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Sample records for cellular factors required

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. Interactome Analysis of the Influenza A Virus Transcription/Replication Machinery Identifies Protein Phosphatase 6 as a Cellular Factor Required for Efficient Virus Replication

    PubMed Central

    York, Ashley; Hutchinson, Edward C.

    2014-01-01

    ABSTRACT The negative-sense RNA genome of influenza A virus is transcribed and replicated by the viral RNA-dependent RNA polymerase (RdRP). The viral RdRP is an important host range determinant, indicating that its function is affected by interactions with cellular factors. However, the identities and the roles of most of these factors remain unknown. Here, we employed affinity purification followed by mass spectrometry to identify cellular proteins that interact with the influenza A virus RdRP in infected human cells. We purified RdRPs using a recombinant influenza virus in which the PB2 subunit of the RdRP is fused to a Strep-tag. When this tagged subunit was purified from infected cells, copurifying proteins included the other RdRP subunits (PB1 and PA) and the viral nucleoprotein and neuraminidase, as well as 171 cellular proteins. Label-free quantitative mass spectrometry revealed that the most abundant of these host proteins were chaperones, cytoskeletal proteins, importins, proteins involved in ubiquitination, kinases and phosphatases, and mitochondrial and ribosomal proteins. Among the phosphatases, we identified three subunits of the cellular serine/threonine protein phosphatase 6 (PP6), including the catalytic subunit PPP6C and regulatory subunits PPP6R1 and PPP6R3. PP6 was found to interact directly with the PB1 and PB2 subunits of the viral RdRP, and small interfering RNA (siRNA)-mediated knockdown of the catalytic subunit of PP6 in infected cells resulted in the reduction of viral RNA accumulation and the attenuation of virus growth. These results suggest that PP6 interacts with and positively regulates the activity of the influenza virus RdRP. IMPORTANCE Influenza A viruses are serious clinical and veterinary pathogens, causing substantial health and economic impacts. In addition to annual seasonal epidemics, occasional global pandemics occur when viral strains adapt to humans from other species. To replicate efficiently and cause disease, influenza

  8. 47 CFR 22.901 - Cellular service requirements and limitations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 2 2010-10-01 2010-10-01 false Cellular service requirements and limitations... 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...

  9. Binding of cellular export factor REF/Aly by Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein is not required for efficient KSHV lytic replication.

    PubMed

    Li, Da-Jiang; Verma, Dinesh; Swaminathan, Sankar

    2012-09-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein is expressed early during lytic KSHV replication, enhances expression of many KSHV genes, and is essential for virus production. ORF57 is a member of a family of proteins conserved among all human and many animal herpesviruses that are multifunctional regulators of gene expression and act posttranscriptionally to increase accumulation of their target mRNAs. The mechanism of ORF57 action is complex and may involve effects on mRNA transcription, stability, and export. ORF57 directly binds to REF/Aly, a cellular RNA-binding protein component of the TREX complex that mediates RNA transcription and export. We analyzed the effects of an ORF57 mutation known to abrogate REF/Aly binding and demonstrate that the REF-binding mutant is impaired in activation of viral mRNAs and noncoding RNAs confined to the nucleus. Although the inability to bind REF leads to decreased ORF57 activity in enhancing gene expression, there is no demonstrable effect on nuclear export of viral mRNA or the ability of ORF57 to support KSHV replication and virus production. These data indicate that REF/Aly-ORF57 interaction is not essential for KSHV lytic replication but may contribute to target RNA stability independent of effects on RNA export, suggesting a novel role for REF/Aly in viral RNA metabolism.

  10. Identification of cellular factors binding to acetylated HIV-1 integrase.

    PubMed

    Allouch, Awatef; Cereseto, Anna

    2011-11-01

    The viral protein integrase (IN) catalyzes the integration of the HIV-1 cDNA into the host cellular genome. We have recently demonstrated that IN is acetylated by a cellular histone acetyltransferase, p300, which modifies three lysines located in the C-terminus of the viral factor (Cereseto et al. in EMBO J 24:3070-3081, 2005). This modification enhances IN catalytic activity, as demonstrated by in vitro assays. Consistently, mutations introduced in the targeted lysines greatly decrease the efficiency of HIV-1 integration. Acetylation was proven to regulate protein functions by modulating protein-protein interactions. HIV-1 to efficiently complete its replication steps, including the integration reaction, requires interacting with numerous cellular factors. Therefore, we sought to investigate whether acetylation might modulate the interaction between IN and the cellular factors. To this aim we performed a yeast two-hybrid screening that differs from the screenings so far performed (Rain et al. in Methods 47:291-297, 2009; Studamire and Goff in Retrovirology 5:48, 2008) for using as bait IN constitutively acetylated. From this analysis we have identified thirteen cellular factors involved in transcription, chromatin remodeling, nuclear transport, RNA binding, protein synthesis regulation and microtubule organization. To validate these interactions, binding assays were performed showing that acetylation increases the affinity of IN with specific factors. Nevertheless, few two-hybrid hits bind with the same affinity the acetylated and the unmodified IN. These results further underlie the relevance of IN post-translational modification by acetylation in HIV-1 replication cycle.

  11. Drak Is Required for Actomyosin Organization During Drosophila Cellularization

    PubMed Central

    Chougule, Ashish B.; Hastert, Mary C.; Thomas, Jeffrey H.

    2016-01-01

    The generation of force by actomyosin contraction is critical for a variety of cellular and developmental processes. Nonmuscle myosin II is the motor that drives actomyosin contraction, and its activity is largely regulated by phosphorylation of the myosin regulatory light chain. During the formation of the Drosophila cellular blastoderm, actomyosin contraction drives constriction of microfilament rings, modified cytokinesis rings. Here, we find that Drak is necessary for most of the phosphorylation of the myosin regulatory light chain during cellularization. We show that Drak is required for organization of myosin II within the microfilament rings. Proper actomyosin contraction of the microfilament rings during cellularization also requires Drak activity. Constitutive activation of myosin regulatory light chain bypasses the requirement for Drak, suggesting that actomyosin organization and contraction are mediated through Drak’s regulation of myosin activity. Drak is also involved in the maintenance of furrow canal structure and lateral plasma membrane integrity during cellularization. Together, our observations suggest that Drak is the primary regulator of actomyosin dynamics during cellularization. PMID:26818071

  12. Tumor Necrosis Factor (TNF) Signaling, but Not TWEAK (TNF-like Weak Inducer of Apoptosis)-triggered cIAP1 (Cellular Inhibitor of Apoptosis Protein 1) Degradation, Requires cIAP1 RING Dimerization and E2 Binding

    PubMed Central

    Feltham, Rebecca; Moulin, Maryline; Vince, James E.; Mace, Peter D.; Wong, Wendy Wei-Lynn; Anderton, Holly; Day, Catherine L.; Vaux, David L.; Silke, John

    2010-01-01

    Cellular inhibitor of apoptosis (cIAP) proteins, cIAP1 and cIAP2, are important regulators of tumor necrosis factor (TNF) superfamily (SF) signaling and are amplified in a number of tumor types. They are targeted by IAP antagonist compounds that are undergoing clinical trials. IAP antagonist compounds trigger cIAP autoubiquitylation and degradation. The TNFSF member TWEAK induces lysosomal degradation of TRAF2 and cIAPs, leading to elevated NIK levels and activation of non-canonical NF-κB. To investigate the role of the ubiquitin ligase RING domain of cIAP1 in these pathways, we used cIAP-deleted cells reconstituted with cIAP1 point mutants designed to interfere with the ability of the RING to dimerize or to interact with E2 enzymes. We show that RING dimerization and E2 binding are required for IAP antagonists to induce cIAP1 degradation and protect cells from TNF-induced cell death. The RING functions of cIAP1 are required for full TNF-induced activation of NF-κB, however, delayed activation of NF-κB still occurs in cIAP1 and -2 double knock-out cells. The RING functions of cIAP1 are also required to prevent constitutive activation of non-canonical NF-κB by targeting NIK for proteasomal degradation. However, in cIAP double knock-out cells TWEAK was still able to increase NIK levels demonstrating that NIK can be regulated by cIAP-independent pathways. Finally we show that, unlike IAP antagonists, TWEAK was able to induce degradation of cIAP1 RING mutants. These results emphasize the critical importance of the RING of cIAP1 in many signaling scenarios, but also demonstrate that in some pathways RING functions are not required. PMID:20356846

  13. 47 CFR 22.901 - Cellular service requirements and limitations.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... limitations. The licensee of each cellular system is responsible for ensuring that its cellular system operates in compliance with this section. (a) Each cellular system must provide either mobile service... cellular services, each cellular system may incorporate any technology that meets all applicable...

  14. 47 CFR 22.901 - Cellular service requirements and limitations.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... limitations. The licensee of each cellular system is responsible for ensuring that its cellular system operates in compliance with this section. (a) Each cellular system must provide either mobile service... cellular services, each cellular system may incorporate any technology that meets all applicable...

  15. 47 CFR 22.901 - Cellular service requirements and limitations.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... limitations. The licensee of each cellular system is responsible for ensuring that its cellular system operates in compliance with this section. (a) Each cellular system must provide either mobile service... cellular services, each cellular system may incorporate any technology that meets all applicable...

  16. 47 CFR 22.901 - Cellular service requirements and limitations.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... limitations. The licensee of each cellular system is responsible for ensuring that its cellular system operates in compliance with this section. (a) Each cellular system must provide either mobile service... cellular services, each cellular system may incorporate any technology that meets all applicable...

  17. Translation Factors Specify Cellular Metabolic State.

    PubMed

    Mata, Juan

    2016-08-16

    In this issue of Cell Reports, Shah et al. present evidence that a subcomplex of the eIF3 translation initiation factor regulates translation of mRNAs encoding components of the mitochondrial electron transport chain and glycolytic enzymes, thus linking translational control with energy metabolism. PMID:27533178

  18. Cellular Reprogramming Using Defined Factors and MicroRNAs.

    PubMed

    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.

  19. 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

  20. The Major Cellular Sterol Regulatory Pathway Is Required for Andes Virus Infection

    PubMed Central

    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

    2014-01-01

    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. PMID:24516383

  1. 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

  2. Cellular antiviral responses against influenza A virus are countered at the posttranscriptional level by the viral NS1A protein via its binding to a cellular protein required for the 3' end processing of cellular pre-mRNAS.

    PubMed

    Noah, Diana L; Twu, Karen Y; Krug, Robert M

    2003-03-15

    The influenza A virus NS1 protein (NS1A protein) binds and inhibits the function of the 30-kDa subunit of CPSF, a cellular factor that is required for the 3'-end processing of cellular pre-mRNAs. Here we generate a recombinant influenza A/Udorn/72 virus that encodes an NS1A protein containing a mutated binding site for the 30-kDa subunit of CPSF. This mutant virus is substantially attenuated, indicating that this binding site in the NS1A protein is required for efficient virus replication. Using this mutant virus, we show that NS1A binding to CPSF mediates the viral posttranscriptional countermeasure against the initial cellular antiviral response--the interferon-alpha/beta (IFN-alpha/beta)-independent activation of the transcription of cellular antiviral genes, which requires the interferon regulatory factor-3 (IRF-3) transcription factor that is activated by virus infection. Whereas the posttranscriptional processing of these cellular antiviral pre-mRNAs is inhibited in cells infected by wild-type influenza A virus, functional antiviral mRNAs are produced in cells infected by the mutant virus. These results establish that the binding of 30-kDa CPSF to the NS1A protein is largely responsible for the posttranscriptional inhibition of the processing of these cellular antiviral pre-mRNAs. Mutation of this binding site in the NS1A protein also affects a second cellular antiviral response: in cells infected by the mutant virus, IFN-beta mRNA is produced earlier and in larger amounts.

  3. 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

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

    PubMed

    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

  5. The GARP complex is required for cellular sphingolipid homeostasis

    PubMed Central

    Fröhlich, Florian; Petit, Constance; Kory, Nora; Christiano, Romain; Hannibal-Bach, Hans-Kristian; Graham, Morven; Liu, Xinran; Ejsing, Christer S; Farese, Robert V; Walther, Tobias C

    2015-01-01

    Sphingolipids are abundant membrane components and important signaling molecules in eukaryotic cells. Their levels and localization are tightly regulated. However, the mechanisms underlying this regulation remain largely unknown. In this study, we identify the Golgi-associated retrograde protein (GARP) complex, which functions in endosome-to-Golgi retrograde vesicular transport, as a critical player in sphingolipid homeostasis. GARP deficiency leads to accumulation of sphingolipid synthesis intermediates, changes in sterol distribution, and lysosomal dysfunction. A GARP complex mutation analogous to a VPS53 allele causing progressive cerebello-cerebral atrophy type 2 (PCCA2) in humans exhibits similar, albeit weaker, phenotypes in yeast, providing mechanistic insights into disease pathogenesis. Inhibition of the first step of de novo sphingolipid synthesis is sufficient to mitigate many of the phenotypes of GARP-deficient yeast or mammalian cells. Together, these data show that GARP is essential for cellular sphingolipid homeostasis and suggest a therapeutic strategy for the treatment of PCCA2. DOI: http://dx.doi.org/10.7554/eLife.08712.001 PMID:26357016

  6. Serum factors in older individuals change cellular clock properties

    PubMed Central

    Pagani, Lucia; Schmitt, Karen; Meier, Fides; Izakovic, Jan; Roemer, Konstanze; Viola, Antoine; Cajochen, Christian; Wirz-Justice, Anna; Brown, Steven A.; Eckert, Anne

    2011-01-01

    Human aging is accompanied by dramatic changes in daily sleep–wake behavior: Activity shifts to an earlier phase, and the consolidation of sleep and wake is disturbed. Although this daily circadian rhythm is brain-controlled, its mechanism is encoded by cell-autonomous circadian clocks functioning in nearly every cell of the body. In fact, human clock properties measured in peripheral cells such as fibroblasts closely mimic those measured physiologically and behaviorally in the same subjects. To understand better the molecular mechanisms by which human aging affects circadian clocks, we characterized the clock properties of fibroblasts cultivated from dermal biopsies of young and older subjects. Fibroblast period length, amplitude, and phase were identical in the two groups even though behavior was not, thereby suggesting that basic clock properties of peripheral cells do not change during aging. Interestingly, measurement of the same cells in the presence of human serum from older donors shortened period length and advanced the phase of cellular circadian rhythms compared with treatment with serum from young subjects, indicating that a circulating factor might alter human chronotype. Further experiments demonstrated that this effect is caused by a thermolabile factor present in serum of older individuals. Thus, even though the molecular machinery of peripheral circadian clocks does not change with age, some age-related circadian dysfunction observed in vivo might be of hormonal origin and therefore might be pharmacologically remediable. PMID:21482780

  7. Cellular Defense and Sensory Cell Survival Require Distinct Functions of ebi in Drosophila

    PubMed Central

    Lim, Young-Mi; Yagi, Yoshimasa; Tsuda, Leo

    2015-01-01

    The innate immune response and stress-induced apoptosis are well-established signaling pathways related to cellular defense. NF-κB and AP-1 are redox-sensitive transcription factors that play important roles in those pathways. Here we show that Ebi, a Drosophila homolog of the mammalian co-repressor molecule transducin β-like 1 (TBL1), variously regulates the expression of specific genes that are targets of redox-sensitive transcription factors. In response to different stimuli, Ebi activated gene expression to support the acute immune response in fat bodies, whereas Ebi repressed genes that are involved in apoptosis in photoreceptor cells. Thus, Ebi seems to act as a regulatory switch for genes that are activated or repressed in response to different external stimuli. Our results offer clear in vivo evidence that the Ebi-containing co-repressor complex acts in a distinct manner to regulate transcription that is required for modulating the output of various processes during Drosophila development. PMID:26524764

  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

    2014-12-01

    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.

  9. 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

    2014-12-01

    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. PMID:25344604

  10. E2F Transcription Factor 1 Regulates Cellular and Organismal Senescence by Inhibiting Forkhead Box O Transcription Factors*

    PubMed Central

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

    2014-01-01

    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. PMID:25344604

  11. 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.

  12. 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. PMID:27558729

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

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 1 2014-10-01 2014-10-01 false Additional assistance capability requirements for wireline, cellular, and PCS telecommunications carriers. 1.20007 Section 1.20007 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL PRACTICE AND PROCEDURE Grants by Random Selection Communications Assistance for Law Enforcement Act...

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

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 1 2013-10-01 2013-10-01 false Additional assistance capability requirements for wireline, cellular, and PCS telecommunications carriers. 1.20007 Section 1.20007 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL PRACTICE AND PROCEDURE Grants by Random Selection Communications Assistance for Law Enforcement Act...

  15. Organic growth factor requirements of some yeasts.

    PubMed

    Madan, M; Gulati, N

    1980-01-01

    Some sporogenous yeasts (Brettanomyces bruxellensis, Debaryomyces hansenii, Hansenula ciferrii, Hansenula polymorpha, Pichia polymorpha, Saccharomycopsis guttulata, and Saccharomyces chevalieri), isolated from various fruits have been examined for their organic growth factor requisites. H. ciferrii was completely deficient in thiamine, biotin, inositol, riboflavin, niacin, and partially deficient in pantothenic acid. It required an external supply of 0.1-1.0 ppm thiamine, 0.01-0.1 ppm biotin, 10.0 ppm inositol, 0.10 ppm niacin and riboflavin for its optimum growth. H. polymorpha showed partial deficiency only in xanthine. P. polymorpha gave indications of partial deficiencies in thiamine and biotin. S. guttulata was completely deficient in biotin, and partially deficient in adenine sulphate. It required 0.01 ppm biotin for optimum growth. S chevalieri was completely deficient in pyridoxine and partially deficient in thiamine. It required 0.1 ppm pyridoxine for maximum growth. D. hansenii and B bruxellensis were auxoautotrophic for the various growth factors studied. PMID:7242379

  16. 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.

  17. Analysis of murine cellular receptors for tumor-killing factor

    SciTech Connect

    Ohsawa, F.; Natori, S.

    1987-01-01

    Receptors for tumor-killing factor (TKF) on the surface of murine cells were analyzed using radioiodinated TKF. Not only sensitive cells but also insensitive cells were found to have specific receptors. Among the sensitive cells, no clear relation was observed between the number of receptors on the cell surface and sensitivity to TKF. Compounds affecting microfilaments (cytochalasin B and D) and microtubules (colchicine and Colcemid) significantly inhibited cytolysis of sensitive cells induced by receptor-bound TKF. It is concluded that internalization of receptor-bound TKF is a prerequisite for triggering cytolysis.

  18. 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.

  19. 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.

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

    PubMed

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

    2007-12-01

    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

  1. 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

  2. Tumor Necrosis Factor Receptor 2: Its Contribution to Acute Cellular Rejection and Clear Cell Renal Carcinoma

    PubMed Central

    Wang, Jun; Al-Lamki, Rafia S.

    2013-01-01

    Tumor necrosis factor receptor 2 (TNFR2) is a type I transmembrane glycoprotein and one of the two receptors that orchestrate the complex biological functions of tumor necrosis factor (TNF, also designed TNF-α). Accumulating experimental evidence suggests that TNFR2 plays an important role in renal disorders associated with acute cellular rejection and clear cell renal carcinoma but its exact role in these settings is still not completely understood. This papers reviews the factors that may mediate TNFR2 induction in acute cellular rejection and clear cell renal carcinoma and its contribution to these conditions and discusses its therapeutic implications. A greater understanding of the function of TNFR2 may lead to the development of new anti-TNF drugs. PMID:24350291

  3. 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.

  4. 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

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

    PubMed

    He, Bing; Martin, Adam; Wieschaus, Eric

    2016-07-01

    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

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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

    2014-01-01

    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. PMID:24269685

  11. 14 CFR 121.647 - Factors for computing fuel required.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 3 2011-01-01 2011-01-01 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...

  12. 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...

  13. 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.

  14. A threshold of endogenous stress is required to engage cellular response to protect against mutagenesis.

    PubMed

    Saintigny, Yannick; Chevalier, François; Bravard, Anne; Dardillac, Elodie; Laurent, David; Hem, Sonia; Dépagne, Jordane; Radicella, J Pablo; Lopez, Bernard S

    2016-07-11

    Endogenous stress represents a major source of genome instability, but is in essence difficult to apprehend. Incorporation of labeled radionuclides into DNA constitutes a tractable model to analyze cellular responses to endogenous attacks. Here we show that incorporation of [(3)H]thymidine into CHO cells generates oxidative-induced mutagenesis, but, with a peak at low doses. Proteomic analysis showed that the cellular response differs between low and high levels of endogenous stress. In particular, these results confirmed the involvement of proteins implicated in redox homeostasis and DNA damage signaling pathways. Induced-mutagenesis was abolished by the anti-oxidant N-acetyl cysteine and plateaued, at high doses, upon exposure to L-buthionine sulfoximine, which represses cellular detoxification. The [(3)H]thymidine-induced mutation spectrum revealed mostly base substitutions, exhibiting a signature specific for low doses (GC > CG and AT > CG). Consistently, the enzymatic activity of the base excision repair protein APE-1 is induced at only medium or high doses. Collectively, the data reveal that a threshold of endogenous stress must be reached to trigger cellular detoxification and DNA repair programs; below this threshold, the consequences of endogenous stress escape cellular surveillance, leading to high levels of mutagenesis. Therefore, low doses of endogenous local stress can jeopardize genome integrity more efficiently than higher doses.

  15. A threshold of endogenous stress is required to engage cellular response to protect against mutagenesis

    PubMed Central

    Saintigny, Yannick; Chevalier, François; Bravard, Anne; Dardillac, Elodie; Laurent, David; Hem, Sonia; Dépagne, Jordane; Radicella, J. Pablo; Lopez, Bernard S.

    2016-01-01

    Endogenous stress represents a major source of genome instability, but is in essence difficult to apprehend. Incorporation of labeled radionuclides into DNA constitutes a tractable model to analyze cellular responses to endogenous attacks. Here we show that incorporation of [3H]thymidine into CHO cells generates oxidative-induced mutagenesis, but, with a peak at low doses. Proteomic analysis showed that the cellular response differs between low and high levels of endogenous stress. In particular, these results confirmed the involvement of proteins implicated in redox homeostasis and DNA damage signaling pathways. Induced-mutagenesis was abolished by the anti-oxidant N-acetyl cysteine and plateaued, at high doses, upon exposure to L-buthionine sulfoximine, which represses cellular detoxification. The [3H]thymidine-induced mutation spectrum revealed mostly base substitutions, exhibiting a signature specific for low doses (GC > CG and AT > CG). Consistently, the enzymatic activity of the base excision repair protein APE-1 is induced at only medium or high doses. Collectively, the data reveal that a threshold of endogenous stress must be reached to trigger cellular detoxification and DNA repair programs; below this threshold, the consequences of endogenous stress escape cellular surveillance, leading to high levels of mutagenesis. Therefore, low doses of endogenous local stress can jeopardize genome integrity more efficiently than higher doses. PMID:27406380

  16. Embryonic MicroRNA-369 Controls Metabolic Splicing Factors and Urges Cellular Reprograming.

    PubMed

    Konno, Masamitsu; Koseki, Jun; Kawamoto, Koichi; Nishida, Naohiro; Matsui, Hidetoshi; Dewi, Dyah Laksmi; Ozaki, Miyuki; Noguchi, Yuko; Mimori, Koshi; Gotoh, Noriko; Tanuma, Nobuhiro; Shima, Hiroshi; Doki, Yuichiro; Mori, Masaki; Ishii, Hideshi

    2015-01-01

    Noncoding microRNAs inhibit translation and lower the transcript stability of coding mRNA, however miR-369 s, in aberrant silencing genomic regions, stabilizes target proteins under cellular stress. We found that in vitro differentiation of embryonic stem cells led to chromatin methylation of histone H3K4 at the miR-369 region on chromosome 12qF in mice, which is expressed in embryonic cells and is critical for pluripotency. Proteomic analyses revealed that miR-369 stabilized translation of pyruvate kinase (Pkm2) splicing factors such as HNRNPA2B1. Overexpression of miR-369 stimulated Pkm2 splicing and enhanced induction of cellular reprogramming by induced pluripotent stem cell factors, whereas miR-369 knockdown resulted in suppression. Furthermore, immunoprecipitation analysis showed that the Argonaute complex contained the fragile X mental retardation-related protein 1 and HNRNPA2B1 in a miR-369-depedent manner. Our findings demonstrate a unique role of the embryonic miR-369-HNRNPA2B1 axis in controlling metabolic enzyme function, and suggest a novel pathway linking epigenetic, transcriptional, and metabolic control in cell reprogramming. PMID:26176628

  17. Embryonic MicroRNA-369 Controls Metabolic Splicing Factors and Urges Cellular Reprograming

    PubMed Central

    Konno, Masamitsu; Koseki, Jun; Kawamoto, Koichi; Nishida, Naohiro; Matsui, Hidetoshi; Dewi, Dyah Laksmi; Ozaki, Miyuki; Noguchi, Yuko; Mimori, Koshi; Gotoh, Noriko; Tanuma, Nobuhiro; Shima, Hiroshi; Doki, Yuichiro

    2015-01-01

    Noncoding microRNAs inhibit translation and lower the transcript stability of coding mRNA, however miR-369 s, in aberrant silencing genomic regions, stabilizes target proteins under cellular stress. We found that in vitro differentiation of embryonic stem cells led to chromatin methylation of histone H3K4 at the miR-369 region on chromosome 12qF in mice, which is expressed in embryonic cells and is critical for pluripotency. Proteomic analyses revealed that miR-369 stabilized translation of pyruvate kinase (Pkm2) splicing factors such as HNRNPA2B1. Overexpression of miR-369 stimulated Pkm2 splicing and enhanced induction of cellular reprogramming by induced pluripotent stem cell factors, whereas miR-369 knockdown resulted in suppression. Furthermore, immunoprecipitation analysis showed that the Argonaute complex contained the fragile X mental retardation-related protein 1 and HNRNPA2B1 in a miR-369-depedent manner. Our findings demonstrate a unique role of the embryonic miR-369-HNRNPA2B1 axis in controlling metabolic enzyme function, and suggest a novel pathway linking epigenetic, transcriptional, and metabolic control in cell reprogramming. PMID:26176628

  18. 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

  19. Viral and cellular factors involved in Phloem transport of plant viruses.

    PubMed

    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

  20. Krüppel-like factor 4 negatively regulates cellular antiviral immune response

    PubMed Central

    Luo, Wei-Wei; Lian, Huan; Zhong, Bo; Shu, Hong-Bing; Li, Shu

    2016-01-01

    Viral infection triggers activation of the transcription factors NF-κB and IRF3, which collaborate to induce the expression of type I interferons (IFNs) and elicit innate antiviral response. In this report, we identified Krüppel-like factor 4 (KLF4) as a negative regulator of virus-triggered signaling. Overexpression of KLF4 inhibited virus-induced activation of ISRE and IFN-β promoter in various types of cells, while knockdown of KLF4 potentiated viral infection-triggered induction of IFNB1 and downstream genes and attenuated viral replication. In addition, KLF4 was found to be localized in the cytosol and nucleus, and viral infection promoted the translocation of KLF4 from cytosol to nucleus. Upon virus infection, KLF4 was bound to the promoter of IFNB gene and inhibited the recruitment of IRF3 to the IFNB promoter. Our study thus suggests that KLF4 negatively regulates cellular antiviral response. PMID:25531393

  1. Aiolos transcription factor controls cell death in T cells by regulating Bcl-2 expression and its cellular localization.

    PubMed Central

    Romero, F; Martínez-A, C; Camonis, J; Rebollo, A

    1999-01-01

    We searched for proteins that interact with Ras in interleukin (IL)-2-stimulated or IL-2-deprived cells, and found that the transcription factor Aiolos interacts with Ras. The Ras-Aiolos interaction was confirmed in vitro and in vivo by co-immunoprecipitation. Indirect immunofluorescence shows that IL-2 controls the cellular distribution of Aiolos and induces its tyrosine phosphorylation, required for dissociation from Ras. We also identified functional Aiolos-binding sites in the Bcl-2 promoter, which are able to activate the luciferase reporter gene. Mutation of Aiolos-binding sites within the Bcl-2 promoter inhibits transactivation of the reporter gene luciferase, suggesting direct control of Bcl-2 expression by Aiolos. Co-transfection experiments confirm that Aiolos induces Bcl-2 expression and prevents apoptosis in IL-2-deprived cells. We propose a model for the regulation of Bcl-2 expression via Aiolos. PMID:10369681

  2. hCLE/C14orf166, a cellular protein required for viral replication, is incorporated into influenza virus particles

    PubMed Central

    Rodriguez-Frandsen, Ariel; de Lucas, Susana; Pérez-González, Alicia; Pérez-Cidoncha, Maite; Roldan-Gomendio, Alejandro; Pazo, Alejandra; Marcos-Villar, Laura; Landeras-Bueno, Sara; Ortín, Juan; Nieto, Amelia

    2016-01-01

    The influenza A virus polymerase associates with a number of cellular transcription-related factors, including the RNA polymerase II (RNAP II). We previously described that the cellular protein hCLE/C14orf166 interacts with and stimulates influenza virus polymerase as well as RNAP II activities. Here we show that, despite the considerable cellular shut-off observed in infected cells, which includes RNAP II degradation, hCLE protein levels increase throughout infection in a virus replication-dependent manner. Human and avian influenza viruses of various subtypes increase hCLE levels, but other RNA or DNA viruses do not. hCLE colocalises and interacts with viral ribonucleoproteins (vRNP) in the nucleus, as well as in the cytoplasm late in infection. Furthermore, biochemical analysis of purified virus particles and immunoelectron microscopy of infected cells show hCLE in virions, in close association with viral vRNP. These findings indicate that hCLE, a cellular protein important for viral replication, is one of the very few examples of transcription factors that are incorporated into particles of an RNA-containing virus. PMID:26864902

  3. 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

  4. 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

  5. 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.

  6. cables1 is required for embryonic neural development: molecular, cellular, and behavioral evidence from the zebrafish.

    PubMed

    Groeneweg, Jolijn W; White, Yvonne A R; Kokel, David; Peterson, Randall T; Zukerberg, Lawrence R; Berin, Inna; Rueda, Bo R; Wood, Antony W

    2011-01-01

    In vitro studies have suggested that the Cables1 gene regulates epithelial cell proliferation, whereas other studies suggest a role in promoting neural differentiation. In efforts to clarify the functions of Cables1 in vivo, we conducted gain- and loss-of-function studies targeting its ortholog (cables1) in the zebrafish embryo. Similar to rodents, zebrafish cables1 mRNA expression is detected most robustly in embryonic neural tissues. Antisense knockdown of cables1 leads to increased numbers of apoptotic cells, particularly in brain tissue, in addition to a distinct behavioral phenotype, characterized by hyperactivity in response to stimulation. Apoptosis and the behavioral abnormality could be rescued by co-expression of a morpholino-resistant cables1 construct. Suppression of p53 expression in cables1 morphants partially rescued both apoptosis and the behavioral phenotype, suggesting that the phenotype of cables1 morphants is due in part to p53-dependent apoptosis. Alterations in the expression patterns of several neural transcription factors were observed in cables1 morphants during early neurulation, suggesting that cables1 is required for early neural differentiation. Ectopic overexpression of cables1 strongly disrupted embryonic morphogenesis, while overexpression of a cables1 mutant lacking the C-terminal cyclin box had little effect, suggesting functional importance of the cyclin box. Lastly, marked reductions in p35, but not Cdk5, were observed in cables1 morphants. Collectively, these data suggest that cables1 is important for neural differentiation during embryogenesis, in a mechanism that likely involves interactions with the Cdk5/p35 kinase pathway.

  7. Sirtuin 6 promotes transforming growth factor-β1/H2O2/HOCl-mediated enhancement of hepatocellular carcinoma cell tumorigenicity by suppressing cellular senescence.

    PubMed

    Feng, Xin-Xia; Luo, Jing; Liu, Mei; Yan, Wei; Zhou, Zhen-Zhen; Xia, Yu-Jia; Tu, Wei; Li, Pei-Yuan; Feng, Zuo-Hua; Tian, De-An

    2015-05-01

    Sirtuin 6 (SIRT6) can function as a tumor suppressor by suppressing aerobic glycolysis and apoptosis resistance. However, the negative effect of SIRT6 on cellular senescence implies that it may also have the potential to promote tumor development. Here we report that the upregulation of SIRT6 expression was required for transforming growth factor (TGF)-β1 and H2O2/HOCl reactive oxygen species (ROS) to promote the tumorigenicity of hepatocellular carcinoma (HCC) cells. Transforming growth factor-β1/H2O2/HOCl could upregulate SIRT6 expression in HCC cells by inducing the sustained activation of ERK and Smad pathways. Sirtuin 6 in turn abrogated the inducing effect of TGF-β1/H2O2/HOCl on cellular senescence of HCC cells, and was required for the ERK pathway to efficiently suppress the expression of p16 and p21. Sirtuin 6 altered the effect of Smad and p38 MAPK pathways on cellular senescence, and contributed to the inhibitory effect of the ERK pathway on cellular senescence. However, SIRT6 was inefficient in antagonizing the promoting effect of TGF-β1/H2O2 HOCl on aerobic glycolysis and anoikis resistance. Intriguingly, if SIRT6 expression was inhibited, the promoting effect of TGF-β1/H2O2/HOCl on aerobic glycolysis and anoikis resistance was not sufficient to enhance the tumorigenicity of HCC cells. Suppressing the upregulation of SIRT6 enabled TGF-β1/H2O2/HOCl to induce cellular senescence, thereby abrogating the enhancement of HCC cell tumorigenicity by TGF-β1/H2O2/HOCl. These results suggest that SIRT6 is required for TGF-β1/H2O2/HOCl to enhance the tumorigenicity of HCC cells, and that targeting the ERK pathway to suppress the upregulation of SIRT6 might be a potential approach in comprehensive strategies for the therapy of HCC.

  8. 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

  9. 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

  10. Cellular and biochemical mechanisms, risk factors and management of preterm birth: state of the art.

    PubMed

    Vitale, S G; Marilli, I; Rapisarda, A M; Rossetti, D; Belluomo, G; Iapichino, V; Stancanelli, F; Cianci, A

    2014-12-01

    Preterm birth (PTB) is usually defined as a delivery before 37 completed weeks or 259 days of gestation. World Health Organization estimates a worldwide incidence of PTB of 9.6%. Infants born preterm are at higher risks than infants born at term for mortality, and acute and chronic morbidity. Major causes of PTB are the following: spontaneous preterm labor with intact membranes (50%), labor induction or caesarean delivery for maternal or fetal indications (30%), and preterm premature rupture of membranes or PPROM (20%). The aim of this review is to analyze this medical condition, focusing on cellular and biochemical mechanisms, maternal risk factors and role of inflammation and infections in preterm premature rupture of membranes (PPROM) and PTB. Moreover we will discuss about the proper therapeutic strategies for its management. Although different methods have been introduced to predict the advent of preterm labour in asymptomatic women, possibilities for real primary prevention are rare. An early estimation of potential risk factors is pivotal in the secondary prevention of PTB. Finally most efforts so far have been tertiary interventions. These measures have reduced perinatal morbidity and mortality. Advances in primary and secondary care will be needed to prevent prematurity-related illness in infants and children.

  11. Cellular requirements for renal allograft rejection in the athymic nude rat

    PubMed Central

    1989-01-01

    This study has examined the ability of adoptively transferred CD4+ and CD8+ T cells to mediate rejection of a fully allogeneic DA renal graft in the PVG nude rat. Transfer, at the time of transplantation, of naive CD4+ T cells caused rapid graft rejection and primed CD4+ cells were several times more potent. In contrast, naive or specifically sensitized CD8+ cells were entirely ineffective at mediating renal allograft rejection. Whereas nonrejecting grafts showed only a mild cellular infiltrate, rejecting grafts in CD4+ reconstituted animals showed a substantial infiltrate and many of the infiltrating cells had a phenotype (MRC OX8+, MRC OX19-), consistent with NK cells. Experiments using a mAb (HIS 41) against an allotypic determinant of the leukocyte common antigen confirmed that the majority (greater than 80%) of the cellular infiltrate in rejecting grafts derived from the host rather than from the CD4+ inoculum. Infiltrating mononuclear cells, obtained from rejecting allografts 7 d after transplantation in CD4+-injected PVG nude hosts, showed high levels of in vitro cytotoxicity against not only kidney donor strain Con A blasts but also third-party allogeneic Con A blasts, as well as against both NK and LAK susceptible targets. When splenocytes from nontransplanted nude PVG rats were tested in vitro they also demonstrated high levels of lytic activity against both NK and LAK susceptible targets as well as allogeneic Con A blasts, which were not susceptible to lysis by spleen cells from euthymic rats. These findings suggest that injected CD4+ cells may cause renal allograft rejection by the recruitment of extrathymically derived, widely alloreactive cells into the kidney in this model of graft rejection. PMID:2659723

  12. Identification of small peptides inhibiting the integrase-LEDGF/p75 interaction through targeting the cellular co-factor.

    PubMed

    Cavalluzzo, Claudia; Christ, Frauke; Voet, Arnout; Sharma, Ajendra; Singh, Brajendra Kumar; Zhang, Kam Y J; Lescrinier, Eveline; De Maeyer, Marc; Debyser, Zeger; Van der Eycken, Erik

    2013-10-01

    The integration of the viral DNA into the host genome is one of the essential steps in the HIV replication cycle. This process is mediated by the viral enzyme integrase (IN) and lens epithelium-derived growth factor (LEDGF/p75). LEDGF/p75 has been identified as a crucial cellular co-factor of integration that acts by tethering IN to the cellular chromatin. Recently, circular peptides were identified that bind to the C-terminal domain of IN and disrupt the interaction with LEDGF/p75. Starting from the circular peptides, we identified a short peptidic sequence able to inhibit the LEDGF/p75-IN interaction at low μM concentration through its binding to the IN binding site of LEDGF/p75. This discovery can lead to the synthesis of peptidomimetics with high anti-HIV activity targeting the cellular co-factor LEDGF/p75 and not the viral protein IN.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes

    PubMed Central

    Nakase, Ikuhiko; Kobayashi, Nahoko Bailey; Takatani-Nakase, Tomoka; Yoshida, Tetsuhiko

    2015-01-01

    Exosomes are approximately 100-nm vesicles that consist of a lipid bilayer of cellular membranes secreted in large quantities from various types of normal and disease-related cells. Endocytosis has been reported as a major pathway for the cellular uptake of exosomes; however, the detailed mechanisms of their cellular uptake are still unknown. Here, we demonstrate the active induction of macropinocytosis (accompanied by actin reorganisation, ruffling of plasma membrane, and engulfment of large volumes of extracellular fluid) by stimulation of cancer-related receptors and show that the epidermal growth factor (EGF) receptor significantly enhances the cellular uptake of exosomes. We also demonstrate that oncogenic K-Ras-expressing MIA PaCa-2 cells exhibit intensive macropinocytosis that actively transports extracellular exosomes into the cells compared with wild-type K-Ras-expressing BxPC-3 cells. Furthermore, encapsulation of the ribosome-inactivating protein saporin with EGF in exosomes using our simple electroporation method produces superior cytotoxicity via the enhanced cellular uptake of exosomes. Our findings contribute to the biological, pharmaceutical, and medical research fields in terms of understanding the macropinocytosis-mediated cellular uptake of exosomes with applications for exosomal delivery systems. PMID:26036864

  20. Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes.

    PubMed

    Nakase, Ikuhiko; Kobayashi, Nahoko Bailey; Takatani-Nakase, Tomoka; Yoshida, Tetsuhiko

    2015-06-03

    Exosomes are approximately 100-nm vesicles that consist of a lipid bilayer of cellular membranes secreted in large quantities from various types of normal and disease-related cells. Endocytosis has been reported as a major pathway for the cellular uptake of exosomes; however, the detailed mechanisms of their cellular uptake are still unknown. Here, we demonstrate the active induction of macropinocytosis (accompanied by actin reorganisation, ruffling of plasma membrane, and engulfment of large volumes of extracellular fluid) by stimulation of cancer-related receptors and show that the epidermal growth factor (EGF) receptor significantly enhances the cellular uptake of exosomes. We also demonstrate that oncogenic K-Ras-expressing MIA PaCa-2 cells exhibit intensive macropinocytosis that actively transports extracellular exosomes into the cells compared with wild-type K-Ras-expressing BxPC-3 cells. Furthermore, encapsulation of the ribosome-inactivating protein saporin with EGF in exosomes using our simple electroporation method produces superior cytotoxicity via the enhanced cellular uptake of exosomes. Our findings contribute to the biological, pharmaceutical, and medical research fields in terms of understanding the macropinocytosis-mediated cellular uptake of exosomes with applications for exosomal delivery systems.

  1. Cellular Requirements for Systemic Control of Salmonella enterica Serovar Typhimurium Infections in Mice

    PubMed Central

    Bedoui, Sammy

    2014-01-01

    The rational design of vaccines requires an understanding of the contributions of individual immune cell subsets to immunity. With this understanding, targeted vaccine delivery approaches and adjuvants can be developed to maximize vaccine efficiency and to minimize side effects (S. H. E. Kaufmann et al., Immunity 33:555–577, 2010; T. Ben-Yedidia and R. Arnon, Hum. Vaccines 1:95–101, 2005). We have addressed the contributions of different immune cell subsets and their ability to contribute to the control and clearance of the facultative intracellular pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) in a murine model. Using a systematic and reproducible model of experimental attenuated S. Typhimurium infection, we show that distinct lymphocyte deficiencies lead to one of four different infection outcomes: clearance, chronic infection, early death, or late death. Our study demonstrates a high level of functional redundancy in the ability of different lymphocyte subsets to provide interferon gamma (IFN-γ), a critical cytokine in Salmonella immunity. Whereas early control of the infection was entirely dependent on IFN-γ but not on any particular lymphocyte subset, clearance of the infection critically required CD4+ T cells but appeared to be independent of IFN-γ. These data reinforce the idea of a bimodal immune response against Salmonella: an early T cell-independent but IFN-γ-dependent phase and a late T cell-dependent phase that may be IFN-γ independent. PMID:25225248

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

    PubMed

    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

  3. Nickel requirement and factor F430 content of methanogenic bacteria.

    PubMed Central

    Diekert, G; Konheiser, U; Piechulla, K; Thauer, R K

    1981-01-01

    Methanobacterium thermoautotrophicum has been reported to require nickel for growth and to contain high concentrations of a nickel tetrapyrrole designated factor F430. In this communication it is shown that all methanogenic bacteria investigated incorporated nickel during growth and also synthesized factor F430. This was also true for Methanobrevibacter smithii, which is dependent on acetate as a carbon source, and for Methanosarcina barkeri growing on acetate or methanol as energy sources. Other bacteria, including Acetobacterium woodii and Clostridium thermoaceticum, contained no factor F430. It is further shown that two yellow nickel-containing degradation products were formed from factor F430 when heated at pH 7. This finding explains why several forms of factor F430 were found in methanogenic bacteria when a heat step was employed in the purification procedure. PMID:7298577

  4. Glutamate Dehydrogenase Is Required by Mycobacterium bovis BCG for Resistance to Cellular Stress

    PubMed Central

    Gallant, James L.; Viljoen, Albertus J.; van Helden, Paul D.; Wiid, Ian J. F.

    2016-01-01

    We recently reported on our success to generate deletion mutants of the genes encoding glutamate dehydrogenase (GDH) and glutamine oxoglutarate aminotransferase (GOGAT) in M. bovis BCG, despite their in vitro essentiality in M. tuberculosis. We could use these mutants to delineate the roles of GDH and GOGAT in mycobacterial nitrogen metabolism by using M. bovis BCG as a model for M. tuberculosis specifically. Here, we extended our investigation towards the involvement of GDH and GOGAT in other aspects of M. bovis BCG physiology, including the use of glutamate as a carbon source and resistance to known phagosomal stresses, as well as in survival inside macrophages. We find that gdh is indispensable for the utilization of glutamate as a major carbon source, in low pH environments and when challenged with nitric oxide. On the other hand, the gltBD mutant had increased viability under low pH conditions and was unaffected by a challenge with nitric oxide. Strikingly, GDH was required to sustain M. bovis BCG during infection of both murine RAW 264.7 and bone-marrow derived and macrophages, while GOGAT was not. We conclude that the catabolism of glutamate in slow growing mycobacteria may be a crucial function during infection of macrophage cells and demonstrate a novel requirement for M. bovis BCG GDH in the protection against acidic and nitrosative stress. These results provide strong clues on the role of GDH in intracellular survival of M. tuberculosis, in which the essentiality of the gdh gene complicates knock out studies making the study of the role of this enzyme in pathogenesis difficult. PMID:26824899

  5. Internalization and cellular pools of never growth factor in pheochromocytoma (PC12) cells

    SciTech Connect

    Neet, K.E.; Kasaian, M.

    1987-05-01

    Nerve Growth Factor (NGF) binds to a cell surface receptor on responsive neuronal cells to initiate cell maintenance and/or differentiation regimes. The purpose of these studies was to define quantitatively the fate of NGF in PC12 cells with respect to various cellular compartments in a single series of biochemical experiments. Different binding methodologies were evaluated in suspension and on plates. 50 pM SVI-NGF was bound to rat PC12 cells in suspension for 30 min at 37, followed by various methods and combinations of methods to remove subsets of bound ligand. Distinction could be made between NGF bound to fast vs. slow cell surface receptors, NGF bound to slow receptors at the cell surface vs. cell interior, and detergent-soluble vs. cytoskeletally-attached NGF. These treatments defined the relative size of five pools, including the fast receptor (65%), two intracellular compartments (12% and 3%) susceptible to nonionic detergent, and a detergent-stable intracellular pool of ligand (16%). At 37 the cold chase stable and the acid stable pools were about the same size because of rapid internalization, but the slow receptor was measurable at 4. Inhibitors were used to define the route of NGF through the cell from the plasma membrane to degradation. Chloroquine caused accumulation of NGF only in pools that were not associated with the cytoskeleton, implicating this compartment in supplying ligand to the lysosome. Results with cytochalasin B and colchicine and suggested both microfilament and microtubule pathways in NGF degradation. A model for the movement of NGF through the cell was developed based on these observations.

  6. 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

  7. 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.

  8. 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

  9. Distinct cellular origin and genetic requirement of Hedgehog-Gli in postnatal rhabdomyosarcoma genesis.

    PubMed

    Rajurkar, M; Huang, H; Cotton, J L; Brooks, J K; Sicklick, J; McMahon, A P; Mao, J

    2014-11-13

    Dysregulation of the Hedgehog (Hh)-Gli signaling pathway is implicated in a variety of human cancers, including basal cell carcinoma (BCC), medulloblastoma (MB) and embryonal rhabdhomyosarcoma (eRMS), three principle tumors associated with human Gorlin syndrome. However, the cells of origin of these tumors, including eRMS, remain poorly understood. In this study, we explore the cell populations that give rise to Hh-related tumors by specifically activating Smoothened (Smo) in both Hh-producing and -responsive cell lineages in postnatal mice. Interestingly, we find that unlike BCC and MB, eRMS originates from the stem/progenitor populations that do not normally receive active Hh signaling. Furthermore, we find that the myogenic lineage in postnatal mice is largely Hh quiescent and that Pax7-expressing muscle satellite cells are not able to give rise to eRMS upon Smo or Gli1/2 overactivation in vivo, suggesting that Hh-induced skeletal muscle eRMS arises from Hh/Gli quiescent non-myogenic cells. In addition, using the Gli1 null allele and a Gli3 repressor allele, we reveal a specific genetic requirement for Gli proteins in Hh-induced eRMS formation and provide molecular evidence for the involvement of Sox4/11 in eRMS cell survival and differentiation. PMID:24276242

  10. Potency assay development for cellular therapy products: an ISCT review of the requirements and experiences in the industry.

    PubMed

    Bravery, Christopher A; Carmen, Jessica; Fong, Timothy; Oprea, Wanda; Hoogendoorn, Karin H; Woda, Juliana; Burger, Scott R; Rowley, Jon A; Bonyhadi, Mark L; Van't Hof, Wouter

    2013-01-01

    The evaluation of potency plays a key role in defining the quality of cellular therapy products (CTPs). Potency can be defined as a quantitative measure of relevant biologic function based on the attributes that are linked to relevant biologic properties. To achieve an adequate assessment of CTP potency, appropriate in vitro or in vivo laboratory assays and properly controlled clinical data need to be created. The primary objective of a potency assay is to provide a mechanism by which the manufacturing process and the final product for batch release are scrutinized for quality, consistency and stability. A potency assay also provides the basis for comparability assessment after process changes, such as scale-up, site transfer and new starting materials (e.g., a new donor). Potency assays should be in place for early clinical development, and validated assays are required for pivotal clinical trials. Potency is based on the individual characteristics of each individual CTP, and the adequacy of potency assays will be evaluated on a case-by-case basis by regulatory agencies. We provide an overview of the expectations and challenges in development of potency assays specific for CTPs; several real-life experiences from the cellular therapy industry are presented as illustrations. The key observation and message is that aggressive early investment in a solid potency evaluation strategy can greatly enhance eventual CTP deployment because it can mitigate the risk of costly product failure in late-stage development.

  11. A Proteomic-Based Workflow Using Purified Respiratory Syncytial Virus Particles to Identify Cellular Factors as Drug Targets.

    PubMed

    Huong, Tra Nguyen; Tan, Boon Huan; Sugrue, Richard J

    2016-01-01

    The identification of cellular factors that play a role in respiratory syncytial virus (RSV) replication is an alternative strategy in the identification of druggable cellular protein that are essential for RSV replication. In this regard experimental strategies that are able to screen relevant proteins from the vast array of proteins in the cellular milieu will facilitate the identification of potential drug targets. In this chapter we describe a procedure where RSV particles are purified from cells that are permissive for RSV infection, and the protein composition of the purified virus particles characterized using a proteomics-based strategy. This procedure revealed that actin, several actin-binding proteins, and the chaperones HSP70 and HSP90 also co-purified with the virus particles. The relevance of the HSP90 protein to virus replication was then further validated using imaging, gene silencing and by using an established small molecule HSP90 inhibitor. PMID:27464695

  12. Cellular factors associated with latency and spontaneous Epstein-Barr virus reactivation in B-lymphoblastoid cell lines.

    PubMed

    Davies, Michael L; Xu, Shushen; Lyons-Weiler, James; Rosendorff, Adam; Webber, Steven A; Wasil, Laura R; Metes, Diana; Rowe, David T

    2010-04-25

    EBV-immortalized B-lymphoblastoid cell lines are used as models for cellular transformation and as antigen-presenting cells in immunological assays. LCLs vary in surface markers and other phenotypic properties, but it is not known how this heterogeneity relates to the EBV life cycle. To explore correlations, we examined 62 LCLs for cellular and viral phenotypes. LCLs generated from pediatric and adult donors could similarly be categorized as either low in EBV copy number or fluctuating within a high range. High-copy status accompanied higher lytic viral gene expression and lower latent gene expression. Inhibiting lytic EBV replication did not affect cellular phenotype or lytic switch protein expression, indicating that an LCL's lytic permissivity was a stable property. Among the cellular genes overexpressed in permissive LCLs were unfolded protein response genes and plasma cell markers. Among genes overexpressed in non-permissive LCLs were transcription factors involved in maintaining B cell lineage, in particular EBF1. This study suggests previously undetected mechanisms by which cellular pathways influence the lytic reactivation of EBV. PMID:20153012

  13. Induction of Cellular Senescence by Insulin-like Growth Factor Binding Protein-5 through a p53-dependent Mechanism

    PubMed Central

    Kim, Kwang Seok; Seu, Young Bae; Baek, Suk-Hwan; Kim, Mi Jin; Kim, Keuk Jun; Kim, Jung Hye

    2007-01-01

    The insulin-like growth factor (IGF) signaling pathway plays a crucial role in the regulation of cell growth, differentiation, apoptosis, and aging. IGF-binding proteins (IGFBPs) are important members of the IGF axis. IGFBP-5 is up-regulated during cellular senescence in human dermal fibroblasts and endothelial cells, but the function of IGFBP-5 in cellular senescence is unknown. Here we show that IGFBP-5 plays important roles in the regulation of cellular senescence. Knockdown of IGFBP-5 in old human umbilical endothelial cells (HUVECs) with IGFBP-5 micro-RNA lentivirus caused partial reduction of a variety of senescent phenotypes, such as changes in cell morphology, increases in cell proliferation, and decreases in senescence-associated β-galactosidase (SA-β-gal) staining. In addition, treatment with IGFBP-5 protein or up-regulation of IGFBP-5 in young cells accelerates cellular senescence, as confirmed by cell proliferation and SA-β-gal staining. Premature senescence induced by IGFBP-5 up-regulation in young cells was rescued by knockdown of p53, but not by knockdown of p16. Furthermore, atherosclerotic arteries exhibited strong IGFBP-5–positive staining along intimal plaques. These results suggest that IGFBP-5 plays a role in the regulation of cellular senescence via a p53-dependent pathway and in aging-associated vascular diseases. PMID:17804819

  14. Analysis of the interactions of viral and cellular factors with human cytomegalovirus lytic origin of replication, oriLyt

    PubMed Central

    Kagele, Dominique; Rossetto, Cyprian C.; Elorza, Margret; Pari, Gregory S.

    2011-01-01

    Human cytomegalovirus transient lytic DNA replication relies on the cis-acting element oriLyt, six viral-encoded core proteins, the proposed DNA replication initiator protein UL84, IE2, IRS1 and the gene products from the UL112/113 loci. In an effort to elucidate cellular and viral-encoded factors that may play a role in oriLyt-dependent replication we used DNA-affinity purification and mass spectrometry to isolate and identify several previously unknown cellular and viral factors that interact with HCMV oriLyt DNA. These proteins include the multifunctional hnRNP-K, BUB3, HMGB1, PTB-1, UL83, UL112/113, and IRS1. Chromatin immunoprecipitation (ChIP) assays confirmed an interaction of several of these factors with oriLyt. Co-immunoprecipitation experiments detected an interaction between UL84 and hnRNP-K in infected and transfected cells. Knockdown of hnRNP K expression by siRNA inhibited the amplification of oriLyt in the transient assay. Together, these data suggest a possible regulatory role in DNA replication for several previously unidentified viral and cellular factors. PMID:22236369

  15. The Cellular Factor NXP2/MORC3 Is a Positive Regulator of Influenza Virus Multiplication

    PubMed Central

    Ver, Lorena S.; Marcos-Villar, Laura; Landeras-Bueno, Sara; Nieto, Amelia

    2015-01-01

    ABSTRACT Transcription and replication of influenza A virus are carried out in the nuclei of infected cells in the context of viral ribonucleoproteins (RNPs). The viral polymerase responsible for these processes is a protein complex composed of the PB1, PB2, and PA proteins. We previously identified a set of polymerase-associated cellular proteins by proteomic analysis of polymerase-containing intracellular complexes expressed and purified from human cells. Here we characterize the role of NXP2/MORC3 in the infection cycle. NXP2/MORC3 is a member of the Microrchidia (MORC) family that is associated with the nuclear matrix and has RNA-binding activity. Influenza virus infection led to a slight increase in NXP2/MORC3 expression and its partial relocalization to the cytoplasm. Coimmunoprecipitation and immunofluorescence experiments indicated an association of NXP2/MORC3 with the viral polymerase and RNPs during infection. Downregulation of NXP2/MORC3 by use of two independent short hairpin RNAs (shRNAs) reduced virus titers in low-multiplicity infections. Consistent with these findings, analysis of virus-specific RNA in high-multiplicity infections indicated a reduction of viral RNA (vRNA) and mRNA after NXP2/MORC3 downregulation. Silencing of NXP2/MORC3 in a recombinant minireplicon system in which virus transcription and replication are uncoupled showed reductions in cat mRNA and chloramphenicol acetyltransferase (CAT) protein accumulation but no alterations in cat vRNA levels, suggesting that NXP2/MORC3 is important for influenza virus transcription. IMPORTANCE Influenza virus infections appear as yearly epidemics and occasional pandemics of respiratory disease, with high morbidity and occasional mortality. Influenza viruses are intracellular parasites that replicate and transcribe their genomic ribonucleoproteins in the nuclei of infected cells, in a complex interplay with host cell factors. Here we characterized the role of the human NXP2/MORC3 protein, a member

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

    PubMed

    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

  17. Nuclear transcription factor Y and its roles in cellular processes related to human disease.

    PubMed

    Ly, Luong Linh; Yoshida, Hideki; Yamaguchi, Masamitsu

    2013-01-01

    Nuclear transcription factor Y (NF-Y) is an example of a transcriptional regulation factor in eukaryotes consisting of three different subunits, NF-YA, NF-YB and NF-YC, which are all necessary for formation of NF-Y complexes and binding to CCAAT boxes in promoters of its target genes. Highly conserved between human and Drosophila, NF-Y regulates transcription of various genes related to the cell cycle and various human diseases. Drosophila models have been widely used as tools for studying genetics and developmental biology and more recently for analyzing the functions of human disease genes, including those responsible for developmental and neurological disorders, cancer, cardiovascular disease and metabolic and storage diseases, as well as genes required for function of the visual, auditory and immune systems. In this review, in vivo findings from Drosophila models relevant to the roles of NF-Y in various human diseases are summarized. Recent studies have demonstrated novel contributions of dNF-Y to apoptosis and apoptosis-induced proliferation, and in photoreceptor cell differentiation during the development of the Drosophila compound eye.

  18. Serratia marcescens suppresses host cellular immunity via the production of an adhesion-inhibitory factor against immunosurveillance cells.

    PubMed

    Ishii, Kenichi; Adachi, Tatsuo; Hamamoto, Hiroshi; Sekimizu, Kazuhisa

    2014-02-28

    Injection of a culture supernatant of Serratia marcescens into the bloodstream of the silkworm Bombyx mori increased the number of freely circulating immunosurveillance cells (hemocytes). Using a bioassay with live silkworms, serralysin metalloprotease was purified from the culture supernatant and identified as the factor responsible for this activity. Serralysin inhibited the in vitro attachment of both silkworm hemocytes and murine peritoneal macrophages. Incubation of silkworm hemocytes or murine macrophages with serralysin resulted in degradation of the cellular immune factor BmSPH-1 or calreticulin, respectively. Furthermore, serralysin suppressed in vitro phagocytosis of bacteria by hemocytes and in vivo bacterial clearance in silkworms. Disruption of the ser gene in S. marcescens attenuated its host killing ability in silkworms and mice. These findings suggest that serralysin metalloprotease secreted by S. marcescens suppresses cellular immunity by decreasing the adhesive properties of immunosurveillance cells, thereby contributing to bacterial pathogenesis.

