two EVA astronauts. Feedback from the analysis of the data was used to further refine the primitives and modifiers used. The project was continued during the summer of 1996, during which data on human errors was also collected and analyzed. Additional data from the STS-71 mission was also collected. Analysis of variance techniques for categorical data was used to determine which factors may affect the primitive times and how much of an effect they have. Probability distributions for the various task were also generated. Further analysis of the modifiers and interactions is planned.

  2. Human factors: should your medical devices require intensive care?

    PubMed

    Hyman, William A

    2010-06-01

    The design of a medical device should include active consideration of the way in which real users will actually use the device in the real environment of use. This consideration must include making the way in which the user interfaces with the device, for example, the buttons, the displays, the connections, such that the user can complete their tasks without error and without undue burden. This consideration of realistic user needs is addressed by the discipline of human factors. Copyright 2010 Elsevier Inc. All rights reserved.

  3. Factors affecting riboflavin requirements of oral contraceptive users and nonusers.

    PubMed

    Roe, D A; Bogusz, S; Sheu, J; McCormick, D B

    1982-03-01

    Riboflavin depletion has been identified in women on oral contraceptives (OC) but change in riboflavin nutriture has not been consistently demonstrated in all OC user groups studied. Discrepant findings in reports have been attributed to differences of pill formulation or riboflavin intake. Aims of this study were to compare the riboflavin requirements of healthy OC users and nonusers on diets prepared in a metabolic unit. A single daily menu and meal pattern was used. The basic diet providing riboflavin at a level of 0.6 mg/1000 kcal was used in the period of acclimation and period 1. In periods 2 and 3, the riboflavin content of the diet was increased to 0.8 and 1.0 mg/1000 kcal, respectively. The riboflavin status of subjects was monitored by erythrocyte glutathione reductase assay and urinary riboflavin excretion. Eight women on OC and 10 nonusers participated. Erythrocyte glutathione reductase assay values and urinary riboflavin excretion showed intersubject and interperiod differences but no significant group differences (OC versus non-OC) in erythrocyte glutathione reductase values or in urinary riboflavin per g creatinine. It was concluded that when dietary intake is controlled, OC do not significantly influence riboflavin status. Riboflavin needs were related to energy requirements of the subjects.

  4. Ebola Virus Modulates Transforming Growth Factor β Signaling and Cellular Markers of Mesenchyme-Like Transition in Hepatocytes

    PubMed Central

    Wahl-Jensen, Victoria; Safronetz, David; Trost, Brett; Hoenen, Thomas; Arsenault, Ryan; Feldmann, Friederike; Traynor, Dawn; Postnikova, Elena; Kusalik, Anthony; Napper, Scott; Blaney, Joseph E.; Feldmann, Heinz; Jahrling, Peter B.

    2014-01-01

    ABSTRACT Ebola virus (EBOV) causes a severe hemorrhagic disease in humans and nonhuman primates, with a median case fatality rate of 78.4%. Although EBOV is considered a public health concern, there is a relative paucity of information regarding the modulation of the functional host response during infection. We employed temporal kinome analysis to investigate the relative early, intermediate, and late host kinome responses to EBOV infection in human hepatocytes. Pathway overrepresentation analysis and functional network analysis of kinome data revealed that transforming growth factor (TGF-β)-mediated signaling responses were temporally modulated in response to EBOV infection. Upregulation of TGF-β signaling in the kinome data sets correlated with the upregulation of TGF-β secretion from EBOV-infected cells. Kinase inhibitors targeting TGF-β signaling, or additional cell receptors and downstream signaling pathway intermediates identified from our kinome analysis, also inhibited EBOV replication. Further, the inhibition of select cell signaling intermediates identified from our kinome analysis provided partial protection in a lethal model of EBOV infection. To gain perspective on the cellular consequence of TGF-β signaling modulation during EBOV infection, we assessed cellular markers associated with upregulation of TGF-β signaling. We observed upregulation of matrix metalloproteinase 9, N-cadherin, and fibronectin expression with concomitant reductions in the expression of E-cadherin and claudin-1, responses that are standard characteristics of an epithelium-to-mesenchyme-like transition. Additionally, we identified phosphorylation events downstream of TGF-β that may contribute to this process. From these observations, we propose a model for a broader role of TGF-β-mediated signaling responses in the pathogenesis of Ebola virus disease. IMPORTANCE Ebola virus (EBOV), formerly Zaire ebolavirus, causes a severe hemorrhagic disease in humans and nonhuman

  5. Cellular dysfunction in the diabetic fibroblast: impairment in migration, vascular endothelial growth factor production, and response to hypoxia.

    PubMed

    Lerman, Oren Z; Galiano, Robert D; Armour, Mary; Levine, Jamie P; Gurtner, Geoffrey C

    2003-01-01

    Although it is known that systemic diseases such as diabetes result in impaired wound healing, the mechanism for this impairment is not understood. Because fibroblasts are essential for wound repair, we compared the in vitro behavior of fibroblasts cultured from diabetic, leptin receptor-deficient (db/db) mice with wild-type fibroblasts from mice of the same genetic background in processes important during tissue repair. Adult diabetic mouse fibroblast migration exhibited a 75% reduction in migration compared to normal fibroblasts (P < 0.001) and was not significantly stimulated by hypoxia (1% O(2)), whereas wild-type fibroblast migration was up-regulated nearly twofold in hypoxic conditions (P < 0.05). Diabetic fibroblasts produced twice the amount of pro-matrix metalloproteinase-9 as normal fibroblasts, as measured by both gelatin zymography and enzyme-linked immunosorbent assay (P < 0.05). Adult diabetic fibroblasts exhibited a sevenfold impairment in vascular endothelial growth factor (VEGF) production (4.5 +/- 1.3 pg/ml versus 34.8 +/- 3.3 pg/ml, P < 0.001) compared to wild-type fibroblasts. Moreover, wild-type fibroblast production of VEGF increased threefold in response to hypoxia, whereas diabetic fibroblast production of VEGF was not up-regulated in hypoxic conditions (P < 0.001). To address the question whether these differences resulted from chronic hyperglycemia or absence of the leptin receptor, fibroblasts were harvested from newborn db/db mice before the onset of diabetes (4 to 5 weeks old). These fibroblasts showed no impairments in VEGF production under basal or hypoxic conditions, confirming that the results from db/db fibroblasts in mature mice resulted from the diabetic state and were not because of alterations in the leptin-leptin receptor axis. Markers of cellular viability including proliferation and senescence were not significantly different between diabetic and wild-type fibroblasts. We conclude that, in vitro, diabetic fibroblasts show

  6. The pioneer factor OCT4 requires the chromatin remodeller BRG1 to support gene regulatory element function in mouse embryonic stem cells

    PubMed Central

    King, Hamish W; Klose, Robert J

    2017-01-01

    Pioneer transcription factors recognise and bind their target sequences in inaccessible chromatin to establish new transcriptional networks throughout development and cellular reprogramming. During this process, pioneer factors establish an accessible chromatin state to facilitate additional transcription factor binding, yet it remains unclear how different pioneer factors achieve this. Here, we discover that the pluripotency-associated pioneer factor OCT4 binds chromatin to shape accessibility, transcription factor co-binding, and regulatory element function in mouse embryonic stem cells. Chromatin accessibility at OCT4-bound sites requires the chromatin remodeller BRG1, which is recruited to these sites by OCT4 to support additional transcription factor binding and expression of the pluripotency-associated transcriptome. Furthermore, the requirement for BRG1 in shaping OCT4 binding reflects how these target sites are used during cellular reprogramming and early mouse development. Together this reveals a distinct requirement for a chromatin remodeller in promoting the activity of the pioneer factor OCT4 and regulating the pluripotency network. DOI: http://dx.doi.org/10.7554/eLife.22631.001 PMID:28287392

  7. Transcription factor motif quality assessment requires systematic comparative analysis

    PubMed Central

    Kibet, Caleb Kipkurui; Machanick, Philip

    2016-01-01

    Transcription factor (TF) binding site prediction remains a challenge in gene regulatory research due to degeneracy and potential variability in binding sites in the genome. Dozens of algorithms designed to learn binding models (motifs) have generated many motifs available in research papers with a subset making it to databases like JASPAR, UniPROBE and Transfac. The presence of many versions of motifs from the various databases for a single TF and the lack of a standardized assessment technique makes it difficult for biologists to make an appropriate choice of binding model and for algorithm developers to benchmark, test and improve on their models. In this study, we review and evaluate the approaches in use, highlight differences and demonstrate the difficulty of defining a standardized motif assessment approach. We review scoring functions, motif length, test data and the type of performance metrics used in prior studies as some of the factors that influence the outcome of a motif assessment. We show that the scoring functions and statistics used in motif assessment influence ranking of motifs in a TF-specific manner. We also show that TF binding specificity can vary by source of genomic binding data. We also demonstrate that information content of a motif is not in isolation a measure of motif quality but is influenced by TF binding behaviour. We conclude that there is a need for an easy-to-use tool that presents all available evidence for a comparative analysis. PMID:27092243

  8. Identification of multiple cellular uptake pathways of polystyrene nanoparticles and factors affecting the uptake: relevance for drug delivery systems.

    PubMed

    Firdessa, Rebuma; Oelschlaeger, Tobias A; Moll, Heidrun

    2014-01-01

    Nanoparticles may address challenges by human diseases through improving diagnosis, vaccination and treatment. The uptake mechanism regulates the type of threat a particle poses on the host cells and how a cell responds to it. Hence, understanding the uptake mechanisms and cellular interactions of nanoparticles at the cellular and subcellular level is a prerequisite for their effective biomedical applications. The present study shows the uptake mechanisms of polystyrene nanoparticles and factors affecting their uptake in bone marrow-derived macrophages, 293T kidney epithelial cells and L929 fibroblasts. Labeling with the endocytic marker FM4-64 and transmission electron microscopy studies show that the nanoparticles were internalized rapidly via endocytosis and accumulated in intracellular vesicles. Soon after their internalizations, nanoparticles trafficked to organelles with acidic pH. Analysis of the ultrastructural morphology of the plasma membrane invaginations or extravasations provides clear evidence for the involvement of several uptake routes in parallel to internalize a given type of nanoparticles by mammalian cells, highlighting the complexity of the nanoparticle-cell interactions. Blocking the specific endocytic pathways by different pharmacological inhibitors shows similar outcomes. The potential to take up nanoparticles varies highly among different cell types in a particle sizes-, time- and energy-dependent manner. Furthermore, infection and the activation status of bone marrow-derived macrophages significantly affect the uptake potential of the cells, indicating the need to understand the diseases' pathogenesis to establish effective and rational drug-delivery systems. This study enhances our understanding of the application of nanotechnology in biomedical sciences.

  9. The HTLV-1 HBZ protein inhibits cyclin D1 expression through interacting with the cellular transcription factor CREB.

    PubMed

    Ma, Yunyun; Zheng, Shangen; Wang, Yuanyuan; Zang, Wenqiao; Li, Min; Wang, Na; Li, Ping; Jin, Jing; Dong, Ziming; Zhao, Guoqiang

    2013-10-01

    Human T cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that can cause adult T-cell leukemia (ATL) and other diseases. The HTLV-1 bZIP factor (HBZ), which is encoded by an mRNA of the opposite polarity of the viral genomic RNA, interacts with several transcription factors and is involved in T cell proliferation, viral gene transcription and cellular transformation. Cyclin D1 is a pivotal regulatory protein involved in cell cycle progression, and its depressed expression correlates with cell cycle prolongation or arrested at the G1/S transition. In our present study, we observed that HBZ expression suppressed cyclin D1 level. To investigate the role of HBZ on cyclin D1 depression, we transduced HBZ with lentivirus vector into 293T cells, CEM cells and Jurkat cells. The results of Western blot, RT-PCR and luciferase assays showed that transcriptional activity of the cyclin D1 promoter was suppressed by the bZIP domain of HBZ (HBZ-bZIP) through cyclic AMP response element (CRE) site. Immunoprecipitation and GST pull-down assays showed the binding of HBZ-bZIP to CRE-binding protein (CREB), which confirmed that the cyclin D1 promoter activity inhibition via the CRE-site was mediated by HBZ-bZIP. The results suggested that HBZ suppressed cyclin D1 transcription through interactions with CREB and along with other viral protein, HBZ may play a causal role for leukemogenesis.

  10. Purification and characterization of FBI-1, a cellular factor that binds to the human immunodeficiency virus type 1 inducer of short transcripts.

    PubMed

    Pessler, F; Pendergrast, P S; Hernandez, N

    1997-07-01

    The human immunodeficiency virus (HIV-1) promoter directs the synthesis of two classes of RNA molecules, short transcripts and full-length transcripts. The synthesis of short transcripts depends on a bipartite DNA element, the inducer of short transcripts (IST), located in large part downstream of the HIV-1 start site of transcription. IST does not require any viral product for function and is thought to direct the assembly of transcription complexes that are incapable of efficient elongation. Nothing is known, however, about the biochemical mechanisms that mediate IST function. Here, we report the identification and purification of a factor that binds specifically to the IST. This factor, FBI-1, recognizes a large bipartite binding site that coincides with the bipartite IST element. It is constituted at least in part by an 86-kDa polypeptide that can be specifically cross-linked to IST. FBI-1 also binds to promoter and attenuation regions of a number of cellular and viral transcription units that are regulated by a transcription elongation block. This observation, together with the observation that the binding of FBI-1 to IST mutants correlates with the ability of these mutants to direct IST function, suggests that FBI-1 may be involved in the establishment of abortive transcription complexes.

  11. The infectivity of prM-containing partially mature West Nile virus does not require the activity of cellular furin-like proteases.

    PubMed

    Mukherjee, Swati; Lin, Tsai-Yu; Dowd, Kimberly A; Manhart, Carolyn J; Pierson, Theodore C

    2011-11-01

    Cleavage of the flavivirus prM protein by a cellular furin-like protease is a hallmark of virion maturation. While this cleavage is a required step in the viral life cycle, it can be inefficient. Virions that retain uncleaved prM may be infectious. We investigated whether cleavage by furin of prM on partially mature West Nile virus (WNV) during virus entry contributes to infectivity. Using quantitative assays of WNV infection, we found that virions incorporating considerable amounts of uncleaved prM protein were insensitive to treatment of cells with a potent inhibitor of furin activity. Thus, partially mature WNV does not require furin-like proteases for infectivity.

  12. Biotinidase deficiency: factors responsible for the increased biotin requirement.

    PubMed

    Baumgartner, E R; Suormala, T; Wick, H; Bausch, J; Bonjour, J P

    1985-01-01

    Inability to recycle biotin from endogenous biocytin in congenital biotinidase deficiency is associated with increased requirement of exogenous free biotin. We have observed that severe biotin depletion with clinical and biochemical consequences occurs within 12 days after birth in a newborn patient and within 15-20 days after withdrawal of biotin supplementation in four other patients. Our studies have shown that: Urinary loss of biotin and biocytin are major causes for this rapid biotin depletion. Intestinal absorption of biotin seems to be normal at least at the loading dose of 1.5 micrograms/kg. At normal or subnormal plasma biotin concentrations biocytin is found in low concentrations (below 1 nmol l-1) in plasma of patients but at much higher concentrations in urine (100-600 nmol l-1). An oral load of biocytin results in patients in unchanged biotin levels but in a marked rise of biocytin in plasma followed by rapid renal excretion of biocytin whereas in controls biotin levels in plasma increase rapidly and biocytin remains below detection levels.

  13. Stereospecific Inhibitory Effects of CCG-1423 on the Cellular Events Mediated by Myocardin-Related Transcription Factor A

    PubMed Central

    Watanabe, Bunta; Minami, Saki; Ishida, Hideaki; Yoshioka, Ryuzo; Nakagawa, Yoshiaki; Morita, Tsuyoshi; Hayashi, Ken’ichiro

    2015-01-01

    CCG-1423 suppresses several pathological processes including cancer cell migration, tissue fibrosis, and the development of atherosclerotic lesions. These suppressions are caused by inhibition of myocardin-related transcription factor A (MRTF-A), which is a critical factor for epithelial–mesenchymal transition (EMT). CCG-1423 can therefore be a potent inhibitor for EMT. CCG-1423 and related compounds, CCG-100602 and CCG-203971 possess similar biological activities. Although these compounds are comprised of two stereoisomers, the differences in their biological activities remain to be assessed. To address this issue, we stereoselectively synthesized optically pure isomers of these compounds and validated their biological activities. The S-isomer of CCG-1423 rather than the R-isomer exhibited modestly but significantly higher inhibitory effects on the cellular events triggered by MRTF-A activation including serum response factor-mediated gene expression and cell migration of fibroblasts and B16F10 melanoma cells. Accordingly, the S-isomer of CCG-1423 more potently blocked the serum-induced nuclear import of MRTF-A than the R-isomer. No such difference was observed in cells treated with each of two stereoisomers of CCG-100602 or CCG-203971. We previously reported that the N-terminal basic domain (NB), which functions as a nuclear localization signal of MRTF-A, is a binding site for CCG-1423. Consistent with the biological activities of two stereoisomers of CCG-1423, docking simulation demonstrated that the S-isomer of CCG-1423 was more likely to bind to NB than the R-isomer. This is a first report demonstrating the stereospecific biological activities of CCG-1423. PMID:26295164

  14. The BAR domain of amphiphysin is required for cleavage furrow tip–tubule formation during cellularization in Drosophila embryos

    PubMed Central

    Su, Jing; Chow, Brenda; Boulianne, Gabrielle L.; Wilde, Andrew

    2013-01-01

    De novo formation of cells in the Drosophila embryo is achieved when each nucleus is surrounded by a furrow of plasma membrane. Remodeling of the plasma membrane during cleavage furrow ingression involves the exocytic and endocytic pathways, including endocytic tubules that form at cleavage furrow tips (CFT-tubules). The tubules are marked by amphiphysin but are otherwise poorly understood. Here we identify the septin family of GTPases as new tubule markers. Septins do not decorate CFT-tubules homogeneously: instead, novel septin complexes decorate different CFT-tubules or different domains of the same CFT-tubule. Using these new tubule markers, we determine that all CFT-tubule formation requires the BAR domain of amphiphysin. In contrast, dynamin activity is preferentially required for the formation of the subset of CFT-tubules containing the septin Peanut. The absence of tubules in amphiphysin-null embryos correlates with faster cleavage furrow ingression rates. In contrast, upon inhibition of dynamin, longer tubules formed, which correlated with slower cleavage furrow ingression rates. These data suggest that regulating the recycling of membrane within the embryo is important in supporting timely furrow ingression. PMID:23447705

  15. Specific requirement of the chromatin modifier mSin3B in cell cycle exit and cellular differentiation.

    PubMed

    David, Gregory; Grandinetti, Kathryn B; Finnerty, Patricia M; Simpson, Natalie; Chu, Gerald C; Depinho, Ronald A

    2008-03-18

    The Sin3-histone deacetylase (HDAC) corepressor complex is conserved from yeast to humans. Mammals possess two highly related Sin3 proteins, mSin3A and mSin3B, which serve as scaffolds tethering HDAC enzymatic activity, and numerous sequence-specific transcription factors to enable local chromatin regulation at specific gene targets. Despite broad overlapping expression of mSin3A and mSin3B, mSin3A is cell-essential and vital for early embryonic development. Here, genetic disruption of mSin3B reveals a very different phenotype characterized by the survival of cultured cells and lethality at late stages of embryonic development with defective differentiation of multiple lineages-phenotypes that are strikingly reminiscent of those associated with loss of retinoblastoma family members or E2F transcriptional repressors. Additionally, we observe that, whereas mSin3B(-/-) cells cycle normally under standard growth conditions, they show an impaired ability to exit the cell cycle with limiting growth factors. Correspondingly, mSin3B interacts physically with the promoters of known E2F target genes, and its deficiency is associated with derepression of these gene targets in vivo. Together, these results reveal a critical role for mSin3B in the control of cell cycle exit and terminal differentiation in mammals and establish contrasting roles for the mSin3 proteins in the growth and development of specific lineages.

  16. The potential of robotic technology applied to meet requirements for tools to support microsurgery and cellular surgery.

    PubMed

    Brett, P N; Ma, X; Tritto, G

    2004-05-01

    Microsurgery and processes involving cell manipulation or cell surgery are clinical practices where the operator works at or beyond the threshold of human dexterity. Current tools available are conventional in their design, and this limits consistency and the level of reliability and achievement. Surgical robotic devices have been explored to improve precision in minimal access surgical procedures to augment control of tool points in tissues, and have enabled feedback of sensory data from which the operator is able to deduce information on the tool at the working site. In this paper, relevant technologies are described that can be harnessed to improve perception of tool point interaction with tissues at the working site and to improve tool control at the small scale required in clinical practice for microsurgery and for cell surgery.

  17. Cellular VPS4 Is Required for Efficient Entry and Egress of Budded Virions of Autographa californica Multiple Nucleopolyhedrovirus

    PubMed Central

    Li, Zhaofei

    2012-01-01

    Membrane budding is essential for the egress of many enveloped viruses, and this process shares similarities with the biogenesis of multivesicular bodies (MVBs). In eukaryotic cells, the budding of intraluminal vesicles (IVLs) is mediated by the endosomal sorting complex required for transport (ESCRT) machinery and some viruses require ESCRT machinery components or functions to bud from host cells. Baculoviruses, such as Autographa californica multiple nucleopolyhedrovirus (AcMNPV), enter host cells by clathrin-mediated endocytosis. Viral DNA replication and nucleocapsid assembly occur within the nucleus. Some progeny nucleocapsids are subsequently trafficked to, and bud from, the plasma membrane, forming budded virions (BV). To determine whether the host ESCRT machinery is important or necessary for AcMNPV replication, we cloned a cDNA of Spodoptera frugiperda VPS4, a key regulator for disassembly and recycling of ESCRT III. We then examined viral infection and budding in the presence of wild-type (WT) or dominant negative (DN) forms of VPS4. First, we used a viral complementation system, in combination with fluorescent tags, to examine the effects of transiently expressed WT or DN VPS4 on viral entry. We found that dominant negative VPS4 substantially inhibited virus entry. Entering virus was observed within aberrant compartments containing the DN VPS4 protein. We next used recombinant bacmids expressing WT or DN VPS4 proteins to examine virus egress. We found that production of infectious AcMNPV BV was substantially reduced by expression of DN VPS4 but not by WT VPS4. Together, these results indicate that a functional VPS4 is necessary for efficient AcMNPV BV entry into, and egress from, insect cells. PMID:22072775

  18. Ume6 Is Required for the MATa/MATα Cellular Identity and Transcriptional Silencing in Kluyveromyces lactis

    PubMed Central

    Barsoum, E.; Sjöstrand, J. O. O.; Åström, S. U.

    2010-01-01

    To explore the similarities and differences of regulatory circuits among budding yeasts, we characterized the role of the unscheduled meiotic gene expression 6 (UME6) gene in Kluyveromyces lactis. We found that Ume6 was required for transcriptional silencing of the cryptic mating-type loci HMLα and HMRa. Chromatin immunoprecipitation (ChIP) suggested that Ume6 acted directly by binding the cis-regulatory silencers of these loci. Unexpectedly, a MATa ume6 strain was mating proficient, whereas a MATα ume6 strain was sterile. This observation was explained by the fact that ume6 derepressed HMLα2 only weakly, but derepressed HMRa1 strongly. Consistently, two a/α-repressed genes (MTS1 and STE4) were repressed in the MATα ume6 strain, but were expressed in the MATa ume6 strain. Surprisingly, ume6 partially suppressed the mating defect of a MATa sir2 strain. MTS1 and STE4 were repressed in the MATa sir2 ume6 double-mutant strain, indicating that the suppression acted downstream of the a1/α2-repressor. We show that both STE12 and the MATa2/HMRa2 genes were overexpressed in the MATa sir2 ume6 strain. Consistent with the idea that this deregulation suppressed the mating defect, ectopic overexpression of Ste12 and a2 in a MATa sir2 strain resulted in efficient mating. In addition, Ume6 served as a block to polyploidy, since ume6/ume6 diploids mated as pseudo a-strains. Finally, Ume6 was required for repression of three meiotic genes, independently of the Rpd3 and Sin3 corepressors. PMID:20139343

  19. Cellular entry via an actin and clathrin-dependent route is required for Lv2 restriction of HIV-2

    SciTech Connect

    Harrison, I.P.; McKnight, A.

    2011-06-20

    Lv2 is a human factor that restricts infection of some HIV-2 viruses after entry into particular target cells. HIV-2 MCR is highly susceptible to Lv2 whereas HIV-2 MCN is not. The block is after reverse transcription but prior to nuclear entry. The viral determinants for this restriction have been mapped to the HIV-2 envelope and the capsid genes. Our model of Lv2 restriction suggests that the route taken into a cell is important in determining whether a productive infection occurs. Here we characterised the infectious routes used by MCN and MCR using chemical compounds and molecular techniques to distinguish between potential pathways. Our results suggest that susceptible MCR can enter restrictive HeLa{sup CD4} cells via two pathways; a clathrin/AP2 mediated endocytic route that is sensitive to Lv2 restriction and an alternative, non-clathrin mediated route, which results in more efficient infection.

  20. Endobronchial allergen challenge in asthma. Demonstration of cellular source of granulocyte macrophage colony-stimulating factor by in situ hybridization.

    PubMed Central

    Broide, D H; Firestein, G S

    1991-01-01

    Airway inflammation is thought to play an important role in the pathogenesis of asthma. We have used in situ hybridization and an immunoassay to determine whether granulocyte macrophage colony-stimulating factor (GM-CSF) (a cytokine capable of eosinophil activation) is present in the airway of asthmatics (n = 6) who have 37.0 +/- 15.1% airway eosinophilia after endobronchial allergen challenge. Levels of immunoreactive GM-CSF (less than 4 pg/ml pre-allergen versus 180.5 +/- 46.9 pg/ml post-allergen) increased significantly 24 h after endobronchial allergen stimulation. The cellular source of bronchoalveolar lavage (BAL) GM-CSF, as determined by in situ hybridization and immunoperoxidase staining, was derived predominantly from UCHL-1 positive BAL lymphocytes, as well as from a smaller population of alveolar macrophages. Before local endobronchial allergen challenge, less than 1% of lymphocytes and alveolar macrophages recovered by BAL expressed GM-CSF mRNA, whereas after allergen stimulation 92.6 +/- 3.4% of lymphocytes and 17.5 +/- 22.7% of alveolar macrophages expressed GM-CSF mRNA. This study provides evidence that in an experimental model of allergen-induced asthma, activation of the immune and inflammatory response (BAL lymphocyte and alveolar macrophage production of GM-CSF) is temporally associated with an inflammatory cell influx of eosinophils into the airway. Images PMID:1885766

  1. [Anti-HIV effects of IFN-tau in human macrophages: role of cellular antiviral factors and interleukin-6].

    PubMed

    Maneglier, B; Rogez-Kreuz, C; Dereuddre-Bosquet, N; Martal, J; Devillier, P; Dormont, D; Clayette, P

    2008-01-01

    Tau interferon (IFN-tau) was shown to inhibit human immunodeficiency virus (HIV) replication in vitro more strongly than human IFN-alpha, particularly in human macrophages. IFN-tau efficiently inhibited the early steps of HIV biological cycle, decreasing intracellular HIV RNA and inhibiting the initiation of the reverse transcription of viral RNA into proviral DNA. In this study, the in vitro immunomodulatory effects of IFN-tau were explored in human macrophages. We found that IFN-tau increased the synthesis of the cellular antiviral factors, such as 2',5'-oligoadenylate synthetase/RNase L and MxA protein. These results suggested that IFN-tau induces the same antiviral pathways in macrophages as other type I IFNs. We found that IFN-tau increased the production of interleukins (IL)-10 and IL-6, but not of IL-1ss or TNF-alpha, in not infected and in in vitro HIV-1/Ba-L-infected macrophages. We also found that the neutralization of IL-6 biological activity in the cell culture supernatants of IFN-tau-treated macrophages led to a decrease in the antiretroviral effects of IFN-tau towards HIV RNA. In conclusion, anti-HIV effects of IFN-tau are mediated by several modes of action, mediated either directly by IFN-tau or via other cytokines, such as IL-6, also known to be induced by IFN-alpha.

  2. SAC1p is an integral membrane protein that influences the cellular requirement for phospholipid transfer protein function and inositol in yeast

    PubMed Central

    1993-01-01

    Mutations in the SAC1 gene exhibit allele-specific genetic interactions with yeast actin structural gene defects and effect a bypass of the cellular requirement for the yeast phosphatidylinositol/phosphatidylcholine transfer protein (SEC14p), a protein whose function is essential for sustained Golgi secretory function. We report that SAC1p is an integral membrane protein that localizes to the yeast Golgi complex and to the yeast ER, but does not exhibit a detectable association with the bulk of the yeast F-actin cytoskeleton. The data also indicate that the profound in vivo effects on Golgi secretory function and the organization of the actin cytoskeleton observed in sac1 mutants result from loss of SAC1p function. This cosuppression of actin and SEC14p defects is a unique feature of sac1 alleles as mutations in other SAC genes that result in a suppression of actin defects do not result in phenotypic suppression of SEC14p defects. Finally, we report that sac1 mutants also exhibit a specific inositol auxotrophy that is not exhibited by the other sac mutant strains. This sac1-associated inositol auxotrophy is not manifested by measurable defects in de novo inositol biosynthesis, nor is it the result of some obvious defect in the ability of sac1 mutants to utilize inositol for phosphatidylinositol biosynthesis. Thus, sac1 mutants represent a novel class of inositol auxotroph in that these mutants appear to require elevated levels of inositol for growth. On the basis of the collective data, we suggest that SAC1p dysfunction exerts its pleiotropic effects on yeast Golgi function, the organization of the actin cytoskeleton, and the cellular requirement for inositol, through altered metabolism of inositol glycerophospholipids. PMID:8314848

  3. Making Bunyaviruses Talk: Interrogation Tactics to Identify Host Factors Required for Infection

    PubMed Central

    Riblett, Amber M.; Doms, Robert W.

    2016-01-01

    The identification of host cellular genes that act as either proviral or antiviral factors has been aided by the development of an increasingly large number of high-throughput screening approaches. Here, we review recent advances in which these new technologies have been used to interrogate host genes for the ability to impact bunyavirus infection, both in terms of technical advances as well as a summary of biological insights gained from these studies. PMID:27187446

  4. Glucose-dependent anaplerosis in cancer cells is required for cellular redox balance in the absence of glutamine

    PubMed Central

    Cetinbas, Naniye Mallı; Sudderth, Jessica; Harris, Robert C.; Cebeci, Aysun; Negri, Gian L.; Yılmaz, Ömer H.; DeBerardinis, Ralph J.; Sorensen, Poul H.

