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Sample records for lytic cycle inducing

  1. Oncogenic Herpesvirus Utilizes Stress-Induced Cell Cycle Checkpoints for Efficient Lytic Replication

    PubMed Central

    Turunen, Mikko; Diaz, Raquel; Lyly, Lauri; Pekkonen, Pirita; Rantala, Juha; Ojala, Krista; Sarek, Grzegorz; Teesalu, Mari; Denisova, Oxana; Peltonen, Karita; Julkunen, Ilkka; Varjosalo, Markku; Kainov, Denis; Kallioniemi, Olli; Laiho, Marikki; Taipale, Jussi; Hautaniemi, Sampsa; Ojala, Päivi M.

    2016-01-01

    Kaposi’s sarcoma herpesvirus (KSHV) causes Kaposi’s sarcoma and certain lymphoproliferative malignancies. Latent infection is established in the majority of tumor cells, whereas lytic replication is reactivated in a small fraction of cells, which is important for both virus spread and disease progression. A siRNA screen for novel regulators of KSHV reactivation identified the E3 ubiquitin ligase MDM2 as a negative regulator of viral reactivation. Depletion of MDM2, a repressor of p53, favored efficient activation of the viral lytic transcription program and viral reactivation. During lytic replication cells activated a p53 response, accumulated DNA damage and arrested at G2-phase. Depletion of p21, a p53 target gene, restored cell cycle progression and thereby impaired the virus reactivation cascade delaying the onset of virus replication induced cytopathic effect. Herpesviruses are known to reactivate in response to different kinds of stress, and our study now highlights the molecular events in the stressed host cell that KSHV has evolved to utilize to ensure efficient viral lytic replication. PMID:26891221

  2. Oncogenic Herpesvirus Utilizes Stress-Induced Cell Cycle Checkpoints for Efficient Lytic Replication.

    PubMed

    Balistreri, Giuseppe; Viiliäinen, Johanna; Turunen, Mikko; Diaz, Raquel; Lyly, Lauri; Pekkonen, Pirita; Rantala, Juha; Ojala, Krista; Sarek, Grzegorz; Teesalu, Mari; Denisova, Oxana; Peltonen, Karita; Julkunen, Ilkka; Varjosalo, Markku; Kainov, Denis; Kallioniemi, Olli; Laiho, Marikki; Taipale, Jussi; Hautaniemi, Sampsa; Ojala, Päivi M

    2016-02-01

    Kaposi's sarcoma herpesvirus (KSHV) causes Kaposi's sarcoma and certain lymphoproliferative malignancies. Latent infection is established in the majority of tumor cells, whereas lytic replication is reactivated in a small fraction of cells, which is important for both virus spread and disease progression. A siRNA screen for novel regulators of KSHV reactivation identified the E3 ubiquitin ligase MDM2 as a negative regulator of viral reactivation. Depletion of MDM2, a repressor of p53, favored efficient activation of the viral lytic transcription program and viral reactivation. During lytic replication cells activated a p53 response, accumulated DNA damage and arrested at G2-phase. Depletion of p21, a p53 target gene, restored cell cycle progression and thereby impaired the virus reactivation cascade delaying the onset of virus replication induced cytopathic effect. Herpesviruses are known to reactivate in response to different kinds of stress, and our study now highlights the molecular events in the stressed host cell that KSHV has evolved to utilize to ensure efficient viral lytic replication. PMID:26891221

  3. Inhibition of class I histone deacetylases by romidepsin potently induces Epstein-Barr virus lytic cycle and mediates enhanced cell death with ganciclovir.

    PubMed

    Hui, Kwai Fung; Cheung, Arthur Kwok Leung; Choi, Chung King; Yeung, Po Ling; Middeldorp, Jaap M; Lung, Maria Li; Tsao, Sai Wah; Chiang, Alan Kwok Shing

    2016-01-01

    Pan-histone deacetylase (HDAC) inhibitors, which inhibit 11 HDAC isoforms, are widely used to induce Epstein-Barr virus (EBV) lytic cycle in EBV-associated cancers in vitro and in clinical trials. Here, we hypothesized that inhibition of one or several specific HDAC isoforms by selective HDAC inhibitors could potently induce EBV lytic cycle in EBV-associated malignancies such as nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). We found that inhibition of class I HDACs, particularly HDAC-1, -2 and -3, was sufficient to induce EBV lytic cycle in NPC and GC cells in vitro and in vivo. Among a panel of selective HDAC inhibitors, the FDA-approved HDAC inhibitor romidepsin was found to be the most potent lytic inducer, which could activate EBV lytic cycle at ∼0.5 to 5 nM (versus ∼800 nM achievable concentration in patients' plasma) in more than 75% of cells. Upregulation of p21(WAF1) , which is negatively regulated by class I HDACs, was observed before the induction of EBV lytic cycle. The upregulation of p21(WAF1) and induction of lytic cycle were abrogated by a specific inhibitor of PKC-δ but not the inhibitors of PI3K, MEK, p38 MAPK, JNK or ATM pathways. Interestingly, inhibition of HDAC-1, -2 and -3 by romidepsin or shRNA knockdown could confer susceptibility of EBV-positive epithelial cells to the treatment with ganciclovir (GCV). In conclusion, we demonstrated that inhibition of class I HDACs by romidepsin could potently induce EBV lytic cycle and mediate enhanced cell death with GCV, suggesting potential application of romidepsin for the treatment of EBV-associated cancers. PMID:26205347

  4. Lytic Cycle of Toxoplasma gondii

    PubMed Central

    Black, Michael W.; Boothroyd, John C.

    2000-01-01

    Toxoplasma gondii is an obligate intracellular pathogen within the phylum Apicomplexa. This protozoan parasite is one of the most widespread, with a broad host range including many birds and mammals and a geographic range that is nearly worldwide. While infection of healthy adults is usually relatively mild, serious disease can result in utero or when the host is immunocompromised. This sophisticated eukaryote has many specialized features that make it well suited to its intracellular lifestyle. In this review, we describe the current knowledge of how the asexual tachyzoite stage of Toxoplasma attaches to, invades, replicates in, and exits the host cell. Since this process is closely analogous to the way in which viruses reproduce, we refer to it as the Toxoplasma “lytic cycle.” PMID:10974128

  5. Chlorpyrifos Induces the Expression of the Epstein-Barr Virus Lytic Cycle Activator BZLF-1 via Reactive Oxygen Species

    PubMed Central

    Zhao, Ling; Xie, Fei; Wang, Ting-ting; Liu, Meng-yu; Li, Jia-la; Shang, Lei; Deng, Zi-xuan; Zhao, Peng-xiang; Ma, Xue-mei

    2015-01-01

    Organophosphate pesticides (OPs) are among the most widely used synthetic chemicals for the control of a wide variety of pests, and reactive oxygen species (ROS) caused by OPs may be involved in the toxicity of various pesticides. Previous studies have demonstrated that a reactivation of latent Epstein-Barr virus (EBV) could be induced by oxidative stress. In this study, we investigated whether OPs could reactivate EBV through ROS accumulation. The Raji cells were treated with chlorpyrifos (CPF), one of the most commonly used OPs. Oxidative stress indicators and the expression of the EBV immediate-early gene BZLF-1 were determined after CPF treatment. Our results show that CPF induces oxidative stress as evidenced by decreased malondialdehyde (MDA) level, accompanied by an increase in ROS production, DNA damage, glutathione (GSH) level, and superoxide dismutase (SOD) and catalase (CAT) activity. Moreover, CPF treatment significantly enhances the expression of BZLF-1, and the increased BZLF-1 expression was ameliorated by N-acetylcysteine (NAC) incubation. These results suggest that OPs could contribute to the reactivation of the EBV lytic cycle through ROS induction, a process that may play an important role in the development of EBV-associated diseases. PMID:26257840

  6. Chlorpyrifos Induces the Expression of the Epstein-Barr Virus Lytic Cycle Activator BZLF-1 via Reactive Oxygen Species.

    PubMed

    Zhao, Ling; Xie, Fei; Wang, Ting-ting; Liu, Meng-yu; Li, Jia-la; Shang, Lei; Deng, Zi-xuan; Zhao, Peng-xiang; Ma, Xue-mei

    2015-01-01

    Organophosphate pesticides (OPs) are among the most widely used synthetic chemicals for the control of a wide variety of pests, and reactive oxygen species (ROS) caused by OPs may be involved in the toxicity of various pesticides. Previous studies have demonstrated that a reactivation of latent Epstein-Barr virus (EBV) could be induced by oxidative stress. In this study, we investigated whether OPs could reactivate EBV through ROS accumulation. The Raji cells were treated with chlorpyrifos (CPF), one of the most commonly used OPs. Oxidative stress indicators and the expression of the EBV immediate-early gene BZLF-1 were determined after CPF treatment. Our results show that CPF induces oxidative stress as evidenced by decreased malondialdehyde (MDA) level, accompanied by an increase in ROS production, DNA damage, glutathione (GSH) level, and superoxide dismutase (SOD) and catalase (CAT) activity. Moreover, CPF treatment significantly enhances the expression of BZLF-1, and the increased BZLF-1 expression was ameliorated by N-acetylcysteine (NAC) incubation. These results suggest that OPs could contribute to the reactivation of the EBV lytic cycle through ROS induction, a process that may play an important role in the development of EBV-associated diseases. PMID:26257840

  7. Valpromide Inhibits Lytic Cycle Reactivation of Epstein-Barr Virus

    PubMed Central

    Gorres, Kelly L.; Daigle, Derek; Mohanram, Sudharshan; McInerney, Grace E.; Lyons, Danielle E.

    2016-01-01

    ABSTRACT Reactivation of Epstein-Barr virus (EBV) from latency into the lytic phase of its life cycle allows the virus to spread among cells and between hosts. Valproic acid (VPA) inhibits initiation of the lytic cycle in EBV-infected B lymphoma cells. While VPA blocks viral lytic gene expression, it induces expression of many cellular genes, because it is a histone deacetylase (HDAC) inhibitor. Here we show, using derivatives of VPA, that blockade of EBV reactivation is separable from HDAC inhibition. Valpromide (VPM), an amide derivative of valproic acid that is not an HDAC inhibitor, prevented expression of two EBV genes, BZLF1 and BRLF1, that mediate lytic reactivation. VPM also inhibited expression of a viral late gene, but not early genes, when BZLF1 was exogenously expressed. Unlike VPA, VPM did not activate lytic expression of Kaposi’s sarcoma-associated herpesvirus. Expression of cellular immediate-early genes, such as FOS and EGR1, is kinetically upstream of the EBV lytic cycle. VPM did not activate expression of these cellular immediate-early genes but decreased their level of expression when induced by butyrate, an HDAC inhibitor. VPM did not alter expression of several other cellular immediate-early genes, including STAT3, which were induced by the HDAC inhibitors in cells refractory to lytic induction. Therefore, VPM selectively inhibits both viral and cellular gene expression. VPA and VPM represent a new class of antiviral agents. The mechanism by which VPA and VPM block EBV reactivation may be related to their anticonvulsant activity. PMID:26933051

  8. The human cytomegalovirus lytic cycle is induced by 1,25-dihydroxyvitamin D3 in peripheral blood monocytes and in the THP-1 monocytic cell line.

    PubMed

    Wu, Shu-En; Miller, William E

    2015-09-01

    Human cytomegalovirus (HCMV) resides in a latent form in hematopoietic progenitors and undifferentiated cells within the myeloid lineage. Maturation and differentiation along the myeloid lineage triggers lytic replication. Here, we used peripheral blood monocytes and the monocytic cell line THP-1 to investigate the effects of 1,25-dihydroxyvitamin D3 on HCMV replication. Interestingly, 1,25-dihydroxyvitamin D3 induces lytic replication marked by upregulation of HCMV gene expression and production of infectious virus. Moreover, we demonstrate that the effects of 1,25-dihydroxyvitamin D3 correlate with maturation/differentiation of the monocytes and not by directly stimulating the MIEP. These results are somewhat surprising as 1,25-dihydroxyvitamin D3 typically boosts immunity to bacteria and viruses rather than driving the infectious life cycle as it does for HCMV. Defining the signaling pathways kindled by 1,25-dihydroxyvitamin D3 will lead to a better understanding of the underlying molecular mechanisms that determine the fate of HCMV once it infects cells in the myeloid lineage. PMID:25965798

  9. Genipin as a novel chemical activator of EBV lytic cycle.

    PubMed

    Son, Myoungki; Lee, Minjung; Ryu, Eunhyun; Moon, Aree; Jeong, Choon-Sik; Jung, Yong Woo; Park, Gyu Hwan; Sung, Gi-Ho; Cho, Hyosun; Kang, Hyojeung

    2015-02-01

    Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus that causes acute infection and establishes life-long latency. EBV causes several human cancers, including Burkitt's lymphoma, nasopharyngeal and gastric carcinoma. Antiviral agents can be categorized as virucides, antiviral chemotherapeutic agents, and immunomodulators. Most antiviral agents affect actively replicating viruses, but not their latent forms. Novel antiviral agents must be active on both the replicating and the latent forms of the virus. Gardenia jasminoides is an evergreen flowering plant belonging to the Rubiaceae family and is most commonly found growing wild in Vietnam, Southern China, Taiwan, Japan, Myanmar, and India. Genipin is an aglycone derived from an iridoid glycoside called geniposide, which is present in large quantities in the fruit of G. jasminoides. In this study, genipin was evaluated for its role as an antitumor and antiviral agent that produces inhibitory effects against EBV and EBV associated gastric carcinoma (EBVaGC). In SNU719 cells, one of EBVaGCs, genipin caused significant cytotoxicity (70 μM), induced methylation on EBV C promoter and tumor suppressor gene BCL7A, arrested cell-cycle progress (S phases), upregulated EBV latent/lytic genes in a dose-dependent manner, stimulated EBV progeny production, activated EBV F promoter for EBV lytic activation, and suppressed EBV infection. These results indicated that genipin could be a promising candidate for antiviral and antitumor agents against EBV and EBVaGC. PMID:25626372

  10. Identification of Novel Small Organic Compounds with Diverse Structures for the Induction of Epstein-Barr Virus (EBV) Lytic Cycle in EBV-Positive Epithelial Malignancies

    PubMed Central

    Choi, Chung King; Ho, Dona N.; Hui, Kwai Fung; Kao, Richard Y.; Chiang, Alan K. S.

    2015-01-01

    Phorbol esters, which are protein kinase C (PKC) activators, and histone deacetylase (HDAC) inhibitors, which cause enhanced acetylation of cellular proteins, are the main classes of chemical inducers of Epstein-Barr virus (EBV) lytic cycle in latently EBV-infected cells acting through the PKC pathway. Chemical inducers which induce EBV lytic cycle through alternative cellular pathways may aid in defining the mechanisms leading to lytic cycle reactivation and improve cells’ responsiveness towards lytic induction. We performed a phenotypic screening on a chemical library of 50,240 novel small organic compounds to identify novel class(es) of strong inducer(s) of EBV lytic cycle in gastric carcinoma (GC) and nasopharyngeal carcinoma (NPC) cells. Five hit compounds were selected after three successive rounds of increasingly stringent screening. All five compounds are structurally diverse from each other and distinct from phorbol esters or HDAC inhibitors. They neither cause hyperacetylation of histone proteins nor significant PKC activation at their working concentrations, suggesting that their biological mode of action are distinct from that of the known chemical inducers. Two of the five compounds with rapid lytic-inducing action were further studied for their mechanisms of induction of EBV lytic cycle. Unlike HDAC inhibitors, lytic induction by both compounds was not inhibited by rottlerin, a specific inhibitor of PKCδ. Interestingly, both compounds could cooperate with HDAC inhibitors to enhance EBV lytic cycle induction in EBV-positive epithelial cancer cells, paving way for the development of strategies to increase cells’ responsiveness towards lytic reactivation. One of the two compounds bears structural resemblance to iron chelators and the other strongly activates the MAPK pathways. These structurally diverse novel organic compounds may represent potential new classes of chemicals that can be used to investigate any alternative mechanism(s) leading to EBV

  11. PKC theta and p38 MAPK activate the EBV lytic cycle through autophagy induction.

    PubMed

    Gonnella, Roberta; Granato, Marisa; Farina, Antonella; Santarelli, Roberta; Faggioni, Alberto; Cirone, Mara

    2015-07-01

    PKC activation by combining TPA with sodium butyrate (T/B) represents the most effective and widely used strategy to induce the Epstein-Barr virus (EBV) lytic cycle. The results obtained in this study show that novel PKCθ is involved in such process and that it acts through the activation of p38 MAPK and autophagy induction. Autophagy, a mechanism of cellular defense in stressful conditions, is manipulated by EBV to enhance viral replication. Besides promoting the EBV lytic cycle, the activation of p38 and autophagy resulted in a pro-survival effect, as indicated by p38 or ATG5 knocking down experiments. However, this pro-survival role was counteracted by a pro-death activity of PKCθ, due to the dephosphorylation of AKT. In conclusion, this study reports, for the first time, that T/B activates a PKCθ-p38 MAPK axis in EBV infected B cells, that promotes the viral lytic cycle and cell survival and dephosphorylates AKT, balancing cell life and cell death. PMID:25827954

  12. Effect of Pinus massoniana Lamb. bark extract on lytic cycle of Epstein-Barr virus.

    PubMed

    Xu, Shuxia; Zhang, Shimin; Wang, Xuedong; Gao, Yaqian; Qin, Xing; Wu, Kun

    2012-10-01

    Pinus massoniana bark extract (PMBE) at a concentration of 60 microg/mL or more inhibits the expression of Epstein-Barr virus (EBV) lytic proteins, such as Rta, Zta, and EA-D. EBV lytic cycle was blocked by inhibiting the transcription of immediate-early genes. The results suggest that the PMBE has anti-EBV activity. Thus, the extract is potentially useful in preventing the lytic development of EBV in vitro. PMID:23214264

  13. Immediate Early and Early Lytic Cycle Proteins Are Frequent Targets of the Epstein-Barr Virus–induced Cytotoxic T Cell Response

    PubMed Central

    Steven, N.M.; Annels, N.E.; Kumar, A.; Leese, A.M.; Kurilla, M.G.; Rickinson, A.B.

    1997-01-01

    Epstein-Barr virus (EBV), a human γ-herpesvirus, can establish both nonproductive (latent) and productive (lytic) infections. Although the CD8+ cytotoxic T lymphocyte (CTL) response to latently infected cells is well characterized, very little is known about T cell controls over lytic infection; this imbalance in our understanding belies the importance of virus-replicative lesions in several aspects of EBV disease pathogenesis. The present work shows that the primary CD8+ CTL response to EBV in infectious mononucleosis patients contains multiple lytic antigen-specific reactivities at levels at least as high as those seen against latent antigens; similar reactivities are also detectable in CTL memory. Clonal analysis revealed individual responses to the two immediate early proteins BZLF1 and BRLF1, and to three (BMLF1, BMRF1, and BALF2) of the six early proteins tested. In several cases, the peptide epitope and HLA-restricting determinant recognized by these CTLs has been defined, one unusual feature being the number of responses restricted through HLA-C alleles. The work strongly suggests that EBVreplicative lesions are subject to direct CTL control in vivo and that immediate early and early proteins are frequently the immunodominant targets. This contrasts with findings in α- and β-herpesvirus systems (herpes simplex, cytomegalovirus) where viral interference with the antigen-processing pathway during lytic infection renders immediate early and early proteins much less immunogenic. The unique capacity of γ-herpesvirus to amplify the viral load in vivo through a latent growth-transforming infection may have rendered these agents less dependent upon viral replication as a means of successfully colonizing their hosts. PMID:9151898

  14. Dynamic chromatin environment of key lytic cycle regulatory regions of the Epstein-Barr virus genome.

    PubMed

    Ramasubramanyan, Sharada; Osborn, Kay; Flower, Kirsty; Sinclair, Alison J

    2012-02-01

    The ability of Epstein-Barr virus (EBV) to establish latency allows it to evade the immune system and to persist for the lifetime of its host; one distinguishing characteristic is the lack of transcription of the majority of viral genes. Entry into the lytic cycle is coordinated by the viral transcription factor, Zta (BZLF1, ZEBRA, and EB1), and downstream effectors, while viral genome replication requires the concerted action of Zta and six other viral proteins at the origins of lytic replication. We explored the chromatin context at key EBV lytic cycle promoters (BZLF1, BRLF1, BMRF1, and BALF5) and the origins of lytic replication during latency and lytic replication. We show that a repressive heterochromatin-like environment (trimethylation of histone H3 at lysine 9 [H3K9me3] and lysine 27 [H3K27me3]), which blocks the interaction of some transcription factors with DNA, encompasses the key early lytic regulatory regions. Epigenetic silencing of the EBV genome is also imposed by DNA methylation during latency. The chromatin environment changes during the lytic cycle with activation of histones H3, H4, and H2AX occurring at both the origins of replication and at the key lytic regulatory elements. We propose that Zta is able to reverse the effects of latency-associated repressive chromatin at EBV early lytic promoters by interacting with Zta response elements within the H3K9me3-associated chromatin and demonstrate that these interactions occur in vivo. Since the interaction of Zta with DNA is not inhibited by DNA methylation, it is clear that Zta uses two routes to overcome epigenetic silencing of its genome. PMID:22090141

  15. DNA Damage Signaling Is Induced in the Absence of Epstein-Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA.

    PubMed

    Wang'ondu, Ruth; Teal, Stuart; Park, Richard; Heston, Lee; Delecluse, Henri; Miller, George

    2015-01-01

    Epstein Barr virus (EBV), like other oncogenic viruses, modulates the activity of cellular DNA damage responses (DDR) during its life cycle. Our aim was to characterize the role of early lytic proteins and viral lytic DNA replication in activation of DNA damage signaling during the EBV lytic cycle. Our data challenge the prevalent hypothesis that activation of DDR pathways during the EBV lytic cycle occurs solely in response to large amounts of exogenous double stranded DNA products generated during lytic viral DNA replication. In immunofluorescence or immunoblot assays, DDR activation markers, specifically phosphorylated ATM (pATM), H2AX (γH2AX), or 53BP1 (p53BP1), were induced in the presence or absence of viral DNA amplification or replication compartments during the EBV lytic cycle. In assays with an ATM inhibitor and DNA damaging reagents in Burkitt lymphoma cell lines, γH2AX induction was necessary for optimal expression of early EBV genes, but not sufficient for lytic reactivation. Studies in lytically reactivated EBV-positive cells in which early EBV proteins, BGLF4, BGLF5, or BALF2, were not expressed showed that these proteins were not necessary for DDR activation during the EBV lytic cycle. Expression of ZEBRA, a viral protein that is necessary for EBV entry into the lytic phase, induced pATM foci and γH2AX independent of other EBV gene products. ZEBRA mutants deficient in DNA binding, Z(R183E) and Z(S186E), did not induce foci of pATM. ZEBRA co-localized with HP1β, a heterochromatin associated protein involved in DNA damage signaling. We propose a model of DDR activation during the EBV lytic cycle in which ZEBRA induces ATM kinase phosphorylation, in a DNA binding dependent manner, to modulate gene expression. ATM and H2AX phosphorylation induced prior to EBV replication may be critical for creating a microenvironment of viral and cellular gene expression that enables lytic cycle progression. PMID:25950714

  16. Induction of epstein-barr virus (EBV) lytic cycle in vitro causes lipid peroxidation, protein oxidation and DNA damage in lymphoblastoid B cell lines

    PubMed Central

    2011-01-01

    Background We investigated the oxidative modifications of lipids, proteins and DNA, potential molecular targets of oxidative stress, in two lymphoblastoid cell lines: B95-8 and Raji, after EBV lytic cycle induction. Conjugated dienes level was measured as biomarker of lipid peroxidation. Malondialdehyde adduct and protein carbonyl levels, as well as protein thiol levels were measured as biomarkers of protein oxidation. DNA fragmentation was evaluated as biomarker of DNA oxidation. Results After 48 h (peak of lytic cycle), a significant increase in conjugated dienes level was observed in B95-8 and Raji cell lines (p = 0.0001 and p = 0.019 respectively). Malondialdehyde adduct, protein carbonyl levels were increased in B95-8 and Raji cell lines after EBV lytic cycle induction as compared to controls (MDA-adduct: p = 0.008 and p = 0.006 respectively; Carbonyl: p = 0.003 and p = 0.0039 respectively). Proteins thiol levels were decreased by induction in B95-8 and Raji cell lines (p = 0.046; p = 0.002 respectively). DNA fragmentation was also detected in B95-8 and Raji cell lines after EBV lytic cycle induction as compared to controls. Conclusion The results of this study demonstrate the presence of increased combined oxidative modifications in lipids, proteins in B95-8 and Raji cells lines after EBV lytic cycle induction. These results suggest that lipid peroxidation, protein oxidation and DNA fragmentation are generally induced during EBV lytic cycle induction and probably contribute to the cytopathic effect of EBV. PMID:21722381

  17. Modulation of Lactobacillus casei bacteriophage A2 lytic/lysogenic cycles by binding of Gp25 to the early lytic mRNA.

    PubMed

    Carrasco, Begoña; Escobedo, Susana; Alonso, Juan C; Suárez, Juan E

    2016-01-01

    The genetic switch of Lactobacillus casei bacteriophage A2 is regulated by the CI protein, which represses the early lytic promoter PR and Cro that abolishes expression from the lysogenic promoter PL . Lysogens contain equivalent cI and cro-gp25 mRNA concentrations, i.e., CI only partially represses P(R), predicting a lytic cycle dominance. However, A2 generates stable lysogens. This may be due to Gp25 binding to the cro-gp25 mRNA between the ribosomal binding site and the cro start codon, which abolishes its translation. Upon lytic cycle induction, CI is partially degraded, cro-gp25 mRNA levels increase, and Cro accumulates, launching viral progeny production. The concomitant concentration increase of Gp25 restricts cro mRNA translation, which, together with the low but detectable levels of CI late during the lytic cycle, promotes reentry of part of the cell population into the lysogenic cycle, thus explaining the low proportion of L. casei lysogens that become lysed (∼ 1%). A2 shares its genetic switch structure with many other Firmicutes phages. The data presented may constitute a model of how these phages make the decision for lysis versus lysogeny. PMID:26417647

  18. Titanium dioxide-assisted photocatalytic induction of prophages to lytic cycle.

    PubMed

    Skorb, Ekaterina V; Andreeva, Daria V; Raiski, Andrey P; Belyasova, Natalya A; Möhwald, Helmuth; Sviridov, Dmitry V

    2011-12-01

    The investigations on the kinetics of photocatalytic inactivation of bacteriophages, lactic bacteria and lysogenic lactic bacteria have shown that the rate of bacterial inactivation is ca. 10 times less than the inactivation of bacteriophages. Titania-assisted photorelease of bacteriophages from lysogenic bacteria proves that photogenerated reactive oxygen species affect the deoxyribonucleic acid (DNA) of bacteria before their deactivation. On this basis a novel photocatalytic method of a prophage induction to the lytic cycle and detection of lysogenic bacteria is proposed. PMID:22057553

  19. Microtubule depolymerization activates the Epstein-Barr virus lytic cycle through protein kinase C pathways in nasopharyngeal carcinoma cells.

    PubMed

    Liu, Yi-Ru; Huang, Sheng-Yen; Chen, Jen-Yang; Wang, Lily Hui-Ching

    2013-12-01

    Elevated levels of antibodies against Epstein-Barr virus (EBV) and the presence of viral DNA in plasma are reliable biomarkers for the diagnosis of nasopharyngeal carcinoma (NPC) in high-prevalence areas, such as South-East Asia. The presence of these viral markers in the circulation suggests that a minimal level of virus reactivation may have occurred in an infected individual, although the underlying mechanism of reactivation remains to be elucidated. Here, we showed that treatment with nocodazole, which provokes the depolymerization of microtubules, induces the expression of two EBV lytic cycle proteins, Zta and EA-D, in EBV-positive NPC cells. This effect was independent of mitotic arrest, as viral reactivation was not abolished in cells synchronized at interphase. Notably, the induction of Zta by nocodazole was mediated by transcriptional upregulation via protein kinase C (PKC). Pre-treatment with inhibitors for PKC or its downstream signalling partners p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) abolished the nocodazole-mediated induction of Zta and EA-D. Interestingly, the effect of nocodazole, as well as colchicine and vinblastine, on lytic gene expression occurred only in NPC epithelial cells but not in cells derived from lymphocytes. These results establish a novel role of microtubule integrity in controlling the EBV life cycle through PKC and its downstream pathways, which represents a tissue-specific mechanism for controlling the life-cycle switch of EBV. PMID:24062531

  20. Direct positive selection for improved nitroreductase variants using SOS triggering of bacteriophage lambda lytic cycle.

    PubMed

    Guise, C P; Grove, J I; Hyde, E I; Searle, P F

    2007-04-01

    Expression of prodrug-activating enzymes that convert non-toxic substrates to cytotoxic derivatives is a promising strategy for cancer gene therapy. However, their catalytic activity with unnatural, prodrug substrates is often suboptimal. Efforts to improve these enzymes have been limited by the inability to select directly for increased prodrug activation. We have focussed on developing variants of Escherichia coli (E. coli) nitroreductase (NTR) with improved ability to activate the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954), and describe here a novel, direct, positive selection for improved enzymes that exploits the alternative life cycles of bacteriophage lambda. In lambda lysogens of E. coli, the activation of the prodrug CB1954 by NTR triggers the SOS response to DNA damage, switching integrated lambda prophages into lytic cycle. This provides a direct, positive selection for phages encoding improved NTR variants, as, upon limiting exposure of lysogenized E. coli to CB1954, only those encoding the most active enzyme variants are triggered into lytic cycle, allowing their selective recovery. We exemplify the selection by isolating highly improved 'turbo-NTR' variants from a library of 6.8 x 10(5) clones, conferring up to 50-fold greater sensitivity to CB1954 than the wild type. Carcinoma cells infected with adenovirus expressing T41Q/N71S/F124T-NTR were sensitized to CB1954 concentrations 40- to 80-fold lower than required with WT-NTR. PMID:17301844

  1. Linking the lytic and lysogenic bacteriophage cycles to environmental conditions, host physiology and their variability in coastal lagoons.

    PubMed

    Maurice, C F; Bouvier, C; de Wit, R; Bouvier, T

    2013-09-01

    Changes in environmental conditions and prokaryote physiology can strongly affect the dynamics of both the lysogenic and lytic bacteriophage replication cycles in aquatic systems. However, it remains unclear whether it is the nature, amplitude or frequency of these changes that alter the phage replication cycles. We performed an annual survey of three Mediterranean lagoons with contrasting levels of chlorophyll a concentration and salinity to explore how these cues and their variability influence either replication cycle. The lytic cycle was always detected and showed seasonal patterns, whereas the lysogenic cycle was often undetected and highly variable. The lytic cycle was influenced by environmental and prokaryotic physiological cues, increasing with concentrations of dissolved organic carbon, chlorophyll a, and the proportion of respiring cells, and decreasing with the proportion of damaged cells. In contrast, lysogeny was not explained by the magnitude of any environmental or physiological parameter, but increased with the amplitude of change in prokaryote physiology. Our study suggests that both cycles are regulated by distinct factors: the lytic cycle is dependent on environmental parameters and host physiology, while lysogeny is dependent on the variability of prokaryote physiology. This could lead to the contrasting patterns observed between both cycles in aquatic systems. PMID:23581698

  2. Calcium Signaling throughout the Toxoplasma gondii Lytic Cycle: A STUDY USING GENETICALLY ENCODED CALCIUM INDICATORS.

    PubMed

    Borges-Pereira, Lucas; Budu, Alexandre; McKnight, Ciara A; Moore, Christina A; Vella, Stephen A; Hortua Triana, Miryam A; Liu, Jing; Garcia, Celia R S; Pace, Douglas A; Moreno, Silvia N J

    2015-11-01

    Toxoplasma gondii is an obligate intracellular parasite that invades host cells, creating a parasitophorous vacuole where it communicates with the host cell cytosol through the parasitophorous vacuole membrane. The lytic cycle of the parasite starts with its exit from the host cell followed by gliding motility, conoid extrusion, attachment, and invasion of another host cell. Here, we report that Ca(2+) oscillations occur in the cytosol of the parasite during egress, gliding, and invasion, which are critical steps of the lytic cycle. Extracellular Ca(2+) enhances each one of these processes. We used tachyzoite clonal lines expressing genetically encoded calcium indicators combined with host cells expressing transiently expressed calcium indicators of different colors, and we measured Ca(2+) changes in both parasites and host simultaneously during egress. We demonstrated a link between cytosolic Ca(2+) oscillations in the host and in the parasite. Our approach also allowed us to measure two new features of motile parasites, which were enhanced by Ca(2+) influx. This is the first study showing, in real time, Ca(2+) signals preceding egress and their direct link with motility, an essential virulence trait. PMID:26374900

  3. Artemisinin derivatives inhibit Toxoplasma gondii in vitro at multiple steps in the lytic cycle

    PubMed Central

    D'Angelo, John G.; Bordón, Claudia; Posner, Gary H.; Yolken, Robert; Jones-Brando, Lorraine

    2009-01-01

    Objectives We sought to improve upon the usefulness of artemisinins as anti-Toxoplasma agents by synthesizing new unsaturated, carba derivatives and then testing them for in vitro efficacy against three steps of the lytic cycle of Toxoplasma gondii tachyzoites. Methods Novel derivatives of ART were synthesized and then tested for in vitro antiparasitic activity using T. gondii tachyzoites constitutively expressing β-galactosidase and human fibroblast host cells. Compounds were evaluated for parasite growth inhibition and cytotoxicity, inhibition of replication and inhibition of parasite invasion of host cells. Results Five of the seven new derivatives, 3a–c, 3e and 3f, effectively inhibited T. gondii growth (IC50 = 1.0–4.4 µM); however, only three of these proved to be relatively non-cytotoxic (TD50 ≥ 200 µM). The same five derivatives also inhibited tachyzoite replication, and attachment to and invasion of host cells as effectively as or better than the parent compound ART. In addition, one of the derivatives incapable of inhibiting growth, deoxy-3a, was found to inhibit parasite invasion. Conclusions These new artemisinin derivatives have the ability to inhibit multiple steps of T. gondii's lytic cycle. Synthetic unsaturated, carba derivatives of ART have potential as therapeutic agents for the prevention and treatment of toxoplasmosis in humans. PMID:18988681

  4. Effect of Metals on the Lytic Cycle of the Coccolithovirus, EhV86

    PubMed Central

    Gledhill, Martha; Devez, Aurélie; Highfield, Andrea; Singleton, Chloe; Achterberg, Eric P.; Schroeder, Declan

    2012-01-01

    In this study we show that metals, and in particular copper (Cu), can disrupt the lytic cycle in the Emiliania huxleyi – EhV86 host–virus system. E. huxleyi lysis rates were reduced at high total Cu concentrations (> approximately 500 nM) in the presence and absence of EDTA (ethylenediaminetetraacetic acid) in acute short term exposure experiments. Zinc (Zn), cadmium (Cd), and cobalt (Co) were not observed to affect the lysis rate of EhV86 in these experiments. The cellular glutathione (GSH) content increased in virus infected cells, but not as a result of metal exposure. In contrast, the cellular content of phytochelatins (PCs) increased only in response to metal exposure. The increase in glutathione content is consistent with increases in the production of reactive oxygen species (ROS) on viral lysis, while increases in PC content are likely linked to metal homeostasis and indicate that metal toxicity to the host was not affected by viral infection. We propose that Cu prevents lytic production of EhV86 by interfering with virus DNA (deoxyribonucleic acid) synthesis through a transcriptional block, which ultimately suppresses the formation of ROS. PMID:22536202

  5. The FIKK kinase of Toxoplasma gondii is not essential for the parasite's lytic cycle.

    PubMed

    Skariah, S; Walwyn, O; Engelberg, K; Gubbels, M-J; Gaylets, C; Kim, N; Lynch, B; Sultan, A; Mordue, D G

    2016-05-01

    FIKK kinases are a novel family of kinases unique to the Apicomplexa. While most apicomplexans encode a single FIKK kinase, Plasmodium falciparum expresses 21 and piroplasms do not encode a FIKK kinase. FIKK kinases share a conserved C-terminal catalytic domain, but the N-terminal region is highly variable and contains no known functional domains. To date, FIKK kinases have been primarily studied in P. falciparum and Plasmodium berghei. Those that have been studied are exported from the parasite and associate with diverse locations in the infected erythrocyte cytosol or membrane. Deletion of individual P. falciparum FIKK kinases indicates that they may play a role in modification of the infected erythrocyte. The current study characterises the single FIKK gene in Toxoplasma gondii to evaluate the importance of the FIKK kinase in an apicomplexan that has a single FIKK kinase. The TgFIKK gene encoded a protein of approximately 280kDa. Endogenous tagging of the FIKK protein with Yellow Fluorescent Protein showed that the FIKK protein exclusively localised to the posterior end of tachyzoites. A Yellow Fluorescent Protein-tagged FIKK and a Ty-tagged FIKK both co-localised with T. gondii membrane occupation and recognition nexus protein to the basal complex and were localised apical to inner membrane complex protein-5 and Centrin2. Deletion of TgFIKK, surprisingly, had no detectable effect on the parasite's lytic cycle in vitro in human fibroblast cells or in acute virulence in vivo. Thus, our results clearly show that while the FIKK kinase is expressed in tachyzoites, it is not essential for the lytic cycle of T. gondii. PMID:26859096

  6. Metabolic Cooperation of Glucose and Glutamine Is Essential for the Lytic Cycle of Obligate Intracellular Parasite Toxoplasma gondii.

    PubMed

    Nitzsche, Richard; Zagoriy, Vyacheslav; Lucius, Richard; Gupta, Nishith

    2016-01-01

    Toxoplasma gondii is a widespread protozoan parasite infecting nearly all warm-blooded organisms. Asexual reproduction of the parasite within its host cells is achieved by consecutive lytic cycles, which necessitates biogenesis of significant energy and biomass. Here we show that glucose and glutamine are the two major physiologically important nutrients used for the synthesis of macromolecules (ATP, nucleic acid, proteins, and lipids) in T. gondii, and either of them is sufficient to ensure the parasite survival. The parasite can counteract genetic ablation of its glucose transporter by increasing the flux of glutamine-derived carbon through the tricarboxylic acid cycle and by concurrently activating gluconeogenesis, which guarantee a continued biogenesis of ATP and biomass for host-cell invasion and parasite replication, respectively. In accord, a pharmacological inhibition of glutaminolysis or oxidative phosphorylation arrests the lytic cycle of the glycolysis-deficient mutant, which is primarily a consequence of impaired invasion due to depletion of ATP. Unexpectedly, however, intracellular parasites continue to proliferate, albeit slower, notwithstanding a simultaneous deprivation of glucose and glutamine. A growth defect in the glycolysis-impaired mutant is caused by a compromised synthesis of lipids, which cannot be counterbalanced by glutamine but can be restored by acetate. Consistently, supplementation of parasite cultures with exogenous acetate can amend the lytic cycle of the glucose transport mutant. Such plasticity in the parasite's carbon flux enables a growth-and-survival trade-off in assorted nutrient milieus, which may underlie the promiscuous survival of T. gondii tachyzoites in diverse host cells. Our results also indicate a convergence of parasite metabolism with cancer cells. PMID:26518878

  7. Detergent sclerosants at sub-lytic concentrations induce endothelial cell apoptosis through a caspase dependent pathway.

    PubMed

    Cooley-Andrade, Osvaldo; Cheung, Kelvin; Chew, An-Ning; Connor, David Ewan; Parsi, Kurosh

    2016-07-01

    To investigate the apoptotic effects of detergent sclerosants sodium tetradecylsulphate (STS) and polidocanol (POL) on endothelial cells at sub-lytic concentrations. Human umbilical vein endothelial cells (HUVECs) were isolated and labelled with antibodies to assess for apoptosis and examined with confocal microscopy and flow cytometry. Isolated HUVECs viability was assessed using propidium iodide staining. Early apoptosis was determined by increased phosphatidylserine exposure by lactadherin binding. Caspase 3, 8, 9 and Bax activation as well as inhibitory assays with Pan Caspase (Z-VAD-FMK) and Bax (BI-6C9) were assessed to identify apoptotic pathways. Porimin activation was used to assess cell membrane permeability. Cell lysis reached almost 100 % with STS at 0.3 % and with POL at 0.6 %. Apoptosis was seen with both STS and POL at concentrations ranging from 0.075 to 0.15 %. PS exposure increased with both STS and POL and exhibited a dose-dependent trend. Active Caspase 3, 8 and 9 but not Bax were increased in HUVECs stimulated with low concentrations of both STS and POL. Inhibitory assays demonstrated Caspase 3, 8, 9 inhibition at low concentrations (0.075 to 0.6 %) with both STS and POL. Both agents increased the activation of porimin at all concentrations. Both sclerosants induced endothelial cell (EC) apoptosis at sub-lytic concentrations through a caspase-dependant pathway. Both agents induced EC oncosis. PMID:27225250

  8. Inhibition of Epstein-Barr virus lytic cycle by an ethyl acetate subfraction separated from Polygonum cuspidatum root and its major component, emodin.

    PubMed

    Yiu, Ching-Yi; Chen, Shih-Ying; Yang, Tsai-Hsiu; Chang, Che-Jung; Yeh, Dong-Bor; Chen, Yi-Jie; Lin, Tsuey-Pin

    2014-01-01

    Polygonum cuspidatum is widely used as a medicinal herb in Asia. In this study, we examined the ethyl acetate subfraction F3 obtained from P. cuspidatum root and its major component, emodin, for their capacity to inhibit the Epstein-Barr virus (EBV) lytic cycle. The cell viability was determined by the MTT [3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide] method. The expression of EBV lytic proteins was analyzed by immunoblot, indirect immunofluorescence and flow cytometric assays. Real-time quantitative PCR was used to assess the EBV DNA replication and the transcription of lytic genes, including BRLF1 and BZLF1. Results showed that the F3 and its major component emodin inhibit the transcription of EBV immediate early genes, the expression of EBV lytic proteins, including Rta, Zta, and EA-D and reduces EBV DNA replication, showing that F3 and emodin are potentially useful as an anti-EBV drug. PMID:24448066

  9. Zebularine reactivates silenced E-cadherin but unlike 5-Azacytidine does not induce switching from latent to lytic Epstein-Barr virus infection in Burkitt's lymphoma Akata cells

    PubMed Central

    Rao, Sieta P; Rechsteiner, Markus P; Berger, Christoph; Sigrist, Jürg A; Nadal, David; Bernasconi, Michele

    2007-01-01

    Epigenetic silencing of regulatory genes by aberrant methylation contributes to tumorigenesis. DNA methyltransferase inhibitors (DNMTI) represent promising new drugs for anti-cancer therapies. The DNMTI 5-Azacytidine is effective against myelodysplastic syndrome, but induces switching of latent to lytic Epstein-Barr virus (EBV) in vitro and results in EBV DNA demethylation with the potential of induction of lytic EBV in vivo. This is of considerable concern given that recurrent lytic EBV has been linked with an increased incidence of EBV-associated lymphomas. Based on the distinct properties of action we hypothesized that the newer DNMTI Zebularine might differ from 5-Azacytidine in its potential to induce switching from latent to lytic EBV. Here we show that both 5-Azacytidine and Zebularine are able to induce expression of E-cadherin, a cellular gene frequently silenced by hypermethylation in cancers, and thus demonstrate that both DNMTI are active in our experimental setting consisting of EBV-harboring Burkitt's lymphoma Akata cells. Quantification of mRNA expression of EBV genes revealed that 5-Azacytidine induces switching from latent to lytic EBV and, in addition, that the immediate-early lytic infection progresses to early and late lytic infection. Furthermore, 5-Azacytidine induced upregulation of the latent EBV genes LMP2A, LMP2B, and EBNA2 in a similar fashion as observed following switching of latent to lytic EBV upon cross-linking of the B-cell receptor. In striking contrast, Zebularine did not exhibit any effect neither on lytic nor on latent EBV gene expression. Thus, Zebularine might be safer than 5-Azacytidine for the treatment of cancers in EBV carriers and could also be applied against EBV-harboring tumors, since it does not induce switching from latent to lytic EBV which may result in secondary EBV-associated malignancies. PMID:17214905

  10. STAT3 Regulates Lytic Activation of Kaposi's Sarcoma-Associated Herpesvirus

    PubMed Central

    Li, Xiaofan; Barbachano-Guerrero, Arturo

    2015-01-01

    ABSTRACT Lytic activation of Kaposi's sarcoma-associated herpesvirus (KSHV) from latency is a critical contributor to pathogenesis and progression of KSHV-mediated disease. Development of targeted treatment strategies and improvement of lytic-phase-directed oncolytic therapies, therefore, hinge on gaining a better understanding of latency-to-lytic-phase transition. A key observation in that regard, also common to other herpesviruses, is the partial permissiveness of latently infected cells to lytic-cycle-inducing agents. Here, we address the molecular basis of why only some KSHV-infected cells respond to lytic stimuli. Since cellular signal transducer and activator of transcription 3 (STAT3) is constitutively active in KSHV-associated cancers, KSHV activates STAT3, and STAT3 has been found to regulate lytic activation of Epstein-Barr virus (EBV)-infected cells, we asked if STAT3 contributes similarly to the life cycle of KSHV. We found that high levels of STAT3 correlate with the refractory state at the single-cell level under conditions of both spontaneous and induced lytic activation; importantly, STAT3 also regulates lytic susceptibility. Further, knockdown of STAT3 suppresses the cellular transcriptional corepressor Krüppel-associated box domain-associated protein 1 (KAP1; also known as TRIM28), and suppression of KAP1 activates lytic genes, including the viral lytic switch RTA, thereby linking STAT3 via KAP1 to regulation of the balance between lytic and latent cells. These findings, taken together with those from EBV-infected and, more recently, herpes simplex virus 1 (HSV-1)-infected cells, cement the contribution of host STAT3 to persistence of herpesviruses and simultaneously reveal an important lead to devise strategies to improve lytic-phase-directed therapies for herpesviruses. IMPORTANCE Lytic activation of the cancer-causing Kaposi's sarcoma-associated herpesvirus (KSHV) is vital to its life cycle and causation of disease. Like other herpesviruses

  11. The Epstein-Barr Virus Immediate-Early Gene Product, BRLF1, Interacts with the Retinoblastoma Protein during the Viral Lytic Cycle

    PubMed Central

    Zacny, Valerie L.; Wilson, Julie; Pagano, Joseph S.

