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Sample records for bacteriophage t4 endonuclease

  1. Restoration of ultraviolet-induced unscheduled DNA synthesis of xeroderma pigmentosum cells by the concomitant treatment with bacteriophage T4 endonuclease V and HVJ (Sendai virus)

    International Nuclear Information System (INIS)

    Ultraviolet (uv)-induced unscheduled DNA synthesis of xeroderma pigmentosum cells, belonging to complementation groups, A, B, C, D, and E, was restored to the normal level by concomitant treatment of the cells with T4 endonuclease V and uv-inactivated HVJ (Sendai virus). The present results suggest that T4 endonuclease molecules were inserted effectively into the cells by the interaction of HVJ with the cell membranes, the enzyme was functional on human chromosomal DNA which had been damaged by uv irradiation in the viable cells, all the studied groups of xeroderma pigmentosum (variant was not tested) were defective in the first step (incision) of excision repair

  2. Structural studies on metal-containing enzymes. T4 endonuclease VII and D. gigas formate dehydrogenase

    NARCIS (Netherlands)

    Raaijmakers, H.C.A.

    2001-01-01

    Many biological processes require metal ions, and many of these metal-ion functions involve metalloproteins. The metal ions in metalloproteins are often critical to the protein's function, structure, or stability. This thesis focuses on two of these proteins, bacteriophage T4 endonuclease VII (EndoV

  3. Genetic diversity among five T4-like bacteriophages

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    Bertrand Claire

    2006-05-01

    Full Text Available Abstract Background Bacteriophages are an important repository of genetic diversity. As one of the major constituents of terrestrial biomass, they exert profound effects on the earth's ecology and microbial evolution by mediating horizontal gene transfer between bacteria and controlling their growth. Only limited genomic sequence data are currently available for phages but even this reveals an overwhelming diversity in their gene sequences and genomes. The contribution of the T4-like phages to this overall phage diversity is difficult to assess, since only a few examples of complete genome sequence exist for these phages. Our analysis of five T4-like genomes represents half of the known T4-like genomes in GenBank. Results Here, we have examined in detail the genetic diversity of the genomes of five relatives of bacteriophage T4: the Escherichia coli phages RB43, RB49 and RB69, the Aeromonas salmonicida phage 44RR2.8t (or 44RR and the Aeromonas hydrophila phage Aeh1. Our data define a core set of conserved genes common to these genomes as well as hundreds of additional open reading frames (ORFs that are nonconserved. Although some of these ORFs resemble known genes from bacterial hosts or other phages, most show no significant similarity to any known sequence in the databases. The five genomes analyzed here all have similarities in gene regulation to T4. Sequence motifs resembling T4 early and late consensus promoters were observed in all five genomes. In contrast, only two of these genomes, RB69 and 44RR, showed similarities to T4 middle-mode promoter sequences and to the T4 motA gene product required for their recognition. In addition, we observed that each phage differed in the number and assortment of putative genes encoding host-like metabolic enzymes, tRNA species, and homing endonucleases. Conclusion Our observations suggest that evolution of the T4-like phages has drawn on a highly diverged pool of genes in the microbial world. The T4

  4. Expression of the bacteriophage T4 denV structural gene in Escherichia coli

    International Nuclear Information System (INIS)

    The expression of the T4 denV gene, which previously had been cloned in plasmid constructs downstream of the bacteriophage lambda hybrid promoter-operator oLpR, was analyzed under a variety of growth parameters. Expression of the denV gene product, endonuclease V, was confirmed in DNA repair-deficient Escherichia coli (uvrA recA) by Western blot analyses and by enhancements of resistance to UV irradiation

  5. den V gene of bacteriophage T4 determines a DNA glycosylase specific for pyrimidine dimers in DNA.

    OpenAIRE

    Seawell, P C; Smith, C A; Ganesan, A K

    1980-01-01

    Endonuclease V of bacteriophage T4 has been described as an enzyme, coded for by the denV gene, that incises UV-irradiated DNA. It has recently been proposed that incision of irradiated DNA by this enzyme and the analogous "correndonucleases" I and II of Micrococcus luteus requires the sequential action of a pyrimidine dimer-specific DNA glycosylase and an apyrimidinic/apurinic endonuclease. In support of this two-step mechanism, we found that our preparations of T4 endonuclease V contained a...