    2016-01-01

    Cancer cells have altered metabolism compared to normal cells, including dependence on glutamine (GLN) for survival, known as GLN addiction. However, some cancer cell lines do not require GLN for survival and the basis for this discrepancy is not well understood. GLN is a precursor for antioxidants such as glutathione (GSH) and NADPH, and GLN deprivation is therefore predicted to deplete antioxidants and increase reactive oxygen species (ROS). Using diverse human cancer cell lines we show that this occurs only in cells that rely on GLN for survival. Thus, the preference for GLN as a dominant antioxidant source defines GLN addiction. We show that despite increased glucose uptake, GLN addicted cells do not metabolize glucose via the TCA cycle when GLN is depleted, as revealed by 13C-glucose labeling. In contrast, GLN independent cells can compensate by diverting glucose-derived pyruvate into the TCA cycle. GLN addicted cells exhibit reduced PDH activity, increased PDK1 expression, and PDK inhibition partially rescues GLN starvation-induced ROS and cell death. Finally, we show that combining GLN starvation with pro-oxidants selectively kills GLN addicted cells. These data highlight a major role for GLN in maintaining redox balance in cancer cells that lack glucose-dependent anaplerosis. PMID:27605385

  5. HTLV-1 Rex is required for viral spread and persistence in vivo but is dispensable for cellular immortalization in vitro.

    PubMed

    Ye, Jianxin; Silverman, Lee; Lairmore, Michael D; Green, Patrick L

    2003-12-01

    Human T-cell leukemia virus type 1 (HTLV-1) is associated with leukemia/lymphoma and neurologic disorders. Although the viral transcriptional activator Tax is the critical viral oncoprotein, Rex, which regulates the expression of the viral structural and enzymatic genes, is essential for efficient viral replication. Herein, we investigate the contribution of Rex in HTLV-1 immortalization of primary T cells in vitro and viral survival in an infectious rabbit animal model. A Rex-deficient HTLV-1 (HTLVRex-) was constructed and characterized for viral gene expression, protein production, and immortalization capacity. Cells transiently transfected with the HTLVRex- proviral clone produced low detectable levels of p19 Gag. 729HTLVRex- stable transfectants produced functional Tax, but undetectable levels of Rex or p19 Gag. Coculture of irradiated 729HTLVRex- cells with peripheral blood mononuclear cells (PBMCs) resulted in sustained interleukin-2 (IL-2)-dependent growth of primary T lymphocytes. These cells carried the HTLVRex- genome and expressed tax/rex mRNA but produced no detectable Rex or p19 Gag. Rabbits inoculated with irradiated 729HTLVRex- cells or 729HTLVRex- cells transiently transfected with a Rex cDNA expression plasmid failed to become persistently infected or mount a detectable antibody response to the viral gene products. Together, our results provide the first direct evidence that Rex and its function to modulate viral gene expression and virion production is not required for in vitro immortalization by HTLV-1. However, Rex is critical for efficient infection of cells and persistence in vivo.

  6. N-myc downstream regulated 1 (NDRG1) is regulated by eukaryotic initiation factor 3a (eIF3a) during cellular stress caused by iron depletion.

    PubMed

    Lane, Darius J R; Saletta, Federica; Suryo Rahmanto, Yohan; Kovacevic, Zaklina; Richardson, Des R

    2013-01-01

    Iron is critical for cellular proliferation and its depletion leads to a suppression of both DNA synthesis and global translation. These observations suggest that iron depletion may trigger a cellular "stress response". A canonical response of cells to stress is the formation of stress granules, which are dynamic cytoplasmic aggregates containing stalled pre-initiation complexes that function as mRNA triage centers. By differentially prioritizing mRNA translation, stress granules allow for the continued and selective translation of stress response proteins. Although the multi-subunit eukaryotic initiation factor 3 (eIF3) is required for translation initiation, its largest subunit, eIF3a, may not be essential for this activity. Instead, eIF3a is a vital constituent of stress granules and appears to act, in part, by differentially regulating specific mRNAs during iron depletion. Considering this, we investigated eIF3a's role in modulating iron-regulated genes/proteins that are critically involved in proliferation and metastasis. In this study, eIF3a was down-regulated and recruited into stress granules by iron depletion as well as by the classical stress-inducers, hypoxia and tunicamycin. Iron depletion also increased expression of the metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), and a known downstream repressed target of eIF3a, namely the cyclin-dependent kinase inhibitor, p27(kip1). To determine if eIF3a regulates NDRG1 expression, eIF3a was inducibly over-expressed or ablated. Importantly, eIF3a positively regulated NDRG1 expression and negatively regulated p27(kip1) expression during iron depletion. This activity of eIF3a could be due to its recruitment to stress granules and/or its ability to differentially regulate mRNA translation during cellular stress. Additionally, eIF3a positively regulated proliferation, but negatively regulated cell motility and invasion, which may be due to the eIF3a-dependent changes in expression of NDRG1 and p27

  7. An Algorithm Measuring Donor Cell-Free DNA in Plasma of Cellular and Solid Organ Transplant Recipients That Does Not Require Donor or Recipient Genotyping

    PubMed Central

    Gordon, Paul M. K.; Khan, Aneal; Sajid, Umair; Chang, Nicholas; Suresh, Varun; Dimnik, Leo; Lamont, Ryan E.; Parboosingh, Jillian S.; Martin, Steven R.; Pon, Richard T.; Weatherhead, Jene; Wegener, Shelly; Isaac, Debra; Greenway, Steven C.

    2016-01-01

    Cell-free DNA (cfDNA) has significant potential in the diagnosis and monitoring of clinical conditions. However, accurately and easily distinguishing the relative proportion of DNA molecules in a mixture derived from two different sources (i.e., donor and recipient tissues after transplantation) is challenging. In human cellular transplantation, there is currently no useable method to detect in vivo engraftment, and blood-based non-invasive tests for allograft rejection in solid organ transplantation are either non-specific or absent. Elevated levels of donor cfDNA have been shown to correlate with solid organ rejection, but complex methodology limits implementation of this promising biomarker. We describe a cost-effective method to quantify donor cfDNA in recipient plasma using a panel of high-frequency single nucleotide polymorphisms, next-generation (semiconductor) sequencing, and a novel mixture model algorithm. In vitro, our method accurately and rapidly determined donor:recipient DNA admixture. For in vivo testing, donor cfDNA was serially quantified in an infant with a urea cycle disorder after receiving six daily infusions of donor liver cells. Donor cfDNA isolated from 1 to 2 ml of recipient plasma was detected as late as 24 weeks after infusion suggesting engraftment. The percentage of circulating donor cfDNA was also assessed in pediatric and adult heart transplant recipients undergoing routine endomyocardial biopsy with levels observed to be stable over time and generally measuring <1% in cases without moderate or severe cellular rejection. Unlike existing non-invasive methods used to define the proportion of donor cfDNA in solid organ transplant patients, our assay does not require sex mismatch, donor genotyping, or whole-genome sequencing and potentially has broad application to detect cellular engraftment or allograft injury after transplantation. PMID:27713880

  8. NK cells control HIV-1 infection of macrophages through soluble factors and cellular contacts in the human decidua.

    PubMed

    Quillay, H; El Costa, H; Duriez, M; Marlin, R; Cannou, C; Madec, Y; de Truchis, C; Rahmati, M; Barré-Sinoussi, F; Nugeyre, M T; Menu, E

    2016-06-06

    During the first trimester of pregnancy, HIV-1 in utero transmission is rare despite the permissivity of the placenta and the decidua (the uterine mucosa during pregnancy) to infection. In the decidua from the first trimester of pregnancy, macrophages (dMs) are the HIV-1 main target cells. Decidual natural killer (dNK) cells account for 70 % of decidual leukocytes. They display distinct phenotype and functions compared to peripheral NK cells. At the periphery, NK cells are involved in the control of HIV-1 infection. In this study, we investigate whether human decidual natural killer (dNK) cells control dM HIV-1 infection. Autologous cocultures of infected dMs with dNK cells reveal that dNK cells strongly inhibit dM HIV-1 infection. The addition of dNK cells to dMs at different times after infection suggests that the control occurs before the complete establishment of the infection. Double chamber cocultures show that cellular contacts are necessary for an optimal control of infection. Nevertheless, soluble factors secreted by dMs and dNK cells in double chamber cocultures partially inhibit dM HIV-1 infection, indicating that soluble factors have also a role in the control of infection. IFN-γ secretion is increased in infected and uninfected cocultures. We show that IFN-γ is involved in the control of dM HIV-1 infection by dNK cells. These results demonstrate that human dNK cells inhibit efficiently HIV-1 infection in dMs in vitro, and highlight the role of innate immune determinants in the control of HIV-1 transmission.

  9. The heat-shock factor is not activated in mammalian cells exposed to cellular phone frequency microwaves.

    PubMed

    Laszlo, Andrei; Moros, Eduardo G; Davidson, Teri; Bradbury, Matt; Straube, William; Roti Roti, Joseph

    2005-08-01

    There has been considerable interest in the biological effects of exposure to radiofrequency electromagnetic radiation, given the explosive growth of cellular telephone use, with the possible induction of malignancy being a significant concern. Thus the determination of whether nonthermal effects of radiofrequency electromagnetic radiation contribute to the process leading to malignancy is an important task. One proposed pathway to malignancy involves the induction of the stress response by exposures to cell phone frequency microwaves. The first step in the induction of the stress response is the activation of the DNA-binding activity of the specific transcription factor involved in this response, the heat-shock factor (HSF). The DNA-binding activity of HSF in hamster, mouse and human cells was determined after acute and continuous exposures to frequency domain multiple access (FDMA)- or code domain multiple access (CDMA)-modulated microwaves at low (0.6 W/kg) or high (approximately 5 W/kg) SARs at frequencies used for mobile communication. The DNA-binding activity of HSF was monitored using a gel shift assay; the calibration of this assay indicated that an increase of approximately 10% in the activation of the DNA-binding activity of HSF after a 1 degrees C increase in temperature could be detected. We failed to detect any increase in the DNA-binding ability of HSF in cultured mammalian cells as a consequence of any exposure tested, within the sensitivity of our assay. Our results do not support the notion that the stress response is activated as a consequence of exposure to microwaves of frequencies associated with mobile communication devices.

  10. Cellular zinc is required for intestinal epithelial barrier maintenance via the regulation of claudin-3 and occludin expression.

    PubMed

    Miyoshi, Yuka; Tanabe, Soichi; Suzuki, Takuya

    2016-07-01

    Intracellular zinc is required for a variety of cell functions, but its precise roles in the maintenance of the intestinal tight junction (TJ) barrier remain unclear. The present study investigated the essential roles of intracellular zinc in the preservation of intestinal TJ integrity and the underlying molecular mechanisms. Depletion of intracellular zinc in both intestinal Caco-2 cells and mouse colons through the application of a cell-permeable zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) induced a disruption of the TJ barrier, as indicated by increased FITC-labeled dextran flux and decreased transepithelial electrical resistance. The TPEN-induced TJ disruption is associated with downregulation of two TJ proteins, occludin and claudin-3. Biotinylation of cell surface proteins revealed that the zinc depletion induced the proteolysis of occludin but not claudin-3. Occludin proteolysis was sensitive to the inhibition of calpain activity, and increased calpain activity was observed in the zinc-depleted cells. Although quantitative PCR analysis and promoter reporter assay have demonstrated that the zinc depletion-induced claudin-3 downregulation occurred at transcriptional levels, a site-directed mutation in the egr1 binding site in the claudin-3 promoter sequence induced loss of both the basal promoter activity and the TPEN-induced decreases. Reduced egr1 expression by a specific siRNA also inhibited claudin-3 expression and transepithelial electrical resistance maintenance in cells. This study shows that intracellular zinc has an essential role in the maintenance of the intestinal epithelial TJ barrier through regulation of occludin proteolysis and claudin-3 transcription. Copyright © 2016 the American Physiological Society.

  11. The Src homology 3 binding domain is required for lysophosphatidic acid 3 receptor-mediated cellular viability in melanoma cells.

    PubMed

    Jia, Wei; Tran, Sterling K; Ruddick, Caitlin A; Murph, Mandi M

    2015-01-28

    The LPA3 receptor is a G protein-coupled receptor that binds extracellular lysophosphatidic acid and mediates intracellular signaling cascades. Although we previously reported that receptor inhibition using siRNA or chemical inhibition obliterates the viability of melanoma cells, the mechanism was unclear. Herein we hypothesized that amino acids comprising the Src homology 3 (SH3) ligand binding motif, R/K-X-X-V/P-X-X-P or (216)-KTNVLSP-(222), within the third intracellular loop of LPA3 were critical in mediating this outcome. Therefore, we performed site-directed mutagenesis of the lysine, valine and proline, replacing these amino acids with alanines, and evaluated the changes in viability, proliferation, ERK1/2 signaling and calcium in response to lysophosphatidic acid. Our results show that enforced LPA3 expression in SK-MEL-2 cells enhanced their resiliency by allowing these cells to oppose any loss of viability during growth in serum-free medium for up to 96 h, in contrast to parental SK-MEL-2 cells, which show a significant decline in viability. Similarly, site-directed alanine substitutions of valine and proline, V219A/P222A or 2aa-SK-MEL-2 cells, did not significantly alter viability, but adding a further alanine to replace the lysine, K216A/V219A/P222A or 3aa-SK-MEL-2 cells, obliterated this function. In addition, an inhibitor of the LPA3 receptor had no impact on the parental SK-MEL-2, 2aa-SK-MEL-2 or 3aa-SK-MEL-2 cells, but significantly reduced viability among wt-LPA3-SK-MEL-2 cells. Taken together, the data suggest that the SH3 ligand binding domain of LPA3 is required to mediate viability in melanoma cells. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  12. Secondary IgG responses to type III pneumococcal polysaccharide. II. Different cellular requirements for induction and elicitation.

    PubMed

    Braley-Mullen, H

    1976-04-01

    Mice primed with a thymus- (T) dependent form of Type III pneumococcal polysaccharide (S3), i.e., S3 coupled to erythrocytes (S3-RBC) produce S3-specific IgG antibody after secondary challenge with either S3 or S3-RBC. The production of IgG antibody by mice challenged with S3 was shown to be T independent since secondary responses were enhanced when mice were treated with anti-lymphocyte serum (ALS) at the time of secondary challenge with S3 and T-depleted spleen cells responded as well as unfractionated spleen cells to S3 in an adoptive transfer system. Secondary S3-specific IgG responses in mice challenged with S3-RBC were shown to be T dependent by the same criteria. The results obtained by using S3 as the antigen indicate that IgG-producing B cells (B lambda cells) can recognize and respond to antigen in the absence of helper T cells. On the other hand, T cells were required for the induction of S3-specific memory B lambda cells since mice depleted of T cells by treatment with ALS at the time of priming with S3-RBC failed to produce S3-specific IgG antibody after secondary challenge with either S3-specific IgG antibody after secondary chall-nge with either S3 or S3rbc. Since RBC-specific memory cells were induced in T-deprived mice the results suggest that T cell regulation of IgG antibody production may vary for different antigens.

  13. HTLV-1 Rex is required for viral spread and persistence in vivo but is dispensable for cellular immortalization in vitro

    PubMed Central

    Ye, Jianxin; Silverman, Lee; Lairmore, Michael D.; Green, Patrick L.

    2010-01-01

    Human T-cell leukemia virus type 1 (HTLV-1) is associated with leukemia/lymphoma and neurologic disorders. Although the viral transcriptional activator Tax is the critical viral oncoprotein, Rex, which regulates the expression of the viral structural and enzymatic genes, is essential for efficient viral replication. Herein, we investigate the contribution of Rex in HTLV-1 immortalization of primary T cells in vitro and viral survival in an infectious rabbit animal model. A Rex-deficient HTLV-1 (HTLVRex−) was constructed and characterized for viral gene expression, protein production, and immortalization capacity. Cells transiently transfected with the HTLVRex− proviral clone produced low detectable levels of p19 Gag. 729HTLVRex− stable transfectants produced functional Tax, but undetectable levels of Rex or p19 Gag. Coculture of irradiated 729HTLVRex− cells with peripheral blood mononuclear cells (PBMCs) resulted in sustained interleukin-2 (IL-2)–dependent growth of primary T lymphocytes. These cells carried the HTLVRex− genome and expressed tax/rex mRNA but produced no detectable Rex or p19 Gag. Rabbits inoculated with irradiated 729HTLVRex− cells or 729HTLVRex− cells transiently transfected with a Rex cDNA expression plasmid failed to become persistently infected or mount a detectable antibody response to the viral gene products. Together, our results provide the first direct evidence that Rex and its function to modulate viral gene expression and virion production is not required for in vitro immortalization by HTLV-1. However, Rex is critical for efficient infection of cells and persistence in vivo. PMID:12907436

  14. Cellular Internalization of Fibroblast Growth Factor-12 Exerts Radioprotective Effects on Intestinal Radiation Damage Independently of FGFR Signaling

    SciTech Connect

    Nakayama, Fumiaki; Umeda, Sachiko; Yasuda, Takeshi; Fujita, Mayumi; Asada, Masahiro; Meineke, Viktor; Imamura, Toru; Imai, Takashi

    2014-02-01

    Purpose: Several fibroblast growth factors (FGFs) were shown to inhibit radiation-induced tissue damage through FGF receptor (FGFR) signaling; however, this signaling was also found to be involved in the pathogenesis of several malignant tumors. In contrast, FGF12 cannot activate any FGFRs. Instead, FGF12 can be internalized readily into cells using 2 cell-penetrating peptide domains (CPP-M, CPP-C). Therefore, this study focused on clarifying the role of FGF12 internalization in protection against radiation-induced intestinal injury. Methods and Materials: Each FGF or peptide was administered intraperitoneally to BALB/c mice in the absence of heparin 24 hours before or after total body irradiation with γ rays at 9 to 12 Gy. Several radioprotective effects were examined in the jejunum. Results: Administration of FGF12 after radiation exposure was as effective as pretreatment in significantly promoting intestinal regeneration, proliferation of crypt cells, and epithelial differentiation. Two domains, comprising amino acid residues 80 to 109 and 140 to 169 of FGF12B, were identified as being responsible for the radioprotective activity, so that deletion of both domains from FGF12B resulted in a reduction in activity. Interestingly, these regions included the CPP-M and CPP-C domains, respectively; however, CPP-C by itself did not show an antiapoptotic effect. In addition, FGF1, prototypic FGF, possesses a domain corresponding to CPP-M, whereas it lacks CPP-C, so the fusion of FGF1 with CPP-C (FGF1/CPP-C) enhanced cellular internalization and increased radioprotective activity. However, FGF1/CPP-C reduced in vitro mitogenic activity through FGFRs compared with FGF1, implying that FGFR signaling might not be essential for promoting the radioprotective effect of FGF1/CPP-C. In addition, internalized FGF12 suppressed the activation of p38α after irradiation, resulting in reduced radiation-induced apoptosis. Conclusions: These findings indicate that FGF12 can protect the

  15. Cellular responses required for oxidative stress tolerance, colonization, and lesion formation by the necrotrophic fungus Alternaria alternata in citrus.

    PubMed

    Lin, Ching-Hsuan; Yang, Siwy Ling; Chung, Kuang-Ren

    2011-03-01

    The pathogenic capability of the tangerine pathotype of Alternaria alternata relies on the production of host-selective ACT toxin. Inoculation of A. alternata in leaves of the citrus quickly induced rapid lipid peroxidation, accumulation of hydrogen peroxide (H(2)O(2)), and cell death, indicative of host defensive response. We previously demonstrated an essential role of the A. alternata AaAP1 gene, encoding a redox-responsive YAP1-like transcription factor, to contribute to fungal pathogenicity. The AaAP1 null mutant fails to incite necrotic lesions. In this study, we show further that the fungal mutant defective at the AaAP1 locus displayed reduced activities for glutathione-S-transferase, glutathione peroxidase, glutathione reductase, and ligninolytic peroxidase, yet retained normal production of ACT toxin. In contrast to the wild-type progenitor and the genetically reverted strain, the mutant strain was unable to detoxify H(2)O(2) effectively and was killed upon exposure to H(2)O(2). The mutant strain induced lower levels of H(2)O(2) accumulation in citrus leaves, compared to those induced by the wild-type or by the genetically reverted strain. Upon exposure to H(2)O(2), A. alternata apparently changed expression of a wide array of the genes regulated by AaAP1. Thus, the impairment of the AaAP1 null mutants to incite necrotic lesions is apparently a consequence of their inability to alleviate the toxicity of ROS, and circumvention of plant defenses is important for the disease process.

  16. The coronin-like protein POD-1 is required for anterior–posterior axis formation and cellular architecture in the nematode Caenorhabditis elegans

    PubMed Central

    Rappleye, Chad A.; Paredez, Alexander R.; Smith, Carol W.; McDonald, Kent L.; Aroian, Raffi V.

    1999-01-01

    Establishment of anterior–posterior (a-p) polarity in the Caenorhabditis elegans embryo depends on filamentous (F-) actin. Previously, we isolated an F-actin-binding protein that was enriched in the anterior cortex of the one-cell embryo and was hypothesized to link developmental polarity to the actin cytoskeleton. Here, we identify this protein, POD-1, as a new member of the coronin family of actin-binding proteins. We have generated a deletion within the pod-1 gene. Elimination of POD-1 from early embryos results in a loss of physical and molecular asymmetries along the a-p axis. For example, PAR-1 and PAR-3, which themselves are polarized and required for a-p polarity, are delocalized in pod-1 mutant embryos. However, unlike loss of PAR proteins, loss of POD-1 gives rise to the formation of abnormal cellular structures, namely large vesicles of endocytic origin, membrane protrusions, unstable cell divisions, a defective eggshell, and deposition of extracellular material. We conclude that, analogous to coronin, POD-1 plays an important role in intracellular trafficking and organizing specific aspects of the actin cytoskeleton. We propose models to explain how the role of POD-1 in basic cellular processes could be linked to the generation of polarity along the embryonic a-p axis. PMID:10557211

  17. An integrated transcriptomics-guided genome-wide promoter analysis and next-generation proteomics approach to mine factor(s) regulating cellular differentiation

    PubMed Central

    Mandal, Kamal; Bader, Samuel L.; Kumar, Pankaj; Malakar, Dipankar; Campbell, David S.; Pradhan, Bhola Shankar; Sarkar, Rajesh K.; Wadhwa, Neerja; Sensharma, Souvik; Jain, Vaibhav; Moritz, Robert L.

    2017-01-01

    Abstract Differential next-generation-omics approaches aid in the visualization of biological processes and pave the way for divulging important events and/or interactions leading to a functional output at cellular or systems level. To this end, we undertook an integrated Nextgen transcriptomics and proteomics approach to divulge differential gene expression of infant and pubertal rat Sertoli cells (Sc).Unlike, pubertal Sc, infant Sc are immature and fail to support spermatogenesis. We found exclusive association of 14 and 19 transcription factor binding sites to infantile and pubertal states of Sc, respectively, using differential transcriptomics-guided genome-wide computational analysis of relevant promoters employing 220 Positional Weight Matrices from the TRANSFAC database. Proteomic SWATH-MS analysis provided extensive quantification of nuclear and cytoplasmic protein fractions revealing 1,670 proteins differentially located between the nucleus and cytoplasm of infant Sc and 890 proteins differentially located within those of pubertal Sc. Based on our multi-omics approach, the transcription factor YY1 was identified as one of the lead candidates regulating differentiation of Sc.YY1 was found to have abundant binding sites on promoters of genes upregulated during puberty. To determine its significance, we generated transgenic rats with Sc specific knockdown of YY1 that led to compromised spermatogenesis. PMID:28065881

  18. An integrated transcriptomics-guided genome-wide promoter analysis and next-generation proteomics approach to mine factor(s) regulating cellular differentiation.

    PubMed

    Mandal, Kamal; Bader, Samuel L; Kumar, Pankaj; Malakar, Dipankar; Campbell, David S; Pradhan, Bhola Shankar; Sarkar, Rajesh K; Wadhwa, Neerja; Sensharma, Souvik; Jain, Vaibhav; Moritz, Robert L; Majumdar, Subeer S

    2017-04-01

    Differential next-generation-omics approaches aid in the visualization of biological processes and pave the way for divulging important events and/or interactions leading to a functional output at cellular or systems level. To this end, we undertook an integrated Nextgen transcriptomics and proteomics approach to divulge differential gene expression of infant and pubertal rat Sertoli cells (Sc).Unlike, pubertal Sc, infant Sc are immature and fail to support spermatogenesis. We found exclusive association of 14 and 19 transcription factor binding sites to infantile and pubertal states of Sc, respectively, using differential transcriptomics-guided genome-wide computational analysis of relevant promoters employing 220 Positional Weight Matrices from the TRANSFAC database. Proteomic SWATH-MS analysis provided extensive quantification of nuclear and cytoplasmic protein fractions revealing 1,670 proteins differentially located between the nucleus and cytoplasm of infant Sc and 890 proteins differentially located within those of pubertal Sc. Based on our multi-omics approach, the transcription factor YY1 was identified as one of the lead candidates regulating differentiation of Sc.YY1 was found to have abundant binding sites on promoters of genes upregulated during puberty. To determine its significance, we generated transgenic rats with Sc specific knockdown of YY1 that led to compromised spermatogenesis. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  19. The human leukemia oncogene bcr-abl abrogates the anchorage requirement but not the growth factor requirement for proliferation.

    PubMed Central

    Renshaw, M W; McWhirter, J R; Wang, J Y

    1995-01-01

    Proliferation of normal cells in a multicellular organism requires not only growth factors but also the proper attachment to the extracellular matrix. A hallmark of neoplastic transformation is the loss of anchorage dependence which usually accompanies the loss of growth factor requirement. The Bcr-Abl tyrosine kinase of human leukemias is shown here to abrogate only the anchorage, not the growth factor, requirement. Bcr-Abl-transformed cells grow in soft agar but do not proliferate in serum-free media. Bcr-Abl does not activate the mitogenic pathway, as indicated by its inability to induce enhancers such as the serum response element or the tetradecanoyl phorbol acetate response element (TRE). However, Bcr-Abl can alleviate the anchorage requirement for the induction of the TRE enhancer; i.e., it allows serum to activate the TRE in detached cells. This activity is dependent on the association of an active Bcr-Abl tyrosine kinase with the actin filaments. Despite its association with the adapter protein Grb2, Bcr-Abl's effect on the TRE enhancer is not blocked by dominant negative Ras or Raf. The finding that Bcr-Abl tyrosine kinase abrogates only anchorage dependence may have important implications on the pathogenesis of chronic myelogenous leukemia. PMID:7862122

  20. Protective effects of a Modified Vaccinia Ankara-based vaccine candidate against Crimean-Congo Haemorrhagic Fever virus require both cellular and humoral responses.

    PubMed

    Dowall, Stuart D; Graham, Victoria A; Rayner, Emma; Hunter, Laura; Watson, Robert; Taylor, Irene; Rule, Antony; Carroll, Miles W; Hewson, Roger

    2016-01-01

    Crimean-Congo Haemorrhagic Fever (CCHF) is a severe tick-borne disease, endemic in many countries in Africa, the Middle East, Eastern Europe and Asia. There is no approved vaccine currently available against CCHF. The most promising candidate, which has previously been shown to confer protection in the small animal model, is a modified Vaccinia Ankara virus vector expressing the CCHF viral glycoprotein (MVA-GP). It has been shown that MVA-GP induces both humoral and cellular immunogenicity. In the present study, sera and T-lymphocytes were passively and adoptively transferred into recipient mice prior to challenge with CCHF virus. Results demonstrated that mediators from both arms of the immune system were required to demonstrate protective effects against lethal challenge.

  1. Protective effects of a Modified Vaccinia Ankara-based vaccine candidate against Crimean-Congo Haemorrhagic Fever virus require both cellular and humoral responses

    PubMed Central

    Dowall, Stuart D.; Graham, Victoria A.; Rayner, Emma; Hunter, Laura; Watson, Robert; Taylor, Irene; Rule, Antony; Carroll, Miles W.; Hewson, Roger

    2016-01-01

    Crimean-Congo Haemorrhagic Fever (CCHF) is a severe tick-borne disease, endemic in many countries in Africa, the Middle East, Eastern Europe and Asia. There is no approved vaccine currently available against CCHF. The most promising candidate, which has previously been shown to confer protection in the small animal model, is a modified Vaccinia Ankara virus vector expressing the CCHF viral glycoprotein (MVA-GP). It has been shown that MVA-GP induces both humoral and cellular immunogenicity. In the present study, sera and T-lymphocytes were passively and adoptively transferred into recipient mice prior to challenge with CCHF virus. Results demonstrated that mediators from both arms of the immune system were required to demonstrate protective effects against lethal challenge. PMID:27272940

  2. Regulation of Tacaribe Mammarenavirus Translation: Positive 5' and Negative 3' Elements and Role of Key Cellular Factors.

    PubMed

    Foscaldi, Sabrina; D'Antuono, Alejandra; Noval, María Gabriela; de Prat Gay, Gonzalo; Scolaro, Luis; Lopez, Nora

    2017-07-15

    Mammarenaviruses are enveloped viruses with a bisegmented negative-stranded RNA genome that encodes the nucleocapsid protein (NP), the envelope glycoprotein precursor (GPC), the RNA polymerase (L), and a RING matrix protein (Z). Viral proteins are synthesized from subgenomic mRNAs bearing a capped 5' untranslated region (UTR) and lacking 3' poly(A) tail. We analyzed the translation strategy of Tacaribe virus (TCRV), a prototype of the New World mammarenaviruses. A virus-like transcript that carries a reporter gene in place of the NP open reading frame and transcripts bearing modified 5' and/or 3' UTR were evaluated in a cell-based translation assay. We found that the presence of the cap structure at the 5' end dramatically increases translation efficiency and that the viral 5' UTR comprises stimulatory signals while the 3' UTR,specifically the presence of a terminal C+G-rich sequence and/or a stem-loop structure, down-modulates translation. Additionally, translation was profoundly reduced in eukaryotic initiation factor (eIF) 4G-inactivated cells, whereas depletion of intracellular levels of eIF4E had less impact on virus-like mRNA translation than on a cell-like transcript. Translation efficiency was independent of NP expression or TCRV infection. Our results indicate that TCRV mRNAs are translated using a cap-dependent mechanism, whose efficiency relies on the interplay between stimulatory signals in the 5' UTR and a negative modulatory element in the 3' UTR. The low dependence on eIF4E suggests that viral mRNAs may engage yet-unknown noncanonical host factors for a cap-dependent initiation mechanism.IMPORTANCE Several members of the Arenaviridae family cause serious hemorrhagic fevers in humans. In the present report, we describe the mechanism by which Tacaribe virus, a prototypic nonpathogenic New World mammarenavirus, regulates viral mRNA translation. Our results highlight the impact of untranslated sequences and key host translation factors on this process

  3. Sustained glucagon-like peptide-2 infusion is required for intestinal adaptation, and cessation reverses increased cellularity in rats with intestinal failure

    PubMed Central

    Koopmann, Matthew C.; Chen, Xueyan; Holst, Jens J.