    1998-01-01

    Retinoblastoma protein (Rb) is a key regulator of cellular proliferation, controlling entry into G1/S in the cell cycle, largely through its action in binding the cellular transcription factor E2F, which activates genes important in DNA synthesis. Small DNA tumor viruses encode gene products which can functionally inactivate Rb, promoting cellular proliferation and viral DNA synthesis. In this study, the Epstein-Barr virus (EBV) immediate-early lytic gene product, BRLF1 (R), is shown to bind Rb in vivo, shortly after induction of the viral lytic cycle in EBV-infected Akata cells. Furthermore, the temporal kinetics of R-Rb interaction correlate with displacement of E2F1 from Rb. Mapping of the domains required for the interaction of R and Rb proteins reveals that R binds specifically to the N terminus of Rb, outside the Rb pocket, and that the first 200 amino acids of R are required for this interaction. The interaction of R and Rb may initiate cell cycle progression and facilitate viral DNA synthesis during lytic replication. PMID:9733844

  12. Efficient lytic induction of kaposi's sarcoma-associated herpesvirus (KSHV) by the anthracyclines

    PubMed Central

    Kim, Hyeongki; Choi, Miri; Lee, So-Young; Kim, Chonsaeng; Lee, Sang Jun; Song, Moon Jung; Kang, Hyojeung; Back, Sung Hoon; Han, Sang-Bae; Cho, Sungchan

    2014-01-01

    Lytic induction of latent Kaposi's sarcoma-associated herpesvirus (KSHV) has been considered as a therapeutic option for efficient treatment of several KSHV-associated malignancies. Here, we developed a robust high-throughput screening system that allows an easy and quantitative measurement of lytic induction of latent KSHV and discovered three anthracyclines as potent inducers from screen of FDA-approved drugs. Lytic induction of latent KSHV by three compounds was verified by the significant induction of lytic genes and subsequent production of infectious KSHV. Importantly, lytic induction by three compounds was much more efficient than that by sodium butyrate, a well-characterized inducer of KSHV lytic cycle. Mechanistically, the anthracyclines caused lytic induction of KSHV through apoptosis induced by their DNA intercalation rather than topoisomerase II inhibition. Consequently, our results clearly demonstrated a role of anthracyclines as effective lytic inducers of KSHV and also provided a molecular basis of their use for efficient treatment of diseases associated with KSHV infection. PMID:25237786

  13. Efficient lytic induction of Kaposi's sarcoma-associated herpesvirus (KSHV) by the anthracyclines.

    PubMed

    Kang, Hyunju; Song, Jaehyung; Choi, Kwangman; Kim, Hyeongki; Choi, Miri; Lee, So-Young; Kim, Chonsaeng; Lee, Sang Jun; Song, Moon Jung; Kang, Hyojeung; Back, Sung Hoon; Han, Sang-Bae; Cho, Sungchan

    2014-09-30

    Lytic induction of latent Kaposi's sarcoma-associated herpesvirus (KSHV) has been considered as a therapeutic option for efficient treatment of several KSHV-associated malignancies. Here, we developed a robust high-throughput screening system that allows an easy and quantitative measurement of lytic induction of latent KSHV and discovered three anthracyclines as potent inducers from screen of FDA-approved drugs. Lytic induction of latent KSHV by three compounds was verified by the significant induction of lytic genes and subsequent production of infectious KSHV. Importantly, lytic induction by three compounds was much more efficient than that by sodium butyrate, a well-characterized inducer of KSHV lytic cycle. Mechanistically, the anthracyclines caused lytic induction of KSHV through apoptosis induced by their DNA intercalation rather than topoisomerase II inhibition. Consequently, our results clearly demonstrated a role of anthracyclines as effective lytic inducers of KSHV and also provided a molecular basis of their use for efficient treatment of diseases associated with KSHV infection. PMID:25237786

  14. Propranolol Decreases Proliferation of Endothelial Cells Transformed by Kaposi's Sarcoma-Associated Herpesvirus and Induces Lytic Viral Gene Expression

    PubMed Central

    Hanson, Ryan S.; Manion, Rory D.

    2015-01-01

    Kaposi's sarcoma (KS) is common in Africa, but economic constraints hinder successful treatment in most patients. Propranolol, a generic β-adrenergic antagonist, decreased proliferation of KS-associated herpesvirus (KSHV)-infected cells. Downregulation of cyclin A2 and cyclin-dependent kinase 1 (CDK1) recapitulated this phenotype. Additionally, propranolol induced lytic gene expression in association with downregulation of CDK6. Thus, propranolol has diverse effects on KSHV-infected cells, and this generic drug has potential as a therapeutic agent for KS. PMID:26269192

  15. Molecular Biology of KSHV Lytic Reactivation

    PubMed Central

    Purushothaman, Pravinkumar; Uppal, Timsy; Verma, Subhash C.

    2015-01-01

    Kaposi’s sarcoma-associated herpesvirus (KSHV) primarily persists as a latent episome in infected cells. During latent infection, only a limited number of viral genes are expressed that help to maintain the viral episome and prevent lytic reactivation. The latent KSHV genome persists as a highly ordered chromatin structure with bivalent chromatin marks at the promoter-regulatory region of the major immediate-early gene promoter. Various stimuli can induce chromatin modifications to an active euchromatic epigenetic mark, leading to the expression of genes required for the transition from the latent to the lytic phase of KSHV life cycle. Enhanced replication and transcription activator (RTA) gene expression triggers a cascade of events, resulting in the modulation of various cellular pathways to support viral DNA synthesis. RTA also binds to the origin of lytic DNA replication to recruit viral, as well as cellular, proteins for the initiation of the lytic DNA replication of KSHV. In this review we will discuss some of the pivotal genetic and epigenetic factors that control KSHV reactivation from the transcriptionally restricted latent program. PMID:25594835

  16. Molecular biology of KSHV lytic reactivation.

    PubMed

    Purushothaman, Pravinkumar; Uppal, Timsy; Verma, Subhash C

    2015-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) primarily persists as a latent episome in infected cells. During latent infection, only a limited number of viral genes are expressed that help to maintain the viral episome and prevent lytic reactivation. The latent KSHV genome persists as a highly ordered chromatin structure with bivalent chromatin marks at the promoter-regulatory region of the major immediate-early gene promoter. Various stimuli can induce chromatin modifications to an active euchromatic epigenetic mark, leading to the expression of genes required for the transition from the latent to the lytic phase of KSHV life cycle. Enhanced replication and transcription activator (RTA) gene expression triggers a cascade of events, resulting in the modulation of various cellular pathways to support viral DNA synthesis. RTA also binds to the origin of lytic DNA replication to recruit viral, as well as cellular, proteins for the initiation of the lytic DNA replication of KSHV. In this review we will discuss some of the pivotal genetic and epigenetic factors that control KSHV reactivation from the transcriptionally restricted latent program. PMID:25594835

  17. Herpes simplex virus 1 DNA is in unstable nucleosomes throughout the lytic infection cycle, and the instability of the nucleosomes is independent of DNA replication.

    PubMed

    Lacasse, Jonathan J; Schang, Luis M

    2012-10-01

    Herpes simplex virus 1 (HSV-1) DNA is chromatinized during latency and consequently regularly digested by micrococcal nuclease (MCN) to nucleosome-size fragments. In contrast, MCN digests HSV-1 DNA in lytically infected cells to mostly heterogeneous sizes. Yet HSV-1 DNA coimmunoprecipitates with histones during lytic infections. We have shown that at 5 h postinfection, most nuclear HSV-1 DNA is in particularly unstable nucleoprotein complexes and consequently is more accessible to MCN than DNA in cellular chromatin. HSV-1 DNA was quantitatively recovered at this time in complexes with the biophysical properties of mono- to polynucleosomes following a modified MCN digestion developed to detect potential unstable intermediates. We proposed that most HSV-1 DNA is in unstable nucleosome-like complexes during lytic infections. Physiologically, nucleosome assembly typically associates with DNA replication, although DNA replication transiently disrupts nucleosomes. It therefore remained unclear whether the instability of the HSV-1 nucleoprotein complexes was related to the ongoing viral DNA replication. Here we tested whether HSV-1 DNA is in unstable nucleosome-like complexes before, during, or after the peak of viral DNA replication or when HSV-1 DNA replication is inhibited. HSV-1 DNA was quantitatively recovered in complexes fractionating as mono- to polynucleosomes from nuclei harvested at 2, 5, 7, or 9 h after infection, even if viral DNA replication was inhibited. Therefore, most HSV-1 DNA is in unstable nucleosome-like complexes throughout the lytic replication cycle, and the instability of these complexes is surprisingly independent of HSV-1 DNA replication. The specific accessibility of nuclear HSV-1 DNA, however, varied at different times after infection. PMID:22875975

  18. Regional variation in lytic and lysogenic viral infection in the Southern Ocean and its contribution to biogeochemical cycling.

    PubMed

    Evans, Claire; Brussaard, Corina P D

    2012-09-01

    Lytic and lysogenic viral infection was investigated throughout the Southern Ocean at sites spanning the sub-Antarctic zone, the Antarctic Circumpolar Current, and an Antarctic continental sea. Higher lytic virus activity was recorded in the more productive sub-Antarctic zone than in the iron-limited waters of the Antarctic Circumpolar Current during two transects. Reduced lytic viral activity in the Antarctic Circumpolar Current was combined with a shift toward lysogenic infection, probably resulting from the lower concentration of potential prokaryotic hosts. Superimposed on this variation, lytic viral production was lower in a transect completed in the Drake Passage in autumn (1.8 × 10(8) to 1.5 × 10(9) liter(-1) day(-1)) than over the Greenwich Meridian during summer (5.1 × 10(8) to 2.0 × 10(10) cells liter(-1) day(-1)), indicating that viral activity is linked to the overall seasonal fluctuations in biotic activity. Interestingly, while prokaryotic abundance was lowest in the coastal Weddell Sea, levels of bacterial and lytic viral production (4.3 × 10(8) to 1.7 × 10(10) cells liter(-1) day(-1)) in this area were similar to those of the other zones. This may explain the weak relationship between the distribution of prokaryotes and chlorophyll in the Weddell Sea, as a high turnover of prokaryotic biomass may have been stimulated by the availability of substrates in the form of viral lysate. With estimated carbon and iron releases of 0.02 to 7.5 μg liter(-1) day(-1) and 1.5 to 175.7 pg liter(-1) day(-1), respectively, viral activity in the Southern Ocean is shown to be a major contributor to satisfying the elemental requirements of microbes, notably prokaryotes in the Weddell Sea and phytoplankton in the sub-Antarctic zone. PMID:22798377

  19. Transient activation of human cytomegalovirus lytic gene expression during latency allows cytotoxic T cell killing of latently infected cells

    PubMed Central

    Krishna, B. A.; Lau, B.; Jackson, S. E.; Wills, M. R.; Sinclair, J. H.; Poole, E.

    2016-01-01

    Human cytomegalovirus (HCMV) latency in the myeloid lineage is maintained by repressive histone modifications around the major immediate early promoter (MIEP), which results in inhibition of the lytic viral life cycle. We now show that pharmacological inhibition of histone deacetylases (HDACs) relieves this repression of the MIEP and induces transient expression of the viral lytic immediate early (IE) antigens but, importantly, not full virus reactivation. In turn, these latently infected cells now become targets for IE-specific cytotoxic T cells (CTLs) which are present at high frequency in all normal healthy HCMV positive carriers but would normally be unable to target latent (lytic antigen-negative) cells. This approach of transiently inducing viral lytic gene expression by HDAC inhibition, in otherwise latently infected cells, offers a window of opportunity to target and purge the latent myeloid cell reservoir by making these normally immunologically undetectable cells visible to pre-existing host immune responses to viral lytic antigens. PMID:27091512

  20. Transient activation of human cytomegalovirus lytic gene expression during latency allows cytotoxic T cell killing of latently infected cells.

    PubMed

    Krishna, B A; Lau, B; Jackson, S E; Wills, M R; Sinclair, J H; Poole, E

    2016-01-01

    Human cytomegalovirus (HCMV) latency in the myeloid lineage is maintained by repressive histone modifications around the major immediate early promoter (MIEP), which results in inhibition of the lytic viral life cycle. We now show that pharmacological inhibition of histone deacetylases (HDACs) relieves this repression of the MIEP and induces transient expression of the viral lytic immediate early (IE) antigens but, importantly, not full virus reactivation. In turn, these latently infected cells now become targets for IE-specific cytotoxic T cells (CTLs) which are present at high frequency in all normal healthy HCMV positive carriers but would normally be unable to target latent (lytic antigen-negative) cells. This approach of transiently inducing viral lytic gene expression by HDAC inhibition, in otherwise latently infected cells, offers a window of opportunity to target and purge the latent myeloid cell reservoir by making these normally immunologically undetectable cells visible to pre-existing host immune responses to viral lytic antigens. PMID:27091512

  1. COX-2 induces lytic reactivation of EBV through PGE2 by modulating the EP receptor signaling pathway.

    PubMed

    Gandhi, Jaya; Gaur, Nivedita; Khera, Lohit; Kaul, Rajeev; Robertson, Erle S

    2015-10-01

    Inflammation is one of the predisposing factors known to be associated with Epstein Barr Virus (EBV) mediated tumorigenesis. However it is not well understood whether inflammation in itself plays a role in regulating the life cycle of this infectious agent. COX-2, a key mediator of the inflammatory processes is frequently over-expressed in EBV positive cancer cells. In various tumors, PGE2 is the principle COX-2 regulated downstream product which exerts its effects on cellular processes through the EP1-4 receptors. In this study, we further elucidated how upregulated COX-2 levels can modulate the events in EBV life cycle related to latency-lytic reactivation. Our data suggest a role for upregulated COX-2 on modulation of EBV latency through its downstream effector PGE2. This study demonstrates a role for increased COX-2 levels in modulation of EBV latency. This is important for understanding the pathogenesis of EBV-associated cancers in people with chronic inflammatory conditions. PMID:26057147

  2. Genome-wide analyses of Zta binding to the Epstein-Barr virus genome reveals interactions in both early and late lytic cycles and an epigenetic switch leading to an altered binding profile.

    PubMed

    Ramasubramanyan, Sharada; Kanhere, Aditi; Osborn, Kay; Flower, Kirsty; Jenner, Richard G; Sinclair, Alison J

    2012-12-01

    The Epstein-Barr virus (EBV) genome sustains substantial epigenetic modification involving chromatin remodelling and DNA methylation during lytic replication. Zta (ZEBRA, BZLF1), a key regulator of the EBV lytic cycle, is a transcription and replication factor, binding to Zta response elements (ZREs) in target promoters and EBV lytic origins of replication. In vitro, Zta binding is modulated by DNA methylation; a subset of CpG-containing Zta binding sites (CpG ZREs) is bound only in a DNA methylation-dependent manner. The question of how the dynamic epigenetic environment impacts Zta interaction during the EBV lytic cycle is unknown. To address this, we used chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-Seq) to identify Zta binding sites across the EBV genome before and after viral DNA replication. Replication did not alter the association of Zta across many regions of the EBV genome, but a striking reduction in Zta binding occurred at some loci that contain CpG ZREs. Separating Zta-bound DNA into methylated and nonmethylated fractions, we found that promoters that contain CpG ZREs were enriched in the methylated fraction but that Zta binding to promoters lacking CpG ZREs was not reduced. We hypothesize that the loss of DNA methylation on the EBV genome during the lytic cycle causes the reduced binding to CpG ZREs; this may act as a lytic cycle epigenetic switch. However, the epigenetic changes associated with the replicated EBV genome do not affect the interaction of Zta with many loci that are rich in non-CpG ZREs; this leads to sustained binding at these regions. PMID:23015699

  3. Purely lytic osteosarcoma

    SciTech Connect

    De Santos, L.A.; Eideken, B.

    1982-11-01

    The radiographic features of 42 purely lytic osteosarcomas are presented. Purely lytic osteosarcoma is identified as a lytic lesion of bone with no demonstrable osteoid matrix by conventional radiographic modalities. Purely lytic osteosarcoma represented 13.7% of a group of 305 osteosarcomas. The most common presentation was that of a lytic illdefined lesion with a moderate to large extraosseous mass component. Nine lesions presented with benign radiographic features. The differential diagnosis is outlined. The need for awareness of this type of presentation of osteosarcoma is stressed.

  4. Histone deacetylases and the nuclear receptor corepressor regulate lytic-latent switch gene 50 in murine gammaherpesvirus 68-infected macrophages.

    PubMed

    Goodwin, Megan M; Molleston, Jerome M; Canny, Susan; Abou El Hassan, Mohamed; Willert, Erin K; Bremner, Rod; Virgin, Herbert W

    2010-11-01

    Gammaherpesviruses are important oncogenic pathogens that transit between lytic and latent life cycles. Silencing the lytic gene expression program enables the establishment of latency and a lifelong chronic infection of the host. In murine gammaherpesvirus 68 (MHV68, γHV68), essential lytic switch gene 50 controls the interchange between lytic and latent gene expression programs. However, negative regulators of gene 50 expression remain largely undefined. We report that the MHV68 lytic cycle is silenced in infected macrophages but not fibroblasts and that histone deacetylases (HDACs) mediate silencing. The HDAC inhibitor trichostatin A (TSA) acts on the gene 50 promoter to induce lytic replication of MHV68. HDAC3, HDAC4, and the nuclear receptor corepressor (NCoR) are required for efficient silencing of gene 50 expression. NCoR is critical for transcriptional repression of cellular genes by unliganded nuclear receptors. Retinoic acid, a known ligand for the NCoR complex, derepresses gene 50 expression and enhances MHV68 lytic replication. Moreover, HDAC3, HDAC4, and NCoR act on the gene 50 promoter and are recruited to this promoter in a retinoic acid-responsive manner. We provide the first example of NCoR-mediated, HDAC-dependent regulation of viral gene expression. PMID:20719946

  5. Virus production in phosphorus-limited Micromonas pusilla stimulated by a supply of naturally low concentrations of different phosphorus sources, far into the lytic cycle.

    PubMed

    Maat, Douwe S; van Bleijswijk, Judith D L; Witte, Harry J; Brussaard, Corina P D

    2016-09-01

    Earlier studies show that the proliferation of phytoplankton viruses can be inhibited by depletion of soluble reactive phosphorus (SRP; orthophosphate). In natural marine waters, phytoplankton phosphorus (P) availability is, however, largely determined by the supply rate of SRP (e.g. through remineralization) and potentially by the source of P as well (i.e. the utilization of soluble non-reactive P; SNP). Here we show how a steady low supply of P (mimicking natural P recycling) to virally infected P-limited Micromonas pusilla stimulates virus proliferation. Independent of the degree of P limitation prior to infection (0.32 and 0.97μmax chemostat cultures), SRP supply resulted in 2-fold higher viral burst sizes (viruses lysed per host cell) as compared with no addition (P starvation). Delaying these spikes during the infection cycle showed that the added SRP was utilized for extra M. pusilla virus (MpV) production far into the lytic cycle (18 h post-infection). Moreover, P-limited M. pusilla utilized several SNP compounds with high efficiency and with the same extent of burst size stimulation as for SRP. Finally, addition of virus-free MpV lysate (representing a complex SNP mixture) to newly infected cells enhanced MpV production, implicating host-associated alkaline phosphatase activity, and highlighting its important role in oligotrophic environments. PMID:27316561

  6. Pneumococcal Pneumolysin Induces DNA Damage and Cell Cycle Arrest.

    PubMed

    Rai, Prashant; He, Fang; Kwang, Jimmy; Engelward, Bevin P; Chow, Vincent T K

    2016-01-01

    Streptococcus pneumoniae produces pneumolysin toxin as a key virulence factor against host cells. Pneumolysin is a cholesterol-dependent cytolysin (CDC) toxin that forms lytic pores in host membranes and mediates pneumococcal disease pathogenesis by modulating inflammatory responses. Here, we show that pneumolysin, which is released during bacterial lysis, induces DNA double strand breaks (DSBs), as indicated by ataxia telangiectasia mutated (ATM)-mediated H2AX phosphorylation (γH2AX). Pneumolysin-induced γH2AX foci recruit mediator of DNA damage checkpoint 1 (MDC1) and p53 binding protein 1 (53BP1), to sites of DSBs. Importantly, results show that toxin-induced DNA damage precedes cell cycle arrest and causes apoptosis when DNA-dependent protein kinase (DNA-PK)-mediated non-homologous end joining is inhibited. Further, we observe that cells that were undergoing DNA replication harbored DSBs in greater frequency during pneumolysin treatment. This observation raises the possibility that DSBs might be arising as a result of replication fork breakdown. Additionally, neutralizing the oligomerization domain of pneumolysin with monoclonal antibody suppresses DNA damage and also cell cycle arrest, indicating that pneumolysin oligomerization is important for causing DNA damage. Taken together, this study reveals a previously unidentified ability of pneumolysin to induce cytotoxicity via DNA damage, with implications in the pathophysiology of S. pneumoniae infection. PMID:27026501

  7. Pneumococcal Pneumolysin Induces DNA Damage and Cell Cycle Arrest

    PubMed Central

    Rai, Prashant; He, Fang; Kwang, Jimmy; Engelward, Bevin P.; Chow, Vincent T.K.

    2016-01-01

    Streptococcus pneumoniae produces pneumolysin toxin as a key virulence factor against host cells. Pneumolysin is a cholesterol-dependent cytolysin (CDC) toxin that forms lytic pores in host membranes and mediates pneumococcal disease pathogenesis by modulating inflammatory responses. Here, we show that pneumolysin, which is released during bacterial lysis, induces DNA double strand breaks (DSBs), as indicated by ataxia telangiectasia mutated (ATM)-mediated H2AX phosphorylation (γH2AX). Pneumolysin-induced γH2AX foci recruit mediator of DNA damage checkpoint 1 (MDC1) and p53 binding protein 1 (53BP1), to sites of DSBs. Importantly, results show that toxin-induced DNA damage precedes cell cycle arrest and causes apoptosis when DNA-dependent protein kinase (DNA-PK)-mediated non-homologous end joining is inhibited. Further, we observe that cells that were undergoing DNA replication harbored DSBs in greater frequency during pneumolysin treatment. This observation raises the possibility that DSBs might be arising as a result of replication fork breakdown. Additionally, neutralizing the oligomerization domain of pneumolysin with monoclonal antibody suppresses DNA damage and also cell cycle arrest, indicating that pneumolysin oligomerization is important for causing DNA damage. Taken together, this study reveals a previously unidentified ability of pneumolysin to induce cytotoxicity via DNA damage, with implications in the pathophysiology of S. pneumoniae infection. PMID:27026501

  8. Characterization of the Neospora caninum NcROP40 and NcROP2Fam-1 rhoptry proteins during the tachyzoite lytic cycle.

    PubMed

    Pastor-Fernández, Iván; Regidor-Cerrillo, Javier; Jiménez-Ruiz, Elena; Álvarez-García, Gema; Marugán-Hernández, Virginia; Hemphill, Andrew; Ortega-Mora, Luis M

    2016-01-01

    Virulence factors from the ROP2-family have been extensively studied in Toxoplasma gondii, but in the closely related Neospora caninum only NcROP2Fam-1 has been partially characterized to date. NcROP40 is a member of this family and was found to be more abundantly expressed in virulent isolates. Both NcROP2Fam-1 and NcROP40 were evaluated as vaccine candidates and exerted a synergistic effect in terms of protection against vertical transmission in mouse models, which suggests that they may be relevant for parasite pathogenicity. NcROP40 is localized in the rhoptry bulbs of tachyzoites and bradyzoites, but in contrast to NcROP2Fam-1, the protein does not associate with the parasitophorous vacuole membrane due to the lack of arginine-rich amphipathic helix in its sequence. Similarly to NcROP2Fam-1, NcROP40 mRNA levels are highly increased during tachyzoite egress and invasion. However, NcROP40 up-regulation does not appear to be linked to the mechanisms triggering egress. In contrast to NcROP2Fam-1, phosphorylation of NcROP40 was not observed during egress. Besides, NcROP40 secretion into the host cell was not successfully detected by immunofluorescence techniques. These findings indicate that NcROP40 and NcROP2Fam-1 carry out different functions, and highlight the need to elucidate the role of NcROP40 within the lytic cycle and to explain its relative abundance in tachyzoites. PMID:26521890

  9. Acetylation changes at lysine 5 of histone H4 associated with lytic gene promoters during reactivation of Kaposi's sarcoma-associated herpesvirus.

    PubMed

    Hwang, L R; Cha, S; Jong, J E; Jang, J H; Seo, T

    2014-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is a pathogenic agent of Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease in humans. Similarly to other gammaherpesviruses such as Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS), KSHV displays two alternative life cycles, latent and lytic one. The transactivation from latency to the lytic phase is the result of transcriptional changes in the KSHV genome caused by the replication and transcriptional activator (RTA). During KSHV reactivation, epigenetic modifications of histone protein on the viral genome occur, which regulate the transcriptional activation of a number of lytic genes. The reactivation of EBV from latency to lytic cycle, induced by an immediate-early Zta protein, was shown to be accompanied by acetylation of specific lysines in histone H4. Accordingly, we hypothesized that the RTA-induced transactivation of KSHV could also be accompanied by histone acetylation. To validate this hypothesis, we assayed alterations of acetyl-histone H4-lysine 5 (acH4K5) during the RTA-mediated KSHV reactivation. While the modified histone protein in a total cell lysate was not distinguished between control and RTA-expressed cells, upregulated acH4K5 was detected on several lytic gene promoter regions during KSHV reactivation. Our results clearly indicate that this epigenetic change is related to transcription of genes expressed in the lytic cycle of KSHV. PMID:25283865

  10. Lytic to temperate switching of viral communities

    NASA Astrophysics Data System (ADS)

    Knowles, B.; Silveira, C. B.; Bailey, B. A.; Barott, K.; Cantu, V. A.; Cobián-Güemes, A. G.; Coutinho, F. H.; Dinsdale, E. A.; Felts, B.; Furby, K. A.; George, E. E.; Green, K. T.; Gregoracci, G. B.; Haas, A. F.; Haggerty, J. M.; Hester, E. R.; Hisakawa, N.; Kelly, L. W.; Lim, Y. W.; Little, M.; Luque, A.; McDole-Somera, T.; McNair, K.; de Oliveira, L. S.; Quistad, S. D.; Robinett, N. L.; Sala, E.; Salamon, P.; Sanchez, S. E.; Sandin, S.; Silva, G. G. Z.; Smith, J.; Sullivan, C.; Thompson, C.; Vermeij, M. J. A.; Youle, M.; Young, C.; Zgliczynski, B.; Brainard, R.; Edwards, R. A.; Nulton, J.; Thompson, F.; Rohwer, F.

    2016-03-01

    Microbial viruses can control host abundances via density-dependent lytic predator–prey dynamics. Less clear is how temperate viruses, which coexist and replicate with their host, influence microbial communities. Here we show that virus-like particles are relatively less abundant at high host densities. This suggests suppressed lysis where established models predict lytic dynamics are favoured. Meta-analysis of published viral and microbial densities showed that this trend was widespread in diverse ecosystems ranging from soil to freshwater to human lungs. Experimental manipulations showed viral densities more consistent with temperate than lytic life cycles at increasing microbial abundance. An analysis of 24 coral reef viromes showed a relative increase in the abundance of hallmark genes encoded by temperate viruses with increased microbial abundance. Based on these four lines of evidence, we propose the Piggyback-the-Winner model wherein temperate dynamics become increasingly important in ecosystems with high microbial densities; thus ‘more microbes, fewer viruses’.

  11. Lytic to temperate switching of viral communities.

    PubMed

    Knowles, B; Silveira, C B; Bailey, B A; Barott, K; Cantu, V A; Cobián-Güemes, A G; Coutinho, F H; Dinsdale, E A; Felts, B; Furby, K A; George, E E; Green, K T; Gregoracci, G B; Haas, A F; Haggerty, J M; Hester, E R; Hisakawa, N; Kelly, L W; Lim, Y W; Little, M; Luque, A; McDole-Somera, T; McNair, K; de Oliveira, L S; Quistad, S D; Robinett, N L; Sala, E; Salamon, P; Sanchez, S E; Sandin, S; Silva, G G Z; Smith, J; Sullivan, C; Thompson, C; Vermeij, M J A; Youle, M; Young, C; Zgliczynski, B; Brainard, R; Edwards, R A; Nulton, J; Thompson, F; Rohwer, F

    2016-03-24

    Microbial viruses can control host abundances via density-dependent lytic predator-prey dynamics. Less clear is how temperate viruses, which coexist and replicate with their host, influence microbial communities. Here we show that virus-like particles are relatively less abundant at high host densities. This suggests suppressed lysis where established models predict lytic dynamics are favoured. Meta-analysis of published viral and microbial densities showed that this trend was widespread in diverse ecosystems ranging from soil to freshwater to human lungs. Experimental manipulations showed viral densities more consistent with temperate than lytic life cycles at increasing microbial abundance. An analysis of 24 coral reef viromes showed a relative increase in the abundance of hallmark genes encoded by temperate viruses with increased microbial abundance. Based on these four lines of evidence, we propose the Piggyback-the-Winner model wherein temperate dynamics become increasingly important in ecosystems with high microbial densities; thus 'more microbes, fewer viruses'. PMID:26982729

  12. Frequent recognition of BCRF1, a late lytic cycle protein of Epstein-Barr virus, in the HLA-B*2705 context: evidence for a TAP-independent processing.

    PubMed

    Saulquin, X; Bodinier, M; Peyrat, M A; Hislop, A; Scotet, E; Lang, F; Bonneville, M; Houssaint, E

    2001-03-01

    Using a transient COS transfection assay, allowing a rapid estimation of the dominant CD8(+) T cell responses against a large number of HLA/viral protein combinations within polyclonal cell lines, we searched for HLA-B*2705-restricted CD8 T cell responses to Epstein-Barr virus (EBV) within T cell samples enriched for EBV-reactive cells. Among the 18 EBV proteins tested, only 2, the latent protein EBNA3A and the late lytic protein BCRF1 (viral IL-10), appeared dominant in the B27 context, as they triggered significant TNF and cytolytic responses in some donors. We provide evidence that the B27/BCRF1 epitope (RRLVVTLQC) is located in the signal sequence and that it can be presented in a TAP-independent manner. Using B27/BCRF1 monomeric complexes coated on immunomagnetic beads, we sorted out BCRF1-specific CD8 T cells from 8 of 15 HLA-B27(+) donors. This is, to our knowledge, the first demonstration of a recognition of BCRF1, suggesting that some immune control against EBV exists even during the late stage of the lytic cycle. This result also strengthens the unusual ability of HLA-B*2705 to present peptide in a TAP-independent manner. PMID:11241274

  13. Onset of penicillin-induced bacteriolysis in staphylococci is cell cycle dependent.

    PubMed Central

    Maidhof, H; Johannsen, L; Labischinski, H; Giesbrecht, P

    1989-01-01

    Synchronously growing staphylococci were treated with "lytic" concentrations of penicillin at different stages of their division cycle. Coulter Counter measurements and light microscopy were used to determine the onset of bacteriolysis. Independent of the stage of the division cycle at which penicillin was added, (i) the cells were always able to perform the next cell division; (ii) the following division, however, did not take place; and (iii) instead, at this time, when the onset of the subsequent cell separation was observed in control cultures, lysis of the penicillin-treated cells occurred. These results support a recent model (P. Giesbrecht, H. Labischinski, and J. Wecke, Arch. Microbiol. 141:315-324, 1985) explaining penicillin-induced bacteriolysis of staphylococci as the result of a special morphogenetic mistake during cross wall formation. Images PMID:2703473

  14. Phosphoproteomic Analyses Reveal Signaling Pathways That Facilitate Lytic Gammaherpesvirus Replication

    PubMed Central

    Stahl, James A.; Chavan, Shweta S.; Sifford, Jeffrey M.; MacLeod, Veronica; Voth, Daniel E.; Edmondson, Ricky D.; Forrest, J. Craig

    2013-01-01

    Lytic gammaherpesvirus (GHV) replication facilitates the establishment of lifelong latent infection, which places the infected host at risk for numerous cancers. As obligate intracellular parasites, GHVs must control and usurp cellular signaling pathways in order to successfully replicate, disseminate to stable latency reservoirs in the host, and prevent immune-mediated clearance. To facilitate a systems-level understanding of phosphorylation-dependent signaling events directed by GHVs during lytic replication, we utilized label-free quantitative mass spectrometry to interrogate the lytic replication cycle of murine gammaherpesvirus-68 (MHV68). Compared to controls, MHV68 infection regulated by 2-fold or greater ca. 86% of identified phosphopeptides – a regulatory scale not previously observed in phosphoproteomic evaluations of discrete signal-inducing stimuli. Network analyses demonstrated that the infection-associated induction or repression of specific cellular proteins globally altered the flow of information through the host phosphoprotein network, yielding major changes to functional protein clusters and ontologically associated proteins. A series of orthogonal bioinformatics analyses revealed that MAPK and CDK-related signaling events were overrepresented in the infection-associated phosphoproteome and identified 155 host proteins, such as the transcription factor c-Jun, as putative downstream targets. Importantly, functional tests of bioinformatics-based predictions confirmed ERK1/2 and CDK1/2 as kinases that facilitate MHV68 replication and also demonstrated the importance of c-Jun. Finally, a transposon-mutant virus screen identified the MHV68 cyclin D ortholog as a viral protein that contributes to the prominent MAPK/CDK signature of the infection-associated phosphoproteome. Together, these analyses enhance an understanding of how GHVs reorganize and usurp intracellular signaling networks to facilitate infection and replication. PMID:24068923

  15. Chromatin assembly on herpes simplex virus genomes during lytic infection

    PubMed Central

    Lu, Xu; Triezenberg, Steven J

    2009-01-01

    The human herpes simplex viruses HSV-1 and HSV-2 infect a significant portion of the human population. Both viruses can undergo lytic infection in epithelial cells and establish lifelong latency in neuronal cells. The large HSV-1 DNA genomes have long been considered to be devoid of histones both inside the virion particle and inside the cell during lytic infection, but to be packaged in repressive chromatin during latency. However, recent reports indicate that many histone and non-histone chromosomal proteins can associate with viral DNA during lytic infection and may influence important events during the HSV-1 lytic cycle. In this article, we summarize recent developments in this field and their implications. PMID:19682614

  16. The lytic phase of epstein-barr virus requires a viral genome with 5-methylcytosine residues in CpG sites.

    PubMed

    Kalla, Markus; Göbel, Christine; Hammerschmidt, Wolfgang

    2012-01-01

    Epstein-Barr virus (EBV) is a human herpesvirus which has been studied intensively for its role in certain human tumors. It also serves as a model of herpesviral latency because it establishes an immediate, latent infection in human B cells. When EBV infects quiescent, primary B cells it induces their continuous proliferation to yield growth-transformed B-cell lines in vitro. The lytic or productive phase of EBV's life cycle is induced by the expression of the viral BZLF1 gene in latently infected cells. The BZLF1 protein is a transactivator, which selectively binds to two classes of distinct DNA sequence motifs. One class is similar to the motifs that are bound by members of the AP-1 transcription factor family to which BZLF1 belongs. The second class, which contains CpG motifs, is predominant in viral promoters of early lytic genes and is BZLF1's preferred or exclusive target sequence when methylated. The BZLF1 gene is transiently expressed in newly infected B cells but fails to induce EBV's lytic cycle, potentially because the virion DNA is unmethylated. Here we report that the lack of 5-methylcytosine residues in CpG sites of virion DNA prevents the expression of essential lytic genes indispensable for viral DNA amplification during productive infection. This finding indicates that BZLF1 transactivates these promoters in a methylation-dependent fashion and explains how progeny virus synthesis is abrogated in newly infected B cells. Our data also reveal that viral lytic DNA synthesis precludes CpG methylation of virion DNA during EBV's lytic, productive cycle, which can be overcome by the ectopic expression of a prokaryotic cytosine methyltransferase to yield CpG-methylated virion DNA. Upon infection of B cells, randomly CpG-methylated virion DNA induces high expression of essential lytic genes in contrast to virion DNA free of 5-methylcytosine residues. Our data suggest that unmethylated virion DNA is part of EBV's strategy to prevent the viral lytic phase in

  17. Kaposi's sarcoma-associated herpesvirus ORF6 gene is essential in viral lytic replication.

    PubMed

    Peng, Can; Chen, Jungang; Tang, Wei; Liu, Chunlan; Chen, Xulin

    2014-01-01

    Kaposi's sarcoma associated herpesvirus (KSHV) is associated with Kaposis's sarcoma (KS), primary effusion lymphoma and multicentric Castleman's disease. KSHV encodes at least 8 open reading frames (ORFs) that play important roles in its lytic DNA replication. Among which, ORF6 of KSHV encodes an ssDNA binding protein that has been proved to participate in origin-dependent DNA replication in transient assays. To define further the function of ORF6 in the virus life cycle, we constructed a recombinant virus genome with a large deletion within the ORF6 locus by using a bacterial artificial chromosome (BAC) system. Stable 293T cells carrying the BAC36 (wild type) and BACΔ6 genomes were generated. When monolayers of 293T-BAC36 and 293T-BACΔ6 cells were induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate, infectious virus was detected from the 293T-BAC36 cell supernatants only and not from the 293T- BACΔ6 cell supernatants. DNA synthesis was defective in 293T-BACΔ6 cells. Expression of ORF6 in trans in BACΔ6-containing cells was able to rescue both defects. Our results provide genetic evidence that ORF6 is essential for KSHV lytic replication. The stable 293T cells carrying the BAC36 and BACΔ6 genomes could be used as tools to investigate the detailed functions of ORF6 in the lytic replication of KSHV. PMID:24911362

  18. Lytic Clostridium perfringens Bacteriophage 39-O Genomic

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Screening for bacteriophages lytic for Clostridium perfringens was completed utilizing filtered samples obtained from poultry (intestinal material), soil, sewage and poultry processing drainage water. Following limit dilution cloning and three rounds of plaque purification lytic phage preparations ...

  19. Specific proteins synthesized during the viral lytic cycle in vaccinia virus-infected HeLa cells: analysis by high-resolution, two-dimensional gel electrophoresis

    SciTech Connect

    Carrasco, L.; Bravo, R.

    1986-05-01

    The proteins synthesized in vaccinia-infected HeLa cells have been analyzed at different times after infection by using two-dimensional gel electrophoresis. Vaccinia-infected cells present up to 198 polypeptides (138 acidic, isoelectric focusing; 60 basic, nonequilibrium pH gradient electrophoresis) not detected in control cells. Cells infected in the presence of cycloheximide show 81 additional polypeptides after cycloheximide removal, resulting in a total estimate of 279 proteins induced after vaccinia infection. The glycoproteins made at various time postinfection were also analyzed. At least 13 proteins labeled with (/sup 3/H)glucosamine were detected in vaccinia-infected HeLa cells.

  20. Human NK cell lytic granules and regulation of their exocytosis

    PubMed Central

    Krzewski, Konrad; Coligan, John E.

    2012-01-01

    Natural killer (NK) cells form a subset of lymphocytes that play a key role in immuno-surveillance and host defense against cancer and viral infections. They recognize stressed cells through a variety of germline-encoded activating cell surface receptors and utilize their cytotoxic ability to eliminate abnormal cells. Killing of target cells is a complex, multi-stage process that concludes in the directed secretion of lytic granules, containing perforin and granzymes, at the immunological synapse. Upon delivery to a target cell, perforin mediates generation of pores in membranes of target cells, allowing granzymes to access target cell cytoplasm and induce apoptosis. Therefore, lytic granules of NK cells are indispensable for normal NK cell cytolytic function. Indeed, defects in lytic granule secretion lead or are related to serious and often fatal diseases, such as familial hemophagocytic lymphohistiocytosis (FHL) type 2–5 or Griscelli syndrome type 2. A number of reports highlight the role of several proteins involved in lytic granule release and NK cell-mediated killing of tumor cells. This review focuses on lytic granules of human NK cells and the advancements in understanding the mechanisms controlling their exocytosis. PMID:23162553

  1. Viroporin potential of the lentivirus lytic peptide (LLP) domains of the HIV-1 gp41 protein

    PubMed Central

    Costin, Joshua M; Rausch, Joshua M; Garry, Robert F; Wimley, William C

    2007-01-01

    Background Mechanisms by which HIV-1 mediates reductions in CD4+ cell levels in infected persons are being intensely investigated, and have broad implications for AIDS drug and vaccine development. Virally induced changes in membrane ionic permeability induced by lytic viruses of many families contribute to cytopathogenesis. HIV-1 induces disturbances in plasma membrane ion transport. The carboxyl terminus of TM (gp41) contains potential amphipathic α-helical motifs identified through their structural similarities to naturally occurring cytolytic peptides. These sequences have been dubbed lentiviral lytic peptides (LLP) -1, -2, and -3. Results Peptides corresponding to the LLP domains (from a clade B virus) partition into lipid membranes, fold into α-helices and disrupt model membrane permeability. A peptide corresponding to the LLP-1 domain of a clade D HIV-1 virus, LLP-1D displayed similar activity to the LLP-1 domain of the clade B virus in all assays, despite a lack of amino acid sequence identity. Conclusion These results suggest that the C-terminal domains of HIV-1 Env proteins may form an ion channel, or viroporin. Increased understanding of the function of LLP domains and their role in the viral replication cycle could allow for the development of novel HIV drugs. PMID:18028545

  2. Comparison of human sera reactivities in immunoblots with recombinant human herpesvirus (HHV)-8 proteins associated with the latent (ORF73) and lytic (ORFs 65, K8.1A, and K8.1B) replicative cycles and in immunofluorescence assays with HHV-8-infected BCBL-1 cells.