  19. Tethering Factors Required for Cytokinesis in Arabidopsis1[W

    PubMed Central

    Thellmann, Martha; Rybak, Katarzyna; Thiele, Knut; Wanner, Gerhard; Assaad, Farhah F.

    2010-01-01

    At the end of the cell cycle, the nascent cross wall is laid down within a transient membrane compartment referred to as the cell plate. Tethering factors, which act by capturing vesicles and holding them in the vicinity of their target membranes, are likely to play an important role in the first stages of cell plate assembly. Factors required for cell plate biogenesis, however, remain to be identified. In this study, we used a reverse genetic screen to isolate tethering factors required for cytokinesis in Arabidopsis (Arabidopsis thaliana). We focused on the TRAPPI and TRAPPII (for transport protein particle) tethering complexes, which are thought to be required for the flow of traffic through the Golgi and for trans-Golgi network function, as well as on the GARP complex, thought to be required for the tethering of endocytotic vesicles to the trans-Golgi network. We found weak cytokinesis defects in some TRAPPI mutants and strong cytokinesis defects in all the TRAPPII lines we surveyed. Indeed, four insertion lines at the TRAPPII locus AtTRS120 had canonical cytokinesis-defective seedling-lethal phenotypes, including cell wall stubs and incomplete cross walls. Confocal and electron microscopy showed that in trs120 mutants, vesicles accumulated at the equator of dividing cells yet failed to assemble into a cell plate. This shows that AtTRS120 is required for cell plate biogenesis. In contrast to the TRAPP complexes, we found no conclusive evidence for cytokinesis defects in seven GARP insertion lines. We discuss the implications of these findings for the origin and identity of cell plate membranes. PMID:20713617

  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. Everolimus enhances cellular cytotoxicity of lapatinib via the eukaryotic elongation factor-2 kinase pathway in nasopharyngeal carcinoma cells

    PubMed Central

    Liu, Lin; Wang, Zhi-Hui; Han, Jun; Tang, Con; Chen, Nan; Lin, Zhong; Peng, Pei-Jian

    2016-01-01

    Background Nasopharyngeal carcinoma (NPC) has a high relapse and metastatic rates; hence, development of new therapeutics is an immediate requirement. Lapatinib and everolimus have been demonstrated to be effective in the treatment of several carcinomas. This preclinical study aimed to investigate the effect and mechanism of lapatinib combined with everolimus on NPC cells. Methods The Cell Counting Kit 8 and colony formation assay were used to detect the effect of lapatinib alone or lapatinib combined with everolimus on the growth and proliferation of cells. Apoptosis was tested by flow cytometry and was further confirmed by western blot. The targets of lapatinib and the effects of lapatinib or everolimus on the eukaryotic elongation factor-2 (eEF-2) kinase pathway were analyzed by western blot, which also evaluated autophagy activity. Results Lapatinib inhibited the cellular viability and colony formation in NPC cells. At 24–72 h, the average half maximal inhibitory concentration (IC50) values of lapatinib were ranging from 3 to 5 μM. This study further found that lapatinib induced both apoptosis and autophagy in NPC cells, and this autophagic activity was described as type II programmed cell death via an eEF-2 kinase-dependent pathway. In addition, augmentation of lapatinib-induced autophagy by mammalian target of rapamycin (mTOR) inhibitor everolimus enhanced the cytocidal effect of lapatinib in NPC cells via the mTOR/S6 kinase/eEF-2 kinase pathway. Conclusion This study reveals that everolimus can sensitize NPC cells to lapatinib by the activation of eEF-2 kinase and provides a potential model of combination therapy. PMID:27785067

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

    PubMed

    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

    2015-04-20

    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

  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. EGFR and mutant p53 expand esophageal cellular subpopulation capable of epithelial-to-mesenchymal transition through ZEB transcription factors

    PubMed Central

    Ohashi, Shinya; Natsuizaka, Mitsuteru; Wong, Gabrielle S.; Michaylira, Carmen Z.; Grugan, Katharine D.; Stairs, Douglas B.; Kalabis, Jiri; Vega, Maria E.; Kalman, Ross A.; Nakagawa, Momo; Klein-Szanto, Andres J; Herlyn, Meenhard; Diehl, J. Alan; Rustgi, Anil K.; Nakagawa, Hiroshi

    2010-01-01

    Transforming growth factor (TGF)-β is a potent inducer of epithelial to mesenchymal transition (EMT). However, it remains elusive as to which molecular mechanisms determine the cellular capacity to undergo EMT in response to TGF-β. We have found that both epidermal growth factor receptor (EGFR) overexpression and mutant p53 tumor suppressor genes contribute to enrichment of an EMT-competent cellular subpopulation amongst telomerase-immortalized human esophageal epithelial cells during malignant transformation. EGFR overexpression triggers oncogene-induced senescence, accompanied by induction of cyclin dependent kinase inhibitors p15INK4B, p16INK4A and p21. Interestingly, a subpopulation of cells emerges by negating senescence without loss of EGFR overexpression. Such cell populations express increased levels of zinc finger E-box binding (ZEB) transcription factors ZEB1 and ZEB2, and undergo EMT upon TGF-β stimulation. Enrichment of EMT-competent cells was more evident in the presence of p53 mutation, which diminished EGFR-induced senescence. RNA interference directed against ZEB resulted in induction of p15INK4B and p16INK4A, reactivating the EGFR-dependent senescence program. Importantly, TGF-β-mediated EMT did not take place when cellular senescence programs were activated by either ZEB knockdown or activation of wild-type p53 function. Thus, senescence checkpoint functions activated by EGFR and p53 may be evaded through the induction of ZEB, thereby allowing expansion of an EMT-competent unique cellular subpopulation, providing novel mechanistic insights into the role of ZEB in esophageal carcinogenesis. PMID:20424117

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. Overexpression of factor VIII after AAV delivery is transiently associated with cellular stress in hemophilia A mice

    PubMed Central

    Lange, Amy M; Altynova, Ekaterina S; Nguyen, Giang N; Sabatino, Denise E

    2016-01-01

    Factor VIII (FVIII) is a large glycoprotein that is challenging to express both in vitro and in vivo. Several studies suggest that high levels of FVIII expression can lead to cellular stress. After gene transfer, transgene expression is restricted to a subset of cells and the increased FVIII load per cell may impact activation of the unfolded protein response. We sought to determine whether increased FVIII expression in mice after adeno-associated viral liver gene transfer would affect the unfolded protein response and/or immune response to the transgene. The FVIII gene was delivered as B-domain deleted single chain or two chain (light and heavy chains) at a range of doses in hemophilia A mice. A correlation between FVIII expression and anti-FVIII antibody titers was observed. Analysis of key components of the unfolded protein response, binding immunoglobulin protein (BiP), and C/EBP homologous protein (CHOP), showed transient unfolded protein response activation in the single chain treated group expressing >200% of FVIII but not after two chain delivery. These studies suggest that supraphysiological single chain FVIII expression may increase the likelihood of a cellular stress response but does not alter liver function. These data are in agreement with the observed long-term expression of FVIII at therapeutic levels after adeno-associated viral delivery in hemophilia A dogs without evidence of cellular toxicity. PMID:27738645

  14. 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.

  15. Low neural exosomal levels of cellular survival factors in Alzheimer’s disease

    PubMed Central

    Goetzl, Edward J; Boxer, Adam; Schwartz, Janice B; Abner, Erin L; Petersen, Ronald C; Miller, Bruce L; Carlson, Olga D; Mustapic, Maja; Kapogiannis, Dimitrios

    2015-01-01

    Transcription factors that mediate neuronal defenses against diverse stresses were quantified in plasma neural-derived exosomes of Alzheimer’s disease or frontotemporal dementia patients and matched controls. Exosomal levels of low-density lipoprotein receptor-related protein 6, heat-shock factor-1, and repressor element 1-silencing transcription factor all were significantly lower in Alzheimer’s disease patients than controls (P < 0.0001). In frontotemporal dementia, the only significant difference was higher levels of repressor element 1-silencing transcription factor than in controls. Exosomal transcription factors were diminished 2–10 years before clinical diagnosis of Alzheimer’s disease. Low exosomal levels of survival proteins may explain decreased neuronal resistance to Alzheimer’s disease neurotoxic proteins. PMID:26273689

  16. 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.

  17. Biomimetic hybrid porous scaffolds immobilized with platelet derived growth factor-BB promote cellularization and vascularization in tissue engineering.

    PubMed

    Murali, Ragothaman; Ponrasu, Thangavel; Cheirmadurai, Kalirajan; Thanikaivelan, Palanisamy

    2016-02-01

    Development of hybrid scaffolds with synergistic combination of growth factor is a promising approach to promote early in vivo wound repair and tissue regeneration. Here, we show the rapid wound healing in Wistar albino rats using biomimetic collagen-poly(dialdehyde) guar gum based hybrid porous scaffolds covalently immobilized with platelet derived growth factor-BB. The immobilized platelet derived growth factor in the hybrid scaffolds not only enhance the total protein, collagen, hexosamine, and uronic acid contents in the granulation tissue but also provide stronger tissues. The wound closure analysis reveal that the complete epithelialization period is 15.4 ± 0.9 days for collagen-poly(dialdehyde) guar gum-platelet derived growth factor hybrid scaffolds, whereas it is significantly higher for control, collagen, collagen- poly(dialdehyde) guar gum and povidine-iodine treated groups. Further, the histological evaluation shows that the immobilized platelet derived growth factor in the hybrid scaffolds induced a more robust cellular and vascular response in the implanted site. Hence, we demonstrate that the collagen-poly(dialdehyde) guar gum hybrid scaffolds loaded with platelet derived growth factor stimulates chemotactic effects in the implanted site to promote rapid tissue regeneration and wound repair without the assistance of antibacterial agents.

  18. Role of vascular endothelial growth factor in the stimulation of cellular invasion and signaling of breast cancer cells.

    PubMed

    Price, D J; Miralem, T; Jiang, S; Steinberg, R; Avraham, H

    2001-03-01

    The expression of vascular endothelial growth factor (VEGF) by breast tumors has been previously correlated with a poor prognosis in the pathogenesis of breast cancer. Furthermore, VEGF secretion is a prerequisite for tumor development. Although most of the effects of VEGF have been shown to be attributable to the stimulation of endothelial cells, we present evidence here that breast tumor cells are capable of responding to VEGF. We show that VEGF stimulation of T-47D breast cancer cells leads to changes in cellular signaling and invasion. VEGF increases the cellular invasion of T-47D breast cancer cells on Matrigel/ fibronectin-coated transwell membranes by a factor of two. Northern analysis for the expression of the known VEGF receptors shows the presence of moderate levels of Flt-1 and low levels of Flk-1/KDR mRNAs in a variety of breast cancer cell lines. T-47D breast cancer cells bind 125I-labeled VEGF with a Kd of 13 x 10(-9) M. VEGF induces the activation of the extracellular regulated kinases 1,2 as well as activation of phosphatidylinositol 3'-kinase, Akt, and Forkhead receptor L1. These findings in T-47D breast cancer cells strongly suggest an autocrine role for VEGF contributing to the tumorigenic phenotype.

  19. 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. PMID:27173747

  20. Cleavage of interferon regulatory factor 7 by enterovirus 71 3C suppresses cellular responses.

    PubMed

    Lei, Xiaobo; Xiao, Xia; Xue, Qinghua; Jin, Qi; He, Bin; Wang, Jianwei

    2013-02-01

    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

  1. 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.

  2. 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 Central

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

    1994-01-01

    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. Images PMID:7966567

  3. 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

  4. Identification of TRAPPC8 as a Host Factor Required for Human Papillomavirus Cell Entry

    PubMed Central

    Ishii, Yoshiyuki; Nakahara, Tomomi; Kataoka, Michiyo; Kusumoto-Matsuo, Rika; Mori, Seiichiro; Takeuchi, Takamasa; Kukimoto, Iwao

    2013-01-01

    Human papillomavirus (HPV) is a non-enveloped virus composed of a circular DNA genome and two capsid proteins, L1 and L2. Multiple interactions between its capsid proteins and host cellular proteins are required for infectious HPV entry, including cell attachment and internalization, intracellular trafficking and viral genome transfer into the nucleus. Using two variants of HPV type 51, the Ma and Nu strains, we have previously reported that MaL2 is required for efficient pseudovirus (PsV) transduction. However, the cellular factors that confer this L2 dependency have not yet been identified. Here we report that the transport protein particle complex subunit 8 (TRAPPC8) specifically interacts with MaL2. TRAPPC8 knockdown in HeLa cells yielded reduced levels of reporter gene expression when inoculated with HPV51Ma, HPV16, and HPV31 PsVs. TRAPPC8 knockdown in HaCaT cells also showed reduced susceptibility to infection with authentic HPV31 virions, indicating that TRAPPC8 plays a crucial role in native HPV infection. Immunofluorescence microscopy revealed that the central region of TRAPPC8 was exposed on the cell surface and colocalized with inoculated PsVs. The entry of Ma, Nu, and L2-lacking PsVs into cells was equally impaired in TRAPPC8 knockdown HeLa cells, suggesting that TRAPPC8-dependent endocytosis plays an important role in HPV entry that is independent of L2 interaction. Finally, expression of GFP-fused L2 that can also interact with TRAPPC8 induced dispersal of the Golgi stack structure in HeLa cells, a phenotype also observed by TRAPPC8 knockdown. These results suggest that during viral intracellular trafficking, binding of L2 to TRAPPC8 inhibits its function resulting in Golgi destabilization, a process that may assist HPV genome escape from the trans-Golgi network. PMID:24244674

  5. Tumor Necrosis Factor-α -and Interleukin-1-Induced Cellular Responses: Coupling Proteomic and Genomic Information

    PubMed Central

    Ott, Lee W.; Resing, Katheryn A.; Sizemore, Alecia W.; Heyen, Joshua W.; Cocklin, Ross R.; Pedrick, Nathan M.; Woods, H. Cary; Chen, Jake Y.; Goebl, Mark G.; Witzmann, Frank A.; Harrington, Maureen A.

    2010-01-01

    The pro-inflammatory cytokines, Tumor Necrosis Factor-alpha (TNFα) and Interleukin-1 (IL-1) mediate the innate immune response. Dysregulation of the innate immune response contributes to the pathogenesis of cancer, arthritis, and congestive heart failure. TNFα- and IL-1-induced changes in gene expression are mediated by similar transcription factors; however, TNFα and IL-1 receptor knock-out mice differ in their sensitivities to a known initiator (lipopolysaccharide, LPS) of the innate immune response. The contrasting responses to LPS indicate that TNFα and IL-1 regulate different processes. A large-scale proteomic analysis of TNFα- and IL-1-induced responses was undertaken to identify processes uniquely regulated by TNFα and IL-1. When combined with genomic studies, our results indicate that TNFα, but not IL-1, mediates cell cycle arrest. PMID:17503796

  6. Functional interaction between the human cytomegalovirus 86-kilodalton IE2 protein and the cellular transcription factor CREB.

    PubMed Central

    Lang, D; Gebert, S; Arlt, H; Stamminger, T

    1995-01-01

    The 86-kDa IE2 protein (IE86) of human cytomegalovirus (HCMV) has been described as a promiscuous transactivator of viral, as well as cellular, gene expression. Investigation of the mechanism used by IE86 to activate gene expression from the early UL112/113 promoter of HCMV revealed the existence of three binding sites for IE86 located between nucleotides -290 and -120 relative to the transcriptional start site (H. Arlt, D. Lang, S. Gebert, and T. Stamminger, J. Virol. 68:4117-4125, 1994). As shown previously, deletion of these target sites resulted in a reduction of IE86-mediated transactivation by approximately 70%. The remaining promoter, however, could still be stimulated about 40-fold, indicating the presence of an additional responsive element within these sequences. Here, we provide evidence that a binding site for the cellular transcription factor CREB can also act as a target for IE86 transactivation. By DNase I protection analysis, a binding sequence for CREB could be detected between nucleotides -78 and -56 within the respective promoter region. After in vitro mutagenesis of this CREB-binding site within the context of the entire UL112/113 promoter, a marked reduction in transactivation levels was evident. Moreover, when individual CREB-binding sites were positioned upstream of a minimal, TATA box-containing UL112/113 promoter, they were able to confer strong IE86 responsiveness, whereas a mutated sequence did not exert any effect. In far Western blot and pull-down experiments, a direct interaction of IE86 with the cellular transcription factor CREB could be observed. The in vivo relevance of this in vitro interaction was confirmed by using various GAL4 fusion proteins in the presence or absence of IE86 which revealed a strong activation only in the presence of both a GAL4-CREB fusion and IE86. This shows that at least one specific member of the ATF/CREB family of transcription factors is involved in mediating transactivation by the HCMV IE86 protein

  7. 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

  8. Eukaryotic translation initiation factor 4GI is a cellular target for NS1 protein, a translational activator of influenza virus.

    PubMed

    Aragón, T; de la Luna, S; Novoa, I; Carrasco, L; Ortín, J; Nieto, A

    2000-09-01

    Influenza virus NS1 protein is an RNA-binding protein whose expression alters several posttranscriptional regulatory processes, like polyadenylation, splicing, and nucleocytoplasmic transport of cellular mRNAs. In addition, NS1 protein enhances the translational rate of viral, but not cellular, mRNAs. To characterize this effect, we looked for targets of NS1 influenza virus protein among cellular translation factors. We found that NS1 coimmunoprecipitates with eukaryotic initiation factor 4GI (eIF4GI), the large subunit of the cap-binding complex eIF4F, either in influenza virus-infected cells or in cells transfected with NS1 cDNA. Affinity chromatography studies using a purified His-NS1 protein-containing matrix showed that the fusion protein pulls down endogenous eIF4GI from COS-1 cells and labeled eIF4GI translated in vitro, but not the eIF4E subunit of the eIF4F factor. Similar in vitro binding experiments with eIF4GI deletion mutants indicated that the NS1-binding domain of eIF4GI is located between residues 157 and 550, in a region where no other component of the translational machinery is known to interact. Moreover, using overlay assays and pull-down experiments, we showed that NS1 and eIF4GI proteins interact directly, in an RNA-independent manner. Mapping of the eIF4GI-binding domain in the NS1 protein indicated that the first 113 N-terminal amino acids of the protein, but not the first 81, are sufficient to bind eIF4GI. The first of these mutants has been previously shown to act as a translational enhancer, while the second is defective in this activity. Collectively, these and previously published data suggest a model where NS1 recruits eIF4GI specifically to the 5' untranslated region (5' UTR) of the viral mRNA, allowing for the preferential translation of the influenza virus messengers.

  9. 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. PMID:9403792

  10. 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.

  11. 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

  12. Viral abrogation of lymphocyte mitogenesis: induction of a soluble factor inhibitory to cellular proliferation.

    PubMed Central

    Israel, E; Beiss, B; Wainberg, M A

    1980-01-01

    PHA and Con A-driven mitogenesis of mouse C3H lymphocytes can be inhibited by co-incubation with a variety of different virus particles. These effects appear independent of infection, and can be obtained using UV-inactivated virus. Viruses may be added to spleen cell cultures as late as 46 h after co-incubation with mitogen, and still achieve significant inhibition of proliferative responsiveness. The described inhibition is apparently mediated, in part at least, by a soluble factor which is induced in splenic cultures following interaction with virus particles. This factor is apparently a product of macrophages. It does not posess interferon activity, but does have the ability to inhibit lectin- and alloantigen-driven mitogenesis, as measured in fresh cultures of splenic lymphocytes and in the mixed lymphocyte culture (MLC) reaction, respectively. Moreover, addition of virus to splenic cultures can apparently activate suppressor lymphocytes with the ability to inhibit proliferative responsiveness of fresh lymphocyte suspensions in the presence of Con A. PMID:6448221

  13. 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

  14. The Grp170 nucleotide exchange factor executes a key role during ERAD of cellular misfolded clients

    PubMed Central

    Inoue, Takamasa; Tsai, Billy

    2016-01-01

    When a protein misfolds in the endoplasmic reticulum (ER), it retrotranslocates to the cytosol and is degraded by the proteasome via a pathway called ER-associated degradation (ERAD). To initiate ERAD, ADP-BiP is often recruited to the misfolded client, rendering it soluble and translocation competent. How the misfolded client is subsequently released from BiP so that it undergoes retrotranslocation, however, remains enigmatic. Here we demonstrate that the ER-resident nucleotide exchange factor (NEF) Grp170 plays an important role during ERAD of the misfolded glycosylated client null Hong Kong (NHK). As a NEF, Grp170 triggers nucleotide exchange of BiP to generate ATP-BiP. ATP-BiP disengages from NHK, enabling it to retrotranslocate to the cytosol. We demonstrate that Grp170 binds to Sel1L, an adapter of the transmembrane Hrd1 E3 ubiquitin ligase postulated to be the retrotranslocon, and links this interaction to Grp170’s function during ERAD. More broadly, Grp170 also promotes degradation of the nonglycosylated transthyretin (TTR) D18G misfolded client. Our findings thus establish a general function of Grp170 during ERAD and suggest that positioning this client-release factor at the retrotranslocation site may afford a mechanism to couple client release from BiP and retrotranslocation. PMID:27030672

  15. Direct combinatorial interaction between a herpes simplex virus regulatory protein and a cellular octamer-binding factor mediates specific induction of virus immediate-early gene expression.

    PubMed Central

    O'Hare, P; Goding, C R; Haigh, A

    1988-01-01

    We provide evidence for a novel mechanism of transcriptional regulation in which the immediate-early (IE) transactivating protein of herpes simplex virus, Vmw65, is assembled into a specific DNA-binding complex together with a cellular octamer-binding factor (TRF). The assembly of Vmw65/TRF complex requires not only the core TRF recognition site, but also flanking sequences which are dispensable for TRF binding alone. We show from functional analyses that TRF binding by a motif is required but not sufficient to confer induction on a heterologous promoter, and it is the ability of the motif to allow TRF/Vmw65 complex assembly which correlates with functional activity. Thus, for the induction of HSV IE expression, Vmw65 forms a complex with TRF by recognition of the specific subset of appropriately flanked TRF binding sites present in each of the IE genes. This mechanism may provide a paradigm for the selective utilization of the same transcription factor in differential gene expression. Images PMID:2854058

  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. 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

  19. RNA export factor Ddx19 is required for nuclear import of the SRF coactivator MKL1

    PubMed Central

    Rajakylä, Eeva Kaisa; Viita, Tiina; Kyheröinen, Salla; Huet, Guillaume; Treisman, Richard; Vartiainen, Maria K.

    2015-01-01

    Controlled transport of macromolecules between the cytoplasm and nucleus is essential for homeostatic regulation of cellular functions. For instance, gene expression entails coordinated nuclear import of transcriptional regulators to activate transcription and nuclear export of the resulting messenger RNAs for cytoplasmic translation. Here we link these two processes by reporting a novel role for the mRNA export factor Ddx19/Dbp5 in nuclear import of MKL1, the signal-responsive transcriptional activator of SRF. We show that Ddx19 is not a general nuclear import factor, and that its specific effect on MKL1 nuclear import is separate from its role in mRNA export. Both helicase and nuclear pore-binding activities of Ddx19 are dispensable for MKL1 nuclear import, but RNA binding is required. Mechanistically, Ddx19 operates by modulating the conformation of MKL1, which affects its interaction with Importin-β for efficient nuclear import. Thus, Ddx19 participates in mRNA export, translation and nuclear import of a key transcriptional regulator. PMID:25585691

  20. 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

  1. Early nucleosome deposition on, and replication of, HSV DNA requires cell factor PCNA

    PubMed Central

    Sanders, Iryna; Boyer, Mark; Fraser, Nigel W.

    2015-01-01

    Herpes Simplex Virus (HSV) is a double stranded DNA virus that can cause lytic infections in epithelial cells of the skin and latent infections in neuronal cells of the peripheral nervous system. After virion attachment to the cell membrane, the capsid enters the cytoplasm and is transported to the nucleus. Following docking at the nuclear pore, the HSV DNA, and contents of the virion, are injected into the nucleus. The viral DNA that enters the nucleus is devoid of histones, but begins to be covered with them soon after entry. The covering of histones, in the form of nucleosomes, reaches a maximum during the early stages of infection and drops off during late infection (after DNA replication). However during latency the genome is saturated with nucleosomes. In this study, we examine the role of cell Proliferating Cell Nuclear Antigen (PCNA) a cellular DNA polymerase accessory protein (processivity factor), and cell DNA polymerases in histone deposition during the early stages of HSV infection. Using SiRNA knockdown, and a cytosine arabinoside (araC) chemical inhibitor, we conclude that PCNA is important for viral replication and histone deposition. However, cell DNA polymerases that bind PCNA do not appear to be required for these processes and PCNA does not appear to bind to the viral DNA polymerase (which has its own viral processivity factor). PMID:25672886

  2. 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.

  3. 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

  4. Effect of photo-immobilization of epidermal growth factor on the cellular behaviors

    SciTech Connect

    Ogiwara, Kazutaka; Nagaoka, Masato; Cho, Chong-Su; Akaike, Toshihiro . E-mail: takaike@bio.titech.ac.jp

    2006-06-23

    We constructed photo-reactive epidermal growth factor (EGF) bearing p-azido phenylalanine at the C-terminal (HEGFP) by genetic engineering to investigate the possibility of immobilized EGF as a novel artificial extracellular matrix (ECM). The constructed recombinant protein was immobilized to glass surface by ultraviolet irradiation. A431 cells adhered both to HEGFP-immobilized and collagen-coated surfaces. Interaction between immobilized HEGFP and EGF receptors in the A431 cells was independent of Mg{sup 2+} although integrin-mediated cell adhesion to natural ECMs is dependent on Mg{sup 2+}. Phosphorylation of EGF receptors in A431 cells was induced by immobilized HEGFP as same as soluble EGF. DNA uptake of hepatocytes decreased by immobilized HEGFP whereas it increased by soluble EGF. Liver-specific functions of hepatocytes were maintained for 3 days by immobilized HEGFP whereas they were not maintained by soluble EGF, indicating that immobilized HEGFP follows different signal transduction pathway from soluble EGF.

  5. 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.

  6. Cellular intrinsic factors involved in the resistance of squamous cell carcinoma to photodynamic therapy.

    PubMed

    Gilaberte, Yolanda; Milla, Laura; Salazar, Nerea; Vera-Alvarez, Jesús; Kourani, Omar; Damian, Alejandra; Rivarola, Viviana; Roca, Maria José; Espada, Jesús; González, Salvador; Juarranz, Angeles

    2014-09-01

    Photodynamic therapy (PDT) is widely used to treat non-melanoma skin cancer. However, some patients affected with squamous cell carcinoma (SCC) do not respond adequately to PDT with methyl-δ-aminolevulinic acid (MAL-PDT) and the tumors acquire an infiltrative phenotype and became histologically more aggressive, less differentiated, and more fibroblastic. To search for potential factors implicated in SCC resistance to PDT, we have used the SCC-13 cell line (parental) and resistant SCC-13 cells obtained by repeated MAL-PDT treatments (5th and 10th PDT-resistant generations). Xenografts assays in immunodeficient mice showed that the tumors generated by resistant cells were bigger than those induced by parental cells. Comparative genomic hybridization array (aCGH) showed that the three cell types presented amplicons in 3p12.1 CADM2, 7p11.2 EFGR, and 11q13.3 CCND1 genes. The 5th and 10th PDT-resistant cells showed an amplicon in 5q11.2 MAP3K1, which was not present in parental cells. The changes detected by aCGH on CCND1, EFGR, and MAP3K1 were confirmed in extracts of SCC-13 cells by reverse-transcriptase PCR and by western blot, and by immunohistochemistry in human biopsies from persistent tumors after MAL-PDT. Our data suggest that genomic imbalances related to CCND1, EFGR, and particularly MAP3K1 seem to be involved in the development of the resistance of SCC to PDT.

  7. Cellular and extracellular factors in early root resorption repair in the rat.

    PubMed

    Jäger, Andreas; Kunert, Dominique; Friesen, Therese; Zhang, Dongliang; Lossdörfer, Stefan; Götz, Werner

    2008-08-01

    The aim of this study was to investigate the role of extracellular matrix components, such as collagen type I, fibronectin, and osteopontin (OPN) during cementum repair following experimentally induced tooth movement, and to characterize the cells taking part in the regenerative process. The upper right first molars were moved mesially in 21 three-month-old male Wistar rats using a coil spring with a force of 0.5 N. After 9 days, the appliance was removed and the animals were killed in groups of three immediately after withdrawal of the force and 5, 7, 10, 12, 14, and 17 days later. Three rats served as non-experimental control animals. The maxillae were prepared and processed for histological analysis. Together with the disappearance of the multinucleated odontoclasts from the resorption lacunae, signs of repair were visible 5 days after the release of the orthodontic force. The first signs of cementum repair were seen on day 10. The newly produced cementum was of the acellular extrinsic fibre type (AEFC) and reattachment was achieved with the principal periodontal ligament (PDL) fibres orientated almost perpendicular to the root surface. The initial interface formed between the old and new cementum, as well as the new AEFC, was characterized by a strong immunoreaction with OPN and collagen I antibody, but only a weak immunoreaction with the fibronectin antibody. Only a small number of mononuclear cells, which were involved in the repair process, showed a positive immunoreaction with the osteoblastic lineage markers runt-related transcription factor 2 and osteocalcin. These same cells stained sparsely with muscle segment homeobox homologue 2, but not with the E11 antibody. Thus, most of the cells associated with this reparative activity on the surface of the lacunae were differentiated PDL cells of the fibroblastic phenotype. Cells with a defined osteoblastic phenotype seemed to be of minor importance in this repair process. PMID:18632841

  8. 40 CFR 63.1431 - Process vent annual epoxides emission factor plan requirements.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... factor plan requirements. 63.1431 Section 63.1431 Protection of Environment ENVIRONMENTAL PROTECTION... Polyether Polyols Production § 63.1431 Process vent annual epoxides emission factor plan requirements. (a) Applicability of emission factor plan requirements. An owner or operator electing to comply with an...

  9. Role of cellular caspases, nuclear factor-kappa B and interferon regulatory factors in Bluetongue virus infection and cell fate

    PubMed Central

    2010-01-01

    Background Bluetongue virus (BTV) infection causes haemorrhagic disease in ruminants and induces cell death. The pathogenesis in animals and in cell culture has been linked to BTV-induced apoptosis. Results In this report, we investigated BTV-induced apoptosis in cell culture in depth and show that both extrinsic (caspase-8 activation) and intrinsic (caspase-9 activation) pathways play roles in BTV apoptosis. Further, by using chemical inhibitors and knock-out cell lines, we show that these pathways act independently of each other in BTV infected cells. In addition to activation of caspase-8, -9 and executioner caspase-3, we also identified that BTV infection causes the activation of caspase-7, which results in the cleavage of poly (ADP-ribose) polymerase (PARP). BTV-induced cell death appears to be due to apoptosis rather than necrosis, as the HMBG-1 was not translocated from the nucleus. We also examined if NF-κB response is related to BTV-induced apoptosis as in reovirus. Our data suggests that NF-κB response is not linked to the induction of apoptosis. It is controlled by the degradation of only IκBα but not IκBβ, resulting in a rapid transient response during BTV infection. This was supported using an NF-κB dependent luciferase reporter gene assay, which demonstrated early response, that appeared to be suppressed by the late stage of BTV replication. Furthermore, virus titres were higher in the presence of NF-κB inhibitor (SN50), indicating that NF-κB has a role in initiating an antiviral environment. In addition, we show that BTV infection induces the translocation of interferon regulatory factors (IRF-3 and IRF-7) into the nucleus. The induction of IRF responses, when measured by IRF dependent luciferase reporter gene assay, revealed that the IRF responses, like NF-κB response, were also at early stage of infection and mirrored the timing of NF-κB induction. Conclusion BTV triggers a wide range of caspase activities resulting in cell apoptosis

  10. 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

  11. Role of Viral RNA and Co-opted Cellular ESCRT-I and ESCRT-III Factors in Formation of Tombusvirus Spherules Harboring the Tombusvirus Replicase

    PubMed Central

    Kovalev, Nikolay; de Castro Martín, Isabel Fernández; Pogany, Judit; Barajas, Daniel; Pathak, Kunj; Risco, Cristina

    2016-01-01

    ABSTRACT Plus-stranded RNA viruses induce membrane deformations in infected cells in order to build viral replication complexes (VRCs). Tomato bushy stunt virus (TBSV) co-opts cellular ESCRT (endosomal sorting complexes required for transport) proteins to induce the formation of vesicle (spherule)-like structures in the peroxisomal membrane with tight openings toward the cytosol. In this study, using a yeast (Saccharomyces cerevisiae) vps23Δ bro1Δ double-deletion mutant, we showed that the Vps23p ESCRT-I protein (Tsg101 in mammals) and Bro1p (ALIX) ESCRT-associated protein, both of which bind to the viral p33 replication protein, play partially complementary roles in TBSV replication in cells and in cell extracts. Dual expression of dominant-negative versions of Arabidopsis homologs of Vps23p and Bro1p inhibited tombusvirus replication to greater extent than individual expression in Nicotiana benthamiana leaves. We also demonstrated the critical role of Snf7p (CHMP4), Vps20p, and Vps24p ESCRT-III proteins in tombusvirus replication in yeast and in vitro. Electron microscopic imaging of vps23Δ yeast revealed the lack of tombusvirus-induced spherule-like structures, while crescent-like structures are formed in ESCRT-III deletion yeasts replicating TBSV RNA. In addition, we also showed that the length of the viral RNA affects the sizes of spherules formed in N. benthamiana cells. The 4.8-kb genomic RNA is needed for the formation of spherules 66 nm in diameter, while spherules formed during the replication of the ∼600-nucleotide (nt)-long defective interfering RNA in the presence of p33 and p92 replication proteins are 42 nm. We propose that the viral RNA serves as a “measuring string” during VRC assembly and spherule formation. IMPORTANCE Plant positive-strand RNA viruses, similarly to animal positive-strand RNA viruses, replicate in membrane-bound viral replicase complexes in the cytoplasm of infected cells. Identification of cellular and viral factors

  12. A biphasic endothelial stress-survival mechanism regulates the cellular response to vascular endothelial growth factor A

    SciTech Connect

    Latham, Antony M.; Odell, Adam F.; Mughal, Nadeem A.; Issitt, Theo; Ulyatt, Clare; Walker, John H.; Homer-Vanniasinkam, Shervanthi; Ponnambalam, Sreenivasan

    2012-11-01

    Vascular endothelial growth factor A (VEGF-A) is an essential cytokine that regulates endothelial function and angiogenesis. VEGF-A binding to endothelial receptor tyrosine kinases such as VEGFR1 and VEGFR2 triggers cellular responses including survival, proliferation and new blood vessel sprouting. Increased levels of a soluble VEGFR1 splice variant (sFlt-1) correlate with endothelial dysfunction in pathologies such as pre-eclampsia; however the cellular mechanism(s) underlying the regulation and function of sFlt-1 are unclear. Here, we demonstrate the existence of a biphasic stress response in endothelial cells, using serum deprivation as a model of endothelial dysfunction. The early phase is characterized by a high VEGFR2:sFlt-1 ratio, which is reversed in the late phase. A functional consequence is a short-term increase in VEGF-A-stimulated intracellular signaling. In the late phase, sFlt-1 is secreted and deposited at the extracellular matrix. We hypothesized that under stress, increased endothelial sFlt-1 levels reduce VEGF-A bioavailability: VEGF-A treatment induces sFlt-1 expression at the cell surface and VEGF-A silencing inhibits sFlt-1 anchorage to the extracellular matrix. Treatment with recombinant sFlt-1 inhibits VEGF-A-stimulated in vitro angiogenesis and sFlt-1 silencing enhances this process. In this response, increased VEGFR2 levels are regulated by the phosphatidylinositol-3-kinase and PKB/Akt signaling pathways and increased sFlt-1 levels by the ERK1/2 signaling pathway. We conclude that during serum withdrawal, cellular sensing of environmental stress modulates sFlt-1 and VEGFR2 levels, regulating VEGF-A bioavailability and ensuring cell survival takes precedence over cell proliferation and migration. These findings may underpin an important mechanism contributing to endothelial dysfunction in pathological states. -- Highlights: Black-Right-Pointing-Pointer Endothelial cells mount a stress response under conditions of low serum. Black

  13. 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

  14. In Vivo Cardiac Cellular Reprogramming Efficacy Is Enhanced by Angiogenic Preconditioning of the Infarcted Myocardium With Vascular Endothelial Growth Factor

    PubMed Central

    Mathison, Megumi; P. Gersch, Robert; Nasser, Ahmed; Lilo, Sarit; Korman, Mallory; Fourman, Mitchell; Hackett, Neil; Shroyer, Kenneth; Yang, Jianchang; Ma, Yupo; Crystal, Ronald G.; Rosengart, Todd K.

    2012-01-01

    Background In situ cellular reprogramming offers the possibility of regenerating functional cardiomyocytes directly from scar fibroblasts, obviating the challenges of cell implantation. We hypothesized that pretreating scar with gene transfer of the angiogenic vascular endothelial growth factor (VEGF) would enhance the efficacy of this strategy. Methods and Results Gata4, Mef2c, and Tbx5 (GMT) administration via lentiviral transduction was demonstrated to transdifferentiate rat fibroblasts into (induced) cardiomyocytes in vitro by cardiomyocyte marker studies. Fisher 344 rats underwent coronary ligation and intramyocardial administration of an adenovirus encoding all 3 major isoforms of VEGF (AdVEGF‐All6A+) or an AdNull control vector (n=12/group). Lentivirus encoding GMT or a GFP control was administered to each animal 3 weeks later, followed by histologic and echocardiographic analyses. GMT administration reduced the extent of fibrosis by half compared with GFP controls (12±2% vs 24±3%, P<0.01) and reduced the number of myofibroblasts detected in the infarct zone by 4‐fold. GMT‐treated animals also demonstrated greater density of cardiomyocyte‐specific marker beta myosin heavy chain 7+ cells compared with animals receiving GFP with or without VEGF (P<0.01). Ejection fraction was significantly improved after GMT vs GFP administration (12±3% vs −7±3%, P<0.01). Eight (73%) GFP animals but no GMT animals demonstrated decreased ejection fraction during this interval (P<0.01). Also, improvement in ejection fraction was 4‐fold greater in GMT/VEGF vs GMT/null animals (17±2% vs 4±1%, P<0.05). Conclusions VEGF administration to infarcted myocardium enhances the efficacy of GMT‐mediated cellular reprogramming in improving myocardial function and reducing the extent of myocardial fibrosis compared with the use of GMT or VEGF alone. PMID:23316332

  15. RhoD activated by fibroblast growth factor induces cytoneme-like cellular protrusions through mDia3C

    PubMed Central

    Koizumi, Kazuhisa; Takano, Kazunori; Kaneyasu, Akiko; Watanabe-Takano, Haruko; Tokuda, Emi; Abe, Tomoyuki; Watanabe, Naoki; Takenawa, Tadaomi; Endo, Takeshi

    2012-01-01

    The small GTPase RhoD regulates actin cytoskeleton to collapse actin stress fibers and focal adhesions, resulting in suppression of cell migration and cytokinesis. It also induces alignment of early endosomes along actin filaments and reduces their motility. We show here that a constitutively activated RhoD generated two types of actin-containing thin peripheral cellular protrusions distinct from Cdc42-induced filopodia. One was longer, almost straight, immotile, and sensitive to fixation, whereas the other was shorter, undulating, motile, and resistant to fixation. Moreover, cells expressing wild-type RhoD extended protrusions toward fibroblast growth factor (FGF) 2/4/8–coated beads. Stimulation of wild-type RhoD-expressing cells with these FGFs also caused formation of cellular protrusions. Nodules moved through the RhoD-induced longer protrusions, mainly toward the cell body. Exogenously expressed FGF receptor was associated with these moving nodules containing endosome-like vesicles. These results suggest that the protrusions are responsible for intercellular communication mediated by FGF and its receptor. Accordingly, the protrusions are morphologically and functionally equivalent to cytonemes. RhoD was activated by FGF2/4/8. Knockdown of RhoD interfered with FGF-induced protrusion formation. Activated RhoD specifically bound to mDia3C and facilitated actin polymerization together with mDia3C. mDia3C was localized to the tips or stems of the protrusions. In addition, constitutively activated mDia3C formed protrusions without RhoD or FGF stimulation. Knockdown of mDia3 obstructed RhoD-induced protrusion formation. These results imply that RhoD activated by FGF signaling forms cytoneme-like protrusions through activation of mDia3C, which induces actin filament formation. PMID:23034183

  16. 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…

  17. The Cryptococcus neoformans Rim101 Transcription Factor Directly Regulates Genes Required for Adaptation to the Host

    PubMed Central

    O'Meara, Teresa R.; Xu, Wenjie; Selvig, Kyla M.; O'Meara, Matthew J.; Mitchell, Aaron P.

    2014-01-01

    The Rim101 protein is a conserved pH-responsive transcription factor that mediates important interactions between several fungal pathogens and the infected host. In the human fungal pathogen Cryptococcus neoformans, the Rim101 protein retains conserved functions to allow the microorganism to respond to changes in pH and other host stresses. This coordinated cellular response enables this fungus to effectively evade the host immune response. Preliminary studies suggest that this conserved transcription factor is uniquely regulated in C. neoformans both by the canonical pH-sensing pathway and by the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. Here we present comparative transcriptional data that demonstrate a strong concordance between the downstream effectors of PKA and Rim101. To define Rim101-dependent gene expression during a murine lung infection, we used nanoString profiling of lung tissue infected with a wild-type or rim101Δ mutant strain. In this setting, we demonstrated that Rim101 controls the expression of multiple cell wall-biosynthetic genes, likely explaining the enhanced immunogenicity of the rim101Δ mutant. Despite its divergent upstream regulation, the C. neoformans Rim101 protein recognizes a conserved DNA binding motif. Using these data, we identified direct targets of this transcription factor, including genes involved in cell wall regulation. Therefore, the Rim101 protein directly controls cell wall changes required for the adaptation of C. neoformans to its host environment. Moreover, we propose that integration of the cAMP/PKA and pH-sensing pathways allows C. neoformans to respond to a broad range of host-specific signals. PMID:24324006

  18. 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.

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

    PubMed Central

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

    1997-01-01

    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. PMID:9199312

  20. Functional evidence for ligand-dependent dissociation of thyroid hormone and retinoic acid receptors from an inhibitory cellular factor.

    PubMed Central

    Casanova, J; Helmer, E; Selmi-Ruby, S; Qi, J S; Au-Fliegner, M; Desai-Yajnik, V; Koudinova, N; Yarm, F; Raaka, B M; Samuels, H H

    1994-01-01

    The ligand-binding domains of thyroid hormone (L-triiodothyronine [T3]) receptors (T3Rs), all-trans retinoic acid (RA) receptors (RARs), and 9-cis RA receptors (RARs and RXRs) contain a series of heptad motifs thought to be important for dimeric interactions. Using a chimera containing amino acids 120 to 392 of chicken T3R alpha (cT3R alpha) positioned between the DNA-binding domain of the yeast GAL4 protein and the potent 90-amino-acid transactivating domain of the herpes simplex virus VP16 protein (GAL4-T3R-VP16), we provide functional evidence that binding of ligand releases T3Rs and RARs from an inhibitory cellular factor. GAL4-T3R-VP16 does not bind T3 and does not activate transcription from a GAL4 reporter when expressed alone but is able to activate transcription when coexpressed with unliganded T3R or RAR. This activation is reversed by T3 or RA, suggesting that these receptors compete with GAL4-T3R-VP16 for a cellular inhibitor and that ligand reverses this effect by dissociating T3R or RAR from the inhibitor. A chimera containing the entire ligand-binding domain of cT3R alpha (amino acids 120 to 408) linked to VP16 [GAL4-T3R(408)-VP16] is activated by unliganded receptor as well as by T3. In contrast, GAL4-T3R containing the amino acid 120 to 408 ligand-binding region without the VP16 domain is activated only by T3. The highly conserved ninth heptad, which is involved in heterodimerization, appears to participate in the receptor-inhibitor interaction, suggesting that the inhibitor is a related member of the receptor gene family. In striking contrast to T3R and RAR, RXR activates GAL4-T3R-VP16 only with its ligand, 9-cis RA, but unliganded RXR does not appear to be the inhibitor suggested by these studies. Further evidence that an orphan receptor may be the inhibitor comes from our finding that COUP-TF inhibits activation of GAL4-T3R-VP16 by unliganded T3R and the activation of GAL4-T3R by T3. These and other results suggest that an inhibitory factor

  1. Stemness factor Sall4 is required for DNA damage response in embryonic stem cells

    PubMed Central

    Xiong, Jianhua; Todorova, Dilyana; Su, Ning-Yuan; Kim, Jinchul; Lee, Pei-Jen; Shen, Zhouxin; Briggs, Steven P.

    2015-01-01

    Mouse embryonic stem cells (ESCs) are genetically more stable than somatic cells, thereby preventing the passage of genomic abnormalities to their derivatives including germ cells. The underlying mechanisms, however, remain largely unclear. In this paper, we show that the stemness factor Sall4 is required for activating the critical Ataxia Telangiectasia Mutated (ATM)–dependent cellular responses to DNA double-stranded breaks (DSBs) in mouse ESCs and confer their resistance to DSB-induced cytotoxicity. Sall4 is rapidly mobilized to the sites of DSBs after DNA damage. Furthermore, Sall4 interacts with Rad50 and stabilizes the Mre11–Rad50–Nbs1 complex for the efficient recruitment and activation of ATM. Sall4 also interacts with Baf60a, a member of the SWI/SNF (switch/sucrose nonfermentable) ATP-dependent chromatin-remodeling complex, which is responsible for recruiting Sall4 to the site of DNA DSB damage. Our findings provide novel mechanisms to coordinate stemness of ESCs with DNA damage response, ensuring genomic stability during the expansion of ESCs. PMID:25733712

  2. Tyrosine-599 of the c-Mpl receptor is required for Shc phosphorylation and the induction of cellular differentiation.

    PubMed Central

    Alexander, W S; Maurer, A B; Novak, U; Harrison-Smith, M

    1996-01-01

    Interaction of thrombopoietin (TPO) with its receptor, c-Mpl, triggers cell growth and differentiation responses controlling primitive haemopoietic cell production and megakaryocytopoiesis. To examine the important receptor domains and signal transduction pathways involved in these cellular responses, c-Mpl cytoplasmic domain truncation and tyrosine substitution mutants were generated. In the myelomonocytic leukaemia cell lines WEHI3B-D+ and M1, ectopic expression of the wild-type c-Mpl receptor induced TPO-dependent cellular differentiation characterized by increased cell migration through agar and acquisition of the morphology and molecular markers of macrophages. Consistent with the concept that proliferative and differentiation signals emanate from distinct receptor domains, the C-terminal 33 amino acids of c-Mpl were dispensable for a proliferative response in Ba/F3 cells but proved critical for WEHI3B-D+ and M1 differentiation. Finer mapping revealed that substitution of Tyr599 by phenylalanine within this c-Mpl domain was sufficient to abolish the normal differentiation response. Moreover, in contrast to the normal c-Mpl receptor, this same mplY599F mutant was also incapable of stimulating TPO-dependent Shc phosphorylation, the association of Shc with Grb2 or c-Mpl and of inducing c-fos expression. Thus activation of components of the Ras signalling cascade, initiated by interaction of Shc with c-Mpl Tyr599, may play a decisive role in specific differentiation signals emanating from the c-Mpl receptor. Images PMID:8978680

  3. The S2 Cu(I) site in CupA from Streptococcus pneumoniae is required for cellular copper resistance†

    PubMed Central

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

    2015-01-01

    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. PMID:26346139

  4. 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.

  5. 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. PMID:20888758

  6. Carboxy-terminal truncations of epidermal growth factor (EGF) receptor affect diverse EGF-induced cellular responses.

    PubMed

    Li, W; Hack, N; Margolis, B; Ullrich, A; Skorecki, K; Schlessinger, J

    1991-08-01

    The binding of epidermal growth factor (EGF) to its receptor induces tyrosine phosphorylation of phospholipase C gamma (PLC gamma), which appears to be necessary for its activation leading to phosphatidyl inositol (PI) hydrolysis. Moreover, EGF-receptor (EGF-R) activation and autophosphorylation results in binding of PLC gamma to the tyrosine phosphorylated carboxy-terminus of the receptor. To gain further insights into the mechanisms and interactions regulating these processes, we have analyzed transfected NIH-3T3 cells expressing two EGF-R carboxy-terminal deletion mutants (CD63 and CD126) with reduced capacity to stimulate PI hydrolysis, Ca2+ rises, and DNA synthesis. In fact, the CD126 mutant lacking 126 carboxy-terminal amino acids, including four tyrosine autophosphorylation sites, was unable to stimulate PI hydrolysis or Ca2+ rise in response to EGF. Surprisingly, EGF binding to the cell lines expressing CD63 or CD126 mutants was followed by similar stimulation of tyrosine phosphorylation of PLC gamma. Our results suggest that although necessary, tyrosine phosphorylation of PLC gamma may not be sufficient for stimulation and PI hydrolysis. It is clear, however, that the carboxy-terminal region of EGF-R is involved in regulation of interactions with cellular targets and therefore plays a crucial role in postreceptor signaling pathways.

  7. 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

  8. The E-domain region of mechano-growth factor inhibits cellular apoptosis and preserves cardiac function during myocardial infarction.

    PubMed

    Mavrommatis, Evangelos; Shioura, Krystyna M; Los, Tamara; Goldspink, Paul H

    2013-09-01

    Insulin-like growth factor-1 (IGF-1) isoforms are expressed via alternative splicing. Expression of the minor isoform IGF-1Eb [also known as mechano-growth factor (MGF)] is responsive to cell stress. Since IGF-1 isoforms differ in their E-domain regions, we are interested in determining the biological function of the MGF E-domain. To do so, a synthetic peptide analog was used to gain mechanistic insight into the actions of the E-domain. Treatment of H9c2 cells indicated a rapid cellular uptake mechanism that did not involve IGF-1 receptor activation but resulted in a nuclear localization. Peptide treatment inhibited the intrinsic apoptotic pathway in H9c2 cells subjected to cell stress with sorbitol by preventing the collapse of the mitochondrial membrane potential and inhibition of caspase-3 activation. Therefore, we administered the peptide at the time of myocardial infarction (MI) in mice. At 2 weeks post-MI cardiac function, gene expression and cell death were assayed. A significant decline in both systolic and diastolic function was evident in untreated mice based on PV loop analysis. Delivery of the E-peptide ameliorated the decline in function and resulted in significant preservation of cardiac contractility. Associated with these changes were an inhibition of pathologic hypertrophy and significantly fewer apoptotic nuclei in the viable myocardium of E-peptide-treated mice post-MI. We conclude that administration of the MGF E-domain peptide may provide a means of modulating local tissue IGF-1 autocrine/paracrine actions to preserve cardiac function, prevent cell death, and pathologic remodeling in the heart.

  9. 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.

  10. Cellular distribution of the new growth factor pleiotrophin (HB-GAM) mRNA in developing and adult rat tissues.

    PubMed

    Vanderwinden, J M; Mailleux, P; Schiffmann, S N; Vanderhaeghen, J J

    1992-09-01

    Pleiotrophin (PTN), also known as HB-GAM, belongs to an emerging cytokine family unrelated to other growth factors. We report here the first comprehensive study using in situ hybridization on the cellular distribution of this new heparin-binding growth factor mRNA in rat tissues. PTN mRNA was developmentally expressed in many--but not all--neuroectodermal and mesodermal lineages, whilst no PTN mRNA was detected in endoderm, ectoderm and trophoblast. PTN mRNA was found in the nervous system throughout development, with a post-natal peak of expression. In the adult nervous system, significant expression persisted in hippocampal CA1 pyramidal neurons and in cortical neurons, but also in different non-neuronal cells types in various locations (olfactory nerve, cerebellar astrocytes, pituicytes, Schwann cells surrounding the neurons in sensory ganglia). PTN mRNA was also found during development in the mesenchyme of lung, gut, kidney and reproductive tract, in bone and cartilage progenitors, in dental pulp, in myoblasts, and in several other sites. Expression was differently regulated in each location, but usually faded around birth. In the adult, PTN mRNA was still present in the meninges, the iris, the Leydig cells of the testis and in the uterus. PTN mRNA was also strongly expressed in the basal layers of the tongue epithelium, which is the only epithelium and ectodermal derivative to express PTN mRNA, and this only after birth. PTN is known to be a growth factor for perinatal brain neurons and a mitogen for fibroblasts in vitro. Recently, trophic effects on epithelial cells and a role as a tumour growth factor have been reported. The mechanisms of regulation and the functions of PTN are however still uncertain. Its expression pattern during development suggests important roles in growth and differentiation. Moreover, the presence of PTN mRNA in several adult tissues and the up-regulation of PTN mRNA expression in the gravid uterus indicate that PTN also has

  11. 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

  12. 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.

  13. 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

  14. Maintenance of Multipotency in Human Dermal Fibroblasts Treated with Xenopus laevis Egg Extract Requires Exogenous Fibroblast Growth Factor-2

    PubMed Central

    Kole, Denis; Ambady, Sakthikumar; Page, Raymond L.

    2014-01-01

    Abstract Direct reprogramming of a differentiated somatic cell into a developmentally more plastic cell would offer an alternative to applications in regenerative medicine that currently depend on either embryonic stem cells (ESCs), adult stem cells, or induced pluripotent stem cells (iPSCs). Here we report the potential of select Xenopus laevis egg extract fractions, in combination with exogenous fibroblast growth factor-2 (FGF2), to affect life span, morphology, gene expression, protein translation, and cellular localization of OCT4 and NANOG transcription factors, and the developmental potential of human dermal fibroblasts in vitro. A gradual change in morphology is accompanied by translation of embryonic transcription factors and their nuclear localization and a life span exceeding 60 population doublings. Cells acquire the ability to follow adipogenic, neuronal, and osteogenic differentiation under appropriate induction conditions in vitro. Analysis of active extract fractions reveals that Xenopus egg protein and RNAs as well as exogenously supplemented FGF2 are required and sufficient for induction and maintenance of this phenotypic change. Factors so far identified in the active fractions include FGF2 itself, transforming growth factor-β, maskin, and nucleoplasmin. Identification of critical factors needed for reprogramming may allow for nonviral, chemically defined derivation of human-induced multipotent cells that can be maintained by exogenous FGF2. PMID:24405062

  15. 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.

  16. The rice transcription factor IDEF1 directly binds to iron and other divalent metals for sensing cellular iron status.

    PubMed

    Kobayashi, Takanori; Itai, Reiko Nakanishi; Aung, May Sann; Senoura, Takeshi; Nakanishi, Hiromi; Nishizawa, Naoko K

    2012-01-01

    Iron is essential for most living organisms and its availability often determines survival and proliferation. The Oryza sativa (rice) transcription factor IDEF1 plays a crucial role in regulating iron deficiency-induced genes involved in iron homeostasis. In the present report, we found characteristic histidine-asparagine repeat and proline-rich regions in IDEF1 and its homolog in Hordeum vulgare (barley), HvIDEF1. An immobilized metal ion affinity chromatography assay revealed that IDEF1 and HvIDEF1 bind to various divalent metals, including Fe(2+) and Ni(2+) . Recombinant IDEF1 protein expressed in Escherichia coli contained mainly Fe and Zn. This metal-binding activity of IDEF1 was almost abolished by deletion of the histidine-asparagine and proline-rich regions, but DNA-binding and trans-activation functions were not impaired by the deletion. Transgenic rice plants constitutively overexpressing IDEF1 without these metal-binding domains failed to cause pleiotropic effects conferred by overexpression of full-length IDEF1, including a low germination rate, impaired seedling growth, tolerance to iron deficiency in hydroponic culture, and enhanced expression of various iron deficiency-inducible genes. Impairment of the transcriptional regulation of IDEF1 by deletion of the metal-binding domains occurred primarily at an early stage of iron deficiency. These results suggest that the histidine-asparagine and proline-rich regions in rice IDEF1 directly bind to divalent metals and sense the cellular metal ion balance caused by changes in iron availability. PMID:21880076

  17. 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...

  18. Human factor requirements of helmet trackers for HMDs

    NASA Astrophysics Data System (ADS)

    Martinsen, Gary L.; Havig, Paul R.; Post, David L.; Reis, George A.; Simpson, Matthew A.

    2003-09-01

    A helmet tracker is a critical element in the path that delivers targeting and other sensor data to the user of a helmet-mounted display (HMD) in a military aircraft. The original purpose of an HMD was to serve as a helmet-mounted sight and provide a means to fully utilize the capabilities of off-boresight munitions. Recently, the role of the HMD has evolved from being strictly a targeting tool to providing detailed flight path and situation awareness information. These changes, however, have placed even greater value on the visual information that is transferred through the helmet tracker to the HMD. Specifically, the timeliness and accuracy of the information, which is of critical importance when the HMD is used as a targeting aid, is of even greater importance when the HMD is used to display flight reference information. This is especially relevant since it has been proposed to build new military aircraft without a physical head-up display (HUD) and display HUD information virtually with an HMD. In this paper, we review the current state of helmet tracker technology with respect to use in military aviation. We also identify the parameters of helmet trackers that offer the greatest risk when using an HMD to provide information beyond targeting data to the user. Finally, we discuss the human factors limitations of helmet tracker systems for delivering both targeting and flight reference information to a military pilot.

  19. 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

  20. Epithelial Adhesion Mediated by Pilin SpaC Is Required for Lactobacillus rhamnosus GG-Induced Cellular Responses

    PubMed Central

    Ardita, Courtney S.; Mercante, Jeffrey W.; Kwon, Young Man; Luo, Liping; Crawford, Madelyn E.; Powell, Domonica N.; Jones, Rheinallt M.

