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Sample records for lytic granule-mediated apoptosis

  1. Inhibition of p38 MAP kinase pathway induces apoptosis and prevents Epstein Barr virus reactivation in Raji cells exposed to lytic cycle inducing compounds

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    Di Renzo Livia

    2009-03-01

    Full Text Available Abstract Background EBV lytic cycle activators, such as phorbol esters, anti-immunoglobulin, transforming growth factor β (TGFβ, sodium butyrate, induce apoptosis in EBV-negative but not in EBV-positive Burkitt's lymphoma (BL cells. To investigate the molecular mechanisms allowing EBV-infected cells to be protected, we examined the expression of viral and cellular antiapoptotic proteins as well as the activation of signal transduction pathways in BL-derived Raji cells exposed to lytic cycle inducing agents. Results Our data show that, following EBV activation, the latent membrane protein 1 (LMP1 and the cellular anti-apoptotic proteins MCL-1 and BCL-2 were quickly up-regulated and that Raji cells remained viable even when exposed simultaneously to P(BU2, sodium butyrate and TGFβ. We report here that inhibition of p38 pathway, during EBV activation, led to a three fold increment of apoptosis and largely prevented lytic gene expression. Conclusion These findings indicate that, during the switch from the latent to the lytic phase of EBV infection, p38 MAPK phosphorylation plays a key role both for protecting the host cells from apoptosis as well as for inducing viral reactivation. Because Raji cells are defective for late antigens expression, we hypothesize that the increment of LMP1 gene expression in the early phases of EBV lytic cycle might contribute to the survival of the EBV-positive cells.

  2. Phage lytic enzymes: a history

    Institute of Scientific and Technical Information of China (English)

    David; Trudil

    2015-01-01

    There are many recent studies regarding the efficacy of bacteriophage-related lytic enzymes: the enzymes of ‘bacteria-eaters’ or viruses that infect bacteria. By degrading the cell wall of the targeted bacteria, these lytic enzymes have been shown to efficiently lyse Gram-positive bacteria without affecting normal flora and non-related bacteria. Recent studies have suggested approaches for lysing Gram-negative bacteria as well(Briersa Y, et al., 2014). These enzymes include: phage-lysozyme, endolysin, lysozyme, lysin, phage lysin, phage lytic enzymes, phageassociated enzymes, enzybiotics, muralysin, muramidase, virolysin and designations such as Ply, PAE and others. Bacteriophages are viruses that kill bacteria, do not contribute to antimicrobial resistance, are easy to develop, inexpensive to manufacture and safe for humans, animals and the environment. The current focus on lytic enzymes has been on their use as anti-infectives in humans and more recently in agricultural research models. The initial translational application of lytic enzymes, however, was not associated with treating or preventing a specifi c disease but rather as an extraction method to be incorporated in a rapid bacterial detection assay(Bernstein D, 1997).The current review traces the translational history of phage lytic enzymes–from their initial discovery in 1986 for the rapid detection of group A streptococcus in clinical specimens to evolving applications in the detection and prevention of disease in humans and in agriculture.

  3. Lytic and non-lytic permeabilization of cardiolipin-containing lipid bilayers induced by cytochrome C.

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    Jian Xu

    Full Text Available The release of cytochrome c (cyt c from mitochondria is an important early step during cellular apoptosis, however the precise mechanism by which the outer mitochondrial membrane becomes permeable to these proteins is as yet unclear. Inspired by our previous observation of cyt c crossing the membrane barrier of giant unilamellar vesicle model systems, we investigate the interaction of cyt c with cardiolipin (CL-containing membranes using the innovative droplet bilayer system that permits electrochemical measurements with simultaneous microscopy observation. We find that cyt c can permeabilize CL-containing membranes by induction of lipid pores in a dose-dependent manner, with membrane lysis eventually observed at relatively high (µM cyt c concentrations due to widespread pore formation in the membrane destabilizing its bilayer structure. Surprisingly, as cyt c concentration is further increased, we find a regime with exceptionally high permeability where a stable membrane barrier is still maintained between droplet compartments. This unusual non-lytic state has a long lifetime (>20 h and can be reversibly formed by mechanically separating the droplets before reforming the contact area between them. The transitions between behavioural regimes are electrostatically driven, demonstrated by their suppression with increasing ionic concentrations and their dependence on CL composition. While membrane permeability could also be induced by cationic PAMAM dendrimers, the non-lytic, highly permeable membrane state could not be reproduced using these synthetic polymers, indicating that details in the structure of cyt c beyond simply possessing a cationic net charge are important for the emergence of this unconventional membrane state. These unexpected findings may hold significance for the mechanism by which cyt c escapes into the cytosol of cells during apoptosis.

  4. Involvement of Noxa in mediating cellular ER stress responses to lytic virus infection

    OpenAIRE

    2011-01-01

    Noxa is a Bcl-2 homology domain-containing pro-apoptotic mitochondrial protein. Noxa mRNA and protein expression are upregulated by dsRNA or virus, and ectopic Noxa expression enhances cellular sensitivity to virus or dsRNA-induced apoptosis. Here we demonstrate that Noxa null baby mouse kidney (BMK) cells are deficient in normal cytopathic response to lytic viruses, and that reconstitution of the knockout cells with wild type Noxa restored normal cytopathic responses. Noxa regulation by viru...

  5. Lytic to temperate switching of viral communities

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    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’.

  6. Lytic clavicular lesions in fibromatosis colli

    Energy Technology Data Exchange (ETDEWEB)

    Sartoris, D.J.; Parker, B.R.; Mochizuki, R.M.

    1983-06-01

    Two patients with fibromatosis colli (congenital torticollis) presented with lytic lesions in the clavicle at the insertion of the fibrosed clavicular head of the sternocleidomastoid muscle. Biopsy of one lesion showed intraosseous fibrosis. These lesions are probably not uncommon but radiographs are rarely performed in uncomplicated cases.

  7. Viral reproductive strategies: How can lytic viruses be evolutionarily competitive?

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    Komarova, Natalia L

    2007-12-21

    Viral release strategies can be roughly classified as lytic (the ones that accumulate inside the host cell and exit in a burst, killing the cell), and budding (the ones that are produced and released from the host cell gradually). Here we study the evolutionary competition between the two strategies. If all the parameters, such as the rate of viral production, cell life-span and the neutralizing capacity of the antibodies, were the same for lytic and budding viruses, the budding life-strategy would have a large evolutionary advantage. The question arises what makes lytic viruses evolutionarily competitive. We propose that it is the different removal capacity of the antibodies against budding and lytic virions. The latter exit the cell in a large burst such that the antibodies are "flooded" and a larger proportion of virions can escape the immune system and spread to new cells. We create two spatial models of virus-antibody interaction and show that for realistic parameter values, the effect of antibody flooding can indeed take place. We also argue that the lytic life cycle, including a relatively large burst-size, has evolved to promote survival in the face of antibody attack. According to the calculations, in the absence of efficient antibodies, the optimal burst size of lytic viruses would be only a few virus particles, as opposed to the observed 10(2)-10(5) viral particles. Similarly, there is an evolutionary pressure to extend the life-span as a response to antibody action.

  8. Painful Lytic Lesions of the Foot : A Case Report

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    R Vaishya

    2015-03-01

    Full Text Available The presence of lytic lesions in the bones of foot raises a number of diagnostic possibilities ranging from infection, inflammatory pathology to neoplastic conditions. Although the radiological picture is not pathognomonic of any pathology, clinical history and histopathological examination can help to clinch the diagnosis. We present a case of multiple lytic lesions of the foot and discuss possible differential diagnoses. The patient was diagnosed as a case of madura foot and the lesions responded to surgical debridement and anti-fungal treatment with a good functional outcome. Madura foot is an uncommon, chronic granulomatous fungal or bacterial infection with a predilection in people who walk barefoot. Although known for a specific geographical distribution, madura foot should be kept as a possible diagnosis in patients presenting with lytic lesions of the foot due to population emigration across the world.

  9. Involvement of Noxa in mediating cellular ER stress responses to lytic virus infection.

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    Rosebeck, Shaun; Sudini, Kuladeep; Chen, Tiannan; Leaman, Douglas W

    2011-09-01

    Noxa is a Bcl-2 homology domain-containing pro-apoptotic mitochondrial protein. Noxa mRNA and protein expression are upregulated by dsRNA or virus, and ectopic Noxa expression enhances cellular sensitivity to virus or dsRNA-induced apoptosis. Here we demonstrate that Noxa null baby mouse kidney (BMK) cells are deficient in normal cytopathic response to lytic viruses, and that reconstitution of the knockout cells with wild-type Noxa restored normal cytopathic responses. Noxa regulation by virus mirrored its regulation by proteasome inhibitors or ER stress inducers and the ER stress response inhibitor salubrinal protected cells against viral cytopathic effects. Noxa mRNA and protein were synergistically upregulated by IFN or dsRNA when combined with ER stress inducers, leading to Noxa/Mcl-1 interaction, activation of Bax and pro-apoptotic caspases, degradation of Mcl-1, loss of mitochondrial membrane potential and initiation of apoptosis. These data highlight the importance of ER stress in augmenting the expression of Noxa following viral infection.

  10. Cortex Peptidoglycan Lytic Activity in Germinating Bacillus anthracis Spores▿

    OpenAIRE

    2008-01-01

    Bacterial endospore dormancy and resistance properties depend on the relative dehydration of the spore core, which is maintained by the spore membrane and its surrounding cortex peptidoglycan wall. During spore germination, the cortex peptidoglycan is rapidly hydrolyzed by lytic enzymes packaged into the dormant spore. The peptidoglycan structures in both dormant and germinating Bacillus anthracis Sterne spores were analyzed. The B. anthracis dormant spore peptidoglycan was similar to that fo...

  11. A green-light inducible lytic system for cyanobacterial cells.

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    Miyake, Kotone; Abe, Koichi; Ferri, Stefano; Nakajima, Mitsuharu; Nakamura, Mayumi; Yoshida, Wataru; Kojima, Katsuhiro; Ikebukuro, Kazunori; Sode, Koji

    2014-01-01

    Cyanobacteria are an attractive candidate for the production of biofuel because of their ability to capture carbon dioxide by photosynthesis and grow on non-arable land. However, because huge quantities of water are required for cultivation, strict water management is one of the greatest issues in algae- and cyanobacteria-based biofuel production. In this study, we aim to construct a lytic cyanobacterium that can be regulated by a physical signal (green-light illumination) for future use in the recovery of biofuel related compounds. We introduced T4 bacteriophage-derived lysis genes encoding holin and endolysin under the control of the green-light regulated cpcG2 promoter in Synechocystis sp. PCC 6803. When cells harboring the lysis genes were illuminated with both red and green light, we observed a considerable decrease in growth rate, a significant increase in cellular phycocyanin released in the medium, and a considerable fraction of dead cells. These effects were not observed when these cells were illuminated with only red light, or when cells not containing the lysis genes were grown under either red light or red and green light. These results indicate that our constructed green-light inducible lytic system was clearly induced by green-light illumination, resulting in lytic cells that released intracellular phycocyanin into the culture supernatant. This property suggests a future possibility to construct photosynthetic genetically modified organisms that are unable to survive under sunlight exposure. Expression of the self-lysis system with green-light illumination was also found to greatly increase the fragility of the cell membrane, as determined by subjecting the induced cells to detergent, osmotic-shock, and freeze-thaw treatments. A green-light inducible lytic system was constructed in Synechocystis sp. PCC 6803. The engineered lytic cyanobacterial cells should be beneficial for the recovery of biofuels and related compounds from cells with minimal effort

  12. Structural characterization of Lytic Polysaccharide MonoOxygenases

    DEFF Research Database (Denmark)

    Frandsen, Kristian Erik Høpfner

    Lytic polysaccharide monooxygenases (LPMOs) are a new class of copper-containingmetalloenzymes that have been found to oxidatively degrade polysaccharides (and recently alsooligosaccharides). They dependent on redox partners to provide them with electrons and they utilizemolecular oxygen to cleave......) and their interaction with substratehave been structurally characterized. A number of structures of LsAA9A have been obtained in complexwith a range of cellulosic- and hemicellulosic substrates and with the active site Cu in different redox state.Two of the LsAA9A structures with the active site Cu in essentially a Cu...

  13. Characterization of newly isolated lytic bacteriophages active against Acinetobacter baumannii.

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    Merabishvili, Maia; Vandenheuvel, Dieter; Kropinski, Andrew M; Mast, Jan; De Vos, Daniel; Verbeken, Gilbert; Noben, Jean-Paul; Lavigne, Rob; Vaneechoutte, Mario; Pirnay, Jean-Paul

    2014-01-01

    Based on genotyping and host range, two newly isolated lytic bacteriophages, myovirus vB_AbaM_Acibel004 and podovirus vB_AbaP_Acibel007, active against Acinetobacter baumannii clinical strains, were selected from a new phage library for further characterization. The complete genomes of the two phages were analyzed. Both phages are characterized by broad host range and essential features of potential therapeutic phages, such as short latent period (27 and 21 min, respectively), high burst size (125 and 145, respectively), stability of activity in liquid culture and low frequency of occurrence of phage-resistant mutant bacterial cells. Genomic analysis showed that while Acibel004 represents a novel bacteriophage with resemblance to some unclassified Pseudomonas aeruginosa phages, Acibel007 belongs to the well-characterized genus of the Phikmvlikevirus. The newly isolated phages can serve as potential candidates for phage cocktails to control A. baumannii infections.

  14. Characterization of newly isolated lytic bacteriophages active against Acinetobacter baumannii.

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    Maia Merabishvili

    Full Text Available Based on genotyping and host range, two newly isolated lytic bacteriophages, myovirus vB_AbaM_Acibel004 and podovirus vB_AbaP_Acibel007, active against Acinetobacter baumannii clinical strains, were selected from a new phage library for further characterization. The complete genomes of the two phages were analyzed. Both phages are characterized by broad host range and essential features of potential therapeutic phages, such as short latent period (27 and 21 min, respectively, high burst size (125 and 145, respectively, stability of activity in liquid culture and low frequency of occurrence of phage-resistant mutant bacterial cells. Genomic analysis showed that while Acibel004 represents a novel bacteriophage with resemblance to some unclassified Pseudomonas aeruginosa phages, Acibel007 belongs to the well-characterized genus of the Phikmvlikevirus. The newly isolated phages can serve as potential candidates for phage cocktails to control A. baumannii infections.

  15. Lytic polysaccharide monooxygenases disrupt the cellulose fibers structure

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    Villares, Ana; Moreau, Céline; Bennati-Granier, Chloé; Garajova, Sona; Foucat, Loïc; Falourd, Xavier; Saake, Bodo; Berrin, Jean-Guy; Cathala, Bernard

    2017-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are a class of powerful oxidative enzymes that breakdown recalcitrant polysaccharides such as cellulose. Here we investigate the action of LPMOs on cellulose fibers. After enzymatic treatment and dispersion, LPMO-treated fibers show intense fibrillation. Cellulose structure modifications visualized at different scales indicate that LPMO creates nicking points that trigger the disintegration of the cellulose fibrillar structure with rupture of chains and release of elementary nanofibrils. Investigation of LPMO action using solid-state NMR provides direct evidence of modification of accessible and inaccessible surfaces surrounding the crystalline core of the fibrils. The chains breakage likely induces modifications of the cellulose network and weakens fibers cohesion promoting their disruption. Besides the formation of new initiation sites for conventional cellulases, this work provides the first evidence of the direct oxidative action of LPMOs with the mechanical weakening of the cellulose ultrastructure. LPMOs can be viewed as promising biocatalysts for enzymatic modification or degradation of cellulose fibers. PMID:28071716

  16. Increased Lytic Efficiency of Bovine Macrophages Trained with Killed Mycobacteria

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    Juste, Ramon A.; Alonso-Hearn, Marta; Garrido, Joseba M.; Abendaño, Naiara; Sevilla, Iker A.; Gortazar, Christian; de la Fuente, José; Dominguez, Lucas

    2016-01-01

    Innate immunity is evolutionarily conserved in multicellular organisms and was considered to lack memory until very recently. One of its more characteristic mechanisms is phagocytosis, the ability of cells to engulf, process and eventually destroy any injuring agent. We report the results of an ex vivo experiment in bovine macrophages in which improved clearance of Mycobacterium bovis (M. bovis) was induced by pre-exposure to a heat killed M. bovis preparation. The effects were independent of humoral and cellular adaptive immune responses and lasted up to six months. Specifically, our results demonstrate the existence of a training effect in the lytic phase of phagocytosis that can be activated by killed mycobacteria, thus suggesting a new mechanism of vaccine protection. These findings are compatible with the recently proposed concept of trained immunity, which was developed to explain the observation that innate immune responses provide unspecific protection against pathogens including other than those that originally triggered the immune response. PMID:27820836

  17. Structural characterization of Lytic Polysaccharide MonoOxygenases

    DEFF Research Database (Denmark)

    Frandsen, Kristian Erik Høpfner

    Lytic polysaccharide monooxygenases (LPMOs) are a new class of copper-containingmetalloenzymes that have been found to oxidatively degrade polysaccharides (and recently alsooligosaccharides). They dependent on redox partners to provide them with electrons and they utilizemolecular oxygen to cleave......) and their interaction with substratehave been structurally characterized. A number of structures of LsAA9A have been obtained in complexwith a range of cellulosic- and hemicellulosic substrates and with the active site Cu in different redox state.Two of the LsAA9A structures with the active site Cu in essentially a Cu......(II) state show differences in thenature of the Cu-ligand with and without cellulosic substrate bound and provide structural insight into themechanistic action of LPMOs. Interestingly, for an LsAA9A complex structure with a hemicellulosicsubstrate (xylooligosaccharide) a corresponding difference...

  18. 5-hydroxymethylation of the EBV genome regulates the latent to lytic switch.

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    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.

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

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

    2016-06-02

    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.

  20. killerFLIP: a novel lytic peptide specifically inducing cancer cell death.

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    Pennarun, B; Gaidos, G; Bucur, O; Tinari, A; Rupasinghe, C; Jin, T; Dewar, R; Song, K; Santos, M T; Malorni, W; Mierke, D; Khosravi-Far, R

    2013-10-31

    One of the objectives in the development of effective cancer therapy is induction of tumor-selective cell death. Toward this end, we have identified a small peptide that, when introduced into cells via a TAT cell-delivery system, shows a remarkably potent cytoxicity in a variety of cancer cell lines and inhibits tumor growth in vivo, whereas sparing normal cells and tissues. This fusion peptide was named killerFLIP as its sequence was derived from the C-terminal domain of c-FLIP, an anti-apoptotic protein. Using structure activity analysis, we determined the minimal bioactive core of killerFLIP, namely killerFLIP-E. Structural analysis of cells using electron microscopy demonstrated that killerFLIP-E triggers cell death accompanied by rapid (within minutes) plasma membrane permeabilization. Studies of the structure of the active core of killerFLIP (-E) indicated that it possesses amphiphilic properties and self-assembles into micellar structures in aqueous solution. The biochemical properties of killerFLIP are comparable to those of cationic lytic peptides, which participate in defense against pathogens and have also demonstrated anticancer properties. We show that the pro-cell death effects of killerFLIP are independent of its sequence similarity with c-FLIPL as killerFLIP-induced cell death was largely apoptosis and necroptosis independent. A killerFLIP-E variant containing a scrambled c-FLIPL motif indeed induced similar cell death, suggesting the importance of the c-FLIPL residues but not of their sequence. Thus, we report the discovery of a promising synthetic peptide with novel anticancer activity in vitro and in vivo.

  1. Properties of Brucella-phages lytic for non-smooth Brucella strains.

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    Corbel, M J

    1984-01-01

    A series of host-range mutants has been selected for brucella-phage R. Two of these mutants designated R/O and R/C have been used for typing purposes. Phage R/O is lytic for non-smooth strains of Brucella abortus and for B. ovis. It is genetically unstable however and produces mutants lytic for smooth B. obortus and B. suis. Phage R/C is lytic for non-smooth B. abortus and for B. ovis and B. canis. It is much more stable than phages R or R/O and shows little or no lytic activity on smooth Brucella strains. It has been effective in differentiating B. canis from B. suis in tests on a limited number of strains. In their properties, all of the brucella-phages of the R series resemble their parent phage.

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

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    Dhananjay M Nawandar

    2015-10-01

    Full Text Available 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.

  3. Enhancement of Lytic Activity by Leptin Is Independent From Lipid Rafts in Murine Primary Splenocytes.

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    Collin, Aurore; Noacco, Audrey; Talvas, Jérémie; Caldefie-Chézet, Florence; Vasson, Marie-Paule; Farges, Marie-Chantal

    2017-01-01

    Leptin, a pleiotropic adipokine, is known as a regulator of food intake, but it is also involved in inflammation, immunity, cell proliferation, and survival. Leptin receptor is integrated inside cholesterol-rich microdomains called lipid rafts, which, if disrupted or destroyed, could lead to a perturbation of lytic mechanism. Previous studies also reported that leptin could induce membrane remodeling. In this context, we studied the effect of membrane remodeling in lytic activity modulation induced by leptin. Thus, primary mouse splenocytes were incubated with methyl-β-cyclodextrin (β-MCD), a lipid rafts disrupting agent, cholesterol, a major component of cell membranes, or ursodeoxycholic acid (UDCA), a membrane stabilizer agent for 1 h. These treatments were followed by splenocyte incubation with leptin (absence, 10 and 100 ng/ml). Unlike β-MCD or cholesterol, UDCA was able to block leptin lytic induction. This result suggests that leptin increased the lytic activity of primary spleen cells against syngenic EO771 mammary cancer cells independently from lipid rafts but may involve membrane fluidity. Furthermore, natural killer cells were shown to be involved in the splenocyte lytic activity. To our knowledge it is the first publication in primary culture that provides the link between leptin lytic modulation and membrane remodeling. J. Cell. Physiol. 232: 101-109, 2017. © 2016 Wiley Periodicals, Inc.

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

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    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. © 2016 Authors; published by Portland Press Limited.

  5. Efficacy of lytic Staphylococcus aureus bacteriophage against multidrug-resistant Staphylococcus aureus in mice.

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    Oduor, Joseph Michael Ochieng'; Onkoba, Nyamongo; Maloba, Fredrick; Arodi, Washingtone Ouma; Nyachieo, Atunga

    2016-11-24

    The use of bacteriophages as an alternative treatment method against multidrug-resistant bacteria has not been explored in Kenya. This study sought to determine the efficacy of environmentally obtained lytic bacteriophage against multidrug-resistant Staphylococcus aureus (MDRSA) bacterium in mice. Staphylococcus aureus bacterium and S. aureus-specific lytic phage were isolated from sewage and wastewater collected within Nairobi County, Kenya. Thirty mice were randomly assigned into three groups: MDRSA infection group (n = 20), phage-infection group (n = 5), and non-infection group (n = 5). The MDRSA infection group was further subdivided into three groups: clindamycin treatment (8 mg/kg; n = 5), lytic phage treatment (108 PFU/mL (n = 5), and a combination treatment of clindamycin and lytic phage (n = 5). Treatments were done at either 24 or 72 hours post-infection (p.i), and data on efficacy, bacterial load, and animal physical health were collected. Treatment with phage was more effective (100%) than with clindamycin (62.25% at 24 hours p.i and 87.5% at 72 hours p.i.) or combination treatment (75% at 24 hours p.i. and 90% at 72 hours p.i.) (p aureus lytic bacteriophage has therapeutic potential against MDRSA bacterium in mice.

  6. TRIM5α Promotes Ubiquitination of Rta from Epstein–Barr Virus to Attenuate Lytic Progression

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    Huang, Hsiang-Hung; Chen, Chien-Sin; Wang, Wen-Hung; Hsu, Shih-Wei; Tsai, Hsiao-Han; Liu, Shih-Tung; Chang, Li-Kwan

    2017-01-01

    Replication and transcription activator (Rta), a key protein expressed by Epstein–Barr virus (EBV) during the immediate-early stage of the lytic cycle, is responsible for the activation of viral lytic genes. In this study, GST-pulldown and coimmunoprecipitation assays showed that Rta interacts in vitro and in vivo with TRIM5α, a host factor known to be involved in the restriction of retroviral infections. Confocal microscopy results revealed that Rta colocalizes with TRIM5α in the nucleus during lytic progression. The interaction involves 190 amino acids in the N-terminal of Rta and the RING domain in TRIM5α, and it was further found that TRIM5α acts as an E3 ubiquitin ligase to promote Rta ubiquitination. Overexpression of TRIM5α reduced the transactivating capabilities of Rta, while reducing TRIM5α expression enhanced EBV lytic protein expression and DNA replication. Taken together, these results point to a critical role for TRIM5α in attenuating EBV lytic progression through the targeting of Rta for ubiquitination, and suggest that the restrictive capabilities of TRIM5α may go beyond retroviral infections. PMID:28105027

  7. A Herpesviral Lytic Protein Regulates the Structure of Latent Viral Chromatin

    Directory of Open Access Journals (Sweden)

    Priya Raja

    2016-05-01

    Full Text Available Latent infections by viruses usually involve minimizing viral protein expression so that the host immune system cannot recognize the infected cell through the viral peptides presented on its cell surface. Herpes simplex virus (HSV, for example, is thought to express noncoding RNAs such as latency-associated transcripts (LATs and microRNAs (miRNAs as the only abundant viral gene products during latent infection. Here we describe analysis of HSV-1 mutant viruses, providing strong genetic evidence that HSV-infected cell protein 0 (ICP0 is expressed during establishment and/or maintenance of latent infection in murine sensory neurons in vivo. Studies of an ICP0 nonsense mutant virus showed that ICP0 promotes heterochromatin and latent and lytic transcription, arguing that ICP0 is expressed and functional. We propose that ICP0 promotes transcription of LATs during establishment or maintenance of HSV latent infection, much as it promotes lytic gene transcription. This report introduces the new concept that a lytic viral protein can be expressed during latent infection and can serve dual roles to regulate viral chromatin to optimize latent infection in addition to its role in epigenetic regulation during lytic infection. An additional implication of the results is that ICP0 might serve as a target for an antiviral therapeutic acting on lytic and latent infections.

  8. Inhibition of the Epstein-Barr virus lytic cycle by moronic acid.

    Science.gov (United States)

    Chang, Fang-Rong; Hsieh, Yi-Chung; Chang, Yung-Fu; Lee, Kuo-Hsiung; Wu, Yang-Chang; Chang, Li-Kwan

    2010-03-01

    Epstein-Barr virus (EBV) expresses two transcription factors, Rta and Zta, during the immediate-early stage of the lytic cycle to activate the transcription of viral lytic genes. Our immunoblotting and flow cytometry analyses find that moronic acid, found in galls of Rhus chinensis and Brazilian propolis, at 10microM inhibits the expression of Rta, Zta, and an EBV early protein, EA-D, after lytic induction with sodium butyrate. This study also finds that moronic acids inhibits the capacity of Rta to activate a promoter that contains an Rta-response element, indicating that moronic acid interferes with the function of Rta. On the other hand, moronic acid does not appear to influence with the transactivation function of Zta. Therefore, the lack of expression of Zta and EA-D after moronic acid treatment is attributable to the inhibition of the transactivation functions of Rta. Because the expression of Zta, EA-D and many EBV lytic genes depends on Rta, the treatment of P3HR1 cells with moronic acid substantially reduces the numbers of EBV particles produced by the cells after lytic induction. This study suggests that moronic acid is a new structural lead for anti-EBV drug development.

  9. The Lytic SA Phage Demonstrate Bactericidal Activity against Mastitis Causing Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Hamza Ameer

    2016-01-01

    Full Text Available Staphylococcus aureus is the major causative agent of mastitis among dairy animals as it causes intramammary gland infection. Due to antibiotic resistance and contamination of antibiotics in the milk of diseased animals; alternative therapeutic agents are required to cure mastitis. Lytic bacteriophages and their gene products can be potential therapeutic agents against bacteria as they are host specific and less harmful than antibiotics. In this study, Staphylococcus aureus were isolated from milk samples of the infected animals and identified biochemically. SA phage was isolated from sewage water showing lytic activity against Staphylococcus aureus isolates. The highest lytic activity of bacteriophages was observed at 37°C and pH 7, and the most suitable storage condition was at 4°C. SA phage efficiently reduced bacterial growth in the bacterial reduction assay. The characterization and bacterial growth reduction activity of the bacteriophages against Staphylococcus aureus signifies their underlying potential of phage therapy against mastitis.

  10. In vitro cytocidal effect of lytic peptides on several transformed mammalian cell lines.

    Science.gov (United States)

    Jaynes, J M; Julian, G R; Jeffers, G W; White, K L; Enright, F M

    1989-01-01

    Several types of transformed mammalian cells, derived from established cell lines, were found to be lysed in vitro by three novel lytic peptides (SB-37, SB-37*, and Shiva-1). This is in contrast with the behavior of normal cells, where the observed lytic activity of the peptides is greatly reduced. Based on experiments utilizing compounds which disrupt the cytoskeleton (colchicine and cytochalasin-D), it is surmised that alterations in the cytoskeleton of transformed cells increase their sensitivity to the cytolytic activity exerted by the peptides, primarily by causing a loss of osmotic integrity. Thus, a stable and regenerative cytoskeletal system, as that possessed by normal cells, would seem requisite to withstanding the lytic effects of the peptides.

  11. In vitro cytocidal effect of novel lytic peptides on Plasmodium falciparum and Trypanosoma cruzi.

    Science.gov (United States)

    Jaynes, J M; Burton, C A; Barr, S B; Jeffers, G W; Julian, G R; White, K L; Enright, F M; Klei, T R; Laine, R A

    1988-10-01

    Plasmodium falciparum and Trypanosoma cruzi were killed by two novel lytic peptides (SB-37 and Shiva-1) in vitro. Human erythrocytes infected with P. falciparum, and Vero cells infected with T. cruzi, were exposed to these peptides. The result, in both cases, was a significant decrease in the level of parasite infection. Furthermore, the peptides had a marked cytocidal effect on trypomastigote stages of T. cruzi in media, whereas host eukaryotic cells were unaffected by the treatments. In view of the worldwide prevalence of these protozoan diseases and the lack of completely suitable treatments, lytic peptides may provide new and unique chemotherapeutic agents for the treatment of these infections.

  12. Listeria monocytogenes has a functional chitinolytic system and an active lytic polysaccharide monooxygenase

    DEFF Research Database (Denmark)

    Paspaliari, Dafni Katerina; Loose, Jennifer S. M.; Larsen, Marianne Halberg

    2015-01-01

    B) and a multi-modular lytic polysaccharide monooxygenase (LmLPMO10). These enzymes have been related to virulence and their role in chitin metabolism is poorly understood. It is thus of interest to functionally characterize the individual enzymes in order to shed light on their roles in vivo. Our results......Chitinases and chitin-active lytic polysaccharide monooxygenases (LPMOs) are most commonly associated with chitin metabolism, but are also reported as virulence factors in pathogenic bacteria. Listeria monocytogenes, a well-known virulent bacterium, possesses two chitinases (ChiA and Chi...

  13. Undetectable bacterial resistance to phage lytic proteins from the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88

    Science.gov (United States)

    The increase in antibiotic resistance world-wide revitalized the interest in the use of phage lysins to combat pathogenic bacteria. In this work, we tested for the emergence of resistant Staphylococcus aureus to any of three phage lytic proteins constructs. The investigated cell wall lytic enzymes w...

  14. Regional Variation in Lytic and Lysogenic Viral Infection in the Southern Ocean and Its Contribution to Biogeochemical Cycling

    NARCIS (Netherlands)

    Evans, C.; Brussaard, C.P.D.

    2012-01-01

    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-limite

  15. Influence of heavy metals on biosynthesis, activity of lytic enzymes and growthstimulating factor of Streptomyces recifensis var. lyticus P-29

    Directory of Open Access Journals (Sweden)

    Т. P. Kilochok

    2005-02-01

    Full Text Available Influence of heavy metals on growth, biosynthesis, lytic action and growthstimulating activity enzymes complex of Streptomyces recifensis var. lyticus was studied. It was showed that salt of plumbum' has positive influence as on biosynthesis hydrolases (lytic endopeptidases, proteinases, amylases as well increase growthstimulating activity of preparation relatively the yeast

  16. Hantavirus-infection confers resistance to cytotoxic lymphocyte-mediated apoptosis.

    Directory of Open Access Journals (Sweden)

    Shawon Gupta

    2013-03-01

    Full Text Available Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS and hantavirus cardio-pulmonary syndrome (HCPS; also called hantavirus pulmonary syndrome (HPS, both human diseases with high case-fatality rates. Endothelial cells are the main targets for hantaviruses. An intriguing observation in patients with HFRS and HCPS is that on one hand the virus infection leads to strong activation of CD8 T cells and NK cells, on the other hand no obvious destruction of infected endothelial cells is observed. Here, we provide an explanation for this dichotomy by showing that hantavirus-infected endothelial cells are protected from cytotoxic lymphocyte-mediated induction of apoptosis. When dissecting potential mechanisms behind this phenomenon, we discovered that the hantavirus nucleocapsid protein inhibits the enzymatic activity of both granzyme B and caspase 3. This provides a tentative explanation for the hantavirus-mediated block of cytotoxic granule-mediated apoptosis-induction, and hence the protection of infected cells from cytotoxic lymphocytes. These findings may explain why infected endothelial cells in hantavirus-infected patients are not destroyed by the strong cytotoxic lymphocyte response.

  17. Hantavirus-infection confers resistance to cytotoxic lymphocyte-mediated apoptosis.

    Science.gov (United States)

    Gupta, Shawon; Braun, Monika; Tischler, Nicole D; Stoltz, Malin; Sundström, Karin B; Björkström, Niklas K; Ljunggren, Hans-Gustaf; Klingström, Jonas

    2013-03-01

    Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardio-pulmonary syndrome (HCPS; also called hantavirus pulmonary syndrome (HPS)), both human diseases with high case-fatality rates. Endothelial cells are the main targets for hantaviruses. An intriguing observation in patients with HFRS and HCPS is that on one hand the virus infection leads to strong activation of CD8 T cells and NK cells, on the other hand no obvious destruction of infected endothelial cells is observed. Here, we provide an explanation for this dichotomy by showing that hantavirus-infected endothelial cells are protected from cytotoxic lymphocyte-mediated induction of apoptosis. When dissecting potential mechanisms behind this phenomenon, we discovered that the hantavirus nucleocapsid protein inhibits the enzymatic activity of both granzyme B and caspase 3. This provides a tentative explanation for the hantavirus-mediated block of cytotoxic granule-mediated apoptosis-induction, and hence the protection of infected cells from cytotoxic lymphocytes. These findings may explain why infected endothelial cells in hantavirus-infected patients are not destroyed by the strong cytotoxic lymphocyte response.

  18. Effectiveness of lytic bacteriophages in reducing E. coli O157:H7 populations introduced through cross-contamination on fresh cut lettuce

    Science.gov (United States)

    Previous research has shown that lytic bacteriophages (phages) can kill E. coli O157:H7 on produce surfaces. 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) at 10^8 PFU/m...

  19. Complete Genome Sequence of a Lytic Siphoviridae Bacteriophage Infecting Several Serovars of Salmonella enterica

    Science.gov (United States)

    Paradiso, Rubina; Lombardi, Serena; Iodice, Maria Grazia; Riccardi, Marita Georgia; Orsini, Massimiliano; Bolletti Censi, Sergio; Galiero, Giorgio

    2016-01-01

    The bacteriophage 100268_sal2 was isolated from water buffalo feces in southern Italy, exhibiting lytic activity against several subspecies of Salmonella enterica. This bacteriophage belongs to the Siphoviridae family and has a 125,114-bp double-stranded DNA (ds-DNA) genome containing 188 coding sequences (CDSs). PMID:27688334

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

    Directory of Open Access Journals (Sweden)

    Giuseppe Balistreri

    2016-02-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  2. Crystal structure and mechanism of the lytic transglycosylase MltA from Escherichia coli

    NARCIS (Netherlands)

    van Straaten, Karin

    2006-01-01

    This thesis describes the determination and analysis of the 3D-structure of the lytic transglycosylase MltA from Escherichia coli by X-ray crystallography. This work aims to further increase our knowledge of the molecular details of the cleaving mechanism and the typical 1,6- anhydromuropeptide prod

  3. Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase

    DEFF Research Database (Denmark)

    Lo Leggio, Leila; Simmons, Thomas J.; Poulsen, Jens-Christian Navarro

    2015-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here...... substrate to maltose by β-amylase. The detailed structure of the enzyme's active site yields insights into the mechanism of action of this important class of enzymes....

  4. Pain relief with percutaneous trochanteroplasty in a patient with bilateral trochanteric myelomatous lytic lesions.

    Science.gov (United States)

    Wahezi, Sayed E; Silva, Kyle; Najafi, Shervin

    2015-01-01

    Multiple myeloma is a hematologic malignancy associated with destructive bone loss. Lytic lesions, a hallmark of this cancer, can result in significant morbidity because of associated pain and structural osseous compromise. Osteoplasty has demonstrated efficacy in the treatment of myelomatous pain within the axial skeleton; however, there is limited evidence supporting the utility of osteoplasty to treat extra-spinal lesions. We describe a 67 year-old woman with stable IgA lambda multiple myeloma with sentinel bilateral greater trochanteric lytic lesions that was referred to our interventional pain management clinic for evaluation of bilateral lateral hip pain. Conservative treatment options including physical therapy, non-steroidal anti-inflammatory drugs (NSAIDs), oral opiates, and local corticosteroid injections to bilateral trochanteric bursae failed to offer pain relief. The patient underwent minimally invasive percutaneous trochanteroplasty with concomitant core biopsy of her bilateral trochanteric lytic lesions. The intended goals of this novel procedure were to determine the cause of the suspected lytic lesions, provide pain relief, and offer structural stability by safely implanting bone cement as part of a fracture prevention strategy. At 12 month follow-up, the patient's pain improved by 70% and she no longer required the use of pain medication. The patient also displayed a significant improvement in her day-to-day functioning and quality of life.

  5. The importance of lytic and nonlytic immune responses in viral infections

    DEFF Research Database (Denmark)

    Wodarz, Dominik; Christensen, Jan Pravsgaard; Thomsen, Allan Randrup

    2002-01-01

    Antiviral immune effector mechanisms can be divided broadly into lytic and nonlytic components. We use mathematical models to investigate the fundamental question of which type of response is required to combat different types of viral infection. According to our model, the relative roles...

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

    Science.gov (United States)

    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.

  7. Crystal structure and mechanism of the lytic transglycosylase MltA from Escherichia coli

    NARCIS (Netherlands)

    van Straaten, Karin

    2006-01-01

    This thesis describes the determination and analysis of the 3D-structure of the lytic transglycosylase MltA from Escherichia coli by X-ray crystallography. This work aims to further increase our knowledge of the molecular details of the cleaving mechanism and the typical 1,6- anhydromuropeptide prod

  8. Characterization of the lytic-lysogenic switch of the lactococcal bacteriophage Tuc2009

    NARCIS (Netherlands)

    Kenny, JG; Leach, S; de la Hoz, AB; Venema, G; Kok, J; Fitzgerald, GF; Nauta, A; Alonso, JC; van Sinderen, D; Kenny, John G.; Hoz, Ana B. de la; Fitzgerald, Gerald F.; Alonso, Juan C.

    2006-01-01

    Tuc2009 is a temperate bacteriophage of Lactococcus lactis subsp. cremoris UC509 which encodes a CI- and Cro-type lysogenic-lytic switch region. A helix-swap of the 0 helices of the closely related Cl-type proteins from the lactococcal phages r1t and Tuc2009 revealed the crucial elements involved in

  9. STUDIES ON THE BACTERIOPHAGE OF D'HERELLE : I. IS THE LYTIC PRINCIPLE VOLATILE?

    Science.gov (United States)

    Bronfenbrenner, J J; Korb, C

    1925-01-01

    The lytic principle concerned in the phenomenon of transmissible lysis is not volatile. The results which have been taken to indicate volatility are, in our opinion, to be attributed to the transfer to the distillate of minute droplets of the original active filtrate.

  10. A comparative study on the activity of fungal lytic polysaccharide monooxygenases for the depolymerization of cellulose in soybean spent flakes

    DEFF Research Database (Denmark)

    Pierce, Brian; Wittrup Agger, Jane; Zhang, Zhenghong

    2017-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes capable of the oxidative breakdown of polysaccharides. They are of industrial interest due to their ability to enhance the enzymatic depolymerization of recalcitrant substrates by glycoside hydrolases. In this paper, twenty-...

  11. Lytic and lysogenic infection of diverse Escherichia coli and Shigella strains with a verocytotoxigenic bacteriophage.

    Science.gov (United States)

    James, C E; Stanley, K N; Allison, H E; Flint, H J; Stewart, C S; Sharp, R J; Saunders, J R; McCarthy, A J

    2001-09-01

    A verocytotoxigenic bacteriophage isolated from a strain of enterohemorrhagic Escherichia coli O157, into which a kanamycin resistance gene (aph3) had been inserted to inactivate the verocytotoxin gene (vt2), was used to infect Enterobacteriaceae strains. A number of Shigella and E. coli strains were susceptible to lysogenic infection, and a smooth E. coli isolate (O107) was also susceptible to lytic infection. The lysogenized strains included different smooth E. coli serotypes of both human and animal origin, indicating that this bacteriophage has a substantial capacity to disseminate verocytotoxin genes. A novel indirect plaque assay utilizing an E. coli recA441 mutant in which phage-infected cells can enter only the lytic cycle, enabling detection of all infective phage, was developed.

  12. Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase.

    Science.gov (United States)

    O'Dell, William B; Agarwal, Pratul K; Meilleur, Flora

    2017-01-16

    Lytic polysaccharide monooxygenases have attracted vast attention owing to their abilities to disrupt glycosidic bonds via oxidation instead of hydrolysis and to enhance enzymatic digestion of recalcitrant substrates including chitin and cellulose. We have determined high-resolution X-ray crystal structures of an enzyme from Neurospora crassa in the resting state and of a copper(II) dioxo intermediate complex formed in the absence of substrate. X-ray crystal structures also revealed "pre-bound" molecular oxygen adjacent to the active site. An examination of protonation states enabled by neutron crystallography and density functional theory calculations identified a role for a conserved histidine in promoting oxygen activation. These results provide a new structural description of oxygen activation by substrate free lytic polysaccharide monooxygenases and provide insights that can be extended to reactivity in the enzyme-substrate complex. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Participation of the lytic replicon in bacteriophage P1 plasmid maintenance.

    OpenAIRE

    1989-01-01

    P1 bacteriophage carries at least two replicons: a plasmid replicon and a viral lytic replicon. Since the isolated plasmid replicon can maintain itself stably at the low copy number characteristic of intact P1 prophage, it has been assumed that this replicon is responsible for driving prophage replication. We provide evidence that when replication from the plasmid replicon is prevented, prophage replication continues, albeit at a reduced rate. The residual plasmid replication is due to incomp...

  14. How Cancer Cells Become Resistant to Cationic Lytic Peptides: It's the Sugar!

    Science.gov (United States)

    Pierce, Joshua G

    2017-02-16

    In this issue of Cell Chemical Biology, Ishikawa et al. (2017) demonstrate that the loss of cell-surface anionic saccharides can impart resistance toward anticancer peptides. This study provides the first insight into potential resistance mechanisms toward cationic lytic peptides and highlights the important, yet previously unappreciated, role cell-surface glycans can play in cellular resistance mechanisms. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Regulation of the Spore Cortex Lytic Enzyme SleB in Bacillus anthracis

    OpenAIRE

    2014-01-01

    Bacillus anthracis is the causative agent of the disease anthrax and poses a threat due to its potential to be used as a biological weapon. The spore form of this bacterium is an extremely resistant structure, making spore decontamination exceptionally challenging. During spore germination, nutrient germinants interact with Ger receptors, triggering a cascade of events. A crucial event in this process is degradation of the cortex peptidoglycan by germination-specific lytic enzymes (GSLEs),...

  16. A novel inhibitory mechanism of mitochondrion-dependent apoptosis by a herpesviral protein.

    Directory of Open Access Journals (Sweden)

    Pinghui Feng

    2007-12-01

    Full Text Available Upon viral infection, cells undergo apoptosis as a defense against viral replication. Viruses, in turn, have evolved elaborate mechanisms to subvert apoptotic processes. Here, we report that a novel viral mitochondrial anti-apoptotic protein (vMAP of murine gamma-herpesvirus 68 (gammaHV-68 interacts with Bcl-2 and voltage-dependent anion channel 1 (VDAC1 in a genetically separable manner. The N-terminal region of vMAP interacted with Bcl-2, and this interaction markedly increased not only Bcl-2 recruitment to mitochondria but also its avidity for BH3-only pro-apoptotic proteins, thereby suppressing Bax mitochondrial translocation and activation. In addition, the central and C-terminal hydrophobic regions of vMAP interacted with VDAC1. Consequently, these interactions resulted in the effective inhibition of cytochrome c release, leading to the comprehensive inhibition of mitochondrion-mediated apoptosis. Finally, vMAP gene was required for efficient gammaHV-68 lytic replication in normal cells, but not in mitochondrial apoptosis-deficient cells. These results demonstrate that gammaHV-68 vMAP independently targets two important regulators of mitochondrial apoptosis-mediated intracellular innate immunity, allowing efficient viral lytic replication.

  17. A novel inhibitory mechanism of mitochondrion-dependent apoptosis by a herpesviral protein.

    Science.gov (United States)

    Feng, Pinghui; Liang, Chengyu; Shin, Young C; Xiaofei, E; Zhang, Weijun; Gravel, Robyn; Wu, Ting-ting; Sun, Ren; Usherwood, Edward; Jung, Jae U

    2007-12-01

    Upon viral infection, cells undergo apoptosis as a defense against viral replication. Viruses, in turn, have evolved elaborate mechanisms to subvert apoptotic processes. Here, we report that a novel viral mitochondrial anti-apoptotic protein (vMAP) of murine gamma-herpesvirus 68 (gammaHV-68) interacts with Bcl-2 and voltage-dependent anion channel 1 (VDAC1) in a genetically separable manner. The N-terminal region of vMAP interacted with Bcl-2, and this interaction markedly increased not only Bcl-2 recruitment to mitochondria but also its avidity for BH3-only pro-apoptotic proteins, thereby suppressing Bax mitochondrial translocation and activation. In addition, the central and C-terminal hydrophobic regions of vMAP interacted with VDAC1. Consequently, these interactions resulted in the effective inhibition of cytochrome c release, leading to the comprehensive inhibition of mitochondrion-mediated apoptosis. Finally, vMAP gene was required for efficient gammaHV-68 lytic replication in normal cells, but not in mitochondrial apoptosis-deficient cells. These results demonstrate that gammaHV-68 vMAP independently targets two important regulators of mitochondrial apoptosis-mediated intracellular innate immunity, allowing efficient viral lytic replication.

  18. Suppression of injuries caused by a lytic RNA virus (mengovirus) and their uncoupling from viral reproduction by mutual cell/virus disarmament.

    Science.gov (United States)

    Mikitas, Olga V; Ivin, Yuri Y; Golyshev, Sergey A; Povarova, Natalia V; Galkina, Svetlana I; Pletjushkina, Olga Y; Nadezhdina, Elena S; Gmyl, Anatoly P; Agol, Vadim I

    2012-05-01

    Viruses often elicit cell injury (cytopathic effect [CPE]), a major cause of viral diseases. CPE is usually considered to be a prerequisite for and/or consequence of efficient viral growth. Recently, we proposed that viral CPE may largely be due to host defensive and viral antidefensive activities. This study aimed to check the validity of this proposal by using as a model HeLa cells infected with mengovirus (MV). As we showed previously, infection of these cells with wild-type MV resulted in necrosis, whereas a mutant with incapacitated antidefensive ("security") viral leader (L) protein induced apoptosis. Here, we showed that several major morphological and biochemical signs of CPE (e.g., alterations in cellular and nuclear shape, plasma membrane, cytoskeleton, chromatin, and metabolic activity) in cells infected with L(-) mutants in the presence of an apoptosis inhibitor were strongly suppressed or delayed for long after completion of viral reproduction. These facts demonstrate that the efficient reproduction of a lytic virus may not directly require development of at least some pathological alterations normally accompanying infection. They also imply that L protein is involved in the control of many apparently unrelated functions. The results also suggest that the virus-activated program with competing necrotic and apoptotic branches is host encoded, with the choice between apoptosis and necrosis depending on a variety of intrinsic and extrinsic conditions. Implementation of this defensive suicidal program could be uncoupled from the viral reproduction. The possibility of such uncoupling has significant implications for the pathogenesis and treatment of viral diseases.

  19. Murine gamma-herpesvirus 68 hijacks MAVS and IKKbeta to initiate lytic replication.

    Directory of Open Access Journals (Sweden)

    Xiaonan Dong

    2010-07-01

    Full Text Available Upon viral infection, the mitochondrial antiviral signaling (MAVS-IKKbeta pathway is activated to restrict viral replication. Manipulation of immune signaling events by pathogens has been an outstanding theme of host-pathogen interaction. Here we report that the loss of MAVS or IKKbeta impaired the lytic replication of gamma-herpesvirus 68 (gammaHV68, a model herpesvirus for human Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus. gammaHV68 infection activated IKKbeta in a MAVS-dependent manner; however, IKKbeta phosphorylated and promoted the transcriptional activation of the gammaHV68 replication and transcription activator (RTA. Mutational analyses identified IKKbeta phosphorylation sites, through which RTA-mediated transcription was increased by IKKbeta, within the transactivation domain of RTA. Moreover, the lytic replication of recombinant gammaHV68 carrying mutations within the IKKbeta phosphorylation sites was greatly impaired. These findings support the conclusion that gammaHV68 hijacks the antiviral MAVS-IKKbeta pathway to promote viral transcription and lytic infection, representing an example whereby viral replication is coupled to host immune activation.

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

    Science.gov (United States)

    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.

  1. KSHV Targeted Therapy: An Update on Inhibitors of Viral Lytic Replication

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    Natacha Coen

    2014-11-01

    Full Text Available Kaposi’s sarcoma-associated herpesvirus (KSHV is the causative agent of Kaposi’s sarcoma, primary effusion lymphoma and multicentric Castleman’s disease. Since the discovery of KSHV 20 years ago, there is still no standard treatment and the management of virus-associated malignancies remains toxic and incompletely efficacious. As the majority of tumor cells are latently infected with KSHV, currently marketed antivirals that target the virus lytic cycle have shown inconsistent results in clinic. Nevertheless, lytic replication plays a major role in disease progression and virus dissemination. Case reports and retrospective studies have pointed out the benefit of antiviral therapy in the treatment and prevention of KSHV-associated diseases. As a consequence, potent and selective antivirals are needed. This review focuses on the anti-KSHV activity, mode of action and current status of antiviral drugs targeting KSHV lytic cycle. Among these drugs, different subclasses of viral DNA polymerase inhibitors and compounds that do not target the viral DNA polymerase are being discussed. We also cover molecules that target cellular kinases, as well as the potential of new drug targets and animal models for antiviral testing.

  2. Host transcript accumulation during lytic KSHV infection reveals several classes of host responses.

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    Sanjay Chandriani

    Full Text Available Lytic infection by Kaposi's sarcoma-associated herpesvirus (KSHV is associated with an extensive shutoff of host gene expression, mediated chiefly by accelerated mRNA turnover due to expression of the viral SOX protein. We have previously identified a small number of host mRNAs that can escape SOX-mediated degradation. Here we present a detailed, transcriptome-wide analysis of host shutoff, with careful microarray normalization to allow rigorous determination of the magnitude and extent of transcript loss. We find that the extent of transcript reduction represents a continuum of susceptibilities of transcripts to virus-mediated shutoff. Our results affirm that the levels of over 75% of host transcripts are substantially reduced during lytic infection, but also show that another approximately 20% of cellular mRNAs declines only slightly (less than 2-fold during the course of infection. Approximately 2% of examined cellular genes are strongly upregulated during lytic infection, most likely due to transcriptional induction of mRNAs that display intrinsic SOX-resistance.

  3. Isolation and characterization of lytic phages TSE1-3 against Enterobacter cloacae

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    Khawaja Komal Ameer

    2016-01-01

    Full Text Available The emergence of antibiotic resistant bacterial pathogens is becoming a major challenge for patient care. The utilization of alternative therapies for infectious diseases other than antibiotics is an urgent need of today medical practice. The utilization of lytic bacteriophages and their gene products as therapeutic agents against antibiotic resistant bacteria is one of the convincing alternative approaches. Here we present the isolation and characterization of three lytic bacteriophages TSE1-3 against Enterobacter cloacae from sewage effluent. The isolates maintained antibacterial activity for 10 hours of incubation followed by the development of phage resistance. Their stability at different temperatures and pH, established their possible application in phage therapy. The highest activity of the phages was observed at 37°C and pH 7.0, while they gave lytic activity up to 60°C. The latent period of all the TSE phages was 20 minutes, while the burst size was 360 for TSE1, 270 for TSE2 and 311 for TSE3. The phages were harboring double-stranded DNA larger than 12kb in size. Further research into the phages genome and proteins, animal experiments, delivery parameters and clinical trials may lead to their utilization in phage therapy.

  4. Effects of inducing or inhibiting apoptosis on Sindbis virus replication in mosquito cells.

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    Wang, Hua; Blair, Carol D; Olson, Ken E; Clem, Rollie J

    2008-11-01

    Sindbis virus (SINV) is a mosquito-borne virus in the genus Alphavirus, family Togaviridae. Like most alphaviruses, SINVs exhibit lytic infection (apoptosis) in many mammalian cell types, but are generally thought to cause persistent infection with only moderate cytopathic effects in mosquito cells. However, there have been several reports of apoptotic-like cell death in mosquitoes infected with alphaviruses or flaviviruses. Given that apoptosis has been shown to be an antiviral response in other systems, we have constructed recombinant SINVs that express either pro-apoptotic or anti-apoptotic genes in order to test the effects of inducing or inhibiting apoptosis on SINV replication in mosquito cells. Recombinant SINVs expressing the pro-apoptotic genes reaper (rpr) from Drosophila or michelob_x (mx) from Aedes aegypti caused extensive apoptosis in cells from the mosquito cell line C6/36, thus changing the normal persistent infection observed with SINV to a lytic infection. Although the infected cells underwent apoptosis, high levels of virus replication were still observed during the initial infection. However, virus production subsequently decreased compared with persistently infected cells, which continued to produce high levels of virus over the next several days. Infection of C6/36 cells with SINV expressing the baculovirus caspase inhibitor P35 inhibited actinomycin D-induced caspase activity and protected infected cells from actinomycin D-induced apoptosis, but had no observable effect on virus replication. This study is the first to test directly whether inducing or inhibiting apoptosis affects arbovirus replication in mosquito cells.

  5. Noncanonical microRNAs and endogenous siRNAs in lytic infection of murine gammaherpesvirus.

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    Jing Xia

    Full Text Available MicroRNA (miRNA and endogenous small interfering RNA (endo-siRNA are two essential classes of small noncoding RNAs (sncRNAs in eukaryotes. The class of miRNA is diverse and there exist noncanonical miRNAs that bypass the canonical miRNA biogenesis pathway. In order to identify noncanonical miRNAs and endo-siRNAs responding to virus infection and study their potential function, we sequenced small-RNA species from cells lytically infected with murine gammaherpesvirus 68 (MHV68. In addition to three novel canonical miRNAs in mouse, two antisense miRNAs in virus and 25 novel noncanonical miRNAs, including miRNAs derived from transfer RNAs, small nucleolar RNAs and introns, in the host were identified. These noncanonical miRNAs exhibited features distinct from that of canonical miRNAs in lengths of hairpins, base pairings and first nucleotide preference. Many of the novel miRNAs are conserved in mammals. Besides several known murine endo-siRNAs detected by the sequencing profiling, a novel locus in the mouse genome was identified to produce endo-siRNAs. This novel endo-siRNA locus is comprised of two tandem inverted B4 short interspersed nuclear elements (SINEs. Unexpectedly, the SINE-derived endo-siRNAs were found in a variety of sequencing data and virus-infected cells. Moreover, a murine miRNA was up-regulated more than 35 fold in infected than in mock-treated cells. The putative targets of the viral and the up-regulated murine miRNAs were potentially involved in processes of gene transcription and protein phosphorylation, and localized to membranes, suggesting their potential role in manipulating the host basal immune system during lytic infection. Our results extended the number of noncanonical miRNAs in mammals and shed new light on their potential functions of lytic infection of MHV68.

  6. Induction of Epstein-Barr Virus Lytic Replication by Recombinant Adenoviruses Expressing the Zebra Gene with EBV Specific Promoters

    Institute of Scientific and Technical Information of China (English)

    Lu CHEN; Juan YIN; Yi CHEN; Jiang ZHONG

    2005-01-01

    The latent Epstein-Barr virus (EBV) is found in the cells of many tumors. For example, EBV is detectable in almost all cases, and in almost all tumor cells, of non-keratinizing nasopharyngeal carcinoma.Activating the latent virus, which will result in its lytic replication and the death of tumor cells, is a potential approach for the treatment of EBV-associated cancers. In this study, three recombinant adenoviruses were constructed to express the Zebra gene, an EBV gene responsible for switching from the latent state to lytic replication. EBV-specific promoters were used in order to limit Zebra expression in EBV-positive cells, and reduce the potential side effects. The EBV promoters used were Cp, Zp and a dual promoter combining both promoters, CpZp. The Zebra protein was detected in HEK293 cells as well as the EBV-positive D98-HR1 cells infected with recombinant viruses. An EBV lytic replication early antigen, EA-D, was also detected in infected D98-HR1, implying the initiation of lytic replication. In the cell viability assay, Zebra-expressing adenoviruses had little effect on EBV-negative HeLa cells, while significantly reducing the cell viability and proliferation of D98-HR1 cells. The results indicate that EBV virus promoters can be used in adenovirus vectors to express the Zebra gene and induce EBV lytic replication in D98-HR1 cells.

  7. Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase.

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    Lo Leggio, Leila; Simmons, Thomas J; Poulsen, Jens-Christian N; Frandsen, Kristian E H; Hemsworth, Glyn R; Stringer, Mary A; von Freiesleben, Pernille; Tovborg, Morten; Johansen, Katja S; De Maria, Leonardo; Harris, Paul V; Soong, Chee-Leong; Dupree, Paul; Tryfona, Theodora; Lenfant, Nicolas; Henrissat, Bernard; Davies, Gideon J; Walton, Paul H

    2015-01-22

    Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here the activity, spectroscopy and three-dimensional structure of a starch-active LPMO, a representative of the new CAZy AA13 family. We demonstrate that these enzymes generate aldonic acid-terminated malto-oligosaccharides from retrograded starch and boost significantly the conversion of this recalcitrant substrate to maltose by β-amylase. The detailed structure of the enzyme's active site yields insights into the mechanism of action of this important class of enzymes.

  8. Regulation of latency to lytic life cycle:multiple tricks by KSHV RTA

    Institute of Scientific and Technical Information of China (English)

    Jiemin Wong

    2010-01-01

    @@ Higher Education Press and Springer-Verlag Berlin Heidelberg 2010The herpesviruses are large enveloped DNA viruses that infect a wide spectrum hosts including human being. A key characteristic of all herpesviruses is their ability to establish life-time latency within the infected host and to periodically reactivate and enter the iytic replication to produce infectious virus progeny. During latency the 120-300 kb double-stranded DNA genomes of these viruses are maintained as multiple copies of circular episomes within the nuclei of the host cells. Lytic replication is marked by an increase in viral gene expression and the production of infectious virus progeny.

  9. Percutaneous aspiration biopsy in cervical spine lytic lesions. Indications and technique

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    Tampieri, D.; Weill, A.; Melanson, D.; Ethier, R. (Montreal Neurological Inst. and Hospital, PQ (Canada). Dept. of Neuroradiology)

    1991-02-01

    We describe the technique and the results of the percutaneous aspiration biopsy (PAB) in a series of 9 patients presenting with neck pain and different degrees of myelopathy, in whom the cervical spine X-ray demonstrated lytic lesions of unknown origin. PAB is a useful, relatively safe technique, and leads to histological diagnosis between metastatic and inflammatory processes. Furthermore, in inflammatory lesions with negative hemoculture, PAB may help in detecting the micro-organism responsible and therefore allow a better antibiotic treatment. (orig.).

  10. Epinecidin-1, an antimicrobial peptide from fish (Epinephelus coioides) which has an antitumor effect like lytic peptides in human fibrosarcoma cells.

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    Lin, Wei-Ju; Chien, Yi-Lun; Pan, Chia-Yu; Lin, Tai-Lang; Chen, Jyh-Yih; Chiu, Shu-Jun; Hui, Cho-Fat

    2009-02-01

    Epinecidin-1, a synthetic 21-mer antimicrobial peptide originally identified from grouper (Epinephelus coioides), specifically exhibited high antimicrobial activities against both Gram-negative and Gram-positive bacteria. In the current study we report on the in vitro cytotoxicity of the peptide, an important factor before it can be considered for further applications in cancer therapy. The cytotoxicity of epinecidin-1 was investigated against several cancer cells (A549, HA59T/VGH, HeLa, HepG2, HT1080, RAW264.7, and U937) and normal cells (AML-12, NIH3T3, and WS-1) with the MTT assay, and the inhibition of cancer cell growth was confirmed by a soft agar assay and scanning electron microscopy. However, cell variations were detected with AO/EtBr staining, while apoptosis and necrosis gene expressions in HT1080 cells after treatment with the epinecidin-1 peptide and Nec-1 showed that epinecidin-1 had an anti-necrosis function in HT1080 cells. The data presented here indicate that epinecidin-1 has in vitro antitumor activity against the HT1080 cell line, and functions like lytic peptides. In addition, our results suggest that epinecidin-1 may prove to be an effective chemotherapeutic agent for human fibrosarcoma cells in the future.

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

    Science.gov (United States)

    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

  12. Protozoacidal Trojan-Horse: use of a ligand-lytic peptide for selective destruction of symbiotic protozoa within termite guts.

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    Sethi, Amit; Delatte, Jennifer; Foil, Lane; Husseneder, Claudia

    2014-01-01

    For novel biotechnology-based termite control, we developed a cellulose bait containing freeze-dried genetically engineered yeast which expresses a protozoacidal lytic peptide attached to a protozoa-recognizing ligand. The yeast acts as a 'Trojan-Horse' that kills the cellulose-digesting protozoa in the termite gut, which leads to the death of termites, presumably due to inefficient cellulose digestion. The ligand targets the lytic peptide specifically to protozoa, thereby increasing its protozoacidal efficiency while protecting non-target organisms. After ingestion of the bait, the yeast propagates in the termite's gut and is spread throughout the termite colony via social interactions. This novel paratransgenesis-based strategy could be a good supplement for current termite control using fortified biological control agents in addition to chemical insecticides. Moreover, this ligand-lytic peptide system could be used for drug development to selectively target disease-causing protozoa in humans or other vertebrates.

  13. Genomic sequence and evolution of marine cyanophage P60: a new insight on lytic and lysogenic phages.

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    Chen, Feng; Lu, Jingrang

    2002-05-01

    The genome of cyanophage P60, a lytic virus which infects marine Synechococcus WH7803, was completely sequenced. The P60 genome contained 47,872 bp with 80 potential open reading frames that were mostly similar to the genes found in lytic phages like T7, phi-YeO3-12, and SIO1. The DNA replication system, consisting of primase-helicase and DNA polymerase, appeared to be more conserved in podoviruses than in siphoviruses and myoviruses, suggesting that DNA replication genes could be the critical elements for lytic phages. Strikingly high sequence similarities in the regions coding for nucleotide metabolism were found between cyanophage P60 and marine unicellular cyanobacteria.

  14. Epstein-Barr virus (EBV Rta-mediated EBV and Kaposi's sarcoma-associated herpesvirus lytic reactivations in 293 cells.

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    Yen-Ju Chen

    Full Text Available Epstein-Barr virus (EBV Rta belongs to a lytic switch gene family that is evolutionarily conserved in all gamma-herpesviruses. Emerging evidence indicates that cell cycle arrest is a common means by which herpesviral immediate-early protein hijacks the host cell to advance the virus's lytic cycle progression. To examine the role of Rta in cell cycle regulation, we recently established a doxycycline (Dox-inducible Rta system in 293 cells. In this cell background, inducible Rta modulated the levels of signature G1 arrest proteins, followed by induction of the cellular senescence marker, SA-β-Gal. To delineate the relationship between Rta-induced cell growth arrest and EBV reactivation, recombinant viral genomes were transferred into Rta-inducible 293 cells. Somewhat unexpectedly, we found that Dox-inducible Rta reactivated both EBV and Kaposi's sarcoma-associated herpesvirus (KSHV, to similar efficacy. As a consequence, the Rta-mediated EBV and KSHV lytic replication systems, designated as EREV8 and ERKV, respectively, were homogenous, robust, and concurrent with cell death likely due to permissive lytic replication. In addition, the expression kinetics of EBV lytic genes in Dox-treated EREV8 cells was similar to that of their KSHV counterparts in Dox-induced ERKV cells, suggesting that a common pathway is used to disrupt viral latency in both cell systems. When the time course was compared, cell cycle arrest was achieved between 6 and 48 h, EBV or KSHV reactivation was initiated abruptly at 48 h, and the cellular senescence marker was not detected until 120 h after Dox treatment. These results lead us to hypothesize that in 293 cells, Rta-induced G1 cell cycle arrest could provide (1 an ideal environment for virus reactivation if EBV or KSHV coexists and (2 a preparatory milieu for cell senescence if no viral genome is available. The latter is hypothetical in a transient-lytic situation.

  15. Epstein-Barr virus (EBV) Rta-mediated EBV and Kaposi's sarcoma-associated herpesvirus lytic reactivations in 293 cells.

    Science.gov (United States)

    Chen, Yen-Ju; Tsai, Wan-Hua; Chen, Yu-Lian; Ko, Ying-Chieh; Chou, Sheng-Ping; Chen, Jen-Yang; Lin, Su-Fang

    2011-03-10

    Epstein-Barr virus (EBV) Rta belongs to a lytic switch gene family that is evolutionarily conserved in all gamma-herpesviruses. Emerging evidence indicates that cell cycle arrest is a common means by which herpesviral immediate-early protein hijacks the host cell to advance the virus's lytic cycle progression. To examine the role of Rta in cell cycle regulation, we recently established a doxycycline (Dox)-inducible Rta system in 293 cells. In this cell background, inducible Rta modulated the levels of signature G1 arrest proteins, followed by induction of the cellular senescence marker, SA-β-Gal. To delineate the relationship between Rta-induced cell growth arrest and EBV reactivation, recombinant viral genomes were transferred into Rta-inducible 293 cells. Somewhat unexpectedly, we found that Dox-inducible Rta reactivated both EBV and Kaposi's sarcoma-associated herpesvirus (KSHV), to similar efficacy. As a consequence, the Rta-mediated EBV and KSHV lytic replication systems, designated as EREV8 and ERKV, respectively, were homogenous, robust, and concurrent with cell death likely due to permissive lytic replication. In addition, the expression kinetics of EBV lytic genes in Dox-treated EREV8 cells was similar to that of their KSHV counterparts in Dox-induced ERKV cells, suggesting that a common pathway is used to disrupt viral latency in both cell systems. When the time course was compared, cell cycle arrest was achieved between 6 and 48 h, EBV or KSHV reactivation was initiated abruptly at 48 h, and the cellular senescence marker was not detected until 120 h after Dox treatment. These results lead us to hypothesize that in 293 cells, Rta-induced G1 cell cycle arrest could provide (1) an ideal environment for virus reactivation if EBV or KSHV coexists and (2) a preparatory milieu for cell senescence if no viral genome is available. The latter is hypothetical in a transient-lytic situation.

  16. Simian virus 40 late proteins possess lytic properties that render them capable of permeabilizing cellular membranes.

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    Daniels, Robert; Rusan, Nasser M; Wilbuer, Anne-Kathrin; Norkin, Leonard C; Wadsworth, Patricia; Hebert, Daniel N

    2006-07-01

    Many nonenveloped viruses have evolved an infectious cycle that culminates in the lysis or permeabilization of the host to enable viral release. How these viruses initiate the lytic event is largely unknown. Here, we demonstrated that the simian virus 40 progeny accumulated at the nuclear envelope prior to the permeabilization of the nuclear, endoplasmic reticulum, and plasma membranes at a time which corresponded with the release of the progeny. The permeabilization of these cellular membranes temporally correlated with late protein expression and was not observed upon the inhibition of their synthesis. To address whether one or more of the late proteins possessed an inherent capacity to induce membrane permeabilization, we examined the permeability of Escherichia coli that separately expressed the late proteins. VP2 and VP3, but not VP1, caused the permeabilization of bacterial membranes. Additionally, VP3 expression resulted in bacterial cell lysis. These findings demonstrate that VP3 possesses an inherent lytic property that is independent of eukaryotic signaling or cell death pathways.

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

    Science.gov (United States)

    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.

  18. CTCF interacts with the lytic HSV-1 genome to promote viral transcription

    Science.gov (United States)

    Lang, Fengchao; Li, Xin; Vladimirova, Olga; Hu, Benxia; Chen, Guijun; Xiao, Yu; Singh, Vikrant; Lu, Danfeng; Li, Lihong; Han, Hongbo; Wickramasinghe, J. M. A. S. P.; Smith, Sheryl T.; Zheng, Chunfu; Li, Qihan; Lieberman, Paul M.; Fraser, Nigel W.; Zhou, Jumin

    2017-01-01

    CTCF is an essential chromatin regulator implicated in important nuclear processes including in nuclear organization and transcription. Herpes Simplex Virus-1 (HSV-1) is a ubiquitous human pathogen, which enters productive infection in human epithelial and many other cell types. CTCF is known to bind several sites in the HSV-1 genome during latency and reactivation, but its function has not been defined. Here, we report that CTCF interacts extensively with the HSV-1 DNA during lytic infection by ChIP-seq, and its knockdown results in the reduction of viral transcription, viral genome copy number and virus yield. CTCF knockdown led to increased H3K9me3 and H3K27me3, and a reduction of RNA pol II occupancy on viral genes. Importantly, ChIP-seq analysis revealed that there is a higher level of CTD Ser2P modified RNA Pol II near CTCF peaks relative to the Ser5P form in the viral genome. Consistent with this, CTCF knockdown reduced the Ser2P but increased Ser5P modified forms of RNA Pol II on viral genes. These results suggest that CTCF promotes HSV-1 lytic transcription by facilitating the elongation of RNA Pol II and preventing silenced chromatin on the viral genome. PMID:28045091

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

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    Garry Robert F

    2007-11-01

    Full Text Available Abstract 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.

  20. Parosteal osteosarcoma dedifferentiating into telangiectatic osteosarcoma: importance of lytic changes and fluid cavities at imaging

    Energy Technology Data Exchange (ETDEWEB)

    Azura, M. [Istituto Ortopedico Rizzoli, Musculoskeletal Oncological Surgery Department, Bologna (Italy); University of Malaya, Department of Orthopaedic Surgery, Kuala Lumpur (Malaysia); Vanel, D. [Radiology, Istituto Ortopedico Rizzoli, Bologna (Italy); Istituti Rizzoli, Anatomia Patologica, Bologna (Italy); Alberghini, M. [Pathology, Istituto Ortopedico Rizzoli, Bologna (Italy); Picci, P.; Staals, E.; Mercuri, M. [Istituto Ortopedico Rizzoli, Musculoskeletal Oncological Surgery Department, Bologna (Italy)

    2009-07-15

    This study was performed to assess the imaging findings in cases of parosteal osteosarcoma dedifferentiated into telangiectatic osteosarcoma. Parosteal osteosarcoma is a low-grade well-differentiated malignant tumor. Dedifferentiation into a more aggressive lesion is frequent and usually visible on imaging as a central lytic area in a sclerotic mass. Only one case of differentiation into a telangiectatic osteosarcoma has been reported. As it has practical consequences, with a need for aggressive chemotherapy, we looked for this rather typical imaging pattern. Review of 199 cases of surface osteosarcomas (including 86 parosteal, of which 23 were dedifferentiated) revealed lesions suggesting a possible telangiectatic osteosarcoma on imaging examinations in five cases (cavities with fluid). Histology confirmed three cases (the two other only had hematoma inside a dedifferentiated tumor). There were three males, aged 24, 28, and 32. They had radiographs and CT, and two an MR examination. Lesions involved the distal femur, proximal tibia, and proximal humerus. The parosteal osteosarcoma was a sclerotic, regular mass, attached to the cortex. A purely lytic mass, partially composed of fluid cavities was easily detected on CT and MR. It involved the medullary cavity twice, and remained outside the bone once. Histology confirmed the two components in each case. Two patients died of pulmonary metastases and one is alive. Knowledge of this highly suggestive pattern should help guide the initial biopsy to diagnose the two components of the tumor, and guide aggressive treatment. (orig.)

  1. Overexpression of antimicrobial lytic peptides protects grapevine from Pierce's disease under greenhouse but not field conditions.

    Science.gov (United States)

    Li, Zhijian T; Hopkins, Donald L; Gray, Dennis J

    2015-10-01

    Pierce's disease (PD) caused by Xylella fastidiosa prevents cultivation of grapevine (Vitis vinifera) and susceptible hybrids in the southeastern United States and poses a major threat to the grape industry of California and Texas. Genetic resistance is the only proven control of X. fastidiosa. Genetic engineering offers an alternative to heretofore ineffective conventional breeding in order to transfer only PD resistance traits into elite cultivars. A synthetic gene encoding lytic peptide LIMA-A was introduced into V. vinifera and a Vitis hybrid to assess in planta inhibition of X. fastidiosa. Over 1050 independent transgenic plant lines were evaluated in the greenhouse, among which nine lines were selected and tested under naturally-inoculated field conditions. These selected plant lines in the greenhouse remain disease-free for 10 years, to date, even with multiple manual pathogen inoculations. However, all these lines in the field, including a grafted transgenic rootstock, succumbed to PD within 7 years. We conclude that in planta production of antimicrobial lytic peptides does not provide durable PD resistance to grapevine under field conditions.