    2010-01-01

    Glucagon-like peptide-2 (GLP-2) is a nutrient-dependent, proglucagon-derived hormone that is a proposed treatment for human short bowel syndrome (SBS). The objective was to determine how the timing, duration, and cessation of GLP-2 administration affect intestinal adaptation and enterocyte kinetics in a rat model of human SBS that results in intestinal failure requiring total parenteral nutrition (TPN). Rats underwent 60% jejunoileal resection plus cecectomy and jugular vein cannulation and were maintained exclusively with TPN for 18 days in these treatments: TPN control (no GLP-2); sustained GLP-2 (1–18 days); early GLP-2 (1–7 days, killed at 7 or 18 days); and delayed GLP-2 (12–18 days). Body weight gain was similar across groups, and plasma bioactive GLP-2 was significantly increased with coinfusion of GLP-2 (100 μg·kg−1·day−1) with TPN. GLP-2-treated rats showed significant increases in duodenum and jejunum mucosal dry mass, protein, DNA, and sucrase activity compared with TPN control. The increased jejunum cellularity reflected significantly decreased apoptosis and increased crypt mitosis and crypt fission due to GLP-2. When GLP-2 infusion stopped at 7 days, these effects were reversed at 18 days. Sustained GLP-2 infusion significantly increased duodenum length and decreased 18-day mortality to 0% from 37.5% deaths in TPN control (P = 0.08). Colon proglucagon expression quantified by real-time RT-qPCR was increased in TPN controls and attenuated by GLP-2 infusion; jejunal expression of the GLP-2 receptor did not differ among groups. In summary, early, sustained GLP-2 infusion reduces mortality, induces crypt fission, and is required for intestinal adaptation, whereas cessation of GLP-2 reverses gains in mucosal cellularity in a rat model of intestinal failure. PMID:20864657

  4. Glia cell stimulating factor (GSF): a new lymphokine. Part 1. Cellular sources and partial purification of murine GSF, role of cytoskeleton and protein synthesis in its production.

    PubMed

    Fontana, A; Dubs, R; Merchant, R; Balsiger, S; Grob, P J

    1982-01-01

    The effect of activated-lymphocyte supernatant on glia cells was investigated. When treated in vitro with Concanavalin A (ConA), murine spleen cells released a soluble product, termed glia cell stimulating factor (GSF), which stimulated RNA and DNA synthesis in cultured murine glia cells. Furthermore, GSF appeared to promote the maturation of undifferentiated glia cells to astrocytes having a high content of glial fibrillary acidic protein. GSF secretion occurred after a lag period of 16 hours and proceeded at a constant rate for more than 48 hours. This GSF produced by ConA-stimulated murine lymphocytes has an apparent molecular weight between 60,000 and 80,000. Antigenic stimulation of primed lymph node cells with BGG resulted in a similar GSF production. Cellular sources of mitogen-induced GSF were investigated by using isolated lymphoid populations. GSF release by ConA-activated pure T-lymphocytes reconstituted with peritoneal macrophages was equivalent to that of unseparated spleen cells, whereas GSF production by T-lymphocytes alone was low. Macrophages alone did not elaborate detectable levels of GSF. GSF was also secreted by enriched -B-lymphocytes populations stimulated by Protein A. Formation of GSF was suppressed when cytochalasin B or cyclo-heximide was added to the cultures, while colchicine failed to have any effect. DNA synthesis is not required for GSF production as determined by resistence to treatment with mitomycin C. The data indicate that the GSF production and secretion mechanism is much like that described for other lymphokines.

  5. 34 CFR 373.6 - What are the priorities and other factors and requirements for competitions?

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 34 Education 2 2013-07-01 2013-07-01 false What are the priorities and other factors and... SPECIAL DEMONSTRATION PROGRAMS General § 373.6 What are the priorities and other factors and requirements.... (ii) Any of the additional factors listed in paragraph (c) of this section that the Secretary may...

  6. Genetic and cellular dissection of the activation of AM14 rheumatoid factor B cells in a mouse model of lupus

    PubMed Central

    Sang, Allison; Zheng, Ying Yi; Choi, Seung-Chul; Zeumer, Leilani; Morel, Laurence

    2015-01-01

    The RF-specific AM14 tg BCR has been used as a model to dissect the mechanisms of B cell tolerance to ICs containing nucleic acids. We have shown previously that AM14 RF B cells break tolerance in the TC mouse model of lupus through the dual engagement of the AM14 BCR and TLR9. In this study, we showed that neither the expression of Sle1 or Sle2 susceptibility loci alone was sufficient to activate AM14 RF B cells, suggesting that the production of antichromatin IgG2aa autoAg mediated by Sle1 and an intrinsically higher B cell activation mediated by Sle2 were required. We also showed that the B6 genetic background enhanced the selection of AM14 RF B cells to the MZB cell compartment regardless of the expression of the Sle loci and therefore, of their activation into AFCs. Furthermore, some AM14 RF B cells were selected into the B-1a compartment, where they did not differentiate into AFCs. Therefore, it is unlikely that the selection of AM14 RF B cells to the MZB or B-1a cell compartments in TC.AM14a mice is responsible for their breach of tolerance. Finally, we showed that the presence of expression of Sle1 in non-tg cells, most likely T cells, is necessary for the activation of AM14 RF B cells into AFCs. Overall, these results suggest a threshold model of activation of AM14 RF B cells on the B6 background with additive genetic and cellular contribution of multiple sources. PMID:25957308

  7. Genetic and cellular dissection of the activation of AM14 rheumatoid factor B cells in a mouse model of lupus.

    PubMed

    Sang, Allison; Zheng, Ying Yi; Choi, Seung-Chul; Zeumer, Leilani; Morel, Laurence

    2015-08-01

    The RF-specific AM14 tg BCR has been used as a model to dissect the mechanisms of B cell tolerance to ICs containing nucleic acids. We have shown previously that AM14 RF B cells break tolerance in the TC mouse model of lupus through the dual engagement of the AM14 BCR and TLR9. In this study, we showed that neither the expression of Sle1 or Sle2 susceptibility loci alone was sufficient to activate AM14 RF B cells, suggesting that the production of antichromatin IgG2a(a) autoAg mediated by Sle1 and an intrinsically higher B cell activation mediated by Sle2 were required. We also showed that the B6 genetic background enhanced the selection of AM14 RF B cells to the MZB cell compartment regardless of the expression of the Sle loci and therefore, of their activation into AFCs. Furthermore, some AM14 RF B cells were selected into the B-1a compartment, where they did not differentiate into AFCs. Therefore, it is unlikely that the selection of AM14 RF B cells to the MZB or B-1a cell compartments in TC.AM14(a) mice is responsible for their breach of tolerance. Finally, we showed that the presence of expression of Sle1 in non-tg cells, most likely T cells, is necessary for the activation of AM14 RF B cells into AFCs. Overall, these results suggest a threshold model of activation of AM14 RF B cells on the B6 background with additive genetic and cellular contribution of multiple sources. © Society for Leukocyte Biology.

  8. CELLULAR BIOAVAILABILITY OF NATURAL HORMONES AND ENVIRONMENTAL CONTAMINANTS AS A FUNCTION OF SERUM AND CYTOSOLIC BINDING FACTORS

    EPA Science Inventory

    Environmental contaminants have been reported to function as hormone mimics in various wildlife species. To investigate a potential mechanism for the interaction of contaminants with the endocrine system, we evaluated the cellular bioavailability of numerous chemicals. Hormone bi...

  9. CELLULAR BIOAVAILABILITY OF NATURAL HORMONES AND ENVIRONMENTAL CONTAMINANTS AS A FUNCTION OF SERUM AND CYTOSOLIC BINDING FACTORS

    EPA Science Inventory

    Environmental contaminants have been reported to function as hormone mimics in various wildlife species. To investigate a potential mechanism for the interaction of contaminants with the endocrine system, we evaluated the cellular bioavailability of numerous chemicals. Hormone bi...

  10. [Persistent human papillomavirus infection in the etiology of cervical carcinoma: the role of immunological, genetic, viral and cellular factors].

    PubMed

    Zivadinović, Radomir; Petrić, Aleksandra; Lilić, Goran; Lilić, Vekoslav; Djordjević, Biljana

    2014-01-01

    The aim of this paper was to present the role of human papillomavirus (HPV) in cervical carcinogenesis from several aspects. By explaining the HPV virus lifecycle and structure, its effect on cervical cell cycle and subversion of immune response can be better understood. Early E region of the viral genome encodes proteins that are directly involved in carcinogenesis. The E6 protein binds to p53 protein (product of tumor-suppressor gene) blocking and degrading it, which in turn prevents cell cycle arrest and apoptosis induction. E6 is also capable of telomerase activation, which leads to cell immortalization; it also reacts with host proto-oncogene c-jun, responsible for transcription, shortens G1 phase and speeds up the transition from G1 to S phase of the cells infected by HPV. E7 forms bonds with retinoblastoma protein (product of tumor-suppressor gene) and inactivates it. It can inactivate cyclin inhibitors p21, p27, and abrogate the mitotic spindle checkpoint with the loss of protective effect of pRB and p53. The immune system cannot initiate early immunological reaction since the virus is non-lytic, while the concentration of viral proteins--antigens is low and has a basal intracellular position. Presentation through Langerhans cells (LC) is weak, because the number of these cells is low due to the effect of HPV. E7 HPV reduces the expression of E-cadherin, which is responsible for LC adhesion to HPV-transformed keratinocytes. Based on these considerations, it may be concluded that the process of cervical carcinogenesis includes viral, genetic, cellular, molecular-biological, endocrine, exocrine and immunological factors.

  11. Fibroblast growth factor 2 retargeted adenovirus has redirected cellular tropism: evidence for reduced toxicity and enhanced antitumor activity in mice.

    PubMed

    Gu, D L; Gonzalez, A M; Printz, M A; Doukas, J; Ying, W; D'Andrea, M; Hoganson, D K; Curiel, D T; Douglas, J T; Sosnowski, B A; Baird, A; Aukerman, S L; Pierce, G F

    1999-06-01

    Adenovirus (Ad) have been used as vectors to deliver genes to a wide variety of tissues. Despite achieving high expression levels in vivo, Ad vectors display normal tissue toxicity, transient expression, and antivector immune responses that limit therapeutic potential. To circumvent these problems, several retargeting strategies to abrogate native tropism and redirect Ad uptake through defined receptors have been attempted. Despite success in cell culture, in vivo results have generally not shown sufficient selectivity for target tissues. We have previously identified (C. K. Goldman et al., Cancer Res., 57: 1447-1451, 1997) the fibroblast growth factor (FGF) ligand and receptor families as conferring sufficient specificity and binding affinity to be useful for targeting DNA in vivo. In the present studies, we retargeted Ad using basic FGF (FGF2) as a targeting ligand. Cellular uptake is redirected through high-affinity FGF receptors (FGFRs) and not the more ubiquitous lower-affinity Ad receptors. Initial in vitro experiments demonstrated a 10- to 100-fold increase in gene expression in numerous FGFR positive (FGFR+) cell lines using FGF2-Ad when compared with Ad. To determine whether increased selectivity could be detected in vivo, FGF2-Ad was administered i.v. to normal mice. FGF2-Ad demonstrates markedly decreased hepatic toxicity and liver transgene expression compared with Ad treatment. Importantly, FGF2-Ad encoding the herpes simplex virus thymidine kinase (TK) gene transduces Ad-resistant FGFR+ tumor cells both ex vivo and in vivo, which results in substantially enhanced survival (180-260%) when the prodrug ganciclovir is administered. Because FGFRs are up-regulated on many types of malignant or injured cells, this broadly useful method to redirect native Ad tropism and to increase the potency of gene expression may offer significant therapeutic advantages.

  12. Expression and cellular localization of the transcription factor NeuroD1 in the developing and adult rat pineal gland.

    PubMed

    Castro, Analía E; Benitez, Sergio G; Farias Altamirano, Luz E; Savastano, Luis E; Patterson, Sean I; Muñoz, Estela M

    2015-05-01

    Circadian rhythms govern many aspects of mammalian physiology. The daily pattern of melatonin synthesis and secretion is one of the classic examples of circadian oscillations. It is mediated by a class of neuroendocrine cells known as pinealocytes which are not yet fully defined. An established method to evaluate functional and cytological characters is through the expression of lineage-specific transcriptional regulators. NeuroD1 is a basic helix-loop-helix transcription factor involved in the specification and maintenance of both endocrine and neuronal phenotypes. We have previously described developmental and adult regulation of NeuroD1 mRNA in the rodent pineal gland. However, the transcript levels were not influenced by the elimination of sympathetic input, suggesting that any rhythmicity of NeuroD1 might be found downstream of transcription. Here, we describe NeuroD1 protein expression and cellular localization in the rat pineal gland during development and the daily cycle. In embryonic and perinatal stages, protein expression follows the mRNA pattern and is predominantly nuclear. Thereafter, NeuroD1 is mostly found in pinealocyte nuclei in the early part of the night and in cytoplasm during the day, a rhythm maintained into adulthood. Additionally, nocturnal nuclear NeuroD1 levels are reduced after sympathetic disruption, an effect mimicked by the in vivo administration of α- and β-adrenoceptor blockers. NeuroD1 phosphorylation at two sites, Ser(274) and Ser(336) , associates with nuclear localization in pinealocytes. These data suggest that NeuroD1 influences pineal phenotype both during development and adulthood, in an autonomic and phosphorylation-dependent manner.

  13. Regulation of Ras Exchange Factors and Cellular Localization of Ras Activation by Lipid Messengers in T Cells

    PubMed Central

    Jun, Jesse E.; Rubio, Ignacio; Roose, Jeroen P.

    2013-01-01

    The Ras-MAPK signaling pathway is highly conserved throughout evolution and is activated downstream of a wide range of receptor stimuli. Ras guanine nucleotide exchange factors (RasGEFs) catalyze GTP loading of Ras and play a pivotal role in regulating receptor-ligand induced Ras activity. In T cells, three families of functionally important RasGEFs are expressed: RasGRF, RasGRP, and Son of Sevenless (SOS)-family GEFs. Early on it was recognized that Ras activation is critical for T cell development and that the RasGEFs play an important role herein. More recent work has revealed that nuances in Ras activation appear to significantly impact T cell development and selection. These nuances include distinct biochemical patterns of analog versus digital Ras activation, differences in cellular localization of Ras activation, and intricate interplays between the RasGEFs during distinct T cell developmental stages as revealed by various new mouse models. In many instances, the exact nature of these nuances in Ras activation or how these may result from fine-tuning of the RasGEFs is not understood. One large group of biomolecules critically involved in the control of RasGEFs functions are lipid second messengers. Multiple, yet distinct lipid products are generated following T cell receptor (TCR) stimulation and bind to different domains in the RasGRP and SOS RasGEFs to facilitate the activation of the membrane-anchored Ras GTPases. In this review we highlight how different lipid-based elements are generated by various enzymes downstream of the TCR and other receptors and how these dynamic and interrelated lipid products may fine-tune Ras activation by RasGEFs in developing T cells. PMID:24027568

  14. Transcription Factors Mat2 and Znf2 Operate Cellular Circuits Orchestrating Opposite- and Same-Sex Mating in Cryptococcus neoformans

    PubMed Central

    Lin, Xiaorong; Jackson, Jennifer C.; Feretzaki, Marianna; Xue, Chaoyang; Heitman, Joseph

    2010-01-01

    Cryptococcus neoformans is a human fungal pathogen that undergoes a dimorphic transition from a unicellular yeast to multicellular hyphae during opposite sex (mating) and unisexual reproduction (same-sex mating). Opposite- and same-sex mating are induced by similar environmental conditions and involve many shared components, including the conserved pheromone sensing Cpk1 MAPK signal transduction cascade that governs the dimorphic switch in C. neoformans. However, the homeodomain cell identity proteins Sxi1α/Sxi2a encoded by the mating type locus that are essential for completion of sexual reproduction following cell–cell fusion during opposite-sex mating are dispensable for same-sex mating. Therefore, identification of downstream targets of the Cpk1 MAPK pathway holds the key to understanding molecular mechanisms governing the two distinct developmental fates. Thus far, homology-based approaches failed to identify downstream transcription factors which may therefore be species-specific. Here, we applied insertional mutagenesis via Agrobacterium-mediated transformation and transcription analysis using whole genome microarrays to identify factors involved in C. neoformans differentiation. Two transcription factors, Mat2 and Znf2, were identified as key regulators of hyphal growth during same- and opposite-sex mating. Mat2 is an HMG domain factor, and Znf2 is a zinc finger protein; neither is encoded by the mating type locus. Genetic, phenotypic, and transcriptional analyses of Mat2 and Znf2 provide evidence that Mat2 is a downstream transcription factor of the Cpk1 MAPK pathway whereas Znf2 functions as a more terminal hyphal morphogenesis determinant. Although the components of the MAPK pathway including Mat2 are not required for virulence in animal models, Znf2, as a hyphal morphology determinant, is a negative regulator of virulence. Further characterization of these elements and their target circuits will reveal genes controlling biological processes central

  15. Transcription factors Mat2 and Znf2 operate cellular circuits orchestrating opposite- and same-sex mating in Cryptococcus neoformans.

    PubMed

    Lin, Xiaorong; Jackson, Jennifer C; Feretzaki, Marianna; Xue, Chaoyang; Heitman, Joseph

    2010-05-13

    Cryptococcus neoformans is a human fungal pathogen that undergoes a dimorphic transition from a unicellular yeast to multicellular hyphae during opposite sex (mating) and unisexual reproduction (same-sex mating). Opposite- and same-sex mating are induced by similar environmental conditions and involve many shared components, including the conserved pheromone sensing Cpk1 MAPK signal transduction cascade that governs the dimorphic switch in C. neoformans. However, the homeodomain cell identity proteins Sxi1alpha/Sxi2a encoded by the mating type locus that are essential for completion of sexual reproduction following cell-cell fusion during opposite-sex mating are dispensable for same-sex mating. Therefore, identification of downstream targets of the Cpk1 MAPK pathway holds the key to understanding molecular mechanisms governing the two distinct developmental fates. Thus far, homology-based approaches failed to identify downstream transcription factors which may therefore be species-specific. Here, we applied insertional mutagenesis via Agrobacterium-mediated transformation and transcription analysis using whole genome microarrays to identify factors involved in C. neoformans differentiation. Two transcription factors, Mat2 and Znf2, were identified as key regulators of hyphal growth during same- and opposite-sex mating. Mat2 is an HMG domain factor, and Znf2 is a zinc finger protein; neither is encoded by the mating type locus. Genetic, phenotypic, and transcriptional analyses of Mat2 and Znf2 provide evidence that Mat2 is a downstream transcription factor of the Cpk1 MAPK pathway whereas Znf2 functions as a more terminal hyphal morphogenesis determinant. Although the components of the MAPK pathway including Mat2 are not required for virulence in animal models, Znf2, as a hyphal morphology determinant, is a negative regulator of virulence. Further characterization of these elements and their target circuits will reveal genes controlling biological processes central

  16. Recovery from an Acute Infection in C. elegans Requires the GATA Transcription Factor ELT-2

    PubMed Central

    Head, Brian; Aballay, Alejandro

    2014-01-01

    The mechanisms involved in the recognition of microbial pathogens and activation of the immune system have been extensively studied. However, the mechanisms involved in the recovery phase of an infection are incompletely characterized at both the cellular and physiological levels. Here, we establish a Caenorhabditis elegans-Salmonella enterica model of acute infection and antibiotic treatment for studying biological changes during the resolution phase of an infection. Using whole genome expression profiles of acutely infected animals, we found that genes that are markers of innate immunity are down-regulated upon recovery, while genes involved in xenobiotic detoxification, redox regulation, and cellular homeostasis are up-regulated. In silico analyses demonstrated that genes altered during recovery from infection were transcriptionally regulated by conserved transcription factors, including GATA/ELT-2, FOXO/DAF-16, and Nrf/SKN-1. Finally, we found that recovery from an acute bacterial infection is dependent on ELT-2 activity. PMID:25340560

  17. Recovery from an acute infection in C. elegans requires the GATA transcription factor ELT-2.

    PubMed

    Head, Brian; Aballay, Alejandro

    2014-10-01

    The mechanisms involved in the recognition of microbial pathogens and activation of the immune system have been extensively studied. However, the mechanisms involved in the recovery phase of an infection are incompletely characterized at both the cellular and physiological levels. Here, we establish a Caenorhabditis elegans-Salmonella enterica model of acute infection and antibiotic treatment for studying biological changes during the resolution phase of an infection. Using whole genome expression profiles of acutely infected animals, we found that genes that are markers of innate immunity are down-regulated upon recovery, while genes involved in xenobiotic detoxification, redox regulation, and cellular homeostasis are up-regulated. In silico analyses demonstrated that genes altered during recovery from infection were transcriptionally regulated by conserved transcription factors, including GATA/ELT-2, FOXO/DAF-16, and Nrf/SKN-1. Finally, we found that recovery from an acute bacterial infection is dependent on ELT-2 activity.

  18. Serum levels, tissue expression and cellular secretion of macrophage migration inhibitory factor in limited and diffuse systemic sclerosis.

    PubMed

    Corallo, Claudio; Paulesu, Luana; Cutolo, Maurizio; Ietta, Francesca; Carotenuto, Claudio; Mannelli, Chiara; Romagnoli, Roberta; Nuti, Ranuccio; Giordano, Nicola

    2015-01-01

    To investigate serum levels, tissue/cellular expression of macrophage migration inhibitory factor (MIF) in patients with limited (lSSc) and diffuse (dSSc) systemic sclerosis. 10 lSSc-patients, 10 dSSc-patients and 10 controls were enrolled. MIF serum levels were assayed by ELISA. MIF and its receptors CD74/CD44 were evaluated by immunohistochemistry on skin biopsies from patients with dSSc, lSSc (affected and not-affected skin) and controls. MIF levels were assessed (ELISA) in supernatants of healthy dermal microvascular endothelial cells (MVECs) and in control (CTR), non-affected SSc (NA) and affected (SSc) fibroblasts treated for 48 h with 10% control serum and 10% SSc-serum. MIF supernatant (ELISA) and mRNA (quantitative real-time PCR) levels were determined in SSc dermal fibroblasts and in control dermal fibroblasts untreated or stimulated at 6 h-24 h-48 h with bleomycin (50 mU/ml). Serum MIF was significantly higher in dSSc (18.7±4.1 ng/ml, p<0.001) and in lSSc (10.4±4.4 ng/ml, p<0.001) patients respect to controls (2.6±1.4 ng/ml). Enhanced MIF immunoreactivity was found in keratinocytes, fibroblasts, endothelium, sebaceous/sweat glands from lSSc/dSSc affected skin. Faint MIF immunoreactivity was found in control skin and not-affected skin of lSSc patients. No differences were found in CD74/CD44 receptors' analysis among control and dSSc/lSSc affected and non-affected skin. MVECs and fibroblasts (CTR, NA and SSc) produced significantly more MIF, when stimulated with SSc serum respect to control-serum (p<0.001). Finally, MIF mRNA levels significantly increased at 6h (p<0.001) and decreased at 48 h (p<0.001) in control fibroblasts treated with bleomycin compared to control untreated. Simultaneously, MIF supernatant protein levels increased after 48 h (p<0.01) in bleomycin-treated fibroblasts respect to untreated ones. These results suggest that MIF could be implicated in the pathogenesis of SSc, probably acting as protective factor against the SSc stressful

  19. A Mechanism to Enhance Cellular Responsivity to Hormone Action: Krüppel-Like Factor 9 Promotes Thyroid Hormone Receptor-β Autoinduction During Postembryonic Brain Development

    PubMed Central

    Hu, Fang; Knoedler, Joseph R.

    2016-01-01

    Thyroid hormone (TH) receptor (TR)-β (trb) is induced by TH (autoinduced) in Xenopus tadpoles during metamorphosis. We previously showed that Krüppel-like factor 9 (Klf9) is rapidly induced by TH in the tadpole brain, associates in chromatin with the trb upstream region in a developmental stage and TH-dependent manner, and forced expression of Klf9 in the Xenopus laevis cell line XTC-2 accelerates and enhances trb autoinduction. Here we investigated whether Klf9 can promote trb autoinduction in tadpole brain in vivo. Using electroporation-mediated gene transfer, we transfected plasmids into premetamorphic tadpole brain to express wild-type or mutant forms of Klf9. Forced expression of Klf9 increased baseline trb mRNA levels in thyroid-intact but not in goitrogen-treated tadpoles, supporting that Klf9 enhances liganded TR action. As in XTC-2 cells, forced expression of Klf9 enhanced trb autoinduction in tadpole brain in vivo and also increased TH-dependent induction of the TR target genes klf9 and thbzip. Consistent with our previous mutagenesis experiments conducted in XTC-2 cells, the actions of Klf9 in vivo required an intact N-terminal region but not a functional DNA binding domain. Forced expression of TRβ in tadpole brain by electroporation-mediated gene transfer increased baseline and TH-induced TR target gene transcription, supporting a role for trb autoinduction during metamorphosis. Our findings support that Klf9 acts as an accessory transcription factor for TR at the trb locus during tadpole metamorphosis, enhancing trb autoinduction and transcription of other TR target genes, which increases cellular responsivity to further TH action on developmental gene regulation programs. PMID:26886257

  20. Viral and Host Factors Required for Avian H5N1 Influenza A Virus Replication in Mammalian Cells

    PubMed Central

    Zhang, Hong; Hale, Benjamin G.; Xu, Ke; Sun, Bing

    2013-01-01

    Following the initial and sporadic emergence into humans of highly pathogenic avian H5N1 influenza A viruses in Hong Kong in 1997, we have come to realize the potential for avian influenza A viruses to be transmitted directly from birds to humans. Understanding the basic viral and cellular mechanisms that contribute to infection of mammalian species with avian influenza viruses is essential for developing prevention and control measures against possible future human pandemics. Multiple physical and functional cellular barriers can restrict influenza A virus infection in a new host species, including the cell membrane, the nuclear envelope, the nuclear environment, and innate antiviral responses. In this review, we summarize current knowledge on viral and host factors required for avian H5N1 influenza A viruses to successfully establish infections in mammalian cells. We focus on the molecular mechanisms underpinning mammalian host restrictions, as well as the adaptive mutations that are necessary for an avian influenza virus to overcome them. It is likely that many more viral and host determinants remain to be discovered, and future research in this area should provide novel and translational insights into the biology of influenza virus-host interactions. PMID:23752648

  1. Cellular Analogs of Operant Behavior

    DTIC Science & Technology

    1990-12-31

    activity rather than by a cellular reinforcement process. We have always required as critical evidence of cellular reinforcement that noncontingent or... reinforcement process. We have always required as critical evidence of cellular reinforcement that noncortingent or random presentations of the positive...the burst- ing of hippocampal pyramidal cells. One approach is to attempt to reinforce hippocamp- al bursting with a nonspecific depolarizing agent

  2. Girdin/GIV is upregulated by cyclic tension, propagates mechanical signal transduction, and is required for the cellular proliferation and migration of MG-63 cells

    SciTech Connect

    Hu, Jiang-Tian; Li, Yan; Yu, Bing; Gao, Guo-Jie; Zhou, Ting; Li, Song

    2015-08-21

    To explore how Girdin/GIV is regulated by cyclic tension and propagates downstream signals to affect cell proliferation and migration. Human osteoblast-like MG-63 cells were exposed to cyclic tension force at 4000 μstrain and 0.5 Hz for 6 h, produced by a four-point bending system. Cyclic tension force upregulated Girdin and Akt expression and phosphorylation in cultured MG-63 cells. Girdin and Akt each promoted the phosphorylation of the other under stimulated tension. In vitro MTT and transwell assays showed that Girdin and Akt are required for cell proliferation and migration during cellular quiescence. Moreover, STAT3 was determined to be essential for Girdin expression under stimulated tension force in the physiological condition, as well as for osteoblast proliferation and migration during quiescence. These findings suggest that the STAT3/Girdin/Akt pathway activates in osteoblasts in response to mechanical stimulation and may play a significant role in triggering osteoblast proliferation and migration during orthodontic treatment. - Highlights: • Tension force upregulates Girdin and Akt expression and phosphorylation. • Girdin and Akt promotes the phosphorylation of each other under tension stimulation. • Girdin and Akt are required for MG-63 cell proliferation and migration. • STAT3 is essential for Girdin expression after application of the tension forces.

  3. Long-Term Channel Block Is Required to Inhibit Cellular Transformation by Human Ether-à-Go-Go–Related Gene (hERG1) Potassium Channels

    PubMed Central

    Pier, David M.; Shehatou, George S. G.; Giblett, Susan; Pullar, Christine E.; Trezise, Derek J.; Pritchard, Catrin A.; Challiss, R. A. John

    2014-01-01

    Both human ether-à-go-go–related gene (hERG1) and the closely related human ether-à-go-go (hEAG1) channel are aberrantly expressed in a large proportion of human cancers. In the present study, we demonstrate that transfection of hERG1 into mouse fibroblasts is sufficient to induce many features characteristic of malignant transformation. An important finding of this work is that this transformation could be reversed by chronic incubation (for 2–3 weeks) with the hERG channel blocker dofetilide (100 nM), whereas more acute applications (for 1–2 days) were ineffective. The hERG1 expression resulted in a profound loss of cell contact inhibition, multiple layers of overgrowing cells, and high saturation densities. Cells also changed from fibroblast-like to a more spindle-shaped morphology, which was associated with a smaller cell size, a dramatic increase in cell polarization, a reduction in the number of actin stress fibers, and less punctate labeling of focal adhesions. Analysis of single-cell migration and scratch-wound closure clearly demonstrated that hERG1-expressing cells migrated more rapidly than vector-transfected control cells. In contrast to previous studies on hEAG1, there were no increases in rates of proliferation, or loss of growth factor dependency; however, hERG1-expressing cells were capable of substrate-independent growth. Allogeneic transplantation of hERG1-expressing cells into nude mice resulted in an increased incidence of tumors. In contrast to hEAG1, the mechanism of cellular transformation is dependent on ion conduction. Trafficking-deficient and conduction-deficient hERG1 mutants also prevented cellular transformation. These results provide evidence that hERG1 expression is sufficient to induce cellular transformation by a mechanism distinct from hEAG1. The most important conclusion of this study is that selective hERG1 channel blockers have therapeutic potential in the treatment of hERG1-expressing cancers. PMID:24830940

  4. Fibroblast Growth Factor 2 Is Required for Epithelial Recovery, but Not for Pulmonary Fibrosis, in Response to Bleomycin

    PubMed Central

    Guzy, Robert D.; Stoilov, Ivan; Elton, Timothy J.; Mecham, Robert P.

    2015-01-01

    The pathogenesis of pulmonary fibrosis involves lung epithelial injury and aberrant proliferation of fibroblasts, and results in progressive pulmonary scarring and declining lung function. In vitro, fibroblast growth factor (FGF) 2 promotes myofibroblast differentiation and proliferation in cooperation with the profibrotic growth factor, transforming growth factor-β1, but the in vivo requirement for FGF2 in the development of pulmonary fibrosis is not known. The bleomycin model of lung injury and pulmonary fibrosis was applied to Fgf2 knockout (Fgf2−/−) and littermate control mice. Weight loss, mortality, pulmonary fibrosis, and histology were analyzed after a single intranasal dose of bleomycin. Inflammation was evaluated in bronchoalveolar lavage (BAL) fluid, and epithelial barrier integrity was assessed by measuring BAL protein and Evans Blue dye permeability. Fgf2 is expressed in mouse and human lung epithelial and inflammatory cells, and, in response to bleomycin, Fgf2−/− mice have significantly increased mortality and weight loss. Analysis of BAL fluid and histology show that pulmonary fibrosis is unaltered, but Fgf2−/− mice fail to efficiently resolve inflammation, have increased BAL cellularity, and, importantly, deficient recovery of epithelial integrity. Fgf2−/− mice similarly have deficient recovery of club cell secretory protein+ bronchial epithelium in response to naphthalene. We conclude that FGF2 is not required for bleomycin-induced pulmonary fibrosis, but rather is essential for epithelial repair and maintaining epithelial integrity after bleomycin-induced lung injury in mice. These data identify that FGF2 acts as a protective growth factor after lung epithelial injury, and call into question the role of FGF2 as a profibrotic growth factor in vivo. PMID:24988442

  5. Fibroblast growth factor 2 is required for epithelial recovery, but not for pulmonary fibrosis, in response to bleomycin.