    PubMed

    Zhu, L; Wang, R; Sweat, A; Goldstein, E; Horvat, R; Chandran, B

    1999-04-10

    The development of reliable, sensitive, and specific serological methods for the detection of human herpesvirus-8 (HHV-8) antibodies is critical for a thorough understanding of HHV-8 prevalence and pathogenesis. To evaluate the potential usefulness of HHV-8 proteins in measuring the responses against both latent and lytic antigens, we selected 1 latent [open reading frame (ORF) 73] antigen and 3 HHV-8 lytic antigens (ORFs 65, K8.1A, and K8.1B) previously identified as immunogenic [Virology (1998) 243, 208-217]. Full-length genomic ORF 73 and full-length ORFs 65, K8.1A, and K8.1B from the cDNA clones were cloned, expressed in bacterial and baculovirus-insect cell expression systems, and purified as GST fusion proteins. These recombinant proteins were used in Western blot reactions to test sera from 104 human immunodeficiency virus (HIV)+/Kaposi's sarcoma (KS)+ homosexual men, 77 HIV+/KS- homosexual men, and 84 age-matched HIV-/KS- men. These sera were also tested in immunofluorescence assays (IFAs) with uninduced and 12-O-tetradecanoylphorbol-13-acetate-induced B cell lymphoma-1 cells to detect antibodies against latency-associated nuclear antigens (LANA) and antibodies against lytic antigens (cytoplasmic fluorescence). These sera exhibited differential reactivities reflecting different titers of antibodies against HHV-8 proteins, and variable reactivities were seen more commonly with the sera from HIV-/KS- adult men. In the Western blot assay, 89% (93 of 104) of HIV+/KS + sera, 60% (46 of 77) of HIV+/KS- sera, and 7% (6 of 84) HIV+/KS- sera were reactive with both latent and lytic recombinant antigens. Western blot reactions with ORF 73 protein were more sensitive than LANA-IFA results. The lytic IFA and lytic Western blot (ORFs 65 and K8.1A) assays were more sensitive than the ORF 73 Western blots and LANA-IFA. With an exception of 2 sera from the HIV-/KS- group, all sera positive for lytic IFA antibodies and ORF 65 and K8.1A antibodies were also positive for

  3. Signal transducer and activator of transcription 3 limits Epstein-Barr virus lytic activation in B lymphocytes.

    PubMed

    Hill, Erik R; Koganti, Siva; Zhi, Jizu; Megyola, Cynthia; Freeman, Alexandra F; Palendira, Umaimainthan; Tangye, Stuart G; Farrell, Paul J; Bhaduri-McIntosh, Sumita

    2013-11-01

    Lytic activation of Epstein-Barr virus (EBV) is central to its life cycle and to most EBV-related diseases. However, not every EBV-infected B cell is susceptible to lytic activation. This lack of uniform susceptibility to lytic activation also directly impacts the success of viral oncolytic therapy for EBV cancers, yet determinants of susceptibility to lytic induction signals are not well understood. To determine if host factors influence susceptibility to EBV lytic activation, we developed a technique to separate lytic from refractory cells and reported that EBV lytic activation occurs preferentially in cells with lower levels of signal transducer and activator of transcription 3 (STAT3). Using this tool to detect single cells, we now extend the correlation between STAT3 and lytic versus refractory states to EBV-infected circulating B cells in patients with primary EBV infection, leading us to investigate whether STAT3 controls susceptibility to EBV lytic activation. In loss-of-function and gain-of-function studies in EBV-positive B lymphoma and lymphoblastoid cells, we found that the levels of functional STAT3 regulate susceptibility to EBV lytic activation. This prompted us to identify a pool of candidate cellular genes that might be regulated by STAT3 to limit EBV lytic activation. From this pool, we confirmed increases in transcript levels in refractory cells of a set of genes known to participate in transcription repression. Taken together, our findings place STAT3 at a critical crossroads between EBV latency and lytic activation, processes fundamental to EBV lymphomagenesis. PMID:23966384

  4. Development of a lytic peptide derived from BH3-only proteins.

    PubMed

    Liu, Q; Zhao, H; Jiang, Y; Wu, M; Tian, Y; Wang, D; Lao, Y; Xu, N; Li, Z

    2016-01-01

    Despite great advances in cancer therapy, drug resistance is a difficult hurdle to overcome that requires development of anticancer agents with novel and effective modes of action. In a number of studies, lytic peptides have shown remarkable ability to eliminate cancer cells through a different way from traditional treatments. Lytic peptides are positively charged, amphiphilic, and are efficient at binding and disrupting the negatively charged cell membrane of cancer cells. In this study, we described the anticancer properties of a lytic peptide that was developed on the basis of the alignment of amphiphilic BH3 peptides. Our results demonstrated that the positive charge and conformation constraint were favourable for efficient cancer cell elimination. Artificial BCL-2 homology 3 peptides (ABH3) exhibited effective anticancer effects against a series of cancer cell lines in vitro and in HeLa human cervical tumour xenografts in vivo. ABH3 induced cell death in an apoptosis-independent manner through the lytic properties of the peptide that caused disruption of cell membrane. Our results showed that charge tuning and conformation constraining in a lytic peptide could be applied to optimise the anticancer activity of lytic peptides. These results also suggest that ABH3 may be a promising beginning for the development of additional lytic peptides as anticancer reagents. PMID:27551502

  5. Development of a lytic peptide derived from BH3-only proteins

    PubMed Central

    Liu, Q; Zhao, H; Jiang, Y; Wu, M; Tian, Y; Wang, D; Lao, Y; Xu, N; Li, Z

    2016-01-01

    Despite great advances in cancer therapy, drug resistance is a difficult hurdle to overcome that requires development of anticancer agents with novel and effective modes of action. In a number of studies, lytic peptides have shown remarkable ability to eliminate cancer cells through a different way from traditional treatments. Lytic peptides are positively charged, amphiphilic, and are efficient at binding and disrupting the negatively charged cell membrane of cancer cells. In this study, we described the anticancer properties of a lytic peptide that was developed on the basis of the alignment of amphiphilic BH3 peptides. Our results demonstrated that the positive charge and conformation constraint were favourable for efficient cancer cell elimination. Artificial BCL-2 homology 3 peptides (ABH3) exhibited effective anticancer effects against a series of cancer cell lines in vitro and in HeLa human cervical tumour xenografts in vivo. ABH3 induced cell death in an apoptosis-independent manner through the lytic properties of the peptide that caused disruption of cell membrane. Our results showed that charge tuning and conformation constraining in a lytic peptide could be applied to optimise the anticancer activity of lytic peptides. These results also suggest that ABH3 may be a promising beginning for the development of additional lytic peptides as anticancer reagents. PMID:27551502

  6. 5-hydroxymethylation of the EBV genome regulates the latent to lytic switch

    PubMed Central

    Wille, Coral K.; Nawandar, Dhananjay M.; Henning, Amanda N.; Ma, Shidong; Oetting, Kayla M.; Lee, Dennis; Lambert, Paul; Johannsen, Eric C.; Kenney, Shannon C.

    2015-01-01

    Latent Epstein–Barr virus (EBV) infection and cellular hypermethylation are hallmarks of undifferentiated nasopharyngeal carcinoma (NPC). However, EBV infection of normal oral epithelial cells is confined to differentiated cells and is lytic. Here we demonstrate that the EBV genome can become 5-hydroxymethylated and that this DNA modification affects EBV lytic reactivation. We show that global 5-hydroxymethylcytosine (5hmC)-modified DNA accumulates during normal epithelial-cell differentiation, whereas EBV+ NPCs have little if any 5hmC-modified DNA. Furthermore, we find that increasing cellular ten–eleven translocation (TET) activity [which converts methylated cytosine (5mC) to 5hmC] decreases methylation, and increases 5hmC modification, of lytic EBV promoters in EBV-infected cell lines containing highly methylated viral genomes. Conversely, inhibition of endogenous TET activity increases lytic EBV promoter methylation in an EBV-infected telomerase-immortalized normal oral keratinocyte (NOKs) cell line where lytic viral promoters are largely unmethylated. We demonstrate that these cytosine modifications differentially affect the ability of the two EBV immediate-early proteins, BZLF1 (Z) and BRLF1 (R), to induce the lytic form of viral infection. Although methylation of lytic EBV promoters increases Z-mediated and inhibits R-mediated lytic reactivation, 5hmC modification of lytic EBV promoters has the opposite effect. We also identify a specific CpG-containing Z-binding site on the BRLF1 promoter that must be methylated for Z-mediated viral reactivation and show that TET-mediated 5hmC modification of this site in NOKs prevents Z-mediated viral reactivation. Decreased 5-hydroxymethylation of cellular and viral genes may contribute to NPC formation. PMID:26663912

  7. 5-hydroxymethylation of the EBV genome regulates the latent to lytic switch.

    PubMed

    Wille, Coral K; Nawandar, Dhananjay M; Henning, Amanda N; Ma, Shidong; Oetting, Kayla M; Lee, Dennis; Lambert, Paul; Johannsen, Eric C; Kenney, Shannon C

    2015-12-29

    Latent Epstein-Barr virus (EBV) infection and cellular hypermethylation are hallmarks of undifferentiated nasopharyngeal carcinoma (NPC). However, EBV infection of normal oral epithelial cells is confined to differentiated cells and is lytic. Here we demonstrate that the EBV genome can become 5-hydroxymethylated and that this DNA modification affects EBV lytic reactivation. We show that global 5-hydroxymethylcytosine (5hmC)-modified DNA accumulates during normal epithelial-cell differentiation, whereas EBV+ NPCs have little if any 5hmC-modified DNA. Furthermore, we find that increasing cellular ten-eleven translocation (TET) activity [which converts methylated cytosine (5mC) to 5hmC] decreases methylation, and increases 5hmC modification, of lytic EBV promoters in EBV-infected cell lines containing highly methylated viral genomes. Conversely, inhibition of endogenous TET activity increases lytic EBV promoter methylation in an EBV-infected telomerase-immortalized normal oral keratinocyte (NOKs) cell line where lytic viral promoters are largely unmethylated. We demonstrate that these cytosine modifications differentially affect the ability of the two EBV immediate-early proteins, BZLF1 (Z) and BRLF1 (R), to induce the lytic form of viral infection. Although methylation of lytic EBV promoters increases Z-mediated and inhibits R-mediated lytic reactivation, 5hmC modification of lytic EBV promoters has the opposite effect. We also identify a specific CpG-containing Z-binding site on the BRLF1 promoter that must be methylated for Z-mediated viral reactivation and show that TET-mediated 5hmC modification of this site in NOKs prevents Z-mediated viral reactivation. Decreased 5-hydroxymethylation of cellular and viral genes may contribute to NPC formation. PMID:26663912

  8. Differentiation-Dependent KLF4 Expression Promotes Lytic Epstein-Barr Virus Infection in Epithelial Cells

    PubMed Central

    Nawandar, Dhananjay M.; Wang, Anqi; Makielski, Kathleen; Lee, Denis; Ma, Shidong; Barlow, Elizabeth; Reusch, Jessica; Jiang, Ru; Wille, Coral K.; Greenspan, Deborah; Greenspan, John S.; Mertz, Janet E.; Hutt-Fletcher, Lindsey; Johannsen, Eric C.; Lambert, Paul F.; Kenney, Shannon C.

    2015-01-01

    Epstein-Barr virus (EBV) is a human herpesvirus associated with B-cell and epithelial cell malignancies. EBV lytically infects normal differentiated oral epithelial cells, where it causes a tongue lesion known as oral hairy leukoplakia (OHL) in immunosuppressed patients. However, the cellular mechanism(s) that enable EBV to establish exclusively lytic infection in normal differentiated oral epithelial cells are not currently understood. Here we show that a cellular transcription factor known to promote epithelial cell differentiation, KLF4, induces differentiation-dependent lytic EBV infection by binding to and activating the two EBV immediate-early gene (BZLF1 and BRLF1) promoters. We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs) cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture. Furthermore, we show that endogenous KLF4 expression is required for efficient lytic viral reactivation in response to phorbol ester and sodium butyrate treatment in several different EBV-infected epithelial cell lines, and that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, is highly synergistic for inducing lytic EBV infection. We confirm that both KLF4 and BLIMP1 are expressed in differentiated, but not undifferentiated, epithelial cells in normal tongue tissue, and show that KLF4 and BLIMP1 are both expressed in a patient-derived OHL lesion. In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells. Thus, KLF4, together with BLIMP1, plays a critical role in mediating lytic EBV reactivation in epithelial cells. PMID:26431332

  9. Parvovirus infection-induced cell death and cell cycle arrest

    PubMed Central

    Chen, Aaron Yun; Qiu, Jianming

    2011-01-01

    The cytopathic effects induced during parvovirus infection have been widely documented. Parvovirus infection-induced cell death is often directly associated with disease outcomes (e.g., anemia resulting from loss of erythroid progenitors during parvovirus B19 infection). Apoptosis is the major form of cell death induced by parvovirus infection. However, nonapoptotic cell death, namely necrosis, has also been reported during infection of the minute virus of mice, parvovirus H-1 and bovine parvovirus. Recent studies have revealed multiple mechanisms underlying the cell death during parvovirus infection. These mechanisms vary in different parvoviruses, although the large nonstructural protein (NS)1 and the small NS proteins (e.g., the 11 kDa of parvovirus B19), as well as replication of the viral genome, are responsible for causing infection-induced cell death. Cell cycle arrest is also common, and contributes to the cytopathic effects induced during parvovirus infection. While viral NS proteins have been indicated to induce cell cycle arrest, increasing evidence suggests that a cellular DNA damage response triggered by an invading single-stranded parvoviral genome is the major inducer of cell cycle arrest in parvovirus-infected cells. Apparently, in response to infection, cell death and cell cycle arrest of parvovirus-infected cells are beneficial to the viral cell lifecycle (e.g., viral DNA replication and virus egress). In this article, we will discuss recent advances in the understanding of the mechanisms underlying parvovirus infection-induced cell death and cell cycle arrest. PMID:21331319

  10. Induced natural convection thermal cycling device

    DOEpatents

    Heung, Leung Kit

    2002-08-13

    A device for separating gases, especially isotopes, by thermal cycling of a separation column using a pressure vessel mounted vertically and having baffled sources for cold and heat. Coils at the top are cooled with a fluid such as liquid nitrogen. Coils at the bottom are either electrical resistance coils or a tubular heat exchange. The sources are shrouded with an insulated "top hat" and simultaneously opened and closed at the outlets to cool or heat the separation column. Alternatively, the sources for cold and heat are mounted separately outside the vessel and an external loop is provided for each circuit.

  11. The Epstein-Barr Virus BDLF4 Gene Is Required for Efficient Expression of Viral Late Lytic Genes

    PubMed Central

    Watanabe, Takahiro; Narita, Yohei; Yoshida, Masahiro; Sato, Yoshitaka; Goshima, Fumi; Kimura, Hiroshi

    2015-01-01

    Epstein-Barr virus (EBV) is a gammaherpesvirus, associated with infectious mononucleosis and various types of malignancy. We focused here on the BDLF4 gene of EBV and identified it as a lytic gene, expressed with early kinetics. Viral late gene expression of the BDLF4 knockout strain was severely restricted; this could be restored by an exogenous supply of BDLF4. These results indicate that BDLF4 is important for the EBV lytic replication cycle, especially in late gene expression. PMID:26202235

  12. Prediction of thermal cycling induced matrix cracking

    NASA Technical Reports Server (NTRS)

    Mcmanus, Hugh L.

    1992-01-01

    Thermal fatigue has been observed to cause matrix cracking in laminated composite materials. A method is presented to predict transverse matrix cracks in composite laminates subjected to cyclic thermal load. Shear lag stress approximations and a simple energy-based fracture criteria are used to predict crack densities as a function of temperature. Prediction of crack densities as a function of thermal cycling is accomplished by assuming that fatigue degrades the material's inherent resistance to cracking. The method is implemented as a computer program. A simple experiment provides data on progressive cracking of a laminate with decreasing temperature. Existing data on thermal fatigue is also used. Correlations of the analytical predictions to the data are very good. A parametric study using the analytical method is presented which provides insight into material behavior under cyclical thermal loads.

  13. NF-κB-Mediated Modulation of Inducible Nitric Oxide Synthase Activity Controls Induction of the Epstein-Barr Virus Productive Cycle by Transforming Growth Factor Beta 1▿†

    PubMed Central

    Oussaief, Lassad; Ramírez, Vanessa; Hippocrate, Aurélie; Arbach, Hratch; Cochet, Chantal; Proust, Alexis; Raphaël, Martine; Khelifa, Ridha; Joab, Irène

    2011-01-01

    Transforming growth factor beta 1 (TGF-β1) signal transduction has been implicated in many second-messenger pathways, including the NF-κB pathway. We provide evidence of a novel TGF-β1-mediated pathway that leads to extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, which in turn induces expression of an Epstein-Barr virus (EBV) protein, ZEBRA, that is responsible for the induction of the viral lytic cycle. This pathway includes two unexpected steps, both of which are required to control ERK 1/2 phosphorylation: first, a quick and transient activation of NF-κB, and second, downregulation of inducible nitric oxide synthase (iNOS) activity that requires the participation of NF-κB activity. Although necessary, NF-κB alone is not sufficient to produce downregulation of iNOS, suggesting that another uncharacterized event(s) is involved in this pathway. Dissection of the steps involved in the switch from the EBV latent cycle to the lytic cycle will be important to understand how virus-host relationships modulate the innate immune system. PMID:21507981

  14. Chromatin structure regulates human cytomegalovirus gene expression during latency, reactivation and lytic infection.

    PubMed

    Sinclair, John

    2010-01-01

    Infection of cells with human cytomegalovirus (HCMV) has two potential outcomes. For instance, infection of fibroblasts results in extensive viral gene expression, viral DNA replication and release of progeny virus. In contrast, in undifferentiated myeloid cells, the lytic transcription programme of HCMV is effectively suppressed and cells undergo latent infection. It is now accepted that the suppression of viral lytic gene expression observed during latency in myeloid cells is a result of the inability of undifferentiated cell types to support robust viral immediate early (IE) gene expression--crucial genes responsible for driving the lytic cycle. The repression of IE gene expression in undifferentiated myeloid cells, at least in part, results from specific post-translational modifications of histones associated with the viral major immediate early promoter (MIEP). In cells of the early myeloid lineage, the histone modifications present on the MIEP impart on it a repressive chromatin structure preventing transcriptional activity. Reactivation of HCMV lytic infection is correlated to changes in histone modifications around the MIEP resulting in a chromatin structure conducive to transcriptional activity. These changes are intimately linked with the differentiation of myeloid cells - a phenomenon known to reactivate latent virus in vivo. Chromatin structure of the viral MIEP, therefore, plays a crucial role in latency and reactivation of this persistent human herpesvirus. Whether chromatin-mediated regulation of viral lytic gene expression also occurs, is only beginning to be addressed. However, recent work suggests that all classes of lytic HCMV promoters are subjected to regulation by post-translational modification of their associated histones throughout the time course of infection. Incoming viral genomes appear to be the targets of intrinsic cellular defence mechanisms which attempt to silence viral gene expression through chromatinisation. Viral functions

  15. Accelerated Tumor Growth Mediated by Sub-lytic Levels of Antibody-Induced Complement Activation is Associated with Activation of the PI3K/AKT Survival Pathway

    PubMed Central

    Wu, Xiaohong; Ragupathi, Govind; Panageas, Katherine; Hong, Feng; Livingston, Philip O.

    2013-01-01

    Purpose We addressed the possibility that low levels of tumor cell bound antibodies targeting gangliosides might accelerate tumor growth. Experimental Design To test this hypothesis, we treated mice with a range of mAb doses against GM2, GD2, GD3 and CD20 after challenge with tumors expressing these antigens and tested the activity of the same mAbs in-vitro. We also explored the mechanisms behind the complement-mediated tumor growth acceleration that we observed and an approach to overcome it. Results Serologically detectable levels of IgM-mAb against GM2 are able to delay or prevent tumor growth of high GM2-expressing cell lines both in-vitro and in a SCID mouse model, while very low levels of this mAb resulted in slight but consistent acceleration of tumor growth in both settings. Surprisingly, this is not restricted to IgM antibodies targeting GM2 but consistent against IgG-mAb targeting GD3 as well. These findings were mirrored by in-vitro studies with antibodies against these antigens as well as GD2 and CD20 (with Rituxan), and shown to be complement-dependent in all cases. Complement-mediated accelerated growth of cultured tumor cell lines initiated by low mAb levels was associated with activation of the PI3K/AKT survival pathway and significantly elevated levels of both p-AKT and p-PRAS40. This complement-mediated PI3K-activation and accelerated tumor growth in-vitro and in-vivo are eliminated by PI3K-inhibitors NVP-BEZ235 and Wortmannin. These PI3K-inhibitors also significantly increased efficacy of high doses of these 4 mAbs. Conclusion Our findings suggest that manipulation of the PI3K/AKT pathway and its signaling network can significantly increase the potency of passively administered mAbs and vaccine-induced-antibodies targeting a variety of tumor-cell-surface-antigens. PMID:23833306

  16. Cell cycle control of polyomavirus-induced transformation.

    PubMed Central

    Chen, H H; Fluck, M M

    1993-01-01

    The cell cycle dependence of polyomavirus transformation was analyzed in infections of nonpermissive Fischer rat (FR3T3) cells released from G0. A 5- to 100-fold (average, ca. 20-fold) difference in relative frequency of transformation was found for cells infected in the early G1 phase of the cell cycle compared with cells infected in G2. Differences in the relative level of early viral gene expression in those two cell populations were equivalent to those obtained for transformation frequencies. The difference in transformation potential was accounted for only in part by a cell cycle control of viral adsorption (2- to 15-fold effect). Furthermore, in cells infected in the early G1 phase, viral gene expression was induced as a big synchronous burst of large transcripts of variable sizes, delayed till the G1 phase of the cell cycle after that in which infection took place. Thus, the results demonstrate that the abortive infection cycle of G0-released FR3T3 cells is cell cycle regulated at least at two steps: adsorption and another early step, nuclear transport, decapsidation, up to or including the transcription of the viral early genes. The cell cycle regulation of these steps results in a similar regulation of the abortive and stable transformation processes, although it is more pronounced for the latter. A model implicating c-fos and c-jun is proposed. Images PMID:8383223

  17. Abortive infection mechanisms and prophage sequences significantly influence the genetic makeup of emerging lytic lactococcal phages.

    PubMed

    Labrie, Simon J; Moineau, Sylvain

    2007-02-01

    In this study, we demonstrated the remarkable genome plasticity of lytic lactococcal phages that allows them to rapidly adapt to the dynamic dairy environment. The lytic double-stranded DNA phage ul36 was used to sequentially infect a wild-type strain of Lactococcus lactis and two isogenic derivatives with genes encoding two phage resistance mechanisms, AbiK and AbiT. Four phage mutants resistant to one or both Abi mechanisms were isolated. Comparative analysis of their complete genomes, as well as morphological observations, revealed that phage ul36 extensively evolved by large-scale homologous and nonhomologous recombination events with the inducible prophage present in the host strain. One phage mutant exchanged as much as 79% of its genome compared to the core genome of ul36. Thus, natural phage defense mechanisms and prophage elements found in bacterial chromosomes contribute significantly to the evolution of the lytic phage population. PMID:17041060

  18. Requirement of E. coli DNA synthesis functions for the lytic replication of bacteriophage P1.

    PubMed

    Hay, N; Cohen, G

    1983-11-01

    P1 lytic growth was examined in a number of different temperature sensitive mutants of E. coli that affect chromosomal replication. Growth was analyzed by measurements of phage burst sizes and specific DNA synthesis. Efficient P1 growth required each of the bacterial elongation functions dnaE (polC), dnaZ (sub units of E. coli polymerase III holoenzyme), and dnaG (primase) but was not dependent on the elongation function dnaB (mobile promoter). Of two initiation functions tested the dnaA function was found to be dispensable for normal growth whereas the dnaC function was essential. Temperature shift experiments with different dnaC mutants showed that the initiation component of the dnaC function was needed continuously throughout at least the first half of the lytic cycle, while the dnaC elongation activity was probably required during the entire cycle for normal phage yields. In two respects the dependence of P1 lytic growth on E. coli DNA synthesis functions was significantly different from that reported for P1 plasmid replication (Scott and Vapnek, 1980). Thus, lytic replication was far more dependent on a functional polC gene product than was plasmid replication and did not require the bacterial dnaB product. PMID:6359668

  19. Anti-TNFα therapy for inflammatory bowel diseases is associated with Epstein-Barr virus lytic activation.

    PubMed

    Lapsia, Sameer; Koganti, Siva; Spadaro, Salvatore; Rajapakse, Ramona; Chawla, Anupama; Bhaduri-McIntosh, Sumita

    2016-02-01

    Anti-TNFα therapy, known to suppress T-cell immunity, is increasingly gaining popularity for treatment of autoimmune diseases including inflammatory bowel diseases (IBD). T-cell suppression increases the risk of B-cell EBV-lymphoproliferative diseases and lymphomas. Since EBV-lytic activation is essential for development of EBV-lymphomas and there have been reports of EBV-lymphomas in patients treated with anti-TNFα therapy, we investigated if patients treated with anti-TNFα antibodies demonstrate greater EBV-lytic activity in blood. Peripheral blood mononuclear cells from 10 IBD patients solely on anti-TNFα therapy compared to 3 control groups (10 IBD patients not on immunosuppressive therapy, 10 patients with abdominal pain but without IBD, and 10 healthy subjects) were examined for the percentage of T-cells, EBV load and EBV-lytic transcripts. Patients on anti-TNFα therapy had significantly fewer T-cells, greater EBV load, and increased levels of transcripts from EBV-lytic genes of all kinetic classes compared to controls. Furthermore, exposure of EBV-infected B-cell lines to anti-TNFα antibodies resulted in increased levels of BZLF1 mRNA; BZLF1 encodes for ZEBRA, the viral latency-to-lytic cycle switch. Thus, IBD patients treated with anti-TNFα antibodies have greater EBV loads likely due to enhanced EBV-lytic gene expression and anti-TNFα antibodies may be sufficient to activate the EBV lytic cycle. Findings from this pilot study lay the groundwork for additional scientific and clinical investigation into the effects of anti-TNFα therapy on the life cycle of EBV, a ubiquitous oncovirus that causes lymphomas in the setting of immunocompromise. PMID:26307954

  20. Enhancement of autophagy during lytic replication by the Kaposi's sarcoma-associated herpesvirus replication and transcription activator.

    PubMed

    Wen, Hui-Ju; Yang, Zhilong; Zhou, You; Wood, Charles

    2010-08-01

    Autophagy is one of two major degradation systems in eukaryotic cells. The degradation mechanism of autophagy is required to maintain the balance between the biosynthetic and catabolic processes and also contributes to defense against invading pathogens. Recent studies suggest that a number of viruses can evade or subvert the host cell autophagic pathway to enhance their own replication. Here, we investigated the effect of autophagy on the KSHV (Kaposi's sarcoma-associated herpesvirus) life cycle. We found that the inhibition of autophagy reduces KSHV lytic reactivation from latency, and an enhancement of autophagy can be detected during KSHV lytic replication. In addition, RTA (replication and transcription activator), an essential viral protein for KSHV lytic reactivation, is able to enhance the autophagic process, leading to an increase in the number of autophagic vacuoles, an increase in the level of the lipidated LC3 protein, and the formation of autolysosomes. Moreover, the inhibition of autophagy affects RTA-mediated lytic gene expression and viral DNA replication. These results suggest that RTA increases autophagy activation to facilitate KSHV lytic replication. This is the first report demonstrating that autophagy is involved in the lytic reactivation of KSHV. PMID:20484505

  1. Hair cycle control by leptin as a new anagen inducer.

    PubMed

    Sumikawa, Yasuyuki; Inui, Shigeki; Nakajima, Takeshi; Itami, Satoshi

    2014-01-01

    Our purpose is to clarify the physiological role of leptin in hair cycle as leptin reportedly causes activation of Stat3, which is indispensable for hair cycling. While hair follicles in dorsal skin of 5-week-old C57/BL6 mice had progressed to late anagen phase, those in dorsal skin of 5-week-old leptin receptor deficient db/db mice remained in the first telogen and later entered the anagen at postnatal day 40, indicating that deficiency in leptin receptor signalling delayed the second hair cycle progression. Next, we shaved dorsal hairs on wild-type mice at postnatal 7 weeks and injected skin with mouse leptin or a mock. After 20 days, although mock injection showed no effect, hair growth occurred around leptin injection area. Human leptin fragment (aa22-56) had similar effects. Although the hair cycle of ob/ob mice was similar to that of wild-type mice, injection of mouse leptin on ob/ob mice at postnatal 7 weeks induced anagen transition. Immunohistochemically, leptin is expressed in hair follicles from catagen to early anagen in wild-type mice, suggesting that leptin is an anagen inducer in vivo. Phosphorylation of Erk, Jak2 and Stat3 in human keratinocytes was stimulated by leptin and leptin fragment. In addition, RT-PCR and ELISA showed that the production of leptin by human dermal papilla cells increased under hypoxic condition, suggesting that hypoxia in catagen/telogen phase promotes leptin production, preparing for entry into the next anagen. In conclusion, leptin, a well-known adipokine, acts as an anagen inducer and represents a new player in hair biology. PMID:24237265

  2. The novel Shewanella putrefaciens-infecting bacteriophage Spp001: genome sequence and lytic enzymes.

    PubMed

    Han, Feng; Li, Meng; Lin, Hong; Wang, Jingxue; Cao, Limin; Khan, Muhammad Naseem

    2014-06-01

    Shewanella putrefaciens has been identified as a specific spoilage organism commonly found in chilled fresh fish, which contributes to the spoilage of fish products. Limiting S. putrefaciens growth can extend the shelf-life of chilled fish. Endolysins, which are lytic enzymes produced by bacteriophages, have been considered an alternative to control bacterial growth, and have been useful in various applications, including food preservation. We report here, for the first time, the complete genome sequence of a novel phage Spp001, which lyses S. putrefaciens Sp225. The Spp001 genome comprises a 54,789-bp DNA molecule with 67 open reading frames and an average total G + C content of 49.42 %. In silico analysis revealed that the Spp001 open reading frames encode various putative functional proteins, including an endolysin (ORF 62); however, no sequence for genes encoding the holin polypeptides, which work in concert with endolysins, was identified. To examine further the lytic activity of Spp001, we analyzed the lytic enzyme-containing fraction from phages released at the end of the phage lytic cycle in S. putrefaciens, using diffusion and turbidimetric assays. The results show that the partially purified extract contained endolysin, as indicated by a high hydrolytic activity towards bacterial peptidoglycan decrease in the OD590 value by 0.160 in 15 min. The results will allow further investigation of the purification of natural Spp001 endolysin, the extension of Spp001 host range, and the applications of the phage-encoded enzymes. PMID:24740748

  3. Human papillomavirus promotes Epstein-Barr virus maintenance and lytic reactivation in immortalized oral keratinocytes.

    PubMed

    Makielski, Kathleen R; Lee, Denis; Lorenz, Laurel D; Nawandar, Dhananjay M; Chiu, Ya-Fang; Kenney, Shannon C; Lambert, Paul F

    2016-08-01

    Epstein-Barr virus and human papillomaviruses are human tumor viruses that infect and replicate in upper aerodigestive tract epithelia and cause head and neck cancers. The productive phases of both viruses are tied to stratified epithelia highlighting the possibility that these viruses may affect each other's life cycles. Our lab has established an in vitro model system to test the effects of EBV and HPV co-infection in stratified squamous oral epithelial cells. Our results indicate that HPV increases maintenance of the EBV genome in the co-infected cells and promotes lytic reactivation of EBV in upper layers of stratified epithelium. Expression of the HPV oncogenes E6 and E7 were found to be necessary and sufficient to account for HPV-mediated lytic reactivation of EBV. Our findings indicate that HPV increases the capacity of epithelial cells to support the EBV life cycle, which could in turn increase EBV-mediated pathogenesis in the oral cavity. PMID:27179345

  4. The role of microRNAs in Epstein-Barr virus latency and lytic reactivation.

    PubMed

    Forte, Eleonora; Luftig, Micah A

    2011-12-01

    Oncogenic viruses reprogram host gene expression driving proliferation, ensuring survival, and evading the immune response. The recent appreciation of microRNAs (miRNAs) as small non-coding RNAs that broadly regulate gene expression has provided new insight into this complex scheme of host control. This review highlights the role of viral and cellular miRNAs during the latent and lytic phases of the EBV life cycle. PMID:21835261

  5. The role of microRNAs in Epstein-Barr virus latency and lytic reactivation

    PubMed Central

    Forte, Eleonora; Luftig, Micah A.

    2016-01-01

    Oncogenic viruses reprogram host gene expression driving proliferation, ensuring survival, and evading the immune response. The recent appreciation of microRNAs (miRNAs) as small non-coding RNAs that broadly regulate gene expression has provided new insight into this complex scheme of host control. This review highlights the role of viral and cellular miRNAs during the latent and lytic phases of the EBV life cycle. PMID:21835261

  6. Modeling and remote sensing of human induced water cycle change

    NASA Astrophysics Data System (ADS)

    Pokhrel, Yadu N.

    2016-04-01

    The global water cycle has been profoundly affected by human land-water management especially during the last century. Since the changes in water cycle can affect the functioning of a wide range of biophysical and biogeochemical processes of the Earth system, it is essential to account for human land-water management in land surface models (LSMs) which are used for water resources assessment and to simulate the land surface hydrologic processes within Earth system models (ESMs). During the last two decades, noteworthy progress has been made in modeling human impacts on the water cycle but sufficient advancements have not yet been made, especially in representing human factors in large-scale LSMs toward integrating them into ESMs. In this study, an integrated modeling framework of continental-scale water cycle, with explicit representation of climate and human induced forces (e.g., irrigation, groundwater pumping) is developed and used to reconstruct the observed water cycle changes in the past and to attribute the observed changes to climatic and human factors. The new model builds upon two different previously developed models: a global LSM called the Human Impacts and GroundWater in the MATSIRO (HiGW-MAT) and a high-resolution regional groundwater model called the LEAF-Hydro-Flood. The model is used to retro-simulate the hydrologic stores and fluxes in close dialogue with in-situ and GRACE satellite based observations at a wide range of river basin scales around the world, with a particular focus on the changes in groundwater dynamics in northwest India, Pakistan, and the High Plains and Central Valley aquifers in the US.

  7. [Lytic phages and prophages of Streptococcus suis--a review].

    PubMed

    Tang, Fang; Lu, Chengping

    2015-04-01

    Streptococcus suis (S. suis) is an important zoonosis and pathogen that can carry prophages. In this review, we focus on the recent advances in our understanding of lytic phage and lysogenic phage of S. suis, including the morphology of S. suis lytic phage, the functions of lysin and terminase large subunit encoded by S. suis lytic phage, comparative genomics of S. suis prophages, lysogenic. conversion between S. suis lytic phage and prophage. Furthermore, prospective evolution of interactions between phage and host was discussed. PMID:26211312

  8. Irradiation-induced changes in nuclear shape and cell cycle

    SciTech Connect

    Iwata, M.; Sasaki, H.; Kishino, Y.; Tsuboi, T.; Sugishita, T.; Hosokawa, T.

    1982-03-01

    Using human uterine cervical carcinoma cells transplanted in nude mice and mice leukemia L5178Y cells, changes in the cell cycle following irradiation were observed by flow cytometry (FCM), and changes in the cell nuclei during the course of irradiation were measured by FCM. Experiments in vivo as well as in vitro caused accumulation of cells in the G2 to M populations, resulting in the so-called G2 block phenomenon as revealed by FCM analysis of DNA distributions. The radiation-induced changes of nuclear shapes were dependent on abnormal mitoses, which occurred more frequently in the G2 to M phases. Therefore it is suggested that the G2 block phenomenon plays an important role in radiation-induced cell death because the process of cell death by irradiation has been shown to proceed via these abnormal mitoses.

  9. Non-lytic, actin-based exit of intracellular parasites from C. elegans intestinal cells.

    PubMed

    Estes, Kathleen A; Szumowski, Suzannah C; Troemel, Emily R

    2011-09-01

    The intestine is a common site for invasion by intracellular pathogens, but little is known about how pathogens restructure and exit intestinal cells in vivo. The natural microsporidian parasite N. parisii invades intestinal cells of the nematode C. elegans, progresses through its life cycle, and then exits cells in a transmissible spore form. Here we show that N. parisii causes rearrangements of host actin inside intestinal cells as part of a novel parasite exit strategy. First, we show that N. parisii infection causes ectopic localization of the normally apical-restricted actin to the basolateral side of intestinal cells, where it often forms network-like structures. Soon after this actin relocalization, we find that gaps appear in the terminal web, a conserved cytoskeletal structure that could present a barrier to exit. Reducing actin expression creates terminal web gaps in the absence of infection, suggesting that infection-induced actin relocalization triggers gap formation. We show that terminal web gaps form at a distinct stage of infection, precisely timed to precede spore exit, and that all contagious animals exhibit gaps. Interestingly, we find that while perturbations in actin can create these gaps, actin is not required for infection progression or spore formation, but actin is required for spore exit. Finally, we show that despite large numbers of spores exiting intestinal cells, this exit does not cause cell lysis. These results provide insight into parasite manipulation of the host cytoskeleton and non-lytic escape from intestinal cells in vivo. PMID:21949650

  10. Non-Lytic, Actin-Based Exit of Intracellular Parasites from C. elegans Intestinal Cells

    PubMed Central

    Estes, Kathleen A.; Szumowski, Suzannah C.; Troemel, Emily R.

    2011-01-01

    The intestine is a common site for invasion by intracellular pathogens, but little is known about how pathogens restructure and exit intestinal cells in vivo. The natural microsporidian parasite N. parisii invades intestinal cells of the nematode C. elegans, progresses through its life cycle, and then exits cells in a transmissible spore form. Here we show that N. parisii causes rearrangements of host actin inside intestinal cells as part of a novel parasite exit strategy. First, we show that N. parisii infection causes ectopic localization of the normally apical-restricted actin to the basolateral side of intestinal cells, where it often forms network-like structures. Soon after this actin relocalization, we find that gaps appear in the terminal web, a conserved cytoskeletal structure that could present a barrier to exit. Reducing actin expression creates terminal web gaps in the absence of infection, suggesting that infection-induced actin relocalization triggers gap formation. We show that terminal web gaps form at a distinct stage of infection, precisely timed to precede spore exit, and that all contagious animals exhibit gaps. Interestingly, we find that while perturbations in actin can create these gaps, actin is not required for infection progression or spore formation, but actin is required for spore exit. Finally, we show that despite large numbers of spores exiting intestinal cells, this exit does not cause cell lysis. These results provide insight into parasite manipulation of the host cytoskeleton and non-lytic escape from intestinal cells in vivo. PMID:21949650

  11. A DNA-damage-induced cell cycle checkpoint in Arabidopsis.

    PubMed Central

    Preuss, S B; Britt, A B

    2003-01-01

    Although it is well established that plant seeds treated with high doses of gamma radiation arrest development as seedlings, the cause of this arrest is unknown. The uvh1 mutant of Arabidopsis is defective in a homolog of the human repair endonuclease XPF, and uvh1 mutants are sensitive to both the toxic effects of UV and the cytostatic effects of gamma radiation. Here we find that gamma irradiation of uvh1 plants specifically triggers a G(2)-phase cell cycle arrest. Mutants, termed suppressor of gamma (sog), that suppress this radiation-induced arrest and proceed through the cell cycle unimpeded were recovered in the uvh1 background; the resulting irradiated plants are genetically unstable. The sog mutations fall into two complementation groups. They are second-site suppressors of the uvh1 mutant's sensitivity to gamma radiation but do not affect the susceptibility of the plant to UV radiation. In addition to rendering the plants resistant to the growth inhibitory effects of gamma radiation, the sog1 mutation affects the proper development of the pollen tetrad, suggesting that SOG1 might also play a role in the regulation of cell cycle progression during meiosis. PMID:12750343

  12. Lytic bacteriophages reduce Escherichia coli O157

    PubMed Central

    Ferguson, Sean; Roberts, Cheryl; Handy, Eric; Sharma, Manan

    2013-01-01

    The role of lytic bacteriophages in preventing cross contamination of produce has not been evaluated. A cocktail of three lytic phages specific for E. coli O157:H7 (EcoShield™) or a control (phosphate buffered saline, PBS) was applied to lettuce by either; (1) immersion of lettuce in 500 ml of EcoShield™ 8.3 log PFU/ml or 9.8 log PFU/ml for up to 2 min before inoculation with E. coli O157:H7; (2) spray-application of EcoShield™ (9.3 log PFU/ml) to lettuce after inoculation with E. coli O157:H7 (4.10 CFU/cm2) following exposure to 50 μg/ml chlorine for 30 sec. After immersion studies, lettuce was spot-inoculated with E. coli O157:H7 (2.38 CFU/cm2). Phage-treated, inoculated lettuce pieces were stored at 4°C for and analyzed for E. coli O157:H7 populations for up to 7 d. Immersion of lettuce in 9.8 log PFU/ml EcoShield™ for 2 min significantly (p < 0.05) reduced E. coli O157:H7 populations after 24 h when stored at 4°C compared with controls. Immersion of lettuce in suspensions containing high concentrations of EcoShield™ (9.8 log PFU/ml) resulted in the deposition of high concentrations (7.8 log log PFU/cm2) of bacteriophages on the surface of fresh cut lettuce, potentially contributing to the efficacy of the lytic phages on lettuce. Spraying phages on to inoculated fresh cut lettuce after being washed in hypochlorite solution was significantly more effective in reducing E. coli O157:H7 populations (2.22 log CFU/cm2) on day 0 compared with control treatments (4.10 log CFU/cm2). Both immersion and spray treatments provided protection from E. coli O157:H7 contamination on lettuce, but spray application of lytic bacteriophages to lettuce was more effective in immediately reducing E. coli O157:H7 populations fresh cut lettuce. PMID:23819106

  13. Prediction of thermal cycling induced cracking in polmer matrix composites

    NASA Technical Reports Server (NTRS)

    Mcmanus, Hugh L.

    1994-01-01

    The work done in the period August 1993 through February 1994 on the 'Prediction of Thermal Cycling Induced Cracking In Polymer Matrix Composites' program is summarized. Most of the work performed in this period, as well as the previous one, is described in detail in the attached Master's thesis, 'Analysis of Thermally Induced Damage in Composite Space Structures,' by Cecelia Hyun Seon Park. Work on a small thermal cycling and aging chamber was concluded in this period. The chamber was extensively tested and calibrated. Temperatures can be controlled very precisely, and are very uniform in the test chamber. Based on results obtained in the previous period of this program, further experimental progressive cracking studies were carried out. The laminates tested were selected to clarify the differences between the behaviors of thick and thin ply layers, and to explore other variables such as stacking sequence and scaling effects. Most specimens tested were made available from existing stock at Langley Research Center. One laminate type had to be constructed from available prepreg material at Langley Research Center. Specimens from this laminate were cut and prepared at MIT. Thermal conditioning was carried out at Langley Research Center, and at the newly constructed MIT facility. Specimens were examined by edge inspection and by crack configuration studies, in which specimens were sanded down in order to examine the distribution of cracks within the specimens. A method for predicting matrix cracking due to decreasing temperatures and/or thermal cycling in all plies of an arbitrary laminate was implemented as a computer code. The code also predicts changes in properties due to the cracking. Extensive correlations between test results and code predictions were carried out. The computer code was documented and is ready for distribution.