  2. Preliminary survey of local bacteriophages with lytic activity against multi-drug resistant bacteria.

    Science.gov (United States)

    Latz, Simone; Wahida, Adam; Arif, Assuda; Häfner, Helga; Hoß, Mareike; Ritter, Klaus; Horz, Hans-Peter

    2016-10-01

    Bacteriophages (phages) represent a potential alternative for combating multi-drug resistant bacteria. Because of their narrow host range and the ever emergence of novel pathogen variants the continued search for phages is a prerequisite for optimal treatment of bacterial infections. Here we performed an ad hoc survey in the surroundings of a University hospital for the presence of phages with therapeutic potential. To this end, 16 aquatic samples of different origins and locations were tested simultaneously for the presence of phages with lytic activity against five current, but distinct strains each from the ESKAPE-group (i.e., Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae). Phages could be isolated for 70% of strains, covering all bacterial species except S. aureus. Apart from samples from two lakes, freshwater samples were largely devoid of phages. By contrast, one liter of hospital effluent collected at a single time point already contained phages active against two-thirds of tested strains. In conclusion, phages with lytic activity against nosocomial pathogens are unevenly distributed across environments with the prime source being the immediate hospital vicinity. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Global mRNA degradation during lytic gammaherpesvirus infection contributes to establishment of viral latency.

    Directory of Open Access Journals (Sweden)

    Justin M Richner

    2011-07-01

    Full Text Available During a lytic gammaherpesvirus infection, host gene expression is severely restricted by the global degradation and altered 3' end processing of mRNA. This host shutoff phenotype is orchestrated by the viral SOX protein, yet its functional significance to the viral lifecycle has not been elucidated, in part due to the multifunctional nature of SOX. Using an unbiased mutagenesis screen of the murine gammaherpesvirus 68 (MHV68 SOX homolog, we isolated a single amino acid point mutant that is selectively defective in host shutoff activity. Incorporation of this mutation into MHV68 yielded a virus with significantly reduced capacity for mRNA turnover. Unexpectedly, the MHV68 mutant showed little defect during the acute replication phase in the mouse lung. Instead, the virus exhibited attenuation at later stages of in vivo infections suggestive of defects in both trafficking and latency establishment. Specifically, mice intranasally infected with the host shutoff mutant accumulated to lower levels at 10 days post infection in the lymph nodes, failed to develop splenomegaly, and exhibited reduced viral DNA levels and a lower frequency of latently infected splenocytes. Decreased latency establishment was also observed upon infection via the intraperitoneal route. These results highlight for the first time the importance of global mRNA degradation during a gammaherpesvirus infection and link an exclusively lytic phenomenon with downstream latency establishment.

  4. Investigating the lytic activity and structural properties of Staphylococcus aureus phenol soluble modulin (PSM) peptide toxins.

    Science.gov (United States)

    Laabei, Maisem; Jamieson, W David; Yang, Yi; van den Elsen, Jean; Jenkins, A Toby A

    2014-12-01

    The ubiquitous bacterial pathogen, Staphylococcus aureus, expresses a large arsenal of virulence factors essential for pathogenesis. The phenol-soluble modulins (PSMs) are a family of cytolytic peptide toxins which have multiple roles in staphylococcal virulence. To gain an insight into which specific factors are important in PSM-mediated cell membrane disruption, the lytic activity of individual PSM peptides against phospholipid vesicles and T cells was investigated. Vesicles were most susceptible to lysis by the PSMα subclass of peptides (α1-3 in particular), when containing between 10 and 30mol% cholesterol, which for these vesicles is the mixed solid ordered (so)-liquid ordered (lo) phase. Our results show that the PSMβ class of peptides has little effect on vesicles at concentrations comparable to that of the PSMα class and exhibited no cytotoxicity. Furthermore, within the PSMα class, differences emerged with PSMα4 showing decreased vesicle and cytotoxic activity in comparison to its counterparts, in contrast to previous studies. In order to understand this, peptides were studied using helical wheel projections and circular dichroism measurements. The degree of amphipathicity, alpha-helicity and properties such as charge and hydrophobicity were calculated, allowing a structure-function relationship to be inferred. The degree of alpha-helicity of the peptides was the single most important property of the seven peptides studied in predicting their lytic activity. These results help to redefine this class of peptide toxins and also highlight certain membrane parameters required for efficient lysis.

  5. Effect of metals on the lytic cycle of the coccolithovirus, EhV86.

    Directory of Open Access Journals (Sweden)

    Martha eGledhill

    2012-04-01

    Full Text Available 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. Numbers of virus particles produced per E. huxleyi cell and E. huxleyi lysis rates were reduced by Cu at total metal concentrations over 500 nM in the presence of EDTA (ethylenediaminetetraacetic acid, and 250 nM in the absence of EDTA 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 gluthatione content is consistent with increases in the production of reactive oxygen species (ROS on viral infection, 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, a biochemical response required for successful virus infection.

  6. PARTIAL CHARACTERIZATION OF A LYTIC METHICILLIN RESISTANT-STAPHYLOCOCCUS AUREUS BACTERIOPHAGE

    Directory of Open Access Journals (Sweden)

    Sulaiman Al-Yousef

    2014-12-01

    Full Text Available A marked increase in the infection incidence caused by methicillin-resistant Staphylococcus aureus (MRSA strains has been noted in medical practice in recent years. This study was conducted to study the biological and characterize of MRSA-phage. Methicillin resistance of Staphylococcus aureus was detected and confirmed by determining of the MIC of oxacillin by the standard agar dilution method. Phage was biologically purified using single plaque technique, then phage characterization were studied using host range, adsorption time, particle morphology and its structural protein. MRSA phage showing lytic nature was purified by repeated plating after picking of single isolated plaques. This phage is active against all 11 isolates either of S. aureus or MRSA tested as hosts. Phage produced clear plaques indicating their lytic nature. This phage was concentrated employing polyethylene glycol (PEG-NaCl precipitation method. Morphologically, MRSA Phage has a hexagonal head having a long non-contractile tail, indicating his icosahedral nature. Adsorption studies showed 100% adsorption of MRSA-Phage after 35 minutes of exposure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE experimentation indicated that the phage particles contain one major structural protein (about 30 Kda.

  7. Preparation and characterization of polyclonal antibody against Kaposi's sarcoma-associated herpesvirus lytic gene encoding RTA.

    Science.gov (United States)

    Fan, Weifei; Tang, Qiao; Shen, Chenyou; Qin, Di; Lu, Chun; Yan, Qin

    2015-11-01

    Replication and transcription activator (RTA) is a critical lytic protein encoded by Kaposi's sarcoma-associated herpesvirus (KSHV). To prepare rabbit polyclonal antibody against RTA, three antigenic polypeptides of KSHV RTA were initially synthesized. The fragment of RTA was cloned into p3FlagBsd to construct the recombinant plasmid, pRTA-Flag. 293 T and EA.hy926 cells were transfected with pRTA-Flag to obtain RTA-Flag fusion protein, which was detected using anti-Flag antibody. Next, New Zealand white rabbits were immunized with keyhole limpet hemocyanin-conjugated peptides to generate polyclonal antibodies against RTA. Enzyme-linked immunosorbent assays were performed to characterize the polyclonal antibodies, and the titers of the polyclonal antibodies against RTA were greater than 1:11,000. Western blotting and immunofluorescence assay revealed that the prepared antibody reacted specifically with the RTA-Flag fusion protein as well as the native viral protein in KSHV-infected primary effusion lymphoma cells. Collectively, our work successfully constructed the recombinant expression vector, pRTA-Flag, and prepared the polyclonal antibody against RTA, which was valuable for investigating the biochemical and biological functions of the critical KSHV lytic gene.

  8. Apoptosis in Critical Conditions

    Directory of Open Access Journals (Sweden)

    A. M. Golubev

    2006-01-01

    Full Text Available Apoptosis is a variant of programmed cell death. This term was introduced by Kerr et al. in 1972, but information on the important role of apoptosis of some cells in critical conditions has recently appeared. The review of literature considers the basic mechanisms of induction, development, and regulation of apoptosis. Based on a literature update, the authors analyze the role of apoptosis in the pathogenesis of various critical conditions: acute lung lesion (neutrophilic and epithelial hypotheses, sepsis, myocardial infarction, and ischemic stroke (apoptosis of tubular epithelial cells, hepatic dysfunction in sepsis, myopathies in critical conditions. The data of studies dealing with the effects of inhaled and non-inhaled anesthetics on the apoptosis of neurons of the brain and lymphocytes are given. The review of literature presents the options of therapeutic apoptosis modulation by pharmacological methods.  

  9. Bronchogenic adenocarcinoma presenting as a synchronous solitary lytic skull lesion with ischaemic stroke--case report and literature review.

    LENUS (Irish Health Repository)

    O'Connell, David

    2011-01-01

    The authors describe a rare case of metastatic bronchogenic adenocarcinoma in a 55-year-old man presenting with concomittant solitary lytic skull lesion and ischaemic stroke. Metastatic bronchogenic carcinoma is known to present as lytic skull lesions. Primary brain tumours are also known to cause ischaemic brain injury. An underlying stroke risk may be exagerated by cranial tumour surgery. Patients with brain tumours are well known to be predisposed to an increased risk of developing thromboembolic disease. It is unusual to see metastatic bronchogenic adenocarcinoma presenting as ischaemic stroke with a background of concomittant cerebral metastasis. The aetio-pathogenesis of this rare occurrence is discussed with a review of literature.

  10. MID2 can substitute for MID1 and control exocytosis of lytic granules in cytotoxic T cells

    DEFF Research Database (Denmark)

    Boding, Lasse; Hansen, Ann K; Meroni, Germana;

    2015-01-01

    We have recently shown that the E3 ubiquitin ligase midline 1 (MID1) is upregulated in murine cytotoxic lymphocytes (CTL), where it controls exocytosis of lytic granules and the killing capacity. Accordingly, CTL from MID1 knock-out (MID1(-/-)) mice have a 25-30% reduction in exocytosis of lytic...... granules and cytotoxicity compared to CTL from wild-type (WT) mice. We wondered why the MID1 gene knock-out did not affect exocytosis and cytotoxicity more severely and speculated whether MID2, a close homologue of MID1, might partially compensate for the loss of MID1 in MID1(-/-) CTL. Here, we showed...

  11. The use of lytic bacteriophages to reduce E. coli O157:H7 on fresh cut lettuce introduced through cross-contamination

    Science.gov (United States)

    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) at 108 PFU/ml or a control (phosphate buffered saline, PBS) was applied to lettuce by either 1) spraying on to lettuce piec...

  12. Phage lysin LysK can be truncated to its CHAP domain and retain lytic activity against live antibiotic-resistant staphylococci.

    Science.gov (United States)

    Horgan, Marianne; O'Flynn, Gary; Garry, Jennifer; Cooney, Jakki; Coffey, Aidan; Fitzgerald, Gerald F; Ross, R Paul; McAuliffe, Olivia

    2009-02-01

    A truncated derivative of the phage endolysin LysK containing only the CHAP (cysteine- and histidine-dependent amidohydrolase/peptidase) domain exhibited lytic activity against live clinical staphylococcal isolates, including methicillin-resistant Staphylococcus aureus. This is the first known report of a truncated phage lysin which retains high lytic activity against live staphylococcal cells.

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

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    Renata Urban-Chmiel

    Full Text Available 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.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.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.The results obtained indicate the need for further research aimed at isolating and characterizing

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

    Science.gov (United States)

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

    2015-01-01

    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. 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). 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. The results obtained indicate the need for further research aimed at isolating and characterizing bacteriophages

  15. Hepatocyte growth factor pathway upregulation in the bone marrow microenvironment in multiple myeloma is associated with lytic bone disease

    DEFF Research Database (Denmark)

    Kristensen, Ida B; Christensen, Jacob H; Lyng, Maria Bibi

    2013-01-01

    Lytic bone disease (LBD) in multiple myeloma (MM) is caused by osteoclast hyperactivation and osteoblast inhibition. Based on in vitro studies, the hepatocyte growth factor (HGF) pathway is thought to be central in osteoblast inhibition. We evaluated the gene expression of the HGF pathway in vivo...

  16. Lytic infection of Lactococcus lactis by bacteriophages Tuc2009 and c2 triggers alternative transcriptional host responses.

    Science.gov (United States)

    Ainsworth, Stuart; Zomer, Aldert; Mahony, Jennifer; van Sinderen, Douwe

    2013-08-01

    Here we present an entire temporal transcriptional profile of Lactococcus lactis subsp. cremoris UC509.9 undergoing lytic infection with two distinct bacteriophages, Tuc2009 and c2. Furthermore, corresponding high-resolution whole-phage genome tiling arrays of both bacteriophages were performed throughout lytic infection. Whole-genome microarrays performed at various time points postinfection demonstrated a rather modest impact on host transcription. The majority of changes in the host transcriptome occur during late infection stages; few changes in host gene transcription occur during the immediate and early infection stages. Alterations in the L. lactis UC509.9 transcriptome during lytic infection appear to be phage specific, with relatively few differentially transcribed genes shared between cells infected with Tuc2009 and those infected with c2. Despite the apparent lack of a coordinated general phage response, three themes common to both infections were noted: alternative transcription of genes involved in catabolic flux and energy production, differential transcription of genes involved in cell wall modification, and differential transcription of genes involved in the conversion of ribonucleotides to deoxyribonucleotides. The transcriptional profiles of both bacteriophages during lytic infection generally correlated with the findings of previous studies and allowed the confirmation of previously predicted promoter sequences. In addition, the host transcriptional response to lysogenization with Tuc2009 was monitored along with tiling array analysis of Tuc2009 in the lysogenic state. Analysis identified 44 host genes with altered transcription during lysogeny, 36 of which displayed levels of transcription significantly reduced from those for uninfected cells.

  17. Probing the structure of glucan lyases – the lytic members of GH31 - by sequence analysis, circular dichroism and proteolysis

    DEFF Research Database (Denmark)

    Ernst, Heidi; Lo Leggio, Leila; Yu, Shukun

    2005-01-01

    Glucan lyase (GL) is a polysaccharide lyase with unique characteristics. It is involved in an alternative pathway for the degradation of alpha-glucans, the anhydrofructose pathway. Sequence similarity suggests that this lytic enzyme belongs to glycoside hydrolase family 31, for which until very r...

  18. Lytic Infection of Lactococcus lactis by Bacteriophages Tuc2009 and c2 Triggers Alternative Transcriptional Host Responses

    NARCIS (Netherlands)

    Ainsworth, S.; Zomer, A.L.; Mahony, J.; Sinderen, D. van

    2013-01-01

    Here we present an entire temporal transcriptional profile of Lactococcus lactis subsp. cremoris UC509.9 undergoing lytic infection with two distinct bacteriophages, Tuc2009 and c2. Furthermore, corresponding high-resolution whole-phage genome tiling arrays of both bacteriophages were performed thro

  19. In vivo dynamics of EBNA1-oriP interaction during latent and lytic replication of Epstein-Barr virus.

    Science.gov (United States)

    Daikoku, Tohru; Kudoh, Ayumi; Fujita, Masatoshi; Sugaya, Yutaka; Isomura, Hiroki; Tsurumi, Tatsuya

    2004-12-24

    The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is required for maintenance of the viral genome DNA during the latent phase of EBV replication but continues to be synthesized after the induction of viral productive replication. An EBV genome-wide chromatin immunoprecipitation assay revealed that EBNA1 constantly binds to oriP of the EBV genome during not only latent but also lytic infection. Although the total levels of EBNA1 proved constant throughout the latter, the levels of the oriP-bound form were increased as lytic infection proceeded. EBV productive DNA replication occurs at discrete sites in nuclei, called replication compartments, where viral replication proteins are clustered. Confocal laser microscopic analyses revealed that whereas EBNA1 was distributed broadly in nuclei as fine punctate dots during the latent phase of infection, the protein became redistributed to the viral replication compartments and localized as distinct spots within and/or nearby the compartments after the induction of lytic replication. Taking these findings into consideration, oriP regions of the EBV genome might be organized by EBNA1 into replication domains that may set up scaffolding for lytic replication and transcription.

  20. Epiphyseal involvement in Erdheim-Chester disease: radiographic and scintigraphic findings in a case with lytic lesions

    Energy Technology Data Exchange (ETDEWEB)

    Ruiz-Hernandez, G.; Tajahuerce-Romera, G.M.; Latorre-Ibanez, M.D.; Lara-Pomares, A. [Servicio de Medicina Nuclear, Hospital Provincial de Castellon (Spain); Vila-Fayos, V. [Servicio de Reumatologia, Hospital Comarcal de Vinaroz (Spain)

    2000-08-01

    We reported a symmetric increase of activity in lower links secondary to Erdheim-Chester disease and demonstrated by bone scans and radiographs. An inusual scintigraphic and radiographic appearance with epiphyseal involvement and lytic lesions is described. Differential diagnosis of bone scan and radiographic findings is discussed. (orig.)

  1. Oxidative cleavage and hydrolytic boosting of cellulose in soybean spent flakes by Trichoderma reesei Cel61A lytic polysaccharide monooxygenase

    DEFF Research Database (Denmark)

    Pierce, Brian; Wittrup Agger, Jane; Wichmann, Jesper

    2017-01-01

    The auxiliary activity family 9 (AA9) copper-dependent lytic polysaccharide monooxygenase (LPMO) from Trichoderma reesei (EG4; TrCel61A) was investigated for its ability to oxidize the complex polysaccharides from soybean. The substrate specificity of the enzyme was assessed against a variety...

  2. Lytic Infection of Lactococcus lactis by Bacteriophages Tuc2009 and c2 Triggers Alternative Transcriptional Host Responses

    NARCIS (Netherlands)

    Ainsworth, S.; Zomer, A.L.; Mahony, J.; Sinderen, D. van

    2013-01-01

    Here we present an entire temporal transcriptional profile of Lactococcus lactis subsp. cremoris UC509.9 undergoing lytic infection with two distinct bacteriophages, Tuc2009 and c2. Furthermore, corresponding high-resolution whole-phage genome tiling arrays of both bacteriophages were performed

  3. High resolution crystal structures of the Escherichia coli lytic transglycosylase Slt70 and its complex with a peptidoglycan fragment

    NARCIS (Netherlands)

    Asselt, Erik J. van; Thunnissen, Andy-Mark W.H.; Dijkstra, Bauke W.

    1999-01-01

    The 70 kDa soluble lytic transglycosylase (Slt70) from Escherichia coli is an exo-muramidase, that catalyses the cleavage of the glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine residues in peptidoglycan, the main structural component of the bacterial cell wall. This cleavage is

  4. A subset of replication proteins enhances origin recognition and lytic replication by the Epstein-Barr virus ZEBRA protein.

    Directory of Open Access Journals (Sweden)

    Ayman El-Guindy

    Full Text Available ZEBRA is a site-specific DNA binding protein that functions as a transcriptional activator and as an origin binding protein. Both activities require that ZEBRA recognizes DNA motifs that are scattered along the viral genome. The mechanism by which ZEBRA discriminates between the origin of lytic replication and promoters of EBV early genes is not well understood. We explored the hypothesis that activation of replication requires stronger association between ZEBRA and DNA than does transcription. A ZEBRA mutant, Z(S173A, at a phosphorylation site and three point mutants in the DNA recognition domain of ZEBRA, namely Z(Y180E, Z(R187K and Z(K188A, were similarly deficient at activating lytic DNA replication and expression of late gene expression but were competent to activate transcription of viral early lytic genes. These mutants all exhibited reduced capacity to interact with DNA as assessed by EMSA, ChIP and an in vivo biotinylated DNA pull-down assay. Over-expression of three virally encoded replication proteins, namely the primase (BSLF1, the single-stranded DNA-binding protein (BALF2 and the DNA polymerase processivity factor (BMRF1, partially rescued the replication defect in these mutants and enhanced ZEBRA's interaction with oriLyt. The findings demonstrate a functional role of replication proteins in stabilizing the association of ZEBRA with viral DNA. Enhanced binding of ZEBRA to oriLyt is crucial for lytic viral DNA replication.

  5. Probing the structure of glucan lyases – the lytic members of GH31 - by sequence analysis, circular dichroism and proteolysis

    DEFF Research Database (Denmark)

    Ernst, Heidi; Lo Leggio, Leila; Yu, Shukun

    2005-01-01

    Glucan lyase (GL) is a polysaccharide lyase with unique characteristics. It is involved in an alternative pathway for the degradation of alpha-glucans, the anhydrofructose pathway. Sequence similarity suggests that this lytic enzyme belongs to glycoside hydrolase family 31, for which until very r...

  6. Isolation and characterization of lytic vibriophage against Vibrio cholerae O1 from environmental water samples in Kelantan, Malaysia.

    Science.gov (United States)

    Al-Fendi, Ali; Shueb, Rafidah Hanim; Ravichandran, Manickam; Yean, Chan Yean

    2014-10-01

    Water samples from a variety of sources in Kelantan, Malaysia (lakes, ponds, rivers, ditches, fish farms, and sewage) were screened for the presence of bacteriophages infecting Vibrio cholerae. Ten strains of V. cholerae that appeared to be free of inducible prophages were used as the host strains. Eleven bacteriophage isolates were obtained by plaque assay, three of which were lytic and further characterized. The morphologies of the three lytic phages were similar with each having an icosahedral head (ca. 50-60 nm in diameter), a neck, and a sheathed tail (ca. 90-100 nm in length) characteristic of the family Myoviridae. The genomes of the lytic phages were indistinguishable in length (ca. 33.5 kb), nuclease sensitivity (digestible with DNase I, but not RNase A or S1 nuclease), and restriction enzyme sensitivity (identical banding patterns with HindIII, no digestion with seven other enzymes). Testing for infection against 46 strains of V. cholerae and 16 other species of enteric bacteria revealed that all three isolates had a narrow host range and were only capable of infecting V. cholerae O1 El Tor Inaba. The similar morphologies, indistinguishable genome characteristics, and identical host ranges of these lytic isolates suggests that they represent one phage, or several very closely related phages, present in different water sources. These isolates are good candidates for further bio-phage-control studies. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. "Lytic" lesions in autologous bone grafts: demonstration of medullary air pockets on post mortem computed tomography.

    Science.gov (United States)

    Rotman, A; Hamilton, K; O'Donnell, C

    2007-12-01

    Donor bone grafts are an important aspect of orthopaedic surgery. The use of plain film as a pathological screening tool before donor bone dispatch has revealed "lytic" lesions in proximal humeri. Donor demographics did not support the diagnosis of myeloma and subsequent computed tomography (CT) scans of these bones identified the lesions as air, not pathology. In total, 27 long bones were scanned and 100% (27/27 cases) exhibited air within the trabecular bone. Three distinct patterns were found: ovoid, linear/branching, and broad channel. A longitudinal course of CT scans was performed to identify at which stage air appeared within the bone. Pre-retrieval, preprocessing, and postprocessing scans revealed that air originated between the retrieval and preprocessing stages of donor bone preparation. There may be multiple aetiology of this phenomenon, including bone retrieval and natural decomposition.

  8. Multiple Lytic Origins of Replication Are Required for Optimal Gammaherpesvirus Fitness In Vitro and In Vivo.

    Directory of Open Access Journals (Sweden)

    Christine Sattler

    2016-03-01

    Full Text Available An unresolved question in herpesvirus biology is why some herpesviruses contain more than one lytic origin of replication (oriLyt. Using murine gammaherpesvirus 68 (MHV-68 as model virus containing two oriLyts, we demonstrate that loss of either of the two oriLyts was well tolerated in some situations but not in others both in vitro and in vivo. This was related to the cell type, the organ or the route of inoculation. Depending on the cell type, different cellular proteins, for example Hexim1 and Rbbp4, were found to be associated with oriLyt DNA. Overexpression or downregulation of these proteins differentially affected the growth of mutants lacking either the left or the right oriLyt. Thus, multiple oriLyts are required to ensure optimal fitness in different cell types and tissues.

  9. Lytic polysaccharide monooxygenases: a crystallographer's view on a new class of biomass-degrading enzymes

    Directory of Open Access Journals (Sweden)

    Kristian E. H. Frandsen

    2016-11-01

    Full Text Available Lytic polysaccharide monooxygenases (LPMOs are a new class of microbial copper enzymes involved in the degradation of recalcitrant polysaccharides. They have only been discovered and characterized in the last 5–10 years and have stimulated strong interest both in biotechnology and in bioinorganic chemistry. In biotechnology, the hope is that these enzymes will finally help to make enzymatic biomass conversion, especially of lignocellulosic plant waste, economically attractive. Here, the role of LPMOs is likely to be in attacking bonds that are not accessible to other enzymes. LPMOs have attracted enormous interest since their discovery. The emphasis in this review is on the past and present contribution of crystallographic studies as a guide to functional understanding, with a final look towards the future.

  10. Isolation and characterization of a T7-like lytic phage for Pseudomonas fluorescens

    Directory of Open Access Journals (Sweden)

    Neubauer Peter

    2008-10-01

    Full Text Available Abstract Background Despite the proven relevance of Pseudomonas fluorescens as a spoilage microorganism in milk, fresh meats and refrigerated food products and the recognized potential of bacteriophages as sanitation agents, so far no phages specific for P. fluorescens isolates from dairy industry have been closely characterized in view of their lytic efficiency. Here we describe the isolation and characterization of a lytic phage capable to infect a variety of P. fluorescens strains isolated from Portuguese and United States dairy industries. Results Several phages were isolated which showed a different host spectrum and efficiency of lysis. One of the phages, phage ϕIBB-PF7A, was studied in detail due to its efficient lysis of a wide spectrum of P. fluorescens strains and ribotypes. Phage ϕIBB-PF7A with a head diameter of about 63 nm and a tail size of about 13 × 8 nm belongs morphologically to the Podoviridae family and resembles a typical T7-like phage, as analyzed by transmission electron microscopy (TEM. The phage growth cycle with a detected latent period of 15 min, an eclipse period of 10 min, a burst size of 153 plaque forming units per infected cell, its genome size of approximately 42 kbp, and the size and N-terminal sequence of one of the protein bands, which gave similarity to the major capsid protein 10A, are consistent with this classification. Conclusion The isolated T7-like phage, phage ϕIBB-PF7A, is fast and efficient in lysing different P. fluorescens strains and may be a good candidate to be used as a sanitation agent to control the prevalence of spoilage causing P. fluorescens strains in dairy and food related environments.

  11. In vitro management of hospital Pseudomonas aeruginosa biofilm using indigenous T7-like lytic phage.

    Science.gov (United States)

    Ahiwale, Sangeeta; Tamboli, Nilofer; Thorat, Kiran; Kulkarni, Rajendra; Ackermann, Hans; Kapadnis, Balasaheb

    2011-02-01

    Pseudomonas aeruginosa, a human pathogen capable of forming biofilm and contaminating medical settings, is responsible for 65% mortality in the hospitals all over the world. This study was undertaken to isolate lytic phages against biofilm forming Ps. aeruginosa hospital isolates and to use them for in vitro management of biofilms in the microtiter plate. Multidrug resistant strains of Ps. aeruginosa were isolated from the hospital environment in and around Pimpri-Chinchwad, Maharashtra by standard microbiological methods. Lytic phages against these strains were isolated from the Pavana river water by double agar layer plaque assay method. A wide host range phage bacterial virus Ps. aeruginosa phage (BVPaP-3) was selected. Electron microscopy revealed that BVPaP-3 phage is a T7-like phage and is a relative of phage species gh-1. A phage at MOI-0.001 could prevent biofilm formation by Ps. aeruginosa hospital strain-6(HS6) on the pegs within 24 h. It could also disperse pre-formed biofilms of all hospital isolates (HS1-HS6) on the pegs within 24 h. Dispersion of biofilm was studied by monitoring log percent reduction in cfu and log percent increase in pfu of respective bacterium and phage on the peg as well as in the well. Scanning electron microscopy confirmed that phage BVPaP-3 indeed causes biofilm reduction and bacterial cell killing. Laboratory studies prove that BVPaP-3 is a highly efficient phage in preventing and dispersing biofilms of Ps. aeruginosa. Phage BVPaP-3 can be used as biological disinfectant to control biofilm problem in medical devices.

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

    Science.gov (United States)

    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. Copyright © 2016 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  13. Trypanosome lytic factor, an antimicrobial high-density lipoprotein, ameliorates Leishmania infection.

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    Marie Samanovic

    2009-01-01

    Full Text Available Innate immunity is the first line of defense against invading microorganisms. Trypanosome Lytic Factor (TLF is a minor sub-fraction of human high-density lipoprotein that provides innate immunity by completely protecting humans from infection by most species of African trypanosomes, which belong to the Kinetoplastida order. Herein, we demonstrate the broader protective effects of human TLF, which inhibits intracellular infection by Leishmania, a kinetoplastid that replicates in phagolysosomes of macrophages. We show that TLF accumulates within the parasitophorous vacuole of macrophages in vitro and reduces the number of Leishmania metacyclic promastigotes, but not amastigotes. We do not detect any activation of the macrophages by TLF in the presence or absence of Leishmania, and therefore propose that TLF directly damages the parasite in the acidic parasitophorous vacuole. To investigate the physiological relevance of this observation, we have reconstituted lytic activity in vivo by generating mice that express the two main protein components of TLFs: human apolipoprotein L-I and haptoglobin-related protein. Both proteins are expressed in mice at levels equivalent to those found in humans and circulate within high-density lipoproteins. We find that TLF mice can ameliorate an infection with Leishmania by significantly reducing the pathogen burden. In contrast, TLF mice were not protected against infection by the kinetoplastid Trypanosoma cruzi, which infects many cell types and transiently passes through a phagolysosome. We conclude that TLF not only determines species specificity for African trypanosomes, but can also ameliorate an infection with Leishmania, while having no effect on T. cruzi. We propose that TLFs are a component of the innate immune system that can limit infections by their ability to selectively damage pathogens in phagolysosomes within the reticuloendothelial system.

  14. Broad-range lytic bacteriophages that kill Staphylococcus aureus local field strains.

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    Abatángelo, Virginia; Peressutti Bacci, Natalia; Boncompain, Carina A; Amadio, Ariel A; Carrasco, Soledad; Suárez, Cristian A; Morbidoni, Héctor R

    2017-01-01

    Staphylococcus aureus is a very successful opportunistic pathogen capable of causing a variety of diseases ranging from mild skin infections to life-threatening sepsis, meningitis and pneumonia. Its ability to display numerous virulence mechanisms matches its skill to display resistance to several antibiotics, including β-lactams, underscoring the fact that new anti-S. aureus drugs are urgently required. In this scenario, the utilization of lytic bacteriophages that kill bacteria in a genus -or even species- specific way, has become an attractive field of study. In this report, we describe the isolation, characterization and sequencing of phages capable of killing S. aureus including methicillin resistant (MRSA) and multi-drug resistant S. aureus local strains from environmental, animal and human origin. Genome sequencing and bio-informatics analysis showed the absence of genes encoding virulence factors, toxins or antibiotic resistance determinants. Of note, there was a high similarity between our set of phages to others described in the literature such as phage K. Considering that reported phages were obtained in different continents, it seems plausible that there is a commonality of genetic features that are needed for optimum, broad host range anti-staphylococcal activity of these related phages. Importantly, the high activity and broad host range of one of our phages underscores its promising value to control the presence of S. aureus in fomites, industry and hospital environments and eventually on animal and human skin. The development of a cocktail of the reported lytic phages active against S. aureus-currently under way- is thus, a sensible strategy against this pathogen.

  15. Revisiting the Cellulosimicrobium cellulans yeast-lytic β-1,3-glucanases toolbox: A review

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    Ferrer Pau

    2006-03-01

    Full Text Available Abstract Cellulosimicrobium cellulans (also known with the synonyms Cellulomonas cellulans, Oerskovia xanthineolytica, and Arthrobacter luteus is an actinomycete that excretes yeast cell wall lytic enzyme complexes containing endo-β-1,3-glucanases [EC 3.2.1.39 and 3.2.1.6] as key constituents. Three genes encoding endo-β-1,3-glucanases from two C. cellulans strains have been cloned and characterised over the past years. The βglII and βglIIA genes from strain DSM 10297 (also known as O. xanthineolytica LL G109 encoded proteins of 40.8 and 28.6 kDa, respectively, whereas the β-1,3-glucanase gene from strain ATCC 21606 (also known as A. luteus 73–14 encoded a 54.5 kDa protein. Alignment of their deduced amino acid sequences reveal that βglII and βglIIA have catalytic domains assigned to family 16 of glycosyl hydrolases, whereas the catalytic domain from the 54.5 kDa glucanase belongs to family 64. Notably, both βglII and the 54.5 kDa β-1,3-glucanase are multidomain proteins, having a lectin-like C-terminal domain that has been assigned to family 13 of carbohydrate binding modules, and that confers to β-1,3-glucanases the ability to lyse viable yeast cells. Furthermore, βglII may also undergo posttranslational proteolytic processing of its C-terminal domain, resulting in a truncated enzyme retaining its glucanase activity but with very low yeast-lytic activity. In this review, the diversity in terms of structural and functional characteristics of the C. cellulans β-1,3-glucanases has been compiled and compared.

  16. Lytic Characteristics and Identification of Two Alga-lysing Bacterial Strains

    Institute of Scientific and Technical Information of China (English)

    PEI Haiyan; HU Wenrong

    2006-01-01

    All previously reported bacterial species which are capable of lysing harmful algae have been isolated from coastal environments in which harmful algae blooms have occurred. Due to the low concentration of alga-lysing bacteria in an algal bloom, it is difficult to isolate the alga-lysing bacteria by existing methods. In this paper, two algae-lysing bacterial strains,P01 and P03, have been isolated from a biosystem immobilized on a sponge that was highly effective in removing algae and microcystins. Their lysing modes and effects on Microcystis aeruginosa have been studied. The results show that the degradation processes of these two strains for M. aeruginosa accorded with a first-order reaction model when the chlorophylla concentration was in the range from 0 to 1000 μg L-1. The degradation rate constants were 0.106 7, 0.127 4 and 0.279 2 for P01and0.0683, 0.0744 and 0.02897 for P03, when the bacterial densities were 8.6 × 105, 8.6 × 106 and 8.6 × 107cells mL 1, respectively. Moreover, the two bacterial strains had favourable lytic effects not only on M. aeruginosa, but also on Chlorella and Scene-desmus. Their lytic effect on M. aeruginosa did not require physical cell to cell contact, but proceeded by the production of an extracellular product. The bacterial strains were identified as Bacillus species by PCR amplification of the 16S rRNA gene, BLAST analysis, and comparison with sequences in the GenBank nucleotide database.

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

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

    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.

  18. Delta-9 tetrahydrocannabinol (THC inhibits lytic replication of gamma oncogenic herpesviruses in vitro

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    Friedman Herman

    2004-09-01

    Full Text Available Abstract Background The major psychoactive cannabinoid compound of marijuana, delta-9 tetrahydrocannabinol (THC, has been shown to modulate immune responses and lymphocyte function. After primary infection the viral DNA genome of gamma herpesviruses persists in lymphoid cell nuclei in a latent episomal circular form. In response to extracellular signals, the latent virus can be activated, which leads to production of infectious virus progeny. Therefore, we evaluated the potential effects of THC on gamma herpesvirus replication. Methods Tissue cultures infected with various gamma herpesviruses were cultured in the presence of increasing concentrations of THC and the amount of viral DNA or infectious virus yield was compared to those of control cultures. The effect of THC on Kaposi's Sarcoma Associated Herpesvirus (KSHV and Epstein-Barr virus (EBV replication was measured by the Gardella method and replication of herpesvirus saimiri (HVS of monkeys, murine gamma herpesvirus 68 (MHV 68, and herpes simplex type 1 (HSV-1 was measured by yield reduction assays. Inhibition of the immediate early ORF 50 gene promoter activity was measured by the dual luciferase method. Results Micromolar concentrations of THC inhibit KSHV and EBV reactivation in virus infected/immortalized B cells. THC also strongly inhibits lytic replication of MHV 68 and HVS in vitro. Importantly, concentrations of THC that inhibit virus replication of gamma herpesviruses have no effect on cell growth or HSV-1 replication, indicating selectivity. THC was shown to selectively inhibit the immediate early ORF 50 gene promoter of KSHV and MHV 68. Conclusions THC specifically targets viral and/or cellular mechanisms required for replication and possibly shared by these gamma herpesviruses, and the endocannabinoid system is possibly involved in regulating gamma herpesvirus latency and lytic replication. The immediate early gene ORF 50 promoter activity was specifically inhibited by THC

  19. Remodelling of cortical actin where lytic granules dock at natural killer cell immune synapses revealed by super-resolution microscopy.

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    Alice C N Brown

    2011-09-01

    Full Text Available Natural Killer (NK cells are innate immune cells that secrete lytic granules to directly kill virus-infected or transformed cells across an immune synapse. However, a major gap in understanding this process is in establishing how lytic granules pass through the mesh of cortical actin known to underlie the NK cell membrane. Research has been hampered by the resolution of conventional light microscopy, which is too low to resolve cortical actin during lytic granule secretion. Here we use two high-resolution imaging techniques to probe the synaptic organisation of NK cell receptors and filamentous (F-actin. A combination of optical tweezers and live cell confocal microscopy reveals that microclusters of NKG2D assemble into a ring-shaped structure at the centre of intercellular synapses, where Vav1 and Grb2 also accumulate. Within this ring-shaped organisation of NK cell proteins, lytic granules accumulate for secretion. Using 3D-structured illumination microscopy (3D-SIM to gain super-resolution of ~100 nm, cortical actin was detected in a central region of the NK cell synapse irrespective of whether activating or inhibitory signals dominate. Strikingly, the periodicity of the cortical actin mesh increased in specific domains at the synapse when the NK cell was activated. Two-colour super-resolution imaging revealed that lytic granules docked precisely in these domains which were also proximal to where the microtubule-organising centre (MTOC polarised. Together, these data demonstrate that remodelling of the cortical actin mesh occurs at the central region of the cytolytic NK cell immune synapse. This is likely to occur for other types of cell secretion and also emphasises the importance of emerging super-resolution imaging technology for revealing new biology.

  20. Remodelling of cortical actin where lytic granules dock at natural killer cell immune synapses revealed by super-resolution microscopy.

    Science.gov (United States)

    Brown, Alice C N; Oddos, Stephane; Dobbie, Ian M; Alakoskela, Juha-Matti; Parton, Richard M; Eissmann, Philipp; Neil, Mark A A; Dunsby, Christopher; French, Paul M W; Davis, Ilan; Davis, Daniel M

    2011-09-01

    Natural Killer (NK) cells are innate immune cells that secrete lytic granules to directly kill virus-infected or transformed cells across an immune synapse. However, a major gap in understanding this process is in establishing how lytic granules pass through the mesh of cortical actin known to underlie the NK cell membrane. Research has been hampered by the resolution of conventional light microscopy, which is too low to resolve cortical actin during lytic granule secretion. Here we use two high-resolution imaging techniques to probe the synaptic organisation of NK cell receptors and filamentous (F)-actin. A combination of optical tweezers and live cell confocal microscopy reveals that microclusters of NKG2D assemble into a ring-shaped structure at the centre of intercellular synapses, where Vav1 and Grb2 also accumulate. Within this ring-shaped organisation of NK cell proteins, lytic granules accumulate for secretion. Using 3D-structured illumination microscopy (3D-SIM) to gain super-resolution of ~100 nm, cortical actin was detected in a central region of the NK cell synapse irrespective of whether activating or inhibitory signals dominate. Strikingly, the periodicity of the cortical actin mesh increased in specific domains at the synapse when the NK cell was activated. Two-colour super-resolution imaging revealed that lytic granules docked precisely in these domains which were also proximal to where the microtubule-organising centre (MTOC) polarised. Together, these data demonstrate that remodelling of the cortical actin mesh occurs at the central region of the cytolytic NK cell immune synapse. This is likely to occur for other types of cell secretion and also emphasises the importance of emerging super-resolution imaging technology for revealing new biology.

  1. Ubiquitination in apoptosis signaling

    NARCIS (Netherlands)

    van de Kooij, L.W.

    2014-01-01

    The work described in this thesis focuses on ubiquitination and protein degradation, with an emphasis on how these processes regulate apoptosis signaling. More specifically, our aims were: 1. To increase the understanding of ubiquitin-mediated regulation of apoptosis signaling. 2. To identify the E3

  2. Calpains, mitochondria, and apoptosis.

    Science.gov (United States)

    Smith, Matthew A; Schnellmann, Rick G

    2012-10-01

    Mitochondrial activity is critical for efficient function of the cardiovascular system. In response to cardiovascular injury, mitochondrial dysfunction occurs and can lead to apoptosis and necrosis. Calpains are a 15-member family of Ca(2+)-activated cysteine proteases localized to the cytosol and mitochondria, and several have been shown to regulate apoptosis and necrosis. For example, in endothelial cells, Ca(2+) overload causes mitochondrial calpain 1 cleavage of the Na(+)/Ca(2+) exchanger leading to mitochondrial Ca(2+) accumulation. Also, activated calpain 1 cleaves Bid, inducing cytochrome c release and apoptosis. In renal cells, calpains 1 and 2 promote apoptosis and necrosis by cleaving cytoskeletal proteins, which increases plasma membrane permeability and cleavage of caspases. Calpain 10 cleaves electron transport chain proteins, causing decreased mitochondrial respiration and excessive activation, or inhibition of calpain 10 activity induces mitochondrial dysfunction and apoptosis. In cardiomyocytes, calpain 1 activates caspase 3 and poly-ADP ribose polymerase during tumour necrosis factor-α-induced apoptosis, and calpain 1 cleaves apoptosis-inducing factor after Ca(2+) overload. Many of these observations have been elucidated with calpain inhibitors, but most calpain inhibitors are not specific for calpains or a specific calpain family member, creating more questions. The following review will discuss how calpains affect mitochondrial function and apoptosis within the cardiovascular system.

  3. Hyperthermia-induced apoptosis

    NARCIS (Netherlands)

    Nijhuis, E.H.A.

    2008-01-01

    This thesis describes a number of studies that investigated several aspects of heat-induced apoptosis in human lymphoid malignancies. Cells harbour both pro- and anti-apoptotic proteins and the balance between these proteins determines whether a cell is susceptible to undergo apoptosis. In this

  4. Ubiquitination in apoptosis signaling

    NARCIS (Netherlands)

    van de Kooij, L.W.

    2014-01-01

    The work described in this thesis focuses on ubiquitination and protein degradation, with an emphasis on how these processes regulate apoptosis signaling. More specifically, our aims were: 1. To increase the understanding of ubiquitin-mediated regulation of apoptosis signaling. 2. To identify the E3

  5. Testing protozoacidal activity of ligand-lytic peptides against termite gut protozoa in vitro (protozoa culture) and in vivo (microinjection into termite hindgut).

    Science.gov (United States)

    Husseneder, Claudia; Sethi, Amit; Foil, Lane; Delatte, Jennifer

    2010-12-29

    We are developing a novel approach to subterranean termite control that would lead to reduced reliance on the use of chemical pesticides. Subterranean termites are dependent on protozoa in the hindguts of workers to efficiently digest wood. Lytic peptides have been shown to kill a variety of protozoan parasites (Mutwiri et al. 2000) and also protozoa in the gut of the Formosan subterranean termite, Coptotermes formosanus (Husseneder and Collier 2009). Lytic peptides are part of the nonspecific immune system of eukaryotes, and destroy the membranes of microorganisms (Leuschner and Hansel 2004). Most lytic peptides are not likely to harm higher eukaryotes, because they do not affect the electrically neutral cholesterol-containing cell membranes of higher eukaryotes (Javadpour et al. 1996). Lytic peptide action can be targeted to specific cell types by the addition of a ligand. For example, Hansel et al. (2007) reported that lytic peptides conjugated with cancer cell membrane receptor ligands could be used to destroy breast cancer cells, while lytic peptides alone or conjugated with non-specific peptides were not effective. Lytic peptides also have been conjugated to human hormones that bind to receptors on tumor cells for targeted destruction of prostate and testicular cancer cells (Leuschner and Hansel 2004). In this article we present techniques used to demonstrate the protozoacidal activity of a lytic peptide (Hecate) coupled to a heptapeptide ligand that binds to the surface membrane of protozoa from the gut of the Formosan subterranean termite. These techniques include extirpation of the gut from termite workers, anaerobic culture of gut protozoa (Pseudotrichonympha grassii, Holomastigotoides hartmanni,Spirotrichonympha leidyi), microscopic confirmation that the ligand marked with a fluorescent dye binds to the termite gut protozoa and other free-living protozoa but not to bacteria or gut tissue. We also demonstrate that the same ligand coupled to a lytic

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

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    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.

  7. Activation and Repression of Epstein-Barr Virus and Kaposi's Sarcoma-Associated Herpesvirus Lytic Cycles by Short- and Medium-Chain Fatty Acids

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    Gorres, Kelly L.; Daigle, Derek; Mohanram, Sudharshan

    2014-01-01

    ABSTRACT The lytic cycles of Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are induced in cell culture by sodium butyrate (NaB), a short-chain fatty acid (SCFA) histone deacetylase (HDAC) inhibitor. Valproic acid (VPA), another SCFA and an HDAC inhibitor, induces the lytic cycle of KSHV but blocks EBV lytic reactivation. To explore the hypothesis that structural differences between NaB and VPA account for their functional effects on the two related viruses, we investigated the capacity of 16 structurally related short- and medium-chain fatty acids to promote or prevent lytic cycle reactivation. SCFAs differentially affected EBV and KSHV reactivation. KSHV was reactivated by all SCFAs that are HDAC inhibitors, including phenylbutyrate. However, several fatty acid HDAC inhibitors, such as isobutyrate and phenylbutyrate, did not reactivate EBV. Reactivation of KSHV lytic transcripts could not be blocked completely by any fatty acid tested. In contrast, several medium-chain fatty acids inhibited lytic activation of EBV. Fatty acids that blocked EBV reactivation were more lipophilic than those that activated EBV. VPA blocked activation of the BZLF1 promoter by NaB but did not block the transcriptional function of ZEBRA. VPA also blocked activation of the DNA damage response that accompanies EBV lytic cycle activation. Properties of SCFAs in addition to their effects on chromatin are likely to explain activation or repression of EBV. We concluded that fatty acids stimulate the two related human gammaherpesviruses to enter the lytic cycle through different pathways. IMPORTANCE Lytic reactivation of EBV and KSHV is needed for persistence of these viruses and plays a role in carcinogenesis. Our direct comparison highlights the mechanistic differences in lytic reactivation between related human oncogenic gammaherpesviruses. Our findings have therapeutic implications, as fatty acids are found in the diet and produced by the human microbiota

  8. Kaposi's sarcoma herpesvirus microRNAs target caspase 3 and regulate apoptosis.

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    Guillaume Suffert

    2011-12-01

    Full Text Available Kaposi's sarcoma herpesvirus (KSHV encodes a cluster of twelve micro (miRNAs, which are abundantly expressed during both latent and lytic infection. Previous studies reported that KSHV is able to inhibit apoptosis during latent infection; we thus tested the involvement of viral miRNAs in this process. We found that both HEK293 epithelial cells and DG75 cells stably expressing KSHV miRNAs were protected from apoptosis. Potential cellular targets that were significantly down-regulated upon KSHV miRNAs expression were identified by microarray profiling. Among them, we validated by luciferase reporter assays, quantitative PCR and western blotting caspase 3 (Casp3, a critical factor for the control of apoptosis. Using site-directed mutagenesis, we found that three KSHV miRNAs, miR-K12-1, 3 and 4-3p, were responsible for the targeting of Casp3. Specific inhibition of these miRNAs in KSHV-infected cells resulted in increased expression levels of endogenous Casp3 and enhanced apoptosis. Altogether, our results suggest that KSHV miRNAs directly participate in the previously reported inhibition of apoptosis by the virus, and are thus likely to play a role in KSHV-induced oncogenesis.

  9. Inhibitor of apoptosis proteins and apoptosis

    Institute of Scientific and Technical Information of China (English)

    Yunbo Wei; Tingjun Fan; Miaomiao Yu

    2008-01-01

    Apoptosis is a physiological cell death process that plays a critical role in development, homeostasis, and immune defense of multicellular animals. Inhibitor of apoptosis proteins (IAPs) constitute a family of proteins that possess between one and three baculovirus IAP repeats. Some of them also have a really interesting new gene finger domain, and can prevent cell death by binding and inhibiting active caspases, but are regulated by IAP antagonists. Some evidence also indicates that IAP can modulate the cell cycle and signal transduction. The three main factors, IAPs, IAP antagonists, and caspases, are involved in regulating the progress of apoptosis in many species. Many studies and assumptions have been focused on the anfractuous interactions between these three main factors to explore their real functional model in order to develop potential anticancer drugs.In this review, we describe the classification, molecular structures, and properties of IAPs and discuss the mechanisms of apoptosis. We also discuss the promising significance of clinical applications of IAPs in the diagnosis and treatment of malignancy.

  10. Epstein-Barr virus evades CD4+ T cell responses in lytic cycle through BZLF1-mediated downregulation of CD74 and the cooperation of vBcl-2.

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    Jianmin Zuo

    2011-12-01

    Full Text Available Evasion of immune T cell responses is crucial for viruses to establish persistence in the infected host. Immune evasion mechanisms of Epstein-Barr virus (EBV in the context of MHC-I antigen presentation have been well studied. In contrast, viral interference with MHC-II antigen presentation is less well understood, not only for EBV but also for other persistent viruses. Here we show that the EBV encoded BZLF1 can interfere with recognition by immune CD4+ effector T cells. This impaired T cell recognition occurred in the absence of a reduction in the expression of surface MHC-II, but correlated with a marked downregulation of surface CD74 on the target cells. Furthermore, impaired CD4+ T cell recognition was also observed with target cells where CD74 expression was downregulated by shRNA-mediated inhibition. BZLF1 downregulated surface CD74 via a post-transcriptional mechanism distinct from its previously reported effect on the CIITA promoter. In addition to being a chaperone for MHC-II αβ dimers, CD74 also functions as a surface receptor for macrophage Migration Inhibitory Factor and enhances cell survival through transcriptional upregulation of Bcl-2 family members. The immune-evasion function of BZLF1 therefore comes at a cost of induced toxicity. However, during EBV lytic cycle induced by BZLF1 expression, this toxicity can be overcome by expression of the vBcl-2, BHRF1, at an early stage of lytic infection. We conclude that by inhibiting apoptosis, the vBcl-2 not only maintains cell viability to allow sufficient time for synthesis and accumulation of infectious virus progeny, but also enables BZLF1 to effect its immune evasion function.

  11. Increased CD8+ T cell response to Epstein-Barr virus lytic antigens in the active phase of multiple sclerosis.

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    Daniela F Angelini

    Full Text Available It has long been known that multiple sclerosis (MS is associated with an increased Epstein-Barr virus (EBV seroprevalence and high immune reactivity to EBV and that infectious mononucleosis increases MS risk. This evidence led to postulate that EBV infection plays a role in MS etiopathogenesis, although the mechanisms are debated. This study was designed to assess the prevalence and magnitude of CD8+ T-cell responses to EBV latent (EBNA-3A, LMP-2A and lytic (BZLF-1, BMLF-1 antigens in relapsing-remitting MS patients (n = 113 and healthy donors (HD (n = 43 and to investigate whether the EBV-specific CD8+ T cell response correlates with disease activity, as defined by clinical evaluation and gadolinium-enhanced magnetic resonance imaging. Using HLA class I pentamers, lytic antigen-specific CD8+ T cell responses were detected in fewer untreated inactive MS patients than in active MS patients and HD while the frequency of CD8+ T cells specific for EBV lytic and latent antigens was higher in active and inactive MS patients, respectively. In contrast, the CD8+ T cell response to cytomegalovirus did not differ between HD and MS patients, irrespective of the disease phase. Marked differences in the prevalence of EBV-specific CD8+ T cell responses were observed in patients treated with interferon-β and natalizumab, two licensed drugs for relapsing-remitting MS. Longitudinal studies revealed expansion of CD8+ T cells specific for EBV lytic antigens during active disease in untreated MS patients but not in relapse-free, natalizumab-treated patients. Analysis of post-mortem MS brain samples showed expression of the EBV lytic protein BZLF-1 and interactions between cytotoxic CD8+ T cells and EBV lytically infected plasma cells in inflammatory white matter lesions and meninges. We therefore propose that inability to control EBV infection during inactive MS could set the stage for intracerebral viral reactivation and disease relapse.

  12. Caspases: An apoptosis mediator

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    Tapan Kumar Palai

    2015-03-01

    Full Text Available The process of programmed cell death, or apoptosis, is generally characterized by distinct morphological characteristics and energy - dependent biochemical mechanisms. Apoptosis is a widely conserved phenomenon helping many processes, including normal cell turnover, proper development and functioning of the immune system, hormone dependent atrophy etc. Inappropriate apoptosis (either low level or high level leads to many developmental abnormalities like, neurodegenerative diseases, ischemic damage, autoimmune disorders and many types of cancer. To use cells for therapeutic purposes through generating cell lines, it is critical to study the cell cycle machinery and signalling pathways that controls cell death and apoptosis. Apoptotic pathways provide a fundamental protective mechanism that decreases cellular sensitivity to damaging events and allow proper developmental process in multi-cellular organisms. Major mediator of apoptosis is a family of proteins known as caspases. There are mainly fourteen types of caspases but out of them only ten caspasese have got essential role in controlling the process of apoptosis. These ten caspases have been categorized into either initiator caspases (caspase 2, 8, 9, 10 or executioner caspases (caspase 3, 6, 7. Although various types of caspases have been identified so far, the exact mechanisms of action of these groups of proteins is still to be fully understood. The aim of this review is to provide a detail overview of role of different caspases in regulating the process of apoptosis.

  13. DNA fragmentation in apoptosis

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Cleavage of chromosomal DNA into oligonucleosomal size fragments is an integral part of apoptosis. Elegant biochemical work identified the DNA fragmentation factor (DFF) as a major apoptotic endonuclease for DNA fragmentation in vitro. Genetic studies in mice support the importance of DFF in DNA fragmentation and possibly in apoptosis in vivo. Recent work also suggests the existence of additional endonucleases for DNA degradation. Understanding the roles of individual endonucleases in apoptosis, and how they might coordinate to degrade DNA in different tissues during normal development and homeostasis, as well as in various diseased states, will be a major research focus in the near future.

  14. Utility of lytic bacteriophage in the treatment of multidrug-resistant Pseudomonas aeruginosa septicemia in mice

    Directory of Open Access Journals (Sweden)

    Vinodkumar C

    2008-07-01

    Full Text Available Drug resistance is the major cause of increase in morbidity and mortality in neonates. One thousand six hundred forty-seven suspected septicemic neonates were subjected for microbiological analysis over a period of 5 years. Forty-two P. aeruginosa were isolated and the antibiogram revealed that 28 P. aeruginosa were resistant to almost all the common drugs used (multidrug-resistant. The emergence of antibiotic-resistant bacterial strains is one of the most critical problems of modern medicine. As a result, a novel and most effective approaches for treating infection caused by multidrug-resistant bacteria are urgently required. In this context, one intriguing approach is to use bacteriophages (viruses that kill bacteria in the treatment of infection caused by drug-resistant bacteria. In the present study, the utility of lytic bacteriophages to rescue septicemic mice with multidrug-resistant (MDR P. aeruginosa infection was evaluated. MDR P. aeruginosa was used to induce septicemia in mice by intraperitoneal (i.p. injection of 10 7 CFU. The resulting bacteremia was fatal within 48 hrs. The phage strain used in this study had lytic activity against a wide range of clinical isolates of MDR P. aeruginosa. A single i.p. injection of 3 x 10 9 PFU of the phage strain, administered 45 min after the bacterial challenge, was sufficient to rescue 100% of the animals. Even when treatment was delayed to the point where all animals were moribund, approximately 50% of them were rescued by a single injection of this phage preparation. The ability of this phage to rescue septicemic mice was demonstrated to be due to the functional capabilities of the phage and not to a nonspecific immune effect. The rescue of septicemic mice could be affected only by phage strains able to grow in vitro on the bacterial host used to infect the animals and when such strains are heat-inactivated, they lose their ability to rescue the infected mice. Multidrug-resistant bacteria have

  15. Survival of Salmonella Newport on Whole and Fresh-Cut Cucumbers Treated with Lytic Bacteriophages.

    Science.gov (United States)

    Sharma, Manan; Dashiell, Gwendolyn; Handy, Eric T; East, Cheryl; Reynnells, Russell; White, Chanelle; Nyarko, Esmond; Micallef, Shirley; Hashem, Fawzy; Millner, Patricia D

    2017-04-01

    Salmonella enterica associated with consumption of cucumbers ( Cucumis sativus ) has led to foodborne outbreaks in the United States. Whole and fresh-cut cucumbers are susceptible to S. enterica contamination during growing, harvesting, and postharvest handling. The application of lytic bacteriophages specific for S. enterica was evaluated to reduce Salmonella populations on cucumbers. Unwaxed cucumbers ('Lisboa' variety, or mini-cucumbers purchased at retail) were inoculated with Salmonella Newport (5 log CFU per cucumber) and were sprayed with 3.2 mL of phosphate-buffered saline (control) or 10 log PFU/ml of SalmoFresh, a Salmonella-specific bacteriophage preparation (phage), to deliver 4.76 × 10(7) PFU/cm(2). Cucumbers were stored at 10 or 22°C for 7 days. Inoculated mini-cucumbers were sliced with a sterile knife to investigate Salmonella transfer to mesocarp, and cut pieces were stored at 4°C for 2 days. Populations (log CFU per cucumber) of Salmonella Newport on phage-treated whole cucumbers were significantly (P cucumbers (4.27 ± 0.37) on day 0. Populations on phage-treated cucumbers stored at 10°C were 1.72 ± 0.77 and 1.56 ± 0.46, which were significantly lower than those on control-treated cucumbers (3.20 ± 0.48 and 2.33 ± 0.25) on days 1 and 4, respectively. Between days 0 and 1, populations on control-treated cucumbers stored at 10 and 22°C declined by 1.07 and 2.47 log CFU per cucumber, respectively. At 22°C, Salmonella Newport populations declined by 2.37 log CFU per cucumber between days 0 and 1. Phage application to whole cucumbers before slicing did not reduce the transfer of Salmonella Newport to fresh-cut slices. Lytic phage application may be a potential intervention to reduce Salmonella populations on whole cucumbers.

  16. A rapid quantitative activity assay shows that the Vibrio cholerae colonization factor GbpA is an active lytic polysaccharide monooxygenase

    NARCIS (Netherlands)

    Loose, Jennifer S. M.; Forsberg, Zarah; Fraaije, Marco W.; Eijsink, Vincent G. H.; Vaaje-Kolstad, Gustav

    2014-01-01

    The discovery of the copper-dependent lytic polysaccharide monooxygenases (LPMOs) has revealed new territory for chemical and biochemical analysis. These unique mononuclear copper enzymes are abundant, suggesting functional diversity beyond their established roles in the depolymerization of biomass

  17. A rapid quantitative activity assay shows that the Vibrio cholerae colonization factor GbpA is an active lytic polysaccharide monooxygenase

    NARCIS (Netherlands)

    Loose, Jennifer S. M.; Forsberg, Zarah; Fraaije, Marco W.; Eijsink, Vincent G. H.; Vaaje-Kolstad, Gustav

    2014-01-01

    The discovery of the copper-dependent lytic polysaccharide monooxygenases (LPMOs) has revealed new territory for chemical and biochemical analysis. These unique mononuclear copper enzymes are abundant, suggesting functional diversity beyond their established roles in the depolymerization of biomass

  18. Inhibition of Epstein-Barr Virus Lytic Cycle by an Ethyl Acetate Subfraction Separated from Polygonum cuspidatum Root and Its Major Component, Emodin

    Directory of Open Access Journals (Sweden)

    Ching-Yi Yiu

    2014-01-01

    Full Text Available 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.

  19. A novel role of IL-17–producing lymphocytes in mediating lytic bone disease in multiple myeloma

    Science.gov (United States)

    Noonan, Kimberly; Marchionni, Luigi; Anderson, Judy; Pardoll, Drew; Roodman, G. David

    2010-01-01

    Osteoclast (OC)–mediated lytic bone disease remains a cause of major morbidity in multiple myeloma. Here we demonstrate the critical role of interleukin-17–producing marrow infiltrating lymphocytes (MILs) in OC activation and development of bone lesions in myeloma patients. Unlike MILs from normal bone marrow, myeloma MILs possess few regulatory T cells (Tregs) and demonstrate an interleukin-17 phenotype that enhances OC activation. In univariate analyses of factors mediating bone destruction, levels of cytokines that selectively induce and maintain the Th17 phenotype tightly correlated with the extent of bone disease in myeloma. In contrast, MILs activated under conditions that skew toward a Th1 phenotype significantly reduced formation of mature OC. These findings demonstrate that interleukin-17 T cells are critical to the genesis of myeloma bone disease and that immunologic manipulations shifting MILs from a Th17 to a Th1 phenotype may profoundly diminish lytic bone lesions in multiple myeloma. PMID:20664052

  20. The algae-lytic ability of bacterium DC10 and the influence of environmental factors on the ability

    Institute of Scientific and Technical Information of China (English)

    SHI Shunyu; LIU Yongding; SHEN Yinwu; LI Genbao

    2005-01-01

    A lysing-bacterium DC10, isolated from Dianchi Lake of Yunnan Province, was characterized to be Pseudomonas sp. It was able to lyse some algae well, such as Microcystis viridis, Selenastrum capricornutum, and so on. In this study, it was shown that the bacterium lysed the algae by releasing a substance; the best lytic effects were achieved at Iow temperatures and in the dark. Different concentrations of CaCI2 and NaNO3 influenced the lytic effects;the ability to lyse algae decreased in the following order: pH 4 > pH 9 > pH 7 > pH 5.5. It was significant to develop a special technology with this kind of bacterium for controlling the bloomforming planktonic microalgae.

  1. Apoptosis in Pneumovirus Infection

    Directory of Open Access Journals (Sweden)

    Reinout A. Bem

    2013-01-01

    Full Text Available Pneumovirus infections cause a wide spectrum of respiratory disease in humans and animals. The airway epithelium is the major site of pneumovirus replication. Apoptosis or regulated cell death, may contribute to the host anti-viral response by limiting viral replication. However, apoptosis of lung epithelial cells may also exacerbate lung injury, depending on the extent, the timing and specific location in the lungs. Differential apoptotic responses of epithelial cells versus innate immune cells (e.g., neutrophils, macrophages during pneumovirus infection can further contribute to the complex and delicate balance between host defense and disease pathogenesis. The purpose of this manuscript is to give an overview of the role of apoptosis in pneumovirus infection. We will examine clinical and experimental data concerning the various pro-apoptotic stimuli and the roles of apoptotic epithelial and innate immune cells during pneumovirus disease. Finally, we will discuss potential therapeutic interventions targeting apoptosis in the lungs.

  2. Phosphoproteomic Analysis of KSHV-Infected Cells Reveals Roles of ORF45-Activated RSK during Lytic Replication.

    Directory of Open Access Journals (Sweden)

    Denis Avey

    2015-07-01

    Full Text Available Kaposi's Sarcoma-Associated Herpesvirus (KSHV is an oncogenic virus which has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK mitogen-activated protein kinase (MAPK pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of ERK/MAPK signaling during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed the consequences of knocking out these substrates on ORF45/RSK-dependent regulation of gene expression and KSHV progeny virion production. Finally, we show data to support that ORF45 regulates the translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5' UTR. Altogether, these data shed light on the mechanisms by which KSHV ORF45

  3. Lytic enzyme production optimization using low-cost substrates and its application in the clarification of xanthan gum culture broth

    Science.gov (United States)

    da Silva, Cíntia Reis; Silva, Marilia Lordelo Cardoso; Kamida, Helio Mitoshi; Goes-Neto, Aristoteles; Koblitz, Maria Gabriela Bello

    2014-01-01

    Lytic enzymes are widely used in industrial biotechnology as they are able to hydrolyze the bacterial cell wall. One application of these enzymes is the clarification of the culture broth for the production of xanthan gum, because of its viability in viscous media and high specificity. The screening process for filamentous fungi producing lytic enzymes, the optimization of production of these enzymes by the selected microorganism, and the optimization of the application of the enzymes produced in the clarification of culture broth are presented in this article. Eleven fungal isolates were tested for their ability to produce enzymes able to increase the transmittance of the culture broth containing cells of Xanthomonas campestris. To optimize the secretion of lytic enzymes by the selected microorganism the following variables were tested: solid substrate, initial pH, incubation temperature, and addition of inducer (gelatin). Thereafter, secretion of the enzymes over time of incubation was assessed. To optimize the clarification process a central composite rotational design was applied in which the pH of the reaction medium, the dilution of the broth, and the reaction temperature were evaluated. The isolate identified as Aspergillus tamarii was selected for increasing the transmittance of the broth from 2.1% to 54.8%. The best conditions for cultivation of this microorganism were: use of coconut husk as solid substrate, with 90% moisture, at 30°C for 20 days. The lytic enzymes produced thereby were able to increase the transmittance of the culture broth from 2.1% to 70.6% at 65°C, without dilution and without pH adjustment. PMID:25473487

  4. Morphological diversity of cultured cold-active lytic bacteriophages isolated from the Napahai plateau wetland in China

    Institute of Scientific and Technical Information of China (English)

    Xiuling Ji; Chunjing Zhang; Anxiu Kuang; Jiankai Li; Yinshan Cui; Kunhao Qin; Lianbing Lin; Benxu Cheng; Qi Zhang; Yunlin Wei

    2015-01-01

    Dear Editor,Viruses are the most abundant,diverse,and ubiquitous entities(approximately 1031)on Earth.They play major roles in horizontal gene transfer,the regulation of bacterial community structures,as well as nutrient and energy cycles of marine ecosystems(Danovaro et al.,2008).In particular,lytic bacteriophages(phages)can infect and kill bacteria without harming human or animal

  5. In vitro and in vivo analyses of the Bacillus anthracis spore cortex lytic protein SleL

    OpenAIRE

    2012-01-01

    The bacterial endospore is the most resilient biological structure known. Multiple protective integument layers shield the spore core and promote spore dehydration and dormancy. Dormancy is broken when a spore germinates and becomes a metabolically active vegetative cell. Germination requires the breakdown of a modified layer of peptidoglycan (PG) known as the spore cortex. This study reports in vitro and in vivo analyses of the Bacillus anthracis SleL protein. SleL is a spore cortex lytic en...

  6. Potential antiviral lignans from the roots of Saururus chinensis with activity against Epstein-Barr virus lytic replication.

    Science.gov (United States)

    Cui, Hui; Xu, Bo; Wu, Taizong; Xu, Jun; Yuan, Yan; Gu, Qiong

    2014-01-24

    Epstein-Barr virus (EBV) is a member of the γ-herpes virus subfamily and has been implicated in the pathogenesis of several human malignancies. Bioassay-guided fractionation was conducted on an EtOAc-soluble extract of the roots of Saururus chinensis and monitored using an EBV lytic replication assay. This led to the isolation of 19 new (1-19) and nine known (20-28) lignans. The absolute configurations of the new lignans were established by Mosher's ester, ECD, and computational methods. Eight lignans, including three sesquineolignans (19, 23, and 24) and five dineolignans (3, 4, 26, 27, and 28), exhibited inhibitory effects toward EBV lytic replication with EC50 values from 1.09 to 7.55 μM and SI values from 3.3 to 116.4. In particular, manassantin B (27) exhibited the most promising inhibition, with an EC50 of 1.72 μM, low cytotoxicity, CC50 > 200 μM, and SI > 116.4. This is the first study demonstrating that lignans possess anti-EBV lytic replication activity.

  7. Advanced lytic lesion is a poor mobilization factor in peripheral blood stem cell collection in patients with multiple myeloma.

    Science.gov (United States)

    Jung, Sung-Hoon; Yang, Deok-Hwan; Ahn, Jae-Sook; Kim, Yeo-Kyeoung; Kim, Hyeoung-Joon; Lee, Je-Jung

    2014-12-01

    This study examined the incidence and predictors of peripheral blood stem cell (PBSC) mobilization failure in patients with multiple myeloma (MM). Retrospective data for 104 patients who received granulocyte colony-stimulating factor (G-CSF) alone or with cyclophosphamide as mobilization regimens were analyzed. The rates of mobilization failure using two definitions of failure (mobilization failure were evaluated using logistic regression analysis which included age, advanced osteolytic lesions, bone marrow cellularity before mobilization, platelet count, body mass index before mobilization, and mobilization method. Lytic bone lesions were assessed using a conventional skeletal survey, and advanced osteolytic lesions were defined as lytic lesions in more than three skeletal sites regardless of the number of lytic lesions. On multivariate analysis, advanced osteolytic lesions [hazard ratio (HR) = 10.95, P = 0.001] and age ≥60 years (HR = 5.45, P = 0.016) were associated with a PBSC yield mobilization (HR = 4.72, P = 0.005), and G-CSF only mobilization (HR 10.52, P mobilization failure in MM patients.

  8. Biomimetic aqueous-core lipid nanoballoons integrating a multiple emulsion formulation: a suitable housing system for viable lytic bacteriophages.

    Science.gov (United States)

    Balcão, Victor M; Glasser, Cássia A; Chaud, Marco V; del Fiol, Fernando S; Tubino, Matthieu; Vila, Marta M D C

    2014-11-01

    The emergence of antibiotic-resistant bacterial strains and the weak penetration of antibiotics into bacterial biofilms put an emphasis in the need for safe and effective alternatives for antimicrobial treatments. The application of strictly lytic bacteriophages (or phages) has been proposed as an alternative (or complement) to conventional antibiotics, allowing release of the natural predators of bacteria directly to the site of infection. In the present research effort, production of bacteriophage derivatives (starting from lytic phage particle isolates), encompassing full stabilization of their three-dimensional structure, has been attempted via housing said bacteriophage particles within lipid nanovesicles integrating a multiple water-in-oil-in-water (W/O/W) emulsion. As a proof-of-concept for the aforementioned strategy, bacteriophage particles with broad lytic spectrum were entrapped within the aqueous core of lipid nanoballoons integrating a W/O/W multiple emulsion. Long-term storage of the multiple emulsions produced did not lead to leaching of phage particles, thus proving the effectiveness of the encapsulation procedure.

  9. Isolation and characterization of five lytic bacteriophages infecting a Vibrio strain closely related to Vibrio owensii.

    Science.gov (United States)

    Yu, Yan-Ping; Gong, Ting; Jost, Günter; Liu, Wen-Hua; Ye, De-Zan; Luo, Zhu-Hua

    2013-11-01

    Vibrio owensii is a potential bacterial pathogen in marine aquaculture system. In this study, five lytic phages specific against Vibrio strain B8D, closely related to V. owensii, were isolated from seawater of an abalone farm. The phages were characterized with respect to morphology, genome size, growth phenotype, as well as thermal, and pH stability. All phages were found to belong to the family Siphoviridae with long noncontractile tails and terminal fibers. Restriction analysis indicated that the five phages were dsDNA viruses with molecular weights ranging from c. 30 to 48 kb. One-step growth experiments revealed that the phages were heterogeneous in latent periods (10-70 min), rise periods (40-70 min), and burst sizes [23-331 plaque-forming units (PFU) per infected cell] at the same host strain. All phages were thermal stable and were tolerant to a wide range of pH. The results indicated that these phages could be potential candidates of a phage cocktail for biological control of V. owensii in aquaculture systems.

  10. Determination of lytic enzyme activities of indigenous Trichoderma isolates from Pakistan.

    Science.gov (United States)

    Asad, Saeed Ahmad; Tabassum, Ayesha; Hameed, Abdul; Hassan, Fayyaz Ul; Afzal, Aftab; Khan, Sabaz Ali; Ahmed, Rafiq; Shahzad, Muhammad

    2015-01-01

    This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, β-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. β-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract.

  11. Lytic phages obscure the cost of antibiotic resistance in Escherichia coli

    Science.gov (United States)

    Tazzyman, Samuel J; Hall, Alex R

    2015-01-01

    The long-term persistence of antibiotic-resistant bacteria depends on their fitness relative to other genotypes in the absence of drugs. Outside the laboratory, viruses that parasitize bacteria (phages) are ubiquitous, but costs of antibiotic resistance are typically studied in phage-free experimental conditions. We used a mathematical model and experiments with Escherichia coli to show that lytic phages strongly affect the incidence of antibiotic resistance in drug-free conditions. Under phage parasitism, the likelihood that antibiotic-resistant genetic backgrounds spread depends on their initial frequency, mutation rate and intrinsic growth rate relative to drug-susceptible genotypes, because these parameters determine relative rates of phage-resistance evolution on different genetic backgrounds. Moreover, the average cost of antibiotic resistance in terms of intrinsic growth in the antibiotic-free experimental environment was small relative to the benefits of an increased mutation rate in the presence of phages. This is consistent with our theoretical work indicating that, under phage selection, typical costs of antibiotic resistance can be outweighed by realistic increases in mutability if drug resistance and hypermutability are genetically linked, as is frequently observed in clinical isolates. This suggests the long-term distribution of antibiotic resistance depends on the relative rates at which different lineages adapt to other types of selection, which in the case of phage parasitism is probably extremely common, as well as costs of resistance inferred by classical in vitro methods. PMID:25268496

  12. Characterization and function of kuruma shrimp lysozyme possessing lytic activity against Vibrio species.

    Science.gov (United States)

    Hikima, Sonomi; Hikima, Jun ichi; Rojtinnakorn, Jiraporn; Hirono, Ikuo; Aoki, Takashi

    2003-10-16

    Lysozyme cDNA was isolated from a kuruma shrimp, Marsupenaeus japonicus, hemocyte cDNA library. The cDNA consists of 1055 base pairs (bp) and encodes a chicken-type (c-type) lysozyme with a deduced amino acid sequence of 156 residues. The kuruma shrimp lysozyme has a high identity (79.7%) with pacific white shrimp lysozyme, and low to moderate identities (33.3-43.0%) with lysozymes of insects and vertebrates. Comparisons with other c-type lysozymes from invertebrates and vertebrates showed that the two catalytic residues (Glu58 and Asp75) and the eight cysteine residue motif were completely conserved. Two novel insertion sequences were also observed in the kuruma and pacific white shrimp lysozyme amino acid sequences. Interestingly, phylogenetic analysis revealed that the kuruma shrimp lysozyme was more closely related to vertebrate c-type lysozymes. Expression of the cDNA in insect cells, using a baculovirus expression system, yielded a recombinant lysozyme with optimum activity at pH 7.5 and 50 degrees C, as evaluated by a lysoplate assay. The kuruma shrimp lysozyme displayed lytic activities against several Vibrio species and fish pathogens, including Vibrio penaeicida (a pathogenic bacteria to the kuruma shrimp) and suggested that shrimp lysozyme affects a greater variety of pathogens.