    PubMed

    Guzy, Robert D; Stoilov, Ivan; Elton, Timothy J; Mecham, Robert P; Ornitz, David M

    2015-01-01

    The pathogenesis of pulmonary fibrosis involves lung epithelial injury and aberrant proliferation of fibroblasts, and results in progressive pulmonary scarring and declining lung function. In vitro, fibroblast growth factor (FGF) 2 promotes myofibroblast differentiation and proliferation in cooperation with the profibrotic growth factor, transforming growth factor-β1, but the in vivo requirement for FGF2 in the development of pulmonary fibrosis is not known. The bleomycin model of lung injury and pulmonary fibrosis was applied to Fgf2 knockout (Fgf2(-/-)) and littermate control mice. Weight loss, mortality, pulmonary fibrosis, and histology were analyzed after a single intranasal dose of bleomycin. Inflammation was evaluated in bronchoalveolar lavage (BAL) fluid, and epithelial barrier integrity was assessed by measuring BAL protein and Evans Blue dye permeability. Fgf2 is expressed in mouse and human lung epithelial and inflammatory cells, and, in response to bleomycin, Fgf2(-/-) mice have significantly increased mortality and weight loss. Analysis of BAL fluid and histology show that pulmonary fibrosis is unaltered, but Fgf2(-/-) mice fail to efficiently resolve inflammation, have increased BAL cellularity, and, importantly, deficient recovery of epithelial integrity. Fgf2(-/-) mice similarly have deficient recovery of club cell secretory protein(+) bronchial epithelium in response to naphthalene. We conclude that FGF2 is not required for bleomycin-induced pulmonary fibrosis, but rather is essential for epithelial repair and maintaining epithelial integrity after bleomycin-induced lung injury in mice. These data identify that FGF2 acts as a protective growth factor after lung epithelial injury, and call into question the role of FGF2 as a profibrotic growth factor in vivo.

  6. Use of a Generalized Additive Model to Investigate Key Abiotic Factors Affecting Microcystin Cellular Quotas in Heavy Bloom Areas of Lake Taihu

    PubMed Central

    Tao, Min; Xie, Ping; Chen, Jun; Qin, Boqiang; Zhang, Dawen; Niu, Yuan; Zhang, Meng; Wang, Qing; Wu, Laiyan

    2012-01-01

    Lake Taihu is the third largest freshwater lake in China and is suffering from serious cyanobacterial blooms with the associated drinking water contamination by microcystin (MC) for millions of citizens. So far, most studies on MCs have been limited to two small bays, while systematic research on the whole lake is lacking. To explain the variations in MC concentrations during cyanobacterial bloom, a large-scale survey at 30 sites across the lake was conducted monthly in 2008. The health risks of MC exposure were high, especially in the northern area. Both Microcystis abundance and MC cellular quotas presented positive correlations with MC concentration in the bloom seasons, suggesting that the toxic risks during Microcystis proliferations were affected by variations in both Microcystis density and MC production per Microcystis cell. Use of a powerful predictive modeling tool named generalized additive model (GAM) helped visualize significant effects of abiotic factors related to carbon fixation and proliferation of Microcystis (conductivity, dissolved inorganic carbon (DIC), water temperature and pH) on MC cellular quotas from recruitment period of Microcystis to the bloom seasons, suggesting the possible use of these factors, in addition to Microcystis abundance, as warning signs to predict toxic events in the future. The interesting relationship between macrophytes and MC cellular quotas of Microcystis (i.e., high MC cellular quotas in the presence of macrophytes) needs further investigation. PMID:22384128

  7. Use of a generalized additive model to investigate key abiotic factors affecting microcystin cellular quotas in heavy bloom areas of Lake Taihu.

    PubMed

    Tao, Min; Xie, Ping; Chen, Jun; Qin, Boqiang; Zhang, Dawen; Niu, Yuan; Zhang, Meng; Wang, Qing; Wu, Laiyan

    2012-01-01

    Lake Taihu is the third largest freshwater lake in China and is suffering from serious cyanobacterial blooms with the associated drinking water contamination by microcystin (MC) for millions of citizens. So far, most studies on MCs have been limited to two small bays, while systematic research on the whole lake is lacking. To explain the variations in MC concentrations during cyanobacterial bloom, a large-scale survey at 30 sites across the lake was conducted monthly in 2008. The health risks of MC exposure were high, especially in the northern area. Both Microcystis abundance and MC cellular quotas presented positive correlations with MC concentration in the bloom seasons, suggesting that the toxic risks during Microcystis proliferations were affected by variations in both Microcystis density and MC production per Microcystis cell. Use of a powerful predictive modeling tool named generalized additive model (GAM) helped visualize significant effects of abiotic factors related to carbon fixation and proliferation of Microcystis (conductivity, dissolved inorganic carbon (DIC), water temperature and pH) on MC cellular quotas from recruitment period of Microcystis to the bloom seasons, suggesting the possible use of these factors, in addition to Microcystis abundance, as warning signs to predict toxic events in the future. The interesting relationship between macrophytes and MC cellular quotas of Microcystis (i.e., high MC cellular quotas in the presence of macrophytes) needs further investigation.

  8. SDF-1 signaling via the CXCR4-TCR heterodimer requires PLC-β3 and PLC-γ1 for distinct cellular responses 1

    PubMed Central

    Kremer, Kimberly N.; Clift, Ian C.; Miamen, Alexander G.; Bamidele, Adebowale O.; Qian, Nan-Xin; Humphreys, Troy D.; Hedin, Karen E.

    2011-01-01

    The CXCR4 chemokine receptor is a G protein-coupled receptor (GPCR) that signals in T lymphocytes by forming a heterodimer with the T cell antigen receptor (TCR). CXCR4 and TCR functions are consequently highly cross-regulated, affecting T cell immune activation, cytokine secretion, and T cell migration. The CXCR4-TCR heterodimer stimulates T cell migration and activation of the ERK MAP kinase and downstream AP-1-dependent cytokine transcription in response to SDF-1, the sole chemokine ligand of CXCR4. These responses require Gi-type G proteins as well as TCR ITAM domains and the ZAP-70 tyrosine kinase, thus indicating that the CXCR4-TCR heterodimer signals to integrate GPCR-associated and TCR-associated signaling molecules in response to SDF-1. Yet, the phospholipase C (PLC) isozymes responsible for coupling the CXCR4-TCR heterodimer to distinct downstream cellular responses are incompletely characterized. Here, we demonstrate that PLC activity is required for SDF-1 to induce ERK activation, migration, and CXCR4 endocytosis in human T cells. SDF-1 signaling via the CXCR4-TCR heterodimer uses PLC-β3 to activate the Ras-ERK pathway and increase intracellular Ca2+ concentrations, while PLC-γ1 is dispensable for these outcomes. In contrast, PLC-γ1, but not PLC-β3, is required for SDF-1-mediated migration, via a mechanism independent of LAT. These results increase understanding of the signaling mechanisms employed by the CXCR4-TCR heterodimer, characterize new roles for PLC-β3 and PLC-γ1 in T cells, and suggest that multiple PLCs may also be activated downstream of other chemokine receptors in order to distinctly regulate migration versus other signaling functions. PMID:21705626

  9. Cellular and molecular mechanisms that mediate basal and tumour necrosis factor-α-induced regulation of myosin light chain kinase gene activity

    PubMed Central

    Ye, Dongmei; Ma, Thomas Y

    2008-01-01

    The patients with Crohn's disease (CD) have a ‘leaky gut’ manifested by an increase in intestinal epithelial tight junction (TJ) permeability. Tumour necrosis factor-α (TNF-α) is a proto-typical pro-inflammatory cytokine that plays a central role in intestinal inflammation of CD. An important pro-inflammatory action of TNF-α is to cause a functional opening of intestinal TJ barrier. Previous studies have shown that TNF-α increase in TJ permeability was regulated by an increase in myosin light chain kinase (MLCK) gene activity and protein expression. The major aim of this study was to elucidate the cellular and molecular mechanisms that mediate basal and TNF-α-induced increase in MLCK gene activity. By progressive 5′ deletion, minimal MLCK promoter was localized between −313 to +118 on MLCK promoter. A p53 binding site located within minimal promoter region was identified as an essential determinant for basal promoter activity. A 4 bp start site and a 5 bp downstream promoter element were required for MLCK gene activity. TNF-α-induced increase in MLCK promoter activity was mediated by NF-κB activation. There were eight κB binding sites on MLCK promoter. The NF-κB1 site at +48 to +57 mediated TNF-α-induced increase in MLCK promoter activity. The NF-κB2 site at −325 to −316 had a repressive role on promoter activity. The opposite effects on promoter activity were due to differences in the NF-κB dimer type binding to the κB sites. p50/p65 dimer preferentially binds to the NF-κB1 site and up-regulates promoter activity; while p50/p50 dimer preferentially binds to the NF-κB2 site and down-regulates promoter activity. In conclusion, we have identified the minimal MLCK promoter region, essential molecular determinants and molecular mechanisms that mediate basal and TNF-α-induced modulation of MLCK promoter activity in Caco-2 intestinal epithelial cells. These studies provide novel insight into the cellular and molecular mechanisms that regulate

  10. A Cellular Biophysics Textbook

    NASA Astrophysics Data System (ADS)

    Wilder, Alan Joseph

    2011-12-01

    In the past two decades, great advances have been made in understanding of the biophysical mechanisms of the protein machines that carry out the fundamental processes of the cell. It is now known that all major eukaryotic cellular processes require a complicated assemblage of proteins acting via a series of concerted motions. In order to grasp current understanding of cellular mechanisms, the new generation of cell biologists needs to be trained in the general characteristics of these cellular properties and the methods with which to study them. This cellular biophysics textbook, to be used in conjunction with the cellular biophysics course (MCB143) at UC-Davis, provides a great tool in the instruction of the new generation of cellular biologists. It provides a hierarchical view of the cell, from atoms to protein machines and explains in depth the mechanisms of cytoskeletal force generators as an example of these principles.

  11. Half pint/Puf68 is required for negative regulation of splicing by the SR splicing factor Transformer2.

    PubMed

    Wang, Shanzhi; Wagner, Eric J; Mattox, William

    2013-08-01

    The SR family of proteins plays important regulatory roles in the control of alternative splicing in a wide range of organisms. These factors affect splicing through both positive and negative controls of splice site recognition by pre-spliceosomal factors. Recent studies indicate that the Drosophila SR factor Transformer 2 (Tra2) activates and represses splicing through distinct and separable effector regions of the protein. While the interactions of its Arg-Ser-rich activator region have been well studied, cofactors involved in splicing repression have yet to be found. Here we use a luciferase-based splicing reporter assay to screen for novel proteins necessary for Tra2-dependent repression of splicing. This approach identified Half pint, also known as Puf68, as a co-repressor required for Tra2-mediated autoregulation of the M1 intron. In vivo, Half pint is required for Tra2-dependent repression of M1 splicing but is not necessary for Tra2-dependent activation of doublesex splicing. Further experiments indicate that the effect of Hfp is sequence-specific and that it associates with these target transcripts in cells. Importantly, known M1 splicing regulatory elements are sufficient to sensitize a heterologous intron to Hfp regulation. Two alternative proteins deriving from Hfp transcripts, Hfp68, and Hfp58, were found to be expressed in vivo but differed dramatically in their effect on M1 splicing. Comparison of the cellular localization of these forms in S2 cells revealed that Hfp68 is predominantly localized to the nucleus while Hfp58 is distributed across both the nucleus and cytoplasm. This accords with their observed effects on splicing and suggests that differential compartmentalization may contribute to the specificity of these isoforms. Together, these studies reveal a function for Half pint in splicing repression and demonstrate it to be specifically required for Tra2-dependent intron inclusion.

  12. Half Pint/Puf68 is required for negative regulation of splicing by the SR factor Transformer2

    PubMed Central

    Wang, Shanzhi; Wagner, Eric J; Mattox, William

    2013-01-01

    The SR family of proteins plays important regulatory roles in the control of alternative splicing in a wide range of organisms. These factors affect splicing through both positive and negative controls of splice site recognition by pre-spliceosomal factors. Recent studies indicate that the Drosophila SR factor Transformer 2 (Tra2) activates and represses splicing through distinct and separable effector regions of the protein. While the interactions of its Arg-Ser-rich activator region have been well studied, cofactors involved in splicing repression have yet to be found. Here we use a luciferase-based splicing reporter assay to screen for novel proteins necessary for Tra2-dependent repression of splicing. This approach identified Half pint, also known as Puf68, as a co-repressor required for Tra2-mediated autoregulation of the M1 intron. In vivo, Half pint is required for Tra2-dependent repression of M1 splicing but is not necessary for Tra2-dependent activation of doublesex splicing. Further experiments indicate that the effect of Hfp is sequence-specific and that it associates with these target transcripts in cells. Importantly, known M1 splicing regulatory elements are sufficient to sensitize a heterologous intron to Hfp regulation. Two alternative proteins deriving from Hfp transcripts, Hfp68, and Hfp58, were found to be expressed in vivo but differed dramatically in their effect on M1 splicing. Comparison of the cellular localization of these forms in S2 cells revealed that Hfp68 is predominantly localized to the nucleus while Hfp58 is distributed across both the nucleus and cytoplasm. This accords with their observed effects on splicing and suggests that differential compartmentalization may contribute to the specificity of these isoforms. Together, these studies reveal a function for Half pint in splicing repression and demonstrate it to be specifically required for Tra2-dependent intron inclusion. PMID:23880637

  13. Stem cell-dependent formation of a functional anterior regeneration pole in planarians requires Zic and Forkhead transcription factors.

    PubMed

    Vogg, Matthias C; Owlarn, Suthira; Pérez Rico, Yuvia A; Xie, Jianlei; Suzuki, Yoko; Gentile, Luca; Wu, Wei; Bartscherer, Kerstin

    2014-06-15

    Planarians can regenerate their head within days. This process depends on the direction of adult stem cells to wound sites and the orchestration of their progenitors to commit to appropriate lineages and to arrange into patterned tissues. We identified a zinc finger transcription factor, Smed-ZicA, as a downstream target of Smed-FoxD, a Forkhead transcription factor required for head regeneration. Smed-zicA and Smed-FoxD are co-expressed with the Wnt inhibitor notum and the Activin inhibitor follistatin in a cluster of cells at the anterior-most tip of the regenerating head - the anterior regeneration pole - and in surrounding stem cell progeny. Depletion of Smed-zicA and Smed-FoxD by RNAi abolishes notum and follistatin expression at the pole and inhibits head formation downstream of initial polarity decisions. We suggest a model in which ZicA and FoxD transcription factors synergize to control the formation of Notum- and Follistatin-producing anterior pole cells. Pole formation might constitute an early step in regeneration, resulting in a signaling center that orchestrates cellular events in the growing tissue.

  14. Requirement of epidermal growth factor receptor for hyperplasia induced by E5, a high-risk human papillomavirus oncogene.

    PubMed

    Genther Williams, Sybil M; Disbrow, Gary L; Schlegel, Richard; Lee, Daekee; Threadgill, David W; Lambert, Paul F

    2005-08-01

    Multicellular organisms rely on complex networks of signaling cascades for development, homeostasis, and responses to the environment. These networks involve diffusible signaling molecules, their receptors, and a variety of downstream effectors. Alterations in the expression or function of any one of these factors can contribute to disease, including cancer. Many viruses have been implicated in cancer, and some of these modulate cellular signal transduction cascades to carry out their life cycles. High-risk human papillomaviruses (HPVs), the causative agents of most cervical and anogenital cancers, encode three oncogenes. One of these, E5, has been postulated to transform cells in tissue culture by modulating growth factor receptors. In this study, we generate and characterize transgenic mice in which the E5 gene of the most common high-risk HPV, HPV16, is targeted to the basal layer of the stratified squamous epithelium. In these mice, E5 alters the growth and differentiation of stratified epithelia and induces epithelial tumors at a high frequency. Through the analysis of these mice, we show a requirement of the epidermal growth factor receptor for the hyperplastic properties of E5.

  15. Irregular Bedtime and Nocturnal Cellular Phone Usage as Risk Factors for Being Involved in Bullying: A Cross-Sectional Survey of Japanese Adolescents

    PubMed Central

    Tochigi, Mamoru; Nishida, Atsushi; Shimodera, Shinji; Oshima, Norihito; Inoue, Ken; Okazaki, Yuji; Sasaki, Tsukasa

    2012-01-01

    Purpose A number of studies have tried to identify risk factors for being involved in bullying in order to help developing preventive measures; however, to our knowledge, no study has investigated the effect of nocturnal lifestyle behavior such as sleep pattern or cellular phone usage. In the present study, we aimed to investigate the relationship between school bullying and sleep pattern or nocturnal cellular phone usage in adolescents. The effect of school size on school bullying was also examined. Methods Data from the cross-sectional survey of psychopathologies conducted for 19,436 Japanese students from 45 public junior high schools (7th–9th grade) and 28 senior high schools (10th–12th grade) were analyzed. Results Bullying status was significantly associated with irregular bedtime (OR = 1.23 and 1.41 for pure bullies and bully-victims, respectively) and e-mail exchange or calling after lights-out (OR = 1.53 and 1.31 for pure bullies and bully-victims, respectively) after controlling domestic violence and substance usage. In addition, school size was significantly associated with the increased risk of bullying in junior high school students (OR = 1.13 for bully-victims). Conclusions The present results suggested that sleep pattern and nocturnal cellular phone usage might be risk factors for being involved in school bullying in adolescents. Although further accumulation of data is needed, progressive trend towards nocturnal lifestyle and increasing usage of cellular phone might impair the well-being of adolescents. School-based interventions for lifestyle including sleep pattern and cellular phone usage may be encouraged to reduce school bullying. PMID:23029211

  16. USP17 is required for clathrin mediated endocytosis of epidermal growth factor receptor

    PubMed Central

    Jaworski, Jakub; de la Vega, Michelle; Fletcher, Sarah J.; McFarlane, Cheryl; Greene, Michelle K.; Smyth, Andrew W.; Van Schaeybroeck, Sandra; Johnston, James A.; Scott, Christopher J.; Rappoport, Joshua Z.; Burrows, James F.

    2014-01-01

    Previously we have shown that expression of the deubiquitinating enzyme USP17 is required for cell proliferation and motility. More recently we reported that USP17 deubiquitinates RCE1 isoform 2 and thus regulates the processing of ‘CaaX’ motif proteins. Here we now show that USP17 expression is induced by epidermal growth factor and that USP17 expression is required for clathrin mediated endocytosis of epidermal growth factor receptor. In addition, we show that USP17 is required for the endocytosis of transferrin, an archetypal substrate for clathrin mediated endocytosis, and that USP17 depletion impedes plasma membrane recruitment of the machinery required for clathrin mediated endocytosis. Thus, our data reveal that USP17 is necessary for epidermal growth factor receptor and transferrin endocytosis via clathrin coated pits, indicate this is mediated via the regulation of the recruitment of the components of the endocytosis machinery and suggest USP17 may play a general role in receptor endocytosis. PMID:25026282

  17. Postures and Motions Library Development for Verification of Ground Crew Human Factors Requirements

    NASA Technical Reports Server (NTRS)

    Stambolian, Damon; Henderson, Gena; Jackson, Mariea Dunn; Dischinger, Charles

    2013-01-01

    Spacecraft and launch vehicle ground processing activities require a variety of unique human activities. These activities are being documented in a primitive motion capture library. The library will be used by human factors engineering analysts to infuse real to life human activities into the CAD models to verify ground systems human factors requirements. As the primitive models are being developed for the library, the project has selected several current human factors issues to be addressed for the Space Launch System (SLS) and Orion launch systems. This paper explains how the motion capture of unique ground systems activities is being used to verify the human factors engineering requirements for ground systems used to process the SLS and Orion vehicles, and how the primitive models will be applied to future spacecraft and launch vehicle processing.

  18. Two distinct types of cellular mechanisms in the development of delayed hypersensitivity in mice: requirement of either mast cells or macrophages for elicitation of the response.

    PubMed Central

    Torii, I; Morikawa, S; Harada, T; Kitamura, Y

    1993-01-01

    Using mast cell-deficient mutant W/Wv mice and their normal counterpart we re-evaluated the significance of participation of mast cells in allergic inflammatory response. W/Wv mice developed immediate hypersensitivity (IH) footpad reaction (FPR) to a somewhat lesser degree than the normal mice, suggesting that the mast cell might amplify the response. To exert classical tuberculin (tbc) delayed-type hypersensitivity (DTH) mast cells were not an essential cellular component. Vasoactive amines were essential to develop the response, but it did not necessarily originate from mast cells. When mice were immunized with methylated human serum albumin (MHSA) emulsified in incomplete Freund's adjuvant (IFA), mast cells were required to elicit DTH FPR. This was confirmed by the lack of the response in W/Wv mice, and the restoration of FPR by local transplantation of mature mast cells into mutant mice. This mast cell-dependent (MD) DTH was different from tbc DTH as follows: mast cell dependency, macrophage dependency as revealed by ferritin sensitivity, kinetics of sensitization, effect of host's age and histopathology. Thus we concluded that there are two types of DTH in mice; one is macrophage-dependent tbc and the other is mast cell-dependent DTH. The correspondence of the DTH to the Jones-Mote (JM) DTH is discussed, although the dominance of mast cells in MD DTH lesion was not observed. Images Figure 2 Figure 4 PMID:8478030

  19. The Role of WD40-Repeat Protein 62 (MCPH2) in Brain Growth: Diverse Molecular and Cellular Mechanisms Required for Cortical Development.

    PubMed

    Shohayeb, Belal; Lim, Nicholas Rui; Ho, Uda; Xu, Zhiheng; Dottori, Mirella; Quinn, Leonie; Ng, Dominic Chi Hiung

    2017-09-22

    Genetic disruptions of spindle/centrosome-associated WD40-repeat protein 62 (WDR62) are causative for autosomal recessive primary microcephaly (MCPH) and a broader range of cortical malformations. Since the identification of WDR62 as encoded by the MCPH2 locus in 2010, recent studies that have deleted/depleted WDR62 in various animal models of cortical development have highlighted conserved functions in brain growth. Here, we provide a timely review of our current understanding of WDR62 contributions in the self-renewal, expansion and fate specification of neural stem and progenitor cells that are critical for neocortical development. Recent studies have revealed multiple functions for WDR62 in the regulation of spindle organization, mitotic progression and the duplication and biased inheritance of centrosomes during asymmetric divisions. We also discuss recently elaborated WDR62 interaction partners that include Aurora and c-Jun N-terminal kinases as part of complex signalling mechanisms that may define its neural functions. These studies provide new insights into the molecular and cellular processes that are required for brain formation and implicated in the genesis of primary microcephaly.

  20. [The electromagnetic fields of cellular phones and the health of children and of teenagers (the situation requiring to take an urgent measure)].

    PubMed

    Grigor'ev, Iu G

    2005-01-01

    The problem of the influence of electromagnetic fields (EMF) of cellular phones (CP) on the health of children and teenagers is considered in this article. The results of the researchs indicate the increased sensitivity of the children and of the teenagers to this kind of radiation. Direct indicators of electromagnetic influence can be infringement of sleep, decrease of the memory, fatigue, breach of a blood-brain barrier permeability, changes in nervous cells of a brain. As the remote consequences the development of tumors of a brain and acoustic nerve are predicted. However all these results require the realization of independent repeated researches. WHO (World Health Organization) recommends to use "Precautionary principle" with the purposes of decreasing of the risk. Russian National Committee of Non-Ionizing Radiation Protection recommended to limit the use of CP by children and teenagers under 16 years old (2002, February 2004). The corresponding recommendations were included into SunPin H2.1.8/2.2.4.1190-03/(2003).

  1. Cellular processing of the interleukin-2 fusion toxin DAB486-IL-2 and efficient delivery of diphtheria fragment A to the cytosol of target cells requires Arg194.

    PubMed

    Williams, D P; Wen, Z; Watson, R S; Boyd, J; Strom, T B; Murphy, J R

    1990-11-25

    We have used site-directed mutagenesis to examine the role played by Arg191, Arg193, and Arg194 of the fusion toxin DAB486-IL-2 in the intoxication of high affinity interleukin-2 receptor-bearing T-lymphocytes. These arginine residues are positioned in the proteolytically sensitive 14-amino acid loop subtended by the disulfide bond between Cys187 and Cys202 in this fusion toxin. DAB486-IL-2 was formed by the genetic substitution of the native diphtheria toxin receptor binding domain with human interleukin-2 (Williams, D.P., Parker, K., Bacha, P., Bishai, W., Borowski, M., Genbauffe, F., Strom, T.B., and Murphy, J.R. (1987) Protein Eng. 1, 493-498). We demonstrate that substitution of Arg194 with Gly results in a 1000-fold loss of DAB486-IL-2 potency. Since trypsin "nicking" of the Gly194 mutant restores biologic activity, we conclude that Arg194 is required for the cellular processing of the fusion toxin which results in the release of fragment A into the cytosol.

  2. Receptor complementation and mutagenesis reveal SR-BI as an essential HCV entry factor and functionally imply its intra- and extra-cellular domains.

    PubMed

    Dreux, Marlène; Dao Thi, Viet Loan; Fresquet, Judith; Guérin, Maryse; Julia, Zélie; Verney, Géraldine; Durantel, David; Zoulim, Fabien; Lavillette, Dimitri; Cosset, François-Loïc; Bartosch, Birke

    2009-02-01

    HCV entry into cells is a multi-step and slow process. It is believed that the initial capture of HCV particles by glycosaminoglycans and/or lipoprotein receptors is followed by coordinated interactions with the scavenger receptor class B type I (SR-BI), a major receptor of high-density lipoprotein (HDL), the CD81 tetraspanin, and the tight junction protein Claudin-1, ultimately leading to uptake and cellular penetration of HCV via low-pH endosomes. Several reports have indicated that HDL promotes HCV entry through interaction with SR-BI. This pathway remains largely elusive, although it was shown that HDL neither associates with HCV particles nor modulates HCV binding to SR-BI. In contrast to CD81 and Claudin-1, the importance of SR-BI has only been addressed indirectly because of lack of cells in which functional complementation assays with mutant receptors could be performed. Here we identified for the first time two cell types that supported HCVpp and HCVcc entry upon ectopic SR-BI expression. Remarkably, the undetectable expression of SR-BI in rat hepatoma cells allowed unambiguous investigation of human SR-BI functions during HCV entry. By expressing different SR-BI mutants in either cell line, our results revealed features of SR-BI intracellular domains that influence HCV infectivity without affecting receptor binding and stimulation of HCV entry induced by HDL/SR-BI interaction. Conversely, we identified positions of SR-BI ectodomain that, by altering HCV binding, inhibit entry. Finally, we characterized alternative ectodomain determinants that, by reducing SR-BI cholesterol uptake and efflux functions, abolish HDL-mediated infection-enhancement. Altogether, we demonstrate that SR-BI is an essential HCV entry factor. Moreover, our results highlight specific SR-BI determinants required during HCV entry and physiological lipid transfer functions hijacked by HCV to favor infection.

  3. Evaluation of cellular determinants required for in vitro xenotropic murine leukemia virus-related virus entry into human prostate cancer and noncancerous cells.

    PubMed

    Bhosle, Sushma; Suppiah, Suganthi; Molinaro, Ross; Liang, Yuying; Arnold, Rebecca; Diehl, William; Makarova, Natalia; Blackwell, Jerry; Petros, John; Liotta, Dennis; Hunter, Eric; Ly, Hinh

    2010-07-01

    The newly identified retrovirus-the xenotropic murine leukemia virus-related virus (XMRV)-has recently been shown to be strongly associated with familial prostate cancer in humans (A. Urisman et al., PLoS Pathog. 2:e25, 2006). While that study showed evidence of XMRV infection exclusively in the prostatic stromal fibroblasts, a recent study found XMRV protein antigens mainly in malignant prostate epithelial cells (R. Schlaberg et al., Proc. Natl. Acad. Sci. U. S. A. 106:16351-16356, 2009). To help elucidate the mechanisms behind XMRV infection, we show that prostatic fibroblast cells express Xpr1, a known receptor of XMRV, but its expression is absent in other cell lines of the prostate (i.e., epithelial and stromal smooth muscle cells). We also show that certain amino acid residues located within the predicted extracellular loop (ECL3 and ECL4) sequences of Xpr1 are required for efficient XMRV entry. Although we found strong evidence to support XMRV infection of prostatic fibroblast cell lines via Xpr1, we learned that XMRV was indeed capable of infecting cells that did not necessarily express Xpr1, such as those of the prostatic epithelial and smooth muscle origins. Further studies suggest that the expression of Xpr1 and certain genotypes of the RNASEL gene, which could restrict XMRV infection, may play important roles in defining XMRV tropisms in certain cell types. Collectively, our data reveal important cellular determinants required for XMRV entry into different human prostate cells in vitro, which may provide important insights into the possible role of XMRV as an etiologic agent in human prostate cancer.

  4. Fatty acid synthase as a factor required for exercise-induced cognitive enhancement and dentate gyrus cellular proliferation.

    PubMed

    Chorna, Nataliya E; Santos-Soto, Iván J; Carballeira, Nestor M; Morales, Joan L; de la Nuez, Janneliz; Cátala-Valentin, Alma; Chornyy, Anatoliy P; Vázquez-Montes, Adrinel; De Ortiz, Sandra Peña

    2013-01-01

    Voluntary running is a robust inducer of adult hippocampal neurogenesis. Given that fatty acid synthase (FASN), the key enzyme for de novo fatty acid biosynthesis, is critically involved in proliferation of embryonic and adult neural stem cells, we hypothesized that FASN could mediate both exercise-induced cell proliferation in the subgranular zone (SGZ) of the dentate gyrus (DG) and enhancement of spatial learning and memory. In 20 week-old male mice, voluntary running-induced hippocampal-specific upregulation of FASN was accompanied also by hippocampal-specific accumulation of palmitate and stearate saturated fatty acids. In experiments addressing the functional role of FASN in our experimental model, chronic intracerebroventricular (i.c.v.) microinfusions of C75, an irreversible FASN inhibitor, and significantly impaired exercise-mediated improvements in spatial learning and memory in the Barnes maze. Unlike the vehicle-injected mice, the C75 group adopted a non-spatial serial escape strategy and displayed delayed escape latencies during acquisition and memory tests. Furthermore, pharmacologic blockade of FASN function with C75 resulted in a significant reduction, compared to vehicle treated controls, of the number of proliferative cells in the DG of running mice as measured by immunoreactive to Ki-67 in the SGZ. Taken together, our data suggest that FASN plays an important role in exercise-mediated cognitive enhancement, which might be associated to its role in modulating exercise-induced stimulation of neurogenesis.