  6. Re-initiation repair in bacteriophage T4

    International Nuclear Information System (INIS)

    Irradiation of bacteriophage T4 with ultraviolet light induces the formation of pyrimidine dimers in its DNA. These dimers hamper replication of DNA and, to a lesser extent, transcription of DNA after its infection of bacteria. A number of pathways enable phage T4 to multiply dimer-containing DNA. One of these pathways has been named replication repair and is described in this thesis. The properties of two phage strains, unable to perform replication repair, have been studied to obtain a picture of the repair process. The mutations in these strains that affect replication repair have been located on the genomic map of T4. (Auth.)

  7. Specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells in vivo

    International Nuclear Information System (INIS)

    The specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells was examined using an in vivo assay system with hemagglutinating virus of Japan (Sendai virus) inactivated by uv light. A clear dose response was observed between the level of uv-induced unscheduled DNA synthesis of xeroderma pigmentosum cells and the amount of T4 endonuclease V activity added. The T4 enzyme was unstable in human cells, and its half-life was 3 hr. Fractions derived from an extract of Escherichia coli infected with T4v1, a mutant defective in the endonuclease V gene, showed no ability to restore the uv-induced unscheduled DNA synthesis of xeroderma pigmentosum cells. However, fractions derived from an extract of T4D-infected E. coli with endonuclease V activity were effective. The T4 enzyme was effective in xeroderma pigmentosum cells on DNA damaged by uv light but not in cells damaged by 4-nitroquinoline 1-oxide. The results of these experiments show that the T4 enzyme has a specific action on human cell DNA in vivo. Treatment with the T4 enzyme increased the survival of group A xeroderma pigmentosum cells after uv irradiation

  8. A promiscuous DNA packaging machine from bacteriophage T4.

    Science.gov (United States)

    Zhang, Zhihong; Kottadiel, Vishal I; Vafabakhsh, Reza; Dai, Li; Chemla, Yann R; Ha, Taekjip; Rao, Venigalla B

    2011-01-01

    Complex viruses are assembled from simple protein subunits by sequential and irreversible assembly. During genome packaging in bacteriophages, a powerful molecular motor assembles at the special portal vertex of an empty prohead to initiate packaging. The capsid expands after about 10%-25% of the genome is packaged. When the head is full, the motor cuts the concatemeric DNA and dissociates from the head. Conformational changes, particularly in the portal, are thought to drive these sequential transitions. We found that the phage T4 packaging machine is highly promiscuous, translocating DNA into finished phage heads as well as into proheads. Optical tweezers experiments show that single motors can force exogenous DNA into phage heads at the same rate as into proheads. Single molecule fluorescence measurements demonstrate that phage heads undergo repeated initiations, packaging multiple DNA molecules into the same head. These results suggest that the phage DNA packaging machine has unusual conformational plasticity, powering DNA into an apparently passive capsid receptacle, including the highly stable virus shell, until it is full. These features probably led to the evolution of viral genomes that fit capsid volume, a strikingly common phenomenon in double-stranded DNA viruses, and will potentially allow design of a novel class of nanocapsid delivery vehicles. PMID:21358801

  9. A promiscuous DNA packaging machine from bacteriophage T4.

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    Zhihong Zhang

    Full Text Available Complex viruses are assembled from simple protein subunits by sequential and irreversible assembly. During genome packaging in bacteriophages, a powerful molecular motor assembles at the special portal vertex of an empty prohead to initiate packaging. The capsid expands after about 10%-25% of the genome is packaged. When the head is full, the motor cuts the concatemeric DNA and dissociates from the head. Conformational changes, particularly in the portal, are thought to drive these sequential transitions. We found that the phage T4 packaging machine is highly promiscuous, translocating DNA into finished phage heads as well as into proheads. Optical tweezers experiments show that single motors can force exogenous DNA into phage heads at the same rate as into proheads. Single molecule fluorescence measurements demonstrate that phage heads undergo repeated initiations, packaging multiple DNA molecules into the same head. These results suggest that the phage DNA packaging machine has unusual conformational plasticity, powering DNA into an apparently passive capsid receptacle, including the highly stable virus shell, until it is full. These features probably led to the evolution of viral genomes that fit capsid volume, a strikingly common phenomenon in double-stranded DNA viruses, and will potentially allow design of a novel class of nanocapsid delivery vehicles.