  13. Hemoglobin is a co-factor of human trypanosome lytic factor.

    Directory of Open Access Journals (Sweden)

    Justin Widener

    2007-09-01

    Full Text Available Trypanosome lytic factor (TLF is a high-density lipoprotein (HDL subclass providing innate protection to humans against infection by the protozoan parasite Trypanosoma brucei brucei. Two primate-specific plasma proteins, haptoglobin-related protein (Hpr and apolipoprotein L-1 (ApoL-1, have been proposed to kill T. b. brucei both singularly or when co-assembled into the same HDL. To better understand the mechanism of T. b. brucei killing by TLF, the protein composition of TLF was investigated using a gentle immunoaffinity purification technique that avoids the loss of weakly associated proteins. HDL particles recovered by immunoaffinity absorption, with either anti-Hpr or anti-ApoL-1, were identical in protein composition and specific activity for T. b. brucei killing. Here, we show that TLF-bound Hpr strongly binds Hb and that addition of Hb stimulates TLF killing of T. b. brucei by increasing the affinity of TLF for its receptor, and by inducing Fenton chemistry within the trypanosome lysosome. These findings suggest that TLF in uninfected humans may be inactive against T. b. brucei prior to initiation of infection. We propose that infection of humans by T. b. brucei causes hemolysis that triggers the activation of TLF by the formation of Hpr-Hb complexes, leading to enhanced binding, trypanolytic activity, and clearance of parasites.

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

    Science.gov (United States)

    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.

  15. Production of four Neurospora crassa lytic polysaccharide monooxygenases in Pichia pastoris monitored by a fluorimetric assay.

    Science.gov (United States)

    Kittl, Roman; Kracher, Daniel; Burgstaller, Daniel; Haltrich, Dietmar; Ludwig, Roland

    2012-10-26

    Recent studies demonstrate that enzymes from the glycosyl hydrolase family 61 (GH61) show lytic polysaccharide monooxygenase (PMO) activity. Together with cellobiose dehydrogenase (CDH) an enzymatic system capable of oxidative cellulose cleavage is formed, which increases the efficiency of cellulases and put PMOs at focus of biofuel research. Large amounts of purified PMOs, which are difficult to obtain from the native fungal producers, are needed to study their reaction kinetics, structure and industrial application. In addition, a fast and robust enzymatic assay is necessary to monitor enzyme production and purification. Four pmo genes from Neurospora crassa were expressed in P. pastoris under control of the AOX1 promoter. High yields were obtained for the glycosylated gene products PMO-01867, PMO-02916 and PMO-08760 (>300 mg L-1), whereas the yield of non-glycosylated PMO-03328 was moderate (~45 mg L-1). The production and purification of all four enzymes was specifically followed by a newly developed, fast assay based on a side reaction of PMO: the production of H2O2 in the presence of reductants. While ascorbate is a suitable reductant for homogeneous PMO preparations, fermentation samples require the specific electron donor CDH. P. pastoris is a high performing expression host for N. crassa PMOs. The pmo genes under control of the native signal sequence are correctly processed and active. The novel CDH-based enzyme assay allows fast determination of PMO activity in fermentation samples and is robust against interfering matrix components.

  16. Isolation and characterisation of lytic bacteriophages of Klebsiella pneumoniae and Klebsiella oxytoca.

    Science.gov (United States)

    Karumidze, Natia; Kusradze, Ia; Rigvava, Sophio; Goderdzishvili, Marine; Rajakumar, Kumar; Alavidze, Zemphira

    2013-03-01

    Klebsiella bacteria have emerged as an increasingly important cause of community-acquired nosocomial infections. Extensive use of broad-spectrum antibiotics in hospitalised patients has led to both increased carriage of Klebsiella and the development of multidrug-resistant strains that frequently produce extended-spectrum β-lactamases and/or other defences against antibiotics. Many of these strains are highly virulent and exhibit a strong propensity to spread. In this study, six lytic Klebsiella bacteriophages were isolated from sewage-contaminated river water in Georgia and characterised as phage therapy candidates. Two of the phages were investigated in greater detail. Biological properties, including phage morphology, nucleic acid composition, host range, growth phenotype, and thermal and pH stability were studied for all six phages. Limited sample sequencing was performed to define the phylogeny of the K. pneumoniae- and K. oxytoca-specific bacteriophages vB_Klp_5 and vB_Klox_2, respectively. Both of the latter phages had large burst sizes, efficient rates of adsorption and were stable under different adverse conditions. Phages reported in this study are double-stranded DNA bacterial viruses belonging to the families Podoviridae and Siphoviridae. One or more of the six phages was capable of efficiently lysing ~63 % of Klebsiella strains comprising a collection of 123 clinical isolates from Georgia and the United Kingdom. These phages exhibit a number of properties indicative of potential utility in phage therapy cocktails.

  17. Characterisation and genome sequence of the lytic Acinetobacter baumannii bacteriophage vB_AbaS_Loki

    Science.gov (United States)

    Wand, Matthew E.; Briers, Yves; Lavigne, Rob; Sutton, J. Mark; Reynolds, Darren M.

    2017-01-01

    Acinetobacter baumannii has emerged as an important nosocomial pathogen in healthcare and community settings. While over 100 of Acinetobacter phages have been described in the literature, relatively few have been sequenced. This work describes the characterisation and genome annotation of a new lytic Acinetobacter siphovirus, vB_AbaS_Loki, isolated from activated sewage sludge. Sequencing revealed that Loki encapsulates a 41,308 bp genome, encoding 51 predicted open reading frames. Loki is most closely related to Acinetobacter phage IME_AB3 and more distantly related to Burkholderia phage KL1, Paracoccus phage vB_PmaS_IMEP1 and Pseudomonas phages vB_Pae_Kakheti25, vB_PaeS_SCH_Ab26 and PA73. Loki is characterised by a narrow host range, among the 40 Acinetobacter isolates tested, productive infection was only observed for the propagating host, A. baumannii ATCC 17978. Plaque formation was found to be dependent upon the presence of Ca2+ ions and adsorption to host cells was abolished upon incubation with a mutant of ATCC 17978 encoding a premature stop codon in lpxA. The complete genome sequence of vB_AbaS_Loki was deposited in the European Nucleotide Archive (ENA) under the accession number LN890663. PMID:28207864

  18. Syntaxin 8 is required for efficient lytic granule trafficking in cytotoxic T lymphocytes.

    Science.gov (United States)

    Bhat, Shruthi S; Friedmann, Kim S; Knörck, Arne; Hoxha, Cora; Leidinger, Petra; Backes, Christina; Meese, Eckart; Keller, Andreas; Rettig, Jens; Hoth, Markus; Qu, Bin; Schwarz, Eva C

    2016-07-01

    Cytotoxic T lymphocytes (CTL) eliminate pathogen-infected and cancerous cells mainly by polarized secretion of lytic granules (LG, containing cytotoxic molecules like perforin and granzymes) at the immunological synapse (IS). Members of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family are involved in trafficking (generation, transport and fusion) of vesicles at the IS. Syntaxin 8 (Stx8) is expressed in LG and colocalizes with the T cell receptor (TCR) upon IS formation. Here, we report the significance of Stx8 for human CTL cytotoxicity. We found that Stx8 mostly localized in late, recycling endosomal and lysosomal compartments with little expression in early endosomal compartments. Down-regulation of Stx8 by siRNA resulted in reduced cytotoxicity. We found that following perforin release of the pre-existing pool upon target cell contact, Stx8 down-regulated CTL regenerate perforin pools less efficiently and thus release less perforin compared to control CTL. CD107a degranulation, real-time and end-point population cytotoxicity assays, and high resolution microscopy support our conclusion that Stx8 is required for proper and timely sorting and trafficking of cytotoxic molecules to functional LG through the endosomal pathway in human CTL.

  19. Determination of lytic enzyme activities of indigenous Trichoderma isolates from Pakistan

    Science.gov (United States)

    Asad, Saeed Ahmad; Tabassum, Ayesha; Hameed, Abdul; Hassan, Fayyaz ul; Afzal, Aftab; Khan, Sabaz Ali; Ahmed, Rafiq; Shahzad, Muhammad

    2015-01-01

    Abstract This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, β-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. β-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract. PMID:26691463

  20. Parachlamydia acanthamoeba is endosymbiotic or lytic for Acanthamoeba polyphaga depending on the incubation temperature.

    Science.gov (United States)

    Greub, Gilbert; La Scola, Bernard; Raoult, Didier

    2003-06-01

    Parachlamydiaceae are potential emerging pathogens that naturally infect free-living amoebae. We investigated the affects of incubation temperature on the growth and cytopathic effect of P. acanthamoeba in Acanthamoeba polyphaga. A. polyphaga were infected with P. acanthamoeba and incubated at different temperatures for ten days. Bacterial growth was quantified by real-time PCR. Cytopathic effects were determined by counting the number of cysts and viable amoebae (unstained with trypan blue) in Nageotte counting chambers. Uninfected amoebae cultures were used as negative control. At 32, 35, and 37 degrees C, we observed a significant decrease in the number of viable A. polyphaga that contrasted with the delayed and smaller decrease in the number of living A. polyphaga observed at 25, 28, and 30 degrees C. Higher incubation temperature, which is associated with amoebal lysis, surprisingly was not associated with increased growth rate. P. acanthamoeba is lytic for A. polyphaga at 32-37 degrees C but endosymbiotic at 25-30 degrees C. This suggests that A. polyphaga may be a reservoir of endosymbionts at the lower temperature of the nasal mucosa, which may be liberated by lysis at higher temperature, for instance, when the amoeba is inhaled and reaches the lower respiratory tract.

  1. Identification of a membrane-bound prepore species clarifies the lytic mechanism of actinoporins

    CERN Document Server

    Morante, Koldo; Gil-Cartón, David; Redondo-Morata, Lorena; Sot, Jesús; Scheuring, Simon; Valle, Mikel; González-Mañas, Juan Manuel; Tsumoto, Kouhei; Caaveiro, Jose M M

    2016-01-01

    Pore-forming toxins (PFT) are cytolytic proteins belonging to the molecular warfare apparatus of living organisms. The assembly of the functional transmembrane pore requires several intermediate steps ranging from a water-soluble monomeric species to the multimeric ensemble inserted in the cell membrane. The non-lytic oligomeric intermediate known as prepore plays an essential role in the mechanism of insertion of the class of $\\beta$-PFT. However, in the class of $\\alpha$-PFT like the actinoporins produced by sea anemones, evidence of membrane-bound prepores is still lacking. We have employed single-particle cryo-electron microscopy (cryo-EM) and atomic force microscopy (AFM) to identify, for the first time, a prepore species of the actinoporin fragaceatoxin C (FraC) bound to lipid vesicles. The size of the prepore coincides that of the functional pore, except for the transmembrane region, which is absent in the prepore. Biochemical assays indicated that, in the prepore species, the N-terminus is not inserte...

  2. Characterization of potential lytic bacteriophage against Vibrio alginolyticus and its therapeutic implications on biofilm dispersal.

    Science.gov (United States)

    Sasikala, Dakshinamurthy; Srinivasan, Pappu

    2016-12-01

    Vibrio alginolyticus is a leading cause of vibriosis, presenting opportunistic infections to humans associated with raw seafood contamination. At present, phage therapy that acts as an alternative sanitizing agent is explored for targeting V. alginolyticus. The study outcome revealed that the phage VP01 with its extreme lytic effect showed a high potential impact on the growth of V. alginolyticus as well as biofilm formation. Electron microscopy revealed the phage resemblance to Myoviridae, based on its morphology. Further study clarified that the phage VP01 possesses a broad host spectrum and amazing phage sensitivity at different pH, high thermal stability, and high burst size of 415 PFU/cell. In addition, the investigation of phage co-culturing against this pathogen resulted in a significant growth reduction even at less MOIs 0.1 and 1. These results suggest that the phage could be a promising candidate for the control of V. alginolyticus infections. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Use of lytic bacteriophages to reduce Salmonella Enteritidis in experimentally contaminated chicken cuts

    Directory of Open Access Journals (Sweden)

    L Fiorentin

    2005-12-01

    Full Text Available Reducing Salmonella contamination in poultry is of major importance to prevent the introduction of this microorganism into the food chain. Salmonellae may spread during storage time (shelf life whenever pre-harvest control fails or post-harvest contamination occurs. Therefore, preventive measures should also be used in the post-harvest level of poultry production in order to control salmonellae. Chicken skin samples were experimentally contaminated by immersing whole legs (thighs and drumsticks in a suspension containing 10(6 colony forming units per milliliter (CFU/mL of Salmonella Enteritidis phage type 4 (SE PT4 at the slaughter day. One day later, samples from one group were immersed in a suspension pool containing 10(9 CFU/mL of each of three wild salmonella-lytic bacteriophages previously isolated from feces of free-range chickens. Salmonella counting was performed at three-day intervals in the chicken legs stored at 5°C and showed a significant reduction (P<0.05 of SE PT4 in bacteriophage-treated cuts on days 3, 6 and 9 post-treatment. These findings suggest that the use of bacteriophages may reduce SE PT4 in chicken skin. Further studies are encouraged and might demonstrate the potential of this approach as an efficient and safe technique to be routinelly used for Salmonella control in chicken products.

  4. Chromatin Modulation of Herpesvirus Lytic Gene Expression: Managing Nucleosome Density and Heterochromatic Histone Modifications

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    Thomas M. Kristie

    2016-03-01

    Full Text Available Like their cellular hosts, herpesviruses are subject to the regulatory impacts of chromatin assembled on their genomes. Upon infection, these viruses are assembled into domains of chromatin with heterochromatic signatures that suppress viral gene expression or euchromatic characteristics that promote gene expression. The organization and modulation of these chromatin domains appear to be intimately linked to the coordinated expression of the different classes of viral genes and thus ultimately play an important role in the progression of productive infection or the establishment and maintenance of viral latency. A recent report from the Knipe laboratory (J. S. Lee, P. Raja, and D. M. Knipe, mBio 7:e02007-15, 2016 contributes to the understanding of the dynamic modulation of chromatin assembled on the herpes simplex virus genome by monitoring the levels of characteristic heterochromatic histone modifications (histone H3 lysine 9 and 27 methylation associated with a model viral early gene during the progression of lytic infection. Additionally, this study builds upon previous observations that the viral immediate-early protein ICP0 plays a role in reducing the levels of heterochromatin associated with the early genes.

  5. Lytic phages obscure the cost of antibiotic resistance in Escherichia coli.

    Science.gov (United States)

    Tazzyman, Samuel J; Hall, Alex R

    2015-03-17

    The long-term persistence of antibiotic-resistant bacteria depends on their fitness relative to other genotypes in the absence of drugs. Outside the laboratory, viruses that parasitize bacteria (phages) are ubiquitous, but costs of antibiotic resistance are typically studied in phage-free experimental conditions. We used a mathematical model and experiments with Escherichia coli to show that lytic phages strongly affect the incidence of antibiotic resistance in drug-free conditions. Under phage parasitism, the likelihood that antibiotic-resistant genetic backgrounds spread depends on their initial frequency, mutation rate and intrinsic growth rate relative to drug-susceptible genotypes, because these parameters determine relative rates of phage-resistance evolution on different genetic backgrounds. Moreover, the average cost of antibiotic resistance in terms of intrinsic growth in the antibiotic-free experimental environment was small relative to the benefits of an increased mutation rate in the presence of phages. This is consistent with our theoretical work indicating that, under phage selection, typical costs of antibiotic resistance can be outweighed by realistic increases in mutability if drug resistance and hypermutability are genetically linked, as is frequently observed in clinical isolates. This suggests the long-term distribution of antibiotic resistance depends on the relative rates at which different lineages adapt to other types of selection, which in the case of phage parasitism is probably extremely common, as well as costs of resistance inferred by classical in vitro methods.

  6. Characterisation and genome sequence of the lytic Acinetobacter baumannii bacteriophage vB_AbaS_Loki.

    Science.gov (United States)

    Turner, Dann; Wand, Matthew E; Briers, Yves; Lavigne, Rob; Sutton, J Mark; Reynolds, Darren M

    2017-01-01

    Acinetobacter baumannii has emerged as an important nosocomial pathogen in healthcare and community settings. While over 100 of Acinetobacter phages have been described in the literature, relatively few have been sequenced. This work describes the characterisation and genome annotation of a new lytic Acinetobacter siphovirus, vB_AbaS_Loki, isolated from activated sewage sludge. Sequencing revealed that Loki encapsulates a 41,308 bp genome, encoding 51 predicted open reading frames. Loki is most closely related to Acinetobacter phage IME_AB3 and more distantly related to Burkholderia phage KL1, Paracoccus phage vB_PmaS_IMEP1 and Pseudomonas phages vB_Pae_Kakheti25, vB_PaeS_SCH_Ab26 and PA73. Loki is characterised by a narrow host range, among the 40 Acinetobacter isolates tested, productive infection was only observed for the propagating host, A. baumannii ATCC 17978. Plaque formation was found to be dependent upon the presence of Ca2+ ions and adsorption to host cells was abolished upon incubation with a mutant of ATCC 17978 encoding a premature stop codon in lpxA. The complete genome sequence of vB_AbaS_Loki was deposited in the European Nucleotide Archive (ENA) under the accession number LN890663.

  7. Caspase-1 activity is required for UVB-induced apoptosis of human keratinocytes.

    Science.gov (United States)

    Sollberger, Gabriel; Strittmatter, Gerhard E; Grossi, Serena; Garstkiewicz, Martha; Auf dem Keller, Ulrich; French, Lars E; Beer, Hans-Dietmar

    2015-05-01

    Caspase-1 has a crucial role in innate immunity as the protease activates the proinflammatory cytokine prointerleukin(IL)-1β. Furthermore, caspase-1 induces pyroptosis, a lytic form of cell death that supports inflammation. Activation of caspase-1 occurs in multi-protein complexes termed inflammasomes, which assemble upon sensing of stress signals. In the skin and in skin-derived keratinocytes, UVB irradiation induces inflammasome-dependent IL-1 secretion and sunburn. Here we present evidence that caspase-1 and caspase-4 are required for UVB-induced apoptosis. In UVB-irradiated human primary keratinocytes, apoptosis occurs significantly later than inflammasome activation but depends on caspase-1 activity. However, it proceeds independently of inflammasome activation. By a proteomics approach, we identified the antiapoptotic Bap31 as a putative caspase-1 substrate. Caspase-1-dependent apoptosis is possibly a recent process in evolution as it was not detected in mice. These results suggest a protective role of caspase-1 in keratinocytes during UVB-induced skin cancer development through the induction of apoptosis.

  8. Using lytic bacteriophages to eliminate or significantly reduce contamination of food by foodborne bacterial pathogens.

    Science.gov (United States)

    Sulakvelidze, Alexander

    2013-10-01

    Bacteriophages (also called 'phages') are viruses that kill bacteria. They are arguably the oldest (3 billion years old, by some estimates) and most ubiquitous (total number estimated to be 10(30) -10(32) ) known organisms on Earth. Phages play a key role in maintaining microbial balance in every ecosystem where bacteria exist, and they are part of the normal microflora of all fresh, unprocessed foods. Interest in various practical applications of bacteriophages has been gaining momentum recently, with perhaps the most attention focused on using them to improve food safety. That approach, called 'phage biocontrol', typically includes three main types of applications: (i) using phages to treat domesticated livestock in order to reduce their intestinal colonization with, and shedding of, specific bacterial pathogens; (ii) treatments for decontaminating inanimate surfaces in food-processing facilities and other food establishments, so that foods processed on those surfaces are not cross-contaminated with the targeted pathogens; and (iii) post-harvest treatments involving direct applications of phages onto the harvested foods. This mini-review primarily focuses on the last type of intervention, which has been gaining the most momentum recently. Indeed, the results of recent studies dealing with improving food safety, and several recent regulatory approvals of various commercial phage preparations developed for post-harvest food safety applications, strongly support the idea that lytic phages may provide a safe, environmentally-friendly, and effective approach for significantly reducing contamination of various foods with foodborne bacterial pathogens. However, some important technical and nontechnical problems may need to be addressed before phage biocontrol protocols can become an integral part of routine food safety intervention strategies implemented by food industries in the USA.

  9. Characterization of four lytic transducing bacteriophages of luminescent Vibrio harveyi isolated from shrimp (Penaeus monodon) hatcheries.

    Science.gov (United States)

    Thiyagarajan, Sanjeevi; Chrisolite, Bagthasingh; Alavandi, Shankar V; Poornima, Modem; Kalaimani, Natarajan; Santiago, T Chinnappan

    2011-12-01

    Four lytic bacteriophages designated as φVh1, φVh2, φVh3, and φVh4 were isolated from commercial shrimp hatcheries, possessing broad spectrum of infectivity against luminescent Vibrio harveyi isolates, considering their potential as biocontrol agent of luminescent bacterial disease in shrimp hatcheries, and were characterized by electron microscopy, genomic analysis, restriction enzyme analysis (REA), and pulsed-field gel electrophoresis (PFGE). Three phages φVh1, φVh2, and φVh4 had an icosahedral head of 60-115 nm size with a long, noncontractile tail of 130-329 × 1-17 nm, belonged to the family Siphoviridae. φVh3 had an icosahedral head (72 ± 5 nm) with a short tail (27 × 12 nm) and belonged to Podoviridae. REA with DraI and PFGE of genomic DNA digested with ScaI and XbaI and cluster analysis of their banding patterns indicated that φVh3 was distinct from the other three siphophages. PFGE-based genome mean size of the four bacteriophages φVh1, φVh2, φVh3, and φVh4 was estimated to be about 85, 58, 64, and 107 kb, respectively. These phages had the property of generalized transduction as demonstrated by transduction with plasmid pHSG 396 with frequencies ranging from 4.1 × 10(-7) to 2 × 10(-9) per plaque-forming unit, suggesting a potential ecological role in gene transfer among aquatic vibrios.

  10. Chemical modification of methionines in a cobra venom cytotoxin differentiates between lytic and binding domains.

    Science.gov (United States)

    Stevens-Truss, R; Hinman, C L

    1996-08-01

    Cytotoxin-III from Naja naja atra (CTX) was chemically modified at either or both of its two methionine residues: Over 50% oxidation of methionine-26 occurred with a 1:1 molar ratio of chloramine-T:methionine; at a 5:1 molar ratio, methionine-26 was almost completely oxidized, while methionine-24 was modified only 26%; at a 10:1 molar ratio, both methionines were completely oxidized. Each oxidized derivative demonstrated a lower toxicity toward T-cells than toward heart cells. Conversely, binding to heart cells was affected more than binding to T-cells. Cyanogen bromide cleaved native CTX at both methionines, excising phenyl-alanine-25 and methionine-26 and converting methionine-24 to homoserine lactone. This treatment of CTX eliminated cytotoxicity toward both heart and T-cells, but had only a modest effect upon T-cell binding, as had 50% oxidation of methionine-26, suggesting that CTX lytic and binding regions may be distinct. A selective loss in heart cell binding following oxidation of methionine-24 further suggests that different parts of CTX may interact with the two types of target cells. Perturbation of the relatively flat hydrophobic surface of the CTX' triple-stranded beta-sheet could result from the introduction of negative charge due to methionine-24 oxidation. Alternatively, amino acid side chain participation in a CTX binding domain may be altered by the potential formation of a new hydrogen bond between tyrosine-51 and methionine-24 sulfoxide, as revealed by computer modeling of the completely oxidized CTX derivative.

  11. Natural killer lytic-associated molecule plays a role in controlling tumor dissemination and metastasis

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    Richard Glenn Hoover

    2012-12-01

    Full Text Available Natural killer lytic-associated molecule (NKLAM is an E3 ubiquitin ligase that plays a major role in the cytolytic activity of NK cells. NKLAM is rapidly synthesized and then targeted to the granule membranes of NK cells upon NK activation. Previous studies have shown an essential role for NKLAM in NK killing activity in vitro. These findings were extended to an in vivo model of NK-mediated tumor killing in which NKLAM-deficient knockout (KO mice injected with B16 melanoma cells were found to have significantly higher numbers of pulmonary tumor nodules than wild type (WT mice. To further investigate the role of NKLAM and NK function in tumor immunity in vivo, we utilized additional tumor models to compare tumor development and progression in NKLAM KO and WT mice. Primary tumor growth, dissemination, and metastasis of RMA-S lymphoma cells and E0771 breast cancer cells were evaluated. Both tumor cell lines were stably transfected with constructs that allow expression of green fluorescent protein (GFP, which serves as a tumor-specific marker. Intravenous injection of NK-sensitive RMA-S lymphoma cells resulted in greater dissemination of lymphoma cells in NKLAM KO mice than in WT mice. Lymphoma cells were found in the lymph nodes and bone marrow of NKLAM KO mice two weeks after injection; few detectable tumor cells remained in WT mice. E0771 syngeneic breast cancer cells were injected into the mammary pads of NKLAM KO and WT mice. Primary tumor growth was greater in NKLAM KO than in WT mice. More significantly, there were four to five fold more tumor cells in the blood and lungs of NKLAM KO than in WT mice two weeks after injection of tumor cells into the mammary pad. These results indicate that NKLAM plays a role in tumor development in vivo, especially in controlling tumor dissemination and metastasis to distant sites.

  12. Analyzing Activities of Lytic Polysaccharide Monooxygenases by Liquid Chromatography and Mass Spectrometry.

    Science.gov (United States)

    Westereng, Bjørge; Arntzen, Magnus Ø; Agger, Jane Wittrup; Vaaje-Kolstad, Gustav; Eijsink, Vincent G H

    2017-01-01

    Lytic polysaccharide monooxygenases perform oxidative cleavage of glycosidic bonds in various polysaccharides. The majority of LMPOs studied so far possess activity on either cellulose or chitin and analysis of these activities is therefore the main focus of this review. Notably, however, the number of LPMOs that are active on other polysaccharides is increasing. The products generated by LPMOs from cellulose are either oxidized in the downstream end (at C1) or upstream end (at C4), or at both ends. These modifications only result in small structural changes, which makes both chromatographic separation and product identification by mass spectrometry challenging. The changes in physicochemical properties that are associated with oxidation need to be considered when choosing analytical approaches. C1 oxidation leads to a sugar that is no longer reducing but instead has an acidic functionality, whereas C4 oxidation leads to products that are inherently labile at high and low pH and that exist in a keto-gemdiol equilibrium that is strongly shifted toward the gemdiol in aqueous solutions. Partial degradation of C4-oxidized products leads to the formation of native products, which could explain why some authors claim to have observed glycoside hydrolase activity for LPMOs. Notably, apparent glycoside hydrolase activity may also be due to small amounts of contaminating glycoside hydrolases since these normally have much higher catalytic rates than LPMOs. The low catalytic turnover rates of LPMOs necessitate the use of sensitive product detection methods, which limits the analytical possibilities considerably. Modern liquid chromatography and mass spectrometry have become essential tools for evaluating LPMO activity, and this chapter provides an overview of available methods together with a few novel tools. The methods described constitute a suite of techniques for analyzing oxidized carbohydrate products, which can be applied to LPMOs as well as other carbohydrate

  13. Antibacterial efficacy of lytic bacteriophages against antibiotic-resistant Klebsiella species.

    Science.gov (United States)

    Karamoddini, M Khajeh; Fazli-Bazzaz, B S; Emamipour, F; Ghannad, M Sabouri; Jahanshahi, A R; Saed, N; Sahebkar, A

    2011-07-07

    Bacterial resistance to antibiotics is a leading and highly prevalent problem in the treatment of infectious diseases. Bacteriophages (phages) appear to be effective and safe alternatives for the treatment of resistant infections because of their specificity for bacterial species and lack of infectivity in eukaryotic cells. The present study aimed to isolate bacteriophages against Klebsiella spp. and evaluate their efficacy against antibiotic-resistant species. Seventy-two antibiotic-resistant Klebsiella spp. were isolated from samples of patients who referred to the Ghaem Hospital (Mashhad, Iran). Lytic bacteriophages against Klebsiella spp. were isolated from wastewater of the septic tank of the same hospital. Bactericidal activity of phages against resistant Klebsiella spp. was tested in both liquid (tube method; after 1 and 24 h of incubation) and solid (double-layer agar plate method; after 24 h of incubation) phases. In each method, three different concentrations of bacteriophages (low: 10(7) PFU/mL) were used. Bacteriophages showed promising bactericidal activity at all assessed concentrations, regardless of the test method and duration of incubation. Overall, bactericidal effects were augmented at higher concentrations. In the tube method, higher activity was observed after 24 h of incubation compared to the 1-h incubation. The bactericidal effects were also higher in the tube method compared to the double-layer agar plate method after 24 h of incubation. The findings of the present study suggest that bacteriophages possess effective bactericidal activity against resistant Klebsiella spp. These bactericidal activities are influenced by phage concentration, duration of incubation, and test method.

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

    Science.gov (United States)

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

    2015-06-09

    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.

  15. Antibacterial Efficacy of Lytic Bacteriophages against Antibiotic-Resistant Klebsiella Species

    Directory of Open Access Journals (Sweden)

    M. Khajeh Karamoddini

    2011-01-01

    Full Text Available Bacterial resistance to antibiotics is a leading and highly prevalent problem in the treatment of infectious diseases. Bacteriophages (phages appear to be effective and safe alternatives for the treatment of resistant infections because of their specificity for bacterial species and lack of infectivity in eukaryotic cells. The present study aimed to isolate bacteriophages against Klebsiella spp. and evaluate their efficacy against antibiotic-resistant species. Seventy-two antibiotic-resistant Klebsiella spp. were isolated from samples of patients who referred to the Ghaem Hospital (Mashhad, Iran. Lytic bacteriophages against Klebsiella spp. were isolated from wastewater of the septic tank of the same hospital. Bactericidal activity of phages against resistant Klebsiella spp. was tested in both liquid (tube method; after 1 and 24 h of incubation and solid (double-layer agar plate method; after 24 h of incubation phases. In each method, three different concentrations of bacteriophages (low: 107 PFU/mL were used. Bacteriophages showed promising bactericidal activity at all assessed concentrations, regardless of the test method and duration of incubation. Overall, bactericidal effects were augmented at higher concentrations. In the tube method, higher activity was observed after 24 h of incubation compared to the 1-h incubation. The bactericidal effects were also higher in the tube method compared to the double-layer agar plate method after 24 h of incubation. The findings of the present study suggest that bacteriophages possess effective bactericidal activity against resistant Klebsiella spp. These bactericidal activities are influenced by phage concentration, duration of incubation, and test method.

  16. Cyclin-dependent kinase activity controls the onset of the HCMV lytic cycle.

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    Martin Zydek

    Full Text Available The onset of human cytomegalovirus (HCMV lytic infection is strictly synchronized with the host cell cycle. Infected G0/G1 cells support viral immediate early (IE gene expression and proceed to the G1/S boundary where they finally arrest. In contrast, S/G2 cells can be infected but effectively block IE gene expression and this inhibition is not relieved until host cells have divided and reentered G1. During latent infection IE gene expression is also inhibited, and for reactivation to occur this block to IE gene expression must be overcome. It is only poorly understood which viral and/or cellular activities maintain the block to cell cycle or latency-associated viral IE gene repression and whether the two mechanisms may be linked. Here, we show that the block to IE gene expression during S and G2 phase can be overcome by both genotoxic stress and chemical inhibitors of cellular DNA replication, pointing to the involvement of checkpoint-dependent signaling pathways in controlling IE gene repression. Checkpoint-dependent rescue of IE expression strictly requires p53 and in the absence of checkpoint activation is mimicked by proteasomal inhibition in a p53 dependent manner. Requirement for the cyclin dependent kinase (CDK inhibitor p21 downstream of p53 suggests a pivotal role for CDKs in controlling IE gene repression in S/G2 and treatment of S/G2 cells with the CDK inhibitor roscovitine alleviates IE repression independently of p53. Importantly, CDK inhibiton also overcomes the block to IE expression during quiescent infection of NTera2 (NT2 cells. Thus, a timely block to CDK activity not only secures phase specificity of the cell cycle dependent HCMV IE gene expression program, but in addition plays a hitherto unrecognized role in preventing the establishment of a latent-like state.

  17. Lytic effects of normal serum on isolated postonchospheral and metacestode stages of Taenia taeniaeformis.

    Science.gov (United States)

    Conder, G A; Picone, J; Geary, A M; deHoog, J; Williams, J F

    1983-06-01

    Postonchospheral stages of Taenia taeniaeformis liberated from rat livers by enzymatic digestion at 1 to 10 days postinfection (DPI) and metacestodes dissected from infected livers at 22, 34, and 69 DPI were exposed in vitro to immune rat serum (IRS) and to normal serum from rats (NRS), human beings (NHS), or guinea pigs (NGS). The onset of rapid and destructive tegumental changes in all organisms exposed to any of the sera was demonstrated to be complement-dependent because the reaction was: (a) inhibited by treatment of serum at 56 C for 30 min; (b) inhibited by prior incubation of serum with zymosan or with complement-fixing, soluble products derived from larvae of T. taeniaeformis maintained in vitro (IVP); and (c) abolished by the addition of EDTA. Lytic effects occurred on exposure to agammaglobulinemic sheep serum, and lysis in the presence of IRS and NRS was shown to result in consumption of available hemolytic complement. Surface changes consisted of vesiculation in the microvillar or microthrix layers followed by sloughing of the tegument, eventually leading to collapse of the cystic bladder and cessation of flame cell activity, or, in the case of early postonchospheral forms, complete disintegration of the organism. When IVP was added to NHS, reduction of hemolytic complement activity was associated with the electrophoretic conversion of C3, and Factor B, but there was little or no consumption of C1. The observations support the hypothesis that complement-mediated effector mechanisms must be counteracted to ensure survival of parasites in vivo, and that the capacity for release of soluble nonspecific complement-fixing factors by taeniid larvae may have an important role to play in this process.

  18. LYMPHOCYTE APOPTOSIS IN PSORIASIS

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    О. M. Kapuler

    2006-01-01

    Full Text Available Abstract. Forty-two patients with progressive vulgar psoriasis (PASI = 19.7 ± 1.5 and 40 healthy volunteers were under investigation. Psoriatic patients were characterized by increased number of CD4+ CD95+ peripheral blood T lymphocytes, which correlates with clinical psoriatic score, and by increased levels of soluble Fas (sFas in serum, as compared to controls (resp., 1868.1 ± 186.8 pg/ml vs. 1281.4 ± 142.5 pg/ml, PLSD = 0.019. The levels of spontaneous lymphocyte apoptosis and anti-Fas (Mab-induced apoptosis in psoriatic patients did not differ from the controls. However, apoptosis induced by “oxidative stress” (50 M Н202, 4 hrs was depressed in the patients. Moreover, a simultaneous assessment of cell cycle structure (metachromatic staining with Acridine Orange, apoptosis and Fas receptor expression (AnnV-FITC/antiFas mAbs-PE staining following a short-term mitogenic stimulation (PHA-P, 5 µg/ml, 24 hrs were performed. We found no marked differences in mitogenic reactivity, activation-induced apoptosis, and activation-induced Fas receptor expression when studying lymphocytes from healthy donors and psoriatic patients. However, PHA-activated lymphocytes from psoriatic patients displayed a significantly decreased ratio of AnnV+CD95+ to the total AnnV+ subpopulation, thus suggesting a decreased role of Fas-dependent mechanisms of apoptosis during the cell activation. The data obtained confirm a view, that an abnormal lymphocyte “apoptotic reactivity”, which plays a crucial role in the mechanisms of autoimmunity, may also of importance in the pathogenesis of psoriasis.

  19. Apoptosis - Methods and Protocols

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    CarloAlberto Redi

    2010-03-01

    Full Text Available Apoptosis - Methods and ProtocolsSecond edition, 2009; Peter Erhardt and Ambrus Toth (Eds; Springer Protocols - Methods in molecular biology, vol. 559; Humana press, Totowa, New Jersey (USA; Pages: 400; €88.35; ISBN: 978-1-60327-016-8The editors rightly begin the preface telling us that: “The ability to detect and quantify apoptosis, to understand its biochemistry and to identify its regulatory genes and proteins is crucial to biomedical research”. Nowadays this is a grounding concept of biology and medicine. What is particularly remarkable...

  20. The biochemistry of apoptosis.

    Science.gov (United States)

    Hengartner, M O

    2000-10-12

    Apoptosis--the regulated destruction of a cell--is a complicated process. The decision to die cannot be taken lightly, and the activity of many genes influence a cell's likelihood of activating its self-destruction programme. Once the decision is taken, proper execution of the apoptotic programme requires the coordinated activation and execution of multiple subprogrammes. Here I review the basic components of the death machinery, describe how they interact to regulate apoptosis in a coordinated manner, and discuss the main pathways that are used to activate cell death.

  1. A paradigm linking herpesvirus immediate-early gene expression apoptosis and myalgic encephalomyelitis chronic fatigue syndrome

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    A Martin Lerner

    2011-02-01

    Full Text Available A Martin Lerner1, Safedin Beqaj21Department of Medicine, William Beaumont Hospital, Royal Oak, MI, USA; 2DCL Medical Laboratories, Indianapolis, IN, USAAbstract: There is no accepted science to relate herpesviruses (Epstein–Barr virus [EBV], human cytomegalovirus [HCMV], and human herpesvirus 6 [HHV6] as causes of myalgic encephalomyelitis (ME/chronic fatigue syndrome (CFS. ME/CFS patients have elevated serum immunoglobulin (IgG serum antibody titers to EBV, HCMV, and HHV6, but there is no herpesvirus DNA-emia, herpesvirus antigenemia, or uniformly elevated IgM serum antibody titers to the complete virions. We propose that herpesvirus EBV, HCMV, and HHV6 immediate-early gene expression in ME/CFS patients leads to host cell dysregulation and host cell apoptosis without lytic herpesvirus replication. Specific antiviral nucleosides, which alleviate ME/CFS, namely valacyclovir for EBV ME/CFS and valganciclovir for HCMV/HHV6 ME/CFS, inhibit herpesvirus DNA polymerases and/or thymidine kinase functions, thus inhibiting lytic virus replication. New host cell recruitment thus ceases. In the absence of new herpesvirus, nonpermissive herpesvirus replication stops, and ME/CFS recovery ensues.Keywords: ME/CFS, Epstein–Barr virus (EBV, human cytomegalovirus (HCMV, HHV6, abortive replication

  2. Genome wide nucleosome mapping for HSV-1 shows nucleosomes are deposited at preferred positions during lytic infection.

    Science.gov (United States)

    Oh, Jaewook; Sanders, Iryna F; Chen, Eric Z; Li, Hongzhe; Tobias, John W; Isett, R Benjamin; Penubarthi, Sindura; Sun, Hao; Baldwin, Don A; Fraser, Nigel W

    2015-01-01

    HSV is a large double stranded DNA virus, capable of causing a variety of diseases from the common cold sore to devastating encephalitis. Although DNA within the HSV virion does not contain any histone protein, within 1 h of infecting a cell and entering its nucleus the viral genome acquires some histone protein (nucleosomes). During lytic infection, partial micrococcal nuclease (MNase) digestion does not give the classic ladder band pattern, seen on digestion of cell DNA or latent viral DNA. However, complete digestion does give a mono-nucleosome band, strongly suggesting that there are some nucleosomes present on the viral genome during the lytic infection, but that they are not evenly positioned, with a 200 bp repeat pattern, like cell DNA. Where then are the nucleosomes positioned? Here we perform HSV-1 genome wide nucleosome mapping, at a time when viral replication is in full swing (6 hr PI), using a microarray consisting of 50mer oligonucleotides, covering the whole viral genome (152 kb). Arrays were probed with MNase-protected fragments of DNA from infected cells. Cells were not treated with crosslinking agents, thus we are only mapping tightly bound nucleosomes. The data show that nucleosome deposition is not random. The distribution of signal on the arrays suggest that nucleosomes are located at preferred positions on the genome, and that there are some positions that are not occupied (nucleosome free regions -NFR or Nucleosome depleted regions -NDR), or occupied at frequency below our limit of detection in the population of genomes. Occupancy of only a fraction of the possible sites may explain the lack of a typical MNase partial digestion band ladder pattern for HSV DNA during lytic infection. On average, DNA encoding Immediate Early (IE), Early (E) and Late (L) genes appear to have a similar density of nucleosomes.

  3. Prospective study of the clinical performance of three BACTEC media in a modern emergency department: Plus Aerobic/F, Plus Anaerobic/F, and Anaerobic Lytic/F.

    Science.gov (United States)

    Rocchetti, Andrea; Di Matteo, Luigi; Bottino, Paolo; Foret, Benjamin; Gamalero, Elisa; Calabresi, Alessandra; Guido, Gianluca; Casagranda, Ivo

    2016-11-01

    The performance of 3 blood culture bottles (BACTEC Plus Aerobic/F, Plus Anaerobic/F, and Anaerobic Lytic/F) were analyzed with clinical specimens collected from 688 Emergency Department patients. A total of 270 strains belonging to 33 species were identified, with E. coli and S. aureus as the most frequently detected. Overall recovery rate (RR) of bacteria and yeast was equivalent in the Plus Aerobic/F vials (208 of 270 isolates; 77.0%) and Anaerobic Lytic/F vials (206 isolates; 76.3%) and significantly better than in the Plus Anaerobic/F vials (189 isolates; 70.0%). Median time to detection (TTD) was earliest with the Anaerobic Lytic/F vials (12.0h) compared with the Plus Aerobic/F (14.6h) and Plus Anaerobic/F vials (15.4h). Positivity rate (PR) was similar for Anaerobic Lytic/F vials (76.9%) and Plus Aerobic/F vials (76.5%), but better if compared with Plus Anaerobic/F vials (69.4%). The PR and TTD for the combination of Plus Aerobic/F with Anaerobic Lytic/F (94.5% and 12.3h, respectively) was significantly better than with Plus Aerobic/F with Plus Anaerobic/F (87.8% and 14.1h).

  4. The phage lytic proteins from the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 display multiple active catalytic domains and do not trigger staphylococcal resistance.

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    Lorena Rodríguez-Rubio

    Full Text Available 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.

  5. Rare presentation of pediatric acute promyelocytic leukemia as multiple lytic bone lesions: Case report and review of literature

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    Manjusha Nair

    2014-01-01

    Full Text Available Acute promyelocytic leukemia (APL is an uncommon malignancy in the pediatric population, accounting for only 5-10% of pediatric acute myeloid leukemias, and for this disease to present with bone lesions at diagnosis is extremely unusual. We wish to convey that very rarely, in a pediatric cancer patient presenting with multiple extensive lytic bone lesions, the diagnosis can be APL. The treatment protocol and prognostic implications are vastly different. Histopathology is the gold standard in arriving at a correct diagnosis and delivering proper treatment in such cases. This patient had excellent response to chemotherapy.

  6. Cello-Oligosaccharide Oxidation Reveals Differences between Two Lytic Polysaccharide Monooxygenases (Family GH61) from Podospora anserina

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    Bey, Mathieu; Zhou, Simeng; Poidevin, Laetitia; Henrissat, Bernard; Coutinho, Pedro M.; Berrin, Jean-Guy; Sigoillot, Jean-Claude

    2013-01-01

    The genome of the coprophilic ascomycete Podospora anserina encodes 33 different genes encoding copper-dependent lytic polysaccharide monooxygenases (LPMOs) from glycoside hydrolase family 61 (GH61). In this study, two of these enzymes (P. anserina GH61A [PaGH61A] and PaGH61B), which both harbored a family 1 carbohydrate binding module, were successfully produced in Pichia pastoris. Synergistic cooperation between PaGH61A or PaGH61B with the cellobiose dehydrogenase (CDH) of Pycnoporus cinnab...

  7. Primary intraosseous atypical inflammatory meningioma presenting as a lytic skull lesion: Case report with review of literature

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    Sangita Bohara

    2016-01-01

    Full Text Available Primary extradural meningiomas of the skull comprise 1% of all meningiomas, and lytic skull meningiomas are still rarer and are said to be more aggressive. We present a case of 38-year-old male with an extradural tumor which on histopathological examination showed features of inflammatory atypical meningioma (WHO Grade II. The intense inflammatory nature of osteolytic primary intraosseous meningioma has not been reported before. This entity deserves special mention because of the need for adjuvant therapy and proper follow-up.

  8. [Isolation of protoplasts from vegetable tissues using extracellular lytic enzymes from fusarium oxysporum f.sp. melonis].

    Science.gov (United States)

    Alconada, T M; Martínez, M J

    1995-01-01

    Fusarium oxysporum f.sp. melonis, a pathogen of melon (Cucumis melo L.), was grown in shaken cultures at 26 degrees C in a mineral salts medium containing glucose, xylan and apple pectin as carbon sources. The extracellular enzymic complex obtained from these cultures showed lytic activity on plant tissues, causing maceration of melon fruits, potato tubers and carrot roots. Protoplasts were isolated from melon fruits when the maceration was carried out under appropriate osmotic conditions. This fact suggest a possible relationship between the enzymes produced by Fusarium oxysporum f.sp. melonis and their pathogenicity on melon plants.

  9. Apoptosis and inflammation

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    C. Haanen

    1995-01-01

    Full Text Available During the last few decades it has been recognized that cell death is not the consequence of accidental injury, but is the expression of a cell suicide programme. Kerr et al. (1972 introduced the term apoptosis. This form of cell death is under the influence of hormones, growth factors and cytokines, which depending upon the receptors present on the target cells, may activate a genetically controlled cell elimination process. During apoptosis the cell membrane remains intact and the cell breaks into apoptotic bodies, which are phagocytosed. Apoptosis, in contrast to necrosis, is not harmful to the host and does not induce any inflammatory reaction. The principal event that leads to inflammatory disease is cell damage, induced by chemical/physical injury, anoxia or starvation. Cell damage means leakage of cell contents into the adjacent tissues, resulting in the capillary transmigration of granulocytes to the injured tissue. The accumulation of neutrophils and release of enzymes and oxygen radicals enhances the inflammatory reaction. Until now there has been little research into the factors controlling the accumulation and the tissue load of granulocytes and their histotoxic products in inflammatory processes. Neutrophil apoptosis may represent an important event in the control of intlamtnation. It has been assumed that granulocytes disintegrate to apoptotic bodies before their fragments are removed by local macrophages. Removal of neutrophils from the inflammatory site without release of granule contents is of paramount importance for cessation of inflammation. In conclusion, apoptotic cell death plays an important role in inflammatory processes and in the resolution of inflammatory reactions. The facts known at present should stimulate further research into the role of neutrophil, eosinophil and macrophage apoptosis in inflammatory diseases.

  10. Abortive lytic reactivation of KSHV in CBF1/CSL deficient human B cell lines.

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    Barbara A Scholz

    Full Text Available Since Kaposi's sarcoma associated herpesvirus (KSHV establishes a persistent infection in human B cells, B cells are a critical compartment for viral pathogenesis. RTA, the replication and transcription activator of KSHV, can either directly bind to DNA or use cellular DNA binding factors including CBF1/CSL as DNA adaptors. In addition, the viral factors LANA1 and vIRF4 are known to bind to CBF1/CSL and modulate RTA activity. To analyze the contribution of CBF1/CSL to reactivation in human B cells, we have successfully infected DG75 and DG75 CBF1/CSL knock-out cell lines with recombinant KSHV.219 and selected for viral maintenance by selective medium. Both lines maintained the virus irrespective of their CBF1/CSL status. Viral reactivation could be initiated in both B cell lines but viral genome replication was attenuated in CBF1/CSL deficient lines, which also failed to produce detectable levels of infectious virus. Induction of immediate early, early and late viral genes was impaired in CBF1/CSL deficient cells at multiple stages of the reactivation process but could be restored to wild-type levels by reintroduction of CBF1/CSL. To identify additional viral RTA target genes, which are directly controlled by CBF1/CSL, we analyzed promoters of a selected subset of viral genes. We show that the induction of the late viral genes ORF29a and ORF65 by RTA is strongly enhanced by CBF1/CSL. Orthologs of ORF29a in other herpesviruses are part of the terminase complex required for viral packaging. ORF65 encodes the small capsid protein essential for capsid shell assembly. Our study demonstrates for the first time that in human B cells viral replication can be initiated in the absence of CBF1/CSL but the reactivation process is severely attenuated at all stages and does not lead to virion production. Thus, CBF1/CSL acts as a global hub which is used by the virus to coordinate the lytic cascade.

  11. Genomic analysis of Bacillus subtilis lytic bacteriophage ϕNIT1 capable of obstructing natto fermentation carrying genes for the capsule-lytic soluble enzymes poly-γ-glutamate hydrolase and levanase.

    Science.gov (United States)

    Ozaki, Tatsuro; Abe, Naoki; Kimura, Keitarou; Suzuki, Atsuto; Kaneko, Jun

    2017-01-01

    Bacillus subtilis strains including the fermented soybean (natto) starter produce capsular polymers consisting of poly-γ-glutamate and levan. Capsular polymers may protect the cells from phage infection. However, bacteriophage ϕNIT1 carries a γ-PGA hydrolase gene (pghP) that help it to counteract the host cell's protection strategy. ϕNIT had a linear double stranded DNA genome of 155,631-bp with a terminal redundancy of 5,103-bp, containing a gene encoding an active levan hydrolase. These capsule-lytic enzyme genes were located in the possible foreign gene cluster regions between central core and terminal redundant regions, and were expressed at the late phase of the phage lytic cycle. All tested natto origin Spounavirinae phages carried both genes for capsule degrading enzymes similar to ϕNIT1. A comparative genomic analysis revealed the diversity among ϕNIT1 and Bacillus phages carrying pghP-like and levan-hydrolase genes, and provides novel understanding on the acquisition mechanism of these enzymatic genes.

  12. An Epstein-Barr Virus (EBV) mutant with enhanced BZLF1 expression causes lymphomas with abortive lytic EBV infection in a humanized mouse model.

    Science.gov (United States)

    Ma, Shi-Dong; Yu, Xianming; Mertz, Janet E; Gumperz, Jenny E; Reinheim, Erik; Zhou, Ying; Tang, Weihua; Burlingham, William J; Gulley, Margaret L; Kenney, Shannon C

    2012-08-01

    Immunosuppressed patients are at risk for developing Epstein-Barr Virus (EBV)-positive lymphomas that express the major EBV oncoprotein, LMP1. Although increasing evidence suggests that a small number of lytically infected cells may promote EBV-positive lymphomas, the impact of enhanced lytic gene expression on the ability of EBV to induce lymphomas is unclear. Here we have used immune-deficient mice, engrafted with human fetal hematopoietic stem cells and thymus and liver tissue, to compare lymphoma formation following infection with wild-type (WT) EBV versus infection with a "superlytic" (SL) mutant with enhanced BZLF1 (Z) expression. The same proportions (2/6) of the WT and SL virus-infected animals developed B-cell lymphomas by day 60 postinfection; the remainder of the animals had persistent tumor-free viral latency. In contrast, all WT and SL virus-infected animals treated with the OKT3 anti-CD3 antibody (which inhibits T-cell function) developed lymphomas by day 29. Lymphomas in OKT3-treated animals (in contrast to lymphomas in the untreated animals) contained many LMP1-expressing cells. The SL virus-infected lymphomas in both OKT3-treated and untreated animals contained many more Z-expressing cells (up to 30%) than the WT virus-infected lymphomas, but did not express late viral proteins and thus had an abortive lytic form of EBV infection. LMP1 and BMRF1 (an early lytic viral protein) were never coexpressed in the same cell, suggesting that LMP1 expression is incompatible with lytic viral reactivation. These results show that the SL mutant induces an "abortive" lytic infection in humanized mice that is compatible with continued cell growth and at least partially resistant to T-cell killing.

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

    Science.gov (United States)

    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.

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

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    benmansour Riadh

    2011-07-01

    Full Text Available Abstract 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.

  15. Isolation, Characterization, and Bioinformatic Analyses of Lytic Salmonella Enteritidis Phages and Tests of Their Antibacterial Activity in Food.

    Science.gov (United States)

    Han, Han; Wei, Xiaoting; Wei, Yi; Zhang, Xiufeng; Li, Xuemin; Jiang, Jinzhong; Wang, Ran

    2017-02-01

    Salmonella Enteritidis remains a major threat for food safety. To take efforts to develop phage-based biocontrol for S. Enteritidis contamination in food, in this study, the phages against S. Enteritidis were isolated from sewage samples, characterized by host range assays, DNA restriction enzyme pattern analyses, and transmission electron microscope observations, and tested for antibacterial activity in food; some potent phages were further characterized by bioinformatic analyses. Results showed that based on the plaque quality and host range, seven lytic phages targeting S. Enteritidis were selected, considered as seven distinct phages through DNA physical maps, and classified as Myoviridae or Siphoviridae family by morphologic observations; the combined use of such seven strain phages as a "food additive" could succeed in controlling the artificial S. Enteritidis contamination in the different physical forms of food at a range of temperatures; by bioinformatic analyses, both selected phage BPS11Q3 and BPS15Q2 seemed to be newfound obligate lytic phage strains with no indications for any potentially harmful genes in their genomes. In conclusion, our results showed a potential of isolated phages as food additives for controlling S. Enteritidis contamination in some salmonellosis outbreak-associated food vehicles, and there could be minimized potential risk associated with using BPS11Q3 and BPS15Q2 in food.

  16. Interactions of a lytic peptide with supported lipid bilayers investigated by time-resolved evanescent wave-induced fluorescence spectroscopy

    Science.gov (United States)

    Rapson, Andrew C.; Gee, Michelle L.; Clayton, Andrew H. A.; Smith, Trevor A.

    2016-12-01

    We report investigations, using time-resolved and polarised evanescent wave-induced fluorescence methods, into the location, orientation and mobility of a fluorescently labelled form of the antimicrobial peptide, melittin, when it interacts with vesicles and supported lipid bilayers (SLBs). This melittin analogue, termed MK14-A430, was found to penetrate the lipid headgroup structure in pure, ordered-phase DPPC membranes but was located near the headgroup-water region when cholesterol was included. MK14-A430 formed lytic pores in SLBs, and an increase in pore formation with incubation time was observed through an increase in polarity and mobility of the probe. When associated with the Cholesterol-containing SLB, the probe displayed polarity and mobility that indicated a population distributed near the lipid headgroup-water interface with MK14-A430 arranged predominantly in a surface-aligned state. This study indicates that the lytic activity of MK14-A430 occurred through a pore-forming mechanism. The lipid headgroup environment experienced by the fluorescent label, where MK14-A430 displayed pore information, indicated that pore formation was best described by the toroidal pore model.

  17. A comparative study on the activity of fungal lytic polysaccharide monooxygenases for the depolymerization of cellulose in soybean spent flakes.

    Science.gov (United States)

    Pierce, Brian C; Agger, Jane Wittrup; Zhang, Zhenghong; Wichmann, Jesper; Meyer, Anne S

    2017-09-08

    Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes capable of the oxidative breakdown of polysaccharides. They are of industrial interest due to their ability to enhance the enzymatic depolymerization of recalcitrant substrates by glycoside hydrolases. In this paper, twenty-four lytic polysaccharide monooxygenases (LPMOs) expressed in Trichoderma reesei were evaluated for their ability to oxidize the complex polysaccharides in soybean spent flakes, an abundant and industrially relevant substrate. TrCel61A, a soy-polysaccharide-active AA9 LPMO from T. reesei, was used as a benchmark in this evaluation. In total, seven LPMOs demonstrated activity on pretreated soy spent flakes, with the products from enzymatic treatments evaluated using mass spectrometry and high performance anion exchange chromatography. The hydrolytic boosting effect of the top-performing enzymes was evaluated in combination with endoglucanase and beta-glucosidase. Two enzymes (TrCel61A and Aspte6) showed the ability to release more than 36% of the pretreated soy spent flake glucose - a greater than 75% increase over the same treatment without LPMO addition. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Application of an Impedimetric Technique for the Detection of Lytic Infection of Salmonella spp. by Specific Phages

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    Lara R. P. Amorim

    2009-01-01

    Full Text Available This study was performed to evaluate the adaption of the impedimetric method to detect the lytic infection by Salmonella-specific bacteriophages and to provide a higher selectivity to this rapid method in detecting Salmonella spp. by using specific agents. Three bacteriophages and twelve strains of Salmonella spp. were tested. Each of the twelve strains was used separately to inoculate TSB together with each one of the phages. The inoculum concentration was between 106 and 107 cfu/mL, at a cell: phage ratio of 1 : 100. From the sample analysis, based on conductance (G measurements (37°C, the infection could be detected, by observation of both detection-time delay and distinct curve trends. The main conclusions were that kinetic detection by impedance microbiology with phage typing constitutes a method of determining whether a test microorganism is sensitive to the bacteriophage and a method to evaluate whether a lytic bacteriophage is present in a sample, by affecting bacterial growth rate/metabolic change.

  19. Effective inhibition of lytic development of bacteriophages λ, P1 and T4 by starvation of their host, Escherichia coli

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    Węgrzyn Alicja

    2007-02-01

    Full Text Available Abstract Background Bacteriophage infections of bacterial cultures cause serious problems in genetic engineering and biotechnology. They are dangerous not only because of direct effects on the currently infected cultures, i.e. their devastation, but also due to a high probability of spreading the phage progeny throughout a whole laboratory or plant, which causes a real danger for further cultivations. Therefore, a simple method for quick inhibition of phage development after detection of bacterial culture infection should be very useful. Results Here, we demonstrate that depletion of a carbon source from the culture medium, which provokes starvation of bacterial cells, results in rapid inhibition of lytic development of three Escherichia coli phages, λ, P1 and T4. Since the effect was similar for three different phages, it seems that it may be a general phenomenon. Moreover, similar effects were observed in flask cultures and in chemostats. Conclusion Bacteriophage lytic development can be inhibited efficiently by carbon source limitation in bacterial cultures. Thus, if bacteriophage contamination is detected, starvation procedures may be recommended to alleviate deleterious effects of phage infection on the culture. We believe that this strategy, in combination with the use of automated and sensitive bacteriophage biosensors, may be employed in the fermentation laboratory practice to control phage outbreaks in bioprocesses more effectively.

  20. Apoptosis and survival

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    Manjul Tiwari

    2011-01-01

    Full Text Available The term apoptosis first appeared in the biomedical literature in 1972, to delineate a structurally distinctive mode of cell death responsible for cell loss within living tissues. The cardinal morphological features are cell shrinkage, accompanied by transient but violent bubbling and blebbing from the surface, and culminating in separation of the cell into a cluster of membrane-bounded bodies. Changes in several cell surface molecules also ensure that, in tissues, apoptotic cells are immediately recognised and phagocytosed by their neighbours. However, it is important to note that apoptosis is only one form of cell death and the particular death pathway that is the most important determinant for cancer therapy is not necessarily that which has the fastest kinetics, as is the bias in many laboratories, but rather that which displays the most sensitive dose-response relationship.

  1. Fullerene and apoptosis

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    M. A. Orlova

    2013-01-01

    Full Text Available Fullerene derivatives superfamily attracts a serious attention as antiviral and anticancer agents and drug delivery carriers as well. A large number of such fullerene С60 derivatives obtained to date. However, there is an obvious deficit of information about causes and mechanisms of immediately and long-term consequences of their effects in vivo which is a true obstacle on the way leading to practical medical use of them. First, this concerns their impact on the proliferation, apoptosis and necrosis regulation. Fullerene nanoparticle functionalization type, their sizes and surface nanopathology are of great importance to further promoting of either cytoprotective or cytotoxic effects. This lecture provides modern concept analysis regarding fullerenes effects on apoptosis pathway in normal and tumor cells.

  2. Apoptosis: una muerte silenciosa

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    Isis Casadelvalle Pérez

    2006-01-01

    Full Text Available La apoptosis o muerte celular programada es un tipo de muerte presente en todas las células eucarióticas. Es un proceso ordenado y esencial del desarrollo normal y de mantenimiento de la homeostasis de un organismo. En el presente trabajo se resumen las principales características fisiológicas, bioquímicas y moleculares de la muerte por apoptosis, evento que ocurre de forma apagada o silenciosa, o sea, sin daño celular aparente diferenciándose claramente del proceso de necrosis celular. En ese proceso se destaca la mitocondria, como organelo celular donde mediado por la activación de las caspasas se inicia el paso hacia la muerte celular programada. En el momento actual, la apoptosis ha cobrado un verdadero valor para la mejor comprensión de los procesos biológicos normales en los que este evento está involucrado y que con anterioridad no era tomado en cuenta. En este sentido, se comentan las principales técnicas de detección de muerte celular programada y se aclara que la elección de algunas de ellas depende del modelo de estudio. Tambi én se dan a conocer algunas de las patologías generales en las que este proceso representa un papel determinante y se discute acerca de cómo algunas alteraciones en los mecanismos de regulación de la apoptosis inducen la aparici ón de varias enfermedades, incluyendo aquellos desórdenes en los que ocurre acumulación celular (cáncer, alteración cardiaca, neurodegeneración y SIDA. El estudio y caracterización de este complejo mecanismo ha cambiado profundamente la comprensión de numerosas patologías en los organismos eucariotas.

  3. Screening of the Human Kinome Identifies MSK1/2-CREB1 as an Essential Pathway Mediating Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication during Primary Infection

    Science.gov (United States)

    Cheng, Fan; Sawant, Tanvee Vinod; Lan, Ke; Lu, Chun; Jung, Jae U.

    2015-01-01

    ABSTRACT Viruses often hijack cellular pathways to facilitate infection and replication. Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus etiologically associated with Kaposi's sarcoma, a vascular tumor of endothelial cells. Despite intensive studies, cellular pathways mediating KSHV infection and replication are still not well defined. Using an antibody array approach, we examined cellular proteins phosphorylated during primary KSHV infection of primary human umbilical vein endothelial cells. Enrichment analysis identified integrin/mitogen-activated protein kinase (integrin/MAPK), insulin/epidermal growth factor receptor (insulin/EGFR), and JAK/STAT as the activated networks during primary KSHV infection. The transcriptional factor CREB1 (cyclic AMP [cAMP]-responsive element-binding protein 1) had the strongest increase in phosphorylation. While knockdown of CREB1 had no effect on KSHV entry and trafficking, it drastically reduced the expression of lytic transcripts and proteins and the production of infectious virions. Chemical activation of CREB1 significantly enhanced viral lytic replication. In contrast, CREB1 neither influenced the expression of the latent gene LANA nor affected KSHV infectivity. Mechanistically, CREB1 was not activated through the classic cAMP/protein kinase A (cAMP/PKA) pathway or via the AKT, MK2, and RSK pathways. Rather, CREB1 was activated by the mitogen- and stress-activated protein kinases 1 and 2 (MSK1/2). Consequently, chemical inhibition or knockdown of MSKs significantly inhibited the KSHV lytic replication program; however, it had a minimal effect on LANA expression and KSHV infectivity. Together, these results identify the MSK1/2-CREB1 proteins as novel essential effectors of KSHV lytic replication during primary infection. The differential effect of the MSK1/2-CREB1 pathway on the expression of viral latent and lytic genes might control the robustness of viral lytic replication, and therefore the

  4. Sphingolipids and mitochondrial apoptosis.

    Science.gov (United States)

    Patwardhan, Gauri A; Beverly, Levi J; Siskind, Leah J

    2016-04-01

    The sphingolipid family of lipids modulate several cellular processes, including proliferation, cell cycle regulation, inflammatory signaling pathways, and cell death. Several members of the sphingolipid pathway have opposing functions and thus imbalances in sphingolipid metabolism result in deregulated cellular processes, which cause or contribute to diseases and disorders in humans. A key cellular process regulated by sphingolipids is apoptosis, or programmed cell death. Sphingolipids play an important role in both extrinsic and intrinsic apoptotic pathways depending on the stimuli, cell type and cellular response to the stress. During mitochondrial-mediated apoptosis, multiple pathways converge on mitochondria and induce mitochondrial outer membrane permeabilization (MOMP). MOMP results in the release of intermembrane space proteins such as cytochrome c and Apaf1 into the cytosol where they activate the caspases and DNases that execute cell death. The precise molecular components of the pore(s) responsible for MOMP are unknown, but sphingolipids are thought to play a role. Here, we review evidence for a role of sphingolipids in the induction of mitochondrial-mediated apoptosis with a focus on potential underlying molecular mechanisms by which altered sphingolipid metabolism indirectly or directly induce MOMP. Data available on these mechanisms is reviewed, and the focus and limitations of previous and current studies are discussed to present important unanswered questions and potential future directions.

  5. Role of Calpain in Apoptosis

    Directory of Open Access Journals (Sweden)

    Hamid Reza Momeni

    2011-01-01

    Full Text Available Apoptosis, a form of programmed cell death that occurs under physiologicalas well as pathological conditions, is characterized by morphological and biochemicalfeatures. While the importance of caspases in apoptosis is established,several noncaspase proteases (Ca2+-dependent proteases such as calpain mayplay a role in the execution of apoptosis. The calpain family consists of twomajor isoforms, calpain I and calpain II which require μM and mM Ca2+ concentrationsto initiate their activity. An increase in intracellular Ca2+ level isthought to trigger a cascade of biochemical processes including calpain activation.Once activated, calpains degrade membrane, cytoplasmic and nuclear substrates,leading to the breakdown of cellular architecture and finally apoptosis.The activation of calpain has been implicated in neuronal apoptosis followingspinal cord injuries and neurodegenerative diseases. This review focuses oncalpain with an emphasis on its key role in the proteolysis of cellular proteinsubstrates following apoptosis.

  6. Decalcified allograft in repair of lytic lesions of bone: A study to evolve bone bank in developing countries

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    Anil Kumar Gupta

    2016-01-01

    Full Text Available Background: The quest for ideal bone graft substitutes still haunts orthopedic researchers. The impetus for this search of newer bone substitutes is provided by mismatch between the demand and supply of autogenous bone grafts. Bone banking facilities such as deep frozen and freeze-dried allografts are not so widely available in most of the developing countries. To overcome the problem, we have used partially decalcified, ethanol preserved, and domestic refrigerator stored allografts which are economical and needs simple technology for procurement, preparation, and preservation. The aim of the study was to assess the radiological and functional outcome of the partially decalcified allograft (by weak hydrochloric acid in patients of benign lytic lesions of bone. Through this study, we have also tried to evolve, establish, and disseminate the concept of the bone bank. Materials and Methods: 42 cases of lytic lesions of bone who were treated by decalcified (by weak hydrochloric acid, ethanol preserved, allografts were included in this prospective study. The allograft was obtained from freshly amputated limbs or excised femoral heads during hip arthroplasties under strict aseptic conditions. The causes of lytic lesions were unicameral bone cyst ( n = 3, aneurysmal bone cyst ( n = 3, giant cell tumor ( n = 9, fibrous dysplasia ( n = 12, chondromyxoid fibroma, chondroma, nonossifying fibroma ( n = 1 each, tubercular osteomyelitis ( n = 7, and chronic pyogenic osteomyelitis ( n = 5. The cavity of the lesion was thoroughly curetted and compactly filled with matchstick sized allografts. Results: Quantitative assessment based on the criteria of Sethi et al. (1993 was done. There was complete assimilation in 27 cases, partial healing in 12 cases, and failure in 3 cases. Functional assessment was also done according to which there were 29 excellent results, 6 good, and 7 cases of failure (infection, recurrence, and nonunion of pathological fracture. We

  7. Apoptosis and DNA Methylation

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Huan X.; Hackett, James A. [MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Nestor, Colm [MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Breakthrough Research Unit, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Dunican, Donncha S.; Madej, Monika; Reddington, James P. [MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Pennings, Sari [Queen' s Medical Research Institute, University of Edinburgh, Edinburgh EH16 4TJ (United Kingdom); Harrison, David J. [Breakthrough Research Unit, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Meehan, Richard R., E-mail: Richard.Meehan@hgu.mrc.ac.uk [MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Breakthrough Research Unit, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU (United Kingdom)

    2011-04-01

    Epigenetic mechanisms assist in maintaining gene expression patterns and cellular properties in developing and adult tissues. The molecular pathology of disease states frequently includes perturbation of DNA and histone methylation patterns, which can activate apoptotic pathways associated with maintenance of genome integrity. This perspective focuses on the pathways linking DNA methyltransferases and methyl-CpG binding proteins to apoptosis, and includes new bioinformatic analyses to characterize the evolutionary origin of two G/T mismatch-specific thymine DNA glycosylases, MBD4 and TDG.

  8. Apoptosis and DNA Methylation

    Directory of Open Access Journals (Sweden)

    Richard R. Meehan

    2011-04-01

    Full Text Available Epigenetic mechanisms assist in maintaining gene expression patterns and cellular properties in developing and adult tissues. The molecular pathology of disease states frequently includes perturbation of DNA and histone methylation patterns, which can activate apoptotic pathways associated with maintenance of genome integrity. This perspective focuses on the pathways linking DNA methyltransferases and methyl-CpG binding proteins to apoptosis, and includes new bioinformatic analyses to characterize the evolutionary origin of two G/T mismatch-specific thymine DNA glycosylases, MBD4 and TDG.

  9. The loss of immunodominant epitopes affects interferon-γ production and lytic activity of the human influenza virus-specific cytotoxic T lymphocyte response in vitro

    NARCIS (Netherlands)

    E.G.M. Berkhoff (Eufemia); M.M. Geelhoed-Mieras (Martina); E.J. Verschuren (Esther); C.A. van Baalen (Carel); R.A. Gruters (Rob); R.A.M. Fouchier (Ron); A.D.M.E. Osterhaus (Albert); G.F. Rimmelzwaan (Guus)

    2007-01-01

    textabstractIn the present study, we examined the effect of the loss of the human leucocyte antigen (HLA)-B*3501-restricted nucleoprotein (NP)418-426epitope on interferon (IFN)-γ-production and lytic activity of the human cytotoxic T lymphocyte (CTL) response in vitro. Extensive amino acid variation

  10. Bacteriophage formulated into a range of semisolid and solid dosage forms maintain lytic capacity against isolated cutaneous and opportunistic oral bacteria.

    Science.gov (United States)

    Brown, Teagan L; Thomas, Tereen; Odgers, Jessica; Petrovski, Steve; Spark, Marion Joy; Tucci, Joseph

    2017-03-01

    Resistance of bacteria to antimicrobial agents is of grave concern. Further research into the development of bacteriophage as therapeutic agents against bacterial infections may help alleviate this problem. To formulate bacteriophage into a range of semisolid and solid dosage forms and investigate the capacity of these preparations to kill bacteria under laboratory conditions. Bacteriophage suspensions were incorporated into dosage forms such as creams, ointments, pastes, pessaries and troches. These were applied to bacterial lawns in order to ascertain lytic capacity. Stability of these formulations containing phage was tested under various storage conditions. A range of creams and ointments were able to support phage lytic activity against Propionibacterium acnes. Assessment of the stability of these formulations showed that storage at 4 °C in light-protected containers resulted in optimal phage viability after 90 days. Pessaries/suppositories and troches were able to support phage lytic activity against Rhodococcus equi. We report here the in-vitro testing of semisolid and solid formulations of bacteriophage lytic against a range of bacteria known to contribute to infections of the epithelia. This study provides a basis for the future formulation of diverse phage against a range of bacteria that infect epithelial tissues. © 2016 Royal Pharmaceutical Society.

  11. Enhancement of lytic activity of leukemic cells by CD8+ cytotoxic T lymphocytes generated against a WT1 peptide analogue.

    Science.gov (United States)

    Al Qudaihi, Ghofran; Lehe, Cynthia; Negash, Muna; Al-Alwan, Monther; Ghebeh, Hazem; Mohamed, Said Yousuf; Saleh, Abu-Jafar Mohammed; Al-Humaidan, Hind; Tbakhi, Abdelghani; Dickinson, Anne; Aljurf, Mahmoud; Dermime, Said

    2009-02-01

    The Wilms tumor antigen 1 (WT1) antigen is over-expressed in human leukemias, making it an attractive target for immunotherapy. Most WT1-specific Cytotoxic T Lymphocytes (CTLs) described so far displayed low avidity, limiting its function. To improve the immunogenicity of CTL epitopes, we replaced the first-amino-acid of two known immunogenic WT1-peptides (126 and 187) with a tyrosine. This modification enhances 126Y analogue-binding ability, triggers significant number of IFN-gamma-producing T cells (P = 0.0003), induces CTL that cross-react with the wild-type peptide, exerts a significant lytic activity against peptide-loaded-targets (P = 0.0006) and HLA-A0201-matched-leukemic cells (P = 0.0014). These data support peptide modification as a feasible approach for the development of a leukemia-vaccine.

  12. Oxidative cleavage and hydrolytic boosting of cellulose in soybean spent flakes by Trichoderma reesei Cel61A lytic polysaccharide monooxygenase.

    Science.gov (United States)

    Pierce, Brian C; Agger, Jane Wittrup; Wichmann, Jesper; Meyer, Anne S

    2017-03-01

    The auxiliary activity family 9 (AA9) copper-dependent lytic polysaccharide monooxygenase (LPMO) from Trichoderma reesei (EG4; TrCel61A) was investigated for its ability to oxidize the complex polysaccharides from soybean. The substrate specificity of the enzyme was assessed against a variety of substrates, including both soy spent flake, a by-product of the soy food industry, and soy spent flake pretreated with sodium hydroxide. Products from enzymatic treatments were analyzed using mass spectrometry and high performance anion exchange chromatography. We demonstrate that TrCel61A is capable of oxidizing cellulose from both pretreated soy spent flake and phosphoric acid swollen cellulose, oxidizing at both the C1 and C4 positions. In addition, we show that the oxidative activity of TrCel61A displays a synergistic effect capable of boosting endoglucanase activity, and thereby substrate depolymerization of soy cellulose, by 27%. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Effect of temperature on the production of cellulases, xylanases and lytic enzymes by selected Trichoderma reesei mutants

    Directory of Open Access Journals (Sweden)

    Piotr Janas

    2014-08-01

    Full Text Available The effect of temperature in the rangę of 26-38°C on the production of cellulases, xylanases and lytic enzymes by four mutant strains of Trichoderma reesei was analysed. On the basis of these investigations three thermosensitive strains (M-7. RUT C 30 and VTT-D-78085 which showed reduced excretion of the above mentioned enzymes as well as protein and a thermoresistant mutant (VTT-D-79I24 which grew within a temperature range of 26-34°C were characterized. Higher temperature caused an increase in the level of xylanolytic enzymes produced by the four mutants. In addition. it effected the complex composition of cellulolytic enzymes secreted by VTT-D-79l 24 (i.c. increased and reduced excertion of (β-glucosidase and β-1,4-endoglucanase respectively.

  14. Lipid mobilization and acid phosphatase activity in lytic compartments during conidium dormancy and appressorium formation of Colletotrichum graminicola.