  5. Fatty Acid Synthase as a Factor Required for Exercise-Induced Cognitive Enhancement and Dentate Gyrus Cellular Proliferation

    PubMed Central

    Chorna, Nataliya E.; Santos-Soto, Iván J.; Carballeira, Nestor M.; Morales, Joan L.; de la Nuez, Janneliz; Cátala-Valentin, Alma; Chornyy, Anatoliy P.; Vázquez-Montes, Adrinel; De Ortiz, Sandra Peña

    2013-01-01

    Voluntary running is a robust inducer of adult hippocampal neurogenesis. Given that fatty acid synthase (FASN), the key enzyme for de novo fatty acid biosynthesis, is critically involved in proliferation of embryonic and adult neural stem cells, we hypothesized that FASN could mediate both exercise-induced cell proliferation in the subgranular zone (SGZ) of the dentate gyrus (DG) and enhancement of spatial learning and memory. In 20 week-old male mice, voluntary running-induced hippocampal-specific upregulation of FASN was accompanied also by hippocampal-specific accumulation of palmitate and stearate saturated fatty acids. In experiments addressing the functional role of FASN in our experimental model, chronic intracerebroventricular (i.c.v.) microinfusions of C75, an irreversible FASN inhibitor, and significantly impaired exercise-mediated improvements in spatial learning and memory in the Barnes maze. Unlike the vehicle-injected mice, the C75 group adopted a non-spatial serial escape strategy and displayed delayed escape latencies during acquisition and memory tests. Furthermore, pharmacologic blockade of FASN function with C75 resulted in a significant reduction, compared to vehicle treated controls, of the number of proliferative cells in the DG of running mice as measured by immunoreactive to Ki-67 in the SGZ. Taken together, our data suggest that FASN plays an important role in exercise-mediated cognitive enhancement, which might be associated to its role in modulating exercise-induced stimulation of neurogenesis. PMID:24223732

  6. The interaction of hepatitis A virus (HAV) with soluble forms of its cellular receptor 1 (HAVCR1) share the physiological requirements of infectivity in cell culture

    PubMed Central

    Silberstein, Erica; Konduru, Krishnamurthy; Kaplan, Gerardo G

    2009-01-01

    Background Hepatitis A virus (HAV), an atypical Picornaviridae that causes acute hepatitis in humans, usurps the HAV cellular receptor 1 (HAVCR1) to infect cells. HAVCR1 is a class 1 integral membrane glycoprotein that contains two extracellular domains: a virus-binding immunoglobulin-like (IgV) domain and a mucin-like domain that extends the IgV from the cell membrane. Soluble forms of HAVCR1 bind, alter, and neutralize cell culture-adapted HAV, which is attenuated for humans. However, the requirements of the HAV-HAVCR1 interaction have not been fully characterized, and it has not been determined whether HAVCR1 also serves as a receptor for wild-type (wt) HAV. Here, we used HAV soluble receptor neutralization and alteration assays to study the requirements of the HAV-HAVCR1 interaction and to determine whether HAVCR1 is also a receptor for wt HAV. Results Treatment of HAV with a soluble form of HAVCR1 that contained the IgV and two-thirds of the mucin domain fused to the Fc fragment of human IgG1 (D1 muc-Fc), altered particles at 37°C but left a residual level of unaltered particles at 4°C. The kinetics of neutralization of HAV by D1 muc-Fc was faster at 37°C than at 4°C. Alteration of HAV particles by D1 muc-Fc required Ca, which could not be replaced by Li, Na, Mg, Mn, or Zn. Neutralization of HAV by D1 muc-Fc occurred at pH 5 to 8 but was more efficient at pH 6 to 7. D1 muc-Fc neutralized wt HAV as determined by a cell culture system that allows the growth of wt HAV. Conclusion The interaction of HAV with soluble forms of HAVCR1 shares the temperature, Ca, and pH requirements for infectivity in cell culture and therefore mimics the cell entry process of HAV. Since soluble forms of HAVCR1 also neutralized wt HAV, this receptor may play a significant role in pathogenesis of HAV. PMID:19860892

  7. Factors determining insulin requirements in women with type 1 diabetes mellitus during pregnancy: a review

    PubMed Central

    McIntyre, Harold David; Callaway, Leonie

    2014-01-01

    Most women with type 1 diabetes mellitus (T1DM) have increased insulin requirements during pregnancy. However, a minority of women have a fall in insulin requirements. When this occurs in late gestation, it often provokes concern regarding possible compromise of the feto-placental unit. In some centres, this is considered as an indication for delivery, including premature delivery. There are, however, many other factors that affect insulin requirements in pregnancy in women with type 1 diabetes mellitus and the decline in insulin requirements may represent a variant of normal pregnancy. If there is no underlying pathological process, expedited delivery in these women is not warranted and confers increased risks to the newborn. We will explore the factors affecting insulin requirements in gestation in this review. We will also discuss some novel concepts regarding beta-cell function in pregnancy. PMID:27512424

  8. Epstein-Barr virus-encoded EBNA1 enhances RNA polymerase III-dependent EBER expression through induction of EBER-associated cellular transcription factors.

    PubMed

    Owen, Thomas J; O'Neil, John D; Dawson, Christopher W; Hu, Chunfang; Chen, Xiaoyi; Yao, Yunhong; Wood, Victoria H J; Mitchell, Louise E; White, Robert J; Young, Lawrence S; Arrand, John R

    2010-09-15

    Epstein-Barr Virus (EBV)-encoded RNAs (EBERs) are non-polyadenylated RNA molecules transcribed from the EBV genome by RNA polymerase III (pol III). EBERs are the most abundant viral latent gene products, although the precise mechanisms by which EBV is able to achieve such high levels of EBER expression are not fully understood. Previously EBV has been demonstrated to induce transcription factors associated with EBER expression, including pol III transcription factors and ATF-2. We have recently demonstrated that EBV-encoded nuclear antigen-1 (EBNA1) induces cellular transcription factors, and given these findings, we investigated the role of EBNA1 in induction of EBER-associated transcription factors. Our data confirm that in epithelial cells EBNA1 can enhance cellular pol III transcription. Transient expression of EBNA1 in Ad/AH cells stably expressing the EBERs led to induction of both EBER1 and EBER2 and conversely, expression of a dominant negative EBNA1 led to reduced EBER expression in EBV-infected Ad/AH cells. EBNA1 can induce transcription factors used by EBER genes, including TFIIIC, ATF-2 and c-Myc. A variant chromatin precipitation procedure showed that EBNA1 is associated with the promoters of these genes but not with the promoters of pol III-transcribed genes, including the EBERs themselves. Using shRNA knock-down, we confirm the significance of both ATF-2 and c-Myc in EBER expression. Further, functional induction of a c-Myc fusion protein led to increased EBER expression, providing c-Myc binding sites upstream of EBER1 were intact. In vivo studies confirm elevated levels of the 102 kD subunit of TFIIIC in the tumour cells of EBV-positive nasopharyngeal carcinoma biopsies. Our findings reveal that EBNA1 is able to enhance EBER expression through induction of cellular transcription factors and add to the repertoire of EBNA1's transcription-regulatory properties.

  9. Cellular Transcription Factors Induced in Trigeminal Ganglia during Dexamethasone-Induced Reactivation from Latency Stimulate Bovine Herpesvirus 1 Productive Infection and Certain Viral Promoters

    PubMed Central

    Workman, Aspen; Eudy, James; Smith, Lynette; Frizzo da Silva, Leticia; Sinani, Devis; Bricker, Halie; Cook, Emily; Doster, Alan

    2012-01-01

    Bovine herpesvirus 1 (BHV-1), an alphaherpesvirinae subfamily member, establishes latency in sensory neurons. Elevated corticosteroid levels, due to stress, reproducibly triggers reactivation from latency in the field. A single intravenous injection of the synthetic corticosteroid dexamethasone (DEX) to latently infected calves consistently induces reactivation from latency. Lytic cycle viral gene expression is detected in sensory neurons within 6 h after DEX treatment of latently infected calves. These observations suggested that DEX stimulated expression of cellular genes leads to lytic cycle viral gene expression and productive infection. In this study, a commercially available assay—Bovine Gene Chip—was used to compare cellular gene expression in the trigeminal ganglia (TG) of calves latently infected with BHV-1 versus DEX-treated animals. Relative to TG prepared from latently infected calves, 11 cellular genes were induced more than 10-fold 3 h after DEX treatment. Pentraxin three, a regulator of innate immunity and neurodegeneration, was stimulated 35- to 63-fold after 3 or 6 h of DEX treatment. Two transcription factors, promyelocytic leukemia zinc finger (PLZF) and Slug were induced more than 15-fold 3 h after DEX treatment. PLZF or Slug stimulated productive infection 20- or 5-fold, respectively, and Slug stimulated the late glycoprotein C promoter more than 10-fold. Additional DEX-induced transcription factors also stimulated productive infection and certain viral promoters. These studies suggest that DEX-inducible cellular transcription factors and/or signaling pathways stimulate lytic cycle viral gene expression, which subsequently leads to successful reactivation from latency in a small subset of latently infected neurons. PMID:22190728

  10. Novel vascular endothelial growth factor blocker improves cellular viability and reduces hypobaric hypoxia-induced vascular leakage and oedema in rat brain.

    PubMed

    Saraswat, Deepika; Nehra, Sarita; Chaudhary, Kamal; CVS, Siva Prasad

    2015-05-01

    Vascular endothelial growth factor (VEGF) is an important cerebral angiogenic and permeability factor under hypoxia. There is a need to find effective molecules that may ameliorate hypoxia-induced cerebral oedema. In silico identification of novel candidate molecules that block VEGF-A site were identified and validated with a Ramachandran plot. The active site residues of VEGF-A were detected by Pocketfinder, CASTp, and DogSiteScorer. Based on in silico data, three VEGF-A blocker (VAB) candidate molecules (VAB1, VAB2, and VAB3) were checked for improvement in cellular viability and regulation of VEGF levels in N2a cells under hypoxia (0.5% O2 ). Additionally, the best candidate molecule's efficacy was assessed in male Sprague-Dawley rats for its ameliorative effect on cerebral oedema and vascular leakage under hypobaric hypoxia 7260 m. All experimental results were compared with the commercially available VEGF blocker sunitinib. Vascular endothelial growth factor-A blocker 1 was found most effective in increasing cellular viability and maintaining normal VEGF levels under hypoxia (0.5% oxygen) in N2a cells. Vascular endothelial growth factor-A blocker 1 effectively restored VEGF levels, decreased cerebral oedema, and reduced vascular leakage under hypobaric hypoxia when compared to sunitinib-treated rats. Vascular endothelial growth factor-A blocker 1 may be a promising candidate molecule for ameliorating hypobaric hypoxia-induced vasogenic oedema by regulating VEGF levels. © 2015 Wiley Publishing Asia Pty Ltd.

  11. No Effect of the Transforming Growth Factor {beta}1 Promoter Polymorphism C-509T on TGFB1 Gene Expression, Protein Secretion, or Cellular Radiosensitivity

    SciTech Connect

    Reuther, Sebastian; Metzke, Elisabeth; Bonin, Michael; Petersen, Cordula; Dikomey, Ekkehard; Raabe, Annette

    2013-02-01

    Purpose: To study whether the promoter polymorphism (C-509T) affects transforming growth factor {beta}1 gene (TGFB1) expression, protein secretion, and/or cellular radiosensitivity for both human lymphocytes and fibroblasts. Methods and Materials: Experiments were performed with lymphocytes taken either from 124 breast cancer patients or 59 pairs of normal monozygotic twins. We used 15 normal human primary fibroblast strains as controls. The C-509T genotype was determined by polymerase chain reaction-restriction fragment length polymorphism or TaqMan single nucleotide polymorphism (SNP) genotyping assay. The cellular radiosensitivity of lymphocytes was measured by G0/1 assay and that of fibroblasts by colony assay. The amount of extracellular TGFB1 protein was determined by enzyme-linked immunosorbent assay, and TGFB1 expression was assessed via microarray analysis or reverse transcription-polymerase chain reaction. Results: The C-509T genotype was found not to be associated with cellular radiosensitivity, neither for lymphocytes (breast cancer patients, P=.811; healthy donors, P=.181) nor for fibroblasts (P=.589). Both TGFB1 expression and TGFB1 protein secretion showed considerable variation, which, however, did not depend on the C-509T genotype (protein secretion: P=.879; gene expression: lymphocytes, P=.134, fibroblasts, P=.605). There was also no general correlation between TGFB1 expression and cellular radiosensitivity (lymphocytes, P=.632; fibroblasts, P=.573). Conclusion: Our data indicate that any association between the SNP C-509T of TGFB1 and risk of normal tissue toxicity cannot be ascribed to a functional consequence of this SNP, either on the level of gene expression, protein secretion, or cellular radiosensitivity.

  12. Prion protein is required for tumor necrosis factor alpha (TNFα)-triggered nuclear factor kappa B (NF-κB) signaling and cytokine production.

    PubMed

    Wu, Gui-Ru; Mu, Tian-Chen; Gao, Zhen-Xing; Wang, Jun; Sy, Man-Sun; Li, Chao-Yang

    2017-09-12

    The expression of normal cellular prion protein (PrP) is required for the pathogenesis of prion diseases. However, the physiological functions of PrP remain ambiguous. Here, we identified PrP as being critical for tumor necrosis factor (TNF)α-triggered signaling in a human melanoma cell line, M2, and a pancreatic ductal cell adenocarcinoma cell line, BxPC-3. In M2 cells, TNFα upregulates the expression of p-I-kappa-B-kinase α/β (p-IKKα/β), p-p65, and p-JNK, but downregulates the IκBα protein, all of which are downstream signaling intermediates in the TNF receptor signaling cascade. When PRNP is deleted in M2 cells, the effects of TNFα are no longer detectable. More importantly, p-p65 and p-JNK responses are restored when PRNP is reintroduced into the PRNP null cells. TNFα also activates NF-κB and increases TNFα production in wildtype M2 cells, but not in PrP-null M2 cells. Similar results are obtained in the BxPC-3 cells. Moreover, TNFα activation of NF-κB requires ubiquitination of receptor-interacting serine/threonine kinase 1 (RIP1) and TNF receptor-associated factor 2 (TRAF2). TNFα treatment increases the binding between PrP and the deubiquitinase tumor suppressor cylindromatosis (CYLD), in these treated cells, binding of CYLD to RIP1 and TRAF2 is reduced. We conclude that PrP traps CYLD, preventing it from binding and deubiquitinating RIP1 and TRAF2. Our findings reveal that PrP enhances the responses to TNFα, promoting proinflammatory cytokine production, which may contribute to inflammation and tumorigenesis. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  13. The Cys-Rich Region of Hepatitis A Virus Cellular Receptor 1 Is Required for Binding of Hepatitis A Virus and Protective Monoclonal Antibody 190/4

    PubMed Central

    Thompson, Peter; Lu, Jinhua; Kaplan, Gerardo G.

    1998-01-01

    The hepatitis A virus cellular receptor 1 (HAVcr-1) cDNA codes for a class I integral membrane glycoprotein, termed havcr-1, of unknown natural function which serves as an African green monkey kidney (AGMK) cell receptor for HAV. The extracellular domain of havcr-1 has an N-terminal Cys-rich region that displays homology with sequences of members of the immunoglobulin superfamily, followed by a Thr/Ser/Pro (TSP)-rich region characteristic of mucin-like O-glycosylated proteins. The havcr-1 glycoprotein contains four putative N-glycosylation sites, two in the Cys-rich region and two in the TSP-rich region. To characterize havcr-1 and define region(s) involved in HAV receptor function, we expressed the TSP-rich region in Escherichia coli fused to glutathione S-transferase and generated antibodies (Ab) in rabbits (anti-GST2 Ab). Western blot analysis with anti-GST2 Ab detected 62- and 65-kDa bands in AGMK cells and 59-, 62-, and 65-kDa bands in dog cells transfected with the HAVcr-1 cDNA (cr5 cells) but not in dog cells transfected with the vector alone (DR2 cells). Treatment of AGMK and cr5 cell extracts with peptide-N-glycosidase F resulted in the collapse of the havcr-1-specific bands into a single band of 56 kDa, which indicated that different N-glycosylated forms of havcr-1 were expressed in these cells. Treatment of AGMK and cr5 cells with tunicamycin reduced binding of protective monoclonal Ab (MAb) 190/4, which suggested that N-glycans are required for binding of MAb 190/4 to havcr-1. To test this hypothesis, havcr-1 mutants lacking the N-glycosylation motif at the first site (mut1), second site (mut2), and both (mut3) sites were constructed and transfected into dog cells. Binding of MAb 190/4 and HAV to mut1 and mut3 cells was highly reduced, while binding to mut2 cells was not affected and binding to dog cells expressing an havcr-1 construct containing a deletion of the Cys-rich region (d1− cells) was undetectable. HAV-infected cr5 and mut2 cells but not

  14. The Cys-rich region of hepatitis A virus cellular receptor 1 is required for binding of hepatitis A virus and protective monoclonal antibody 190/4.

    PubMed

    Thompson, P; Lu, J; Kaplan, G G

    1998-05-01

    The hepatitis A virus cellular receptor 1 (HAVcr-1) cDNA codes for a class I integral membrane glycoprotein, termed havcr-1, of unknown natural function which serves as an African green monkey kidney (AGMK) cell receptor for HAV. The extracellular domain of havcr-1 has an N-terminal Cys-rich region that displays homology with sequences of members of the immunoglobulin superfamily, followed by a Thr/Ser/Pro (TSP)-rich region characteristic of mucin-like O-glycosylated proteins. The havcr-1 glycoprotein contains four putative N-glycosylation sites, two in the Cys-rich region and two in the TSP-rich region. To characterize havcr-1 and define region(s) involved in HAV receptor function, we expressed the TSP-rich region in Escherichia coli fused to glutathione S-transferase and generated antibodies (Ab) in rabbits (anti-GST2 Ab). Western blot analysis with anti-GST2 Ab detected 62- and 65-kDa bands in AGMK cells and 59-, 62-, and 65-kDa bands in dog cells transfected with the HAVcr-1 cDNA (cr5 cells) but not in dog cells transfected with the vector alone (DR2 cells). Treatment of AGMK and cr5 cell extracts with peptide-N-glycosidase F resulted in the collapse of the havcr-1-specific bands into a single band of 56 kDa, which indicated that different N-glycosylated forms of havcr-1 were expressed in these cells. Treatment of AGMK and cr5 cells with tunicamycin reduced binding of protective monoclonal Ab (MAb) 190/4, which suggested that N-glycans are required for binding of MAb 190/4 to havcr-1. To test this hypothesis, havcr-1 mutants lacking the N-glycosylation motif at the first site (mut1), second site (mut2), and both (mut3) sites were constructed and transfected into dog cells. Binding of MAb 190/4 and HAV to mut1 and mut3 cells was highly reduced, while binding to mut2 cells was not affected and binding to dog cells expressing an havcr-1 construct containing a deletion of the Cys-rich region (d1- cells) was undetectable. HAV-infected cr5 and mut2 cells but not mut1

  15. Beyond transcription factors: The role of chromatin modifying enzymes in regulating transcription required for memory

    PubMed Central

    Barrett, Ruth M.; Wood, Marcelo A.

    2008-01-01

    One of the alluring aspects of examining chromatin modifications in the role of modulating transcription required for long-term memory processes is that these modifications may provide transient and potentially stable epigenetic marks in the service of activating and/or maintaining transcriptional processes. These, in turn, may ultimately participate in the molecular mechanisms required for neuronal changes subserving long-lasting changes in behavior. As an epigenetic mechanism of transcriptional control, chromatin modification has been shown to participate in maintaining cellular memory (e.g., cell fate) and may underlie the strengthening and maintenance of synaptic connections required for long-term changes in behavior. Epigenetics has become central to several fields of neurobiology, where researchers have found that regulation of chromatin modification has a significant role in epilepsy, drug addiction, depression, neurodegenerative diseases, and memory. In this review, we will discuss the role of chromatin modifying enzymes in memory processes, as well as how recent studies in yeast genetics and cancer biology may impact the way we think about how chromatin modification and chromatin remodeling regulate neuronal function. PMID:18583646

  16. 34 CFR 373.6 - What are the priorities and other factors and requirements for competitions?

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 34 Education 2 2011-07-01 2010-07-01 true What are the priorities and other factors and requirements for competitions? 373.6 Section 373.6 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF SPECIAL EDUCATION AND REHABILITATIVE SERVICES, DEPARTMENT OF...

  17. 34 CFR 373.6 - What are the priorities and other factors and requirements for competitions?

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 34 Education 2 2012-07-01 2012-07-01 false What are the priorities and other factors and requirements for competitions? 373.6 Section 373.6 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF SPECIAL EDUCATION AND REHABILITATIVE SERVICES, DEPARTMENT OF...

  18. 34 CFR 373.6 - What are the priorities and other factors and requirements for competitions?

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 34 Education 2 2014-07-01 2013-07-01 true What are the priorities and other factors and requirements for competitions? 373.6 Section 373.6 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF SPECIAL EDUCATION AND REHABILITATIVE SERVICES, DEPARTMENT OF...

  19. 34 CFR 373.6 - What are the priorities and other factors and requirements for competitions?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 34 Education 2 2010-07-01 2010-07-01 false What are the priorities and other factors and requirements for competitions? 373.6 Section 373.6 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF SPECIAL EDUCATION AND REHABILITATIVE SERVICES, DEPARTMENT OF EDUCATION...

  20. Cultured fibroblast monolayers secrete a protein that alters the cellular binding of somatomedin-C/insulinlike growth factor I

    SciTech Connect

    Clemmons, D.R.; Elgin, R.G.; Han, V.K.; Casella, S.J.; D'Ercole, A.J.; Van Wyk, J.J.

    1986-05-01

    We studied somatomedin-C/insulinlike growth factor (Sm-C/IGF-I) binding to human fibroblasts in both adherent monolayers and in suspension cultures. The addition of Sm-C/IGF-I in concentrations between 0.5 and 10 ng/ml to monolayers cultures resulted in a paradoxical increase in /sup 125/I-Sm-C/IGF-I binding and concentrations between 25 and 300 ng/ml were required to displace the labeled peptide. The addition of unlabeled insulin resulted in no displacement of labeled Sm-C/IGF-I from the adherent cells. When fibroblast suspensions were used Sm-C/IGF-I concentrations between 1 and 10 ng/ml caused displacement, the paradoxical increase in /sup 125/I-Sm-C/IGF-I binding was not detected, and insulin displaced 60% of the labeled peptide. Affinity cross-linking to fibroblast monolayers revealed a 43,000-mol wt /sup 125/I-Sm-C-binding-protein complex that was not detected after cross-linking to suspended cells. The 43,000-mol wt complex was not detected after cross-linking to smooth muscle cell monolayers, and binding studies showed that /sup 125/I-Sm-C/IGF-I was displaced greater than 90% by Sm-C/IGF-I using concentrations between 0.5 and 10 ng/ml. Because fibroblast-conditioned medium contains the 43,000-mol wt complex, smooth muscle cells were incubated with conditioned medium for 24 h prior to initiation of the binding studies. /sup 125/I-Sm-C/IGF-I-binding increased 1.6-fold compared to control cultures and after cross-linking the 43,000-mol wt complex could be detected on the smooth muscle cell surface. Human fibroblast monolayers secrete a protein that binds /sup 125/I-Sm-C/IGF-I which can be transferred to the smooth muscle cell surface and alters 125I-Sm-C/IGF-I binding.

  1. Molecular and cellular basis for the unique functioning of Nrf1, an indispensable transcription factor for maintaining cell homoeostasis and organ integrity.

    PubMed

    Zhang, Yiguo; Xiang, Yuancai

    2016-04-15

    The consensuscis-regulatory AP-1 (activator protein-1)-like AREs (antioxidant-response elements) and/or EpREs (electrophile-response elements) allow for differential recruitment of Nrf1 [NF-E2 (nuclear factor-erythroid 2)-related factor 1], Nrf2 and Nrf3, together with each of their heterodimeric partners (e.g. sMaf, c-Jun, JunD or c-Fos), to regulate different sets of cognate genes. Among them, NF-E2 p45 and Nrf3 are subject to tissue-specific expression in haemopoietic and placental cell lineages respectively. By contrast, Nrf1 and Nrf2 are two important transcription factors expressed ubiquitously in various vertebrate tissues and hence may elicit putative combinational or competitive functions. Nevertheless, they have de facto distinct biological activities because knockout of their genes in mice leads to distinguishable phenotypes. Of note, Nrf2 is dispensable during development and growth, albeit it is accepted as a master regulator of antioxidant, detoxification and cytoprotective genes against cellular stress. Relative to the water-soluble Nrf2, less attention has hitherto been drawn to the membrane-bound Nrf1, even though it has been shown to be indispensable for embryonic development and organ integrity. The biological discrepancy between Nrf1 and Nrf2 is determined by differences in both their primary structures and topovectorial subcellular locations, in which they are subjected to distinct post-translational processing so as to mediate differential expression of ARE-driven cytoprotective genes. In the present review, we focus on the molecular and cellular basis for Nrf1 and its isoforms, which together exert its essential functions for maintaining cellular homoeostasis, normal organ development and growth during life processes. Conversely, dysfunction of Nrf1 results in spontaneous development of non-alcoholic steatohepatitis, hepatoma, diabetes and neurodegenerative diseases in animal models. © 2016 The Author(s). Published by Portland Press Limited

  2. Stem Cell Therapies as an Emerging Paradigm in Stroke (STEPS): bridging basic and clinical science for cellular and neurogenic factor therapy in treating stroke.

    PubMed

    2009-02-01

    Investigators developing cellular therapy for stroke face many challenges. Preclinical models used for cellular therapy studies should be relevant to human stroke and predictive of benefit despite differences in stroke size, cerebrovascular anatomy, immune status, and neurological responses. Translating preclinical testing to human trials is compounded by consideration of delivery method and translation of dosing with cell survival. Many issues must be approached in designing clinical trials of cellular therapy for stroke, including appropriate outcome measures, controlling for confounding factors such as rehabilitation therapy, and possible surrogate outcomes using imaging such as MRI and newer imaging techniques. It is also important to establish standardized clinical protocols and clinical database registries in advance of early proof-of-concept studies. Investigators must adopt a standardized nomenclature and characterization schema for cell products to accurately define potency and determine clinical outcome from early proof-of-concept studies. The Stem Cell Therapies as an Emerging Paradigm in Stroke (STEPS) meeting was organized to bring together clinical and basic researchers with industry and regulatory representatives to assess the critical issues in the field and to create a framework to guide future investigations.

  3. Cellular density, a major factor involved in PDT cytotoxic responses: Study on three different lines of human retinoblastoma grafted on nude mice.

    PubMed

    Thomas, Carole D; Poyer, Florent; Maillard, Philippe; Chauvin, Benoit; Lupu, Mihaela; Mispelter, Joël

    2015-06-01

    PDT represents a very localized and non-mutagen antitumoral treatment using a photosensitive molecule (porphyrin family) light activated. The first way of cell damage is a direct one, active on the very site where ROSs have been produced. The second one is indirect by activating and transmitting the processes of cellular death signaling. In order to seek for a better characterization of the photo-biology involved in in vivo PDT and to better understand the differences on the treatment outcome, we have used three different human retinoblastomas xenografted on mice. Mice were treated according to the double targeting protocol exposed in a previous paper. One i.v. dose (0.6 mg/kg) of PS was followed by a second dose, separated by a 3 h interval (double targeting PDT). As a consequence both cancer cells and blood vessels were targeted. The treatment was repeated two times, at 4 days interval. First of all, sodium MRI revealed qualitative differences in the sodium average content of the three retinoblastoma lines before treatment. After the PDT treatments the tumor responses were different between the lines as revealed by sodium MRI and later on by histology. We have put into evidence that PDT is accompanied by a bystander effect that may propagate the cellular death triggered by the initial photoreaction. This effect is highly dependent on the cellular density of the tissue; therefore this factor is to be taken into account in clinical PDT protocols. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Hunting for huntingtin associated factors: Identification and characterization of huntingtin expanded polyglutamine aggregate associated factors and their impact on Huntington disease model cellular toxicity

    DTIC Science & Technology

    2016-05-20

    Hunting for huntingtin associated factors: Identification and characterization of Huntingtin expanded polyglutamine aggregate- associated ... associated factors: Identification and characterization of huntingtin expanded polyglutamine aggregate- associated factors and their impact on Huntington...Signature] Maggie P. Wear Maggie P. Wear [May 20, 2016] vii ABSTRACT Hunting for huntingtin associated factors: Identification and

  5. Risk factors for uterine atony/postpartum hemorrhage requiring treatment after vaginal delivery.

    PubMed

    Wetta, Luisa A; Szychowski, Jeff M; Seals, Samantha; Mancuso, Melissa S; Biggio, Joseph R; Tita, Alan T N

    2013-07-01

    We sought to identify risk factors for uterine atony or hemorrhage. We conducted a secondary analysis of a 3-arm double-blind randomized trial of different dose regimens of oxytocin to prevent uterine atony after vaginal delivery. The primary outcome was uterine atony or hemorrhage requiring treatment. In all, 21 potential risk factors were evaluated. Logistic regression was used to identify independent risk factors using 2 complementary predefined model selection strategies. Among 1798 women randomized to 10, 40, or 80 U of prophylactic oxytocin after vaginal delivery, treated uterine atony occurred in 7%. Hispanic (odds ratio [OR], 2.1; 95% confidence interval [CI], 1.3-3.4), non-Hispanic white (OR, 1.6; 95% CI, 1.0-2.5), preeclampsia (OR, 3.2; 95% CI, 2.0-4.9), and chorioamnionitis (OR, 2.8; 95% CI, 1.6-5.0) were consistent independent risk factors. Other risk factors based on the specified selection strategies were obesity, induction/augmentation of labor, twins, hydramnios, anemia, and arrest of descent. Amnioinfusion appeared to be protective against uterine atony (OR, 0.53; 95% CI, 0.29-0.98). Independent risk factors for uterine atony requiring treatment include Hispanic and non-Hispanic white ethnicity, preeclampsia, and chorioamnionitis. Copyright © 2013 Mosby, Inc. All rights reserved.