  14. Phage lytic proteins: biotechnological applications beyond clinical antimicrobials

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Most bacteriophages encode two types of cell wall lytic proteins: Endolysins (lysins) and virion-associated peptidoglycan hydrolases. Both enzymes have the ability to degrade the peptidoglycan of Gram positive bacteria resulting in cell lysis when they are applied externally. Bacteriophage lytic p...

  15. Quantitative analysis of HSV gene expression during lytic infection

    PubMed Central

    Turner, Anne-Marie W.; Arbuckle, Jesse H.; Kristie, Thomas M.

    2014-01-01

    Herpes Simplex Virus (HSV) is a human pathogen that establishes latency and undergoes periodic reactivation, resulting in chronic recurrent lytic infection. HSV lytic infection is characterized by an organized cascade of three gene classes, however successful transcription and expression of the first, the immediate early class, is critical to the overall success of viral infection. This initial event of lytic infection is also highly dependent on host cell factors. This unit uses RNA interference and small molecule inhibitors to examine the role of host and viral proteins in HSV lytic infection. Methods detailing isolation of viral and host RNA and genomic DNA, followed by quantitative real-time PCR, allow characterization of impacts on viral transcription and replication respectively. Western blot can be used to confirm quantitative PCR results. This combination of protocols represents a starting point for researchers interested in virus-host interactions during HSV lytic infection. PMID:25367270

  16. The Impacts of Climate-Induced Drought on Biogeochemical Cycles

    NASA Astrophysics Data System (ADS)

    Peng, C.

    2014-12-01

    Terrestrial ecosystems and, in particular, forests exert strong controls on the global biogeochemical cycles and influence regional hydrology and climatology directly through water and surface energy budgets. Recent studies indicated that forest mortality caused by rising temperature and drought from around the world have unexpectedly increased in the past decade and they collectively illustrate the vulnerability of many forested ecosystems to rapid increases in tree mortality due to warmer temperatures and more severe drought. Persistent changes in tree mortality rates can alter forest structure, composition, and ecosystem services (such as albedo and carbon sequestration). Quantifying potential impacts of tree mortality on ecosystem processes requires research into mortality effects on carbon, energy, and water budgets at both site and regional levels. Despite recent progress, the uncertainty around mortality responses still limits our ability to predict the likelihood and anticipate the impacts of tree die-off. Studies are needed that explore tree death physiology for a wide variety of functional types, connect patterns of mortality with climate events, and quantify the impacts on carbon, energy, and water flux. In this presentation, I will highlight recent research progress, and identify key research needs and future challenges to predict the consequence and impacts of drought-induced large-scale forest mortality on biogeochemical cycles. I will focus on three main forest ecosystems (tropic rainforest in Amazon, temperate forest in Western USA, and boreal forest in Canada) as detailed case studies.

  17. Interannual Variations of MLS Carbon Monoxide Induced by Solar Cycle

    NASA Technical Reports Server (NTRS)

    Lee, Jae N.; Wu, Dong L.; Ruzmaikin, Alexander

    2013-01-01

    More than eight years (2004-2012) of carbon monoxide (CO) measurements from the Aura Microwave Limb Sounder (MLS) are analyzed. The mesospheric CO, largely produced by the carbon dioxide (CO2) photolysis in the lower thermosphere, is sensitive to the solar irradiance variability. The long-term variation of observed mesospheric MLS CO concentrations at high latitudes is likely driven by the solar-cycle modulated UV forcing. Despite of different CO abundances in the southern and northern hemispheric winter, the solar-cycle dependence appears to be similar. This solar signal is further carried down to the lower altitudes by the dynamical descent in the winter polar vortex. Aura MLS CO is compared with the Solar Radiation and Climate Experiment (SORCE) total solar irradiance (TSI) and also with the spectral irradiance in the far ultraviolet (FUV) region from the SORCE Solar-Stellar Irradiance Comparison Experiment (SOLSTICE). Significant positive correlation (up to 0.6) is found between CO and FUVTSI in a large part of the upper atmosphere. The distribution of this positive correlation in the mesosphere is consistent with the expectation of CO changes induced by the solar irradiance variations.

  18. Epstein-Barr Virus BZLF1-Mediated Downregulation of Proinflammatory Factors Is Essential for Optimal Lytic Viral Replication

    PubMed Central

    Li, Yuqing; Long, Xubing; Huang, Lu; Yang, Mengtian; Yuan, Yan; Wang, Yan; Delecluse, Henri-Jacques

    2015-01-01

    ABSTRACT Elevated secretion of inflammatory factors is associated with latent Epstein-Barr virus (EBV) infection and the pathology of EBV-associated diseases; however, knowledge of the inflammatory response and its biological significance during the lytic EBV cycle remains elusive. Here, we demonstrate that the immediate early transcriptional activator BZLF1 suppresses the proinflammatory factor tumor necrosis factor alpha (TNF-α) by binding to the promoter of TNF-α and preventing NF-κB activation. A BZLF1Δ207-210 mutant with a deletion of 4 amino acids (aa) in the protein-protein binding domain was not able to inhibit the proinflammatory factors TNF-α and gamma interferon (IFN-γ) and reduced viral DNA replication with complete transcriptional activity during EBV lytic gene expression. TNF-α depletion restored the viral replication mediated by BZLF1Δ207-210. Furthermore, a combination of TNF-α- and IFN-γ-neutralizing antibodies recovered BZLF1Δ207-210-mediated viral replication, indicating that BZLF1 attenuates the antiviral response to aid optimal lytic replication primarily through the inhibition of TNF-α and IFN-γ secretion during the lytic cycle. These results suggest that EBV BZLF1 attenuates the proinflammatory responses to facilitate viral replication. IMPORTANCE The proinflammatory response is an antiviral and anticancer strategy following the complex inflammatory phenotype. Latent Epstein-Barr virus (EBV) infection strongly correlates with an elevated secretion of inflammatory factors in a variety of severe diseases, while the inflammatory responses during the lytic EBV cycle have not been established. Here, we demonstrate that BZLF1 acts as a transcriptional suppressor of the inflammatory factors TNF-α and IFN-γ and confirm that BZLF1-facilitated escape from the TNF-α and IFN-γ response during the EBV lytic life cycle is required for optimal viral replication. This finding implies that the EBV lytic cycle employs a distinct strategy to

  19. Prediction of thermal cycling induced cracking in polymer matrix composites

    NASA Technical Reports Server (NTRS)

    Mcmanus, Hugh L.

    1993-01-01

    This report summarizes the work done in the period February 1993 through July 1993 on the 'Prediction of Thermal Cycling Induced Cracking In Polymer Matrix Composites' program. An oral presentation of this work was given to Langley personnel in September of 1993. This document was prepared for archival purposes. Progress studies have been performed on the effects of spatial variations in material strength. Qualitative agreement was found with observed patterns of crack distribution. These results were presented to NASA Langley personnel in November 1992. The analytical methodology developed by Prof. McManus in the summer of 1992 (under an ASEE fellowship) has been generalized. A method for predicting matrix cracking due to decreasing temperatures and/or thermal cycling in all plies of an arbitrary laminate has been implemented as a computer code. The code also predicts changes in properties due to the cracking. Experimental progressive cracking studies on a variety of laminates were carried out at Langley Research Center. Results were correlated to predictions using the new methods. Results were initially mixed. This motivated an exploration of the configuration of cracks within laminates. A crack configuration study was carried out by cutting and/or sanding specimens in order to examine the distribution of cracks within the specimens. These investigations were supplemented by dye-penetrant enhanced X-ray photographs. The behavior of thin plies was found to be different from the behavior of thicker plies (or ply groups) on which existing theories are based. Significant edge effects were also noted, which caused the traditional metric of microcracking (count of cracks on a polished edge) to be very inaccurate in some cases. With edge and configuration taken into account, rough agreement with predictions was achieved. All results to date were reviewed with NASA Langley personnel in September 1993.

  20. Epstein-Barr virus latency type and spontaneous reactivation predict lytic induction levels.

    PubMed

    Phan, An T; Fernandez, Samantha G; Somberg, Jessica J; Keck, Kristin M; Miranda, Jj L

    2016-05-20

    The human Epstein-Barr virus (EBV) evades the immune system by entering a transcriptionally latent phase in B cells. EBV in tumor cells expresses distinct patterns of genes referred to as latency types. Viruses in tumor cells also display varying levels of lytic transcription resulting from spontaneous reactivation out of latency. We measured this dynamic range of lytic transcription with RNA deep sequencing and observed no correlation with EBV latency types among genetically different viruses, but type I cell lines reveal more spontaneous reactivation than isogenic type III cultures. We further determined that latency type and spontaneous reactivation levels predict the relative amount of induced reactivation generated by cytotoxic chemotherapy drugs. Our work has potential implications for personalizing medicine against EBV-transformed malignancies. Identifying latency type or measuring spontaneous reactivation may provide predictive power in treatment contexts where viral production should be either avoided or coerced. PMID:27091426

  1. Diversity of phage infection types and associated terminology: the problem with 'Lytic or lysogenic'.

    PubMed

    Hobbs, Zack; Abedon, Stephen T

    2016-04-01

    Bacteriophages, or phages, are viruses of members of domain Bacteria. These viruses play numerous roles in shaping the diversity of microbial communities, with impact differing depending on what infection strategies specific phages employ. From an applied perspective, these especially are communities containing undesired or pathogenic bacteria that can be modified through phage-mediated bacterial biocontrol, that is, through phage therapy. Here we seek to categorize phages in terms of their infection strategies as well as review or suggest more descriptive, accurate or distinguishing terminology. Categories can be differentiated in terms of (1) whether or not virion release occurs (productive infections versus lysogeny, pseudolysogeny and/or the phage carrier state), (2) the means of virion release (lytic versus chronic release) and (3) the degree to which phages are genetically equipped to display lysogenic cycles (temperate versus non-temperate phages). We address in particular the use or overuse of what can be a somewhat equivocal phrase, 'Lytic or lysogenic', especially when employed as a means of distinguishing among phages types. We suggest that the implied dichotomy is inconsistent with both modern as well as historical understanding of phage biology. We consider, therefore, less ambiguous terminology for distinguishing between 'Lytic' versus 'Lysogenic' phage types. PMID:26925588

  2. Enhancement of Zta-activated lytic transcription of Epstein-Barr virus by Ku80.

    PubMed

    Chen, Chien-Chang; Yang, Ya-Chun; Wang, Wen-Hung; Chen, Chien-Sin; Chang, Li-Kwan

    2011-03-01

    Zta, encoded by the BZLF1 gene of Epstein-Barr virus (EBV), is a transcription factor that is expressed during the immediate-early stage of the lytic cycle. The expression of Zta is crucial to viral lytic development. Earlier studies showed that Ku80 is a binding partner of Zta in ZKO-293 cells and is co-purified with Zta. This study verifies the interaction between Ku80 and Zta by using glutathione S-transferase-pull-down and co-immunoprecipitation assays, and also by indirect immunofluorescence analysis. This investigation also reveals that Ku80 binds to Zta on Zta-response elements in the BHLF1 promoter, enhancing the promoter activity. This study also reveals that the interaction between Zta and Ku80 involves the C-terminal region of Zta and the 425 aa N-terminal region of Ku80. The interaction between these two proteins and the enhancement of transcription that is activated by Zta suggest that Ku80 is important to EBV lytic development. PMID:21123545

  3. Cycle-Induced Flow and Surfactant Transport in an Alveolus

    NASA Astrophysics Data System (ADS)

    Wei, H. H.

    2002-11-01

    The flow and transport in an alveolus are of fundamental importance to partial liquid ventilation, surfactant transport, pulmonary drug administration, cell-cell signaling pathways and gene therapy. We model the system in which an alveolus is partially filled with liquid in the presence of surfactants. Assuming a circular interface due to sufficiently strong surface tension, we can apply two-dimensional bipolar coordinates to describe the system. We then combine analytical and numerical techniques to solve the Stokes flow and the surfactant concentration. In the absence of surfactants, there is no steady streaming because of reversibility of the Stokes flow. The presence of surfactants however induces a non-trivial cycle-averaged surfactant concentration gradient along the interface that generates steady streaming. The steady streaming patterns (e.g., number of vortices) depend on the parameters, especially on the ratio of inspiration to expiration periods (I:E ratio). Either smaller or larger I:E ratio exhibits two primary vortices but the direction of primary vortices for small I:E is opposite to large I:E. Extension to soluble surfactants is also discussed. For sufficiently high surfactant bulk concentration, the surfactant transport is sorption-controlled and soluble surfactants diminish the size of steady vortices near the alveolar opening. For the estimated steady velocity u 10-5 cm/s, the corresponding Peclet number is 10-7/ D_m. Therefore, for Dm <= 10-7 cm^2/s, the convective transport dominates.

  4. Lytic Promoters Express Protein during Herpes Simplex Virus Latency

    PubMed Central

    Russell, Tiffany A.; Tscharke, David C.

    2016-01-01

    Herpes simplex virus (HSV) has provided the prototype for viral latency with previously well-defined acute or lytic and latent phases. More recently, the deep quiescence of HSV latency has been questioned with evidence that lytic genes can be transcribed in this state. However, to date the only evidence that these transcripts might be translated has come from immunological studies that show activated T cells persist in the nervous system during latency. Here we use a highly sensitive Cre-marking model to show that lytic and latent phases are less clearly defined in two significant ways. First, around half of the HSV spread leading to latently infected sites occurred beyond the initial acute infection and second, we show direct evidence that lytic promoters can drive protein expression during latency. PMID:27348812

  5. Lytic Action of the Truncated yncE Gene in Escherichia coli.

    PubMed

    Li, Jianhua; Xiong, Kun; Zou, Lingyun; Chen, Zhijin; Wang, Yiran; Hu, Xiaomei; Rao, Xiancai; Cong, Yanguang

    2016-04-01

    We recently found lytic action of the truncated yncE gene. When the truncated yncE gene of Salmonella enterica serovar Paratyphi A was expressed in Escherichia coli DH5α under the control of the Ara promoter, bacterial growth was markedly inhibited. In the present study, we characterized this lytic action. The N-terminal 103 aa of YncE, containing a signal peptide, was demonstrated to be essential for inhibition. Microscopic observation showed that the bacterial envelope of E. coli was damaged by the expression of truncated yncE, resulting in the release of cytoplasmic content and the formation of bacterial ghosts. The addition of MgSO4 or spermine, which is the stabilizer of bacterial membrane structure, dramatically reversed the cell lysis induced by the toxic truncated YncE. In contrast, the lytic action was significantly enhanced by the addition of SDS or EDTA. Our data indicated that the toxic truncated YncE could cause cell lysis by the disruption of the bacterial membrane. PMID:26687463

  6. In vitro model for lytic replication, latency, and transformation of an oncogenic alphaherpesvirus.

    PubMed

    Schermuly, Julia; Greco, Annachiara; Härtle, Sonja; Osterrieder, Nikolaus; Kaufer, Benedikt B; Kaspers, Bernd

    2015-06-01

    Marek's disease virus (MDV) is an alphaherpesvirus that causes deadly T-cell lymphomas in chickens and serves as a natural small animal model for virus-induced tumor formation. In vivo, MDV lytically replicates in B cells that transfer the virus to T cells in which the virus establishes latency. MDV also malignantly transforms CD4+ T cells with a T(reg) signature, ultimately resulting in deadly lymphomas. No in vitro infection system for primary target cells of MDV has been available due to the short-lived nature of these cells in culture. Recently, we characterized cytokines and monoclonal antibodies that promote survival of cultured chicken B and T cells. We used these survival stimuli to establish a culture system that allows efficient infection of B and T cells with MDV. We were able to productively infect with MDV B cells isolated from spleen, bursa or blood cultured in the presence of soluble CD40L. Virus was readily transferred from infected B to T cells stimulated with an anti-TCRαVβ1 antibody, thus recapitulating the in vivo situation in the culture dish. Infected T cells could then be maintained in culture for at least 90 d in the absence of TCR stimulation, which allowed the establishment of MDV-transformed lymphoblastoid cell lines (LCL). The immortalized cells had a signature comparable to MDV-transformed CD4+ α/β T cells present in tumors. In summary, we have developed a novel in vitro system that precisely reflects the life cycle of an oncogenic herpesivrus in vivo and will allow us to investigate the interaction between virus and target cells in an easily accessible system. PMID:26039998

  7. In vitro model for lytic replication, latency, and transformation of an oncogenic alphaherpesvirus

    PubMed Central

    Schermuly, Julia; Greco, Annachiara; Härtle, Sonja; Osterrieder, Nikolaus; Kaufer, Benedikt B.; Kaspers, Bernd

    2015-01-01

    Marek’s disease virus (MDV) is an alphaherpesvirus that causes deadly T-cell lymphomas in chickens and serves as a natural small animal model for virus-induced tumor formation. In vivo, MDV lytically replicates in B cells that transfer the virus to T cells in which the virus establishes latency. MDV also malignantly transforms CD4+ T cells with a Treg signature, ultimately resulting in deadly lymphomas. No in vitro infection system for primary target cells of MDV has been available due to the short-lived nature of these cells in culture. Recently, we characterized cytokines and monoclonal antibodies that promote survival of cultured chicken B and T cells. We used these survival stimuli to establish a culture system that allows efficient infection of B and T cells with MDV. We were able to productively infect with MDV B cells isolated from spleen, bursa or blood cultured in the presence of soluble CD40L. Virus was readily transferred from infected B to T cells stimulated with an anti-TCRαVβ1 antibody, thus recapitulating the in vivo situation in the culture dish. Infected T cells could then be maintained in culture for at least 90 d in the absence of TCR stimulation, which allowed the establishment of MDV-transformed lymphoblastoid cell lines (LCL). The immortalized cells had a signature comparable to MDV-transformed CD4+ α/β T cells present in tumors. In summary, we have developed a novel in vitro system that precisely reflects the life cycle of an oncogenic herpesivrus in vivo and will allow us to investigate the interaction between virus and target cells in an easily accessible system. PMID:26039998

  8. Molecular markers and cell cycle inhibitors show the importance of cell cycle progression in nematode-induced galls and syncytia.

    PubMed Central

    de Almeida Engler, J; De Vleesschauwer, V; Burssens, S; Celenza, J L; Inzé, D; Van Montagu, M; Engler, G; Gheysen, G

    1999-01-01

    Root knot and cyst nematodes induce large multinucleated cells, designated giant cells and syncytia, respectively, in plant roots. We have used molecular markers to study cell cycle progression in these specialized feeding cells. In situ hybridization with two cyclin-dependent kinases and two cyclins showed that these genes were induced very early in galls and syncytia and that the feeding cells progressed through the G2 phase. By using cell cycle blockers, DNA synthesis and progression through the G2 phase, or mitosis, were shown to be essential for gall and syncytium establishment. When mitosis was blocked, further gall development was arrested. This result demonstrates that cycles of endoreduplication or other methods of DNA amplification are insufficient to drive giant cell expansion. On the other hand, syncytium development was much less affected by a mitotic block; however, syncytium expansion was inhibited. PMID:10330466

  9. Lytic viral infection of bacterioplankton in deep waters of the western Pacific Ocean

    NASA Astrophysics Data System (ADS)

    Li, Y.; Luo, T.; Sun, J.; Cai, L.; Liang, Y.; Jiao, N.; Zhang, R.

    2014-05-01

    As the most abundant biological entities in the ocean, viruses influence host mortality and nutrient recycling mainly through lytic infection. Yet, the ecological characteristics of virioplankton and viral impacts on host mortality and biogeochemical cycling in the deep sea are largely unknown. In the present study, viral abundance and lytic infection were investigated throughout the water column in the western Pacific Ocean. Both the prokaryotic and viral abundance and production showed a significantly decreasing trend from epipelagic to meso- and bathypelagic waters. Viral abundance decreased from 0.36-1.05 × 1010 particles L-1 to 0.43-0.80 × 109 particles L-1, while the virus : prokaryote ratio varied from 7.21 to 16.23 to 2.45-23.40, at the surface and 2000 m, respectively. Lytic viral production rates in surface and 2000 m waters were, on average, 1.03 × 1010 L-1 day-1 and 5.74 × 108 L-1 day-1. Relatively high percentages of prokaryotic cells lysed by viruses at 1000 and 2000 m were observed, suggesting a significant contribution of viruses to prokaryotic mortality in the deep ocean. The carbon released by viral lysis in deep western Pacific Ocean waters was from 0.03 to 2.32 μg C L-1 day-1. Our findings demonstrated a highly dynamic and active viral population in these deep waters and suggested that virioplankton play an important role in the microbial loop and subsequently biogeochemical cycling in deep oceans.

  10. Lytic viral infection of bacterioplankton in deep waters of the western Pacific Ocean

    NASA Astrophysics Data System (ADS)

    Li, Y.; Luo, T.; Sun, J.; Cai, L.; Jiao, N.; Zhang, R.

    2013-12-01

    As the most abundant biological entities in the ocean, viruses can influence host mortality and nutrients recycling mainly through lytic infection. Yet ecological characteristics of virioplankton and viral impacts on host mortality and biogeochemical cycling in the deep sea are largely unknown. In present study, viral abundance and lytic infection was investigated throughout the water column in the western Pacific Ocean. Both the prokaryotic and viral abundance and production showed a significantly decreasing trend from epipelagic to meso- and bathypelagic waters. Viral abundance decreased from 0.36-1.05 × 1010 particles L-1 to 0.43-0.80 × 109 particles L-1, while the virus : prokaryote ratio varied from 7.21-16.23 to 2.45-23.40, at surface and 2000 m depth, respectively. The lytic viral production rates in surface and 2000 m waters were, averagely, 1.03 × 1010 L-1 day-1 and 5.74 × 108 L-1 day-1, respectively. Relatively high percentages of prokaryotic cells lysed by virus in 1000 m and 2000 m were observed, suggesting a significant contribution of viruses to prokaryotic mortality in deep ocean. The carbon released by viral lysis in deep western Pacific Ocean waters was from 0.03 to 2.32 μg C L-1 day-1. Our findings demonstrated a highly dynamic and active viral population in the deep western Pacific Ocean and suggested that virioplankton play an important role in the microbial loop and subsequently biogeochemical cycling in deep oceans.

  11. 'Ca. Liberibacter asiaticus' carries an excision plasmid prophage and a chromosomally integrated prophage that becomes lytic in plant infections.

    PubMed

    Zhang, Shujian; Flores-Cruz, Zomary; Zhou, Lijuan; Kang, Byung-Ho; Fleites, Laura A; Gooch, Mark D; Wulff, Nelson A; Davis, Michael J; Duan, Yong-Ping; Gabriel, Dean W

    2011-04-01

    Huanglongbing (HLB), also known as citrus greening, is a lethal disease of citrus caused by several species of 'Candidatus Liberibacter', a psyllid-transmitted, phloem-limited, alpha proteobacteria. 'Ca. Liberibacter asiaticus' is widespread in Florida citrus. The recently published 'Ca. L. asiaticus' psy62 genome, derived from a psyllid, revealed a prophage-like region of DNA in the genome, but phage have not been associated with 'Ca. L. asiaticus' to date. In the present study, shotgun sequencing and a fosmid DNA library of curated 'Ca. L. asiaticus' UF506, originally derived from citrus symptomatic for HLB, revealed two largely homologous, circular phage genomes, SC1 and SC2. SC2 encoded putative adhesin and peroxidase genes that had not previously been identified in 'Ca. L. asiaticus' and which may be involved in lysogenic conversion. SC2 also appeared to lack lytic cycle genes and replicated as a prophage excision plasmid, in addition to being found integrated in tandem with SC1 in the UF506 chromosome. By contrast, SC1 carried suspected lytic cycle genes and was found in nonintegrated, lytic cycle forms only in planta. Phage particles associated with 'Ca. L. asiaticus' were found in the phloem of infected periwinkles by transmission electron microscopy. In psyllids, both SC1 and SC2 were found only as prophage. PMID:21190436

  12. Adenosine induces G2/M cell-cycle arrest by inhibiting cell mitosis progression.

    PubMed

    Jia, Kun-Zhi; Tang, Bo; Yu, Lu; Cheng, Wei; Zhang, Rong; Zhang, Jian-Fa; Hua, Zi-Chun

    2010-01-01

    Cellular adenosine accumulates under stress conditions. Few papers on adenosine are concerned with its function in the cell cycle. The cell cycle is the essential mechanism by which all living things reproduce and the target machinery when cells encounter stresses, so it is necessary to examine the relationship between adenosine and the cell cycle. In the present study, adenosine was found to induce G-2/M cell-cycle arrest. Furthermore, adenosine was found to modulate the expression of some important proteins in the cell cycle, such as cyclin B and p21, and to inhibit the transition of metaphase to anaphase in mitosis. PMID:19947935

  13. Gene Expression of Lytic Endopeptidases AlpA and AlpB from Lysobacter sp. XL1 in Pseudomonads.

    PubMed

    Tsfasman, Irina M; Lapteva, Yulia S; Krasovskaya, Ludmila A; Kudryakova, Irina V; Vasilyeva, Natalia V; Granovsky, Igor E; Stepnaya, Olga A

    2015-01-01

    Development of an efficient expression system for (especially secreted) bacterial lytic enzymes is a complicated task due to the specificity of their action. The substrate for such enzymes is peptidoglycan, the main structural component of bacterial cell walls. For this reason, expression of recombinant lytic proteins is often accompanied with lysis of the producing bacterium. This paper presents data on the construction of an inducible system for expression of the lytic peptidases AlpA and AlpB from Lysobacter sp. XL1 in Pseudomonas fluorescens Q2-87, which provides for the successful secretion of these proteins into the culture liquid. In this system, the endopeptidase gene under control of the T7lac promoter was integrated into the bacterial chromosome, as well as the Escherichia coli lactose operon repressor protein gene. The T7 pol gene under lac promoter control, which encodes the phage T7 RNA polymerase, is maintained in Pseudomonas cells on the plasmids. Media and cultivation conditions for the recombinant strains were selected to enable the production of AlpA and AlpB by a simple purification protocol. Production of recombinant lytic enzymes should contribute to the development of new-generation antimicrobial drugs whose application will not be accompanied by selection of resistant microorganisms. PMID:26138026

  14. Metrological characterization of a cycle-ergometer to optimize the cycling induced by functional electrical stimulation on patients with stroke.

    PubMed

    Comolli, Lorenzo; Ferrante, Simona; Pedrocchi, Alessandra; Bocciolone, Marco; Ferrigno, Giancarlo; Molteni, Franco

    2010-05-01

    Functional electrical stimulation (FES) is a well established method in the rehabilitation of stroke patients. Indeed, a bilateral movement such as cycling induced by FES would be crucial for these patients who had an unilateral motor impairment and had to recover an equivalent use of limbs. The aim of this study was to develop a low-cost meteorologically qualified cycle-ergometer, optimized for patients with stroke. A commercial ergometer was instrumented with resistive strain gauges and was able to provide the torque produced at the right and left crank, independently. The developed system was integrated with a stimulator, obtaining a novel FES cycling device able to control in real-time the movement unbalance. A dynamic calibration of the sensors was performed and a total torque uncertainty was computed. The system was tested on a healthy subject and on a stroke patient. Results demonstrated that the proposed sensors could be successfully used during FES cycling sessions where the maximum torque produced is about 9Nm, an order of magnitude less than the torque produced during voluntary cycling. This FES cycling system will assist in future investigations on stroke rehabilitation by means of FES and in new exercise regimes designed specifically for patients with unilateral impairments. PMID:20171923

  15. Discovery and industrial applications of lytic polysaccharide mono-oxygenases.

    PubMed

    Johansen, Katja S

    2016-02-01

    The recent discovery of copper-dependent lytic polysaccharide mono-oxygenases (LPMOs) has opened up a vast area of research covering several fields of application. The biotech company Novozymes A/S holds patents on the use of these enzymes for the conversion of steam-pre-treated plant residues such as straw to free sugars. These patents predate the correct classification of LPMOs and the striking synergistic effect of fungal LPMOs when combined with canonical cellulases was discovered when fractions of fungal secretomes were evaluated in industrially relevant enzyme performance assays. Today, LPMOs are a central component in the Cellic CTec enzyme products which are used in several large-scale plants for the industrial production of lignocellulosic ethanol. LPMOs are characterized by an N-terminal histidine residue which, together with an internal histidine and a tyrosine residue, co-ordinates a single copper atom in a so-called histidine brace. The mechanism by which oxygen binds to the reduced copper atom has been reported and the general mechanism of copper-oxygen-mediated activation of carbon is being investigated in the light of these discoveries. LPMOs are widespread in both the fungal and the bacterial kingdoms, although the range of action of these enzymes remains to be elucidated. However, based on the high abundance of LPMOs expressed by microbes involved in the decomposition of organic matter, the importance of LPMOs in the natural carbon-cycle is predicted to be significant. In addition, it has been suggested that LPMOs play a role in the pathology of infectious diseases such as cholera and to thus be relevant in the field of medicine. PMID:26862199

  16. TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

    SciTech Connect

    Ding, Li; Huang, Yong; Du, Qian; Dong, Feng; Zhao, Xiaomin; Zhang, Wenlong; Xu, Xingang; Tong, Dewen

    2014-03-07

    Highlights: • TGEV N protein reduces cell viability by inducing cell cycle arrest and apoptosis. • TGEV N protein induces cell cycle arrest and apoptosis by regulating p53 signaling. • TGEV N protein plays important roles in TGEV-induced cell cycle arrest and apoptosis. - Abstract: Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressed cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence.

  17. Phage lytic proteins: biotechnological applications beyond clinical antimicrobials.

    PubMed

    Rodríguez-Rubio, Lorena; Gutiérrez, Diana; Donovan, David M; Martínez, Beatriz; Rodríguez, Ana; García, Pilar

    2016-06-01

    Most bacteriophages encode two types of cell wall lytic proteins: endolysins (lysins) and virion-associated peptidoglycan hydrolases. Both enzymes have the ability to degrade the peptidoglycan of Gram-positive bacteria resulting in cell lysis when they are applied externally. Bacteriophage lytic proteins have a demonstrated potential in treating animal models of infectious diseases. There has also been an increase in the study of these lytic proteins for their application in areas such as food safety, pathogen detection/diagnosis, surfaces disinfection, vaccine development and nanotechnology. This review summarizes the more recent developments, outlines the full potential of these proteins to develop new biotechnological tools and discusses the feasibility of these proposals. PMID:25603721

  18. The Phage Lytic Proteins from the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA88 Display Multiple Active Catalytic Domains and Do Not Trigger Staphylococcal Resistance

    PubMed Central

    Rodríguez-Rubio, Lorena; Martínez, Beatriz; Rodríguez, Ana; Donovan, David M.; Götz, Friedrich; García, Pilar

    2013-01-01

    The increase in antibiotic resistance world-wide revitalized the interest in the use of phage lysins to combat pathogenic bacteria. In this work, we analyzed the specific cleavage sites on the staphylococcal peptidoglycan produced by three phage lytic proteins. The investigated cell wall lytic enzymes were the endolysin LysH5 derived from the S. aureus bacteriophage vB_SauS-phi-IPLA88 (phi-IPLA88) and two fusion proteins between lysostaphin and the virion-associated peptidoglycan hydrolase HydH5 (HydH5SH3b and HydH5Lyso). We determined that all catalytic domains present in these proteins were active. Additionally, we tested for the emergence of resistant Staphylococcus aureus to any of the three phage lytic proteins constructs. Resistant S. aureus could not be identified after 10 cycles of bacterial exposure to phage lytic proteins either in liquid or plate cultures. However, a quick increase in lysostaphin resistance (up to 1000-fold in liquid culture) was observed. The lack of resistant development supports the use of phage lytic proteins as future therapeutics to treat staphylococcal infections. PMID:23724076

  19. Temperature Cycles Induce Early Maturation in Channel Catfish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A major impediment in improvement of channel catfish by selective breeding is that a high percent of fish do not spawn until the third year. The conditions that lead to sexual maturation in fish have not been established. Size, nutritional state and number of seasonal cycles have all been suggeste...

  20. Advantage of combining resin with lytic BACTEC blood culture media.

    PubMed

    Rohner, P; Pepey, B; Auckenthaler, R

    1997-10-01

    The BACTEC 9240 (Becton Dickinson, Sparks, Md.) automated blood culture system is based on the continuous monitoring of CO2 production by means of a fluorescent sensor attached to the bottom of a culture vial. We compared two media for this system, resin-containing Plus aerobic/F and Lytic anaerobic/F. Sets of Plus aerobic/F and Lytic anaerobic/F vials inoculated with similar volumes (9 +/- 2.5 ml) were evaluated. In the laboratory, the vials were introduced into the system in accordance with the recommendations of the manufacturer and incubated at 35 degrees C for 5 days. A total of 10,914 sets consisting of two bottles each were obtained from 3,674 patients (2.97 cultures per patient). Of these, 1,233 (11%) were culture positive, including 1,074 (10%) yielding at least one pathogen, and 178 (2%) were contaminated. A total of 1,135 isolates were considered clinically relevant in 624 septic episodes; we isolated 894 from Plus aerobic/F and 852 from Lytic anaerobic/F (P = 0.06 [not significant]). More S. aureus isolates (P = 0.05), Pseudomonas spp. (P < 0.0001), other gram-negative bacteria (P = 0.004), and yeasts (P < 0.0001) were isolated from Plus aerobic/F medium, but more streptococci (P < 0.0001), E. coli (P = 0.02) strains and anaerobes (P < 0.0001) were detected with Lytic anaerobic/F medium. Lytic anaerobic/F vials were significantly (P < 0.0001) more often positive at least 6 h before Plus aerobic/F vials (n = 112 versus 52, respectively). Significantly more (P < 0.0001) Plus aerobic/F vials (n = 210; 1.9%) than Lytic anaerobic/F vials (n = 42; 0.4%) were unconfirmed positives. Plus aerobic/F and Lytic anaerobic/F proved to be a valuable pair of blood culture media. Plus aerobic/F performs better for patients under antibiotic treatment, due to the antimicrobial-neutralizing effect of resins. For patients without antibiotic therapy, more microorganisms could be isolated from Lytic anaerobic/F due to cell lysis. PMID:9316921

  1. Advantage of combining resin with lytic BACTEC blood culture media.

    PubMed Central

    Rohner, P; Pepey, B; Auckenthaler, R

    1997-01-01

    The BACTEC 9240 (Becton Dickinson, Sparks, Md.) automated blood culture system is based on the continuous monitoring of CO2 production by means of a fluorescent sensor attached to the bottom of a culture vial. We compared two media for this system, resin-containing Plus aerobic/F and Lytic anaerobic/F. Sets of Plus aerobic/F and Lytic anaerobic/F vials inoculated with similar volumes (9 +/- 2.5 ml) were evaluated. In the laboratory, the vials were introduced into the system in accordance with the recommendations of the manufacturer and incubated at 35 degrees C for 5 days. A total of 10,914 sets consisting of two bottles each were obtained from 3,674 patients (2.97 cultures per patient). Of these, 1,233 (11%) were culture positive, including 1,074 (10%) yielding at least one pathogen, and 178 (2%) were contaminated. A total of 1,135 isolates were considered clinically relevant in 624 septic episodes; we isolated 894 from Plus aerobic/F and 852 from Lytic anaerobic/F (P = 0.06 [not significant]). More S. aureus isolates (P = 0.05), Pseudomonas spp. (P < 0.0001), other gram-negative bacteria (P = 0.004), and yeasts (P < 0.0001) were isolated from Plus aerobic/F medium, but more streptococci (P < 0.0001), E. coli (P = 0.02) strains and anaerobes (P < 0.0001) were detected with Lytic anaerobic/F medium. Lytic anaerobic/F vials were significantly (P < 0.0001) more often positive at least 6 h before Plus aerobic/F vials (n = 112 versus 52, respectively). Significantly more (P < 0.0001) Plus aerobic/F vials (n = 210; 1.9%) than Lytic anaerobic/F vials (n = 42; 0.4%) were unconfirmed positives. Plus aerobic/F and Lytic anaerobic/F proved to be a valuable pair of blood culture media. Plus aerobic/F performs better for patients under antibiotic treatment, due to the antimicrobial-neutralizing effect of resins. For patients without antibiotic therapy, more microorganisms could be isolated from Lytic anaerobic/F due to cell lysis. PMID:9316921

  2. Inhibition of autophagy in EBV-positive Burkitt's lymphoma cells enhances EBV lytic genes expression and replication.

    PubMed

    De Leo, A; Colavita, F; Ciccosanti, F; Fimia, G M; Lieberman, P M; Mattia, E

    2015-01-01

    Autophagy, an important degradation system involved in maintaining cellular homeostasis, serves also to eliminate pathogens and process their fragments for presentation to the immune system. Several viruses have been shown to interact with the host autophagic machinery to suppress or make use of this cellular catabolic pathway to enhance their survival and replication. Epstein Barr virus (EBV) is a γ-herpes virus associated with a number of malignancies of epithelial and lymphoid origin in which establishes a predominantly latent infection. Latent EBV can periodically reactivate to produce infectious particles that allow the virus to spread and can lead to the death of the infected cell. In this study, we analyzed the relationship between autophagy and EBV reactivation in Burkitt's lymphoma cells. By monitoring autophagy markers and EBV lytic genes expression, we demonstrate that autophagy is enhanced in the early phases of EBV lytic activation but decreases thereafter concomitantly with increased levels of EBV lytic proteins. In a cell line defective for late antigens expression, we found an inverse correlation between EBV early antigens expression and autophagosomes formation, suggesting that early after activation, the virus is able to suppress autophagy. We report here for the first time that inhibition of autophagy by Bafilomycin A1 or shRNA knockdown of Beclin1 gene, highly incremented EBV lytic genes expression as well as intracellular viral DNA and viral progeny yield. Taken together, these findings indicate that EBV activation induces the autophagic response, which is soon inhibited by the expression of EBV early lytic products. Moreover, our findings open the possibility that pharmacological inhibitors of autophagy may be used to enhance oncolytic viral therapy of EBV-related lymphomas. PMID:26335716

  3. Inhibition of autophagy in EBV-positive Burkitt's lymphoma cells enhances EBV lytic genes expression and replication

    PubMed Central

    De Leo, A; Colavita, F; Ciccosanti, F; Fimia, G M; Lieberman, P M; Mattia, E

    2015-01-01

    Autophagy, an important degradation system involved in maintaining cellular homeostasis, serves also to eliminate pathogens and process their fragments for presentation to the immune system. Several viruses have been shown to interact with the host autophagic machinery to suppress or make use of this cellular catabolic pathway to enhance their survival and replication. Epstein Barr virus (EBV) is a γ-herpes virus associated with a number of malignancies of epithelial and lymphoid origin in which establishes a predominantly latent infection. Latent EBV can periodically reactivate to produce infectious particles that allow the virus to spread and can lead to the death of the infected cell. In this study, we analyzed the relationship between autophagy and EBV reactivation in Burkitt's lymphoma cells. By monitoring autophagy markers and EBV lytic genes expression, we demonstrate that autophagy is enhanced in the early phases of EBV lytic activation but decreases thereafter concomitantly with increased levels of EBV lytic proteins. In a cell line defective for late antigens expression, we found an inverse correlation between EBV early antigens expression and autophagosomes formation, suggesting that early after activation, the virus is able to suppress autophagy. We report here for the first time that inhibition of autophagy by Bafilomycin A1 or shRNA knockdown of Beclin1 gene, highly incremented EBV lytic genes expression as well as intracellular viral DNA and viral progeny yield. Taken together, these findings indicate that EBV activation induces the autophagic response, which is soon inhibited by the expression of EBV early lytic products. Moreover, our findings open the possibility that pharmacological inhibitors of autophagy may be used to enhance oncolytic viral therapy of EBV-related lymphomas. PMID:26335716

  4. Drought-induced Changes in Dryland Soil Biogeochemical Cycles

    NASA Astrophysics Data System (ADS)

    Belnap, J.; Darrouzet-Nardi, A.; Duniway, M.; Ferrenberg, S.; Hoover, D. L.; Reed, S.

    2015-12-01

    Approximately 41% of Earth´s terrestrial surface consists of drylands and they are an important biome on all continents. Although dryland biota would be expected to be drought adapted, they can be surprisingly vulnerable to extended dry periods with subsequent consequences for biogeochemical cycles. Biological soil crusts, constituting up to 70% of the living cover in these regions, are important in these cycles. They fix both N and C, providing a significant percentage of regional and global inputs. However, extended drought reduces both types of inputs, as biocrusts are only metabolically active when wet, yet losses continue even when soils are dry. In addition, extended droughts can result in their mortality. The amount of net soil C exchange of biocrusted soils is controversial, but in SE Utah, soil C uptake only occurred when only when soils were wet. As soils are infrequently wet, annual balances were negative during the 2 year study and with future extended droughts or increased temperatures that reduce soil moisture, these losses will become even greater. As with C, N fixation also requires biocrusts be wet and thus inputs decline with extended drought or higher temperatures that both reduce input and result in lichen and cyanobacterial mortality. And similarly, N losses continue even when soils are dry. Loss of biocrust mosses can profoundly alter N cycles. Desert plants are also affected by drought: in plots where experimental drought was imposed, plants had lower photosynthetic rates and higher leaf C:N, which will likely affect productivity and decomposition rates and thus have further impacts on soil biogeochemical cycles.

  5. Disulfide Sensitivity in the Env Protein Underlies Lytic Inactivation of HIV-1 by Peptide Triazole Thiols.

    PubMed

    Bailey, Lauren D; Kalyana Sundaram, Ramalingam Venkat; Li, Huiyuan; Duffy, Caitlin; Aneja, Rachna; Rosemary Bastian, Arangassery; Holmes, Andrew P; Kamanna, Kantharaju; Rashad, Adel A; Chaiken, Irwin

    2015-12-18

    We investigated the mode of action underlying lytic inactivation of HIV-1 virions by peptide triazole thiol (PTT), in particular the relationship between gp120 disulfides and the C-terminal cysteine-SH required for virolysis. Obligate PTT dimer obtained by PTT SH cross-linking and PTTs with serially truncated linkers between pharmacophore isoleucine-ferrocenyltriazole-proline-tryptophan and cysteine-SH were synthesized. PTT variants showed loss of lytic activity but not binding and infection inhibition upon SH blockade. A disproportionate loss of lysis activity vs binding and infection inhibition was observed upon linker truncation. Molecular docking of PTT onto gp120 argued that, with sufficient linker length, the peptide SH could approach and disrupt several alternative gp120 disulfides. Inhibition of lysis by gp120 mAb 2G12, which binds at the base of the V3 loop, as well as disulfide mutational effects, argued that PTT-induced disruption of the gp120 disulfide cluster at the base of the V3 loop is an important step in lytic inactivation of HIV-1. Further, PTT-induced lysis was enhanced after treating virus with reducing agents dithiothreitol and tris (2-carboxyethyl)phosphine. Overall, the results are consistent with the view that the binding of PTT positions the peptide SH group to interfere with conserved disulfides clustered proximal to the CD4 binding site in gp120, leading to disulfide exchange in gp120 and possibly gp41, rearrangement of the Env spike, and ultimately disruption of the viral membrane. The dependence of lysis activity on thiol-disulfide interaction may be related to intrinsic disulfide exchange susceptibility in gp120 that has been reported previously to play a role in HIV-1 cell infection. PMID:26458166

  6. UV-induced changes in cell cycle and gene expression within rabbit lens epithelial cells

    SciTech Connect

    Sidjanin, D.; Grdina, D.; Woloschak, G.E.