    Science.gov (United States)

    Schadeck, R J; Leite, B; de Freitas Buchi, D

    1998-12-01

    Colletotrichum graminicola, a pathogen of sorghum and corn, was investigated prior and during germination as to certain aspects of acid phosphatase activity and lipid mobilization. Ungerminated conidia cytoplasm was filled with lipid deposits, which were mobilized during the germination process. Cytochemical ultrastructural examination showed that conidia vacuoles exhibit acid phosphatase activity, which is suggestive of lytic activity. Lipid bodies, stored in the ungerminated conidia cytoplasm, were internalized by vacuoles in a process analogous to microautophagy and were apparently digested inside them. The lipid bodies disappeared and vacuoles became enlarged in conidial cells during germination. Appressoria also showed acid phosphatase activity in multiple heterogeneous vesicles which were, in most cases, juxtaposed with lipid bodies. These results suggest that the vacuolar system plays an important role during C. graminicola germination and that the initial stages of lipid metabolization are taking place inside the vacuoles.

  15. Lytic polysaccharide monooxygenases: a crystallographer’s view on a new class of biomass-degrading enzymes

    Science.gov (United States)

    Frandsen, Kristian E. H.; Lo Leggio, Leila

    2016-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are a new class of microbial copper enzymes involved in the degradation of recalcitrant polysaccharides. They have only been discovered and characterized in the last 5–10 years and have stimulated strong interest both in biotechnology and in bioinorganic chemistry. In biotechnology, the hope is that these enzymes will finally help to make enzymatic biomass conversion, especially of lignocellulosic plant waste, economically attractive. Here, the role of LPMOs is likely to be in attacking bonds that are not accessible to other enzymes. LPMOs have attracted enormous interest since their discovery. The emphasis in this review is on the past and present contribution of crystallographic studies as a guide to functional understanding, with a final look towards the future. PMID:27840684

  16. Apoptosis Resistance in Endometriosis

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    Liselotte Mettler

    2011-08-01

    Full Text Available Introduction: In a cytological analysis of endometriotic lesions neither granulocytes nor cytotoxic T-cells appear in an appreciable number. Based on this observation we aimed to know, whether programmed cell death plays an essential role in the destruction of dystopic endometrium. Disturbances of the physiological mechanisms of apoptosis, a persistence of endometrial tissue could explain the disease. Another aspect of this consideration is the proliferation competence of the dystopic mucous membrane. Methods: Endometriotic lesions of 15 patients were examined through a combined measurement of apoptosis activity with the TUNEL technique (terminal deoxyribosyltransferase mediated dUTP Nick End Labeling and the proliferation activity (with the help of the Ki-67-Antigens using the monoclonal antibody Ki-S5. Results: Twelve out of 15 women studied showed a positive apoptotic activity of 3-47% with a proliferation activity of 2-25% of epithelial cells. Therefore we concluded that the persistence of dystopic endometrium requires proliferative epithelial cells from middle to lower endometrial layers. Conclusion: A dystopia misalignment of the epithelia of the upper layers of the functionalism can be rapidly eliminated by apoptotic procedures.

  17. RTA Occupancy of the Origin of Lytic Replication during Murine Gammaherpesvirus 68 Reactivation from B Cell Latency

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    Alexis L. Santana

    2017-02-01

    Full Text Available RTA, the viral Replication and Transcription Activator, is essential for rhadinovirus lytic gene expression upon de novo infection and reactivation from latency. Lipopolysaccharide (LPS/toll-like receptor (TLR4 engagement enhances rhadinovirus reactivation. We developed two new systems to examine the interaction of RTA with host NF-kappaB (NF-κB signaling during murine gammaherpesvirus 68 (MHV68 infection: a latent B cell line (HE-RIT inducible for RTA-Flag expression and virus reactivation; and a recombinant virus (MHV68-RTA-Bio that enabled in vivo biotinylation of RTA in BirA transgenic mice. LPS acted as a second stimulus to drive virus reactivation from latency in the context of induced expression of RTA-Flag. ORF6, the gene encoding the single-stranded DNA binding protein, was one of many viral genes that were directly responsive to RTA induction; expression was further increased upon treatment with LPS. However, NF-κB sites in the promoter of ORF6 did not influence RTA transactivation in response to LPS in HE-RIT cells. We found no evidence for RTA occupancy of the minimal RTA-responsive region of the ORF6 promoter, yet RTA was found to complex with a portion of the right origin of lytic replication (oriLyt-R that contains predicted RTA recognition elements. RTA occupancy of select regions of the MHV-68 genome was also evaluated in our novel in vivo RTA biotinylation system. Streptavidin isolation of RTA-Bio confirmed complex formation with oriLyt-R in LPS-treated primary splenocytes from BirA mice infected with MHV68 RTA-Bio. We demonstrate the utility of reactivation-inducible B cells coupled with in vivo RTA biotinylation for mechanistic investigations of the interplay of host signaling with RTA.

  18. Novel bacteriophage lysin with broad lytic activity protects against mixed infection by Streptococcus pyogenes and methicillin-resistant Staphylococcus aureus.

    Science.gov (United States)

    Gilmer, Daniel B; Schmitz, Jonathan E; Euler, Chad W; Fischetti, Vincent A

    2013-06-01

    Methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pyogenes (group A streptococcus [GrAS]) cause serious and sometimes fatal human diseases. They are among the many Gram-positive pathogens for which resistance to leading antibiotics has emerged. As a result, alternative therapies need to be developed to combat these pathogens. We have identified a novel bacteriophage lysin (PlySs2), derived from a Streptococcus suis phage, with broad lytic activity against MRSA, vancomycin-intermediate S. aureus (VISA), Streptococcus suis, Listeria, Staphylococcus simulans, Staphylococcus epidermidis, Streptococcus equi, Streptococcus agalactiae (group B streptococcus [GBS]), S. pyogenes, Streptococcus sanguinis, group G streptococci (GGS), group E streptococci (GES), and Streptococcus pneumoniae. PlySs2 has an N-terminal cysteine-histidine aminopeptidase (CHAP) catalytic domain and a C-terminal SH3b binding domain. It is stable at 50 °C for 30 min, 37 °C for >24 h, 4°C for 15 days, and -80 °C for >7 months; it maintained full activity after 10 freeze-thaw cycles. PlySs2 at 128 μg/ml in vitro reduced MRSA and S. pyogenes growth by 5 logs and 3 logs within 1 h, respectively, and exhibited a MIC of 16 μg/ml for MRSA. A single, 2-mg dose of PlySs2 protected 92% (22/24) of the mice in a bacteremia model of mixed MRSA and S. pyogenes infection. Serially increasing exposure of MRSA and S. pyogenes to PlySs2 or mupirocin resulted in no observed resistance to PlySs2 and resistance to mupirocin. To date, no other lysin has shown such notable broad lytic activity, stability, and efficacy against multiple, leading, human bacterial pathogens; as such, PlySs2 has all the characteristics to be an effective therapeutic.

  19. RTA Occupancy of the Origin of Lytic Replication during Murine Gammaherpesvirus 68 Reactivation from B Cell Latency

    Science.gov (United States)

    Santana, Alexis L.; Oldenburg, Darby G.; Kirillov, Varvara; Malik, Laraib; Dong, Qiwen; Sinayev, Roman; Marcu, Kenneth B.; White, Douglas W.; Krug, Laurie T.

    2017-01-01

    RTA, the viral Replication and Transcription Activator, is essential for rhadinovirus lytic gene expression upon de novo infection and reactivation from latency. Lipopolysaccharide (LPS)/toll-like receptor (TLR)4 engagement enhances rhadinovirus reactivation. We developed two new systems to examine the interaction of RTA with host NF-kappaB (NF-κB) signaling during murine gammaherpesvirus 68 (MHV68) infection: a latent B cell line (HE-RIT) inducible for RTA-Flag expression and virus reactivation; and a recombinant virus (MHV68-RTA-Bio) that enabled in vivo biotinylation of RTA in BirA transgenic mice. LPS acted as a second stimulus to drive virus reactivation from latency in the context of induced expression of RTA-Flag. ORF6, the gene encoding the single-stranded DNA binding protein, was one of many viral genes that were directly responsive to RTA induction; expression was further increased upon treatment with LPS. However, NF-κB sites in the promoter of ORF6 did not influence RTA transactivation in response to LPS in HE-RIT cells. We found no evidence for RTA occupancy of the minimal RTA-responsive region of the ORF6 promoter, yet RTA was found to complex with a portion of the right origin of lytic replication (oriLyt-R) that contains predicted RTA recognition elements. RTA occupancy of select regions of the MHV-68 genome was also evaluated in our novel in vivo RTA biotinylation system. Streptavidin isolation of RTA-Bio confirmed complex formation with oriLyt-R in LPS-treated primary splenocytes from BirA mice infected with MHV68 RTA-Bio. We demonstrate the utility of reactivation-inducible B cells coupled with in vivo RTA biotinylation for mechanistic investigations of the interplay of host signaling with RTA. PMID:28212352

  20. CTCF and Rad21 act as host cell restriction factors for Kaposi's sarcoma-associated herpesvirus (KSHV lytic replication by modulating viral gene transcription.

    Directory of Open Access Journals (Sweden)

    Da-Jiang Li

    2014-01-01

    Full Text Available Kaposi's sarcoma-associated herpesvirus (KSHV is a human herpesvirus that causes Kaposi's sarcoma and is associated with the development of lymphoproliferative diseases. KSHV reactivation from latency and virion production is dependent on efficient transcription of over eighty lytic cycle genes and viral DNA replication. CTCF and cohesin, cellular proteins that cooperatively regulate gene expression and mediate long-range DNA interactions, have been shown to bind at specific sites in herpesvirus genomes. CTCF and cohesin regulate KSHV gene expression during latency and may also control lytic reactivation, although their role in lytic gene expression remains incompletely characterized. Here, we analyze the dynamic changes in CTCF and cohesin binding that occur during the process of KSHV viral reactivation and virion production by high resolution chromatin immunoprecipitation and deep sequencing (ChIP-Seq and show that both proteins dissociate from viral genomes in kinetically and spatially distinct patterns. By utilizing siRNAs to specifically deplete CTCF and Rad21, a cohesin component, we demonstrate that both proteins are potent restriction factors for KSHV replication, with cohesin knockdown leading to hundred-fold increases in viral yield. High-throughput RNA sequencing was used to characterize the transcriptional effects of CTCF and cohesin depletion, and demonstrated that both proteins have complex and global effects on KSHV lytic transcription. Specifically, both proteins act as positive factors for viral transcription initially but subsequently inhibit KSHV lytic transcription, such that their net effect is to limit KSHV RNA accumulation. Cohesin is a more potent inhibitor of KSHV transcription than CTCF but both proteins are also required for efficient transcription of a subset of KSHV genes. These data reveal novel effects of CTCF and cohesin on transcription from a relatively small genome that resemble their effects on the cellular

  1. Cross talk between EBV and telomerase: the role of TERT and NOTCH2 in the switch of latent/lytic cycle of the virus.

    Science.gov (United States)

    Giunco, S; Celeghin, A; Gianesin, K; Dolcetti, R; Indraccolo, S; De Rossi, A

    2015-05-28

    Epstein-Barr virus (EBV)-associated malignancies, as well as lymphoblastoid cell lines (LCLs), obtained in vitro by EBV infection of B cells, express latent viral proteins and maintain their ability to grow indefinitely through inappropriate activation of telomere-specific reverse transcriptase (TERT), the catalytic component of telomerase. Our previous studies demonstrated that high levels of TERT expression in LCLs prevent the activation of EBV lytic cycle, which is instead triggered by TERT silencing. As lytic infection promotes the death of EBV-positive tumor cells, understanding the mechanism(s) by which TERT affects the latent/lytic status of EBV may be important for setting new therapeutic strategies. BATF, a transcription factor activated by NOTCH2, the major NOTCH family member in B cells, negatively affects the expression of BZLF1, the master regulator of viral lytic cycle. We therefore analyzed the interplay between TERT, NOTCH and BATF in LCLs and found that high levels of endogenous TERT are associated with high NOTCH2 and BATF expression levels. In addition, ectopic expression of TERT in LCLs with low levels of endogenous telomerase was associated with upregulation of NOTCH2 and BATF at both mRNA and protein levels. By contrast, infection of LCLs with retroviral vectors expressing functional NOTCH2 did not alter TERT transcript levels. Luciferase reporter assays, demonstrated that TERT significantly activated NOTCH2 promoter in a dose-dependent manner. We also found that NF-κB pathway is involved in TERT-induced NOTCH2 activation. Lastly, pharmacologic inhibition of NOTCH signaling triggers the EBV lytic cycle, leading to the death of EBV-infected cells. Overall, these results indicate that TERT contributes to preserve EBV latency in B cells mainly through the NOTCH2/BAFT pathway, and suggest that NOTCH2 inhibition may represent an appealing therapeutic strategy against EBV-associated malignancies.

  2. Role of protein kinase C in TBT-induced inhibition of lytic function and MAPK activation in human natural killer cells.

    Science.gov (United States)

    Abraha, Abraham B; Rana, Krupa; Whalen, Margaret M

    2010-11-01

    Human natural killer (NK) cells are lymphocytes that destroy tumor and virally infected cells. Previous studies have shown that exposure of NK cells to tributyltin (TBT) greatly diminishes their ability to destroy tumor cells (lytic function) while activating mitogen-activated protein kinases (MAPK) (p44/42, p38, and JNK) in NK cells. The signaling pathway that regulates NK lytic function appears to include activation of protein kinase C(PKC) as well as MAPK activity. TBT-induced activation of MAPKs would trigger a portion of the NK lytic signaling pathway, which would then leave the NK cell unable to trigger this pathway in response to a subsequent encounter with a target cell. In the present study we evaluated the involvement of PKC in inhibition of NK lysis of tumor cells and activation of MAPKs caused by TBT exposure. TBT caused a 2–3-fold activation of PKC at concentrations ranging from 50 to 300 nM (16–98 ng/ml),indicating that activation of PKC occurs in response to TBT exposure. This would then leave the NK cell unable to respond to targets. Treatment with the PKC inhibitor, bisindolylmaleimide I, caused an 85% decrease in the ability of NK cells to lyse tumor cells, validating the involvement of PKC in the lytic signaling pathway. The role of PKC in the activation of MAPKs by TBT was also investigated using bisindolylmaleimide I. The results indicated that, in NK cells where PKC activation was blocked, there was no activation of the MAPK, p44/42 in response to TBT.However, TBT-induced activation of the MAPKs, p38 and JNK did not require PKC activation. These results indicate the pivotal role of PKC in the TBT-induced loss of NK lytic function including activation of p44/42 by TBT in NK cells.

  3. Apoptosis : Target of cancer therapy

    NARCIS (Netherlands)

    Ferreira, CG; Epping, M; Kruyt, FAE; Giaccone, G

    2002-01-01

    Recent knowledge on apoptosis has made it possible to devise novel approaches, which exploit this process to treat cancer. In this review, we discuss in detail approaches to induce tumor cell apoptosis, their mechanism of action, stage of development, and possible drawbacks. Finally, the obstacles y

  4. Apoptosis : Target of cancer therapy

    NARCIS (Netherlands)

    Ferreira, CG; Epping, M; Kruyt, FAE; Giaccone, G

    2002-01-01

    Recent knowledge on apoptosis has made it possible to devise novel approaches, which exploit this process to treat cancer. In this review, we discuss in detail approaches to induce tumor cell apoptosis, their mechanism of action, stage of development, and possible drawbacks. Finally, the obstacles y

  5. Cardiovascular molecular imaging of apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Wolters, S.L.; Reutelingsperger, C.P.M. [Maastricht University, Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht (Netherlands); Corsten, M.F.; Hofstra, L. [Maastricht University, Department of Cardiology, Cardiovascular Research Institute Maastricht, P.O. Box 616, Maastricht (Netherlands); Narula, J. [University of California Irvine, Department of Cardiology, Irvine (United States)

    2007-06-15

    Molecular imaging strives to visualise processes at the molecular and cellular level in vivo. Understanding these processes supports diagnosis and evaluation of therapeutic efficacy on an individual basis and thereby makes personalised medicine possible. Apoptosis is a well-organised mode of cell suicide that plays a role in cardiovascular diseases (CVD). Apoptosis is associated with loss of cardiomyocytes following myocardial infarction, atherosclerotic plaque instability, congestive heart failure and allograft rejection of the transplanted heart. Thus, apoptosis constitutes an attractive target for molecular imaging of CVD. Our current knowledge about the molecular players and mechanisms underlying apoptosis offers a rich palette of potential molecular targets for molecular imaging. However, only a few have been successfully developed so far. This review highlights aspects of the molecular machinery and biochemistry of apoptosis relevant to the development of molecular imaging probes. It surveys the role of apoptosis in four major areas of CVD and portrays the importance and future perspectives of apoptosis imaging. The annexin A5 imaging protocol is emphasised since it is the most advanced protocol to measure apoptosis in both preclinical and clinical studies. (orig.)

  6. Caspase Family Proteases and Apoptosis

    Institute of Scientific and Technical Information of China (English)

    Ting-Jun FAN; Li-Hui HAN; Ri-Shan CONG; Jin LIANG

    2005-01-01

    Apoptosis, or programmed cell death, is an essential physiological process that plays a critical role in development and tissue homeostasis. The progress of apoptosis is regulated in an orderly way by a series of signal cascades under certain circumstances. The caspase-cascade system plays vital roles in the induction, transduction and amplification of intracellular apoptotic signals. Caspases, closely associated with apoptosis, are aspartate-specific cysteine proteases and members of the interleukin-1β-converting enzyme family. The activation and function of caspases, involved in the delicate caspase-cascade system, are regulated by various kinds of molecules, such as the inhibitor of apoptosis protein, Bcl-2 family proteins, calpain,and Ca2+. Based on the latest research, the members of the caspase family, caspase-cascade system and caspase-regulating molecules involved in apoptosis are reviewed.

  7. Apoptosis Evaluation by Electrochemical Techniques.

    Science.gov (United States)

    Yin, Jian; Miao, Peng

    2016-03-01

    Apoptosis has close relevance to pathology, pharmacology, and toxicology. Accurate and convenient detection of apoptosis would be beneficial for biological study, clinical diagnosis, and drug development. Based on distinct features of apoptotic cells, a diversity of analytical techniques have been exploited for sensitive analysis of apoptosis, such as surface plasmon resonance, electrochemical methods, flow cytometry, and some imaging assays. Among them, the features of simplicity, easy operation, low cost, and high sensitivity make electrochemical techniques powerful tools to investigate electron-transfer processes of in vitro biological systems. In this contribution, a general overview of current knowledge on various technical approaches for apoptosis evaluation is provided. Furthermore, recently developed electrochemical biosensors for detecting apoptotic cells and their advantages over traditional methods are summarized. One of the main considerations focuses on designing the recognition elements based on various biochemical events during apoptosis.

  8. Apoptosis in Primary Hyperparathyroidism.

    Science.gov (United States)

    Segiet, Oliwia Anna; Mielańczyk, Łukasz; Piecuch, Adam; Michalski, Marek; Tyczyński, Szczepan; Brzozowa-Zasada, Marlena; Deska, Mariusz; Wojnicz, Romuald

    2017-03-31

    Primary hyperparathyroidism (PHPT) is defined by inappropriate elevation of parathormone, caused by parathyroid hyperplasia, also known as multi-gland disease (MGD), parathyroid adenoma (PA), or parathyroid carcinoma (PC). Although several studies have already been conducted, there is a lack of a definite diagnostic marker, which could unambiguously distinguish MGD from PA or PC. The accurate and prompt diagnosis has the key meaning for effective treatment and follow-up. This review paper presents the role of apoptosis in PHPT. The comparison of the expression of Fas, TRAIL, BCL-2 family members, p53 in MGD, PA, and PC, among others, was described. The expression of described factors varies among proliferative lesions of parathyroid gland; therefore, these could serve as additional markers to assist in the diagnosis.

  9. Cl- channels in apoptosis

    DEFF Research Database (Denmark)

    Wanitchakool, Podchanart; Ousingsawat, Jiraporn; Sirianant, Lalida

    2016-01-01

    , and cystic fibrosis transmembrane conductance regulator (CFTR) in cellular apoptosis. LRRC8A-E has been identified as a volume-regulated anion channel expressed in many cell types. It was shown to be required for regulatory and apoptotic volume decrease (RVD, AVD) in cultured cell lines. Its presence also......(-) channels or as regulators of other apoptotic Cl(-) channels, such as LRRC8. CFTR has been known for its proapoptotic effects for some time, and this effect may be based on glutathione release from the cell and increase in cytosolic reactive oxygen species (ROS). Although we find that CFTR is activated...... by cell swelling, it is possible that CFTR serves RVD/AVD through accumulation of ROS and activation of independent membrane channels such as ANO6. Thus activation of ANO6 will support cell shrinkage and induce additional apoptotic events, such as membrane phospholipid scrambling....

  10. Apoptosis and necrosis in vaccinia virus-infected HeLa G and BSC-40 cells.

    Science.gov (United States)

    Liskova, Jana; Knitlova, Jarmila; Honner, Richard; Melkova, Zora

    2011-09-01

    In most cells, vaccinia virus (VACV) infection is considered to cause a lytic cell death, an equivalent of necrosis. However, upon infection of the epithelial cell lines HeLa G and BSC-40 with VACV strain Western Reserve (WR), we have previously observed an increased activation of and activity attributable to caspases, a typical sign of apoptosis. In this paper, we have further analyzed the type of cell death in VACV-infected cells HeLa G and BSC-40. In a cell-based flow cytometric assay, we showed a specific activation of caspase-2 and 4 in HeLa G and BSC-40 cells infected with VACV, strain WR, while we did not find any effects of inhibitors of calpain and cathepsin D and E. The actual activity of the two caspases, but also of caspase-3, was then confirmed in lysates of infected HeLa G, but not in BSC-40 cells. Accordingly, poly(ADP)-ribose polymerase (PARP) cleavage was found increased only in infected HeLa G cells. Consequently, we have determined morphological features of apoptosis and/or activity of the executioner caspase-3 in infected HeLa G cells in situ, while only a background apoptosis was observed in infected BSC-40 cells. Finally, vaccination strains Dryvax and Praha were found to induce apoptosis in both HeLa G and BSC-40 cells, as characterized morphologically and by PARP cleavage. These findings may be important for understanding the differences in VACV-host interactions and post-vaccination complications in different individuals.

  11. Abortive lytic Epstein–Barr virus replication in tonsil-B lymphocytes in infectious mononucleosis and a subset of the chronic fatigue syndrome

    Directory of Open Access Journals (Sweden)

    Lerner AM

    2012-11-01

    Full Text Available A Martin Lerner,1 Safedin Beqaj21Department of Medicine, Oakland University William Beaumont School of Medicine, Rochester, MI, USA; 2Pathology Inc, Torrance, CA, USAAbstract: A systematic 2001–2007 review of 142 chronic fatigue syndrome (CFS patients identified 106 CFS patients with elevated serum IgG antibodies to the herpesviruses Epstein–Barr virus (EBV, cytomegalovirus, or human herpesvirus (HHV 6 in single or multiple infections, with no other co-infections detected. We named these 106 patients group-A CFS. Eighty-six of these 106 group-A CFS patients (81% had elevated EBV early antibody, early antigen (diffuse, serum titers. A small group of six patients in the group-A EBV subset of CFS, additionally, had repetitive elevated-serum titers of antibody to the early lytic replication-encoded proteins, EBV dUTPase, and EBV DNA polymerase. The presence of these serum antibodies to EBV dUTPase and EBV DNA polymerase indicated EBV abortive lytic replication in these 6 CFS patients. None of 20 random control people (age- and sex-matched, with blood drawn at a commercial laboratory had elevated serum titers of antibody to EBV dUTPase or EBV DNA polymerase (P < 0.01. This finding needs verification in a larger group of EBV CFS subset patients, but if corroborated, it may represent a molecular marker for diagnosing the EBV subset of CFS. We review evidence that EBV abortive lytic replication with unassembled viral proteins in the blood may be the same in infectious mononucleosis (IM and a subset of CFS. EBV-abortive lytic replication in tonsil plasma cells is dominant in IM. No complete lytic virion is in the blood of IM or CFS patients. Complications of CFS and IM include cardiomyopathy and encephalopathy. Circulating abortive lytic-encoded EBV proteins (eg, EBV dUTPase, EBV DNA polymerase, and others may be common to IM and CFS. The intensity and duration of the circulating EBV-encoded proteins might differentiate the IM and EBV subsets of CFS

  12. Posterior lumbar interbody fusion for lytic spondylolisthesis: restoration of sagittal balance using insert-and-rotate interbody spacers.

    Science.gov (United States)

    Sears, William

    2005-01-01

    The role of surgical correction of sagittal plane deformity in cases of lytic spondylolisthesis remains controversial. While some early evidence is emerging of the possible short- and long-term benefits of restoring spinal balance, many surgeons have been concerned about the associated risks. The insert-and-rotate posterior lumbar interbody fusion (PLIF) technique, first described by Jaslow in 1946, may enable surgeons to safely and effectively correct sagittal balance through a single posterior approach. To determine whether the focal kyphosis and subluxation associated with a lytic lumbosacral spondylolisthesis can be safely and effectively corrected using a single-stage posterior distraction/reduction technique and insert-and-rotate interbody fusion spacers. A prospective, single cohort, observational study of the clinical outcomes and retrospective radiological review, in a series of 18 consecutive patients with lytic spondylolisthesis Grades I to IV, operated between September 2000 and December 2002. Mean age of 50.2 years (range, 15.5 to 77.8 years). Principal indication for surgery was relief of radicular pain secondary to foraminal stenosis in 16 of 18 patients, and back pain was the principal symptom in 2 patients. Mean preoperative slip was 30.2% (range, 9% to 78%). Mean preoperative focal lordosis was 10.6 degrees (range, -12 to 33 degrees). Minimum 12-month follow-up was available on all patients except one, who died of unrelated causes after his 6-month visit. Patients completed Visual Analogue Pain Score (VAS), Low Back Outcome Score (LBOS), Short Form (SF)-12 and patient satisfaction questionnaires. Pre- and postoperative measurements of the percentage slip and lumbar lordosis of the involved segments were available on 13 patients. SURGICAL METHODS: Decompressive laminectomy was followed by reduction of the spondylolisthesis with the aid of intervertebral disc space spreaders and supplementary pedicle screw instrumentation. The vertebral bodies were

  13. Human Herpesvirus 6B Downregulates Expression of Activating Ligands during Lytic Infection To Escape Elimination by Natural Killer Cells.

    Science.gov (United States)

    Schmiedel, Dominik; Tai, Julie; Levi-Schaffer, Francesca; Dovrat, Sarah; Mandelboim, Ofer

    2016-11-01

    The Herpesviridae family consists of eight viruses, most of which infect a majority of the human population. One of the less-studied members is human herpesvirus 6 (HHV-6) (Roseolovirus), which causes a mild, well-characterized childhood disease. Primary HHV-6 infection is followed by lifelong latency. Reactivation frequently occurs in immunocompromised patients, such as those suffering from HIV infection or cancer or following transplantation, and causes potentially life-threatening complications. In this study, we investigated the mechanisms that HHV-6 utilizes to remain undetected by natural killer (NK) cells, which are key participants in the innate immune response to infections. We revealed viral mechanisms which downregulate ligands for two powerful activating NK cell receptors: ULBP1, ULBP3, and MICB, which trigger NKG2D, and B7-H6, which activates NKp30. Accordingly, this downregulation impaired the ability of NK cells to recognize HHV-6-infected cells. Thus, we describe for the first time immune evasion mechanisms of HHV-6 that protect lytically infected cells from NK elimination. Human herpesvirus 6 (HHV-6) latently infects a large portion of the human population and can reactivate in humans lacking a functional immune system, such as cancer or AIDS patients. Under these conditions, it can cause life-threatening diseases. To date, the actions and interplay of immune cells, and particularly cells of the innate immune system, during HHV-6 infection are poorly defined. In this study, we aimed to understand how cells undergoing lytic HHV-6 infection interact with natural killer (NK) cells, innate lymphocytes constituting the first line of defense against viral intruders. We show that HHV-6 suppresses the expression of surface proteins that alert the immune cells by triggering two major receptors on NK cells, NKG2D and NKp30. As a consequence, HHV-6 can replicate undetected by the innate immune system and potentially spread infection throughout the body. This

  14. Protooncogenes as mediators of apoptosis.

    Science.gov (United States)

    Teng, C S

    2000-01-01

    Apoptosis has been well established as a vital biological phenomenon that is important in the maintenance of cellular homeostasis. Three major protooncogene families and their encoded proteins function as mediators of apoptosis in various cell types and are the subject of this chapter. Protooncogenic proteins such as c-Myc/Max, c-Fos/c-Jun, and Bcl-2/Bax utilize a synergetic effect to enhance their roles in the pro- or antiapoptotic action. These family members activate and repress the expression of their target genes, control cell cycle progression, and execute programmed cell death. Repression or overproduction of these protooncogenic proteins induces apoptosis, which may vary as a result of either cell type specificity or the nature of the apoptotic stimuli. The proapoptotic and antiapoptotic proteins exert their effects in the membrane of cellular organelles. Here they generate cell-type-specific signals that activate the caspase family of proteases and their regulators for the execution of apoptosis.

  15. Antibodies against lytic and latent Kaposi's sarcoma-associated herpes virus antigens and lymphoma in the European EpiLymph case–control study

    Science.gov (United States)

    Benavente, Y; Mbisa, G; Labo, N; Casabonne, D; Becker, N; Maynadie, M; Foretova, L; Cocco, P L; Nieters, A; Staines, A; Bofetta, P; Brennan, P; Whitby, D; de Sanjosé, S

    2011-01-01

    Background: Kaposi's sarcoma-associated herpes virus is associated with primary effusion lymphoma and multicentric Castleman's disease. Methods: Seropositivity to lytic and latent Kaposi's sarcoma herpes virus (KSHV) antigens were examined in 2083 lymphomas and 2013 controls from six European countries. Results: Antibodies against KSHV latent and lytic antigens were detectable in 4.5% and 3.4% of controls, respectively, and 3.6% of cases (P>0.05). The KSHV seropositivity was associated with splenic marginal zone lymphoma (SMZL) (odds ratio (OR)=4.11, 95% confidence interval (CI)=1.57–10.83) and multiple myeloma (OR=0.31, 95% CI=0.11–0.85). Conclusion: The KSHV is unlikely to contribute importantly to lymphomagenesis among immunocompetent subjects. However, the observed association with SMZL may underline a chronic antigen mechanism in its aetiology. PMID:21952625

  16. Invertebrate Iridovirus Modulation of Apoptosis

    Institute of Scientific and Technical Information of China (English)

    Trevor Williams; Nllesh S. Chitnis; Sh(a)n L. Bilimoria

    2009-01-01

    Programmed cell death (apoptosis) is a key host response to virus infection. Viruses that can modulate host apoptotic responses are likely to gain important opportunities for transmission. Here we review recent studies that demonstrate that particles of Invertebrate iridescent virus 6 (IIV-6) (Iridoviridae, genus Iridovirus), or an IIV-6 virion protein extract, are capable of inducing apoptosis in lepidopteran and coleopteran cells, at concentrations 1000-fold lower than that required to shut-off host macromolecular synthesis. Induction of apoptosis depends on endocytosis of one or more heat-sensitive virion component(s). Studies with a JNK inh ibitor(SP600125) indicated that the JNK signaling pathway is significantly involved in apoptosis in IIV-6 infections of Choristoneurafumiferana ceils. The genome of IIV-6 codes for an inhibitor of apoptosis iap gene (193R) that encodes a protein of 208 aa with 15% identity and 28% similarity in its amino acid sequence to IAP-3 from Cydia pomonella ganulovirus (CpGV). Transcription of IIV-6 iap did not require prior DNA or protein synthesis, indicating that it is an immediate-early class gene. Transient expression and gene knockdown studies have confirmed the functional nature of the IIV-6 iap gene. We present a tentative model for IIV-6 induction and inhibition of apoptosis in insect cells and discuss the potential applications of these findings in insect pest control.

  17. [Apoptosis: cellular and clinical aspects].

    Science.gov (United States)

    Løvschall, H; Mosekilde, L

    1997-04-01

    Removal of damaged cells is essential for the maintenance of life in multicellular organisms. The process of self destruction, apoptosis, eliminates surplus or damaged cells as part of the pathophysiological defence system. Apoptosis is essential in structural and functional organogenesis during embryological development. The physiological regulation of tissue kinetics is a product of both cell proliferation and cell death. Internal and external regulatory stimuli regulate the balance between apoptosis and mitosis by genetic interaction. Apoptosis is characterized by condensation of chromatine as a result of DNA degradation, formation of blebs in the plasma and nuclear membranes, condensation of cytoplasma, formation of vesicular apoptotic bodies, and phagocytosis by neighbouring cells without inflammatory response. A number of observations indicate that programmed cell death plays an important role in the regulation of cytofunctional homeostasis and defense against accumulation of damaged cells, eg with DNA alterations. Dysregulation of the apoptotic gene program, eg by mutations, may not only lead to loss or degeneration of tissue, but also to hyperproliferative and tumorigenic disorders. New evidence indicates that apoptosis regulation is important both in aging processes and diseases such as: neuropathies, immunopathies, viral infections, cancer, etc. Pharmacological intervention designed to modulate apoptosis seems to raise new possibilities in the treatment of disease.

  18. Kaposi's Sarcoma-Associated Herpesvirus K-bZIP Protein Is Necessary for Lytic Viral Gene Expression, DNA Replication, and Virion Production in Primary Effusion Lymphoma Cell Lines▿ †

    OpenAIRE

    Lefort, Sylvain; Flamand, Louis

    2009-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of three human proliferative disorders, namely, Kaposi's sarcoma, primary effusion lymphomas (PEL), and multicentric Castleman's disease. Lytic DNA replication of KSHV, which is essential for viral propagation, requires the binding of at least two KSHV proteins, replication and transactivation activator (RTA) and K-bZIP, on the lytic origin of replication. Moreover, K-bZIP physically interacts with RTA and represses its tra...

  19. Isolation and characterization of glacier VMY22, a novel lytic cold-active bacteriophage of Bacillus cereus

    Institute of Scientific and Technical Information of China (English)

    Xiuling; Ji; Chunjing; Zhang; Yuan; Fang; Qi; Zhang; Lianbing; Lin; Bing; Tang; Yunlin; Wei

    2015-01-01

    As a unique ecological system with low temperature and low nutrient levels, glaciers are considered a "living fossil" for the research of evolution. In this work, a lytic cold-active bacteriophage designated VMY22 against Bacillus cereus MYB41-22 was isolated from Mingyong Glacier in China, and its characteristics were studied. Electron microscopy revealed that VMY22 has an icosahedral head(59.2 nm in length, 31.9 nm in width) and a tail(43.2 nm in length). Bacteriophage VMY22 was classified as a Podoviridae with an approximate genome size of 18 to 20 kb. A one-step growth curve revealed that the latent and the burst periods were 70 and 70 min, respectively, with an average burst size of 78 bacteriophage particles per infected cell. The pH and thermal stability of bacteriophage VMY22 were also investigated. The maximum stability of the bacteriophage was observed to be at pH 8.0 and it was comparatively stable at p H 5.0–9.0. As VMY22 is a cold-active bacteriophage with low production temperature, its characterization and the relationship between MYB41-22 and Bacillus cereus bacteriophage deserve further study.

  20. Changes in coagulation and lytic activity of the blood and tissues at the pelvic trauma during anticoagulant therapy

    Directory of Open Access Journals (Sweden)

    A. P. Vlasov

    2014-01-01

    Full Text Available The purpose of our study was exploration of coagulation and lytic activity in blood and tissues during anticoagulation therapy in the early posttraumatic period in patients with pelvic bone fracture. The study was based on experiment researches using methods allowing to estimate coagulation activity in different tissues (skeletal muscles, liver, kidneys, heart, lungs and blood at pelvic trauma during anticoagulation therapy. It was established that at pelvic trauma using anticoagulation therapy (fraxiparine leads to hemostatic system modification in the early posttraumatic period. We observed fast decrease of a hypercoagulability in a blood plasma (organism level and growth fibrinolytic activity. In liver, kidneys, heart and lungs tissues (organ level we also registered correction the hemostatic disorders. However, the rate of these recovery processes in tissues is lower than in the blood. Especially low it was in skeletal muscles in the area of injury. Thus, it is proved that anticoagulant therapy at a pelvic trauma affects on the extrinsic coagulation pathway less than on the intrinsic coagulation pathway. The established regularity explains the risks of coagulation abnormalities in the early posttraumatic period during anticoagulation treatment.

  1. Acquisition of intact polar lipids from the Prymnesiophyte Phaeocystis globosa by its lytic virus PgV-07T

    Directory of Open Access Journals (Sweden)

    D. S. Maat

    2013-07-01

    Full Text Available Recent studies showed changes in phytoplankton lipid composition during viral infection and have indicated roles for specific lipids in the mechanisms of algal virus-host interaction. To investigate the generality of these findings and obtain a better understanding of the allocation of specific lipids to viruses, we studied the intact polar lipid (IPL composition of virally infected and non-infected cultures of the Prymnesiophyte Phaeocystis globosa G(A and its lytic virus PgV-07T. The P. globosa IPL composition was relatively stable over a diel cycle and not strongly affected by viral infection. Glycolipids, phospholipids and betaine lipids were present in both the host and virus, although specific groups such as the diacylglyceryl-hydroxymethyltrimethyl-β-alanines and the sulfoquinovosyldiacylglycerols, were present in a lower proportion or were not detected in the virus. Viral glycosphingolipids (vGSLs, which have been shown to play a role in the infection strategy of the virus EhV-86, infecting the Prymnesiophyte Emiliania huxleyi CCMP374, were not encountered. Our results show that the involvement of lipids in virus-algal host interactions can be very different amongst virus-algal host systems.

  2. Cello-oligosaccharide oxidation reveals differences between two lytic polysaccharide monooxygenases (family GH61) from Podospora anserina.

    Science.gov (United States)

    Bey, Mathieu; Zhou, Simeng; Poidevin, Laetitia; Henrissat, Bernard; Coutinho, Pedro M; Berrin, Jean-Guy; Sigoillot, Jean-Claude

    2013-01-01

    The genome of the coprophilic ascomycete Podospora anserina encodes 33 different genes encoding copper-dependent lytic polysaccharide monooxygenases (LPMOs) from glycoside hydrolase family 61 (GH61). In this study, two of these enzymes (P. anserina GH61A [PaGH61A] and PaGH61B), which both harbored a family 1 carbohydrate binding module, were successfully produced in Pichia pastoris. Synergistic cooperation between PaGH61A or PaGH61B with the cellobiose dehydrogenase (CDH) of Pycnoporus cinnabarinus on cellulose resulted in the formation of oxidized and nonoxidized cello-oligosaccharides. A striking difference between PaGH61A and PaGH61B was observed through the identification of the products, among which were doubly and triply oxidized cellodextrins, which were released only by the combination of PaGH61B with CDH. The mass spectrometry fragmentation patterns of these oxidized products could be consistent with oxidation at the C-6 position with a geminal diol group. The different properties of PaGH61A and PaGH61B and their effect on the interaction with CDH are discussed in regard to the proposed in vivo function of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis.

  3. Characterization of the Newly Isolated Lytic Bacteriophages KTN6 and KT28 and Their Efficacy against Pseudomonas aeruginosa Biofilm.

    Directory of Open Access Journals (Sweden)

    Katarzyna Danis-Wlodarczyk

    Full Text Available We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90% in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants.

  4. In vitro characterization and in vivo properties of Salmonellae lytic bacteriophages isolated from free-range layers

    Directory of Open Access Journals (Sweden)

    L Fiorentin

    2004-06-01

    Full Text Available Occurrence of food poisoning related to Salmonella-contaminated eggs and chicken meat has been frequent in humans. Salmonella Enteritidis (SE and Salmonella Typhimurium (ST are included among the most important paratyphoid salmonellae associated with chicken meat and eggs. Elimination of Salmonella at the pre-harvest stage can play a significant role in preventing the introduction of this pathogen into the food chain and consequently in the reduction of food poisoning in humans. Bactericidal bacteriophages may provide a natural, nontoxic, feasible and non-expensive component of the multi-factorial approach for a pre-harvest control of Salmonella in poultry. Five bacteriophages lytic for SE PT4 and ST were obtained from 107 samples of feces of free-range layers in Brazil. All bacteriophages were characterized in vitro and in vivo, showing head and tail morphology and dsDNA as nucleic acids. Results of "in vivo" studies suggested that bacteriophages do not remain in Salmonella-free birds longer than one day, whereas they multiply in Salmonella-infected birds for longer periods. Besides, selection for phage-resistant SE PT4 did not seem to occur in the short term. Isolated bacteriophages will be investigated for their potential for pre-harvest biocontrol of SE PT4 in poultry.

  5. Characterization of the Newly Isolated Lytic Bacteriophages KTN6 and KT28 and Their Efficacy against Pseudomonas aeruginosa Biofilm.

    Science.gov (United States)

    Danis-Wlodarczyk, Katarzyna; Olszak, Tomasz; Arabski, Michal; Wasik, Slawomir; Majkowska-Skrobek, Grazyna; Augustyniak, Daria; Gula, Grzegorz; Briers, Yves; Jang, Ho Bin; Vandenheuvel, Dieter; Duda, Katarzyna Anna; Lavigne, Rob; Drulis-Kawa, Zuzanna

    2015-01-01

    We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants.

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. S-Layered Aneurinibacillus and Bacillus spp. Are Susceptible to the Lytic Action of Pseudomonas aeruginosa Membrane Vesicles

    Science.gov (United States)

    Kadurugamuwa, J. L.; Mayer, A.; Messner, P.; Sára, M.; Sleytr, U. B.; Beveridge, T. J.

    1998-01-01

    When S-layered strains of Bacillus stearothermophilus and Aneurinibacillus thermoaerophilus, possessing S-layers of different lattice type and lattice constant as well as S-(glyco)protein chemistry, and isogenic S-layerless variants were subjected to membrane vesicles (MVs) from P. aeruginosa during plaque assays on plates or CFU measurements on cell suspensions, all bacterial types lysed. Electron microscopy of negative stains, thin sections, and immunogold-labelled MV preparations revealed that the vesicles adhered to all bacterial surfaces, broke open, and digested the underlying peptidoglycan-containing cell wall of all cell types. Reassembled S-layer did not appear to be affected by MVs, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the S-(glyco)proteins remained intact. meso-Diaminopimelic acid, as a peptidoglycan breakdown product, was found in all culture supernatants after MV attack. These results suggest that even though MVs are much larger than the channels which penetrate these proteinaceous arrays, S-layers on gram-positive bacteria do not form a defensive barrier against the lytic action of MVs. The primary mode of attack is by the liberation from the MVs of a peptidoglycan hydrolase, which penetrates through the S-layer to digest the underlying peptidoglycan-containing cell wall. The S-layer is not affected by MV protease. PMID:9573179

  8. In vitro design of a novel lytic bacteriophage cocktail with therapeutic potential against organisms causing diabetic foot infections.

    Science.gov (United States)

    Mendes, João J; Leandro, Clara; Mottola, Carla; Barbosa, Raquel; Silva, Filipa A; Oliveira, Manuela; Vilela, Cristina L; Melo-Cristino, José; Górski, Andrzej; Pimentel, Madalena; São-José, Carlos; Cavaco-Silva, Patrícia; Garcia, Miguel

    2014-08-01

    In patients with diabetes mellitus, foot infections pose a significant risk. These are complex infections commonly caused by Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii, all of which are potentially susceptible to bacteriophages. Here, we characterized five bacteriophages that we had determined previously to have antimicrobial and wound-healing potential in chronic S. aureus, P. aeruginosa and A. baumannii infections. Morphological and genetic features indicated that the bacteriophages were lytic members of the family Myoviridae or Podoviridae and did not harbour any known bacterial virulence genes. Combinations of the bacteriophages had broad host ranges for the different target bacterial species. The activity of the bacteriophages against planktonic cells revealed effective, early killing at 4 h, followed by bacterial regrowth to pre-treatment levels by 24 h. Using metabolic activity as a measure of cell viability within established biofilms, we found significant cell impairment following bacteriophage exposure. Repeated treatment every 4 h caused a further decrease in cell activity. The greatest effects on both planktonic and biofilm cells occurred at a bacteriophage : bacterium input multiplicity of 10. These studies on both planktonic cells and established biofilms allowed us to better evaluate the effects of a high input multiplicity and a multiple-dose treatment protocol, and the findings support further clinical development of bacteriophage therapy. © 2014 The Authors.

  9. (-)-Epigallocatechin-3-gallate inhibition of Epstein-Barr virus spontaneous lytic infection involves ERK1/2 and PI3-K/Akt signaling in EBV-positive cells.

    Science.gov (United States)

    Liu, Sufang; Li, Hongde; Chen, Lin; Yang, Lifang; Li, Lili; Tao, Yongguan; Li, Wei; Li, Zijian; Liu, Haidan; Tang, Min; Bode, Ann M; Dong, Zigang; Cao, Ya

    2013-03-01

    Epstein-Barr virus (EBV) reactivation into the lytic cycle plays certain roles in the development of EBV-associated diseases, including nasopharyngeal carcinoma and lymphoma. In this study, we investigated the effects of the tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) on EBV spontaneous lytic infection and the mechanism(s) involved in EBV-positive cells. We found that EGCG could effectively inhibit the constitutive lytic infection of EBV at the DNA, gene transcription and protein levels by decreasing the phosphorylation and activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt. By using cellular signaling pathway-specific inhibitors, we also explored the signaling mechanisms underlying the inhibitory effects of EGCG on EBV spontaneous lytic infection in cell models. Results show that specific inhibitors of Mitogen-Activated Protein Kinase Kinase (MEK) (PD98059) and phosphatidylinositol 3-kinase [PI3-K (LY294002)] markedly downregulated gene transcription and expression of BZLF1 and BMRF1 indicating that the MEK/ERK1/2 and PI3-K/Akt pathways are involved in the EBV spontaneous lytic cycle cascade. Therefore, one of the mechanisms by which EGCG inhibits EBV spontaneous lytic infection appears to involve the suppression of the activation of MEK/ERK1/2 and PI3-K/Akt signaling.

  10. Viral control of mitochondrial apoptosis.

    Directory of Open Access Journals (Sweden)

    Lorenzo Galluzzi

    2008-05-01

    Full Text Available Throughout the process of pathogen-host co-evolution, viruses have developed a battery of distinct strategies to overcome biochemical and immunological defenses of the host. Thus, viruses have acquired the capacity to subvert host cell apoptosis, control inflammatory responses, and evade immune reactions. Since the elimination of infected cells via programmed cell death is one of the most ancestral defense mechanisms against infection, disabling host cell apoptosis might represent an almost obligate step in the viral life cycle. Conversely, viruses may take advantage of stimulating apoptosis, either to kill uninfected cells from the immune system, or to induce the breakdown of infected cells, thereby favoring viral dissemination. Several viral polypeptides are homologs of host-derived apoptosis-regulatory proteins, such as members of the Bcl-2 family. Moreover, viral factors with no homology to host proteins specifically target key components of the apoptotic machinery. Here, we summarize the current knowledge on the viral modulation of mitochondrial apoptosis, by focusing in particular on the mechanisms by which viral proteins control the host cell death apparatus.

  11. Placental apoptosis in recurrent miscarriage

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    Tarek A. Atia

    2017-09-01

    Full Text Available Apoptosis is an interactive and dynamic biological process involved in all phases of embryogenesis. We aimed to study the effect of placental apoptosis on recurrent miscarriage (RM. Placental tissue samples were collected from 40 women with RM (study group and 30 women with sporadic spontaneous abortion (control group. Samples were prepared and stained immunohistochemically with markers for both the apoptotic protein (p53 and anti-apoptotic Bcl-2 antibodies. Our results showed that expression of the apoptotic (p53 protein was significantly increased in the placental tissues of the RM group (p = 0.003. By contrast, the expression of anti-apoptotic (Bcl-2 antibodies was significantly increased in the placental tissues of the control group (p = 0.025. We concluded that placental apoptosis plays a crucial role in pregnancy continuation. However, increased p53 expression in placental tissue in early pregnancy could negatively affect pregnancy continuation.

  12. Functional Elucidation of Nemopilema nomurai and Cyanea nozakii Nematocyst Venoms’ Lytic Activity Using Mass Spectrometry and Zymography

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    Yang Yue

    2017-01-01

    Full Text Available Background: Medusozoans utilize explosively discharging penetrant nematocysts to inject venom into prey. These venoms are composed of highly complex proteins and peptides with extensive bioactivities, as observed in vitro. Diverse enzymatic toxins have been putatively identified in the venom of jellyfish, Nemopilema nomurai and Cyanea nozakii, through examination of their proteomes and transcriptomes. However, functional examination of putative enzymatic components identified in proteomic approaches to elucidate potential bioactivities is critically needed. Methods: In this study, enzymatic toxins were functionally identified using a combined approach consisting of in gel zymography and liquid chromatography tandem mass spectrometry (LC-MS/MS. The potential roles of metalloproteinases and lipases in hemolytic activity were explored using specific inhibitors. Results: Zymography indicated that nematocyst venom possessed protease-, lipase- and hyaluronidase-class activities. Further, proteomic approaches using LC-MS/MS indicated sequence homology of proteolytic bands observed in zymography to extant zinc metalloproteinase-disintegrins and astacin metalloproteinases. Moreover, pre-incubation of the metalloproteinase inhibitor batimastat with N. nomurai nematocyst venom resulted in an approximate 62% reduction of hemolysis compared to venom exposed sheep erythrocytes, suggesting that metalloproteinases contribute to hemolytic activity. Additionally, species within the molecular mass range of 14–18 kDa exhibited both egg yolk and erythrocyte lytic activities in gel overlay assays. Conclusion: For the first time, our findings demonstrate the contribution of jellyfish venom metalloproteinase and suggest the involvement of lipase species to hemolytic activity. Investigations of this relationship will facilitate a better understanding of the constituents and toxicity of jellyfish venom.

  13. Herpesviral ICP0 Protein Promotes Two Waves of Heterochromatin Removal on an Early Viral Promoter during Lytic Infection

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    Jennifer S. Lee

    2016-01-01

    Full Text Available Herpesviruses must contend with host cell epigenetic silencing responses acting on their genomes upon entry into the host cell nucleus. In this study, we confirmed that unchromatinized herpes simplex virus 1 (HSV-1 genomes enter primary human foreskin fibroblasts and are rapidly subjected to assembly of nucleosomes and association with repressive heterochromatin modifications such as histone 3 (H3 lysine 9-trimethylation (H3K9me3 and lysine 27-trimethylation (H3K27me3 during the first 1 to 2 h postinfection. Kinetic analysis of the modulation of nucleosomes and heterochromatin modifications over the course of lytic infection demonstrates a progressive removal that coincided with initiation of viral gene expression. We obtained evidence for three phases of heterochromatin removal from an early gene promoter: an initial removal of histones and heterochromatin not dependent on ICP0, a second ICP0-dependent round of removal of H3K9me3 that is independent of viral DNA synthesis, and a third phase of H3K27me3 removal that is dependent on ICP0 and viral DNA synthesis. The presence of ICP0 in transfected cells is also sufficient to promote removal of histones and H3K9me3 modifications of cotransfected genes. Overall, these results show that ICP0 promotes histone removal, a reduction of H3K9me3 modifications, and a later indirect reduction of H3K27me3 modifications following viral early gene expression and DNA synthesis. Therefore, HSV ICP0 promotes the reversal of host epigenetic silencing mechanisms by several mechanisms.

  14. An antisense RNA in a lytic cyanophage links psbA to a gene encoding a homing endonuclease.

    Science.gov (United States)

    Millard, Andrew D; Gierga, Gregor; Clokie, Martha R J; Evans, David J; Hess, Wolfgang R; Scanlan, David J

    2010-09-01

    Cyanophage genomes frequently possess the psbA gene, encoding the D1 polypeptide of photosystem II. This protein is believed to maintain host photosynthetic capacity during infection and enhance phage fitness under high-light conditions. Although the first documented cyanophage-encoded psbA gene contained a group I intron, this feature has not been widely reported since, despite a plethora of new sequences becoming available. In this study, we show that in cyanophage S-PM2, this intron is spliced during the entire infection cycle. Furthermore, we report the widespread occurrence of psbA introns in marine metagenomic libraries, and with psbA often adjacent to a homing endonuclease (HE). Bioinformatic analysis of the intergenic region between psbA and the adjacent HE gene F-CphI in S-PM2 showed the presence of an antisense RNA (asRNA) connecting these two separate genetic elements. The asRNA is co-regulated with psbA and F-CphI, suggesting its involvement with their expression. Analysis of scaffolds from global ocean survey datasets shows this asRNA to be commonly associated with the 3' end of cyanophage psbA genes, implying that this potential mechanism of regulating marine 'viral' photosynthesis is evolutionarily conserved. Although antisense transcription is commonly found in eukaryotic and increasingly also in prokaryotic organisms, there has been no indication for asRNAs in lytic phages so far. We propose that this asRNA also provides a means of preventing the formation of mobile group I introns within cyanophage psbA genes.

  15. Reduction of Salmonella on chicken meat and chicken skin by combined or sequential application of lytic bacteriophage with chemical antimicrobials.

    Science.gov (United States)

    Sukumaran, Anuraj T; Nannapaneni, Rama; Kiess, Aaron; Sharma, Chander Shekhar

    2015-08-17

    The effectiveness of recently approved Salmonella lytic bacteriophage preparation (SalmoFresh™) in reducing Salmonella in vitro and on chicken breast fillets was examined in combination with lauric arginate (LAE) or cetylpyridinium chloride (CPC). In another experiment, a sequential spray application of this bacteriophage (phage) solution on Salmonella inoculated chicken skin after a 20s dip in chemical antimicrobials (LAE, CPC, peracetic acid, or chlorine) was also examined in reducing Salmonella counts on chicken skin. The application of phage in combination with CPC or LAE reduced S. Typhimurium, S. Heidelberg, and S. Enteritidis up to 5 log units in vitro at 4 °C. On chicken breast fillets, phage in combination with CPC or LAE resulted in significant (pSalmonella ranging from 0.5 to 1.3 log CFU/g as compared to control up to 7 days of refrigerated storage. When phage was applied sequentially with chemical antimicrobials, all the treatments resulted in significant reductions of Salmonella. The application of chlorine (30 ppm) and PAA (400 ppm) followed by phage spray (10(9)PFU/ml) resulted in highest Salmonella reductions of 1.6-1.7 and 2.2-2.5l og CFU/cm(2), respectively. In conclusion, the surface applications of phage in combination with LAE or CPC significantly reduced Salmonella counts on chicken breast fillets. However, higher reductions in Salmonella counts were achieved on chicken skin by the sequential application of chemical antimicrobials followed by phage spray. The sequential application of chlorine, PAA, and phage can provide additional hurdles to reduce Salmonella on fresh poultry carcasses or cut up parts.

  16. Hemorrhagic, coagulant and fibrino(geno)lytic activities of crude venom and fractions from mapanare (Bothrops colombiensis) snakes.

    Science.gov (United States)

    Girón, María E; Salazar, Ana M; Aguilar, Irma; Pérez, John C; Sánchez, Elda E; Arocha-Piñango, Carmen L; Rodríguez-Acosta, Alexis; Guerrero, Belsy

    2008-01-01

    Bothrops colombiensis venom from two similar geographical locations were tested for their hemostatic functions and characterized by gel-filtration chromatography and SDS-PAGE electrophoresis. The snakes were from Caucagua and El Guapo towns of the Venezuelan state of Miranda. Fibrino(geno)lytic, procoagulant, hemorrhagic, lethal activities, gel-filtration chromatography and SDS-PAGE profiles were analyzed and compared for both venoms. The highest hemorrhagic activity of 5.3 mug was seen in El Guapo venom while Caucagua venom had the lowest LD(50) of 5.8 mg/kg. Both venoms presented similar thrombin-like activity. El Guapo showed a factor Xa-like activity two times higher than Caucagua. Differences were observed in kallikrein-like and t-PA activities, being highest in El Guapo. Caucagua venom showed the maximum fibrin lysis. Both crude venom runs on Sephadex G-100 chromatography gave fraction SII with the high fibrinolytic activity. Proteases presented in SII fractions and eluted from Benzamidine-Sepharose (not bound to the column) provoked a fast degradation of fibrinogen alpha chains and a slower degradation of beta chains, which could possibly be due to a higher content of alpha fibrinogenases in these venoms. The fibrinogenolytic activity was decreased by metalloprotease inhibitors. The results suggested that metalloproteases in SII fractions were responsible for the fibrinolytic activity. The analysis of samples for fibrin-zymography of SII fractions showed an active band with a molecular mass of approximately 30 kDa. These results reiterate the importance of using pools of venoms for antivenom immunization, to facilitate the neutralization of the maximum potential number of toxins.

  17. Cardiomyocytic apoptosis and heart failure

    Institute of Scientific and Technical Information of China (English)

    Quanzhou Feng

    2008-01-01

    Heart failure is a major disease seriously threatening human health.Once left ventricular dysfunction develops,cardiac function usually deteriorates and progresses to congestive heart failure in several months or years even if no factors which accelerate the deterioration repeatedly exist.Mechanism through which cardiac function continually deteriorates is still unclear.Cardiomyocytic apoptosis can occur in acute stage of ischemic heart diseases and the compensated stage of cardiac dysfunction.In this review,we summarize recent advances in understanding the role of cardiomyocytic apoptosis in heart failure.

  18. An Epstein-Barr Virus-Encoded Protein Complex Requires an Origin of Lytic Replication In Cis to Mediate Late Gene Transcription.

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    Reza Djavadian

    2016-06-01

    Full Text Available Epstein-Barr virus lytic replication is accomplished by an intricate cascade of gene expression that integrates viral DNA replication and structural protein synthesis. Most genes encoding structural proteins exhibit "true" late kinetics-their expression is strictly dependent on lytic DNA replication. Recently, the EBV BcRF1 gene was reported to encode a TATA box binding protein homolog, which preferentially recognizes the TATT sequence found in true late gene promoters. BcRF1 is one of seven EBV genes with homologs found in other β- and γ-, but not in α-herpesviruses. Using EBV BACmids, we systematically disrupted each of these "βγ" genes. We found that six of them, including BcRF1, exhibited an identical phenotype: intact viral DNA replication with loss of late gene expression. The proteins encoded by these six genes have been found by other investigators to form a viral protein complex that is essential for activation of TATT-containing reporters in EBV-negative 293 cells. Unexpectedly, in EBV infected 293 cells, we found that TATT reporter activation was weak and non-specific unless an EBV origin of lytic replication (OriLyt was present in cis. Using two different replication-defective EBV genomes, we demonstrated that OriLyt-mediated DNA replication is required in cis for TATT reporter activation and for late gene expression from the EBV genome. We further demonstrate by fluorescence in situ hybridization that the late BcLF1 mRNA localizes to EBV DNA replication factories. These findings support a model in which EBV true late genes are only transcribed from newly replicated viral genomes.

  19. The Molecular Switch of Telomere Phages: High Binding Specificity of the PY54 Cro Lytic Repressor to a Single Operator Site

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    Jens Andre Hammerl

    2015-06-01

    Full Text Available Temperate bacteriophages possess a molecular switch, which regulates the lytic and lysogenic growth. The genomes of the temperate telomere phages N15, PY54 and ɸKO2 harbor a primary immunity region (immB comprising genes for the prophage repressor, the lytic repressor and a putative antiterminator. The roles of these products are thought to be similar to those of the lambda proteins CI, Cro and Q, respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ɸKO2 are also reminiscent of lambda-like phages. By contrast, in silico analyses revealed the presence of only one operator (O\\(_{\\rm{R}}\\3 in PY54. The purified PY54 Cro protein was used for EMSA studies demonstrating that it exclusively binds to a 16-bp palindromic site (O\\(_{\\rm{R}}\\3 upstream of the prophage repressor gene. The O\\(_{\\rm{R}}\\3 operator sequences of PY54 and ɸKO2/N15 only differ by their peripheral base pairs, which are responsible for Cro specificity. PY54 cI and cro transcription is regulated by highly active promoters initiating the synthesis of a homogenious species of leaderless mRNA. The location of the PY54 Cro binding site and of the identified promoters suggests that the lytic repressor suppresses cI transcription but not its own synthesis. The results indicate an unexpected diversity of the growth regulation mechanisms in lambda-related phages.

  20. Salivary production of IgA and IgG to human herpes virus 8 latent and lytic antigens by patients in whom Kaposi's sarcoma has regressed.

    Science.gov (United States)

    Mbopi-Keou, Francois-Xavier; Legoff, Jerome; Piketty, Christophe; Hocini, Hakim; Malkin, Jean-Elie; Inoue, Naoki; Scully, Crispian M; Porter, Stephen R; Teo, Chong-Gee; Belec, Laurent

    2004-01-23

    IgG and IgA antibodies with specificities to a latent and a lytic antigen of human herpes virus 8 (HHV-8) were detectable in the saliva and serum of eight patients whose Kaposi's sarcoma had regressed, seven of whom were HIV-1 infected. The measurement of antibody-specific activity and secretion rate, and the detection of secretory IgA all indicate anti-HHV-8 antibody activity in saliva. The specific humoral responses possibly influence mucosal replication of HHV-8, and in turn, that of HIV.

  1. A Lytic Polysaccharide Monooxygenase with Broad Xyloglucan Specificity from the Brown-Rot Fungus Gloeophyllum trabeum and Its Action on Cellulose-Xyloglucan Complexes

    OpenAIRE

    KOJIMA, Yuka; Várnai, Anikó; Ishida, Takuya; Sunagawa, Naoki; Petrovic, Dejan M.; Igarashi, Kiyohiko; Jellison, Jody; GOODELL, BARRY; Alfredsen, Gry; Westereng, Bjørge; Vincent G H Eijsink; Yoshida, Makoto

    2016-01-01

    ABSTRACT Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and...

  2. Open kyphoplasty in the treatment of a painful vertebral lytic lesion with spinal cord compression caused by multiple myeloma: A case report

    OpenAIRE

    Pan, Jun; QIAN, ZHONG-LAI; Sun, Zhi-Yong; Yang, Hui-Lin

    2013-01-01

    Multiple myeloma is a fatal hematological malignancy, with the most common localization being the spine. A 72-year-old male patient presented with progressive back pain and dysfunction of ambulation. Spinal computed tomography (CT) and magnetic resonance imaging (MRI) showed spinal cord compression at the T9-T10 level due to an extensive epidural mass in the spinal canal, a large lytic mass of T7-T12 with extraosseous extension and involvement of T9 and T10 vertebral pedicle and posterior wal...

  3. Clinical Manifestations of Kaposi Sarcoma Herpesvirus Lytic Activation: Multicentric Castleman Disease (KSHV–MCD and the KSHV Inflammatory Cytokine Syndrome

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    Mark N. Polizzotto

    2012-03-01

    Full Text Available Soon after the discovery of Kaposi sarcoma (KS-associated herpesvirus (KSHV, it was appreciated that this virus was associated with most cases of multicentric Castleman disease (MCD arising in patients infected with human immunodeficiency virus. It has subsequently been recognized that KSHV–MCD is a distinct entity from other forms of MCD. Like MCD that is unrelated to KSHV, the clinical presentation of KSHV–MCD is dominated by systemic inflammatory symptoms including fevers, cachexia, and laboratory abnormalities including cytopenias, hypoalbuminemia, hyponatremia, and elevated C-reactive protein. Pathologically KSHV–MCD is characterized by polyclonal, IgM-lambda restricted plasmacytoid cells in the intrafollicular areas of affected lymph nodes. A portion of these cells are infected with KSHV and a sizable subset of these cells express KSHV lytic genes including a viral homolog of interleukin-6 (vIL-6. Patients with KSHV–MCD generally have elevated KSHV viral loads in their peripheral blood. Production of vIL-6 and induction of human (h IL-6 both contribute to symptoms, perhaps in combination with overproduction of IL-10 and other cytokines. Until recently, the prognosis of patients with KSHV–MCD was poor. Recent therapeutic advances targeting KSHV-infected B cells with the anti-CD20 monoclonal antibody rituximab and utilizing KSHV enzymes to target KSHV-infected cells have substantially improved patient outcomes. Recently another KSHV-associated condition, the KSHV inflammatory cytokine syndrome (KICS has been described. Its clinical manifestations resemble those of KSHV–MCD but lymphadenopathy is not prominent and the pathologic nodal changes of KSHV–MCD are absent. Patients with KICS exhibit elevated KSHV viral loads and elevation of vIL-6, homolog of human interleukin-6 and IL-10 comparable to those seen in KSHV–MCD; the cellular origin of these is a matter of investigation. KICS may contribute to the inflammatory symptoms

  4. Induction of lytic pathways in T cell clones derived from wild-type or protein tyrosine kinase Fyn mutant mice.

    Science.gov (United States)

    Lancki, D W; Fields, P; Qian, D; Fitch, F W

    1995-08-01

    detected in CD8+ clones derived from fyn-/- mutant mice. Thus, Fyn is not required for expression of these components of antigen specific lysis by CD8+ alloreactive CTL clones. It appears that CD8+ clones that use multiple lytic mechanisms may selectively employ the perforin or Fas-based pathway depending on properties of the target cell or stimulus.(ABSTRACT TRUNCATED AT 400 WORDS)

  5. Lytic HSV-1 infection induces the multifunctional transcription factor Early Growth Response-1 (EGR-1 in rabbit corneal cells

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    McFerrin Harris E

    2011-05-01

    Full Text Available Abstract Background Herpes simplex virus type-1 (HSV-1 infections can cause a number of diseases ranging from simple cold sores to dangerous keratitis and lethal encephalitis. The interaction between virus and host cells, critical for viral replication, is being extensively investigated by many laboratories. In this study, we tested the hypothesis that HSV-1 lytic infection triggers the expression of important multi-functional transcription factor Egr1. The mechanisms of induction are mediated, at least in part, by signaling pathways such as NFκB and CREB. Methods SIRC, VERO, and 293HEK cell lines were infected with HSV-1, and the Egr-1 transcript and protein were detected by RT-PCR and Western blot, respectively. The localization and expression profile of Egr-1 were investigated further by immunofluorescence microscopy analyses. The recruitment of transcription factors to the Egr-1 promoter during infection was studied by chromatin immunoprecipitation (ChIP. Various inhibitors and dominant-negative mutant were used to assess the mechanisms of Egr-1 induction and their effects were addressed by immunofluorescence microscopy. Results Western blot analyses showed that Egr-1 was absent in uninfected cells; however, the protein was detected 24-72 hours post treatment, and the response was directly proportional to the titer of the virus used for infection. Using recombinant HSV-1 expressing EGFP, Egr-1 was detected only in the infected cells. ChIP assays demonstrated that NFкB and cAMP response element binding protein (CREB were recruited to the Egr-1 promoter upon infection. Additional studies showed that inhibitors of NFкB and dominant-negative CREB repressed the Egr-1 induction by HSV-1 infection. Conclusion Collectively, these results demonstrate that Egr-1 is expressed rapidly upon HSV-1 infection and that this novel induction could be due to the NFкB/CREB-mediated transactivation. Egr-1 induction might play a key role in the viral gene

  6. Saffold virus is able to productively infect primate and rodent cell lines and induces apoptosis in these cells.

    Science.gov (United States)

    Xu, Yishi; Victorio, Carla Bianca Luena; Ng, Qimei; Tan, Yee Joo; Chua, Kaw Bing

    2014-02-01

    Saffold virus (SAFV), a newly discovered human cardiovirus of the Picornaviridae family, causes widespread infection among children, as shown by previous seroprevalence studies. To determine the host cell range of SAFV and its cytopathogenicity, eight mammalian cell lines that were available in the laboratory were screened for productive SAFV infection by a laboratory-adapted SAFV of genotype 3. Five of the cell lines (Neuro2A, CHO-K1, NIH/3T3, Vero and HEp-2) were found to be permissible. The time required for SAFV to induce complete lysis as a cytopathic effect (CPE) in these permissibly infected cells and the resultant end point virus titer differed for each cell type. HEp-2 exhibited the shortest time frame to reach full CPE compared to the others. All infected cell lines produced a high virus titer at 72 h post-infection. In addition to causing lytic cell death, SAFV also induced apoptotic cell death in host cells through both extrinsic and intrinsic pathways, although the apoptotic events in HEp-2 cells appeared to have been blocked between the early and late stages. In conclusion, laboratory-adapted SAFV is able to productively infect a number of mammalian cell lines and induce apoptosis in the infected host cells. However, apoptosis in HEp-2 cells is blocked before the end stage.

  7. Signal transduction pathways in mast cell granule-mediated endothelial cell activation

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    Luqi Chi

    2003-01-01

    Full Text Available Background: We have previously shown that incubation of human endothelial cells with mast cell granules results in potentiation of lipopolysaccharide-induced production of interleukin-6 and interleukin-8.

  8. Produção, purificação, clonagem e aplicação de enzimas líticas Production, purification, cloning and application of lytic enzymes

    Directory of Open Access Journals (Sweden)

    Luciana Francisco Fleuri

    2005-10-01

    Full Text Available Lytic enzymes such as beta-1,3 glucanases, proteases and chitinases are able to hydrolyse, respectively, beta-1,3 glucans, mannoproteins and chitin, as well as the cell walls of many yeast species. Lytic enzymes are useful in a great variety of applications including the preparation of protoplasts; the extraction of proteins, enzymes, pigments and functional carbohydrates; pre-treatment for the mechanical rupture of cells; degradation of residual yeast cell mass for the preparation of animal feed; analysis of the yeast cell wall structure and composition; study of the yeast cell wall synthesis and the control of pathogenic fungi. This review presents the most important aspects with respect to lytic enzymes, especially their production, purification, cloning and application.

  9. A decay-accelerating factor-binding strain of coxsackievirus B3 requires the coxsackievirus-adenovirus receptor protein to mediate lytic infection of rhabdomyosarcoma cells.

    Science.gov (United States)

    Shafren, D R; Williams, D T; Barry, R D

    1997-12-01

    The composition of the cellular receptor complex for coxsackievirus B3 (CVB3) has been an area of much contention for the last 30 years. Recently, two individual components of a putative CVB3 cellular receptor complex have been identified as (i) decay-accelerating factor (DAF) and (ii) the coxsackievirus-adenovirus receptor protein (CAR). The present study elucidates the individual roles of DAF and CAR in cell entry of CVB3 Nancy. First, we confirm that the DAF-binding phenotype of CVB3 correlates to the presence of key amino acids located in the viral capsid protein, VP2. Second, using antibody blockade, we show that complete protection of permissive cells from infection by high input multiplicities of CVB3 requires a combination of both anti-DAF and anti-CAR antibodies. Finally, it is shown that expression of the CAR protein on the surface of nonpermissive DAF-expressing RD cells renders them highly susceptible to CVB3-mediated lytic infection. Therefore, although the majority of CVB3 Nancy attaches to the cell via DAF, only virus directly interacting with the CAR protein mediates lytic infection. The role of DAF in CVB3 cell infection may be analogous to that recently described for coxsackievirus A21 (D. R. Shafren, D. J. Dorahy, R. A. Ingham, G. F. Burns, and R. D. Barry, J. Virol. 71:4736-4743, 1997), in that DAF may act as a CVB3 sequestration site, enhancing viral presentation to the functional CAR protein.

  10. The HSV-1 Latency-Associated Transcript Functions to Repress Latent Phase Lytic Gene Expression and Suppress Virus Reactivation from Latently Infected Neurons.

    Science.gov (United States)

    Nicoll, Michael P; Hann, William; Shivkumar, Maitreyi; Harman, Laura E R; Connor, Viv; Coleman, Heather M; Proença, João T; Efstathiou, Stacey

    2016-04-01

    Herpes simplex virus 1 (HSV-1) establishes life-long latent infection within sensory neurons, during which viral lytic gene expression is silenced. The only highly expressed viral gene product during latent infection is the latency-associated transcript (LAT), a non-protein coding RNA that has been strongly implicated in the epigenetic regulation of HSV-1 gene expression. We have investigated LAT-mediated control of latent gene expression using chromatin immunoprecipitation analyses and LAT-negative viruses engineered to express firefly luciferase or β-galactosidase from a heterologous lytic promoter. Whilst we were unable to determine a significant effect of LAT expression upon heterochromatin enrichment on latent HSV-1 genomes, we show that reporter gene expression from latent HSV-1 genomes occurs at a greater frequency in the absence of LAT. Furthermore, using luciferase reporter viruses we have observed that HSV-1 gene expression decreases during long-term latent infection, with a most marked effect during LAT-negative virus infection. Finally, using a fluorescent mouse model of infection to isolate and culture single latently infected neurons, we also show that reactivation occurs at a greater frequency from cultures harbouring LAT-negative HSV-1. Together, our data suggest that the HSV-1 LAT RNA represses HSV-1 gene expression in small populations of neurons within the mouse TG, a phenomenon that directly impacts upon the frequency of reactivation and the maintenance of the transcriptionally active latent reservoir.

  11. Genetically Engineered Yeast Expressing a Lytic Peptide from Bee Venom (Melittin) Kills Symbiotic Protozoa in the Gut of Formosan Subterranean Termites.

    Science.gov (United States)

    Husseneder, Claudia; Donaldson, Jennifer R; Foil, Lane D

    2016-01-01

    The Formosan subterranean termite, Coptotermes formosanus Shiraki, is a costly invasive urban pest in warm and humid regions around the world. Feeding workers of the Formosan subterranean termite genetically engineered yeast strains that express synthetic protozoacidal lytic peptides has been shown to kill the cellulose digesting termite gut protozoa, which results in death of the termite colony. In this study, we tested if Melittin, a natural lytic peptide from bee venom, could be delivered into the termite gut via genetically engineered yeast and if the expressed Melittin killed termites via lysis of symbiotic protozoa in the gut of termite workers and/or destruction of the gut tissue itself. Melittin expressing yeast did kill protozoa in the termite gut within 56 days of exposure. The expressed Melittin weakened the gut but did not add a synergistic effect to the protozoacidal action by gut necrosis. While Melittin could be applied for termite control via killing the cellulose-digesting protozoa in the termite gut, it is unlikely to be useful as a standalone product to control insects that do not rely on symbiotic protozoa for survival.

  12. A novel Pseudomonas aeruginosa bacteriophage, Ab31, a chimera formed from temperate phage PAJU2 and P. putida lytic phage AF: characteristics and mechanism of bacterial resistance.