  6. Epstein-Barr virus-encoded EBNA1 enhances RNA polymerase III-dependent EBER expression through induction of EBER-associated cellular transcription factors

    PubMed Central

    2010-01-01

    Background Epstein-Barr Virus (EBV)-encoded RNAs (EBERs) are non-polyadenylated RNA molecules transcribed from the EBV genome by RNA polymerase III (pol III). EBERs are the most abundant viral latent gene products, although the precise mechanisms by which EBV is able to achieve such high levels of EBER expression are not fully understood. Previously EBV has been demonstrated to induce transcription factors associated with EBER expression, including pol III transcription factors and ATF-2. We have recently demonstrated that EBV-encoded nuclear antigen-1 (EBNA1) induces cellular transcription factors, and given these findings, we investigated the role of EBNA1 in induction of EBER-associated transcription factors. Results Our data confirm that in epithelial cells EBNA1 can enhance cellular pol III transcription. Transient expression of EBNA1 in Ad/AH cells stably expressing the EBERs led to induction of both EBER1 and EBER2 and conversely, expression of a dominant negative EBNA1 led to reduced EBER expression in EBV-infected Ad/AH cells. EBNA1 can induce transcription factors used by EBER genes, including TFIIIC, ATF-2 and c-Myc. A variant chromatin precipitation procedure showed that EBNA1 is associated with the promoters of these genes but not with the promoters of pol III-transcribed genes, including the EBERs themselves. Using shRNA knock-down, we confirm the significance of both ATF-2 and c-Myc in EBER expression. Further, functional induction of a c-Myc fusion protein led to increased EBER expression, providing c-Myc binding sites upstream of EBER1 were intact. In vivo studies confirm elevated levels of the 102 kD subunit of TFIIIC in the tumour cells of EBV-positive nasopharyngeal carcinoma biopsies. Conclusions Our findings reveal that EBNA1 is able to enhance EBER expression through induction of cellular transcription factors and add to the repertoire of EBNA1's transcription-regulatory properties. PMID:20843307

  7. Paradoxical interplay of viral and cellular functions.

    PubMed

    Domingo, Esteban

    2011-03-01

    Some cellular editing functions can restrict the replication of some viruses but contribute to completion of the life cycle of others. A recent study has identified an isoform of the adenosine deaminase acting on RNA type 1 (ADAR 1) as required for embryogenesis, and as a restriction factor for a number of important RNA virus pathogens. The dual implication of key cellular functions in the innate immunity against viruses, or, paradoxically, as mediators of virus replication is interpreted in the light of the concept of virus-host coevolution and tinkering proposed for general evolution by François Jacob decades ago.

  8. Human Factors Engineering Requirements for the International Space Station - Successes and Challenges

    NASA Technical Reports Server (NTRS)

    Whitmore, M.; Blume, J.

    2003-01-01

    Advanced technology coupled with the desire to explore space has resulted in increasingly longer human space missions. Indeed, any exploration mission outside of Earth's neighborhood, in other words, beyond the moon, will necessarily be several months or even years. The International Space Station (ISS) serves as an important advancement toward executing a successful human space mission that is longer than a standard trip around the world or to the moon. The ISS, which is a permanently occupied microgravity research facility orbiting the earth, will support missions four to six months in duration. In planning for the ISS, the NASA developed an agency-wide set of human factors standards for the first time in a space exploration program. The Man-Systems Integration Standard (MSIS), NASA-STD-3000, a multi-volume set of guidelines for human-centered design in microgravity, was developed with the cooperation of human factors experts from various NASA centers, industry, academia, and other government agencies. The ISS program formed a human factors team analogous to any major engineering subsystem. This team develops and maintains the human factors requirements regarding end-to-end architecture design and performance, hardware and software design requirements, and test and verification requirements. It is also responsible for providing program integration across all of the larger scale elements, smaller scale hardware, and international partners.

  9. A minimum of two distinct heritable factors are required to explain correlation structures in proliferating lymphocytes

    PubMed Central

    Markham, John F.; Wellard, Cameron J.; Hawkins, Edwin D.; Duffy, Ken R.; Hodgkin, Philip D.

    2010-01-01

    During the adaptive immune response, lymphocyte populations undergo a characteristic three-phase process: expansion through a series of cell divisions; cessation of expansion; and, finally, most of the accumulated lymphocytes die by apoptosis. The data used, thus far, to inform understanding of these processes, both in vitro and in vivo, are taken from flow cytometry experiments. One significant drawback of flow cytometry is that individual cells cannot be tracked, so that it is not possible to investigate interdependencies in the fate of cells within a family tree. This deficit in experimental information has recently been overcome by Hawkins et al. (Hawkins et al. 2009 Proc. Natl Acad. Sci. USA 106, 13 457–13 462 (doi:10.1073/pnas.0905629106)), who reported on time-lapse microscopy experiments in which B-cells were stimulated through the TLR-9 receptor. Cells stimulated in this way do not aggregate, so that data regarding family trees can be recorded. In this article, we further investigate the Hawkins et al. data. Our conclusions are striking: in order to explain the familial correlation structure in division times, death times and propensity to divide, a minimum of two distinct heritable factors are necessary. As the data show that two distinct factors are necessary, we develop a stochastic model that has two heritable factors and demonstrate that it can reproduce the key features of the data. This model shows that two heritable factors are sufficient. These deductions have a clear impact upon biological understanding of the adaptive immune response. They also necessitate changes to the fundamental premises behind the tools developed by statisticians to draw deductions from flow cytometry data. Finally, they affect the mathematical modelling paradigms that are used to study these systems, as these are widely developed based on assumptions of cellular independence that are not accurate. PMID:20053654

  10. Conversion from human haematopoietic stem cells to keratinocytes requires keratinocyte secretory factors.

    PubMed

    Fujita, Y; Inokuma, D; Abe, R; Sasaki, M; Nakamura, H; Shimizu, T; Shimizu, H

    2012-08-01

    Recent studies have reported that bone-marrow-derived stem cells (BMSCs), including haematopoietic stem cells (HSCs) and mesenchymal stromal cells, differentiate in order to regenerate various cellular lineages. Based on these findings, it is known that BMSCs can be used clinically to treat various disorders, such as myocardial infarction and neurotraumatic injuries. However, the mechanism of HSC conversion into organ cells is incompletely understood. The mechanism is suspected to involve direct cell-cell interaction between BMSCs, damaged organ cells, and paracrine-regulated soluble factors from the organ, but to date, there have been no investigations into which of these are essential for keratinocyte differentiation from HSCs. To elucidate the mechanism and necessary conditions for HSC differentiation into keratinocytes in vitro. We cultured human (h)HSCs under various conditions to try to elucidate the mechanism and necessary conditions for hHSCs to differentiate into keratinocytes. hHSCs cocultured with mouse keratinocytes induced expression of human keratin 14 and transglutaminase I. Only 0.1% of the differentiated keratinocytes possessed multiple nuclei indicating cell fusion. Coculture of hHSCs with fixed murine keratinocytes (predicted to stabilize cellular components) failed to induce conversion into keratinocytes. Conversely, keratinocyte-conditioned medium from both human and mouse keratinocytes was found to mediate hHSC conversion into keratinocytes. Human HSCs are capable of differentiation into keratinocytes, and cell fusion is extremely rare. This differentiating is mediated by the plasma environment rather than by direct cell-cell interactions. © The Author(s). CED © 2012 British Association of Dermatologists.

  11. Activation of the exchange factor Ras-GRF by calcium requires an intact Dbl homology domain.

    PubMed

    Freshney, N W; Goonesekera, S D; Feig, L A

    1997-04-21

    Ras-GRF is a guanine nucleotide exchange factor that activates Ras proteins. Its activity on Ras in cells is enhanced upon calcium influx. Activation follows calcium-induced binding of calmodulin to an IQ motif near the N-terminus of Ras-GRF. Ras-GRF also contains a Dbl homology (DH) domain C-terminal to the IQ motif. In many proteins, DH domains act as exchange factors for Rho-GTPase family members. However, we failed to detect exchange activity of this domain on well characterized Rho family members. Instead, we found that mutations analogous to those that block exchange activity of Dbl prevented Ras-GRF activation by calcium/ calmodulin in vivo. All DH domains are followed immediately by a pleckstrin homology (PH) domain. We found that a mutation at a conserved site within the PH domain following the DH domain also prevented Ras-GRF activation by calcium in vivo. These results suggest that in addition to playing a role as activators of Rho proteins, DH domains can also contribute to the coupling of cellular signals to Ras activation.

  12. Human immunodeficiency virus type 1 Tat increases the expression of cleavage and polyadenylation specificity factor 73-kilodalton subunit modulating cellular and viral expression.

    PubMed

    Calzado, Marco A; Sancho, Rocío; Muñoz, Eduardo

    2004-07-01

    The human immunodeficiency virus type 1 (HIV-1) Tat protein, which is essential for HIV gene expression and viral replication, is known to mediate pleiotropic effects on various cell functions. For instance, Tat protein is able to regulate the rate of transcription of host cellular genes and to interact with the signaling machinery, leading to cellular dysfunction. To study the effect that HIV-1 Tat exerts on the host cell, we identified several genes that were up- or down-regulated in tat-expressing cell lines by using the differential display method. HIV-1 Tat specifically increases the expression of the cleavage and polyadenylation specificity factor (CPSF) 73-kDa subunit (CPSF3) without affecting the expression of the 160- and 100-kDa subunits of the CPSF complex. This complex comprises four subunits and has a key function in the 3'-end processing of pre-mRNAs by a coordinated interaction with other factors. CPSF3 overexpression experiments and knockdown of the endogenous CPSF3 by mRNA interference have shown that this subunit of the complex is an important regulatory protein for both viral and cellular gene expression. In addition to the known CPSF3 function in RNA polyadenylation, we also present evidence that this protein exerts transcriptional activities by repressing the mdm2 gene promoter. Thus, HIV-1-Tat up-regulation of CPSF3 could represent a novel mechanism by which this virus increases mRNA processing, causing an increase in both cell and viral gene expression.

  13. Factors influencing the transfection efficiency and cellular uptake mechanisms of Pluronic P123-modified polypropyleneimine/pDNA polyplexes in multidrug resistant breast cancer cells.

    PubMed

    Gu, Jijin; Hao, Junguo; Fang, Xiaoling; Sha, Xianyi

    2016-04-01

    Generally, the major obstacles for efficient gene delivery are cellular internalization and endosomal escape of nucleic acid such as plasmid DNA (pDNA) or small interfering RNA (siRNA). We previously developed Pluronic P123 modified polypropyleneimine (PPI)/pDNA (P123-PPI/pDNA) polyplexes as a gene delivery system. The results showed that P123-PPI/pDNA polyplexes revealed higher transfection efficiency than PPI/pDNA polyplexes in multidrug resistant breast cancer cells. As a continued effort, the present investigation on the factors influencing the transfection efficiency, cellular uptake mechanisms, and intracellular fate of P123-PPI/pDNA polyplexes is reported. The presence of P123 was the main factor influencing the transfection efficiency of P123-PPI/pDNA polyplexes in MCF-7/ADR cells, but other parameters, such as N/P ratio, FBS concentration, incubation time and temperature were important as well. The endocytic inhibitors against clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME), and macropinocytosis were involved in the internalization to investigate their effects on the cellular uptake and transfection efficiency of P123-PPI/pDNA polyplexes in vitro. The data showed that the internalization of P123-PPI/pDNA polyplexes was obtained from both CME and CvME. Colocalization experiments with TRITC-transferrin (CME indicator), Alexa Fluor 555-CTB (CvME indicator), monoclonal anti-α-tubulin (microtubule indicator), and LysoTracker Green (Endosome/lysosome indicator) were carried out to confirm the internalization routes. The results showed that both CME and CvME played vital roles in the effective transfection of P123-PPI/pDNA polyplexes. Endosome/lysosome system and skeleton, including actin filament and microtubule, were necessary for the transportation after internalization.

  14. Early in-situ cellularization of a supramolecular vascular graft is modified by synthetic stromal cell-derived factor-1α derived peptides.

    PubMed

    Muylaert, Dimitri E P; van Almen, Geert C; Talacua, Hanna; Fledderus, Joost O; Kluin, Jolanda; Hendrikse, Simone I S; van Dongen, Joost L J; Sijbesma, Eline; Bosman, Anton W; Mes, Tristan; Thakkar, Shraddha H; Smits, Anthal I P M; Bouten, Carlijn V C; Dankers, Patricia Y W; Verhaar, Marianne C

    2016-01-01

    In an in-situ approach towards tissue engineered cardiovascular replacement grafts, cell-free scaffolds are implanted that engage in endogenous tissue formation. Bioactive molecules can be incorporated into such grafts to facilitate cellular recruitment. Stromal cell derived factor 1α (SDF1α) is a powerful chemoattractant of lymphocytes, monocytes and progenitor cells and plays an important role in cellular signaling and tissue repair. Short SDF1α-peptides derived from its receptor-activating domain are capable of activating the SDF1α-specific receptor CXCR4. Here, we show that SDF1α-derived peptides can be chemically modified with a supramolecular four-fold hydrogen bonding ureido-pyrimidinone (UPy) moiety, that allows for the convenient incorporation of the UPy-SDF1α-derived peptides into a UPy-modified polymer scaffold. We hypothesized that a UPy-modified material bioactivated with these UPy-SDF1α-derived peptides can retain and stimulate circulating cells in an anti-inflammatory, pro-tissue formation signaling environment. First, the early recruitment of human peripheral blood mononuclear cells to the scaffolds was analyzed in vitro in a custom-made mesofluidic device applying physiological pulsatile fluid flow. Preferential adhesion of lymphocytes with reduced expression of inflammatory factors TNFα, MCP1 and lymphocyte activation marker CD25 was found in the bioactivated scaffolds, indicating a reduction in inflammatory signaling. As a proof of concept, in-vivo implantation of the bioactivated scaffolds as rat abdominal aorta interposition grafts showed increased cellularity by CD68+ cells after 7 days. These results indicate that a completely synthetic, cell-free biomaterial can attract and stimulate specific leukocyte populations through supramolecular incorporation of short bioactive SDF1α derived peptides.

  15. Sensitivity of wild type and mutant ras alleles to Ras specific exchange factors: Identification of factor specific requirements.

    PubMed

    Nielsen, K H; Gredsted, L; Broach, J R; Willumsen, B M

    2001-04-19

    We have investigated the productive interaction between the four mammalian Ras proteins (H-, N-, KA- and KB-Ras) and their activators, the mammalian exchange factors mSos1, GRF1 and GRP, by using a modified Saccharomyces cerevisiae whose growth is dependent on activation of a mammalian Ras protein by its activator. All four mammalian Ras proteins were activated with similar efficiencies by the individual exchange factors. The H-Ras mutant V103E, which is competent for membrane localization, nucleotide binding, intrinsic and stimulated GTPase activity as well as intrinsic exchange, was defective for activation by all factors tested, suggesting that the integrity of this residue is necessary for catalyzed exchange. However, when other H-Ras mutants were studied, some distinct sensitivities to the exchange factors were observed. GRP-mediated, but not mSos1-mediated, exchange was blocked in additional mutants, suggesting different structural requirements for GRP. Analysis of Ras-mediated gene activation in murine fibroblasts confirmed these results.

  16. Inwardly rectifying K+ (Kir) channels in Drosophila. A crucial role of cellular milieu factors Kir channel function.

    PubMed

    Döring, Frank; Wischmeyer, Erhard; Kühnlein, Ronald P; Jäckle, Herbert; Karschin, Andreas

    2002-07-12

    Three cDNAs encoding inwardly rectifying potassium (Kir) channels were isolated from Drosophila melanogaster. The protein sequences of Drosophila KirI (dKirI) and dKirII are moderately (<44%) and dKirIII sequence is weakly (<27%) identical to human Kir channel subunits. During fly development, five dKir channel transcripts derived from three genes are differentially expressed. Whole mount in situ hybridizations revealed dKirI transcripts absent from embryos, but dKirII and dKirIII are expressed in the embryonic hind gut and in Malpighian tubules, respectively, thus covering the entire osmoregulatory system of the developing fly. In the head of adult flies, predominantly dKirII transcripts were detected. When expressed in Xenopus oocytes, dKir channel activity was only observed after amino acid substitutions in their cytosolic tails (e.g. exchange of a unique valine in the NH(2) terminus). In contrast, heterologous expression of wild type dKirI and dKirII in Drosophila S2 cells readily evoked typical inwardly rectifying K(+) currents, which were weakly sensitive to Ba(2+). Thus, the specific milieu of insect cells provides a crucial cellular environment for proper function of dKir channels.

  17. Analysis of Epidermal Growth Factor Receptor Related Gene Expression Changes in a Cellular and Animal Model of Parkinson’s Disease

    PubMed Central

    Kim, In-Su; Koppula, Sushruta; Park, Shin-Young; Choi, Dong-Kug

    2017-01-01

    We employed transcriptome analysis of epidermal growth factor receptor related gene expression changes in cellular and animal models of Parkinson’s disease (PD). We used a well-known Parkinsonian toxin 1-methyl-4-phenylpyridine (MPP+) to induce neuronal apoptosis in the human neuroblastoma SH-SY5Y cell line. The MPP+-treatment of SH-SY5Y cells was capable of inducing neuro-apoptosis, but it remains unclear what kinds of transcriptional genes are affected by MPP+ toxicity. Therefore the pathways that were significantly perturbed in MPP+ treated human neuroblastoma SH-SY5Y cells were identified based on genome-wide gene expression data at two time points (24 and 48 h). We found that the Epidermal Growth Factor Receptor (EGFR) pathway-related genes showed significantly differential expression at all time points. The EGFR pathway has been linked to diverse cellular events such as proliferation, differentiation, and apoptosis. Further, to evaluate the functional significance of the altered EGFR related gene expression observed in MPP+-treated SH-SY5Y cells, the EGFR related GJB2 (Cx26) gene expression was analyzed in an MPP+-intoxicated animal PD model. Our findings identify that the EGFR signaling pathway and its related genes, such as Cx26, might play a significant role in dopaminergic (DAergic) neuronal cell death during the process of neuro-apoptosis and therefore can be focused on as potential targets for therapeutic intervention. PMID:28212331

  18. Otx but not Mitf transcription factors are required for zebrafish retinal pigment epithelium development.

    PubMed

    Lane, Brandon M; Lister, James A

    2012-01-01

    Otx and Mitf transcription factors have been implicated in the development of the retinal pigmented epithelium (RPE), but the relationship between these factors and their specific roles in the development of the RPE have not been fully defined. The role of the three Otx transcription factors (Otx1a, Otx1b, and Otx2) and two Mitf transcription factors (Mitfa and Mitfb) in the development of the zebrafish RPE was explored in these experiments. The loss of Otx activity through morpholino knockdown produced variable eye defects, ranging from delayed RPE pigmentation to severe coloboma, depending on the combination of Otx factors that were targeted. Expression analysis through in situ hybridization demonstrates that otx transcription factors are necessary for the proper expression of mitfa and mitfb while Mitf transcription factors are not required for the expression of otx genes. Surprisingly, the loss of Mitf activity in mitfa, mitfb, or double mitf mutant zebrafish had no effect on RPE pigmentation or development. Moreover, histological analysis revealed that retinal lamination is unaffected in mitf mutants, as well as in otx morphants, even in regions lacking RPE. Otx and Mitf combined loss of function experiments suggest that mitfa and mitfb may still influence zebrafish RPE development. This is further supported by the ability of mitfa to induce pigmentation in the zebrafish retina when misexpressed. These findings suggest that one or more Otx targets in addition to mitfa and mitfb, possibly another mitf family member, are necessary for development of the RPE in zebrafish.

  19. An analysis of thermal response factors and how to reduce their computational time requirement

    NASA Technical Reports Server (NTRS)

    Wiese, M. R.

    1982-01-01

    Te RESFAC2 version of the Thermal Response Factor Program (RESFAC) is the result of numerous modifications and additions to the original RESFAC. These modifications and additions have significantly reduced the program's computational time requirement. As a result of this work, the program is more efficient and its code is both readable and understandable. This report describes what a thermal response factor is; analyzes the original matrix algebra calculations and root finding techniques; presents a new root finding technique and streamlined matrix algebra; supplies ten validation cases and their results.

  20. Meeting Human Reliability Requirements through Human Factors Design, Testing, and Modeling

    SciTech Connect

    R. L. Boring

    2007-06-01

    In the design of novel systems, it is important for the human factors engineer to work in parallel with the human reliability analyst to arrive at the safest achievable design that meets design team safety goals and certification or regulatory requirements. This paper introduces the System Development Safety Triptych, a checklist of considerations for the interplay of human factors and human reliability through design, testing, and modeling in product development. This paper also explores three phases of safe system development, corresponding to the conception, design, and implementation of a system.

  1. The coordinate cellular response to insulin-like growth factor-I (IGF-I) and insulin-like growth factor-binding protein-2 (IGFBP-2) is regulated through vimentin binding to receptor tyrosine phosphatase β (RPTPβ).

    PubMed

    Shen, Xinchun; Xi, Gang; Wai, Christine; Clemmons, David R

    2015-05-01

    Insulin-like growth factor-binding protein-2 (IGFBP-2) functions coordinately with IGF-I to stimulate cellular proliferation and differentiation. IGFBP-2 binds to receptor tyrosine phosphatase β (RPTPβ), and this binding in conjunction with IGF-I receptor stimulation induces RPTPβ polymerization leading to phosphatase and tensin homolog inactivation, AKT stimulation, and enhanced cell proliferation. To determine the mechanism by which RPTPβ polymerization is regulated, we analyzed the protein(s) that associated with RPTPβ in response to IGF-I and IGFBP-2 in vascular smooth muscle cells. Proteomic experiments revealed that IGF-I stimulated the intermediate filament protein vimentin to bind to RPTPβ, and knockdown of vimentin resulted in failure of IGFBP-2 and IGF-I to stimulate RPTPβ polymerization. Knockdown of IGFBP-2 or inhibition of IGF-IR tyrosine kinase disrupted vimentin/RPTPβ association. Vimentin binding to RPTPβ was mediated through vimentin serine phosphorylation. The serine threonine kinase PKCζ was recruited to vimentin in response to IGF-I and inhibition of PKCζ activation blocked these signaling events. A cell-permeable peptide that contained the vimentin phosphorylation site disrupted vimentin/RPTPβ association, and IGF-I stimulated RPTPβ polymerization and AKT activation. Integrin-linked kinase recruited PKCζ to SHPS-1-associated vimentin in response to IGF-I and inhibition of integrin-linked kinase/PKCζ association reduced vimentin serine phosphorylation. PKCζ stimulation of vimentin phosphorylation required high glucose and vimentin/RPTPβ-association occurred only during hyperglycemia. Disruption of vimetin/RPTPβ in diabetic mice inhibited RPTPβ polymerization, vimentin serine phosphorylation, and AKT activation in response to IGF-I, whereas nondiabetic mice showed no difference. The induction of vimentin phosphorylation is important for IGFBP-2-mediated enhancement of IGF-I-stimulated proliferation during hyperglycemia, and it

  2. Identification of the DNA damage-responsive element of RNR2 and evidence that four distinct cellular factors bind it.

    PubMed Central

    Elledge, S J; Davis, R W

    1989-01-01

    The RNR2 gene encodes the small subunit of ribonucleotide reductase, the enzyme that catalyzes the first step in the pathway for the production of the deoxyribonucleotides needed for DNA synthesis. Transcription of this gene is induced approximately 20-fold in response to environmental stimuli that damage DNA or block DNA replication. Deletion and subcloning analysis identified two, and possibly three, upstream activating sequences (UAS) and one repressing (URS) element in the RNR2 regulatory region. A 42-base-pair (bp) fragment from this region was found to be necessary for proper regulation of RNR2 and to be capable of conferring DNA damage inducibility upon a heterologous promoter. This fragment contained both positively and negatively acting sequences. Four DNA-binding factors interacted with the RNR2 regulatory region. One factor was identified as the GRF1 protein, the product of the RAP1 gene. GRF1 bound to the UAS2 element of RNR2, which was found to be directly adjacent to the 42-bp fragment. UAS2 activity was repressed by the 42-bp fragment. Three other factors bound to the 42-bp fragment; one of these factors, RRF3, had a second binding site in the RNR2 promoter. These factors are likely to mediate the response of RNR2 to DNA damage. Images PMID:2685561

  3. Dual-function radiation sensitizers and bioreductive drugs: factors affecting cellular uptake and sensitizing efficiency in analogues of RSU 1069.

    PubMed

    Walling, J; Stratford, I J; Adams, G E; Stephens, M A

    1988-04-01

    Alkyl aziridine analogues of the hypoxic cell radiosensitizer RSU 1069 have been synthesized and one of these, RB 7040, containing the tetramethyl substituted aziridine, is a more efficient sensitizer in vitro than RSU 1069 (Ahmed et al., 1986). The extent to which variation in drug uptake can influence the sensitizing efficiency of RSU 1069 and its analogues has been investigated by determining the cellular uptake of these weakly basic sensitizers as a function of the pH of the extracellular medium (pHe) over the range 5.4-8.4. Following exposure of V79 cells to these agents for 1 h at room temperature, the ratio of intra- to extracellular concentration (Ci/Ce) was near unity at pH 5.4. Increasing pHe to 8.4 resulted in no change in the ratio Ci/Ce for RSU 1069 (pKa = 6.04). In contrast, the values of Ci/Ce increased three-fold for RSU 1165 (pKa = 7.38) and eleven-fold for RB 7040 (pKa = 8.45). Radiosensitization by RSU 1069 showed little dependence on pHe over the range studied, whereas increasing pH caused an apparent increase in sensitizing efficiency of both RSU 1165 and RB 7040. However, when the enhancement ratios for sensitization were normalized to take account of the effect of extracellular pH on drug uptake, efficiency of sensitization was independent of pHe. This study suggests that changes in basicity (pKa) may have wider potential for therapeutic exploitation on the basis of selective tumour uptake for this type of agent.

  4. Role of Myeloperoxidase Oxidants in the Modulation of Cellular Lysosomal Enzyme Function: A Contributing Factor to Macrophage Dysfunction in Atherosclerosis?

    PubMed Central

    Ismael, Fahd O.; Barrett, Tessa J.; Sheipouri, Diba; Brown, Bronwyn E.; Davies, Michael J.; Hawkins, Clare L.

    2016-01-01

    Low-density lipoprotein (LDL) is the major source of lipid within atherosclerotic lesions. Myeloperoxidase (MPO) is present in lesions and forms the reactive oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). These oxidants modify LDL and have been strongly linked with the development of atherosclerosis. In this study, we examined the effect of HOCl, HOSCN and LDL pre-treated with these oxidants on the function of lysosomal enzymes responsible for protein catabolism and lipid hydrolysis in murine macrophage-like J774A.1 cells. In each case, the cells were exposed to HOCl or HOSCN or LDL pre-treated with these oxidants. Lysosomal cathepsin (B, L and D) and acid lipase activities were quantified, with cathepsin and LAMP-1 protein levels determined by Western blotting. Exposure of J774A.1 cells to HOCl or HOSCN resulted in a significant decrease in the activity of the Cys-dependent cathepsins B and L, but not the Asp-dependent cathepsin D. Cathepsins B and L were also inhibited in macrophages exposed to HOSCN-modified, and to a lesser extent, HOCl-modified LDL. No change was seen in cathepsin D activity or the expression of the cathepsin proteins or lysosomal marker protein LAMP-1. The activity of lysosomal acid lipase was also decreased on treatment of macrophages with each modified LDL. Taken together, these results suggest that HOCl, HOSCN and LDL modified by these oxidants could contribute to lysosomal dysfunction and thus perturb the cellular processing of LDL, which could be important during the development of atherosclerosis. PMID:27997605

  5. Effects of the breed, sex and age on cellular content and growth factor release from equine pure-platelet rich plasma and pure-platelet rich gel

    PubMed Central

    2013-01-01

    Background There is no information on the effects of the breed, gender and age on the cellular content and growth factor (GF) release from equine pure-platelet rich plasma (P-PRP) and pure-platelet rich gel (P-PRG). The objectives of this study were: 1) to compare the cellular composition of P-PRP with whole blood and platelet poor plasma (PPP); 2) to compare the concentration of transforming GF beta 1 (TGF-β1) and platelet derived GF isoform BB (PDGF-BB) between P-PRP treated with non-ionic detergent (P-PRP+NID), P-PRG (activated with calcium gluconate -CG-), PPP+NID, PPP gel (PPG), and plasma and; 3) to evaluate and to correlate the effect of the breed, gender and age on the cellular and GF concentration for each blood component. Forty adult horses, 20 Argentinean Creole Horses (ACH) and, 20 Colombian Creole Horses (CCH) were included. Data were analyzed by parametric (i.e.: t-test, one way ANOVA) and non parametric (Kruskal-Wallis test, Wilcoxon test) tests. Correlation analysis was also performed by using the Spearman and Pearson tests. A p ≤ 0.05 was set as significant for all tests. All the blood components were compared for platelet (PLT), leukocyte (WBC), TGF-β1 and PDGF-BB concentrations. The effect of the breed, gender and age on these variables was analyzed. A P ≤ 0.05 was accepted as significant for all the tests. Results PLT counts were 1.8 and 0.6 times higher in P-PRP than in whole blood and PPP, respectively; WBC counts were 0.5 and 0.1 times lower in P-PRP, in comparison with whole blood and PPP, respectively. TGF-β1 and PDGF-BB concentrations were 2.3 and 262 times higher, respectively, in P-PRG than in plasma, and 0.59 and 0.48 times higher, respectively, in P-PRG than in PPG. P-PRG derived from CCH females or young horses presented significantly (P < 0.001) higher PDGF-BB concentrations than P-PRG derived from ACH males or older horses. Conclusions Our results indicated that P-PRP obtained by a manual method was affected by

  6. Effects of the breed, sex and age on cellular content and growth factor release from equine pure-platelet rich plasma and pure-platelet rich gel.

    PubMed

    Giraldo, Carlos E; López, Catalina; Álvarez, María E; Samudio, Ismael J; Prades, Marta; Carmona, Jorge U

    2013-02-12

    There is no information on the effects of the breed, gender and age on the cellular content and growth factor (GF) release from equine pure-platelet rich plasma (P-PRP) and pure-platelet rich gel (P-PRG). The objectives of this study were: 1) to compare the cellular composition of P-PRP with whole blood and platelet poor plasma (PPP); 2) to compare the concentration of transforming GF beta 1 (TGF-β1) and platelet derived GF isoform BB (PDGF-BB) between P-PRP treated with non-ionic detergent (P-PRP+NID), P-PRG (activated with calcium gluconate -CG-), PPP+NID, PPP gel (PPG), and plasma and; 3) to evaluate and to correlate the effect of the breed, gender and age on the cellular and GF concentration for each blood component. Forty adult horses, 20 Argentinean Creole Horses (ACH) and, 20 Colombian Creole Horses (CCH) were included. Data were analyzed by parametric (i.e.: t-test, one way ANOVA) and non parametric (Kruskal-Wallis test, Wilcoxon test) tests. Correlation analysis was also performed by using the Spearman and Pearson tests. A p ≤ 0.05 was set as significant for all tests. All the blood components were compared for platelet (PLT), leukocyte (WBC), TGF-β1 and PDGF-BB concentrations. The effect of the breed, gender and age on these variables was analyzed. A P ≤ 0.05 was accepted as significant for all the tests. PLT counts were 1.8 and 0.6 times higher in P-PRP than in whole blood and PPP, respectively; WBC counts were 0.5 and 0.1 times lower in P-PRP, in comparison with whole blood and PPP, respectively. TGF-β1 and PDGF-BB concentrations were 2.3 and 262 times higher, respectively, in P-PRG than in plasma, and 0.59 and 0.48 times higher, respectively, in P-PRG than in PPG. P-PRG derived from CCH females or young horses presented significantly (P < 0.001) higher PDGF-BB concentrations than P-PRG derived from ACH males or older horses. Our results indicated that P-PRP obtained by a manual method was affected by intrinsic factors such as the breed

  7. Fibronectin Growth Factor-Binding Domains Are Required for Fibroblast Survival

    PubMed Central

    Lin, Fubao; Ren, Xiang-Dong; Pan, Zhi; Macri, Lauren; Zong, Wei-Xing; Tonnesen, Marcia G.; Rafailovich, Miriam; Bar-Sagi, Dafna; Clark, Richard A.F.