    1994-11-01

    Damage to lens epithelial cells is a probable initiation process in cataract formation induced by ultraviolet radiation. These experiments investigated the ability of 254 nm radiation on cell cycle progression and gene expression in rabbit lens epithelial cell line N/N1003A. No changes in expression of c-fos, c-jun, alpha- tubulin, or vimentin was observed following UV exposure. Using flow cytometry, an accumulation of cells in G1/S phase of the cell cycle 1 hr following exposure. The observed changes in gene expression, especially the decreased histone transcripts reported here may play a role in UV induced inhibition of cell cycle progression.

  7. Computation Molecular Kinetics Model of HZE Induced Cell Cycle Arrest

    NASA Technical Reports Server (NTRS)

    Cucinotta, Francis A.; Ren, Lei

    2004-01-01

    Cell culture models play an important role in understanding the biological effectiveness of space radiation. High energy and charge (HZE) ions produce prolonged cell cycle arrests at the G1/S and G2/M transition points in the cell cycle. A detailed description of these phenomena is needed to integrate knowledge of the expression of DNA damage in surviving cells, including the determination of relative effectiveness factors between different types of radiation that produce differential types of DNA damage and arrest durations. We have developed a hierarchical kinetics model that tracks the distribution of cells in various cell phase compartments (early G1, late G1, S, G2, and M), however with transition rates that are controlled by rate-limiting steps in the kinetics of cyclin-cdk's interactions with their families of transcription factors and inhibitor molecules. The coupling of damaged DNA molecules to the downstream cyclin-cdk inhibitors is achieved through a description of the DNA-PK and ATM signaling pathways. For HZE irradiations we describe preliminary results, which introduce simulation of the stochastic nature of the number of direct particle traversals per cell in the modulation of cyclin-cdk and cell cycle population kinetics. Comparison of the model to data for fibroblast cells irradiated photons or HZE ions are described.

  8. Detection and characterization of a lytic Pediococcus bacteriophage from the fermenting cucumber brine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Of the twelve lytic bacteriophages recovered from five different fermenting cucumber tanks that were inoculated with Pediococcus sp. LA0281, a lytic phage, 'ps05, was characterized in the present study. The plaques were mostly clear and round-shaped on the lawn of starter strain, indicating lytic ph...

  9. A systemic non-lytic state and local thrombolytic failure of anistreplase (anisoylated plasminogen streptokinase activator complex, APSAC) in acute myocardial infarction.

    PubMed Central

    Brügemann, J; van der Meer, J; Takens, B H; Hillege, H; Lie, K I

    1990-01-01

    The relation between coronary thrombolysis and coagulation variables after administration of anistreplase (anisoylated plasminogen streptokinase activator complex, APSAC) was studied in patients with an acute myocardial infarction. Fifty eight consecutive patients with acute myocardial infarction were given 30 U of anistreplase intravenously within 4 hours of the onset of symptoms. A fall in the plasma concentration fibrinogen to less than 1.0 g/l 90 minutes after administration of anistreplase was considered to reflect a systemic lytic state. Coronary angiography was performed 48 hours after thrombolytic treatment. The overall patency rate was 74% (43/58). Patency rates were significantly different in patients with a systemic lytic (83% (43/52)) and a systemic non-lytic state (0% (0/6)). The absence of a systemic lytic state after anistreplase administration seemed to be highly predictive of the failure of coronary thrombolysis. Coagulation studies showed evidence of inhibition of anistreplase induced fibrinolytic activity which may explain the failure of thrombolytic treatment in patients with evidence of a systemic non-lytic state. PMID:2271340

  10. Percutaneous pediculoplasty: polymethylmethacrylate injection into lytic vertebral pedicle lesions.

    PubMed

    Gailloud, Philippe; Beauchamp, Norman J; Martin, Jean-Baptiste; Murphy, Kieran J

    2002-05-01

    Two patients with painful lytic lesions of the vertebral pedicle were treated with percutaneous pediculoplasty. This technique, similar to vertebroplasty but with increased procedural risks because of the immediate vicinity of neural structures, was performed under high-quality biplane fluoroscopic guidance. For lesions involving both the pedicle and the vertebral body, a double-needle unipedicular technique is advocated. PMID:11997361

  11. Climatically induced sedimentary cycles in Pliocene deep-water carbonates

    SciTech Connect

    Gardulski, A.F. )

    1991-03-01

    Two DSDP sites (86 and 94) on the Campeche ramp in the southern Gulf of Mexico penetrated more than 100 m of Pliocene pelagic ooze. The ooze is primarily carbonate, with a much smaller volcanic ash component than occurs in some Pleistocene sediments at these sites. Cores recovered from these holes display variations in carbonate mineralogy as well as total carbonate and sand abundances that are correlated with the oxygen isotope stratigraphy. Diagenetic loss of Mg-calcite is complete by the base of the Pleistocene, but aragonite, especially high-Sr aragonite forming algal needles that were transported off the shelf to the slope, persists through upper Pliocene cores. Variations in oxygen isotope ratios in planktonic foraminifera occur throughout the Pliocene, although the amplitude of those cycles is smaller than for the Pleistocene, with its more dramatic glacial-interglacial contrasts. As in overlying Pleistocene slope sediments, cooler intervals correspond with greater abundances of aragonite in the upper Pliocene section, reflecting a shift of the shallow, productive shelf seaward across the ramp surface during times of relatively low sea level. However, the aragonite abundances in the Pliocene are reduced on average compared to the Pleistocene. This difference is due in part to diagenetic loss, but also it likely reflects the overall higher sea level that apparently characterized Pliocene oceans, trapping more algal aragonite landward. Although sea level and climatic fluctuations were indeed less extreme in the Pliocene, they were still sufficient to generate sedimentary cycles in deep-water carbonates.

  12. The menstrual cycle and susceptibility to coriolis-induced sickness.

    PubMed

    Cheung, B; Heskin, R; Hofer, K; Gagnon, M

    2001-01-01

    Survey studies on motion sickness susceptibility suggest that females tend to report greater severity in illness and higher incidence of vomiting than males. Menstruation is said to be a contributing factor. A recent study suggested that females were least susceptible to seasickness during ovulation in a "round the world" yacht race. Sixteen subjects (18-36 years old) were exposed to Coriolis cross-coupling stimulation in the laboratory. They were tested once during permenstruation (Day 1-5), ovulation (Day 12-15) and premenstruation (Day 24-28), based on a normalized 28-day cycle, in a randomised design. Physiological measurements of motion sickness included forearm and calf cutaneous blood flow. Subjective evaluation of sickness symptoms was based on Graybiel's diagnostic criteria and Golding's rating method. Our results indicated that under controlled laboratory conditions, different phases of the menstrual cycle appear to have no influence on subjective symptoms of motion sickness or on cutaneous blood flow increase in the forearm and calf. The lack of commonality between the types and levels of hormones that are released during motion sickness and those that are involved in different menstrual phases appears to support our findings. PMID:11847456

  13. Starspot-Induced Radial Velocity Jitter During a Stellar Cycle

    NASA Astrophysics Data System (ADS)

    Korhonen, Heidi; Andersen, Jan Marie; Järvinen, Silva

    2014-04-01

    Late-type stars exhibit cool regions on their surface, the stellar equivalent of sunspots. These dark starspots can also mimic the radial velocity variations caused by orbiting planets, making it at times difficult to distinguish between planets and activity signatures. The amount of spots on the Sun and other cool stars changes cyclically during an activity cycle, which has length varying from about a year to longer than the solar 11 years. In this work we investigate the influence of varying amount of starspots on the sparsely sampled radial velocity observations - which are the norm in the radial velocity studies searching for exoplanets on wide orbits. We study two simulated cases: one with a random spot configuration, and one where the spot occurrence is concentrated. In addition we use Doppler images of young solar analogue V889 Her as a high activity case.

  14. Toll-like receptor 4 is involved in the cell cycle modulation and required for effective human cytomegalovirus infection in THP-1 macrophages

    SciTech Connect

    Arcangeletti, Maria-Cristina; Germini, Diego; Rodighiero, Isabella; Mirandola, Prisco; De Conto, Flora; Medici, Maria-Cristina; Gatti, Rita; Chezzi, Carlo; Calderaro, Adriana

    2013-05-25

    Suitable host cell metabolic conditions are fundamental for the effective development of the human cytomegalovirus (HCMV) lytic cycle. Indeed, several studies have demonstrated the ability of this virus to interfere with cell cycle regulation, mainly by blocking proliferating cells in G1 or G1/S. In the present study, we demonstrate that HCMV deregulates the cell cycle of THP-1 macrophages (a cell line irreversibly arrested in G0) by pushing them into S and G2 phases. Moreover, we show that HCMV infection of THP-1 macrophages leads to Toll-like receptor 4 (TLR4) activation. Since various studies have indicated TLR4 to be involved in promoting cell proliferation, here we investigate the possible role of TLR4 in the observed HCMV-induced cell cycle perturbation. Our data strongly support TLR4 as a mediator of HCMV-triggered cell cycle activation in THP-1 macrophages favouring, in turn, the development of an efficient viral lytic cycle. - Highlights: ► We studied HCMV infection impact on THP-1 macrophage cell cycle. ► We analysed the role played by Toll-like receptor (TLR) 4 upon HCMV infection. ► HCMV pushes THP-1 macrophages (i.e. resting cells) to re-enter the cell cycle. ► TLR4 pathway inhibition strongly affects the effectiveness of HCMV replication. ► TLR4 pathway inhibition significantly decreases HCMV-induced cell cycle re-entry.

  15. Metabolic changes induced by sustained exhaustive cycling and diet manipulation.

    PubMed

    Brouns, F; Saris, W H; Beckers, E; Adlercreutz, H; van der Vusse, G J; Keizer, H A; Kuipers, H; Menheere, P; Wagenmakers, A J; ten Hoor, F

    1989-05-01

    Thirteen highly trained subjects were studied concerning the effect of consuming a normal carbohydrate-rich diet (N) on energy exchange, substrate metabolism, and performance. Six of these subjects performed the same protocol receiving N supplemented with a high-maltodextrin, low-fructose beverage (Mf). The studies were performed in random order. The subjects performed 2 days of sustained exhausting cycling, preceded and followed by a standardized resting day, in a respiration chamber, allowing continuous gas analysis, weighed food and fluid intake procedures, collection of excretes, and drawing of blood samples at 7:00 AM, 12:00 AM (halfway exercise) and 3:00 PM at exhaustion. Muscle biopsies were taken prior to, 45 min after, and 24 h after exercise (energy expenditure 25.2-26.6 MJ.day-1). The results showed that while consuming a normal diet, the cyclists developed a negative energy balance (-9 MJ.day-1) and regulated their hormone levels in such a way that fat oxidation and protein breakdown were increased and CHO oxidation became depressed. When supplemented with Mf, the subjects showed increased blood glucose, insulin and decreased glucagon levels. Fat metabolism was significantly depressed as indicated by the levels of blood fatty acids, glycerol, and ketones. A significant glycogen sparing, as well as supercompensation within 24 h of recovery, was observed after Mf supplementation. The normal CHO-rich diet, available ad libitum, was insufficient to fully restore glycogen within 24 h. The changes in substrate availability and glycogen depletion were accompanied by a significant performance improvement, 126% when cycling a final 90% Wmax bout, when supplemented with Mf compared with N. PMID:2663743

  16. Martian induced magnetosphere variations with solar activity cycle

    NASA Astrophysics Data System (ADS)

    Fedorov, Andrey; Ronan, Modolo; Jarninen, Riku; Mazelle, Christian; Barabash, Stas

    2014-05-01

    During the last 6 years of ESA Mars Express mission we have accumulated plasma data taken inside and around the Martian induced magnetosphere corresponding to the increasing branch of solar activity. This data allows to make an enhanced study of the magnetosphere variations as a response of the solar activity level. Since Mars Express has no onboard magnetometer, we used the hybrid models of the Martian plasma environment to get a proper frame to make an adequate statistics of the magnetospheric response. In this paper we present a spatial distribution of the planetary plasma in the planetary wake as well as the ionsospheric escape as a function of the solar activity.

  17. De Novo Herpes Simplex Virus VP16 Expression Gates a Dynamic Programmatic Transition and Sets the Latent/Lytic Balance during Acute Infection in Trigeminal Ganglia.

    PubMed

    Sawtell, Nancy M; Thompson, Richard L

    2016-09-01

    The life long relationship between herpes simplex virus and its host hinges on the ability of the virus to aggressively replicate in epithelial cells at the site of infection and transport into the nervous system through axons innervating the infection site. Interaction between the virus and the sensory neuron represents a pivot point where largely unknown mechanisms lead to a latent or a lytic infection in the neuron. Regulation at this pivot point is critical for balancing two objectives, efficient widespread seeding of the nervous system and host survival. By combining genetic and in vivo in approaches, our studies reveal that the balance between latent and lytic programs is a process occurring early in the trigeminal ganglion. Unexpectedly, activation of the latent program precedes entry into the lytic program by 12 -14hrs. Importantly, at the individual neuronal level, the lytic program begins as a transition out of this acute stage latent program and this escape from the default latent program is regulated by de novo VP16 expression. Our findings support a model in which regulated de novo VP16 expression in the neuron mediates entry into the lytic cycle during the earliest stages of virus infection in vivo. These findings support the hypothesis that the loose association of VP16 with the viral tegument combined with sensory axon length and transport mechanisms serve to limit arrival of virion associated VP16 into neuronal nuclei favoring latency. Further, our findings point to specialized features of the VP16 promoter that control the de novo expression of VP16 in neurons and this regulation is a key component in setting the balance between lytic and latent infections in the nervous system. PMID:27607440

  18. Triaxial MMG investigation during FES-induced cycle movements

    NASA Astrophysics Data System (ADS)

    Gelain, M. C.; Nogueira-Neto, G. N.; Nohama, P.; Gewehr, P. M.

    2016-04-01

    Spinal cord injury is damage to the spinal cord that results in loss of functions, causes muscle, joint and bone deficits, moreover it increases the muscle tone level. FEScycling is an alternative rehabilitation process to conserve the musculoskeletal system affected by the injury in the functional state. The aim of this study is to get the legs bilaterally synchronized by FES and compare MMG signal responses. The preliminary protocol consists of five consecutive contractions. The first and the last contractions were discarded and the three intermediate contractions were analyzed. A ratio between MMG values of muscle responses during propulsion and return of pedalling phases was calculated for each cycle and each leg. The applied FES profile consisted of pulse and burst on time of 250 us and 5 ms and frequencies of 1000 Hz and 20 Hz, respectively. Results showed that greater MMG RMS values tend to occur during the propulsion phase than in the return phase. MMG Z axis of RLL and MMG X axis of LLL were the axes that presented the greatest ratios. The MMG RMS signal showed that greater values tend to occur during the propulsion phase than in the return phase of the same thigh.

  19. MCAF1 and synergistic activation of the transcription of Epstein-Barr virus lytic genes by Rta and Zta.

    PubMed

    Chang, Li-Kwan; Chuang, Jian-Ying; Nakao, Mitsuyoshi; Liu, Shih-Tung

    2010-08-01

    Epstein-Barr virus (EBV) expresses two transcription factors, Rta and Zta, during the immediate-early stage of the lytic cycle. The two proteins often collaborate to activate the transcription of EBV lytic genes synergistically. This study demonstrates that Rta and Zta form a complex via an intermediary protein, MCAF1, on Zta response element (ZRE) in vitro. The interaction among these three proteins in P3HR1 cells is also verified via coimmunoprecipitation, CHIP analysis and confocal microscopy. The interaction between Rta and Zta in vitro depends on the region between amino acid 562 and 816 in MCAF1. In addition, overexpressing MCAF1 enhances and introducing MCAF1 siRNA into the cells markedly reduces the level of the synergistic activation in 293T cells. Moreover, the fact that the synergistic activation depends on ZRE but not on Rta response element (RRE) originates from the fact that Rta and Zta are capable of activating the BMRF1 promoter synergistically after an RRE but not ZREs in the promoter are mutated. The binding of Rta-MCAF1-Zta complex to ZRE but not RRE also explains why Rta and Zta do not use RRE to activate transcription synergistically. Importantly, this study elucidates the mechanism underlying synergistic activation, which is important to the lytic development of EBV. PMID:20385599

  20. Treatment-induced cell cycle kinetics dictate tumor response to chemotherapy

    PubMed Central

    Hallett, Robin M.; Huang, Cheng; Motazedian, Ali; Auf der Mauer, Stefanie; Pond, Gregory R.; Hassell, John A.; Nordon, Robert E.; Draper, Jonathan S.

    2015-01-01

    Chemotherapy fails to provide durable cure for the majority of cancer patients. To identify mechanisms associated with chemotherapy resistance, we identified genes differentially expressed before and after chemotherapeutic treatment of breast cancer patients. Treatment response resulted in either increased or decreased cell cycle gene expression. Tumors in which cell cycle gene expression was increased by chemotherapy were likely to be chemotherapy sensitive, whereas tumors in which cell cycle gene transcripts were decreased by chemotherapy were resistant to these agents. A gene expression signature that predicted these changes proved to be a robust and novel index that predicted the response of patients with breast, ovarian, and colon tumors to chemotherapy. Investigations in tumor cell lines supported these findings, and linked treatment induced cell cycle changes with p53 signaling and G1/G0 arrest. Hence, chemotherapy resistance, which can be predicted based on dynamics in cell cycle gene expression, is associated with TP53 integrity. PMID:25749523

  1. Chemotherapy Drug Induced Discoordination of Mitochondrial Life Cycle Detected by Cardiolipin Fluctuation.

    PubMed

    Chao, Yu-Jen; Chan, Jui-Fen; Hsu, Yuan-Hao Howard

    2016-01-01

    Chemotherapy drugs have been prescribed for the systemic treatment of cancer. We selected three chemotherapy drugs, including methotrexate, mitomycine C and vincristine to inhibit the proliferation of HT1080 human fibrosarcoma cells in S, G2 and M phases of the cell cycle respectively. These chemotherapy drugs showed significant toxicity and growth inhibition to the cancer cells measured by MTT assay. After treated with a 50% inhibitory dosage for 48 hours, these cancer cells showed significant accumulation of cardiolipin (CL), which was a reverse trend of the nutritional deficiency induced arrest at G1 phase. The quantity of each CL species was further semi-quantitated by HPLC-ion trap mass spectrometer. Methotraxate treatment caused unique increases of acyl chain length on CL, which were the opposite of the serum starvation, mitomycine C and vincristine treatments. Although mitomycine C and vincristine have different mechanisms to induce cell cycle arrest, these two drugs displayed similar effects on decreasing chain length of CL. Continuation of CL synthesis during cell cycle arrest indicated the chemotherapy drugs resulting in the discoordination of the mitochondrial life cycle from the cell cycle and thus caused the accumulation of CL. These finding reveals that the pre-remodeling nascent CL accumulates during the methotraxate induced arrest; however, the post-remodeling mature CL accumulates during the mitomycine C and vincristine induced arrest after the synthesis phase. PMID:27627658

  2. Cycling-Induced Changes in the Entropy Profiles of Lithium Cobalt Oxide Electrodes

    DOE PAGESBeta

    Hudak, N. S.; Davis, L. E.; Nagasubramanian, G.

    2014-12-09

    Entropy profiles of lithium cobalt oxide (LiCoO2) electrodes were measured at various stages in the cycle life to examine performance degradation and cycling-induced changes, or lack thereof, in thermodynamics. LiCoO2 electrodes were cycled at C/2 rate in half-cells (vs. lithium anodes) up to 20 cycles or C/5 rate in full cells (vs. MCMB anodes) up to 500 cycles. The electrodes were then subjected to entropy measurements (∂E/∂T, where E is open-circuit potential and T is temperature) in half-cells at regular intervals over the approximate range 0.5 ≤ x ≤ 1 in LixCoO2. Despite significant losses in capacity upon cycling, neithermore » cycling rate resulted in any change to the overall shape of the entropy profile relative to an uncycled electrode, indicating retention of the basic LiCoO2 structure, lithium insertion mechanism, and thermodynamics. This confirms that cycling-induced performance degradation in LiCoO2 electrodes is primarily caused by kinetic barriers that increase with cycling. In the case of electrodes cycled at C/5, there was a subtle, quantitative, and gradual change in the entropy profile in the narrow potential range of the hexagonal-to-monoclinic phase transition. The observed change is indicative of a decrease in the intralayer lithium ordering that occurs at these potentials, and it demonstrates that a cyclinginduced structural disorder accompanies the kinetic degradation mechanisms.« less

  3. Cycling-Induced Changes in the Entropy Profiles of Lithium Cobalt Oxide Electrodes

    SciTech Connect

    Hudak, N. S.; Davis, L. E.; Nagasubramanian, G.

    2014-12-09

    Entropy profiles of lithium cobalt oxide (LiCoO2) electrodes were measured at various stages in the cycle life to examine performance degradation and cycling-induced changes, or lack thereof, in thermodynamics. LiCoO2 electrodes were cycled at C/2 rate in half-cells (vs. lithium anodes) up to 20 cycles or C/5 rate in full cells (vs. MCMB anodes) up to 500 cycles. The electrodes were then subjected to entropy measurements (∂E/∂T, where E is open-circuit potential and T is temperature) in half-cells at regular intervals over the approximate range 0.5 ≤ x ≤ 1 in LixCoO2. Despite significant losses in capacity upon cycling, neither cycling rate resulted in any change to the overall shape of the entropy profile relative to an uncycled electrode, indicating retention of the basic LiCoO2 structure, lithium insertion mechanism, and thermodynamics. This confirms that cycling-induced performance degradation in LiCoO2 electrodes is primarily caused by kinetic barriers that increase with cycling. In the case of electrodes cycled at C/5, there was a subtle, quantitative, and gradual change in the entropy profile in the narrow potential range of the hexagonal-to-monoclinic phase transition. The observed change is indicative of a decrease in the intralayer lithium ordering that occurs at these potentials, and it demonstrates that a cyclinginduced structural disorder accompanies the kinetic degradation mechanisms.

  4. Members Only: Hypoxia-Induced Cell-Cycle Activation in Cardiomyocytes.

    PubMed

    Sharma, Arun; Wu, Sean M

    2015-09-01

    A low level of cardiomyocyte turnover occurs in the adult mammalian heart, but the source of these new cells remains unknown. Kimura et al., 2015 utilized a novel hypoxia-induced fate mapping system to identify a rare population of adult cardiomyocytes undergoing cell-cycle entry and expansion in healthy adult hearts and following ischemic injury. PMID:26331604

  5. Astaxanthin Inhibits Proliferation and Induces Apoptosis and Cell Cycle Arrest of Mice H22 Hepatoma Cells.

    PubMed

    Shao, Yiye; Ni, Yanbo; Yang, Jing; Lin, Xutao; Li, Jun; Zhang, Lixia

    2016-01-01

    BACKGROUND It is widely recognized that astaxanthin (ASX), a member of the carotenoid family, has strong biological activities including antioxidant, anti-inflammation, and immune-modulation activities. Previous studies have confirmed that ASX can effectively inhibit hepatoma cells in vitro. MATERIAL AND METHODS MTT was used to assay proliferation of mice H22 cells, and flow cytometry was used to determine apoptosis and cell cycle arrest of H22 cells in vitro and in vivo. Moreover, anti-tumor activity of ASX was observed in mice. RESULTS ASX inhibited the proliferation of H22 cells, promoted cell necrosis, and induced cell cycle arrest in G2 phase in vitro and in vivo. CONCLUSIONS This study indicated that ASX can inhibit proliferation and induce apoptosis and cell cycle arrest in mice H22 hepatoma cells in vitro and in vivo. PMID:27333866

  6. Astaxanthin Inhibits Proliferation and Induces Apoptosis and Cell Cycle Arrest of Mice H22 Hepatoma Cells

    PubMed Central

    Shao, Yiye; Ni, Yanbo; Yang, Jing; Lin, Xutao; Li, Jun; Zhang, Lixia

    2016-01-01

    Background It is widely recognized that astaxanthin (ASX), a member of the carotenoid family, has strong biological activities including antioxidant, anti-inflammation, and immune-modulation activities. Previous studies have confirmed that ASX can effectively inhibit hepatoma cells in vitro. Material/Methods MTT was used to assay proliferation of mice H22 cells, and flow cytometry was used to determine apoptosis and cell cycle arrest of H22 cells in vitro and in vivo. Moreover, anti-tumor activity of ASX was observed in mice. Results ASX inhibited the proliferation of H22 cells, promoted cell necrosis, and induced cell cycle arrest in G2 phase in vitro and in vivo. Conclusions This study indicated that ASX can inhibit proliferation and induce apoptosis and cell cycle arrest in mice H22 hepatoma cells in vitro and in vivo. PMID:27333866

  7. Cordycepin enhances Epstein-Barr virus lytic infection and Epstein-Barr virus-positive tumor treatment efficacy by doxorubicin.

    PubMed

    Du, Yinping; Yu, Jieshi; Du, Li; Tang, Jun; Feng, Wen-Hai

    2016-07-01

    The consistent latent presence of Epstein-Barr virus (EBV) in tumor cells offers potential for virus-targeted therapies. The switch from the latent form of EBV to the lytic form in tumor cells can lead to tumor cell lysis. In this study, we report that a natural small molecule compound, cordycepin, can induce lytic EBV infection in tumor cells. Subsequently, we demonstrate that cordycepin can enhance EBV reactivating capacity and EBV-positive tumor cell killing ability of low dose doxorubicin. The combination of cordycepin and doxorubicin phosphorylates CCAAT/enhancer binding protein β (C/EBPβ) through protein kinase C (PKC)-p38 mitogen activated protein kinases (p38 MAPK) signaling pathway, and C/EBPβ is required for the activation of lytic EBV infection. Most importantly, an in vivo experiment demonstrates that the combination of cordycepin and doxorubicin is more effective in inhibiting tumor growth in SCID mice than is doxorubicin alone. Our findings establish that cordycepin can enhance the efficacy of conventional chemotherapy for treatment of EBV-positive tumors. PMID:27063964

  8. Using Drosophila Larval Imaginal Discs to Study Low-Dose Radiation-Induced Cell Cycle Arrest

    PubMed Central

    Yan, Shian-Jang; Li, Willis X.

    2012-01-01

    Under genotoxic stress, activation of cell cycle checkpoint responses leads to cell cycle arrest, which allows cells to repair DNA damage before continuing to cycle. Drosophila larval epithelial sacs, called imaginal discs, are an excellent in vivo model system for studying radiation-induced cell cycle arrest. Larval imaginal discs go into cell cycle arrest after being subjected to low-dose irradiation, are subject to easy genetic manipulation, are not crucial for survival of the organism, and can be dissected easily for further molecular or cellular analysis. In this chapter, we describe methods for assessing low-dose irradiation-induced cell cycle arrest. Mitotic cells are identified by immunofluorescence staining for the mitotic marker phosphorylated histone H3 (phospho-histone H3 or pH3). When wandering third-instar control larvae, without transgene expression, are exposed to 500 rads of X-ray or γ-ray irradiation, the number of pH3-positive cells in wing imaginal discs is reduced from hundreds before irradiation to approximately 30 after irradiation, with an equal distribution between the anterior and posterior compartments (Yan et al., 2011, FASEB J). Using the GAL4/UAS system, RNAi, cDNA, or microRNA sponge transgenes can be expressed in the posterior compartment of the wing disc using drivers such as engrailed (en)-Gal4, while the anterior compartment serves as an internal control. This approach makes it possible to do genome-wide genetic screening for molecules involved in radiation-induced cell cycle arrest. PMID:21870287

  9. Using Drosophila larval imaginal discs to study low-dose radiation-induced cell cycle arrest.

    PubMed

    Yan, Shian-Jang; Li, Willis X

    2011-01-01

    Under genotoxic stress, activation of cell cycle checkpoint responses leads to cell cycle arrest, which allows cells to repair DNA damage before continuing to cycle. Drosophila larval epithelial sacs, called imaginal discs, are an excellent in vivo model system for studying radiation-induced cell cycle arrest. Larval imaginal discs go into cell cycle arrest after being subjected to low-dose irradiation, are subject to easy genetic manipulation, are not crucial for survival of the organism, and can be dissected easily for further molecular or cellular analysis. In this chapter, we describe methods for assessing low-dose irradiation-induced cell cycle arrest. Mitotic cells are identified by immunofluorescence staining for the mitotic marker phosphorylated histone H3 (phospho-histone H3 or pH3). When wandering third-instar control larvae, without transgene expression, are exposed to 500 rads of X-ray or γ-ray irradiation, the number of pH3-positive cells in wing imaginal discs is reduced from hundreds before irradiation to approximately 30 after irradiation, with an equal distribution between the anterior and posterior compartments (Yan et al., 2011, FASEB J). Using the GAL4/UAS system, RNAi, cDNA, or microRNA sponge transgenes can be expressed in the posterior compartment of the wing disc using drivers such as engrailed (en)-Gal4, while the anterior compartment serves as an internal control. This approach makes it possible to do genome-wide genetic screening for molecules involved in radiation-induced cell cycle arrest. PMID:21870287

  10. Polydatin inhibits growth of lung cancer cells by inducing apoptosis and causing cell cycle arrest.

    PubMed

    Zhang, Yusong; Zhuang, Zhixiang; Meng, Qinghui; Jiao, Yang; Xu, Jiaying; Fan, Saijun

    2014-01-01

    Polydatin (PD), a small natural compound from Polygonum cuspidatum, has a number of biological functions. However, the anticancer activity of PD has been poorly investigated. In the present study, thiazolyl blue tetrazolium bromide assay was used to evaluate the inhibitory effect of PD on cell growth. Cell cycle distribution and apoptosis were investigated by flow cytometry. In addition, the expression of several proteins associated with apoptosis and cell cycle were analyzed by western blot analysis. The results demonstrated that PD significantly inhibits the proliferation of A549 and NCI-H1975 lung cancer cell lines and causes dose-dependent apoptosis. Cell cycle analysis revealed that PD induces S phase cell cycle arrest. Western blot analysis showed that the expression of Bcl-2 decreased as that of Bax increased, and the expression of cyclin D1 was also suppressed. The results suggest that PD has potential therapeutic applications in the treatment of lung cancer. PMID:24348867

  11. Gravitational effects of process-induced dislocations in silicon. [during thermal cycling

    NASA Technical Reports Server (NTRS)

    Porter, W. A.; Parker, D. L.

    1974-01-01

    Matters pertaining to semiconductor device fabrication were studied in terms of the influence of gravity on the production of dislocations in silicon wafers during thermal cycling in a controlled ambient where no impurities are present and oxidation is minimal. Both n-type and p-type silicon wafers having a diameter of 1.25 in to 1.5 in, with fixed orientation and resistivity values, were used. The surface dislocation densities were measured quantitatively by the Sirtl etch technique. The results show two significant features of the plastic flow phenomenon as it is related to gravitational stress: (1) the density of dislocations generated during a given thermal cycle is directly related to the duration of the cycle; and (2) the duration of the thermal cycle required to produce a given dislocation density is inversely related to the equilibrium temperature. Analysis of the results indicates that gravitational stress is instrumental in process-induced defect generation.

  12. Dux4 induces cell cycle arrest at G1 phase through upregulation of p21 expression

    SciTech Connect

    Xu, Hongliang; Wang, Zhaoxia; Jin, Suqin; Hao, Hongjun; Zheng, Lemin; Zhou, Boda; Zhang, Wei; Lv, He; Yuan, Yun

    2014-03-28

    Highlights: • Dux4 induced TE671 cell proliferation defect and G1 phase arrest. • Dux4 upregulated p21 expression without activating p53. • Silencing p21 rescued Dux4 mediated proliferation defect and cell cycle arrest. • Sp1 binding site was required for Dux4-induced p21 promoter activation. - Abstract: It has been implicated that Dux4 plays crucial roles in development of facioscapulohumeral dystrophy. But the underlying myopathic mechanisms and related down-stream events of this retrogene were far from clear. Here, we reported that overexpression of Dux4 in a cell model TE671 reduced cell proliferation rate, and increased G1 phase accumulation. We also determined the impact of Dux4 on p53/p21 signal pathway, which controls the checkpoint in cell cycle progression. Overexpression of Dux4 increased p21 mRNA and protein level, while expression of p53, phospho-p53 remained unchanged. Silencing p21 rescued Dux4 mediated proliferation defect and cell cycle arrest. Furthermore, we demonstrated that enhanced Dux4 expression increased p21 promoter activity and elevated expression of Sp1 transcription factor. Mutation of Sp1 binding site decreased dux4 induced p21 promoter activation. Chromatin immunoprecipitation (ChIP) assays confirmed the Dux4-induced binding of Sp1 to p21 promoter in vivo. These results suggest that Dux4 might induce proliferation inhibition and G1 phase arrest through upregulation of p21.

  13. Difference of cell cycle arrests induced by lidamycin in human breast cancer cells.

    PubMed

    Liu, Xia; He, Hongwei; Feng, Yun; Zhang, Min; Ren, Kaihuan; Shao, Rongguang

    2006-02-01

    Lidamycin (LDM) is a member of the enediyne antibiotic family. It is undergoing phase I clinical trials in China as a potential chemotherapeutic agent. In the present study, we investigated the mechanism by which LDM induced cell cycle arrest in human breast cancer cells. The results showed that LDM induced G1 arrest in p53 wild-type MCF-7 cells at low concentrations, and caused both G1 and G2/M arrests at higher concentrations. In contrast, LDM induced only G2/M arrest in p53-mutant MCF-7/DOX cells. Western blotting analysis indicated that LDM-induced G1 and G2/M arrests in MCF-7 cells were associated with an increase of p53 and p21, and a decrease of phosphorylated retinoblastoma tumor suppressor protein, cyclin-dependent kinase (Cdk), Cdc2 and cyclin B1 protein levels. However, LDM-induced G2/M arrest in MCF-7/DOX cells was correlated with the reduction of cyclin B1 expression. Further study indicated that the downregulation of cyclin B1 by LDM in MCF-7 cells was associated with decreasing cyclin B1 mRNA levels and promoting protein degradation, whereas it was only due to inducing cyclin B1 protein degradation in MCF-7/DOX cells. In addition, activation of checkpoint kinases Chk1 or Chk2 maybe contributed to LDM-induced cell cycle arrest. Taken together, we provide the first evidence that LDM induces different cell cycle arrests in human breast cancer cells, which are dependent on drug concentration and p53 status. These findings are helpful in understanding the molecular anti-cancer mechanisms of LDM and support its clinical trials. PMID:16428935

  14. Impact of HIV-1 Membrane Cholesterol on Cell-Independent Lytic Inactivation and Cellular Infectivity.

    PubMed

    Kalyana Sundaram, Ramalingam Venkat; Li, Huiyuan; Bailey, Lauren; Rashad, Adel A; Aneja, Rachna; Weiss, Karl; Huynh, James; Bastian, Arangaserry Rosemary; Papazoglou, Elisabeth; Abrams, Cameron; Wrenn, Steven; Chaiken, Irwin

    2016-01-26

    Peptide triazole thiols (PTTs) have been found previously to bind to HIV-1 Env spike gp120 and cause irreversible virus inactivation by shedding gp120 and lytically releasing luminal capsid protein p24. Since the virions remain visually intact, lysis appears to occur via limited membrane destabilization. To better understand the PTT-triggered membrane transformation involved, we investigated the role of envelope cholesterol on p24 release by measuring the effect of cholesterol depletion using methyl beta-cyclodextrin (MβCD). An unexpected bell-shaped response of PTT-induced lysis to [MβCD] was observed, involving lysis enhancement at low [MβCD] vs loss of function at high [MβCD]. The impact of cholesterol depletion on PTT-induced lysis was reversed by adding exogenous cholesterol and other sterols that support membrane rafts, while sterols that do not support rafts induced only limited reversal. Cholesterol depletion appears to cause a reduced energy barrier to lysis as judged by decreased temperature dependence with MβCD. Enhancement/replenishment responses to [MβCD] also were observed for HIV-1 infectivity, consistent with a similar energy barrier effect in the membrane transformation of virus cell fusion. Overall, the results argue that cholesterol in the HIV-1 envelope is important for balancing virus stability and membrane transformation, and that partial depletion, while increasing infectivity, also makes the virus more fragile. The results also reinforce the argument that the lytic inactivation and infectivity processes are mechanistically related and that membrane transformations occurring during lysis can provide an experimental window to investigate membrane and protein factors important for HIV-1 cell entry. PMID:26713837

  15. Cell cycle re-entry sensitizes podocytes to injury induced death.

    PubMed

    Hagen, Manuel; Pfister, Eva; Kosel, Andrea; Shankland, Stuart; Pippin, Jeffrey; Amann, Kerstin; Daniel, Christoph

    2016-07-17

    Podocytes are terminally differentiated renal cells, lacking the ability to regenerate by proliferation. However, during renal injury, podocytes re-enter into the cell cycle but fail to divide. Earlier studies suggested that re-entry into cell cycle results in loss of podocytes, but a direct evidence for this is lacking. Therefore, we established an in vitro model to test the consequences of re-entry into the cell cycle on podocyte survival. A mouse immortalized podocyte cell line was differentiated to non-permissive podocytes and stimulated with e.g. growth factors. Stimulated cells were analyzed for mRNA-expression or stained for cell cycle analysis using flow cytometry and immunocytofluorescence microscopy. After stimulation to re-entry into cell cycle, podocytes were stressed with puromycin aminonucleoside (PAN) and analyzed for survival. During permissive stage more than 40% of immortalized podocytes were in the S-phase. In contrast, S-phase in non-permissive differentiated podocytes was reduced to 5%. Treatment with b-FGF dose dependently induced re-entry into cell cycle increasing the number of podocytes in the S-phase to 10.7% at an optimal bFGF dosage of 10 ng/ml. Forty eight hours after stimulation with bFGF the number of bi-nucleated podocytes significantly increased. A secondary injury stimulus significantly reduced podocyte survival preferentially in bi-nucleated podocytes In conclusion, stimulation of podocytes using bFGF was able to induce re-entry of podocytes into the cell cycle and to sensitize the cells for cell death by secondary injuries. Therefore, this model is appropriate for testing new podocyte protective substances that can be used for therapy. PMID:27232327

  16. Antirepression system associated with the life cycle switch in the temperate podoviridae phage SPC32H.

    PubMed

    Kim, Minsik; Ryu, Sangryeol

    2013-11-01

    Prophages switch from lysogenic to lytic mode in response to the host SOS response. The primary factor that governs this switch is a phage repressor, which is typically a host RecA-dependent autocleavable protein. Here, in an effort to reveal the mechanism underlying the phenotypic differences between the Salmonella temperate phages SPC32H and SPC32N, whose genome sequences differ by only two nucleotides, we identified a new class of Podoviridae phage lytic switch antirepressor that is structurally distinct from the previously reported Sipho- and Myoviridae phage antirepressors. The SPC32H repressor (Rep) is not cleaved by the SOS response but instead is inactivated by a small antirepressor (Ant), the expression of which is negatively controlled by host LexA. A single nucleotide mutation in the consensus sequence of the LexA-binding site, which overlaps with the ant promoter, results in constitutive Ant synthesis and consequently induces SPC32N to enter the lytic cycle. Numerous potential Ant homologues were identified in a variety of putative prophages and temperate Podoviridae phages, indicating that antirepressors may be widespread among temperate phages in the order Caudovirales to mediate a prudent prophage induction. PMID:23986584

  17. Antirepression System Associated with the Life Cycle Switch in the Temperate Podoviridae Phage SPC32H

    PubMed Central

    Kim, Minsik

    2013-01-01

    Prophages switch from lysogenic to lytic mode in response to the host SOS response. The primary factor that governs this switch is a phage repressor, which is typically a host RecA-dependent autocleavable protein. Here, in an effort to reveal the mechanism underlying the phenotypic differences between the Salmonella temperate phages SPC32H and SPC32N, whose genome sequences differ by only two nucleotides, we identified a new class of Podoviridae phage lytic switch antirepressor that is structurally distinct from the previously reported Sipho- and Myoviridae phage antirepressors. The SPC32H repressor (Rep) is not cleaved by the SOS response but instead is inactivated by a small antirepressor (Ant), the expression of which is negatively controlled by host LexA. A single nucleotide mutation in the consensus sequence of the LexA-binding site, which overlaps with the ant promoter, results in constitutive Ant synthesis and consequently induces SPC32N to enter the lytic cycle. Numerous potential Ant homologues were identified in a variety of putative prophages and temperate Podoviridae phages, indicating that antirepressors may be widespread among temperate phages in the order Caudovirales to mediate a prudent prophage induction. PMID:23986584

  18. Raman spectrum reveals the cell cycle arrest of Triptolide-induced leukemic T-lymphocytes apoptosis

    NASA Astrophysics Data System (ADS)

    Zhang, Daosen; Feng, Yanyan; Zhang, Qinnan; Su, Xin; Lu, Xiaoxu; Liu, Shengde; Zhong, Liyun

    2015-04-01

    Triptolide (TPL), a traditional Chinese medicine extract, possesses anti-inflammatory and anti-tumor properties. Though some research results have implicated that Triptolide (TPL) can be utilized in the treatment of leukemia, it remains controversial about the mechanism of TPL-induced leukemic T-lymphocytes apoptosis. In this study, combining Raman spectroscopic data, principal component analysis (PCA) and atomic force microscopy (AFM) imaging, both the biochemical changes and morphological changes during TPL-induced cell apoptosis were presented. In contrast, the corresponding data during Daunorubicin (DNR)-induced cell apoptosis was also exhibited. The obtained results showed that Raman spectral changes during TPL-induced cell apoptosis were greatly different from DNR-induced cell apoptosis in the early stage of apoptosis but revealed the high similarity in the late stage of apoptosis. Moreover, above Raman spectral changes were respectively consistent with the morphological changes of different stages during TPL-induced apoptosis or DNR-induced apoptosis, including membrane shrinkage and blebbing, chromatin condensation and the formation of apoptotic bodies. Importantly, it was found that Raman spectral changes with TPL-induced apoptosis or DNR-induced apoptosis were respectively related with the cell cycle G1 phase arrest or G1 and S phase arrest.

  19. Yet another way that phage λ manipulates its Escherichia coli host: λrexB is involved in the lysogenic-lytic switch.

    PubMed

    Engelberg-Kulka, Hanna; Kumar, Sathish

    2015-05-01

    The life cycle of phage λ has been studied extensively. Of particular interest has been the process leading to the decision of the phage to switch from lysogenic to lytic cycle. The principal participant in this process is the λcI repressor, which is cleaved under conditions of DNA damage. Cleaved λcI no longer acts as a repressor, allowing phage λ to switch from its lysogenic to lytic cycle. The well-known mechanism responsible for λcI cleavage is the SOS response. We have recently reported that the Escherichia coli toxin-antitoxin mazEF pathway inhibits the SOS response; in fact, the SOS response is permitted only in E. coli strains deficient in the expression of the mazEF pathway. Moreover, in strains lysogenic for prophage λ, the SOS response is enabled by the presence of λrexB. λRexB had previously been found to inhibit the degradation of the antitoxin MazE, thereby preventing the toxic action of MazF. Thus, phage λ rexB gene not only safeguards the prophage state by preventing death of its E. coli host but is also indirectly involved in the lysogenic-lytic switch. PMID:25684601

  20. The Arctic freshwater cycle during a naturally and an anthropogenically induced warm climate

    NASA Astrophysics Data System (ADS)

    Davies, Frazer J.; Renssen, Hans; Goosse, Hugues

    2014-04-01

    The Arctic freshwater cycle plays an important role in regulating regional and global climate. Current observations suggest that an intensification of the high-northern latitude hydrological cycle has caused a freshening of the Arctic and sub-Arctic seas, increasing the potential of weakening overturning strength in the Nordic seas, and reducing temperatures. It is not known if this freshening is a manifestation of the current anthropogenic warming and if the Arctic freshwater cycle has exhibited similar changes in the past, in particular as a response to naturally induced periods of warming, for example during the mid-Holocene hypsithermal. Thus, we have used an earth model of intermediate complexity, LOVECLIM, to investigate the response of the Arctic freshwater cycle, during two warm periods that evolved under different sets of forcings, the mid-Holocene and the twenty-first century. A combination of proxy reconstructions and modelling studies have shown these two periods to exhibit similar surface temperature anomalies, compared to the pre-industrial period, however, it has yet to be determined if the Arctic freshwater cycle and thus, the transport and redistribution of freshwater to the Arctic and the sub-Arctic seas, during these two warm periods, is comparable. Here we provide an overview that shows that the response of the Arctic freshwater cycle during the first half of the twenty-first century can be interpreted as an `extreme' mid-Holocene hydrological cycle. Whilst for the remainder of the twenty-first century, the Arctic freshwater cycle and the majority of its components will likely transition into what can only be described as truly anthropogenic in nature.