    Directory of Open Access Journals (Sweden)

    Libera Latino

    Full Text Available A novel temperate bacteriophage of Pseudomonas aeruginosa, phage vB_PaeP_Tr60_Ab31 (alias Ab31 is described. Its genome is composed of structural genes related to those of lytic P. putida phage AF, and regulatory genes similar to those of temperate phage PAJU2. The virion structure resembles that of phage AF and other lytic Podoviridae (S. enterica Epsilon 15 and E. coli phiv10 with similar tail spikes. Ab31 was able to infect P. aeruginosa strain PA14 and two genetically related strains called Tr60 and Tr162, out of 35 diverse strains from cystic fibrosis patients. Analysis of resistant host variants revealed different phenotypes, including induction of pigment and alginate overproduction. Whole genome sequencing of resistant variants highlighted the existence of a large deletion of 234 kbp in two strains, encompassing a cluster of genes required for the production of CupA fimbriae. Stable lysogens formed by Ab31 in strain Tr60, permitted the identification of the insertion site. During colonization of the lung in cystic fibrosis patients, P. aeruginosa adapts by modifying its genome. We suggest that bacteriophages such as Ab31 may play an important role in this adaptation by selecting for bacterial characteristics that favor persistence of bacteria in the lung.

  13. Functional characterization of a novel lytic phage EcSw isolated from Sus scrofa domesticus and its potential for phage therapy.

    Science.gov (United States)

    Easwaran, Maheswaran; Paudel, Sarita; De Zoysa, Mahanama; Shin, Hyun-Jin

    2015-06-01

    In this study, multi-drug resistant Escherichia coli Sw1 (E. coli Sw1) and active lytic phage EcSw was isolated from feces samples of Sus scrofa domesticus (piglet) suffering from diarrhea. Transmission electron microscopy (TEM) indicated that isolated EcSw belongs to the Myoviridae family with an icosahedral head (80 ± 4) and a long tail (180 ± 5 nm). The EcSw phage genome size was estimated to be approximately 75 Kb of double-stranded DNA (dsDNA). Phage dynamic studies show that the latent period and burst size of EcSw were approximately 20 min and 28 PFU per cell, respectively. Interestingly, the EcSw phage can tolerate a wide range of environmental conditions, such as temperature, pH and ions (Ca(2+) and Mg(2+)). Furthermore, genome sequence analysis revealed that the lytic genes of the EcSw phage are notably similar to those of enterobacteria phages. In addition, phage-antibiotic synergy has notable effects compared with the effects of phages or antibiotics alone. Inhibition of E. coli Sw1 and 0157:H7 strains showed that the limitations of host specificity and infectivity of EcSw. Even though, it has considerable potential for phage therapy for handling the problem of the emergence of multidrug resistant pathogens.

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

    Science.gov (United States)

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

    2012-09-01

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

  15. Analysis of nanomechanical properties of Borrelia burgdorferi spirochetes under the influence of lytic factors in an in vitro model using atomic force microscopy.

    Science.gov (United States)

    Tokarska-Rodak, Małgorzata; Kozioł-Montewka, Maria; Skrzypiec, Krzysztof; Chmielewski, Tomasz; Mendyk, Ewaryst; Tylewska-Wierzbanowska, Stanisława

    2015-11-12

    Atomic force microscopy (AFM) is an experimental technique which recently has been used in biology, microbiology, and medicine to investigate the topography of surfaces and in the evaluation of mechanical properties of cells. The aim of this study was to evaluate the influence of the complement system and specific anti-Borrelia antibodies in in vitro conditions on the modification of nanomechanical features of B. burgdorferi B31 cells. In order to assess the influence of the complement system and anti-Borrelia antibodies on B. burgdorferi s.s. B31 spirochetes, the bacteria were incubated together with plasma of identified status. The samples were applied on the surface of mica disks. Young's modulus and adhesive forces were analyzed with a NanoScope V, MultiMode 8 AFM microscope (Bruker) by the PeakForce QNM technique in air using NanoScope Analysis 1.40 software (Bruker). The average value of flexibility of spirochetes' surface expressed by Young's modulus was 10185.32 MPa, whereas the adhesion force was 3.68 nN. AFM is a modern tool with a broad spectrum of observational and measurement abilities. Young's modulus and the adhesion force can be treated as parameters in the evaluation of intensity and changes which take place in pathogenic microorganisms under the influence of various lytic factors. The visualization of the changes in association with nanomechanical features provides a realistic portrayal of the lytic abilities of the elements of the innate and adaptive human immune system.

  16. Apoptosis in irradiated murine tumors.

    Science.gov (United States)

    Stephens, L C; Ang, K K; Schultheiss, T E; Milas, L; Meyn, R E

    1991-09-01

    Early radiation responses of transplantable murine ovarian (OCaI) and hepatocellular (HCaI) carcinomas were examined at 6, 24, 48, 96, and 144 h after single photon doses of 25, 35, or 45 Gy. Previous studies using tumor growth delay and tumor radiocurability assays had shown OCaI tumors to be relatively radiosensitive and HCaI tumors to be radioresistant. At 6 h, approximately 20% of nuclei in OCaI tumors showed aberrations characteristic of cell death by apoptosis. This contrasted to an incidence of 3% in HCaI tumors. Mitotic activity was eliminated in OCaI tumors but was only transiently suppressed in HCaI tumors. At 24-96 h, OCaI tumors continued to display apoptosis and progressive necrosis, whereas HCaI tumors responded by exhibiting marked pleomorphism. Factors other than mitotic activity may influence tumor radiosensitivity, and one of these may be susceptibility to induction of apoptosis (programmed cell death), because this was a prominent early radiation response by the radiosensitive OCaI tumors.

  17. Proteasome inhibitors induce apoptosis and reduce viral replication in primary effusion lymphoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Saji, Chiaki [Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812 (Japan); Higashi, Chizuka; Niinaka, Yasufumi [Faculty of Medicine, University of Yamanashi, Chuoh-shi 409-3898 (Japan); Yamada, Koji [Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812 (Japan); Noguchi, Kohji [Faculty of Pharmacy, Keio University, 1-5-30 Shiba-koen, Minato-ku, Tokyo 105-8512 (Japan); Fujimuro, Masahiro, E-mail: fuji2@mb.kyoto-phu.ac.jp [Department of Cell Biology, Kyoto Pharmaceutical University, Misasagi-Shichonocho 1, Yamashinaku, Kyoto 607-8412 (Japan)

    2011-12-02

    Highlights: Black-Right-Pointing-Pointer Constitutive NF-{kappa}B signaling is essential for the survival and growth of PEL cells. Black-Right-Pointing-Pointer NF-{kappa}B signaling is upregulated by the proteasome-dependent degradation of I{kappa}B{alpha}. Black-Right-Pointing-Pointer Proteasome inhibitors suppress NF-{kappa}B signaling and induce apoptosis in PEL cells through stabilization of I{kappa}B{alpha}. Black-Right-Pointing-Pointer Proteasome inhibitors suppress viral replication in PEL cells during lytic KSHV infection. -- Abstract: Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV). This study provides evidence that proteasomal activity is required for both survival of PEL cells stably harboring the KSHV genome and viral replication of KSHV. We evaluated the cytotoxic effects of proteasome inhibitors on PEL cells. The proteasome inhibitors MG132, lactacystin, and proteasome inhibitor I dramatically inhibited cell proliferation and induced apoptosis of PEL cells through the accumulation of p21 and p27. Furthermore, proteasome inhibitors induced the stabilization of NF-{kappa}B inhibitory molecule (I{kappa}B{alpha}) and suppressed the transcriptional activity of NF-{kappa}B in PEL cells. The NF-{kappa}B specific inhibitor BAY11-7082 also induced apoptosis in PEL cells. The constitutive activation of NF-{kappa}B signaling is essential for the survival and growth of B cell lymphoma cells, including PEL cells. NF-{kappa}B signaling is upregulated by proteasome-dependent degradation of I{kappa}B{alpha}. The suppression of NF-{kappa}B signaling by proteasome inhibitors may contribute to the induction of apoptosis in PEL cells. In addition, proteasome activity is required for KSHV replication in KSHV latently infected PEL cells. MG132 reduced the production of progeny virus from PEL cells at low concentrations, which do not affect PEL cell growth. These findings suggest that proteasome

  18. Apoptosis and congestive heart failure.

    Science.gov (United States)

    Feuerstein, G; Ruffolo, R R; Yue, T L

    1997-10-01

    Congestive heart failure (CHF) is the final clinical manifestation of a variety of cardiac (myopathies), coronary (atherosclerosis), and systemic diseases (diabetes, hypertension). Regardless of the origin of the cardiac insult, left ventricular dysfunction resulting in decreased cardiac output elicits a series of adaptational processes that attempt to compensate for some of the decrement in myocardial function. One of the key manifestations of these compensatory processes is cardiac hypertrophy, which is characterized by a marked increase in myocyte size and an increase in contractile proteins. The benefits resulting from these compensatory adaptational mechanisms, however, are only transient, and within a period of months to years, the changes induced in the myocardium fail to sustain cardiac output at a level that is sufficient to meet the demands of the body; subsequently, physical performance is impaired. Typically, progressive dilation and thinning of the left ventricle occur along with progression of CHF. The mechanisms responsible for the thinning of ventricular tissue and loss of left ventricular mass are poorly understood; traditionally, such loss has been attributed to tissue necrosis based on the morphologic observation of dead cardiac myocytes. Very recently, there have been data suggesting that apoptosis, a form of programmed cell death (PCD), occurs in the heart and may be responsible, at least in part, for the progression of CHF and the chronic loss of left ventricular function and mass. Evidence for a role of apoptosis/PCD in the progression of heart failure has been obtained from a variety of observations, including in vitro studies of cardiac myocytes in culture, experimental animal models of cardiac injury, and cardiac tissue obtained from patients with CHF. Thus, apoptosis/PCD may be a critical mechanism involved in the progressive loss of cardiac myocytes, which ultimately results in end-stage heart failure. In this brief review, the evidence

  19. Molecular signal transduction in vascular cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Apoptosis is a form of genetically programmed cell death, which plays a key role in regulation of cellularity in a variety of tissue and cell types including the cardiovascular tissues. Under both physiological and pathophysiological conditions, various biophysiological and biochemical factors, including mechanical forces, reactive oxygen and nitrogen species, cytokines, growth factors, oxidized lipoproteins, etc., may influence apoptosis of vascular cells. The Fas/Fas ligand/caspase death-signaling pathway, Bcl-2 protein family/mitochondria, the tumor suppressive gene p53, and the proto-oncogene c-myc may be activated in atherosclerotic lesions, and mediates vascular apoptosis during the development of atherosclerosis. Abnormal expression and dysfunction of these apoptosis-regulating genes may attenuate or accelerate vascular cell apoptosis and affect the integrity and stability of atherosclerotic plaques. Clarification of the molecular mechanism that regulates apoptosis may help design a new strategy for treatment of atherosclerosis and its major complication, the acute vascular syndromes.

  20. Apoptosis and Molecular Targeting Therapy in Cancer

    Directory of Open Access Journals (Sweden)

    Mohamed Hassan

    2014-01-01

    Full Text Available Apoptosis is the programmed cell death which maintains the healthy survival/death balance in metazoan cells. Defect in apoptosis can cause cancer or autoimmunity, while enhanced apoptosis may cause degenerative diseases. The apoptotic signals contribute into safeguarding the genomic integrity while defective apoptosis may promote carcinogenesis. The apoptotic signals are complicated and they are regulated at several levels. The signals of carcinogenesis modulate the central control points of the apoptotic pathways, including inhibitor of apoptosis (IAP proteins and FLICE-inhibitory protein (c-FLIP. The tumor cells may use some of several molecular mechanisms to suppress apoptosis and acquire resistance to apoptotic agents, for example, by the expression of antiapoptotic proteins such as Bcl-2 or by the downregulation or mutation of proapoptotic proteins such as BAX. In this review, we provide the main regulatory molecules that govern the main basic mechanisms, extrinsic and intrinsic, of apoptosis in normal cells. We discuss how carcinogenesis could be developed via defective apoptotic pathways or their convergence. We listed some molecules which could be targeted to stimulate apoptosis in different cancers. Together, we briefly discuss the development of some promising cancer treatment strategies which target apoptotic inhibitors including Bcl-2 family proteins, IAPs, and c-FLIP for apoptosis induction.

  1. Apoptosis and Molecular Targeting Therapy in Cancer

    Science.gov (United States)

    Hassan, Mohamed; Watari, Hidemichi; AbuAlmaaty, Ali; Ohba, Yusuke; Sakuragi, Noriaki

    2014-01-01

    Apoptosis is the programmed cell death which maintains the healthy survival/death balance in metazoan cells. Defect in apoptosis can cause cancer or autoimmunity, while enhanced apoptosis may cause degenerative diseases. The apoptotic signals contribute into safeguarding the genomic integrity while defective apoptosis may promote carcinogenesis. The apoptotic signals are complicated and they are regulated at several levels. The signals of carcinogenesis modulate the central control points of the apoptotic pathways, including inhibitor of apoptosis (IAP) proteins and FLICE-inhibitory protein (c-FLIP). The tumor cells may use some of several molecular mechanisms to suppress apoptosis and acquire resistance to apoptotic agents, for example, by the expression of antiapoptotic proteins such as Bcl-2 or by the downregulation or mutation of proapoptotic proteins such as BAX. In this review, we provide the main regulatory molecules that govern the main basic mechanisms, extrinsic and intrinsic, of apoptosis in normal cells. We discuss how carcinogenesis could be developed via defective apoptotic pathways or their convergence. We listed some molecules which could be targeted to stimulate apoptosis in different cancers. Together, we briefly discuss the development of some promising cancer treatment strategies which target apoptotic inhibitors including Bcl-2 family proteins, IAPs, and c-FLIP for apoptosis induction. PMID:25013758

  2. Apoptosis in cancer: from pathogenesis to treatment

    Directory of Open Access Journals (Sweden)

    Wong Rebecca SY

    2011-09-01

    Full Text Available Abstract Apoptosis is an ordered and orchestrated cellular process that occurs in physiological and pathological conditions. It is also one of the most studied topics among cell biologists. An understanding of the underlying mechanism of apoptosis is important as it plays a pivotal role in the pathogenesis of many diseases. In some, the problem is due to too much apoptosis, such as in the case of degenerative diseases while in others, too little apoptosis is the culprit. Cancer is one of the scenarios where too little apoptosis occurs, resulting in malignant cells that will not die. The mechanism of apoptosis is complex and involves many pathways. Defects can occur at any point along these pathways, leading to malignant transformation of the affected cells, tumour metastasis and resistance to anticancer drugs. Despite being the cause of problem, apoptosis plays an important role in the treatment of cancer as it is a popular target of many treatment strategies. The abundance of literature suggests that targeting apoptosis in cancer is feasible. However, many troubling questions arise with the use of new drugs or treatment strategies that are designed to enhance apoptosis and critical tests must be passed before they can be used safely in human subjects.

  3. The cellular decision between apoptosis and autophagy

    Institute of Scientific and Technical Information of China (English)

    Yong-Jun Fan; Wei-Xing Zong

    2013-01-01

    Apoptosis and autophagy are important molecular processes that maintain organismal and cellular homeostasis,respectively.While apoptosis fulfills its role through dismantling damaged or unwanted cells,autophagy maintains cellular homeostasis through recycling selective intracellular organelles and molecules.Yet in some conditions,autophagy can lead to cell death.Apoptosis and autophagy can be stimulated by the same stresses.Emerging evidence indicates an interplay between the core proteins in both pathways,which underlies the molecular mechanism of the crosstalk between apoptosis and autophagy.This review summarizes recent literature on molecules that regulate both the apoptotic and autophagic processes.

  4. Mycobacterium tuberculosis effectors interfering host apoptosis signaling.

    Science.gov (United States)

    Liu, Minqiang; Li, Wu; Xiang, Xiaohong; Xie, Jianping

    2015-07-01

    Tuberculosis remains a serious human public health concern. The coevolution between its pathogen Mycobacterium tuberculosis and human host complicated the way to prevent and cure TB. Apoptosis plays subtle role in this interaction. The pathogen endeavors to manipulate the apoptosis via diverse effectors targeting key signaling nodes. In this paper, we summarized the effectors pathogen used to subvert the apoptosis, such as LpqH, ESAT-6/CFP-10, LAMs. The interplay between different forms of cell deaths, such as apoptosis, autophagy, necrosis, is also discussed with a focus on the modes of action of effectors, and implications for better TB control.

  5. Lytic activity of the virion-associated peptidoglycan hydrolase HydH5 of Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88

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    Donovan David M

    2011-06-01

    Full Text Available Abstract Background Staphylococcus aureus is a food-borne pathogen and the most common cause of infections in hospitalized patients. The increase in the resistance of this pathogen to antibacterials has made necessary the development of new anti-staphylococcal agents. In this context, bacteriophage lytic enzymes such as endolysins and structural peptidoglycan (PG hydrolases have received considerable attention as possible antimicrobials against gram-positive bacteria. Results S. aureus bacteriophage vB_SauS-phiIPLA88 (phiIPLA88 contains a virion-associated muralytic enzyme (HydH5 encoded by orf58, which is located in the morphogenetic module. Comparative bioinformatic analysis revealed that HydH5 significantly resembled other peptidoglycan hydrolases encoded by staphylococcal phages. The protein consists of 634 amino acid residues. Two putative lytic domains were identified: an N-terminal CHAP (cysteine, histidine-dependent amidohydrolase/peptidase domain (135 amino acid residues, and a C-terminal LYZ2 (lysozyme subfamily 2 domain (147 amino acid residues. These domains were also found when a predicted three-dimensional structure of HydH5 was made which provided the basis for deletion analysis. The complete HydH5 protein and truncated proteins containing only each catalytic domain were overproduced in E. coli and purified from inclusion bodies by subsequent refolding. Truncated and full-length HydH5 proteins were all able to bind and lyse S. aureus Sa9 cells as shown by binding assays, zymogram analyses and CFU reduction analysis. HydH5 demonstrated high antibiotic activity against early exponential cells, at 45°C and in the absence of divalent cations (Ca2+, Mg2+, Mn2+. Thermostability assays showed that HydH5 retained 72% of its activity after 5 min at 100°C. Conclusions The virion-associated PG hydrolase HydH5 has lytic activity against S. aureus, which makes it attractive as antimicrobial for food biopreservation and anti

  6. Murine Gammaherpesvirus 68 ORF48 Is an RTA-Responsive Gene Product and Functions in both Viral Lytic Replication and Latency during In Vivo Infection.

    Science.gov (United States)

    Qi, Jing; Han, Chuanhui; Gong, Danyang; Liu, Ping; Zhou, Sheng; Deng, Hongyu

    2015-06-01

    Replication and transcription activator (RTA) of gammaherpesvirus is an immediate early gene product and regulates the expression of many downstream viral lytic genes. ORF48 is also conserved among gammaherpesviruses; however, its expression regulation and function remained largely unknown. In this study, we characterized the transcription unit of ORF48 from murine gammaherpesvirus 68 (MHV-68) and analyzed its transcriptional regulation. We showed that RTA activates the ORF48 promoter via an RTA-responsive element (48pRRE). RTA binds to 48pRRE directly in vitro and also associates with ORF48 promoter in vivo. Mutagenesis of 48pRRE in the context of the viral genome demonstrated that the expression of ORF48 is activated by RTA through 48pRRE during de novo infection. Through site-specific mutagenesis, we generated an ORF48-null virus and examined the function of ORF48 in vitro and in vivo. The ORF48-null mutation remarkably reduced the viral replication efficiency in cell culture. Moreover, through intranasal or intraperitoneal infection of laboratory mice, we showed that ORF48 is important for viral lytic replication in the lung and establishment of latency in the spleen, as well as viral reactivation from latency. Collectively, our study identified ORF48 as an RTA-responsive gene and showed that ORF48 is important for MHV-68 replication both in vitro and in vivo. The replication and transcription activator (RTA), conserved among gammaherpesviruses, serves as a molecular switch for the virus life cycle. It works as a transcriptional regulator to activate the expression of many viral lytic genes. However, only a limited number of such downstream genes have been uncovered for MHV-68. In this study, we identified ORF48 as an RTA-responsive gene of MHV-68 and mapped the cis element involved. By constructing a mutant virus that is deficient in ORF48 expression and through infection of laboratory mice, we showed that ORF48 plays important roles in different stages of

  7. Stem cell ageing and apoptosis.

    Science.gov (United States)

    Fulle, Stefania; Centurione, Lucia; Mancinelli, Rosa; Sancilio, Silvia; Manzoli, Francesco Antonio; Di Pietro, Roberta

    2012-01-01

    Ageing has been defined as the process of deterioration of many body functions over the lifespan of an individual. In spite of the number of different theories about ageing, there is a general consensus in identifying ageing effects in a reduced capacity to regenerate injured tissues or organs and an increased propensity to infections and cancer. In recent years the stem cell theory of ageing has gained much attention. Adult stem cells residing in mammalian tissues are essential for tissue homeostasis and repair throughout adult life. With advancing age, the highly regulated molecular signalling necessary to ensure proper cellular, tissue, and organ homeostasis loses coordination and leads, as a consequence, to a compromised potential of regeneration and repair of damaged cells and tissues. Although a complete comprehension of the molecular mechanisms involved in stem cell ageing and apoptosis is far to be reached, recent studies are beginning to unravel the processes involved in stem cell ageing, particularly in adult skeletal muscle stem cells, namely satellite cells. Thus, the focus of this review is to analyse the relationship between stem cell ageing and apoptosis with a peculiar attention to human satellite cells as compared to haematopoietic stem cells. Undoubtedly, the knowledge of age-related changes of stem cells will help in understanding the ageing process itself and will provide novel therapeutic challenges for improved tissue regeneration.

  8. Application of zinc chloride precipitation method for rapid isolation and concentration of infectious Pectobacterium spp. and Dickeya spp. lytic bacteriophages from surface water and plant and soil extracts.

    Science.gov (United States)

    Czajkowski, Robert; Ozymko, Zofia; Lojkowska, Ewa

    2016-01-01

    This is the first report describing precipitation of bacteriophage particles with zinc chloride as a method of choice to isolate infectious lytic bacteriophages against Pectobacterium spp. and Dickeya spp. from environmental samples. The isolated bacteriophages are ready to use to study various (ecological) aspects of bacteria-bacteriophage interactions. The method comprises the well-known precipitation of phages from aqueous extracts of the test material by addition of ZnCl2, resuscitation of bacteriophage particles in Ringer's buffer to remove the ZnCl2 excess and a soft agar overlay assay with the host bacterium to isolate infectious individual phage plaques. The method requires neither an enrichment step nor other steps (e. g., PEG precipitation, ultrafiltration, or ultracentrifugation) commonly used in other procedures and results in isolation of active viable bacteriophage particles.

  9. Lysis to Kill: Evaluation of the Lytic Abilities, and Genomics of Nine Bacteriophages Infective for Gordonia spp. and Their Potential Use in Activated Sludge Foam Biocontrol.

    Directory of Open Access Journals (Sweden)

    Zoe A Dyson

    Full Text Available Nine bacteriophages (phages infective for members of the genus Gordonia were isolated from wastewater and other natural water environments using standard enrichment techniques. The majority were broad host range phages targeting more than one Gordonia species. When their genomes were sequenced, they all emerged as double stranded DNA Siphoviridae phages, ranging from 17,562 to 103,424 bp in size, and containing between 27 and 127 genes, many of which were detailed for the first time. Many of these phage genomes diverged from the expected modular genome architecture of other characterized Siphoviridae phages and contained unusual lysis gene arrangements. Whole genome sequencing also revealed that infection with lytic phages does not appear to prevent spontaneous prophage induction in Gordonia malaquae lysogen strain BEN700. TEM sample preparation techniques were developed to view both attachment and replication stages of phage infection.

  10. Conversion of α-chitin substrates with varying particle size and crystallinity reveals substrate preferences of the chitinases and lytic polysaccharide monooxygenase of Serratia marcescens.

    Science.gov (United States)

    Nakagawa, Yuko S; Eijsink, Vincent G H; Totani, Kazuhide; Vaaje-Kolstad, Gustav

    2013-11-20

    Industrial depolymerization of chitinous biomass generally requires numerous steps and the use of deleterious substances. Enzymatic methods provide an alternative, but fundamental knowledge that could direct potential development of industrial enzyme cocktails is scarce. We have studied the contribution of monocomponent chitinases (ChiA, -B, and -C) and the lytic polysaccharide monooxygenase (LPMO) from Serratia marcescens on depolymerization of α-chitin substrates with varying particle size and crystallinity that were generated using a converge mill. For all chitinases activity was positively correlated to a decline in particle size and crystallinity. Especially ChiC, the only nonprocessive endochitinase from the S. marcescens chitinolytic machinery, benefited from mechanical pretreatment. Combining the chitinases revealed clear synergies for all substrates tested. CBP21, the chitin-active LPMO from S. marcescens, increased solubilization of substrates with high degrees of crystallinity when combined with each of the three chitinases, but this synergy was reduced upon decline in crystallinity.

  11. Reduction of Salmonella on chicken breast fillets stored under aerobic or modified atmosphere packaging by the application of lytic bacteriophage preparation SalmoFreshTM.

    Science.gov (United States)

    Sukumaran, Anuraj T; Nannapaneni, Rama; Kiess, Aaron; Sharma, Chander Shekhar

    2016-03-01

    The present study evaluated the efficacy of recently approved Salmonella lytic bacteriophage preparation (SalmoFresh™) in reducing Salmonella on chicken breast fillets, as a surface and dip application. The effectiveness of phage in combination with modified atmosphere packaging (MAP) and the ability of phage preparation in reducing Salmonella on chicken breast fillets at room temperature was also evaluated. Chicken breast fillets inoculated with a cocktail of Salmonella Typhimurium, S. Heidelberg, and S. Enteritidis were treated with bacteriophage (10(9) PFU/mL) as either a dip or surface treatment. The dip-treated samples were stored at 4°C aerobically and the surface-treated samples were stored under aerobic and MAP conditions (95% CO2/5% O2) at 4°C for 7 d. Immersion of Salmonella-inoculated chicken breast fillets in bacteriophage solution reduced Salmonella (P chicken breast fillets when the samples were surface treated with phage and stored under MAP conditions. The Salmonella counts were reduced by 1.2, 1.1, and 1.2 log CFU/g on d 0, 1, and 7 of storage, respectively. Bacteriophage surface application on chicken breast fillets stored at room temperature reduced the Salmonella counts by 0.8, 0.9, and 0.4 log CFU/g after 0, 4, and 8 h, respectively, compared to the untreated positive control. These findings indicate that lytic phage preparation was effective in reducing Salmonella on chicken breast fillets stored under aerobic and modified atmosphere conditions.

  12. Co-therapy using lytic bacteriophage and linezolid: effective treatment in eliminating methicillin resistant Staphylococcus aureus (MRSA from diabetic foot infections.

    Directory of Open Access Journals (Sweden)

    Sanjay Chhibber

    Full Text Available BACKGROUND: Staphylococcus aureus remains the predominant pathogen in diabetic foot infections and prevalence of methicillin resistant S.aureus (MRSA strains further complicates the situation. The incidence of MRSA in infected foot ulcers is 15-30% and there is an alarming trend for its increase in many countries. Diabetes acts as an immunosuppressive state decreasing the overall immune functioning of body and to worsen the situation, wounds inflicted with drug resistant strains represent a morbid combination in diabetic patients. Foot infections caused by MRSA are associated with an increased risk of amputations, increased hospital stay, increased expenses and higher infection-related mortality. Hence, newer, safer and effective treatment strategies are required for treating MRSA mediated diabetic foot infections. The present study focuses on the use of lytic bacteriophage in combination with linezolid as an effective treatment strategy against foot infection in diabetic population. METHODOLOGY: Acute hindpaw infection with S.aureus ATCC 43300 was established in alloxan induced diabetic BALB/c mice. Therapeutic efficacy of a well characterized broad host range lytic bacteriophage, MR-10 was evaluated alone as well as in combination with linezolid in resolving the course of hindpaw foot infection in diabetic mice. The process of wound healing was also investigated. RESULTS AND CONCLUSIONS: A single administration of phage exhibited efficacy similar to linezolid in resolving the course of hindpaw infection in diabetic animals. However, combination therapy using both the agents was much more effective in arresting the entire infection process (bacterial load, lesion score, foot myeloperoxidase activity and histopathological analysis. The entire process of tissue healing was also hastened. Use of combined agents has been known to decrease the frequency of emergence of resistant mutants, hence this approach can serve as an effective strategy in

  13. Consumption of purple sweet potato leaves modulates human immune response: T-lymphocyte functions, lytic activity of natural killer cell and antibody production

    Institute of Scientific and Technical Information of China (English)

    Chiao-Ming Chen; Sing-Chung Li; Ya-Ling Lin; Ching-Yun Hsu; Ming-Jer Shieh; Jen-Fang Liu

    2005-01-01

    AIM: To study the immunological effects of physiological doses of purple sweet potato leaves (PSPL).METHODS: The randomized crossover study (two periods,each lasting for 2 wk) involved 16 healthy non-smoking adults of normal weight. The 6-wk study consisted of a run-in (wk 1) PSPL diet (daily consumption of 200 g PSPL) or a control diet (low polyphenols, with the amount of carotenoids adjusted to the same level as that of PSPL) (wk 2-3), washout diet (wk 4), and switched diet (wk 5-6). Fasting blood was collected weekly in the morning. T-lymphocyte function was assessed via the proliferation and secretion of immunoreactive cytokines.Salivary IgA secretion and the specific cytotoxic activities of cytotoxic T lymphocytes and natural killer (NK) cells were determined.RESULTS: The plasma β-carotene level increased with time in both groups, while the plasma polyphenol level decreased in the control group, and no significant difference was detected between the two groups.Although plasma polyphenol levels did not significantly increase in the PSPL group at the end of the study, they were significantly elevated in urine. PSPL consumption produced a significant increase in proliferation responsiveness of peripheral blood mononuclear cells (PBMC) and their secretion of immunoreactive IL-2 and IL-4. As well, lytic activity in NK cells was elevated in a time-dependent fashion. Salivary TgA secretion significantly decreased in control group after 2 wk, and returned to baseline following dietary switch to PSPL.CONCLUSION: Consumption of PSPL modulates various immune functions including increased proliferation responsiveness of PBMC, secretion of cytokines IL-2 and IL-4, and the lytic activity of NK cells. The responsible determinants of PSPL remain to be elucidated, as does the biological significance of the present observations.

  14. Fungal cell-wall lytic enzymes, antifungal metabolite(s) production, and characterization from Streptomyces exfoliatus MT9 for controlling fruit-rotting fungi.

    Science.gov (United States)

    Choudhary, Bharti; Nagpure, Anand; Gupta, Rajinder K

    2014-12-01

    An antifungal actinomycete strain MT9 was isolated from Loktak Lake, Manipur, India and its cultural characteristics, fatty acid methyl ester, 16S rRNA gene analysis suggests that strain MT9 is identical to Streptomyces exfoliatus. Strain MT9 displayed strong and broad-spectrum antagonism towards several fruit-rotting fungi by mycelial growth suppression. Crude fungal cell-wall lytic enzymes, i.e., chitinase, β-1,3-glucanase, and protease produced by S. exfoliatus MT9 were optimally active at pH 8.0 and 50 °C, pH 5.0 and 60 °C, pH 9.0 and 70 °C, respectively. All three mycolytic enzymes had good stability over a wide pH range of 5.0-10.0, with protease being more thermostable than both chitinase and β-1,3-glucanase. Interestingly zymogram analysis revealed that S. exfoliatus MT9 secretes six distinct chitinase isoenzymes with approximate molecular weights of 9.42, 13.93, 27.87, 36.43, 54.95, 103.27 kDa, six active protease isoenzymes with apparent molecular weights of 12.45, 30.20, 37.45, 46.32, 52.46, 131.46 kDa, and an active band of 119.39 kDa as β-1,3-glucanase enzyme. Extracellular fluid and its organic solvent extracts also exhibited inhibitory activity to various fruit-rotting fungi. The MIC value of n-butanol extract was 2-25 µg/ml against tested fruit-rotting fungi. Antifungal secondary metabolite(s) was found to be polyene in nature. To the best of our knowledge, this is the first report on extracellular production of fungal cell-wall lytic enzymes and antifungal metabolites by bioactive S. exfoliatus MT9 under submerged fermentation.

  15. Targeted induction of apoptosis for cancer therapy

    NARCIS (Netherlands)

    Bremer, Edwin

    2006-01-01

    Introduction to the thesis Programmed cell death, known as apoptosis, is an essential cellular homeostasis mechanism that ensures correct development and function of multi-cellular organisms. The pivotal importance of correct execution of apoptosis is apparent from the many human diseases with aberr

  16. Apoptosis in mammalian oocytes: a review.

    Science.gov (United States)

    Tiwari, Meenakshi; Prasad, Shilpa; Tripathi, Anima; Pandey, Ashutosh N; Ali, Irfan; Singh, Arvind K; Shrivastav, Tulsidas G; Chaube, Shail K

    2015-08-01

    Apoptosis causes elimination of more than 99% of germ cells from cohort of ovary through follicular atresia. Less than 1% of germ cells, which are culminated in oocytes further undergo apoptosis during last phases of oogenesis and depletes ovarian reserve in most of the mammalian species including human. There are several players that induce apoptosis directly or indirectly in oocytes at various stages of meiotic cell cycle. Premature removal of encircling granulosa cells from immature oocytes, reduced levels of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate, increased levels of calcium (Ca(2+)) and oxidants, sustained reduced level of maturation promoting factor, depletion of survival factors, nutrients and cell cycle proteins, reduced meiotic competency, increased levels of proapoptotic as well as apoptotic factors lead to oocyte apoptosis. The BH3-only proteins also act as key regulators of apoptosis in oocyte within the ovary. Both intrinsic (mitochondria-mediated) as well as extrinsic (cell surface death receptor-mediated) pathways are involved in oocyte apoptosis. BID, a BH3-only protein act as a bridge between both apoptotic pathways and its cleavage activates cell death machinery of both the pathways inside the follicular microenvironment. Oocyte apoptosis leads to the depletion of ovarian reserve that directly affects reproductive outcome of various mammals including human. In this review article, we highlight some of the important players and describe the pathways involved during oocyte apoptosis in mammals.

  17. Crosstalk between apoptosis and inflammation in atherosclerosis

    NARCIS (Netherlands)

    Westra, Marijke Marianne

    2010-01-01

    In this thesis the role of several apoptosis regulating proteins in the development of atherosclerosis and atherosclerotic plaque stability is investigated. Apoptosis of different cell types in atherosclerotic plaques, such as macrophages and smooth muscle cells may inhibit or promote plaque develop

  18. Crosstalk between apoptosis and inflammation in atherosclerosis

    NARCIS (Netherlands)

    Westra, Marijke Marianne

    2010-01-01

    In this thesis the role of several apoptosis regulating proteins in the development of atherosclerosis and atherosclerotic plaque stability is investigated. Apoptosis of different cell types in atherosclerotic plaques, such as macrophages and smooth muscle cells may inhibit or promote plaque

  19. Targeted induction of apoptosis for cancer therapy

    NARCIS (Netherlands)

    Bremer, Edwin

    2006-01-01

    Introduction to the thesis Programmed cell death, known as apoptosis, is an essential cellular homeostasis mechanism that ensures correct development and function of multi-cellular organisms. The pivotal importance of correct execution of apoptosis is apparent from the many human diseases with

  20. Hepatitis C virus infection and apoptosis

    Institute of Scientific and Technical Information of China (English)

    Richard Fischer; Thomas Baumert; Hubert E Blum

    2007-01-01

    Apoptosis is central for the control and elimination of viral infections. In chronic hepatitis C virus (HCV) infection,enhanced hepatocyte apoptosis and upregulation of the death inducing ligands CD95/Fas, TRAIL and TNFα occur.Nevertheless, HCV infection persists in the majority of patients. The impact of apoptosis in chronic HCV infection is not well understood. It may be harmful by triggering liver fibrosis, or essential in interferon (IFN)induced HCV elimination. For virtually all HCV proteins,pro- and anti-apoptotic effects have been described,especially for the core and NS5A protein. To date, it is not known which HCV protein affects apoptosis in vivo and whether the infectious virions act pro- or antiapoptotic. With the availability of an infectious tissue culture system, we now can address pathophysiologically relevant issues. This review focuses on the effect of HCV infection and different HCV proteins on apoptosis and of the corresponding signaling cascades.

  1. Study of apoptosis in human liver cancers

    Institute of Scientific and Technical Information of China (English)

    Chang-Min Shan; Juan Li

    2002-01-01

    AIM: To investigate the action of apoptosis in occurrence ofliver cacinornas in vivo and the biological effect of Solanumlyratum Thumb on BEL-7404 cell line inducing apoptosis invitro.METHODS: The apoptosis in the liver carcinoma wasdetected with terminal deoxynucl neotidyl transferasemediated dUTP nick end labelling (TUNEL); the cancer cellscultured in DMED medium were treated with extract ofSolanum lyratum Thumb and observed under microscope,and their DNA was assayed by gel electrophoresis.RESULTS: In vivo apoptotic cells in the cancer adjacenttissues inceased; in vitro treatment of liver cancers withextract of Solanum lyratum Thumb could induce the cells tomanifest a typical apoptotic morphology. Their DNA wasfractured and a characteristic ladder pattem could be foundusing electrophoresis.CONCLUSION: In vivo the apoptosis of carcinomas waslower; maybe the cells divided quickly and then the cancersoccurred. In the cancer adjacent tissues, the apoptosispricked up, and in vitro Solarium lyratum Thumb couldinduce the apoptosis of BEL-7404 cells.

  2. APOPTOSIS AFTER SPINAL CORD INJURY IN RATS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To confirm the role played by apoptosis in spinal cord injury. Methods 36 rats models of spinal cord injury were made by Allen method. Histological examinations using HE staining and in situ end-labeling were used to observe apoptosis in spinal cord tissues from 1h to 21d after injury. Results HE staining sections showed hemorrhage and necrosis, neuronal degeneration and gliai cell proliferation. In situ end-labeling sections showed the appearance of apoptosis in both gray and white matter as well as in both central and surrounding region. The number of apoptotic cells increased from 12h after injury, increased to the peak at 4d and declined to normal at 21d. Conclu sion The results suggest that apoptosis, especially glial apoptosis, plays a role in the pathogenesis of spinal cord in jury.

  3. Cancer Therapy Due to Apoptosis: Galectin-9

    Directory of Open Access Journals (Sweden)

    Koji Fujita

    2017-01-01

    Full Text Available Dysregulation of apoptosis is a major hallmark in cancer biology that might equip tumors with a higher malignant potential and chemoresistance. The anti-cancer activities of lectin, defined as a carbohydrate-binding protein that is not an enzyme or antibody, have been investigated for over a century. Recently, galectin-9, which has two distinct carbohydrate recognition domains connected by a linker peptide, was noted to induce apoptosis in thymocytes and immune cells. The apoptosis of these cells contributes to the development and regulation of acquired immunity. Furthermore, human recombinant galectin-9, hG9NC (null, which lacks an entire region of the linker peptide, was designed to resist proteolysis. The hG9NC (null has demonstrated anti-cancer activities, including inducing apoptosis in hematological, dermatological and gastrointestinal malignancies. In this review, the molecular characteristics, history and apoptosis-inducing potential of galectin-9 are described.

  4. Autophagy and apoptosis: where do they meet?

    Science.gov (United States)

    Mukhopadhyay, Subhadip; Panda, Prashanta Kumar; Sinha, Niharika; Das, Durgesh Nandini; Bhutia, Sujit Kumar

    2014-04-01

    Autophagy and apoptosis are two important cellular processes with complex and intersecting protein networks; as such, they have been the subjects of intense investigation. Recent advances have elucidated the key players and their molecular circuitry. For instance, the discovery of Beclin-1's interacting partners has resulted in the identification of Bcl-2 as a central regulator of autophagy and apoptosis, which functions by interacting with both Beclin-1 and Bax/Bak respectively. When localized to the endoplasmic reticulum and mitochondria, Bcl-2 inhibits autophagy. Cellular stress causes the displacement of Bcl-2 from Beclin-1 and Bax, thereby triggering autophagy and apoptosis, respectively. The induction of autophagy or apoptosis results in disruption of complexes by BH3-only proteins and through post-translational modification. The mechanisms linking autophagy and apoptosis are not fully defined; however, recent discoveries have revealed that several apoptotic proteins (e.g., PUMA, Noxa, Nix, Bax, XIAP, and Bim) modulate autophagy. Moreover, autophagic proteins that control nucleation and elongation regulate intrinsic apoptosis through calpain- and caspase-mediated cleavage of autophagy-related proteins, which switches the cellular program from autophagy to apoptosis. Similarly, several autophagic proteins are implicated in extrinsic apoptosis. This highlights a dual cellular role for autophagy. On one hand, autophagy degrades damaged mitochondria and caspases, and on the other hand, it provides a membrane-based intracellular platform for caspase processing in the regulation of apoptosis. In this review, we highlight the crucial factors governing the crosstalk between autophagy and apoptosis and describe the mechanisms controlling cell survival and cell death.

  5. A new approach to comparing anti-CD20 antibodies: importance of the lipid rafts in their lytic efficiency

    Directory of Open Access Journals (Sweden)

    Mariam Hammadi

    2010-06-01

    Full Text Available Mariam Hammadi, Jacques-Olivier Pers, Christian Berthou, Pierre Youinou, Anne BordronCentre Hospitalier Universitaire EA2216 and IFR148, Université de Bretagne Occidentale and Université Européenne de Bretagne, BP824, 29609 Brest cedex, FranceAbstract: The view that B lymphocytes are pathogenic in diverse pathological settings is supported by the efficacy of B-cell-ablative therapy in lymphoproliferative disorders, autoimmune diseases and graft rejection. Anti-B-cell antibodies (Abs directed against CD20 have therefore been generated, and of these, rituximab was the first anti-CD20 monoclonal Ab (mAb to be applied. Rituximab-mediated apoptosis, complement-dependent cytotoxicity and Ab-dependent cellular cytotoxicity differ from one disease to another, and, for the same disease, from one patient to another. This knowledge has prompted the development of new anti-CD20 mAbs in the hope of improving B-cell depletion. The inclusion of CD20/anti-CD20 complexes in large lipid rafts (LRs enhances the results of some, but not all, anti-CD20 mAbs, and it may be possible to include smaller LRs. Lipid contents of membrane may be abnormal in malignant B-cells, and could explain resistance to treatment. The function of these mAbs and the importance of LRs warrant further investigation. A detailed understanding of them will increase results for B-cell depletion in lymphoproliferative diseases.Keywords: anti-CD20 antibodies, lymphocyte B, lipid rafts, B-cell disorders

  6. Posttraumatic Chondrocyte Apoptosis in the Murine Xiphoid

    Science.gov (United States)

    Davis, Christopher G.; Eisner, Eric; McGlynn, Margaret; Shelton, John M.; Richardson, James

    2013-01-01

    Objective. To demonstrate posttraumatic chondrocyte apoptosis in the murine xiphoid after a crush-type injury and to ultimately determine the pathway (i.e., intrinsic or extrinsic) by which chondrocytes undergo apoptosis in response to mechanical injury. Design. The xiphoids of adult female wild-type mice were injured with the use of a modified Kelly clamp. Postinjury xiphoid cartilage was analyzed via 3 well-described independent means of assessing apoptosis in chondrocytes: hematoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and activated caspase-3 staining. Results. Injured specimens contained many chondrocytes with evidence of apoptosis, which is characterized by cell shrinkage, chromatin condensation, nuclear fragmentation, and the liberation of apoptotic bodies. There was a statistically significant increase in the number of chondrocytes undergoing apoptosis in the injured specimens as compared with the uninjured specimens. Conclusions. Chondrocytes can be stimulated to undergo apoptosis as a result of mechanical injury. These experiments involving predominantly cartilaginous murine xiphoid in vivo establish a baseline for future investigations that employ the genetic and therapeutic modulation of chondrocyte apoptosis in response to mechanical injury. PMID:26069679

  7. Cytochrome c and insect cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    Kai-Yu Liu; Hong Yang; Jian-Xin Peng; Hua-Zhu Hong

    2012-01-01

    The role ofcytochrome c in insect cell apoptosis has drawn considerable attention and has been subject to considerable controversy.In Drosophila,the majority of studies have demonstrated that cytochrome c may not be involved in apoptosis,although there are conflicting reports.Cytochrome c is not released from mitochondria into the cytosol and activation of the initiator caspase Dronc or effector caspase Drice is not associated with cytochrome c during apoptosis in Drosophila SL2 cells or BG2 cells.Cytochrome c failed to induce caspase activation and promote caspase activation in Drosophila cell lysates,but remarkably caused caspase activation in extracts from human cells.Knockdown of cytochrome c does not protect cells from apoptosis and over-expression of cytochrome c also does not promote apoptosis.Structural analysis has revealed that cytochrome c is not required for Dapaf-1 complex assembly.In Lepidoptera,the involvement of cytochrome c in apoptosis has been demonstrated by the accumulating evidence.Cytochrome c release from mitochondria into cytosol has been observed in different cell lines such as Spodoptera frugiperda Sf9,Spodoptera litura S1-1 and Lymantria dispar LdFB.Silencing of cytochrome c expression significantly affected apoptosis and activation of caspase and the addition of cytochrome c to cell-free extracts results in caspase activation,suggesting the activation of caspase is dependent on cytochrome c.Although Apaf- 1 has not been identified in Lepidoptera,the inhibitor of apoptosome formation can inhibit apoptosis and caspase activation.Cytochrome c may be exclusively required for Lepidoptera apoptosis.

  8. Metadherin facilitates podocyte apoptosis in diabetic nephropathy

    Science.gov (United States)

    Liu, Wen-Ting; Peng, Fen-Fen; Li, Hong-Yu; Chen, Xiao-Wen; Gong, Wang-Qiu; Chen, Wen-Jing; Chen, Yi-Hua; Li, Pei-Lin; Li, Shu-Ting; Xu, Zhao-Zhong; Long, Hai-Bo

    2016-01-01

    Apoptosis, one of the major causes of podocyte loss, has been reported to have a vital role in diabetic nephropathy (DN) pathogenesis, and understanding the mechanisms underlying the regulation of podocyte apoptosis is crucial. Metadherin (MTDH) is an important oncogene, which is overexpressed in most cancers and responsible for apoptosis, metastasis, and poor patient survival. Here we show that the expression levels of Mtdh and phosphorylated p38 mitogen-activated protein kinase (MAPK) are significantly increased, whereas those of the microRNA-30 family members (miR-30s) are considerably reduced in the glomeruli of DN rat model and in high glucose (HG)-induced conditionally immortalized mouse podocytes (MPC5). These levels are positively correlated with podocyte apoptosis rate. The inhibition of Mtdh expression, using small interfering RNA, but not Mtdh overexpression, was shown to inhibit HG-induced MPC5 apoptosis and p38 MAPK pathway, and Bax and cleaved caspase 3 expression. This was shown to be similar to the effects of p38 MAPK inhibitor (SB203580). Furthermore, luciferase assay results demonstrated that Mtdh represents the target of miR-30s. Transient transfection experiments, using miR-30 microRNA (miRNA) inhibitors, led to the increase in Mtdh expression and induced the apoptosis of MPC5, whereas the treatment with miR-30 miRNA mimics led to the reduction in Mtdh expression and apoptosis of HG-induced MPC5 cells in comparison with their respective controls. Our results demonstrate that Mtdh is a potent modulator of podocyte apoptosis, and that it represents the target of miR-30 miRNAs, facilitating podocyte apoptosis through the activation of HG-induced p38 MAPK-dependent pathway. PMID:27882943

  9. Hypoxia-inducible factor-1α plays roles in Epstein-Barr virus's natural life cycle and tumorigenesis by inducing lytic infection through direct binding to the immediate-early BZLF1 gene promoter.

    Science.gov (United States)

    Kraus, Richard J; Yu, Xianming; Cordes, Blue-Leaf A; Sathiamoorthi, Saraniya; Iempridee, Tawin; Nawandar, Dhananjay M; Ma, Shidong; Romero-Masters, James C; McChesney, Kyle G; Lin, Zhen; Makielski, Kathleen R; Lee, Denis L; Lambert, Paul F; Johannsen, Eric C; Kenney, Shannon C; Mertz, Janet E

    2017-06-01

    When confronted with poor oxygenation, cells adapt by activating survival signaling pathways, including the oxygen-sensitive transcriptional regulators called hypoxia-inducible factor alphas (HIF-αs). We report here that HIF-1α also regulates the life cycle of Epstein-Barr virus (EBV). Incubation of EBV-positive gastric carcinoma AGS-Akata and SNU-719 and Burkitt lymphoma Sal and KemIII cell lines with a prolyl hydroxylase inhibitor, L-mimosine or deferoxamine, or the NEDDylation inhibitor MLN4924 promoted rapid and sustained accumulation of both HIF-1α and lytic EBV antigens. ShRNA knockdown of HIF-1α significantly reduced deferoxamine-mediated lytic reactivation. HIF-1α directly bound the promoter of the EBV primary latent-lytic switch BZLF1 gene, Zp, activating transcription via a consensus hypoxia-response element (HRE) located at nt -83 through -76 relative to the transcription initiation site. HIF-1α did not activate transcription from the other EBV immediate-early gene, BRLF1. Importantly, expression of HIF-1α induced EBV lytic-gene expression in cells harboring wild-type EBV, but not in cells infected with variants containing base-pair substitution mutations within this HRE. Human oral keratinocyte (NOK) and gingival epithelial (hGET) cells induced to differentiate by incubation with either methyl cellulose or growth in organotypic culture accumulated both HIF-1α and Blimp-1α, another cellular factor implicated in lytic reactivation. HIF-1α activity also accumulated along with Blimp-1α during B-cell differentiation into plasma cells. Furthermore, most BZLF1-expressing cells observed in lymphomas induced by EBV in NSG mice with a humanized immune system were located distal to blood vessels in hypoxic regions of the tumors. Thus, we conclude that HIF-1α plays central roles in both EBV's natural life cycle and EBV-associated tumorigenesis. We propose that drugs that induce HIF-1α protein accumulation are good candidates for development of a lytic

  10. MicroRNA-1 promotes apoptosis of hepatocarcinoma cells by targeting apoptosis inhibitor-5 (API-5).

    Science.gov (United States)

    Li, Dong; Liu, Yu; Li, Hua; Peng, Jing-Jing; Tan, Yan; Zou, Qiang; Song, Xiao-Feng; Du, Min; Yang, Zheng-Hui; Tan, Yong; Zhou, Jin-Jun; Xu, Tao; Fu, Zeng-Qiang; Feng, Jian-Qiong; Cheng, Peng; chen, Tao; Wei, Dong; Su, Xiao-Mei; Liu, Huan-Yi; Qi, Zhong-Chun; Tang, Li-Jun; Wang, Tao; Guo, Xin; Hu, Yong-He; Zhang, Tao

    2015-01-02

    Although microRNA-1 (miR-1) is a known liver cancer suppressor, the role of miR-1 in apoptosis of hepatoma cells has remained largely unknown. Our study shows that ectopic miR-1 overexpression induced apoptosis of liver hepatocellular carcinoma (HepG2) cells. Apoptosis inhibitor 5 (API-5) was found to be a potential regulator of miR-1 induced apoptosis, using a bioinformatics approach. Furthermore, an inverse relationship between miR-1 and API-5 expression was observed in human liver cancer tissues and adjacent normal liver tissues. Negative regulation of API-5 expression by miR-1 was demonstrated to promote apoptosis of HepG2 cells. Our study provides a novel regulatory mechanism of miR-1 in the apoptosis of hepatoma cells.

  11. Inhibition of Reaper-induced apoptosis by interaction with inhibitor of apoptosis proteins (IAPs)

    OpenAIRE

    1997-01-01

    IAPs comprise a family of inhibitors of apoptosis found in viruses and animals. In vivo binding studies demonstrated that both baculovirus and Drosophila IAPs physically interact with an apoptosis-inducing protein of Drosophila, Reaper (RPR), through their baculovirus IAP repeat (BIR) region. Expression of IAPs blocked RPR-induced apoptosis and resulted in the accumulation of RPR in punctate perinuclear locations which coincided with IAP localization. When expressed alone, RPR rapidly disappe...

  12. Targeting apoptosis in acute tubular injury.

    Science.gov (United States)

    Ortiz, Alberto; Justo, Pilar; Sanz, Ana; Lorz, Corina; Egido, Jesús

    2003-10-15

    Recent research has shown that apoptosis and its regulatory mechanisms contribute to cell number regulation in acute renal failure. Acute tubular necrosis is the most frequent form of parenchymal acute renal failure. The main causes are ischemia-reperfusion, sepsis and nephrotoxic drugs. Exogenous factors such as nephrotoxic drugs and bacterial products, and endogenous factors such as lethal cytokines promote tubular cell apoptosis. Such diverse stimuli engage intracellular death pathways that in some cases are stimulus-specific. We now review the role of apoptosis in acute renal failure, the potential molecular targets of therapeutic intervention, the therapeutic weapons to modulate the activity of these targets and the few examples of therapeutic intervention on apoptosis.

  13. [Apoptosis and thymocyte development (epithelial cells as inducers of thymocyte apoptosis)].

    Science.gov (United States)

    Iarilin, A A; Bulanova, E G; Sharova, N I; Budagian, V M

    1998-01-01

    Apoptosis, together with proliferation, is a main factor of selection of the clones of developing T-lymphocytes: the clones not supported by positive selection are subject to apoptosis and apoptosis accounts for discarding of potentially autoaggressive clones, i.e., for negative selection in the thymus and peripheral lymphoid tissue. Realization of apoptosis at different stages of the development of T-lymphocytes depends to a varying extent on Fas, Bcl-2, p53, and other regulators. The dendritic cells are the main cell type, the contact with determines apoptosis of T-lymphocytes. A possible role of the epithelial cells was shown in few models (on murine cells) and was not practically studied. We obtained a line of epithelial cells of the human thymus cells HTSC, cocultivation with which induces apoptosis of immature thymocytes and blood T-cells activated by mitogens. Development of apoptosis is suppressed by inhibitors of protein and RNA synthesis, chelators Ca2+, ions Zn2+, and factors destroying the cytoskeleton components. In this model, interaction of pairs of molecules CD4-HLA class II and LFA-1-ICAM-1. When in contact with the HTSC cells, the thymocytes of mice mutant for Fas-receptor (line MRL.lpr) are subject to apoptosis, but when this receptor is present, it affects the development of apoptosis.

  14. Research of BH3 domain protein inducing cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    FENG Wan-yu; LIU Yang; ZHANG Zhi-cheng

    2008-01-01

    Objective BH3 domain protein plays an important role in control mechanism of cell apoptosis. The article mainly discusses its mechanism of promoting cell apoptosis and control. Methods The article analyzed and evaluated the mechanism of BH3 domain protein promoting cell apoptosis by internal and overseas literature. Results Activation of BH3 domain protein could promote the increase of mitochondrial membrane permeability, then it would start mitoehondrial apoptosis pathway, and at the last the cell apoptosis. Conclusions BH3 domain protein is the necessary condition of starting cell apoptosis. Its activation can cause cell apoptosis.

  15. Apoptosis in amphibian organs during metamorphosis

    Science.gov (United States)

    Ishizuya-Oka, Atsuko; Hasebe, Takashi; Shi, Yun-Bo

    2012-01-01

    During amphibian metamorphosis, the larval tissues/organs rapidly degenerate to adapt from the aquatic to the terrestrial life. At the cellular level, a large quantity of apoptosis occurs in a spatiotemporally-regulated fashion in different organs to ensure timely removal of larval organs/tissues and the development of adult ones for the survival of the individuals. Thus, amphibian metamorphosis provides us a good opportunity to understand the mechanisms regulating apoptosis. To investigate this process at the molecular level, a number of thyroid hormone (TH) response genes have been isolated from several organs of Xenopus laevis tadpoles and their expression and functional analyses are now in progress using modern molecular and genetic technologies. In this review, we will first summarize when and where apoptosis occurs in typical larva-specific and larval-to-adult remodeling amphibian organs to highlight that the timing of apoptosis is different in different tissues/organs, even though all are induced by the same circulating TH. Next, to discuss how TH spatiotemporally regulates the apoptosis, we will focus on apoptosis of the X. laevis small intestine, one of the best characterized remodeling organs. Functional studies of TH response genes using transgenic frogs and culture techniques have shown that apoptosis of larval epithelial cells can be induced by TH either cell-autonomously or indirectly through interactions with extracellular matrix (ECM) components of the underlying basal lamina. Here, we propose that multiple intra- and extracellular apoptotic pathways are coordinately controlled by TH to ensure massive but well-organized apoptosis, which is essential for the proper progression of amphibian metamorphosis. PMID:20238476

  16. Effect of sevoflurane on human neutrophil apoptosis.

    LENUS (Irish Health Repository)

    Tyther, R

    2012-02-03

    BACKGROUND AND OBJECTIVE: Both chronic occupational exposure to volatile anaesthetic agents and acute in vitro exposure of neutrophils to isoflurane have been shown to inhibit the rate of apoptosis of human neutrophils. It is possible that inhibition of neutrophil apoptosis arises through delaying mitochondrial membrane potential collapse. We assessed mitochondrial depolarization and apoptosis in unexposed neutrophils and neutrophils exposed to sevoflurane in vivo. METHODS: A total of 20 mL venous blood was withdrawn pre- and postinduction of anaesthesia, the neutrophils isolated and maintained in culture. At 1, 12 and 24 h in culture, the percentage of neutrophil apoptosis was assessed by dual staining with annexin V-FITC and propidium iodide. Mitochondrial depolarization was measured using the dual emission styryl dye JC-1. RESULTS: Apoptosis was significantly inhibited in neutrophils exposed to sevoflurane in vivo at 24 (exposed: 38 (12)% versus control: 28 (11)%, P = 0.001), but not at 1 or 12 h, in culture. Mitochondrial depolarization was not delayed in neutrophils exposed to sevoflurane. CONCLUSIONS: The most important findings are that sevoflurane inhibits neutrophil apoptosis in vivo and that inhibition is not mediated primarily by an effect on mitochondrial depolarization.

  17. [Protein kinase C activation induces platelet apoptosis].

    Science.gov (United States)

    Zhao, Li-Li; Chen, Meng-Xing; Zhang, Ming-Yi; Dai, Ke-Sheng

    2013-10-01

    Platelet apoptosis elucidated by either physical or chemical compound or platelet storage occurs wildly, which might play important roles in controlling the numbers and functions of circulated platelets, or in the development of some platelet-related diseases. However, up to now, a little is known about the regulatory mechanisms of platelet apoptosis. Protein kinase C (PKC) is highly expressed in platelets and plays central roles in regulating platelet functions. Although there is evidence indicating that PKC is involved in the regulation of apoptosis of nucleated cells, it is still unclear whether PKC plays a role in platelet apoptosis. The aim of this study was to investigate the role of PKC in platelet apoptosis. The effects of PKC on mitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) exposure, and caspase-3 activation of platelets were analyzed by flow cytometry and Western blot. The results showed that the ΔΨm depolarization in platelets was induced by PKC activator in time-dependent manner, and the caspase-3 activation in platelets was induced by PKC in concentration-dependent manner. However, the platelets incubated with PKC inhibitor did not results in ΔΨm depolarization and PS exposure. It is concluded that the PKC activation induces platelet apoptosis through influencing the mitochondrial functions and activating caspase 3. The finds suggest a novel mechanism for PKC in regulating platelet numbers and functions, which has important pathophysiological implications for thrombosis and hemostasis.

  18. Epithelial Cell Apoptosis and Lung Remodeling

    Institute of Scientific and Technical Information of China (English)

    Kazuyoshi Kuwano

    2007-01-01

    Lung epithelium is the primary site of lung damage in various lung diseases. Epithelial cell apoptosis has been considered to be initial event in various lung diseases. Apoptosis signaling is classically composed of two principle pathways. One is a direct pathway from death receptor ligation to caspase cascade activation and cell death. The other pathway triggered by stresses such as drugs, radiation, infectious agents and reactive oxygen species is mediated by mitochondria. Endoplasmic reticulum has also been shown to be the organelle to mediate apoptosis.Epithelial cell death is followed by remodeling processes, which consist of epithelial and fibroblast activation,cytokine production, activation of coagulation pathway, neoangiogenesis, re-epithelialization and fibrosis.Epithelial and mesenchymal interaction plays important roles in these processes. Further understanding of apoptosis signaling and its regulation by novel strategies may lead to effective treatments against various lung diseases. We review the recent advances in the understanding of apoptosis signaling and discuss the involvement of apoptosis in lung remodeling.

  19. Host-pathogen interactions during apoptosis

    Indian Academy of Sciences (India)

    Seyed E Hasnain; Rasheeda Begum; K V A Ramaiah; Sudhir Sahdev; E M Shajil; Tarvinder K Taneja; Manjari Mohan; M Athar; Nand K Sah; M Krishnaveni

    2003-04-01

    Host pathogen interaction results in a variety of responses, which include phagocytosis of the pathogen, release of cytokines, secretion of toxins, as well as production of reactive oxygen species (ROS). Recent studies have shown that many pathogens exert control on the processes that regulate apoptosis in the host. The induction of apoptosis upon infection results from a complex interaction of parasite proteins with cellular host proteins. Abrogation of host cell apoptosis is often beneficial for the pathogen and results in a successful host invasion. However, in some cases, it has been shown that induction of apoptosis in the infected cells significantly imparts protection to the host from the pathogen. There is a strong correlation between apoptosis and the host protein translation machinery: the pathogen makes all possible efforts to modify this process so as to inhibit cell suicide and ensure that it can survive and, in some cases, establish latent infection. This review discusses the significance of various pathways/steps during virus-mediated modulation of host cell apoptosis.

  20. Molecular Mechanism of Apoptosis and Necrosis

    Directory of Open Access Journals (Sweden)

    Gulfidan Coskun

    2011-06-01

    Full Text Available Organismal homeostasis depends on an intricate balance between cell death and renewal. Apoptosis is a process of programmed cell death that plays a critical role in some normal and pathologic conditions beginning from embryologic development and ends at death. Apoptosis is initiated by morphological changes at the cell membrane, surface organels and nucleus. Apoptosis starts with death signals coming from outside or inside of the cell and continue to activate the mechanisms of apoptosis via cell death receptor or mitochondrial pathways. During apoptosis a group proteases are activated which cause DNA fragmentation, cytoplasmic shrinkage and membrane blebbing. Apoptotic cells divide into apoptotic bodies and then these apoptotic bodies are removed from tissue by phagocytes and adjacent cells In contrast to the “programmed” nature of apoptosis, necrotic cell death has always been believed to be a random, uncontrolled process that leads to death of the cell. Also necrosis, which is an other type of cell death, came to be used to describe pathologic cell death which cause inflamation. [Archives Medical Review Journal 2011; 20(3.000: 145-158

  1. Epstein-Barr virus interactions with the Bcl-2 protein family and apoptosis in human tumor cells

    Institute of Scientific and Technical Information of China (English)

    Qin FU; Chen HE; Zheng-rong MAO

    2013-01-01

    Epstein-Barr virus (EBV),a human gammaherpesvirus carried by more than 90% of the world's population,is associated with malignant tumors such as Burkitt's lymphoma (BL),Hodgkin lymphoma,post-transplant lymphoma,extra-nodal natural killer/T cell lymphoma,and nasopharyngeal and gastric carcinomas in immune-compromised patients.In the process of infection,EBV faces challenges:the host cell environment is harsh,and the survival and apoptosis of host cells are precisely regulated.Only when host cells receive sufficient survival signals may they immortalize.To establish efficiently a lytic or long-term latent infection,EBV must escape the host cell immunologic mechanism and resist host cell apoptosis by interfering with multiple signaling pathways.This review details the apoptotic pathway disrupted by EBV in EBV-infected cells and describes the interactions of EBV gene products with host cellular factors as well as the function of these factors,which decide the fate of the host cell.The relationships between other EBV-encoded genes and proteins of the B-cell leukemia/lymphoma (Bcl) family are unknown.Still,EBV seems to contribute to establishing its own latency and the formation of tumors by modifying events that impact cell survival and proliferation as well as the immune response of the infected host.We discuss potential therapeutic drugs to provide a foundation for further studies of tumor pathogenesis aimed at exploiting novel therapeutic strategies for EBV-associated diseases.

  2. Exo-exo synergy between Cel6A and Cel7A from Hypocrea jecorina: Role of carbohydrate binding module and the endo-lytic character of the enzymes.

    Science.gov (United States)

    Badino, Silke F; Christensen, Stefan J; Kari, Jeppe; Windahl, Michael S; Hvidt, Søren; Borch, Kim; Westh, Peter

    2017-08-01

    Synergy between cellulolytic enzymes is essential in both natural and industrial breakdown of biomass. In addition to synergy between endo- and exo-lytic enzymes, a lesser known but equally conspicuous synergy occurs among exo-acting, processive cellobiohydrolases (CBHs) such as Cel7A and Cel6A from Hypocrea jecorina. We studied this system using microcrystalline cellulose as substrate and found a degree of synergy between 1.3 and 2.2 depending on the experimental conditions. Synergy between enzyme variants without the carbohydrate binding module (CBM) and its linker was strongly reduced compared to the wild types. One plausible interpretation of this is that exo-exo synergy depends on the targeting role of the CBM. Many earlier works have proposed that exo-exo synergy was caused by an auxiliary endo-lytic activity of Cel6A. However, biochemical data from different assays suggested that the endo-lytic activity of both Cel6A and Cel7A were 10(3) -10(4) times lower than the common endoglucanase, Cel7B, from the same organism. Moreover, the endo-lytic activity of Cel7A was 2-3-fold higher than for Cel6A, and we suggest that endo-like activity of Cel6A cannot be the main cause for the observed synergy. Rather, we suggest the exo-exo synergy found here depends on different specificities of the enzymes possibly governed by their CBMs. Biotechnol. Bioeng. 2017;114: 1639-1647. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  3. Technological application of an extracellular cell lytic enzyme in xanthan gum clarification Aplicação tecnológica de uma enzima celulolítica para clarificação de goma xantana

    Directory of Open Access Journals (Sweden)

    Suresh Shastry

    2005-03-01

    Full Text Available An extracellular cell lytic enzyme from Pseudomonas sp. was active on heat killed cells of Xanthomonas campestris. The lytic activity caused enzymatic digestion of X.campestris xanthan gum. Digestion was effective for highly viscous native xanthan 2.0% (w/v and 2.5% (w/v commercial Sigma xanthan. Scanning electron microscopy and SDS-PAGE observations confirmed the cell lytic action on X.campestris cells.Uma enzima extracelular celulolítica produzida por Pseudomonas sp. foi ativa sobre células de Xanthomonas campestris mortas pelo calor. A atividade lítica causou a digestão enzimática de goma xantana de X. campestris. A digestão foi eficiente tanto para xantana nativa altamante viscosa (2,0% w/v como para xantana comercial Sigma (2,5% w/v. Observações por microscopia eletrônica de varredura demonstraram a ação celulolítica sobre células de X. campestris.

  4. Evidence of a dominant lineage of Vibrio cholerae-specific lytic bacteriophages shed by cholera patients over a 10-year period in Dhaka, Bangladesh.

    Science.gov (United States)

    Seed, Kimberley D; Bodi, Kip L; Kropinski, Andrew M; Ackermann, Hans-Wolfgang; Calderwood, Stephen B; Qadri, Firdausi; Camilli, Andrew

    2011-01-01

    Lytic bacteriophages are hypothesized to contribute to the seasonality and duration of cholera epidemics in Bangladesh. However, the bacteriophages contributing to this phenomenon have yet to be characterized at a molecular genetic level. In this study, we isolated and sequenced the genomes of 15 bacteriophages from stool samples from cholera patients spanning a 10-year surveillance period in Dhaka, Bangladesh. Our results indicate that a single novel bacteriophage type, designated ICP1 (for the International Centre for Diarrhoeal Disease Research, Bangladesh cholera phage 1) is present in all stool samples from cholera patients, while two other bacteriophage types, one novel (ICP2) and one T7-like (ICP3), are transient. ICP1 is a member of the Myoviridae family and has a 126-kilobase genome comprising 230 open reading frames. Comparative sequence analysis of ICP1 and related isolates from this time period indicates a high level of genetic conservation. The ubiquitous presence of ICP1 in cholera patients and the finding that the O1 antigen of lipopolysaccharide (LPS) serves as the ICP1 receptor suggest that ICP1 is extremely well adapted to predation of human-pathogenic V. cholerae O1.

  5. Unique functional properties of conserved arginine residues in the lentivirus lytic peptide domains of the C-terminal tail of HIV-1 gp41.

    Science.gov (United States)

    Kuhlmann, Anne-Sophie; Steckbeck, Jonathan D; Sturgeon, Timothy J; Craigo, Jodi K; Montelaro, Ronald C

    2014-03-14

    A previous study from our laboratory reported a preferential conservation of arginine relative to lysine in the C-terminal tail (CTT) of HIV-1 envelope (Env). Despite substantial overall sequence variation in the CTT, specific arginines are highly conserved in the lentivirus lytic peptide (LLP) motifs and are scarcely substituted by lysines, in contrast to gp120 and the ectodomain of gp41. However, to date, no explanation has been provided to explain the selective incorporation and conservation of arginines over lysines in these motifs. Herein, we address the functions in virus replication of the most conserved arginines by performing conservative mutations of arginine to lysine in the LLP1 and LLP2 motifs. The presence of lysine in place of arginine in the LLP1 motif resulted in significant impairment of Env expression and consequently virus replication kinetics, Env fusogenicity, and incorporation. By contrast, lysine exchanges in LLP2 only affected the level of Env incorporation and fusogenicity. Our findings demonstrate that the conservative lysine substitutions significantly affect Env functional properties indicating a unique functional role for the highly conserved arginines in the LLP motifs. These results provide for the first time a functional explanation to the preferred incorporation of arginine, relative to lysine, in the CTT of HIV-1 Env. We propose that these arginines may provide unique functions for Env interaction with viral or cellular cofactors that then influence overall Env functional properties.

  6. The structure of a LysM domain from E. coli membrane-bound lytic murein transglycosylase D (MltD).

    Science.gov (United States)

    Bateman, A; Bycroft, M

    2000-06-16

    The LysM domain is a widespread protein module. It was originally identified in enzymes that degrade bacterial cell walls but is also present in many other bacterial proteins. Several proteins that contain the domain, such as Staphylococcal IgG binding proteins and Escherichia coli intimin, are involved in bacterial pathogenesis. LysM domains are also found in some eukaryotic proteins, apparently as a result of horizontal gene transfer from bacteria. The available evidence suggests that the LysM domain is a general peptidoglycan-binding module. We have determined the structure of this domain from E. coli membrane-bound lytic murein transglycosylase D. The LysM domain has a betaalphaalphabeta secondary structure with the two helices packing onto the same side of an anti- parallel beta sheet. The structure shows no similarity to other bacterial cell surface domains. A potential binding site in a shallow groove on surface of the protein has been identified. Copyright 2000 Academic Press.

  7. Disruption of the cell wall lytic enzyme CwlO affects the amount and molecular size of poly-γ-glutamic acid produced by Bacillus subtilis (natto).

    Science.gov (United States)

    Mitsui, Nobuo; Murasawa, Hisashi; Sekiguchi, Junichi

    2011-01-01

    Poly-γ-glutamic acid (γPGA), a polymer of glutamic acid, is a component of the viscosity substance of natto, a traditional Japanese food made from soybeans fermented with Bacillus subtilis (natto). Here we investigate the effects of the cell wall lytic enzymes belonging to the D,L-endopeptidases (LytE, LytF, CwlO and CwlS) on γPGA production by B. subtilis (natto). γPGA levels in a cwlO disruptant were about twofold higher than that of the wild-type strain, whereas disruption of the lytE, lytF and cwlS genes had little effect on γPGA production. The molecular size of γPGA in the cwlO disruptant was larger than that of the wild-type strain. A complementary strain was constructed by insertion of the entire cwlO gene into the amyE locus of the CwlO mutant genome, and γPGA production was restored to wild-type levels in this complementary strain. These results indicated that the peptidoglycan degradation enzyme, CwlO, plays an important role in γPGA production and affects the molecular size of γPGA.

  8. Isolation and characterization of φkm18p, a novel lytic phage with therapeutic potential against extensively drug resistant Acinetobacter baumannii.

    Directory of Open Access Journals (Sweden)

    Gwan-Han Shen

    Full Text Available AIMS: To isolate phages against extensively drug resistant Acinetobacter baumannii (XDRAB and characterize the highest lytic capability phage as a model to evaluate the potential on phage therapy. METHODS AND RESULTS: Eight phages were isolated from hospital sewage and showed narrow host spectrum. Phage φkm18p was able to effectively lyse the most XDRAB. It has a dsDNA genome of 45 kb in size and hexagonal head of about 59 nm in diameter and no tail. Bacterial population decreased quickly from 10(8 CFU ml(-1 to 10(3 CFU ml(-1 in 30 min by φkm18p. The 185 kDa lysis protein encoded by φkm18p genome was detected when the extracted protein did not boil before SDS-PAGE; it showed that the lysis protein is a complex rather than a monomer. Phage φkm18p improved human lung epithelial cells survival rates when they were incubated with A. baumannii. Combination of phages (φkm18p, φTZ1 and φ314 as a cocktail could lyse all genotype-varying XDRAB isolates. CONCLUSION: Infections with XDRAB are extremely difficult to treat and development of a phage cocktails therapy could be a therapeutic alternative in the future. Phage φkm18p is a good candidate for inclusion in phage cocktails.

  9. The bacteriophage P1 hot gene, encoding a homolog of the E. coli DNA polymerase III theta subunit, is expressed during both lysogenic and lytic growth stages.

    Science.gov (United States)

    Chikova, Anna K; Schaaper, Roel M

    2007-11-01

    The bacteriophage P1 hot gene product is a homolog of the theta subunit of E. coli DNA polymerase III. Previous studies with hot cloned on a plasmid have shown that Hot protein can substitute for theta, as evidenced by its stabilizing effect on certain dnaQ mutator mutants carrying an unstable pol III proofreading subunit (epsilon subunit). These results are consistent with Hot, like theta, being a replication protein involved in stabilizing the intrinsically unstable epsilon proofreading function. However, the function of hot for the viral life cycle is less clear. In the present study, we show that the hot gene is not essential. Based on its promoter structure, hot has been previously classified as a "late" phage gene, a property that is not easily reconciled with a presumed replication function. Here, we clarify this issue by demonstrating that P1 hot is actively expressed both during the lysogenic state and in the early stages of a lytic induction, in addition to its expression in the late stage of phage development. The results indicate that P1 hot has a complex expression pattern, compatible with a model in which Hot may affect the host replication machinery to benefit overall phage replication.