    2014-01-01

    Lactobacillus rhamnosus GG is a widely used probiotic, and the strain's salutary effects on the intestine have been extensively documented. We previously reported that strain GG can modulate inflammatory signaling, as well as epithelial migration and proliferation, by activating NADPH oxidase 1-catalyzed generation of reactive oxygen species (ROS). However, how strain GG induces these responses is unknown. Here, we report that strain GG's probiotic benefits are dependent on the bacterial-epithelial interaction mediated by the SpaC pilin subunit. By comparing strain GG to an isogenic mutant that lacks SpaC (strain GGΩspaC), we establish that SpaC is necessary for strain GG to adhere to gut mucosa, that SpaC contributes to strain GG-induced epithelial generation of ROS, and that SpaC plays a role in strain GG's capacity to stimulate extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling in enterocytes. In addition, we show that SpaC is required for strain GG-mediated stimulation of cell proliferation and protection against radiologically inflicted intestinal injury. The identification of a critical surface protein required for strain GG to mediate its probiotic influence advances our understanding of the molecular basis for the symbiotic relationship between some commensal bacteria of the gut lumen and enterocytes. Further insights into this relationship are critical for the development of novel approaches to treat intestinal diseases. PMID:24928883

  1. Epithelial adhesion mediated by pilin SpaC is required for Lactobacillus rhamnosus GG-induced cellular responses.

    PubMed

    Ardita, Courtney S; Mercante, Jeffrey W; Kwon, Young Man; Luo, Liping; Crawford, Madelyn E; Powell, Domonica N; Jones, Rheinallt M; Neish, Andrew S

    2014-08-01

    Lactobacillus rhamnosus GG is a widely used probiotic, and the strain's salutary effects on the intestine have been extensively documented. We previously reported that strain GG can modulate inflammatory signaling, as well as epithelial migration and proliferation, by activating NADPH oxidase 1-catalyzed generation of reactive oxygen species (ROS). However, how strain GG induces these responses is unknown. Here, we report that strain GG's probiotic benefits are dependent on the bacterial-epithelial interaction mediated by the SpaC pilin subunit. By comparing strain GG to an isogenic mutant that lacks SpaC (strain GGΩspaC), we establish that SpaC is necessary for strain GG to adhere to gut mucosa, that SpaC contributes to strain GG-induced epithelial generation of ROS, and that SpaC plays a role in strain GG's capacity to stimulate extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling in enterocytes. In addition, we show that SpaC is required for strain GG-mediated stimulation of cell proliferation and protection against radiologically inflicted intestinal injury. The identification of a critical surface protein required for strain GG to mediate its probiotic influence advances our understanding of the molecular basis for the symbiotic relationship between some commensal bacteria of the gut lumen and enterocytes. Further insights into this relationship are critical for the development of novel approaches to treat intestinal diseases.

  2. GAP Activity, but Not Subcellular Targeting, Is Required for Arabidopsis RanGAP Cellular and Developmental Functions[OPEN

    PubMed Central

    Boruc, Joanna; Griffis, Anna H.N.; Rodrigo-Peiris, Thushani; Zhou, Xiao; Tilford, Bailey; Van Damme, Daniël; Meier, Iris

    2015-01-01

    The Ran GTPase activating protein (RanGAP) is important to Ran signaling involved in nucleocytoplasmic transport, spindle organization, and postmitotic nuclear assembly. Unlike vertebrate and yeast RanGAP, plant RanGAP has an N-terminal WPP domain, required for nuclear envelope association and several mitotic locations of Arabidopsis thaliana RanGAP1. A double null mutant of the two Arabidopsis RanGAP homologs is gametophyte lethal. Here, we created a series of mutants with various reductions in RanGAP levels by combining a RanGAP1 null allele with different RanGAP2 alleles. As RanGAP level decreases, the severity of developmental phenotypes increases, but nuclear import is unaffected. To dissect whether the GAP activity and/or the subcellular localization of RanGAP are responsible for the observed phenotypes, this series of rangap mutants were transformed with RanGAP1 variants carrying point mutations abolishing the GAP activity and/or the WPP-dependent subcellular localization. The data show that plant development is differentially affected by RanGAP mutant allele combinations of increasing severity and requires the GAP activity of RanGAP, while the subcellular positioning of RanGAP is dispensable. In addition, our results indicate that nucleocytoplasmic trafficking can tolerate both partial depletion of RanGAP and delocalization of RanGAP from the nuclear envelope. PMID:26091693

  3. A Mechanism to Enhance Cellular Responsivity to Hormone Action: Krüppel-Like Factor 9 Promotes Thyroid Hormone Receptor-β Autoinduction During Postembryonic Brain Development.

    PubMed

    Hu, Fang; Knoedler, Joseph R; Denver, Robert J

    2016-04-01

    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.

  4. Dual Role of the Saccharomyces cerevisiae TEA/ATTS Family Transcription Factor Tec1p in Regulation of Gene Expression and Cellular Development

    PubMed Central

    Köhler, Tim; Wesche, Stefanie; Taheri, Naimeh; Braus, Gerhard H.; Mösch, Hans-Ulrich

    2002-01-01

    In Saccharomyces cerevisiae, the transcription factors Tec1p and Ste12p are required for haploid invasive and diploid pseudohyphal growth. Tec1p and Ste12p have been postulated to regulate these developmental processes primarily by cooperative binding to filamentous and invasion-responsive elements (FREs), which are combined enhancer elements that consist of a Tec1p-binding site (TCS) and an Ste12p-binding site (PRE). They are present in the promoter regions of target genes, e.g., FLO11. Here, we show that Tec1p efficiently activates target gene expression and cellular development in the absence of Ste12p. We further demonstrate that TCS elements alone are sufficient to mediate Tec1p-driven gene expression by a mechanism termed TCS control that is operative even when Ste12p is absent. Mutational analysis of TEC1 revealed that TCS control, FLO11 expression, and haploid invasive growth require the C terminus of Tec1p. In contrast, the Ste12p-dependent FRE control mechanism is sufficiently executed by the N-terminal portion of Tec1p, which contains the TEA/ATTS DNA-binding domain. Our study suggests that regulation of haploid invasive and diploid pseudohyphal growth by Ste12p and Tec1p is not only executed by combinatorial control but involves additional control mechanisms in which Ste12p activates TEC1 expression via clustered PREs and where Tec1p regulates expression of target genes, e.g., FLO11, by TCS control. PMID:12455687

  5. 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.

  6. 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

  7. The cellular RNA helicase UAP56 is required for prevention of double-stranded RNA formation during influenza A virus infection.

    PubMed

    Wisskirchen, Christian; Ludersdorfer, Thomas H; Müller, Dominik A; Moritz, Eva; Pavlovic, Jovan

    2011-09-01

    The cellular DEAD box RNA helicase UAP56 plays a pivotal role in the efficient transcription/replication of influenza A virus. UAP56 is recruited by the nucleoprotein (NP) of influenza A viruses, and recent data revealed that the RNA helicase is required for the nuclear export of a subset of spliced and unspliced viral mRNAs. The fact that influenza viruses do not produce detectable amounts of double-stranded RNA (dsRNA) intermediates during transcription/replication suggests the involvement of cellular RNA helicases. Hence, we examined whether the RNA-unwinding activity of UAP56 or its paralog URH49 plays a role in preventing the accumulation of dsRNA during infection. First, our data showed that not only UAP56 but also its paralog URH49 can interact with NPs of avian and human influenza A viruses. The small interfering RNA (siRNA)-mediated depletion of either RNA helicase reduced the transport of M1 and hemagglutinin (HA) mRNAs and, to a lesser extent, NP and NS1 mRNAs into the cytoplasm. Moreover, we found that virus infection of UAP56-depleted cells leads to the rapid accumulation of dsRNA in the perinuclear region. In parallel, we observed a robust virus-mediated activation of dsRNA-dependent protein kinase R (PKR), indicating that the cellular RNA helicase UAP56 may be recruited by influenza virus to prevent dsRNA formation. The accumulation of dsRNA was blocked when actinomycin D or cycloheximide was used to inhibit viral transcription/replication or translation, respectively. In summary, we demonstrate that UAP56 is utilized by influenza A viruses to prevent the formation of dsRNA and, hence, the activation of the innate immune response.

  8. Glucose-dependent anaplerosis in cancer cells is required for cellular redox balance in the absence of glutamine.

    PubMed

    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 (13)C-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

  9. Glucose-dependent anaplerosis in cancer cells is required for cellular redox balance in the absence of glutamine.

    PubMed

    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 (13)C-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.

  10. 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

  11. Glucose-dependent anaplerosis in cancer cells is required for cellular redox balance in the absence of glutamine

    NASA Astrophysics Data System (ADS)

    Cetinbas, Naniye Mallı; Sudderth, Jessica; Harris, Robert C.; Cebeci, Aysun; Negri, Gian L.; Yılmaz, Ömer H.; Deberardinis, Ralph J.; Sorensen, Poul H.

    2016-09-01

    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.

  12. Timed interactions between viral and cellular replication factors during the initiation of SV40 in vitro DNA replication

    PubMed Central

    Taneja, Poonam; Nasheuer, Heinz-Peter; Hartmann, Hella; Grosse, Frank; Fanning, Ellen; Weisshart, Klaus

    2007-01-01

    The initiation of SV40 (simian virus 40) DNA replication requires the co-operative interactions between the viral Tag (large T-antigen), RPA (replication protein A) and Pol (DNA polymerase α-primase) on the template DNA. Binding interfaces mapped on these enzymes and expressed as peptides competed with the mutual interactions of the native proteins. Prevention of the genuine interactions was accomplished only prior to the primer synthesis step and blocked the assembly of a productive initiation complex. Once the complex was engaged in the synthesis of an RNA primer and its extension, the interfering effects of the peptides ceased, suggesting a stable association of the replication factors during the initiation phase. Specific antibodies were still able to disrupt preformed interactions and inhibited primer synthesis and extension activities, underlining the crucial role of specific protein–protein contacts during the entire initiation process. PMID:17666013

  13. The LIM domain-containing Dbm1 GTPase-activating protein is required for normal cellular morphogenesis in Saccharomyces cerevisiae.

    PubMed Central

    Chen, G C; Zheng, L; Chan, C S

    1996-01-01

    Normal cell growth in the yeast Saccharomyces cerevisiae involves the selection of genetically determined bud sites where most growth is localized. Previous studies have shown that BEM2, which encodes a GTPase-activating protein (GAP) that is specific for the Rho-type GTPase Rho1p in vitro, is required for proper bud site selection and bud emergence. We show here that DBM1, which encodes another putative Rho-type GAP with two tandemly arranged cysteine-rich LIM domains, also is needed for proper bud site selection, as haploid cells lacking Dbm1p bud predominantly in a bipolar, rather than the normal axial, manner. Furthermore, yeast cells lacking both Bem2p and Dbm1p are inviable. The nonaxial budding defect of dbm1 mutants can be rescued partially by overproduction of Bem3p and is exacerbated by its absence. Since Bem3p has previously been shown to function as a GAP for Cdc42p, and also less efficiently for Rho1p, our results suggest that Dbm1p, like Bem2p and Bem3p, may function in vivo as a GAP for Cdc42p and/or Rho1p. Both LIM domains of Dbm1p are essential for its normal function. Point mutations that alter single conserved cysteine residues within either LIM domain result in mutant forms of Dbm1p that can no longer function in bud site selection but instead are capable of rescuing the inviability of bem2 mutants at 35 degrees C. PMID:8657111

  14. 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. PMID:27151944

  15. 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

  16. 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.

  17. 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

  18. 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

  19. A murine host cell factor required for nicking of the dimer bridge of MVM recognizes two CG nucleotides displaced by 10 basepairs.

    PubMed

    Liu, Q; Astell, C R

    1996-10-01

    During replication of the minute virus of mice (MVM) genome, a dimer replicative form (RF) intermediate is resolved into two monomer RF molecules in such a way as to retain a unique sequence within the left hand hairpin terminus of the viral genome. Although the proposed mechanism for resolution of the dimer RF remains uncertain, it likely involves site-specific nicking of the dimer bridge. The RF contains two double-stranded copies of the viral genome joined by the extended 3' hairpin. Minor sequence asymmetries within the 3' hairpin allow the two halves of the dimer bridge to be distinguished. The A half contains the sequence [sequence: see text], whereas the B half contains the sequence [sequence: see text]. Using an in vitro assay, we show that only the B half of the MVM dimer bridge is nicked site-specifically when incubated with crude NS-1 protein (expressed in insect cells) and mouse LA9 cellular extract. When highly purified NS-1, the major nonstructural protein of MVM, is used in this nicking reaction, there is an absolute requirement for the LA9 cellular extract, suggesting a cellular factor (or factors) is (are) required. A series of mutations were created in the putative host factor binding region (HFBR) on the B half of the MVM dimer bridge adjacent to the NS-1 binding site. Nicking assays of these B half mutants showed that two CG motifs displaced by 10 nucleotides are important for nicking. Gel mobility shift assays demonstrated that a host factor(s) can bind to the HFBR of the B half of the dimer bridge and efficient binding depends on the presence of both CG motifs. Competitor DNA containing the wild-type HFBR sequence is able to specifically inhibit nicking of the B half, indicating that the host factor(s) bound to the HFBR is(are) essential for site-specific nicking to occur.

  20. Regulon and promoter analysis of the E. coli heat-shock factor, sigma32, reveals a multifaceted cellular response to heat stress.

    PubMed

    Nonaka, Gen; Blankschien, Matthew; Herman, Christophe; Gross, Carol A; Rhodius, Virgil A

    2006-07-01

    The heat-shock response (HSR), a universal cellular response to heat, is crucial for cellular adaptation. In Escherichia coli, the HSR is mediated by the alternative sigma factor, sigma32. To determine its role, we used genome-wide expression analysis and promoter validation to identify genes directly regulated by sigma32 and screened ORF overexpression libraries to identify sigma32 inducers. We triple the number of genes validated to be transcribed by sigma32 and provide new insights into the cellular role of this response. Our work indicates that the response is propagated as the regulon encodes numerous global transcriptional regulators, reveals that sigma70 holoenzyme initiates from 12% of sigma32 promoters, which has important implications for global transcriptional wiring, and identifies a new role for the response in protein homeostasis, that of protecting complex proteins. Finally, this study suggests that the response protects the cell membrane and responds to its status: Fully 25% of sigma32 regulon members reside in the membrane and alter its functionality; moreover, a disproportionate fraction of overexpressed proteins that induce the response are membrane localized. The intimate connection of the response to the membrane rationalizes why a major regulator of the response resides in that cellular compartment.

  1. Transcription factors of Lotus: regulation of isoflavonoid biosynthesis requires coordinated changes in transcription factor activity.

    PubMed

    Shelton, Dale; Stranne, Maria; Mikkelsen, Lisbeth; Pakseresht, Nima; Welham, Tracey; Hiraka, Hideki; Tabata, Satoshi; Sato, Shusei; Paquette, Suzanne; Wang, Trevor L; Martin, Cathie; Bailey, Paul

    2012-06-01

    Isoflavonoids are a class of phenylpropanoids made by legumes, and consumption of dietary isoflavonoids confers benefits to human health. Our aim is to understand the regulation of isoflavonoid biosynthesis. Many studies have shown the importance of transcription factors in regulating the transcription of one or more genes encoding enzymes in phenylpropanoid metabolism. In this study, we coupled bioinformatics and coexpression analysis to identify candidate genes encoding transcription factors involved in regulating isoflavonoid biosynthesis in Lotus (Lotus japonicus). Genes encoding proteins belonging to 39 of the main transcription factor families were examined by microarray analysis of RNA from leaf tissue that had been elicited with glutathione. Phylogenetic analyses of each transcription factor family were used to identify subgroups of proteins that were specific to L. japonicus or closely related to known regulators of the phenylpropanoid pathway in other species. R2R3MYB subgroup 2 genes showed increased expression after treatment with glutathione. One member of this subgroup, LjMYB14, was constitutively overexpressed in L. japonicus and induced the expression of at least 12 genes that encoded enzymes in the general phenylpropanoid and isoflavonoid pathways. A distinct set of six R2R3MYB subgroup 2-like genes was identified. We suggest that these subgroup 2 sister group proteins and those belonging to the main subgroup 2 have roles in inducing isoflavonoid biosynthesis. The induction of isoflavonoid production in L. japonicus also involves the coordinated down-regulation of competing biosynthetic pathways by changing the expression of other transcription factors. PMID:22529285

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

    PubMed Central

    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.

    2016-01-01

    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. Significance There is no cure for Alzheimer’s disease (AD) and

  3. 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.

  4. Titration of cellular export factors, but not heteromultimerization, is the molecular mechanism of trans-dominant HTLV-1 rex mutants.

    PubMed

    Heger, P; Rosorius, O; Hauber, J; Stauber, R H

    1999-07-15

    The HTLV-1 Rex protein is an essential shuttle protein required for nuclear export of unspliced and incompletely-spliced viral RNAs. Several trans-dominant (TD) mutant Rex proteins have been reported, however, the mechanism of trans-dominance is not known. We compared TD Rex mutants and found that a natural occurring Rex mutant, Rexp21, lacking the RNA binding domain, was highly TD and inhibited also HIV-1 Rev function. Using fusions to the green fluorescent protein (GFP) we observed that Rexp21-GFP displayed a cytoplasmic localization but was actively shuttling between the nucleus and the cytoplasm in live human cells. The presence of Rexp21-GFP inhibited the nuclear export of Rex and HIV-1 Rev as assayed by cotransfection and microinjection experiments. However, Rex-GFP or Rexp21-GFP did not form heteromultimers with nuclear Rex mutants in vivo. In contrast, shuttling was essential for trans-dominance. Thus, we propose that TD Rex mutants do not function by retaining WT Rex in the nucleus by protein-protein interactions, as demonstrated for Rev, but to titrate factors essential for Rex/Rev export. Our findings demonstrate differences between the regulatory proteins Rex and Rev and implicate a novel strategy to generate highly TD Rex mutants also applicable to other proteins.

  5. Herpes simplex virus virion host shutoff protein requires a mammalian factor for efficient in vitro endoribonuclease activity.

    PubMed

    Lu, P; Jones, F E; Saffran, H A; Smiley, J R

    2001-02-01

    The virion host shutoff protein (vhs) of herpes simplex virus (HSV) triggers global shutoff of host protein synthesis and accelerated mRNA turnover during virus infection and induces endoribonucleolytic cleavage of exogenous RNA substrates when it is produced in a rabbit reticulocyte (RRL) in vitro translation system. Although vhs induces RNA turnover in the absence of other HSV gene products, it is not yet known whether cellular factors are required for its activity. As one approach to addressing this question, we expressed vhs in the budding yeast Saccharomyces cerevisiae. Expression of vhs inhibited colony formation, and the severity of this effect varied with the carbon source. The biological relevance of this effect was assessed by examining the activity of five mutant forms of vhs bearing previously characterized in-frame linker insertions. The results indicated a complete concordance between the growth inhibition phenotype in yeast and mammalian host cell shutoff. Despite these results, expression of vhs did not trigger global mRNA turnover in vivo, and cell extracts of yeast expressing vhs displayed little if any vhs-dependent endoribonuclease activity. However, activity was readily detected when such extracts were mixed with RRL. These data suggest that the vhs-dependent endoribonuclease requires one or more mammalian macromolecular factors for efficient activity.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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. PMID:23880637

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. Cellular apoptosis susceptibility (CAS) is linked to integrin β1 and required for tumor cell migration and invasion in hepatocellular carcinoma (HCC)

    PubMed Central

    Winkler, Juliane; Roessler, Stephanie; Sticht, Carsten; DiGuilio, Amanda L.; Drucker, Elisabeth; Holzer, Kerstin; Eiteneuer, Eva; Herpel, Esther; Breuhahn, Kai; Gretz, Norbert; Schirmacher, Peter; Ori, Alessandro; Singer, Stephan

    2016-01-01

    Importins and exportins represent an integral part of the nucleocytoplasmic transport machinery with fundamental importance for eukaryotic cell function. A variety of malignancies including hepatocellular carcinoma (HCC) show de-regulation of nuclear transport factors such as overexpression of the exportin Cellular Apoptosis Susceptibility (CAS). The functional implications of CAS in hepatocarcinogenesis remain, however, poorly understood. Here we integrated proteomics, transcriptomics and functional assays with patient data to further characterize the role of CAS in HCC. By analyzing ∼ 1700 proteins using quantitative mass spectrometry in HCC cells we found that CAS depletion by RNAi leads to de-regulation of integrins, particularly down-regulation of integrin β1. Consistent with this finding, CAS knockdown resulted in substantially reduced migration and invasion of HCC cell lines as analyzed by 2D ‘scratch’ and invasion chamber assays, respectively. Supporting the potential in vivo relevance, high expression levels of CAS in HCC tissue samples were associated with macroangioinvasion and poorer patient outcome. Our data suggest a previously unanticipated link between CAS and integrin signaling which correlates with an aggressive HCC phenotype. PMID:27015362

  16. Multiple Regions of Kaposi’s Sarcoma-Associated Herpesvirus ORF59 RNA are Required for Its Expression Mediated by Viral ORF57 and Cellular RBM15

    PubMed Central

    Massimelli, Maria Julia; Majerciak, Vladimir; Kang, Jeong-Gu; Liewehr, David J.; Steinberg, Seth M.; Zheng, Zhi-Ming

    2015-01-01

    KSHV ORF57 (MTA) promotes RNA stability of ORF59, a viral DNA polymerase processivity factor. Here, we show that the integrity of both ORF59 RNA ends is necessary for ORF57-mediated ORF59 expression and deletion of both 5’ and 3’ regions, or one end region with a central region, of ORF59 RNA prevents ORF57-mediated translation of ORF59. The ORF59 sequence between nt 96633 and 96559 resembles other known MTA-responsive elements (MREs). ORF57 specifically binds to a stem-loop region from nt 96596–96572 of the MRE, which also binds cellular RBM15. Internal deletion of the MRE from ORF59 led to poor export, but accumulation of nuclear ORF59 RNA in the presence of ORF57 or RBM15. Despite of being translatable in the presence of ORF57, this deletion mutant exhibits translational defect in the presence of RBM15. Together, our results provide novel insight into the roles of ORF57 and RBM15 in ORF59 RNA accumulation and protein translation. PMID:25690794

  17. 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.

  18. Tumor Cellular Proteasome Inhibition and Growth Suppression by 8-Hydroxyquinoline and Clioquinol Requires Their Capabilities to Bind Copper and Transport Copper into Cells

    PubMed Central

    Zhai, Shumei; Yang, Lei; Cui, Qiuzhi Cindy; Sun, Ying; Dou, Q. Ping; Yan, Bing

    2009-01-01

    We have previously reported that when mixed with copper, 8-hydroxyquinoline (8-OHQ) and its analog clioquinol (CQ) inhibited the proteasomal activity and proliferation in cultured human cancer cells. CQ treatment of high copper-containing human tumor xenografts also caused cancer suppression, associated with proteasome inhibition in vivo. However, the nature of copper dependence of these events has not been elucidated experimentally. In the current study, by using chemical probe molecules that mimic structures of 8-OHQ and CQ, but have no copper binding capability, we dissected the complex cellular processes elicited by 8-OHQ-Cu or CQ-Cu mixture and revealed that copper-binding to 8-OHQ or CQ is required for transportation of copper complex into human breast cancer cells and the consequent proteasome-inhibitory, growth-suppressive and apoptosis-inducing activities. In contrast, the non-copper-binding analogs of 8-OHQ or CQ blocked the very first step – copper binding in this chain of events mediated by 8-OHQ-Cu or CQ-Cu. PMID:19809836

  19. ERp57 as a novel cellular factor controlling prion protein biosynthesis: Therapeutic potential of protein disulfide isomerases.

    PubMed

    Sepulveda, Martin; Rozas, Pablo; Hetz, Claudio; Medinas, Danilo B

    2016-01-01

    Disturbance of endoplasmic reticulum (ER) proteostasis is observed in Prion-related disorders (PrDs). The protein disulfide isomerase ERp57 is a stress-responsive ER chaperone up-regulated in the brain of Creutzfeldt-Jakob disease patients. However, the actual role of ERp57 in prion protein (PrP) biogenesis and the ER stress response remained poorly defined. We have recently addressed this question using gain- and loss-of-function approaches in vitro and animal models, observing that ERp57 regulates steady-state levels of PrP. Our results revealed that ERp57 modulates the biosynthesis and maturation of PrP but, surprisingly, does not contribute to the global cellular reaction against ER stress in neurons. Here we discuss the relevance of ERp57 as a possible therapeutic target in PrDs and other protein misfolding disorders. PMID:26864548

  20. 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. PMID:26741268

  1. 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.

  2. Human Immunodeficiency Virus Type 1 Tat Increases the Expression of Cleavage and Polyadenylation Specificity Factor 73-Kilodalton Subunit Modulating Cellular and Viral Expression

    PubMed Central

    Calzado, Marco A.; Sancho, Rocío; Muñoz, Eduardo

    2004-01-01

    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. PMID:15194760

  3. Immunohistochemical analysis of factors related to apoptosis and cellular proliferation in relation to inflammation in dentigerous and odontogenic keratocyst

    PubMed Central

    Sreedhar, Gadiputi; Raju, M. Vijaya; Metta, Kiran Kumar; Manjunath, SM; Shetty, Sujan; Agarwal, Rakesh Kumar

    2014-01-01

    Background: The effect of inflammation on pathogenesis and biological behavior of odontogenic keratocyst (OKC) and dentigerous cyst (DC) is not completely understood. Hence, we aimed to analyze the effect of inflammation on biological behavior of OKC and DC using a proliferative and anti-apoptotic marker, i.e., proliferative cellular nuclear antigen (PCNA) and Bcl-2, respectively. Materials and Methods: Immunohistochemical staining was performed using anti-PCNA and Bcl-2 antibody in 10 cases each of classical OKC, inflamed OKC and classical DC and inflamed DC. Results: Inflamed OKC and DC showed a significant increase in PCNA expression and decrease in Bcl-2 expression when compared with non-inflamed cyst. Correlation between inflammation and proliferative and anti-apoptotic activity was found to be statistically non-significant. Conclusion: Inflammation is responsible for change in behavior of neoplastic epithelium of OKC and hence should be treated meticulously, whereas in DC it is responsible for changes in the epithelial lining. PMID:24678208

  4. 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

  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. Fat-specific protein 27 modulates nuclear factor of activated T cells 5 and the cellular response to stress

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. 21 CFR 1311.115 - Additional requirements for two-factor authentication.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 9 2013-04-01 2013-04-01 false Additional requirements for two-factor authentication. 1311.115 Section 1311.115 Food and Drugs DRUG ENFORCEMENT ADMINISTRATION, DEPARTMENT OF JUSTICE... criteria of FIPS 140-2 Security Level 1, as incorporated by reference in § 1311.08, for...

  8. 21 CFR 1311.115 - Additional requirements for two-factor authentication.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 9 2012-04-01 2012-04-01 false Additional requirements for two-factor authentication. 1311.115 Section 1311.115 Food and Drugs DRUG ENFORCEMENT ADMINISTRATION, DEPARTMENT OF JUSTICE... criteria of FIPS 140-2 Security Level 1, as incorporated by reference in § 1311.08, for...

  9. Genetic and nongenetic factors associated with warfarin dose requirements in Egyptian patients

    PubMed Central

    Shahin, Mohamed Hossam A.; Khalifa, Sherief I.; Gong, Yan; Hammad, Lamiaa N.; Sallam, Mohamed T.H.; Shafey, Mostafa El; Ali, Shawky S.; Mohamed, Mohamed-Eslam F.; Langaee, Taimour; Johnson, Julie A.

    2011-01-01

    Background and objective Warfarin is a commonly used oral anticoagulant with a narrow therapeutic index and various genetic and clinical factors that influence interpatient variability in dose requirements. This study investigated the impact of genetic and nongenetic factors on warfarin dose requirements in Egyptians. Methods DNA was extracted from 207 patients taking warfarin for more than 2 months and genotyped for VKORC1 (3673 G> A), CYP2C9 *2*3*4*5*8, CYP4F2 (V33M; rs2108622), APOE (rs429358, rs7412), and CALU (rs339097) gene polymorphisms. Linear regression modeling was conducted to identify the genetic and nongenetic factors that independently influence warfarin dose requirements. Results VKORC1 3673 AA or GA genotype (P < 0.0001), one or two variant alleles of CYP2C9 gene (P= 0.0004), APOE ε2 haplotype (P = 0.01), and increasing age (P < 0.0001) were all associated with lower warfarin dose, whereas smoking (P = 0.025) and pulmonary embolism (P = 0.0059) showed association with higher warfarin doses. These factors explained 31% of the warfarin dose variability. This is the first independent confirmation of the association of the CALU rs339097 variant with higher warfarin dose requirement, although inclusion of this single nucleotide polymorphism in the multiple regression model failed to achieve significance (P = 0.066). CYP4F2 (V33M) polymorphism was not significant (P = 0.314), despite its high frequency in the studied population (42%). Conclusion The study shows that VKORC1, CYP2C9 polymorphisms, APOE ε2 variant, and several clinical/ demographic variables are important determinants of warfarin dose requirements in Egyptian patients. The percentage of variability explained by these factors is lower than in those of European ancestry, but similar to the variability explained in Asians and African ancestry. PMID:21228733

  10. Factor VIII C1 domain spikes 2092-2093 and 2158-2159 comprise regions that modulate cofactor function and cellular uptake.

    PubMed

    Bloem, Esther; van den Biggelaar, Maartje; Wroblewska, Aleksandra; Voorberg, Jan; Faber, Johan H; Kjalke, Marianne; Stennicke, Henning R; Mertens, Koen; Meijer, Alexander B

    2013-10-11

    The C1 domain of factor VIII (FVIII) has been implicated in binding to multiple constituents, including phospholipids, von Willebrand factor, and low-density lipoprotein receptor-related protein (LRP). We have previously described a human monoclonal antibody called KM33 that blocks these interactions as well as cellular uptake by LRP-expressing cells. To unambiguously identify the apparent "hot spot" on FVIII to which this antibody binds, we have employed hydrogen-deuterium exchange mass spectrometry. The results showed that KM33 protects FVIII regions 2091-2104 and 2157-2162 from hydrogen-deuterium exchange. These comprise the two C1 domain spikes 2092-2093 and 2158-2159. Spike 2092-2093 has been demonstrated recently to contribute to assembly with lipid membranes with low phosphatidylserine (PS) content. Therefore, spike 2158-2159 might serve a similar role. This was assessed by replacement of Arg-2159 for Asn, which introduces a motif for N-linked glycosylation. Binding studies revealed that the purified, glycosylated R2159N variant had lost its interaction with antibody KM33 but retained substantial binding to von Willebrand factor and LRP. Cellular uptake of the R2159N variant was reduced both by LRP-expressing U87-MG cells and by human monocyte-derived dendritic cells. FVIII activity was virtually normal on membranes containing 15% PS but reduced at low PS content. These findings suggest that the C1 domain spikes 2092-2093 and 2158-2159 together modulate FVIII membrane assembly by a subtle, PS-dependent mechanism. These findings contribute evidence in favor of an increasingly important role of the C1 domain in FVIII biology. PMID:24009077

  11. Factor VIII C1 Domain Spikes 2092–2093 and 2158–2159 Comprise Regions That Modulate Cofactor Function and Cellular Uptake

    PubMed Central

    Bloem, Esther; van den Biggelaar, Maartje; Wroblewska, Aleksandra; Voorberg, Jan; Faber, Johan H.; Kjalke, Marianne; Stennicke, Henning R.; Mertens, Koen; Meijer, Alexander B.

    2013-01-01

    The C1 domain of factor VIII (FVIII) has been implicated in binding to multiple constituents, including phospholipids, von Willebrand factor, and low-density lipoprotein receptor-related protein (LRP). We have previously described a human monoclonal antibody called KM33 that blocks these interactions as well as cellular uptake by LRP-expressing cells. To unambiguously identify the apparent “hot spot” on FVIII to which this antibody binds, we have employed hydrogen-deuterium exchange mass spectrometry. The results showed that KM33 protects FVIII regions 2091–2104 and 2157–2162 from hydrogen-deuterium exchange. These comprise the two C1 domain spikes 2092–2093 and 2158–2159. Spike 2092–2093 has been demonstrated recently to contribute to assembly with lipid membranes with low phosphatidylserine (PS) content. Therefore, spike 2158–2159 might serve a similar role. This was assessed by replacement of Arg-2159 for Asn, which introduces a motif for N-linked glycosylation. Binding studies revealed that the purified, glycosylated R2159N variant had lost its interaction with antibody KM33 but retained substantial binding to von Willebrand factor and LRP. Cellular uptake of the R2159N variant was reduced both by LRP-expressing U87-MG cells and by human monocyte-derived dendritic cells. FVIII activity was virtually normal on membranes containing 15% PS but reduced at low PS content. These findings suggest that the C1 domain spikes 2092–2093 and 2158–2159 together modulate FVIII membrane assembly by a subtle, PS-dependent mechanism. These findings contribute evidence in favor of an increasingly important role of the C1 domain in FVIII biology. PMID:24009077

  12. Motif affinity and mass spectrometry proteomic approach for the discovery of cellular AMPK targets: identification of mitochondrial fission factor as a new AMPK substrate.

    PubMed

    Ducommun, Serge; Deak, Maria; Sumpton, David; Ford, Rebecca J; Núñez Galindo, Antonio; Kussmann, Martin; Viollet, Benoit; Steinberg, Gregory R; Foretz, Marc; Dayon, Loïc; Morrice, Nicholas A; Sakamoto, Kei

    2015-05-01

    AMP-activated protein kinase (AMPK) is a key cellular energy sensor and regulator of metabolic homeostasis. Although it is best known for its effects on carbohydrate and lipid metabolism, AMPK is implicated in diverse cellular processes, including mitochondrial biogenesis, autophagy, and cell growth and proliferation. To further our understanding of energy homeostasis through AMPK-dependent processes, the design and application of approaches to identify and characterise novel AMPK substrates are invaluable. Here, we report an affinity proteomicstrategy for the discovery and validation of AMPK targets using an antibody to isolate proteins containing the phospho-AMPK substrate recognition motif from hepatocytes that had been treated with pharmacological AMPK activators. We identified 57 proteins that were uniquely enriched in the activator-treated hepatocytes, but were absent in hepatocytes lacking AMPK. We focused on two candidates, cingulin and mitochondrial fission factor (MFF), and further characterised/validated them as AMPK-dependent targets by immunoblotting with phosphorylation site-specific antibodies. A small-molecule AMPK activator caused transient phosphorylation of endogenous cingulin at S137 in intestinal Caco2 cells. Multiple splice-variants of MFF appear to express in hepatocytes and we identified a common AMPK-dependent phospho-site (S129) in all the 3 predominant variants spanning the mass range and a short variant-specific site (S146). Collectively, our proteomic-based approach using a phospho-AMPK substrate antibody in combination with genetic models and selective AMPK activators will provide a powerful and reliable platform for identifying novel AMPK-dependent cellular targets.

  13. 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.

  14. Risk Factors for Uterine Atony/Postpartum Hemorrhage Requiring Treatment after Vaginal Delivery

    PubMed Central

    Wetta, Luisa A; Szychowski, Jeff M; Seals, Ms. Samantha; Mancuso, Melissa S; Biggio, Joseph R; Tita, Alan TN

    2013-01-01

    Objective To identify risk factors for uterine atony or hemorrhage. Study Design 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. Twenty-one potential risk factors were evaluated. Logistic regression was used to identify independent risk factors using 2 complementary pre-defined model selection strategies. Results Among 1798 women randomized to 10, 40 or 80U prophylactic oxytocin after vaginal delivery, treated uterine atony occurred in 7%. Hispanic (OR 2.1; 95% CI 1.3–3.4) and non-Hispanic whites (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). Conclusion Independent risk factors for uterine atony requiring treatment include Hispanic and non-Hispanic white ethnicity, preeclampsia and chorioamnionitis. PMID:23507549

  15. 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

  16. Cellular Factor XIIIA Transglutaminase Localizes in Caveolae and Regulates Caveolin-1 Phosphorylation, Homo-oligomerization and c-Src Signaling in Osteoblasts.

    PubMed

    Wang, Shuai; Kaartinen, Mari T

    2015-11-01

    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

  17. 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.

  18. Activation of nuclear factor-kappaB and not activator protein-1 in cellular response to nickel compounds.

    PubMed Central

    Huang, Yi; Davidson, Gerard; Li, Jingxia; Yan, Yan; Chen, Fei; Costa, Max; Chen, Lung Chi; Huang, Chuanshu

    2002-01-01

    The predominant exposure route for nickel compounds is by inhalation, and several studies have indicated the correlation between nickel exposure and respiratory cancers. The tumor-promoting effects of nickel compounds are thought to be associated with their transactivation of transcription factors. We have investigated the possible activation of activator protein-1 (AP-1) and nuclear factor KB (NF-kappaB) in mouse C141 epidermal cells and fibroblasts 3T3 and B82, and human bronchoepithelial BEAS-2B cells in response to nickel compound exposure. Our results show that NF-kappaB activity is induced by nickel exposure in 3T3 and BEAS-2B cells. Conversely, similar nickel treatment of these cells did not induce AP-1 activity, suggesting that nickel tumorigenesis occurs through NF-kappaB and not AP-1. We also investigated the role of NF-kappaB in the induction of Cap43 by nickel compounds using dominant negative mutant Ikappabeta kinase b-KM BEAS-2B transfectants. PMID:12426142

  19. Expression of the Human Endogenous Retrovirus HTDV/HERV-K Is Enhanced by Cellular Transcription Factor YY1

    PubMed Central

    Knössl, Michael; Löwer, Roswitha; Löwer, Johannes

    1999-01-01

    The human endogenous retrovirus HTDV/HERV-K, which resides in moderate copy numbers in the human genome, is expressed in a cell-type-specific manner, predominantly in teratocarcinoma cells. We have analyzed the regulatory potential of the 5′ enhancer of the HERV-K long terminal repeat. Protein extracts of HERV-K-expressing teratocarcinoma cell lines (GH and Tera2) and nonexpressing HeLa and HepG2 cells form different protein complexes on the enhancer sequence as detected by electrophoretic mobility shift assays (EMSA). Using competition EMSAs, DNase I footprinting, and supershift experiments, we localized the binding site of these complexes to a 20-bp sequence within the enhancer and showed that the transcription factor YY1 is one component of the HERV-K enhancer complex. Replacement of the YY1 binding site with unrelated sequences reduced expression of the luciferase gene as a reporter in transient-transfection assays. PMID:9882329

  20. 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.

  1. 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.

  2. Cellular transcription factor Oct-1 interacts with the Epstein-Barr virus BRLF1 protein to promote disruption of viral latency.

    PubMed

    Robinson, Amanda R; Kwek, Swee Sen; Hagemeier, Stacy R; Wille, Coral K; Kenney, Shannon C

    2011-09-01

    The Epstein-Barr virus (EBV) latent-to-lytic switch is an essential part of the viral life cycle, but the cellular factors that promote viral reactivation are not well defined. In this report, we demonstrate that the cellular transcription factor Oct-1 cooperates with the EBV immediate-early protein BRLF1 (R, Rta) to induce lytic viral reactivation. We show that cotransfected Oct-1 enhances the ability of BRLF1 to activate lytic gene expression in 293 cells stably infected with a BRLF1-defective EBV mutant (BRLF1-stop) and that Oct-1 increases BRLF1-mediated activation of lytic EBV promoters in reporter gene assays. We find that Oct-1 interacts directly with BRLF1 in vitro and that a mutant BRLF1 protein (the M140A mutant) attenuated for the ability to interact with Oct-1 in vitro is also resistant to Oct-1-mediated transcriptional enhancement in 293 BRLF1-stop cells. Furthermore, we show that cotransfected Oct-1 augments BRLF1 binding to a variety of lytic EBV promoters in chromatin immunoprecipitation (ChIP) assays (including the BZLF1, BMRF1, and SM promoters) and that BRLF1 tethers Oct-1 to lytic EBV promoters. In addition, we demonstrate that an Oct-1 mutant defective in DNA binding (the S335D mutant) still retains the ability to enhance BRLF1 transcriptional effects. Finally, we show that knockdown of endogenous Oct-1 expression reduces the level of constitutive lytic EBV gene expression in both EBV-positive B-cell and EBV-positive epithelial cell lines. These results suggest that Oct-1 acts as a positive regulator of EBV lytic gene expression and that this effect is at least partially mediated through its interaction with the viral protein BRLF1. PMID:21697476

  3. The SEB-1 Transcription Factor Binds to the STRE Motif in Neurospora crassa and Regulates a Variety of Cellular Processes Including the Stress Response and Reserve Carbohydrate Metabolism

    PubMed Central

    Freitas, Fernanda Zanolli; Virgilio, Stela; Cupertino, Fernanda Barbosa; Kowbel, David John; Fioramonte, Mariana; Gozzo, Fabio Cesar; Glass, N. Louise; Bertolini, Maria Célia

    2016-01-01

    When exposed to stress conditions, all cells induce mechanisms resulting in an attempt to adapt to stress that involve proteins which, once activated, trigger cell responses by modulating specific signaling pathways. In this work, using a combination of pulldown assays and mass spectrometry analyses, we identified the Neurospora crassa SEB-1 transcription factor that binds to the Stress Response Element (STRE) under heat stress. Orthologs of SEB-1 have been functionally characterized in a few filamentous fungi as being involved in stress responses; however, the molecular mechanisms mediated by this transcription factor may not be conserved. Here, we provide evidences for the involvement of N. crassa SEB-1 in multiple cellular processes, including response to heat, as well as osmotic and oxidative stress. The Δseb-1 strain displayed reduced growth under these conditions, and genes encoding stress-responsive proteins were differentially regulated in the Δseb-1 strain grown under the same conditions. In addition, the SEB-1-GFP protein translocated from the cytosol to the nucleus under heat, osmotic, and oxidative stress conditions. SEB-1 also regulates the metabolism of the reserve carbohydrates glycogen and trehalose under heat stress, suggesting an interconnection between metabolism control and this environmental condition. We demonstrated that SEB-1 binds in vivo to the promoters of genes encoding glycogen metabolism enzymes and regulates their expression. A genome-wide transcriptional profile of the Δseb-1 strain under heat stress was determined by RNA-seq, and a broad range of cellular processes was identified that suggests a role for SEB-1 as a protein interconnecting these mechanisms. PMID:26994287

  4. Comprehensive Identification of Krüppel-Like Factor Family Members Contributing to the Self-Renewal of Mouse Embryonic Stem Cells and Cellular Reprogramming

    PubMed Central

    Azami, Takuya; Khoa, Le Tran Phuc; Yanagisawa, Jun; Takahashi, Satoru; Ema, Masatsugu

    2016-01-01

    Pluripotency is maintained in mouse embryonic stem (ES) cells and is induced from somatic cells by the activation of appropriate transcriptional regulatory networks. Krüppel-like factor gene family members, such as Klf2, Klf4 and Klf5, have important roles in maintaining the undifferentiated state of mouse ES cells as well as in cellular reprogramming, yet it is not known whether other Klf family members exert self-renewal and reprogramming functions when overexpressed. In this study, we examined whether overexpression of any representative Klf family member, such as Klf1–Klf10, would be sufficient for the self-renewal of mouse ES cells. We found that only Klf2, Klf4, and Klf5 produced leukemia inhibitory factor (LIF)-independent self-renewal, although most KLF proteins, if not all, have the ability to occupy the regulatory regions of Nanog, a critical Klf target gene. We also examined whether overexpression of any of Klf1-Klf10 would be sufficient to convert epiblast stem cells into a naïve pluripotent state and found that Klf5 had such reprogramming ability, in addition to Klf2 and Klf4. We also delineated the functional domains of the Klf2 protein for LIF-independent self-renewal and reprogramming. Interestingly, we found that both the N-terminal transcriptional activation and C-terminal zinc finger domains were indispensable for this activity. Taken together, our comprehensive analysis provides new insight into the contribution of Klf family members to mouse ES self-renewal and cellular reprogramming. PMID:26943822

  5. Exosite-mediated substrate recognition of factor IX by factor XIa. The factor XIa heavy chain is required for initial recognition of factor IX.

    PubMed

    Ogawa, Taketoshi; Verhamme, Ingrid M; Sun, Mao-Fu; Bock, Paul E; Gailani, David

    2005-06-24

    Studies of the mechanisms of blood coagulation zymogen activation demonstrate that exosites (sites on the activating complex distinct from the protease active site) play key roles in macromolecular substrate recognition. We investigated the importance of exosite interactions in recognition of factor IX by the protease factor XIa. Factor XIa cleavage of the tripeptide substrate S2366 was inhibited by the active site inhibitors p-aminobenzamidine (Ki 28 +/- 2 microM) and aprotinin (Ki 1.13 +/- 0.07 microM) in a classical competitive manner, indicating that substrate and inhibitor binding to the active site was mutually exclusive. In contrast, inhibition of factor XIa cleavage of S2366 by factor IX (Ki 224 +/- 32 nM) was characterized by hyperbolic mixed-type inhibition, indicating that factor IX binds to free and S2366-bound factor XIa at exosites. Consistent with this premise, inhibition of factor XIa activation of factor IX by aprotinin (Ki 0.89 +/- 0.52 microM) was non-competitive, whereas inhibition by active site-inhibited factor IXa beta was competitive (Ki 0.33 +/- 0.05 microM). S2366 cleavage by isolated factor XIa catalytic domain was competitively inhibited by p-aminobenzamidine (Ki 38 +/- 14 microM) but was not inhibited by factor IX, consistent with loss of factor IX-binding exosites on the non-catalytic factor XI heavy chain. The results support a model in which factor IX binds initially to exosites on the factor XIa heavy chain, followed by interaction at the active site with subsequent bond cleavage, and support a growing body of evidence that exosite interactions are critical determinants of substrate affinity and specificity in blood coagulation reactions. PMID:15829482

  6. Transforming growth factor β-mediated suppression of antitumor T cells requires FoxP1 transcription factor expression.

    PubMed

    Stephen, Tom L; Rutkowski, Melanie R; Allegrezza, Michael J; Perales-Puchalt, Alfredo; Tesone, Amelia J; Svoronos, Nikolaos; Nguyen, Jenny M; Sarmin, Fahmida; Borowsky, Mark E; Tchou, Julia; Conejo-Garcia, Jose R

    2014-09-18

    Tumor-reactive T cells become unresponsive in advanced tumors. Here we have characterized a common mechanism of T cell unresponsiveness in cancer driven by the upregulation of the transcription factor Forkhead box protein P1 (Foxp1), which prevents CD8⁺ T cells from proliferating and upregulating Granzyme-B and interferon-γ in response to tumor antigens. Accordingly, Foxp1-deficient lymphocytes induced rejection of incurable tumors and promoted protection against tumor rechallenge. Mechanistically, Foxp1 interacted with the transcription factors Smad2 and Smad3 in preactivated CD8⁺ T cells in response to microenvironmental transforming growth factor-β (TGF-β), and was essential for its suppressive activity. Therefore, Smad2 and Smad3-mediated c-Myc repression requires Foxp1 expression in T cells. Furthermore, Foxp1 directly mediated TGF-β-induced c-Jun transcriptional repression, which abrogated T cell activity. Our results unveil a fundamental mechanism of T cell unresponsiveness different from anergy or exhaustion, driven by TGF-β signaling on tumor-associated lymphocytes undergoing Foxp1-dependent transcriptional regulation.

  7. TRANSFORMING GROWTH FACTOR-BETA MEDIATED SUPPRESSION OF ANTI-TUMOR T CELLS REQUIRES FOXP1 TRANSCRIPTION FACTOR EXPRESSION

    PubMed Central

    Stephen, Tom L.; Rutkowski, Melanie R.; Allegrezza, Michael J.; Perales-Puchalt, Alfredo; Tesone, Amelia J.; Svoronos, Nikolaos; Nguyen, Jenny M.; Sarmin, Fahmida; Borowsky, Mark E.; Tchou, Julia; Conejo-Garcia, Jose R.

    2014-01-01

    SUMMARY Tumor-reactive T cells become unresponsive in advanced tumors. Here we have characterized a common mechanism of T cell unresponsiveness in cancer driven by the up-regulation of the transcription factor Forkhead box protein P1 (Foxp1), which prevents CD8+ T cells from proliferating and up-regulating Granzyme-B and interferon-γ (IFN-γ) in response to tumor antigens. Accordingly, Foxp1-deficient lymphocytes induced rejection of incurable tumors, and promoted protection against tumor re-challenge. Mechanistically, Foxp1 interacted with the transcription factors Smad2 and Smad3 in pre-activated CD8+ T cells in response to microenvironmental transforming growth factor-β (TGF-β), and was essential for its suppressive activity. Therefore, Smad2 and Smad3-mediated c-Myc repression requires Foxp1 expression in T cells. Furthermore, Foxp1 directly mediated TGF-β-induced c-Jun transcriptional repression, which abrogated T cell activity. Our results unveil a fundamental mechanism of T cell unresponsiveness different from anergy or exhaustion, driven by TGF-β signaling on tumor-associated lymphocytes undergoing Foxp1-dependent transcriptional regulation. PMID:25238097

  8. 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.

  9. OGFOD1, a novel modulator of eukaryotic translation initiation factor 2alpha phosphorylation and the cellular response to stress.

    PubMed

    Wehner, Karen A; Schütz, Sylvia; Sarnow, Peter

    2010-04-01

    Cells possess mechanisms that permit survival and recovery from stress, several of which regulate the phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha). We identified the human OGFOD1 protein as a novel stress granule component that regulates the phosphorylation of eIF2alpha and the resumption of translation in cells recovering from arsenite-induced stress. Coimmunoprecipitation studies revealed that OGFOD1 associates with a small subset of stress granule proteins (G3BP1, USP10, Caprin1, and YB-1) and the ribosome in both unstressed and stressed cells. Overexpression of OGFOD1 led to increased abundance of phosphorylated eIF2alpha, both in unstressed cells and in cells exposed to arsenite-induced stress, and to accelerated apoptosis during stress. Conversely, knockdown of OGFOD1 resulted in smaller amounts of phosphorylated eIF2alpha and a faster accumulation of polyribosomes in cells recovering from stress. Finally, OGFOD1 interacted with both eIF2alpha and the eIF2alpha kinase heme-regulated inhibitor (HRI), which was identified as a novel stress granule resident. These findings argue that OGFOD1 plays important proapoptotic roles in the regulation of translation and HRI-mediated phosphorylation of eIF2alpha in cells subjected to arsenite-induced stress.

  10. Requirement of the eukaryotic translation initiation factor 4F complex in hepatitis E virus replication.

    PubMed

    Zhou, Xinying; Xu, Lei; Wang, Yijin; Wang, Wenshi; Sprengers, Dave; Metselaar, Herold J; Peppelenbosch, Maikel P; Pan, Qiuwei

    2015-12-01

    Hepatitis E virus (HEV) infection, one of the foremost causes of acute hepatitis, is becoming a health problem of increasing magnitude. As other viruses, HEV exploits elements from host cell biochemistry, but we understand little as to which components of the human hepatocellular machinery are perverted for HEV multiplication. It is, however, known that the eukaryotic translation initiation factors 4F (eIF4F) complex, the key regulator of the mRNA-ribosome recruitment phase of translation initiation, serves as an important component for the translation and replication of many viruses. Here we aim to investigate the role of three subunits of the eIF4F complex: eukaryotic translation initiation factor 4A (eIF4A), eukaryotic translation initiation factor 4G (eIF4G) and eukaryotic translation initiation factor 4E (eIF4E) in HEV replication. We found that efficient replication of HEV requires eIF4A, eIF4G and eIF4E. Consistently, the negative regulatory factors of this complex: programmed cell death 4 (PDCD4) and eIF4E-binding protein 1 (4E-BP1) exert anti-HEV activities, which further illustrates the requirement for eIF4A and eIF4E in supporting HEV replication. Notably, phosphorylation of eIF4E induced by MNK1/2 activation is not involved in HEV replication. Although ribavirin and interferon-α (IFN-α), the most often-used off-label drugs for treating hepatitis E, interact with this complex, their antiviral activities are independent of eIF4E. In contrast, eIF4E silencing provokes enhanced anti-HEV activity of these compounds. Thus, HEV replication requires eIF4F complex and targeting essential elements of this complex provides important clues for the development of novel antiviral therapy against HEV.

  11. Plasmin-dependent elimination of the growth-factor-like domain in urokinase causes its rapid cellular uptake and degradation.

    PubMed Central

    Poliakov, A; Tkachuk, V; Ovchinnikova, T; Potapenko, N; Bagryantsev, S; Stepanova, V

    2001-01-01

    Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) act in concert to mediate pericellular proteolysis and to stimulate intracellular signalling responsible for cell migration and proliferation. uPA is composed of three domains, a proteolytic domain (PD), a kringle domain (KD) and a growth-factor-like domain (GFD), the last of which mediates the interaction with uPAR. We demonstrate that uPA, associated with the surface of U937 cells, undergoes plasmin-mediated cleavage of the Lys(46)-Ser(47) bond with elimination of the GFD. Using recombinant forms of uPA, we show that a uPA variant lacking the GFD (r-uPADeltaGFD) and unable to associate with uPAR is rapidly cleared from the cell surface. Binding and internalization of r-uPADeltaGFD are markedly decreased in the presence of 39 kDa receptor-associated protein (RAP), the antagonist of several endocytic receptors of the low-density lipoprotein receptor family, suggesting that this protein clearance pathway is used for r-uPADeltaGFD. In contrast with rapidly internalized r-uPADeltaGFD, the intact recombinant single-chain urokinase with wild-type structure (r-uPAwt) bound to uPAR is retained on the cell surface. Soluble uPAR protects uPA from cleavage by plasmin that results in the elimination of GFD, suggesting that uPAR might protect cell-bound urokinase from plasmin-mediated cleavage between the GFD and KD and subsequent degradation. PMID:11311125

  12. Regulation of gene expression by NFAT transcription factors in hibernating ground squirrels is dependent on the cellular environment.

    PubMed

    Zhang, Yichi; Storey, Kenneth B

    2016-09-01

    Calcineurin is a calmodulin-stimulated phosphatase that regulates the nuclear translocation of nuclear factor of activated T cell (NFAT) c1-4 through dephosphorylation. We believe that this mechanism plays various roles in the remodeling and maintenance of Ictidomys tridecemlineatus skeletal muscle. During hibernation, bouts of torpor and arousal take place, and squirrels do not lose muscle mass despite being inactive. Protein expression of Ca(2+) signaling proteins were studied using immunoblotting. A DNA-protein interaction ELISA technique was created to test the binding of NFATs in the nucleus to DNA probes containing the NFAT response element under environmental conditions reflective of those during hibernation. Calcineurin protein levels increased by 3.08-fold during torpor (compared to euthermic control), whereas calpain1 levels also rose by 3.66-fold during torpor. Calmodulin levels were elevated upon entering torpor. NFATc4 binding to DNA showed a 1.4-fold increase during torpor, and we found that this binding was further enhanced when 600 nM of Ca(2+) was supplemented. We also found that decreasing the temperature of ELISAs resulted in progressive decreases in the binding of NFATs c1, c3, and c4 to DNA. In summary, calmodulin and calpain1 appear to activate calcineurin and NFATc4 during torpor. NFAT binding to target promoters is affected by intranuclear [Ca(2+)] and environmental temperatures. Therefore, Ca(2+) signaling and temperature changes play key roles in regulation of the NFAT-calcineurin pathway in skeletal muscle of hibernating 13-lined ground squirrels over the torpor-arousal cycle, and they may contribute to the avoidance of disuse-induced muscle atrophy that occurs naturally in these animals.

  13. Regulation of gene expression by NFAT transcription factors in hibernating ground squirrels is dependent on the cellular environment.

    PubMed

    Zhang, Yichi; Storey, Kenneth B

    2016-09-01

    Calcineurin is a calmodulin-stimulated phosphatase that regulates the nuclear translocation of nuclear factor of activated T cell (NFAT) c1-4 through dephosphorylation. We believe that this mechanism plays various roles in the remodeling and maintenance of Ictidomys tridecemlineatus skeletal muscle. During hibernation, bouts of torpor and arousal take place, and squirrels do not lose muscle mass despite being inactive. Protein expression of Ca(2+) signaling proteins were studied using immunoblotting. A DNA-protein interaction ELISA technique was created to test the binding of NFATs in the nucleus to DNA probes containing the NFAT response element under environmental conditions reflective of those during hibernation. Calcineurin protein levels increased by 3.08-fold during torpor (compared to euthermic control), whereas calpain1 levels also rose by 3.66-fold during torpor. Calmodulin levels were elevated upon entering torpor. NFATc4 binding to DNA showed a 1.4-fold increase during torpor, and we found that this binding was further enhanced when 600 nM of Ca(2+) was supplemented. We also found that decreasing the temperature of ELISAs resulted in progressive decreases in the binding of NFATs c1, c3, and c4 to DNA. In summary, calmodulin and calpain1 appear to activate calcineurin and NFATc4 during torpor. NFAT binding to target promoters is affected by intranuclear [Ca(2+)] and environmental temperatures. Therefore, Ca(2+) signaling and temperature changes play key roles in regulation of the NFAT-calcineurin pathway in skeletal muscle of hibernating 13-lined ground squirrels over the torpor-arousal cycle, and they may contribute to the avoidance of disuse-induced muscle atrophy that occurs naturally in these animals. PMID:27344571

  14. 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.

  15. 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

  16. miR-28-3p is a cellular restriction factor that inhibits human T cell leukemia virus, type 1 (HTLV-1) replication and virus infection.

    PubMed

    Bai, Xue Tao; Nicot, Christophe

    2015-02-27

    Human T cell leukemia virus, type 1 (HTLV-1) replication and spread are controlled by different viral and cellular factors. Although several anti-HIV cellular microRNAs have been described, such a regulation for HTLV-1 has not been reported. In this study, we found that miR-28-3p inhibits HTLV-1 virus expression and its replication by targeting a specific site within the genomic gag/pol viral mRNA. Because miR-28-3p is highly expressed in resting T cells, which are resistant to HTLV-1 infection, we investigated a potential protective role of miR-28-3p against de novo HTLV-1 infection. To this end, we developed a new sensitive and quantitative assay on the basis of the detection of products of reverse transcription. We demonstrate that miR-28-3p does not prevent virus receptor interaction or virus entry but, instead, induces a post-entry block at the reverse transcription level. In addition, we found that HTLV-1, subtype 1A isolates corresponding to the Japanese strain ATK-1 present a natural, single-nucleotide polymorphism within the miR-28-3p target site. As a result of this polymorphism, the ATK-1 virus sequence was not inhibited by miR-28. Interestingly, genetic studies on the transmission of the virus has shown that the ATK-1 strain, which carries a Thr-to-Cys transition mutation, is transmitted efficiently between spouses, suggesting that miR-28 may play an important role in HTLV-1 transmission. PMID:25568327

  17. Cellular Uptake and Cytotoxic Effect of Epidermal Growth Factor Receptor Targeted and Plitidepsin Loaded Co-Polymeric Polymersomes on Colorectal Cancer Cell Lines.