  10. Adsorption of T4 bacteriophages on planar indium tin oxide surface via controlled surface tailoring.

    Science.gov (United States)

    Liana, Ayu Ekajayanthi; Chia, Ed Win; Marquis, Christopher P; Gunawan, Cindy; Gooding, J Justin; Amal, Rose

    2016-04-15

    The work investigates the influence of surface physicochemical properties of planar indium tin oxide (ITO) as a model substrate on T4 bacteriophage adsorption. A comparative T4 bacteriophage adsorption study shows a significant difference in bacteriophage adsorption observed on chemically modified planar ITO when compared to similarly modified particulate ITO, which infers that trends observed in virus-particle interaction studies are not necessarily transferrable to predict virus-planar surface adsorption behaviour. We also found that ITO surfaces modified with methyl groups, (resulting in increased surface roughness and hydrophobicity) remained capable of adsorbing T4 bacteriophage. The adsorption of T4 onto bare, amine and carboxylic functionalised planar ITO suggests the presence of a unique binding behaviour involving specific functional groups on planar ITO surface beyond the non-specific electrostatic interactions that dominate phage to particle interactions. The paper demonstrates the significance of physicochemical properties of surfaces on bacteriophage-surface interactions.

  11. The effect of bacteriophages T4 and HAP1 on in vitro melanoma migration

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    Boratyński Janusz

    2009-01-01

    Full Text Available Abstract Background The antibacterial activity of bacteriophages has been described rather well. However, knowledge about the direct interactions of bacteriophages with mammalian organisms and their other, i.e. non-antibacterial, activities in mammalian systems is quite scarce. It must be emphasised that bacteriophages are natural parasites of bacteria, which in turn are parasites or symbionts of mammals (including humans. Bacteriophages are constantly present in mammalian bodies and the environment in great amounts. On the other hand, the perspective of the possible use of bacteriophage preparations for antibacterial therapies in cancer patients generates a substantial need to investigate the effects of phages on cancer processes. Results In these studies the migration of human and mouse melanoma on fibronectin was inhibited by purified T4 and HAP1 bacteriophage preparations. The migration of human melanoma was also inhibited by the HAP1 phage preparation on matrigel. No response of either melanoma cell line to lipopolysaccharide was observed. Therefore the effect of the phage preparations cannot be attributed to lipopolysaccharide. No differences in the effects of T4 and HAP1 on melanoma migration were observed. Conclusion We believe that these observations are of importance for any further attempts to use bacteriophage preparations in antibacterial treatment. The risk of antibiotic-resistant hospital infections strongly affects cancer patients and these results suggest the possibility of beneficial phage treatment. We also believe that they will contribute to the general understanding of bacteriophage biology, as bacteriophages, extremely ubiquitous entities, are in permanent contact with human organisms.

  12. Biochemical characterization of a thermostable HNH endonuclease from deep-sea thermophilic bacteriophage GVE2.

    Science.gov (United States)

    Zhang, Likui; Huang, Yanchao; Xu, Dandan; Yang, Lixiang; Qian, Kaicheng; Chang, Guozhu; Gong, Yong; Zhou, Xiaojian; Ma, Kesen

    2016-09-01

    His-Asn-His (HNH) proteins are a very common family of small nucleic acid-binding proteins that are generally associated with endonuclease activity and are found in all kingdoms of life. Although HNH endonucleases from mesophiles have been widely investigated, the biochemical functions of HNH endonucleases from thermophilic bacteriophages remain unknown. Here, we characterized the biochemical properties of a thermostable HNH endonuclease from deep-sea thermophilic bacteriophage GVE2. The recombinant GVE2 HNH endonuclease exhibited non-specific cleavage activity at high temperature. The optimal temperature of the GVE2 HNH endonuclease for cleaving DNA was 60-65 °C, and the enzyme retained its DNA cleavage activity even after heating at 100 °C for 30 min, suggesting the enzyme is a thermostable endonuclease. The GVE2 HNH endonuclease cleaved DNA over a wide pH spectrum, ranging from 5.5 to 9.0, and the optimal pH for the enzyme activity was 8.0-9.0. Furthermore, the GVE2 HNH endonuclease activity was dependent on a divalent metal ion. While the enzyme is inactive in the presence of Cu(2+), the GVE2 HNH endonuclease displayed cleavage activity of varied efficiency with Mn(2+), Mg(2+), Ca(2+), Fe(2+), Co(2+), Zn(2+), and Ni(2+). The GVE2 HNH endonuclease activity was inhibited by NaCl. This study provides the basis for determining the role of this endonuclease in life cycle of the bacteriophage GVE2 and suggests the potential application of the enzyme in molecular biology and biotechnology. PMID:27131500