  10. Crystallization of a fungal lytic polysaccharide monooxygenase expressed from glycoengineered Pichia pastoris for X-ray and neutron diffraction

    Energy Technology Data Exchange (ETDEWEB)

    O' Dell, William B.; Swartz, Paul D.; Weiss, Kevin L.; Meilleur, Flora

    2017-01-19

    Lytic polysaccharide monooxygenases (LPMOs) are carbohydrate-disrupting enzymes secreted by bacteria and fungi that break glycosidic bondsviaan oxidative mechanism. Fungal LPMOs typically act on cellulose and can enhance the efficiency of cellulose-hydrolyzing enzymes that release soluble sugars for bioethanol production or other industrial uses. The enzyme PMO-2 fromNeurospora crassa(NcPMO-2) was heterologously expressed inPichia pastoristo facilitate crystallographic studies of the fungal LPMO mechanism. Diffraction resolution and crystal morphology were improved by expressingNcPMO-2 from a glycoengineered strain ofP. pastorisand by the use of crystal seeding methods, respectively. These improvements resulted in high-resolution (1.20 Å) X-ray diffraction data collection at 100 K and the production of a largeNcPMO-2 crystal suitable for room-temperature neutron diffraction data collection to 2.12 Å resolution.

  11. Identification of lytic bacteriophage MmP1, assigned to a new member of T7-like phages infecting Morganella morganii.

    Science.gov (United States)

    Zhu, Junmin; Rao, Xiancai; Tan, Yinling; Xiong, Kun; Hu, Zhen; Chen, Zhijin; Jin, Xiaolin; Li, Shu; Chen, Yao; Hu, Fuquan

    2010-09-01

    MmP1 (Morganella morganii phage 1) is a lytic bacteriophage newly isolated from the host bacterium M. morganii. The entire genome was sequenced, and final assembly yielded a 38,234bp linear double-stranded DNA (dsDNA) with a G+C content of 46.5%. In the MmP1 genome, 49 putative genes, 10 putative promoters and 2 predicted sigma-independent terminators were determined through bioinformatic analysis. A striking feature of the MmP1 genome is its high degree of similarity to the T7 group of phages. All of the 49 predicted genes exist on the same DNA strand, and functions were assigned to 35 genes based on the similarity of the homologues deposited in GenBank, which share 30-80% identity to their counterparts in T7-like phages. The analyses of MmP1 using CoreGenes, phylogenetic tree of RNA polymerase and structural proteins have demonstrated that bacteriophage MmP1 should be assigned as a new member of T7-like phages but as a relatively distant member of this family. This is the first report that a T7-like phage adaptively parasitizes in M. morganii, and this will advance our understanding of biodiversity and adaptive evolution of T7-like phages.

  12. Metformin induces apoptosis of pancreatic cancer cells

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    AIM: To assess the role and mechanism of mefformin in inducing apoptosis of pancreatic cancer cells. METHODS: The human pancreatic cancer cell lines ASPC-1, BxPc-3, PANC-1 and SW1990 were exposed to mefformin. The inhibition of cell proliferation and colony formation via apoptosis induction and S phase arrest in pancreatic cancer cell lines of mefformin was tested.RESULTS: In each pancreatic cancer cell line tested, metformin inhibited cell proliferation in a dose dependent manner in MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assays). Flow cytometric analysis showed that metformin reduced the number of cells in G1 and increased the percentage of cells in S phase as well as the apoptotic fraction. Enzymelinked immunosorbent assay (EUSA) showed that metformin induced apaptosis in all pancreatic cancer cell lines. In Western blot studies, metformin induced oly-ADP-ribose polymerase(PARP) cleavage (an indicator of aspase activation) in all pancreatic cancer cell lines. The general caspase inhibitor (VAD-fmk) completely abolished metformin-induced PARP cleavage and apoptosis in ASPC-1 BxPc-3 and PANC-1, the caspase-8 specific inhibitor (IETD-fmk) and the caspase-9 specific inhibitor (LEHD-fmk) only partially abrogated metformin-induced apoptosis and PARP cleavage in BxPc-3 and PANC-1 cells. We also observed that metformin treatment ramatically reduced epidermal growth factor receptor (EGFR) and phosphorylated mitogen activated protein kinase (P-MAPK) in both a time- and dose-dependent manner in all cell lines tested.CONCLUSION: Metformin significantly inhibits cell proliferation and apoptosis in all pancreatic cell lines. And the metformin-induced apoptosis is associated with PARP leavage, activation of caspase-3, -8, and -9 in a time- and dose-dependent manner. Hence, both caspase-8 and -9-initiated apoptotic signaling pathways contribute to metforrnin-induced apoptosis in pancreatic cell lines.

  13. [Depression and treatment. Apoptosis, neuroplasticity and antidepressants].

    Science.gov (United States)

    Arantes-Gonçalves, Filipe; Coelho, Rui

    2006-01-01

    Depression's neurobiology begins to be better understood. The last decade data considers neuroplasticity and stress as implicated factors on the pathophisiology of depression. Because antidepressants have a lag-time on their action it is possible that inhibition of neurotransmitters recaptation is not sufficient to explain long term changes. For that purpose, neurogenesis increase, nervous fibers sprouting, new synapses and stabilization of the old ones can be responsible for those changes. AMPc-MAPcinases-CREB-BDNF cellular cascade can play a significant role in the mechanisms of dendritic restructuration, hippocampal neurogenesis increase and nervous cells survival. The aim of this article is to discuss if apoptosis could play a key role as an ethiopathogenic factor on the patogenesis of depression. It was done a medline search for references with apoptosis, stress, neuroplasticity, depression and antidepressants key-words. It were found 101 original or review references about these subjects. Stress plays a key role in the etiopathogeny of depression. Its deletery effects on apoptosis and neuroplasticity can be changed by antidepressants. Neurogenesis' increase is necessary for their action. This increase is reached with chronic antidepressant treatment and not with other psychotropic drugs which means some pharmacological specificity of antidepressants. AMPc, CREB, BDNF and Bcl-2 can be considered as target genes in antidepressant synthesis. At the level of this neurotrophic factors apoptosis might be included in the neuroplastic model of depression and play a prominent role in etiopathogeny of depression. To confirm that, we need more research on the field to know which are the mechanisms that trigger apoptosis and its biological significance. In relation to the last one, we can say that is possible to be physiological apoptosis in deteriorated neurons death which cannot make strong connections and pathological apoptosis because of stress via, namely, HPA axis.

  14. Chondrocyte Apoptosis in the Pathogenesis of Osteoarthritis

    Science.gov (United States)

    Hwang, Hyun Sook; Kim, Hyun Ah

    2015-01-01

    Apoptosis is a highly-regulated, active process of cell death involved in development, homeostasis and aging. Dysregulation of apoptosis leads to pathological states, such as cancer, developmental anomalies and degenerative diseases. Osteoarthritis (OA), the most common chronic joint disease in the elderly population, is characterized by progressive destruction of articular cartilage, resulting in significant disability. Because articular cartilage depends solely on its resident cells, the chondrocytes, for the maintenance of extracellular matrix, the compromising of chondrocyte function and survival would lead to the failure of the articular cartilage. The role of subchondral bone in the maintenance of proper cartilage matrix has been suggested as well, and it has been proposed that both articular cartilage and subchondral bone interact with each other in the maintenance of articular integrity and physiology. Some investigators include both articular cartilage and subchondral bone as targets for repairing joint degeneration. In late-stage OA, the cartilage becomes hypocellular, often accompanied by lacunar emptying, which has been considered as evidence that chondrocyte death is a central feature in OA progression. Apoptosis clearly occurs in osteoarthritic cartilage; however, the relative contribution of chondrocyte apoptosis in the pathogenesis of OA is difficult to evaluate, and contradictory reports exist on the rate of apoptotic chondrocytes in osteoarthritic cartilage. It is not clear whether chondrocyte apoptosis is the inducer of cartilage degeneration or a byproduct of cartilage destruction. Chondrocyte death and matrix loss may form a vicious cycle, with the progression of one aggravating the other, and the literature reveals that there is a definite correlation between the degree of cartilage damage and chondrocyte apoptosis. Because current treatments for OA act only on symptoms and do not prevent or cure OA, chondrocyte apoptosis would be a valid

  15. Sodium nitroprusside induces apoptosis of rabbit chondrocytes

    Science.gov (United States)

    Liang, Qian; Wang, Xiao-Ping; Chen, Tong-Sheng

    2013-02-01

    Osteoarthritis (OA) is characterized by a slowly progressing degradation of the matrix and destruction of articular cartilage. Apoptosis of chondrocyte is accounted for the mechanism of OA. Nitric oxide (NO), as a stimulus, has been shown to induce chondrocyte apoptosis by activating the matrix metalloproteinases (MMPs), increasing the expression of cyclooxygenase 2 (COX-2) and the level of prostaglandin E2 (PGE2), inhibiting the proteoglycan synthesis and type II collagen expression. In this study, sodium nitroprusside (SNP) was administered to be the NO donor to explore the mechanism of NO-induced apoptosis of rabbit chondrocytes obtained from six weeks old New Zealand rabbits. CCK-8 assay revealed the inhibitory effect of SNP on cell viability. We used flow cytometry (FCM) to assess the form of cell death by Annexin-V/propidium iodide (PI) double staining, and evaluate the change of mitochondrial membrane potential (ΔΨm). We found that the SNP induced chondrocyte apoptosis in a dose- and time-dependent manner and an observable reduction of ΔΨm. In conclusion, our findings indicate that SNP induces apoptosis of rabbit chondrocytes via a mitochondria-mediated pathway.

  16. Apoptosis of beta cells in diabetes mellitus.

    Science.gov (United States)

    Anuradha, Rachakatla; Saraswati, Mudigonda; Kumar, Kishore G; Rani, Surekha H

    2014-11-01

    Diabetes mellitus is a multifactorial metabolic disorder characterized by hyperglycemia. Apoptosis in beta cells has been observed in response to diverse stimuli, such as glucose, cytokines, free fatty acids, leptin, and sulfonylureas, leading to the activation of polyol, hexosamine, and diacylglycerol/protein kinase-C (DAG/PKC) pathways that mediate oxidative and nitrosative stress causing the release of different cytokines. Cytokines induce the expression of Fas and tumor necrosis factor-alpha (TNF-α) by activating the transcription factor, nuclear factor-κb, and signal transducer and activator of transcription 1 (STAT-1) in the β cells in the extrinsic pathway of apoptosis. Cytokines produced in beta cells also induce proapoptotic members of the intrinsic pathway of apoptosis. The genetic alterations in apoptosis signaling machinery and the pathogenesis of diabetes include Fas, FasL, Akt, caspases, calpain-10, and phosphatase and tensin homolog (Pten). The other gene products that are involved in diabetes are nitric oxide synthase-2 (NOS2), small ubiquitin-like modifier (SUMO), apolipoprotein CIII (ApoCIII), forkhead box protein O1 (FOXO1), and Kruppel-like zinc finger protein Gli-similar 3 (GLIS3). The gene products having antiapoptotic nature are Bcl-2 and Bcl-XL. Epigenetic mechanisms play an important role in type I and type II diabetes. Further studies on the apoptotic genes and gene products in diabetics may be helpful in pharmacogenomics and individualized treatment along with antioxidants targeting apoptosis in diabetes.

  17. NMR exposure sensitizes tumor cells to apoptosis.

    Science.gov (United States)

    Ghibelli, L; Cerella, C; Cordisco, S; Clavarino, G; Marazzi, S; De Nicola, M; Nuccitelli, S; D'Alessio, M; Magrini, A; Bergamaschi, A; Guerrisi, V; Porfiri, L M

    2006-03-01

    NMR technology has dramatically contributed to the revolution of image diagnostic. NMR apparatuses use combinations of microwaves over a homogeneous strong (1 Tesla) static magnetic field. We had previously shown that low intensity (0.3-66 mT) static magnetic fields deeply affect apoptosis in a Ca2+ dependent fashion (Fanelli et al., 1999 FASEBJ., 13;95-102). The rationale of the present study is to examine whether exposure to the static magnetic fields of NMR can affect apoptosis induced on reporter tumor cells of haematopoietic origin. The impressive result was the strong increase (1.8-2.5 fold) of damage-induced apoptosis by NMR. This potentiation is due to cytosolic Ca2+ overload consequent to NMR-promoted Ca2+ influx, since it is prevented by intracellular (BAPTA-AM) and extracellular (EGTA) Ca2+ chelation or by inhibition of plasma membrane L-type Ca2+ channels. Three-days follow up of treated cultures shows that NMR decrease long term cell survival, thus increasing the efficiency of cytocidal treatments. Importantly, mononuclear white blood cells are not sensitised to apoptosis by NMR, showing that NMR may increase the differential cytotoxicity of antitumor drugs on tumor vs normal cells. This strong, differential potentiating effect of NMR on tumor cell apoptosis may have important implications, being in fact a possible adjuvant for antitumor therapies.

  18. Measuring Apoptosis by Microscopy and Flow Cytometry.

    Science.gov (United States)

    Hollville, Emilie; Martin, Seamus J

    2016-02-02

    Apoptosis is a mode of programmed cell death that plays an important role during development and in the maintenance of tissue homeostasis. Numerous physiological as well as pathological stimuli trigger apoptosis such as engagement of Fas, TRAIL, or TNF receptors, growth factor deprivation, hypoxia, or exposure to cytotoxic drugs. Apoptosis is coordinated from within by members of the caspase family of cysteine proteases that, upon activation, trigger a series of morphological changes including cell shrinkage, extensive plasma membrane blebbing, chromatin condensation, DNA hydrolysis, and nuclear fragmentation. These dramatic structural and biochemical alterations result not only in the controlled dismantling of the cell, but also in the efficient recognition and removal of apoptotic cells by phagocytes. Necrosis, which is typically nonprogrammed or imposed upon the cell by overwhelming membrane or organelle damage, is characterized by rapid plasma membrane rupture followed by organelle and cell swelling. Necrosis is often provoked by infectious agents or a severe departure from physiological conditions. This unit describes protocols for the measurement of apoptosis and for distinguishing apoptosis from necrosis.

  19. Apoptosis in Drosophila: which role for mitochondria?

    Science.gov (United States)

    Clavier, Amandine; Rincheval-Arnold, Aurore; Colin, Jessie; Mignotte, Bernard; Guénal, Isabelle

    2016-03-01

    It is now well established that the mitochondrion is a central regulator of mammalian cell apoptosis. However, the importance of this organelle in non-mammalian apoptosis has long been regarded as minor, mainly because of the absence of a crucial role for cytochrome c in caspase activation. Recent results indicate that the control of caspase activation and cell death in Drosophila occurs at the mitochondrial level. Numerous proteins, including RHG proteins and proteins of the Bcl-2 family that are key regulators of Drosophila apoptosis, constitutively or transiently localize in mitochondria. These proteins participate in the cell death process at different levels such as degradation of Diap1, a Drosophila IAP, production of mitochondrial reactive oxygen species or stimulation of the mitochondrial fission machinery. Here, we review these mitochondrial events that might have their counterpart in human.

  20. Apoptosis: Therapeutic target in neurodegeneration and sepsis

    Directory of Open Access Journals (Sweden)

    Gonzalo Arboleda

    2005-12-01

    Full Text Available Cellular apoptosis has been considered as themain physiological mechanism underlyingneuronal demise associated to neurodegenerativediseases. Apoptosis has also been described inparenquimal and microvascular endotheliumin the acute phase of sepsis during multi-organicdysfunction. Therefore, strategies aimed toMuerte celular: blanco terapéutico enneurodegeneración y sepsisGonzalo Arboleda*, Luisa M. Matheus†prevent apoptosis (anti-apoptotic represent avaluable tool for prevention and/or retardationof the appearance of clinical symptoms in thesedisorders, which generate a large morbilitymortality,social and economic burden worldwide.The present review is aim to show that antiapoptoticstrategies hold a great therapeuticpotential. In this sense, we will review some ofthese potential therapies such as caspaseinhibitors, activated protein C, Bcl-2 family andthe PI3K/Akt signalling pathway.

  1. Death penalty for keratinocytes: apoptosis versus cornification.

    Science.gov (United States)

    Lippens, S; Denecker, G; Ovaere, P; Vandenabeele, P; Declercq, W

    2005-11-01

    Homeostasis implies a balance between cell growth and cell death. This balance is essential for the development and maintenance of multicellular organisms. Homeostasis is controlled by several mechanisms including apoptosis, a process by which cells condemned to death are completely eliminated. However, in some cases, total destruction and removal of dead cells is not desirable, as when they fulfil a specific function such as formation of the skin barrier provided by corneocytes, also known as terminally differentiated keratinocytes. In this case, programmed cell death results in accumulation of functional cell corpses. Previously, this process has been associated with apoptotic cell death. In this overview, we discuss differences and similarities in the molecular regulation of epidermal programmed cell death and apoptosis. We conclude that despite earlier confusion, apoptosis and cornification occur through distinct molecular pathways, and that possibly antiapoptotic mechanisms are implicated in the terminal differentiation of keratinocytes.

  2. Apoptosis and Necrosis in the Liver

    Science.gov (United States)

    Guicciardi, Maria Eugenia; Malhi, Harmeet; Mott, Justin L.; Gores, Gregory J.

    2013-01-01

    Because of its unique function and anatomical location, the liver is exposed to a multitude of toxins and xenobiotics, including medications and alcohol, as well as to infection by hepatotropic viruses, and therefore, is highly susceptible to tissue injury. Cell death in the liver occurs mainly by apoptosis or necrosis, with apoptosis also being the physiologic route to eliminate damaged or infected cells and to maintain tissue homeostasis. Liver cells, especially hepatocytes and cholangiocytes, are particularly susceptible to death receptor-mediated apoptosis, given the ubiquitous expression of the death receptors in the organ. In a quite unique way, death receptor-induced apoptosis in these cells is mediated by both mitochondrial and lysosomal permeabilization. Signaling between the endoplasmic reticulum and the mitochondria promotes hepatocyte apoptosis in response to excessive free fatty acid generation during the metabolic syndrome. These cell death pathways are partially regulated by microRNAs. Necrosis in the liver is generally associated with acute injury (i.e., ischemia/reperfusion injury) and has been long considered an unregulated process. Recently, a new form of “programmed” necrosis (named necroptosis) has been described: the role of necroptosis in the liver has yet to be explored. However, the minimal expression of a key player in this process in the liver suggests this form of cell death may be uncommon in liver diseases. Because apoptosis is a key feature of so many diseases of the liver, therapeutic modulation of liver cell death holds promise. An updated overview of these concepts is given in this article. PMID:23720337

  3. Dimerization of two novel apoptosis-inducing proteins and its function in regulating cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    刘青珍; 甘淼; 齐义鹏; 李凌云; 齐兵

    2003-01-01

    Asy (apoptosis/saibousi Yutsudo) is a novel apoptosis-inducing gene found in 1999 by Yutsudo group in Japan. In 2000, Qi Bing et al. cloned another novel gene, named hap (homologue of ASY protein), which encoded the ASY interact ing protein, from human lung cell line (WI-38) cDNA library by using yeast two-h ybrid system. It has been proved that ASY formed homodimer in yeast and human ce ll line, ASY and HAP formed heterodimer in yeast cells, and both induced cell ap optosis in human tumor cell lines Sao2 and CGL4. This paper showed that HAP coul d form homodimer in yeast cells by yeast two-hybrid system; HAP and ASY could pr oduce heterodimer in human cell line by cross-immunoprecipitation test; by using apoptosis-testing technologies such as AnnexinV, TUNEL, DNA ladder and Flow Cyt ometry, the cell apoptosis in human normal or tumor cell lines transfected with hap or asy individually or cotransfected by the both was qualified or quantified . It was firstly demonstrated that ASY or HAP induced cell apoptosis not only in human tumor cell lines, but also in human normal cell lines. Moreover, we prove d that the heterodimer between ASY and HAP decreased apoptosis-inducing activity from the homodimer of ASY or HAP. It revealed that by choosing to form heterodi mer or homodimer between ASY and / or HAP is an important mechanism of regulatin g apoptosis in human cell lines.

  4. Apoptosis de fibroblastos gingivales en periodontitis

    OpenAIRE

    Roger Mauricio Arce; Oscar Tamayo; Armando Cortés

    2007-01-01

    Introducción: Los fibroblastos gingivales humanos (FGH) tienen un papel importante en la enfermedad periodontal, pues alteran su normal funcionamiento en respuesta a estímulos pro-inflamatorios. Se cree que los fibroblastos se pueden eliminar anormalmente por medio de apoptosis en periodontitis. El propósito de este estudio es determinar y cuantificar la apoptosis de FGH en biopsias del periodonto de individuos sanos y con enfermedad periodontal. Métodos: Se realizó un estudio clínico descrip...

  5. Apoptosis of gingival fibroblasts in periodontitis.

    OpenAIRE

    Roger Mauricio Arce; Oscar Tamayo; Armando Cortés

    2009-01-01

    Introducción: Los fibroblastos gingivales humanos (FGH) tienen un papel importante en la enfermedad periodontal, pues alteran su normal funcionamiento en respuesta a estímulos pro-inflamatorios. Se cree que los fibroblastos se pueden eliminar anormalmente por medio de apoptosis en periodontitis. El propósito de este estudio es determinar y cuantificar la apoptosis de FGH en biopsias del periodonto de individuos sanos y con enfermedad periodontal. Métodos: Se realizó un estudio clínico descri...

  6. The relationship between prostate cancer and apoptosis

    Directory of Open Access Journals (Sweden)

    Zeki Arı

    2011-03-01

    Full Text Available Prostate is the largest accessory gland of male genitaltract and the beginning part of male urethra. Prostatecancer is the most common internal malignancy inmales. Prostate cancer is ranked as second in death fromto cancer. A malignant disease is known as uncontrolledproliferation of cells. Beside excessive proliferation, decreasedapoptosis was also observed contribute to thedevelopment of malignancy. Apoptosis (programmedcell death plays an important role in many diseases andfree radical damage, triggers by cytokines and inflammatoryinjury. This review has been prepared to show theinteresting link between apoptosis and cancer and toprovide collective source to who want to do research onthis subject. J Clin Exp Invest 2011; 2(1: 124-131

  7. Clinical Manifestations of Kaposi Sarcoma Herpesvirus (KSHV Lytic Activation: Multicentric Castleman Disease (KSHV-MCD and the KSHV Inflammatory Cytokine Syndrome (KICS

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    Mark N. Polizzotto

    2012-03-01

    Full Text Available Soon after the discovery of Kaposi sarcoma associated herpesvirus (KSHV, it was appreciated that this virus was associated with most cases of multicentric Castleman disease (MCD arising in patients infected with human immunodeficiency virus (HIV. It has subsequently been recognized that KSHV-MCD is a distinct entity from other forms of MCD. Like MCD that is unrelated to KSHV, the clinical presentation of KSHV-MCD is dominated by systemic inflammatory symptoms including fevers, cachexia and laboratory abnormalities including cytopenias, hypoalbuminemia, hyponatremia, and elevated C-reactive protein. Pathologically KSHV-MCD is characterized by polyclonal, IgM-lambda restricted plasmacytoid cells in the intrafollicular areas of affected lymph nodes. A portion of these cells are infected with KSHV and a sizable subset of these cells express KSHV lytic genes including a viral homolog of interleukin-6 (vIL-6. Patients with KSHV-MCD generally have elevated KSHV viral loads in their peripheral blood. Production of vIL-6 and induction of human (h IL-6 both contribute to symptoms, perhaps in combination with overproduction of IL-10 and other cytokines. Until recently, the prognosis of patients with KSHV-MCD was poor. Recent therapeutic advances targeting KSHV-infected B cells with the anti-CD20 monoclonal antibody rituximab and utilizing KSHV enzymes to target KSHV-infected cells have substantially improved patient outcomes. Recently another KSHV-associated condition, the KSHV inflammatory cytokine syndrome (KICS has been described. Its clinical manifestations resemble those of KSHV-MCD but lymphadenopathy is not prominent and the pathologic nodal changes of KSHV-MCD are absent. Patients with KICS exhibit elevated KSHV viral loads and elevation of vIL-6, hIL-6 and IL-10 comparable to those seen in KSHV-MCD; the cellular origin of these is a matter of investigation. KICS may contribute to the inflammatory symptoms seen in some patients with severe Kaposi

  8. Characterization of the replication, transfer, and plasmid/lytic phage cycle of the Streptomyces plasmid-phage pZL12.

    Science.gov (United States)

    Zhong, Li; Cheng, Qiuxiang; Tian, Xinli; Zhao, Liqian; Qin, Zhongjun

    2010-07-01

    We report here the isolation and recombinational cloning of a large plasmid, pZL12, from endophytic Streptomyces sp. 9R-2. pZL12 comprises 90,435 bp, encoding 112 genes, 30 of which are organized in a large operon resembling bacteriophage genes. A replication locus (repA) and a conjugal transfer locus (traA-traC) were identified in pZL12. Surprisingly, the supernatant of a 9R-2 liquid culture containing partially purified phage particles infected 9R-2 cured of pZL12 (9R-2X) to form plaques, and a phage particle (phiZL12) was observed by transmission electron microscopy. Major structural proteins (capsid, portal, and tail) of phiZL12 virions were encoded by pZL12 genes. Like bacteriophage P1, linear phiZL12 DNA contained ends from a largely random pZL12 sequence. There was also a hot end sequence in linear phiZL12. phiZL12 virions efficiently infected only one host, 9R-2X, but failed to infect and form plaques in 18 other Streptomyces strains. Some 9R-2X spores rescued from lysis by infection of phiZL12 virions contained a circular pZL12 plasmid, completing a cycle comprising autonomous plasmid pZL12 and lytic phage phiZL12. These results confirm pZL12 as the first example of a plasmid-phage in Streptomyces.

  9. KSHV miRNAs Decrease Expression of Lytic Genes in Latently Infected PEL and Endothelial Cells by Targeting Host Transcription Factors

    Directory of Open Access Journals (Sweden)

    Karlie Plaisance-Bonstaff

    2014-10-01

    Full Text Available Kaposi’s sarcoma-associated herpesvirus (KSHV microRNAs are encoded in the latency-associated region. Knockdown of KSHV miR-K12-3 and miR-K12-11 increased expression of lytic genes in BC-3 cells, and increased virus production from latently infected BCBL-1 cells. Furthermore, iSLK cells infected with miR-K12-3 and miR-K12-11 deletion mutant viruses displayed increased spontaneous reactivation and were more sensitive to inducers of reactivation than cells infected with wild type KSHV. Predicted binding sites for miR-K12-3 and miR-K12-11 were found in the 3’UTRs of the cellular transcription factors MYB, Ets-1, and C/EBPα, which activate RTA, the KSHV replication and transcription activator. Targeting of MYB by miR-K12-11 was confirmed by cloning the MYB 3’UTR downstream from the luciferase reporter. Knockdown of miR‑K12-11 resulted in increased levels of MYB transcript, and knockdown of miR-K12-3 increased both C/EBPα and Ets-1 transcripts. Thus, miR-K12-11 and miR-K12-3 contribute to maintenance of latency by decreasing RTA expression indirectly, presumably via down‑regulation of MYB, C/EBPα and Ets-1, and possibly other host transcription factors.

  10. Ocean acidification and viral replication cycles: Frequency of lytically infected and lysogenic cells during a mesocosm experiment in the NW Mediterranean Sea

    Science.gov (United States)

    Tsiola, Anastasia; Pitta, Paraskevi; Giannakourou, Antonia; Bourdin, Guillaume; Marro, Sophie; Maugendre, Laure; Pedrotti, Maria Luiza; Gazeau, Frédéric

    2017-02-01

    The frequency of lytically infected and lysogenic cells (FLIC and FLC, respectively) was estimated during an in situ mesocosm experiment studying the impact of ocean acidification on the plankton community of a low nutrient low chlorophyll (LNLC) system in the north-western Mediterranean Sea (Bay of Villefranche, France) in February/March 2013. No direct effect of elevated partial pressure of CO2 (pCO2) on viral replication cycles could be detected. FLC variability was negatively correlated to heterotrophic bacterial and net community production as well as the ambient bacterial abundance, confirming that lysogeny is a prevailing life strategy under unfavourable-for-the-hosts conditions. Further, the phytoplankton community, assessed by chlorophyll a concentration and the release of >0.4 μm transparent exopolymeric particles, was correlated with the occurrence of lysogeny, indicating a possible link between photosynthetic processes and bacterial growth. Higher FLC was found occasionally at the highest pCO2-treated mesocosm in parallel to subtle differences in the phytoplankton community. This observation suggests that elevated pCO2 could lead to short-term alterations in lysogenic dynamics coupled to phytoplankton-derived processes. Correlation of FLIC with any environmental parameter could have been obscured by the sampling time or the synchronization of lysis to microbial processes not assessed in this experiment. Furthermore, alterations in microbial and viral assemblage composition and gene expression could be a confounding factor. Viral-induced modifications in organic matter flow affect bacterial growth and could interact with ocean acidification with unpredictable ecological consequences.

  11. An improved system for the surface immobilisation of proteins on Bacillus thuringiensis vegetative cells and spores through a new spore cortex-lytic enzyme anchor.

    Science.gov (United States)

    Shao, Xiaohu; Ni, Hong; Lu, Ting; Jiang, Mengtian; Li, Hua; Huang, Xinfeng; Li, Lin

    2012-02-15

    An improved surface-immobilisation system was engineered to target heterologous proteins onto vegetative cells and spores of Bacillus thuringiensis plasmid-free recipient strain BMB171. The sporulation-dependent spore cortex-lytic enzyme from B. thuringiensis YBT-1520, SceA, was expressed in vegetative cells and used as the surface anchoring motif. Green fluorescent protein (GFP) and a Bacillus endo-β-1,3-1,4-glucanase (BglS) were used as the fusion partners to test the binding efficiency and the functional activities of immobilised surface proteins. The surface localisation of the SceA-GFP fusion protein on vegetative cells and spores was confirmed by Western blot, immunofluorescence microscopy and flow cytometry. The GFP fluorescence intensity from both vegetative cells and spores was measured and compared to a previously characterised surface display system using a peptidoglycan hydrolase anchor (Mbg). Results demonstrated comparable efficiency of SceA- and Mbg-mediated immobilisation on vegetative cells but a more efficient immobilisation on spores using the SceA anchor, suggesting SceA has greater potential for spore-based applications. The SceA protein was then applied to target BglS onto vegetative cells and spores, and the surface immobilisation was verified by the substantial whole-cell enzymatic activity and enhanced whole-spore enzymatic activity compared to vegetative cells. A dually active B. thuringiensis vegetative cell and spore display system could prove especially valuable for the development of regenerable and heat-stable biocatalysts that function under adverse environmental conditions, for example, an effective feed additive for improved digestion and nutrient absorption by livestock.

  12. Edwardsiella tarda Ivy, a lysozyme inhibitor that blocks the lytic effect of lysozyme and facilitates host infection in a manner that is dependent on the conserved cysteine residue.

    Science.gov (United States)

    Wang, Chong; Hu, Yong-hua; Sun, Bo-guang; Li, Jun; Sun, Li

    2013-10-01

    Edwardsiella tarda is a Gram-negative bacterial pathogen with a broad host range that includes fish and humans. In this study, we examined the activity and function of the lysozyme inhibitor Ivy (named IvyEt) identified in the pathogenic E. tarda strain TX01. IvyEt possesses the Ivy signature motif CKPHDC in the form of (82)CQPHNC(87) and contains several highly conserved residues, including a tryptophan (W55). For the purpose of virulence analysis, an isogenic TX01 mutant, TXivy, was created. TXivy bears an in-frame deletion of the ivyEt gene. A live infection study in a turbot (Scophthalmus maximus) model showed that, compared to TX01, TXivy exhibited attenuated overall virulence, reduced tissue dissemination and colonization capacity, an impaired ability to replicate in host macrophages, and decreased resistance against the bactericidal effect of host serum. To facilitate functional analysis, recombinant IvyEt (rIvy) and three mutant proteins, i.e., rIvyW55A, rIvyC82S, and rIvyH85D, which bear Ala, Ser, and Asp substitutions at W55, C82, and H85, respectively, were prepared. In vitro studies showed that rIvy, rIvyW55A, and rIvyH85D were able to block the lytic effect of lysozyme on a Gram-positive bacterium, whereas rIvyC82S could not do so. Likewise, rIvy, but not rIvyC82S, inhibited the serum-facilitated killing effect of lysozyme on E. tarda. In vivo analysis showed that rIvy, but not rIvyC82S, restored the lost pathogenicity of TXivy and enhanced the infectivity of TX01. Together these results indicate that IvyEt is a lysozyme inhibitor and a virulence factor that depends on the conserved C82 for biological activity.

  13. Inhibitory and lytic effects of phenothiazine derivatives and related tricyclic neuroleptic compounds, on Entamoeba histolytica HK9 and HM1 trophozoites.

    Science.gov (United States)

    Ondarza, R N; Hernández, E; Iturbe, A; Hurtado, G

    2000-08-01

    It has been shown previously that tricyclic neuroleptics like clomipramine and chlorpromazine have lethal effects on Leishmania donovani and L. major, and other studies indicate that the phenothiazine inhibitors of trypanothione reductase are potential anti-trypanosomal and anti-leishmanial drugs. With this in mind and our original observation on the presence of trypanothione in Entamoeba histolytica HK9, we examined the possible inhibitory effects of various phenothiazine and tricyclic derivatives on this human parasite. We found that drugs like clomipramine (KD002), the most potent in vitro inhibitor of trypanothione reductase among 30 tricyclic compounds tested, at 25 microM after 24 h of culture under aerobic conditions, caused a substantial decrease in the number of E. histolytica HK9 trophozoites, from approx. 15 x 10(6) to 5.37 x 10(6) cells, and at 100 microM to 0.8 x 10(6) cells. A substantial inhibitory effect on cell proliferation could also be demonstrated with metronidazol (used clinically against amoebiasis). Under similar experimental conditions other tricyclic and phenothiazine derivatives (OFKs), designed originally to inhibit the trypanothione reductase of trypanosomatides, had an inhibitory effect of 16 to 95%. For comparison, similar results were obtained using clomipramine and a phenothiazine derivative (OFK006) with Trypanosoma cruzi and Crithidia luciliae, except that with the latter the inhibitory effect of clomipramine was less dramatic. Experiments comparing two E. histolytica strains showed that normal cell proliferation under anaerobiosis was higher in strain HK9 than in HM1, which is highly virulent, but that metronidazol and clomipramine were less effective against HM1. Two other drugs tested, diphenydramine (KD005) and a phenothiazine derivative (OFK008), also had significant but lower inhibitory effects on both strains. The inhibitory activity on cell proliferation and the lytic effects on this human parasite by the tricyclic

  14. Stress response and apoptosis in pro- and antiinflammatory macrophages.

    Science.gov (United States)

    Malyshev, I Yu; Kruglov, S V; Bakhtina, L Yu; Malysheva, E V; Zubin, M; Norkin, M

    2004-08-01

    We showed that stress response and apoptosis in macrophages depend on the phenotype of their secretory activity and specific biological and physical characteristics of the factor inducing stress-response or apoptosis.

  15. Thiol redox state in apoptosis : physiological and toxicant modulation

    OpenAIRE

    Nobel, Stefan

    1997-01-01

    Apoptosis is a physiological type of cell death used to regulate the number of cells during development and im adult organs. However, apoptosis can also be inappropriately activated or inhibited under pathological conditions. One of the critical mechanisms of apoptosis is the activity of cysteine proteases belonging to the caspase family. The present study was designed to investigate the role of oxidative stress in apoptosis and how the apoptotic death program might be regul...

  16. A novel method for detection of apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Zagariya, Alexander M., E-mail: zagariya@uic.edu

    2012-04-15

    There are two different Angiotensin II (ANG II) peptides in nature: Human type (ANG II) and Bovine type (ANG II*). These eight amino acid peptides differ only at position 5 where Valine is replaced by Isoleucine in the Bovine type. They are present in all species studied so far. These amino acids are different by only one atom of carbon. This difference is so small, that it will allow any of ANG II, Bovine or Human antibodies to interact with all species and create a universal method for apoptosis detection. ANG II concentrations are found at substantially higher levels in apoptotic, compared to non-apoptotic, tissues. ANG II accumulation can lead to DNA damage, mutations, carcinogenesis and cell death. We demonstrate that Bovine antiserum can be used for universal detection of apoptosis. In 2010, the worldwide market for apoptosis detection reached the $20 billion mark and significantly increases each year. Most commercially available methods are related to Annexin V and TUNNEL. Our new method based on ANG II is more widely known to physicians and scientists compared to previously used methods. Our approach offers a novel alternative for assessing apoptosis activity with enhanced sensitivity, at a lower cost and ease of use.

  17. Calpain Activator Dibucaine Induces Platelet Apoptosis

    Directory of Open Access Journals (Sweden)

    Jun Liu

    2011-03-01

    Full Text Available Calcium-dependent calpains are a family of cysteine proteases that have been demonstrated to play key roles in both platelet glycoprotein Ibα shedding and platelet activation and altered calpain activity is associated with thrombotic thrombocytopenic purpura. Calpain activators induce apoptosis in several types of nucleated cells. However, it is not clear whether calpain activators induce platelet apoptosis. Here we show that the calpain activator dibucaine induced several platelet apoptotic events including depolarization of the mitochondrial inner transmembrane potential, up-regulation of Bax and Bak, down-regulation of Bcl-2 and Bcl-XL, caspase-3 activation and phosphatidylserine exposure. Platelet apoptosis elicited by dibucaine was not affected by the broad spectrum metalloproteinase inhibitor GM6001. Furthermore, dibucaine did not induce platelet activation as detected by P-selectin expression and PAC-1 binding. However, platelet aggregation induced by ristocetin or α-thrombin, platelet adhesion and spreading on von Willebrand factor were significantly inhibited in platelets treated with dibucaine. Taken together, these data indicate that dibucaine induces platelet apoptosis and platelet dysfunction.

  18. Epac inhibits apoptosis of human leukocytes

    NARCIS (Netherlands)

    Grandoch, M.; Bujok, V.; Fleckenstein, D.; Schmidt, M.; Fischer, J. W.; Weber, A. -A.

    2009-01-01

    cAMP is known to participate in the regulation of apoptosis in leukocytes. Depending on the cell type, pro- and antiapoptotic effects of cAMP have been described. Thus far, most of the cAMP-dependent effects have been attributed to the activation of PKA. However, Epac proteins (direct cAMP targets a

  19. Signaling Pathways in Cardiac Myocyte Apoptosis

    Science.gov (United States)

    Xia, Peng; Liu, Yuening

    2016-01-01

    Cardiovascular diseases, the number 1 cause of death worldwide, are frequently associated with apoptotic death of cardiac myocytes. Since cardiomyocyte apoptosis is a highly regulated process, pharmacological intervention of apoptosis pathways may represent a promising therapeutic strategy for a number of cardiovascular diseases and disorders including myocardial infarction, ischemia/reperfusion injury, chemotherapy cardiotoxicity, and end-stage heart failure. Despite rapid growth of our knowledge in apoptosis signaling pathways, a clinically applicable treatment targeting this cellular process is currently unavailable. To help identify potential innovative directions for future research, it is necessary to have a full understanding of the apoptotic pathways currently known to be functional in cardiac myocytes. Here, we summarize recent progress in the regulation of cardiomyocyte apoptosis by multiple signaling molecules and pathways, with a focus on the involvement of these pathways in the pathogenesis of heart disease. In addition, we provide an update regarding bench to bedside translation of this knowledge and discuss unanswered questions that need further investigation. PMID:28101515

  20. EpCAM-targeted induction of apoptosis

    NARCIS (Netherlands)

    Bremer, Edwin; Helfrich, Wijnand

    2008-01-01

    EpCAM is a well-established pancarcinoma-associated target antigen that has been used in a variety of therapeutic approaches. Of particular appeal are those strategies that aim to retarget and locally activate immune effector mechanisms involving apoptosis. Cancer cells typically employ various stra

  1. EpCAM-targeted induction of apoptosis.

    Science.gov (United States)

    Bremer, Edwin; Helfrich, Wijnand

    2008-05-01

    EpCAM is a well-established pancarcinoma-associated target antigen that has been used in a variety of therapeutic approaches. Of particular appeal are those strategies that aim to retarget and locally activate immune effector mechanisms involving apoptosis. Cancer cells typically employ various strategies to evade recognition and elimination by immune effector cells, including low or absent expression of MHCI molecules and active elimination of tumor infiltrating immune cells. In addition, cancer cells show an increased resistance towards endogenous pro-apoptotic stimuli due to aberrancies in their apoptotic machinery. However, compelling evidence indicates that cancer cells are often reliant on these molecular aberrations for continued cell survival. This pivotal role of immune evasion and apoptosis resistance has fueled the quest for therapeutic strategies that can selectively retarget and reactivate immune effector cells or molecules, whereby the balance of cellular fate of cancer cells is selectively tipped towards apoptosis. Here we review and discuss the perspectives for EpCAM-targeted apoptosis induction in cancer by EpCAM-selective bispecific antibodies and TRAIL fusion proteins.

  2. Spermine inhibits Endoplasmic Reticulum Stress - induced Apoptosis: a New Strategy to Prevent Cardiomyocyte Apoptosis

    Directory of Open Access Journals (Sweden)

    Can Wei

    2016-02-01

    Full Text Available Background/Aims: Endoplasmic reticulum stress (ERS plays an important role in the progression of acute myocardial infarction (AMI, in part by mediating apoptosis. Polyamines, including putrescine, spermidine, and spermine, are polycations with anti-oxidative, anti-aging, and cell growth-promoting activities. This study aimed to determine the mechanisms by which spermine protects against ERS-induced apoptosis in rats following AMI. Methods and Results: AMI was established by ligation of the left anterior descending coronary artery (LAD in rats, and exogenous spermine was administered by intraperitoneal injection (2.5 mg/ml daily for 7 days pre-AMI. Spermine treatment limited infarct size, attenuated cardiac troponin I and creatinine kinase-MB release, improved cardiac function, and decreased ERS and apoptosis related protein expression. Isolated cardiomyocytes subjected to hypoxia showed significant increase in reactive oxygen species (ROS and the expression of apoptosis and ERS related proteins; these effects occurred through PERK and eIF2α phosphorylation. The addition of spermine attenuated cardiomyocyte apoptosis, suppressed the production of ROS, and inhibited ERS related pathways. Conclusions: Spermine was an effective pre-treatment strategy to attenuate cardiac ERS injury in rats, and the cardioprotective mechanism occurring through inhibition of ROS production and down regulation of the PERK-eIF2α pathway. These findings provide a novel target for the prevention of apoptosis in the setting of AMI.

  3. Noscapine induces apoptosis in human glioma cells by an apoptosis-inducing factor-dependent pathway.

    Science.gov (United States)

    Newcomb, Elizabeth W; Lukyanov, Yevgeniy; Smirnova, Iva; Schnee, Tona; Zagzag, David

    2008-07-01

    Previously, we identified noscapine as a small molecule inhibitor of the hypoxia-inducible factor-1 pathway in hypoxic human glioma cells and human umbilical vein endothelial cells. Noscapine is a nontoxic ingredient in cough medicine currently used in clinical trials for patients with non-Hodgkin's lymphoma or chronic lymphocytic leukemia to assess antitumor efficacy. Here, we have evaluated the sensitivity of four human glioma cell lines to noscapine-induced apoptosis. Noscapine was a potent inhibitor of proliferation and inducer of apoptosis. Induction of apoptosis was associated with activation of the c-jun N-terminal kinase signaling pathway concomitant with inactivation of the extracellular signal regulated kinase signaling pathway and phosphorylation of the antiapoptotic protein Bcl-2. Noscapine-induced apoptosis was associated with the release of mitochondrial proteins apoptosis-inducing factor (AIF) and/or cytochrome c. In some glioma cell lines, only AIF release occurred without cytochrome c release or poly (ADP-ribose) polymerase cleavage. Knock-down of AIF decreased noscapine-induced apoptosis. Our results suggest the potential importance of noscapine as a novel agent for use in patients with glioblastoma owing to its low toxicity profile and its potent anticancer activity.

  4. Activation of PI3K/AKT and ERK MAPK signal pathways is required for the induction of lytic cycle replication of Kaposi's Sarcoma-associated herpesvirus by herpes simplex virus type 1

    Directory of Open Access Journals (Sweden)

    Lv Zhigang

    2011-10-01

    Full Text Available Abstract Background Kaposi's sarcoma-associated herpesvirus (KSHV is causally linked to several acquired immunodeficiency syndrome-related malignancies, including Kaposi's sarcoma (KS, primary effusion lymphoma (PEL and a subset of multicentric Castleman's disease. Regulation of viral lytic replication is critical to the initiation and progression of KS. Recently, we reported that herpes simplex virus type 1 (HSV-1 was an important cofactor that activated lytic cycle replication of KSHV. Here, we further investigated the possible signal pathways involved in HSV-1-induced reactivation of KSHV. Results By transfecting a series of dominant negative mutants and protein expressing constructs and using pharmacologic inhibitors, we found that either Janus kinase 1 (JAK1/signal transducer and activator of transcription 3 (STAT3 or JAK1/STAT6 signaling failed to regulate HSV-1-induced KSHV replication. However, HSV-1 infection of BCBL-1 cells activated phosphatidylinositol 3-kinase (PI3K/protein kinase B (PKB, also called AKT pathway and inactivated phosphatase and tensin homologue deleted on chromosome ten (PTEN and glycogen synthase kinase-3β (GSK-3β. PTEN/PI3K/AKT/GSK-3β pathway was found to be involved in HSV-1-induced KSHV reactivation. Additionally, extracellular signal-regulated protein kinase (ERK mitogen-activated protein kinase (MAPK pathway also partially contributed to HSV-1-induced KSHV replication. Conclusions HSV-1 infection stimulated PI3K/AKT and ERK MAPK signaling pathways that in turn contributed to KSHV reactivation, which provided further insights into the molecular mechanism controlling KSHV lytic replication, particularly in the context of HSV-1 and KSHV co-infection.

  5. Lytic efficacy of apoli protein E2 (ApoE2) and recombinant tissue plasminogen activator (rt-PA) treatment with 120 kHz ultrasound in an in-vitro human clot model

    Science.gov (United States)

    Meunier, Jason M.; Cheng, Jason Y.; Clark, Joseph F.; Shaw, George J.

    2005-04-01

    Currently, the only FDA approved therapy for acute ischemic stroke is recombinant tissue plasminogen activator (rt-PA). However rt-PA has substantial side effects such as hemorrhage. This has led to interest in other potential therapies. For example, ultrasound (US) increases the lytic efficacy of rt-PA. Also, apolipoprotein E2 (ApoE2) increases rt-PA activity. This suggests combining US, ApoE2 and rt-PA to improve thrombolysis, but the efficacy is not known. Here, the lytic efficacy of apoE2, rt-PA and 120 kHz US is measured in a human clot model. Whole blood was obtained from volunteers, after local institutional approval. Clots were formed in 1.7 mm micropipettes, and placed in a water tank that allowed microscopic video imaging during US and thrombolytic exposure. Clots were treated with rt-PA ([rt-PA]=3.15 μg/ml), rt-PA and apoE2 ([apoE2]=9.8 μg/ml), or rt-PA, apoE2 and 120 kHz US (0.35 MPa, PRF=1667 Hz, 80% duty cycle) for 15 min at 37°C in human plasma. Clot lysis was visually recorded and the lysis depth (LD) determined from these data using an image analysis algorithm. LD was linear with time for all treatments (R2>=0.81), allowing the determination of a lytic rate (LR). LR was found to be 0.35+/-0.03, 1.55+/-0.11, and 0.75+/-0.04 μm/min for the rt-PA, rt-PA and apoE2, and US treated groups respectively. The thrombolytic efficacy of rt-PA is enhanced by ApoE2. The interaction of 120 kHz with apoE2 and rt-PA showed a reduced lytic efficacy compared with rt-PA and apoE2 treatment alone. It is possible that US interferes with the ApoE2-mediated activation of rt-PA.

  6. Paclitaxel induces apoptosis in human gastric carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Hai-Bo Zhou; Ju-Ren Zhu

    2003-01-01

    AIM: To investigate the apoptosis in gastric cancer cells induced by paclitaxel, and the relation between this apoptosis and expression of Bcl-2 and Bax.METHODS: In in vitro experiments, MTT assay was used to determine the cell growth inhibitory rate. Transmission electron microscope and TUNEL staining method were used to quantitatively and qualitively detect the apoptosis status of gastric cancer cell line SGC-7901 before and after the paditaxel treatment. Immunohistochemical staining was used to detect the expression of apoptosis-regulated gene Bcl-2and Bax.RESULTS: Paclitaxel inhibited the growth of gastric cancer cell line SGC-7901 in a dose-and time-dependent manner.Paclitaxel induced SGC-7901 cells to undergo apoptosis with typically apoptotic characteristics, including morphological changes of chromatin condensation, chromatin crescent formation, nucleus fragmentation and apoptotic body formation. Paclitaxel could reduce the expression of apoptosis-regulated gene Bcl-2, and improve the expression of apoptosis-regulated gene Bax.CONCLUSION: Paclitaxel is able to induce the apoptosis in gastric cancer. This apoptosis may be mediated by downexpression of apoptosis-regulated gene Bcl-2 and upexpression of apoptosis-regulated gene Bax.

  7. Comparison of BACTEC MYCO/F LYTIC and WAMPOLE ISOLATOR 10 (lysis-centrifugation) systems for detection of bacteremia, mycobacteremia, and fungemia in a developing country.

    Science.gov (United States)

    Archibald, L K; McDonald, L C; Addison, R M; McKnight, C; Byrne, T; Dobbie, H; Nwanyanwu, O; Kazembe, P; Reller, L B; Jarvis, W R

    2000-08-01

    In less-developed countries, studies of bloodstream infections (BSI) have been hindered because of the difficulty and costs of culturing blood for bacteria, mycobacteria, and fungi. During two study periods (study period I [1997] and study period II [1998]), we cultured blood from patients in Malawi by using the BACTEC MYCO/F LYTIC (MFL), ISOLATOR 10 (Isolator), Septi-Chek AFB (SC-AFB), and Septi-Chek bacterial (SC-B) systems. During study period I, blood was inoculated at 5 ml into an MFL bottle, 10 ml into an Isolator tube for lysis and centrifugation, and 10 ml into an SC-B bottle. Next, 0.5-ml aliquots of Isolator concentrate were inoculated into an SC-AFB bottle and onto Middlebrook 7H11 agar slants, chocolate agar slants, and Inhibitory Mold Agar (IMA) slants. During study period II, the SC-B and chocolate agar cultures were discontinued. MFL growth was detected by fluorescence caused by shining UV light (lambda = 365 nm) onto the indicator on the bottom of the bottle. During study period I, 251 blood cultures yielded 44 bacterial isolates. For bacteremia, the MFL was similar to the Isolator concentrate on chocolate agar (34 of 44 versus 27 of 44; P, not significant [NS]), but more sensitive than the SC-B bottle (34 of 44 versus 24 of 44; P = 0.05). For both study periods combined, 486 blood cultures yielded 37 mycobacterial and 13 fungal isolates. For mycobacteremia, the sensitivities of the MFL and Isolator concentrate in the SC-AFB bottle were similar (30 of 37 versus 29 of 37; P, NS); the MFL bottle was more sensitive than the concentrate on Middlebrook agar (30 of 37 versus 15 of 37; P = 0.002). For fungemia, the MFL bottle was as sensitive as the SC-B bottle or Isolator concentrate on chocolate agar or IMA slants. We conclude that the MFL bottle, inoculated with just 5 ml of blood and examined under UV light, provides a sensitive and uncomplicated method for comprehensive detection of BSI in less-developed countries.

  8. In vivo activation of toll-like receptor-9 induces an age-dependent abortive lytic cycle reactivation of murine gammaherpesvirus-68.

    Science.gov (United States)

    Ptaschinski, Catherine; Wilmore, Joel; Fiore, Nancy; Rochford, Rosemary

    2010-12-01

    Infection of mice with murine gammaherpesvirus-68 (γHV-68) serves as a model to understand the pathogenesis of persistent viral infections, including the potential for co-infections to modulate viral latency. We have previously found that infection of neonates (8-day-old mice) with γHV-68 resulted in a high level of persistence of the virus in the lungs as well as the spleen, in contrast to infection of adult mice, for which long-term latency was only readily detected in the spleen. In this study we investigated whether stimulation of toll-like receptor (TLR)9 would modulate viral latency in mice infected with γHV-68 in an age-dependent manner. Pups and adult mice were injected with the synthetic TLR9 ligand CpG ODN at 30 dpi, at which time long-term latency has been established. Three days after CpG injection, the lungs and spleens were removed, and a limiting dilution assay was done to determine the frequency of latently infected cells. RNA was extracted to measure viral transcripts using a ribonuclease protection assay. We observed that CpG injection resulted in an increase in the frequency of latently-infected cells in both the lungs and spleens of infected pups, but only in the spleens of infected adult mice. No preformed virus was detected, suggesting that TLR9 stimulation did not trigger complete viral reactivation. When we examined viral gene expression in these same tissues, we observed expression only of the immediate early lytic genes, rta and K3, but not the early DNA polymerase gene or late gB transcript indicative of an abortive reactivation in the spleen. Additionally, mice infected as pups had greater numbers of germinal center B cells in the spleen following CpG injection, whereas CpG stimulated the expansion of follicular zone B cells in adult mice. These data suggest that stimulation of TLR9 differentially modulates gammaherpesvirus latency via an age-dependent mechanism.

  9. Characterization, sequencing and comparative genomic analysis of vB_AbaM-IME-AB2, a novel lytic bacteriophage that infects multidrug-resistant Acinetobacter baumannii clinical isolates.

    Science.gov (United States)

    Peng, Fan; Mi, Zhiqiang; Huang, Yong; Yuan, Xin; Niu, Wenkai; Wang, Yahui; Hua, Yuhui; Fan, Huahao; Bai, Changqing; Tong, Yigang

    2014-07-05

    With the use of broad-spectrum antibiotics, immunosuppressive drugs, and glucocorticoids, multidrug-resistant Acinetobacter baumannii (MDR-AB) has become a major nosocomial pathogen species. The recent renaissance of bacteriophage therapy may provide new treatment strategies for combatting drug-resistant bacterial infections. In this study, we isolated a lytic bacteriophage vB_AbaM-IME-AB2 has a short latent period and a small burst size, which clear its host's suspension quickly, was selected for characterization and a complete genomic comparative study. The isolated bacteriophage vB_AbaM-IME-AB2 has an icosahedral head and displays morphology resembling Myoviridae family. Gel separation assays showed that the phage particle contains at least nine protein bands with molecular weights ranging 15-100 kDa. vB_AbaM-IME-AB2 could adsorb its host cells in 9 min with an adsorption rate more than 99% and showed a short latent period (20 min) and a small burst size (62 pfu/cell). It could form clear plaques in the double-layer assay and clear its host's suspension in just 4 hours. Whole genome of vB_AbaM-IME-AB2 was sequenced and annotated and the results showed that its genome is a double-stranded DNA molecule consisting of 43,665 nucleotides. The genome has a G + C content of 37.5% and 82 putative coding sequences (CDSs). We compared the characteristics and complete genome sequence of all known Acinetobacter baumannii bacteriophages. There are only three that have been sequenced Acinetobacter baumannii phages AB1, AP22, and phiAC-1, which have a relatively high similarity and own a coverage of 65%, 50%, 8% respectively when compared with our phage vB_AbaM-IME-AB2. A nucleotide alignment of the four Acinetobacter baumannii phages showed that some CDSs are similar, with no significant rearrangements observed. Yet some sections of these strains of phage are nonhomologous. vB_AbaM-IME-AB2 was a novel and unique A. baumannii bacteriophage. These findings suggest a common

  10. ING function in apoptosis in diverse model systems.

    Science.gov (United States)

    Shah, Sitar; Smith, Heather; Feng, Xiaolan; Rancourt, Derrick E; Riabowol, Karl

    2009-02-01

    Genetic studies in model organisms have shown that programmed cell death (apoptosis) plays a significant role during development, where a deficiency in apoptosis results in severe and diverse diseases. Dysregulation of apoptosis also contributes to a variety of human diseases, such as cancer and autoimmune diseases. ING family proteins (ING1-ING5) are involved in many cellular processes, and appear to play a significant role in apoptosis. Loss or downregulation of ING protein function is frequently observed in different tumour types, many of which are resistant to apoptosis, thus warranting their classification as type II tumour suppressors. Several different in vitro and in vivo models have explored the role of ING proteins in regulating apoptosis. In this review, we discuss the progress that has been made in understanding ING protein function in apoptosis using in vitro studies and Mus musculus, Xenopus laevis, and Caenorhabditis elegans experimental models, with an emphasis on ING1 and ING3.

  11. Cold-inducible RNA-binding protein inhibits neuron apoptosis through the suppression of mitochondrial apoptosis.

    Science.gov (United States)

    Zhang, Hai-Tao; Xue, Jing-Hui; Zhang, Zhi-Wen; Kong, Hai-Bo; Liu, Ai-Jun; Li, Shou-Chun; Xu, Dong-Gang

    2015-10-01

    Cold-inducible RNA-binding protein (CIRP) is induced by mild hypothermia in several mammals, but the precise mechanism by which CIRP mediates hypothermia-induced neuroprotection remains unknown. We aimed to investigate the molecular mechanisms by which CIRP protects the nervous system during mild hypothermia. Rat cortical neurons were isolated and cultured in vitro under mild hypothermia (32°C). Apoptosis was measured by annexin V and propidium iodide staining, visualized by flow cytometry. Neuron ultrastructure was visualized by transmission electron microscopy. CIRP overexpression and knockdown were achieved via infection with pL/IRES/GFP-CIRP and pL/shRNA/F-CIRP-A lentivirus. RT(2) Profiler PCR Array Pathway Analysis and western blotting were used to evaluate the effects of CIRP overexpresion/knockdown on the neurons׳ transcriptome. Neuron late apoptosis was significantly reduced at day 7 of culture by 12h hypothermia, but neuron ultrastructure remained relatively intact. RT(2) Profiler PCR Array Pathway Analysis of 84 apoptosis pathway-associated factors revealed that mild hypothermia and CIRP overexpression induce similar gene expression profiles, specifically alterations of genes implicated in the mitochondrial apoptosis pathway. Mild hypothermia-treated neurons up-regulated 12 and down-regulated 38 apoptosis pathway-associated genes. CIRP-overexpressing neurons up-regulated 15 and down-regulated 46 genes. CIRP-knocked-down hypothermia-treated cells up-regulated 9 and down-regulated 40 genes. Similar results were obtained at the protein level. In conclusion, CIRP may inhibit neuron apoptosis through the suppression of the mitochondria apoptosis pathway during mild hypothermia.

  12. Lytic spondylolisthesis in helicopter pilots.

    Science.gov (United States)

    Froom, P; Froom, J; Van Dyk, D; Caine, Y; Ribak, J; Margaliot, S; Floman, Y

    1984-06-01

    Trauma to the back from the force of chronic stress is thought to be an etiologic factor in isthmic spondylolisthesis (SLL). The relationship of first degree spondylolisthesis to low back pain (LBP) is controversial. We compare the prevalence of SLL in helicopter pilots who are subject to strong vibrational forces, with other airforce personnel. Helicopter pilots had more than a four times higher prevalence of SLL (4.5%) than did cadets (1.0%) and transport pilots (0.9%). Low back pain was more frequent in pilots with SLL than in those without this lesion but in no case was the pain disabling or the defect progressive. We conclude that SLL may be induced by vibrational forces and although SLL is associated with LBP, the pain was little clinical significance.

  13. Autophagy and apoptosis: rivals or mates?

    Institute of Scientific and Technical Information of China (English)

    Yan Cheng; Jin-Ming Yang

    2013-01-01

    Autophagy,a cellular process of "self-eating" by which intracellular components are degraded within the lysosome,is an evolutionarily conserved response to various stresses.Autophagy is associated with numerous patho-physiological conditions,and dysregulation of autophagy contributes to the pathogenesis of a variety of human diseases including cancer.Depending on context,activation of autophagy may promote either cell survival or death,two major events that determine pathological process of many illnesses.Importantly,the activity of autophagy is often associated with apoptosis,another critical cellular process determining cellular fate.A better understanding of biology of autophagy and its implication in human health and disorder,as well as the relationship between autophagy and apoptosis,has the potential of facilitating the development of autophagy-based therapeutic interventions for human diseases such as cancer.

  14. Ordering the multiple pathways of apoptosis.

    Science.gov (United States)

    Park, D S; Stefanis, L; Greene, L A

    1997-11-01

    Apoptosis plays an important role in development, homeostasis, and disease. Current work has suggested that apoptosis can be evoked by multiple stimuli that, in turn, initiate distinct death pathways. Recently, exciting advances have been made in the understanding of biochemical pathways that regulate apoptotic processes. These pathways contain both evolutionarily conserved elements and components that are dependent on the death stimulus and cell context. Accordingly, this review focuses on the compositions and relative ordering of the apoptotic pathways in four different death paradigms: activation of receptors of the Fas ligand, destruction by cytotoxic T lymphocytes, exposure to DNA damaging agents, and loss of support by neurotrophic factors. These examples illustrate the conservation and divergence in the ways that death pathways are composed and ordered. (Trends Cardiovasc Med 1997;7:294-301). © 1997, Elsevier Science Inc.

  15. THE CONSEQUENCES OF APOPTOSIS IN AUTOIMMUNITY

    Science.gov (United States)

    Lleo, Ana; Selmi, Carlo; Invernizzi, Pietro; Podda, Mauro; Gershwin, M. Eric

    2008-01-01

    The clearance of apoptotic cells is a highly regulated mechanism, normally associated with anti-inflammatory response. During early stages of apoptosis the cell is promptly recognized and engulfed by professional phagocytes or tissue cells to avoid the outflow of intracellular content and limit the immunological reaction against released antigens. However, increasing evidences suggest that impairment in the uptake of apoptotic cell debris is linked to the development of autoimmunity. In fact, autoantigens have been demonstrated to be content within apoptotic bodies and apoptotic cells seems to be critical in the presentation of antigens, activation of innate immunity and regulation of macrophage cytokine secretion. We herein review the known mechanisms for regulating the uptake of the products of apoptosis in the development of autoimmunity. PMID:18513925

  16. Apoptosis and Its Suppression in Hepatocytes Culture

    OpenAIRE

    Mukwena, Nyaradzo T.; Al-Rubeai, Mohamed

    2004-01-01

    In order to achieve the goal of developing extracorporeal liver support devices, it is necessary to optimise bioprocess environment such that viability and function are maximised. Optimising culture medium composition and controlling the constitution of the cellular microenvironment within the bioreactor have for many years been considered vital to achieving these aims. Coupled to this is the need to understand apoptosis, the prime suspect in the demise of animal cultures, including those of ...

  17. Safrole oxide inhibits angiogenesis by inducing apoptosis.

    Science.gov (United States)

    Zhao, Jing; Miao, Junying; Zhao, Baoxiang; Zhang, Shangli; Yin, Deling

    2005-06-01

    Our previous studies indicate that 3, 4-(methylenedioxy)-1-(2', 3'-epoxypropyl)-benzene (safrole oxide), a newly synthesized compound, induces apoptosis in vascular endothelial cells (VECs) and A549 lung cancer cells. To our knowledge, the inhibition of angiogenesis by safrole oxide has not been reported yet. We report here that cultured rat aorta treated with safrole oxide exhibited a significant microvessel reduction as determined by counting the number of microvessels in a phase contrast microscope. There were more microvessels formed in the presence of A549 lung cancer cells in rat aorta model, while a dramatic inhibition of angiogenesis was obtained by adding 220-450 micromol l(-1) of safrole oxide to the growth medium (Psafrole oxide produced only some abortive endothelial cells but not microvessels. Furthermore, safrole oxide induced antiangiogenic effect in the chorioallantoic membranes (CAM) as a dose dependent manner. Eggs treated with 2-11 micromol 100 microl(-1) per egg of the safrole oxide for 48 h exhibited a significant reduction in blood vessel area of the CAM, a process likely mediated by apoptosis as demonstrated by DNA fragmentation. Our results suggest that safrole oxide has antiangiogenic activity and this effect might occur by induction of cellular apoptosis.

  18. Apoptosis de fibroblastos gingivales en periodontitis

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    Roger Mauricio Arce

    2007-09-01

    Full Text Available Introducción: Los fibroblastos gingivales humanos (FGH tienen un papel importante en la enfermedad periodontal, pues alteran su normal funcionamiento en respuesta a estímulos pro-inflamatorios. Se cree que los fibroblastos se pueden eliminar anormalmente por medio de apoptosis en periodontitis. El propósito de este estudio es determinar y cuantificar la apoptosis de FGH en biopsias del periodonto de individuos sanos y con enfermedad periodontal. Métodos: Se realizó un estudio clínico descriptivo de corte transversal en personas con diagnóstico de salud periodontal (S, gingivitis (G y periodontitis crónica (PC. Se tomaron biopsias escisionales y se hicieron tinciones inmunohistoquímicas (hematoxilina-eosina, caspasa-3 y vimentina. Las placas se interpretaron por histopatología y se digitalizaron para cuantificar las células apoptóticas. Todos los datos se analizaron con un software estadístico para encontrar diferencias significativas (p0.5, r²=0.02; mientras que para las células inflamatorias se encontró una relación proporcional significativa (p<0.05, r²=0.2018. Conclusiones: Los resultados permiten concluir que tanto los fibroblastos gingivales como las células inflamatorias presentan apoptosis manifiesta por la expresión de caspasa-3, y ésta se incrementa significativamente en gingivitis y enfermedad periodontal.

  19. Hormonal regulation of apoptosis an ovarian perspective.

    Science.gov (United States)

    Hsu, S Y; Hsueh, A J

    1997-07-01

    Using the ovary as a model system for studying the hormonal regulation of apoptosis, recent studies have revealed that the survival of growing follicles is under the regulation of a complex array of hormones through endocrine, paracrine, autocrine, or juxtacrine mechanism in a development-dependent manner. More effort is needed, however, to identify tissue-specific factors required for the survival of ovarian somatic and germ cells at specific stage of development. New insights based on characterization of conserved apoptotic effectors, both extracellular and intracellular, have suggested that apoptosis in ovarian cells may be mediated by apoptotic programs common to other cells but using specific members of the death domain proteins as well as ced-9/Bcl-2 and ced-3/ICE caspase families of genes. Future studies may provide new therapeutic modalities for different ovarian diseases caused by aberrant regulation of apoptosis in ovarian cells, including premature ovarian failure and polycystic ovarian syndrome. (Trends Endocrinol Metab 1997;8:207-213). (c) 1997, Elsevier Science Inc.

  20. Control of apoptosis by asymmetric cell division.

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    Julia Hatzold

    2008-04-01

    Full Text Available Asymmetric cell division and apoptosis (programmed cell death are two fundamental processes that are important for the development and function of multicellular organisms. We have found that the processes of asymmetric cell division and apoptosis can be functionally linked. Specifically, we show that asymmetric cell division in the nematode Caenorhabditis elegans is mediated by a pathway involving three genes, dnj-11 MIDA1, ces-2 HLF, and ces-1 Snail, that directly control the enzymatic machinery responsible for apoptosis. Interestingly, the MIDA1-like protein GlsA of the alga Volvox carteri, as well as the Snail-related proteins Snail, Escargot, and Worniu of Drosophila melanogaster, have previously been implicated in asymmetric cell division. Therefore, C. elegans dnj-11 MIDA1, ces-2 HLF, and ces-1 Snail may be components of a pathway involved in asymmetric cell division that is conserved throughout the plant and animal kingdoms. Furthermore, based on our results, we propose that this pathway directly controls the apoptotic fate in C. elegans, and possibly other animals as well.

  1. Alcohol and Apoptosis: Friends or Foes?

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    Ana Rodriguez

    2015-11-01

    Full Text Available Alcohol abuse causes 79,000 deaths stemming from severe organ damage in the United States every year. Clinical manifestations of long-term alcohol abuse on the cardiac muscle include defective contractility with the development of dilated cardiomyopathy and low-output heart failure; which has poor prognosis with less than 25% survival for more than three years. In contrast, low alcohol consumption has been associated with reduced risk of cardiovascular disease, however the mechanism of this phenomenon remains elusive. The aim of this study was to determine the significance of apoptosis as a mediating factor in cardiac function following chronic high alcohol versus low alcohol exposure. Adult rats were provided 5 mM (low alcohol, 100 mM (high alcohol or pair-fed non-alcohol controls for 4–5 months. The hearts were dissected, sectioned and stained with cresyl violet or immunohistochemically for caspase-3, a putative marker for apoptosis. Cardiomyocytes were isolated to determine the effects of alcohol exposure on cell contraction and relaxation. High alcohol animals displayed a marked thinning of the left ventricular wall combined with elevated caspase-3 activity and decreased contractility. In contrast, low alcohol was associated with increased contractility and decreased apoptosis suggesting an overall protective mechanism induced by low levels of alcohol exposure.

  2. Keratinocyte Apoptosis is Decreased in Psoriatic Epidermis

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    Fatma Eskioğlu

    2009-12-01

    Full Text Available Background and Design: Abnormal differentiation and hyperproliferation of keratinocytes are the hallmarks of psoriasis vulgaris. Although psoriasis vulgaris is generally accepted as a disease of decreased keratinocyte apoptosis, the results are contradictory. The aim of the current study is to investigate whether decreased keratinocyte apoptosis contributes to the formation of a thickened epidermis as increased keratinocyte proliferation. Material and Method: Forty-three untreated psoriasis vulgaris patients and 20 healthy control subjects were included into the study. Biopsy specimens taken from the enrollee were evaluated by immunohistochemical staining for Ki-67 expressions to show the proliferation of keratinocytes and by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL method to show the apoptotic keratinocytes. Results: Apoptotic index (percentage of the TUNEL positive cells was significantly lower in psoriatic epidermis (0.33±0.64 than in normal epidermis (0.75±0.85; whereas Ki-67 index (percentage of positively staining cells for Ki-67 was significantly higher in psoriatic epidermis (30.86±10.49 than in normal epidermis (11.65±2.98, (p=0.021 and p=0.00; respectively. Conclusion: Decreased keratinocyte apoptosis also contribute to increased epidermal thickness in psoriasis as well as increased keratinocyte proliferation.

  3. Apoptosis and oxidative stress in neurodegenerative diseases.

    Science.gov (United States)

    Radi, Elena; Formichi, Patrizia; Battisti, Carla; Federico, Antonio

    2014-01-01

    Neurodegenerative disorders affect almost 30 million individuals leading to disability and death. These disorders are characterized by pathological changes in disease-specific areas of the brain and degeneration of distinct neuron subsets. Despite the differences in clinical manifestations and neuronal vulnerability, the pathological processes appear similar, suggesting common neurodegenerative pathways. Apoptosis seems to play a key role in the progression of several neurologic disorders like Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis as demonstrated by studies on animal models and cell lines. On the other hand, research on human brains reported contradictory results. However, many dying neurons have been detected in brains of patients with neurodegenerative diseases, and these conditions are often associated with significant cell loss accompanied by typical morphological features of apoptosis such as chromatin condensation, DNA fragmentation, and activation of cysteine-proteases, caspases. Cell death and neurodegenerative conditions have been linked to oxidative stress and imbalance between generation of free radicals and antioxidant defenses. Multiple sclerosis, stroke, and neurodegenerative diseases have been associated with reactive oxygen species and nitric oxide. Here we present an overview of the involvement of neuronal apoptosis and oxidative stress in the most important neurodegenerative diseases, mainly focusing the attention on several genetic disorders, discussing the interaction between primary genetic abnormalities and the apoptotic pathways.

  4. Radiosensitivity of tumor cells. Oncogenes and apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Peltenburg, L. T. C. [Leiden Univ., Leiden (Netherlands). Dept. of Clinical Oncology

    2000-12-01

    The success of treatment of cancer patients by radiotherapy largely depends on tumor radiosensitivity. Several molecular factors that determine the sensitivity of tumor cells to ionizing radiation have been identified during the last couple of years. Some of these factors are known as oncogenes and tumor suppressor genes. This review focuses on the influence of some of these molecular factors on a major determinant of radiosensitivity: i. e. programmed cell death or apoptosis. The crucial molecular step in ionizing radiation-induced apoptosis is the release of mitochondrial cytochrome c into the cell's cytosol. The ways the tumor suppressor protein p53, as well as the oncogenes ras and raf, c-myc and Bcl-2 can influence this process at different stages are presented. As will be discussed, the result of activation of an oncoprotein on tumor radiosensitivity depends on its mechanism of action and on the presence of other (oncogenic) factors, since complex interactions among many molecular factors determine the delicate balance between cell proliferation and cell death. The ongoing identification and characterization of factors influencing apoptosis will eventually make it possible to predict tumor radiosensitivity and thereby improve cancer treatment.

  5. Neuronal apoptosis: signal and cell diversity

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    Lina Vanessa Becerra

    2009-12-01

    Full Text Available Programmed cell death occurs as a physiological process during development. In the brain and spinal cord this event determines the number and location of the different cell types. In adulthood, programmed cell death or apoptosis is more restricted but it may play a major role in different acute and chronic pathological entities. However, in contrast to other tissues where apoptosis has been widely documented from a morphological point of view, in the central nervous system complete anatomical evidence of apoptosis is scanty. In spite of this there is consensus about the activation of different signal systems associated to programmed cell death. In the present article we attempt to summarize the main apoptotic pathways so far identified in nervous tissue. Considering that apoptotic pathways are multiple, the neuronal cell types are highly diverse and specialized and that neuronal response to injury and survival depends upon tissue context, (i.e., preservation of connectivity, glial integrity and cell matrix, blood supply and trophic factors availability what is relevant for the apoptotic process in a sector of the brain may not be important in another.

  6. Resveratrol induces apoptosis in pancreatic cancer cells

    Institute of Scientific and Technical Information of China (English)

    ZHOU Jia-hua; CHENG Hai-yan; YU Ze-qian; HE Dao-wei; PAN Zheng; YANG De-tong

    2011-01-01

    Background Pancreatic cancer is one of the most lethal human cancers with a very low survival rate of 5 years.Conventional cancer treatments including surgery, radiation, chemotherapy or combinations of these show little effect on this disease. Several proteins have been proved critical to the development and the progression of pancreatic cancer.The aim of this study was to investigate the effect of resveratrol on apoptosis in pancreatic cancer cells.Methods Several pancreatic cancer cell lines were screened by resveratrol, and its toxicity was tested by normal pancreatic cells. Western blotting was then performed to analyze the molecular mechanism of resveratrol induced apoptosis of pancreatic cancer cell lines.Results In the screened pancreatic cancer cell lines, capan-2 and colo357 showed high sensitivity to resveratrol induced apoptosis. Resveratrol exhibited insignificant toxicity to normal pancreatic cells. In resveratrol sensitive cells,capan-2 and colo357, the activation of caspase-3 was detected and showed significant caspase-3 activation upon resveratrol treatment; p53 and p21 were also detected up-regulated upon resveratrol treatment.Conclusion Resveratrol provides a promising anti-tumor stratagy to fight against pancreatic cancer.