  1. Structure of the Bacteriophage [phi]KZ Lytic Transglycosylase gp144

    SciTech Connect

    Fokine, Andrei; Miroshnikov, Konstantin A.; Shneider, Mikhail M.; Mesyanzhinov, Vadim V.; Rossmann, Michael G.

    2008-04-02

    Lytic transglycosylases are enzymes that act on the peptidoglycan of bacterial cell walls. They cleave the glycosidic linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anhydromuramoyl product. The x-ray structure of the lytic transglycosylase gp144 from the Pseudomonas bacteriophage {phi}KZ has been determined to 2.5-{angstrom} resolution. This protein is probably employed by the bacteriophage in the late stage of the virus reproduction cycle to destroy the bacterial cell wall to release the phage progeny. {phi}KZ gp144 is a 260-residue {alpha}-helical protein composed of a 70-residue N-terminal cell wall-binding domain and a C-terminal catalytic domain. The fold of the N-terminal domain is similar to the peptidoglycan-binding domain from Streptomyces albus G d-Ala-d-Ala carboxypeptidase and to the N-terminal prodomain of human metalloproteinases that act on extracellular matrices. The C-terminal catalytic domain of gp144 has a structural similarity to the catalytic domain of the transglycosylase Slt70 from Escherichia coli and to lysozymes. The gp144 catalytic domain has an elongated groove that can bind at least five sugar residues at sites A-E. As in other lysozymes, the peptidoglycan cleavage (catalyzed by Glu{sup 115} in gp144) occurs between sugar-binding subsites D and E. The x-ray structure of the {phi}KZ transglycosylase complexed with the chitotetraose (N-acetylglucosamine){sub 4} has been determined to 2.6-{angstrom} resolution. The N-acetylglucosamine residues of the chitotetraose bind in sites A-D.

  2. Evidence for climate variations induced by the 11-year solar and cosmic rays cycles

    NASA Astrophysics Data System (ADS)

    Bruckman, William; Ramos, Elio

    2010-02-01

    We analyzed data from PSMSL monthly mean sea level seeking correlations between sea level fluctuations and the solar and cosmic rays 11 year cycle. The data reveals decadal variability that could be causally connected to the solar and cosmic rays cycle, since these periodic changes are correlated. It is also found that the solar (cosmic rays) cycle correlates (anti-correlates) with the mean global surface temperature anomaly. A probable explanation of the above correlations is that the solar intensity and cosmic rays variations induce oscillations in the average temperature and precipitation, with corresponding changes in the continental water and snow accumulation. Thus, for instance, a higher than average snow and water over land, and lower temperatures produce oceans thermal contraction and lower mass, implicating lower mean sea level.

  3. The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases.

    PubMed

    Frandsen, Kristian E H; Simmons, Thomas J; Dupree, Paul; Poulsen, Jens-Christian N; Hemsworth, Glyn R; Ciano, Luisa; Johnston, Esther M; Tovborg, Morten; Johansen, Katja S; von Freiesleben, Pernille; Marmuse, Laurence; Fort, Sébastien; Cottaz, Sylvain; Driguez, Hugues; Henrissat, Bernard; Lenfant, Nicolas; Tuna, Floriana; Baldansuren, Amgalanbaatar; Davies, Gideon J; Lo Leggio, Leila; Walton, Paul H

    2016-04-01

    Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that oxidatively break down recalcitrant polysaccharides such as cellulose and chitin. Since their discovery, LPMOs have become integral factors in the industrial utilization of biomass, especially in the sustainable generation of cellulosic bioethanol. We report here a structural determination of an LPMO-oligosaccharide complex, yielding detailed insights into the mechanism of action of these enzymes. Using a combination of structure and electron paramagnetic resonance spectroscopy, we reveal the means by which LPMOs interact with saccharide substrates. We further uncover electronic and structural features of the enzyme active site, showing how LPMOs orchestrate the reaction of oxygen with polysaccharide chains. PMID:26928935

  4. On the catalytic mechanisms of lytic polysaccharide monooxygenases.

    PubMed

    Walton, Paul H; Davies, Gideon J

    2016-04-01

    Lytic polysaccharide monooxygenases (LPMOs) are recently discovered copper-containing oxygenases. LPMOs oxidise recalcitrant polysaccharides such as chitin and cellulose, thereby making these substrates more tractable to canonical chitinase or cellulase action. As such, LPMOs are attracting much attention not only for their capacity to greatly increase the efficiency of production of cellulosic-based biofuels, but also for the new questions they pose about the mechanisms of biological oxidation of recalcitrant substrates. This review draws together the current thinking on the catalytic mechanisms of LPMOs and other copper catalysed oxygenations and provides a blueprint for further investigation into the mechanisms of action of these intriguing enzymes. PMID:27094791

  5. Cell cycle control in isoproterenol-induced murine salivary acinar cell proliferation.

    PubMed

    Zeng, T; Yamamoto, H; Bowen, E; Broverman, R L; Nguyen, K H; Humphreys-Beher, M G

    1996-11-01

    The eukaryotic cell cycle is a summary of a complex network of signal transduction pathways resulting in both DNA replication and cell division. Cyclin-dependent kinases (CDKs) control the cell cycle in all eukaryotes, whereas other proteins, known as cyclins, act as their regulatory subunits. Chronic injection with isoproterenol (ISO) can induce acinar cell proliferation in rodent salivary glands. Cyclins and CDK proteins from control and ISO-treated murine parotid acinar cells were detected by using Western blotting techniques. By comparing the expression of these cell cycle regulatory kinases in the parotid acinar cell transition from a quiescent state to a hypertrophic state, we found rapid increases in the protein levels of all CDKs, cyclin D and proliferating cell nuclear antigen (PCNA). The highest protein levels for CDKs and cyclins appeared at about 72 hr of ISO stimulation and were coincident with the highest rate of increase in gland wet weight. After 72 hr, the increase of both cell cycle protein and gland wet weight began to subside. By using a co-immunoprecipitation method, the following cell cycle regulators (CDK-cyclin complexes) were detected, CDK4-cyclin D, CDK2-cyclin E, CDK2-cyclin A, and cdc2-cyclin B, along with an increase in kinase activity over control untreated animals. Additionally, we detected significant decreases in the newly isolated CDK inhibitor (CKI) p27kip but not Wee 1 kinase. The increased levels of CKI correlated with a decrease in kinase activity of CDK/cyclin complexes by 144 hr of chronic isoproterenol treatment. Our data suggest that the holoenzymes for cell cycle control (cyclin-CDK complexes) function as a final regulatory mechanism leading to salivary gland acinar cell proliferation. The gradual decline in protein levels of the CDKs and cyclins after 3 days of chronic treatment further indicates that ISO-induced proliferation of parotid acinar cells is self-limiting and non-tumorigenic. PMID:9375366

  6. PEA15 Regulates the DNA Damage-Induced Cell Cycle Checkpoint and Oncogene-Directed Transformation

    PubMed Central

    Nagarajan, Arvindhan; Dogra, Shaillay Kumar; Liu, Alex Y.; Green, Michael R.

    2014-01-01

    Regulation of the DNA damage response and cell cycle progression is critical for maintaining genome integrity. Here, we report that in response to DNA damage, COPS5 deubiquitinates and stabilizes PEA15 in an ATM kinase-dependent manner. PEA15 expression oscillates throughout the cell cycle, and the loss of PEA15 accelerates cell cycle progression by activating CDK6 expression via the c-JUN transcription factor. Cells lacking PEA15 exhibit a DNA damage-induced G2/M checkpoint defect due to increased CDC25C activity and, consequentially, higher cyclin-dependent kinase 1 (CDK1)/cyclin B activity, and accordingly they have an increased rate of spontaneous mutagenesis. We find that oncogenic RAS inhibits PEA15 expression and that ectopic PEA15 expression blocks RAS-mediated transformation, which can be partially rescued by ectopic expression of CDK6. Finally, we show that PEA15 expression is downregulated in colon, breast, and lung cancer samples. Collectively, our results demonstrate that tumor suppressor PEA15 is a regulator of genome integrity and is an integral component of the DNA damage response pathway that regulates cell cycle progression, the DNA-damage-induced G2/M checkpoint, and cellular transformation. PMID:24710276

  7. Suppression of the antiferroelectric phase during polarization cycling of an induced ferroelectric phase

    SciTech Connect

    Liu, Xiaoming; Tan, Xiaoli

    2015-08-17

    The ceramic Pb{sub 0.99}Nb{sub 0.02}[(Zr{sub 0.57}Sn{sub 0.43}){sub 0.92}Ti{sub 0.08}]{sub 0.98}O{sub 3} can exist in either an antiferroelectric or a ferroelectric phase at room temperature, depending on the thermal and electrical history. The antiferroelectric phase can be partially recovered from the induced ferroelectric phase when the applied field reverses polarity. Therefore, polarization cycling of the ferroelectric phase in the ceramic under bipolar fields at room temperature is accompanied with repeated phase transitions. In this letter, the stability of the recovered antiferroelectric phase upon electrical cycling of the ceramic is investigated. Ex-situ X-ray diffraction reveals that bipolar cycling suppresses the antiferroelectric phase; this is indirectly supported by piezoelectric coefficient d{sub 33} measurements. It is speculated that the accumulated charged point defects during polarization cycling stabilize the polar ferroelectric phase. The findings presented are important to the fundamental studies of electric fatigue and field-induced phase transitions in ferroelectrics.

  8. PEA15 regulates the DNA damage-induced cell cycle checkpoint and oncogene-directed transformation.

    PubMed

    Nagarajan, Arvindhan; Dogra, Shaillay Kumar; Liu, Alex Y; Green, Michael R; Wajapeyee, Narendra

    2014-06-01

    Regulation of the DNA damage response and cell cycle progression is critical for maintaining genome integrity. Here, we report that in response to DNA damage, COPS5 deubiquitinates and stabilizes PEA15 in an ATM kinase-dependent manner. PEA15 expression oscillates throughout the cell cycle, and the loss of PEA15 accelerates cell cycle progression by activating CDK6 expression via the c-JUN transcription factor. Cells lacking PEA15 exhibit a DNA damage-induced G2/M checkpoint defect due to increased CDC25C activity and, consequentially, higher cyclin-dependent kinase 1 (CDK1)/cyclin B activity, and accordingly they have an increased rate of spontaneous mutagenesis. We find that oncogenic RAS inhibits PEA15 expression and that ectopic PEA15 expression blocks RAS-mediated transformation, which can be partially rescued by ectopic expression of CDK6. Finally, we show that PEA15 expression is downregulated in colon, breast, and lung cancer samples. Collectively, our results demonstrate that tumor suppressor PEA15 is a regulator of genome integrity and is an integral component of the DNA damage response pathway that regulates cell cycle progression, the DNA-damage-induced G2/M checkpoint, and cellular transformation. PMID:24710276

  9. Hydrogen cycling-induced phase segregation in AB{sub 5}-type intermetallics

    SciTech Connect

    Mordkovich, V.Z.

    1998-12-31

    A study of hydrogen cycling-induced phase segregation in AB{sub 5}-type intermetallics is presented. Influence of temperature for the alloy compositions YNi{sub 4}Al, LaNi{sub 5}, Ce{sub 0.5}La{sub 0.5}Ni{sub 5} and Ce{sub 0.7}La{sub 0.3}Ni{sub 5} is shown. Morphology of the nanostructured particles formed is studied and discussed.

  10. Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) contains hypoxia response elements: relevance to lytic induction by hypoxia.

    PubMed

    Haque, Muzammel; Davis, David A; Wang, Victoria; Widmer, Isabelle; Yarchoan, Robert

    2003-06-01

    Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), also known as human herpesvirus 8, is an etiologic agent of KS, primary effusion lymphoma (PEL), and multicentric Castleman's disease. We recently demonstrated that hypoxia can induce lytic replication of KSHV in PEL cell lines. Hypoxia induces the accumulation of hypoxia-inducible factors (HIF), and we hypothesized that the KSHV genome may respond to hypoxia through functional hypoxia response elements (HREs). Here, we demonstrate the presence of at least two promoters within the KSHV genome that are activated by hypoxia or hypoxia mimics. One is in the promoter region of the gene for Rta, the main lytic switch gene, and the other is within the promoter region of ORF34, a lytic gene of unknown function. The ORF34 promoter contains three putative consensus HREs oriented in the direction of the gene. Dissection and site-directed mutagenesis studies confirmed that one of the HREs of the ORF34 promoter is functional. Under conditions of hypoxia, the ORF34 promoter was strongly upregulated by HIF-1 alpha and HIF-2 alpha. By contrast, the promoter of the gene for Rta appeared to be preferentially upregulated by HIF-2 alpha. Reverse transcription-PCR analysis revealed that specific messages for ORF34 and ORF50 are upregulated in BCBL-1 cells exposed to hypoxia. An HIF-1 binding and competition assay demonstrated that the HRE sequence from the ORF34 promoter can compete for HIF-1 alpha binding to an erythropoietin HRE oligonucleotide while a mutant sequence cannot. Thus, we demonstrated that a viral gene can be activated by hypoxia through activation of a functional viral HRE. To our knowledge, this is the first example of a functional HRE in a viral promoter. PMID:12767996

  11. Behavior and Properties of Mature Lytic Granules at the Immunological Synapse of Human Cytotoxic T Lymphocytes

    PubMed Central

    Ming, Min; Schirra, Claudia; Becherer, Ute; Stevens, David R.; Rettig, Jens

    2015-01-01

    Killing of virally infected cells or tumor cells by cytotoxic T lymphocytes requires targeting of lytic granules to the junction between the CTL and its target. We used whole-cell patch clamp to measure the cell capacitance at fixed intracellular [Ca2+] to study fusion of lytic granules in human CTLs. Expression of a fluorescently labeled human granzyme B construct allowed identification of lytic granule fusion using total internal reflection fluorescence microscopy. In this way capacitance steps due to lytic granule fusion were identified. Our goal was to determine the size of fusing lytic granules and to describe their behavior at the plasma membrane. On average, 5.02 ± 3.09 (mean ± s.d.) lytic granules were released per CTL. The amplitude of lytic granule fusion events was ~ 3.3 fF consistent with a diameter of about 325 nm. Fusion latency was biphasic with time constants of 15.9 and 106 seconds. The dwell time of fusing lytic granules was exponentially distributed with a mean dwell time of 28.5 seconds. Fusion ended in spite of the continued presence of granules at the immune synapse. The mobility of fusing granules at the membrane was indistinguishable from that of lytic granules which failed to fuse. While dwelling at the plasma membrane lytic granules exhibit mobility consistent with docking interspersed with short periods of greater mobility. The failure of lytic granules to fuse when visible in TIRF at the membrane may indicate that a membrane-confined reaction is rate limiting. PMID:26296096

  12. Salidroside induces cell-cycle arrest and apoptosis in human breast cancer cells

    SciTech Connect

    Hu, Xiaolan; Zhang, Xianqi; Qiu, Shuifeng; Yu, Daihua; Lin, Shuxin

    2010-07-16

    Research highlights: {yields} Salidroside inhibits the growth of human breast cancer cells. {yields} Salidroside induces cell-cycle arrest of human breast cancer cells. {yields} Salidroside induces apoptosis of human breast cancer cell lines. -- Abstract: Recently, salidroside (p-hydroxyphenethyl-{beta}-D-glucoside) has been identified as one of the most potent compounds isolated from plants of the Rhodiola genus used widely in traditional Chinese medicine, but pharmacokinetic data on the compound are unavailable. We were the first to report the cytotoxic effects of salidroside on cancer cell lines derived from different tissues, and we found that human breast cancer MDA-MB-231 cells (estrogen receptor negative) were sensitive to the inhibitory action of low-concentration salidroside. To further investigate the cytotoxic effects of salidroside on breast cancer cells and reveal possible ER-related differences in response to salidroside, we used MDA-MB-231 cells and MCF-7 cells (estrogen receptor-positive) as models to study possible molecular mechanisms; we evaluated the effects of salidroside on cell growth characteristics, such as proliferation, cell cycle duration, and apoptosis, and on the expression of apoptosis-related molecules. Our results demonstrated for the first time that salidroside induces cell-cycle arrest and apoptosis in human breast cancer cells and may be a promising candidate for breast cancer treatment.

  13. Isolation and Characterization of Lytic Properties of Bacteriophages Specific for M. haemolytica Strains

    PubMed Central

    Urban-Chmiel, Renata; Wernicki, Andrzej; Stęgierska, Diana; Dec, Marta; Dudzic, Anna; Puchalski, Andrzej

    2015-01-01

    Aim of Study The objective of this study was isolation and morphological characterization of temperate bacteriophages obtained from M. haemolytica strains and evaluation of their lytic properties in vitro against M. haemolytica isolated from the respiratory tract of calves. Material and Methods The material for the study consisted of the reference strain M. haemolytica serotype 1 (ATCC®) BAA-410™, reference serotypes A1, A2, A5, A6, A7, A9 and A11, and wild-type isolates of M. haemolytica. Bacteriophages were induced from an overnight bacterial starter culture of all examined M. haemolytica strains treated with mitomycin C. The lytic properties and host ranges were determined by plaque assays. The morphology of the bacteriophages was examined in negative-stained smears with 5% uranyl acetate solution using a transmission electron microscope. The genetic analysis of the bacteriophages was followed by restriction analysis of bacteriophage DNA. This was followed by analysis of genetic material by polymerase chain reaction (PCR). Results Eight bacteriophages were obtained, like typical of the families Myoviridae, Siphoviridae and Podoviridae. Most of the bacteriophages exhibited lytic properties against the M. haemolytica strains. Restriction analysis revealed similarities to the P2-like phage obtained from the strain M. haemolytica BAA-410. The most similar profiles were observed in the case of bacteriophages φA1 and φA5. All of the bacteriophages obtained were characterized by the presence of additional fragments in the restriction profiles with respect to the P2-like reference phage. In the analysis of PCR products for the P2-like reference phage phi-MhaA1-PHL101 (DQ426904) and the phages of the M. haemolytica serotypes, a 734-bp phage PCR product was obtained. The primers were programmed in Primer-Blast software using the structure of the sequence DQ426904 of reference phage PHL101. Conclusions The results obtained indicate the need for further research aimed

  14. Pfaffosidic Fraction from Hebanthe paniculata Induces Cell Cycle Arrest and Caspase-3-Induced Apoptosis in HepG2 Cells

    PubMed Central

    da Silva, Tereza Cristina; Cogliati, Bruno; Latorre, Andréia Oliveira; Akisue, Gokithi; Nagamine, Márcia Kazumi; Haraguchi, Mitsue; Hansen, Daiane; Sanches, Daniel Soares; Dagli, Maria Lúcia Zaidan

    2015-01-01

    Hebanthe paniculata roots (formerly Pfaffia paniculata and popularly known as Brazilian ginseng) show antineoplastic, chemopreventive, and antiproliferative properties. Functional properties of these roots and their extracts are usually attributed to the pfaffosidic fraction, which is composed mainly by pfaffosides A–F. However, the therapeutic potential of this fraction in cancer cells is not yet entirely understood. This study aimed to analyze the antitumoral effects of the purified pfaffosidic fraction or saponinic fraction on the human hepatocellular carcinoma HepG2 cell line. Cellular viability, proliferation, and apoptosis were evaluated, respectively, by MTT assay, BrdU incorporation, activated caspase-3 immunocytochemistry, and DNA fragmentation assay. Cell cycle was analyzed by flow cytometry and the cell cycle-related proteins were analyzed by quantitative PCR and Western blot. The cells exposed to pfaffosidic fraction had reduced viability and cellular growth, induced G2/M at 48 h or S at 72 h arrest, and increased sub-G1 cell population via cyclin E downregulation, p27KIP1 overexpression, and caspase-3-induced apoptosis, without affecting the DNA integrity. Antitumoral effects of pfaffosidic fraction from H. paniculata in HepG2 cells originated by multimechanisms of action might be associated with cell cycle arrest in the S phase, by CDK2 and cyclin E downregulation and p27KIP1 overexpression, besides induction of apoptosis through caspase-3 activation. PMID:26075002

  15. A virally encoded small peptide regulates RTA stability and facilitates Kaposi's sarcoma-associated herpesvirus lytic replication.

    PubMed

    Jaber, Tareq; Yuan, Yan

    2013-03-01

    In both mammalian and viral genomes, a large proportion of sequences are transcribed and annotated as noncoding RNAs. A polyadenylated RNA of 3.0 kb (T3.0) is transcribed from the opposite strand of the open reading frame 50 (ORF50) DNA template in the genome of Kaposi's sarcoma-associated herpesvirus (KSHV) and has been annotated previously as a noncoding RNA. ORF50 encodes the replication and transcription activator (RTA), which controls the switch of the virus between the latent and lytic phases of the life cycle. Here we show that T3.0 encodes a small peptide of 48 amino acids (designated viral small peptide 1 [vSP-1]). vSP-1 interacts with RTA at the protein abundance regulatory signal (PARS) motifs, and the association prevents RTA from being subjected to degradation through the ubiquitin-proteasome pathway. As a consequence, vSP-1 facilitates KSHV gene expression and lytic replication. This finding reveals a novel mechanism of gene regulation in the viral life cycle. PMID:23302891

  16. Sialomucin and lytic susceptibility of rat mammary tumor ascites cells.

    PubMed

    Moriarty, J; Skelly, C M; Bharathan, S; Moody, C E; Sherblom, A P

    1990-11-01

    The potential role of cell surface sialomucin in preventing natural killer (NK)-mediated lysis of tumor cell targets has been addressed by comparing the properties of 2 NK-resistant [ascites (ASC) and short-term cultured (STC)] and 2 NK-susceptible [tunicamycin-treated (TUN) and long-term cultured (LTC)] preparations of 13762 MAT-B1 rat mammary tumor cells. Both the ASC and STC cell preparations contain elevated levels of the sialomucin ASGP-1 relative to TUN and LTC preparations as determined by [3H]glucosamine labeling and by binding of peanut agglutinin. The major difference in the susceptibility to NK-mediated lysis appeared to be due to the differences in the susceptibility to lysis by lytic granules, rather than to differences in the ability to bind or trigger effector cells, since TUN and LTC cells were approximately 10-fold more sensitive to lysis by lytic granules than were ASC and STC cells. All preparations inhibited the lysis of the susceptible target YAC-1 by normal rat splenocytes, indicating an ability to bind these effector cells. Triggering of effectors, as monitored either by incorporation of 32P into phosphatidylinositol or by transmethylation of phosphatidylcholine, was similar for the positive control YAC-1, STC, TUN, and LTC, whereas ASC appeared to be defective in triggering effectors. These results suggest that tumor sialomucin blocks the final phase of lysis, but not the initial recognition of tumor cells by NK effectors. PMID:2208144

  17. LHRH-Conjugated Lytic Peptides Directly Target Prostate Cancer Cells

    PubMed Central

    Yates, Clayton; Sharp, Starlette; Jones, Jacqueline; Topps, Daphne; Coleman, Mathew; Aneja, Ritu; Jaynes, Jesse; Turner, Timothy

    2010-01-01

    Prostate cancer is the second leading cause of cancer deaths among men. For patients with hormone-refractory disease, few treatments are available once the tumor has metastasized beyond the prostate. In the present study, two conjugated lytic peptide sequences (named JCHLHRH and JC21LHRH) were designed to target luteinizing hormone-releasing hormone receptors (LHRH-R). Our results indicate that human prostate cancer cell lines were sensitive to both LHRH-conjugated and non-conjugated lytic peptides, with IC50 concentrations for LNCaP cells, 4.4 and 9.1 µM; for DU-145 cells, 4.8 and 5.7 µM; and for PC-3 cells, 4.4 and 8.2 µM, respectively. JCHLHRH and JC21LHRH were nontoxic to normal primary human prostate epithelial cells or to bone marrow stromal cells in co-culture. There were morphological changes in PC-3 cells after 3 hr of exposure to either peptide; after 6 hr, there were significant reductions in cell numbers. Exposure of PC-3 cells for 24 hr to either JCHLHRH or JC21LHRH blocked their growth over 3 days. Since JCHLHRH and JC21LHRH have specificity for and anti-proliferative activity against tumor cells, and low toxicity for normal prostate cells, these peptides could serve as a new type of therapy for prostate cancer. PMID:20869347

  18. Molecular Basis for Lytic Bacteriophage Resistance in Enterococci

    PubMed Central

    Duerkop, Breck A.; Huo, Wenwen; Bhardwaj, Pooja

    2016-01-01

    ABSTRACT The human intestine harbors diverse communities of bacteria and bacteriophages. Given the specificity of phages for their bacterial hosts, there is growing interest in using phage therapies to combat the rising incidence of multidrug-resistant bacterial infections. A significant barrier to such therapies is the rapid development of phage-resistant bacteria, highlighting the need to understand how bacteria acquire phage resistance in vivo. Here we identify novel lytic phages in municipal raw sewage that kill Enterococcus faecalis, a Gram-positive opportunistic pathogen that resides in the human intestine. We show that phage infection of E. faecalis requires a predicted integral membrane protein that we have named PIPEF (for phage infection protein from E. faecalis). We find that PIPEF is conserved in E. faecalis and harbors a 160-amino-acid hypervariable region that determines phage tropism for distinct enterococcal strains. Finally, we use a gnotobiotic mouse model of in vivo phage predation to show that the sewage phages temporarily reduce E. faecalis colonization of the intestine but that E. faecalis acquires phage resistance through mutations in PIPEF. Our findings define the molecular basis for an evolutionary arms race between E. faecalis and the lytic phages that prey on them. They also suggest approaches for engineering E. faecalis phages that have altered host specificity and that can subvert phage resistance in the host bacteria. PMID:27578757

  19. THE GENOME SEQUENCE OF BACTERIOPHAGE CpV1 LYTIC FOR CLOSTRIDIUM PERFRINGENS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Application of bacteriophages and their lytic enzymes to control Clostri-dium perfringens is one potential approach to reduce the pathogen on poultry farms and in poultry-processing facilities. We have established a collection of 30 bacteriophages lytic for C. perfringens. These were isolated from s...

  20. A Stress-Induced Small RNA Modulates Alpha-Rhizobial Cell Cycle Progression

    PubMed Central

    Robledo, Marta; Frage, Benjamin; Wright, Patrick R.; Becker, Anke

    2015-01-01

    Mechanisms adjusting replication initiation and cell cycle progression in response to environmental conditions are crucial for microbial survival. Functional characterization of the trans-encoded small non-coding RNA (trans-sRNA) EcpR1 in the plant-symbiotic alpha-proteobacterium Sinorhizobium meliloti revealed a role of this class of riboregulators in modulation of cell cycle regulation. EcpR1 is broadly conserved in at least five families of the Rhizobiales and is predicted to form a stable structure with two defined stem-loop domains. In S. meliloti, this trans-sRNA is encoded downstream of the divK-pleD operon. ecpR1 belongs to the stringent response regulon, and its expression was induced by various stress factors and in stationary phase. Induced EcpR1 overproduction led to cell elongation and increased DNA content, while deletion of ecpR1 resulted in reduced competitiveness. Computationally predicted EcpR1 targets were enriched with cell cycle-related mRNAs. Post-transcriptional repression of the cell cycle key regulatory genes gcrA and dnaA mediated by mRNA base-pairing with the strongly conserved loop 1 of EcpR1 was experimentally confirmed by two-plasmid differential gene expression assays and compensatory changes in sRNA and mRNA. Evidence is presented for EcpR1 promoting RNase E-dependent degradation of the dnaA mRNA. We propose that EcpR1 contributes to modulation of cell cycle regulation under detrimental conditions. PMID:25923724

  1. Sonoporation induces apoptosis and cell cycle arrest in human promyelocytic leukemia cells.

    PubMed

    Zhong, Wenjing; Sit, Wai Hung; Wan, Jennifer M F; Yu, Alfred C H

    2011-12-01

    Despite being a transient biophysical phenomenon, sonoporation is known to disturb the homeostasis of living cells. This work presents new evidence on how sonoporation may lead to antiproliferation effects including cell-cycle arrest and apoptosis through disrupting various cell signaling pathways. Our findings were obtained from sonoporation experiments conducted on HL-60 human promyelocytic leukemia cells (with 1% v/v microbubbles; 1 MHz ultrasound; 0.3 or 0.5MPa peak negative pressure; 10% duty cycle; 1 kHz pulse repetition frequency; 1 min exposure period). Membrane resealing in these sonoporated cells was first verified using scanning electron microscopy. Time-lapse flow cytometry analysis of cellular deoxyribonucleic acid (DNA) contents was then performed at four post-sonoporation time points (4 h, 8 h, 12 h and 24 h). Results indicate that an increasing trend in the apoptotic cell population can be observed for at least 12 h after sonoporation, whilst viable sonoporated cells are found to temporarily accumulate in the G(2)/M (gap-2/mitosis) phase of the cell cycle. Further analysis using western blotting reveals that sonoporation-induced apoptosis involves cleavage of poly adenosine diphosphate ribose polymerase (PARP) proteins: a pro-apoptotic hallmark related to loss of DNA repair functionality. Also, mitochondrial signaling seems to have taken part in triggering this cellular event as the expression of two complementary regulators for mitochondrial release of pro-apoptotic molecules, Bcl-2 (B-cell lymphoma 2) and Bax (Bcl-2-associated X), are seen to be imbalanced in sonoporated cells. Furthermore, sonoporation is found to induce cell-cycle arrest through perturbing the expression of various cyclin and Cdk (cyclin-dependent kinase) checkpoint proteins that play an enabling role in cell-cycle progression. These bioeffects should be taken into account when using sonoporation for therapeutic purposes. PMID:22033133

  2. Contributions of Four Cortex Lytic Enzymes to Germination of Bacillus anthracis Spores▿

    PubMed Central

    Heffron, Jared D.; Lambert, Emily A.; Sherry, Nora; Popham, David L.

    2010-01-01

    Bacterial spores remain dormant and highly resistant to environmental stress until they germinate. Completion of germination requires the degradation of spore cortex peptidoglycan by germination-specific lytic enzymes (GSLEs). Bacillus anthracis has four GSLEs: CwlJ1, CwlJ2, SleB, and SleL. In this study, the cooperative action of all four GSLEs in vivo was investigated by combining in-frame deletion mutations to generate all possible double, triple, and quadruple GSLE mutant strains. Analyses of mutant strains during spore germination and outgrowth combined observations of optical density loss, colony-producing ability, and quantitative identification of spore cortex fragments. The lytic transglycosylase SleB alone can facilitate enough digestion to allow full spore viability and generates a variety of small and large cortex fragments. CwlJ1 is also sufficient to allow completion of nutrient-triggered germination independently and is a major factor in Ca2+-dipicolinic acid (DPA)-triggered germination, but its enzymatic activity remains unidentified because its products are large and not readily released from the spore's integuments. CwlJ2 contributes the least to overall cortex digestion but plays a subsidiary role in Ca2+-DPA-induced germination. SleL is an N-acetylglucosaminidase that plays the major role in hydrolyzing the large products of other GSLEs into small, rapidly released muropeptides. As the roles of these enzymes in cortex degradation become clearer, they will be targets for methods to stimulate premature germination of B. anthracis spores, greatly simplifying decontamination measures. PMID:19966006

  3. The diurnal and solar cycle variation of the charge exchange induced hydrogen escape flux

    NASA Technical Reports Server (NTRS)

    Maher, L. J.; Tinsley, B. A.

    1978-01-01

    On the basis of ion temperature and density data at specific points and times in June 1969 provided by the OGO 6 satellite, and altitude profiles of the ion and electron temperature and concentration provided by the Arecibo radar facility over the period February 1972-April 1974, the diurnal and solar cycle variation of the charge-exchange-induced hydrogen escape flux was investigated. It was calculated that for low to moderate solar activity at Arecibo, the diurnal ratio of the maximum-to-minimum charge-exchange-induced hydrogen escape flux was approximately 6 with a peak around noon and a minimum somewhere between 0100 and 0300 h LT. This study of a limited amount of OGO 6 and Arecibo data seems to indicate that the charge-exchange-induced hydrogen escape flux increases as the F(10.7) flux increases for low to moderate solar activity.

  4. Springs-neaps cycles in daily total seabed light: Daylength-induced changes

    NASA Astrophysics Data System (ADS)

    Roberts, E. M.; Bowers, D. G.; Davies, A. J.

    2014-04-01

    In shallow, tidal seas, daily total seabed light is determined largely by the interaction of the solar elevation cycle, the tidal cycle in water depth, and any temporal variability in turbidity. Since tidal range, times of low water, and often turbidity vary in regular ways over the springs-neaps cycle, daily total seabed light exhibits cycles of the same periodicity. Corresponding cycles are likely to be induced in the daily total primary production of benthic algae and plants, particularly those light-limited specimens occupying the lower reaches of a sub-tidal population. Consequently, this effect is an important control on the growth patterns, depth distribution and survival of, for example, macroalgal forests and seagrass meadows. Seasonal changes in daylength exert an important additional control on these cycles, as they alter the fraction of the tidal and turbidity cycles occurring within daylight hours. Bowers et al. (1997) modelled this phenomenon numerically and predicted that for a site with low water at about midday and midnight at neaps tides, 6 am and 6 pm at springs, daily total seabed light peaks at neaps in winter, but the ‘sense' of the cycle ‘switches' so that it peaks at springs in summer - the longer daylength permits the morning and evening low water springs to contribute substantially to the daily total. Observations for such a site in North Wales (UK), presented in this paper, show that no such ‘switch' occurs, and neaps tides host the largest daily totals throughout the year. The predicted ‘switch' is not observed because turbidity increases generally at spring tides, and specifically at low water springs, both of which were not accounted for in the model. Observations at a second site in Brittany (France), diametrically opposite in terms of the times of low water at neaps and at springs, indicate a peak at springs throughout the year. Analytical tools are developed to calculate the percentage of daily total sea surface irradiance

  5. Unprecedented evidence for high viral abundance and lytic activity in coral reef waters of the South Pacific Ocean.

    PubMed

    Payet, Jérôme P; McMinds, Ryan; Burkepile, Deron E; Vega Thurber, Rebecca L

    2014-01-01

    Despite nutrient-depleted conditions, coral reef waters harbor abundant and diverse microbes; as major agents of microbial mortality, viruses are likely to influence microbial processes in these ecosystems. However, little is known about marine viruses in these rapidly changing ecosystems. Here we examined spatial and short-term temporal variability in marine viral abundance (VA) and viral lytic activity across various reef habitats surrounding Moorea Island (French Polynesia) in the South Pacific. Water samples were collected along four regional cross-reef transects and during a time-series in Opunohu Bay. Results revealed high VA (range: 5.6 × 10(6)-3.6 × 10(7) viruses ml(-1)) and lytic viral production (range: 1.5 × 10(9)-9.2 × 10(10) viruses l(-1) d(-1)). Flow cytometry revealed that viral assemblages were composed of three subsets that each displayed distinct spatiotemporal relationships with nutrient concentrations and autotrophic and heterotrophic microbial abundances. The results highlight dynamic shifts in viral community structure and imply that each of these three subsets is ecologically important and likely to infect distinct microbial hosts in reef waters. Based on viral-reduction approach, we estimate that lytic viruses were responsible for the removal of ca. 24-367% of bacterial standing stock d(-1) and the release of ca. 1.0-62 μg of organic carbon l(-1) d(-1) in reef waters. Overall, this work demonstrates the highly dynamic distribution of viruses and their critical roles in controlling microbial mortality and nutrient cycling in coral reef water ecosystems. PMID:25295032

  6. Unprecedented evidence for high viral abundance and lytic activity in coral reef waters of the South Pacific Ocean

    PubMed Central

    Payet, Jérôme P.; McMinds, Ryan; Burkepile, Deron E.; Vega Thurber, Rebecca L.

    2014-01-01

    Despite nutrient-depleted conditions, coral reef waters harbor abundant and diverse microbes; as major agents of microbial mortality, viruses are likely to influence microbial processes in these ecosystems. However, little is known about marine viruses in these rapidly changing ecosystems. Here we examined spatial and short-term temporal variability in marine viral abundance (VA) and viral lytic activity across various reef habitats surrounding Moorea Island (French Polynesia) in the South Pacific. Water samples were collected along four regional cross-reef transects and during a time-series in Opunohu Bay. Results revealed high VA (range: 5.6 × 106–3.6 × 107 viruses ml-1) and lytic viral production (range: 1.5 × 109–9.2 × 1010 viruses l-1 d-1). Flow cytometry revealed that viral assemblages were composed of three subsets that each displayed distinct spatiotemporal relationships with nutrient concentrations and autotrophic and heterotrophic microbial abundances. The results highlight dynamic shifts in viral community structure and imply that each of these three subsets is ecologically important and likely to infect distinct microbial hosts in reef waters. Based on viral-reduction approach, we estimate that lytic viruses were responsible for the removal of ca. 24–367% of bacterial standing stock d-1 and the release of ca. 1.0–62 μg of organic carbon l-1 d-1 in reef waters. Overall, this work demonstrates the highly dynamic distribution of viruses and their critical roles in controlling microbial mortality and nutrient cycling in coral reef water ecosystems. PMID:25295032

  7. Commitment to Lysogeny Is Preceded by a Prolonged Period of Sensitivity to the Late Lytic Regulator Q in Bacteriophage λ

    PubMed Central

    Svenningsen, Sine Lo

    2014-01-01

    A key event in development is the irreversible commitment to a particular cell fate, which may be concurrent with or delayed with respect to the initial cell fate decision. In this work, we use the paradigmatic bacteriophage λ lysis-lysogeny decision circuit to study the timing of commitment. The lysis-lysogeny decision is made based on the expression trajectory of CII. The chosen developmental strategy is manifested by repression of the pR and pL promoters by CI (lysogeny) or by antitermination of late gene expression by Q (lysis). We found that expression of Q in trans from a plasmid at the time of infection resulted in a uniform lytic decision. Furthermore, expression of Q up to 50 min after infection results in lysis of the majority of cells which initially chose lysogenic development. In contrast, expression of Q in cells containing a single chromosomal prophage had no effect on cell growth, indicating commitment to lysogeny. Notably, if the prophage was present in 10 plasmid-borne copies, Q expression resulted in lytic development, suggesting that the cellular phage chromosome number is the critical determinant of the timing of lysogenic commitment. Based on our results, we conclude that (i) the lysogenic decision made by the CI-Cro switch soon after infection can be overruled by ectopic Q expression at least for a time equivalent to one phage life cycle, (ii) the presence of multiple λ chromosomes is a prerequisite for a successful Q-mediated switch from lysogenic to lytic development, and (iii) phage chromosomes within the same cell can reach different decisions. PMID:25092034

  8. Binding Specificities of the Telomere Phage ϕKO2 Prophage Repressor CB and Lytic Repressor Cro.

    PubMed

    Hammerl, Jens Andre; Jäckel, Claudia; Lanka, Erich; Roschanski, Nicole; Hertwig, Stefan

    2016-01-01

    Temperate bacteriophages possess a genetic switch which regulates the lytic and lysogenic cycle. The genomes of the temperate telomere phages N15, PY54, and ϕKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor (cI or cB), the lytic repressor (cro) and a putative antiterminator (q). The roles of these products are thought to be similar to those of the lambda proteins CI (CI prophage repressor), Cro (Cro repressor), and Q (antiterminator Q), respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ϕKO2 are reminiscent of lambda-like phages. We determined binding sites of the ϕKO2 prophage repressor CB and lytic repressor Cro on the ϕKO2 genome in detail by electrophoretic mobility shift assay (EMSA) studies. Unexpectedly, ϕKO2 CB and Cro revealed different binding specificities. CB was bound to three OR operators in the intergenic region between cB and cro, two OL operators between cB and the replication gene repA and even to operators of N15. Cro bound exclusively to the 16 bp operator site OR3 upstream of the ϕKO2 prophage repressor gene. The ϕKO2 genes cB and cro are regulated by several strong promoters overlapping with the OR operators. The data suggest that Cro represses cB transcription but not its own synthesis, as already reported for PY54 Cro. Thus, not only PY54, but also phage ϕKO2 possesses a genetic switch that diverges significantly from the switch of lambda-like phages. PMID:27527206

  9. Binding Specificities of the Telomere Phage ϕKO2 Prophage Repressor CB and Lytic Repressor Cro

    PubMed Central

    Hammerl, Jens Andre; Jäckel, Claudia; Lanka, Erich; Roschanski, Nicole; Hertwig, Stefan

    2016-01-01

    Temperate bacteriophages possess a genetic switch which regulates the lytic and lysogenic cycle. The genomes of the temperate telomere phages N15, PY54, and ϕKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor (cI or cB), the lytic repressor (cro) and a putative antiterminator (q). The roles of these products are thought to be similar to those of the lambda proteins CI (CI prophage repressor), Cro (Cro repressor), and Q (antiterminator Q), respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ϕKO2 are reminiscent of lambda-like phages. We determined binding sites of the ϕKO2 prophage repressor CB and lytic repressor Cro on the ϕKO2 genome in detail by electrophoretic mobility shift assay (EMSA) studies. Unexpectedly, ϕKO2 CB and Cro revealed different binding specificities. CB was bound to three OR operators in the intergenic region between cB and cro, two OL operators between cB and the replication gene repA and even to operators of N15. Cro bound exclusively to the 16 bp operator site OR3 upstream of the ϕKO2 prophage repressor gene. The ϕKO2 genes cB and cro are regulated by several strong promoters overlapping with the OR operators. The data suggest that Cro represses cB transcription but not its own synthesis, as already reported for PY54 Cro. Thus, not only PY54, but also phage ϕKO2 possesses a genetic switch that diverges significantly from the switch of lambda-like phages. PMID:27527206

  10. Commitment to lysogeny is preceded by a prolonged period of sensitivity to the late lytic regulator Q in bacteriophage λ.

    PubMed

    Svenningsen, Sine Lo; Semsey, Szabolcs

    2014-10-01

    A key event in development is the irreversible commitment to a particular cell fate, which may be concurrent with or delayed with respect to the initial cell fate decision. In this work, we use the paradigmatic bacteriophage λ lysis-lysogeny decision circuit to study the timing of commitment. The lysis-lysogeny decision is made based on the expression trajectory of CII. The chosen developmental strategy is manifested by repression of the pR and pL promoters by CI (lysogeny) or by antitermination of late gene expression by Q (lysis). We found that expression of Q in trans from a plasmid at the time of infection resulted in a uniform lytic decision. Furthermore, expression of Q up to 50 min after infection results in lysis of the majority of cells which initially chose lysogenic development. In contrast, expression of Q in cells containing a single chromosomal prophage had no effect on cell growth, indicating commitment to lysogeny. Notably, if the prophage was present in 10 plasmid-borne copies, Q expression resulted in lytic development, suggesting that the cellular phage chromosome number is the critical determinant of the timing of lysogenic commitment. Based on our results, we conclude that (i) the lysogenic decision made by the CI-Cro switch soon after infection can be overruled by ectopic Q expression at least for a time equivalent to one phage life cycle, (ii) the presence of multiple λ chromosomes is a prerequisite for a successful Q-mediated switch from lysogenic to lytic development, and (iii) phage chromosomes within the same cell can reach different decisions. PMID:25092034

  11. Induction of protease release of the resistant diatom Chaetoceros didymus in response to lytic enzymes from an algicidal bacterium.