    2011-01-01

    Fibronectin (FN) is required for embryogenesis, morphogenesis, and wound repair, and its Arg–Gly–Asp-containing central cell-binding domain (CCBD) is essential for mesenchymal cell survival and growth. Here, we demonstrate that FN contains three growth factor-binding domains (FN-GFBDs) that bind platelet-derived growth factor-BB (PDGF-BB), a potent fibroblast survival and mitogenic factor. These sites bind PDGF-BB with dissociation constants of 10–100 nm. FN-null cells cultured on recombinant CCBD (FNIII8–11) without a FN-GFBD demonstrated minimal metabolism and underwent autophagy at 24 hours, followed by apoptosis at 72 hours, even in the presence of PDGF-BB. In contrast, FN-null cells plated on FNIII8–11 contiguous with FN-GFBD survived without, and proliferated with, PDGF-BB. FN-null cell survival on FNIII8–11 and noncontiguous arrays of FN-GFBDs required these domains to be adsorbed on the same surface, suggesting the existence of a mesenchymal cell-extracellular matrix synapse. Thus, fibroblast survival required GF stimulation in the presence of a FN-GFBD, as well as adhesion to FN through the CCBD. The findings that fibroblast survival is dependent on FN-GFBD underscore the critical importance of pericellular matrix for cell survival and have significant implications for cutaneous wound healing and regeneration. PMID:20811396

  8. Biomedical and Human Factors Requirements for a Manned Earth Orbiting Station

    NASA Technical Reports Server (NTRS)

    Helvey, W.; Martell, C.; Peters, J.; Rosenthal, G.; Benjamin, F.; Albright, G.

    1964-01-01

    The primary objective of this study is to determine which biomedical and human factors measurements must be made aboard a space station to assure adequate evaluation of the astronaut's health and performance during prolonged space flights. The study has employed, where possible, a medical and engineering systems analysis to define the pertinent life sciences and space station design parameters and their influence on a measurement program. The major areas requiring evaluation in meeting the study objectives include a definition of the space environment, man's response to the environment, selection of measurement and data management techniques, experimental program, space station design requirements, and a trade-off analysis with final recommendations. The space environment factors that are believed to have a significant effect on man were evaluated. This includes those factors characteristic of the space environment (e. g. weightlessness, radiation) as well as those created within the space station (e. g. toxic contaminants, capsule atmosphere). After establishing the general features of the environment, an appraisal was made of the anticipated response of the astronaut to each of these factors. For thoroughness, the major organ systems and functions of the body were delineated, and a determination was made of their anticipated response to each of the environmental categories. A judgment was then made on the medical significance or importance of each response, which enabled a determination of which physiological and psychological effects should be monitored. Concurrently, an extensive list of measurement techniques and methods of data management was evaluated for applicability to the space station program. The various space station configurations and design parameters were defined in terms of the biomedical and human factors requirements to provide the measurements program. Research design of experimental programs for various station configurations, mission

  9. Fat-specific protein 27 modulates nuclear factor of activated T cells 5 and the cellular response to stress

    USDA-ARS?s Scientific Manuscript database

    Fat-specific protein 27 (FSP27), a member of the cell death-inducing DNA fragmentation factor a-like effector (Cide) family, is highly expressed in adipose tissues and is a lipid droplet (LD)-associated protein that induces the accumulation of LDs. Using a yeast two-hybrid system to examine potentia...

  10. Regulation of the hypertonic stress response and other cellular functions by the Rel-like transcription factor NFAT5.

    PubMed

    Aramburu, José; Drews-Elger, Katherine; Estrada-Gelonch, Anaïs; Minguillón, Jordi; Morancho, Beatriz; Santiago, Verónica; López-Rodríguez, Cristina

    2006-11-30

    Stress, be it from environmental factors or intrinsic to the cell as result of growth and metabolism, can be harmful to cells. Mammalian cells have developed numerous mechanisms to respond to diverse forms of stress. These mechanisms combine signaling cascades and activation of gene expression programs to orchestrate an adaptive response that will allow the cell to survive and resume its normal functioning. In this review we will focus on the transcription factor NFAT5, a fundamental regulator of the response to osmotic stress in mammalian cells. Identified in 1999, NFAT5 is the latest addition to the Rel family, which comprises the NF-kappaB and NFATc proteins. Though in some of its structural and functional features NFAT5 is a hybrid between these two major groups of Rel proteins, it has unique characteristics that make it stand on its own as a third type of Rel transcription factor. Since its discovery, NFAT5 has been studied mostly in the context of the hypertonicity stress response. The advent of mouse models deficient in NFAT5 and other recent advances have confirmed a fundamental osmoprotective role for this factor in mammals, but also revealed features that suggest it may have a wider range of functions.

  11. Foxm1 transcription factor is required for the initiation of lung tumorigenesis by oncogenic Kras(G12D.).

    PubMed

    Wang, I-C; Ustiyan, V; Zhang, Y; Cai, Y; Kalin, T V; Kalinichenko, V V

    2014-11-13

    Lung cancer is the leading cause of deaths in cancer patients in the United States. Identification of new molecular targets is clearly needed to improve therapeutic outcomes of this devastating human disease. Activating mutations in K-Ras oncogene and increased expression of FOXM1 protein are associated with poor prognosis in patients with non-small-cell lung cancer. Transgenic expression of activated Kras(G12D) in mouse respiratory epithelium is sufficient to induce lung adenocarcinomas; however, transcriptional mechanisms regulated by K-Ras during the initiation of lung cancer remain poorly understood. Foxm1 transcription factor, a downstream target of K-Ras, stimulates cellular proliferation during embryogenesis, organ repair and tumor growth, but its role in tumor initiation is unknown. In the present study, we used transgenic mice expressing Kras(G12D) under control of Sftpc promoter to demonstrate that Foxm1 was induced in type II epithelial cells before the formation of lung tumors. Conditional deletion of Foxm1 from Kras(G12D)-expressing respiratory epithelium prevented the initiation of lung tumors in vivo. The loss of Foxm1 inhibited expression of K-Ras target genes critical for the nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) pathways, including Ikbkb, Nfkb1, Nfkb2, Rela, Jnk1, N-Myc, Pttg1 and Cdkn2a. Transgenic overexpression of activated FOXM1 mutant was sufficient to induce expression of these genes in alveolar type II cells. FOXM1 directly bound to promoter regions of Ikbkb, Nfkb2, N-Myc, Pttg1 and Cdkn2a, indicating that these genes are direct FOXM1 targets. FOXM1 is required for K-Ras-mediated lung tumorigenesis by activating genes critical for the NF-κB and JNK pathways.

  12. Risk factors for drainage-requiring ascites after refractory peritonitis in peritoneal dialysis patients.

    PubMed

    Lee, Cheng-Chia; Tu, Kun-Hua; Chen, Hsiao-Hui; Chang, Ming-Yang; Hung, Cheng-Chieh

    2016-10-01

    Refractory peritonitis remains a thorny issue for patients with chronic peritoneal dialysis (PD). Shortly after catheter removal, some patients develop persistent peritoneal inflammation and ascites formation, which require percutaneous drainage for symptom relief. Our study aimed at finding the risk factors for this kind of event. A total of 47 PD patients complicated with refractory peritonitis who underwent catheter removal between January 2009 and December 2011 were enrolled in this study. Data were compared between patients with and without the development of symptomatic ascites requiring drainage during hospitalization. Among the 47 refractory peritonitis patients, 15 patients developed symptomatic ascites that needed further drainage shortly after catheter removal during hospitalization. The following factors were associated with an increased risk: longer dialysis duration, higher peritoneal Kt/V urea, and a significant rise in serum C-reactive protein (CRP) level after catheter removal. These patients had a prolonged hospital stay (62 vs 21 days, P < 0.001) and a significantly higher risk of recurrent loculated ascites during subsequent 6 months of follow-up (33.3 vs 6.2 %, P = 0.022) compared with patients who did not develop ascites requiring drainage during hospitalization. A significant portion of patients with refractory PD peritonitis experienced ascites requiring drainage shortly after catheter removal, which led to a prolonged hospitalization. Whether routine drain placement at the time of catheter removal for this high-risk group would be of benefit warrants further prospective studies.

  13. Identification and sequence of an accessory factor required for activation of the human interferon gamma receptor.

    PubMed

    Soh, J; Donnelly, R J; Kotenko, S; Mariano, T M; Cook, J R; Wang, N; Emanuel, S; Schwartz, B; Miki, T; Pestka, S

    1994-03-11

    Human chromosomes 6 and 21 are both necessary to confer sensitivity to human interferon gamma (Hu-IFN-gamma), as measured by induction of class I human leukocyte antigen (HLA) and protection against encephalomyocarditis virus (EMCV) infection. Whereas human chromosome 6 encodes the Hu-IFN-gamma receptor, human chromosome 21 encodes accessory factors for generating biological activity through the Hu-IFN-gamma receptor. Probes from a genomic clone were used to identity cDNA clones expressing a species-specific accessory factor. These cDNA clones are able to substitute for human chromosome 21 to reconstitute the Hu-IFN-gamma receptor-mediated induction of class I HLA antigens. However, the factor encoded by the cDNA does not confer full antiviral protection against EMCV, confirming that an additional factor encoded on human chromosome 21 is required for reconstitution of antiviral activity against EMCV. We conclude that this accessory factor belongs to a family of such accessory factors responsible for different actions of IFN-gamma.

  14. Murine thymic NK cells are distinct from ILC1s and have unique transcription factor requirements.

    PubMed

    Gabrielli, Sara; Sun, Mengxi; Bell, April; Zook, Erin C; de Pooter, Renee F; Zamai, Loris; Kee, Barbara L

    2017-03-09

    Group 1 innate lymphoid cells include natural killer (NK) cells and ILC1s, which mediate the response to intracellular pathogens. Thymic NK (tNK) cells were described with hybrid features of immature NK cells and ILC1 but whether these cells are related to NK cells or ILC1 has not been fully investigated. We report that murine tNK cells expressed the NK-cell associated transcription factor EOMES and developed independent of the essential ILC1 factor TBET, confirming their placement within the NK lineage. Moreover, tNK cells resemble NK cells rather than ILC1 in their requirements for the E protein transcription factor inhibitor ID2. We provide further insight into the mechanisms governing tNK-cell development by showing that the transcription factor ETS1 prevented tNK cell acquisition of the conventional NK-cell maturation markers CD11b and KLRG1. Our data reveal few ILC1 in the thymus and clarify the identity and developmental requirements of tNK cells. This article is protected by copyright. All rights reserved.

  15. Lethal Factor, but Not Edema Factor, Is Required to Cause Fatal Anthrax in Cynomolgus Macaques after Pulmonary Spore Challenge

    PubMed Central

    Hutt, Julie A.; Lovchik, Julie A.; Drysdale, Melissa; Sherwood, Robert L.; Brasel, Trevor; Lipscomb, Mary F.; Lyons, C. Rick

    2015-01-01

    Inhalational anthrax is caused by inhalation of Bacillus anthracis spores. The ability of B. anthracis to cause anthrax is attributed to the plasmid-encoded A/B-type toxins, edema toxin (edema factor and protective antigen) and lethal toxin (lethal factor and protective antigen), and a poly-d-glutamic acid capsule. To better understand the contribution of these toxins to the disease pathophysiology in vivo, we used B. anthracis Ames strain and isogenic toxin deletion mutants derived from the Ames strain to examine the role of lethal toxin and edema toxin after pulmonary spore challenge of cynomolgus macaques. Lethal toxin, but not edema toxin, was required to induce sustained bacteremia and death after pulmonary challenge with spores delivered via bronchoscopy. After intravenous challenge with bacilli to model the systemic phase of infection, lethal toxin contributed to bacterial proliferation and subsequent host death to a greater extent than edema toxin. Deletion of protective antigen resulted in greater loss of virulence after intravenous challenge with bacilli than deletion of lethal toxin or edema toxin alone. These findings are consistent with the ability of anti–protective antigen antibodies to prevent anthrax and suggest that lethal factor is the dominant toxin that contributes to the escape of significant numbers of bacilli from the thoracic cavity to cause anthrax after inhalation challenge with spores. PMID:25285720

  16. Lethal factor, but not edema factor, is required to cause fatal anthrax in cynomolgus macaques after pulmonary spore challenge.

    PubMed

    Hutt, Julie A; Lovchik, Julie A; Drysdale, Melissa; Sherwood, Robert L; Brasel, Trevor; Lipscomb, Mary F; Lyons, C Rick

    2014-12-01

    Inhalational anthrax is caused by inhalation of Bacillus anthracis spores. The ability of B. anthracis to cause anthrax is attributed to the plasmid-encoded A/B-type toxins, edema toxin (edema factor and protective antigen) and lethal toxin (lethal factor and protective antigen), and a poly-d-glutamic acid capsule. To better understand the contribution of these toxins to the disease pathophysiology in vivo, we used B. anthracis Ames strain and isogenic toxin deletion mutants derived from the Ames strain to examine the role of lethal toxin and edema toxin after pulmonary spore challenge of cynomolgus macaques. Lethal toxin, but not edema toxin, was required to induce sustained bacteremia and death after pulmonary challenge with spores delivered via bronchoscopy. After intravenous challenge with bacilli to model the systemic phase of infection, lethal toxin contributed to bacterial proliferation and subsequent host death to a greater extent than edema toxin. Deletion of protective antigen resulted in greater loss of virulence after intravenous challenge with bacilli than deletion of lethal toxin or edema toxin alone. These findings are consistent with the ability of anti-protective antigen antibodies to prevent anthrax and suggest that lethal factor is the dominant toxin that contributes to the escape of significant numbers of bacilli from the thoracic cavity to cause anthrax after inhalation challenge with spores.

  17. The master transcription factor Spo0A is required for poly(3-hydroxybutyrate) (PHB) accumulation and expression of genes involved in PHB biosynthesis in Bacillus thuringiensis.

    PubMed

    Chen, Hui-Ju; Tsai, Teng-Kuan; Pan, Shih-Chuan; Lin, Jer-Sheng; Tseng, Chi-Ling; Shaw, Gwo-Chyuan

    2010-03-01

    Bacillus thuringiensis is a gram-positive spore-forming bacterium that can accumulate poly(3-hydroxybutyrate) (PHB) as a carbon and energy storage substance in response to nutritional stress. The regulatory mechanism for PHB biosynthesis in B. thuringiensis and diverse Bacillus species is still poorly understood. We now report that disruption of the sigH gene or the gene encoding the master sporulation transcription factor Spo0A severely impaired PHB accumulation in B. thuringiensis. Complementation of the spo0A mutation with the spo0A gene restored PHB accumulation. We have found that the requirement of Spo0A for PHB accumulation is independent of the transition state regulator AbrB and of loss of sporulation ability. We also show that Spo0A is required for the expression of three genes involved in PHB biosynthesis. These findings have uncovered a new role of Spo0A in the regulation of stationary-phase-associated cellular events.

  18. Inhibition of Cellular Protein Secretion by Norwalk Virus Nonstructural Protein p22 Requires a Mimic of an Endoplasmic Reticulum Export Signal

    PubMed Central

    Sharp, Tyler M.; Guix, Susana; Katayama, Kazuhiko; Crawford, Sue E.; Estes, Mary K.

    2010-01-01

    Protein trafficking between the endoplasmic reticulum (ER) and Golgi apparatus is central to cellular homeostasis. ER export signals are utilized by a subset of proteins to rapidly exit the ER by direct uptake into COPII vesicles for transport to the Golgi. Norwalk virus nonstructural protein p22 contains a YXΦESDG motif that mimics a di-acidic ER export signal in both sequence and function. However, unlike normal ER export signals, the ER export signal mimic of p22 is necessary for apparent inhibition of normal COPII vesicle trafficking, which leads to Golgi disassembly and antagonism of Golgi-dependent cellular protein secretion. This is the first reported function for p22. Disassembly of the Golgi apparatus was also observed in cells replicating Norwalk virus, which may contribute to pathogenesis by interfering with cellular processes that are dependent on an intact secretory pathway. These results indicate that the ER export signal mimic is critical to the antagonistic function of p22, shown herein to be a novel antagonist of ER/Golgi trafficking. This unique and well-conserved human norovirus motif is therefore an appealing target for antiviral drug development. PMID:20976190

  19. Cellular organization of pre-mRNA splicing factors in several tissues. Changes in the uterus by hormone action.

    PubMed

    George-Téllez, R; Segura-Valdez, M L; González-Santos, L; Jiménez-García, L F

    2002-05-01

    In the mammalian cell nucleus, splicing factors are distributed in nuclear domains known as speckles or splicing factor compartments (SFCs). In cultured cells, these domains are dynamic and reflect transcriptional and splicing activities. We used immunofluorescence and confocal microscopy to monitor whether splicing factors in differentiated cells display similar features. Speckled patterns are observed in rat hepatocytes, beta-cells, bronchial and intestine epithelia and also in three cell types of the uterus. Moreover, the number, distribution and sizes of the speckles vary among them. In addition, we studied variations in the circular form (shape) of speckles in uterine cells that are transcriptionally modified by a hormone action. During proestrus of the estral cycle, speckles are irregular in shape while in diestrus I they are circular. Experimentally, in castrated rats luminal epithelial cells show a pattern where speckles are dramatically rounded, but they recover their irregular shape rapidly after an injection of estradiol. The same results were observed in muscle and gland epithelial cells of the uterus. We concluded that different speckled patterns are present in various cells types in differentiated tissues and that these patterns change in the uterus depending upon the presence or absence of hormones such as estradiol.

  20. Induction of cellular transcription factors in trigeminal ganglia of mice by corneal scarification, herpes simplex virus type 1 infection, and explantation of trigeminal ganglia.

    PubMed Central

    Valyi-Nagy, T; Deshmane, S; Dillner, A; Fraser, N W

    1991-01-01

    In a mouse model for herpes simplex virus type 1 (HSV-1) latency in which the virus was inoculated via the eye after corneal scarification, HSV-1 replicated in corneal epithelial cells and infected the nerve cell endings. HSV-1 reached the trigeminal ganglia by fast axonal transport between 2 and 10 days postinfection (p.i.) and established a latent infection in neuronal cells or replicated and spread to nonneuronal cells. By using in situ hybridization, we showed that cellular transcription factors are stimulated by HSV-1 infection in trigeminal ganglia. This stimulation is biphasic, peaking at 1 and 3 to 4 days p.i. The first peak involves c-jun and oct-1 expression in neurons, and the second involves c-jun, c-fos, and oct-1 expression in neurons and nonneuronal cells. Corneal scarification, alone or followed by infection with UV-inactivated HSV-1, induced monophasic c-jun and oct-1 expression in some neurons of the trigeminal ganglia, with a peak at 1 day p.i. Corneal infection without prior scarification induced c-jun, c-fos, and oct-1 expression in some neuronal and nonneuronal cells of the trigeminal ganglia 2 to 9 days p.i. Explanation of ganglia from latently infected animals resulted in reactivation of the latent virus. Independently of the presence of latent HSV-1 in explanted ganglia, expression of c-fos, c-jun, and oct-1 was induced first in nonneuronal cells, peaking 6 to 10 h postexplantation, and then in neuronal cells, with a peak at 24 h after explantation when expression of viral replicative genes was first detectable. Since ocular HSV-1 infection, corneal scarification, and explantation of trigeminal ganglia all resulted in induction of expression of cellular transcription factors in ganglia, these factors may play a critical role in the permissiveness of cells for HSV-1 replication during acute infection, latency, and reactivation. Images PMID:1649322

  1. Induction of cellular transcription factors in trigeminal ganglia of mice by corneal scarification, herpes simplex virus type 1 infection, and explantation of trigeminal ganglia.

    PubMed

    Valyi-Nagy, T; Deshmane, S; Dillner, A; Fraser, N W

    1991-08-01

    In a mouse model for herpes simplex virus type 1 (HSV-1) latency in which the virus was inoculated via the eye after corneal scarification, HSV-1 replicated in corneal epithelial cells and infected the nerve cell endings. HSV-1 reached the trigeminal ganglia by fast axonal transport between 2 and 10 days postinfection (p.i.) and established a latent infection in neuronal cells or replicated and spread to nonneuronal cells. By using in situ hybridization, we showed that cellular transcription factors are stimulated by HSV-1 infection in trigeminal ganglia. This stimulation is biphasic, peaking at 1 and 3 to 4 days p.i. The first peak involves c-jun and oct-1 expression in neurons, and the second involves c-jun, c-fos, and oct-1 expression in neurons and nonneuronal cells. Corneal scarification, alone or followed by infection with UV-inactivated HSV-1, induced monophasic c-jun and oct-1 expression in some neurons of the trigeminal ganglia, with a peak at 1 day p.i. Corneal infection without prior scarification induced c-jun, c-fos, and oct-1 expression in some neuronal and nonneuronal cells of the trigeminal ganglia 2 to 9 days p.i. Explanation of ganglia from latently infected animals resulted in reactivation of the latent virus. Independently of the presence of latent HSV-1 in explanted ganglia, expression of c-fos, c-jun, and oct-1 was induced first in nonneuronal cells, peaking 6 to 10 h postexplantation, and then in neuronal cells, with a peak at 24 h after explantation when expression of viral replicative genes was first detectable. Since ocular HSV-1 infection, corneal scarification, and explantation of trigeminal ganglia all resulted in induction of expression of cellular transcription factors in ganglia, these factors may play a critical role in the permissiveness of cells for HSV-1 replication during acute infection, latency, and reactivation.

  2. Requirement of Src kinase Lyn for induction of DNA synthesis by granulocyte colony-stimulating factor.

    PubMed

    Corey, S J; Dombrosky-Ferlan, P M; Zuo, S; Krohn, E; Donnenberg, A D; Zorich, P; Romero, G; Takata, M; Kurosaki, T

    1998-02-06

    Treatment of cells with granulocyte colony-stimulating factor (G-CSF) leads to tyrosine phosphorylation of cellular proteins. G-CSF stimulates both the activation of protein tyrosine kinases Lyn, Jak1, and Jak2 and the association of these enzymes with the G-CSF receptor. Wild-type, lyn-deficient, and syk-deficient chicken B lymphocyte cell lines were transfected with the human G-CSF receptor, and stable transfectants were studied. G-CSF-dependent tyrosyl phosphorylation of Jak1 and Jak2 occurred in all three cell lines. Wild-type and syk-deficient transfectants responded to G-CSF in a dose-responsive fashion with increased thymidine incorporation, but none of the clones of lyn-deficient transfectants did. Ectopic expression of Lyn, but not that of c-Src, in the lyn-deficient cells restored their mitogenic responsiveness to G-CSF. Ectopic expression in wild-type cells of the kinase-inactive form of Lyn, but not of the kinase-inactive form of Jak2, inhibited thymidine incorporation in response to G-CSF. These studies show that the absence of Lyn results in the loss of mitogenic signaling in the G-CSF signaling pathway and that activation of Jak1 or Jak2 is not sufficient to cause mitogenesis.

  3. Co-repressor activity of scaffold attachment factor B1 requires sumoylation

    SciTech Connect

    Garee, Jason P.; Meyer, Rene; Oesterreich, Steffi

    2011-05-20

    Highlights: {yields} SAFB1 is sumoylated to two lysine residues K231 and K294. {yields} SAFB1 sumoylation is regulated by PIAS1 and SENP1. {yields} Sumoylation of SAFB1 regulates its transcriptional repressor activity. {yields} Mutation of sumoylation sites leads to decreased SAFB1 binding to HDAC3. -- Abstract: Sumoylation is an emerging modification associated with a variety of cellular processes including the regulation of transcriptional activities of nuclear receptors and their coregulators. As SUMO modifications are often associated with transcriptional repression, we examined if sumoylation was involved in modulation of the transcriptional repressive activity of scaffold attachment factor B1. Here we show that SAFB1 is modified by both the SUMO1 and SUMO2/3 family of proteins, on lysine's K231 and K294. Further, we demonstrate that SAFB1 can interact with PIAS1, a SUMO E3 ligase which mediates SAFB1 sumoylation. Additionally, SENP1 was identified as the enzyme desumoylating SAFB1. Mutation of the SAFB1 sumoylation sites lead to a loss of transcriptional repression, at least in part due to decreased interaction with HDAC3, a known transcriptional repressor and SAFB1 binding partner. In summary, the transcriptional repressor SAFB1 is modified by both SUMO1 and SUMO2/3, and this modification is necessary for its full repressive activity.

  4. Predisposing factors and associated symptomatology of British soldiers requiring a mental health assessment.

    PubMed

    Finnegan, A; Finnegan, S; Jackson, C; Simpson, R; Ashford, R

    2010-06-01

    To critically evaluate the predisposing factors and symptomatology that resulted in serving officers and soldiers requiring a Mental Health (MH) assessment. 317 regular Army personnel who required a formal MH assessment completed a survey that detailed the predisposing factors and symptoms leading to the referral. SPSSv10 was used for data management and analysis of the data by descriptive and inferential statistical methods. Three quarter presented with at least two predisposing factors, the commonest being family issues (42%), relationship problems (40%) and general military stress (39%). Up to half of young male Soldiers required a MH assessment as a result of wanting to leave the Army, and were positively associated with self harming ideology. Female soldiers are significantly over represented. No-one reported feeling isolated. The majority of personnel accessing the Army MH Services present with multi-factorial problems and symptoms that should result in colleagues being aware of their distress, and every effort must be made to support these soldiers within unit lines. That no one reported feeling isolated, challenges the perception that soldiers with MH problems are stigmatised. In those young male soldiers who wish to leave the Army there are indicators that significant periods of notice to leave can have a negative impact on MH. It is unclear why females are more likely to require support. If the emerging themes noted in this study are addressed, and the lessons learnt encapsulated within a predictive theoretical model, then the result could be an improvement in operational capability through the early return of Army personnel and Officers to full duty.

  5. Community-associated urinary infections requiring hospitalization: risk factors, microbiological characteristics and patterns of antibiotic resistance.

    PubMed

    Medina-Polo, J; Guerrero-Ramos, F; Pérez-Cadavid, S; Arrébola-Pajares, A; Sopeña-Sutil, R; Benítez-Sala, R; Jiménez-Alcaide, E; García-González, L; Alonso-Isa, M; Lara-Isla, A; Passas-Martínez, J B; Tejido-Sánchez, Á

    2015-03-01

    Although patients with urinary tract infections (UTIs) are usually managed as outpatients, a percentage of them requires hospitalization. To review risk factors and microbiological characteristics of community-associated UTIs (CAUTIs) requiring hospitalization has been our objective. A prospective observational study was carried out from November 2011 to December 2013. Incidence, microbiological characteristics and antibiotic resistance patterns in patients with CAUTIs that required hospitalization were analyzed. Risk factors (including diabetes mellitus, urolithiasis, urinary catheterization) and resistance rates of each pathogen were also analyzed. Four hundred and fifty seven patients were hospitalized in our department with CAUTI. The mean age was 56.2±19.85 years. Of them, 52.1% patients were women, 19.7% had urinary indwelling catheter and 11.4% have had a previous UTI. The most frequently isolated pathogens were Escherichia coli (60.6%), followed by Klebsiella (9.2%), Enterococcus (8.4%) and Pseudomonas (7.2%). Enterobacteriaceae other than E.coli were more prevalent in male and older patients. On the other side the most frequently isolated pathogen in patients with a previous UTI and a urinary catheter was Entercoccus. The resistance rates E. coli against ampicillin/amoxicillin + β lactamase inhibitor was 23.5%, against third-generation cephalosporins 16.6%, against fluoroquinolones 31.3% and 16.7% against aminoglycosides. 11.4% E. coli strains were producers of extended-spectrum Beta-lactamases (ESBL). Finally, the resistance rates of Enterococcus and Pseudomonas against quinolones were of 50.0% and 61.5%, respectively. CAUTIs that require hospitalization are most frequent in older age, male gender, and presence of urinary catheter, with urolithiasis and with previous episodes of UTI. These factors are also related to isolation of pathogens other than E. coli and higher resistance rates. Copyright © 2014 AEU. Publicado por Elsevier España, S.L.U. All

  6. Multiple requirements for SHPTP2 in epidermal growth factor-mediated cell cycle progression.

    PubMed Central

    Bennett, A M; Hausdorff, S F; O'Reilly, A M; Freeman, R M; Neel, B G

    1996-01-01

    Using transient overexpression and microinjection approaches, we examined SHPTP2's function in growth factor signaling. Overexpression of catalytically inactive SHPTP2 (PTP2CS) but not catalytically inactive SHPTP1, inhibited mitogen-activated protein (MAP) kinase activation and Elk-1 transactivation following epidermal growth factor (EGF) stimulation of 293 cells. An SHPTP2 mutant with both C-terminal tyrosyl phosphorylation sites converted to phenylalanine (PTP2YF) was also without effect; moreover, PTP2YF rescued PTP2CS-induced inhibition of EGF-induced Elk-1 transactivation. PTP2CS did not inhibit transactivation by activated Ras, suggesting that SHPTP2 acts upstream of or parallel to Ras. Neither PTP2CS nor PTP2YF inhibited platelet-derived growth factor (PDGF)-induced Elk-1 transactivation. Thus, protein-tyrosine phosphatase activity, but not tyrosyl phosphorylation of SHPTP2, is required for the immediate-early responses to EGF but not to PDGF. To determine whether SHPTP2 is required later in the cell cycle, we assessed S-phase entry in NIH 3T3 cells microinjected with anti-SHPTP2 antibodies or with a glutathione S-transferase (GST) fusion protein encoding both SH2 domains (GST-SH2). Microinjection of anti-SHPTP2 antibodies prior to stimulation inhibited EGF- but no PDGF- or serum-induced S-phase entry. Anti-SHPTP2 antibodies or GST-SH2 fusion protein could inhibit EGF-induced S-phase entry for up to 8 h after EGF addition. Although MAP kinase activation was detected shortly after EGF stimulation, no MAP kinase activation was detected around the restriction point. Therefore, SHPTP2 is absolutely required for immediate-early and late events induced by some, but not all, growth factors, and the immediate-early and late signal transduction pathways regulated by SHPTP2 are distinguishable. PMID:8622663

  7. Risk factors for progressive neuromuscular scoliosis requiring posterior spinal fusion after selective dorsal rhizotomy.