    PubMed

    Goñi-de-Cerio, Felipe; Thevenot, Julie; Oliveira, Hugo; Pérez-Andrés, Encarnación; Berra, Edurne; Masa, Marc; Suárez-Merino, Blanca; Lecommandoux, Sébastien; Heredia, Pedro

    2015-11-01

    Encapsulating chemotherapy drugs in targeted nanodelivery systems is one of the most promising approaches to tackle cancer disease, avoiding side effects of common treatment. In the last decade, several nanocarriers with different nature have been tested, but polypeptide-based copolymers have attracted considerable attention for their biocompatibility, controlled and slow biodegradability as well as their low toxicity. In this work, we synthesized, characterized and evaluated poly(trimethylene carbonate)-bock-poly(L-glutamic acid) derived polymersomes, targeted to epidermal growth factor receptor (EGFR), loaded with plitidepsin and ultimately tested in HT29 and LS174T colorectal cancer cell lines for specificity and efficacy. Furthermore, morphology, physico-chemical properties and plitidepsin loading were carefully investigated. A thorough in vitro cytotoxicity analysis of the unloaded polymersomes was carried out for biocompatibility check, studying viability, cell membrane asymmetry and reactive oxygen species levels. Those cytotoxicity assays showed good biocompatibility for plitidepsin-unloaded polymersomes. Cellular uptake and cytotoxic effect of EGFR targeted and plitidepsin loaded polymersome indicated that colorectal cancer cell lines were.more sensitive to anti-EGFR-drug-loaded than untargeted drug-loaded polymersomes. Also, in both cell lines, the use of untargeted polymersomes greatly reduced plitidepsin cytotoxicity as well as the cellular uptake, indicating that the use of this targeted nanocarrier is a promising approach to tackle colorectal cancer disease and avoid the undesired effects of the usual treatment. Furthermore, in vivo assays support the in vitro conclusions that EGFR targeted polymersomes could be a good drug delivery system. This work provides a proof of concept for the use of encapsulated targeted drugs as future therapeutic treatments for cancer.

  18. miR-28-3p is a cellular restriction factor that inhibits human T cell leukemia virus, type 1 (HTLV-1) replication and virus infection.

    PubMed

    Bai, Xue Tao; Nicot, Christophe

    2015-02-27

    Human T cell leukemia virus, type 1 (HTLV-1) replication and spread are controlled by different viral and cellular factors. Although several anti-HIV cellular microRNAs have been described, such a regulation for HTLV-1 has not been reported. In this study, we found that miR-28-3p inhibits HTLV-1 virus expression and its replication by targeting a specific site within the genomic gag/pol viral mRNA. Because miR-28-3p is highly expressed in resting T cells, which are resistant to HTLV-1 infection, we investigated a potential protective role of miR-28-3p against de novo HTLV-1 infection. To this end, we developed a new sensitive and quantitative assay on the basis of the detection of products of reverse transcription. We demonstrate that miR-28-3p does not prevent virus receptor interaction or virus entry but, instead, induces a post-entry block at the reverse transcription level. In addition, we found that HTLV-1, subtype 1A isolates corresponding to the Japanese strain ATK-1 present a natural, single-nucleotide polymorphism within the miR-28-3p target site. As a result of this polymorphism, the ATK-1 virus sequence was not inhibited by miR-28. Interestingly, genetic studies on the transmission of the virus has shown that the ATK-1 strain, which carries a Thr-to-Cys transition mutation, is transmitted efficiently between spouses, suggesting that miR-28 may play an important role in HTLV-1 transmission.

  19. A genome-wide genetic screen for host factors required for hepatitis C virus propagation

    PubMed Central

    Li, Qisheng; Brass, Abraham L.; Ng, Aylwin; Hu, Zongyi; Xavier, Ramnik J.; Liang, T. Jake; Elledge, Stephen J.

    2009-01-01

    Hepatitis C virus (HCV) infection is a major cause of end-stage liver disease and a leading indication for liver transplantation. Current therapy fails in many instances and is associated with significant side effects. HCV encodes only a few proteins and depends heavily on host factors for propagation. Each of these host dependencies is a potential therapeutic target. To find host factors required by HCV, we completed a genome-wide small interfering RNA (siRNA) screen using an infectious HCV cell culture system. We applied a two-part screening protocol to allow identification of host factors involved in the complete viral lifecycle. The candidate genes found included known or previously identified factors, and also implicate many additional host cell proteins in HCV infection. To create a more comprehensive view of HCV and host cell interactions, we performed a bioinformatic meta-analysis that integrates our data with those of previous functional and proteomic studies. The identification of host factors participating in the complete HCV lifecycle will both advance our understanding of HCV pathogenesis and illuminate therapeutic targets. PMID:19717417

  20. 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

  1. Cellular inhibitor of apoptosis protein-1 (cIAP1) can regulate E2F1 transcription factor-mediated control of cyclin transcription.

    PubMed

    Cartier, Jessy; Berthelet, Jean; Marivin, Arthur; Gemble, Simon; Edmond, Valérie; Plenchette, Stéphanie; Lagrange, Brice; Hammann, Arlette; Dupoux, Alban; Delva, Laurent; Eymin, Béatrice; Solary, Eric; Dubrez, Laurence

    2011-07-29

    The inhibitor of apoptosis protein cIAP1 (cellular inhibitor of apoptosis protein-1) is a potent regulator of the tumor necrosis factor (TNF) receptor family and NF-κB signaling pathways in the cytoplasm. However, in some primary cells and tumor cell lines, cIAP1 is expressed in the nucleus, and its nuclear function remains poorly understood. Here, we show that the N-terminal part of cIAP1 directly interacts with the DNA binding domain of the E2F1 transcription factor. cIAP1 dramatically increases the transcriptional activity of E2F1 on synthetic and CCNE promoters. This function is not conserved for cIAP2 and XIAP, which are cytoplasmic proteins. Chromatin immunoprecipitation experiments demonstrate that cIAP1 is recruited on E2F binding sites of the CCNE and CCNA promoters in a cell cycle- and differentiation-dependent manner. cIAP1 silencing inhibits E2F1 DNA binding and E2F1-mediated transcriptional activation of the CCNE gene. In cells that express a nuclear cIAP1 such as HeLa, THP1 cells and primary human mammary epithelial cells, down-regulation of cIAP1 inhibits cyclin E and A expression and cell proliferation. We conclude that one of the functions of cIAP1 when localized in the nucleus is to regulate E2F1 transcriptional activity.

  2. Molecular cloning of cellular genes encoding retinoblastoma-associated proteins: identification of a gene with properties of the transcription factor E2F.

    PubMed Central

    Shan, B; Zhu, X; Chen, P L; Durfee, T; Yang, Y; Sharp, D; Lee, W H

    1992-01-01

    The retinoblastoma protein interacts with a number of cellular proteins to form complexes which are probably crucial for its normal physiological function. To identify these proteins, we isolated nine distinct clones by direct screening of cDNA expression libraries using purified RB protein as a probe. One of these clones, Ap12, is expressed predominantly at the G1-S boundary and in the S phase of the cell cycle. The nucleotide sequence of Ap12 has features characteristic of transcription factors. The C-terminal region binds to unphosphorylated RB in regions similar to those to which T antigen binds and contains a transactivation domain. A region containing a potential leucine zipper flanked by basic residues is able to bind an E2F recognition sequence specifically. Expression of Ap12 in mammalian cells significantly enhances E2F-dependent transcriptional activity. These results suggest that Ap12 encodes a protein with properties known to be characteristic of transcription factor E2F. Images PMID:1448092

  3. 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.

  4. Human Immunodeficiency Virus Type 1 Tat Protein Activates Transcription Factor NF-κB through the Cellular Interferon-Inducible, Double-Stranded RNA-Dependent Protein Kinase, PKR

    PubMed Central

    Demarchi, Francesca; Gutierrez, Maria Ines; Giacca, Mauro

    1999-01-01

    The transactivator protein of human immunodeficiency virus type 1 (HIV-1) (Tat) is a powerful activator of nuclear factor-κB (NF-κB), acting through degradation of the inhibitor IκB-α (F. Demarchi, F. d’Adda di Fagagna, A. Falaschi, and M. Giacca, J. Virol. 70:4427–4437, 1996). Here, we show that this activity of Tat requires the function of the cellular interferon-inducible protein kinase PKR. Tat-mediated NF-κB activation and transcriptional induction of the HIV-1 long terminal repeat were impaired in murine cells in which the PKR gene was knocked out. Both functions were restored by cotransfection of Tat with the cDNA for PKR. Expression of a dominant-negative mutant of PKR specifically reduced the levels of Tat transactivation in different human cell types. Activation of NF-κB by Tat required integrity of the basic domain of Tat; previous studies have indicated that this domain is necessary for specific Tat-PKR interaction. PMID:10400814

  5. Bone morphogenetic protein 15 and growth differentiation factor 9 expression in the ovary of European sea bass (Dicentrarchus labrax): cellular localization, developmental profiles, and response to unilateral ovariectomy.

    PubMed

    García-López, Ángel; Sánchez-Amaya, María Isabel; Halm, Silke; Astola, Antonio; Prat, Francisco

    2011-12-01

    Vertebrate oocytes actively contribute to follicle development by secreting a variety of growth factors, among which bone morphogenetic protein 15 (BMP15/Bmp15) and growth differentiation factor 9 (GDF9/Gdf9) have been paid particular attention. In the present study, we describe the cellular localization, the developmental profiles, and the response to unilateral ovariectomy (a procedure implying the surgical removal of one of the ovaries) of protein and mRNA steady-state levels of Bmp15 and Gdf9 in the ovary of European sea bass, an important fish species for marine aquaculture industry. In situ hybridization and immunohistochemistry demonstrated that the oocyte is the main production site of Bmp15 and Gdf9 in European sea bass ovary. During oocyte development, Bmp15 protein expression started to be detected only from the lipid vesicle stage onwards but not in primary pre-vitellogenic (i.e. perinucleolar) oocytes as the bmp15 mRNA already did. Gdf9 protein and gdf9 mRNA expression were both detected in primary perinucleolar oocytes and followed similar decreasing patterns thereafter. Unilateral ovariectomy induced a full compensatory growth of the remaining ovary in the 2-month period following surgery (Á. García-López, M.I. Sánchez-Amaya, C.R. Tyler, F. Prat 2011). The compensatory growth elicited different changes in the expression levels of mRNA and protein of both factors, although the involvement of Bmp15 and Gdf9 in the regulatory network orchestrating such process remains unclear at present. Altogether, our results establish a solid base for further studies focused on elucidating the specific functions of Bmp15 and Gdf9 during primary and secondary oocyte growth in European sea bass.

  6. Human factors requirements for telerobotic command and control: The European Space Agency experimental programme

    NASA Technical Reports Server (NTRS)

    Stone, Robert J.

    1991-01-01

    Space Telerobotics research, performed under contract to the European Space Agency (ESA), concerning the execution of human factors experiments, and ultimately leading to the development of a telerobotics test bed, has been carried out since 1985 by a British Consortium consisting of British Aerospace, the United Kingdom Atomic Energy Authority and, more recently, the UK National Advanced Robotics Research Centre. The principal aim of the first study of the series was to derive preliminary requirements for a teleoperation servicing system, with reference to two mission model scenarios. The first scenario introduced the problem of communications time delays, and their likely effect on the ground-based operator in control of a manipulator system on board an unmanned servicing vehicle in Low Earth Orbit. In the second scenario, the operator was located on the NASA Orbiter aft flight deck, supervising the control of a prototype manipulator in the 'servicing' of an experimental payload in the cargo bay area. Human factors analyses centered on defining the requirements for the teleoperator workstation, such as identifying basic ergonomic requirements for workstation and panel layouts, defining teleoperation strategies, developing alphanumeric and graphic screen formats for the supervision or direct control of the manipulator, and the potential applications of expert system technology. The second study for ESA involved an experimental appraisal of some of the important issues highlighted in the first study, for which relevant human factors data did not exist. Of central importance during the second study was the issue of communications time delays and their effect on the manual control of a teleoperated manipulator from a ground-based command and control station.

  7. Requirement of nucleotide exchange factor for Ypt1 GTPase mediated protein transport.

    PubMed

    Jones, S; Litt, R J; Richardson, C J; Segev, N

    1995-09-01

    Small GTPases of the rab family are involved in the regulation of vesicular transport. It is believed that cycling between the GTP- and GDP-bound forms, and accessory factors regulating this cycling are crucial for rab function. However, an essential role for rab nucleotide exchange factors has not yet been demonstrated. In this report we show the requirement of nucleotide exchange factor activity for Ypt1 GTPase mediated protein transport. The Ypt1 protein, a member of the rab family, plays a role in targeting vesicles to the acceptor compartment and is essential for the first two steps of the yeast secretory pathway. We use two YPT1 dominant mutations that contain alterations in a highly conserved GTP-binding domain, N121I and D124N. YPT1-D124N is a novel mutation that encodes a protein with nucleotide specificity modified from guanine to xanthine. This provides a tool for the study of an individual rab GTPase in crude extracts: a xanthosine triphosphate (XTP)-dependent conditional dominant mutation. Both mutations confer growth inhibition and a block in protein secretion when expressed in vivo. The purified mutant proteins do not bind either GDP or GTP. Moreover, they completely inhibit the ability of the exchange factor to stimulate nucleotide exchange for wild type Ypt1 protein, and are potent inhibitors of ER to Golgi transport in vitro at the vesicle targeting step. The inhibitory effects of the Ypt1-D124N mutant protein on both nucleotide exchange activity and protein transport in vitro can be relieved by XTP, indicating that it is the nucleotide-free form of the mutant protein that is inhibitory. These results suggest that the dominant mutant proteins inhibit protein transport by sequestering the exchange factor from the wild type Ypt1 protein, and that this factor has an essential role in vesicular transport.

  8. Neuronal migration in the murine rostral migratory stream requires serum response factor

    PubMed Central

    Alberti, Siegfried; Krause, Sven M.; Kretz, Oliver; Philippar, Ulrike; Lemberger, Thomas; Casanova, Emilio; Wiebel, Franziska F.; Schwarz, Heinz; Frotscher, Michael; Schütz, Günther; Nordheim, Alfred

    2005-01-01

    The central nervous system is fundamentally dependent on guided cell migration, both during development and in adulthood. We report an absolute requirement of the transcription factor serum response factor (SRF) for neuronal migration in the mouse forebrain. Conditional, late-prenatal deletion of Srf causes neurons to accumulate ectopically at the subventricular zone (SVZ), a prime neurogenic region in the brain. SRF-deficient cells of the SVZ exhibit impaired tangential chain migration along the rostral migratory stream into the olfactory bulb. SVZ explants display retarded chain migration in vitro. Regarding target genes, SRF deficiency impairs expression of the β-actin and gelsolin genes, accompanied by reduced cytoskeletal actin fiber density. At the posttranslational level, cofilin, a key regulator of actin dynamics, displays dramatically elevated inhibitory phosphorylation at Ser-3. Our studies indicate that SRF-controlled gene expression directs both the structure and dynamics of the actin microfilament, thereby determining cell-autonomous neuronal migration. PMID:15837932

  9. Differential requirements for Myocyte Enhancer Factor-2 during adult myogenesis in Drosophila

    PubMed Central

    Bryantsev, Anton L.; Baker, Phillip W.; Lovato, TyAnna L.; Jaramillo, MaryAnn S.; Cripps, Richard M.

    2011-01-01

    SUMMARY Identifying the genetic program that leads to formation of functionally and morphologically distinct muscle fibers is one of the major challenges in developmental biology. In Drosophila, the Myocyte Enhancer Factor-2 (MEF2) transcription factor is important for all types of embryonic muscle differentiation. In this study we investigated the role of MEF2 at different stages of adult skeletal muscle formation, where a diverse group of specialized muscles arises. Through stage- and tissue- specific expression of Mef2 RNAi constructs, we demonstrate that MEF2 is critical at the early stages of adult myoblast fusion: mutant myoblasts are attracted normally to their founder cell targets, but are unable to fuse to form myotubes. Interestingly, ablation of Mef2 expression at later stages of development showed MEF2 to be more dispensable for structural gene expression: after myoblast fusion, Mef2 knockdown did not interrupt expression of major structural gene transcripts, and myofibrils were formed. However, the MEF2-depleted fibers showed impaired integrity and a lack of fibrillar organization. When Mef2 RNAi was induced in muscles following eclosion, we found no adverse effects of attenuating Mef2 function. We conclude that in the context of adult myogenesis, MEF2 remains an essential factor, participating in control of myoblast fusion, and myofibrillogenesis in developing myotubes. However, MEF2 does not show a major requirement in the maintenance of muscle structural gene expression. Our findings point to the importance of a diversity of regulatory factors that are required for the formation and function of the distinct muscle fibers found in animals. PMID:22008792

  10. N-Ethylmaleimide–Sensitive Factor b (nsfb) Is Required for Normal Pigmentation of the Zebrafish Retinal Pigment Epithelium

    PubMed Central

    Hanovice, Nicholas J.; Daly, Christina M. S.; Gross, Jeffrey M.

    2015-01-01

    Purpose Despite the number of albinism-causing mutations identified in human patients and animal models, there remain a significant number of cases for which no mutation has been identified, suggesting that our understanding of melanogenesis is incomplete. Previously, we identified two oculocutaneous albinism mutations in zebrafish, au13 and au18. Here, we sought to identify the mutated loci and determine how the affected proteins contribute to normal pigmentation of the retinal pigment epithelium (RPE). Methods Complementation analyses revealed that au13 and au18 belonged to a single complementation group, suggesting that they affected the same locus. Whole-genome sequencing and single nucleotide polymorphism (SNP) analysis was performed to identify putative mutations, which were confirmed by cDNA sequencing and mRNA rescue. Transmission electron microscopy (TEM) and image quantification were used to identify the cellular basis of hypopigmentation. Results Whole-genome sequencing and SNP mapping identified a nonsense mutation in the N-ethylmaleimide–sensitive factor b (nsfb) gene in au18 mutants. Complementary DNA sequencing confirmed the presence of the mutation (C893T), which truncates the nsfb protein by roughly two-thirds (Y297X). No coding sequence mutations were identified in au13, but quantitative PCR revealed a significant decrease in nsfb expression, and nsfb mRNA injection rescued the hypopigmentation phenotype, suggesting a regulatory mutation. In situ hybridization revealed that nsfb is broadly expressed during embryonic development, including in the RPE. Transmission electron microscopy analyses indicated that average melanosome density and maturity were significantly decreased in nsfb mutants. Conclusions au18 and au13 contain mutations in nsfb, which encodes a protein that is required for the maturation of melanosomes in zebrafish RPE. PMID:26618645

  11. Zic2 is an enhancer-binding factor required for embryonic stem cell specification

    PubMed Central

    Luo, Zhuojuan; Gao, Xin; Lin, Chengqi; Smith, Edwin; Marshall, Stacy; Swanson, Selene K.; Florens, Laurence; Washburn, Michael P.; Shilatifard, Ali

    2016-01-01

    SUMMARY The Zinc finger protein of the cerebellum 2 (Zic2) is one of the vertebrate homologs of the Drosophila pair-rule gene odd-paired (opa). Our molecular and biochemical studies demonstrate that Zic2 preferentially binds to transcriptional enhancers and is required for the regulation of gene expression in embryonic stem cells. Detailed genome-wide and molecular studies reveal that Zic2 can function with Mbd3/NuRD in regulating the chromatin state and transcriptional output of genes linked to differentiation. Zic2 is required for proper differentiation of ES cells, similar to what has been previously reported for Mbd3/NuRD. Our study identifies Zic2 as a key factor in the execution of transcriptional fine-tuning with Mbd3/NuRD in ES cells through interactions with enhancers. Our study also points to the role of the Zic family of proteins as enhancer-specific binding factors functioning in development. PMID:25699711

  12. Myoferlin is required for insulin-like growth factor response and muscle growth.

    PubMed

    Demonbreun, Alexis R; Posey, Avery D; Heretis, Konstantina; Swaggart, Kayleigh A; Earley, Judy U; Pytel, Peter; McNally, Elizabeth M

    2010-04-01

    Insulin-like growth factor (IGF) is a potent stimulus of muscle growth. Myoferlin is a membrane-associated protein important for muscle development and regeneration. Myoferlin-null mice have smaller muscles and defective myoblast fusion. To understand the mechanism by which myoferlin loss retards muscle growth, we found that myoferlin-null muscle does not respond to IGF1. In vivo after IGF1 infusion, control muscle increased myofiber diameter by 25%, but myoferlin-null muscle was unresponsive. Myoblasts cultured from myoferlin-null muscle and treated with IGF1 also failed to show the expected increase in fusion to multinucleate myotubes. The IGF1 receptor colocalized with myoferlin at sites of myoblast fusion. The lack of IGF1 responsiveness in myoferlin-null myoblasts was linked directly to IGF1 receptor mistrafficking as well as decreased IGF1 signaling. In myoferlin-null myoblasts, the IGF1 receptor accumulated into large vesicular structures. These vesicles colocalized with a marker of late endosomes/lysosomes, LAMP2, specifying redirection from a recycling to a degradative pathway. Furthermore, ultrastructural analysis showed a marked increase in vacuoles in myoferlin-null muscle. These data demonstrate that IGF1 receptor recycling is required for normal myogenesis and that myoferlin is a critical mediator of postnatal muscle growth mediated by IGF1.-Demonbreun, A. R., Posey, A. D., Heretis, K., Swaggart, K. A., Earley, J. U., Pytel, P., McNally, E. M. Myoferlin is required for insulin-like growth factor response and muscle growth.

  13. Nuclear factor-kappa B directs carcinoembryonic antigen-related cellular adhesion molecule 1 receptor expression in Neisseria gonorrhoeae-infected epithelial cells.

    PubMed

    Muenzner, Petra; Billker, Oliver; Meyer, Thomas F; Naumann, Michael

    2002-03-01

    The human-specific pathogen Neisseria gonorrhoeae expresses opacity-associated (Opa) protein adhesins that bind to various members of the carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) family. In this study, we have analyzed the mechanism underlying N. gonorrhoeae-induced CEACAM up-regulation in epithelial cells. Epithelial cells represent the first barrier for the microbial pathogen. We therefore characterized CEACAM expression in primary human ovarian surface epithelial (HOSE) cells and found that CEACAM1-3 (L, S) and CEACAM1-4 (L, S) splice variants mediate an increased Opa(52)-dependent gonoccocal binding to HOSE cells. Up-regulation of these CEACAM molecules in HOSE cells is a direct process that takes place within 2 h postinfection and depends on close contact between microbial pathogen and HOSE cells. N. gonorrhoeae-triggered CEACAM1 up-regulation involves activation of the transcription factor nuclear factor kappaB (NF-kappaB), which translocates as a p50/p65 heterodimer into the nucleus, and an NF-kappaB-specific inhibitory peptide inhibited CEACAM1-receptor up-regulation in N. gonorrhoeae-infected HOSE cells. Bacterial lipopolysaccharides did not induce NF-kappaB and CEACAM up-regulation, which corresponds to our findings that HOSE cells do not express toll-like receptor 4. The ability of N. gonorrhoeae to up-regulate its epithelial receptor CEACAM1 through NF-kappaB suggests an important mechanism allowing efficient bacterial colonization during the initial infection process. PMID:11751883

  14. Cellular and Tumor Radiosensitivity is Correlated to Epidermal Growth Factor Receptor Protein Expression Level in Tumors Without EGFR Amplification;Epidermal growth factor receptor; Radiotherapy; Squamous cell carcinoma; Biomarker; Local tumor control

    SciTech Connect

    Kasten-Pisula, Ulla; Saker, Jarob; Eicheler, Wolfgang; Krause, Mechthild; Yaromina, Ala; Meyer-Staeckling, Soenke; Scherkl, Benjamin; Kriegs, Malte; Brandt, Burkhard; Grenman, Reidar; Petersen, Cordula; Baumann, Michael; Dikomey, Ekkehard

    2011-07-15

    Purpose: There is conflicting evidence for whether the expression of epidermal growth factor receptor in human tumors can be used as a marker of radioresponse. Therefore, this association was studied in a systematic manner using squamous cell carcinoma (SCC) cell lines grown as cell cultures and xenografts. Methods and Materials: The study was performed with 24 tumor cell lines of different tumor types, including 10 SCC lines, which were also investigated as xenografts on nude mice. Egfr gene dose and the length of CA-repeats in intron 1 were determined by polymerase chain reaction, protein expression in vitro by Western blot and in vivo by enzyme-linked immunosorbent assay, and radiosensitivity in vitro by colony formation. Data were correlated with previously published tumor control dose 50% data after fractionated irradiation of xenografts of the 10 SCC. Results: EGFR protein expression varies considerably, with most tumor cell lines showing moderate and only few showing pronounced upregulation. EGFR upregulation could only be attributed to massive gene amplification in the latter. In the case of little or no amplification, in vitro EGFR expression correlated with both cellular and tumor radioresponse. In vivo EGFR expression did not show this correlation. Conclusions: Local tumor control after the fractionated irradiation of tumors with little or no gene amplification seems to be dependent on in vitro EGFR via its effect on cellular radiosensitivity.

  15. Risk Factors for Refractory and Delayed De novo Otitis Media Requiring Pressure Equalization Tube Insertion

    PubMed Central

    Bowe, Sarah N.; Jatana, Kris R.; Kang, D. Richard

    2016-01-01

    Objective Limited data exists regarding risk factors for otitis media in older children and specifically those for which surgical intervention is performed. This study investigated potential risk factors in this older age group who required pressure equalization tube (PET) insertion. Study design Retrospective cohort study Setting Tertiary care pediatric academic medical center Subjects and methods Children 6–12 years old undergoing PET insertion between October 1, 2010 and September 30, 2011. Data was stratified into two separate age cohorts (6–7 versus 8–12-year-olds) and compared using chi-square analysis. Results A total of 263 patients met study criteria. PET insertion was most common in 6 year-olds (36%, 95/263). Presence of siblings (p=0.03) and history of recurrent upper respiratory tract infection (p<0.01), otalgia (p<0.05), otorrhea (p<0.001), and nasal discharge (p<0.001) were common in the older cohort. No statistical difference was found for history of recurrent acute otitis media, allergy, asthma, or atopy between the two groups (p=0.23–0.92), although the overall prevalence of these conditions was high in both cohorts. Conclusion The 8–12-year-olds had a history of recurrent upper respiratory tract infection and more infectious symptoms than the 6–7-year-olds. Atopy can lead to a heightened susceptibility to upper respiratory tract infections and potential increase in the relative risk of otitis media. In our patient population, while there was no statistically significant difference in history of asthma, allergy, or atopy, the overall prevalence within both cohorts was relatively high. Therefore, this study provides insight into many pertinent and potentially modifiable risk factors for older children requiring PET insertion. PMID:27175444

  16. Characteristics of Active Tuberculosis Patients Requiring Intensive Care Monitoring and Factors Affecting Mortality

    PubMed Central

    Levent, Dalar; Emel, Eryüksel; Pelin, Uysal; Turkay, Akbaş; Aybüke, Kekeçoğlu

    2016-01-01

    Background One to three percent of cases of acute tuberculosis (TB) require monitoring in the intensive care unit (ICU). The purpose of this study is to establish and determine the mortality rate and discuss the causes of high mortality in these cases, and to evaluate the clinical and laboratory findings of TB patients admitted to the pulmonary ICU. Methods The data of patients admitted to the ICU of Yedikule Chest Diseases and Chest Surgery Education and Research Hospital due to active TB were retrospectively evaluated. Demographic characteristics, medical history, and clinical and laboratory findings were evaluated. Results Thirty-five TB patients (27 males) with a median age of 47 years were included, of whom 20 died within 30 days (57%). The Acute Physiology and Chronic Health Evaluation II (APACHE II) and Sequential Organ Failure Assessment (SOFA) scores were significantly higher, and albumin and PaO2/FIO2 levels were significantly lower, and shock, multiple organ failure, the need for invasive mechanical ventilation and drug resistance were more common in the patients who died. The mortality risk was 7.58 times higher in the patients requiring invasive mechanical ventilation. The SOFA score alone was a significant risk factor affecting survival. Conclusion The survival rate is low in cases of tuberculosis treated in an ICU. The predictors of mortality include the requirement of invasive mechanical ventilation and multiple organ failure. Another factor specific to TB patients is the presence of drug resistance, which should be taken seriously in countries where there is a high incidence of the disease. Finding new variables that can be established with new prospective studies may help to decrease the high mortality rate. PMID:27433176

  17. RAS is required for epidermal growth factor-stimulated arachidonic acid release in rat-1 fibroblasts.

    PubMed

    Warner, L C; Hack, N; Egan, S E; Goldberg, H J; Weinberg, R A; Skorecki, K L

    1993-12-01

    Previous studies have provided suggestive evidence for an interaction between ras activation and signalling pathways involved in agonist-stimulated arachidonic acid release in a variety of cell systems. In order to clarify this interaction, we have measured epidermal growth factor (EGF)-stimulated arachidonic acid release in rat-1 fibroblasts transfected with the N-17 dominant negative mutation of ras. Cells transfected with the N-17 ras mutant, display a markedly attenuated arachidonic acid-release response to EGF, compared to sham-transfected and non-transfected cells. In contrast, the response to phorbol myristate acetate (PMA) was not attenuated in the N-17-mutant expressing cells. No differences were detected between sham-transfected and N-17 mutant expressing cells in levels of immunodetectable EGF receptor, cytosolic phospholipase A2 or mitogen-activated protein (MAP) kinase. Attenuation of EGF-stimulated arachidonic acid release in the N-17 mutant expressing cells, was accompanied by a marked diminution in EGF-stimulated tyrosine phosphorylation of MAP kinase. We conclude that the signalling pathway involved in epidermal growth factor-stimulated arachidonic acid release is similar to the signalling pathway for mitogenic responses to epidermal growth factor and requires ras activation, likely followed by a downstream cascade of kinases eventuating in MAP kinase activation.

  18. RNA-directed DNA methylation and plant development require an IWR1-type transcription factor

    PubMed Central

    Kanno, Tatsuo; Bucher, Etienne; Daxinger, Lucia; Huettel, Bruno; Kreil, David P; Breinig, Frank; Lind, Marc; Schmitt, Manfred J; Simon, Stacey A; Gurazada, Sai Guna Ranjan; Meyers, Blake C; Lorkovic, Zdravko J; Matzke, Antonius J M; Matzke, Marjori

    2010-01-01

    RNA-directed DNA methylation (RdDM) in plants requires two RNA polymerase (Pol) II-related RNA polymerases, namely Pol IV and Pol V. A genetic screen designed to reveal factors that are important for RdDM in a developmental context in Arabidopsis identified DEFECTIVE IN MERISTEM SILENCING 4 (DMS4). Unlike other mutants defective in RdDM, dms4 mutants have a pleiotropic developmental phenotype. The DMS4 protein is similar to yeast IWR1 (interacts with RNA polymerase II), a conserved putative transcription factor that interacts with Pol II subunits. The DMS4 complementary DNA partly complements the K1 killer toxin hypersensitivity of a yeast iwr1 mutant, suggesting some functional conservation. In the transgenic system studied, mutations in DMS4 directly or indirectly affect Pol IV-dependent secondary short interfering RNAs, Pol V-mediated RdDM, Pol V-dependent synthesis of intergenic non-coding RNA and expression of many Pol II-driven genes. These data suggest that DMS4 might be a regulatory factor for several RNA polymerases, thus explaining its diverse roles in the plant. PMID:20010803

  19. Nod Factor-Independent Nodulation in Aeschynomene evenia Required the Common Plant-Microbe Symbiotic Toolkit.

    PubMed

    Fabre, Sandrine; Gully, Djamel; Poitout, Arthur; Patrel, Delphine; Arrighi, Jean-François; Giraud, Eric; Czernic, Pierre; Cartieaux, Fabienne

    2015-12-01

    Nitrogen fixation in the legume-rhizobium symbiosis is a crucial area of research for more sustainable agriculture. Our knowledge of the plant cascade in response to the perception of bacterial Nod factors has increased in recent years. However, the discovery that Nod factors are not involved in the Aeschynomene-Bradyrhizobium spp. interaction suggests that alternative molecular dialogues may exist in the legume family. We evaluated the conservation of the signaling pathway common to other endosymbioses using three candidate genes: Ca(2+)/Calmodulin-Dependent Kinase (CCaMK), which plays a central role in cross signaling between nodule organogenesis and infection processes; and Symbiosis Receptor Kinase (SYMRK) and Histidine Kinase1 (HK1), which act upstream and downstream of CCaMK, respectively. We showed that CCaMK, SYMRK, and HK1 are required for efficient nodulation in Aeschynomene evenia. Our results demonstrate that CCaMK and SYMRK are recruited in Nod factor-independent symbiosis and, hence, may be conserved in all vascular plant endosymbioses described so far.

  20. Deficiency in kwashiorkor serum of factors required for optimal lymphocyte transformation in vitro.

    PubMed

    Beatty, D W; Dowdle, E B

    1979-03-01

    Blastogenic responses of normal human peripheral blood lymphocytes cultured in media supplemented with serum from children with kwashiorkor were, on average, 47.7% of those observed when the same cells were cultured in the presence of normal AB serum. Incorporation of radioactive uridine was also diminished in the presence of normal AB serum. Incorporation of radioactive uridine was also diminished in the presence of kwashiorkor serum indicating that lectin-induced RNA synthesis was also affected. The kwashiorkor serum effect was not due to a cytotoxic action nor could it be attributed to the presence of saccharides or other inhibitors of the inducing lectins. Mixing experiments showed that kwashiorkor serum was not inhibitory, but that it lacked factors present in normal serum that are required for optimal lymphocyte blastogenesis. The deficiency of these factors could largely be rectified by supplementing kwashiorkor serum with an ultrafiltrate of normal serum containing components with molecular weights of less than 500 Daltons. We conclude that nutritional deprivation of severity sufficient to cause kwashiorkor leads to a deficiency of low molecular weight lymphocyte growth factors. This lack may contribute to the immunodeficiency associated with the disease.

  1. The RNA Export Factor, Nxt1, Is Required for Tissue Specific Transcriptional Regulation

    PubMed Central

    Jiang, Jianqiao; White-Cooper, Helen

    2013-01-01

    The highly conserved, Nxf/Nxt (TAP/p15) RNA nuclear export pathway is important for export of most mRNAs from the nucleus, by interacting with mRNAs and promoting their passage through nuclear pores. Nxt1 is essential for viability; using a partial loss of function allele, we reveal a role for this gene in tissue specific transcription. We show that many Drosophila melanogaster testis-specific mRNAs require Nxt1 for their accumulation. The transcripts that require Nxt1 also depend on a testis-specific transcription complex, tMAC. We show that loss of Nxt1 leads to reduced transcription of tMAC targets. A reporter transcript from a tMAC-dependent promoter is under-expressed in Nxt1 mutants, however the same transcript accumulates in mutants if driven by a tMAC-independent promoter. Thus, in Drosophila primary spermatocytes, the transcription factor used to activate expression of a transcript, rather than the RNA sequence itself or the core transcription machinery, determines whether this expression requires Nxt1. We additionally find that transcripts from intron-less genes are more sensitive to loss of Nxt1 function than those from intron-containing genes and propose a mechanism in which transcript processing feeds back to increase activity of a tissue specific transcription complex. PMID:23754955

  2. p21(Waf1) is required for cellular senescence but not for cell cycle arrest induced by the HDAC inhibitor sodium butyrate.

    PubMed

    Romanov, V S; Abramova, M V; Svetlikova, S B; Bykova, T V; Zubova, S G; Aksenov, N D; Fornace, A J; Pospelova, T V; Pospelov, V A

    2010-10-01

    Cell senescence is characterized by senescent morphology and permanent loss of proliferative potential. HDAC inhibitors (HDACI) induce senescence and/or apoptosis in many types of tumor cells. Here, we studied the role of cyclin-kinase inhibitor p21(waf1) (Cdkn1n gene) in cell cycle arrest, senescence markers (cell hypertrophy, SA-βGal staining and accumulation of γH2AX foci) in p21(Waf1+/+) versus p21(Waf1-/-) mouse embryonic fibroblast cells transformed with E1A and cHa-Ras oncogenes (mERas). While short treatment with the HDACI sodium butyrate (NaB) induced a reversible G(1) cell cycle arrest in both parental and p21(Waf1-/-) cells, long-term treatment led to dramatic changes in p21(Waf1+/+) cells only: cell cycle arrest became irreversible and cells become hypertrophic, SA-βGal-positive and accumulated γH2AX foci associated with mTORC1 activation. The p21(Waf1+/+) cells lost their ability to migrate into the wound and through a porous membrane. Suppression of migration was accompanied by accumulation of vinculin-staining focal adhesions and Ser3-phosphorylation of cofilin, incapable for F-actin depolymerization. In contrast, the knockout of the p21(Waf1) abolished most of the features of NaB-induced senescence, including irreversibility of cell cycle arrest, hypertrophy, additional focal adhesions and block of migration, γH2AX foci accumulation and SA-βGal staining. Rapamycin, a specific inhibitor of mTORC1 kinase, decreased cellular hypertrophy, canceled coffilin phosphorylation and partially restored cell migration in p21(Waf1+/+) cells. Taken together, our data indicate a new role of p21(Waf1) in cell senescence, which may be connected not only with execution of cell cycle arrest, but also with the development of mTOR-dependent markers of cellular senescence.

  3. AKTIP/Ft1, a New Shelterin-Interacting Factor Required for Telomere Maintenance

    PubMed Central

    Burla, Romina; Carcuro, Mariateresa; Raffa, Grazia D.; Galati, Alessandra; Raimondo, Domenico; Rizzo, Angela; La Torre, Mattia; Micheli, Emanuela; Ciapponi, Laura; Cenci, Giovanni; Cundari, Enrico; Musio, Antonio; Biroccio, Annamaria; Cacchione, Stefano; Gatti, Maurizio; Saggio, Isabella

    2015-01-01

    Telomeres are nucleoprotein complexes that protect the ends of linear chromosomes from incomplete replication, degradation and detection as DNA breaks. Mammalian telomeres are protected by shelterin, a multiprotein complex that binds the TTAGGG telomeric repeats and recruits a series of additional factors that are essential for telomere function. Although many shelterin-associated proteins have been so far identified, the inventory of shelterin-interacting factors required for telomere maintenance is still largely incomplete. Here, we characterize AKTIP/Ft1 (human AKTIP and mouse Ft1 are orthologous), a novel mammalian shelterin-bound factor identified on the basis of its homology with the Drosophila telomere protein Pendolino. AKTIP/Ft1 shares homology with the E2 variant ubiquitin-conjugating (UEV) enzymes and has been previously implicated in the control of apoptosis and in vesicle trafficking. RNAi-mediated depletion of AKTIP results in formation of telomere dysfunction foci (TIFs). Consistent with these results, AKTIP interacts with telomeric DNA and binds the shelterin components TRF1 and TRF2 both in vivo and in vitro. Analysis of AKTIP- depleted human primary fibroblasts showed that they are defective in PCNA recruiting and arrest in the S phase due to the activation of the intra S checkpoint. Accordingly, AKTIP physically interacts with PCNA and the RPA70 DNA replication factor. Ft1-depleted p53-/- MEFs did not arrest in the S phase but displayed significant increases in multiple telomeric signals (MTS) and sister telomere associations (STAs), two hallmarks of defective telomere replication. In addition, we found an epistatic relation for MST formation between Ft1 and TRF1, which has been previously shown to be required for replication fork progression through telomeric DNA. Ch-IP experiments further suggested that in AKTIP-depleted cells undergoing the S phase, TRF1 is less tightly bound to telomeric DNA than in controls. Thus, our results collectively

  4. Cellular iron metabolism.

    PubMed

    Ponka, P

    1999-03-01

    Iron is essential for oxidation-reduction catalysis and bioenergetics, but unless appropriately shielded, iron plays a key role in the formation of toxic oxygen radicals that can attack all biological molecules. Hence, specialized molecules for the acquisition, transport (transferrin), and storage (ferritin) of iron in a soluble nontoxic form have evolved. Delivery of iron to most cells, probably including those of the kidney, occurs following the binding of transferrin to transferrin receptors on the cell membrane. The transferrin-receptor complexes are then internalized by endocytosis, and iron is released from transferrin by a process involving endosomal acidification. Cellular iron storage and uptake are coordinately regulated post-transcriptionally by cytoplasmic factors, iron-regulatory proteins 1 and 2 (IRP-1 and IRP-2). Under conditions of limited iron supply, IRP binding to iron-responsive elements (present in 5' untranslated region of ferritin mRNA and 3' untranslated region of transferrin receptor mRNA) blocks ferritin mRNA translation and stabilizes transferrin receptor mRNA. The opposite scenario develops when iron in the transit pool is plentiful. Moreover, IRP activities/levels can be affected by various forms of "oxidative stress" and nitric oxide. The kidney also requires iron for metabolic processes, and it is likely that iron deficiency or excess can cause disturbed function of kidney cells. Transferrin receptors are not evenly distributed throughout the kidney, and there is a cortical-to-medullary gradient in heme biosynthesis, with greatest activity in the cortex and least in the medulla. This suggests that there are unique iron/heme metabolism features in some kidney cells, but the specific aspects of iron and heme metabolism in the kidney are yet to be explained.

  5. Requirements for fault-tolerant factoring on an atom-optics quantum computer

    NASA Astrophysics Data System (ADS)

    Devitt, Simon J.; Stephens, Ashley M.; Munro, William J.; Nemoto, Kae

    2013-10-01

    Quantum information processing and its associated technologies have reached a pivotal stage in their development, with many experiments having established the basic building blocks. Moving forward, the challenge is to scale up to larger machines capable of performing computational tasks not possible today. This raises questions that need to be urgently addressed, such as what resources these machines will consume and how large will they be. Here we estimate the resources required to execute Shor’s factoring algorithm on an atom-optics quantum computer architecture. We determine the runtime and size of the computer as a function of the problem size and physical error rate. Our results suggest that once the physical error rate is low enough to allow quantum error correction, optimization to reduce resources and increase performance will come mostly from integrating algorithms and circuits within the error correction environment, rather than from improving the physical hardware.

  6. The transcription factor Mef2 is required for normal circadian behavior in Drosophila

    PubMed Central

    Blanchard, Florence J.; Collins, Ben; Cyran, Shawn A.; Hancock, Daniel H.; Taylor, Michael V.; Blau, Justin

    2010-01-01

    The transcription factor Mef2 has well-established roles in muscle development in Drosophila and in the differentiation of many cell types in mammals, including neurons. Here, we describe a role for Mef2 in the Drosophila pacemaker neurons that regulate circadian behavioral rhythms. We found that Mef2 is normally produced in all adult clock neurons and that Mef2 over-expression in clock neurons leads to long period and complex rhythms of adult locomotor behavior. Knocking down Mef2 expression via RNAi or expressing a repressor form of Mef2 caused flies to lose circadian behavioral rhythms. These behavioral changes are correlated with altered molecular clocks in pacemaker neurons: Mef2 over-expression causes the oscillations in individual pacemaker neurons to become desynchronized, while Mef2 knockdown strongly dampens molecular rhythms. Thus, a normal level of Mef2 activity is required in clock neurons to maintain robust and accurate circadian behavioral rhythms. PMID:20427646

  7. JunB is required for endothelial cell morphogenesis by regulating core-binding factor β

    PubMed Central

    Licht, Alexander H.; Pein, Oliver T.; Florin, Lore; Hartenstein, Bettina; Reuter, Hendrik; Arnold, Bernd; Lichter, Peter; Angel, Peter; Schorpp-Kistner, Marina

    2006-01-01

    The molecular mechanism triggering the organization of endothelial cells (ECs) in multicellular tubules is mechanistically still poorly understood. We demonstrate that cell-autonomous endothelial functions of the AP-1 subunit JunB are required for proper endothelial morphogenesis both in vivo in mouse embryos with endothelial-specific ablation of JunB and in in vitro angiogenesis models. By cDNA microarray analysis, we identified core-binding factor β (CBFβ), which together with the Runx proteins forms the heterodimeric core-binding transcription complex CBF, as a novel JunB target gene. In line with our findings, expression of the CBF target MMP-13 was impaired in JunB-deficient ECs. Reintroduction of CBFβ into JunB-deficient ECs rescued the tube formation defect and MMP-13 expression, indicating an important role for CBFβ in EC morphogenesis. PMID:17158955

  8. Myoferlin is required for insulin-like growth factor response and muscle growth

    PubMed Central

    Demonbreun, Alexis R.; Posey, Avery D.; Heretis, Konstantina; Swaggart, Kayleigh A.; Earley, Judy U.; Pytel, Peter; McNally, Elizabeth M.

    2010-01-01

    Insulin-like growth factor (IGF) is a potent stimulus of muscle growth. Myoferlin is a membrane-associated protein important for muscle development and regeneration. Myoferlin-null mice have smaller muscles and defective myoblast fusion. To understand the mechanism by which myoferlin loss retards muscle growth, we found that myoferlin-null muscle does not respond to IGF1. In vivo after IGF1 infusion, control muscle increased myofiber diameter by 25%, but myoferlin-null muscle was unresponsive. Myoblasts cultured from myoferlin-null muscle and treated with IGF1 also failed to show the expected increase in fusion to multinucleate myotubes. The IGF1 receptor colocalized with myoferlin at sites of myoblast fusion. The lack of IGF1 responsiveness in myoferlin-null myoblasts was linked directly to IGF1 receptor mistrafficking as well as decreased IGF1 signaling. In myoferlin-null myoblasts, the IGF1 receptor accumulated into large vesicular structures. These vesicles colocalized with a marker of late endosomes/lysosomes, LAMP2, specifying redirection from a recycling to a degradative pathway. Furthermore, ultrastructural analysis showed a marked increase in vacuoles in myoferlin-null muscle. These data demonstrate that IGF1 receptor recycling is required for normal myogenesis and that myoferlin is a critical mediator of postnatal muscle growth mediated by IGF1.—Demonbreun, A. R., Posey, A. D., Heretis, K., Swaggart, K. A., Earley, J. U., Pytel, P., McNally, E. M. Myoferlin is required for insulin-like growth factor response and muscle growth. PMID:20008164

  9. Comprehensive identification of virulence factors required for respiratory melioidosis using Tn-seq mutagenesis

    PubMed Central

    Gutierrez, Maria G.; Yoder-Himes, Deborah R.; Warawa, Jonathan M.

    2015-01-01

    Respiratory melioidosis is a disease presentation of the biodefense pathogen, Burkholderia pseudomallei, which is frequently associated with a lethal septicemic spread of the bacteria. We have recently developed an improved respiratory melioidosis model to study the pathogenesis of Burkholderia pseudomallei in the lung (intubation-mediated intratracheal [IMIT] inoculation), which more closely models descriptions of human melioidosis, including prominent septicemic spread from the lung and reduced involvement of the upper respiratory tract. We previously demonstrated that the Type 3 Secretion System cluster 3 (T3SS3) is a critical virulence determinant for B. pseudomallei when delivered directly into the lung. We decided to comprehensively identify all virulence determinants required for respiratory melioidosis using the Tn-seq phenotypic screen, as well as to investigate which virulence determinants are required for dissemination to the liver and spleen. While previous studies have used Tn-seq to identify essential genes for in vitro cultured B. pseudomallei, this represents the first study to use Tn-seq to identify genes required for in vivo fitness. Consistent with our previous findings, we identified T3SS3 as the largest genetic cluster required for fitness in the lung. Furthermore, we identified capsular polysaccharide and Type 6 Secretion System cluster 5 (T6SS5) as the two additional major genetic clusters facilitating respiratory melioidosis. Importantly, Tn-seq did not identify additional, novel large genetic systems supporting respiratory melioidosis, although these studies identified additional small gene clusters that may also play crucial roles in lung fitness. Interestingly, other previously identified virulence determinants do not appear to be required for lung fitness, such as lipopolysaccharide. The role of T3SS3, capsule, and T6SS5 in lung fitness was validated by competition studies, but only T3SS3 was found to be important for respiratory

  10. Comprehensive identification of virulence factors required for respiratory melioidosis using Tn-seq mutagenesis.

    PubMed

    Gutierrez, Maria G; Yoder-Himes, Deborah R; Warawa, Jonathan M

    2015-01-01

    Respiratory melioidosis is a disease presentation of the biodefense pathogen, Burkholderia pseudomallei, which is frequently associated with a lethal septicemic spread of the bacteria. We have recently developed an improved respiratory melioidosis model to study the pathogenesis of Burkholderia pseudomallei in the lung (intubation-mediated intratracheal [IMIT] inoculation), which more closely models descriptions of human melioidosis, including prominent septicemic spread from the lung and reduced involvement of the upper respiratory tract. We previously demonstrated that the Type 3 Secretion System cluster 3 (T3SS3) is a critical virulence determinant for B. pseudomallei when delivered directly into the lung. We decided to comprehensively identify all virulence determinants required for respiratory melioidosis using the Tn-seq phenotypic screen, as well as to investigate which virulence determinants are required for dissemination to the liver and spleen. While previous studies have used Tn-seq to identify essential genes for in vitro cultured B. pseudomallei, this represents the first study to use Tn-seq to identify genes required for in vivo fitness. Consistent with our previous findings, we identified T3SS3 as the largest genetic cluster required for fitness in the lung. Furthermore, we identified capsular polysaccharide and Type 6 Secretion System cluster 5 (T6SS5) as the two additional major genetic clusters facilitating respiratory melioidosis. Importantly, Tn-seq did not identify additional, novel large genetic systems supporting respiratory melioidosis, although these studies identified additional small gene clusters that may also play crucial roles in lung fitness. Interestingly, other previously identified virulence determinants do not appear to be required for lung fitness, such as lipopolysaccharide. The role of T3SS3, capsule, and T6SS5 in lung fitness was validated by competition studies, but only T3SS3 was found to be important for respiratory

  11. Myeloid derived hypoxia inducible factor 1-alpha is required for protection against pulmonary Aspergillus fumigatus infection.

    PubMed

    Shepardson, Kelly M; Jhingran, Anupam; Caffrey, Alayna; Obar, Joshua J; Suratt, Benjamin T; Berwin, Brent L; Hohl, Tobias M; Cramer, Robert A

    2014-09-01

    Hypoxia inducible factor 1α (HIF1α) is the mammalian transcriptional factor that controls metabolism, survival, and innate immunity in response to inflammation and low oxygen. Previous work established that generation of hypoxic microenvironments occurs within the lung during infection with the human fungal pathogen Aspergillus fumigatus. Here we demonstrate that A. fumigatus stabilizes HIF1α protein early after pulmonary challenge that is inhibited by treatment of mice with the steroid triamcinolone. Utilizing myeloid deficient HIF1α mice, we observed that HIF1α is required for survival and fungal clearance early following pulmonary challenge with A. fumigatus. Unlike previously reported research with bacterial pathogens, HIF1α deficient neutrophils and macrophages were surprisingly not defective in fungal conidial killing. The increase in susceptibility of the myeloid deficient HIF1α mice to A. fumigatus was in part due to decreased early production of the chemokine CXCL1 (KC) and increased neutrophil apoptosis at the site of infection, resulting in decreased neutrophil numbers in the lung. Addition of recombinant CXCL1 restored neutrophil survival and numbers, murine survival, and fungal clearance. These results suggest that there are unique HIF1α mediated mechanisms employed by the host for protection and defense against fungal pathogen growth and invasion in the lung. Additionally, this work supports the strategy of exploring HIF1α as a therapeutic target in specific immunosuppressed populations with fungal infections.

  12. Human papilloma virus DNAs immortalize normal human mammary epithelial cells and reduce their growth factor requirements

    SciTech Connect

    Band, V.; Zajchowski, D.; Kulesa, V.; Sager, R. )

    1990-01-01

    Human papilloma virus (HPV) types 16 and 18 are most commonly associated with cervical carcinoma in patients and induce immortalization of human keratinocytes in culture. HPV has not been associated with breast cancer. This report describes the immortalization of normal human mammary epithelial cells (76N) by plasmid pHPV18 or pHPV16, each containing the linearized viral genome. Transfectants were grown continuously for more than 60 passages, whereas 76N cells senesce after 18-20 passages. The transfectants also differ from 76N cells in cloning in a completely defined medium called D2 and growing a minimally supplemented defined medium (D3) containing epidermal growth factor. All transfectant tested contain integrated HPV DNA, express HPV RNA, and produce HPV E7 protein. HPV transfectants do not form tumors in a nude mouse assay. It is concluded that products of the HPV genome induce immortalization of human breast epithelial cells and reduce their growth factor requirements. This result raises the possibility that HPV might be involved in breast cancer. Furthermore, other tissue-specific primary epithelial cells that are presently difficult to grown and investigate may also be immortalized by HPV.

  13. Normal Dendrite Growth in Drosophila Motor Neurons Requires the AP-1 Transcription Factor

    PubMed Central

    Hartwig, Cortnie L.; Worrell, Jason; Levine, Richard B.; Ramaswami, Mani; Sanyal, Subhabrata

    2009-01-01

    During learning and memory formation, information flow through networks is regulated significantly through structural alterations in neurons. Dendrites, sites of signal integration, are key targets of activity-mediated modifications. Although local mechanisms of dendritic growth ensure synapse-specific changes, global mechanisms linking neural activity to nuclear gene expression may have profound influences on neural function. Fos, being an immediate-early gene, is ideally suited to be an initial transducer of neural activity, but a precise role for the AP-1 transcription factor in dendrite growth remains to be elucidated. Here we measure changes in the dendritic fields of identified Drosophila motor neurons in vivo and in primary culture to investigate the role of the immediate-early transcription factor AP-1 in regulating endogenous and activity-induced dendrite growth. Our data indicate that (a) increased neural excitability or depolarization stimulates dendrite growth, (b) AP-1 (a Fos, Jun heterodimer) is required for normal motor neuron dendritic growth during development and in response to activity induction, and (c) neuronal Fos protein levels are rapidly but transiently induced in motor neurons following neural activity. Taken together, these results show that AP-1 mediated transcription is important for dendrite growth, and that neural activity influences global dendritic growth through a gene-expression dependent mechanism gated by AP-1. PMID:18548486

  14. Myeloid Derived Hypoxia Inducible Factor 1-alpha Is Required for Protection against Pulmonary Aspergillus fumigatus Infection

    PubMed Central

    Shepardson, Kelly M.; Jhingran, Anupam; Caffrey, Alayna; Obar, Joshua J.; Suratt, Benjamin T.; Berwin, Brent L.; Hohl, Tobias M.; Cramer, Robert A.

    2014-01-01

    Hypoxia inducible factor 1α (HIF1α) is the mammalian transcriptional factor that controls metabolism, survival, and innate immunity in response to inflammation and low oxygen. Previous work established that generation of hypoxic microenvironments occurs within the lung during infection with the human fungal pathogen Aspergillus fumigatus. Here we demonstrate that A. fumigatus stabilizes HIF1α protein early after pulmonary challenge that is inhibited by treatment of mice with the steroid triamcinolone. Utilizing myeloid deficient HIF1α mice, we observed that HIF1α is required for survival and fungal clearance early following pulmonary challenge with A. fumigatus. Unlike previously reported research with bacterial pathogens, HIF1α deficient neutrophils and macrophages were surprisingly not defective in fungal conidial killing. The increase in susceptibility of the myeloid deficient HIF1α mice to A. fumigatus was in part due to decreased early production of the chemokine CXCL1 (KC) and increased neutrophil apoptosis at the site of infection, resulting in decreased neutrophil numbers in the lung. Addition of recombinant CXCL1 restored neutrophil survival and numbers, murine survival, and fungal clearance. These results suggest that there are unique HIF1α mediated mechanisms employed by the host for protection and defense against fungal pathogen growth and invasion in the lung. Additionally, this work supports the strategy of exploring HIF1α as a therapeutic target in specific immunosuppressed populations with fungal infections. PMID:25255025

  15. Whole Genome Sequencing Identifies a Novel Factor Required for Secretory Granule Maturation in Tetrahymena thermophila

    PubMed Central

    Kontur, Cassandra; Kumar, Santosh; Lan, Xun; Pritchard, Jonathan K.; Turkewitz, Aaron P.

    2016-01-01

    Unbiased genetic approaches have a unique ability to identify novel genes associated with specific biological pathways. Thanks to next generation sequencing, forward genetic strategies can be expanded to a wider range of model organisms. The formation of secretory granules, called mucocysts, in the ciliate Tetrahymena thermophila relies, in part, on ancestral lysosomal sorting machinery, but is also likely to involve novel factors. In prior work, multiple strains with defects in mucocyst biogenesis were generated by nitrosoguanidine mutagenesis, and characterized using genetic and cell biological approaches, but the genetic lesions themselves were unknown. Here, we show that analyzing one such mutant by whole genome sequencing reveals a novel factor in mucocyst formation. Strain UC620 has both morphological and biochemical defects in mucocyst maturation—a process analogous to dense core granule maturation in animals. Illumina sequencing of a pool of UC620 F2 clones identified a missense mutation in a novel gene called MMA1 (Mucocyst maturation). The defects in UC620 were rescued by expression of a wild-type copy of MMA1, and disrupting MMA1 in an otherwise wild-type strain phenocopies UC620. The product of MMA1, characterized as a CFP-tagged copy, encodes a large soluble cytosolic protein. A small fraction of Mma1p-CFP is pelletable, which may reflect association with endosomes. The gene has no identifiable homologs except in other Tetrahymena species, and therefore represents an evolutionarily recent innovation that is required for granule maturation. PMID:27317773

  16. Caenorhabditis elegans-based screen identifies Salmonella virulence factors required for conserved host-pathogen interactions.

    PubMed

    Tenor, Jennifer L; McCormick, Beth A; Ausubel, Frederick M; Aballay, Alejandro

    2004-06-01

    A Caenorhabditis elegans-Salmonella enterica host-pathogen model was used to identify both novel and previously known S. enterica virulence factors (HilA, HilD, InvH, SptP, RhuM, Spi4-F, PipA, VsdA, RepC, Sb25, RfaL, GmhA, LeuO, CstA, and RecC), including several related to the type III secretion system (TTSS) encoded in Salmonella pathogenicity island 1 (SPI-1). Mutants corresponding to presumptive novel virulence-related genes exhibited diminished ability to invade epithelial cells and/or to induce polymorphonuclear leukocyte migration in a tissue culture model of mammalian enteropathogenesis. When expressed in C. elegans intestinal cells, the S. enterica TTSS-exported effector protein SptP inhibited a conserved p38 MAPK signaling pathway and suppressed the diminished pathogenicity phenotype of an S. enterica sptP mutant. These results show that C. elegans is an attractive model to study the interaction between Salmonella effector proteins and components of the innate immune response, in part because there is a remarkable overlap between Salmonella virulence factors required for human and nematode pathogenesis.