    PubMed

    Paul, Carsten; Pohnert, Georg

    2013-01-01

    Marine lytic bacteria can have a substantial effect on phytoplankton and are even capable to terminate blooms of microalgae. The bacterium Kordia algicida was reported to lyse cells of the diatom Skeletonema costatum and several other diatoms by a quorum sensing controlled excretion of proteases. However the diatom Chaetoceros didymus is fully resistant against the bacterial enzymes. We show that the growth curve of this diatom is essentially unaffected by addition of bacterial filtrates that are active against other diatoms. By monitoring proteases from the medium using zymography and fluorescence based activity assays we demonstrate that C. didymus responds to the presence of the lytic bacteria with the induced production of algal proteases. These proteases exhibit a substantially increased activity compared to the bacterial counterparts. The induction is also triggered by signals in the supernatant of a K. algicida culture. Size fractionation shows that only the >30 kD fraction of the bacterial exudates acts as an inducing cue. Implications for a potential induced defense of the diatom C. didymus are discussed. PMID:23469204

  12. SPARC expression induces cell cycle arrest via STAT3 signaling pathway in medulloblastoma cells

    SciTech Connect

    Chetty, Chandramu; Dontula, Ranadheer; Gujrati, Meena; Lakka, Sajani S.

    2012-01-13

    Highlights: Black-Right-Pointing-Pointer Ectopic expression of SPARC impaired cell proliferation in medulloblastoma cells. Black-Right-Pointing-Pointer SPARC expression induces STAT3 mediated cell cycle arrest in medulloblastoma cells. Black-Right-Pointing-Pointer SPARC expression significantly inhibited pre-established tumor growth in nude-mice. -- Abstract: Dynamic cell interaction with ECM components has profound influence in cancer progression. SPARC is a component of the ECM, impairs the proliferation of different cell types and modulates tumor cell aggressive features. We previously reported that SPARC expression significantly impairs medulloblastoma tumor growth in vivo. In this study, we demonstrate that expression of SPARC inhibits medulloblastoma cell proliferation. MTT assay indicated a dose-dependent reduction in tumor cell proliferation in adenoviral mediated expression of SPARC full length cDNA (Ad-DsRed-SP) in D425 and UW228 cells. Flow cytometric analysis showed that Ad-DsRed-SP-infected cells accumulate in the G2/M phase of cell cycle. Further, immunoblot and immunoprecipitation analyses revealed that SPARC induced G2/M cell cycle arrest was mediated through inhibition of the Cyclin-B-regulated signaling pathway involving p21 and Cdc2 expression. Additionally, expression of SPARC decreased STAT3 phosphorylation at Tyr-705; constitutively active STAT3 expression reversed SPARC induced G2/M arrest. Ad-DsRed-SP significantly inhibited the pre-established orthotopic tumor growth and tumor volume in nude-mice. Immunohistochemical analysis of tumor sections from mice treated with Ad-DsRed-SP showed decreased immunoreactivity for pSTAT3 and increased immunoreactivity for p21 compared to tumor section from mice treated with mock and Ad-DsRed. Taken together our studies further reveal that STAT3 plays a key role in SPARC induced G2/M arrest in medulloblastoma cells. These new findings provide a molecular basis for the mechanistic understanding of the

  13. Protein PSMD8 may mediate microgravity-induced cell cycle arrest

    NASA Astrophysics Data System (ADS)

    Hang, Xiaoming; Sun, Yeqing; Xu, Dan; Wu, Di; Chen, Xiaoning

    Microgravity environment of space can induce a serial of changes in cells, such as morphology alterations, cytoskeleton disorder and cell cycle disturbance. Our previous study of simulated-microgravity on zebrafish (Danio rerio) embryos demonstrated 26s proteasome non-ATPase regulatory subunit 8 (PSMD8) might be a microgravity sensitive gene. However, functional study on PSMD8 is very limited and it has not been cloned in zebrafish till now. In this study, we tried to clone PSMD8 gene in zebrafish, quantify its protein expression level in zebrafish embryos after simulated microgravity and identify its possible function in cell cycle regulation. A rotary cell culture system (RCCS) designed by national aeronautics and apace administration (NASA) of America was used to simulate microgravity. The full-length of psmd8 gene in zebrafish was cloned. Preliminary analysis on its sequence and phylogenetic tree construction were carried out subsequently. Quantitative analysis by western blot showed that PSMD8 protein expression levels were significantly increased 1.18 and 1.22 times after 24-48hpf and 24-72hpf simulated microgravity, respectively. Moreover, a significant delay on zebrafish embryo development was found in simulated-microgravity exposed group. Inhibition of PSMD8 protein in zebrafish embryonic cell lines ZF4 could block cell cycle in G1 phase, which indicated that PSMD8 may play a role in cell cycle regulation. Interestingly, simulated-microgravity could also block ZF4 cell in G1 phase. Whether it is PSMD8 mediated cell cycle regulation result in the zebrafish embryo development delay after simulated microgravity exposure still needs further study. Key Words: PSMD8; Simulated-microgravity; Cell cycle; ZF4 cell line

  14. Fusarochromanone Induces G1 Cell Cycle Arrest and Apoptosis in COS7 and HEK293 Cells

    PubMed Central

    Gu, Ying; Chen, Xin; Shang, Chaowei; Singh, Karnika; Barzegar, Mansoureh; Mahdavian, Elahe; Salvatore, Brian A.; Jiang, Shanxiang; Huang, Shile

    2014-01-01

    Fusarochromanone (FC101), a mycotoxin produced by the fungus Fusarium equiseti, is frequently observed in the contaminated grains and feedstuffs, which is toxic to animals and humans. However, the underlying molecular mechanism remains to be defined. In this study, we found that FC101 inhibited cell proliferation and induced cell death in COS7 and HEK293 cells in a concentration-dependent manner. Flow cytometric analysis showed that FC101 induced G1 cell cycle arrest and apoptosis in the cells. Concurrently, FC101 downregulated protein expression of cyclin D1, cyclin-dependent kinases (CDK4 and CDK6), and Cdc25A, and upregulated expression of the CDK inhibitors (p21Cip1 and p27Kip1), resulting in hypophosphorylation of Rb. FC101 also inhibited protein expression of Bcl-2, Bcl-xL, Mcl-1 and survivin, and induced expression of BAD, leading to activation of caspase 3 and cleavage of PARP, indicating caspase-dependent apoptosis. However, Z-VAD-FMK, a pan-caspase inhibitor, only partially prevented FC101-induced cell death, implying that FC101 may induce cell death through both caspase-dependent and -independent mechanisms. Our results support the notion that FC101 executes its toxicity at least by inhibiting cell proliferation and inducing cell death. PMID:25384025

  15. Effects of ply thickness on thermal cycle induced damage and thermal strain

    NASA Technical Reports Server (NTRS)

    Tompkins, Stephen S.

    1994-01-01

    An experimental study was conducted to determine the effects of ply thickness in composite laminates on thermally induced cracking and changes in the coefficient of thermal expansion, CTE. A graphite-epoxy composite material, P75/ERL 1962, in thin (1 mil) and thick (5 mils) prepregs was used to make cross-ply laminates, ((0/90)(sub n))s, with equal total thickness (n=2, n=10) and cross-ply laminates with the same total number of plies (n=2). Specimens of each laminate configuration were cycled up to 1500 times between -250 and 250 F. Thermally induced microdamage was assessed as a function of the number of cycles as was the change in CTE. The results showed that laminates fabricated with thin-plies microcracked at significantly different rates and reached significantly different equilibrium crack densities than the laminate fabricated with thick-ply and n=2. The CTE of thin-ply laminates was less affected by thermal cycling and damage than the CTE of thick-ply laminates. These differences are attributed primarily to differences in interply constraints. Observed effects of ply thickness on crack density was qualitatively predicted by a combined shear-lag stress/energy method.

  16. Solar Cycle Variability in Mean Thermospheric Composition and Temperature Induced by Atmospheric Tides

    NASA Astrophysics Data System (ADS)

    Jones, M., Jr.; Forbes, J. M.; Hagan, M. E.

    2015-12-01

    Vertically-propagating atmospheric thermal tides whose origins lie in Earth's lower atmosphere are now widely recognized as one of the dominant "meteorological" drivers of space weather. Many prior research efforts have focused on documenting and understanding the role that dissipating tides play in determining the longitudinal and seasonal variability associated with lower thermospheric winds, temperature, and constituent densities. However, considerably less attention has focused on understanding the potential solar cycle variability in the mean thermospheric state induced by the tides. In this paper we utilize the National Center for Atmospheric Research Thermosphere-Ionosphere-Electrodynamics General Circulation Model (TIE-GCM), forced with observationally-based tides at the model lower boundary from the Climatological Tidal Model of the Thermosphere (CTMT, from Oberheide et al. [2011]), to elucidate how the dissipating tides induce variations of up to 30 K in the zonal-mean thermosphere temperature between solar minimum and maximum. Numerical experiments are performed for the month of September and for solar minimum, medium, and maximum conditions in order to quantify the solar cycle variability associated with the different terms in the thermodynamic energy, major and minor neutral constituent continuity equations. Our analysis indicates that solar cycle variability in neutral temperatures results from a combination of net eddy heat transport effects and tidal modulation of net nitric oxide (NO) cooling. The chemical and dynamical pathways through which dissipating tides affect mean NO cooling differently at solar minimum and maximum are diagnosed.

  17. Severe hypoxia induces complete antifolate resistance in carcinoma cells due to cell cycle arrest

    PubMed Central

    Raz, S; Sheban, D; Gonen, N; Stark, M; Berman, B; Assaraf, Y G

    2014-01-01

    Antifolates have a crucial role in the treatment of various cancers by inhibiting key enzymes in purine and thymidylate biosynthesis. However, the frequent emergence of inherent and acquired antifolate resistance in solid tumors calls for the development of novel therapeutic strategies to overcome this chemoresistance. The core of solid tumors is highly hypoxic due to poor blood circulation, and this hypoxia is considered to be a major contributor to drug resistance. However, the cytotoxic activity of antifolates under hypoxia is poorly characterized. Here we show that under severe hypoxia, gene expression of ubiquitously expressed key enzymes and transporters in folate metabolism and nucleoside homeostasis is downregulated. We further demonstrate that carcinoma cells become completely refractory, even at sub-millimolar concentrations, to all hydrophilic and lipophilic antifolates tested. Moreover, tumor cells retained sensitivity to the proteasome inhibitor bortezomib and the topoisomerase II inhibitor doxorubicin, which are independent of cell cycle. We provide evidence that this antifolate resistance, associated with repression of folate metabolism, is a result of the inability of antifolates to induce DNA damage under hypoxia, and is attributable to a hypoxia-induced cell cycle arrest, rather than a general anti-apoptotic mechanism. Our findings suggest that solid tumors harboring a hypoxic core of cell cycle-arrested cells may display antifolate resistance while retaining sensitivity to the chemotherapeutics bortezomib and doxorubicin. This study bears important implications for the molecular basis underlying antifolate resistance under hypoxia and its rational overcoming in solid tumors. PMID:24556682

  18. Tangeretin induces cell cycle arrest and apoptosis through upregulation of PTEN expression in glioma cells.

    PubMed

    Ma, Li-Li; Wang, Da-Wei; Yu, Xu-Dong; Zhou, Yan-Ling

    2016-07-01

    Tangeretin (TANG), present in peel of citrus fruits, has been shown to various medicinal properties such as chemopreventive and neuroprotective. However, the chemopreventive effect of TANG on glioblastoma cells has not been examined. The present study was designed to explore the anticancer potential of TANG in glioblastoma cells and to investigate the related mechanism. Human glioblastoma U-87MG and LN-18 cells were treated with 45μM concentration of TANG and cell growth was measured by MTT assay. The cell cycle distribution and cell death were measured by flow cytometry. The expression of cell cycle and apoptosis related genes were analyzed by quantitative RT-PCR and western blot. The cells treated with TANG were significantly increased cell growth suppression and cell death effects than vehicle treated cells. Further, TANG treatment increases G2/M arrest and apoptosis by modulating PTEN and cell-cycle regulated genes such as cyclin-D and cdc-2 mRNA and protein expressions. Moreover, the ability of TANG to decrease cell growth and to induce cell death was compromised when PTEN was knockdown by siRNA. Taken together, the chemopreventive effect of TANG is associated with regulation of cell-cycle and apoptosis in glioblastoma, thereby attenuating glioblastoma cell growth. Hence, the present findings suggest that TANG may be a therapeutic agent for glioblastoma treatment. PMID:27261630

  19. Penicillin Induced Persistence in Chlamydia trachomatis: High Quality Time Lapse Video Analysis of the Developmental Cycle

    PubMed Central

    Barlow, David; Wang, Yibing; Salim, Omar; Lambden, Paul R.; Clarke, Ian N.

    2009-01-01

    Background Chlamydia trachomatis is a major human pathogen with a unique obligate intracellular developmental cycle that takes place inside a modified cytoplasmic structure known as an inclusion. Following entry into a cell, the infectious elementary body (EB) differentiates into a non - infectious replicative form known as a reticulate body (RB). RBs divide by binary fission and at the end of the cycle they redifferentiate into EBs. Treatment of C.trachomatis with penicillin prevents maturation of RBs which survive and enlarge to become aberrant RBs within the inclusion in a non - infective persistent state. Persistently infected individuals may be a reservoir for chlamydial infection. The C.trachomatis genome encodes the enzymes for peptidoglycan (PG) biosynthesis but a PG sacculus has never been detected. This coupled to the action of penicillin is known as the chlamydial anomaly. We have applied video microscopy and quantitative DNA assays to the chlamydial developmental cycle to assess the effects of penicillin treatment and establish a framework for investigating penicillin induced chlamydial persistence. Principal Findings Addition of penicillin at the time of cell infection does not prevent uptake and the establishment of an inclusion. EB to RB transition occurs but bacterial cytokinesis is arrested by the second binary fission. RBs continue to enlarge but not divide in the presence of penicillin. The normal developmental cycle can be recovered by the removal of penicillin although the large, aberrant RBs do not revert to the normal smaller size but remain present to the completion of the developmental cycle. Chromosomal and plasmid DNA replication is unaffected by the addition of penicillin but the arrest of bacterial cytokinesis under these conditions results in RBs accumulating multiple copies of the genome. Conclusions We have applied video time lapse microscopy to the study of the chlamydial developmental cycle. Linked with accurate measures of genome

  20. Niclosamide inhibits the proliferation of human osteosarcoma cell lines by inducing apoptosis and cell cycle arrest.

    PubMed

    Li, Zonghuan; Yu, Yifeng; Sun, Shaoxing; Qi, Baiwen; Wang, Weiyang; Yu, Aixi

    2015-04-01

    Niclosamide, used as an antihelminthic, has demonstrated some properties of anticancer effects. However, its role in osteosarcoma remains to be determined. The aim of this study was to determine the effect of niclosamide on human osteosarcoma cell lines. The human MG-63 and U2OS osteosarcoma cell lines were treated with different concentrations of niclosamide. The cell inhibitory rate was calculated by CCK-8 assay. Cell cycle was detected by flow cytometry. Cell apoptosis was determined by Hoechst 33324 staining, flow cytometry and fluorescence microscope, respectively. The expression of bcl-2, bax and pro-caspase-3 were measured by western blotting. Niclosamide exerted an inhibitory effect on the two cell lines in a time- and dose-dependent manner. Niclosamide was found to induce the arrest of S and G2/M phase in U2OS cells and G2/M in MG-63 cells. Moreover, niclosamide induced apoptosis in MG-63 and U2OS cells. The bax/bcl-2 ratio increased while the expression of pro‑caspase-3 decreased significantly in the two cell lines. The results indicated that niclosamide inhibits proliferation, and induces apoptosis and cell cycle arrest in human osteosarcoma cell lines. PMID:25634333

  1. Prion-induced neurotoxicity: Possible role for cell cycle activity and DNA damage response

    PubMed Central

    Bujdoso, Raymond; Landgraf, Matthias; Jackson, Walker S; Thackray, Alana M

    2015-01-01

    Protein misfolding neurodegenerative diseases arise through neurotoxicity induced by aggregation of host proteins. These conditions include Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, motor neuron disease, tauopathies and prion diseases. Collectively, these conditions are a challenge to society because of the increasing aged population and through the real threat to human food security by animal prion diseases. It is therefore important to understand the cellular and molecular mechanisms that underlie protein misfolding-induced neurotoxicity as this will form the basis for designing strategies to alleviate their burden. Prion diseases are an important paradigm for neurodegenerative conditions in general since several of these maladies have now been shown to display prion-like phenomena. Increasingly, cell cycle activity and the DNA damage response are recognised as cellular events that participate in the neurotoxic process of various neurodegenerative diseases, and their associated animal models, which suggests they are truly involved in the pathogenic process and are not merely epiphenomena. Here we review the role of cell cycle activity and the DNA damage response in neurodegeneration associated with protein misfolding diseases, and suggest that these events contribute towards prion-induced neurotoxicity. In doing so, we highlight PrP transgenic Drosophila as a tractable model for the genetic analysis of transmissible mammalian prion disease. PMID:26279981

  2. Diosgenin induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma cells.

    PubMed

    Li, Yongjian; Wang, Xiaorong; Cheng, Silu; Du, Juan; Deng, Zhengting; Zhang, Yani; Liu, Qun; Gao, Jingdong; Cheng, Binbin; Ling, Changquan

    2015-02-01

    Diosgenin is a major compound of Dioscoreaceae plants such as yam, which is used as a drug in Traditional Chinese Medicine, and a common vegetable worldwide. The anticancer effect of diosgenin has been reported in various tumor cells, including leukemia, gastric, colorectal, and breast cancer. However, the activity of diosgenin on hepatocellular carcinoma (HCC) and the underlying mechanism have not been completely investigated. Therefore, we investigated the efficacy and associated mechanisms of diosgenin in HCC cells. Flow cytometric analysis was performed to determine the presence of cell cycle arrest and apopotic cells. Diosgenin significantly inhibited the growth of Bel-7402, SMMC-7721 and HepG2 HCC cells in a concentration-dependent manner. Diosgenin treatment for 24 h induced G2/M cell cycle arrest and apoptosis of hepatoma cells. Diosgenin inhibited Akt phosphorylation and upregulated p21 and p27 expression, but did not alter the expression of p53, suggesting diosgenin-induced upregulation of p21 and p57 is p53-independent in HCC cells. Diosgenin induced HCC cell apoptosis by activating caspase cascades -3, -8 and -9. However, diosgenin did not affect Bcl-2 and Bax levels. In conclusion, results of the present study suggest that diosgenin may be an active anti-HCC agent obtained from natural plants and provide new insights in understanding the mechanisms of diosgenin. PMID:25434486

  3. α-Mangostin Induces Apoptosis and Cell Cycle Arrest in Oral Squamous Cell Carcinoma Cell

    PubMed Central

    Kwak, Hyun-Ho; Park, Bong-Soo

    2016-01-01

    Mangosteen has long been used as a traditional medicine and is known to have antibacterial, antioxidant, and anticancer effects. Although the effects of α-mangostin, a natural compound extracted from the pericarp of mangosteen, have been investigated in many studies, there is limited data on the effects of the compound in human oral squamous cell carcinoma (OSCC). In this study, α-mangostin was assessed as a potential anticancer agent against human OSCC cells. α-Mangostin inhibited cell proliferation and induced cell death in OSCC cells in a dose- and time-dependent manner with little to no effect on normal human PDLF cells. α-Mangostin treatment clearly showed apoptotic evidences such as nuclear fragmentation and accumulation of annexin V and PI-positive cells on OSCC cells. α-Mangostin treatment also caused the collapse of mitochondrial membrane potential and the translocation of cytochrome c from the mitochondria into the cytosol. The expressions of the mitochondria-related proteins were activated by α-mangostin. Treatment with α-mangostin also induced G1 phase arrest and downregulated cell cycle-related proteins (CDK/cyclin). Hence, α-mangostin specifically induces cell death and inhibits proliferation in OSCC cells via the intrinsic apoptosis pathway and cell cycle arrest at the G1 phase, suggesting that α-mangostin may be an effective agent for the treatment of OSCC. PMID:27478478

  4. High-dose irradiation induces cell cycle arrest, apoptosis, and developmental defects during Drosophila oogenesis.

    PubMed

    Shim, Hee Jin; Lee, Eun-Mi; Nguyen, Long Duy; Shim, Jaekyung; Song, Young-Han

    2014-01-01

    Ionizing radiation (IR) treatment induces a DNA damage response, including cell cycle arrest, DNA repair, and apoptosis in metazoan somatic cells. Because little has been reported in germline cells, we performed a temporal analysis of the DNA damage response utilizing Drosophila oogenesis as a model system. Oogenesis in the adult Drosophila female begins with the generation of 16-cell cyst by four mitotic divisions of a cystoblast derived from the germline stem cells. We found that high-dose irradiation induced S and G2 arrests in these mitotically dividing germline cells in a grp/Chk1- and mnk/Chk2-dependent manner. However, the upstream kinase mei-41, Drosophila ATR ortholog, was required for the S-phase checkpoint but not for the G2 arrest. As in somatic cells, mnk/Chk2 and dp53 were required for the major cell death observed in early oogenesis when oocyte selection and meiotic recombination occurs. Similar to the unscheduled DNA double-strand breaks (DSBs) generated from defective repair during meiotic recombination, IR-induced DSBs produced developmental defects affecting the spherical morphology of meiotic chromosomes and dorsal-ventral patterning. Moreover, various morphological abnormalities in the ovary were detected after irradiation. Most of the IR-induced defects observed in oogenesis were reversible and were restored between 24 and 96 h after irradiation. These defects in oogenesis severely reduced daily egg production and the hatch rate of the embryos of irradiated female. In summary, irradiated germline cells induced DSBs, cell cycle arrest, apoptosis, and developmental defects resulting in reduction of egg production and defective embryogenesis. PMID:24551207

  5. Glutathione cycle activity and pyridine nucleotide levels in oxidant-induced injury of cells.

    PubMed Central

    Schraufstätter, I U; Hinshaw, D B; Hyslop, P A; Spragg, R G; Cochrane, C G

    1985-01-01

    Exposure of target cells to a bolus of H2O2 induced cell lysis after a latent period of several hours, which was prevented only when the H2O2 was removed within the first 30 min of injury by addition of catalase. This indicated that early metabolic events take place that are important in the fate of the cell exposed to oxidants. In this study, we described two early and independent events of H2O2-induced injury in P388D1 macrophagelike tumor cells: activation of the glutathione cycle and depletion of cellular NAD. Glutathione cycle and hexose monophosphate shunt (HMPS) were activated within seconds after the addition of H2O2. High HMPS activity maintained glutathione that was largely reduced. However, when HMPS activity was inhibited--by glucose depletion or by incubation at 4 degrees C--glutathione remained in the oxidized state. Total pyridine nucleotide levels were diminished when cells were exposed to H2O2, and the breakdown product, nicotinamide, was recovered in the extracellular medium. Intracellular NAD levels fell by 80% within 20 min of exposure of cells to H2O2. The loss of NADP(H) and stimulation of the HMPS could be prevented when the glutathione cycle was inhibited by either blocking glutathione synthesis with buthionine sulfoximine (BSO) or by inhibiting glutathione reductase with (1,3-bis) 2 chlorethyl-1-nitrosourea. The loss of NAD developed independently of glutathione cycle and HMPS activity, as it also occurred in BSO-treated cells. PMID:3840176

  6. The adverse effects of high fat induced obesity on female reproductive cycle and hormones

    NASA Astrophysics Data System (ADS)

    Donthireddy, Laxminarasimha Reddy

    The prevalence of obesity, an established risk and progression factor for abnormal reproductive cycle and tissue damage in female mice. It leads to earlier puberty, menarche in young females and infertility. There are extensive range of consequences of obesity which includes type-2 diabetes, cardiovascular disease and insulin resistance. Obesity is the interaction between dietary intake, genes, life style and environment. The interplay of hormones estrogen, insulin, and leptin is well known on energy homeostasis and reproduction. The aim of this study is to determine the effect of high fat induced obesity on reproductive cycles and its hormonal abnormalities on mice model. Two week, 3 month and 8 month long normal (WT) and very high fat diet (VHFD) diet course is followed. When mice are fed with very high fat diet, there is a drastic increase in weight within the first week later. There was a significant (p<0.001) increase in leptin levels in 6 month VHFD treated animals. 2 week, 3 month and 6 month time interval pap smear test results showed number of cells, length of estrous cycle and phases of the estrous cycle changes with VHFD mice(n=30) compared to normal diet mice(n=10). These results also indicate that the changes in the reproductive cycles in VHFD treated female mice could be due to the changes in hormones. Histo-pathological analyses of kidney, ovary, liver, pancreas, heart and lungs showed remarkable changes in some tissue on exposure to very high fat. Highly deposited fat packets observed surrounding the hepatocytes and nerve cells.

  7. Thermal cycling induced load on copper-ribbons in crystalline photovoltaic modules

    NASA Astrophysics Data System (ADS)

    Meier, R.; Kraemer, F.; Wiese, S.; Wolter, K.-J.; Bagdahn, J.

    2010-08-01

    Solar module lifetime is limited by the fatigue behavior of its cell interconnectors: the copper-ribbons. Every change in temperature induces thermo-mechanical stresses in the module components due to their thermo-mechanical mismatch. The purpose of this work is to quantify this load on the copper-ribbons between the individual cells of a cell string during a thermal cycling test by measuring cell displacement using digital image correlation and to compare the results to finite element analysis (FEM). Furthermore with help of FEM the influences of different materials were investigated, allowing material and layout optimizations with respect to copper-ribbon loading.

  8. Widespread lytic lesions—A metastatic or vasculitic process?

    PubMed Central

    Khan, Fahd; Srirangan, Srinivasan; El-Miedany, Yasser; Madaan, Sanjeev

    2015-01-01

    Introduction This case highlights the complexities in the initial diagnosis and investigations of widespread lytic lesions initially perceived to be a widespread metastatic process and the consideration of alternative diagnosis. Presentation of case A 57 year-old man with a background of psoriatic arthritis presented to the rheumatology department with lumbar back pain and sensory disturbance over L4/5. Magnetic resonance imaging (MRI) and bone scan identified lesions consistent with bony metastases at L5. The patient previously had a raised prostate specific antigen (PSA) of 10.8 μg/L (normal < 4) but prostate biopsy was benign. Multiple metastatic deposits in the liver and kidneys (confirmed necrotic tissue on biopsy) were identified through further investigations. The initial diagnosis of malignancy was challenged after a positron emission tomography (PET) scan showed lesions highly suggestive of polyarteritis nodosa (PAN) and subsequent magnetic resonance angiogram (MRA) revealed stenosis and aneurysm in the renal artery in keeping with PAN. Therefore what was initially thought to be a widespread metastatic disease process was in fact the manifestation of a systemic vasculitic disease. Discussion PAN is a vasculitis that predominantly involves small to medium-sized vessels. The disease can affect any site in the body, but holds a predisposition for organs such as kidneys, heart and the gastrointestinal tract. Differential diagnosis of PAN should be considered in patients with widespread lytic lesions. Conclusion Due to the pathological nature of PAN and its variable clinical manifestations that add to the challenges of its diagnosis, one must hold a high clinical suspicion, even in urological conditions. PMID:26684863

  9. The immunological generation of a platelet-activating factor and a platet-lytic factor in the rat.

    PubMed Central

    Valone, F H; Whitmer, D I; Pickett, W C; Austen, K F; Goetzl, E J

    1979-01-01

    Antigen challenge of the rat peritoneal cavity which had been prepared with IgGa-rich antiserum generated activities which released [14C]-serotonin from pre-labelled human platelets. After adsorption of these activities onto Amberlite XAD-8 and elution in 80% ethanol, two factors of differing polarity were resolved by chromatography on diethylaminoethyl cellulose in organic solvents. The activity eluting in the 7:1 chloroform:methanol solvent contained a platelet-lytic factor (PLF) assessed by the parallel release of lactic acid dehydrogenase and [14C]-serotonin; the cytotoxicity of this fraction was confirmed by phase-contrast microscopy examination which demonstrated fragmentation of the exposed platelets. The activity eluting in the 1:1 methanol: aqueous 1.0 M ammonium carbonate solvent was a platelet-activating factor (PAF) as defined by release of [14C]-serotonin without lactic acid dehydrogenase. Both the lytic and the activating principles were separable from slow reacting substance of anaphylaxis and polymorphonuclear leucocyte chemotactic activity, and each presented a single activity peak of differing mobility when chromatographed on silica gel H plates. Human eosinophil phospholipase D inactivated the lytic factor by more than 85% in 2 h at 37 degrees without affecting the activity of the activating factor. The release of [14C]-serotonin induced by the PAF was not affected by the absence of calcium from the medium or by elevations in the platelet concentrations of cyclic AMP or cyclic GMP that resulted from pre-incubation of platelets with prostaglandin D2 or sodium ascorbate, respectively. PMID:227784

  10. Cell-wall-bound lytic activity in Chlorella fusca: function and characterization of an endo-mannanase.

    PubMed

    Loos, E; Meindl, D

    1985-12-01

    A cell-wall-degrading activity was solubilized from young cells and from mother cell walls of Chlorella fusca by treatment with LiCl. The cytoplasmic enzyme hexokinase was not detectable in these extracts. The LiCl-solubilized activity increased in the cell cycle parallel to the release of autospores. The enzyme was purified on a chromatofocusing column followed by gel filtration. Sodium dodecyl sulfate/polyacryl amide gel electrophoresis of the purified enzyme revealed a molecular weight of 44 kDa, whereas gel filtration indicated a molecular weight of 25 kDa. Cell-wall-lytic activity and β-1,4-mannanase activity coeluted in gel filtration and were separated from β-D-fucosidase activity. The enzyme degraded isolated cell walls and ivory nut mannan primarily to oligosaccharides with an estimated degree of polymerization ≧6. The soluble degradation products of the cell wall consisted of 92-96% mannose and 4-8% glucose. It is concluded that the cell-wall-lytic activity is caused by an endo-mannanase. In vivo, this enzyme probably degrades the mother cell wall and, after autospore release, remains bound to it as well as to the surface of the daughter cells by ionic forces. The identity of this bound enzyme with a soluble wall-degrading enzyme previously obtained from mother cells is discussed. PMID:24241623

  11. 6-Shogaol induces cell cycle arrest and apoptosis in human hepatoma cells through pleiotropic mechanisms.

    PubMed

    Wu, Jung-Ju; Omar, Hany A; Lee, Ying-Ray; Teng, Yen-Ni; Chen, Pin-Shern; Chen, Yu-Chung; Huang, Hsiao-Shan; Lee, Kuan-Han; Hung, Jui-Hsiang

    2015-09-01

    Shogaols are a group of the active constituents of ginger that have been identified to have various biological activities. The aim of the current study was to investigate the antitumor activity of 6-shogaol in hepatocellular carcinoma (HCC) and the possible involvement of reactive oxygen species as a putative mechanism of action. HCC cell lines, HepG2 and Huh-7, were used to study the in vitro anti-cancer activity of 6-shogaol via the application of various molecular biology techniques. Results showed that 6-shogaol effectively inhibited the cell viability, caused cell cycle arrest at G2/M phase and induced apoptosis in HCC cells as indicated by MTT assay, DAPI nuclear staining, annexin V assay, cell cycle analysis, and activation of caspase-3. Western blot analysis revealed the ability of 6-shogaol to target cancer survival signaling pathways mediated by mitogen-activated protein kinase (MAPK), 5' AMP-activated protein kinase (AMPK) and Akt. In addition, 6-Shogaol induced alteration of cyclin proteins expression and caused cleavage of protein kinase C delta. Furthermore, 6-Shogaol was able to induce the production of reactive oxygen species and endoplasmic reticulum (ER) stress-associated proteins and the consequent activation of autophagy in HepG2 cells. Taken together, the current study highlights evidences that 6-shogaol induces apoptosis, modulates cyclins expression and targets cancer survival signaling pathways in HCC cell lines, at least in part, via the production of reactive oxygen species. These findings support 6-shogaol's clinical promise as a potential candidate for HCC therapy. PMID:26101062

  12. In vitro atrazine-exposure inhibits human natural killer cell lytic granule release

    SciTech Connect

    Rowe, Alexander M.; Brundage, Kathleen M.; Barnett, John B. . E-mail: jbarnett@hsc.wvu.edu

    2007-06-01

    The herbicide atrazine is a known immunotoxicant and an inhibitor of human natural killer (NK) cell lytic function. The precise changes in NK cell lytic function following atrazine exposure have not been fully elucidated. The current study identifies the point at which atrazine exerts its affect on the stepwise process of human NK cell-mediated lyses of the K562 target cell line. Using intracellular staining of human peripheral blood lymphocytes, it was determined that a 24-h in vitro exposure to atrazine did not decrease the level of NK cell lytic proteins granzyme A, granzyme B or perforin. Thus, it was hypothesized that atrazine exposure was inhibiting the ability of the NK cells to bind to the target cell and subsequently inhibit the release of lytic protein from the NK cell. To test this hypothesis, flow cytometry and fluorescent microscopy were employed to analyze NK cell-target cell co-cultures following atrazine exposure. These assays demonstrated no significant decrease in the level of target cell binding. However, the levels of NK intracellular lytic protein retained and the amount of lytic protein released were assessed following a 4-h incubation with K562 target cells. The relative level of intracellular lytic protein was 25-50% higher, and the amount of lytic protein released was 55-65% less in atrazine-treated cells than vehicle-treated cells following incubation with the target cells. These results indicate that ATR exposure inhibits the ability of NK cells to lyse target cells by blocking lytic granule release without affecting the ability of the NK cell to form stable conjugates with target cells.

  13. Conditional inactivation of PDCD2 induces p53 activation and cell cycle arrest.

    PubMed

    Granier, Celine J; Wang, Wei; Tsang, Tiffany; Steward, Ruth; Sabaawy, Hatem E; Bhaumik, Mantu; Rabson, Arnold B

    2014-01-01

    PDCD2 (programmed cell death domain 2) is a highly conserved, zinc finger MYND domain-containing protein essential for normal development in the fly, zebrafish and mouse. The molecular functions and cellular activities of PDCD2 remain unclear. In order to better understand the functions of PDCD2 in mammalian development, we have examined PDCD2 activity in mouse blastocyst embryos, as well as in mouse embryonic stem cells (ESCs) and embryonic fibroblasts (MEFs). We have studied mice bearing a targeted PDCD2 locus functioning as a null allele through a splicing gene trap, or as a conditional knockout, by deletion of exon2 containing the MYND domain. Tamoxifen-induced knockout of PDCD2 in MEFs, as well as in ESCs, leads to defects in progression from the G1 to the S phase of cell cycle, associated with increased levels of p53 protein and p53 target genes. G1 prolongation in ESCs was not associated with induction of differentiation. Loss of entry into S phase of the cell cycle and marked induction of nuclear p53 were also observed in PDCD2 knockout blastocysts. These results demonstrate a unique role for PDCD2 in regulating the cell cycle and p53 activation during early embryonic development of the mouse. PMID:25150276

  14. PDMP sensitizes neuroblastoma to paclitaxel by inducing aberrant cell cycle progression leading to hyperploidy.

    PubMed

    Dijkhuis, Anne-Jan; Klappe, Karin; Jacobs, Susan; Kroesen, Bart-Jan; Kamps, Willem; Sietsma, Hannie; Kok, Jan Willem

    2006-03-01

    The sphingolipid ceramide has been recognized as an important mediator in the apoptotic machinery, and its efficient conversion to glucosylceramide has been associated with multidrug resistance. Therefore, inhibitors of glucosylceramide synthase are explored as tools for treatment of cancer. In this study, we used D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol to sensitize Neuro-2a murine neuroblastoma cells to the microtubule-stabilizing agent paclitaxel. This treatment resulted in a synergistic inhibition of viable cell number increase, which was based on a novel mechanism: (a) After a transient mitotic arrest, cells proceeded through an aberrant cell cycle resulting in hyperploidy. Apoptosis also occurred but to a very limited extent. (b) Hyperploidy was not abrogated by blocking de novo sphingolipid biosynthesis using ISP-1, ruling out involvement of ceramide as a mediator. (c) Cyclin-dependent kinase 1 and 2 activities were synergistically decreased on treatment. In conclusion, instead of inducing apoptosis through ceramide accumulation, D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol by itself affects cell cycle-related proteins in paclitaxel-arrested Neuro-2a cells resulting in aberrant cell cycle progression leading to hyperploidy. PMID:16546973

  15. Conditional inactivation of PDCD2 induces p53 activation and cell cycle arrest

    PubMed Central

    Granier, Celine J.; Wang, Wei; Tsang, Tiffany; Steward, Ruth; Sabaawy, Hatem E.; Bhaumik, Mantu; Rabson, Arnold B.

    2014-01-01

    ABSTRACT PDCD2 (programmed cell death domain 2) is a highly conserved, zinc finger MYND domain-containing protein essential for normal development in the fly, zebrafish and mouse. The molecular functions and cellular activities of PDCD2 remain unclear. In order to better understand the functions of PDCD2 in mammalian development, we have examined PDCD2 activity in mouse blastocyst embryos, as well as in mouse embryonic stem cells (ESCs) and embryonic fibroblasts (MEFs). We have studied mice bearing a targeted PDCD2 locus functioning as a null allele through a splicing gene trap, or as a conditional knockout, by deletion of exon2 containing the MYND domain. Tamoxifen-induced knockout of PDCD2 in MEFs, as well as in ESCs, leads to defects in progression from the G1 to the S phase of cell cycle, associated with increased levels of p53 protein and p53 target genes. G1 prolongation in ESCs was not associated with induction of differentiation. Loss of entry into S phase of the cell cycle and marked induction of nuclear p53 were also observed in PDCD2 knockout blastocysts. These results demonstrate a unique role for PDCD2 in regulating the cell cycle and p53 activation during early embryonic development of the mouse. PMID:25150276

  16. Oridonin inhibits tumor growth in glioma by inducing cell cycle arrest and apoptosis.

    PubMed

    Zhang, X-H; Liu, Y-X; Jia, M; Han, J-S; Zhao, M; Ji, S-P; Li, A-M

    2014-01-01

    Glioma is the most common malignant intracranial tumors. Despite newly developed therapies, these treatments mainly target oncogenic signals, and unfortunately, fail to provide enough survival benefit in both human patients and mouse xenograft models, especially the first-generation therapies. Oridonin is purified from the Chinese herb Rabdosia rubescens and considered to exert extensive anti-cancer effects on human tumorigenesis. In this study, we systemically investigated the role of Oridonin in tumor growth and the underlying mechanisms in human glioma. We found that Oridonin inhibited cell proliferations in a dose- and time-dependent manner in both glioma U87 and U251 cells. Moreover, these anti-cancer effects were also confirmed in a mouse model bearing glioma. Furthermore, cell cycle arrest in S phase was observed in Oridonin-mediated growth inhibition by flow cytometry. Cell cycle arrest in S phase led to eventual cell apoptosis, as revealed by Hoechst 33342 staining and annexin V/PI double-staining. The cell apoptosis might be accomplished through a mitochondrial manner. In all, we were the first to our knowledge to report that Oridonin could exert anti-cancer effects on tumor growth in human glioma by inducing cell cycle arrest and eventual cell apoptosis. The identification of Oridonin as a critical mediator of glioma growth may potentiate Oridonin as a novel therapeutic strategies in glioma treatments. PMID:25553351

  17. SOX10 ablation arrests the cell cycle, induces senescence and suppresses melanomagenesis

    PubMed Central

    Cronin, Julia C.; Watkins-Chow, Dawn E.; Incao, Art; Hasskamp, Joanne H.; Schönewolf, Nicola; Aoude, Lauren G.; Hayward, Nicholas K.; Bastian, Boris C.; Dummer, Reinhard; Loftus, Stacie K.; Pavan, William J.

    2013-01-01

    The transcription factor SOX10 is essential for survival and proper differentiation of neural crest cell lineages, where it plays an important role in the generation and maintenance of melanocytes. SOX10 is also highly expressed in melanoma tumors, but a role in disease progression has not been established. Here we report that melanoma tumor cell lines require wild-type SOX10 expression for proliferation, and SOX10 haploinsufficiency reduces melanoma initiation in the metabotropic glutamate receptor 1 (Grm1Tg) transgenic mouse model. Stable SOX10 knockdown in human melanoma cells arrested cell growth, altered cellular morphology, and induced senescence. Melanoma cells with stable loss of SOX10 were arrested in the G1 phase of the cell cycle, with reduced expression in the melanocyte determining factor MITF, elevated expression of p21WAF1 and p27KIP2, hypophosphorylated RB and reduced levels of its binding partner E2F1. Since cell cycle dysregulation is a core event in neoplastic transformation, the role for SOX10 in maintaining cell cycle control in melanocytes suggests a rational new direction for targeted treatment or prevention of melanoma. PMID:23913827

  18. Methamphetamine Alters the Normal Progression by Inducing Cell Cycle Arrest in Astrocytes

    PubMed Central

    Jackson, Austin R.; Shah, Ankit; Kumar, Anil

    2014-01-01

    Methamphetamine (MA) is a potent psychostimulant with a high addictive capacity, which induces many deleterious effects on the brain. Chronic MA abuse leads to cognitive dysfunction and motor impairment. MA affects many cells in the brain, but the effects on astrocytes of repeated MA exposure is not well understood. In this report, we used Gene chip array to analyze the changes in the gene expression profile of primary human astrocytes treated with MA for 3 days. Range of genes were found to be differentially regulated, with a large number of genes significantly downregulated, including NEK2, TTK, TOP2A, and CCNE2. Gene ontology and pathway analysis showed a highly significant clustering of genes involved in cell cycle progression and DNA replication. Further pathway analysis showed that the genes downregulated by multiple MA treatment were critical for G2/M phase progression and G1/S transition. Cell cycle analysis of SVG astrocytes showed a significant reduction in the percentage of cell in the G2/M phase with a concomitant increase in G1 percentage. This was consistent with the gene array and validation data, which showed that repeated MA treatment downregulated the genes associated with cell cycle regulation. This is a novel finding, which explains the effect of MA treatment on astrocytes and has clear implication in neuroinflammation among the drug abusers. PMID:25290377

  19. Complete Genome Sequences of Lytic Bacteriophages of Xanthomonas arboricola pv. Juglandis

    PubMed Central

    Vasquez, Ignacio; Santos, Leonardo; Segovia, Cristopher; Ayala, Manuel; Alvarado, Romina; Nuñez, Pablo

    2016-01-01

    Three bacteriophages, f20-Xaj, f29-Xaj, and f30-Xaj, with lytic activity against Xanthomonas arboricola pv. juglandis were isolated from walnut trees (VIII Bío Bío Region, Chile). These lytic bacteriophages have double-stranded DNA (dsDNA) genomes of 43,851 bp, 41,865 bp, and 44,262 bp, respectively. These are the first described bacteriophages with lytic activity against X. arboricola pv. juglandis that can be utilized as biocontrol agents. PMID:27257210

  20. Complete Genome Sequences of Lytic Bacteriophages of Xanthomonas arboricola pv. juglandis.

    PubMed

    Retamales, Julio; Vasquez, Ignacio; Santos, Leonardo; Segovia, Cristopher; Ayala, Manuel; Alvarado, Romina; Nuñez, Pablo; Santander, Javier

    2016-01-01

    Three bacteriophages, f20-Xaj, f29-Xaj, and f30-Xaj, with lytic activity against Xanthomonas arboricola pv. juglandis were isolated from walnut trees (VIII Bío Bío Region, Chile). These lytic bacteriophages have double-stranded DNA (dsDNA) genomes of 43,851 bp, 41,865 bp, and 44,262 bp, respectively. These are the first described bacteriophages with lytic activity against X. arboricola pv. juglandis that can be utilized as biocontrol agents. PMID:27257210

  1. The small noncoding RNAs (sncRNAs) of murine gammaherpesvirus 68 (MHV-68) are involved in regulating the latent-to-lytic switch in vivo

    PubMed Central

    Steer, Beatrix; Strehle, Martin; Sattler, Christine; Bund, Dagmar; Flach, Britta; Stoeger, Tobias; Haas, Jürgen G.; Adler, Heiko

    2016-01-01

    The human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV), which are associated with a variety of diseases including tumors, produce various small noncoding RNAs (sncRNAs) such as microRNAs (miRNAs). Like all herpesviruses, they show two stages in their life cycle: lytic replication and latency. During latency, hardly any viral proteins are expressed to avoid recognition by the immune system. Thus, sncRNAs might be exploited since they are less likely to be recognized. Specifically, it has been proposed that sncRNAs might contribute to the maintenance of latency. This has already been shown in vitro, but the respective evidence in vivo is very limited. A natural model system to explore this question in vivo is infection of mice with murine gammaherpesvirus 68 (MHV-68). We used this model to analyze a MHV-68 mutant lacking the expression of all miRNAs. In the absence of the miRNAs, we observed a higher viral genomic load during late latency in the spleens of mice. We propose that this is due to a disturbed regulation of the latent-to-lytic switch, altering the balance between latent and lytic infection. Hence, we provide for the first time evidence that gammaherpesvirus sncRNAs contribute to the maintenance of latency in vivo. PMID:27561205

  2. The small noncoding RNAs (sncRNAs) of murine gammaherpesvirus 68 (MHV-68) are involved in regulating the latent-to-lytic switch in vivo.