  7. Lipid Metabolism, Apoptosis and Cancer Therapy

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    Chunfa Huang

    2015-01-01

    Full Text Available Lipid metabolism is regulated by multiple signaling pathways, and generates a variety of bioactive lipid molecules. These bioactive lipid molecules known as signaling molecules, such as fatty acid, eicosanoids, diacylglycerol, phosphatidic acid, lysophophatidic acid, ceramide, sphingosine, sphingosine-1-phosphate, phosphatidylinositol-3 phosphate, and cholesterol, are involved in the activation or regulation of different signaling pathways. Lipid metabolism participates in the regulation of many cellular processes such as cell growth, proliferation, differentiation, survival, apoptosis, inflammation, motility, membrane homeostasis, chemotherapy response, and drug resistance. Bioactive lipid molecules promote apoptosis via the intrinsic pathway by modulating mitochondrial membrane permeability and activating different enzymes including caspases. In this review, we discuss recent data in the fields of lipid metabolism, lipid-mediated apoptosis, and cancer therapy. In conclusion, understanding the underlying molecular mechanism of lipid metabolism and the function of different lipid molecules could provide the basis for cancer cell death rationale, discover novel and potential targets, and develop new anticancer drugs for cancer therapy.

  8. Apoptosis signal-regulating kinase 1 mediates denbinobin-induced apoptosis in human lung adenocarcinoma cells

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    Pan Shiow-Lin

    2009-05-01

    Full Text Available Abstract In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1 in denbinobin-induced apoptosis in human lung adenocarcinoma (A549 cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN, two antioxidants (N-acetyl-L-cysteine (NAC and glutathione (GSH, a c-Jun N-terminal kinase (JNK inhibitor (SP600125, and an activator protein-1 (AP-1 inhibitor (curcumin. Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis.

  9. Enterohemorrhagic Escherichia coli hemolysin employs outer membrane vesicles to target mitochondria and cause endothelial and epithelial apoptosis.

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    Martina Bielaszewska

    Full Text Available Enterohemorrhagic Escherichia coli (EHEC strains cause diarrhea and hemolytic uremic syndrome resulting from toxin-mediated microvascular endothelial injury. EHEC hemolysin (EHEC-Hly, a member of the RTX (repeats-in-toxin family, is an EHEC virulence factor of increasingly recognized importance. The toxin exists as free EHEC-Hly and as EHEC-Hly associated with outer membrane vesicles (OMVs released by EHEC during growth. Whereas the free toxin is lytic towards human endothelium, the biological effects of the OMV-associated EHEC-Hly on microvascular endothelial and intestinal epithelial cells, which are the major targets during EHEC infection, are unknown. Using microscopic, biochemical, flow cytometry and functional analyses of human brain microvascular endothelial cells (HBMEC and Caco-2 cells we demonstrate that OMV-associated EHEC-Hly does not lyse the target cells but triggers their apoptosis. The OMV-associated toxin is internalized by HBMEC and Caco-2 cells via dynamin-dependent endocytosis of OMVs and trafficked with OMVs into endo-lysosomal compartments. Upon endosome acidification and subsequent pH drop, EHEC-Hly is separated from OMVs, escapes from the lysosomes, most probably via its pore-forming activity, and targets mitochondria. This results in decrease of the mitochondrial transmembrane potential and translocation of cytochrome c to the cytosol, indicating EHEC-Hly-mediated permeabilization of the mitochondrial membranes. Subsequent activation of caspase-9 and caspase-3 leads to apoptotic cell death as evidenced by DNA fragmentation and chromatin condensation in the intoxicated cells. The ability of OMV-associated EHEC-Hly to trigger the mitochondrial apoptotic pathway in human microvascular endothelial and intestinal epithelial cells indicates a novel mechanism of EHEC-Hly involvement in the pathogenesis of EHEC diseases. The OMV-mediated intracellular delivery represents a newly recognized mechanism for a bacterial toxin to

  10. Intracoronary levosimendan during ischemia prevents myocardial apoptosis.

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    Markus eMalmberg

    2012-02-01

    Full Text Available Background. Levosimendan is a calcium-sensitizing inotropic agent that prevents myocardial contractile depression following cardiac surgery. Levosimendan has also anti-apoptotic properties, but the role of this mechanism is not clear. We studied whether levosimendan prevents cardiomyocyte apoptosis and post-operative stunning after either intracoronary administration or intravenous infusion in an experimental model. Methods. Pigs (n=24 were subjected to 40 minutes of global, cardioplegic ischemia under cardiopulmonary bypass and 240 minutes of reperfusion. L-IV group received intravenous infusion of levosimendan (65 μg/kg 40 minutes before ischemia and L-IC group received levosimendan (65 μg/kg during ischemia administered intracoronary. Control group was operated without levosimendan. Echocardiography was performed to all animals. Apoptosis was determined from transmyocardial biopsies taken from left ventricle using TUNEL assay and immunohistochemistry of active caspace-3. Results. Apoptosis was induced after ischemia-reperfusion in all groups (pre L-IV 0.002±0.004 % vs. post L-IV 0.020±0.017 % p=0.02, pre L-IC 0.001±0.004 % vs. post L-IC 0.020±0.017 % p<0.001, pre control 0.007±0.013 % vs. post control 0.062±0.044 % p=0.01. The amount of apoptosis was higher in the controls, compared with the L-IV (p=0.03 and the L-IC (p=0.03 groups. Longitudinal left ventricular contraction was significantly reduced in the L-IC and the control groups when compared to the L-IV group (L-IV 0.75±0.12 mm vs. L-IC 0.53±0.11 mm p=0.003, L-IV vs. control 0.54±0.11 p=0.01. Conclusions. Both intracoronary administration and pre-ischemic intravenous infusion of levosimendan equally prevented apoptosis, but intravenous administration was required for optimal preservation of the post-operative systolic left ventricle function.

  11. Host and Viral Factors in HIV-Mediated Bystander Apoptosis

    Science.gov (United States)

    Garg, Himanshu; Joshi, Anjali

    2017-01-01

    Human immunodeficiency virus (HIV) infections lead to a progressive loss of CD4 T cells primarily via the process of apoptosis. With a limited number of infected cells and vastly disproportionate apoptosis in HIV infected patients, it is believed that apoptosis of uninfected bystander cells plays a significant role in this process. Disease progression in HIV infected individuals is highly variable suggesting that both host and viral factors may influence HIV mediated apoptosis. Amongst the viral factors, the role of Envelope (Env) glycoprotein in bystander apoptosis is well documented. Recent evidence on the variability in apoptosis induction by primary patient derived Envs underscores the role of Env glycoprotein in HIV disease. Amongst the host factors, the role of C-C Chemokine Receptor type 5 (CCR5), a coreceptor for HIV Env, is also becoming increasingly evident. Polymorphisms in the CCR5 gene and promoter affect CCR5 cell surface expression and correlate with both apoptosis and CD4 loss. Finally, chronic immune activation in HIV infections induces multiple defects in the immune system and has recently been shown to accelerate HIV Env mediated CD4 apoptosis. Consequently, those factors that affect CCR5 expression and/or immune activation in turn indirectly regulate HIV mediated apoptosis making this phenomenon both complex and multifactorial. This review explores the complex role of various host and viral factors in determining HIV mediated bystander apoptosis. PMID:28829402

  12. Scavenger receptor BI and HDL regulate thymocyte apoptosis in sepsis

    Science.gov (United States)

    Guo, Ling; Zheng, Zhong; Ai, Junting; Howatt, Deborah A.; Mittelstadt, Paul R.; Thacker, Seth; Daugherty, Alan; Ashwell, Jonathan D.; Remaley, Alan T.; Li, Xiang-An

    2014-01-01

    Objective Thymocyte apoptosis is a major event in sepsis; however, how this process is regulated remains poorly understood. Approach and Results Septic stress induces glucocorticoids (GC) production which triggers thymocyte apoptosis. Here, we used scavenger receptor BI (SR-BI) null mice, which are completely deficient in inducible GC (iGC) in sepsis, to investigate the regulation of thymocyte apoptosis in sepsis. Cecal ligation and puncture (CLP) induced profound thymocyte apoptosis in SR-BI+/+ mice, but no thymocyte apoptosis in SR-BI−/− mice due to lack of iGC. Unexpectedly, supplementation of GC only partly restored thymocyte apoptosis in SR-BI−/− mice. We demonstrated that HDL is a critical modulator for thymocyte apoptosis. SR-BI+/+ HDL significantly enhanced GC-induced thymocyte apoptosis but SR-BI−/− HDL had no such activity. Further study revealed that SR-BI+/+ HDL modulates GC-induced thymocyte apoptosis via promoting glucocorticoid receptor translocation, but SR-BI−/− HDL loses such regulatory activity. To understand why SR-BI−/− HDL loses its regulatory activity, we analyzed HDL cholesterol contents. There was 3-fold enrichment of unesterified cholesterol in SR-BI−/− HDL compared with SR-BI+/+ HDL. Normalization of unesterified cholesterol in SR-BI−/− HDL by probucol administration or LCAT expression restored GC-induced thymocyte apoptosis, and incorporating unesterified cholesterol into SR-BI+/+ HDL rendered SR-BI+/+ HDL dysfunctional. Using lckCre-GRfl/fl mice in whom thymocytes lack CLP-induced thymocyte apoptosis, we showed that lckCre-GRfl/fl mice were significantly more susceptible to CLP-induced septic death than GRfl/fl control mice, suggesting that GC-induced thymocyte apoptosis is required for protection against sepsis. Conclusions The findings in this study reveal a novel regulatory mechanism of thymocyte apoptosis in sepsis by SR-BI and HDL. PMID:24603680

  13. Spironolactone induces apoptosis in human mononuclear cells. Association between apoptosis and cytokine suppression

    DEFF Research Database (Denmark)

    Mikkelsen, Martin; Sønder, S U; Nersting, J;

    2006-01-01

    Spironolactone (SPIR) has been described to suppress accumulation of pro-inflammatory cytokines. Here, the suppression of TNF-alpha in lipopolysaccharide (LPS)-stimulated mononuclear cell cultures was confirmed. However, SPIR was also found to induce apoptosis, prompting the investigations...... of a possible association between the two effects: The apoptosis-inducing and the cytokine-suppressive effects of SPIR correlated with regard to the effective concentration range. Also, pre-incubation experiments demonstrated a temporal separation of the two effects of ... preceding apoptosis. An association between the two effects was also seen when testing several SPIR analogues. Contrary to TNF-alpha, the levels of IL-1beta increased in SPIR-treated cultures. However, the amount of IL-1beta in the supernatants depended upon the order of SPIR and LPS addition, as IL-1beta...

  14. Spironolactone induces apoptosis in human mononuclear cells. Association between apoptosis and cytokine suppression

    DEFF Research Database (Denmark)

    Mikkelsen, Martin; Sønder, S U; Nersting, J;

    2006-01-01

    Spironolactone (SPIR) has been described to suppress accumulation of pro-inflammatory cytokines. Here, the suppression of TNF-alpha in lipopolysaccharide (LPS)-stimulated mononuclear cell cultures was confirmed. However, SPIR was also found to induce apoptosis, prompting the investigations...... of a possible association between the two effects: The apoptosis-inducing and the cytokine-suppressive effects of SPIR correlated with regard to the effective concentration range. Also, pre-incubation experiments demonstrated a temporal separation of the two effects of TNF-alpha suppression...... preceding apoptosis. An association between the two effects was also seen when testing several SPIR analogues. Contrary to TNF-alpha, the levels of IL-1beta increased in SPIR-treated cultures. However, the amount of IL-1beta in the supernatants depended upon the order of SPIR and LPS addition, as IL-1beta...

  15. Artesunate induces AIF-dependent apoptosis in A549 cells

    Science.gov (United States)

    Zhou, Chen-juan; Chen, Tong-Sheng

    2012-03-01

    Artesunate (ART), a semi-synthetic derivative of the sesquiterpene artemisinin extracted from the Chinese herb Artemisia annua, exerts a broad spectrum of clinical activity against human cancers. It has been shown that ART induces cancer cells death through apoptosis pathway. This study investigated whether ART treatment induced reactive oxygen species (ROS)-dependent cell death in the apoptosis fashion in human lung adenocarconoma A549 cell line and the proapoptotic protein apoptosis inducing factor (AIF) is involved in ART-induced apoptosis. Cells treated with ART exhibited typical apoptotic morphology as chromatin condensation, margination and shrunken nucleus. ART treatment also induced a loss of mitochondrial membrane potential and AIF release from mitochondria. Silencing AIF can remarkable attenuated ART-induced apoptosis. Collectively, ART induces apoptosis by caspase-independent intrinsic pathway in A549 cells.

  16. Regulation of apoptosis by the papillomavirus E6 oncogene

    Institute of Scientific and Technical Information of China (English)

    Ting-Ting Li; Li-Na Zhao; Zhi-Guo Liu; Ying Han; Dai-Ming Fan

    2005-01-01

    Infection with human papillomaviruses is strongly associated with the development of multiple cancers including esophageal squamous cell carcinoma. The HPV E6 gene is essential for the oncogenic potential of HPV.The recgulation of apoptosis by oncogene has been relatel to carcinogenesis closely; therefore, the modulation of E6 on cellular apoptosis has become a hot research topic recently. Inactivation of the pro-apoptotic tumor suppressor p53 by E6 is an important mechanism by which E6promotes cell growth; it is expected that inactivation of p53 by E6 should lead to a reduction in cellular apoptosis,numerous studies showed that E6 could in fact sensitize cells to apoptosis. The molecular basis for apoptosis modulation by E6 is poorly understood. In this article, we will present an overview of observations and current understanding of molecular basis for E6-induced apoptosis.

  17. Aspartame-induced apoptosis in PC12 cells.

    Science.gov (United States)

    Horio, Yukari; Sun, Yongkun; Liu, Chuang; Saito, Takeshi; Kurasaki, Masaaki

    2014-01-01

    Aspartame is an artificial sweetner added to many low-calorie foods. The safety of aspartame remains controversial even though there are many studies on its risks. In this study, to understand the physiological effects of trace amounts of artificial sweetners on cells, the effects of aspartame on apoptosis were investigated using a PC12 cell system. In addition, the mechanism of apoptosis induced by aspartame in PC12 cells and effects on apoptotic factors such as cytochrome c, apoptosis-inducing factor, and caspase family proteins were studied by Western blotting and RT-PCR. Aspartame-induced apoptosis in PC12 cells in a dose-dependent manner. In addition, aspartame exposure increased the expressions of caspases 8 and 9, and cytochrome c. These results indicate that aspartame induces apoptosis mainly via mitochondrial pathway involved in apoptosis due to oxigen toxicity.

  18. Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells.

    Science.gov (United States)

    Bhattacharya, Sujoy; Ray, Ramesh M; Johnson, Leonard R

    2014-03-01

    Homeostasis of the gastrointestinal epithelium is dependent upon a balance between cell proliferation and apoptosis. Cyclin-dependent kinases (Cdks) are well known for their role in cell proliferation. Previous studies from our group have shown that polyamine-depletion of intestinal epithelial cells (IEC-6) decreases cyclin-dependent kinase 2 (Cdk2) activity, increases p53 and p21Cip1 protein levels, induces G1 arrest, and protects cells from camptothecin (CPT)-induced apoptosis. Although emerging evidence suggests that members of the Cdk family are involved in the regulation of apoptosis, their roles directing apoptosis of IEC-6 cells are not known. In this study, we report that inhibition of Cdk1, 2, and 9 (with the broad range Cdk inhibitor, AZD5438) in proliferating IEC-6 cells triggered DNA damage, activated p53 signaling, inhibited proliferation, and induced apoptosis. By contrast, inhibition of Cdk2 (with NU6140) increased p53 protein and activity, inhibited proliferation, but had no effect on apoptosis. Notably, AZD5438 sensitized, whereas, NU6140 rescued proliferating IEC-6 cells from CPT-induced apoptosis. However, in colon carcinoma (Caco-2) cells with mutant p53, treatment with either AZD5438 or NU6140 blocked proliferation, albeit more robustly with AZD5438. Both Cdk inhibitors induced apoptosis in Caco-2 cells in a p53-independent manner. In serum starved quiescent IEC-6 cells, both AZD5438 and NU6140 decreased TNF-α/CPT-induced activation of p53 and, consequently, rescued cells from apoptosis, indicating that sustained Cdk activity is required for apoptosis of quiescent cells. Furthermore, AZD5438 partially reversed the protective effect of polyamine depletion whereas NU6140 had no effect. Together, these results demonstrate that Cdks possess opposing roles in the control of apoptosis in quiescent and proliferating cells. In addition, Cdk inhibitors uncouple proliferation from apoptosis in a p53-dependent manner.

  19. Molecular mechanisms of TRAIL-induced apoptosis of cancer cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    @@Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) is a recently identified member of the tumor necrosis factor (TNF) family[1]. Numerous studies indicate that TRAIL can induce apoptosis of cancer cells but not of normal cells, pointing to the possibility of de-veloping TRAIL into a cancer drug[2-4]. This review will summary the molecular mechanisms of TRAIL-induced apoptosis and discuss the questions to be resolved in this field.

  20. Apoptosis and Its Significance in Oral Diseases: An Update

    Directory of Open Access Journals (Sweden)

    Megha Jain

    2013-01-01

    Full Text Available Apoptosis is a well defined mode of cell death which plays an imperative role in the development, regulation, and maintenance of the cell populations in multicellular organisms. Apoptosis is implicated in both health and diseases. Errors in apoptotic mechanisms have been allied to a wide range of pathologies including oral diseases. This review presents an update focused on the role and significance of apoptosis in various oral diseases ranging from reactive to benign and malignant pathologies.

  1. Apoptosis induced by propolis in human hepatocellular carcinoma cell line.

    Science.gov (United States)

    Choi, Y H; Lee, W Y; Nam, S Y; Choi, K C; Park, Y E

    1999-07-01

    Propolis has been reported to exhibit a wide spectrum of activities including antibiotic, antiviral, anti-inflammatory, immunostimulatory and tumor carcinostatic properties. We showed propolis induced apoptosis in a human hepatoma cell line (SNU449) by FITC-Annexin V/PI staining. We also compared the apoptosis inducing effect between Korean and Commercial (Sigma # p-1010) propolis. There was no difference on apoptosis between them.

  2. Chalcones Enhance TRAIL-Induced Apoptosis in Prostate Cancer Cells

    OpenAIRE

    2009-01-01

    Chalcones exhibit chemopreventive and antitumor effects. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a naturally occurring anticancer agent that induces apoptosis in cancer cells and is not toxic to normal cells. We examined the cytotoxic and apoptotic effect of five chalcones in combination with TRAIL on prostate cancer cells. The cytotoxicity was evaluated by the MTT and LDH assays. The apoptosis was determined using flow cytometry with annexin V-FITC. Our study showe...

  3. Apoptosis of gingival fibroblasts in periodontitis.

    Directory of Open Access Journals (Sweden)

    Roger Mauricio Arce

    2009-11-01

    Full Text Available Introducción: Los fibroblastos gingivales humanos (FGH tienen un papel importante en la enfermedad periodontal, pues alteran su normal funcionamiento en respuesta a estímulos pro-inflamatorios. Se cree que los fibroblastos se pueden eliminar anormalmente por medio de apoptosis en periodontitis. El propósito de este estudio es determinar y cuantificar la apoptosis de FGH en biopsias del periodonto de individuos sanos y con enfermedad periodontal. Métodos: Se realizó un estudio clínico descriptivo de corte transversal en personas con diagnóstico de salud periodontal (S, gingivitis (G y periodontitis crónica (PC. Se tomaron biopsias escisionales y se hicieron tinciones inmunohistoquímicas (hematoxilina-eosina, caspasa-3 y vimentina. Las placas se interpretaron por histopatología y se digitalizaron para cuantificar las células apoptóticas. Todos los datos se analizaron con un software estadístico para encontrar diferencias significativas (p Resultados: La población celular total de fibroblastos tuvo un promedio de 430±67.6 en los individuos sanos y una disminución significativamente progresiva en gingivitis (270±37.1 y periodontitis crónica (206.5±69.8 (p0.5, r²=0.02; mientras que para las células inflamatorias se encontró una relación proporcional significativa (p Conclusiones: Los resultados permiten concluir que tanto los fibroblastos gingivales como las células inflamatorias presentan apoptosis manifiesta por la expresión de caspasa-3, y ésta se incrementa significativamente en gingivitis y enfermedad periodontal.

  4. Role of PUMA in methamphetamine-induced neuronal apoptosis.

    Science.gov (United States)

    Chen, Chuanxiang; Qincao, Litao; Xu, Jingtao; Du, Sihao; Huang, Enping; Liu, Chao; Lin, Zhoumeng; Xie, Wei-Bing; Wang, Huijun

    2016-01-05

    Exposure to methamphetamine (METH), a widely used illicit drug, has been shown to cause neuron apoptosis. p53 upregulated modulator of apoptosis (PUMA) is a key mediator in neuronal apoptosis. This study aimed to examine the effects of PUMA in METH-induced neuronal apoptosis. We determined PUMA protein expression in PC12 cells and SH-SY5Y cells after METH exposure using western blot. We also observed the effect of METH on neuronal apoptosis after silencing PUMA expression with siRNA using TUNEL staining and flow cytometry. Additionally, to investigate possible mechanisms of METH-induced PUMA-mediated neuronal apoptosis, we measured the protein expression of apoptotic markers, including cleaved caspase-3, cleaved PARP, Bax, B-cell leukemia/lymphoma-2 (Bcl-2) and cytochrome c (cyto c), after METH treatment with or without PUMA knockdown. Results showed that METH exposure induced cell apoptosis, increased PUMA protein levels, activated caspase-3 and PARP, elevated Bax and reduced Bcl-2 expression, as well as increased the release of cyto c from mitochondria to the cytoplasm in both PC12 and SH-SY5Y cells. All these effects were attenuated or reversed after silencing PUMA. A schematic depicting the role of PUMA in METH-induced mitochondrial apoptotic pathway was proposed. Our results suggest that PUMA plays an important role in METH-triggered apoptosis and it may be a potential target for ameliorating neuronal injury and apoptosis caused by METH.

  5. Shifting the balance of mitochondrial apoptosis: therapeutic perspectives

    Directory of Open Access Journals (Sweden)

    Simone eFulda

    2012-10-01

    Full Text Available Signaling via the intrinsic (mitochondrial pathway of apoptosis represents one of the critical signal transduction cascades that control the regulation of cell death. This pathway is typically altered in human cancers, thereby providing a suitable target for therapeutic intervention. Members of the Bcl-2 family of proteins as well as cell survival signaling cascades such as the PI3K/Akt/mTOR pathway are involved in the regulation of mitochondria-mediated apoptosis. Therefore, further insights into the molecular mechanisms that form the basis for the control of mitochondria-mediated apoptosis will likely open new perspectives to bypass evasion of apoptosis and treatment resistance in human cancers.

  6. Carbamate Pesticide-Induced Apoptosis in Human T Lymphocytes

    Directory of Open Access Journals (Sweden)

    Qing Li

    2015-04-01

    Full Text Available We previously found that carbamate pesticides induced significant apoptosis in human natural killer cells. To investigate whether carbamate pesticides also induce apoptosis in human T lymphocytes, in the present study Jurkat human T cells were treated in vitro with thiram, maneb, carbaryl or ziram. Apoptosis was determined by FITC-Annexin-V/PI staining. To explore the mechanism of apoptosis, intracellular levels of active caspase 3 and mitochondrial cytochrome-c release were determined by flow cytometry. We found that thiram, ziram, maneb and carbaryl also induced apoptosis in a time- and dose-dependent manner in the human T cells. However, the strength of the apoptosis-inducing effect differed among the pesticides, with the: thiram > ziram > maneb > carbaryl. Moreover, thiram significantly increased the intracellular level of active caspase 3 and caspase inhibitors significantly inhibited apoptosis. Thiram also significantly caused mitochondrial cytochrome-c release. These findings indicate that carbamate pesticides can induce apoptosis in human T cells, and the apoptosis is mediated by the activation of caspases and the release of mitochondrial cytochrome-c.

  7. Research Advances on Pathways of Nickel-Induced Apoptosis

    Science.gov (United States)

    Guo, Hongrui; Chen, Lian; Cui, Hengmin; Peng, Xi; Fang, Jing; Zuo, Zhicai; Deng, Junliang; Wang, Xun; Wu, Bangyuan

    2015-01-01

    High concentrations of nickel (Ni) are harmful to humans and animals. Ni targets a number of organs and produces multiple toxic effects. Apoptosis is important in Ni-induced toxicity of the kidneys, liver, nerves, and immune system. Apoptotic pathways mediated by reactive oxygen species (ROS), mitochondria, endoplasmic reticulum (ER), Fas, and c-Myc participate in Ni-induced cell apoptosis. However, the exact mechanism of apoptosis caused by Ni is still unclear. Understanding the mechanism of Ni-induced apoptosis may help in designing measures to prevent Ni toxicity. PMID:26703593

  8. The changing shape of mitochondrial apoptosis.

    Science.gov (United States)

    Wasilewski, Michał; Scorrano, Luca

    2009-08-01

    Mitochondria are key organelles in conversion of energy, regulation of cellular signaling and amplification of programmed cell death. The anatomy of the organelle matches this functional versatility in complexity and is modulated by the concerted action of proteins that impinge on its fusion-fission equilibrium. A growing body of evidence implicates changes in mitochondrial shape in the progression of apoptosis and, therefore, proteins governing such changes are likely candidates for involvement in pathogenetic mechanisms in neurodegeneration and cancer. Here, we discuss the recent advancements in our knowledge about the machinery that regulates mitochondrial shape and on the role of molecular mechanisms controlling mitochondrial morphology during cell death.

  9. Induction of Apoptosis in Protoplasts and Suspension Cultures of Plant Cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Many studies have showed that apoptosis exists in plants. Our study shows that (1) menadione(VK3) induces apoptosis in suspension cultures of carrot cells; (2) heat shock induces apoptosis in suspension cultures of tobacco cells; and (3) ethrel induces apoptosis in carrot protoplasts. Some important indications of apoptosis were observed, including DNA laddering, TUNEL-positive reaction, condensation and degradation of nuclei.

  10. Kinetic expression analysis of the cluster mdv1-mir-M9-M4, genes meq and vIL-8 differs between the lytic and latent phases of Marek's disease virus infection.

    Science.gov (United States)

    Coupeau, D; Dambrine, G; Rasschaert, D

    2012-07-01

    Marek's disease virus (GaHV-2) is an alphaherpesvirus that induces T-cell lymphoma in chickens. The infection includes both lytic and latent stages. GaHV-2 encodes three clusters of microRNAs (miRNAs) located in the internal (I)/terminal (T) repeat (R) regions. We characterized transcripts encompassing the mdv1-mir-M9-M4 and mir-M11-M1 clusters located in the IR(L)/TR(L) region, upstream and downstream from the meq oncogene, respectively. By 5'- and 3'-RACE-PCR and targeted RT-PCR, we showed that mdv1-mir-M9-M4 could be transcribed from an unspliced transcript or from at least 15 alternatively spliced transcripts covering the IR(L)/TR(L) region, encompassing the meq and vIL-8 genes and localizing the mdv1-mir-M9-M4 cluster to the first intron at the 5'-end. However, all these transcripts, whether spliced or unspliced, seemed to start at the same transcriptional start site, their transcription being driven by a single promoter, prmiRM9M4. We demonstrated alternative promoter usage for the meq and vIL-8 genes, depending on the phase of GaHV-2 infection. During the latent phase, the prmiRM9M4 promoter drove transcription of the meq and vIL-8 genes and the mdv1-mir-M9-M4 cluster in the first intron of the corresponding transcripts. By contrast, during the lytic phase, this promoter drove the transcription only of the mdv1-mir-M9-M4 cluster to generate unspliced mRNA, the meq and vIL-8 genes being transcribed principally from their own promoters. Despite the expression of meq and the mdv1-mir-M9-M4 cluster under two different transcriptional processes during the latent and lytic phases, our data provide an explanation for meq expression and mdv1-mir-M4-5P overexpression in miRNA libraries from GaHV-2-infected cells, regardless of the phase of infection.

  11. Triggering of dendritic cell apoptosis by xanthohumol.

    Science.gov (United States)

    Xuan, Nguyen Thi; Shumilina, Ekaterina; Gulbins, Erich; Gu, Shuchen; Götz, Friedrich; Lang, Florian

    2010-07-01

    Xanthohumol, a flavonoid from beer with anticancer activity is known to trigger apoptosis in a variety of tumor cells. Xanthohumol further has anti-inflammatory activity. However, little is known about the effect of xanthohumol on survival and function of immune cells. The present study thus addressed the effect of xanthohumol on dendritic cells (DCs), key players in the regulation of innate and adaptive immunity. To this end, mouse bone marrow-derived DCs were treated with xanthohumol with subsequent assessment of enzymatic activity of acid sphingomyelinase (Asm), ceramide formation determined with anti-ceramide antibodies in FACS and immunohistochemical analysis, caspase activity utilizing FITC conjugated anti-active caspase 8 or caspase 3 antibodies in FACS and by Western blotting, DNA fragmentation by determining the percentage of cells in the sub-G1 phase and cell membrane scrambling by annexin V binding in FACS analysis. As a result, xanthohumol stimulated Asm, enhanced ceramide formation, activated caspases 8 and 3, triggered DNA fragmentation and led to cell membrane scrambling, all effects virtually absent in DCs from gene targeted mice lacking functional Asm or in wild-type cells treated with sphingomyelinase inhibitor amitriptyline. In conclusion, xanthohumol stimulated Asm leading to caspase activation and apoptosis of bone marrow-derived DCs.

  12. The Adipokine Chemerin Induces Apoptosis in Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Diego Rodríguez-Penas

    2015-08-01

    Full Text Available Background: The adipokine chemerin has been associated with cardiovascular disease. We investigated the effects of chemerin on viability and intracellular signalling in murine cardiomyocytes, and the effects of insulin and TNF-α on cardiomyocyte chemerin production. Methods: Hoechst dye vital staining and cell cycle analysis were used to analyse the viability of murine cardiac cells in culture. Western blot was used to explore the phosphorylation of AKT and caspase-9 activity in neonatal rat cardiomyocytes and HL-1 cells. Finally, RT-qPCR, ELISA and western blot were performed to examine chemerin and CMKLR1 expression after insulin and TNF-α treatment in cardiac cells. Results: Chemerin treatment increased apoptosis, reduced phosphorylation of AKT at Thr308 and increased caspase-9 activity in murine cardiomyocytes. Insulin treatment lowered chemerin and CMKLR1 mRNA and protein levels, and the amount of chemerin in the cell media, while TNF-α treatment increased chemerin mRNA and protein levels but decreased expression of the CMKLR1 gene. Conclusion: Chemerin induces apoptosis, reduces AKT phosphorylation and increases the cleavage of caspase-9 in murine cardiomyocytes. The expression of chemerin is regulated by important metabolic (insulin and inflammatory (TNF-α mediators at cardiac level. Our results suggest that chemerin could play a role in the physiopathology of cardiac diseases.

  13. Benzene metabolites induce apoptosis in lymphocytes.

    Science.gov (United States)

    Martínez-Velázquez, M; Maldonado, V; Ortega, A; Meléndez-Zajgla, J; Albores, A

    2006-08-01

    Benzene is an important environmental pollutant with important health implications. Exposure to this aromatic hydrocarbon is associated with hematotoxicity, and bone marrow carcinogenic effects. It has been shown that benzene induces oxidative stress, cell cycle alterations, and programmed cell death in cultured cells. Hepatic metabolism of benzene is thought to be a prerequisite for its bone marrow toxicity. Nevertheless, there are no reports on the cellular effects of reactive intermediates derived from hepatic metabolism of benzene. Thus, the goal of this project was to determine the cellular alterations of benzene metabolites produced by the cultured hepatic cell line HepG2. Supernatants collected from these cells were applied to a culture of freshly isolated lymphocytes. A higher decrease in cell viability was found in cells exposed to these supernatants than to unmetabolized benzene. This viability decrease was due to apoptosis, as determined by Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) assay and internucleosomal fragmentation of DNA. When supernatants were analyzed by HPLC, we found that not all the hydrocarbon was biotransformed, since a 28 microM concentration (37%) remained. The only metabolite found in the culture medium was muconic acid. The present results show that muconic acid derived from benzene metabolism is able to cooperate with the pollutant for the induction of apoptosis in rat lymphocytes.

  14. Helicobacter pylori vacuolating toxin A and apoptosis

    Directory of Open Access Journals (Sweden)

    Rassow Joachim

    2011-11-01

    Full Text Available Abstract VacA, the vacuolating cytotoxin A of Helicobacter pylori, induces apoptosis in epithelial cells of the gastic mucosa and in leukocytes. VacA is released by the bacteria as a protein of 88 kDa. At the outer surface of host cells, it binds to the sphingomyelin of lipid rafts. At least partially, binding to the cells is facilitated by different receptor proteins. VacA is internalized by a clathrin-independent mechanism and initially accumulates in GPI-anchored proteins-enriched early endosomal compartments. Together with early endosomes, VacA is distributed inside the cells. Most of the VacA is eventually contained in the membranes of vacuoles. VacA assembles in hexameric oligomers forming an anion channel of low conductivity with a preference for chloride ions. In parallel, a significant fraction of VacA can be transferred from endosomes to mitochondria in a process involving direct endosome-mitochondria juxtaposition. Inside the mitochondria, VacA accumulates in the mitochondrial inner membrane, probably forming similar chloride channels as observed in the vacuoles. Import into mitochondria is mediated by the hydrophobic N-terminus of VacA. Apoptosis is triggered by loss of the mitochondrial membrane potential, recruitment of Bax and Bak, and release of cytochrome c.

  15. Modulation of neutrophil apoptosis by antimicrobial peptides.

    Science.gov (United States)

    Nagaoka, Isao; Suzuki, Kaori; Niyonsaba, François; Tamura, Hiroshi; Hirata, Michimasa

    2012-01-01

    Peptide antibiotics possess the potent antimicrobial activities against invading microorganisms and contribute to the innate host defense. Human antimicrobial peptides, α-defensins (human neutrophil peptides, HNPs), human β-defensins (hBDs), and cathelicidin (LL-37) not only exhibit potent bactericidal activities against Gram-negative and Gram-positive bacteria, but also function as immunomodulatory molecules by inducing cytokine and chemokine production, and inflammatory and immune cell activation. Neutrophil is a critical effector cell in host defense against microbial infection, and its lifespan is regulated by various pathogen- and host-derived substances. Here, we provided the evidence that HNP-1, hBD-3, and LL-37 cannot only destroy bacteria but also potently modulate (suppress) neutrophil apoptosis, accompanied with the phosphorylation of ERK-1/-2, the downregulation of tBid (an proapoptotic protein) and upregulation of Bcl-xL (an antiapoptotic protein), and the inhibition of mitochondrial membrane potential change and caspase 3 activity, possibly via the actions on the distinct receptors, the P2Y6 nucleotide receptor, the chemokine receptor CCR6, and the low-affinity formyl-peptide receptor FPRL1/the nucleotide receptor P2X7, respectively. Suppression of neutrophil apoptosis results in the prolongation of their lifespan and may be advantageous for the host defense against bacterial invasion.

  16. Role of the Crosstalk between Autophagy and Apoptosis in Cancer

    Directory of Open Access Journals (Sweden)

    Minfei Su

    2013-01-01

    Full Text Available Autophagy and apoptosis are catabolic pathways essential for organismal homeostasis. Autophagy is normally a cell-survival pathway involving the degradation and recycling of obsolete, damaged, or harmful macromolecular assemblies; however, excess autophagy has been implicated in type II cell death. Apoptosis is the canonical programmed cell death pathway. Autophagy and apoptosis have now been shown to be interconnected by several molecular nodes of crosstalk, enabling the coordinate regulation of degradation by these pathways. Normally, autophagy and apoptosis are both tumor suppressor pathways. Autophagy fulfils this role as it facilitates the degradation of oncogenic molecules, preventing development of cancers, while apoptosis prevents the survival of cancer cells. Consequently, defective or inadequate levels of either autophagy or apoptosis can lead to cancer. However, autophagy appears to have a dual role in cancer, as it has now been shown that autophagy also facilitates the survival of tumor cells in stress conditions such as hypoxic or low-nutrition environments. Here we review the multiple molecular mechanisms of coordination of autophagy and apoptosis and the role of the proteins involved in this crosstalk in cancer. A comprehensive understanding of the interconnectivity of autophagy and apoptosis is essential for the development of effective cancer therapeutics.

  17. Epithelial apoptosis: cause or consequence of ulcerative colitis?

    DEFF Research Database (Denmark)

    Seidelin, Jakob Benedict; Nielsen, Ole Haagen

    2009-01-01

    OBJECTIVE: Epithelial apoptosis rates are increased in ulcerative colitis (UC). The increased apoptosis rate could expose mucosal cells to luminal pathogens and thereby be regarded as a primary pathogenic factor in UC. On the other hand, the local inflammatory reaction could cause epithelial...

  18. Resveratrol induces apoptosis in human esophageal carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Hai-Bo Zhou; Yun Yan; Ya-Ni Sun; Ju-Ren Zhu

    2003-01-01

    AIM: To investigate the apoptosis in esophageal cancer cells induced by resveratrol, and the relation between this apoptosis and expression of Bcl-2 and Bax.METHODS: In in vitro experiments, MTr assay was used to determine the cell growth inhibitory rate. Transmission electron microscope and TUNEL staining method were used to quantitatively and qualitively detect the apoptosis status of esophageal cancer cell line EC-9706 before and after the resveratrol treatment. Immunohistochemical staining was used to detect the expression of apoptosis-regulated gene Bcl-2 and Bax.RESULTS: Resveratrol inhibited the growth of esophageal cancer cell line EC-9706 in a dose-and time-dependent manner. Resveratrol induced EC-9706 cells to undergo apoptosis with typically apoptotic characteristics, including morphological changes of chromatin condensation, chromatin crescent formation, nucleus fragmentation and apoptotic body formation. TUNEL assay showed that after the for 24 to 96 hours, the AIs were apparently increased with treated time (P<0.05). Immunohistochemical staining showed that after the treatment of EC-9706 cells with proteins were apparently reduced with treated time (P<0.05)and the PRs of Bax proteins were apparently increased with treated time (P<0.05).CONCLUSION: Resveratrol is able to induce the apoptosisin esophageal cancer. This apoptosis may be mediated by down-regulating the apoptosis-regulated gene Bcl-2 and upregulating the expression of apoptosis-regulated gene bax.

  19. Apoptosis and T cell depletion during feline infectious peritonitis

    NARCIS (Netherlands)

    Horzinek, M.C.; Haagmans, B.L.; Egberink, H.F.

    1996-01-01

    Cats that have succumbed to feline infectious peritonitis, an immune- mediated disease caused by variants of feline coronaviruses, show apoptosis and T-cell depletion in their lymphoid organs. The ascitic fluid that develops in the course of the condition causes apoptosis in vitro but only in activa

  20. Genomic, proteomic and morphological characterization of two novel broad host lytic bacteriophages ΦPD10.3 and ΦPD23.1 infecting pectinolytic Pectobacterium spp. and Dickeya spp.

    Science.gov (United States)

    Czajkowski, Robert; Ozymko, Zofia; de Jager, Victor; Siwinska, Joanna; Smolarska, Anna; Ossowicki, Adam; Narajczyk, Magdalena; Lojkowska, Ewa

    2015-01-01

    Pectinolytic Pectobacterium spp. and Dickeya spp. are necrotrophic bacterial pathogens of many important crops, including potato, worldwide. This study reports on the isolation and characterization of broad host lytic bacteriophages able to infect the dominant Pectobacterium spp. and Dickeya spp. affecting potato in Europe viz. Pectobacterium carotovorum subsp. carotovorum (Pcc), P. wasabiae (Pwa) and Dickeya solani (Dso) with the objective to assess their potential as biological disease control agents. Two lytic bacteriophages infecting stains of Pcc, Pwa and Dso were isolated from potato samples collected from two potato fields in central Poland. The ΦPD10.3 and ΦPD23.1 phages have morphology similar to other members of the Myoviridae family and the Caudovirales order, with a head diameter of 85 and 86 nm and length of tails of 117 and 121 nm, respectively. They were characterized for optimal multiplicity of infection, the rate of adsorption to the Pcc, Pwa and Dso cells, the latent period and the burst size. The phages were genotypically characterized with RAPD-PCR and RFLP techniques. The structural proteomes of both phages were obtained by fractionation of phage proteins by SDS-PAGE. Phage protein identification was performed by liquid chromatography-mass spectrometry (LC-MS) analysis. Pulsed-field gel electrophoresis (PFGE), genome sequencing and comparative genome analysis were used to gain knowledge of the length, organization and function of the ΦPD10.3 and ΦPD23.1 genomes. The potential use of ΦPD10.3 and ΦPD23.1 phages for the biocontrol of Pectobacterium spp. and Dickeya spp. infections in potato is discussed.

  1. Genomic, proteomic and morphological characterization of two novel broad host lytic bacteriophages ΦPD10.3 and ΦPD23.1 infecting pectinolytic Pectobacterium spp. and Dickeya spp.

    Directory of Open Access Journals (Sweden)

    Robert Czajkowski

    Full Text Available Pectinolytic Pectobacterium spp. and Dickeya spp. are necrotrophic bacterial pathogens of many important crops, including potato, worldwide. This study reports on the isolation and characterization of broad host lytic bacteriophages able to infect the dominant Pectobacterium spp. and Dickeya spp. affecting potato in Europe viz. Pectobacterium carotovorum subsp. carotovorum (Pcc, P. wasabiae (Pwa and Dickeya solani (Dso with the objective to assess their potential as biological disease control agents. Two lytic bacteriophages infecting stains of Pcc, Pwa and Dso were isolated from potato samples collected from two potato fields in central Poland. The ΦPD10.3 and ΦPD23.1 phages have morphology similar to other members of the Myoviridae family and the Caudovirales order, with a head diameter of 85 and 86 nm and length of tails of 117 and 121 nm, respectively. They were characterized for optimal multiplicity of infection, the rate of adsorption to the Pcc, Pwa and Dso cells, the latent period and the burst size. The phages were genotypically characterized with RAPD-PCR and RFLP techniques. The structural proteomes of both phages were obtained by fractionation of phage proteins by SDS-PAGE. Phage protein identification was performed by liquid chromatography-mass spectrometry (LC-MS analysis. Pulsed-field gel electrophoresis (PFGE, genome sequencing and comparative genome analysis were used to gain knowledge of the length, organization and function of the ΦPD10.3 and ΦPD23.1 genomes. The potential use of ΦPD10.3 and ΦPD23.1 phages for the biocontrol of Pectobacterium spp. and Dickeya spp. infections in potato is discussed.

  2. Effect of the cortex-lytic enzyme SleC from non-food-borne Clostridium perfringens on the germination properties of SleC-lacking spores of a food poisoning isolate.

    Science.gov (United States)

    Paredes-Sabja, Daniel; Sarker, Mahfuzur R

    2010-11-01

    The hallmark of bacterial spore germination is peptidoglycan cortex hydrolysis by cortex-lytic enzymes. In spores of Clostridium perfringens wild-type strain SM101, which causes food poisoning, the sole essential cortex-lytic enzyme SleC is activated by a unique serine protease CspB. Interestingly, the non-food-borne wild-type strain F4969 encodes a significantly divergent SleC variant (SleCF4969) and 3 serine proteases (CspA, CspB, and CspC). Consequently, in this study we evaluated the functional compatibility of SleCF4969 and SleCSM101 by complementing the germination phenotypes of SM101ΔsleC spores with sleCF4969. Our results show that although pro-SleCF4969 was processed into mature SleCF4969 in the SM101ΔsleC spores, it partially restored spore germination with nutrient medium, with a mixture of ʟ-asparagine and KCl, or with a 1:1 chelate of Ca2+ and dipicolinic acid. While the amount of dipicolinic acid released was lower, the amount of hexosamine-containing material released during germination of SM101ΔsleC(sleCF4969) spores was similar to the amount released during germination of SM101 wild-type spores. The viability of SM101ΔsleC(sleCF4969) spores was 8- and 3-fold lower than that of SM101 and F4969 spores, respectively. Together, these data indicate that the peptidoglycan cortex hydrolysis machinery in the food poisoning isolate SM101 is functionally divergent than that in the non-food-borne isolate F4969.

  3. The Epstein-Barr virus (EBV)-encoded protein kinase, EBV-PK, but not the thymidine kinase (EBV-TK), is required for ganciclovir and acyclovir inhibition of lytic viral production.

    Science.gov (United States)

    Meng, Qiao; Hagemeier, Stacy R; Fingeroth, Joyce D; Gershburg, Edward; Pagano, Joseph S; Kenney, Shannon C

    2010-05-01

    Ganciclovir (GCV) and acyclovir (ACV) are guanine nucleoside analogues that inhibit lytic herpesvirus replication. GCV and ACV must be monophosphorylated by virally encoded enzymes to be converted into nucleotides and incorporated into viral DNA. However, whether GCV and/or ACV phosphorylation in Epstein-Barr virus (EBV)-infected cells is mediated primarily by the EBV-encoded protein kinase (EBV-PK), the EBV-encoded thymidine kinase (EBV-TK), or both is controversial. To examine this question, we constructed EBV mutants containing stop codons in either the EBV-PK or EBV-TK open reading frame and selected for stable 293T clones latently infected with wild-type EBV or each of the mutant viruses. Cells were induced to the lytic form of viral replication with a BZLF1 expression vector in the presence and absence of various doses of GCV and ACV, and infectious viral titers were determined by a green Raji cell assay. As expected, virus production in wild-type EBV-infected 293T cells was inhibited by both GCV (50% inhibitory concentration [IC(50)] = 1.5 microM) and ACV (IC(50) = 4.1 microM). However, the EBV-PK mutant (which replicates as well as the wild-type (WT) virus in 293T cells) was resistant to both GCV (IC(50) = 19.6 microM) and ACV (IC(50) = 36.4 microM). Expression of the EBV-PK protein in trans restored GCV and ACV sensitivity in cells infected with the PK mutant virus. In contrast, in 293T cells infected with the TK mutant virus, viral replication remained sensitive to both GCV (IC(50) = 1.2 microM) and ACV (IC(50) = 2.8 microM), although susceptibility to the thymine nucleoside analogue, bromodeoxyuridine, was reduced. Thus, EBV-PK but not EBV-TK mediates ACV and GCV susceptibilities.

  4. Simplified evaluation of apoptosis using the Muse cell analyzer.

    Science.gov (United States)

    Khan, Asima; Gillis, Katherine; Clor, Julie; Tyagarajan, Kamala

    2012-01-01

    The degree of apoptosis in a cell population is an important parameter of cell health and is characterized by distinct morphological changes. Current methods of accurate detection and measurement of cellular apoptosis require expensive and complicated instrument platforms and expertise. The Muse Cell Analyzer is a unique instrument that enables multidimensional cell health analysis on a single platform. In this study, we used the Muse Cell Analyzer for apoptosis studies using the Muse Annexin V & Dead Cell Assay. The assay is based on the detection of phosphatidylserine (PS) on the surface of apoptotic cells. The results obtained from Muse Cell Analyzer were compared with traditional methods for apoptosis analysis. Our results indicate that Muse Annexin V & Dead Cell Assay and software module enabled the acquisition of accurate and highly precise measurements of cellular apoptosis. The assay is versatile and works with both suspension and adherent cell lines and multiple treatment conditions.

  5. Identification of genes responsive to apoptosis in HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    Wei JIN; Le-feng QU; Ping MIN; Shan CHEN; Hong LI; He LU; Yong-tai HOU

    2004-01-01

    AIM: To identify genes responsive to apoptosis in HL-60 cells treated by homoharringtonine. METHODS: cDNA microarray technology was used to detect gene expression and the result of microarrays for genes (TIEG and VDUP1) was confirmed by Northern analysis. RESULTS: Seventy-five individual mRNAs whose mass changed significantly were identified. Among these genes (25 were up-regulated and 50 were down-regulated), most are known related to oncogenes and tumor suppressor. Some genes were involved in apoptosis signaling pathways.CONCLUSION: TGFβ and TNF apoptosis signaling pathways were initiated during apoptosis in HL-60 cells.TIEG and VDUP1 play important roles in mediating apoptosis.

  6. Apoptosis of Cancer Cells Induced by HAP Nanoparticles

    Institute of Scientific and Technical Information of China (English)

    HU Sheng; LI Shipu; YAN Yuhua; WANG Youfa; CAO Xianying

    2005-01-01

    To confirm apoptosis is one of the hepatoma cells death pathways after HAP nanoparticles absorption, hepatoma cells were collected for ultrathin sections preparation and examined under a transmission electron microscope (TEM) after 1 h incubation with HAP nanoparticle. Apoptosis was detected by TUNEL technique. After absorption, some vacuoles with membrane containing HAP nanoparticles were found in cytoplasma.The nuclear envelope shrinked, and some area pullulated from nucleus. The karyotin became pycnosis and assembled at the edge. An apoptosis body was found. And the data of IOD and numbers of the positive apoptosic signals in nuclear area of slides could illustrate much more apoptosis in the HAP group than those in the control group ( P < 0.001 ). The experimental results indicate that the HAP nanoparticles can induce cancer cells apoptosis.

  7. Apoptosis in skeletal muscle and its relevance to atrophy

    Institute of Scientific and Technical Information of China (English)

    Esther E Dupont-Versteegden

    2006-01-01

    Apoptosis is necessary for maintaining the integrity of proliferative tissues, such as epithelial cells of the gastrointestinal system. The role of apoptosis in post mitotic tissues, such as skeletal muscle, is less well defined. Apoptosis during muscle atrophy occurs in both myonuclei and other muscle cell types. Apoptosis of myonuclei likely contributes to the loss of muscle mass, but the mechanisms underlying this process are largely unknown. Caspase-dependent as well as -independent pathways have been implicated and the mode by which atrophy is induced likely determines the apoptotic mechanisms that are utilized. It remains to be determined whether a decrease in apoptosis will alleviate atrophy and distinct research strategies may be required for different causes of skeletal muscle loss.

  8. Lymphocyte apoptosis: role of uremia and permeability of dialysis membrane.

    Science.gov (United States)

    Soriano, Sagrario; Martín-Malo, Alejandro; Carracedo, Julia; Ramírez, Rafael; Rodríguez, Mariano; Aljama, Pedro

    2005-01-01

    Uremia is associated to host defense mechanism disorders. Lymphocyte apoptosis, which may cause alteration of the immune system, is increased in uremic patients. The aim of the present study was to determine if, in addition to uremia, dialysis membranes with different biocompatibility and permeability have an effect on lymphocyte apoptosis. Cell apoptosis and Fas expression were assessed using flow cytometry in four groups of patients: (1) uremic non-dialyzed (Non-D) patients; (2) hemodialysis (HD) patients on hemophan; (3) low-flux polysulfone, and (4) high-flux polysulfone membrane. Ten healthy volunteers were used as controls. At baseline, lymphocytes from patients on hemophan showed an increase in apoptosis (18.4 +/- 6.9%) as compared with Non-D (7.2 +/- 2.8%; p dialysis patients lymphocyte apoptosis is influenced not only by the biocompatibility but also by the permeability of the dialysis membrane.

  9. Periodontal Ligament Stem Cells Regulate Apoptosis of Neutrophils

    Science.gov (United States)

    Wang, Qing; Ding, Gang; Xu, Xin

    2017-01-01

    Abstract Periodontal ligament stem cells (PDLSCs) are promising cell resource for the cell-based therapy for periodontitis and regeneration of bio-root. In this study, we investigated the effect of PDLSCs on neutrophil, a critical constituent of innate immunity, and the underlying mechanisms. The effect of PDLSCs on the proliferation and apoptosis of resting neutrophils and IL-8 activated neutrophils was tested under cell-cell contact culture and Transwell culture, with or without anti-IL-6 neutralizing antibody. We found that PDLSCs could promote the proliferation and reduce the apoptosis of neutrophils whether under cell-cell contact or Transwell culture. Anti-IL-6 antibody reduced PDLSCs-mediated inhibition of neutrophil apoptosis. IL-6 at the concentration of 10ng/ml and 20ng/ml could inhibit neutrophil apoptosis statistically. Collectively, PDLSCs could reduce the apoptosis of neutrophils via IL-6.

  10. Relationship between osteocyte apoptosis and orbital bone development in rabbits

    Institute of Scientific and Technical Information of China (English)

    MA Jian-min; LI Zhi-hui

    2004-01-01

    Objective To investigate whether the osteocyte apoptosis exists in orbital bones and to discuss its effect on the orbital development.Methods Seven young Newzealand white rabbits were selected as experimental animals.At two-month-old ,all rabbits were killed and then zygomas were made into paraffin and electron microscope sections after they were decalcified.Apoptosis of osteocytes was observed by light microscope and transmission electron microscopes and detected by TUNEL staining.Results The classical apoptosis of osteocytes was found under light and transmission electron microscopes.Apoptosis of osteocytes was diffused irregularly in the zygomatic tissue. Conclusion Osteocyte can apoptosis and it may participate in the development of the bony orbit.

  11. UXT plays dual opposing roles on SARM-induced apoptosis.

    Science.gov (United States)

    Sethurathinam, Shalini; Singh, Laishram Pradeepkumar; Panneerselvam, Porkodi; Byrne, Bernadette; Ding, Jeak Ling

    2013-10-11

    Apoptosis is a vital defense mechanism for the clearance of infected cells. Ubiquitously expressed transcript (UXT), which exists in two isoforms (V1 and V2), interact with both apoptotic and cellular proteins. By yeast two-hybrid analysis, we found that UXT interacts with SARM (sterile α and HEAT armadillo motif-containing protein). Since SARM is a TLR adaptor which induces intrinsic apoptosis following immune activation, we were prompted to query whether UXT and SARM might co-regulate apoptosis. We found that the UXT isoforms elicit dual opposing regulatory effects on SARM-induced apoptosis; while UXT V1, co-expressed with SARM, caused a reduction in caspase 8 activity, UXT V2 strongly increased caspase 8 activity and enhanced SARM-induced apoptosis by activating the extrinsic pathway and depolarizing the mitochondria. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  12. Role of heat shock proteins in cell apoptosis

    Directory of Open Access Journals (Sweden)

    Arleta Kaźmierczuk

    2010-06-01

    Full Text Available Apoptosis is, apart from necrosis and autophagy, one of the possible cell death mechanisms eliminating needless, not normal or infected cells. This process ensures quantitative and qualitative cell control of organisms. Apoptosis is tightly regulated, it requires both activation of a large number of genes and energy input. Up-to-date two main apoptotic pathways have been recognized – external/receptor and internal, processed with the participation of mitochondria. Heat shock proteins HSPs, the molecules known from their chaperone activity and molecular conservatism, play essential functions in the course of apoptosis. Among that proteins family, i.e. HSP100, 90, 70, 60, 40 and small molecular (sHSP, there are agents mainly protective against programmed cell death. However, in some conditions some of these proteins may promote apoptosis. This review describes different key apoptotic proteins interacting with main members of HSP family and the consequence of these events for cell survival or apoptosis.

  13. Role of apoptosis-inducing factor, proline dehydrogenase, and NADPH oxidase in apoptosis and oxidative stress

    Directory of Open Access Journals (Sweden)

    Becker DF

    2012-02-01

    Full Text Available Sathish Kumar Natarajan, Donald F BeckerDepartment of Biochemistry and Redox Biology Center, University of Nebraska-Lincoln, Lincoln, NEAbstract: Flavoproteins catalyze a variety of reactions utilizing flavin mononucleotide or flavin adenine dinucleotide as cofactors. The oxidoreductase properties of flavoenzymes implicate them in redox homeostasis, oxidative stress, and various cellular processes, including programmed cell death. Here we explore three critical flavoproteins involved in apoptosis and redox signaling, ie, apoptosis-inducing factor (AIF, proline dehydrogenase, and NADPH oxidase. These proteins have diverse biochemical functions and influence apoptotic signaling by unique mechanisms. The role of AIF in apoptotic signaling is two-fold, with AIF changing intracellular location from the inner mitochondrial membrane space to the nucleus upon exposure of cells to apoptotic stimuli. In the mitochondria, AIF enhances mitochondrial bioenergetics and complex I activity/assembly to help maintain proper cellular redox homeostasis. After translocating to the nucleus, AIF forms a chromatin degrading complex with other proteins, such as cyclophilin A. AIF translocation from the mitochondria to the nucleus is triggered by oxidative stress, implicating AIF as a mitochondrial redox sensor. Proline dehydrogenase is a membrane-associated flavoenzyme in the mitochondrion that catalyzes the rate-limiting step of proline oxidation. Upregulation of proline dehydrogenase by the tumor suppressor, p53, leads to enhanced mitochondrial reactive oxygen species that induce the intrinsic apoptotic pathway. NADPH oxidases are a group of enzymes that generate reactive oxygen species for oxidative stress and signaling purposes. Upon activation, NADPH oxidase 2 generates a burst of superoxide in neutrophils that leads to killing of microbes during phagocytosis. NADPH oxidases also participate in redox signaling that involves hydrogen peroxide-mediated activation of

  14. Quercetin-induced apoptosis prevents EBV infection

    Science.gov (United States)

    Lee, Minjung; Son, Myoungki; Ryu, Eunhyun; Shin, Yu Su; Kim, Jong Gwang; Kang, Byung Woog; Sung, Gi-Ho; Cho, Hyosun; Kang, Hyojeung

    2015-01-01

    Epstein-Barr virus (EBV) is a human gamma-1 herpesvirus that establishes a lifelong latency in over 90% of the world's population. During latency, virus exists predominantly as a chromatin-associated, multicopy episome in the nuclei of a variety of tumor cells derived from B cells, T cells, natural killer (NK) cells, and epithelial cells. Licorice is the root of Glycyrrhiza uralensis or G. glabra that has traditionally cultivated in eastern part of Asia. Licorice was reported to have anti-viral, anti-inflammatory, anti-atopic, hepatoprotective, anti-neurodegenerative, anti-tumor, anti-diabetic effects and so forth. Quercetin and isoliquiritigenin are produced from licorice and highly similar in molecular structure. They have diverse bioactive effects such as antiviral activity, anti-asthmatic activity, anti-cancer activity, anti-inflammation activity, monoamine-oxidase inhibitor, and etc. To determine anti-EBV and anti-EBVaGC (Epstein-Barr virus associated gastric carcinoma) effects of licorice, we investigated antitumor and antiviral effects of quercetin and isoliquiritigenin against EBVaGC. Although both quercetin and isoliquiritigenin are cytotoxic to SNU719 cells, quercetin induced more apoptosis in SNU719 cells than isoliquiritigenin, more completely eliminated DNMT1 and DNMT3A expressions than isoliquiritigenin, and more strongly affects the cell cycle progression of SNU719 than isoliquiritigenin. Both quercetin and isoliquiritigenin induce signal transductions to stimulate apoptosis, and induce EBV gene transcription. Quercetin enhances frequency of F promoter use, whereas isoliquiritigenin enhances frequency of Q promoter use. Quercetin reduces EBV latency, whereas isoliquiritigenin increases the latency. Quercetin increases more the EBV progeny production, and inhibits more EBV infection than isoliquiritigenin. These results indicate that quercetin could be a promising candidate for antiviral and antitumor agents against EBV and human gastric carcinoma

  15. Endoplasmic reticulum quality control and apoptosis.

    Science.gov (United States)

    Groenendyk, Jody; Michalak, Marek

    2005-01-01

    The ER is one of the most important folding compartments within the cell, as well as an intracellular Ca(2+) storage organelle and it contains a number of Ca(2+) regulated molecular chaperones responsible for the proper folding of glycosylated as well as non-glycosylated proteins. The luminal environment of the ER contains Ca(2+) which is involved in regulating chaperones such as calnexin and calreticulin, as well as apoptotic proteins caspase-12 and Bap31, which may play an important role in determining cellular sensitivity to ER stress and apoptosis. The ER quality control system consists of several molecular chaperones, including calnexin, that assist in properly folding proteins and transporting them through the ER as well as sensing misfolded proteins, attempting to refold them and if this is not possible, targeting them for degradation. Accumulation of misfolded protein in the ER leads to activation of genes responsible for the expression of ER chaperones. The UPR mechanism involves transcriptional activation of chaperones by the membrane-localized transcription factor ATF6, in conjunction with the ER membrane kinase IRE1, as well as translational repression of protein synthesis by another ER membrane kinase PERK. When accumulation of misfolded protein becomes toxic, apoptosis is triggered, potentially with IRE1 involved in signaling via caspase-12. Both the extrinsic and intrinsic apoptotic pathways appear to culminate in the activation of caspases and this results in the recruitment of mitochondria in an essential amplifying manner. Bap31 may direct pro-apoptotic crosstalk between the ER and the mitochondria via Ca(2+) in conjunction with caspase-12 and calnexin. Accordingly, ER stress and the resultant Ca(2+) release must be very carefully regulated because of their effects in virtually all areas of cell function.

  16. Apoptosis in thymus of teleost fish.

    Science.gov (United States)

    Romano, Nicla; Ceccarelli, Giuseppina; Caprera, Cecilia; Caccia, Elisabetta; Baldassini, Maria Rosaria; Marino, Giovanna

    2013-08-01

    The presence and distribution of apoptotic cells during thymus development and in adult were studied by in situ end-labelling of fragmented DNA in three temperate species carp (Cyprinus carpio), sea bass (Dicentrarchus labrax) and dusky grouper (Epinephelus marginatus) and in the adult thymus of three Antarctic species belonging to the genus Trematomus spp. During thymus development some few isolated apoptotic cell (AC) firstly appeared in the central-external part of the organ (carp: 5 days ph; sea bass: 35 days ph grouper: 43 days ph). Initially the cells were isolated and then increased in number and aggregated in small groups in the outer-cortical region of the thymus larvae. The high density of apoptotic cells was observed in the junction between cortex and medulla from its appearance (border between cortex and medulla, BCM). ACs decreased in number in juveniles and adult as well as the ACs average diameter. In late juveniles and in adulthood, the apoptosis were restricted to the cortex. In Antarctic species the thymus is highly adapted to low temperature (high vascularisation to effort the circulation of glycoproteins enriched plasma and strongly compact parenchyma). The apoptosis process was more extended (4-7 fold) as compare with the thymus of temperate species, even if the distribution of ACs was similar in all examined species. Data suggested a common process of T lymphocyte negative-selection in BCM of thymus during the ontogeny. The selection process seems to be still active in adult polar fish, but restricted mainly in the cortex zone.

  17. Quercetin-induced apoptosis prevents EBV infection.

    Science.gov (United States)

    Lee, Minjung; Son, Myoungki; Ryu, Eunhyun; Shin, Yu Su; Kim, Jong Gwang; Kang, Byung Woog; Cho, Hyosun; Kang, Hyojeung

    2015-05-20

    Epstein-Barr virus (EBV) is a human gamma-1 herpesvirus that establishes a lifelong latency in over 90% of the world's population. During latency, virus exists predominantly as a chromatin-associated, multicopy episome in the nuclei of a variety of tumor cells derived from B cells, T cells, natural killer (NK) cells, and epithelial cells. Licorice is the root of Glycyrrhiza uralensis or G. glabra that has traditionally cultivated in eastern part of Asia. Licorice was reported to have anti-viral, anti-inflammatory, anti-atopic, hepatoprotective, anti-neurodegenerative, anti-tumor, anti-diabetic effects and so forth. Quercetin and isoliquiritigenin are produced from licorice and highly similar in molecular structure. They have diverse bioactive effects such as antiviral activity, anti-asthmatic activity, anti-cancer activity, anti-inflammation activity, monoamine-oxidase inhibitor, and etc. To determine anti-EBV and anti-EBVaGC (Epstein-Barr virus associated gastric carcinoma) effects of licorice, we investigated antitumor and antiviral effects of quercetin and isoliquiritigenin against EBVaGC. Although both quercetin and isoliquiritigenin are cytotoxic to SNU719 cells, quercetin induced more apoptosis in SNU719 cells than isoliquiritigenin, more completely eliminated DNMT1 and DNMT3A expressions than isoliquiritigenin, and more strongly affects the cell cycle progression of SNU719 than isoliquiritigenin. Both quercetin and isoliquiritigenin induce signal transductions to stimulate apoptosis, and induce EBV gene transcription. Quercetin enhances frequency of F promoter use, whereas isoliquiritigenin enhances frequency of Q promoter use. Quercetin reduces EBV latency, whereas isoliquiritigenin increases the latency. Quercetin increases more the EBV progeny production, and inhibits more EBV infection than isoliquiritigenin. These results indicate that quercetin could be a promising candidate for antiviral and antitumor agents against EBV and human gastric carcinoma.

  18. Mechanisms and Biomarkers of Apoptosis in Liver Disease and Fibrosis

    Directory of Open Access Journals (Sweden)

    Jayashree Bagchi Chakraborty

    2012-01-01

    Full Text Available Liver fibrosis and cirrhosis are a major cause of morbidity and mortality worldwide. Development of the fibrotic scar is an outcome of chronic liver diseases of varying aetiologies including alcoholic liver disease (ALD nonalcoholic liver disease (NAFLD including non-alcoholic steatohepatitis (NASH viral hepatitis B and C (HBV, HCV. The critical step in the development of scar is activation of hepatic stellate cells (HSCs, which become the primary source of extracellular matrix. Aberrant apoptosis is a feature of chronic liver diseases and is associated with worsening stages of fibrosis. However, apoptosis is also the main mechanism promoting the resolution of fibrosis, and spontaneous or targeted apoptosis of HSC is associated with regression of fibrosis in animal models and patients with chronic liver disease. Given the importance of apoptosis in disease progression and resolution, there is much interest in precisely delineating the mechanisms involved and also developing biomarkers that accurately reflect the underlying pathogenesis. Here, we review the mechanisms driving apoptosis in development of liver disease and use of apoptosis -related biomarkers to aid in clinical diagnosis. Finally, we will also examine the recent literature regarding new insights into mechanisms involved in apoptosis of activated HSCs as possible method of fibrosis regression.

  19. APOPTOSIS OF HYPERPLASIA AND CANCER OF THE GALLBLADDER WITH CALCULAS

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective:To investigate the relation between different extent of proliferation caused by gallstone and gallbladder cancer by counting the proliferation and the apoptosis of the gallbladder cancer for the clinically prevention of the gallbladder carcinoma.Methods:The TUNEL method was used to detect the apoptosis of the specimens and the mean apoptosis indices obtained by quantification of apoptosis cells flurescence by laser scanning confocal microscope were compared among the varible pathological paterns,Results:The mean apoptosis indexed in the mormal and abnormal specimens with cholecystits,simple hyperplasia,low-grade dysplasia,mid-grade dysplasia,high-grade dysplasia and carcinoma were 5.11,5.49,6.32,8.65,12.27,25.24,39.62,119.8,respectively.There was significant difference among the variable pathological patterns and as the lesion progressing,the index went up gradually with the carcinoma had the highest index.Conclusion:the apoptosis indexes increase with the pathological progress during the carcinogenesis of gallbladder cancer caused by lithiasis,which stimulate the epithelium for long time and result in an increasing of the apoptosis;and it may play an important role in the carcinogenesis of gallbladder cancer.

  20. Apoptosis of human pancreatic cancer cells induced by Triptolide

    Institute of Scientific and Technical Information of China (English)

    Guo-Xiong Zhou; Xiao-Ling Ding; Jie-Fei Huang; Hong Zhang; Sheng-Bao Wu; Jian-Ping Cheng; Qun Wei

    2008-01-01

    AIM:To investigate apoptosis in human pancreatic cancer ceils induced by Triptolide (TL),and the relationship between this apoptosis and expression of caspase-3' bcl-2 and bax.METHODS:Human pancreatic cancer cell line SW1990 was cultured in DIEM media for this study.MTT assay was used to determine the cell growth inhibitory rate in vitro.Flow cytometry and TUNEL assay were used to detect the apoptosis of human pancreatic cancer cells before and after TL treatment.RT-PCR was used to detect the expression of apoptosis-associated gene caspase-3' bcl-2 and bax.RESULTS:TL inhibited the growth of human pancreatic cancer cells in a dose-and time-dependent manner.TL induced human pancreatic cancer cells to undergo apoptosis with typically apoptotic characteristics.TUNEL assay showed that after the treatment of human pancreatic cancer cells with 40 ng/mL TL for 12 h and 24 h,the apoptotic rates of human pancreatic cancer cells increased significantly.RT-PCR demonstrated that caspase-3 and bax were significantly up-regulated in SW1990 cells treated with TL while bcl-2 mRNA was not.CONCLUSION:TL is able to induce the apoptosis in human pancreatic cancer cells.This apoptosis may be mediated by up-regulating the expression of apoptosisassociated caspase-3 and bax gene.

  1. Apoptosis as form of natural ovarian cell death

    Directory of Open Access Journals (Sweden)

    Radovanović Anita M.

    2004-01-01

    Full Text Available Different hormones, cytokines, the absence of growth factors, and others, are some of the signals for initiating apoptosis in ovarian cells. Each of them in its own way, trigger apoptosis as a form of death in which the cell actively participates by precisely implementing a genetically programmed sequence of biochemical and morphological changes which lead to selfdestruction. Apoptosis is a physiological form of death, which helps establish a dynamic balance among proiliferation, differenciation, and death of ovarian cells. It has been confirmed so far that follicular cells oocytes, cells of the germinal epithelium, theca cells, and corpus luteum cells die through apoptosis. The physiological deaths of these cells are an integral part of normal ovarian function, both during intrauterine and postnatal life. Namely, during intrauterine ovarian development, about half the total number of germinative cells (future oocytes die through apoptosis and their population is gradually reduced after birth by so-called selection of follicles which will continue further growth (folliculogenesis and the apoptosis of cells of those follicles which will be subjected to atresion. Most ovarian cells die by apoptosis continuously until the end of the reproductive life period of healthy females, and some can continue dieing in this way until the death of the given individual (e.g. germinal epithelium cells.

  2. Apoptosis of circulating lymphocytes during pediatric cardiac surgery

    Science.gov (United States)

    Bocsi, J.; Pipek, M.; Hambsch, J.; Schneider, P.; Tárnok, A.

    2006-02-01

    There is a constant need for clinical diagnostic systems that enable to predict disease course for preventative medicine. Apoptosis, programmed cell death, is the end point of the cell's response to different induction and leads to changes in the cell morphology that can be rapidly detected by optical systems. We tested whether apoptosis of T-cells in the peripheral blood is useful as predictor and compared different preparation and analytical techniques. Surgical trauma is associated with elevated apoptosis of circulating leukocytes. Increased apoptosis leads to partial removal of immune competent cells and could therefore in part be responsible for reduced immune defence. Cardiovascular surgery with but not without cardiopulmonary bypass (CPB) induces transient immunosuppression. Its effect on T-cell apoptosis has not been shown yet. Flow-cytometric data of blood samples from 107 children (age 3-16 yr.) who underwent cardiac surgery with (78) or without (29) CPB were analysed. Apoptotic T-lymphocytes were detected based on light scatter and surface antigen (CD45/CD3) expression (ClinExpImmunol2000;120:454). Results were compared to staining with CD3 antibodies alone and in the absence of antibodies. T-cell apoptosis rate was comparable when detected with CD45/CD3 or CD3 alone, however not in the absence of CD3. Patients with but not without CPB surgery had elevated lymphocyte apoptosis. T-cell apoptosis increased from 0.47% (baseline) to 0.97% (1 day postoperatively). In CPB patients with complication 1.10% significantly higher (ANOVA p=0.01) comparing to CPB patients without complications. Quantitation of circulating apoptotic cells based on light scatter seems an interesting new parameter for diagnosis. Increased apoptosis of circulating lymphocytes and neutrophils further contributes to the immune suppressive response to surgery with CPB. (Support: MP, Deutsche Herzstiftung, Frankfurt, Germany)

  3. Smad2 is Involved in Aggregatibacter actinomycetemcomitans-induced Apoptosis

    Science.gov (United States)

    Yoshimoto, T.; Fujita, T.; Ouhara, K.; Kajiya, M.; Imai, H.; Shiba, H.; Kurihara, H.

    2014-01-01

    Apoptosis is thought to contribute to the progression of periodontitis. It has been suggested that the apoptosis of epithelial cells may contribute to the loss of epithelial barrier function. Smad2, a downstream signaling molecule of TGF-β receptors (TGF-βRs), is critically involved in apoptosis in several cell types. However, the relationship between smad2 and bacteria-induced apoptosis has not yet been elucidated. It is possible that the regulation of apoptosis induced by periodontopathic bacteria may lead to novel preventive therapies for periodontitis. Therefore, in the present study, we investigated the involvement of smad2 phosphorylation in apoptosis of human gingival epithelial cells induced by Aggregatibacter actinomycetemcomitans (Aa). Aa apparently induced the phosphorylation of smad2 in primary human gingival epithelial cells (HGECs) or the human gingival epithelial cell line, OBA9 cells. In addition, Aa induced phosphorylation of the serine residue of the TGF-β type I receptor (TGF-βRI) in OBA9 cells. SB431542 (a TGF-βRI inhibitor) and siRNA transfection for TGF-βRI, which reduced both TGF-βRI mRNA and protein levels, markedly attenuated the Aa-induced phosphorylation of smad2. Furthermore, the disruption of TGF-βRI signaling cascade by SB431542 and siRNA transfection for TGF-βRI abrogated the activation of cleaved caspase-3 expression and repressed apoptosis in OBA9 cells treated with Aa. Thus, Aa induced apoptosis in gingival epithelial cells by activating the TGF-βRI-smad2-caspase-3 signaling pathway. The results of the present study may suggest that the periodontopathic bacteria, Aa, activates the TGF-βR/smad2 signaling pathway in human gingival epithelial cells and induces apoptosis in epithelial cells, which may lead to new therapeutic strategies that modulate the initiation of periodontitis. PMID:25192897

  4. The interplays between autophagy and apoptosis induced by enterovirus 71.

    Directory of Open Access Journals (Sweden)

    Xueyan Xi

    Full Text Available BACKGROUND: Enterovirus 71 (EV71 is the causative agent of human diseases with distinct severity, from mild hand, foot and mouth disease to severe neurological syndromes, such as encephalitis and meningitis. The lack of understanding of viral pathogenesis as well as lack of efficient vaccine and drugs against this virus impedes the control of EV71 infection. EV71 virus induces autophagy and apoptosis; however, the relationship between EV71-induced autophagy and apoptosis as well as the influence of autophagy and apoptosis on virus virulence remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: In this study, it was observed that the Anhui strain of EV71 induced autophagy and apoptosis in human rhabdomyosarcoma (RD-A cells. Additionally, by either applying chemical inhibitors or knocking down single essential autophagic or apoptotic genes, inhibition of EV71 induced autophagy inhibited the apoptosis both at the autophagosome formation stage and autophagy execution stage. However, inhibition of autophagy at the stage of autophagosome and lysosome fusion promoted apoptosis. In reverse, the inhibition of EV71-induced apoptosis contributed to the conversion of microtubule-associated protein 1 light chain 3-I (LC3-I to LC3-II and degradation of sequestosome 1 (SQSTM1/P62. Furthermore, the inhibition of autophagy in the autophagsome formation stage or apoptosis decreased the release of EV71 viral particles. CONCLUSIONS/SIGNIFICANCE: In conclusion, the results of this study not only revealed novel aspect of the interplay between autophagy and apoptosis in EV71 infection, but also provided a new insight to control EV71 infection.