    PubMed

    Ravindra, Vijay M; Christensen, Michael T; Onwuzulike, Kaine; Smith, John T; Halvorson, Kyle; Brockmeyer, Douglas L; Walker, Marion L; Bollo, Robert J

    2017-09-08

    OBJECTIVE Selective dorsal rhizotomy (SDR) via limited laminectomy is an effective treatment of lower-extremity spasticity in the pediatric population. Children with spasticity are also at risk for neuromuscular scoliosis; however, specific risk factors for progressive spinal deformity requiring posterior spinal fusion (PSF) after SDR are unknown. The authors' goal was to identify potential risk factors. METHODS The authors performed a retrospective cohort study of patients who underwent SDR via limited laminectomy between 2003 and 2014 and who had at least 1 year of follow-up. They analyzed demographic, clinical, and radiographic variables to elucidate risk factors for progressive neuromuscular scoliosis. The primary outcome was need for PSF. RESULTS One hundred thirty-four patients underwent SDR and had at least 12 months of follow-up (mean 65 months); 48 patients (36%) had detailed pre- and postoperative radiographic data available. The mean age at surgery was 10 years (SD 5.1 years). Eighty-four patients (63%) were ambulatory before SDR, 109 (82%) underwent a single-level laminectomy, and a mean of 53% of the dorsal rootlets from L-1 to S-1 were sectioned. Fifteen patients (11.2%) subsequently required PSF for progressive deformity. Nonambulatory status (p < 0.001) and a preoperative Cobb angle > 30° (p = 0.003) were significantly associated with PSF on univariate analysis, but no statistically significant correlation was found with any clinical or radiographic variable and PSF after SDR on multivariate regression analysis. CONCLUSIONS Patients with preoperative nonambulatory status and Cobb angle > 30° may be at risk for progressive spinal deformity requiring PSF after SDR. These are well-known risk factors for progressive deformity in children with spasticity in general. Although our analysis suggests SDR via limited laminectomy may not significantly accelerate the development of neuromuscular scoliosis, further case-control studies are critical to

  8. Cellular pathways for viral transport through plasmodesmata.

    PubMed

    Niehl, Annette; Heinlein, Manfred

    2011-01-01

    Plant viruses use plasmodesmata (PD) to spread infection between cells and systemically. Dependent on viral species, movement through PD can occur in virion or non-virion form, and requires different mechanisms for targeting and modification of the pore. These mechanisms are supported by viral movement proteins and by other virus-encoded factors that interact among themselves and with plant cellular components to facilitate virus movement in a coordinated and regulated fashion.

  9. Cellular distribution of transforming growth factor-beta 1 and procollagen types I, III, and IV transcripts in carbon tetrachloride-induced rat liver fibrosis.

    PubMed Central

    Nakatsukasa, H; Nagy, P; Evarts, R P; Hsia, C C; Marsden, E; Thorgeirsson, S S

    1990-01-01

    The cellular distribution and temporal expression of transcripts from transforming growth factor-beta 1 (TGF-beta 1) and procollagen alpha 1(I), alpha 1(III), and alpha 1(IV) genes were studied in carbon tetrachloride (CCl4)-induced rat liver fibrosis by using in situ hybridization technique. During the fibrotic process, TGF-beta 1 and procollagen genes were similarly and predominantly expressed in Desmin-positive perisinusoidal cells (e.g., fat-storing cells and myofibroblasts) and fibroblasts and their expression continued to be higher than those observed in control rats. These transcripts were also observed in inflammatory cells mainly granulocytes and macrophage-like cells at the early stages of liver fibrosis. The production of extracellular matrix along small blood vessels and fibrous septa coincided with the expression of these genes. Expression of TGF-beta 1 and procollagen genes were not detected in hepatocytes throughout the experiment. No significant differences in cellular distribution or time course of gene expression among procollagen alpha 1(I), alpha 1(III), and alpha 1(IV) were observed. Desmin-positive perisinusoidal cells and fibroblasts appeared to play the principal role in synthesis of collagens in CCl4-induced hepatic fibrosis. The simultaneous expression of TGF-beta 1 and procollagen genes in mesenchymal cells, including Desmin-positive perisinusoidal cells, during hepatic fibrosis suggests the possibility that TGF-beta 1 may have an important role in the production of fibrosis. Images PMID:1693377

  10. Basic Fibroblast Growth Factor Inhibits Apoptosis and Promotes Proliferation of Adipose-Derived Mesenchymal Stromal Cells Isolated from Patients with Type 2 Diabetes by Reducing Cellular Oxidative Stress

    PubMed Central

    2017-01-01

    Type 2 diabetes (T2D) is a chronic metabolic disorder affecting increasing number of people in developed countries. Therefore new strategies for treatment of T2D and its complications are of special interest. Nowadays, cellular therapies involving mesenchymal stromal cells that reside in adipose tissue (ASCs) constitute a promising approach; however, there are still many obstacles concerning safety and effectiveness that need to be overcome before ASCs could be engaged for the treatment of diabetes mellitus. One of the challenges is preventing ASCs from deterioration caused by elevated oxidative stress present in diabetes milieu. In the current study we investigated the effect of basic fibroblast growth factor (bFGF) treatment on ASCs isolated from patients with diagnosed T2D. We demonstrate here that cell exposition to bFGF in 5 and 10 ng/mL dosages results in improved morphology, increased proliferative activity, reduced cellular senescence and apoptosis, and decreased oxidative stress, indicating recovery of ASCs' function impaired by T2D. Therefore our results provide a support for bFGF as a potential therapeutic agent for improving stem cell-based approaches for the treatment of diabetes mellitus and its complications. PMID:28168007

  11. Cellular Factor XIIIA Transglutaminase Localizes in Caveolae and Regulates Caveolin-1 Phosphorylation, Homo-oligomerization and c-Src Signaling in Osteoblasts

    PubMed Central

    Wang, Shuai; Kaartinen, Mari T.

    2015-01-01

    Transglutaminases (TGs) are a family of widely distributed enzymes that catalyze protein crosslinking by forming a covalent isopeptide bond between the substrate proteins. We have shown that MC3T3-E1 osteoblasts express Factor XIII-A (FXIII-A), and that the extracellular crosslinking activity of FXIII-A is involved in regulating matrix secretion and deposition. In this study, we have investigated the localization and potential role of intracellular FXIII-A. Conventional immunofluorescence microscopy and TIRF microscopy analyses showed that FXIII-A co-localizes with caveolin-1 in specialized membrane structures, caveolae, in differentiating osteoblasts. The caveolae-disrupting agent methyl-β-cyclodextrin abolished FXIII-A staining and co-localization with caveolin-1 from the osteoblast plasma membrane. The presence of FXIII-A in caveolae was confirmed by preparing caveolae-enriched cellular fractions using sucrose density gradient ultracentrifugation followed by western blotting. Despite this association of FXIII-A with caveolae, there was no detectable transglutaminase activity in caveolae, as measured by monodansylcadaverine incorporation. TG inhibitor NC9—which can alter TG enzyme conformation—localized to caveolae and displaced FXIII-A from these structures when added to the osteoblast cultures. The decreased FXIII-A levels in caveolae after NC9 treatment increased c-Src activation, which resulted in caveolin-1 phosphorylation, homo-oligomerization and Akt phosphorylation, suggesting cellular FXIII-A has a role in regulating c-Src signaling in osteoblasts. PMID:26231113

  12. [Citophotometric expression of the factor of cellular proliferation ki-67 in the goiter colloid and in the papillary carcinoma of the thyroid].

    PubMed

    Souza, Gleim Dias de; Czeczko, Nicolau Gregori; Moreira, Hamilton; Ribas Filho, Jurandir Marcondes; Mafafaia, Osvaldo; Czeczko, Leticia Elizabeth Augustin; Thiele, Edilson Schwansee; Aguiar, Luiz Roberto Farion de

    2009-04-01

    To compare the cytophotometric quantitative expression of Ki-67 cellular proliferation factor in the colloid goiter with papillary carcinoma of the thyroid. The protein Ki-67 was studied with immunohistochemistry in 20 cases of papillary carcinoma of the thyroid and 12 cases of colloid goiter. The immunomarked cell nuclei were quantified through the software SAMBA 4000 and analyzed by software IMMUNO, considering variables index marker and optical density. The coefficient of the Spearman rank correlation and the non-parametric test of Mann-Whitney were estimated. There is significant difference between the goiter colloid and the papillary carcinoma of the thyroid in Ki-67 measurements, being bigger in papillary carcinomas. No difference was found in optical density. The correlation coefficient between the index marker and the optic density was 0,78. In colloid goiter, there was positive and significant association between the index marker and the optic density. For the papillary carcinoma of the thyroid the correlation between index marker and optic density was 0,18 (p = 0,572). There was no association between the index marker and the optic density in the carcinoma papillary of the thyroid. The cytophotometric expression of the Ki-67 showed higher cellular proliferation in the papillary carcinoma of the thyroid in comparison with in the colloid goiter.

  13. Inhibition of cellular proliferation by the Wilms' tumor suppressor WT1 is associated with suppression of insulin-like growth factor I receptor gene expression.

    PubMed Central

    Werner, H; Shen-Orr, Z; Rauscher, F J; Morris, J F; Roberts, C T; LeRoith, D

    1995-01-01

    We have investigated the regulation of the insulin-like growth factor I receptor (IGF-I-R) gene promoter by the Wilms' tumor suppressor WT1 in intact cells. The levels of endogenous IGF-I-R mRNA and the activity of IGF-I-R gene promoter fragments in luciferase reporter constructs were found to be significantly higher in G401 cells (a Wilms' tumor-derived cell line lacking detectable WT1 mRNA) than in 293 cells (a human embryonic kidney cell line which expresses significant levels of WT1 mRNA). To study whether WT1 could suppress the expression of the endogenous IGF-I-R gene, WT1-negative G401 cells were stably transfected with a WT1 expression vector. Expression of WT1 mRNA in G401 cells resulted in a significant decrease in the rate of cellular proliferation, which was associated with a reduction in the levels of IGF-I-R mRNA, promoter activity, and ligand binding and with a reduction in IGF-I-stimulated cellular proliferation, thymidine incorporation, and anchorage-independent growth. These data suggest that a major aspect of the action of the WT1 tumor suppressor is the repression of IGF-I-R gene expression. PMID:7791758

  14. Correlation of transforming growth factor-β messenger RNA (TGF-β mRNA) expression with cellular immunoassays in Triamcinolone-treated captive hybrid striped bass

    USGS Publications Warehouse

    Harms, Craig A.; Ottinger, Christopher A.; Kennedy-Stoskopf, S.

    2000-01-01

    Assessing fish immune status with molecular markers has been hampered by a lack of specific reagents. A quantitative polymerase chain reaction (PCR) method (reverse transcription quantitative–competitive PCR, RT-qcPCR) for measuring transforming growth factor-β (TGF-β) transcription from a broad range of teleost fish has recently been developed. The quantitative PCR now permits monitoring production of this important immunosuppressive cytokine in response to immunomodulating agents and conditions. We examined anterior kidney and spleen mononuclear cells from hybrid striped bass (female striped bass Morone saxatilis× male white bass M. chrysops) for production of TGF-β messenger RNA (mRNA) in response to administration of the synthetic glucocorticoid triamcinolone. We also compared TGF-β transcription with anterior kidney macrophage bactericidal activity and splenic lymphocyte blastogenesis. Anterior kidney mononuclear cell TGF-β mRNA levels decreased, whereas bactericidal activity increased. Spleen TGF-β mRNA levels did not change significantly, and splenic lymphocyte pokeweed mitogen stimulation index increased in triamcinolone-treated fish. Since triamcinolone is used therapeutically as a suppressive immunomodulator, the enhanced immune functions indicated by the cellular immunoassays were unexpected; however, the inverse response of TGF-β production and macrophage bactericidal activity was consistent with the known relationship between TGF-β and macrophage activation in mammals. Induced immunomodulation in hybrid striped bass was detectable by both traditional cellular immunoassays and the new RT-qcPCR for TGF-β.

  15. Pharmaceutical strategic purchasing requirements in Iran: Price interventions and the related effective factors

    PubMed Central

    Bastani, Peivand; Dinarvand, Rasoul; SamadBeik, Mahnaz; Pourmohammadi, Kimia

    2016-01-01

    Objective: Pharmaceutical access for the poor is an essential factor in developing countries that can be improved through strategic purchasing. This study was conducted to identify the elements affecting price in order to enable insurance organizations to put strategic purchasing into practice. Methods: This was a qualitative study conducted through content analysis with an inductive approach applying a five-stage framework analysis (familiarization, identifying a thematic framework, indexing, mapping, and interpretation). Data analysis was started right after transcribing each interview applying ATLAS.ti. Data were saturated after 32 semi-structured interviews by experts. These key informants were selected purposefully and through snowball sampling. Findings: Findings showed that there are four main themes as Pharmaceutical Strategic Purchasing Requirements in Iran as follows essential and structural factors, international factors, economical factors, and legal factors. Moreover, totally 14 related sub-themes were extracted in this area as the main effective variables. Conclusion: It seems that paying adequate attention to the four present themes and 14 sub-themes affecting price can enable health system policy-makers of developing countries like Iran to make the best decisions through strategic purchasing of drugs by the main insurers in order to improve access and health in the country. PMID:26985434

  16. Identification and characterization of an oocyte factor required for development of porcine nuclear transfer embryos

    PubMed Central

    Miyamoto, Kei; Nagai, Kouhei; Kitamura, Naoya; Nishikawa, Tomoaki; Ikegami, Haruka; Binh, Nguyen T.; Tsukamoto, Satoshi; Matsumoto, Mai; Tsukiyama, Tomoyuki; Minami, Naojiro; Yamada, Masayasu; Ariga, Hiroyoshi; Miyake, Masashi; Kawarasaki, Tatsuo; Matsumoto, Kazuya; Imai, Hiroshi

    2011-01-01

    Nuclear reprogramming of differentiated cells can be induced by oocyte factors. Despite numerous attempts, these factors and mechanisms responsible for successful reprogramming remain elusive. Here, we identify one such factor, necessary for the development of nuclear transfer embryos, using porcine oocyte extracts in which some reprogramming events are recapitulated. After incubating somatic nuclei in oocyte extracts from the metaphase II stage, the oocyte proteins that were specifically and abundantly incorporated into the nuclei were identified by mass spectrometry. Among 25 identified proteins, we especially focused on a multifunctional protein, DJ-1. DJ-1 is present at a high concentration in oocytes from the germinal vesicle stage until embryos at the four-cell stage. Inhibition of DJ-1 function compromises the development of nuclear transfer embryos but not that of fertilized embryos. Microarray analysis of nuclear transfer embryos in which DJ-1 function is inhibited shows perturbed expression of P53 pathway components. In addition, embryonic arrest of nuclear transfer embryos injected with anti–DJ-1 antibody is rescued by P53 inhibition. We conclude that DJ-1 is an oocyte factor that is required for development of nuclear transfer embryos. This study presents a means for identifying natural reprogramming factors in mammalian oocytes and a unique insight into the mechanisms underlying reprogramming by nuclear transfer. PMID:21482765

  17. Pharmaceutical strategic purchasing requirements in Iran: Price interventions and the related effective factors.

    PubMed

    Bastani, Peivand; Dinarvand, Rasoul; SamadBeik, Mahnaz; Pourmohammadi, Kimia

    2016-01-01

    Pharmaceutical access for the poor is an essential factor in developing countries that can be improved through strategic purchasing. This study was conducted to identify the elements affecting price in order to enable insurance organizations to put strategic purchasing into practice. This was a qualitative study conducted through content analysis with an inductive approach applying a five-stage framework analysis (familiarization, identifying a thematic framework, indexing, mapping, and interpretation). Data analysis was started right after transcribing each interview applying ATLAS.ti. Data were saturated after 32 semi-structured interviews by experts. These key informants were selected purposefully and through snowball sampling. Findings showed that there are four main themes as Pharmaceutical Strategic Purchasing Requirements in Iran as follows essential and structural factors, international factors, economical factors, and legal factors. Moreover, totally 14 related sub-themes were extracted in this area as the main effective variables. It seems that paying adequate attention to the four present themes and 14 sub-themes affecting price can enable health system policy-makers of developing countries like Iran to make the best decisions through strategic purchasing of drugs by the main insurers in order to improve access and health in the country.

  18. CELLULAR PATHOGENESIS OF DIABETIC GASTROENTEROPATHY

    PubMed Central

    Ördög, Tamás; Hayashi, Yujiro; Gibbons, Simon J.

    2010-01-01

    SUMMARY Gastroenteropathy manifesting in upper gastrointestinal symptoms, delayed gastric emptying, constipation, diarrhea and fecal incontinence occurs frequently in patients with diabetes mellitus and represents a significant health care burden. Current treatments are largely symptomatic and ineffective. Better understanding of the cellular and molecular pathogenesis of these disorders is required for the development of more effective therapies. Recent advances in our understanding of the inherent, high-level complexities of the control systems that execute and regulate gastrointestinal motility, together with the utilization of new experimental models and sophisticated physiological, morphological and molecular techniques have lead to the realization that diabetic gastroenteropathies cannot be ascribed to any singular defect or dysfunction. In fact, these disorders are multifactorial and involve a spectrum of metabolic and dystrophic changes that can potentially affect all key components of motor control including the systemic autonomic and enteric nervous systems, interstitial cells of Cajal and smooth muscle cells. Candidate pathomechanisms are also varied and include imbalance between pro- and anti-oxidative factors, altered trophic stimuli to mature cells and their progenitors, and, possibly, autoimmune factors. The goal of this paper is to review the cellular changes underlying diabetic gastroenteropathies and their potential causes, with particular focus on functional interactions between various cell types. It is proposed that diabetic gastroenteropathies should be considered a form of gastrointestinal neuromuscular dystrophy rather than a “functional” disorder. Future research should identify ways to block cytotoxic factors, support the regeneration of damaged cells and translate the experimental findings into new treatment modalities. PMID:19829287

  19. Large discrepancies in cellular distribution of the tonicity-induced expression of osmoprotective genes and their regulatory transcription factor TonEBP in rat brain.

    PubMed

    Maallem, S; Berod, A; Mutin, M; Kwon, H M; Tappaz, M L

    2006-10-13

    Osmoprotective genes are tonicity-activated genes involved in cellular osmoadaptation to hypertonicity and considered to be regulated by a specific transcription factor called tonicity-responsive enhancer-binding protein (TonEBP). In the brain we had previously established that TonEBP was expressed and tonicity-induced in neurons only. Here we have compared in various brain regions of rats subjected to systemic hypertonicity, the cellular expression of TonEBP through immunocytochemistry and the cellular expression of osmoprotective genes, namely aldose reductase (AR), sodium-dependent myo-inositol transporter (SMIT), betaine/GABA transporter (BGT1) and taurine transporter (TauT), by in situ hybridization using non-radioactive digoxigenin-labeled riboprobes. In neurons where TonEBP was strongly tonicity-induced, AR-mRNA labeling was strongly increased in some subsets (e.g. hippocampus pyramidal cells, cerebellar Purkinje cells and neurons of the hypothalamic magnocellular nuclei) but remained undetectable in some other subsets (e.g. neurons in cerebral cortex). Tonicity-induced AR-mRNA labeling was observed only several hours after the tonicity-induced expression of TonEBP. SMIT-mRNA labeling was tonicity-induced as densely and evenly distributed dots in neuron poor regions (e.g. cerebral cortex layer I and hippocampus stratum lacunosum-moleculare). The tonicity-induced expression of SMIT-mRNA may thus occur in non-neuronal cells, presumably astrocytes, where TonEBP is neither significantly expressed, nor tonicity-induced. In neurons showing a strong tonicity-induced expression of TonEBP, no SMIT-mRNA labeling was observed. BGT1-mRNA and TauT-mRNA labeling could not be detected, even after systemic hypertonicity. The present work reveals large discrepancies between the cellular distribution of the tonicity-induced expression of osmoprotective genes and that of their regulatory transactivator TonEBP. Depending on the cell subsets and the osmoprotective genes, Ton

  20. Extracellular Matrix-Regulated Gene Expression RequiresCooperation of SWI/SNF and Transcription Factors

    SciTech Connect

    Xu, Ren; Spencer, Virginia A.; Bissell, Mina J.

    2006-05-25

    Extracellular cues play crucial roles in the transcriptional regulation of tissue-specific genes, but whether and how these signals lead to chromatin remodeling is not understood and subject to debate. Using chromatin immunoprecipitation (ChIP) assays and mammary-specific genes as models, we show here that extracellular matrix (ECM) molecules and prolactin cooperate to induce histone acetylation and binding of transcription factors and the SWI/SNF complex to the {beta}- and ?-casein promoters. Introduction of a dominant negative Brg1, an ATPase subunit of SWI/SNF complex, significantly reduced both {beta}- and ?-casein expression, suggesting that SWI/SNF-dependent chromatin remodeling is required for transcription of mammary-specific genes. ChIP analyses demonstrated that the ATPase activity of SWI/SNF is necessary for recruitment of RNA transcriptional machinery, but not for binding of transcription factors or for histone acetylation. Coimmunoprecipitation analyses showed that the SWI/SNF complex is associated with STAT5, C/EBP{beta}, and glucocorticoid receptor (GR). Thus, ECM- and prolactin-regulated transcription of the mammary-specific casein genes requires the concerted action of chromatin remodeling enzymes and transcription factors.

  1. Productive Replication of Human Papillomavirus 31 Requires DNA Repair Factor Nbs1

    PubMed Central

    Anacker, Daniel C.; Gautam, Dipendra; Gillespie, Kenric A.; Chappell, William H.

    2014-01-01

    ABSTRACT Activation of the ATM (ataxia telangiectasia-mutated kinase)-dependent DNA damage response (DDR) is necessary for productive replication of human papillomavirus 31 (HPV31). We previously found that DNA repair and homologous recombination (HR) factors localize to sites of HPV replication, suggesting that ATM activity is required to recruit factors to viral genomes that can productively replicate viral DNA in a recombination-dependent manner. The Mre11-Rad50-Nbs1 (MRN) complex is an essential component of the DDR that is necessary for ATM-mediated HR repair and localizes to HPV DNA foci. In this study, we demonstrate that the HPV E7 protein is sufficient to increase levels of the MRN complex and also interacts with MRN components. We have found that Nbs1 depletion blocks productive viral replication and results in decreased localization of Mre11, Rad50, and the principal HR factor Rad51 to HPV DNA foci upon differentiation. Nbs1 contributes to the DDR by acting as an upstream activator of ATM in response to double-strand DNA breaks (DSBs) and as a downstream effector of ATM activity in the intra-S-phase checkpoint. We have found that phosphorylation of ATM and its downstream target Chk2, as well as SMC1 (structural maintenance of chromosome 1), is maintained upon Nbs1 knockdown in differentiating cells. Given that ATM and Chk2 are required for productive replication, our results suggest that Nbs1 contributes to viral replication outside its role as an ATM activator, potentially through ensuring localization of DNA repair factors to viral genomes that are necessary for efficient productive replication. IMPORTANCE The mechanisms that regulate human papillomavirus (HPV) replication during the viral life cycle are not well understood. Our finding that Nbs1 is necessary for productive replication even in the presence of ATM (ataxia telangiectasia-mutated kinase) and Chk2 phosphorylation offers evidence that Nbs1 contributes to viral replication downstream of

  2. Productive replication of human papillomavirus 31 requires DNA repair factor Nbs1.

    PubMed

    Anacker, Daniel C; Gautam, Dipendra; Gillespie, Kenric A; Chappell, William H; Moody, Cary A

    2014-08-01

    Activation of the ATM (ataxia telangiectasia-mutated kinase)-dependent DNA damage response (DDR) is necessary for productive replication of human papillomavirus 31 (HPV31). We previously found that DNA repair and homologous recombination (HR) factors localize to sites of HPV replication, suggesting that ATM activity is required to recruit factors to viral genomes that can productively replicate viral DNA in a recombination-dependent manner. The Mre11-Rad50-Nbs1 (MRN) complex is an essential component of the DDR that is necessary for ATM-mediated HR repair and localizes to HPV DNA foci. In this study, we demonstrate that the HPV E7 protein is sufficient to increase levels of the MRN complex and also interacts with MRN components. We have found that Nbs1 depletion blocks productive viral replication and results in decreased localization of Mre11, Rad50, and the principal HR factor Rad51 to HPV DNA foci upon differentiation. Nbs1 contributes to the DDR by acting as an upstream activator of ATM in response to double-strand DNA breaks (DSBs) and as a downstream effector of ATM activity in the intra-S-phase checkpoint. We have found that phosphorylation of ATM and its downstream target Chk2, as well as SMC1 (structural maintenance of chromosome 1), is maintained upon Nbs1 knockdown in differentiating cells. Given that ATM and Chk2 are required for productive replication, our results suggest that Nbs1 contributes to viral replication outside its role as an ATM activator, potentially through ensuring localization of DNA repair factors to viral genomes that are necessary for efficient productive replication. The mechanisms that regulate human papillomavirus (HPV) replication during the viral life cycle are not well understood. Our finding that Nbs1 is necessary for productive replication even in the presence of ATM (ataxia telangiectasia-mutated kinase) and Chk2 phosphorylation offers evidence that Nbs1 contributes to viral replication downstream of facilitating ATM

  3. Pharmacological modulation of heat shock factor 1 by antiinflammatory drugs results in protection against stress-induced cellular damage.

    PubMed Central

    Lee, B S; Chen, J; Angelidis, C; Jurivich, D A; Morimoto, R I

    1995-01-01

    The activation of heat shock genes by diverse forms of environmental and physiological stress has been implicated in a number of human diseases, including ischemic damage, reperfusion injury, infection, neurodegeneration, and inflammation. The enhanced levels of heat shock proteins and molecular chaperones have broad cytoprotective effects against acute lethal exposures to stress. Here, we show that the potent antiinflammatory drug indomethacin activates the DNA-binding activity of human heat shock transcription factor 1 (HSF1). Perhaps relevant to its pharmacological use, indomethacin pretreatment lowers the temperature threshold of HSF1 activation, such that a complete heat shock response can be attained at temperatures that are by themselves insufficient. The synergistic effect of indomethacin and elevated temperature is biologically relevant and results in the protection of cells against exposure to cytotoxic conditions. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7638169

  4. Discovery of a small molecule that inhibits the interaction of anthrax edema factor with its cellular activator, calmodulin.

    PubMed

    Lee, Young-Sam; Bergson, Pamela; He, Wei Song; Mrksich, Milan; Tang, Wei-Jen

    2004-08-01

    The catalytic efficiency of adenylyl cyclase activity of edema factor (EF) from Bacillus anthracis is enhanced by approximately 1000-fold upon its binding to mammalian protein calmodulin (CaM). A tandem cell-based and protein binding-based screen of a 10,000 member library identified a molecule that inhibits the EF-CaM interaction and therefore the adenylyl cyclase activity. A combination of fluorescence spectroscopy and photolabeling studies showed that the molecule targets the CaM binding region of EF. A series of related compounds were synthesized and evaluated to identify one compound, 4-[4-(4-nitrophenyl)-thiazolylamino]-benzenesulfonamide, that maintained activity against EF but showed minimal toxicity to two cultured cell lines. This compound represents an important reagent to study the role of EF in anthrax pathology and may represent a drug lead against anthrax infection.

  5. Telomere length and telomere repeating factors: Cellular markers for post-traumatic stress disorder-like model.

    PubMed

    Dong, Yuanjun; Zhang, Guiqing; Yuan, Xiuyu; Zhang, Yueqi; Hu, Min

    2016-05-01

    The aim of the present study was to explore the telomere length of peripheral blood leukocytes from a rat model of post-traumatic stress disorder (PTSD), as well as the expression level of telomere-binding protein in the hippocampal CA1 region. The PTSD model was established with 42 adult male Wistar rats. The relative telomere length of the leukocytes was measured by real-time fluorescence quantitative polymerase chain reaction, and the expression levels of telomere repeating factor 1 (TRF1) and telomere repeating factor 2 (TRF2) in the hippocampal CA1 region of the PTSD rat model were determined by immunofluorescence technology. The covariance analysis of repeated measurements by the mixed model approach was used for the telomere length analysis. The comparison of averaged data among groups was performed using least significant difference and analysis of variance. The Student's t test or the Mann-Whitney U test was used for intragroup comparison. The association study among groups was conducted using the Spearman test. The shortening speed of telomere length significantly accelerated in rats after Single Prolonged Stress (SPS) stimulation (P<0.05). The expression levels of TRF1 and TRF2 increased with the progress of PTSD, and the expression peak was shown in day 14, which was significantly different from the control group (P<0.05). The shortening speed of the telomere length of peripheral blood leukocytes accelerated in PTSD rats, and the expression levels of TRF1 and TRF2 increased in hippocampus, both of which were closely associated with the pathological progress of the PTSD-like model and unfavorable prognosis. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Definition of human factor requirements in the invitation to tender and system specifications

    SciTech Connect

    Remond, C.; Pirus, D.

    2006-07-01

    For more than 20 years, EDF has led Human Factors (HF) studies in order to improve its plants, the interfaces used by operating crews in nuclear power plants, the prescriptive operating documents, the organization and the control of the NPPs... The experience feedback of these projects highlights the need for defining, as soon as possible in the projects, the HF requirements to take into account and to list them in the HSI Specifications addressed to the designer or to the supplier. Experience feedback shows that if these requirements (in terms of integration of the HF process in the project and the HF criteria for the design) are not clearly and precisely defined to the designer, the persons in charge of projects can encounter real difficulties for: 1) Defining the resources required to carry out the tests (usability tests during the design; verification and validation tests during or at the end of the design), 2) Plan the HF interventions and studies in the design schedule, 3) Refer to HF standards and requirements during the design phase, 4) Carry out modifications on the products designed before they are commissioned on site in case of differences between these products' design features and the HF requirements. This paper presents the solutions recommended by EDF to cope with these problems, in particular with respect to the Human Factors Engineering (HFE) methodology. This paper insists on the functional needs to take into account by the designer during the design of the product and the need for writing a document as 'functional requirements' which presents the experience feedback of the existing products, the HF studies carried out on site to have in-depth knowledge of the tasks and activities done by the operating crews related to the considered interface modifications, the requirements applicable during the design, etc. This paper insists on the need for being also prescriptive in the specifications on: 1) the iterative character of the design process, 2

  7. Factors associated with postoperative requirement of renal replacement therapy following off-pump coronary bypass surgery.

    PubMed

    Kato, Tomoko S; Machida, Yoichiro; Kuwaki, Kenji; Yamamoto, Taira; Amano, Atsushi

    2017-02-01

    Cardiopulmonary bypass usage provokes a systemic inflammatory response resulting in deterioration of renal function. However, risk factors for requiring renal replacement therapy (RRT) following off-pump coronary artery bypass graft surgery (CABG) have not yet been fully elucidated. We reviewed 718 consecutive patients undergoing elective off-pump CABG at our institution, excluding patients on chronic hemodialysis preoperatively. Sub-analysis of patients with preserved renal function, defined as a creatinine level below a cut-off value of 1.12 mg/dL (obtained by receiver operating characteristic curve), was also performed. Of the 718 patients, 41 (5.7 %) required RRT. There were 556 patients (77.4 %) with preserved renal function preoperatively, and 13 (2.4 %) of these required postoperative RRT. Multivariate analysis revealed that age (years) and preoperative serum creatinine (mg/dL) and brain natriuretic peptide (BNP) levels (pg/dL) were associated with RRT [odds ratios (OR) 1.052, 95 % confidence interval (CI) 9.064 and 1.001, respectively, all p < 0.05] in the total population, whereas low albumin concentration was the only independent predictor for RRT in patients with preserved renal function (OR 0.062, p < 0.0001). When creatinine levels were below 1.5 mg/dL, the predictive power of hypoalbuminemia for RRT requirement overwhelmed that of creatinine or BNP levels. Older age, preoperative elevated creatinine and BNP levels were associated with a requirement for RRT following off-pump CABG. In pat