    PubMed

    Steer, Beatrix; Strehle, Martin; Sattler, Christine; Bund, Dagmar; Flach, Britta; Stoeger, Tobias; Haas, Jürgen G; Adler, Heiko

    2016-01-01

    The human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), which are associated with a variety of diseases including tumors, produce various small noncoding RNAs (sncRNAs) such as microRNAs (miRNAs). Like all herpesviruses, they show two stages in their life cycle: lytic replication and latency. During latency, hardly any viral proteins are expressed to avoid recognition by the immune system. Thus, sncRNAs might be exploited since they are less likely to be recognized. Specifically, it has been proposed that sncRNAs might contribute to the maintenance of latency. This has already been shown in vitro, but the respective evidence in vivo is very limited. A natural model system to explore this question in vivo is infection of mice with murine gammaherpesvirus 68 (MHV-68). We used this model to analyze a MHV-68 mutant lacking the expression of all miRNAs. In the absence of the miRNAs, we observed a higher viral genomic load during late latency in the spleens of mice. We propose that this is due to a disturbed regulation of the latent-to-lytic switch, altering the balance between latent and lytic infection. Hence, we provide for the first time evidence that gammaherpesvirus sncRNAs contribute to the maintenance of latency in vivo. PMID:27561205

  3. The Involvement of Acetaldehyde in Ethanol-Induced Cell Cycle Impairment

    PubMed Central

    Scheer, Marc A.; Schneider, Katrina J.; Finnigan, Rochelle L.; Maloney, Eamon P.; Wells, Mark A.; Clemens, Dahn L.

    2016-01-01

    Background: Hepatocytes metabolize the vast majority of ingested ethanol. This metabolic activity results in hepatic toxicity and impairs the ability of hepatocytes to replicate. Previous work by our group has shown that ethanol metabolism results in a G2/M cell cycle arrest. The intent of these studies was to discern the roles of acetaldehyde and reactive oxygen, two of the major by-products of ethanol metabolism, in the G2/M cell cycle arrest. Methods: To investigate the role of ethanol metabolites in the cell cycle arrest, VA-13 and VL-17A cells were used. These are recombinant Hep G2 cells that express alcohol dehydrogenase or alcohol dehydrogenase and cytochrome P450 2E1, respectively. Cells were cultured with or without ethanol, lacking or containing the antioxidants N-acetylcysteine (NAC) or trolox, for three days. Cellular accumulation was monitored by the DNA content of the cultures. The accumulation of the cyclin-dependent kinase, Cdc2 in the inactive phosphorylated form (p-Cdc2) and the cyclin-dependent kinase inhibitor p21 were determined by immunoblot analysis. Results: Cultures maintained in the presence of ethanol demonstrated a G2/M cell cycle arrest that was associated with a reduction in DNA content and increased levels of p-Cdc2 and p21, compared with cells cultured in its absence. Inclusion of antioxidants in the ethanol containing media was unable to rescue the cells from the cell cycle arrest or these ethanol metabolism-mediated effects. Additionally, culturing the cells in the presence of acetaldehyde alone resulted in increased levels of p-Cdc2 and p21. Conclusions: Acetaldehyde produced during ethanol oxidation has a major role in the ethanol metabolism-mediated G2/M cell cycle arrest, and the concurrent accumulation of p21 and p-Cdc2. Although reactive oxygen species are thought to have a significant role in ethanol-induced hepatocellular damage, they may have a less important role in the inability of hepatocytes to replace dead or damaged

  4. Toxicity of drinking water disinfection byproducts: cell cycle alterations induced by the monohaloacetonitriles.

    PubMed

    Komaki, Yukako; Mariñas, Benito J; Plewa, Michael J

    2014-10-01

    Haloacetonitriles (HANs) are a chemical class of drinking water disinfection byproducts (DBPs) that form from reactions between disinfectants and nitrogen-containing precursors, the latter more prevalent in water sources impacted by algae bloom and municipal wastewater effluent discharge. HANs, previously demonstrated to be genotoxic, were investigated for their effects on the mammalian cell cycle. Treating Chinese hamster ovary (CHO) cells with monoHANs followed by the release from the chemical treatment resulted in the accumulation of abnormally high DNA content in cells over time (hyperploid). The potency for the cell cycle alteration followed the order: iodoacetonitrile (IAN) > bromoacetonitrile (BAN) ≫ chloroacetonitrile (CAN). Exposure to 6 μM IAN, 12 μM BAN and 900 μM CAN after 26 h post-treatment incubation resulted in DNA repair; however, subsequent cell cycle alteration effects were observed. Cell proliferation of HAN-treated cells was suppressed for as long as 43 to 52 h. Enlarged cell size was observed after 52 h post-treatment incubation without the induction of cytotoxicity. The HAN-mediated cell cycle alteration was mitosis- and proliferation-dependent, which suggests that HAN treatment induced mitosis override, and that HAN-treated cells proceeded into S phase and directly into the next cell cycle. Cells with multiples genomes would result in aneuploidy (state of abnormal chromosome number and DNA content) at the next mitosis since extra centrosomes could compromise the assembly of bipolar spindles. There is accumulating evidence of a transient tetraploid state proceeding to aneuploidy in cancer progression. Biological self-defense systems to ensure genomic stability and to eliminate tetraploid cells exist in eukaryotic cells. A key tumor suppressor gene, p53, is oftentimes mutated in various types of human cancer. It is possible that HAN disruption of the normal cell cycle and the generation of aberrant cells with an abnormal number of

  5. Galiellalactone induces cell cycle arrest and apoptosis through the ATM/ATR pathway in prostate cancer cells

    PubMed Central

    García, Víctor; Lara-Chica, Maribel; Cantarero, Irene; Sterner, Olov; Calzado, Marco A.; Muñoz, Eduardo

    2016-01-01

    Galiellalactone (GL) is a fungal metabolite that presents antitumor activities on prostate cancer in vitro and in vivo. In this study we show that GL induced cell cycle arrest in G2/M phase, caspase-dependent apoptosis and also affected the microtubule organization and migration ability in DU145 cells. GL did not induce double strand DNA break but activated the ATR and ATM-mediated DNA damage response (DDR) inducing CHK1, H2AX phosphorylation (fH2AX) and CDC25C downregulation. Inhibition of the ATM/ATR activation with caffeine reverted GL-induced G2/M cell cycle arrest, apoptosis and DNA damage measured by fH2AX. In contrast, UCN-01, a CHK1 inhibitor, prevented GL-induced cell cycle arrest but enhanced apoptosis in DU145 cells. Furthermore, we found that GL did not increase the levels of intracellular ROS, but the antioxidant N-acetylcysteine (NAC) completely prevented the effects of GL on fH2AX, G2/M cell cycle arrest and apoptosis. In contrast to NAC, other antioxidants such as ambroxol and EGCG did not interfere with the activity of GL on cell cycle. GL significantly suppressed DU145 xenograft growth in vivo and induced the expression of fH2AX in the tumors. These findings identify for the first time that GL activates DDR in prostate cancer. PMID:26683224

  6. GnRH-agonist induced depressive and anxiety symptoms during in vitro fertilization-embryo transfer cycles.

    PubMed

    Bloch, Miki; Azem, Foad; Aharonov, Inbar; Ben Avi, Irit; Yagil, Yaron; Schreiber, Shaul; Amit, Ami; Weizman, Abraham

    2011-01-01

    To determine whether the use of a GnRH agonist inducing a hypogonadic state during IVF-ET cycles induces negative mood symptoms, we conducted a prospective randomized study in 108 women comparing two different controlled ovarian stimulation protocols. A significant phase effect was observed for depression and anxiety symptoms during IVF-ET cycles reflecting an increase in symptoms between the hypogonadal phase and the peak in gonadotropin stimulation; however, the hypogonadal phase induced by the GnRH agonist was not associated with a significant increase in any of the studied mood parameters. PMID:20801439

  7. Self-cycling operation increases productivity of recombinant protein in Escherichia coli.

    PubMed

    Storms, Zachary J; Brown, Tobin; Sauvageau, Dominic; Cooper, David G

    2012-09-01

    Self-cycling fermentation (SCF), a cyclical, semi-continuous process that induces cell synchrony, was incorporated into a recombinant protein production scheme. Escherichia coli CY15050, a lac(-) mutant lysogenized with temperature-sensitive phage λ modified to over-express β-galactosidase, was used as a model system. The production scheme was divided into two de-coupled stages. The host cells were cultured under SCF operation in the first stage before being brought to a second stage where protein production was induced. In the first stage, the host strain demonstrated a stable cycling pattern immediately following the first cycle. This reproducible pattern was maintained over the course of the experiments and a significant degree of cell synchrony was obtained. By growing cells using SCF, productivity increased 50% and production time decreased by 40% compared to a batch culture under similar conditions. In addition, synchronized cultures induced from the end of a SCF cycle displayed shorter lysis times and a more complete culture-wide lysis than unsynchronized cultures. Finally, protein synthesis was influenced by the time at which the lytic phase was induced in the cell life cycle. For example, induction of a synchronized culture immediately prior to cell division resulted in the maximum protein productivity, suggesting protein production can be optimized with respect to the cell life cycle using SCF. PMID:22407770

  8. Unprecedented inhibition of tubulin polymerization directed by gold nanoparticles inducing cell cycle arrest and apoptosis

    NASA Astrophysics Data System (ADS)

    Choudhury, Diptiman; Xavier, Paulrajpillai Lourdu; Chaudhari, Kamalesh; John, Robin; Dasgupta, Anjan Kumar; Pradeep, Thalappil; Chakrabarti, Gopal

    2013-05-01

    The effect of gold nanoparticles (AuNPs) on the polymerization of tubulin has not been examined till now. We report that interaction of weakly protected AuNPs with microtubules (MTs) could cause inhibition of polymerization and aggregation in the cell free system. We estimate that single citrate capped AuNPs could cause aggregation of ~105 tubulin heterodimers. Investigation of the nature of inhibition of polymerization and aggregation by Raman and Fourier transform-infrared (FTIR) spectroscopies indicated partial conformational changes of tubulin and microtubules, thus revealing that AuNP-induced conformational change is the driving force behind the observed phenomenon. Cell culture experiments were carried out to check whether this can happen inside a cell. Dark field microscopy (DFM) combined with hyperspectral imaging (HSI) along with flow cytometric (FC) and confocal laser scanning microscopic (CLSM) analyses suggested that AuNPs entered the cell, caused aggregation of the MTs of A549 cells, leading to cell cycle arrest at the G0/G1 phase and concomitant apoptosis. Further, Western blot analysis indicated the upregulation of mitochondrial apoptosis proteins such as Bax and p53, down regulation of Bcl-2 and cleavage of poly(ADP-ribose) polymerase (PARP) confirming mitochondrial apoptosis. Western blot run after cold-depolymerization revealed an increase in the aggregated insoluble intracellular tubulin while the control and actin did not aggregate, suggesting microtubule damage induced cell cycle arrest and apoptosis. The observed polymerization inhibition and cytotoxic effects were dependent on the size and concentration of the AuNPs used and also on the incubation time. As microtubules are important cellular structures and target for anti-cancer drugs, this first observation of nanoparticles-induced protein's conformational change-based aggregation of the tubulin-MT system is of high importance, and would be useful in the understanding of cancer therapeutics

  9. Carrageenan Induces Cell Cycle Arrest in Human Intestinal Epithelial Cells in Vitro1–3

    PubMed Central

    Bhattacharyya, Sumit; Borthakur, Alip; Dudeja, Pradeep K.; Tobacman, Joanne K.

    2016-01-01

    Multiple studies in animal models have shown that the commonly used food additive carrageenan (CGN) induces inflammation and intestinal neoplasia. We performed the first studies to determine the effects of CGN exposure on human intestinal epithelial cells (IEC) in tissue culture and tested the effect of very low concentrations (1–10 mg/L) of undegraded, high-molecular weight CGN. These concentrations of CGN are less than the anticipated exposure of the human colon to CGN from the average Western diet. In the human colonic epithelial cell line NCM460 and in primary human colonic epithelial cells that were exposed to CGN for 1–8 d, we found increased cell death, reduced cell proliferation, and cell cycle arrest compared with unexposed control cells. After 6–8 d of CGN exposure, the percentage of cells reentering G0–G1 significantly decreased and the percentages of cells in S and G2-M phases significantly increased. Increases in activated p53, p21, and p15 followed CGN exposure, consistent with CGN-induced cell cycle arrest. Additional data, including DNA ladder, poly ADP ribose polymerase Western blot, nuclear DNA staining, and activities of caspases 3 and 7, indicated no evidence of increased apoptosis following CGN exposure and were consistent with CGN-induced necrotic cell death. These data document for the first time, to our knowledge, marked adverse effects of low concentrations of CGN on survival of normal human IEC and suggest that CGN exposure may have a role in development of human intestinal pathology. PMID:18287351

  10. Arecoline induced cell cycle arrest, apoptosis, and cytotoxicity to human endothelial cells.

    PubMed

    Tseng, Shuei-Kuen; Chang, Mei-Chi; Su, Cheng-Yao; Chi, Lin-Yang; Chang, Jenny Zwei-Ching; Tseng, Wan-Yu; Yeung, Sin-Yuet; Hsu, Ming-Lun; Jeng, Jiiang-Huei

    2012-08-01

    Betel quid (BQ) chewing is a common oral habit in South Asia and Taiwan. BQ consumption may increase the risk of oral squamous cell carcinoma (OSCC), oral submucous fibrosis (OSF), and periodontitis as well as systemic diseases (atherosclerosis, hypertension, etc.). However, little is known about the toxic effect of BQ components on endothelial cells that play important roles for angiogenesis, carcinogenesis, tissue fibrosis, and cardiovascular diseases. EAhy 926 (EAHY) endothelial cells were exposed to arecoline, a major BQ alkaloid, for various time periods. Cytotoxicity was estimated by 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay. The cell cycle distribution of EAHY cells residing in sub-G0/G1, G0/G1, S-, and G2/M phases was analyzed by propidium iodide staining of cellular DNA content and flow cytometry. Some EAHY cells retracted, became round-shaped in appearance, and even detached from the culture plate after exposure to higher concentrations of arecoline (> 0.4 mM). At concentrations of 0.4 and 0.8 mM, arecoline induced significant cytotoxicity to EAHY cells. At similar concentrations, arecoline induced G2/M cell cycle arrest and increased sub-G0/G1 population, a hallmark of apoptosis. Interestingly, prolonged exposure to arecoline (0.1 mM) for 12 and 21 days significantly suppressed the proliferation of EAHY cells, whereas EAHY cells showed adaptation and survived when exposed to 0.05 mM arecoline. These results suggest that BQ components may contribute to the pathogenesis of OSF and BQ chewing-related cardiovascular diseases via toxicity to oral or systemic endothelial cells, leading to impairment of vascular function. During BQ chewing, endothelial damage may be induced by areca nut components and associate with the pathogenesis of OSF, periodontitis, and cardiovascular diseases. PMID:21847594

  11. Cell Cycle-Dependent Mechanisms Underlie Vincristine-Induced Death of Primary Acute Lymphoblastic Leukemia Cells.

    PubMed

    Kothari, Anisha; Hittelman, Walter N; Chambers, Timothy C

    2016-06-15

    Microtubule-targeting agents (MTA), such as the taxanes and vinca alkaloids, are used to treat a variety of cancers due to their ability to perturb microtubule dynamics. In cell culture, MTAs exert their anticancer effects primarily by causing mitotic arrest and cell death. However, accumulating indirect evidence suggests that MTAs may exert their cytotoxicity in human tumors by interfering with interphase microtubules. In this study, we sought to develop and characterize an experimental system in which to test the hypothesis that MTAs induce cell death during interphase. Primary adult acute lymphoblastic leukemia (ALL) cells treated with vincristine only weakly exhibited colocalization between mitotic and apoptotic markers and major characteristics of mitotic death, such as an increase in cells with 4N DNA content before the appearance of cells with <2N DNA content, suggesting a mixed response. Therefore, we separated ALL cells into distinct phases of the cell cycle by centrifugal elutriation, labeled cells with 5-ethynyl-2'-deoxyuridine (EdU), and then treated each population with vincristine. Cells isolated during G1 underwent cell death without evidence of EdU uptake, indicating that the cytotoxic effects of vincristine took place during G1 Conversely, cells isolated during S or G2-M phases underwent death following mitotic arrest. Thus, vincristine induces distinct death programs in primary ALL cells depending on cell-cycle phase, and cells in G1 are particularly susceptible to perturbation of interphase microtubules. Primary ALL cells may therefore provide a powerful model system in which to study the multimodal mechanisms underlying MTA-induced cell death. Cancer Res; 76(12); 3553-61. ©2016 AACR. PMID:27197148

  12. Low viral predation pressure in cold hypersaline Arctic sediments and limits on lytic replication.

    PubMed

    Colangelo-Lillis, Jesse; Wing, Boswell A; Whyte, Lyle G

    2016-04-01

    Viruses are ubiquitous drivers of microbial ecology and evolution and contribute to biogeochemical cycling. Attention to these attributes has been more substantial for marine viruses than viruses of other environments. Microscopy-based investigation of the viral communities from two cold, hypersaline Arctic springs was undertaken to explore the effects of these conditions on microbe-viral ecology. Sediments and water samples were collected along transects from each spring, from anoxic spring outlets through oxygenated downstream channels. Viral abundance, virus-microbe ratios and modelled virus-microbe contact rates were lower than comparable aqueous and sedimentary environments and most similar to deep subsurface sediments. No individual cell from either spring was visibly infected. Viruses in these springs appear to play a smaller role in controlling microbial populations through lytic activity than in marine water column or surface sedimentary environments. Relief from viral predation indicates the microbial communities are primarily controlled by nutrient limitation. The similarity of these springs to deep subsurface sediments suggests a biogeographic divide in viral replication strategy in marine sediments. PMID:26743115

  13. Exogenous lytic activity of SPN9CC endolysin against gram-negative bacteria.

    PubMed

    Lim, Jeong-A; Shin, Hakdong; Heu, Sunggi; Ryu, Sangryeol

    2014-06-28

    Concerns over drug-resistant bacteria have stimulated interest in developing alternative methods to control bacterial infections. Endolysin, a phage-encoded enzyme that breaks down bacterial peptidoglycan at the terminal stage of the phage reproduction cycle, is reported to be effective for the control of bacterial pathogenic bacteria. Bioinformatic analysis of the SPN9CC bacteriophage genome revealed a gene that encodes an endolysin with a domain structure similar to those of the endolysins produced by the P1 and P22 coliphages. The SPN9CC endolysin was purified with a C-terminal oligo-histidine tag. The endolysin was relatively stable and active over a broad temperature range (from 24°C to 65°C). It showed maximal activity at 50°C, and its optimum pH range was from pH 7.5 to 8.5. The SPN9CC endolysin showed antimicrobial activity against only gram-negative bacteria and functioned by cutting the glycosidic bond of peptidoglycan. Interestingly, the SPN9CC endolysin could lyse intact gram-negative bacteria in the absence of EDTA as an outer membrane permeabilizer. The exogenous lytic activity of the SPN9CC endolysin makes it a potential therapeutic agent against gram-negative bacteria. PMID:24690638

  14. Simulations of airglow variations induced by the CO2 increase and solar cycle variation from 1980 to 1991

    NASA Astrophysics Data System (ADS)

    Huang, Tai-Yin

    2016-09-01

    Airglow intensity and Volume Emission Rate (VER) variations induced by the increase of CO2 gas concentration and F10.7 variation (used as a proxy for the 11-year solar cycle variation) were investigated for the period from 1980 to 1991, encompassing a full solar cycle. Two airglow models are used to simulate the induced variations of O(1S) greenline, O2(0,1) atmospheric band , and OH(8,3) airglow for this study. The results show that both the airglow intensities and peak VERs correlate positively with the F10.7 solar cycle variation and display a small linear trend due to the increase of CO2 gas concentration. The solar-cycle induced airglow intensity variations show that O(1S) greenline has the largest variation (~26%) followed by the O2(0,1) atmospheric band (~23%) and then OH(8,3) airglow (~8%) over the 11 year timespan. The magnitudes of the induced airglow intensity variations by the increase of CO2 gas concentration are about an order of magnitude smaller than those by the F10.7 solar cycle variation. In general, the F10.7 solar cycle variation and CO2 increase do not seem to systematically alter the VER peak altitude of the airglow emissions, though the OH(8,3) VER peak altitude moves up slightly during the years when the F10.7 value falls under 100 SFU.

  15. Respiratory syncytial virus matrix protein induces lung epithelial cell cycle arrest through a p53 dependent pathway.

    PubMed

    Bian, Tao; Gibbs, John D; Örvell, Claes; Imani, Farhad

    2012-01-01

    Respiratory syncytial virus (RSV) is the major cause of viral respiratory infections in children. Our previous study showed that the RSV infection induced lung epithelial cell cycle arrest, which enhanced virus replication. To address the mechanism of RSV-induced cell cycle arrest, we examined the contribution of RSV-matrix (RSV-M) protein. In this report, we show that in both the A549 cell line and primary human bronchial epithelial (PHBE) cells, transfection with RSV-M protein caused the cells to proliferate at a slower rate than in control cells. The cell cycle analysis showed that RSV-M protein induced G1 phase arrest in A549 cells, and G1 and G2/M phase arrest in PHBE cells. Interestingly, RSV-M expression induced p53 and p21 accumulation and decreased phosphorylation of retinoblastoma protein (Rb). Further, induction of cell cycle arrest by RSV-M was not observed in a p53-deficient epithelial cell line (H1299). However, cell cycle arrest was restored after transfection of p53 cDNA into H1299 cells. Taken together, these results indicate that RSV-M protein regulates lung epithelial cell cycle through a p53-dependent pathway, which enhances RSV replication. PMID:22662266

  16. A novel mechanism inducing genome instability in Kaposi's sarcoma-associated herpesvirus infected cells.

    PubMed

    Jackson, Brian R; Noerenberg, Marko; Whitehouse, Adrian

    2014-05-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus associated with multiple AIDS-related malignancies. Like other herpesviruses, KSHV has a biphasic life cycle and both the lytic and latent phases are required for tumorigenesis. Evidence suggests that KSHV lytic replication can cause genome instability in KSHV-infected cells, although no mechanism has thus far been described. A surprising link has recently been suggested between mRNA export, genome instability and cancer development. Notably, aberrations in the cellular transcription and export complex (hTREX) proteins have been identified in high-grade tumours and these defects contribute to genome instability. We have previously shown that the lytically expressed KSHV ORF57 protein interacts with the complete hTREX complex; therefore, we investigated the possible intriguing link between ORF57, hTREX and KSHV-induced genome instability. Herein, we show that lytically active KSHV infected cells induce a DNA damage response and, importantly, we demonstrate directly that this is due to DNA strand breaks. Furthermore, we show that sequestration of the hTREX complex by the KSHV ORF57 protein leads to this double strand break response and significant DNA damage. Moreover, we describe a novel mechanism showing that the genetic instability observed is a consequence of R-loop formation. Importantly, the link between hTREX sequestration and DNA damage may be a common feature in herpesvirus infection, as a similar phenotype was observed with the herpes simplex virus 1 (HSV-1) ICP27 protein. Our data provide a model of R-loop induced DNA damage in KSHV infected cells and describes a novel system for studying genome instability caused by aberrant hTREX. PMID:24788796

  17. Characterization and quenching of friction-induced limit cycles of electro-hydraulic servovalve control systems with transport delay.

    PubMed

    Wang, Yuan-Jay

    2010-10-01

    This paper develops a systematic and straightforward methodology to characterize and quench the friction-induced limit cycle conditions in electro-hydraulic servovalve control systems with transport delay in the transmission line. The nonlinear friction characteristic is linearized by using its corresponding describing function. The delay time in the transmission line, which could accelerate the generation of limit cycles is particularly considered. The stability equation method together with parameter plane method provides a useful tool for the establishment of necessary conditions to sustain a limit cycle directly in the constructed controller coefficient plane. Also, the stable region, the unstable region, and the limit cycle region are identified in the parameter plane. The parameter plane characterizes a clear relationship between limit cycle amplitude, frequency, transport delay, and the controller coefficients to be designed. The stability of the predicted limit cycle is checked by plotting stability curves. The stability of the system is examined when the viscous gain changes with respect to the temperature of the working fluid. A feasible stable region is characterized in the parameter plane to allow a flexible choice of controller gains. The robust prevention of limit cycle is achieved by selecting controller gains from the asymptotic stability region. The predicted results are verified by simulations. It is seen that the friction-induced limit cycles can be effectively predicted, removed, and quenched via the design of the compensator even in the case of viscous gain and delay time variations unconditionally. PMID:20542269

  18. Hexabromocyclododecane Decreases the Lytic Function and ATP Levels of Human Natural Killer Cells

    PubMed Central

    Hinkson, Natasha C.; Whalen, Margaret M.

    2009-01-01

    This study investigates the effect of Hexabromocyclododecane (HBCD) on the lytic function of human natural killer (NK) cells and on ATP levels in NK cells. NK cells are capable of lysing tumor cells, virally infected cells, and antibody-coated cells. HBCD is a, brominated cyclic alkane used primarily as an additive flame retardant. If HBCD interferes with NK cell function, this could increase risk of tumor development and/or viral infection. NK cells were exposed to various concentrations of HBCD for 24 h, 48 h, and 6 days before determining lytic function and ATP levels. ATP levels and lytic function were also determined in NK cells that were exposed to HBCD for 1 h followed by 24 h, 48 h, and 6 days in HBCD-free media. The results indicated that exposure of NK cells to 10 μM HBCD for 24 h causes a very significant decrease in both NK cell lytic function and ATP levels (93.5% and 90.5%, respectively). Exposure of NK cells to 10 μM HBCD for 1 h followed by 24 h in HBCD-free media showed a progressive and persistent loss of lytic function (89.3%) as well as a decrease in ATP levels (46.1%). The results indicate that HBCD exposures decreased lytic function as well as ATP levels. However, a decrease in lytic function was not necessarily accompanied by a similar decrease in ATP. Importantly, these results also indicate that a brief (1 h) exposure to HBCD causes a progressive loss of lytic function over a 6 d period. PMID:19551757

  19. alpha-lytic protease can exist in two separately stable conformations with different His57 mobilities and catalytic activities.

    PubMed

    Haddad, Kristin Coffman; Sudmeier, James L; Bachovchin, Daniel A; Bachovchin, William W

    2005-01-25

    alpha-Lytic protease is a bacterial serine protease widely studied as a model system of enzyme catalysis. Here we report that lyophilization induces a structural change in the enzyme that is not reversed by redissolution in water. The structural change reduces the mobility of the active-site histidine residue and the catalytic activity of the enzyme. The application of mild pressure to solutions of the altered enzyme reverses the lyophilization-induced structural change and restores the mobility of the histidine residue and the enzyme's catalytic activity. This effect of lyophilization permits a unique opportunity for investigating the relationship between histidine ring dynamics and catalytic activity. The results demonstrate that His57 in resting enzymes is more mobile than previously thought, especially when protonated. The histidine motion and its correlation to enzyme activity lend support to the reaction-driven ring flip hypothesis. PMID:15657134

  20. α-Lytic protease can exist in two separately stable conformations with different His57 mobilities and catalytic activities

    PubMed Central

    Haddad, Kristin Coffman; Sudmeier, James L.; Bachovchin, Daniel A.; Bachovchin, William W.

    2005-01-01

    α-Lytic protease is a bacterial serine protease widely studied as a model system of enzyme catalysis. Here we report that lyophilization induces a structural change in the enzyme that is not reversed by redissolution in water. The structural change reduces the mobility of the active-site histidine residue and the catalytic activity of the enzyme. The application of mild pressure to solutions of the altered enzyme reverses the lyophilization-induced structural change and restores the mobility of the histidine residue and the enzyme's catalytic activity. This effect of lyophilization permits a unique opportunity for investigating the relationship between histidine ring dynamics and catalytic activity. The results demonstrate that His57 in resting enzymes is more mobile than previously thought, especially when protonated. The histidine motion and its correlation to enzyme activity lend support to the reaction-driven ring flip hypothesis. PMID:15657134

  1. Static mechanical strain induces capillary endothelial cell cycle re-entry and sprouting.

    PubMed

    Zeiger, A S; Liu, F D; Durham, J T; Jagielska, A; Mahmoodian, R; Van Vliet, K J; Herman, I M

    2016-01-01

    Vascular endothelial cells are known to respond to a range of biochemical and time-varying mechanical cues that can promote blood vessel sprouting termed angiogenesis. It is less understood how these cells respond to sustained (i.e., static) mechanical cues such as the deformation generated by other contractile vascular cells, cues which can change with age and disease state. Here we demonstrate that static tensile strain of 10%, consistent with that exerted by contractile microvascular pericytes, can directly and rapidly induce cell cycle re-entry in growth-arrested microvascular endothelial cell monolayers. S-phase entry in response to this strain correlates with absence of nuclear p27, a cyclin-dependent kinase inhibitor. Furthermore, this modest strain promotes sprouting of endothelial cells, suggesting a novel mechanical 'angiogenic switch'. These findings suggest that static tensile strain can directly stimulate pathological angiogenesis, implying that pericyte absence or death is not necessarily required of endothelial cell re-activation. PMID:27526677

  2. Cryogenic hydrogen-induced air-liquefaction technologies for combined-cycle propulsion applications

    NASA Technical Reports Server (NTRS)

    Escher, William J. D.

    1992-01-01

    Given here is a technical assessment of the realization of cryogenic hydrogen induced air liquefaction technologies in a prospective onboard aerospace vehicle process setting. The technical findings related to the status of air liquefaction technologies are reviewed. Compact lightweight cryogenic heat exchangers, heat exchanger atmospheric constituent fouling alleviation measures, para/ortho-hydrogen shift-conversion catalysts, cryogenic air compressors and liquid air pumps, hydrogen recycling using slush hydrogen as a heat sink, liquid hydrogen/liquid air rocket-type combustion devices, and technically related engine concepts are discussed. Much of the LACE work is related to aerospaceplane propulsion concepts that were developed in the 1960's. Emphasis is placed on the Liquid Air Cycle Engine (LACE).

  3. Curcumin loaded PLGA-poloxamer blend nanoparticles induce cell cycle arrest in mesothelioma cells.

    PubMed

    Mayol, Laura; Serri, Carla; Menale, Ciro; Crispi, Stefania; Piccolo, Maria Teresa; Mita, Luigi; Giarra, Simona; Forte, Maurizio; Saija, Antonina; Biondi, Marco; Mita, Damiano Gustavo

    2015-06-01

    The pharmacological potential of curcumin (CURC) is severely restricted because of its low water solubility/absorption, short half-life and poor bioavailability. To overcome these issues, CURC-loaded nanoparticles (NPs) were produced by a double emulsion technique. In particular, NPs were made up of an amphiphilic blend of poloxamers and PLGA to confer stealth properties to the NPs to take advantage of the enhanced permeability and retention (EPR) effect. Different surface properties of NPs made up of bare PLGA and PLGA/poloxamer blend were confirmed by the different interactions of these NPs with serum proteins and also by their ability to be internalized by mesothelioma cell line. The uptake of PLGA/poloxamer NPs induces a persistent block in G0/G1 phase of the cell cycle up to 72 h, thus overcoming the drug tolerance phenomenon, normally evidenced with free CURC. PMID:25794477

  4. Freeze-Thaw Cycling Induced Isotropic-Nematic Coexistence of Amyloid Fibrils Suspensions.

    PubMed

    Zhao, Jianguo; Bolisetty, Sreenath; Adamcik, Jozef; Han, Jun; Fernández-Ronco, María P; Mezzenga, Raffaele

    2016-03-15

    Amyloid fibrils are charged semiflexible assemblies with very large aspect ratio (length to diameter, L/D). Because of this large aspect ratio, the isotropic (I) and nematic (N) phase coexistence expected from the first-order thermodynamic nature of the I-N phase transition, as predicted from the Onsager's theory, is vanishingly small and, in practice, challenging to experimentally observe. In this study we present a remarkable widening of the I + N biphasic region in β-lactoglobulin fibrils suspension via freeze-thaw (F-T) cycling. The demixing behavior can be induced and controlled by a slow growth of propagation front of the ice crystals, which grow by excluding the amyloid fibrils from the crystal phase and thus concentrating them in the liquid phase. The growth of the ice crystals is accompanied by the formation of concentrated and elongated tactoid-like structure in the liquid phase. During the subsequent thawing cycling, at large tactoid domains, the mismatch in density caused by the presence of amyloid fibrils is sufficient to generate a sedimentation of the N phase at the bottom of the vial, coexisting with an I phase on the top. We reason why, despite the remarkable stability of the coexisting I and N phases observed over several weeks after F-T cycling, the biphasic region is understood to be a nonequilibrium, metastable state. Yet, the results in this study suggest that the F-T treatment is an effective approach to stabilize multiphase coexistence of liquid crystalline phases in colloidal suspensions of anisotropic particles without the need of additives, such as depleting agents, needed to modify interaction potentials. PMID:26907697

  5. Investigation of the indulgence cycles hypothesis of suppression on experimentally induced visual intrusions in dysphoria.

    PubMed

    Williams, Alishia D; Moulds, Michelle L

    2007-11-01

    Within the thought control literature, Wegner [(1989). White bears and other unwanted thoughts: Suppression, obsession, and the psychology of mental control. New York, NY, USA: Penguin Press.] referred to the combined initial suppression and expression phases of thought control as an indulgence cycle which results in the rebound effect typically observed in suppression studies. According to Ironic Process Theory [Wegner, (1994). Ironic processes of mental control. Psychological Review, 101, 34-52.], this rebound leads to further attempts to suppress that are difficult due to the target thought's hyperaccessibility, resulting in a secondary rebound effect owing to a positive feedback system of indulgence cycles. The current study investigated (i) the effects of repeated suppression and opportunities for expression by using a method to index the frequency, duration, and associated levels of distress of an experimentally induced visual intrusion, and (ii) whether any observed effects were differentially linked to depressive symptomatology in an analogue sample of low and high dysphoric participants. Results supported a secondary rebound effect in those participants most successful at suppressing target intrusions. The findings offer an important extension to the emerging literature on the management of intrusive memories in depression. PMID:17692285

  6. Src kinase inhibitors induce apoptosis and mediate cell cycle arrest in lymphoma cells.

    PubMed

    Nowak, Daniel; Boehrer, Simone; Hochmuth, Simone; Trepohl, Bettina; Hofmann, Wencke; Hoelzer, Dieter; Hofmann, Wolf-Karsten; Mitrou, Paris S; Ruthardt, Martin; Chow, Kai Uwe

    2007-10-01

    Src kinases are involved in multiple cellular contexts such as proliferation, adhesion, tumor invasiveness, angiogenesis, cell cycle control and apoptosis. We here demonstrate that three newly developed dual selective Src/Abl kinase inhibitors (SrcK-I) (AZM559756, AZD0530 and AZD0424) are able to induce apoptosis and cell cycle arrest in BCR-ABL, c-KIT and platelet-derived growth factor-negative lymphoma cell lines. Treatment of DOHH-2, WSU-NHL, Raji, Karpas-299, HUT78 and Jurkat cells with SrcK-I revealed that the tested substances were effective on these parameters in the cell lines DOHH-2 and WSU-NHL, whereas the other tested cell lines remained unaffected. Phosphorylation of Lyn and in particular Lck were affected most heavily by treatment with the SrcK-I. Extrinsic as well as intrinsic apoptosis pathways were activated and elicited unique expressional patterns of apoptosis-relevant proteins such as downregulation of survivin, Bcl-XL and c-FLIP. Protein levels of c-abl were downregulated and Akt phosphorylation was decreased by treatment with SrcK-I. Basal expression levels of c-Myc were notably lower in sensitive cell lines as compared with nonsensitive cell lines, possibly providing an explanation for sensitivity versus resistance against these novel substances. This study provides the first basis for establishing novel SrcK-I as weapons in the arsenal against lymphoma cells. PMID:17704648

  7. Quantitative stratigraphic models of rifts based on orbitally induced lake cycles

    SciTech Connect

    Olsen, P.E.; Schlische, R.W.

    1988-02-01

    Orbitally induced lacustrine cycles in the rift sequences of the Newark Supergroup provide a direct measure of sedimentation rates (S) averaged at the 20 thousand year level. For the Triassic, the basins followed a pattern exemplified by the Newark basin: early fluvial fill, followed by lacustrine deposition where S slowly and exponentially decreased. Once lacustrine deposition began, lakes fluctuated with orbital cycles; however, maximum lake depth (MLD) began shallow, rapidly deepened, then slowly and exponentially shallowed toward the Triassic-Jurassic boundary. These observations suggest a model based on the filling of a trough, the simplest of which is a graben where the volumetric sedimentation rate (V) is constant and extension is uniform. First, S equals subsidence until hydrographic closure. Afterward, S = BV/L(B/sup 2/D/sup 2/ + (2BVt(A-D))/L)/sup minus/1/2 (t = time after closure and A, D, B, and L are the final widths, depth, and length of the graben). The observed changes in MLD conform to the predictions of this filing model. Half-graben are more complex, but similar patterns result. Major deviations reflect tectonic events. One dramatic deviation occurred at the Triassic-Jurassic boundary, where S and MLD greatly increased. This coincided with massive tholeitic magmatism, probably reflecting increased extension rates and a marked basin asymmetry. This model explains why Newark hydrocarbon targets are in strata of either the early Late Triassic or Early Jurassic, because those intervals were deposited by the deepest lakes.

  8. Quantitative stratigraphic models of rifts based on orbitally induced lake cycles

    SciTech Connect

    Olsen, P.E.; Schlische, R.W.

    1988-01-01

    Orbitally induced lacustrine cycles in the rift sequences of the Newark Supergroup provide a direct measure of sedimentation rates (S) averaged at the 20 thousand year level. For the Triassic, the basins followed a pattern exemplified by the Newark basin: early fluvial fill, followed by lacustrine deposition where S slowly and exponentially decreased. Once lacustrine deposition began, lakes fluctuated with orbital cycles; however, maximum lake depth (MLD) began shallow, rapidly deepened, then slowly and exponentially shallowed toward the Triassic-Jurassic boundary. These observations suggest a model based on the filling of a trough, the simplest of which is a graben where the volumetric sedimentation rate (V) is constant and extension in uniform. The observed changes in MLD conform to the predictions of this filling model. Half-graben are more complex, but similar patterns result. Major deviations reflect tectonic events. One dramatic deviation occurred at the Triassic-Jurassic boundary, where subsidence and MLD greatly increased. This coincided with massive tholeiitic magmatism, probably reflecting increased extension rates and a marked basin asymmetry. This model explains why Newark hydrocarbon targets are in strata of either the early Late Triassic or Early Jurassic, because those intervals were deposited by the deepest lakes.

  9. Dual CDK4/CDK6 Inhibition Induces Cell Cycle Arrest and Senescence in Neuroblastoma

    PubMed Central

    Rader, JulieAnn; Russell, Mike R.; Hart, Lori S.; Nakazawa, Michael S.; Belcastro, Lili T.; Martinez, Daniel; Li, Yimei; Carpenter, Erica L.; Attiyeh, Edward F.; Diskin, Sharon J.; Kim, Sunkyu; Parasuraman, Sudha; Caponigro, Giordano; Schnepp, Robert W.; Wood, Andrew C.; Pawel, Bruce; Cole, Kristina A.; Maris, John M.

    2013-01-01

    Purpose Neuroblastoma is a pediatric cancer that continues to exact significant morbidity and mortality. Recently, a number of cell cycle proteins, particularly those within the Cyclin D/CDK4/CDK6/RB network, have been shown to exert oncogenic roles in neuroblastoma, suggesting that their therapeutic exploitation might improve patient outcomes. Experimental Procedures We evaluated the effect of dual CDK4/CDK6 inhibition on neuroblastoma viability using LEE011, a highly specific CDK4/6 inhibitor. Results Treatment with LEE011 significantly reduced proliferation in 12 of 17 human neuroblastoma-derived cell lines by inducing cytostasis at nanomolar concentrations (mean IC50 = 307 ± 68 nM in sensitive lines). LEE011 caused cell cycle arrest and cellular senescence that was attributed to dose-dependent decreases in phosphorylated RB and FOXM1, respectively. In addition, responsiveness of neuroblastoma xenografts to LEE011 translated to the in vivo setting in that there was a direct correlation of in vitro IC50 values with degree of subcutaneous xenograft growth delay. While our data indicate that neuroblastomas sensitive to LEE011 were more likely to contain genomic amplification of MYCN (p = 0.01), the identification of additional clinically accessible biomarkers is of high importance. Conclusions Taken together, our data show that LEE011 is active in a large subset of neuroblastoma cell line and xenograft models, and supports the clinical development of this CDK4/6 inhibitor as a therapy for patients with this disease. PMID:24045179

  10. Noise-induced dispersion and breakup of clusters in cell cycle dynamics

    PubMed Central

    Gong, Xue; Moses, Gregory; Neiman, Alexander B.; Young, Todd

    2014-01-01

    We study the effects of random perturbations on collective dynamics of a large ensemble of interacting cells in a model of the cell division cycle. We consider a parameter region for which the unperturbed model possesses asymptotically stable two-cluster periodic solutions. Two biologically motivated forms of random perturbations are considered: bounded variations in growth rate and asymmetric division. We compare the effects of these two dispersive mechanisms with additive Gaussian white noise perturbations. We observe three distinct phases of the response to noise in the model. First, for weak noise there is a linear relationship between the applied noise strength and the dispersion of the clusters. Second, for moderate noise strengths the clusters begin to mix, i.e. individual cells move between clusters, yet the population distribution clearly continues to maintain a two-cluster structure. Third, for strong noise the clusters are destroyed and the population is characterized by a uniform distribution. The second and third phases are separated by an order - disorder phase transition that has the characteristics of a Hopf bifurcation. Furthermore, we show that for the cell cycle model studied, the effects of bounded random perturbations are virtually indistinguishable from those induced by additive Gaussian noise, after appropriate scaling of the variance of noise strength. We then use the model to predict the strength of coupling among the cells from experimental data. In particular, we show that coupling must be rather strong to account for the observed clustering of cells given experimentally estimated noise variance. PMID:24694583