  5. Apoptosis is not required for mammalian neural tube closure.

    Science.gov (United States)

    Massa, Valentina; Savery, Dawn; Ybot-Gonzalez, Patricia; Ferraro, Elisabetta; Rongvaux, Anthony; Cecconi, Francesco; Flavell, Richard; Greene, Nicholas D E; Copp, Andrew J

    2009-05-19

    Apoptotic cell death occurs in many tissues during embryonic development and appears to be essential for processes including digit formation and cardiac outflow tract remodeling. Studies in the chick suggest a requirement for apoptosis during neurulation, because inhibition of caspase activity was found to prevent neural tube closure. In mice, excessive apoptosis occurs in association with failure of neural tube closure in several genetic mutants, but whether regulated apoptosis is also necessary for neural tube closure in mammals is unknown. Here we investigate the possible role of apoptotic cell death during mouse neural tube closure. We confirm the presence of apoptosis in the neural tube before and during closure, and identify a correlation with 3 main events: bending and fusion of the neural folds, postfusion remodeling of the dorsal neural tube and surface ectoderm, and emigration of neural crest cells. Both Casp3 and Apaf1 null embryos exhibit severely reduced apoptosis, yet neurulation proceeds normally in the forebrain and spine. In contrast, the mutant embryos fail to complete neural tube closure in the midbrain and hindbrain. Application of the apoptosis inhibitors z-Vad-fmk and pifithrin-alpha to neurulation-stage embryos in culture suppresses apoptosis but does not prevent initiation or progression of neural tube closure along the entire neuraxis, including the midbrain and hindbrain. Remodeling of the surface ectoderm to cover the closed tube, as well as delamination and migration of neural crest cells, also appear to be normal in the apoptosis-suppressed embryos. We conclude that apoptosis is not required for neural tube closure in the mouse embryo.

  6. Genetic Signatures of HIV-1 Envelope-mediated Bystander Apoptosis

    Science.gov (United States)

    Joshi, Anjali; Lee, Raphael T. C.; Mohl, Jonathan; Sedano, Melina; Khong, Wei Xin; Ng, Oon Tek; Maurer-Stroh, Sebastian; Garg, Himanshu

    2014-01-01

    The envelope (Env) glycoprotein of HIV is an important determinant of viral pathogenesis. Several lines of evidence support the role of HIV-1 Env in inducing bystander apoptosis that may be a contributing factor in CD4+ T cell loss. However, most of the studies testing this phenomenon have been conducted with laboratory-adapted HIV-1 isolates. This raises the question of whether primary Envs derived from HIV-infected patients are capable of inducing bystander apoptosis and whether specific Env signatures are associated with this phenomenon. We developed a high throughput assay to determine the bystander apoptosis inducing activity of a panel of primary Envs. We tested 38 different Envs for bystander apoptosis, virion infectivity, neutralizing antibody sensitivity, and putative N-linked glycosylation sites along with a comprehensive sequence analysis to determine if specific sequence signatures within the viral Env are associated with bystander apoptosis. Our studies show that primary Envs vary considerably in their bystander apoptosis-inducing potential, a phenomenon that correlates inversely with putative N-linked glycosylation sites and positively with virion infectivity. By use of a novel phylogenetic analysis that avoids subtype bias coupled with structural considerations, we found specific residues like Arg-476 and Asn-425 that were associated with differences in bystander apoptosis induction. A specific role of these residues was also confirmed experimentally. These data demonstrate for the first time the potential of primary R5 Envs to mediate bystander apoptosis in CD4+ T cells. Furthermore, we identify specific genetic signatures within the Env that may be associated with the bystander apoptosis-inducing phenotype. PMID:24265318

  7. Glucocorticoid-induced apoptosis and cellular mechanisms of myopathy.

    Science.gov (United States)

    Dirks-Naylor, Amie J; Griffiths, Carrie L

    2009-10-01

    Glucocorticoid-induced myopathy is a common side effect of chronic glucocorticoid therapy. Several mechanisms are currently being examined as ways in which glucocorticoid-induced myopathy occurs. These include apoptotic signaling through mitochondrial-mediated and Fas-mediated apoptosis, the role of the proteosome, the suppression of the IGF-1 signaling, and the role of ceramide in glucocorticoid-induced apoptosis and myopathy. It is difficult to differentiate which mechanism may be the initiating event responsible for the induction of apoptosis; however, all of the mechanisms play a vital role in glucocorticoid-induced myopathy.

  8. Executionary pathway for apoptosis: lessons from mutant mice

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Apoptosis or programmed cell death (PCD) is an evolutionarily conserved cellular process that is essential for normal development and homeostasis of multicellular organisms. Defects in the apoptosis signaling result in many diseases including autoimmune diseases and cancer. The apoptosis signaling pathway was first described genetically in the nematode Caenorhabditis elegans which serves as a framework for the more complex apop totic pathways that exist in mammals. In this review, we will discuss the apoptotic pathways that are emerging in mammals as elucidated by studies of gene-targeted mutant mice.

  9. INHIBITION OF SPONTANEOUS APOPTOSIS IN HUMAN BREAST CANCER

    Institute of Scientific and Technical Information of China (English)

    邵志敏; 江明; 吴炅; 余黎民; 韩企夏; 张延璆; 沈镇宙

    1996-01-01

    Breast tumorigenesis proceeds through an accumulation of specific genetic alteration. Breast malignant transformation is dependent on not only the rate of cell production but also on apoptcsis,a genetically prograined process of autonomous ceil death. We investigated whether breast tumorigenesis involved an altered susceptibility to apoptosis and proliferation by examining normal breast epithelium and breast cancer sampies. We found there is a great inhibition of spontaneous apoptosis in breast cancer ceils compared with normal breast epithelium. The inhibition of apoptosis in breast cancer may contribute to neoplastic transformation.

  10. Stochastic modeling of p53-regulated apoptosis upon radiation damage

    CERN Document Server

    Bhatt, Divesh; Bahar, Ivet

    2011-01-01

    We develop and study the evolution of a model of radiation induced apoptosis in cells using stochastic simulations, and identified key protein targets for effective mitigation of radiation damage. We identified several key proteins associated with cellular apoptosis using an extensive literature survey. In particular, we focus on the p53 transcription dependent and p53 transcription independent pathways for mitochondrial apoptosis. Our model reproduces known p53 oscillations following radiation damage. The key, experimentally testable hypotheses that we generate are - inhibition of PUMA is an effective strategy for mitigation of radiation damage if the treatment is administered immediately, at later stages following radiation damage, inhibition of tBid is more effective.

  11. Differentiation and apoptosis in human immortalized sebocytes.

    Science.gov (United States)

    Wróbel, Anna; Seltmann, Holger; Fimmel, Sabine; Müller-Decker, Karin; Tsukada, Miki; Bogdanoff, Birgit; Mandt, Nathalie; Blume-Peytavi, Ulrike; Orfanos, Constantin E; Zouboulis, Christos C

    2003-02-01

    Increased cell volume, accumulation of lipid droplets in the cytoplasm, and nuclear degeneration are phenomena indicating terminal differentiation of human sebocytes followed by holocrine secretion and cell death. The molecular pathways of natural and induced sebocyte elimination are still unknown, however. In this study, SZ95 sebocytes were found to exhibit DNA fragmentation after a 6 h culture followed by increased lactate dehydrogenase release after 24 h, indicating cell damage. With the help of morphologic studies and using Oil Red detection of cellular lipids, cell enlargement, accumulation of lipid droplets in the cytoplasm, and nuclear fragmentation could be observed under treatment with arachidonic acid. Staurosporine, a potent inhibitor of phospholipid Ca2+-dependent protein kinase, increased externalized phosphatidylserine levels on SZ95 sebocytes, detected by annexin V/propidium iodide flow cytometry, as early as after 1 h, whereas dose-dependent reduction of bcl-2 mRNA and protein expression, enhanced DNA fragmentation, and increased caspase 3 levels, detected by caspase 3 inhibitor/propidium iodide flow cytometry, were found after 6 h of treatment. SZ95 sebocyte death was detected as early as after 6 h of SZ95 sebocyte treatment with high staurosporine concentrations (10(-6)-10(-5) M). 5Alpha-dihydrotestosterone (10(-8)-10(-5) M) did not affect externalized phosphatidylserine levels and DNA fragmentation in SZ95 sebocytes but slightly decreased lactate dehydrogenase cell release. Neither acitretin nor 13-cis retinoic acid (10(-8)-10(-5) M) affected externalized phosphatidylserine levels, DNA fragmentation, and lactate dehydrogenase cell release, despite the increased caspase 3 levels under treatment with 13-cis retinoic acid. The combined staurosporine and 13-cis retinoic acid treatment enhanced DNA fragmentation in SZ95 sebocytes to the same magnitude as in cells only treated with staurosporine. In conclusion, SZ95 sebocytes in vitro undergo apoptosis

  12. Cytokines and Pancreatic β-Cell Apoptosis.

    Science.gov (United States)

    Berchtold, L A; Prause, M; Størling, J; Mandrup-Poulsen, T

    Recommendations are activated by inflammatory cytokines in the pancreatic β-cell to guide the identification of antidiabetic targets. Although there are still scarce human data, the cellular and preclinical studies point to the caspase-dependent intrinsic apoptosis pathway as the prime effector of inflammatory β-cell apoptosis.

  13. Overexpressed TP73 induces apoptosis in medulloblastoma

    Directory of Open Access Journals (Sweden)

    Perlaky Laszlo

    2007-07-01

    Full Text Available Abstract Background Medulloblastoma is the most common malignant brain tumor of childhood. Children who relapse usually die of their disease, which reflects resistance to radiation and/or chemotherapy. Improvements in outcome require a better understanding of the molecular basis of medulloblastoma growth and treatment response. TP73 is a member of the TP53 tumor suppressor gene family that has been found to be overexpressed in a variety of tumors and mediates apoptotic responses to genotoxic stress. In this study, we assessed expression of TP73 RNA species in patient tumor specimens and in medulloblastoma cell lines, and manipulated expression of full-length TAp73 and amino-terminal truncated ΔNp73 to assess their effects on growth. Methods We analyzed medulloblastoma samples from thirty-four pediatric patients and the established medulloblastoma cell lines, Daoy and D283MED, for expression of TP73 RNA including the full-length transcript and the 5'-terminal variants that encode the ΔNp73 isoform, as well as TP53 RNA using quantitative real time-RTPCR. Protein expression of TAp73 and ΔNp73 was quantitated with immunoblotting methods. Clinical outcome was analyzed based on TP73 RNA and p53 protein expression. To determine effects of overexpression or knock-down of TAp73 and ΔNp73 on cell cycle and apoptosis, we analyzed transiently transfected medulloblastoma cell lines with flow cytometric and TUNEL methods. Results Patient medulloblastoma samples and cell lines expressed full-length and 5'-terminal variant TP73 RNA species in 100-fold excess compared to non-neoplastic brain controls. Western immunoblot analysis confirmed their elevated levels of TAp73 and amino-terminal truncated ΔNp73 proteins. Kaplan-Meier analysis revealed trends toward favorable overall and progression-free survival of patients whose tumors display TAp73 RNA overexpression. Overexpression of TAp73 or ΔNp73 induced apoptosis under basal growth conditions in vitro and

  14. Induction of apoptosis and cell proliferation inhibition by paclitaxel in ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-02-18

    Feb 18, 2009 ... phase contrast and fluorescent microscope. ... most intense apoptotic cell formations were observed for P2 dose, which is .... Chemical structure of PAC. ..... human prostate cancer cell apoptosis via alteration in bcl-xL and bak.

  15. Chloroquinone Inhibits Cell Proliferation and Induces Apoptosis in ...

    African Journals Online (AJOL)

    Chloroquinone Inhibits Cell Proliferation and Induces Apoptosis in ... of cell proliferation while an inverted microscope was employed for the analysis of ... μΜ concentration of CQ without affecting normal human skin keratinocyte cell line, K38.

  16. ING1 induces apoptosis through direct effects at the mitochondria

    DEFF Research Database (Denmark)

    Bose, P; Thakur, S; Thalappilly, S

    2013-01-01

    translocates to the mitochondria of primary fibroblasts and established epithelial cell lines in response to apoptosis inducing stimuli, independent of the cellular p53 status. The ability of ING1 to induce apoptosis in various breast cancer cell lines correlates well with its degree of translocation......The ING family of tumor suppressors acts as readers and writers of the histone epigenetic code, affecting DNA repair, chromatin remodeling, cellular senescence, cell cycle regulation and apoptosis. The best characterized member of the ING family, ING1,interacts with the proliferating cell nuclear...... to the mitochondria after UV treatment. Endogenous ING1 protein specifically interacts with the pro-apoptotic BCL2 family member BAX, and colocalizes with BAX in a UV-inducible manner. Ectopic expression of a mitochondria-targeted ING1 construct is more proficient in inducing apoptosis than the wild type ING1 protein...

  17. Chalcones Enhance TRAIL-Induced Apoptosis in Prostate Cancer Cells

    Directory of Open Access Journals (Sweden)

    Ewelina Szliszka

    2009-12-01

    Full Text Available Chalcones exhibit chemopreventive and antitumor effects. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL is a naturally occurring anticancer agent that induces apoptosis in cancer cells and is not toxic to normal cells. We examined the cytotoxic and apoptotic effect of five chalcones in combination with TRAIL on prostate cancer cells. The cytotoxicity was evaluated by the MTT and LDH assays. The apoptosis was determined using flow cytometry with annexin V-FITC. Our study showed that all five tested chalcones: chalcone, licochalcone-A, isobavachalcone, xanthohumol, butein markedly augmented TRAIL-mediated apoptosis and cytotoxicity in prostate cancer cells and confirmed the significant role of chalcones in chemoprevention of prostate cancer.

  18. Apoptosis and the target genes of microRNA-21

    Institute of Scientific and Technical Information of China (English)

    Lindsey E. Becker Buscaglia; Yong Li

    2011-01-01

    MicroRNA-21 (miR-21) is frequently up-regulated in cancer and the majodty of its reported targets are tumor suppressors. Through functional suppression, miR-21 is implicated in practically every walk of oncogenic life: the promotion of cell proliferation, invasion and metastasis, genome instability and mutation, inflammation, replicative immortalization, abnormal metabolism, angiogenesis, and evading apoptosis, immune destruction, and growth suppressors. In particular, miR-21 is strongly involved in apoptosis. In this article, we reviewed the experimentally validated targets of miR-21 and found that two thirds are linked to intrinsic and/or extrinsic pathways of cellular apoptosis. This suggests that miR-21 is an oncogene which plays a key role in resisting programmed cell death in cancer cells and that targeting apoptosis is a viable therapeutic option against cancers expressing miR-21.

  19. Bax, Bak and beyond - mitochondrial performance in apoptosis.

    Science.gov (United States)

    Peña-Blanco, Aida; García-Sáez, Ana J

    2017-07-29

    Bax and Bak are members of the Bcl-2 family and core regulators of the intrinsic pathway of apoptosis. Upon apoptotic stimuli, they are activated and oligomerize at the mitochondrial outer membrane (MOM) to mediate its permeabilization, which is considered a key step in apoptosis. However, the molecular mechanism underlying Bax and Bak function has remained a key question in the field. Here, we review recent structural and biophysical evidence that has changed our understanding of how Bax and Bak promote MOM permeabilization. We also discuss how the spatial regulation of Bcl-2 family preference for binding partners contributes to regulate Bax and Bak activation. Finally, we consider the contribution of mitochondrial composition, dynamics and interaction with other organelles to apoptosis commitment. A new perspective is emerging, in which the control of apoptosis by Bax and Bak goes beyond them and is highly influenced by additional mitochondrial components. © 2017 Federation of European Biochemical Societies.

  20. Effect of Celecoxib on Apoptosis of Endometrial Carcinoma Cell

    Institute of Scientific and Technical Information of China (English)

    SHENG Xiu-jie; FANG Zhao

    2007-01-01

    Objective: To investigate the effect of Celecoxib on proliferation and apoptosis of the endometrial carcinoma cell HEC-1B and the effect on the expression of Fas and Survivin mRNA. Methods: The inhibition on the growth of human endometrial carcinoma cell HEC-1B was investigated by cell culture and MTT experiment when treated with different concentrations of Celecoxib. The cell apoptosis was detected by flow cytometry and DNA Ladder Electrophoresis. The change of the expression of Fas and Survivin mRNA after the treatment of Celecoxib was detected With RT-PCR. Results: Celecoxib could effectively inhibit the growth of HEC-1B cells and induce apoptosis. Survivin mRNA expression was decreased and Fas mRNA expression was increased after treating with Celecoxib. Conclusion: Celecoxib could inhibit HEC-1B cell proliferation and induce its apoptosis.

  1. Ankaferd Blood Stopper induces apoptosis and regulates PAR1 and ...

    African Journals Online (AJOL)

    Mine Mumcuoglu

    2014-12-16

    Dec 16, 2014 ... Besides its widely accepted role in plate- ... important role in cell cycle arrest and apoptosis [17]. ABS has ..... lial cells, fibroblasts, neuronal cells and tumor cells by either inducing or ... hemorrhages and wound healing.

  2. MODERN TECHNOLOGIES AND APPROACHES TO APOPTOSIS STUDIES IN EXPERIMENTAL BIOLOGY

    Directory of Open Access Journals (Sweden)

    I. V. Kudriavtsev

    2012-01-01

    Full Text Available Abstract. This review is focused on analysis of currently used flow cytometric methods designed foridentifying apoptotic cells in various invertebrate and vertebrate species. Apoptosis can be characterized by stage-specific morphological and biochemical changes that are typical to all kinds of eukaryotic cells. In this article, we consider different techniques of apoptosis detection based on assessment of cellular morphology and plasma membrane alterations, activation of intracellular enzymes and components of a caspase cascade, as well as DNA fragmentation and failure of mitochondrial transmembrane potential, as assessed in various animal groups. Apoptosis recognized as a key mechanism aiming at maintenance of cellular homeostasis in multicellular organisms, and such investigations represent a necessary component of fundamental and applied studies in diverse fields of experimental biology and immunology. A broad spectrum of apoptosis markers isused, and the preference is given to optimal approaches, as determined by experimental tasks, and technical opportunities of the laboratory.

  3. Control of Apoptosis in Treatment and Biology of Pancreatic Cancer.

    Science.gov (United States)

    Modi, Shrey; Kir, Devika; Banerjee, Sulagna; Saluja, Ashok

    2016-02-01

    Pancreatic cancer is estimated to be the 12th most common cancer in the United States in 2014 and yet this malignancy is the fourth leading cause of cancer-related death in the United States. Late detection and resistance to therapy are the major causes for its dismal prognosis. Apoptosis is an actively orchestrated cell death mechanism that serves to maintain tissue homoeostasis. Cancer develops from normal cells by accruing significant changes through one or more mechanisms, leading to DNA damage and mutations, which in a normal cell would induce this programmed cell death pathway. As a result, evasion of apoptosis is one of the hallmarks of cancer cells. PDAC is notoriously resistant to apoptosis, thereby explaining its aggressive nature and resistance to conventional treatment modalities. The current review is focus on understanding different intrinsic and extrinsic pathways in pancreatic cancer that may affect apoptosis in this disease.

  4. Induction of Apoptosis by Hypertension Via Endoplasmic Reticulum Stress

    Directory of Open Access Journals (Sweden)

    Yingying Sun

    2015-02-01

    Full Text Available Background/Aims: Endoplasmic reticulum (ER stress is one of the intrinsic apoptosis pathways, and cardiac apoptosis can occur in cardiovascular diseases, such as hypertension. However, the mechanisms by which ER stress leads to apoptosis remain enigmatic, particularly in the progression from cardiac hypertrophy to diastolic heart failure due to hypertension. Methods: We used spontaneously hypertensive rats (SHRs to investigate possible signalling pathways for ER stress. Results: We found that cardiac protein and mRNA levels of glucose-regulated protein 78 were up-regulated. In addition, the CHOP- and caspase-12-dependent pathways, but not that of JNK, were activated in the SHR rats. Conclusions: These results suggest that ER stress can contribute to myocardial apoptosis during hypertensive disease.

  5. Key role of mitochondria in apoptosis of lymphocytes.

    Science.gov (United States)

    Boichuk, S V; Minnebaev, M M; Mustafin, I G

    2001-12-01

    Changes in the mitochondrial potential, expression of phosphatidylserine, parameters of direct and lateral light scattering, and DNA fragmentation during spontaneous and induced apoptosis in peripheral blood lymphocytes were studied by flow cytofluorometry. Dexamethasone and Ca2+ ionophore A23187 served as inductors of apoptosis. A decrease in the mitochondrial potential is an early sign of spontaneous and induced apoptosis. Phosphatidylserine expression on the outer plasma membrane occurred later and inversely depended on the mitochondrial potential. Our results indicate that the involvement of mitochondria in spontaneous and induced apoptosis accompanied by a decrease in the mitochondrial potential is an early and key event of programmed lymphocyte death. The decrease in the mitochondrial potential of lymphocytes induced degradation of their nuclei (DNA fragmentation) and promoted elimination of apoptotic cells (phosphatidylserine expression).

  6. Vaccination with apoptosis colorectal cancer cell pulsed autologous ...

    African Journals Online (AJOL)

    Vaccination with apoptosis colorectal cancer cell pulsed autologous dendritic ... and interferon-γ (IFN-γ) significantly increased after DCs vaccination (P < 0.05). ... Five patients showed a positive skin response to the apoptotic cells loaded DC ...

  7. APOPTOSIS INDUCTION BY THE RECOMBINANT FUSION APOPTOSIS INDUCING FACTOR ON HELA CELLS

    Institute of Scientific and Technical Information of China (English)

    于翠娟; 孟艳玲; 桂俊豪; 赵晶; 金明; 王智; 王成济; 杨安钢

    2003-01-01

    Objective: To obtain the recombinant fusion AIF genes inserted into the eukaryotic expression vector Pires2-EGFP, to observe the expression and location of the fusion AIF genes (3NE: PE(280-358)-AIFΔ1-120, and 4NE: PE(280-364)-AIFΔ1-120), and to detect and compare their apoptosis inducing effects on the transfected HeLa cells. Methods: Full-length human AIF gene was cloned by RT-PCR, and its N-terminal mitochondrial localization sequence (MLS) was replaced by part sequence of Psuedomonas exotoxin A (PE) translocation domain (PEII(280-358/364)), then the recombinant fusion genes were inserted into the Pires2-EGFP eukaryotic expression vector. After these genes were transiently transfected into HeLa cells with LipofectAmine, the expression of the recombinant fusion AIF genes and their effects on HeLa cells were detected by fluorescent microscopy, laser confocal microscopy and electron microscopy. Results: The eukaryotic expression vectors containing the recombinant fusion AIF genes (Pires2-EGFP-PEII(280-358/364)- AIFΔ1- 120) were constructed successfully. It was demonstrated that the fusion AIF protein genes were expressed effectively in the transfected cells, with the GFP comco-expressed in cells by indirect immunofluorescence staining analysis. After transfection, expression of the genes could induce HeLa cells to exhibit the typical apoptosis features: such as plasma membrane blebbing and peripheral chromatin condensation. As compared with control groups, the untreated cells and the void vector transfected cells, the living cell number of the AIF gene transfected cells reduced distinctly. Conclusion: Our data prove that the expression of the recombinant human AIF fusion genes could induce apoptosis in transfected HeLa cells, which provides new strategy for cancer killing.

  8. Determinants of PDT-induced apoptosis

    Science.gov (United States)

    Kessel, David; Luo, Yu; Kim, Hyeong-Reh C.

    2000-03-01

    Photodynamic therapy can initiate cell death by apoptosis or necrosis. Using agents with known patterns of sub-cellular localization, we examined the correlation between sites of photodamage and the mode of cell death, using murine leukemia cells in vitro. Mitochondrial or mitochondrial/lysosomal photodamage caused the rapid release of cytochrome c. This effect was not temperature sensitive, and could be demonstrated immediately after irradiation of photosensitized cells at 10 degrees C. Subsequent warming to 37 degrees C led to a rapid apoptotic response, consistent with the known ability of cytochrome c to trigger the activation of caspase-3. In contrast, lysosomal or lysosomal/membrane photodamage resulted in the release of cathepsins and other proteolytic enzymes. A subsequent incubation at 37 degrees C resulted in mitochondrial degradation, leading to loss of cytochrome c within 30 min. The apoptotic response was both delayed and incomplete, with many dead cells not exhibiting an apoptotic morphology. The latter outcome was traced to photodamage to procaspase-3, an effect not observed with sensitizers that caused mainly mitochondrial photodamage. Studies in a cell-free system demonstrated that agents with lysosomal and/or membrane targets could bring about photoinactivation of caspase-3. These result are consistent with the proposal that photodynamic therapy can both activate and inactivate components of the apoptotic process.

  9. Coronavirus infection, ER stress and Apoptosis

    Directory of Open Access Journals (Sweden)

    TO SING eFUNG

    2014-06-01

    Full Text Available The replication of coronavirus, a family of important animal and human pathogens, is closely associated with the cellular membrane compartments, especially the endoplasmic reticulum (ER. Coronavirus infection of cultured cells was previously shown to cause ER stress and induce the unfolded protein response (UPR, a process that aims to restore the ER homeostasis by global translation shutdown and increasing the ER folding capacity. However under prolonged ER stress, UPR can also induce apoptotic cell death. Accumulating evidence from recent studies has shown that induction of ER stress and UPR may constitute a major aspect of coronavirus-host interaction. Activation of the three branches of UPR modulates a wide variety of signaling pathways, such as mitogen-activated protein (MAP kinases activation, autophagy, apoptosis and innate immune response. ER stress and UPR activation may therefore contribute significantly to the viral replication and pathogenesis during coronavirus infection. In this review, we summarize current knowledge on coronavirus-induced ER stress and UPR activation, with emphasis on their cross-talking to apoptotic signaling.

  10. Social apoptosis in honey bee superorganisms

    Science.gov (United States)

    Page, Paul; Lin, Zheguang; Buawangpong, Ninat; Zheng, Huoqing; Hu, Fuliang; Neumann, Peter; Chantawannakul, Panuwan; Dietemann, Vincent

    2016-01-01

    Eusocial insect colonies form superorganisms, in which nestmates cooperate and use social immunity to combat parasites. However, social immunity may fail in case of emerging diseases. This is the case for the ectoparasitic mite Varroa destructor, which switched hosts from the Eastern honeybee, Apis cerana, to the Western honey bee, Apis mellifera, and currently is the greatest threat to A. mellifera apiculture globally. Here, we show that immature workers of the mite’s original host, A. cerana, are more susceptible to V. destructor infestations than those of its new host, thereby enabling more efficient social immunity and contributing to colony survival. This counterintuitive result shows that susceptible individuals can foster superorganism survival, offering empirical support to theoretical arguments about the adaptive value of worker suicide in social insects. Altruistic suicide of immature bees constitutes a social analogue of apoptosis, as it prevents the spread of infections by sacrificing parts of the whole organism, and unveils a novel form of transgenerational social immunity in honey bees. Taking into account the key role of susceptible immature bees in social immunity will improve breeding efforts to mitigate the unsustainably high colony losses of Western honey bees due to V. destructor infestations worldwide. PMID:27264643

  11. Visualizing Vpr-induced G2 arrest and apoptosis.

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    Tomoyuki Murakami

    Full Text Available Vpr is an accessory protein of human immunodeficiency virus type 1 (HIV-1 with multiple functions. The induction of G2 arrest by Vpr plays a particularly important role in efficient viral replication because the transcriptional activity of the HIV-1 long terminal repeat is most active in G2 phase. The regulation of apoptosis by Vpr is also important for immune suppression and pathogenesis during HIV infection. However, it is not known whether Vpr-induced apoptosis depends on the ability of Vpr to induce G2 arrest, and the dynamics of Vpr-induced G2 arrest and apoptosis have not been visualized. We performed time-lapse imaging to examine the temporal relationship between Vpr-induced G2 arrest and apoptosis using HeLa cells containing the fluorescent ubiquitination-based cell cycle indicator2 (Fucci2. The dynamics of G2 arrest and subsequent long-term mitotic cell rounding in cells transfected with the Vpr-expression vector were visualized. These cells underwent nuclear mis-segregation after prolonged mitotic processes and then entered G1 phase. Some cells subsequently displayed evidence of apoptosis after prolonged mitotic processes and nuclear mis-segregation. Interestingly, Vpr-induced apoptosis was seldom observed in S or G2 phase. Likewise, visualization of synchronized HeLa/Fucci2 cells infected with an adenoviral vector expressing Vpr clearly showed that Vpr arrests the cell cycle at G2 phase, but does not induce apoptosis at S or G2 phase. Furthermore, time-lapse imaging of HeLa/Fucci2 cells expressing SCAT3.1, a caspase-3-sensitive fusion protein, clearly demonstrated that Vpr induces caspase-3-dependent apoptosis. Finally, to examine whether the effects of Vpr on G2 arrest and apoptosis were reversible, we performed live-cell imaging of a destabilizing domain fusion Vpr, which enabled rapid stabilization and destabilization by Shield1. The effects of Vpr on G2 arrest and subsequent apoptosis were reversible. This study is the first to

  12. Drug-induced reactivation of apoptosis abrogates HIV-1 infection.

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    Hartmut M Hanauske-Abel

    Full Text Available HIV-1 blocks apoptosis, programmed cell death, an innate defense of cells against viral invasion. However, apoptosis can be selectively reactivated in HIV-infected cells by chemical agents that interfere with HIV-1 gene expression. We studied two globally used medicines, the topical antifungal ciclopirox and the iron chelator deferiprone, for their effect on apoptosis in HIV-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates. Both medicines activated apoptosis preferentially in HIV-infected cells, suggesting that the drugs mediate escape from the viral suppression of defensive apoptosis. In infected H9 cells, ciclopirox and deferiprone enhanced mitochondrial membrane depolarization, initiating the intrinsic pathway of apoptosis to execution, as evidenced by caspase-3 activation, poly(ADP-ribose polymerase proteolysis, DNA degradation, and apoptotic cell morphology. In isolate-infected peripheral blood mononuclear cells, ciclopirox collapsed HIV-1 production to the limit of viral protein and RNA detection. Despite prolonged monotherapy, ciclopirox did not elicit breakthrough. No viral re-emergence was observed even 12 weeks after drug cessation, suggesting elimination of the proviral reservoir. Tests in mice predictive for cytotoxicity to human epithelia did not detect tissue damage or activation of apoptosis at a ciclopirox concentration that exceeded by orders of magnitude the concentration causing death of infected cells. We infer that ciclopirox and deferiprone act via therapeutic reclamation of apoptotic proficiency (TRAP in HIV-infected cells and trigger their preferential elimination. Perturbations in viral protein expression suggest that the antiretroviral activity of both drugs stems from their ability to inhibit hydroxylation of cellular proteins essential for apoptosis and for viral infection, exemplified by eIF5A. Our findings identify ciclopirox and deferiprone as prototypes of

  13. Cerebral ischemia—induced neuronal apoptosis mediated by nitric oxide

    Institute of Scientific and Technical Information of China (English)

    NomuY

    2002-01-01

    To elucidate the cellular and molecular mechanism of cerebral ischemia-induced neuronal apoptosis mediated by nitric oxide (NO) in the brain,we investigated:(1)cell death in hippocampal CA1 neurons of rats after a rransient four vessel occlusion (4VO)/reperfusion and (2) apoptosis induced by NOC18(NO releaser) using SHSY5Y cells,a human neuroblastoma cell line.We found that 4VO caused expression of inducible type of NO synthase (iNOS) in glial cells and neuronal apoptosis in CA1 region of rats.Next we examined in vitro apoptotic effects of NOC18 on SHSY5Y cells and suggest that NO decrease mitochondrial membrane potential,release cytochrome C from mitochondria,activates caspase-3,degrade inhibitor of caspase-activated DNase(Icad),and activated DNase translocate into nucleus and induce DNA fragmentation.Thus we conclude that the excess amount of NO produced by glial iNOS at cerebral ischemia could be involved in neuronal apoptosis in CA1 region.Regarding NO action on neurons,we further obtained that NO propects neuronal apoptosis in PC12 cells perhaps by nitrosylation of caspase,subsequent reduction of proteolytic activity.Taken together,we suggest that NO seem to exert dual effects(toxic and beneficial) on neuronal apoptosis,the one (toxic);apoptosis-induction throuth the decrease in mitochondrial membrane potentials and cytochrome C release and the othe (beneficial);protection against apoptosis through the inhibition of caspase activity.

  14. Apoptosis of human primary gastric carcinoma cells induced by genistein

    Institute of Scientific and Technical Information of China (English)

    Hai-Bo Zhou; Juan-Juan Chen; Wen-Xia Wang; Jian-Ting Cai; Qin Du

    2004-01-01

    AIM: To investigate the apoptosis in primary gastric cancer cells induced by genistein, and the relationship between this apoptosis and expression of bcl-2 and bax.METHODS: MTT assay was used to determine the cell growth inhibitory rate in vitro. Transmission electron microscope and TUNEL staining were used to quantitatively and qualitatively detect the apoptosis of primary gastric cancer cells before and after genistein treatment. Immunohistochemical staining and RT-PCR were used to detect the expression of apoptosisassociated genes bcl-2 and bax.RESULTS: Genistein inhibited the growth of primary gastric cancer cells in dose-and time-dependent manner. Genistein induced primary gastric cancer cells to undergo apoptosis with typically apoptotic characteristics. TUNEL assay showed that after the treatment of primary gastric cancer cells with genistein for 24 to 96 h, the apoptotic rates of primary gastric cancer cells increased time-dependently. Immunohistochemical staining showed that after the treatment of primary gastric cancer cells with genistein for 24 to 96 h, the positivity rates of Bcl-2 proteins were apparently reduced with time and the positivity rates of Bax proteins were apparently increased with time. After exposed to genistein at 20 μmol/L for 24,48, 72 and 96 respectively, the density of bcl-2 mRNA decreased progressively and the density of bax mRNA increased progressively with elongation of time.CONCLUSION: Genistein is able to induce the apoptosis in primary gastric cancer cells. This apoptosis may be mediated by down-regulating the apoptosis- associated bcl-2 gene and up-regulating the expression of apoptosis-associated bax gene.

  15. Multiple-Valued Immune Network with Apoptosis System

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    Yamaguchi, Takayuki; Tang, Zheng

    In this paper, we describe a new model of immune network based on biological immune response network. We propose an immunity like multiple-valued network with apoptosis mechanism. The model is based on the interaction between B cells and T cells and the biological apoptosis mechanism in human body. With the mechanism, a naturally immune system can be reproduced. The model is also applied to pattern recognition. It gets possible with a conventional model to restricting categories increase of memory patterns.

  16. Novel pathogenesis: regulation of apoptosis by Apelin/APJ system.

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    Liu, Jiaqi; Liu, Meiqing; Chen, Linxi

    2017-06-01

    Apelin is the endogenous peptide APJ receptor, while APJ is a member of the G protein-coupled receptors family. Recent evidence strongly suggests that Apelin/APJ system influences apoptosis in various diseases through different signal pathways. In this review, we discuss the possible mechanisms by which the Apelin/APJ system inhibits apoptosis, including the phosphatidylinositol-3-kinase (PI3K)/Akt, ERK1/2, caspase signaling, and autophagy pathway. We also summarize the role of Apelin/APJ system in apoptosis in myocardial ischemia-reperfusion (I/R) injury, pulmonary artery hypertension, retinal neovascular disease, acute renal injury, skeletal homeostasis, and gastrointestinal diseases. Apelin/APJ system decreases myocardial infarction size and alleviates myocardial I/R injury by inhibiting cardiomyocytes apoptosis. However, Apelin/APJ system improves pulmonary artery hypertension via increasing apoptosis. Apelin/APJ system exerts neuroprotective effect by blocking apoptosis and participates in the recovery of retinal neovascular disease by suppressing apoptosis. Apelin/APJ system also shows anti-apoptotic effect against acute renal injury and plays a role in regulating skeletal homeostasis. In gastrointestinal disease, Apelin/APJ system plays a potential physiological role in gastrointestinal cytoprotection by regulating apoptosis. We hope that a better understanding of the Apelin/APJ system will help to discover new disease pathogenesis and find possible therapeutic targets of the Apelin/APJ system essential for various diseases. © The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  17. Apoptosis in HEp-2 cells infected with Ureaplasma diversum.

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    Amorim, Aline Teixeira; Marques, Lucas Miranda; Santos, Angelita Maria Oliveira Gusmão; Martins, Hellen Braga; Barbosa, Maysa Santos; Rezende, Izadora Souza; Andrade, Ewerton Ferraz; Campos, Guilherme Barreto; Lobão, Tássia Neves; Cortez, Beatriz Araujo; Monezi, Telma Alvez; Machado-Santelli, Glaucia Maria; Timenetsky, Jorge

    2014-09-04

    Bacterial pathogens have many strategies for infecting and persisting in host cells. Adhesion, invasion and intracellular life are important features in the biology of mollicutes. The intracellular location of Ureaplasma diversum may trigger disturbances in the host cell. This includes activation or inhibition of pro and anti-apoptotic factors, which facilitate the development of host damage. The aim of the present study was to associate U. diversum infection in HEp-2 cells and apoptosis induction. Cells were infected for 72hs with four U. diversum clinical isolates and an ATCC strain. The U. diversum invasion was analyzed by Confocal Laser Scanning Microscopy and gentamicin invasion assay. The apoptosis was evaluated using pro-apoptotic and anti-apoptotic gene expression, and FITC Annexin V/Dead Cell Apoptosis Kit. The number of internalized ureaplasma in HEp-2 cells increased significantly throughout the infection. The flow cytometry analysis with fluorochromes to detect membrane depolarization and gene expression for caspase 2, 3 and 9 increased in infected cells after 24 hours. However, after 72 hours a considerable decrease of apoptotic cells was observed. The data suggests that apoptosis may be initially induced by some isolates in association with HEp-2 cells, but over time, there was no evidence of apoptosis in the presence of ureaplasma and HEp-2 cells. The initial increase and then decrease in apoptosis could be related to bacterial pathogen-associated molecular pattern (PAMPS). Moreover, the isolates of U. diversum presented differences in the studied parameters for apoptosis. It was also observed that the amount of microorganisms was not proportional to the induction of apoptosis in HEp-2 cells.

  18. Peptides Regulate Cortical Thymocytes Differentiation, Proliferation, and Apoptosis

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    V. Kh. Khavinson

    2011-01-01

    Full Text Available The processes of differentiation, proliferation, and apoptosis were studied in a cell culture of human cortical thymocytes under the influence of short peptides T-32 (Glu-Asp-Ala and T-38 (Lys-Glu-Asp. Peptides T-32 and T-38 amplified cortical thymocytes differentiation towards regulatory T cells, increased their proliferative activity, and decreased the level of apoptosis. Moreover, peptides under study stimulated proliferative and antiapoptotic activity of the mature regulatory T cells.

  19. Apoptosis and T cell depletion during feline infectious peritonitis

    OpenAIRE

    Horzinek, M.C.; Haagmans, B. L.; Egberink, H F

    1996-01-01

    Cats that have succumbed to feline infectious peritonitis, an immune- mediated disease caused by variants of feline coronaviruses, show apoptosis and T-cell depletion in their lymphoid organs. The ascitic fluid that develops in the course of the condition causes apoptosis in vitro but only in activated T cells. Since feline infectious peritonitis virus does not infect T cells, and viral proteins did not inhibit T-cell proliferation, we postulate that soluble mediators released during the infe...

  20. Apoptosis transcriptional mechanism of feline infectious peritonitis virus infected cells.

    Science.gov (United States)

    Shuid, Ahmad Naqib; Safi, Nikoo; Haghani, Amin; Mehrbod, Parvaneh; Haron, Mohd Syamsul Reza; Tan, Sheau Wei; Omar, Abdul Rahman

    2015-11-01

    Apoptosis has been postulated to play an important role during feline infectious peritonitis virus (FIPV) infection; however, its mechanism is not well characterized. This study is focused on apoptosis and transcriptional profiling of FIPV-infected cells following in vitro infection of CRFK cells with FIPV 79-1146 WSU. Flow cytometry was used to determine mode of cell death in first 42 h post infection (hpi). FIPV infected cells underwent early apoptosis at 9 hpi (p < 0.05) followed by late apoptosis at 12 hpi (p < 0.05) and necrosis from 24 hpi (p < 0.05). Then, next generation sequencing was performed on 9 hpi and control uninfected cells by Illumina analyzer. An aggregate of 4546 genes (2229 down-regulated and 2317 up-regulated) from 17 cellular process, 11 molecular functions and 130 possible biological pathways were affected by FIPV. 131 genes from apoptosis cluster (80 down-regulated and 51 up-regulated) along with increase of apoptosis, p53, p38 MAPK, VEGF and chemokines/cytokines signaling pathways were probably involved in apoptosis process. Six of the de-regulated genes expression (RASSF1, BATF2, MAGEB16, PDCD5, TNFα and TRAF2) and TNFα protein concentration were analyzed by RT-qPCR and ELISA, respectively, at different time-points. Up-regulations of both pro-apoptotic (i.e. PDCD5) and anti-apoptotic (i.e. TRAF2) were detected from first hpi and continuing to deregulate during apoptosis process in the infected cells.

  1. Crizotinib induces PUMA-dependent apoptosis in colon cancer cells.

    Science.gov (United States)

    Zheng, Xingnan; He, Kan; Zhang, Lin; Yu, Jian

    2013-05-01

    Oncogenic alterations in MET or anaplastic lymphoma kinase (ALK) have been identified in a variety of human cancers. Crizotinib (PF02341066) is a dual MET and ALK inhibitor and approved for the treatment of a subset of non-small cell lung carcinoma and in clinical development for other malignancies. Crizotinib can induce apoptosis in cancer cells, whereas the underlying mechanisms are not well understood. In this study, we found that crizotinib induces apoptosis in colon cancer cells through the BH3-only protein PUMA. In cells with wild-type p53, crizotinib induces rapid induction of PUMA and Bim accompanied by p53 stabilization and DNA damage response. The induction of PUMA and Bim is mediated largely by p53, and deficiency in PUMA or p53, but not Bim, blocks crizotinib-induced apoptosis. Interestingly, MET knockdown led to selective induction of PUMA, but not Bim or p53. Crizotinib also induced PUMA-dependent apoptosis in p53-deficient colon cancer cells and synergized with gefitinib or sorafenib to induce marked apoptosis via PUMA in colon cancer cells. Furthermore, PUMA deficiency suppressed apoptosis and therapeutic responses to crizotinib in xenograft models. These results establish a critical role of PUMA in mediating apoptotic responses of colon cancer cells to crizotinib and suggest that mechanisms of oncogenic addiction to MET/ALK-mediated survival may be cell type-specific. These findings have important implications for future clinical development of crizotinib.

  2. Apoptosis of wound fibroblasts induced by oxidative stress.

    Science.gov (United States)

    Takahashi, Atsushi; Aoshiba, Kazutetsu; Nagai, Atsushi

    2002-06-01

    Irreversible lung parenchymal injury is usually healed by fibrosis, which depends on the abilities of fibroblasts to proliferate, migrate into the wound, and survive. Because the lung is frequently exposed to increased oxidative stress, which is thought to mediate apoptosis, we examined whether oxidative stress induces apoptosis in fibroblasts during wound healing. We performed an in vitro scratch wound assay where cultured fibroblast monolayers were exposed to H2O2 (10-500 microM) after artificial wounding. Apoptosis was evaluated by nuclear staining with Hoechst33342 or terminal deoxynucleotidyl transferase (TdT)-mediated nucleotide nick end-labeling (TUNEL). Intracellular oxidants were assessed with the peroxide-sensitive fluorochrome carboxydichlorodihydrofluorescein (CDCF). We found that repopulating fibroblasts at the wound margin, but not quiescent fibroblasts at the intact site, selectively underwent oxidant accumulation and apoptosis in response to H2O2 exposure. Some of the apoptotic cells had incorporated bromodeoxyuridine (BrdU), an indicator of proliferating cells. These results suggest that oxidative stress selectively induces apoptosis in fibroblasts that are stimulated to proliferate and/or migrate into the wound. Fibroblast apoptosis induced by oxidative stress during wound repopulation may be relevant to intractable wound healing.

  3. Noxa couples lysosomal membrane permeabilization and apoptosis during oxidative stress.

    Science.gov (United States)

    Eno, Colins O; Zhao, Guoping; Venkatanarayan, Avinashnarayan; Wang, Bing; Flores, Elsa R; Li, Chi

    2013-12-01

    The exact roles of lysosomal membrane permeabilization (LMP) in oxidative stress-triggered apoptosis are not completely understood. Here, we first studied the temporal relation between LMP and mitochondrial outer membrane permeabilization (MOMP) during the initial stage of apoptosis caused by the oxidative stress inducer H2O2. Despite its essential role in mediating apoptosis, the expression of the BH3-only Bcl-2 protein Noxa was dispensable for LMP. In contrast, MOMP was dependent on Noxa expression and occurred downstream of LMP. When lysosomal membranes were stabilized by the iron-chelating agent desferrioxamine, H2O2-induced increase in DNA damage, Noxa expression, and subsequent apoptosis were abolished by the inhibition of LMP. Importantly, LMP-induced Noxa expression increase was mediated by p53 and seems to be a unique feature of apoptosis caused by oxidative stress. Finally, exogenous iron loading recapitulated the effects of H2O2 on the expression of BH3-only Bcl-2 proteins. Overall, these data reveal a Noxa-mediated signaling pathway that couples LMP with MOMP and ultimate apoptosis during oxidative stress.

  4. Fluidization of tissues by cell division and apoptosis.

    Science.gov (United States)

    Ranft, Jonas; Basan, Markus; Elgeti, Jens; Joanny, Jean-François; Prost, Jacques; Jülicher, Frank

    2010-12-07

    During the formation of tissues, cells organize collectively by cell division and apoptosis. The multicellular dynamics of such systems is influenced by mechanical conditions and can give rise to cell rearrangements and movements. We develop a continuum description of tissue dynamics, which describes the stress distribution and the cell flow field on large scales. In the absence of division and apoptosis, we consider the tissue to behave as an elastic solid. Cell division and apoptosis introduce stress sources that, in general, are anisotropic. By combining cell number balance with dynamic equations for the stress source, we show that the tissue effectively behaves as a viscoelastic fluid with a relaxation time set by the rates of division and apoptosis. If the system is confined in a fixed volume, it reaches a homeostatic state in which division and apoptosis balance. In this state, cells undergo a diffusive random motion driven by the stochasticity of division and apoptosis. We calculate the expression for the effective diffusion coefficient as a function of the tissue parameters and compare our results concerning both diffusion and viscosity to simulations of multicellular systems using dissipative particle dynamics.

  5. Mitochondrial-dependent apoptosis in experimental rodent abdominal aortic aneurysms.

    Science.gov (United States)

    Sinha, Indranil; Sinha-Hikim, Amiya P; Hannawa, Kevin K; Henke, Peter K; Eagleton, Matthew J; Stanley, James C; Upchurch, Gilbert R

    2005-10-01

    While extrinsic mechanisms of apoptosis in abdominal aortic aneurysms (AAAs) are recognized, this project hypothesizes that an intrinsic, mitochondrial-dependent, mechanism of apoptosis also contributes to experimental AAA formation. Rat aortas were perfused with either saline or elastase (N = 5 per group) and harvested 7 days postperfusion. The aortas were placed in gluteraldehyde for subsequent transmission electron microscopy, Bouin's solution for TUNEL, or paraformaldehyde for immunohistochemical staining for caspase-9, caspase-3, and Bid. Abdominal aortic diameters increased 168 +/- 25% (mean +/- SEM) after elastase perfusion. compared with 30 +/- 5% after saline perfusion (P < .001). Apoptosis of aortic smooth muscle cells, macrophages, and neutrophils was evidenced by transmission electron microscopy and TUNEL in the elastase-perfused aneurysmal aortas. Quantitative analysis of the apoptotic cells revealed a significant (P < .01) increase in the number of total apoptotic cells in the elastase-perfused aortas (12 +/- 3 cells per high-power field), compared with that of saline-infused controls (1.3 +/- 0.2). Caspase-9, the key initiator in the mitochondrial-dependent apoptotic pathway, stained positively in only elastase-perfused aortas. Bid staining was not detected in either the elastase-perfused aortas or the saline controls. Apoptosis is evident in multiple cell lines in elastase-perfused aneurysmal aortas, but rarely observed in control aortas. Caspase-9, the key initiator of intrinsic apoptosis, was documented only in elastase-perfused aortas. These results suggest that mitochondrial-dependent apoptosis is associated with abdominal aortic aneurysm formation.

  6. Enhancement of basophil apoptosis by olopatadine and theophylline.

    Science.gov (United States)

    Kawakami, Ayako; Suzukawa, Maho; Koketsu, Rikiya; Komiya, Akiko; Ohta, Ken; Yamamoto, Kazuhiko; Yamaguchi, Masao

    2008-01-01

    Regulation of basophil survival is an important aspect in the pathogenesis of allergic inflammation associated with local accumulation of basophils. However, pharmacologic modulation of basophil survival is largely unknown except for the apoptosis-enhancing effect of glucocorticoids. We tested the effects of two anti-allergic and anti-asthmatic drugs, olopatadine and theophylline, on basophil survival. Basophils were highly purified from normal human peripheral blood. Apoptosis was analyzed by flow cytometry using annexin V staining or another staining method that detected alterations in the mitochondrial transmembrane potential. In addition to the conventional method using annexin V, basophil apoptosis was successfully established by analysis of the mitochondrial transmembrane potential. Olopatadine decreased the number of live basophils, and they induced apoptosis of basophils during culture. The decline in live basophils was induced by olopatadine even when low doses of IL-3 were included in the culture medium. Theophylline also affected basophil apoptosis and induced a decrease in the number of live basophils. Basophil apoptosis was enhanced by both olopatadine and theophylline. This effect may partly explain the pharmacologic basis of why these drugs are effective on allergic diseases.

  7. Wogonin Induces Eosinophil Apoptosis and Attenuates Allergic Airway Inflammation

    Science.gov (United States)

    Dorward, David A.; Sharma, Sidharth; Rennie, Jillian; Felton, Jennifer M.; Alessandri, Ana L.; Duffin, Rodger; Schwarze, Jurgen; Haslett, Christopher; Rossi, Adriano G.

    2015-01-01

    Rationale: Eosinophils are key effector cells in allergic diseases, including allergic rhinitis, eczema, and asthma. Their tissue presence is regulated by both recruitment and increased longevity at inflamed sites. Objectives: To investigate the ability of the flavone wogonin to induce eosinophil apoptosis in vitro and attenuate eosinophil-dominant allergic inflammation in vivo in mice. Methods: Human and mouse eosinophil apoptosis in response to wogonin was investigated by cellular morphology, flow cytometry, mitochondrial membrane permeability, and pharmacological caspase inhibition. Allergic lung inflammation was modeled in mice sensitized and challenged with ovalbumin. Bronchoalveolar lavage (BAL) and lung tissue were examined for inflammation, mucus production, and inflammatory mediator production. Airway hyperresponsiveness to aerosolized methacholine was measured. Measurements and Main Results: Wogonin induced time- and concentration-dependent human and mouse eosinophil apoptosis in vitro. Wogonin-induced eosinophil apoptosis occurred with activation of caspase-3 and was inhibited by pharmacological caspase inhibition. Wogonin administration attenuated allergic airway inflammation in vivo with reductions in BAL and interstitial eosinophil numbers, increased eosinophil apoptosis, reduced airway mucus production, and attenuated airway hyperresponsiveness. This wogonin-induced reduction in allergic airway inflammation was prevented by concurrent caspase inhibition in vivo. Conclusions: Wogonin induces eosinophil apoptosis and attenuates allergic airway inflammation, suggesting that it has therapeutic potential for the treatment of allergic inflammation in humans. PMID:25629436

  8. Signaling pathways of the ING proteins in apoptosis.

    Science.gov (United States)

    Shah, Sitar; Riabowol, Karl

    2009-05-01

    Members of the ING family of type II tumor suppressors reside in different chromatin regulatory complexes and are stoichiometeric members of histone acetyltransferase (HAT) and histone deacetylase (HDAC) complexes. It has been frequently observed that expressing ING proteins promotes apoptosis in both normal and transformed cells of different species. They have also been reported to either rely upon p53, or to add to its ability to promote programmed cell death (apoptosis) although whether ING proteins require p53 to induce apoptosis is now questionable based upon observations using knockout cell lines and animal models. Genetic studies in model organisms, and particularly in Caenorhabditis elegans, have identified different pathways involved in apoptosis during development, in the germ line and in response to various forms of stress including DNA damage. In this review we summarize structural features of the INGs and recent observations made in knockout models of Mus musculus and Caenorhabditis elegans that have helped to further clarify the functions of the ING proteins in biochemical pathways leading to apoptosis. Based upon these observations we propose a model for how ING proteins may act both independently and in concert with p53 to promote apoptosis.

  9. Differential Apoptosis Radiosensitivity of Neural Progenitors in Adult Mouse Hippocampus

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    Yu-Qing Li

    2016-06-01

    Full Text Available Mammalian tissue-specific stem cells and progenitors demonstrate differential DNA damage response. Neural progenitors in dentate gyrus of the hippocampus are known to undergo apoptosis after irradiation. Using a mouse model of hippocampal neuronal development, we characterized the apoptosis sensitivity of the different neural progenitor subpopulations in adult mouse dentate gyrus after irradiation. Two different bromodeoxyuridine incorporation paradigms were used for cell fate mapping. We identified two apoptosis sensitive neural progenitor subpopulations after irradiation. The first represented non-proliferative and non-newborn neuroblasts and immature neurons that expressed doublecortin, calretinin or both. The second consisted of proliferative intermediate neural progenitors. The putative radial glia-like neural stem cells or type-1 cells, regardless of proliferation status, were apoptosis resistant after irradiation. There was no evidence of radiation-induced apoptosis in the absence of the Trp53 (p53 gene but absence of Cdkn1a (p21 did not alter the apoptotic response. Upregulation of nuclear p53 was observed in neuroblasts after irradiation. We conclude that adult hippocampal neural progenitors may demonstrate differential p53-dependent apoptosis sensitivity after irradiation.

  10. Endothelial cell apoptosis correlates with low haptoglobin concentrations in diabetes.

    Science.gov (United States)

    Dalan, Rinkoo; Liu, Xiaofeng; Goh, Liuh Ling; Bing, Sun; Luo, Kathy Qian

    2017-08-01

    The haptoglobin 2-2 genotype is associated with lower haptoglobin concentrations and atherosclerosis in diabetes. Endothelial cell apoptosis contributes significantly to atherosclerosis. We studied endothelial cell apoptosis in diabetes patients with haptoglobin 2-2 and non-haptoglobin 2-2 genotype. Approach and results: We pooled plasma from 10 patients with haptoglobin 2-2 and non-haptoglobin 2-2 genotype and quantified endothelial cell apoptosis using a hemodynamic lab-on-chip system. Then, we conducted similar experiments on individual diabetes plasma samples with the haptoglobin 2-2 ( n = 20) and non-haptoglobin 2-2 genotype ( n = 20). Haptoglobin beta concentrations were measured by Western blot analysis. We looked for association with demographic, metabolic variables, inflammation and oxidative stress. In pooled plasma, endothelial cell apoptosis was higher in haptoglobin 2-2 group (haptoglobin 2-2: 23.18% vs non-haptoglobin 2-2:15.32%). In individual samples, univariate analysis showed that endothelial cell apoptosis correlated with haptoglobin beta concentration [ β = -10.29 (95% confidence interval: -13.44, -7.14), p  0.05). These results show that regardless of the haptoglobin genotype, haptoglobin is associated with prevention of endothelial cell apoptosis in diabetes.

  11. Effect of hepatitis C infection on HIV-induced apoptosis.

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    Tomasz Laskus

    Full Text Available BACKGROUND: Hepatitis C virus (HCV coinfection was reported to negatively affect HIV disease and HIV infection has a deleterious effect on HCV-related liver disease. However, despite common occurrence of HCV/HIV coinfection little is known about the mechanisms of interactions between the two viruses. METHODS: We studied CD4+ and CD8+ T cell and CD19+ B cell apoptosis in 104 HIV-positive patients (56 were also HCV-positive and in 22 HCV/HIV-coinfected patients treated for chronic hepatitis C with pegylated interferon and ribavirin. We also analyzed HCV/HIV coinfection in a Daudi B-cell line expressing CD4 and susceptible to both HCV and HIV infection. Apoptosis was measured by AnnexinV staining. RESULTS: HCV/HIV coinfected patients had lower CD4+ and CD8+ T cell apoptosis and higher CD19+ B cell apoptosis than those with HIV monoinfection. Furthermore, anti-HCV treatment of HCV/HIV coinfected patients was followed by an increase of CD4+ and CD8+ T cell apoptosis and a decrease of CD19+ B cell apoptosis. In the Daudi CD4+ cell line, presence of HCV infection facilitated HIV replication, however, decreased the rate of HIV-related cell death. CONCLUSION: In HCV/HIV coinfected patients T-cells were found to be destroyed at a slower rate than in HIV monoinfected patients. These results suggest that HCV is a molecular-level determinant in HIV disease.

  12. La dicotomía de los virus polioma: ¿Infección lítica o inducción de neoplasias? The paradox of polyomaviruses Lytic infection or tumor induction?

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    Norberto A. Sanjuan

    2004-02-01

    Full Text Available Los virus Polioma murinos provocan infecciones líticas en cultivos de células de ratón y transforman in vitro células de rata a través de la interacción de su oncogén mT con diversos reguladores celulares. Luego de su inoculación en ratones neonatos inducen neoplasias epiteliales y mesenquimáticas. Se ha propuesto que las cepas de polioma más oncogénicas son aquellas que previamente replican más en el ratón. Sin embargo, a nivel de una sola célula la infección lítica y la transformación deberían ser mutuamente excluyentes. En cada neoplasia han sido descriptos 3 tipos celulares según expresen el DNA viral solo o concomitantemente con la proteína mayor de la cápside VP1, o que no contengan DNA viral ni VP-1. En nuestro laboratorio detectamos la existencia de un cuarto tipo celular en las neoplasias, en el que se expresa la totalidad del genoma viral pero no ocurre el ensamblaje, probablemente por alteraciones en la fosforilación de VP-1. Se discuten los mecanismos de migración intracelular de Polioma, la diseminación en el ratón y los factores que podrían estar involucrados en la inducción de neoplasias o en la infección lítica inducidas por el virus.Murine polyomaviruses can produce lytic infections in mouse cell cultures or transform in vitro rat fibroblasts through a complex interaction with key cellular regulators. After infection of newborn mice, some strains of polyomavirus induce epithelial and mesenchymal tumors. It has been described that there is a direct relationship between viral dissemination in the mouse and tumor induction. However, at a single cell level lytic infection and transformation would not be able to coexist. The existence of 3 distinct cell populations in polyoma-induced tumors, classified according to the presence or absence of viral DNA and viral capsid protein VP-1 have been described. We have reported a fourth type of cell in the neoplasms, that can express the early and the late viral

  13. Ultrastructural study shows morphologic features of apoptosis and para-apoptosis in megakaryocytes from patients with idiopathic thrombocytopenic purpura

    NARCIS (Netherlands)

    Houwerzijl, EJ; Blom, NR; van der Want, JJL; Esselink, MT; Koornstra, JJ; Smit, JW; Louwes, H; Vellenga, E; de Wolf, JTM

    2004-01-01

    To investigate whether altered megakaryocyte morphology contributes to reduced platelet production in idiopathic thrombocytopenic purpura (ITP), ultrastructural analysis of megakaryocytes was performed in 11 ITP patients. Ultrastructural abnormalities compatible with (para-)apoptosis were present in

  14. Advance research in apoptosis mediating by death receptor

    Institute of Scientific and Technical Information of China (English)

    JI Yu-bin; LIU Hong-juan; JI Chen-feng; ZHANG He

    2008-01-01

    Apoptosis is one of the main types of programmed cell deaths (PCD) and involves a series of biochemical events that lead to a variety of morphological changes and death. The initial and progress of apoptosis is precisely regulated. This review will summarize current knowledge of the signal transduction pathways of apoptosis. It is now well-established that the apoptotic signals generally involve the extrinsic or intrinsic pathways of apoptosis. The extrinsic pathway originates at the membrane and engages cell surface death receptors whereas the intrinsic pathway predominantly involves mitochondria. In the intrinsic pathway, the cell death signal induced changes of mitochondrial membrane permeability and the loss of membrane potential. Many proteins factors released, and then cytoplasmic cytochrome C and easpase-9 form of apoptosis. The activated caspase-9 cut caspase-3, then cell dead at last. In the case of extrinsic pathway, several death receptors exist including Fas, TNFR-1, DR3, DR4, DR5 and DR6. These death receptors contain an intracellular region of approximately 80 amino acids that is designated as "death domain". The death domain is an important structure that plays a key role in the transduction of apoptotic signals. The interaction between Fas and its ligand (FasL) triggers the formation of a death-inducing signaling complex (DISC), which subsequently recruits and activates caspase-8; this in turn activates other procaspases and culminates in the cleavage of cellular substrates and apoptosis. During the process of tumor cell lines apoptosis Inducted by chemotherapy. It is easy to see the increasing of the Fas receptors and inducing of FasL expression, it can inhibite apoptosis when the blocking Fas / FasL. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a type Ⅱ transmembrane protein belonging to the TNF family of death ligands. TRAIL has been suggested as a safe and tumorselective anticancer agent with low toxicity to normal

  15. Avenanthramides Prevent Osteoblast and Osteocyte Apoptosis and Induce Osteoclast Apoptosis in Vitro in an Nrf2-Independent Manner

    Directory of Open Access Journals (Sweden)

    Gretel G. Pellegrini

    2016-07-01

    Full Text Available Oats contain unique bioactive compounds known as avenanthramides (AVAs with antioxidant properties. AVAs might enhance the endogenous antioxidant cellular response by activation of the transcription factor Nrf2. Accumulation of reactive oxygen species plays a critical role in many chronic and degenerative diseases, including osteoporosis. In this disease, there is an imbalance between bone formation by osteoblasts and bone resorption by osteoclasts, which is accompanied by increased osteoblast/osteocyte apoptosis and decreased osteoclast apoptosis. We investigated the ability of the synthethic AVAs 2c, 2f and 2p, to 1-regulate gene expression in bone cells, 2-affect the viability of osteoblasts, osteocytes and osteoclasts, and the generation of osteoclasts from their precursors, and 3-examine the potential involvement of the transcription factor Nrf2 in these actions. All doses of AVA 2c and 1 and 5 µM dose of 2p up-regulated collagen 1A expression. Lower doses of AVAs up-regulated OPG (osteoprotegerin in OB-6 osteoblastic cells, whereas 100 μM dose of 2f and all concentrations of 2c down-regulated RANKL gene expression in MLO-Y4 osteocytic cells. AVAs did not affect apoptosis of OB-6 osteoblastic cells or MLO-Y4 osteocytic cells; however, they prevented apoptosis induced by the DNA topoisomerase inhibitor etoposide, the glucocorticoid dexamethasone, and hydrogen peroxide. AVAs prevented apoptosis of both wild type (WT and Nrf2 Knockout (KO osteoblasts, demonstrating that AVAs-induced survival does not require Nrf2 expression. Further, KO osteoclast precursors produced more mature osteoclasts than WT; and KO cultures exhibited less apoptotic osteoclasts than WT cultures. Although AVAs did not affect WT osteoclasts, AVA 2p reversed the low apoptosis of KO osteoclasts. These in vitro results demonstrate that AVAs regulate, in part, the function of osteoblasts and osteocytes and prevent osteoblast/osteocyte apoptosis and increase osteoclast

  16. Increased expression of cytokines, soluble cytokine receptors, soluble apoptosis ligand and apoptosis in dengue.

    Science.gov (United States)

    Arias, Julia; Valero, Nereida; Mosquera, Jesús; Montiel, Milagros; Reyes, Eduardo; Larreal, Yraima; Alvarez-Mon, Melchor

    2014-03-01

    Several studies have been performed to determine biomarkers that define the risk factors to developing severe forms of dengue. In this study, the levels of TNF-α, IL-6, IL-1, IL-17, soluble interleukin-1 receptor like 1 protein (sST2), soluble TNF-related apoptosis-inducing ligand (sTRAIL), IL-12 and soluble receptors for TNF (sTNF-RI and sTNF-RII) were determined by ELISA in dengue patients and monocyte/macrophage cultures. Dengue was classified as dengue without warning symptoms (DNWS), with warning symptoms (DWWS) and severe dengue (SD). High values of IL-6, sTNFRI, sTNFRII and sST2 were observed in DWWS and/or SD and IL-12 and sTRAIL in DNWS. TNF-α and IL-17 were increased not associated to the disease severity. High production of TNF-α, IL-1β, IL-12, IL-17, sST2 and sTRAIL and apoptosis expression were observed in dengue monocyte/macrophage cultures. This study shows that beneficial or deleterious biomarkers can be present in dengue regardless the disease severity and that monocytes may be in part the source of studied molecules.

  17. Apoptosis in ovarian cells in postmenopausal women.

    Directory of Open Access Journals (Sweden)

    Maria Laszczyńska

    2007-06-01

    Full Text Available Apoptosis is a natural process which accompanies human ovary from the moment of birth until old age. While it is a well-known process at the reproductive age, it still needs to be thoroughly examined when referring to the postmenopausal age. The study involved 30 postmenopausal women who had their ovaries removed by laparotomy due to nonneoplastic diseases of the uterus. The women were divided into 3 groups depending on the time that had passed since the last menstruation. Group A consisted of women who had their last menstruation no more than 5 years earlier. In group B menopause occurred 5 to 10 years earlier. Group C was composed of patients who had the last menstruation over 10 years earlier. In all the patients concentrations of follitropin (FSH and estradiol (E2 in blood plasma were measured. Ovarian tissue was obtained during surgery. For morphological studies, ovaries were fixed in Bouin's solution and 4% formalin and embedded in paraffin. Morphological analysis was carried out after hematoxylin-eosin (H-E staining. For histochemical detection of apoptotic cells (in situ localization of fragment DNA, the TUNEL method was used. The expression of caspase-3 positive cells was determined immunohistochemically in paraffin-embedded specimens. Comparing to groups A and B, the ovaries in group C contained small number of corpora albicantia located in the medullary part as well as thinned blood vessels and few lymphatic vessels and nerves. In contrast to group A where the number of TUNEL-positive cells was high and caspase-3 expression was observed, no TUNEL-positive nuclei and caspase-3 expression were found in the examined ovaries of group C women.

  18. Enoxacin Directly Inhibits Osteoclastogenesis without Inducing Apoptosis*

    Science.gov (United States)

    Toro, Edgardo J.; Zuo, Jian; Ostrov, David A.; Catalfamo, Dana; Bradaschia-Correa, Vivian; Arana-Chavez, Victor; Caridad, Aliana R.; Neubert, John K.; Wronski, Thomas J.; Wallet, Shannon M.; Holliday, L. Shannon

    2012-01-01

    Enoxacin has been identified as a small molecule inhibitor of binding between the B2-subunit of vacuolar H+-ATPase (V-ATPase) and microfilaments. It inhibits bone resorption by calcitriol-stimulated mouse marrow cultures. We hypothesized that enoxacin acts directly and specifically on osteoclasts by disrupting the interaction between plasma membrane-directed V-ATPases, which contain the osteoclast-selective a3-subunit of V-ATPase, and microfilaments. Consistent with this hypothesis, enoxacin dose-dependently reduced the number of multinuclear cells expressing tartrate-resistant acid phosphatase (TRAP) activity produced by RANK-L-stimulated osteoclast precursors. Enoxacin (50 μm) did not induce apoptosis as measured by TUNEL and caspase-3 assays. V-ATPases containing the a3-subunit, but not the “housekeeping” a1-subunit, were isolated bound to actin. Treatment with enoxacin reduced the association of V-ATPase subunits with the detergent-insoluble cytoskeleton. Quantitative PCR revealed that enoxacin triggered significant reductions in several osteoclast-selective mRNAs, but levels of various osteoclast proteins were not reduced, as determined by quantitative immunoblots, even when their mRNA levels were reduced. Immunoblots demonstrated that proteolytic processing of TRAP5b and the cytoskeletal protein l-plastin was altered in cells treated with 50 μm enoxacin. Flow cytometry revealed that enoxacin treatment favored the expression of high levels of DC-STAMP on the surface of osteoclasts. Our data show that enoxacin directly inhibits osteoclast formation without affecting cell viability by a novel mechanism that involves changes in posttranslational processing and trafficking of several proteins with known roles in osteoclast function. We propose that these effects are downstream to blocking the binding interaction between a3-containing V-ATPases and microfilaments. PMID:22474295

  19. Enoxacin directly inhibits osteoclastogenesis without inducing apoptosis.

    Science.gov (United States)

    Toro, Edgardo J; Zuo, Jian; Ostrov, David A; Catalfamo, Dana; Bradaschia-Correa, Vivian; Arana-Chavez, Victor; Caridad, Aliana R; Neubert, John K; Wronski, Thomas J; Wallet, Shannon M; Holliday, L Shannon

    2012-05-18

    Enoxacin has been identified as a small molecule inhibitor of binding between the B2-subunit of vacuolar H+-ATPase (V-ATPase) and microfilaments. It inhibits bone resorption by calcitriol-stimulated mouse marrow cultures. We hypothesized that enoxacin acts directly and specifically on osteoclasts by disrupting the interaction between plasma membrane-directed V-ATPases, which contain the osteoclast-selective a3-subunit of V-ATPase, and microfilaments. Consistent with this hypothesis, enoxacin dose-dependently reduced the number of multinuclear cells expressing tartrate-resistant acid phosphatase (TRAP) activity produced by RANK-L-stimulated osteoclast precursors. Enoxacin (50 μM) did not induce apoptosis as measured by TUNEL and caspase-3 assays. V-ATPases containing the a3-subunit, but not the "housekeeping" a1-subunit, were isolated bound to actin. Treatment with enoxacin reduced the association of V-ATPase subunits with the detergent-insoluble cytoskeleton. Quantitative PCR revealed that enoxacin triggered significant reductions in several osteoclast-selective mRNAs, but levels of various osteoclast proteins were not reduced, as determined by quantitative immunoblots, even when their mRNA levels were reduced. Immunoblots demonstrated that proteolytic processing of TRAP5b and the cytoskeletal protein L-plastin was altered in cells treated with 50 μM enoxacin. Flow cytometry revealed that enoxacin treatment favored the expression of high levels of DC-STAMP on the surface of osteoclasts. Our data show that enoxacin directly inhibits osteoclast formation without affecting cell viability by a novel mechanism that involves changes in posttranslational processing and trafficking of several proteins with known roles in osteoclast function. We propose that these effects are downstream to blocking the binding interaction between a3-containing V-ATPases and microfilaments.

  20. BIGH3 protein and macrophages in retinal endothelial cell apoptosis.

    Science.gov (United States)

    Mondragon, Albert A; Betts-Obregon, Brandi S; Moritz, Robert J; Parvathaneni, Kalpana; Navarro, Mary M; Kim, Hong Seok; Lee, Chi Fung; LeBaron, Richard G; Asmis, Reto; Tsin, Andrew T

    2015-01-01

    Diabetes is a pandemic disease with a higher occurrence in minority populations. The molecular mechanism to initiate diabetes-associated retinal angiogenesis remains largely unknown. We propose an inflammatory pathway of diabetic retinopathy in which macrophages in the diabetic eye provide TGFβ to retinal endothelial cells (REC) in the retinal microvasculature. In response to TGFβ, REC synthesize and secrete a pro-apoptotic BIGH3 (TGFβ-Induced Gene Human Clone 3) protein, which acts in an autocrine loop to induce REC apoptosis. Rhesus monkey retinal endothelial cells (RhREC) were treated with dMCM (cell media of macrophages treated with high glucose and LDL) and assayed for apoptosis (TUNEL), BIGH3 mRNA (qPCR), and protein (Western blots) expressions. Cells were also treated with ΤGFβ1 and 2 for BIGH3 mRNA and protein expression. Inhibition assays were carried out using antibodies for TGFβ1 and for BIGH3 to block apoptosis and mRNA expression. BIGH3 in cultured RhREC cells were identified by immunohistochemistry (IHC). Distribution of BIGH3 and macrophages in the diabetic mouse retina was examined with IHC. RhRECs treated with dMCM or TGFβ showed a significant increase in apoptosis and BIGH3 protein expression. Recombinant BIGH3 added to RhREC culture medium led to a dose-dependent increase in apoptosis. Antibodies (Ab) directed against BIGH3 and TGFβ, as well as TGFβ receptor blocker resulted in a significant reduction in apoptosis induced by either dMCM, TGFβ or BIGH3. IHC showed that cultured RhREC constitutively expressed BIGH3. Macrophage and BIGH3 protein were co-localized to the inner retina of the diabetic mouse eye. Our results support a novel inflammatory pathway for diabetic retinopathy. This pathway is initiated by TGFβ released from macrophages, which promotes synthesis and release of BIGH3 protein by REC and REC apoptosis.