  17. Specific factors are required for kinase-dependent endocytosis of insulin receptors.

    PubMed Central

    Welsh, J B; Worthylake, R; Wiley, H S; Gill, G N

    1994-01-01

    Mouse B82 cells that support high affinity saturable endocytosis of epidermal growth factor receptors (EGFR) exhibited only low rates of nonsaturable internalization of insulin receptors (InsR). To investigate the defect in endocytosis of InsR in B82 cells, we examined the role of sequence motifs and tyrosine kinase, the two receptor components shown to be required for efficient saturable endocytosis of InsR in Rat 1 cells. Placement of residues encoded by exon 16 of the InsR onto an EGFR truncated to residue 958 restored EGF-induced internalization of this mutant receptor indicating that the sequence codes in exon 16 are recognized by B82 cells. To determine whether the kinase function could be provided in trans, a B82 cell expressing both receptors was established. EGF-activated EGFR kinase was not able to restore insulin-dependent rapid endocytosis to InsR. However, fusion of untransfected Rat1 cells with InsR-expressing B82 cells enabled rapid endocytosis of InsR, indicating that the internalization defect can be complemented. These results indicate that, although internalization codes can function in the context of other receptors, activation of tyrosine kinase receptors requires an additional specific component. Images PMID:7919536

  18. Human mitochondrial transcription factor A is required for the segregation of mitochondrial DNA in cultured cells.

    PubMed

    Kasashima, Katsumi; Sumitani, Megumi; Endo, Hitoshi

    2011-01-15

    The segregation and transmission of the mitochondrial genome in humans are complicated processes but are particularly important for understanding the inheritance and clinical abnormalities of mitochondrial disorders. However, the molecular mechanism of the segregation of mitochondrial DNA (mtDNA) is largely unclear. In this study, we demonstrated that human mitochondrial transcription factor A (TFAM) is required for the segregation of mtDNA in cultured cells. RNAi-mediated knockdown of TFAM in HeLa cells resulted in the enlarged mtDNA, as indicated by the assembly of fluorescent signals stained with PicoGreen. Fluorescent in situ hybridization confirmed the enlarged mtDNA and further showed the existence of increased numbers of mitochondria lacking mtDNA signals in TFAM knockdown cells. By complementation analysis, the C-terminal tail of TFAM, which enhances its affinity with DNA, was found to be required for the appropriate distribution of mtDNA. Furthermore, we found that TFAM knockdown induced asymmetric segregation of mtDNA between dividing daughter cells. These results suggest an essential role for human TFAM in symmetric segregation of mtDNA. PMID:20955698

  19. The matri-cellular proteins 'cysteine-rich, angiogenic-inducer, 61' and 'connective tissue growth factor' are regulated in experimentally-induced sepsis with multiple organ dysfunction.

    PubMed

    Hviid, Claus V B; Erdem, Johanna Samulin; Kunke, David; Ahmed, Shakil M; Kjeldsen, Signe F; Wang, Yun Yong; Attramadal, Håvard; Aasen, Ansgar O

    2012-10-01

    Organ failure is a severe complication in sepsis for which the pathophysiology remains incompletely understood. Recently, the matri-cellular cysteine-rich, angiogenic induced, 61 (Cyr61/CCN1); connective tissue growth factor (Ctgf/CCN2); and nephroblastoma overexpressed gene (Nov/CCN3) (CCN)-protein family have been attributed organ-protective properties. Their expression is sensitive to mediators of sepsis pathophysiology but a potential role in sepsis remains elusive. To provide an initial assessment, 50 rats were subjected to 18 h of cecal-ligation and puncture or sham operation. Hepatic and pulmonary CCN1 mRNA displayed an average 7.4- and 3.3-fold induction, while its cardiac expression was unchanged. The changes coincided with excessive hepatic and pulmonary inflammatory gene activation and a restricted cardiac inflammation. Furthermore, hepatocytes displayed a dosage-dependent CCN1 mRNA response in vitro, supporting a cytokine-mediated CCN1 regulation in sepsis. CCN2 mRNA was 2.2-fold induced in the liver, while 2.0-fold and 1.4-fold repressed in the heart and lung. Meanwhile, it did not respond to TNF-α exposure in vitro, which indicates different means of regulation than for CCN1. Taken together, this study provides the first evidence for multi-organ regulation of CCN1 and CCN2 in early stages of sepsis, and implies the eruption of inflammatory mediators as a potential mechanism behind the observed CCN1 regulation.

  20. Changes in gene expression and cellular localization of insulin-like growth factors 1 and 2 in the ovaries during ovary development of the yellowtail, Seriola quinqueradiata.

    PubMed

    Higuchi, Kentaro; Gen, Koichiro; Izumida, Daisuke; Kazeto, Yukinori; Hotta, Takuro; Takashi, Toshinori; Aono, Hideaki; Soyano, Kiyoshi

    2016-06-01

    A method of controlling the somatic growth and reproduction of yellowtail fish (Seriola quinqueradiata) is needed in order to establish methods for the efficient aquaculture production of the species. However, little information about the hormonal interactions between somatic growth and reproduction is available for marine teleosts. There is accumulating evidence that insulin-like growth factor (IGF), a major hormone related somatic growth, plays an important role in fish reproduction. As the first step toward understanding the physiological role of IGF in the development of yellowtail ovaries, we characterized the expression and cellular localization of IGF-1 and IGF-2 in the ovary during development. We histologically classified the maturity of two-year-old females with ovaries at various developmental stages into the perinucleolar (Pn), yolk vesicle (Yv), primary yolk (Py), secondary yolk and tertiary yolk (Ty) stages, according to the most advanced type of oocyte present. The IGF-1 gene expression showed constitutively high levels at the different developmental stages, although IGF-1 mRNA levels tended to increase from the Py to the Ty stage with vitellogenesis, reaching maximum levels during the Ty stage. The IGF-2 mRNA levels increased as ovarian development advanced. Using immunohistochemistry methods, immunoreactive IGF-1 was mainly detected in the theca cells of ovarian follicles during late secondary oocyte growth, and in part of the granulosa cells of Ty stage oocytes. IGF-2 immunoreactivity was observed in all granulosa cells in layer in Ty stage oocytes. These results indicate that follicular IGFs may be involved in yellowtail reproduction via autocrine/paracrine mechanisms. PMID:26764214

  1. Cellular distribution of vascular endothelial growth factor A (VEGFA) and B (VEGFB) and VEGF receptors 1 and 2 in focal cortical dysplasia type IIB

    PubMed Central

    Boer, Karin; Troost, Dirk; Spliet, Wim G. M.; van Rijen, Peter C.; Gorter, Jan A.

    2008-01-01

    Members of the vascular endothelial growth factor (VEGF) family are key signaling proteins in the induction and regulation of angiogenesis, both during development and in pathological conditions. However, signaling mediated through VEGF family proteins and their receptors has recently been shown to have direct effects on neurons and glial cells. In the present study, we immunocytochemically investigated the expression and cellular distribution of VEGFA, VEGFB, and their associated receptors (VEGFR-1 and VEGFR-2) in focal cortical dysplasia (FCD) type IIB from patients with medically intractable epilepsy. Histologically normal temporal cortex and perilesional regions displayed neuronal immunoreactivity (IR) for VEGFA, VEGFB, and VEGF receptors (VEGFR-1 and VEGFR-2), mainly in pyramidal neurons. Weak IR was observed in blood vessels and there was no notable glial IR within the grey and white matter. In all FCD specimens, VEGFA, VEGFB, and both VEGF receptors were highly expressed in dysplastic neurons. IR in astroglial and balloon cells was observed for VEGFA and its receptors. VEGFR-1 displayed strong endothelial staining in FCD. Double-labeling also showed expression of VEGFA, VEGFB and VEGFR-1 in cells of the microglia/macrophage lineage. The neuronal expression of both VEGFA and VEGFB, together with their specific receptors in FCD, suggests autocrine/paracrine effects on dysplastic neurons. These autocrine/paracrine effects could play a role in the development of FCD, preventing the death of abnormal neuronal cells. In addition, the expression of VEGFA and its receptors in glial cells within the dysplastic cortex indicates that VEGF-mediated signaling could contribute to astroglial activation and associated inflammatory reactions. PMID:18317782

  2. Molecular and cellular analysis of the pH response transcription factor PacC in the fungal symbiont Epichloë festucae.

    PubMed

    Lukito, Yonathan; Chujo, Tetsuya; Scott, Barry

    2015-12-01

    In order to survive and adapt to the environment, it is imperative for fungi to be able to sense and respond to changes in extracellular pH conditions. In ascomycetes, sensing of extracellular pH is mediated by the Pal pathway resulting in activation of the PacC transcription factor at alkaline pH. The role of PacC in regulating fungal virulence and pathogenicity has been described in several pathogenic fungi but to date not in a symbiotic fungus. Epichloë festucae is a biotrophic fungal endophyte that forms a stable mutualistic interaction with Lolium perenne. In this study, pacC deletion (ΔpacC) and dominant active (pacC(C)) mutants were generated in order to study the cellular roles of PacC in E. festucae. Deletion of pacC resulted in increased sensitivity of the mutant to salt-stress but surprisingly did not affect the ability of the mutant to grow under alkaline pH conditions. Alkaline pH was observed to induce conidiation in wild-type E. festucae but not in the ΔpacC mutant. On the other hand the pacC(C) mutant had increased conidiation at neutral pH alone. Null pacC mutants had no effect on the symbiotic interaction with ryegrass plants whereas the pacC(C) mutant increased the tiller number. Examination of the growth of the pacC(C) mutant in the plant revealed the formation of aberrant convoluted hyphal structures and an increase in hyphal breakage, which are possible reasons for the altered host interaction phenotype.

  3. Adrenal Development in Mice Requires GATA4 and GATA6 Transcription Factors.

    PubMed

    Tevosian, Sergei G; Jiménez, Elizabeth; Hatch, Heather M; Jiang, Tianyu; Morse, Deborah A; Fox, Shawna C; Padua, Maria B

    2015-07-01

    The adrenal glands consist of an outer cortex and an inner medulla, and their primary purposes include hormone synthesis and secretion. The adrenal cortex produces a complex array of steroid hormones, whereas the medulla is part of the sympathetic nervous system and produces the catecholamines epinephrine and norepinephrine. In the mouse, GATA binding protein (GATA) 4 and GATA6 transcription factors are coexpressed in several embryonic tissues, including the adrenal cortex. To explore the roles of GATA4 and GATA6 in mouse adrenal development, we conditionally deleted these genes in adrenocortical cells using the Sf1Cre strain of animals. We report here that mice with Sf1Cre-mediated double deletion of Gata4 and Gata6 genes lack identifiable adrenal glands, steroidogenic factor 1-positive cortical cells and steroidogenic gene expression in the adrenal location. The inactivation of the Gata6 gene alone (Sf1Cre;Gata6(flox/flox)) drastically reduced the adrenal size and corticosterone production in the adult animals. Adrenocortical aplasia is expected to result in the demise of the animal within 2 weeks after birth unless glucocorticoids are provided. In accordance, Sf1Cre;Gata4(flox/flox)Gata6(flox/flox) females depend on steroid supplementation to survive after weaning. Surprisingly, Sf1Cre;Gata4(flox/flox)Gata6(flox/flox) males appear to live normal lifespans as vital steroidogenic synthesis shifts to their testes. Our results reveal a requirement for GATA factors in adrenal development and provide a novel tool to characterize the transcriptional network controlling adrenocortical cell fates.

  4. Adrenal Development in Mice Requires GATA4 and GATA6 Transcription Factors

    PubMed Central

    Jiménez, Elizabeth; Hatch, Heather M.; Jiang, Tianyu; Morse, Deborah A.; Fox, Shawna C.

    2015-01-01

    The adrenal glands consist of an outer cortex and an inner medulla, and their primary purposes include hormone synthesis and secretion. The adrenal cortex produces a complex array of steroid hormones, whereas the medulla is part of the sympathetic nervous system and produces the catecholamines epinephrine and norepinephrine. In the mouse, GATA binding protein (GATA) 4 and GATA6 transcription factors are coexpressed in several embryonic tissues, including the adrenal cortex. To explore the roles of GATA4 and GATA6 in mouse adrenal development, we conditionally deleted these genes in adrenocortical cells using the Sf1Cre strain of animals. We report here that mice with Sf1Cre-mediated double deletion of Gata4 and Gata6 genes lack identifiable adrenal glands, steroidogenic factor 1-positive cortical cells and steroidogenic gene expression in the adrenal location. The inactivation of the Gata6 gene alone (Sf1Cre;Gata6flox/flox) drastically reduced the adrenal size and corticosterone production in the adult animals. Adrenocortical aplasia is expected to result in the demise of the animal within 2 weeks after birth unless glucocorticoids are provided. In accordance, Sf1Cre;Gata4flox/floxGata6flox/flox females depend on steroid supplementation to survive after weaning. Surprisingly, Sf1Cre;Gata4flox/floxGata6flox/flox males appear to live normal lifespans as vital steroidogenic synthesis shifts to their testes. Our results reveal a requirement for GATA factors in adrenal development and provide a novel tool to characterize the transcriptional network controlling adrenocortical cell fates. PMID:25933105

  5. Differential Requirements of Cellular and Humoral Immune Responses for Fv2-Associated Resistance to Erythroleukemia and for Regulation of Retrovirus-Induced Myeloid Leukemia Development

    PubMed Central

    Kawabata, Hiroyuki; Matsukuma, Hideaki; Kinoshita, Saori; Chikaishi, Tomomi; Sakamoto, Mayumi; Kawasaki, Yuri

    2013-01-01

    To assess the possible contribution of host immune responses to the exertion of Fv2-associated resistance to Friend virus (FV)-induced disease development, we inoculated C57BL/6 (B6) mice that lacked various subsets of lymphocytes with FV containing no lactate dehydrogenase-elevating virus. Fv2r B6 mice lacking CD4+ T cells developed early polycythemia and fatal erythroleukemia, while B6 mice lacking CD8+ T cells remained resistant. Erythroid progenitor cells infected with spleen focus-forming virus (SFFV) were eliminated, and no polycythemia was observed in B cell-deficient B6 mice, but they later developed myeloid leukemia associated with oligoclonal integration of ecotropic Friend murine leukemia virus. Additional depletion of natural killer and/or CD8+ T cells from B cell-deficient B6 mice resulted in the expansion of SFFV proviruses and the development of polycythemia, indicating that SFFV-infected erythroid cells are not only restricted in their growth but are actively eliminated in Fv2r mice through cellular immune responses. PMID:24109240

  6. Accessory Factors of Cytoplasmic Viral RNA Sensors Required for Antiviral Innate Immune Response

    PubMed Central

    Oshiumi, Hiroyuki; Kouwaki, Takahisa; Seya, Tsukasa

    2016-01-01

    Type I interferon (IFN) induces many antiviral factors in host cells. RIG-I-like receptors (RLRs) are cytoplasmic viral RNA sensors that trigger the signal to induce the innate immune response that includes type I IFN production. RIG-I and MDA5 are RLRs that form nucleoprotein filaments along viral double-stranded RNA, resulting in the activation of MAVS adaptor molecule. The MAVS protein forms a prion-like aggregation structure, leading to type I IFN production. RIG-I and MDA5 undergo post-translational modification. TRIM25 and Riplet ubiquitin ligases deliver a K63-linked polyubiquitin moiety to the RIG-I N-terminal caspase activation and recruitment domains (CARDs) and C-terminal region; the polyubiquitin chain then stabilizes the two-CARD tetramer structure required for MAVS assembly. MDA5 activation is regulated by phosphorylation. RIOK3 is a protein kinase that phosphorylates the MDA5 protein in a steady state, and PP1α/γ dephosphorylate this protein, resulting in its activation. RIG-I and MDA5 require cytoplasmic RNA helicases for their efficient activation. LGP2, another RLR, is an RNA helicase involved in RLR signaling. This protein does not possess N-terminal CARDs and, thus, cannot trigger downstream signaling by itself. Recent studies have revealed that this protein modulates MDA5 filament formation, resulting in enhanced type I IFN production. Several other cytoplasmic RNA helicases are involved in RLR signaling. DDX3, DHX29, DHX36, and DDX60 RNA helicases have been reported to be involved in RLR-mediated type I IFN production after viral infection. However, the underlying mechanism is largely unknown. Future studies are required to reveal the role of RNA helicases in the RLR signaling pathway. PMID:27252702

  7. A novel embryological theory of autism causation involving endogenous biochemicals capable of initiating cellular gene transcription: a possible link between twelve autism risk factors and the autism 'epidemic'.

    PubMed

    King, Chiara R

    2011-05-01

    Human alpha-fetoprotein is a pregnancy-associated protein with an undetermined physiological role. As human alpha-fetoprotein binds retinoids and inhibits estrogen-dependent cancer cell proliferation, and because retinoic acid (a retinol metabolite) and estradiol (an estrogen) can both initiate cellular gene transcription, it is hypothesized here that alpha-fetoprotein functions during critical gestational periods to prevent retinoic acid and maternal estradiol from inappropriately stimulating gene expression in developing brain regions which are sensitive to these chemicals. Prenatal/maternal factors linked to increased autism risk include valproic acid, thalidomide, alcohol, rubella, cytomegalovirus, depression, schizophrenia, obsessive-compulsive disorder, autoimmune disease, stress, allergic reaction, and hypothyroidism. It will be shown how each of these risk factors may initiate expression of genes which are sensitive to retinoic acid and/or estradiol - whether by direct promotion or by reducing production of alpha-fetoprotein. It is thus hypothesized here that autism is not a genetic disorder, but is rather an epigenetic disruption in brain development caused by gestational exposure to chemicals and/or conditions which either inhibit alpha-fetoprotein production or directly promote retinoic acid-sensitive or estradiol-sensitive gene expression. This causation model leads to potential chemical explanations for autistic brain morphology, the distinct symptomatology of Asperger's syndrome, and the differences between high-functioning and low-functioning autism with regard to mental retardation, physical malformation, and sex ratio. It will be discussed how folic acid may cause autism under the retinoic acid/estradiol model, and the history of prenatal folic acid supplementation will be shown to coincide with the history of what is popularly known as the autism epidemic. It is thus hypothesized here that prenatal folic acid supplementation has contributed to the

  8. A novel embryological theory of autism causation involving endogenous biochemicals capable of initiating cellular gene transcription: a possible link between twelve autism risk factors and the autism 'epidemic'.

    PubMed

    King, Chiara R

    2011-05-01

    Human alpha-fetoprotein is a pregnancy-associated protein with an undetermined physiological role. As human alpha-fetoprotein binds retinoids and inhibits estrogen-dependent cancer cell proliferation, and because retinoic acid (a retinol metabolite) and estradiol (an estrogen) can both initiate cellular gene transcription, it is hypothesized here that alpha-fetoprotein functions during critical gestational periods to prevent retinoic acid and maternal estradiol from inappropriately stimulating gene expression in developing brain regions which are sensitive to these chemicals. Prenatal/maternal factors linked to increased autism risk include valproic acid, thalidomide, alcohol, rubella, cytomegalovirus, depression, schizophrenia, obsessive-compulsive disorder, autoimmune disease, stress, allergic reaction, and hypothyroidism. It will be shown how each of these risk factors may initiate expression of genes which are sensitive to retinoic acid and/or estradiol - whether by direct promotion or by reducing production of alpha-fetoprotein. It is thus hypothesized here that autism is not a genetic disorder, but is rather an epigenetic disruption in brain development caused by gestational exposure to chemicals and/or conditions which either inhibit alpha-fetoprotein production or directly promote retinoic acid-sensitive or estradiol-sensitive gene expression. This causation model leads to potential chemical explanations for autistic brain morphology, the distinct symptomatology of Asperger's syndrome, and the differences between high-functioning and low-functioning autism with regard to mental retardation, physical malformation, and sex ratio. It will be discussed how folic acid may cause autism under the retinoic acid/estradiol model, and the history of prenatal folic acid supplementation will be shown to coincide with the history of what is popularly known as the autism epidemic. It is thus hypothesized here that prenatal folic acid supplementation has contributed to the

  9. Cellular Reprogramming

    PubMed Central

    Takahashi, Kazutoshi

    2014-01-01

    Nuclear reprogramming technology was first established more than 50 years ago. It can rejuvenate somatic cells by erasing the epigenetic memories and reconstructing a new pluripotent order. The recent discovery reviewed here that induced pluripotency can be achieved by a small set of transcription factors has opened up unprecedented opportunities in the pharmaceutical industry, the clinic, and laboratories. This technology allows us to access pathological studies by using patient-specific induced pluripotent stem (iPS) cells. In addition, iPS cells are also expected to be a rising star for regenerative medicine, as sources of transplantation therapy. PMID:24492711

  10. Endotoxin Tolerance Inhibits Degradation of Tumor Necrosis Factor Receptor-Associated Factor 3 by Suppressing Pellino 1 Expression and the K48 Ubiquitin Ligase Activity of Cellular Inhibitor of Apoptosis Protein 2.

    PubMed

    Li, Peizhi; Liu, Hongxiang; Zhang, Yiyin; Liao, Rui; He, Kun; Ruan, Xiongzhong; Gong, Jianping

    2016-09-15

    Pellino 1 positively regulates Toll-like receptor 4 signaling by regulating tumor necrosis factor receptor-associated factor 3 (TRAF3) degradation and is suppressed with the induction of endotoxin tolerance. However, the role of TRAF3 in endotoxin tolerance is largely unknown. In this study, we found that lipopolysaccharide (LPS) stimulation decreased TARF3 protein expression in mouse Kupffer cells (KCs) and liver tissues, whereas endotoxin tolerization abrogated this effect. Degradative TRAF3 K48-linked ubiquitination and the cytoplasmic translocation of the MYD88-associated multiprotein complex were significantly inhibited in tolerized KCs, which led to markedly impaired activation of MYD88-dependent JNK and p38 and downregulation of inflammatory cytokines. TRAF3 ablation failed to induce a fully endotoxin-tolerant state in RAW264.7 cells. Pellino 1 knockdown in Raw264.7 cells did not impair induction of cIAP2 in response to LPS but inhibited the K63-linked ubiquitination of cellular inhibitor of apoptosis protein 2 (cIAP2) and K48-linked ubiquitination of TRAF3 protein. We also found upregulation of Pellino 1 and downregulation of TRAF3 in liver tissues of patients with cholangitis. Our findings reveal a novel mechanism that endotoxin tolerance reprograms mitogen-activated protein kinase signaling by suppressing Pellino 1-mediated K63-linked ubiquitination of cIAP2, K48-linked ubiquitination, and degradation of TRAF3. PMID:27377744

  11. Implication of STAT3 signaling in human colonic cancer cells during intestinal trefoil factor 3 (TFF3) -- and vascular endothelial growth factor-mediated cellular invasion and tumor growth.

    PubMed

    Rivat, Christine; Christine, Rivat; Rodrigues, Sylvie; Sylvie, Rodrigues; Bruyneel, Erik; Erik, Bruyneel; Piétu, Geneviève; Geneviève, Piétu; Robert, Amélie; Amélie, Robert; Redeuilh, Gérard; Gérard, Redeuilh; Bracke, Marc; Marc, Bracke; Gespach, Christian; Christian, Gespach; Attoub, Samir; Samir, Attoub

    2005-01-01

    Signal transducer and activator of transcription (STAT) 3 is overexpressed or activated in most types of human tumors and has been classified as an oncogene. In the present study, we investigated the contribution of the STAT3s to the proinvasive activity of trefoil factors (TFF) and vascular endothelial growth factor (VEGF) in human colorectal cancer cells HCT8/S11 expressing VEGF receptors. Both intestinal trefoil peptide (TFF3) and VEGF, but not pS2 (TFF1), activate STAT3 signaling through Tyr(705) phosphorylation of both STAT3alpha and STAT3beta isoforms. Blockade of STAT3 signaling by STAT3beta, depletion of the STAT3alpha/beta isoforms by RNA interference, and pharmacologic inhibition of STAT3alpha/beta phosphorylation by cucurbitacin or STAT3 inhibitory peptide abrogates TFF- and VEGF-induced cellular invasion and reduces the growth of HCT8/S11 tumor xenografts in athymic mice. Differential gene expression analysis using DNA microarrays revealed that overexpression of STAT3beta down-regulates the VEGF receptors Flt-1, neuropilins 1 and 2, and the inhibitor of DNA binding/differentiation (Id-2) gene product involved in the neoplastic transformation. Taken together, our data suggest that TFF3 and the essential tumor angiogenesis regulator VEGF(165) exert potent proinvasive activity through STAT3 signaling in human colorectal cancer cells. We also validate new therapeutic strategies targeting STAT3 signaling by pharmacologic inhibitors and RNA interference for the treatment of colorectal cancer patients.

  12. Fabrication of cellular materials

    NASA Astrophysics Data System (ADS)

    Prud'homme, Robert K.; Aksay, Ilhan A.; Garg, Rajeev

    1996-02-01

    Nature uses cellular materials in applications requiring strength while, simultaneously, minimizing raw materials requirements. Minimizing raw materials is efficient both in terms of the energy expended by the organism to synthesize the structure and in terms of the strength- to-weight ratio of the structure. Wood is the most obvious example of cellular bio-materials, and it is the focus of other presentations in this symposium. The lightweight bone structure of birds is another excellent example where weight is a key criterion. The anchoring foot of the common muscle [Mytilus edulis] whereby it attaches itself to objects is a further example of a biological system that uses a foam to fill space and yet conserve on raw materials. In the case of the muscle the foam is water filled and the foot structure distributes stress over a larger area so that the strength of the byssal thread from which it is suspended is matched to the strength of interfacial attachment of the foot to a substrate. In these examples the synthesis and fabrication of the cellular material is directed by intercellular, genetically coded, biochemical reactions. The resulting cell sizes are microns in scale. Cellular materials at the next larger scale are created by organisms at the next higher level of integration. For example an African tree frog lays her eggs in a gas/fluid foam sack she builds on a branch overhanging a pond. The outside of the foam sack hardens in the sun and prevents water evaporation. The foam structure minimizes the amount of fluid that needs to be incorporated into the sack and minimizes its weight. However, as far as the developing eggs are concerned, they are in an aqueous medium, i.e. the continuous fluid phase of the foam. After precisely six days the eggs hatch, and the solidified outer wall re-liquefies and dumps the emerging tadpoles into the pond below. The bee honeycomb is an example of a cellular material with exquisite periodicity at millimeter length scales. The

  13. Membrane potential mediates the cellular binding of nanoparticles

    NASA Astrophysics Data System (ADS)

    Shin, Edwin H.; Li, Ye; Kumar, Umesh; Sureka, Hursh V.; Zhang, Xianren; Payne, Christine K.

    2013-06-01

    The use of nanoparticles for cellular therapeutic or sensing applications requires nanoparticles to bind, or adhere, to the cell surface. While nanoparticle parameters such as size, shape, charge, and composition are important factors in cellular binding, the cell itself must also be considered. All cells have an electrical potential across the plasma membrane driven by an ion gradient. Under standard conditions the ion gradient will result in a -10 to -100 mV potential across the membrane with a net negative charge on the cytosolic face. Using a combination of flow cytometry and fluorescence microscopy experiments and dissipative particle dynamics simulations, we have found that a decrease in membrane potential leads to decreased cellular binding of anionic nanoparticles. The decreased cellular binding of anionic nanoparticles is a general phenomenon, independent of depolarization method, nanoparticle composition, and cell type. Increased membrane potential reverses this trend resulting in increased binding of anionic nanoparticles. The cellular binding of cationic nanoparticles is minimally affected by membrane potential due to the interaction of cationic nanoparticles with cell surface proteins. The influence of membrane potential on the cellular binding of nanoparticles is especially important when considering the use of nanoparticles in the treatment or detection of diseases, such as cancer, in which the membrane potential is decreased.The use of nanoparticles for cellular therapeutic or sensing applications requires nanoparticles to bind, or adhere, to the cell surface. While nanoparticle parameters such as size, shape, charge, and composition are important factors in cellular binding, the cell itself must also be considered. All cells have an electrical potential across the plasma membrane driven by an ion gradient. Under standard conditions the ion gradient will result in a -10 to -100 mV potential across the membrane with a net negative charge on the

  14. 20 CFR 404.1536 - Treatment required for individuals whose drug addiction or alcoholism is a contributing factor...

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... addiction or alcoholism is a contributing factor material to the determination of disability. 404.1536... Treatment required for individuals whose drug addiction or alcoholism is a contributing factor material to the determination of disability. (a) If we determine that you are disabled and drug addiction...

  15. 20 CFR 416.936 - Treatment required for individuals whose drug addiction or alcoholism is a contributing factor...

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... addiction or alcoholism is a contributing factor material to the determination of disability. 416.936... AGED, BLIND, AND DISABLED Determining Disability and Blindness Drug Addiction and Alcoholism § 416.936 Treatment required for individuals whose drug addiction or alcoholism is a contributing factor material...

  16. 20 CFR 416.936 - Treatment required for individuals whose drug addiction or alcoholism is a contributing factor...

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... addiction or alcoholism is a contributing factor material to the determination of disability. 416.936... AGED, BLIND, AND DISABLED Determining Disability and Blindness Drug Addiction and Alcoholism § 416.936 Treatment required for individuals whose drug addiction or alcoholism is a contributing factor material...

  17. 20 CFR 404.1536 - Treatment required for individuals whose drug addiction or alcoholism is a contributing factor...

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... addiction or alcoholism is a contributing factor material to the determination of disability. 404.1536... Treatment required for individuals whose drug addiction or alcoholism is a contributing factor material to the determination of disability. (a) If we determine that you are disabled and drug addiction...

  18. 20 CFR 416.936 - Treatment required for individuals whose drug addiction or alcoholism is a contributing factor...

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... addiction or alcoholism is a contributing factor material to the determination of disability. 416.936... AGED, BLIND, AND DISABLED Determining Disability and Blindness Drug Addiction and Alcoholism § 416.936 Treatment required for individuals whose drug addiction or alcoholism is a contributing factor material...

  19. 20 CFR 404.1536 - Treatment required for individuals whose drug addiction or alcoholism is a contributing factor...

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... addiction or alcoholism is a contributing factor material to the determination of disability. 404.1536... Treatment required for individuals whose drug addiction or alcoholism is a contributing factor material to the determination of disability. (a) If we determine that you are disabled and drug addiction...

  20. 20 CFR 404.1536 - Treatment required for individuals whose drug addiction or alcoholism is a contributing factor...

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... addiction or alcoholism is a contributing factor material to the determination of disability. 404.1536... Treatment required for individuals whose drug addiction or alcoholism is a contributing factor material to the determination of disability. (a) If we determine that you are disabled and drug addiction...

  1. 20 CFR 416.936 - Treatment required for individuals whose drug addiction or alcoholism is a contributing factor...

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... addiction or alcoholism is a contributing factor material to the determination of disability. 416.936... AGED, BLIND, AND DISABLED Determining Disability and Blindness Drug Addiction and Alcoholism § 416.936 Treatment required for individuals whose drug addiction or alcoholism is a contributing factor material...

  2. 20 CFR 416.936 - Treatment required for individuals whose drug addiction or alcoholism is a contributing factor...

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... addiction or alcoholism is a contributing factor material to the determination of disability. 416.936... AGED, BLIND, AND DISABLED Determining Disability and Blindness Drug Addiction and Alcoholism § 416.936 Treatment required for individuals whose drug addiction or alcoholism is a contributing factor material...

  3. 20 CFR 404.1536 - Treatment required for individuals whose drug addiction or alcoholism is a contributing factor...

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... addiction or alcoholism is a contributing factor material to the determination of disability. 404.1536... Treatment required for individuals whose drug addiction or alcoholism is a contributing factor material to the determination of disability. (a) If we determine that you are disabled and drug addiction...

  4. Activation of hippocampal nuclear factor-kappa B by retrieval is required for memory reconsolidation.

    PubMed

    Boccia, Mariano; Freudenthal, Ramiro; Blake, Mariano; de la Fuente, Veronica; Acosta, Gabriela; Baratti, Carlos; Romano, Arturo

    2007-12-01

    Initially, memory is labile and requires consolidation to become stable. However, several studies support that consolidated memories can undergo a new period of lability after retrieval. The mechanistic differences of this process, termed reconsolidation, with the consolidation process are under debate, including the participation of hippocampus. Up to this point, few reports describe molecular changes and, in particular, transcription factor (TF) involvement in memory restabilization. Increasing evidence supports the participation of the TF nuclear factor-kappaB (NF-kappaB) in memory consolidation. Here, we demonstrate that the inhibition of NF-kappaB after memory reactivation impairs retention of a hippocampal-dependent inhibitory avoidance task in mice. We used two independent disruptive strategies to reach this conclusion. First, we administered intracerebroventricular or intrahippocampal sulfasalazine, an inhibitor of IKK (IkappaB kinase), the kinase that activates NF-kappaB. Second, we infused intracerebroventricular or intrahippocampal kappaB decoy, a direct inhibitor of NF-kappaB consisting of a double-stranded DNA oligonucleotide that contains the kappaB consensus sequence. When injected immediately after memory retrieval, sulfasalazine or kappaB decoy (Decoy) impaired long-term retention. In contrast, a one base mutated kappaB decoy (mDecoy) had no effect. Furthermore, we also found NF-kappaB activation in the hippocampus, with a peak 15 min after memory retrieval. This activation was earlier than that found during consolidation. Together, these results indicate that NF-kappaB is an important transcriptional regulator in memory consolidation and reconsolidation in hippocampus, although the temporal kinetics of activation differs between the two processes.

  5. Initiation on the divergent Type I cadicivirus IRES: factor requirements and interactions with the translation apparatus

    PubMed Central

    Asnani, Mukta; Pestova, Tatyana V.; Hellen, Christopher U.T.

    2016-01-01

    Cadicivirus (CDV) is unique amongst picornaviruses in having a dicistronic genome with internal ribosomal entry sites (IRESs) preceding both open reading frames. Here, we investigated initiation on the 5′-terminal IRES. We report that the 982-nt long 5′UTR comprises 12 domains (d1-d12), five of which (d8-d12, nts 341–950) constitute a divergent Type I IRES. It comprises central elements (the apex of d10, d11 and the following polypyrimidine tract) that are homologous to corresponding elements in canonical Type 1 IRESs, and non-canonical flanking domains (d8, d9 and d12). In vitro reconstitution revealed that as with canonical Type I IRESs, 48S complex formation requires eukaryotic initiation factors (eIFs) 1, 1A, 2, 3, 4A, 4B and 4G, and the poly(C) binding protein 2 (PCBP2), and starts with specific binding of eIF4G/eIF4A to d11. However, in contrast to canonical Type I IRESs, subsequent recruitment of 43S ribosomal complexes does not require direct interaction of their eIF3 constituent with the IRES-bound eIF4G. On the other hand, the CDV IRES forms a 40S/eIF3/IRES ternary complex, with multiple points of contact. These additional interactions with translational components could potentially stimulate recruitment of the 43S complex and alleviate the necessity for direct eIF4G/eIF3 interaction. PMID:26873921

  6. Glucose ingestion induces an increase in intranuclear nuclear factor kappaB, a fall in cellular inhibitor kappaB, and an increase in tumor necrosis factor alpha messenger RNA by mononuclear cells in healthy human subjects.

    PubMed

    Aljada, Ahmad; Friedman, Jay; Ghanim, Husam; Mohanty, Priya; Hofmeyer, Deborah; Chaudhuri, Ajay; Dandona, Paresh

    2006-09-01

    Because hyperglycemia is a major detrimental factor in the prognosis of acute cardiovascular conditions such as acute myocardial infarction (AMI) and stroke, and because an acute glucose challenge in healthy subjects has been shown to induce oxidative stress in mononuclear cells (MNCs), we have now investigated whether glucose induces inflammatory stress at the cellular and molecular level. Glucose ingestion (75 g in 300 mL water) in healthy human subjects resulted in an increase in intranuclear nuclear factor kappaB (NF-kappaB) binding, the reduction of inhibitor kappaB alpha (IkappaBalpha) protein, and an increase in the activity of inhibitor kappaB kinase (IKK) and the expression of IKKalpha and IKKbeta, the enzymes that phosphorylate IkappaBalpha, in MNCs. Glucose intake caused an increase in NF-kappaB binding to NF-kappaB2, NF-kappaB2a, and NF-kappaB3 sequences in the promoter site of tumor necrosis factor alpha (TNF-alpha) gene along with an increase in the expression of TNF-alpha messenger RNA in MNCs. Membranous p47(phox) subunit, an index of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression and activation, also increased after glucose intake. We conclude that glucose intake induces an immediate increase in intranuclear NF-kappaB binding, a fall in IkappaBalpha, an increase in IKKalpha, IKKbeta, IKK activity, and messenger RNA expression of TNF-alpha in MNCs in healthy subjects. These data are consistent with profound acute pro-inflammatory changes in MNCs after glucose intake.

  7. Nutrient requirements and other factors involved in the culture of human kidney cells on microcarrier beads

    NASA Technical Reports Server (NTRS)

    Lewis, Marian L.; Morrison, Dennis R.

    1987-01-01

    The culture of human kidney cells on microcarrier beads in the Bioprocessing Laboratory at the Johnson Space Center is described. These were the first series of studies performed before and during 1983 to determine optimum conditions, including medium type, bead type and density. The composition of several medium types and the molecular weights of some common culture medium supplements and cellular proteins are included. The microgravity cell-to-bead attachment experiment performed on Space Transportation System Flight 8 is described.

  8. Foxm1 transcription factor is required for lung fibrosis and epithelial-to-mesenchymal transition.

    PubMed

    Balli, David; Ustiyan, Vladimir; Zhang, Yufang; Wang, I-Ching; Masino, Alex J; Ren, Xiaomeng; Whitsett, Jeffrey A; Kalinichenko, Vladimir V; Kalin, Tanya V

    2013-01-23

    Alveolar epithelial cells (AECs) participate in the pathogenesis of pulmonary fibrosis, producing pro-inflammatory mediators and undergoing epithelial-to-mesenchymal transition (EMT). Herein, we demonstrated the critical role of Forkhead Box M1 (Foxm1) transcription factor in radiation-induced pulmonary fibrosis. Foxm1 was induced in AECs following lung irradiation. Transgenic expression of an activated Foxm1 transcript in AECs enhanced radiation-induced pneumonitis and pulmonary fibrosis, and increased the expression of IL-1β, Ccl2, Cxcl5, Snail1, Zeb1, Zeb2 and Foxf1. Conditional deletion of Foxm1 from respiratory epithelial cells decreased radiation-induced pulmonary fibrosis and prevented the increase in EMT-associated gene expression. siRNA-mediated inhibition of Foxm1 prevented TGF-β-induced EMT in vitro. Foxm1 bound to and increased promoter activity of the Snail1 gene, a critical transcriptional regulator of EMT. Expression of Snail1 restored TGF-β-induced loss of E-cadherin in Foxm1-deficient cells in vitro. Lineage-tracing studies demonstrated that Foxm1 increased EMT during radiation-induced pulmonary fibrosis in vivo. Foxm1 is required for radiation-induced pulmonary fibrosis by enhancing the expression of genes critical for lung inflammation and EMT.

  9. Splicing factor TRA2B is required for neural progenitor survival.

    PubMed

    Roberts, Jacqueline M; Ennajdaoui, Hanane; Edmondson, Carina; Wirth, Brunhilde; Sanford, Jeremy R; Chen, Bin

    2014-02-01

    Alternative splicing of pre-mRNAs can rapidly regulate the expression of large groups of proteins. The RNA binding protein TRA2B (SFRS10) plays well-established roles in developmentally regulated alternative splicing during Drosophila sexual differentiation. TRA2B is also essential for mammalian embryogenesis and is implicated in numerous human diseases. Precise regulation of alternative splicing is critical to the development and function of the central nervous system; however, the requirements for specific splicing factors in neurogenesis are poorly understood. This study focuses on the role of TRA2B in mammalian brain development. We show that, during murine cortical neurogenesis, TRA2B is expressed in both neural progenitors and cortical projection neurons. Using cortex-specific Tra2b mutant mice, we show that TRA2B depletion results in apoptosis of the neural progenitor cells as well as disorganization of the cortical plate. Thus, TRA2B is essential for proper development of the cerebral cortex. PMID:23818142

  10. Factors Impacting Habitable Volume Requirements: Results from the 2011 Habitable Volume Workshop

    NASA Technical Reports Server (NTRS)

    Simon, M.; Whitmire, A.; Otto, C.; Neubek, D. (Editor)

    2011-01-01

    This report documents the results of the Habitable Volume Workshop held April 18-21, 2011 in Houston, TX at the Center for Advanced Space Studies-Universities Space Research Association. The workshop was convened by NASA to examine the factors that feed into understanding minimum habitable volume requirements for long duration space missions. While there have been confinement studies and analogs that have provided the basis for the guidance found in current habitability standards, determining the adequacy of the volume for future long duration exploration missions is a more complicated endeavor. It was determined that an improved understanding of the relationship between behavioral and psychosocial stressors, available habitable and net habitable volume, and interior layouts was needed to judge the adequacy of long duration habitat designs. The workshop brought together a multi-disciplinary group of experts from the medical and behavioral sciences, spaceflight, human habitability disciplines and design professionals. These subject matter experts identified the most salient design-related stressors anticipated for a long duration exploration mission. The selected stressors were based on scientific evidence, as well as personal experiences from spaceflight and analogs. They were organized into eight major categories: allocation of space; workspace; general and individual control of environment; sensory deprivation; social monotony; crew composition; physical and medical issues; and contingency readiness. Mitigation strategies for the identified stressors and their subsequent impact to habitat design were identified. Recommendations for future research to address the stressors and mitigating design impacts are presented.

  11. Cellular therapy in tuberculosis.

    PubMed

    Parida, Shreemanta K; Madansein, Rajhmun; Singh, Nalini; Padayatchi, Nesri; Master, Iqbal; Naidu, Kantharuben; Zumla, Alimuddin; Maeurer, Markus

    2015-03-01

    Cellular therapy now offer promise of potential adjunct therapeutic options for treatment of drug-resistant tuberculosis (TB). We review here the role of Mesenchymal stromal cells, (MSCs), as well as other immune effector cells in the therapy of infectious diseases with a focus on TB. MSCs represent a population of tissue-resident non-hematopoietic adult progenitor cells which home into injured tissues increase the proliferative potential of broncho-alveolar stem cells and restore lung epithelium. MSCs have been shown to be immune-modulatory and anti-inflammatory mediated via cell-cell contacts as well as soluble factors. We discuss the functional profile of MSCs and their potential use for adjunct cellular therapy of multi-drug resistant TB, with the aim of limiting tissue damage, and to convert unproductive inflammatory responses into effective anti-pathogen directed immune responses. Adjunct cellular therapy could potentially offer salvage therapy options for patients with drug-resistant TB, increase clinically relevant anti-M.tuberculosis directed immune responses and possibly shorten the duration of anti-TB therapy. PMID:25809753

  12. The forkhead transcription factor, FOXP3, is required for normal pituitary gonadotropin expression in mice.

    PubMed

    Jung, Deborah O; Jasurda, Jake S; Egashira, Noboru; Ellsworth, Buffy S

    2012-05-01

    The hypothalamic-pituitary-gonadal axis is central to normal reproductive function. This pathway begins with the release of gonadotropin-releasing hormone in systematic pulses by the hypothalamus. Gonadotropin-releasing hormone is bound by receptors on gonadotroph cells in the anterior pituitary gland and stimulates the synthesis and secretion of luteinizing hormone and, to some extent, follicle-stimulating hormone. Once stimulated by these glycoprotein hormones, the gonads begin gametogenesis and the synthesis of sex hormones. In humans, mutations of the forkhead transcription factor, FOXP3, lead to an autoimmune disorder known as immunodysregulation, polyendocrinopathy, and enteropathy, X-linked syndrome. Mice with a mutation in the Foxp3 gene have a similar autoimmune syndrome and are infertile. To understand why FOXP3 is required for reproductive function, we are investigating the reproductive phenotype of Foxp3 mutant mice (Foxp3(sf/Y)). Although the gonadotroph cells appear to be intact in Foxp3(sf/Y) mice, luteinizing hormone beta (Lhb) and follicle-stimulating hormone beta (Fshb) expression are significantly decreased, demonstrating that these mice exhibit a hypogonadotropic hypogonadism. Hypothalamic expression of gonadotropin-releasing hormone is not significantly decreased in Foxp3(sf/Y) males. Treatment of Foxp3(sf/Y) males with a gonadotropin-releasing hormone receptor agonist does not rescue expression of Lhb or Fshb. Interestingly, we do not detect Foxp3 expression in the pituitary or hypothalamus, suggesting that the infertility seen in Foxp3(sf/Y) males is a secondary effect, possibly due to loss of FOXP3 in immune cells. Pituitary expression of glycoprotein hormone alpha (Cga) and prolactin (Prl) are significantly reduced in Foxp3(sf/Y) males, whereas the precursor for adrenocorticotropic hormone, pro-opiomelanocortin (Pomc), is increased. Human patients diagnosed with IPEX often exhibit thyroiditis due to destruction of the thyroid gland by

  13. SWI/SNF regulates the cellular response to hypoxia.

    PubMed

    Kenneth, Niall S; Mudie, Sharon; van Uden, Patrick; Rocha, Sonia

    2009-02-13

    Hypoxia induces a variety of cellular responses such as cell cycle arrest, apoptosis, and autophagy. Most of these responses are mediated by the hypoxia-inducible factor-1alpha. To induce target genes, hypoxia-inducible factor-1alpha requires a chromatin environment conducive to allow binding to specific sequences. Here, we have studied the role of the chromatin-remodeling complex SWI/SNF in the cellular response to hypoxia. We find that SWI/SNF is required for several of the cellular responses induced by hypoxia. Surprisingly, hypoxia-inducible factor-1alpha is a direct target of the SWI/SNF chromatin-remodeling complex. SWI/SNF components are found associated with the hypoxia-inducible factor-1alpha promoter and modulation of SWI/SNF levels results in pronounced changes in hypoxia-inducible factor-1alpha expression and its ability to transactivate target genes. Furthermore, impairment of SWI/SNF function renders cells resistant to hypoxia-induced cell cycle arrest. These results reveal a previously uncharacterized dependence of hypoxia signaling on the SWI/SNF complex and demonstrate a new level of control over the hypoxia-inducible factor-1alpha system.

  14. Endocardial Cushion Morphogenesis and Coronary Vessel Development Require Chicken Ovalbumin Upstream Promoter-Transcription Factor II

    PubMed Central

    Lin, Fu-Jung; You, Li-Ru; Yu, Cheng-Tai; Hsu, Wen-Hsin; Tsai, Ming-Jer; Tsai, Sophia Y.

    2013-01-01

    Objective Septal defects and coronary vessel anomalies are common congenital heart defects, yet their ontogeny and the underlying genetic mechanisms are not well understood. Here, we investigated the role of chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII, NR2F2) in cardiac organogenesis. Methods and Results We analyzed embryos deficient in COUP-TFII and observed a spectrum of cardiac defects, including atrioventricular septal defect, thin-walled myocardium, and abnormal coronary morphogenesis. We show by expression analysis that COUP-TFII is expressed in the endocardium and the epicardium but not in the myocardium of the ventricle. Using endothelial-specific COUP-TFII mutants and molecular approaches, we show that COUP-TFII deficiency resulted in endocardial cushion hypoplasia. This was attributed to the reduced growth and survival of atrioventricular cushion mesenchymal cells and defective epithelial-mesenchymal transformation (EMT) in the underlying endocardium. In addition, the endocardial EMT defect was accompanied by downregulation of Snai1, one of the master regulators of EMT, and upregulation of vascular endothelial-cadherin. Furthermore, we show that although COUP-TFII does not play a major role in the formation of epicardial cell cysts, it is critically important for the formation of epicardium. Ablation of COUP-TFII impairs epicardial EMT and coronary plexus formation. Conclusion Our results reveal that COUP-TFII plays cell-autonomous roles in the endocardium and the epicardium for endocardial and epicardial EMT, which are required for proper valve and coronary vessel formation during heart development. PMID:22962329

  15. Phenylalanine is required to promote specific developmental responses and prevents cellular damage in response to ultraviolet light in soybean (Glycine max) during the seed-to-seedling transition.

    PubMed

    Sullivan, Joe H; Muhammad, DurreShahwar; Warpeha, Katherine M

    2014-01-01

    UV-radiation elicits a suite of developmental (photomorphogenic) and protective responses in plants, but responses early post-germination have received little attention, particularly in intensively bred plants of economic importance. We examined germination, hypocotyl elongation, leaf pubescence and subcellular responses of germinating and/or etiolated soybean (Glycine max (L.) Merr.) seedlings in response to treatment with discrete wavelengths of UV-A or UV-B radiation. We demonstrate differential responses of germinating/young soybean seedlings to a range of UV wavelengths that indicate unique signal transduction mechanisms regulate UV-initiated responses. We have investigated how phenylalanine, a key substrate in the phenylpropanoid pathway, may be involved in these responses. Pubescence may be a key location for phenylalanine-derived protective compounds, as UV-B irradiation increased pubescence and accumulation of UV-absorbing compounds within primary leaf pubescence, visualized by microscopy and absorbance spectra. Mass spectrometry analysis of pubescence indicated that sinapic esters accumulate in the UV-irradiated hairs compared to unirradiated primary leaf tissue. Deleterious effects of some UV-B wavelengths on germination and seedling responses were reduced or entirely prevented by inclusion of phenylalanine in the growth media. Key effects of phenylalanine were not duplicated by tyrosine or tryptophan or sucrose, nor is the specificity of response due to the absorbance of phenylalanine itself. These results suggest that in the seed-to-seedling transition, phenylalanine may be a limiting factor in the development of initial mechanisms of UV protection in the developing leaf.

  16. Mitochondrial localization of fission yeast manganese superoxide dismutase is required for its lysine acetylation and for cellular stress resistance and respiratory growth

    SciTech Connect

    Takahashi, Hidekazu; Shirai, Atsuko; Matsuyama, Akihisa; Yoshida, Minoru

    2011-03-04

    Research highlights: {yields} Fission yeast manganese superoxide dismutase (MnSOD) is acetylated. {yields} The mitochondrial targeting sequence (MTS) is required for the acetylation of MnSOD. {yields} The MTS is not crucial for MnSOD activity, but is important for respiratory growth. {yields} Posttranslational regulation of MnSOD differs between budding and fission yeast. -- Abstract: Manganese-dependent superoxide dismutase (MnSOD) is localized in the mitochondria and is important for oxidative stress resistance. Although transcriptional regulation of MnSOD has been relatively well studied, much less is known about the protein's posttranslational regulation. In budding yeast, MnSOD is activated after mitochondrial import by manganese ion incorporation. Here we characterize posttranslational modification of MnSOD in the fission yeast Schizosaccharomyces pombe. Fission yeast MnSOD is acetylated at the 25th lysine residue. This acetylation was diminished by deletion of N-terminal mitochondrial targeting sequence, suggesting that MnSOD is acetylated after import into mitochondria. Mitochondrial localization of MnSOD is not essential for the enzyme activity, but is crucial for oxidative stress resistance and growth under respiratory conditions of fission yeast. These results suggest that, unlike the situation in budding yeast, S. pombe MnSOD is already active even before mitochondrial localization; nonetheless, mitochondrial localization is critical to allow the cell to cope with reactive oxygen species generated inside or outside of mitochondria.

  17. Requirement of CD4-positive T cells for cellular recruitment to the lungs of mice in response to a particulate intratracheal antigen.

    PubMed Central

    Curtis, J L; Byrd, P K; Warnock, M L; Kaltreider, H B

    1991-01-01

    To determine whether CD4+ T cells participate in the recruitment of other lymphocyte subsets to the lungs, we examined pulmonary immune responses in C57BL/6 mice treated in vivo with the MAb GK1.5, either intact (which depletes CD4+ cells) or as F(ab')2 fragments (which block CD4 molecules). After intratracheal challenge with sheep erythrocytes, antigen-primed mice treated with intact GK1.5 had marked decreases in lymphocytes and macrophages in bronchoalveolar lavage fluid and minimal parenchymal inflammation, compared to primed mice treated with an isotype-matched irrelevant antibody or with no antibody. At 7 d after challenge, flow cytometric analysis showed that numbers of Thy 1.2+ and B220+ cells, but not of CD8+ cells, were markedly decreased in lavage fluid of CD4-depleted mice. Similar suppression of the pulmonary immune response to intratracheal challenge was found in primed mice injected repeatedly with F(ab')2 fragments of GK1.5, which did not deplete CD4+ T cells, and in athymic mice. These findings indicate that, in response to a single intratracheal antigen challenge, recruitment to the lungs of leukocytes other than CD8+ T cells depends largely on CD4+ T cells, possibly because of signals requiring T cell activation via interactions with antigen-presenting cells. Images PMID:1680880

  18. Combined structural, biochemical and cellular evidence demonstrates that both FGDF motifs in alphavirus nsP3 are required for efficient replication

    PubMed Central

    Schulte, Tim; Liu, Lifeng; Panas, Marc D.; Thaa, Bastian; Dickson, Nicole; Götte, Benjamin; Achour, Adnane

    2016-01-01

    Recent findings have highlighted the role of the Old World alphavirus non-structural protein 3 (nsP3) as a host defence modulator that functions by disrupting stress granules, subcellular phase-dense RNA/protein structures formed upon environmental stress. This disruption mechanism was largely explained through nsP3-mediated recruitment of the host G3BP protein via two tandem FGDF motifs. Here, we present the 1.9 Å resolution crystal structure of the NTF2-like domain of G3BP-1 in complex with a 25-residue peptide derived from Semliki Forest virus nsP3 (nsP3-25). The structure reveals a poly-complex of G3BP-1 dimers interconnected through the FGDF motifs in nsP3-25. Although in vitro and in vivo binding studies revealed a hierarchical interaction of the two FGDF motifs with G3BP-1, viral growth curves clearly demonstrated that two intact FGDF motifs are required for efficient viral replication. Chikungunya virus nsP3 also binds G3BP dimers via a hierarchical interaction, which was found to be critical for viral replication. These results highlight a conserved molecular mechanism in host cell modulation. PMID:27383630

  19. Phenylalanine Is Required to Promote Specific Developmental Responses and Prevents Cellular Damage in Response to Ultraviolet Light in Soybean (Glycine max) during the Seed-to-Seedling Transition

    PubMed Central

    Sullivan, Joe H.; Muhammad, DurreShahwar; Warpeha, Katherine M.

    2014-01-01

    UV-radiation elicits a suite of developmental (photomorphogenic) and protective responses in plants, but responses early post-germination have received little attention, particularly in intensively bred plants of economic importance. We examined germination, hypocotyl elongation, leaf pubescence and subcellular responses of germinating and/or etiolated soybean (Glycine max (L.) Merr.) seedlings in response to treatment with discrete wavelengths of UV-A or UV-B radiation. We demonstrate differential responses of germinating/young soybean seedlings to a range of UV wavelengths that indicate unique signal transduction mechanisms regulate UV-initiated responses. We have investigated how phenylalanine, a key substrate in the phenylpropanoid pathway, may be involved in these responses. Pubescence may be a key location for phenylalanine-derived protective compounds, as UV-B irradiation increased pubescence and accumulation of UV-absorbing compounds within primary leaf pubescence, visualized by microscopy and absorbance spectra. Mass spectrometry analysis of pubescence indicated that sinapic esters accumulate in the UV-irradiated hairs compared to unirradiated primary leaf tissue. Deleterious effects of some UV-B wavelengths on germination and seedling responses were reduced or entirely prevented by inclusion of phenylalanine in the growth media. Key effects of phenylalanine were not duplicated by tyrosine or tryptophan or sucrose, nor is the specificity of response due to the absorbance of phenylalanine itself. These results suggest that in the seed-to-seedling transition, phenylalanine may be a limiting factor in the development of initial mechanisms of UV protection in the developing leaf. PMID:25549094

  20. Phenylalanine is required to promote specific developmental responses and prevents cellular damage in response to ultraviolet light in soybean (Glycine max) during the seed-to-seedling transition.

    PubMed

    Sullivan, Joe H; Muhammad, DurreShahwar; Warpeha, Katherine M

    2014-01-01

    UV-radiation elicits a suite of developmental (photomorphogenic) and protective responses in plants, but responses early post-germination have received little attention, particularly in intensively bred plants of economic importance. We examined germination, hypocotyl elongation, leaf pubescence and subcellular responses of germinating and/or etiolated soybean (Glycine max (L.) Merr.) seedlings in response to treatment with discrete wavelengths of UV-A or UV-B radiation. We demonstrate differential responses of germinating/young soybean seedlings to a range of UV wavelengths that indicate unique signal transduction mechanisms regulate UV-initiated responses. We have investigated how phenylalanine, a key substrate in the phenylpropanoid pathway, may be involved in these responses. Pubescence may be a key location for phenylalanine-derived protective compounds, as UV-B irradiation increased pubescence and accumulation of UV-absorbing compounds within primary leaf pubescence, visualized by microscopy and absorbance spectra. Mass spectrometry analysis of pubescence indicated that sinapic esters accumulate in the UV-irradiated hairs compared to unirradiated primary leaf tissue. Deleterious effects of some UV-B wavelengths on germination and seedling responses were reduced or entirely prevented by inclusion of phenylalanine in the growth media. Key effects of phenylalanine were not duplicated by tyrosine or tryptophan or sucrose, nor is the specificity of response due to the absorbance of phenylalanine itself. These results suggest that in the seed-to-seedling transition, phenylalanine may be a limiting factor in the development of initial mechanisms of UV protection in the developing leaf. PMID:25549094

  1. Structural Design Requirements and Factors of Safety for Spaceflight Hardware: For Human Spaceflight. Revision A

    NASA Technical Reports Server (NTRS)

    Bernstein, Karen S.; Kujala, Rod; Fogt, Vince; Romine, Paul

    2011-01-01

    This document establishes the structural requirements for human-rated spaceflight hardware including launch vehicles, spacecraft and payloads. These requirements are applicable to Government Furnished Equipment activities as well as all related contractor, subcontractor and commercial efforts. These requirements are not imposed on systems other than human-rated spacecraft, such as ground test articles, but may be tailored for use in specific cases where it is prudent to do so such as for personnel safety or when assets are at risk. The requirements in this document are focused on design rather than verification. Implementation of the requirements is expected to be described in a Structural Verification Plan (SVP), which should describe the verification of each structural item for the applicable requirements. The SVP may also document unique verifications that meet or exceed these requirements with NASA Technical Authority approval.

  2. Central Role of Cellular Senescence in TSLP-Induced Airway Remodeling in Asthma

    PubMed Central

    Wu, Jinxiang; Dong, Fangzheng; Wang, Rui-An; Wang, Junfei; Zhao, Jiping; Yang, Mengmeng; Gong, Wenbin; Cui, Rutao; Dong, Liang

    2013-01-01

    Background Airway remodeling is a repair process that occurs after injury resulting in increased airway hyper-responsiveness in asthma. Thymic stromal lymphopoietin (TSLP), a vital cytokine, plays a critical role in orchestrating, perpetuating and amplifying the inflammatory response in asthma. TSLP is also a critical factor in airway remodeling in asthma. Objectives To examine the role of TSLP-induced cellular senescence in airway remodeling of asthma in vitro and in vivo. Methods Cellular senescence and airway remodeling were examined in lung specimens from patients with asthma using immunohischemical analysis. Both small molecule and shRNA approaches that target the senescent signaling pathways were used to explore the role of cellular senescence in TSLP-induced airway remodeling in vitro. Senescence-Associated β-galactosidase (SA-β-Gal) staining, and BrdU assays were used to detect cellular senescence. In addition, the Stat3-targeted inhibitor, WP1066, was evaluated in an asthma mouse model to determine if inhibiting cellular senescence influences airway remodeling in asthma. Results Activation of cellular senescence as evidenced by checkpoint activation and cell cycle arrest was detected in airway epithelia samples from patients with asthma. Furthermore, TSLP-induced cellular senescence was required for airway remodeling in vitro. In addition, a mouse asthma model indicates that inhibiting cellular senescence blocks airway remodeling and relieves airway resistance. Conclusion TSLP stimulation can induce cellular senescence during airway remodeling in asthma. Inhibiting the signaling pathways of cellular senescence overcomes TSLP-induced airway remodeling. PMID:24167583

  3. Endoplasmic Reticulum Stress-Activated Transcription Factor ATF6α Requires the Disulfide Isomerase PDIA5 To Modulate Chemoresistance

    PubMed Central

    Higa, Arisa; Taouji, Said; Lhomond, Stéphanie; Jensen, Devon; Fernandez-Zapico, Martin E.; Simpson, Jeremy C.; Pasquet, Jean-Max; Schekman, Randy

    2014-01-01

    ATF6α, a membrane-anchored transcription factor from the endoplasmic reticulum (ER) that modulates the cellular response to stress as an effector of the unfolded-protein response (UPR), is a key player in the development of tumors of different origin. ATF6α activation has been linked to oncogenic transformation and tumor maintenance; however, the mechanism(s) underlying this phenomenon remains elusive. Here, using a phenotypic small interfering RNA (siRNA) screening, we identified a novel role for ATF6α in chemoresistance and defined the protein disulfide isomerase A5 (PDIA5) as necessary for ATF6α activation upon ER stress. PDIA5 contributed to disulfide bond rearrangement in ATF6α under stress conditions, thereby leading to ATF6α export from the ER and activation of its target genes. Further analysis of the mechanism demonstrated that PDIA5 promotes ATF6α packaging into coat protein complex II (COPII) vesicles and that the PDIA5/ATF6α activation loop is essential to confer chemoresistance on cancer cells. Genetic and pharmacological inhibition of the PDIA5/ATF6α axis restored sensitivity to the drug treatment. This work defines the mechanisms underlying the role of ATF6α activation in carcinogenesis and chemoresistance; furthermore, it identifies PDIA5 as a key regulator ATF6α-mediated cellular functions in cancer. PMID:24636989

  4. Prostaglandin E2 requirement for transforming growth factor beta 1 inhibition of elicited macrophage 14 kDa phospholipase A2 release.

    PubMed Central

    McCord, M.; Bolognese, B.; Marshall, L. A.

    1995-01-01

    1. Cultured elicited-peritoneal macrophages release a soluble type II 14 kDa phospholipase A2 (PLA2) over time, reaching a plateau by 20-24 h of incubation and maintaining these levels over 72 h. Prostaglandin E2 (PGE2) is also produced but does not plateau until 48-72 h. 2. Transforming growth factor beta 1 (TGF beta 1) reduces cellular 14 kDa PLA2 and its subsequent release by approximately half, but does not alter PGE2 production. Co-incubation of TGF beta 1 with indomethacin interfered, in a concentration-dependent manner, with the ability of TGF beta 1 to reduce cellular 14 kDa PLA2 and its subsequent release over 24 h. The regulation of TGF beta 1 was not specific to indomethacin since other non-steroidal anti-inflammatory drugs had the same effect. This suggested that cyclooxygenase activity was essential for TGF beta 1 to exert its effect and indeed, the addition of exogenous PGE2 restored the TGF beta 1 action. 3. PGE2 alone exerted a concentration-dependent negative feedback action on elicited-macrophage 14 kDa PLA2 release. The inhibitory concentration (IC50 = approximately 180 ng PGE2 ml-1) approximated the PGE2 levels measured in the 24 h macrophage conditioned media (85-140 ng PGE2 ml-1) where PLA2 release began to plateau. Further, incubation of cells with indomethacin over 48 h resulted in the enhancement of 14 kDa PLA2 activity compared to that released from untreated cells. Forskolin failed to inhibit 14 kDa PLA2 release, suggesting PGE2 was not acting through an increase in adenylate cyclase. 4. Taken together, the data are consistent with the immunosuppressive aspects reported for both mediators during inflammation and demonstrates the requirement of PGE2 for TGF beta 1 action on the elicited macrophage. Images Figure 3 PMID:8590973

  5. Cellular: Toward personal communications

    NASA Astrophysics Data System (ADS)

    Heffernan, Stuart

    1991-09-01

    The cellular industry is one of the fastest growing segment of the telecommunications industry. With an estimated penetration rate of 20 percent in the near future, cellular is becoming an ubiquitous telecommunications service in the U.S. In this paper we will examine the major advancements in the cellular industry: customer equipment, cellular networks, engineering tools, customer support, and nationwide seamless service.

  6. Standardized ileal digestible lysine requirements of male pigs immunized against gonadotrophin releasing factor.

    PubMed

    Moore, K L; Mullan, B P; Kim, J C; Dunshea, F R

    2016-05-01

    An experiment was conducted to determine the standardized ileal digestible (SID) Lys requirement of entire male and male pigs immunized against gonadotrophin releasing factor (GnRF; immunocastrates). A total of 420 entire male and immunocastrated (IC) male pigs weighing 60.1 kg BW (SEM 0.49) were used in a 2 × 5 factorial experiment with the main effects being gender (entire males or IC males) and 5 concentrations of SID Lys:DE ratio (0.32, 0.43, 0.54, 0.64, or 0.75 g SID Lys/MJ DE). The diets were fed for 6 wk until slaughter at 107.5 kg BW (SEM 5.72). Over the entire period, IC males had a greater ADG ( < 0.001), greater ADFI ( < 0.001), and lower G:F ( < 0.001) compared with entire males. Immunocastrated males had increased plasma urea nitrogen (PUN) concentrations compared with entire males from d 10 to 42 ( < 0.001 for all days). Plasma urea nitrogen concentration also increased as Lys concentrations increased from d 3 to 42 ( < 0.001 for all days). Using the linear-plateau model, the optimal ADG for entire males was achieved at SID Lys concentrations of 0.68, 0.62, 0.54, and 0.58 g/MJ DE whereas optimal G:F was achieved at SID Lys concentrations of 0.72, 0.60, 0.54, and 0.51 g/MJ DE for the time periods d 0 to 14, d 15 to 28, d 29 to 42, and d 0 to 42, respectively. For IC males, optimal ADG was achieved at SID Lys concentrations of 0.64, 0.43, 0.38, and 0.40 g/MJ DE whereas optimal G:F was achieved at SID Lys concentrations of 0.64, 0.43, 0.36, and 0.42 g/MJ DE for the same respective time periods. Using the quadratic polynomial model, maximum ADG for entire males was achieved at SID Lys concentrations of 0.62 and 0.58 g/MJ DE whereas maximum G:F was achieved at SID Lys concentrations of 0.59 and 0.68 g/MJ DE for d 29 to 42 and d 0 to 42, respectively. For IC pigs, maximum ADG was achieved at SID Lys concentrations of 0.69, 0.54, and 0.64 g/MJ DE whereas maximum G:F was achieved at SID Lys concentrations of 0.81, 0.54, and 0.64 g/MJ DE for d 0 to 14, d 29

  7. Platelet-Derived Stromal Cell-Derived Factor-1 Is Required for the Transformation of Circulating Monocytes into Multipotential Cells

    PubMed Central

    Seta, Noriyuki; Okazaki, Yuka; Miyazaki, Hiroshi; Kato, Takashi; Kuwana, Masataka

    2013-01-01

    Background We previously described a primitive cell population derived from human circulating CD14+ monocytes, named monocyte-derived multipotential cells (MOMCs), which are capable of differentiating into mesenchymal and endothelial lineages. To generate MOMCs in vitro, monocytes are required to bind to fibronectin and be exposed to soluble factor(s) derived from circulating CD14− cells. The present study was conducted to identify factors that induce MOMC differentiation. Methods We cultured CD14+ monocytes on fibronectin in the presence or absence of platelets, CD14− peripheral blood mononuclear cells, platelet-conditioned medium, or candidate MOMC differentiation factors. The transformation of monocytes into MOMCs was assessed by the presence of spindle-shaped adherent cells, CD34 expression, and the potential to differentiate in vitro into mesenchymal and endothelial lineages. Results The presence of platelets or platelet-conditioned medium was required to generate MOMCs from monocytes. A screening of candidate platelet-derived soluble factors identified stromal cell-derived factor (SDF)-1 as a requirement for generating MOMCs. Blocking an interaction between SDF-1 and its receptor CXCR4 inhibited MOMC generation, further confirming SDF-1′s critical role in this process. Finally, circulating MOMC precursors were found to reside in the CD14+CXCR4high cell population. Conclusion The interaction of SDF-1 with CXCR4 is essential for the transformation of circulating monocytes into MOMCs. PMID:24066125

  8. What Factors Are Required for Microbes to Grow, Survive, and Die?

    NASA Astrophysics Data System (ADS)

    Kornacki, Jeffrey L.

    This chapter focuses on the impact of extrinsic and intrinsic factors that impact the growth of bacteria and fungi in foods. A bacterium with a generation time of 20 min can grow from 1 cell to over a million in 7 h. Intrinsic factors that impact microbial growth or survival are those properties within the food itself. Examples of such factors are the amount of available (not chemically bound) water (i.e., water activity), the oxidation/reduction potential (ORP) of the food, its pH, and the type of acid present. Extrinsic factors are those applied to the food such as thermal processes and refrigeration. Sometimes extrinsic factors such as heating result in creation of intrinsic factors such as a reduced ORP. The dynamic interaction between intrinsic and extrinsic factors will have a profound effect on the type of microbiota in the ingredient, food, and factory environment. The extrinsic and intrinsic factors that impact microbial survival and growth in food or in factory niches are manifold and can be quite dynamic. This highlights the need for research to better understand the relationship of microbes to their environments. Food processors should exercise appropriate caution (e.g., via challenge studies, appropriate testing, selection, and monitoring of valid CCPs) when formulating new products. Assumptions about microbial behavior in one product may not necessarily apply to another.

  9. Hierarchical recruitment into nascent ribosomes of assembly factors required for 27SB pre-rRNA processing in Saccharomyces cerevisiae

    PubMed Central

    Talkish, Jason; Zhang, Jingyu; Jakovljevic, Jelena; Horsey, Edward W.; Woolford, John L.

    2012-01-01

    To better define the roles of assembly factors required for eukaryotic ribosome biogenesis, we have focused on one specific step in maturation of yeast 60 S ribosomal subunits: processing of 27SB pre-ribosomal RNA. At least 14 assembly factors, the ‘B-factor’ proteins, are required for this step. These include most of the major functional classes of assembly factors: RNA-binding proteins, scaffolding protein, DEAD-box ATPases and GTPases. We have investigated the mechanisms by which these factors associate with assembling ribosomes. Our data establish a recruitment model in which assembly of the B-factors into nascent ribosomes ultimately leads to the recruitment of the GTPase Nog2. A more detailed analysis suggests that this occurs in a hierarchical manner via two largely independent recruiting pathways that converge on Nog2. Understanding recruitment has allowed us to better determine the order of association of all assembly factors functioning in one step of ribosome assembly. Furthermore, we have identified a novel subcomplex composed of the B-factors Nop2 and Nip7. Finally, we identified a means by which this step in ribosome biogenesis is regulated in concert with cell growth via the TOR protein kinase pathway. Inhibition of TOR kinase decreases association of Rpf2, Spb4, Nog1 and Nog2 with pre-ribosomes. PMID:22735702

  10. Activation of the Transcription Factor NF-[Kappa]B by Retrieval Is Required for Long-Term Memory Reconsolidation

    ERIC Educational Resources Information Center

    Maldonado, Hector; Romano, Arturo; Merlo, Emiliano; Freudenthal, Ramiro

    2005-01-01

    Several studies support that stored memories undergo a new period of consolidation after retrieval. It is not known whether this process, termed reconsolidation, requires the same transcriptional mechanisms involved in consolidation. Increasing evidence supports the participation of the transcription factor NF-[Kappa]B in memory. This was…

  11. Factors Affecting Accuracy and Time Requirements of a Glucose Oxidase-Peroxidase Assay for Determination of Glucose

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Accurate and rapid assays for glucose are desirable for analysis of glucose and starch in food and feedstuffs. An established colorimetric glucose oxidase-peroxidase method for glucose was modified to reduce analysis time, and evaluated for factors that affected accuracy. Time required to perform t...

  12. The proliferative and morphologic responses of a colon carcinoma cell line (LIM 1215) require the production of two autocrine factors.

    PubMed Central

    Sizeland, A M; Burgess, A W

    1991-01-01

    The role of autocrine growth factors in tumor cell growth has been difficult to prove. Our results indicate that more than one autocrine factor is required for the autonomous growth of the LIM 1215 colonic carcinoma cell line. Furthermore, the morphologic changes induced by epidermal growth factor (EGF) are also density dependent and appear to require a synergistic autocrine factor. The serum-free proliferation of the colonic carcinoma cell line LIM 1215 depends on cell density and the presence of EGF (A. Sizeland, S. Bol, and A.W. Burgess, Growth Factors 4:129-143, 1991). At cell densities below 10(4)/cm2, conditioned medium (from cells at a density of 10(5)/cm2) was required for the cells to elicit a mitogenic response to exogenous EGF. At higher cell densities (10(5)/cm2), the cells were independent of both exogenous EGF and conditioned medium. In addition, the EGF receptor was found to be phosphorylated on tyrosine in LIM 1215 cells proliferating at high density, suggesting that the autocrine production of transforming growth factor alpha (TGF alpha) and subsequent ligation to the EGF receptor was occurring. The proliferation of cells at high density was partly inhibited by TGF alpha antibodies but was almost completely inhibited by an antisense oligonucleotide to TGF alpha. The antisense inhibition could be overcome by the addition of EGF, indicating that the effect of the antisense TGF alpha oligonucleotide was on the production of autocrine TGF alpha. LIM 1215 cells were also observed to undergo morphologic changes (spreading and actin cable organization) in response to EGF. These changes were density dependent, but they occurred with a cell density dependence different from that of the proliferative response. These results suggest two possibilities: that the morphologic changes and proliferative responses have different sensitivities to the autocrine factors or that the actions of the autocrine factors are mediated through different signal transduction

  13. 21 CFR 1311.115 - Additional requirements for two-factor authentication.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ..., such as a password or response to a challenge question. (2) Something the practitioner is, biometric... modules or one-time-password devices. (c) If one factor is a biometric, the biometric subsystem...

  14. 21 CFR 1311.115 - Additional requirements for two-factor authentication.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ..., such as a password or response to a challenge question. (2) Something the practitioner is, biometric... modules or one-time-password devices. (c) If one factor is a biometric, the biometric subsystem...

  15. Structural basis for the recognition of cellular mRNA export factor REF by herpes viral proteins HSV-1 ICP27 and HVS ORF57.

    PubMed

    Tunnicliffe, Richard B; Hautbergue, Guillaume M; Kalra, Priti; Jackson, Brian R; Whitehouse, Adrian; Wilson, Stuart A; Golovanov, Alexander P

    2011-01-06

    The herpesvirus proteins HSV-1 ICP27 and HVS ORF57 promote viral mRNA export by utilizing the cellular mRNA export machinery. This function is triggered by binding to proteins of the transcription-export (TREX) complex, in particular to REF/Aly which directs viral mRNA to the TAP/NFX1 pathway and, subsequently, to the nuclear pore for export to the cytoplasm. Here we have determined the structure of the REF-ICP27 interaction interface at atomic-resolution and provided a detailed comparison of the binding interfaces between ICP27, ORF57 and REF using solution-state NMR. Despite the absence of any obvious sequence similarity, both viral proteins bind on the same site of the folded RRM domain of REF, via short but specific recognition sites. The regions of ICP27 and ORF57 involved in binding by REF have been mapped as residues 104-112 and 103-120, respectively. We have identified the pattern of residues critical for REF/Aly recognition, common to both ICP27 and ORF57. The importance of the key amino acid residues within these binding sites was confirmed by site-directed mutagenesis. The functional significance of the ORF57-REF/Aly interaction was also probed using an ex vivo cytoplasmic viral mRNA accumulation assay and this revealed that mutants that reduce the protein-protein interaction dramatically decrease the ability of ORF57 to mediate the nuclear export of intronless viral mRNA. Together these data precisely map amino acid residues responsible for the direct interactions between viral adaptors and cellular REF/Aly and provide the first molecular details of how herpes viruses access the cellular mRNA export pathway.

  16. Structural Basis for the Recognition of Cellular mRNA Export Factor REF by Herpes Viral Proteins HSV-1 ICP27 and HVS ORF57

    PubMed Central

    Tunnicliffe, Richard B.; Hautbergue, Guillaume M.; Kalra, Priti; Jackson, Brian R.; Whitehouse, Adrian; Wilson, Stuart A.; Golovanov, Alexander P.

    2011-01-01

    The herpesvirus proteins HSV-1 ICP27 and HVS ORF57 promote viral mRNA export by utilizing the cellular mRNA export machinery. This function is triggered by binding to proteins of the transcription-export (TREX) complex, in particular to REF/Aly which directs viral mRNA to the TAP/NFX1 pathway and, subsequently, to the nuclear pore for export to the cytoplasm. Here we have determined the structure of the REF-ICP27 interaction interface at atomic-resolution and provided a detailed comparison of the binding interfaces between ICP27, ORF57 and REF using solution-state NMR. Despite the absence of any obvious sequence similarity, both viral proteins bind on the same site of the folded RRM domain of REF, via short but specific recognition sites. The regions of ICP27 and ORF57 involved in binding by REF have been mapped as residues 104–112 and 103–120, respectively. We have identified the pattern of residues critical for REF/Aly recognition, common to both ICP27 and ORF57. The importance of the key amino acid residues within these binding sites was confirmed by site-directed mutagenesis. The functional significance of the ORF57-REF/Aly interaction was also probed using an ex vivo cytoplasmic viral mRNA accumulation assay and this revealed that mutants that reduce the protein-protein interaction dramatically decrease the ability of ORF57 to mediate the nuclear export of intronless viral mRNA. Together these data precisely map amino acid residues responsible for the direct interactions between viral adaptors and cellular REF/Aly and provide the first molecular details of how herpes viruses access the cellular mRNA export pathway. PMID:21253573

  17. Reduced growth factor requirement of keloid-derived fibroblasts may account for tumor growth

    SciTech Connect

    Russell, S.B.; Trupin, K.M.; Rodriguez-Eaton, S.; Russell, J.D.; Trupin, J.S.

    1988-01-01

    Keloids are benign dermal tumors that form during an abnormal wound-healing process is genetically susceptible individuals. Although growth of normal and keloid cells did not differ in medium containing 10% (vol/vol) fetal bovine serum, keloid culture grew to significantly higher densities than normal cells in medium containing 5% (vol/vol) fetal bovine serum, keloid cultures grew to significantly higher densities than normal cells in medium containing 5% (vol/vol) plasma or 1% fetal bovine serum. Conditioned medium from keloid cultures did not stimulate growth of normal cells in plasma nor did it contain detectable platelet-derived growth factor or epidermal growth factor. Keloid fibroblasts responded differently than normal adult fibroblasts to transforming growth factor ..beta... Whereas transforming growth factor ..beta.. reduced growth stimulation by epidermal growth factor in cells from normal adult skin or scars, it enhanced the activity of epidermal growth factor in cells from normal adult skin or scars, it enhanced the activity of epidermal growth factor in cells from keloids. Normal and keloid fibroblasts also responded differently to hydrocortisone: growth was stimulated in normal adult cells and unaffected or inhibited in keloid cells. Fetal fibroblasts resembled keloid cells in their ability to grow in plasma and in their response to hydrocortisone. The ability of keloid fibroblasts to grow to higher cell densities in low-serum medium than cells from normal adult skin or from normal early or mature scars suggests that a reduced dependence on serum growth factors may account for their prolonged growth in vivo. Similarities between keloid and fetal cells suggest that keloids may result from the untimely expression of growth-control mechanism that is developmentally regulated.

  18. [Type 2 diabetes mellitus and cardiovascular risk factors: is comprehensive treatment required?].

    PubMed

    Nadal, Josep Franch; Gutiérrez, Pedro Conthe

    2013-09-01

    Diabetes mellitus, especially type 2, is a metabolic disease involving the coexistence of several cardiovascular risk factors. Affected patients are therefore at high cardiovascular risk (2-3 times higher than that of men in the general population and 2-6 times higher than that of women). Cardiovascular disease is the main cause of death in the diabetic population, followed by cancer. Cardiovascular risk cannot be compared between diabetic patients and persons who have already shown one or more manifestations of cardiovascular disease (such as myocardial infarction). Single risk factors should be evaluated in combination with other risk factors and a person's cardiovascular risk should be individually assessed. Cardiovascular risk assessment in patients with diabetes through current calculations methods is complex because their ability to predict risk in individuals is very low. Studies such as that by Steno have demonstrated the validity of a comprehensive strategy to control all the risk factors present in persons with type 2 diabetes mellitus, which can reduce the development of micro- and macrovascular complications and mortality by almost 50%. The present article reviews each of the classical cardiovascular risk factors (hypertension, dyslipidemia, smoking, obesity, sedentariness) in relation to diabetes, as well as their recommended targets and the benefits of their control. In view of the above, a comprehensive approach is recommended to control the multiple risk factors that can coexist in persons with type 2 diabetes mellitus.

  19. [Type 2 diabetes mellitus and cardiovascular risk factors: is comprehensive treatment required?].

    PubMed

    Nadal, Josep Franch; Gutiérrez, Pedro Conthe

    2013-09-01

    Diabetes mellitus, especially type 2, is a metabolic disease involving the coexistence of several cardiovascular risk factors. Affected patients are therefore at high cardiovascular risk (2-3 times higher than that of men in the general population and 2-6 times higher than that of women). Cardiovascular disease is the main cause of death in the diabetic population, followed by cancer. Cardiovascular risk cannot be compared between diabetic patients and persons who have already shown one or more manifestations of cardiovascular disease (such as myocardial infarction). Single risk factors should be evaluated in combination with other risk factors and a person's cardiovascular risk should be individually assessed. Cardiovascular risk assessment in patients with diabetes through current calculations methods is complex because their ability to predict risk in individuals is very low. Studies such as that by Steno have demonstrated the validity of a comprehensive strategy to control all the risk factors present in persons with type 2 diabetes mellitus, which can reduce the development of micro- and macrovascular complications and mortality by almost 50%. The present article reviews each of the classical cardiovascular risk factors (hypertension, dyslipidemia, smoking, obesity, sedentariness) in relation to diabetes, as well as their recommended targets and the benefits of their control. In view of the above, a comprehensive approach is recommended to control the multiple risk factors that can coexist in persons with type 2 diabetes mellitus. PMID:24444518

  20. Superoxide radicals increase transforming growth factor-{beta}1 and collagen release from human lung fibroblasts via cellular influx through chloride channels

    SciTech Connect

    Qi Shufan Hartog, Gertjan J.M. den; Bast, Aalt

    2009-05-15

    Reactive oxygen species (ROS) have been implicated in the pathogenesis of fibrosis. However, it remains unclear which ROS is the major cause. We hypothesize that superoxide elicits specific toxicity to human lung fibroblasts and plays an important role in the development of pulmonary fibrosis. In this study, superoxide generated from xanthine and xanthine oxidase activated lung fibroblasts by increasing the release of TGF-{beta}1 and collagen. This was associated with increased levels of intracellular superoxide. SOD and tempol, by scavenging respectively extracellular and intracellular superoxide, prevented the activation of fibroblasts induced by exposure to exogenous superoxide, whereas catalase did not. Moreover, hydrogen peroxide did not activate fibroblasts. Apparently, superoxide rather than hydrogen peroxide is involved in the regulation of TGF-{beta}1 and collagen release in lung fibroblasts. The chloride channel blocker, DIDS, inhibited the increase of intracellular superoxide levels induced by exogenous superoxide and consequently prevented the activation of fibroblasts. This suggests that the cellular influx of superoxide through chloride channels is essential for superoxide-induced activation of fibroblasts. ERK1/2 and p38 MAPKs are involved in the intracellular pathway leading to superoxide-induced fibroblasts activation. Superoxide possesses until now undiscovered specific pro-fibrotic properties in human lung fibroblasts. This takes place via the cellular influx of superoxide through chloride channels rather than via the formation of hydrogen peroxide.

  1. The mechanisms involved in seed dormancy alleviation by hydrogen cyanide unravel the role of reactive oxygen species as key factors of cellular signaling during germination.

    PubMed

    Oracz, Krystyna; El-Maarouf-Bouteau, Hayat; Kranner, Ilse; Bogatek, Renata; Corbineau, Françoise; Bailly, Christophe

    2009-05-01

    The physiological dormancy of sunflower (Helianthus annuus) embryos can be overcome during dry storage (after-ripening) or by applying exogenous ethylene or hydrogen cyanide (HCN) during imbibition. The aim of this work was to provide a comprehensive model, based on oxidative signaling by reactive oxygen species (ROS), for explaining the cellular mode of action of HCN in dormancy alleviation. Beneficial HCN effect on germination of dormant embryos is associated with a marked increase in hydrogen peroxide and superoxide anion generation in the embryonic axes. It is mimicked by the ROS-generating compounds methylviologen and menadione but suppressed by ROS scavengers. This increase results from an inhibition of catalase and superoxide dismutase activities and also involves activation of NADPH oxidase. However, it is not related to lipid reserve degradation or gluconeogenesis and not associated with marked changes in the cellular redox status controlled by the glutathione/glutathione disulfide couple. The expression of genes related to ROS production (NADPHox, POX, AO1, and AO2) and signaling (MAPK6, Ser/ThrPK, CaM, and PTP) is differentially affected by dormancy alleviation either during after-ripening or by HCN treatment, and the effect of cyanide on gene expression is likely to be mediated by ROS. It is also demonstrated that HCN and ROS both activate similarly ERF1, a component of the ethylene signaling pathway. We propose that ROS play a key role in the control of sunflower seed germination and are second messengers of cyanide in seed dormancy release.

  2. Binding site requirements and differential representation of TGF factors in nuclear ASF-1 activity.

    PubMed

    Lam, E; Lam, Y K

    1995-09-25

    Activating sequence factor 1 (ASF-1) is a nuclear DNA-binding activity that is found in monocots and dicots. It interacts with several TGACG-containing elements that have been characterized from viral and T-DNA genes, the prototypes of which are the as-1 element of the CaMV 35S promoter and the ocs element from the octopine synthase promoter. This class of cis-acting elements can respond to auxin and salicylic acid treatments. Consistent with these observations, we have shown that ASF-1 can interact with promoter elements of an auxin-inducible tobacco gene GNT35, encoding a glutathione S-transferase. Characterization of the nuclear factors that make up ASF-1 activity in vivo will be an important step toward understanding this induction phenomenon. The TGA family of basic-leucine-zipper (bZIP) proteins are good candidates for the ASF-1 nuclear factor. However, there may be as many as seven distinct TGA genes in Arabidopsis, five of which have now been reported. In this study, we expressed the cDNAs that encode four of these five Arabidopsis TGA factors in vitro and compared their DNA-binding behavior using two types of TGACG-containing elements. With specific antisera prepared against three of the five known Arabidopsis TGA factors, we also investigated the relative abundance of these three proteins within the ASF-1 activities of root and leaf nuclear extracts. Our results indicate that these TGA factors bind to DNA with different degrees of cooperativity and their relative affinity toward as-1 also can differ significantly. The results of a supershift assay suggested that only one of the three TGA factors represented a significant component of nuclear ASF-1 activity. Arabidopsis TGA2 comprises approximately 33 and 50% of the ASF-1 activity detected in root and leaf nuclear extracts respectively. These results suggest that each member of the TGA factor family may be differentially regulated and that they may play different roles by virtue of their distinct DNA

  3. Human genome-wide RNAi screen reveals host factors required for enterovirus 71 replication

    PubMed Central

    Wu, Kan Xing; Phuektes, Patchara; Kumar, Pankaj; Goh, Germaine Yen Lin; Moreau, Dimitri; Chow, Vincent Tak Kwong; Bard, Frederic; Chu, Justin Jang Hann

    2016-01-01

    Enterovirus 71 (EV71) is a neurotropic enterovirus without antivirals or vaccine, and its host-pathogen interactions remain poorly understood. Here we use a human genome-wide RNAi screen to identify 256 host factors involved in EV71 replication in human rhabdomyosarcoma cells. Enrichment analyses reveal overrepresentation in processes like mitotic cell cycle and transcriptional regulation. We have carried out orthogonal experiments to characterize the roles of selected factors involved in cell cycle regulation and endoplasmatic reticulum-associated degradation. We demonstrate nuclear egress of CDK6 in EV71 infected cells, and identify CDK6 and AURKB as resistance factors. NGLY1, which co-localizes with EV71 replication complexes at the endoplasmatic reticulum, supports EV71 replication. We confirm importance of these factors for EV71 replication in a human neuronal cell line and for coxsackievirus A16 infection. A small molecule inhibitor of NGLY1 reduces EV71 replication. This study provides a comprehensive map of EV71 host factors and reveals potential antiviral targets. PMID:27748395

  4. A nexus for cellular homeostasis: the interplay between metabolic and signal transduction pathways.

    PubMed

    Gomes, Ana P; Blenis, John

    2015-08-01

    In multicellular organisms, individual cells have evolved to sense external and internal cues in order to maintain cellular homeostasis and survive under different environmental conditions. Cells efficiently adjust their metabolism to reflect the abundance of nutrients, energy and growth factors. The ability to rewire cellular metabolism between anabolic and catabolic processes is crucial for cells to thrive. Thus, cells have developed, through evolution, metabolic networks that are highly plastic and tightly regulated to meet the requirements necessary to maintain cellular homeostasis. The plasticity of these cellular systems is tightly regulated by complex signaling networks that integrate the intracellular and extracellular information. The coordination of signal transduction and metabolic pathways is essential in maintaining a healthy and rapidly responsive cellular state.

  5. The Glucose Transporter (GLUT4) Enhancer Factor Is Required for Normal Wing Positioning in Drosophila

    PubMed Central

    Yazdani, Umar; Huang, Zhiyu; Terman, Jonathan R.

    2008-01-01

    Many of the transcription factors and target genes that pattern the developing adult remain unknown. In the present study, we find that an ortholog of the poorly understood transcription factor, glucose transporter (GLUT4) enhancer factor (Glut4EF, GEF) [also known as the Huntington's disease gene regulatory region-binding protein (HDBP) 1], plays a critical role in specifying normal wing positioning in adult Drosophila. Glut4EF proteins are zinc-finger transcription factors named for their ability to regulate expression of GLUT4 but nothing is known of Glut4EF's in vivo physiological functions. Here, we identify a family of Glut4EF proteins that are well conserved from Drosophila to humans and find that mutations in Drosophila Glut4EF underlie the wing-positioning defects seen in stretch mutants. In addition, our results indicate that previously uncharacterized mutations in Glut4EF are present in at least 11 publicly available fly lines and on the widely used TM3 balancer chromosome. These results indicate that previous observations utilizing these common stocks may be complicated by the presence of Glut4EF mutations. For example, our results indicate that Glut4EF mutations are also present on the same chromosome as two gain-of-function mutations of the homeobox transcription factor Antennapedia (Antp) and underlie defects previously attributed to Antp. In fact, our results support a role for Glut4EF in the modulation of morphogenetic processes mediated by Antp, further highlighting the importance of Glut4EF transcription factors in patterning and morphogenesis. PMID:18245850

  6. The AP-2 Transcription Factor APTF-2 Is Required for Neuroblast and Epidermal Morphogenesis in Caenorhabditis elegans Embryogenesis

    PubMed Central

    Budirahardja, Yemima; Tan, Pei Yi; Weisdepp, Peter; Zaidel-Bar, Ronen

    2016-01-01

    The evolutionarily conserved family of AP-2 transcription factors (TF) regulates proliferation, differentiation, and apoptosis. Mutations in human AP-2 TF have been linked with bronchio-occular-facial syndrome and Char Syndrome, congenital birth defects characterized by craniofacial deformities and patent ductus arteriosus, respectively. How mutations in AP-2 TF cause the disease phenotypes is not well understood. Here, we characterize the aptf-2(qm27) allele in Caenorhabditis elegans, which carries a point mutation in the conserved DNA binding region of AP-2 TF. We show that compromised APTF-2 activity leads to defects in dorsal intercalation, aberrant ventral enclosure and elongation defects, ultimately culminating in the formation of morphologically deformed larvae or complete arrest during epidermal morphogenesis. Using cell lineaging, we demonstrate that APTF-2 regulates the timing of cell division, primarily in ABarp, D and C cell lineages to control the number of neuroblasts, muscle and epidermal cells. Live imaging revealed nuclear enrichment of APTF-2 in lineages affected by the qm27 mutation preceding the relevant morphogenetic events. Finally, we found that another AP-2 TF, APTF-4, is also essential for epidermal morphogenesis, in a similar yet independent manner. Thus, our study provides novel insight on the cellular-level functions of an AP-2 transcription factor in development. PMID:27176626

  7. The AP-2 Transcription Factor APTF-2 Is Required for Neuroblast and Epidermal Morphogenesis in Caenorhabditis elegans Embryogenesis.

    PubMed

    Budirahardja, Yemima; Tan, Pei Yi; Doan, Thang; Weisdepp, Peter; Zaidel-Bar, Ronen

    2016-05-01

    The evolutionarily conserved family of AP-2 transcription factors (TF) regulates proliferation, differentiation, and apoptosis. Mutations in human AP-2 TF have been linked with bronchio-occular-facial syndrome and Char Syndrome, congenital birth defects characterized by craniofacial deformities and patent ductus arteriosus, respectively. How mutations in AP-2 TF cause the disease phenotypes is not well understood. Here, we characterize the aptf-2(qm27) allele in Caenorhabditis elegans, which carries a point mutation in the conserved DNA binding region of AP-2 TF. We show that compromised APTF-2 activity leads to defects in dorsal intercalation, aberrant ventral enclosure and elongation defects, ultimately culminating in the formation of morphologically deformed larvae or complete arrest during epidermal morphogenesis. Using cell lineaging, we demonstrate that APTF-2 regulates the timing of cell division, primarily in ABarp, D and C cell lineages to control the number of neuroblasts, muscle and epidermal cells. Live imaging revealed nuclear enrichment of APTF-2 in lineages affected by the qm27 mutation preceding the relevant morphogenetic events. Finally, we found that another AP-2 TF, APTF-4, is also essential for epidermal morphogenesis, in a similar yet independent manner. Thus, our study provides novel insight on the cellular-level functions of an AP-2 transcription factor in development. PMID:27176626

  8. Cross-resistance to UV radiation of a cisplatin-resistant human cell line: Overexpression of cellular factors that recognize UV-modified DNA

    SciTech Connect

    Chao, C.C.; Huang, S.L.; Huang, H.M.; Lin-Chao, S. )

    1991-04-01

    A human cell line selected for cisplatin resistance (CPR) was irradiated with UV light and showed cross-resistance to UV light. Applying a modified chloramphenicol acetyltransferase assay, we observed that CPR cells acquired enhanced host cell reactivation of a transfected plasmid carrying UV damage. Gel mobility shift analysis indicated that two nuclear factors that recognize UV-modified DNA were overexpressed in CPR cells. In addition, factors that bind UV-modified DNA were independent from the factors that bind cisplatin-modified DNA. The significance of the identified binding factors, possibly DNA repair enzymes, is discussed.

  9. The Transcriptional Activator Krüppel-like Factor-6 Is Required for CNS Myelination

    PubMed Central

    Mariani, John N.; Zhang, Jingya; Liu, Jia; Sawai, Setsu; Chapouly, Candice; Horng, Sam; Kramer, Elisabeth G.; Loo, Hannah; Burlant, Natalie; Nudelman, German; Lee, Young-Min; Braun, David A.; Lu, Q. Richard; Narla, Goutham; Raine, Cedric S.; Friedman, Scott L.; Casaccia, Patrizia; John, Gareth R.

    2016-01-01

    Growth factors of the gp130 family promote oligodendrocyte differentiation, and viability, and myelination, but their mechanisms of action are incompletely understood. Here, we show that these effects are coordinated, in part, by the transcriptional activator Krüppel-like factor-6 (Klf6). Klf6 is rapidly induced in oligodendrocyte progenitors (OLP) by gp130 factors, and promotes differentiation. Conversely, in mice with lineage-selective Klf6 inactivation, OLP undergo maturation arrest followed by apoptosis, and CNS myelination fails. Overlapping transcriptional and chromatin occupancy analyses place Klf6 at the nexus of a novel gp130-Klf-importin axis, which promotes differentiation and viability in part via control of nuclear trafficking. Klf6 acts as a gp130-sensitive transactivator of the nuclear import factor importin-α5 (Impα5), and interfering with this mechanism interrupts step-wise differentiation. Underscoring the significance of this axis in vivo, mice with conditional inactivation of gp130 signaling display defective Klf6 and Impα5 expression, OLP maturation arrest and apoptosis, and failure of CNS myelination. PMID:27213272

  10. The Transcriptional Activator Krüppel-like Factor-6 Is Required for CNS Myelination.

    PubMed

    Laitman, Benjamin M; Asp, Linnéa; Mariani, John N; Zhang, Jingya; Liu, Jia; Sawai, Setsu; Chapouly, Candice; Horng, Sam; Kramer, Elisabeth G; Mitiku, Nesanet; Loo, Hannah; Burlant, Natalie; Pedre, Xiomara; Hara, Yuko; Nudelman, German; Zaslavsky, Elena; Lee, Young-Min; Braun, David A; Lu, Q Richard; Narla, Goutham; Raine, Cedric S; Friedman, Scott L; Casaccia, Patrizia; John, Gareth R

    2016-05-01

    Growth factors of the gp130 family promote oligodendrocyte differentiation, and viability, and myelination, but their mechanisms of action are incompletely understood. Here, we show that these effects are coordinated, in part, by the transcriptional activator Krüppel-like factor-6 (Klf6). Klf6 is rapidly induced in oligodendrocyte progenitors (OLP) by gp130 factors, and promotes differentiation. Conversely, in mice with lineage-selective Klf6 inactivation, OLP undergo maturation arrest followed by apoptosis, and CNS myelination fails. Overlapping transcriptional and chromatin occupancy analyses place Klf6 at the nexus of a novel gp130-Klf-importin axis, which promotes differentiation and viability in part via control of nuclear trafficking. Klf6 acts as a gp130-sensitive transactivator of the nuclear import factor importin-α5 (Impα5), and interfering with this mechanism interrupts step-wise differentiation. Underscoring the significance of this axis in vivo, mice with conditional inactivation of gp130 signaling display defective Klf6 and Impα5 expression, OLP maturation arrest and apoptosis, and failure of CNS myelination.

  11. 21 CFR 1311.115 - Additional requirements for two-factor authentication.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...) separate from the computer to which the practitioner is gaining access. (b) If one factor is a hard token, it must be separate from the computer to which it is gaining access and must meet at least the criteria of FIPS 140-2 Security Level 1, as incorporated by reference in § 1311.08, for...

  12. Th1 Stimulatory Proteins of Leishmania donovani: Comparative Cellular and Protective Responses of rTriose Phosphate Isomerase, rProtein Disulfide Isomerase and rElongation Factor-2 in Combination with rHSP70 against Visceral Leishmaniasis

    PubMed Central

    Jaiswal, Anil Kumar; Khare, Prashant; Joshi, Sumit; Kushawaha, Pramod Kumar; Sundar, Shyam; Dube, Anuradha

    2014-01-01

    In visceral leishmaniasis, the recovery from the disease is always associated with the generation of Th1-type of cellular responses. Based on this, we have previously identified several Th1-stimulatory proteins of Leishmania donovani -triose phosphate isomerase (TPI), protein disulfide isomerase (PDI) and elongation factor-2 (EL-2) etc. including heat shock protein 70 (HSP70) which induced Th1-type of cellular responses in both cured Leishmania patients/hamsters. Since, HSPs, being the logical targets for vaccines aimed at augmenting cellular immunity and can be early targets in the immune response against intracellular pathogens; they could be exploited as vaccine/adjuvant to induce long-term immunity more effectively. Therefore, in this study, we checked whether HSP70 can further enhance the immunogenicity and protective responses of the above said Th1-stimulatory proteins. Since, in most of the studies, immunogenicity of HSP70 of L. donovani was assessed in native condition, herein we generated recombinant HSP70 and tested its potential to stimulate immune responses in lymphocytes of cured Leishmania infected hamsters as well as in the peripheral blood mononuclear cells (PBMCs) of cured patients of VL either individually or in combination with above mentioned recombinant proteins. rLdHSP70 alone elicited strong cellular responses along with remarkable up-regulation of IFN-γ and IL-12 cytokines and extremely lower level of IL-4 and IL-10. Among the various combinations, rLdHSP70 + rLdPDI emerged as superior one augmenting improved cellular responses followed by rLdHSP70 + rLdEL-2. These combinations were further evaluated for its protective potential wherein rLdHSP70 + rLdPDI again conferred utmost protection (∼80%) followed by rLdHSP70 + rLdEL-2 (∼75%) and generated a strong cellular immune response with significant increase in the levels of iNOS transcript as well as IFN-γ and IL-12 cytokines which was further supported by the high level of IgG2 antibody

  13. Th1 stimulatory proteins of Leishmania donovani: comparative cellular and protective responses of rTriose phosphate isomerase, rProtein disulfide isomerase and rElongation factor-2 in combination with rHSP70 against visceral leishmaniasis.

    PubMed

    Jaiswal, Anil Kumar; Khare, Prashant; Joshi, Sumit; Kushawaha, Pramod Kumar; Sundar, Shyam; Dube, Anuradha

    2014-01-01

    In visceral leishmaniasis, the recovery from the disease is always associated with the generation of Th1-type of cellular responses. Based on this, we have previously identified several Th1-stimulatory proteins of Leishmania donovani -triose phosphate isomerase (TPI), protein disulfide isomerase (PDI) and elongation factor-2 (EL-2) etc. including heat shock protein 70 (HSP70) which induced Th1-type of cellular responses in both cured Leishmania patients/hamsters. Since, HSPs, being the logical targets for vaccines aimed at augmenting cellular immunity and can be early targets in the immune response against intracellular pathogens; they could be exploited as vaccine/adjuvant to induce long-term immunity more effectively. Therefore, in this study, we checked whether HSP70 can further enhance the immunogenicity and protective responses of the above said Th1-stimulatory proteins. Since, in most of the studies, immunogenicity of HSP70 of L. donovani was assessed in native condition, herein we generated recombinant HSP70 and tested its potential to stimulate immune responses in lymphocytes of cured Leishmania infected hamsters as well as in the peripheral blood mononuclear cells (PBMCs) of cured patients of VL either individually or in combination with above mentioned recombinant proteins. rLdHSP70 alone elicited strong cellular responses along with remarkable up-regulation of IFN-γ and IL-12 cytokines and extremely lower level of IL-4 and IL-10. Among the various combinations, rLdHSP70 + rLdPDI emerged as superior one augmenting improved cellular responses followed by rLdHSP70 + rLdEL-2. These combinations were further evaluated for its protective potential wherein rLdHSP70 + rLdPDI again conferred utmost protection (∼80%) followed by rLdHSP70 + rLdEL-2 (∼75%) and generated a strong cellular immune response with significant increase in the levels of iNOS transcript as well as IFN-γ and IL-12 cytokines which was further supported by the high level of IgG2 antibody

  14. Beyond Transcription Factors: The Role of Chromatin Modifying Enzymes in Regulating Transcription Required for Memory

    ERIC Educational Resources Information Center

    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…

  15. Cellular Phone Towers

    MedlinePlus

    ... the call. How are people exposed to the energy from cellular phone towers? As people use cell ... where people can be exposed to them. The energy from a cellular phone tower antenna, like that ...

  16. Biosynthesis of Rhizobium meliloti lipooligosaccharide Nod factors: NodA is required for an N-acyltransferase activity

    SciTech Connect

    Atkinson, E.M.; Long, S.R. ); Palcic, M.M.; Hindsgaul, O. )

    1994-08-30

    Rhizobium bacteria synthesize N-acylated [beta]-1,4-N-acetylglucosamine lipooligosaccharides, called Nod factors, which act as morphogenic signal molecules to legume roots during development of nitrogen-fixing nodules. The biosynthesis of Nod factors is genetically dependent upon the nodulation (nod) genes, including the common nod genes nodABC. We used the Rhizobium meliloti NodH sulfotransferase to prepare [sup 35]S-labeled oligosaccharides which served as metabolic tracers for Nod enzyme activities. This approach provides a general method for following chitooligosaccharide modifications. We found nodAB-dependent conversion of N-acetylchitotetraose (chitotetraose) monosulfate into hydrophobic compounds which by chromatographic and chemical tests were equivalent to acylated Nod factors. Sequential incubation of labeled intermediates with Escherichia coli containing either NodA or NodB showed that NodB was required before NodA during Nod factor biosynthesis. The acylation activity was sensitive to oligosaccharide chain length, with chitotetraose serving as a better substrate than chitobiose or chitotriose. We constructed a putative Nod factor intermediate, GlcN-[beta]1,4-(GlcNac)[sub 3], by enzymatic synthesis and labeled it by NodH-mediated sulfation to create a specific metabolic probe. Acylation of this oligosaccharide required only NodA. These results confirm previous reports that NodB is an N-deacetylase and suggest that NodA is an N-acyltransferase. 31 refs., 6 figs.

  17. Cellular signaling protective against noise-induced hearing loss – A role for novel intrinsic cochlear signaling involving corticotropin-releasing factor?

    PubMed

    Vetter, Douglas E

    2015-09-01

    Hearing loss afflicts approximately 15% of the world's population, and crosses all socioeconomic boundaries. While great strides have been made in understanding the genetic components of syndromic and non-syndromic hearing loss, understanding of the mechanisms underlying noise-induced hearing loss (NIHL) have come much more slowly. NIHL is not simply a mechanism by which older individuals loose their hearing. Significantly, the incidence of NIHL is increasing, and is now involving ever younger populations. This may predict future increased occurrences of hearing loss. Current research has shown that even short-term exposures to loud sounds generating what was previously considered temporary hearing loss, actually produces an almost immediate and permanent loss of specific populations of auditory nerve fibers. Additionally, recurrent exposures to intense sound may hasten age-related hearing loss. While NIHL is a significant medical concern, to date, few compounds have delivered significant protection, arguing that new targets need to be identified. In this commentary, we will explore cellular signaling processes taking place in the cochlea believed to be involved in protection against hearing loss, and highlight new data suggestive of novel signaling not previously recognized as occurring in the cochlea, that is perhaps protective of hearing. This includes a recently described local hypothalamic-pituitary-adrenal axis (HPA)-like signaling system fully contained in the cochlea. This system may represent a local cellular stress-response system based on stress hormone release similar to the systemic HPA axis. Its discovery may hold hope for new drug therapies that can be delivered directly to the cochlea, circumventing systemic side effects.

  18. Hierarchical cellular materials

    SciTech Connect

    Gibson, L.J.

    1991-12-31

    In this paper a method for estimating the contributions of both the composite and the cellular microstructures to the overall material properties and the mechanical efficiency of natural cellular solids will be described. The method will be demonstrated by focusing on the Young`s modulus; similar techniques can be used for other material properties. The results suggest efficient microstructures for engineered cellular materials.

  19. Hierarchical cellular materials

    SciTech Connect

    Gibson, L.J.

    1991-01-01

    In this paper a method for estimating the contributions of both the composite and the cellular microstructures to the overall material properties and the mechanical efficiency of natural cellular solids will be described. The method will be demonstrated by focusing on the Young's modulus; similar techniques can be used for other material properties. The results suggest efficient microstructures for engineered cellular materials.

  20. Muscle cell fate choice requires the T-box transcription factor midline in Drosophila.

    PubMed

    Kumar, Ram P; Dobi, Krista C; Baylies, Mary K; Abmayr, Susan M

    2015-03-01

    Drosophila Midline (Mid) is an ortholog of vertebrate Tbx20, which plays roles in the developing heart, migrating cranial motor neurons, and endothelial cells. Mid functions in cell-fate specification and differentiation of tissues that include the ectoderm, cardioblasts, neuroblasts, and egg chambers; however, a role in the somatic musculature has not been described. We identified mid in genetic and molecular screens for factors contributing to somatic muscle morphogenesis. Mid is expressed in founder cells (FCs) for several muscle fibers, and functions cooperatively with the T-box protein H15 in lateral oblique muscle 1 and the segment border muscle. Mid is particularly important for the specification and development of the lateral transverse (LT) muscles LT3 and LT4, which arise by asymmetric division of a single muscle progenitor. Mid is expressed in this progenitor and its two sibling FCs, but is maintained only in the LT4 FC. Both muscles were frequently missing in mid mutant embryos, and LT4-associated expression of the transcription factor Krüppel (Kr) was lost. When present, LT4 adopted an LT3-like morphology. Coordinately, mid misexpression caused LT3 to adopt an LT4-like morphology and was associated with ectopic Kr expression. From these data, we concluded that mid functions first in the progenitor to direct development of LT3 and LT4, and later in the FCs to influence whichever of these differentiation profiles is selected. Mid is the first T-box factor shown to influence LT3 and LT4 muscle identity and, along with the T-box protein Optomotor-blind-related-gene 1 (Org-1), is representative of a new class of transcription factors in muscle specification.

  1. Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens.

    PubMed

    Zheng, Zuyu; Qamar, Synan Abu; Chen, Zhixiang; Mengiste, Tesfaye

    2006-11-01

    Plant WRKY transcription factors are key regulatory components of plant responses to microbial infection. In addition to regulating the expression of defense-related genes, WRKY transcription factors have also been shown to regulate cross-talk between jasmonate- and salicylate-regulated disease response pathways. The two pathways mediate resistance against different types of microbial pathogens, and there are numerous reports of antagonistic interactions between them. Here we show that mutations of the Arabidopsis WRKY33 gene encoding a WRKY transcription factor cause enhanced susceptibility to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola concomitant with reduced expression of the jasmonate-regulated plant defensin PDF1.2 gene. Ectopic over-expression of WRKY33, on the other hand, increases resistance to the two necrotrophic fungal pathogens. The wrky33 mutants do not show altered responses to a virulent strain of the bacterial pathogen Pseudomonas syringae, although the ectopic expression of WRKY33 results in enhanced susceptibility to this pathogen. The susceptibility of WRKY33-over-expressing plants to P. syringae is associated with reduced expression of the salicylate-regulated PR-1 gene. The WRKY33 transcript is induced in response to pathogen infection, or treatment with salicylate or the paraquat herbicide that generates activated oxygen species in exposed cells. WRKY33 is localized to the nucleus of plant cells and recognizes DNA molecules containing the TTGACC W-box sequence. Together, these results indicate that pathogen-induced WRKY33 is an important transcription factor that regulates the antagonistic relationship between defense pathways mediating responses to P. syringae and necrotrophic pathogens.

  2. Nuclear localization of Src-family tyrosine kinases is required for growth factor-induced euchromatinization

    SciTech Connect

    Takahashi, Akinori; Obata, Yuuki; Fukumoto, Yasunori; Nakayama, Yuji; Kasahara, Kousuke; Kuga, Takahisa; Higashiyama, Yukihiro; Saito, Takashi; Yokoyama, Kazunari K.; Yamaguchi, Naoto

    2009-04-15

    Src-family kinases (SFKs), which participate in various signaling events, are found at not only the plasma membrane but also several subcellular compartments, including the nucleus. Nuclear structural changes are frequently observed during transcription, cell differentiation, senescence, tumorigenesis, and cell cycle. However, little is known about signal transduction in the alteration of chromatin texture. Here, we develop a pixel imaging method for quantitatively evaluating chromatin structural changes. Growth factor stimulation increases euchromatic hypocondensation and concomitant heterochromatic hypercondensation in G{sub 1} phase, and the levels reach a plateau by 30 min, sustain for at least 5 h and return to the basal levels after 24 h. Serum-activated SFKs in the nucleus were more frequently detected in the euchromatin areas than the heterochromatin areas. Nuclear expression of kinase-active SFKs, but not unrelated Syk kinase, drastically increases both euchromatinization and heterochromatinization in a manner dependent on the levels of nuclear tyrosine phosphorylation. However, growth factor stimulation does not induce chromatin structural changes in SYF cells lacking SFKs, and reintroduction of one SFK member into SYF cells can, albeit insufficiently, induce chromatin structural changes. These results suggest that nuclear tyrosine phosphorylation by SFKs plays an important role in chromatin structural changes upon growth factor stimulation.

  3. A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen

    PubMed Central

    Kushner, Nicholas; Zhang, Dong; Touzjian, Neal; Essex, Max; Lieberman, Judy; Lu, Yichen

    2003-01-01

    Anthrax protective antigen (PA) is a 735-aa polypeptide that facilitates the exit of anthrax lethal factor (LF) from the endosome to the cytosol where the toxin acts. We recently found, however, that a fusion protein of the detoxified N-terminal domain of lethal factor (LFn) with a foreign peptide could induce CD8 T cell immune responses in the absence of PA. Because CD8 T cells recognize peptides derived from proteins degraded in the cytosol, this result suggests that lethal factor may be capable of entering the cytosol independently of PA. To investigate this further, the intracellular trafficking of an LFn-enhanced green fluorescent protein fusion protein (LFn-GFP) in the presence or absence of PA was examined by using confocal microscopy. LFn-GFP is able to enter the cytosol without PA. Moreover, it efficiently colocalizes with the proteosome 20s subunit, which degrades proteins into peptides for presentation to CD8 T cells by the MHC class I pathway. We further demonstrate that in the presence of an immune adjuvant LFn fusion protein without PA is able to effectively elicit anti-HIV cytotoxic T lymphocyte in inbred mice. These results indicate that LFn may be used without PA in a protein vaccine as a carrier to deliver antigens into the cytosol for efficient induction of T lymphocyte responses. Furthermore, these results enable us to propose a modified molecular mechanism of anthrax lethal toxin. PMID:12740437

  4. Establishment of Centromeric Chromatin by the CENP-A Assembly Factor CAL1 Requires FACT-Mediated Transcription.

    PubMed

    Chen, Chin-Chi; Bowers, Sarion; Lipinszki, Zoltan; Palladino, Jason; Trusiak, Sarah; Bettini, Emily; Rosin, Leah; Przewloka, Marcin R; Glover, David M; O'Neill, Rachel J; Mellone, Barbara G

    2015-07-01

    Centromeres are essential chromosomal structures that mediate accurate chromosome segregation during cell division. Centromeres are specified epigenetically by the heritable incorporation of the centromeric histone H3 variant CENP-A. While many of the primary factors that mediate centromeric deposition of CENP-A are known, the chromatin and DNA requirements of this process have remained elusive. Here, we uncover a role for transcription in Drosophila CENP-A deposition. Using an inducible ectopic centromere system that uncouples CENP-A deposition from endogenous centromere function and cell-cycle progression, we demonstrate that CENP-A assembly by its loading factor, CAL1, requires RNAPII-mediated transcription of the underlying DNA. This transcription depends on the CAL1 binding partner FACT, but not on CENP-A incorporation. Our work establishes RNAPII passage as a key step in chaperone-mediated CENP-A chromatin establishment and propagation. PMID:26151904

  5. Current good manufacturing practices, quality control procedures, quality factors, notification requirements, and records and reports, for infant formula. Final rule.

    PubMed

    2014-06-10

    The Food and Drug Administration (FDA or we) is issuing a final rule that adopts, with some modifications, the interim final rule (IFR) entitled "Current Good Manufacturing Practices, Quality Control Procedures, Quality Factors, Notification Requirements, and Records and Reports, for Infant Formula'' (February 10, 2014). This final rule affirms the IFR's changes to FDA's regulations and provides additional modifications and clarifications. The final rule also responds to certain comments submitted in response to the request for comments in the IFR.

  6. Current good manufacturing practices, quality control procedures, quality factors, notification requirements, and records and reports, for infant formula. Final rule.

    PubMed

    2014-06-10

    The Food and Drug Administration (FDA or we) is issuing a final rule that adopts, with some modifications, the interim final rule (IFR) entitled "Current Good Manufacturing Practices, Quality Control Procedures, Quality Factors, Notification Requirements, and Records and Reports, for Infant Formula'' (February 10, 2014). This final rule affirms the IFR's changes to FDA's regulations and provides additional modifications and clarifications. The final rule also responds to certain comments submitted in response to the request for comments in the IFR. PMID:24922980

  7. SOX9 modulates the expression of key transcription factors required for heart valve development.

    PubMed

    Garside, Victoria C; Cullum, Rebecca; Alder, Olivia; Lu, Daphne Y; Vander Werff, Ryan; Bilenky, Mikhail; Zhao, Yongjun; Jones, Steven J M; Marra, Marco A; Underhill, T Michael; Hoodless, Pamela A

    2015-12-15

    Heart valve formation initiates when endothelial cells of the heart transform into mesenchyme and populate the cardiac cushions. The transcription factor SOX9 is highly expressed in the cardiac cushion mesenchyme, and is essential for heart valve development. Loss of Sox9 in mouse cardiac cushion mesenchyme alters cell proliferation, embryonic survival, and valve formation. Despite this important role, little is known about how SOX9 regulates heart valve formation or its transcriptional targets. Therefore, we mapped putative SOX9 binding sites by ChIP-Seq in E12.5 heart valves, a stage at which the valve mesenchyme is actively proliferating and initiating differentiation. Embryonic heart valves have been shown to express a high number of genes that are associated with chondrogenesis, including several extracellular matrix proteins and transcription factors that regulate chondrogenesis. Therefore, we compared regions of putative SOX9 DNA binding between E12.5 heart valves and E12.5 limb buds. We identified context-dependent and context-independent SOX9-interacting regions throughout the genome. Analysis of context-independent SOX9 binding suggests an extensive role for SOX9 across tissues in regulating proliferation-associated genes including key components of the AP-1 complex. Integrative analysis of tissue-specific SOX9-interacting regions and gene expression profiles on Sox9-deficient heart valves demonstrated that SOX9 controls the expression of several transcription factors with previously identified roles in heart valve development, including Twist1, Sox4, Mecom and Pitx2. Together, our data identify SOX9-coordinated transcriptional hierarchies that control cell proliferation and differentiation during valve formation.

  8. Runx1 Transcription Factor Is Required for Myoblasts Proliferation during Muscle Regeneration

    PubMed Central

    Umansky, Kfir Baruch; Gruenbaum-Cohen, Yael; Tsoory, Michael; Feldmesser, Ester; Goldenberg, Dalia; Brenner, Ori; Groner, Yoram

    2015-01-01

    Following myonecrosis, muscle satellite cells proliferate, differentiate and fuse, creating new myofibers. The Runx1 transcription factor is not expressed in naïve developing muscle or in adult muscle tissue. However, it is highly expressed in muscles exposed to myopathic damage yet, the role of Runx1 in muscle regeneration is completely unknown. Our study of Runx1 function in the muscle’s response to myonecrosis reveals that this transcription factor is activated and cooperates with the MyoD and AP-1/c-Jun transcription factors to drive the transcription program of muscle regeneration. Mice lacking dystrophin and muscle Runx1 (mdx - /Runx1 f/f), exhibit impaired muscle regeneration leading to age-dependent muscle waste, gradual decrease in motor capabilities and a shortened lifespan. Runx1-deficient primary myoblasts are arrested at cell cycle G1 and consequently differentiate. Such premature differentiation disrupts the myoblasts’ normal proliferation/differentiation balance, reduces the number and size of regenerating myofibers and impairs muscle regeneration. Our combined Runx1-dependent gene expression, ChIP-seq, ATAC-seq and histone H3K4me1/H3K27ac modification analyses revealed a subset of Runx1-regulated genes that are co-occupied by MyoD and c-Jun in mdx - /Runx1 f/f muscle. The data provide unique insights into the transcriptional program driving muscle regeneration and implicate Runx1 as an important participant in the pathology of muscle wasting diseases. PMID:26275053

  9. The LysR Transcription Factor, HexS, Is Required for Glucose Inhibition of Prodigiosin Production by Serratia marcescens.

    PubMed

    Stella, Nicholas A; Fender, James E; Lahr, Roni M; Kalivoda, Eric J; Shanks, Robert M Q

    2012-12-01

    Generation of many useful microbe-derived secondary metabolites, including the red pigment prodigiosin of the bacterium Serratia marcescens, is inhibited by glucose. In a previous report, a genetic approach was used to determine that glucose dehydrogenase activity (GDH) is required for inhibiting prodigiosin production and transcription of the prodigiosin biosynthetic operon (pigA-N). However, the transcription factor(s) that regulate this process were not characterized. Here we tested the hypothesis that HexS, a LysR-family transcription factor similar to LrhA of Escherichia coli, is required for inhibition of prodigiosin by growth in glucose. We observed that mutation of the hexS gene in S. marcescens allowed the precocious production of prodigiosin in glucose-rich medium conditions that completely inhibited prodigiosin production by the wild type. Unlike previously described mutants able to generate prodigiosin in glucose-rich medium, hexS mutants exhibited GDH activity and medium acidification similar to the wild type. Glucose inhibittion of pigA expression was shown to be dependent upon HexS, suggesting that HexS is a key transcription factor in secondary metabolite regulation in response to medium pH. These data give insight into the prodigiosin regulatory pathway and could be used to enhance the production of secondary metabolites.

  10. A Putative Transcription Factor MYT1 Is Required for Female Fertility in the Ascomycete Gibberella zeae

    PubMed Central

    Lin, Yang; Son, Hokyoung; Lee, Jungkwan; Min, Kyunghun; Choi, Gyung Ja; Kim, Jin-Cheol; Lee, Yin-Won

    2011-01-01

    Gibberella zeae is an important pathogen of major cereal crops. The fungus produces ascospores that forcibly discharge from mature fruiting bodies, which serve as the primary inocula for disease epidemics. In this study, we characterized an insertional mutant Z39P105 with a defect in sexual development and identified a gene encoding a putative transcription factor designated as MYT1. This gene contains a Myb DNA-binding domain and is conserved in the subphylum Pezizomycotina of Ascomycota. The MYT1 protein fused with green fluorescence protein localized in nuclei, which supports its role as a transcriptional regulator. The MYT1 deletion mutant showed similar phenotypes to the wild-type strain in vegetative growth, conidia production and germination, virulence, and mycotoxin production, but had defect in female fertility. A mutant overexpressing MYT1 showed earlier germination, faster mycelia growth, and reduced mycotoxin production compared to the wild-type strain, suggesting that improper MYT1 expression affects the expression of genes involved in the cell cycle and secondary metabolite production. This study is the first to characterize a transcription factor containing a Myb DNA-binding domain that is specific to sexual development in G. zeae. PMID:21984921

  11. A transcription factor FgSte12 is required for pathogenicity in Fusarium graminearum.

    PubMed

    Gu, Qin; Zhang, Chengqi; Liu, Xin; Ma, Zhonghua

    2015-01-01

    A conserved mitogen-activated protein kinase (MAPK) cascade homologous to the yeast Fus3/Kss1 mating/filamentation pathway is involved in the regulation of vegetative development and pathogenicity in Fusarium graminearum. However, little is known about the downstream transcription factors of this pathway. In Saccharomyces cerevisiae, the homeodomain protein Ste12 is a key transcription factor activated by Fus3/Kss1. In this study, we characterized a Ste12 orthologue FgSte12 in F. graminearum. The FgSTE12 deletion mutant (ΔFgSte12) was impaired in virulence and in the secretion of cellulase and protease, although it did not show recognizable phenotype changes in hyphal growth, conidiation or deoxynivalenol (DON) biosynthesis. In addition, ΔFgSte12 and the FgGPMK1 (a FUS3/KSS1-related MAPK gene) mutant shared several phenotypic traits. Furthermore, we found that FgGpmk1 controls the nuclear localization of FgSte12. Yeast two-hybrid and affinity capture assays indicated that FgSte12 interacts with the FgSte11-Ste7-Gpmk1 complex. Taken together, these results indicate that FgSte12 is a downstream target of FgSte11-Ste7-Gpmk1 and plays an important role in pathogenicity in F. graminearum.

  12. Is phosphorus intake that exceeds dietary requirements a risk factor in bone health?

    PubMed

    Calvo, Mona S; Tucker, Katherine L

    2013-10-01

    Phosphorus intake in excess of the nutrient needs of healthy adults is thought to disrupt hormonal regulation of phosphorus (P), calcium (Ca), and vitamin D, contributing to impaired peak bone mass, bone resorption, and greater risk of fracture. Elevation of extracellular phosphorus due to excessive intake is thought to be the main stimulus disrupting phosphorus homeostasis in healthy individuals, as it is in renal disease even when intake is modest. If high serum phosphorus is the critical link to the effect of high phosphorus intake on bone health, the issue could be addressed through epidemiologic or dietary studies. However, several confounding factors, including problems estimating accurate phosphorus intake, the influence of a low dietary Ca:P ratio, the acidic nature of phosphorus, the rapid rate of absorption and greater phosphorus bioavailability from processed food such as cola drinks, and circadian fluctuation in serum phosphorus, make this question difficult to address using conventional study designs. These confounding factors are considered in this review, exploring whether phosphorus intake exceeding nutrient needs in healthy individuals disrupts phosphorus regulation and negatively affects bone accretion or loss. Specific attention is given to phosphorus intake from processed foods rich in phosphorus additives, which significantly contribute to phosphorus intake.

  13. A transcription factor FgSte12 is required for pathogenicity in Fusarium graminearum.

    PubMed

    Gu, Qin; Zhang, Chengqi; Liu, Xin; Ma, Zhonghua

    2015-01-01

    A conserved mitogen-activated protein kinase (MAPK) cascade homologous to the yeast Fus3/Kss1 mating/filamentation pathway is involved in the regulation of vegetative development and pathogenicity in Fusarium graminearum. However, little is known about the downstream transcription factors of this pathway. In Saccharomyces cerevisiae, the homeodomain protein Ste12 is a key transcription factor activated by Fus3/Kss1. In this study, we characterized a Ste12 orthologue FgSte12 in F. graminearum. The FgSTE12 deletion mutant (ΔFgSte12) was impaired in virulence and in the secretion of cellulase and protease, although it did not show recognizable phenotype changes in hyphal growth, conidiation or deoxynivalenol (DON) biosynthesis. In addition, ΔFgSte12 and the FgGPMK1 (a FUS3/KSS1-related MAPK gene) mutant shared several phenotypic traits. Furthermore, we found that FgGpmk1 controls the nuclear localization of FgSte12. Yeast two-hybrid and affinity capture assays indicated that FgSte12 interacts with the FgSte11-Ste7-Gpmk1 complex. Taken together, these results indicate that FgSte12 is a downstream target of FgSte11-Ste7-Gpmk1 and plays an important role in pathogenicity in F. graminearum. PMID:24832137

  14. ADAM binding protein Eve-1 is required for ectodomain shedding of epidermal growth factor receptor ligands.

    PubMed

    Tanaka, Motonari; Nanba, Daisuke; Mori, Seiji; Shiba, Fumio; Ishiguro, Hiroshi; Yoshino, Koichiro; Matsuura, Nariaki; Higashiyama, Shigeki

    2004-10-01

    A disintegrin and metalloproteases (ADAMs) are implicated in the ectodomain shedding of epidermal growth factor receptor (EGFR) ligands in EGFR transactivation. However, the activation mechanisms of ADAMs remain elusive. To analyze the regulatory mechanisms of ADAM activation, we performed yeast two-hybrid screening using the cytoplasmic domain of ADAM12 as bait, and identified a protein that we designated Eve-1. Two cDNAs were cloned and characterized. They encode alternatively spliced isoforms of Eve-1, called Eve-1a and Eve-1b, that have four and five tandem Src homology 3 (SH3) domains in the carboxyl-terminal region, respectively, and seven proline-rich SH3 domain binding motifs in the amino-terminal region. The short forms of Eve-1, Eve-1c and Eve-1d, translated at Met-371 are human counterparts of mouse Sh3d19. Northern blot analysis demonstrated that Eve-1 is abundantly expressed in skeletal muscle and heart. Western blot analysis revealed the dominant production of Eve-1c in human cancer cell lines. Knockdown of Eve-1 by small interfering RNA in HT1080 cells reduced the shedding of proHB-EGF induced by angiotensin II and 12-O-tetradecanoylphorbol-13-acetate, as well as the shedding of pro-transforming growth factor-alpha, promphiregulin, and proepiregulin by 12-O-tetradecanoylphorbol-13-acetate, suggesting that Eve-1 plays a role in positively regulating the activity of ADAMs in the signaling of EGFR-ligand shedding.

  15. Transforming Growth Factor Beta 3 Is Required for Excisional Wound Repair In Vivo

    PubMed Central

    Le, Mark; Naridze, Rachelle; Morrison, Jasmine; Biggs, Leah C.; Rhea, Lindsey; Schutte, Brian C.; Kaartinen, Vesa; Dunnwald, Martine

    2012-01-01

    Wound healing is a complex process that relies on proper levels of cytokines and growth factors to successfully repair the tissue. Of particular interest are the members of the transforming growth factor family. There are three TGF-ß isoforms–TGF- ß 1, 2, and 3, each isoform showing a unique expression pattern, suggesting that they each play a distinct function during development and repair. Previous studies reported an exclusive role for TGF-ß 3 in orofacial development and a potent anti-scarring effect. However, the role of TGF- ß 3 in excisional wound healing and keratinocyte migration remains poorly understood. We tested the effect of TGF-ß 3 levels on excisional cutaneous wounds in the adult mouse by directly injecting recombinant TGF-ß 3 or neutralizing antibody against TGF-ß 3 (NAB) in the wounds. Our results demonstrate that TGF-ß 3 does not promote epithelialization. However, TGF-ß 3 is necessary for wound closure as wounds injected with neutralizing antibody against TGF-ß 3 showed increased epidermal volume and proliferation in conjunction with a delay in keratinocyte migration. Wild type keratinocytes treated with NAB and Tgfb3-deficient keratinocytes closed an in vitro scratch wound with no delay, suggesting that our in vivo observations likely result from a paracrine effect. PMID:23110169

  16. Hypoxia-inducible factors are required for chemotherapy resistance of breast cancer stem cells.

    PubMed

    Samanta, Debangshu; Gilkes, Daniele M; Chaturvedi, Pallavi; Xiang, Lisha; Semenza, Gregg L

    2014-12-16

    Triple negative breast cancers (TNBCs) are defined by the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 expression, and are treated with cytotoxic chemotherapy such as paclitaxel or gemcitabine, with a durable response rate of less than 20%. TNBCs are enriched for the basal subtype gene expression profile and the presence of breast cancer stem cells, which are endowed with self-renewing and tumor-initiating properties and resistance to chemotherapy. Hypoxia-inducible factors (HIFs) and their target gene products are highly active in TNBCs. Here, we demonstrate that HIF expression and transcriptional activity are induced by treatment of MDA-MB-231, SUM-149, and SUM-159, which are human TNBC cell lines, as well as MCF-7, which is an ER(+)/PR(+) breast cancer line, with paclitaxel or gemcitabine. Chemotherapy-induced HIF activity enriched the breast cancer stem cell population through interleukin-6 and interleukin-8 signaling and increased expression of multidrug resistance 1. Coadministration of HIF inhibitors overcame the resistance of breast cancer stem cells to paclitaxel or gemcitabine, both in vitro and in vivo, leading to tumor eradication. Increased expression of HIF-1α or HIF target genes in breast cancer biopsies was associated with decreased overall survival, particularly in patients with basal subtype tumors and those treated with chemotherapy alone. Based on these results, clinical trials are warranted to test whether treatment of patients with TNBC with a combination of cytotoxic chemotherapy and HIF inhibitors will improve patient survival.

  17. Detection of cis- and trans-acting Factors in DNA Structure-Induced Genetic Instability Using In silico and Cellular Approaches

    PubMed Central

    Wang, Guliang; Zhao, Junhua; Vasquez, Karen M.

    2016-01-01

    Sequences that can adopt alternative DNA structures (i.e., non-B DNA) are very abundant in mammalian genomes, and recent studies have revealed many important biological functions of non-B DNA structures in chromatin remodeling, DNA replication, transcription, and genetic instability. Here, we provide results from an in silico web-based search engine coupled with cell-based experiments to characterize the roles of non-B DNA conformations in genetic instability in eukaryotes. The purpose of this article is to illustrate strategies that can be used to identify and interrogate the biological roles of non-B DNA structures, particularly on genetic instability. We have included unpublished data using a short H-DNA-forming sequence from the human c-MYC promoter region as an example, and identified two different mechanisms of H-DNA-induced genetic instability in yeast and mammalian cells: a DNA replication-related model of mutagenesis; and a replication-independent cleavage model. Further, we identified candidate proteins involved in H-DNA-induced genetic instability by using a yeast genetic screen. A combination of in silico and cellular methods, as described here, should provide further insight into the contributions of non-B DNA structures in biological functions, genetic evolution, and disease development. PMID:27532010

  18. Detection of cis- and trans-acting Factors in DNA Structure-Induced Genetic Instability Using In silico and Cellular Approaches.

    PubMed

    Wang, Guliang; Zhao, Junhua; Vasquez, Karen M

    2016-01-01

    Sequences that can adopt alternative DNA structures (i.e., non-B DNA) are very abundant in mammalian genomes, and recent studies have revealed many important biological functions of non-B DNA structures in chromatin remodeling, DNA replication, transcription, and genetic instability. Here, we provide results from an in silico web-based search engine coupled with cell-based experiments to characterize the roles of non-B DNA conformations in genetic instability in eukaryotes. The purpose of this article is to illustrate strategies that can be used to identify and interrogate the biological roles of non-B DNA structures, particularly on genetic instability. We have included unpublished data using a short H-DNA-forming sequence from the human c-MYC promoter region as an example, and identified two different mechanisms of H-DNA-induced genetic instability in yeast and mammalian cells: a DNA replication-related model of mutagenesis; and a replication-independent cleavage model. Further, we identified candidate proteins involved in H-DNA-induced genetic instability by using a yeast genetic screen. A combination of in silico and cellular methods, as described here, should provide further insight into the contributions of non-B DNA structures in biological functions, genetic evolution, and disease development. PMID:27532010

  19. Detection of cis- and trans-acting Factors in DNA Structure-Induced Genetic Instability Using In silico and Cellular Approaches.

    PubMed

    Wang, Guliang; Zhao, Junhua; Vasquez, Karen M

    2016-01-01

    Sequences that can adopt alternative DNA structures (i.e., non-B DNA) are very abundant in mammalian genomes, and recent studies have revealed many important biological functions of non-B DNA structures in chromatin remodeling, DNA replication, transcription, and genetic instability. Here, we provide results from an in silico web-based search engine coupled with cell-based experiments to characterize the roles of non-B DNA conformations in genetic instability in eukaryotes. The purpose of this article is to illustrate strategies that can be used to identify and interrogate the biological roles of non-B DNA structures, particularly on genetic instability. We have included unpublished data using a short H-DNA-forming sequence from the human c-MYC promoter region as an example, and identified two different mechanisms of H-DNA-induced genetic instability in yeast and mammalian cells: a DNA replication-related model of mutagenesis; and a replication-independent cleavage model. Further, we identified candidate proteins involved in H-DNA-induced genetic instability by using a yeast genetic screen. A combination of in silico and cellular methods, as described here, should provide further insight into the contributions of non-B DNA structures in biological functions, genetic evolution, and disease development.

  20. The Zinc Finger Transcription Factor Sp9 Is Required for the Development of Striatopallidal Projection Neurons.

    PubMed

    Zhang, Qiangqiang; Zhang, Yue; Wang, Chunyang; Xu, Zhejun; Liang, Qifei; An, Lei; Li, Jiwen; Liu, Zhidong; You, Yan; He, Miao; Mao, Ying; Chen, Bin; Xiong, Zhi-Qi; Rubenstein, John L; Yang, Zhengang

    2016-08-01

    Striatal medium-sized spiny neurons (MSNs), composed of striatonigral and striatopallidal neurons, are derived from the lateral ganglionic eminence (LGE). We find that the transcription factor Sp9 is expressed in LGE progenitors that generate nearly all striatal MSNs and that Sp9 expression is maintained in postmitotic striatopallidal MSNs. Sp9-null mice lose most striatopallidal MSNs because of decreased proliferation of striatopallidal MSN progenitors and increased Bax-dependent apoptosis, whereas the development of striatonigral neurons is largely unaffected. ChIP qPCR provides evidence that Ascl1 directly binds the Sp9 promoter. RNA-seq and in situ hybridization reveal that Sp9 promotes expression of Adora2a, P2ry1, Gpr6, and Grik3 in the LGE and striatum. Thus, Sp9 is crucial for the generation, differentiation, and survival of striatopallidal MSNs. PMID:27452460

  1. Transforming growth factor Beta2 is required for valve remodeling during heart development.

    PubMed

    Azhar, Mohamad; Brown, Kristen; Gard, Connie; Chen, Hwudaurw; Rajan, Sudarsan; Elliott, David A; Stevens, Mark V; Camenisch, Todd D; Conway, Simon J; Doetschman, Thomas

    2011-09-01

    Although the function of transforming growth factor beta2 (TGFβ2) in epithelial mesenchymal transition (EMT) is well studied, its role in valve remodeling remains to be fully explored. Here, we used histological, morphometric, immunohistochemical and molecular approaches and showed that significant dysregulation of major extracellular matrix (ECM) components contributed to valve remodeling defects in Tgfb2(-/-) embryos. The data indicated that cushion mesenchymal cell differentiation was impaired in Tgfb2(-/-) embryos. Hyaluronan and cartilage link protein-1 (CRTL1) were increased in hyperplastic valves of Tgfb2(-/-) embryos, indicating increased expansion and diversification of cushion mesenchyme into the cartilage cell lineage during heart development. Finally, Western blot and immunohistochemistry analyses indicate that the activation of SMAD2/3 was decreased in Tgfb2(-/-) embryos during valve remodeling. Collectively, the data indicate that TGFβ2 promotes valve remodeling and differentiation by inducing matrix organization and suppressing cushion mesenchyme differentiation into cartilage cell lineage during heart development.

  2. Molecular and cellular targets.

    PubMed

    Bode, Ann M; Dong, Zigang

    2006-06-01

    Carcinogenesis is a multistage process consisting of initiation, promotion, and progression stages and each stage may be a possible target for chemopreventive agents. A significant outcome of these investigations on the elucidation of molecular and cellular mechanisms is the explication of signal transduction pathways induced by tumor promoters in cancer development. The current belief today is that cancer may be prevented or treated by targeting specific cancer genes, signaling proteins, and transcription factors. The molecular mechanisms explaining how normal cells undergo neoplastic transformation induced by tumor promoters are rapidly being clarified. Accumulating research evidence suggests that many of dietary factors, including tea compounds, may be used alone or in combination with traditional chemotherapeutic agents to prevent or treat cancer. The potential advantage of many natural or dietary compounds seems to focus on their potent anticancer activity combined with low toxicity and very few adverse side effects. This review summarizes some of our recent work regarding the effects of the various tea components on signal transduction pathways involved in neoplastic cell transformation and carcinogenesis. PMID:16688728

  3. Molecular and Cellular Targets

    PubMed Central

    Bode, Ann M.; Dong, Zigang

    2008-01-01

    Carcinogenesis is a multistage process consisting of initiation, promotion and progression stages and each stage may be a possible target for chemopreventive agents. A significant outcome of these investigations on the elucidation of molecular and cellular mechanisms is the explication of signal transduction pathways induced by tumor promoters in cancer development. The current belief today is that cancer may be prevented or treated by targeting specific cancer genes, signaling proteins and transcription factors. The molecular mechanisms explaining how normal cells undergo neoplastic transformation induced by tumor promoters are rapidly being clarified. Accumulating research evidence suggests that many of dietary factors, including tea compounds, may be used alone or in combination with traditional chemotherapeutic agents to prevent or treat cancer. The potential advantage of many natural or dietary compounds seems to focus on their potent anticancer activity combined with low toxicity and very few adverse side effects. This review summarizes some of our recent work regarding the effects of the various tea components on signal transduction pathways involved in neoplastic cell transformation and carcinogenesis. PMID:16688728

  4. Asymmetric Receptor Contact is Required for Tyrosine Autophosphorylation of Fibroblast Growth Factor Receptor in Living Cells

    SciTech Connect

    Bae, J.; Boggon, T; Tomé, F; Mandiyan, V; Lax, I; Schlessinge, J

    2010-01-01

    Tyrosine autophosphorylation of receptor tyrosine kinases plays a critical role in regulation of kinase activity and in recruitment and activation of intracellular signaling pathways. Autophosphorylation is mediated by a sequential and precisely ordered intermolecular (trans) reaction. In this report we present structural and biochemical experiments demonstrating that formation of an asymmetric dimer between activated FGFR1 kinase domains is required for transphosphorylation of FGFR1 in FGF-stimulated cells. Transphosphorylation is mediated by specific asymmetric contacts between the N-lobe of one kinase molecule, which serves as an active enzyme, and specific docking sites on the C-lobe of a second kinase molecule, which serves a substrate. Pathological loss-of-function mutations or oncogenic activating mutations in this interface may hinder or facilitate asymmetric dimer formation and transphosphorylation, respectively. The experiments presented in this report provide the molecular basis underlying the control of transphosphorylation of FGF receptors and other receptor tyrosine kinases.

  5. Automated work packages architecture: An initial set of human factors and instrumentation and controls requirements

    SciTech Connect

    Agarwal, Vivek; Oxstrand, Johanna H.; Le Blanc, Katya L.

    2014-09-01

    The work management process in current fleets of national nuclear power plants is so highly dependent on large technical staffs and quality of work instruction, i.e., paper-based, that this puts nuclear energy at somewhat of a long-term economic disadvantage and increase the possibility of human errors. Technologies like mobile portable devices and computer-based procedures can play a key role in improving the plant work management process, thereby increasing productivity and decreasing cost. Automated work packages are a fundamentally an enabling technology for improving worker productivity and human performance in nuclear power plants work activities because virtually every plant work activity is accomplished using some form of a work package. As part of this year’s research effort, automated work packages architecture is identified and an initial set of requirements identified, that are essential and necessary for implementation of automated work packages in nuclear power plants.

  6. Induction of metallothionein I by phenolic antioxidants requires metal-activated transcription factor 1 (MTF-1) and zinc.

    PubMed Central

    Bi, Yongyi; Palmiter, Richard D; Wood, Kristi M; Ma, Qiang

    2004-01-01

    Phenolic antioxidants, such as tBHQ [2,5-di-(t-butyl)-1,4-hydroquinone], induce Mt1 (metallothionein 1) gene expression and accumulation of MT protein. Induction of Mt1 mRNA does not depend on protein synthesis, and correlates with oxidation-reduction functions of the antioxidants. In the present study, we analysed the biochemical pathway of the induction. Induction depends on the presence of MTF-1 (metal-activated transcription factor 1), a transcription factor that is required for metal-induced transcription of Mt1, but does not require nuclear factor erythroid 2-related factor 2, a tBHQ-activated CNC bZip (cap 'n' collar basic leucine zipper) protein, that is responsible for regulating genes encoding phase II drug-metabolizing enzymes. Moreover, tBHQ induces the expression of MRE-beta Geo, a reporter gene driven by five metal response elements that constitute an optimal MTF-1 binding site. Reconstitution of Mtf1 -null cells with MTF-1 restores induction by both zinc and tBHQ. Unlike activation of phase II genes by tBHQ, induction of Mt1 expression does not occur in the presence of EDTA, when cells are cultured in zinc-depleted medium, or in cells with reduced intracellular 'free' zinc due to overexpression of ZnT1, a zinc-efflux transporter, indicating that induction requires zinc. In addition, fluorescence imaging reveals that tBHQ increases cytoplasmic free zinc concentration by mobilizing intracellular zinc pools. These findings establish that phenolic antioxidants activate Mt1 transcription by a zinc-dependent mechanism, which involves MTF-1 binding to metal regulator elements in the Mt1 gene promoter. PMID:14998373

  7. Silymarin Suppresses Cellular Inflammation By Inducing Reparative Stress Signaling

    PubMed Central

    Lovelace, Erica S.; Wagoner, Jessica; MacDonald, James; Bammler, Theo; Bruckner, Jacob; Brownell, Jessica; Beyer, Richard; Zink, Erika M.; Kim, Young-Mo; Kyle, Jennifer E.; Webb-Robertson, Bobbie-Jo; Waters, Katrina M.; Metz, Thomas O.; Farin, Federico; Oberlies, Nicholas H.; Polyak, Stephen J.

    2016-01-01

    Silymarin, a characterized extract of the seeds of milk thistle (Silybum marianum), suppresses cellular inflammation. To define how this occurs, transcriptional profiling, metabolomics, and signaling studies were performed in human liver and T cell lines. Cellular stress and metabolic pathways were modulated within 4 h of silymarin treatment: activation of Activating Transcription Factor 4 (ATF-4) and adenosine monophosphate protein kinase (AMPK) and inhibition of mammalian target of rapamycin (mTOR) signaling, the latter being associated with induction of DNA-damage-inducible transcript 4 (DDIT4). Metabolomics analyses revealed silymarin suppression of glycolytic, tricarboxylic acid (TCA) cycle, and amino acid metabolism. Anti-inflammatory effects arose with prolonged (i.e. 24 h) silymarin exposure, with suppression of multiple pro-inflammatory mRNAs and signaling pathways including nuclear factor kappa B (NF-κB) and forkhead box O (FOXO). Studies with murine knock out cells revealed that silymarin inhibition of both mTOR and NF-κB was partially AMPK dependent, while silymarin inhibition of mTOR required DDIT4. Other natural products induced similar stress responses, which correlated with their ability to suppress inflammation. Thus, natural products activate stress and repair responses that culminate in an anti-inflammatory cellular phenotype. Natural products like silymarin may be useful as tools to define how metabolic, stress, and repair pathways regulate cellular inflammation. PMID:26186142

  8. Transforming growth factor-beta requires its target plasminogen activator inhibitor-1 for cytostatic activity.

    PubMed

    Kortlever, Roderik M; Nijwening, Jeroen H; Bernards, René

    2008-09-01

    The cytokine transforming growth factor beta (TGFbeta) has strong antiproliferative activity in most normal cells but contributes to tumor progression in the later stages of oncogenesis. It is not fully understood which TGFbeta target genes are causally involved in mediating its cytostatic activity. We report here that suppression of the TGFbeta target gene encoding plasminogen activator inhibitor-1 (PAI-1) by RNA interference leads to escape from the cytostatic activity of TGFbeta both in human keratinocytes (HaCaTs) and primary mouse embryo fibroblasts. Consistent with this, PAI-1 knock-out mouse embryo fibroblasts are also resistant to TGFbeta growth arrest. Conversely, we show that ectopic expression of PAI-1 in proliferating HaCaT cells induces a growth arrest. PAI-1 knockdown does not interfere with canonical TGFbeta signaling as judged by SMAD phosphorylation and induction of bona fide TGFbeta target genes. Instead, knockdown of PAI-1 results in sustained activation of protein kinase B. Significantly, we find that constitutive protein kinase B activity leads to evasion of the growth-inhibitory action of TGFbeta. Our data are consistent with a model in which induction of PAI-1 by TGFbeta is critical for the induction of proliferation arrest.

  9. Connective Tissue Growth Factor Is Required for Normal Follicle Development and Ovulation

    PubMed Central

    Nagashima, Takashi; Kim, Jaeyeon; Li, Qinglei; Lydon, John P.; DeMayo, Francesco J.; Lyons, Karen M.

    2011-01-01

    Connective tissue growth factor (CTGF) is a cysteine-rich protein the synthesis and secretion of which are hypothesized to be selectively regulated by activins and other members of the TGF-β superfamily. To investigate the in vivo roles of CTGF in female reproduction, we generated Ctgf ovarian and uterine conditional knockout (cKO) mice. Ctgf cKO mice exhibit severe subfertility and multiple reproductive defects including disrupted follicle development, decreased ovulation rates, increased numbers of corpus luteum, and smaller but functionally normal uterine horns. Steroidogenesis is disrupted in the Ctgf cKO mice, leading to increased levels of serum progesterone. We show that disrupted follicle development is accompanied by a significant increase in granulosa cell apoptosis. Moreover, despite normal cumulus expansion, Ctgf cKO mice exhibit a significant decrease in oocytes ovulated, likely due to impaired ovulatory process. During analyses of mRNA expression, we discovered that Ctgf cKO granulosa cells show gene expression changes similar to our previously reported granulosa cell-specific knockouts of activin and Smad4, the common TGF-β family intracellular signaling protein. We also discovered a significant down-regulation of Adamts1, a progesterone-regulated gene that is critical for the remodeling of extracellular matrix surrounding granulosa cells of preovulatory follicles. These findings demonstrate that CTGF is a downstream mediator in TGF-β and progesterone signaling cascades and is necessary for normal follicle development and ovulation. PMID:21868453

  10. Expression of corticotropin-releasing factor in inflamed tissue is required for intrinsic peripheral opioid analgesia.

    PubMed Central

    Schafer, M; Mousa, S A; Zhang, Q; Carter, L; Stein, C

    1996-01-01

    Immune cell-derived opioid peptides can activate opioid receptors on peripheral sensory nerves to inhibit inflammatory pain. The intrinsic mechanisms triggering this neuroimmune interaction are unknown. This study investigates the involvement of endogenous corticotropin-releasing factor (CRF) and interleukin-1beta (IL-1). A specific stress paradigm, cold water swim (CWS), produces potent opioid receptor-specific antinociception in inflamed paws of rats. This effect is dose-dependently attenuated by intraplantar but not by intravenous alpha-helical CRF. IL-1 receptor antagonist is ineffective. Similarly, local injection of antiserum against CRF, but not to IL-1, dose-dependently reverses this effect. Intravenous anti-CRF is only inhibitory at 10(4)-fold higher concentrations and intravenous CRF does not produce analgesia. Pretreatment of inflamed paws with an 18-mer 3'-3'-end inverted CRF-antisense oligodeoxynucleotide abolishes CWS-induced antinociception. The same treatment significantly reduces the amount of CRF extracted from inflamed paws and the number of CRF-immunostained cells without affecting gross inflammatory signs. A mismatch oligodeoxynucleotide alters neither the CWS effect nor CRF immunoreactivity. These findings identify locally expressed CRF as the predominant agent to trigger opioid release within inflamed tissue. Endogenous IL-1, circulating CRF or antiinflammatory effects, are not involved. Thus, an intact immune system plays an essential role in pain control, which is important for the understanding of pain in immunosuppressed patients with cancer or AIDS. Images Fig. 4 PMID:8650225

  11. The putative Drosophila transcription factor woc is required to prevent telomeric fusions.

    PubMed

    Raffa, Grazia D; Cenci, Giovanni; Siriaco, Giorgia; Goldberg, Michael L; Gatti, Maurizio

    2005-12-22

    Woc is a Drosophila zinc finger protein that shares homology with the human polypeptides ZNF261 and ZNF198 implicated in mental retardation and leukemia syndromes. We show that mutations in the woc gene cause frequent telomeric fusions in Drosophila brain cells. Woc localizes to all telomeres and most interbands of polytene chromosomes. In interbands, Woc precisely colocalizes with the initiating forms of RNA polymerase II (Pol II). To characterize the role of woc in telomere maintenance, we analyzed its relationships with Su(var)205, cav, atm, and rad50, four genes that prevent telomeric fusions; Su(var)205 and cav encode HP1 and HP1/ORC Associated Protein (HOAP), respectively. woc mutants displayed normal telomeric accumulations of both HP1 and HOAP, and mutations in cav, Su(var)205, atm, and rad50 did not affect Woc localization on polytene chromosome telomeres. Collectively, our results indicate that Woc is a transcription factor with a telomere-capping function independent of those of Su(var)205, cav, atm, and rad50. PMID:16364909

  12. Trefoil factor 1 is required for the commitment programme of mouse oxyntic epithelial progenitors

    PubMed Central

    Karam, S M; Tomasetto, C; Rio, M-C

    2004-01-01

    Background: Trefoil factor 1 (TFF1/pS2) is a major secretory product of the stomach and TFF1 knockout mice constantly develop adenomas and occasional carcinomas in the pyloric antrum. Aim: To analyse the role of TFF1 in the differentiation of gastric epithelial cell lineages using oxyntic mucosae from normal and TFF1 knockout mice. Methods: The various cell lineages were labelled using specific markers of pit, neck, parietal, and enteroendocrine cells. Patterns of TFF1, TFF2, and TFF3 expressions were defined using western blotting, immunohistochemistry, and/or immunogold electron microscopy. Results: In normal mice, starting from postnatal day 1 (P1), TFF1 and TFF2 were produced by mucus secreting cells of the developing epithelium. At P7, TFF3 expression occurred in pit and parietal cells. When oxyntic glands were compartmentalised, at P21 and in older mice, TFF1 and TFF2 were expressed in pit and neck cells, respectively, and TFF3 was no longer in parietal cells but became a feature of zymogenic cells. In TFF1 deficient mice, alteration of oxyntic epithelial differentiation became obvious at P21, showing significant amplification of pit cells at the expense of parietal cells. At the molecular level, lack of TFF1 induced dramatic inhibition of TFF2 expression and more precocious TFF3 expression. Conclusion: In the oxyntic mucosa, all three TFFs are produced in a lineage specific manner and TFF1 is essential in maintaining the normal commitment programme of epithelial progenitors. PMID:15361486

  13. DipM, a new factor required for peptidoglycan remodelling during cell division in Caulobacter crescentus.

    PubMed

    Möll, Andrea; Schlimpert, Susan; Briegel, Ariane; Jensen, Grant J; Thanbichler, Martin

    2010-07-01

    In bacteria, cytokinesis is dependent on lytic enzymes that facilitate remodelling of the cell wall during constriction. In this work, we identify a thus far uncharacterized periplasmic protein, DipM, that is required for cell division and polarity in Caulobacter crescentus. DipM is composed of four peptidoglycan binding (LysM) domains and a C-terminal lysostaphin-like (LytM) peptidase domain. It binds to isolated murein sacculi in vitro, and is recruited to the site of constriction through interaction with the cell division protein FtsN. Mutational analyses showed that the LysM domains are necessary and sufficient for localization of DipM, while its peptidase domain is essential for function. Consistent with a role in cell wall hydrolysis, DipM was found to interact with purified murein sacculi in vitro and to induce cell lysis upon overproduction. Its inactivation causes severe defects in outer membrane invagination, resulting in a significant delay between cytoplasmic compartmentalization and final separation of the daughter cells. Overall, these findings indicate that DipM is a periplasmic component of the C. crescentus divisome that facilitates remodelling of the peptidoglycan layer and, thus, coordinated constriction of the cell envelope during the division process.

  14. A B lymphocyte mitogen is a Brucella abortus virulence factor required for persistent infection

    PubMed Central

    Spera, Juan Manuel; Ugalde, Juan Esteban; Mucci, Juan; Comerci, Diego J.; Ugalde, Rodolfo Augusto

    2006-01-01

    Microbial pathogens with the ability to establish chronic infections have evolved strategies to actively modulate the host immune response. Brucellosis is a disease caused by a Gram-negative intracellular pathogen that if not treated during the initial phase of the infection becomes chronic as the bacteria persist for the lifespan of the host. How this pathogen and others achieve this action is a largely unanswered question. We report here the identification of a Brucella abortus gene (prpA) directly involved in the immune modulation of the host. PrpA belongs to the proline-racemase family and elicits a B lymphocyte polyclonal activation that depends on the integrity of its proline-racemase catalytic site. Stimulation of splenocytes with PrpA also results in IL-10 secretion. Construction of a B. abortus-prpA mutant allowed us to assess the contribution of PrpA to the infection process. Mice infected with B. abortus induced an early and transient nonresponsive status of splenocytes to both Escherichia coli LPS and ConA. This phenomenon was not observed when mice were infected with a B. abortus-prpA mutant. Moreover, the B. abortus-prpA mutant had a reduced capacity to establish a chronic infection in mice. We propose that an early and transient nonresponsive immune condition of the host mediated by this B cell polyclonal activator is required for establishing a successful chronic infection by Brucella. PMID:17053080

  15. Unique Requirement for ESCRT Factors in Flavivirus Particle Formation on the Endoplasmic Reticulum.

    PubMed

    Tabata, Keisuke; Arimoto, Masaru; Arakawa, Masashi; Nara, Atsuki; Saito, Kazunobu; Omori, Hiroko; Arai, Arisa; Ishikawa, Tomohiro; Konishi, Eiji; Suzuki, Ryosuke; Matsuura, Yoshiharu; Morita, Eiji

    2016-08-30

    Flavivirus infection induces endoplasmic reticulum (ER) membrane rearrangements to generate a compartment for replication of the viral genome and assembly of viral particles. Using quantitative mass spectrometry, we identified several ESCRT (endosomal sorting complex required for transport) proteins that are recruited to sites of virus replication on the ER. Systematic small interfering RNA (siRNA) screening revealed that release of both dengue virus and Japanese encephalitis virus was dramatically decreased by single depletion of TSG101 or co-depletion of specific combinations of ESCRT-III proteins, resulting in ≥1,000-fold titer reductions. By contrast, release was unaffected by depletion of some core ESCRTs, including VPS4. Reintroduction of ESCRT proteins to siRNA-depleted cells revealed interactions among ESCRT proteins that are crucial for flavivirus budding. Electron-microscopy studies revealed that the CHMP2 and CHMP4 proteins function directly in membrane deformation at the ER. Thus, a unique and specific subset of ESCRT contributes to ER membrane biogenesis during flavivirus infection. PMID:27545892

  16. Integrated Control Of Hepatic Lipogenesis Vs. Glucose Production Requires FoxO Transcription Factors

    PubMed Central

    Haeusler, Rebecca A.; Hartil, Kirsten; Vaitheesvaran, Bhavapriya; Arrieta–Cruz, Isabel; Knight, Colette M.; Cook, Joshua R.; Kammoun, Helene L.; Febbraio, Mark A.; Gutierrez–Juarez, Roger; Kurland, Irwin J.; Accili, Domenico

    2014-01-01

    Insulin integrates hepatic glucose and lipid metabolism, directing nutrients to storage as glycogen and triglyceride. In type 2 diabetes, levels of the former are low and the latter are exaggerated, posing a pathophysiologic and therapeutic conundrum. A branching model of insulin signaling, with FoxO1 presiding over glucose production and Srebp–1c regulating lipogenesis, provides a potential explanation. Here we illustrate an alternative mechanism that integrates glucose production and lipogenesis under the unifying control of FoxO. Liver–specific ablation of three FoxOs (L–FoxO1,3,4) prevents the induction of glucose–6–phosphatase and the repression of glucokinase during fasting, thus increasing lipogenesis at the expense of glucose production. We document a similar pattern in the early phases of diet-induced insulin resistance, and propose that FoxOs are required to enable the liver to direct nutritionally derived carbons to glucose vs. lipid metabolism. Our data underscore the heterogeneity of hepatic insulin resistance during progression from the metabolic syndrome to overt diabetes, and the conceptual challenge of designing therapies that curtail glucose production without promoting hepatic lipid accumulation. PMID:25307742

  17. Lipopolysaccharide (LPS)-Induced Biliary Epithelial Cell NRas Activation Requires Epidermal Growth Factor Receptor (EGFR).

    PubMed

    Trussoni, Christy E; Tabibian, James H; Splinter, Patrick L; O'Hara, Steven P

    2015-01-01

    Cholangiocytes (biliary epithelial cells) actively participate in microbe-induced proinflammatory responses in the liver and contribute to inflammatory and infectious cholangiopathies. We previously demonstrated that cholangiocyte TLR-dependent NRas activation contributes to proinflammatory/ proliferative responses. We test the hypothesis that LPS-induced activation of NRas requires the EGFR. SV40-transformed human cholangiocytes (H69 cells), or low passage normal human cholangiocytes (NHC), were treated with LPS in the presence or absence of EGFR or ADAM metallopeptidase domain 17 (TACE) inhibitors. Ras activation assays, quantitative RT-PCR, and proliferation assays were performed in cells cultured with or without inhibitors or an siRNA to Grb2. Immunofluorescence for phospho-EGFR was performed on LPS-treated mouse samples and specimens from patients with primary sclerosing cholangitis, primary biliary cirrhosis, hepatitis C, and normal livers. LPS-treatment induced an association between the TLR/MyD88 and EGFR/Grb2 signaling apparatus, NRas activation, and EGFR phosphorylation. NRas activation was sensitive to EGFR and TACE inhibitors and correlated with EGFR phosphorylation. The TACE inhibitor and Grb2 depletion prevented LPS-induced IL6 expression (p<0.05) and proliferation (p<0.01). Additionally, cholangiocytes from LPS-treated mouse livers and human primary sclerosing cholangitis (PSC) livers exhibited increased phospho-EGFR (p<0.01). Moreover, LPS-induced mouse cholangiocyte proliferation was inhibited by concurrent treatment with the EGFR inhibitor, Erlotinib. Our results suggest that EGFR is essential for LPS-induced, TLR4/MyD88-mediated NRas activation and induction of a robust proinflammatory cholangiocyte response. These findings have implications not only for revealing the signaling potential of TLRs, but also implicate EGFR as an integral component of cholangiocyte TLR-induced proinflammatory processes.

  18. Overview of cellular CDMA

    NASA Astrophysics Data System (ADS)

    Lee, William C. Y.

    1991-05-01

    A general description of code division multiple access (CDMA) is presented. This overview of CDMA highlights the potential of increasing capacity in future cellular communications. The author describes the mobile radio environment and its impact on narrowband and wideband propagation. The advantage of having CDMA in cellular systems is discussed, and the concept of radio capacity in cellular is introduced. The power control schemes in CDMA are analyzed in detail.

  19. Molecular and cellular effects of vitamin B12 in brain, myocardium and liver through its role as co-factor of methionine synthase.

    PubMed

    Guéant, Jean-Louis; Caillerez-Fofou, Maatem; Battaglia-Hsu, Shyuefang; Alberto, Jean-Marc; Freund, Jean-Noel; Dulluc, Isabelle; Adjalla, Charles; Maury, Florence; Merle, Carole; Nicolas, Jean-Pierre; Namour, Fares; Daval, Jean-Luc

    2013-05-01

    Vitamin B12 (cobalamin, cbl) is a cofactor of methionine synthase (MTR) in the synthesis of methionine, the precursor of the universal methyl donor S-Adenosylmethionine (SAM), which is involved in epigenomic regulatory mechanisms. We have established a neuronal cell model with stable expression of a transcobalamin-oleosin chimer and subsequent decreased cellular availability of vitamin B12, which produces reduced proliferation, increased apoptosis and accelerated differentiation through PP2A, NGF and TACE pathways. Anti-transcobalamin antibody or impaired transcobalamin receptor expression produce also impaired proliferation in other cells. Consistently, the transcription, protein expression and activity of MTR are increased in proliferating cells of skin and intestinal epitheliums, in rat intestine crypts and in proliferating CaCo2 cells, while MTR activity correlates with DNA methylation in rat intestine villi. Exposure to nitrous oxide in animal models identified impairment of MTR reaction as the most important metabolic cause of neurological manifestations of B12 deficiency. Early vitamin B12 and folate deprivation during gestation and lactation of a 'dam-progeny' rat model developed in our laboratory is associated with long-lasting disabilities of behavior and memory capacities, with persisting hallmarks related to increased apoptosis, impaired neurogenesis and altered plasticity. We found also an epigenomic deregulation of energy metabolism and fatty acids beta-oxidation in myocardium and liver, through imbalanced methylation/acetylation of PGC-1alpha and decreased expression of SIRT1. These nutrigenomic effects display similarities with the molecular mechanisms of fetal programming. Beside deficiency, B12 loading increases the expression of MTR through internal ribosome entry sites (IRES) and down-regulates MDR-1 gene expression. In conclusion, vitamin B12 influences cell proliferation, differentiation and apoptosis in brain. Vitamin B12 and folate combined

  20. The cellular RNA export receptor TAP/NXF1 is required for ICP27-mediated export of herpes simplex virus 1 RNA, but the TREX complex adaptor protein Aly/REF appears to be dispensable.

    PubMed

    Johnson, Lisa A; Li, Ling; Sandri-Goldin, Rozanne M

    2009-07-01

    Herpes simplex virus 1 (HSV-1) protein ICP27 has been shown to shuttle between the nucleus and cytoplasm and to bind viral RNA during infection. ICP27 was found to interact with the cellular RNA export adaptor protein Aly/REF, which is part of the TREX complex, and to relocalize Aly/REF to viral replication sites. ICP27 is exported to the cytoplasm through the export receptor TAP/NXF1, and ICP27 must be able to interact with TAP/NXF1 for efficient export of HSV-1 early and late transcripts. We examined the dynamics of ICP27 movement and its localization with respect to Aly/REF and TAP/NXF1 in living cells during viral infection. Recombinant viruses with a yellow fluorescent protein (YFP) tag on the N or C terminus of ICP27 were constructed. While the N-terminally tagged ICP27 virus behaved like wild-type HSV-1, the C-terminally tagged virus was defective in viral replication and gene expression, and ICP27 was confined to the nucleus, suggesting that the C-terminal YFP tag interfered with ICP27's C-terminal interactions, including the interaction with TAP/NXF1. To assess the role of Aly/REF and TAP/NXF1 in viral RNA export, these factors were knocked down using small interfering RNA. Knockdown of Aly/REF had little effect on the export of ICP27 or poly(A)(+) RNA during infection. In contrast, a decrease in TAP/NXF1 levels severely impaired export of ICP27 and poly(A)(+) RNA. We conclude that TAP/NXF1 is essential for ICP27-mediated export of RNA during HSV-1 infection, whereas Aly/REF may be dispensable.

  1. Aeromonas salmonicida Infection Only Moderately Regulates Expression of Factors Contributing to Toll-Like Receptor Signaling but Massively Activates the Cellular and Humoral Branches of Innate Immunity in Rainbow Trout (Oncorhynchus mykiss).

    PubMed

    Brietzke, Andreas; Korytář, Tomáš; Jaros, Joanna; Köllner, Bernd; Goldammer, Tom; Seyfert, Hans-Martin; Rebl, Alexander

    2015-01-01

    Toll-like receptors (TLRs) are known to detect a defined spectrum of microbial structures. However, the knowledge about the specificity of teleost Tlr factors for distinct pathogens is limited so far. We measured baseline expression profiles of 18 tlr genes and associated signaling factors in four immune-relevant tissues of rainbow trout Oncorhynchus mykiss. Intraperitoneal injection of a lethal dose of Aeromonas salmonicida subsp. salmonicida induced highly increased levels of cytokine mRNAs during a 72-hour postinfection (hpi) period. In contrast, only the fish-specific tlr22a2 and the downstream factor irak1 featured clearly increased transcript levels, while the mRNA concentrations of many other tlr genes decreased. Flow cytometry quantified cell trafficking after infection indicating a dramatic influx of myeloid cells into the peritoneum and a belated low level immigration of lymphoid cells. T and B lymphocytes were differentiated with RT-qPCR revealing that B lymphocytes emigrated from and T lymphocytes immigrated into head kidney. In conclusion, no specific TLR can be singled out as a dominant receptor for A. salmonicida. The recruitment of cellular factors of innate immunity rather than induced expression of pathogen receptors is hence of key importance for mounting a first immune defense against invading A. salmonicida. PMID:26266270

  2. Aeromonas salmonicida Infection Only Moderately Regulates Expression of Factors Contributing to Toll-Like Receptor Signaling but Massively Activates the Cellular and Humoral Branches of Innate Immunity in Rainbow Trout (Oncorhynchus mykiss)

    PubMed Central

    Brietzke, Andreas; Korytář, Tomáš; Jaros, Joanna; Köllner, Bernd; Goldammer, Tom; Seyfert, Hans-Martin; Rebl, Alexander

    2015-01-01

    Toll-like receptors (TLRs) are known to detect a defined spectrum of microbial structures. However, the knowledge about the specificity of teleost Tlr factors for distinct pathogens is limited so far. We measured baseline expression profiles of 18 tlr genes and associated signaling factors in four immune-relevant tissues of rainbow trout Oncorhynchus mykiss. Intraperitoneal injection of a lethal dose of Aeromonas salmonicida subsp. salmonicida induced highly increased levels of cytokine mRNAs during a 72-hour postinfection (hpi) period. In contrast, only the fish-specific tlr22a2 and the downstream factor irak1 featured clearly increased transcript levels, while the mRNA concentrations of many other tlr genes decreased. Flow cytometry quantified cell trafficking after infection indicating a dramatic influx of myeloid cells into the peritoneum and a belated low level immigration of lymphoid cells. T and B lymphocytes were differentiated with RT-qPCR revealing that B lymphocytes emigrated from and T lymphocytes immigrated into head kidney. In conclusion, no specific TLR can be singled out as a dominant receptor for A. salmonicida. The recruitment of cellular factors of innate immunity rather than induced expression of pathogen receptors is hence of key importance for mounting a first immune defense against invading A. salmonicida. PMID:26266270

  3. Purification of Xenopus laevis mitochondrial RNA polymerase and identification of a dissociable factor required for specific transcription.

    PubMed Central

    Bogenhagen, D F; Insdorf, N F

    1988-01-01

    The Xenopus laevis mitochondrial RNA (mtRNA) polymerase was purified to near homogeneity with an overall yield approaching 50%. The major polypeptides in the final fraction were a doublet of proteins of approximately 140 kilodaltons that copurified with the mtRNA polymerase activity. It appeared likely that the smaller polypeptide is a breakdown product of the larger one. The highly purified polymerase was active in nonspecific transcription but required a dissociable factor for specific transcription of X. laevis mtDNA. The factor could be resolved from mtRNA polymerase by hydrophobic chromatography and had a sedimentation coefficient of 3.0 S. The transcription factor eluted from both the hydrophobic column and a Mono Q anion-exchange column as a single symmetrical peak. The mtRNA polymerase and this factor together are necessary and sufficient for active transcription from four promoters located in a noncoding region of the mtDNA genome between the gene for tRNA(Phe) and the displacement loop. Images PMID:2457154

  4. Interactions of cellular proteins involved in the transcriptional regulation of the human immunodeficiency virus.

    PubMed Central

    Garcia, J A; Wu, F K; Mitsuyasu, R; Gaynor, R B

    1987-01-01

    The human immunodeficiency virus (HIV) is a human retrovirus which is the etiologic agent of the acquired immunodeficiency syndrome. To study the cellular factors involved in the transcriptional regulation of this virus, we performed DNase I footprinting of the viral LTR using partially purified HeLa cell extracts. Five regions of the viral LTR appear critical for DNA binding of cellular proteins. These include the negative regulatory, enhancer, SP1, TATA and untranslated regions. Deletion mutagenesis of these binding domains has significant effects on the basal level of transcription and the ability to be induced by the viral tat protein. Mutations of either the negative regulatory or untranslated regions affect factor binding to the enhancer region. In addition, oligonucleotides complementary to several of the binding domains specifically compete for factor binding. These results suggest that interactions between several distinct cellular proteins are required for HIV transcriptional regulation. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 6. PMID:3428273

  5. Interactions of cellular proteins involved in the transcriptional regulation of the human immunodeficiency virus.

    PubMed

    Garcia, J A; Wu, F K; Mitsuyasu, R; Gaynor, R B

    1987-12-01

    The human immunodeficiency virus (HIV) is a human retrovirus which is the etiologic agent of the acquired immunodeficiency syndrome. To study the cellular factors involved in the transcriptional regulation of this virus, we performed DNase I footprinting of the viral LTR using partially purified HeLa cell extracts. Five regions of the viral LTR appear critical for DNA binding of cellular proteins. These include the negative regulatory, enhancer, SP1, TATA and untranslated regions. Deletion mutagenesis of these binding domains has significant effects on the basal level of transcription and the ability to be induced by the viral tat protein. Mutations of either the negative regulatory or untranslated regions affect factor binding to the enhancer region. In addition, oligonucleotides complementary to several of the binding domains specifically compete for factor binding. These results suggest that interactions between several distinct cellular proteins are required for HIV transcriptional regulation.

  6. Analysis of cellular factors influencing the replication of human immunodeficiency virus type I in human macrophages derived from blood of different healthy donors.

    PubMed

    Eisert, V; Kreutz, M; Becker, K; Königs, C; Alex, U; Rübsamen-Waigmann, H; Andreesen, R; von Briesen, H

    2001-07-20

    We analyzed parameters influencing HIV-1 infectibility of cells of the monocyte/macrophage lineage (MO/MAC) isolated from different healthy donors. The proportion of in vitro-infected cells and replication kinetics in different donor MAC ranged from 0.03 to 99% p24 antigen-positive MAC and from undetectable RT activity up to 5 x 10(6) cpm/ml/90 min, respectively. As a quantitative measurement for HIV-1 susceptibility of donor MO/MAC, we determined TCID(50) values of defined virus stocks which varied up to 3000-fold depending on the donor MAC used for titration. As host factors which may influence the viral infection we determined the expression of virus receptors CD4, CCR5, CXCR4, and CCR3 as well as the secretion of the natural ligands of CCR5, which altogether showed no correlation with HIV-1 infectibility of the cells. Moreover, other MO-derived secretory factors which might affect viral infection of these cells could be excluded. Furthermore, expression of maturation-related antigens CD14, CD16, HLA-DR, and MAX.1/CPM was determined. Analysis of the reverse transcription process revealed that restricted HIV-1 infection was reflected by highly reduced or even undetectable full-length HIV-1 DNA formation, although early and intermediate transcripts appeared, suggesting that viral replication is blocked after entry at the level of early reverse transcription.

  7. Loss of α1,6-fucosyltransferase suppressed liver regeneration: implication of core fucose in the regulation of growth factor receptor-mediated cellular signaling.

    PubMed

    Wang, Yuqin; Fukuda, Tomohiko; Isaji, Tomoya; Lu, Jishun; Gu, Wei; Lee, Ho-Hsun; Ohkubo, Yasuhito; Kamada, Yoshihiro; Taniguchi, Naoyuki; Miyoshi, Eiji; Gu, Jianguo

    2015-02-05

    Core fucosylation is an important post-translational modification, which is catalyzed by α1,6-fucosyltransferase (Fut8). Increased expression of Fut8 has been shown in diverse carcinomas including hepatocarcinoma. In this study, we investigated the role of Fut8 expression in liver regeneration by using the 70% partial hepatectomy (PH) model, and found that Fut8 is also critical for the regeneration of liver. Interestingly, we show that the Fut8 activities were significantly increased in the beginning of PH (~4d), but returned to the basal level in the late stage of PH. Lacking Fut8 led to delayed liver recovery in mice. This retardation mainly resulted from suppressed hepatocyte proliferation, as supported not only by a decreased phosphorylation level of epidermal growth factor (EGF) receptor and hepatocyte growth factor (HGF) receptor in the liver of Fut8(-/-) mice in vivo, but by the reduced response to exogenous EGF and HGF of the primary hepatocytes isolated from the Fut8(-/-) mice. Furthermore, an administration of L-fucose, which can increase GDP-fucose synthesis through a salvage pathway, significantly rescued the delayed liver regeneration of Fut8(+/-) mice. Overall, our study provides the first direct evidence for the involvement of Fut8 in liver regeneration.

  8. The hinge region of Escherichia coli ribosomal protein L7/L12 is required for factor binding and GTP hydrolysis.

    PubMed

    Dey, D; Oleinikov, A V; Traut, R R

    1995-01-01

    A variant form of Escherichia coli ribosomal protein L7/L12 that lacked residues 42 to 52 (L7/L12: delta 42-52) in the hinge region was shown previously to be completely inactive in supporting polyphenylalanine synthesis although it bound to L7/L12 deficient core particles with the normal stoichiometry of four copies per particle (Oleinikov AV, Perroud B, Wang B, Traut RR (1993) J Biol Chem, 268, 917-922). The result suggested that the hinge confers flexibility that is required for activity because the resulting bent conformation allows the distal C-terminal domain to occupy a location on the body of the large ribosomal subunit proximal to the base of the L7/L12 stalk where elongation factors bind. Factor binding to the hinge-truncated variant was tested. As an alternative strategy to deleting residues from the hinge, seven amino acid residues within the putative hinge region were replaced by seven consecutive proline residues in an attempt to confer increased rigidity that might reduce or eliminate the bending of the molecule inferred to be functionally important. This variant, L7/L12:(Pro)7, remained fully active in protein synthesis. Whereas the binding of both factors in ribosomes containing L7/L12:delta 42-52 was decreased by about 50%, there was no loss of factor binding in ribosomes containing L7/L12:(Pro)7, as predicted from the retention of protein synthesis activity. The factor:ribosome complexes that contained L7/L12:delta 42-52 had the same low level of GTP hydrolysis as the core particles completely lacking L7/L12 and EF-G did not support translocation measured by the reaction of phe-tRNA bound in the A site with puromycin. It is concluded that the hinge region is required for the functionally productive binding of elongation factors, and the defect in protein synthesis reported previously is due to this defect. The variant produced by the introduction of the putative rigid Pro7 sequence retains sufficient flexibility for full activity.

  9. Cellular manufacturing for clinical applications.

    PubMed

    Sheu, Jonathan; Klassen, Henry; Bauer, Gerhard

    2014-01-01

    Rapid progress has been made in the development of novel cell-based approaches for the potential treatment of retinal degenerative diseases. As a result, one must consider carefully the conditions under which these therapeutics are manufactured if they are to be used in clinical studies or, ultimately, be approved as licensed cellular therapeutics. Here, we describe the principles behind the manufacturing of clinical-grade cellular products, as well as potential methods for large-scale expansion and processing according to Good Manufacturing Practice (GMP) standards sets by the United States Food and Drug Administration. Standards for personnel, materials, procedures, and facilities required for such manufacturing processes are reviewed. We also discuss current and future scale-up methods for the manufacturing of large doses of cellular therapeutics under GMP conditions and compare the use of conventional culture methods such as tissue culture flasks and multi-layered cell factories with novel systems such as closed system hollow-fiber bioreactors. Incorporation of these novel bioreactor systems into GMP facilities may enable us to provide adequate cell numbers for multi-center clinical trials and paves the way for development of cellular therapeutics with the potential to treat very large numbers of patients.

  10. Hijacking cellular garbage cans.

    PubMed

    Welsch, Sonja; Locker, Jacomine Krijnse

    2010-06-25

    Viruses are perfect opportunists that have evolved to modify numerous cellular processes in order to complete their replication cycle in the host cell. An article by Reggiori and coworkers in this issue of Cell Host & Microbe reveals how coronaviruses can divert a cellular quality control pathway that normally functions in degradation of mis-folded proteins to replicate the viral genome. PMID:20542246

  11. Release of platelet-activating factor (PAF) and histamine. II. The cellular origin of human PAF: monocytes, polymorphonuclear neutrophils and basophils.

    PubMed Central

    Camussi, G; Aglietta, M; Coda, R; Bussolino, F; Piacibello, W; Tetta, C

    1981-01-01

    The origin of platelet activating factor (PAF) from human leucocytes was investigated. Purified monocytes release PAF passively at pH 10.6, when challenged with Ionophore A 23187 or under phagocytic stimuli. Pure preparations of polymorphonuclear neutrophils liberate PAF passively, when challenged with C5a, neutrophil cationic proteins (CP), their carboxypeptidase B derived products (C5a des Arg, CP des Arg) or under phagocytic stimuli. Basophil rich buffy coat cells release PAF when challenged with C5a, CP, anti-IgE (in low amount) or Synacthen concomitantly with basophil degranulation and histamine release. Electron microscopy studies, carried out on Synacthen-stimulated basophil rich buffy coat, provide morphological evidence for platelet-basophil interaction. In conclusion our data demonstrate that PAF can be released from different leucocyte populations. However, the stimuli able to trigger such release appear to have some specificity for the cell target. Images Figure 5 PMID:6161885

  12. Genome-wide CRISPR screen reveals novel host factors required for Staphylococcus aureus α-hemolysin-mediated toxicity

    PubMed Central

    Virreira Winter, Sebastian; Zychlinsky, Arturo; Bardoel, Bart W.

    2016-01-01

    Staphylococcus aureus causes a wide variety of infections and antibiotic resistant strains are a major problem in hospitals. One of the best studied virulence factors of S. aureus is the pore-forming toxin alpha hemolysin (αHL) whose mechanism of action is incompletely understood. We performed a genome-wide loss-of-function screen using CRISPR/Cas9 technology to identify host targets required for αHL susceptibility in human myeloid cells. We found gRNAs for ten genes enriched after intoxication with αHL and focused on the top five hits. Besides a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), the host receptor for αHL, we identified three proteins, Sys1 golgi trafficking protein (SYS1), ADP-ribosylation factor 1 (ARFRP1), and tetraspanin-14 (TSPAN14) which regulate the presentation of ADAM10 on the plasma membrane post-translationally. Interestingly, we also showed that cells lacking sphingomyelin synthase 1 (SGMS1) resist αHL intoxication, but have only a slightly reduced ADAM10 surface expression. SGMS1 regulates lipid raft formation, suggesting that αHL requires these membrane microdomains for attachment and cytotoxicity. PMID:27066838

  13. Brain-derived neurotrophic factor is required for normal development of the central respiratory rhythm in mice

    PubMed Central

    Balkowiec, Agnieszka; Katz, David M

    1998-01-01

    Molecular mechanisms underlying maturation of the central respiratory rhythm are largely unknown. Previously, we found that brain-derived neurotrophic factor (BDNF) is required for expression of normal breathing behaviour in newborn mice, raising the possibility that maturation of central respiratory output is dependent on BDNF. Respiratory activity was recorded in vitro from cervical ventral roots (C1 or C4) using the isolated brainstem–spinal cord preparation from postnatal day (P) 0.5–2.0 and P4.5 wild-type mice and mice lacking functional bdnf alleles. Loss of one or both bdnf alleles resulted in an approximately 50 % depression of central respiratory frequency compared with wild-type controls. In addition, respiratory cycle length variability was 214 % higher in bdnf null (bdnf−/−) animals compared with controls at P4.5. In contrast, respiratory burst duration was unaffected by bdnf gene mutation. These derangements of central respiratory rhythm paralleled the ventilatory depression and irregular breathing characteristic of bdnf mutants in vivo, indicating that central deficits can largely account for the abnormalities in resting ventilation produced by genetic loss of BDNF. BDNF is thus the first growth factor identified that is required for normal development of the central respiratory rhythm, including the stabilization of central respiratory output that occurs after birth. PMID:9706001

  14. The chitinase 3-like protein human cartilage glycoprotein 39 inhibits cellular responses to the inflammatory cytokines interleukin-1 and tumour necrosis factor-alpha.

    PubMed Central

    Ling, Hua; Recklies, Anneliese D

    2004-01-01

    Expression of the chitinase 3-like protein HC-gp39 (human cartilage glycoprotein 39) is associated with conditions of increased matrix turnover and tissue remodelling. High levels of this protein have been found in sera and synovial fluids of patients with inflammatory and degenerative arthritis. In order to assess the role of HC-gp39 in matrix degradation induced by inflammatory cytokines, we have examined its effect on the responses of connective tissue cells to TNF-alpha (tumour necrosis factor-alpha) and IL-1 (interleukin-1) with respect to activation of signalling pathways and production of MMPs (matrix metalloproteases) and chemokines. Stimulation of human skin fibroblasts or articular chondrocytes with IL-1 or TNF-alpha in the presence of HC-gp39 resulted in a marked reduction of both p38 mitogen-activated protein kinase and stress-activated protein kinase/Jun N-terminal kinase phosphorylation, whereas nuclear translocation of nuclear factor kappaB proceeded unimpeded. HC-gp39 suppressed the cytokine-induced secretion of MMP1, MMP3 and MMP13, as well as secretion of the chemokine IL-8. The suppressive effects of HC-gp39 were dependent on phosphoinositide 3-kinase activity, and treatment of cells with HC-gp39 resulted in AKT-mediated serine/threonine phosphorylation of apoptosis signal-regulating kinase 1. This process could therefore be responsible for the down-regulation of cytokine signalling by HC-gp39. These results suggest a physiological role for HC-gp39 in limiting the catabolic effects of inflammatory cytokines. PMID:15015934

  15. Expression of steroidogenic factor 1 in the testis requires an E box and CCAAT box in its promoter proximal region.

    PubMed

    Daggett, M A; Rice, D A; Heckert, L L

    2000-03-01

    Steroidogenic factor 1 (SF-1), also known as adrenal 4-binding protein, is a member of the nuclear hormone receptor family that regulates transcription of genes encoding hormones and steroidogenic enzymes important to the function of the hypothalamic-pituitary-gonadal axis. The mammalian Ftz-F1 gene encodes SF-1 and is required for development of adrenal glands and gonads. To better understand the mechanisms regulating this gene in the gonads, we have examined its expression in the testis and characterized the promoter region for SF-1 in two testicular cell types. SF-1 promoter activity was examined in primary cultures of Sertoli cells and cell lines representative of Sertoli and Leydig cells. Deletion mutagenesis of the promoter identified several regions: both 5' and 3' to the transcriptional start sites that are important for transcriptional activity. Two elements, an E box and a CCAAT box, were found to be important for SF-1 transcription in the testis. An oligodeoxynucleotide containing both of these elements bound three specific protein complexes. The binding of one complex required only sequences within the E box and cross-reacted with antibodies against the basic helix-loop-helix ZIP proteins USF1 and USF2. A second specific complex required sequences within both the E box and CCAAT box for efficient binding, while a third complex predominantly interacted with sequences within the CCAAT motif. The presence of multiple protein complexes binding these sites suggests that regulation through these elements may involve interactions with different factors that depend on the state of the cell and its environment.

  16. Replication factor C recruits DNA polymerase delta to sites of nucleotide excision repair but is not required for PCNA recruitment.

    PubMed

    Overmeer, René M; Gourdin, Audrey M; Giglia-Mari, Ambra; Kool, Hanneke; Houtsmuller, Adriaan B; Siegal, Gregg; Fousteri, Maria I; Mullenders, Leon H F; Vermeulen, Wim

    2010-10-01

    Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre- and postincision complexes. The postincision step of NER includes gap-filling DNA synthesis and ligation. However, the exact composition of this NER-associated DNA synthesis complex in vivo and the dynamic interactions of the factors involved are not well understood. Using immunofluorescence, chromatin immunoprecipitation, and live-cell protein dynamic studies, we show that replication factor C (RFC) is implicated in postincision NER in mammalian cells. Small interfering RNA-mediated knockdown of RFC impairs upstream removal of UV lesions and abrogates the downstream recruitment of DNA polymerase delta. Unexpectedly, RFC appears dispensable for PCNA recruitment yet is required for the subsequent recruitment of DNA polymerases to PCNA, indicating that RFC is essential to stably load the polymerase clamp to start DNA repair synthesis at 3' termini. The kinetic studies are consistent with a model in which RFC exchanges dynamically at sites of repair. However, its persistent localization at stalled NER complexes suggests that RFC remains targeted to the repair complex even after loading of PCNA. We speculate that RFC associates with the downstream 5' phosphate after loading; such interaction would prevent possible signaling events initiated by the RFC-like Rad17 and may assist in unloading of PCNA. PMID:20713449

  17. Replication Factor C Recruits DNA Polymerase δ to Sites of Nucleotide Excision Repair but Is Not Required for PCNA Recruitment▿

    PubMed Central

    Overmeer, René M.; Gourdin, Audrey M.; Giglia-Mari, Ambra; Kool, Hanneke; Houtsmuller, Adriaan B.; Siegal, Gregg; Fousteri, Maria I.; Mullenders, Leon H. F.; Vermeulen, Wim

    2010-01-01

    Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre- and postincision complexes. The postincision step of NER includes gap-filling DNA synthesis and ligation. However, the exact composition of this NER-associated DNA synthesis complex in vivo and the dynamic interactions of the factors involved are not well understood. Using immunofluorescence, chromatin immunoprecipitation, and live-cell protein dynamic studies, we show that replication factor C (RFC) is implicated in postincision NER in mammalian cells. Small interfering RNA-mediated knockdown of RFC impairs upstream removal of UV lesions and abrogates the downstream recruitment of DNA polymerase delta. Unexpectedly, RFC appears dispensable for PCNA recruitment yet is required for the subsequent recruitment of DNA polymerases to PCNA, indicating that RFC is essential to stably load the polymerase clamp to start DNA repair synthesis at 3′ termini. The kinetic studies are consistent with a model in which RFC exchanges dynamically at sites of repair. However, its persistent localization at stalled NER complexes suggests that RFC remains targeted to the repair complex even after loading of PCNA. We speculate that RFC associates with the downstream 5′ phosphate after loading; such interaction would prevent possible signaling events initiated by the RFC-like Rad17 and may assist in unloading of PCNA. PMID:20713449

  18. Two distinct factors bind to the rabbit uteroglobin TATA-box region and are required for efficient transcription.

    PubMed Central

    Klug, J; Knapp, S; Castro, I; Beato, M

    1994-01-01

    The rabbit uteroglobin gene is expressed in a variety of epithelial cell types like the lung Clara cells and the glandular and luminal epithelial cells of the endometrium. Expression in Clara cells is on a high constitutive level, whereas expression in the rabbit endometrium is under tight hormonal control. One important element of the rabbit uteroglobin gene mediating its efficient transcription in two epithelial cell lines from human endometrium (Ishikawa) and lung (NCI-H441) is its noncanonical TATA box (TACA). Here, we show that two factors (TATA core factor [TCF] and TATA palindrome factor [TPF]) different from the TATA-box binding protein bind to the DNA major groove at two adjacent sites within the uteroglobin TATA-box region and that one of them (TCF) is specifically expressed in cell lines derived from uteroglobin-expressing tissues. The binding sites for TCF and TPF, respectively, are both required for efficient transcription in Ishikawa and NCI-H441 cells. Mutation of the TACA box, which we show is a poor TATA box in functional terms, to a canonical TATA motif does not affect TCF and TPF binding. Therefore, we suggest that the function of the unusual cytosine could be to reduce rabbit uteroglobin expression in cells lacking TCF and that the interaction of TATA-box binding protein with the weak TACA site is facilitated in TCF- and TPF-positive cells. Images PMID:8065353

  19. Ybp1 is required for the hydrogen peroxide-induced oxidation of the Yap1 transcription factor.

    PubMed

    Veal, Elizabeth A; Ross, Sarah J; Malakasi, Panagiota; Peacock, Emma; Morgan, Brian A

    2003-08-15

    We describe the characterization of Ybp1, a novel protein, in Saccharomyces cerevisiae, that is required for the oxidative stress response to peroxides. Ybp1 is required for H2O2-induced expression of the antioxidant encoding gene TRX2. Our data indicate that the effects of Ybp1 are mediated through the Yap1 transcription factor. Indeed, Ybp1 forms a stress-induced complex with Yap1 in vivo and stimulates the nuclear accumulation of Yap1 in response to H2O2 but not in response to the thiol-oxidizing agent diamide. The H2O2-induced nuclear accumulation of Yap1 is regulated by the oxidation of specific cysteine residues and is dependent on the thiol peroxidase Gpx3. Our data suggest that Ybp1 is required for the H2O2-induced oxidation of Yap1 and acts in the same pathway as Gpx3. Consequently, Ybp1 represents a novel class of stress regulator of Yap1. These data have important implications for the regulation of protein oxidation and stress responses in eukaryotes. PMID:12743123

  20. Activation of the transcription factor NF-kappaB by retrieval is required for long-term memory reconsolidation.

    PubMed

    Merlo, Emiliano; Freudenthal, Ramiro; Maldonado, Héctor; Romano, Arturo

    2005-01-01

    Several studies support that stored memories undergo a new period of consolidation after retrieval. It is not known whether this process, termed reconsolidation, requires the same transcriptional mechanisms involved in consolidation. Increasing evidence supports the participation of the transcription factor NF-kappaB in memory. This was initially demonstrated in the crab Chasmagnathus model of associative contextual memory, in which re-exposure to the training context induces a well characterized reconsolidation process. Here we studied the role of NF-kappaB in reconsolidation. NF-kappaB was specifically activated in trained animals re-exposed to the training context but not to a different context. NF-kappaB was not activated when animals were re-exposed to the context after a weak training protocol insufficient to induce long-term memory. A specific inhibitor of the NF-kappaB pathway, sulfasalazine, impaired reconsolidation when administered 20 min before re-exposure to the training context but was not effective when a different context was used. These findings indicate for the first time that NF-kappaB is activated specifically by retrieval and that this activation is required for memory reconsolidation, supporting the view that this molecular mechanism is required in both consolidation and reconsolidation.

  1. Expression of Steroidogenic Factor 1 in the Testis Requires an Interactive Array of Elements Within Its Proximal Promoter1

    PubMed Central

    Scherrer, Serge P.; Rice, Daren A.; Heckert, Leslie L.

    2006-01-01

    Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor that is important for expression of genes involved in sexual differentiation, testicular and adrenal development, and hormone synthesis and regulation. To better understand the mechanisms required for SF-1 production, we employed transient transfec-tion analysis and electrophoretic mobility shift assays to characterize the elements and proteins required for transcriptional activity of the SF-1 proximal promoter in testicular Sertoli and Leydig cells and adrenocortical cells. Direct comparison of SF-1-promoter activity in testis and adrenal cell types established that a similar set of regulatory elements (an E box, CCAAT box, and Sp1-binding sites) is required for proximal promoter activity in these cells. Further evaluation of the E box and CCAAT box revealed a novel synergism between the two elements and iden-tified functionally important bases within the elements. Importantly, DNA/protein-binding studies uncovered new proteins interacting with the E box and CCAAT box. Thus, in addition to the previously identified USF and NF-Y proteins, newly described complexes, having migration properties that differed between Sertoli and Leydig cells, were observed bound to the E box and CCAAT box. Transient transfection analysis also identified several Sp1/Sp3-binding elements important for expression of SF-1 in the testis, one of which was previously described for expression in the adrenal gland whereas the other two were newly disclosed elements. PMID:12390883

  2. Requirement for neurogenesis to proceed through the division of neuronal progenitors following differentiation of epidermal growth factor and fibroblast growth factor-2-responsive human neural stem cells.

    PubMed

    Ostenfeld, Thor; Svendsen, Clive N

    2004-01-01

    Epidermal growth factor (EGF)- and fibroblast growth factor-2 (FGF-2)-responsive human neural stem cells may provide insight into mechanisms of neural development and have applications in cell-based therapeutics for neurological disease. However, their biology after expansion in vitro is currently poorly understood. Cells grown in either EGF or FGF-2 or a combination of both mitogens displayed characteristically similar levels of transcriptional activation and comparable proliferative profiles with linear cell-cycle kinetics and possessed similar neuronal differentiation capabilities. These data support the view that human neurospheres at later stages of expansion (>10 weeks) are comprised overwhelmingly of a single type of stem cell responsive to both EGF and FGF-2. After mitogen withdrawal and neurosphere plating, bromodeoxyuridine pulse-chase experiments revealed that the stem cells did not undergo differentiation directly into neurons. Instead, most immature neurons arose via the division of emerging progenitor cells in the absence of exogenous EGF or FGF-2. Neurogenesis was abolished by application of high concentrations of either EGF/FGF-2 or the mitotic inhibitor cytosine-b-arabinofuranoside, suggesting that there is an obligatory requirement for at least one round of cell division in the absence of mitogens as a prelude to terminal neuronal differentiation. The differentiation of human neurospheres provides a useful model of human neurogenesis, and the data presented indicate that it proceeds through the division of committed neuronal progenitor cells rather than directly from the neural stem cell. PMID:15342944

  3. The cellular distribution of Na+/H+ exchanger regulatory factor 1 is determined by the PDZ-I domain and regulates the malignant progression of breast cancer

    PubMed Central

    Du, Guifang; Gu, Yanan; Hao, Chengcheng; Yuan, Zhu; He, Junqi; Jiang, Wen G.; Cheng, Shan

    2016-01-01

    The oncogenic role of ectopic expression of Na+/H+ exchanger regulatory factor 1 (NHERF1) was recently suggested. Here, we show that NHERF1 was upregulated in high grades compared with low grades. Increased NHERF1 expression was correlated with poor prognosis and poor survival. NHERF1 expression was higher in the nucleus of cancer cells than in contiguous non- mammary epithelial cells. A novel mutation, namely NHERF1 Y24S, was identified in human breast cancer tissues and shown to correspond to a conserved residue in the PDZ-I domain of NHERF1. Truncation and mutation of the PDZ-I domain of NHERF1 increased the nuclear distribution of the NHERF1 protein, and this redistribution was associated with the malignant phenotype of breast cancer cells, including growth, migration, and adhesion. The present results suggest a role for NHERF1 in the progression of breast cancer mediated by the nuclear distribution of the NHERF1 protein, as determined by the truncation or key site mutation of the PDZ-I domain. PMID:27097111

  4. Functional high-throughput screening identifies the miR-15 microRNA family as cellular restriction factors for Salmonella infection.

    PubMed

    Maudet, Claire; Mano, Miguel; Sunkavalli, Ushasree; Sharan, Malvika; Giacca, Mauro; Förstner, Konrad U; Eulalio, Ana

    2014-08-22

    Increasing evidence suggests an important role for miRNAs in the molecular interplay between bacterial pathogens and host cells. Here we perform a fluorescence microscopy-based screen using a library of miRNA mimics and demonstrate that miRNAs modulate Salmonella infection. Several members of the miR-15 miRNA family were among the 17 miRNAs that more efficiently inhibit Salmonella infection. We discovered that these miRNAs are downregulated during Salmonella infection, through the inhibition of the transcription factor E2F1. Analysis of miR-15 family targets revealed that derepression of cyclin D1 and the consequent promotion of G1/S transition are crucial for Salmonella intracellular proliferation. In addition, Salmonella induces G2/M cell cycle arrest in infected cells, further promoting its replication. Overall, these findings uncover a mechanism whereby Salmonella renders host cells more susceptible to infection by controlling cell cycle progression through the active modulation of host cell miRNAs.

  5. Platelet-derived growth factor-α receptor is the cellular receptor for human cytomegalovirus gHgLgO trimer.

    PubMed

    Kabanova, Anna; Marcandalli, Jessica; Zhou, Tongqing; Bianchi, Siro; Baxa, Ulrich; Tsybovsky, Yaroslav; Lilleri, Daniele; Silacci-Fregni, Chiara; Foglierini, Mathilde; Fernandez-Rodriguez, Blanca Maria; Druz, Aliaksandr; Zhang, Baoshan; Geiger, Roger; Pagani, Massimiliano; Sallusto, Federica; Kwong, Peter D; Corti, Davide; Lanzavecchia, Antonio; Perez, Laurent

    2016-06-06

    Human cytomegalovirus encodes at least 25 membrane glycoproteins that are found in the viral envelope(1). While gB represents the fusion protein, two glycoprotein complexes control the tropism of the virus: the gHgLgO trimer is involved in the infection of fibroblasts, and the gHgLpUL128L pentamer is required for infection of endothelial, epithelial and myeloid cells(2-5). Two reports suggested that gB binds to ErbB1 and PDGFRα (refs 6,7); however, these results do not explain the tropism of the virus and were recently challenged(8,9). Here, we provide a 19 Å reconstruction for the gHgLgO trimer and show that it binds with high affinity through the gO subunit to PDGFRα, which is expressed on fibroblasts but not on epithelial cells. We also provide evidence that the trimer is essential for viral entry in both fibroblasts and epithelial cells. Furthermore, we identify the pentamer, which is essential for infection of epithelial cells, as a trigger for the ErbB pathway. These findings help explain the broad tropism of human cytomegalovirus and indicate that PDGFRα and the viral gO subunit could be targeted by novel anti-viral therapies.

  6. Platelet-derived growth factor-α receptor is the cellular receptor for human cytomegalovirus gHgLgO trimer.

    PubMed

    Kabanova, Anna; Marcandalli, Jessica; Zhou, Tongqing; Bianchi, Siro; Baxa, Ulrich; Tsybovsky, Yaroslav; Lilleri, Daniele; Silacci-Fregni, Chiara; Foglierini, Mathilde; Fernandez-Rodriguez, Blanca Maria; Druz, Aliaksandr; Zhang, Baoshan; Geiger, Roger; Pagani, Massimiliano; Sallusto, Federica; Kwong, Peter D; Corti, Davide; Lanzavecchia, Antonio; Perez, Laurent

    2016-01-01

    Human cytomegalovirus encodes at least 25 membrane glycoproteins that are found in the viral envelope(1). While gB represents the fusion protein, two glycoprotein complexes control the tropism of the virus: the gHgLgO trimer is involved in the infection of fibroblasts, and the gHgLpUL128L pentamer is required for infection of endothelial, epithelial and myeloid cells(2-5). Two reports suggested that gB binds to ErbB1 and PDGFRα (refs 6,7); however, these results do not explain the tropism of the virus and were recently challenged(8,9). Here, we provide a 19 Å reconstruction for the gHgLgO trimer and show that it binds with high affinity through the gO subunit to PDGFRα, which is expressed on fibroblasts but not on epithelial cells. We also provide evidence that the trimer is essential for viral entry in both fibroblasts and epithelial cells. Furthermore, we identify the pentamer, which is essential for infection of epithelial cells, as a trigger for the ErbB pathway. These findings help explain the broad tropism of human cytomegalovirus and indicate that PDGFRα and the viral gO subunit could be targeted by novel anti-viral therapies. PMID:27573107

  7. Potentiation of growth factor signaling by insulin-like growth factor-binding protein-3 in breast epithelial cells requires sphingosine kinase activity.

    PubMed

    Martin, Janet L; Lin, Mike Z; McGowan, Eileen M; Baxter, Robert C

    2009-09-18

    We have investigated the mechanism underlying potentiation of epidermal growth factor receptor (EGFR) and type 1 insulin-like growth factor receptor (IGFR1) signaling by IGF-binding protein-3 (IGFBP-3) in MCF-10A breast epithelial cells, focusing on a possible involvement of the sphingosine kinase (SphK) system. IGFBP-3 potentiated EGF-stimulated EGF receptor activation and DNA synthesis, and this was blocked by inhibitors of SphK activity or small interference RNA-mediated silencing of SphK1, but not SphK2, expression. Similarly, IGFR1 phosphorylation and DNA synthesis stimulated by LR3-IGF-I (an IGF-I analog not bound by IGFBP-3), were enhanced by IGFBP-3, and this was blocked by SphK1 silencing. SphK1 expression and activity were stimulated by IGFBP-3 approximately 2-fold over 24 h. Silencing of sphingosine 1-phosphate receptor 1 (S1P1) or S1P3, but not S1P2, abolished the effect of IGFBP-3 on EGF-stimulated EGFR activation. The effects of IGFBP-3 could be reproduced with exogenous S1P or medium conditioned by cells treated with IGFBP-3, and this was also blocked by inhibition of S1P1 and S1P3. These data indicate that potentiation of growth factor signaling by IGFBP-3 in MCF-10A cells requires SphK1 activity and S1P1/S1P3, suggesting that S1P, the product of SphK activity and ligand for S1P1 and S1P3, is the "missing link" mediating IGF and EGFR transactivation and cell growth stimulation by IGFBP-3.

  8. Turnip Mosaic Virus Genome-Linked Protein VPg Binds C-Terminal Region of Cap-Bound Initiation Factor 4E Orthologue Without Exhibiting Host Cellular Specificity

    PubMed Central

    Okade, Hayato; Fujita, Yuki; Miyamoto, Saori; Tomoo, Koji; Muto, Shinji; Miyoshi, Hiroshi; Natsuaki, Tomohide; Rhoads, Robert E.; Ishida, Toshimasa

    2014-01-01

    To investigate the binding specificity of turnip mosaic virus (TuMV) viral protein-genome linked (VPg) with translation initiation factor 4E, we evaluated here the kinetic parameters for the interactions of human eIF4E, Caenorhabditis elegans IFE-3 and IFE-5 and Arabidopsis eIFiso4E, by surface plasmon resonance (SPR). The results indicated that TuMV VPg does not show a binding preference for Arabidopsis eIFiso4E, even though it is from a host species whereas the other eIF4E orthologues are not. Surprisingly, the effect of m7GTP on both the rate constants and equilibrium binding constants for the interactions of VPg differed for the four eIF4E orthologues. In the case of eIFiso4E and IFE-3, m7GTP increased kon, but for eIF4E and IFE-5, it decreased kon. To provide insight into the structural basis for these differences in VPg binding, tertiary structures of the eIF4E orthologues were predicted on the basis of the previously determined crystal structure of m7GpppA-bound human eIF4E. The results suggested that in cap-bound eIF4E orthologues, the VPg binds to the C-terminal region, which constitutes one side of the entrance to the cap-binding pocket, whereas in the cap-free state, VPg binds to the widely opened cap-binding pocket and its surrounding region. The binding of VPg to the C-terminal region was confirmed by the SPR analyses of N- or C-terminal residues-deleted eIF4E orthologues. PMID:19122207

  9. Insulin-like growth factor-I improves cellular and molecular aspects of healing in a collagenase-induced model of flexor tendinitis.

    PubMed

    Dahlgren, Linda A; van der Meulen, Marjolein C H; Bertram, John E A; Starrak, Greg S; Nixon, Alan J

    2002-09-01

    Flexor tendinitis is a common and debilitating injury of elite and recreational athletes. Healing may be improved through intratendinous injection of insulin-like growth factor-I (IGF-I), which has been shown in vitro to stimulate mitogenesis and enhance tendon matrix production. This study investigated the effects of intratendinous injection of IGF-I on tendon healing in an equine model of flexor tendinitis. Collagenase-induced lesions were created in the tensile region of theflexor digitorum superficialis tendon of both forelimbs of eight horses. Treated tendons were injected with 2 microg rhlGF-I intralesionally every other day for 10 injections, while controls received 0.9% NaCl. Tendon fiber deposition and organization were evaluated serially using ultrasonography throughout the 8 week trial period. Following euthanasia, the tendons were harvested and DNA, hydroxyproline, and glycosaminoglycan content determined, mechanical strength and stiffness evaluated, gene expression and spatial arrangement of collagen types I and III assessed by northern blot and in situ hybridization, and tendon fiber architecture assessed by polarized light microscopy. Local soft tissue swelling was reduced in the IGF-I treated limbs. Similarly, lesion size in IGF-I treated tendons was smaller 3 and 4 weeks after initiation of treatment. Cell proliferation and collagen content of the IGF-I treated tendons were increased compared to controls. Mechanically, IGF-I treated tendons showed a trend toward increased stiffness compared to saline treated controls. Considered together with the decreased soft tissue swelling and improved sonographic healing, these data support the potential use of intralesional IGF-I for treatment of debilitating tendon injuries.

  10. Hox Targets and Cellular Functions

    PubMed Central

    Sánchez-Herrero, Ernesto

    2013-01-01

    Hox genes are a group of genes that specify structures along the anteroposterior axis in bilaterians. Although in many cases they do so by modifying a homologous structure with a different (or no) Hox input, there are also examples of Hox genes constructing new organs with no homology in other regions of the body. Hox genes determine structures though the regulation of targets implementing cellular functions and by coordinating cell behavior. The genetic organization to construct or modify a certain organ involves both a genetic cascade through intermediate transcription factors and a direct regulation of targets carrying out cellular functions. In this review I discuss new data from genome-wide techniques, as well as previous genetic and developmental information, to describe some examples of Hox regulation of different cell functions. I also discuss the organization of genetic cascades leading to the development of new organs, mainly using Drosophila melanogaster as the model to analyze Hox function. PMID:24490109

  11. Cellular Reflectarray Antenna

    NASA Technical Reports Server (NTRS)

    Romanofsky, Robert R.

    2010-01-01

    The cellular reflectarray antenna is intended to replace conventional parabolic reflectors that must be physically aligned with a particular satellite in geostationary orbit. These arrays are designed for specified geographical locations, defined by latitude and longitude, each called a "cell." A particular cell occupies nominally 1,500 square miles (3,885 sq. km), but this varies according to latitude and longitude. The cellular reflectarray antenna designed for a particular cell is simply positioned to align with magnetic North, and the antenna surface is level (parallel to the ground). A given cellular reflectarray antenna will not operate in any other cell.

  12. Complement-Mediated Regulation of Metabolism and Basic Cellular Processes.

    PubMed

    Hess, Christoph; Kemper, Claudia

    2016-08-16

    Complement is well appreciated as a critical arm of innate immunity. It is required for the removal of invading pathogens and works by directly destroying them through the activation of innate and adaptive immune cells. However, complement activation and function is not confined to the extracellular space but also occurs within cells. Recent work indicates that complement activation regulates key metabolic pathways and thus can impact fundamental cellular processes, such as survival, proliferation, and autophagy. Newly identified functions of complement include a key role in shaping metabolic reprogramming, which underlies T cell effector differentiation, and a role as a nexus for interactions with other effector systems, in particular the inflammasome and Notch transcription-factor networks. This review focuses on the contributions of complement to basic processes of the cell, in particular the integration of complement with cellular metabolism and the potential implications in infection and other disease settings. PMID:27533012

  13. Regulation of cellular metabolism: programming and maintaining metabolic homeostasis.

    PubMed

    Wilson, David F

    2013-12-01

    Mitochondrial oxidative phosphorylation is programmed to set and maintain metabolic homeostasis. This is accomplished through an intrinsic program that determines the metabolic [ATP]/[ADP]/[Pi], where [Pi] is the concentration of inorganic phosphate (energy state) and maintains it through a bidirectional sensory/signaling control network that reaches every aspect of cellular metabolism. The program sets the energy state with high precision (to better than one part in 10(9)) and can respond to transient changes in energy demand (ATP use) to more than 100 times the resting rate. Epigenetic and environmental factors are able to "fine tune" the programmed set point over a narrow range to meet the special needs associated with cell differentiation and chronic changes in metabolic requirements. The result is robust, across platform control of metabolism, essential to cellular differentiation and the evolution of complex organisms.

  14. Cellular Manufacturing Internet Performance Support System

    SciTech Connect

    Bohley, M.C.; Schwartz, M.E.

    1998-03-04

    The objective of this project was to develop an Internet-based electronic performance support system (EPSS) for cellular manufacturing providing hardware/software specifications, process descriptions, estimated cost savings, manufacturing simulations, training information, and service resources for government and industry users of Cincinnati Milacron machine tools and products. AlliedSignal Federal Manufacturing and Technologies (ASFM and T) used expertise in the areas of Internet design and multimedia creation to develop a performance support system (PSS) for the Internet with assistance from CM's subject matter experts from engineering, manufacturing, and technical support. Reference information was both created and re-purposed from other existing formats, then made available on the Internet. On-line references on cellular manufacturing operations include: definitions of cells and cellular manufacturing; illustrations on how cellular manufacturing improves part throughput, resource utilization, part quality, and manufacturing flexibility; illustrations on how cellular manufacturing reduces labor and overhead costs; identification of critical factors driving decisions toward cellular manufacturing; a method for identifying process improvement areas using cellular manufacturing; a method for customizing the size of cells for a specific site; a simulation for making a part using cellular manufacturing technology; and a glossary of terms and concepts.

  15. Apoptosis-Inducing-Factor-Dependent Mitochondrial Function Is Required for T Cell but Not B Cell Function.

    PubMed

    Milasta, Sandra; Dillon, Christopher P; Sturm, Oliver E; Verbist, Katherine C; Brewer, Taylor L; Quarato, Giovanni; Brown, Scott A; Frase, Sharon; Janke, Laura J; Perry, S Scott; Thomas, Paul G; Green, Douglas R

    2016-01-19

    The role of apoptosis inducing factor (AIF) in promoting cell death versus survival remains controversial. We report that the loss of AIF in fibroblasts led to mitochondrial electron transport chain defects and loss of proliferation that could be restored by ectopic expression of the yeast NADH dehydrogenase Ndi1. Aif-deficiency in T cells led to decreased peripheral T cell numbers and defective homeostatic proliferation, but thymic T cell development was unaffected. In contrast, Aif-deficient B cells developed and functioned normally. The difference in the dependency of T cells versus B cells on AIF for function and survival correlated with their metabolic requirements. Ectopic Ndi1 expression rescued homeostatic proliferation of Aif-deficient T cells. Despite its reported roles in cell death, fibroblasts, thymocytes and B cells lacking AIF underwent normal death. These studies suggest that the primary role of AIF relates to complex I function, with differential effects on T and B cells.

  16. [Main Cellular Redox Couples].

    PubMed

    Bilan, D S; Shokhina, A G; Lukyanov, S A; Belousov, V V

    2015-01-01

    Most of the living cells maintain the continuous flow of electrons, which provides them by energy. Many of the compounds are presented in a cell at the same time in the oxidized and reduced states, forming the active redox couples. Some of the redox couples, such as NAD+/NADH, NADP+/NADPH, oxidized/reduced glutathione (GSSG/GSH), are universal, as they participate in adjusting of many cellular reactions. Ratios of the oxidized and reduced forms of these compounds are important cellular redox parameters. Modern research approaches allow setting the new functions of the main redox couples in the complex organization of cellular processes. The following information is about the main cellular redox couples and their participation in various biological processes.

  17. Arabidopsis ARF-GTP exchange factor, GNOM, mediates transport required for innate immunity and focal accumulation of syntaxin PEN1.

    PubMed

    Nielsen, Mads Eggert; Feechan, Angela; Böhlenius, Henrik; Ueda, Takashi; Thordal-Christensen, Hans

    2012-07-10

    Penetration resistance to powdery mildew fungi, conferred by localized cell wall appositions (papillae), is one of the best-studied processes in plant innate immunity. The syntaxin PENETRATION (PEN)1 is required for timely appearance of papillae, which contain callose and extracellular membrane material, as well as PEN1 itself. Appearance of membrane material in papillae suggests secretion of exosomes. These are potentially derived from multivesicular bodies (MVBs), supported by our observation that ARA6-labeled organelles assemble at the fungal attack site. However, the trafficking components that mediate delivery of extracellular membrane material are unknown. Here, we show that the delivery is independent of PEN1 function. Instead, we find that application of brefeldin (BF)A blocks the papillary accumulation of GFP-PEN1-labeled extracellular membrane and callose, while impeding penetration resistance. We subsequently provide evidence indicating that the responsible BFA-sensitive ADP ribosylation factor-GTP exchange factor (ARF-GEF) is GNOM. Firstly, analysis of the transheterozygote gnom(B4049/emb30-1) (gnom(B)(/E)) mutant revealed a delay in papilla formation and reduced penetration resistance. Furthermore, a BFA-resistant version of GNOM restored the BFA-sensitive papillary accumulation of GFP-PEN1 and callose. Our data, therefore, provide a link between GNOM and disease resistance. We suggest that papilla formation requires rapid reorganization of material from the plasma membrane mediated by GNOM. The papilla material is subsequently presumed to be sorted into MVBs and directed to the site of fungal attack, rendering the epidermal plant cell inaccessible for the invading powdery mildew fungus.

  18. Identification of a Novel Virulence Factor in Burkholderia cenocepacia H111 Required for Efficient Slow Killing of Caenorhabditis elegans

    PubMed Central

    Huber, Birgit; Feldmann, Friederike; Köthe, Manuela; Vandamme, Peter; Wopperer, Julia; Riedel, Kathrin; Eberl, Leo

    2004-01-01

    Burkholderia cenocepacia H111, which was isolated from a cystic fibrosis patient, employs a quorum-sensing (QS) system, encoded by cep, to control the expression of virulence factors as well as the formation of biofilms. The QS system is thought to ensure that pathogenic traits are expressed only when the bacterial population density is high enough to overwhelm the host before it is able to mount an efficient response. While the wild-type strain effectively kills the nematode Caenorhabditis elegans, the pathogenicity of mutants with defective quorum sensing is attenuated. To date, very little is known about the cep-regulated virulence factors required for nematode killing. Here we report the identification of a cep-regulated gene, whose predicted amino acid sequence is highly similar to the QS-regulated protein AidA of the plant pathogen Ralstonia solanacearum. By use of polyclonal antibodies directed against AidA, it is demonstrated that the protein is expressed in the late-exponential phase and accumulates during growth arrest. We show that B. cenocepacia H111 AidA is essential for slow killing of C. elegans but has little effect on fast killing, suggesting that the protein plays a role in the accumulation of the strain in the nematode gut. Thus, AidA appears to be required for establishing an infection-like process rather than acting as a toxin. Furthermore, evidence is provided that AidA is produced not only by B. cenocepacia but also by many other strains of the Burkholderia cepacia complex. PMID:15557647

  19. Nanostructured cellular networks.

    PubMed

    Moriarty, P; Taylor, M D R; Brust, M

    2002-12-01

    Au nanocrystals spin-coated onto silicon from toluene form cellular networks. A quantitative statistical crystallography analysis shows that intercellular correlations drive the networks far from statistical equilibrium. Spin-coating from hexane does not produce cellular structure, yet a strong correlation is retained in the positions of nanocrystal aggregates. Mechanisms based on Marangoni convection alone cannot account for the variety of patterns observed, and we argue that spinodal decomposition plays an important role in foam formation.

  20. Specific requirement for translation initiation factor 4E or its isoform drives plant host susceptibility to Tobacco etch virus

    PubMed Central

    2014-01-01

    Background In plants, eIF4E translation initiation factors and their eIFiso4E isoforms are essential susceptibility factors for many RNA viruses, including potyviruses. Mutations altering these factors are a major source of resistance to the viruses. The eIF4E allelic series is associated with specific resistance spectra in crops such as Capsicum annum. Genetic evidence shows that potyviruses have a specific requirement for a given 4E isoform that depends on the host plant. For example, Tobacco etch virus (TEV) uses eIF4E1 to infect Capsicum annuum but uses eIFiso4E to infect Arabidopsis thaliana. Here, we investigated how TEV exploits different translation initiation factor isoforms to infect these two plant species. Results A complementation system was set up in Arabidopsis to test the restoration of systemic infection by TEV. Using this system, Arabidopsis susceptibility to TEV was complemented with a susceptible pepper eIF4E1 allele but not with a resistant allele. Therefore, in Arabidopsis, TEV can use the pepper eIF4E1 instead of the endogenous eIFiso4E isoform so is able to switch between translation initiation factor 4E isoform to infect the same host. Moreover, we show that overexpressing the pepper eIF4E1 alleles is sufficient to make Arabidopsis susceptible to an otherwise incompatible TEV strain. Lastly, we show that the resistant eIF4E1 allele is similarly overcome by a resistance-breaking TEV strain as in pepper, confirming that this Arabidopsis TEV-susceptibility complementation system is allele-specific. Conclusion We report here a complementation system in Arabidopsis that makes it possible to assess the role of pepper pvr2-eIF4E alleles in susceptibility to TEV. Heterologous complementation experiments showed that the idiosyncratic properties of the 4E and iso4E proteins create a major checkpoint for viral infection of different hosts. This system could be used to screen natural or induced eIF4E alleles to find and study alleles of interest for

  1. Cellular aging and cancer

    PubMed Central

    Hornsby, Peter J.

    2010-01-01

    Aging is manifest in a variety of changes over time, including changes at the cellular level. Cellular aging acts primarily as a tumor suppressor mechanism, but also may enhance cancer development under certain circumstances. One important process of cellular aging is oncogene-induced senescence, which acts as an important anti-cancer mechanism. Cellular senescence resulting from damage caused by activated oncogenes prevents the growth or potentially neoplastic cells. Moreover, cells that have entered senescence appear to be targets for elimination by the innnate immune system. In another aspect of cellular aging, the absence of telomerase activity in normal tissues results in such cells lacking a telomere maintenance mechanism. One consequence is that in aging there is an increase in cells with shortened telomeres. In the presence of active oncogenes that cause expansion of a neoplastic clone, shortening of telomeres leading to telomere dysfunction prevents the indefinite expansion of the clone because the cells enter crisis. Crisis results from fusions and other defects caused by dysfunctional telomeres and is a terminal state of the neoplastic clone. In this way the absence of telomerase in human cells, while one cause of cellular aging, also acts as an anti-cancer mechanism. PMID:20705476

  2. A Conserved 20S Proteasome Assembly Factor Requires a C-terminal HbYX Motif for Proteasomal Precursor Binding

    PubMed Central

    Kusmierczyk, Andrew R.; Kunjappu, Mary J.; Kim, Roger Y.; Hochstrasser, Mark

    2011-01-01

    Dedicated chaperones facilitate eukaryotic proteasome assembly, yet how they function remains largely unknown. Here we demonstrate that a yeast 20S proteasome assembly factor, Pba1–Pba2, requires a previously overlooked C-terminal HbYX (hydrophobic-tyrosine-X) motif for function. HbYX motifs in proteasome activators open the 20S proteasome entry pore, but Pba1–Pba2 instead binds inactive proteasomal precursors. We discovered an archaeal ortholog of this factor, here named PbaA, that also binds preferentially to proteasomal precursors in a HbYX-dependent fashion using the same proteasomal α-ring surface pockets bound by activators. Remarkably, PbaA and the related PbaB protein can be induced to bind mature 20S proteasomes if the active sites in the central chamber are occupied by inhibitors. Our data suggest an allosteric mechanism in which proteasome active-site maturation determines assembly chaperone binding, potentially shielding assembly intermediates or misassembled complexes from non-productive associations until assembly is complete. PMID:21499243

  3. Transcription factor Sox4 is required for PUMA-mediated apoptosis induced by histone deacetylase inhibitor, TSA.

    PubMed

    Jang, Sang-Min; Kang, Eun-Jin; Kim, Jung-Woong; Kim, Chul-Hong; An, Joo-Hee; Choi, Kyung-Hee

    2013-08-23

    PUMA is a crucial regulator of apoptotic cell death mediated by p53-dependent and p53-independent mechanisms. In many cancer cells, PUMA expression is induced in response to DNA-damaging reagent in a p53-dependent manner. However, few studies have investigated transcription factors that lead to the induction of PUMA expression via p53-independent apoptotic signaling. In this study, we found that the transcription factor Sox4 increased PUMA expression in response to trichostatin A (TSA), a histone deacetylase inhibitor in the p53-null human lung cancer cell line H1299. Ectopic expression of Sox4 led to the induction of PUMA expression at the mRNA and protein levels, and TSA-mediated up-regulation of PUMA transcription was repressed by the knockdown of Sox4. Using luciferase assays and chromatin immunoprecipitation, we also determined that Sox4 recruits p300 on the PUMA promoter region and increases PUMA gene expression in response to TSA treatment. Taken together, these results suggest that Sox4 is required for p53-independent apoptotic cell death mediated by PUMA induction via TSA treatment.

  4. Brain-derived neurotrophic factor (BDNF) is required for the enhancement of hippocampal neurogenesis following environmental enrichment.

    PubMed

    Rossi, Chiara; Angelucci, Andrea; Costantin, Laura; Braschi, Chiara; Mazzantini, Mario; Babbini, Francesco; Fabbri, Maria Elena; Tessarollo, Lino; Maffei, Lamberto; Berardi, Nicoletta; Caleo, Matteo

    2006-10-01

    Neurogenesis continues to occur in the adult mammalian hippocampus and is regulated by both genetic and environmental factors. It is known that exposure to an enriched environment enhances the number of newly generated neurons in the dentate gyrus. However, the mechanisms by which enriched housing produces these effects are poorly understood. To test a role for neurotrophins, we used heterozygous knockout mice for brain-derived neurotrophic factor (BDNF+/-) and mice lacking neurotrophin-4 (NT-4-/-) together with their wild-type littermates. Mice were either reared in standard laboratory conditions or placed in an enriched environment for 8 weeks. Animals received injections of the mitotic marker bromodeoxyuridine (BrdU) to label newborn cells. Enriched wild-type and enriched NT-4-/- mice showed a two-fold increase in hippocampal neurogenesis as assessed by stereological counting of BrdU-positive cells in the dentate gyrus and double labelling for BrdU and the neuronal marker NeuN. Remarkably, this enhancement of hippocampal neurogenesis was not seen in enriched BDNF+/- mice. Failure to up-regulate BDNF accompanied the lack of a neurogenic response in enriched BDNF heterozygous mice. We conclude that BDNF but not NT-4 is required for the environmental induction of neurogenesis. PMID:17040481

  5. Co-operative DNA binding by GAGA transcription factor requires the conserved BTB/POZ domain and reorganizes promoter topology.

    PubMed Central

    Katsani, K R; Hajibagheri, M A; Verrijzer, C P

    1999-01-01

    The POZ domain is a conserved protein-protein interaction motif present in a variety of transcription factors involved in development, chromatin remodelling and human cancers. Here, we study the role of the POZ domain of the GAGA transcription factor in promoter recognition. Natural target promoters for GAGA typically contain multiple GAGA-binding elements. Our results show that the POZ domain mediates strong co-operative binding to multiple sites but inhibits binding to single sites. Protein cross-linking and gel filtration chromatography experiments established that the POZ domain is required for GAGA oligomerization into higher order complexes. Thus, GAGA oligomerization increases binding specificity by selecting only promoters with multiple sites. Electron microscopy revealed that GAGA binds to multiple sites as a large oligomer and induces bending of the promoter DNA. Our results indicate a novel mode of DNA binding by GAGA, in which a large GAGA complex binds multiple GAGA elements that are spread out over a region of a few hundred base pairs. We suggest a model in which the promoter DNA is wrapped around a GAGA multimer in a conformation that may exclude normal nucleosome formation. PMID:9927429

  6. Nuclear factor kappa B-dependent Zif268 expression in hippocampus is required for recognition memory in mice.

    PubMed

    Zalcman, Gisela; Federman, Noel; de la Fuente, Verónica; Romano, Arturo

    2015-03-01

    Long-term memory formation requires gene expression after acquisition of new information. The first step in the regulation of gene expression is the participation of transcription factors (TFs) such as nuclear factor kappa B (NF-кB), which are present before the neuronal activity induced by training. It was proposed that the activation of these types of TFs allows a second step in gene regulation by induction of immediate-early genes (IEGs) whose protein products are, in turn, TFs. Between these IEGs, zif268 has been found to play a critical role in long-term memory formation and reprocessing after retrieval. Here we found in mice hippocampus that, on one hand, NF-кB was activated 45 min after training in a novel object recognition (NOR) task and that inhibiting NF-кB immediately after training by intrahippocampal administration of NF-кB Decoy DNA impaired NOR memory consolidation. On the other hand, Zif268 protein expression was induced 45 min after NOR training and the administration of DNA antisense to its mRNA post-training impaired recognition memory. Finally, we found that the inhibition of NF-кB by NF-кB Decoy DNA reduced significantly the training-induced Zif268 increment, indicating that NF-кB is involved in the regulation of Zif268 expression. Thus, the present results support the involvement of NF-кB activity-dependent Zif268 expression in the hippocampus during recognition memory consolidation.

  7. A Member of the Arabidopsis Mitochondrial Transcription Termination Factor Family Is Required for Maturation of Chloroplast Transfer RNAIle(GAU)

    PubMed Central

    Romani, Isidora; Manavski, Nikolay; Morosetti, Arianna; Tadini, Luca; Maier, Swetlana; Kühn, Kristina; Ruwe, Hannes; Schmitz-Linneweber, Christian; Wanner, Gerhard; Leister, Dario; Kleine, Tatjana

    2015-01-01

    Plastid gene expression is crucial for organelle function, but the factors that control it are still largely unclear. Members of the so-called mitochondrial transcription termination factor (mTERF) family are found in metazoans and plants and regulate organellar gene expression at different levels. Arabidopsis (Arabidopsis thaliana) mTERF6 is localized in chloroplasts and mitochondria, and its knockout perturbs plastid development and results in seedling lethality. In the leaky mterf6-1 mutant, a defect in photosynthesis is associated with reduced levels of photosystem subunits, although corresponding messenger RNA levels are unaffected, whereas translational capacity and maturation of chloroplast ribosomal RNAs (rRNAs) are perturbed in mterf6-1 mutants. Bacterial one-hybrid screening, electrophoretic mobility shift assays, and coimmunoprecipitation experiments reveal a specific interaction between mTERF6 and an RNA sequence in the chloroplast isoleucine transfer RNA gene (trnI.2) located in the rRNA operon. In vitro, recombinant mTERF6 bound to its plastid DNA target site can terminate transcription. At present, it is unclear whether disturbed rRNA maturation is a primary or secondary defect. However, it is clear that mTERF6 is required for the maturation of trnI.2. This points to an additional function of mTERFs. PMID:26152711

  8. A forkhead Transcription Factor Is Wound-Induced at the Planarian Midline and Required for Anterior Pole Regeneration

    PubMed Central

    Scimone, M. Lucila; Lapan, Sylvain W.; Reddien, Peter W.

    2014-01-01

    Planarian regeneration requires positional information to specify the identity of tissues to be replaced as well as pluripotent neoblasts capable of differentiating into new cell types. We found that wounding elicits rapid expression of a gene encoding a Forkhead-family transcription factor, FoxD. Wound-induced FoxD expression is specific to the ventral midline, is regulated by Hedgehog signaling, and is neoblast-independent. FoxD is subsequently expressed within a medial subpopulation of neoblasts at wounds involving head regeneration. Ultimately, FoxD is co-expressed with multiple anterior markers at the anterior pole. Inhibition of FoxD with RNA interference (RNAi) results in the failure to specify neoblasts expressing anterior markers (notum and prep) and in anterior pole formation defects. FoxD(RNAi) animals fail to regenerate a new midline and to properly pattern the anterior blastema, consistent with a role for the anterior pole in organizing pattern of the regenerating head. Our results suggest that wound signaling activates a forkhead transcription factor at the midline and, if the head is absent, FoxD promotes specification of neoblasts at the prior midline for anterior pole regeneration. PMID:24415944

  9. Macromolecular substrate-binding exosites on both the heavy and light chains of factor XIa mediate the formation of the Michaelis complex required for factor IX-activation.

    PubMed

    Sinha, Dipali; Marcinkiewicz, Mariola; Navaneetham, Duraiswamy; Walsh, Peter N

    2007-08-28

    Binding of factor IX (FIX) to an exosite on the heavy chain of factor XIa (FXIa) is essential for the optimal activation of FIX (Sinha, D., Seaman, F. S., and Walsh, P. N. (1987) Biochemistry 26, 3768-3775). To gain further insight into the mechanisms of activation of FIX by FXIa, we have investigated the kinetic properties of FXIa-light chain (FXIa-LC) with its active site occupied by either a reversible inhibitor of serine proteases (p-aminobenzamidine, PAB) or a small peptidyl substrate (S-2366) and have examined FIX cleavage products resulting from activation by FXIa or FXIa-LC. PAB inhibited the hydrolysis of S-2366 by FXIa-LC in a classically competitive fashion. In contrast, PAB was found to be a noncompetitive inhibitor of the activation of the macromolecular substrate FIX. Occupancy of the active site of the FXIa-LC by S-2366 also resulted in noncompetitive inhibition of FIX activation. These results demonstrate the presence of an exosite for FIX binding on the FXIa-LC remote from its active site. Furthermore, examination of the cleavage products of FIX indicated that in the absence of either Ca2+ or the heavy chain of FXIa there was substantial accumulation of the inactive intermediate FIXalpha, indicating a slower rate of cleavage of the scissile bond Arg180-Val181. We conclude that binding to two substrate-binding exosites one on the heavy chain and the other on the light chain of FXIa is required to mediate the formation of the Michaelis complex and efficient cleavages of the two spatially separated scissile bonds of FIX. PMID:17676929

  10. Association of genetic variation in pharmacodynamic factors with methadone dose required for effective treatment of opioid addiction

    PubMed Central

    Levran, Orna; Peles, Einat; Randesi, Matthew; Shu, Xu; Ott, Jurg; Shen, Pei-Hong; Adelson, Miriam; Kreek, Mary Jeanne

    2013-01-01

    Aim The interindividual variability in the dose required for effective methadone maintenance treatment (MMT) for opioid addiction may be influenced in part by genetic variations in genes encoding pharmacodynamic factors of methadone. This study was conducted to identify some of these variants. Materials & methods This study focused on 11 genes encoding components of the opioidergic (OPRM1, POMC and ARRB2), the dopaminergic (ANKK1 and DRD2) and the glutamatergic pathways (GRIN1 and GRIN2A), as well as the neurotrophin system (NGFB, BDNF, NTRK1 and NTRK2). The study includes 227 Israeli patients undergoing stable MMT. Results Out of the 110 variants analyzed, 12 SNPs (in BDNF, NTRK2, OPRM1, DRD2 and ANKK1) were associated with methadone dose (nominal p < 0.05). Of these SNPs, ANKK1 rs7118900 and DRD2 rs2283265 are known to affect gene expression. Logistic regression of five representative SNPs discriminated between individuals requiring a methadone dose of >120 mg/day and <120 mg/day (p = 0.019), and showed moderate sensitivity and specificity (AUC of 0.63 in receiver operating characteristic analysis). Conclusion This data should stimulate further research on the potential influence and clinical significance of these variants on MMT. PMID:23651024

  11. Slam is an outer membrane protein that is required for the surface display of lipidated virulence factors in Neisseria.

    PubMed

    Hooda, Yogesh; Lai, Christine Chieh-Lin; Judd, Andrew; Buckwalter, Carolyn M; Shin, Hyejin Esther; Gray-Owen, Scott D; Moraes, Trevor F

    2016-01-01

    Lipoproteins decorate the surface of many Gram-negative bacterial pathogens, playing essential roles in immune evasion and nutrient acquisition. In Neisseria spp., the causative agents of gonorrhoea and meningococcal meningitis, surface lipoproteins (SLPs) are required for virulence and have been extensively studied as prime candidates for vaccine development. However, the machinery and mechanism that allow for the surface display of SLPs are not known. Here, we describe a transposon (Tn5)-based search for the proteins required to deliver SLPs to the surface of Neisseria meningitidis, revealing a family of proteins that we have named the surface lipoprotein assembly modulator (Slam). N. meningitidis contains two Slam proteins, each exhibiting distinct substrate preferences. The Slam proteins are sufficient to reconstitute SLP transport in laboratory strains of Escherichia coli, which are otherwise unable to efficiently display these lipoproteins on their cell surface. Immunoprecipitation and domain probing experiments suggest that the SLP, TbpB, interacts with Slam during the transit process; furthermore, the membrane domain of Slam is sufficient for selectivity and proper surface display of SLPs. Rather than being a Neisseria-specific factor, our bioinformatic analysis shows that Slam can be found throughout proteobacterial genomes, indicating a conserved but until now unrecognized virulence mechanism. PMID:2