  13. Introns in the genome of bacteriophage T4

    International Nuclear Information System (INIS)

    RNA from T4-infected cells yields multiple end-labeled species when incubated with [α-32P]GTP under self-splicing conditions. One of these corresponds to the previously characterized intron from the T4 td gene and, as shown in this work, the others represent additional group I introns in T4. Two loci distinct from the td gene were found to hybridize to the mixed GTP-labeled T4 RNA probe. These were mapped to the unlinked genes nrdB and sunY. Cloned DNA from the nrdB region that contained the intron was shown to generate characteristic group I splice products with RNA synthesized in vivo or in vitro. The splice junction of the nrdB gene was determined and the nature of the RNA reaction products characterized. In vivo expression of the nrdB gene and the open reading frame within the intron was studied using in-frame lacZ fusions and primer extension analyses. The data suggest that expression of the intron open reading frame is highly regulated during T4 infection. Possible regulatory mechanisms are discussed

  14. Bacteriophage T4 genes sp and 40 apparently are the same.

    OpenAIRE

    Obringer, J; McCreary, P.; Bernstein, H

    1988-01-01

    The bacteriophage T4 spackle gene, which maintains host membrane integrity, mapped at the same position as gene 40 (head morphogenesis). The cloned spackle gene complemented and cross-reactivated a gene 40 mutant. Like the spackle mutant, gene 40 mutants were defective in genetic exclusion. Apparently, genes spackle and 40 are the same gene.

  15. Genetic analysis of the bacteriophage T4-encoded cochaperonin Gp31.

    OpenAIRE

    Richardson, A.; Georgopoulos, C

    1999-01-01

    Previous genetic and biochemical analyses have established that the bacteriophage T4-encoded Gp31 is a cochaperonin that interacts with Escherichia coli's GroEL to ensure the timely and accurate folding of Gp23, the bacteriophage-encoded major capsid protein. The heptameric Gp31 cochaperonin, like the E. coli GroES cochaperonin, interacts with GroEL primarily through its unstructured mobile loop segment. Upon binding to GroEL, the mobile loop adopts a structured, beta-hairpin turn. In this ar...

  16. Assembly and dynamics of the bacteriophage T4 homologous recombination machinery

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    Morrical Scott W

    2010-12-01

    Full Text Available Abstract Homologous recombination (HR, a process involving the physical exchange of strands between homologous or nearly homologous DNA molecules, is critical for maintaining the genetic diversity and genome stability of species. Bacteriophage T4 is one of the classic systems for studies of homologous recombination. T4 uses HR for high-frequency genetic exchanges, for homology-directed DNA repair (HDR processes including DNA double-strand break repair, and for the initiation of DNA replication (RDR. T4 recombination proteins are expressed at high levels during T4 infection in E. coli, and share strong sequence, structural, and/or functional conservation with their counterparts in cellular organisms. Biochemical studies of T4 recombination have provided key insights on DNA strand exchange mechanisms, on the structure and function of recombination proteins, and on the coordination of recombination and DNA synthesis activities during RDR and HDR. Recent years have seen the development of detailed biochemical models for the assembly and dynamics of presynaptic filaments in the T4 recombination system, for the atomic structure of T4 UvsX recombinase, and for the roles of DNA helicases in T4 recombination. The goal of this chapter is to review these recent advances and their implications for HR and HDR mechanisms in all organisms.

  17. Sequence characteristics of T4-like bacteriophage IME08 benome termini revealed by high throughput sequencing

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    An Xiaoping

    2011-04-01

    Full Text Available Abstract Background T4 phage is a model species that has contributed broadly to our understanding of molecular biology. T4 DNA replication and packaging share various mechanisms with human double-stranded DNA viruses such as herpes virus. The literature indicates that T4-like phage genomes have permuted terminal sequences, and are generated by a DNA terminase in a sequence-independent manner; Methods genomic DNA of T4-like bacteriophage IME08 was subjected to high throughput sequencing, and the read sequences with extraordinarily high occurrences were analyzed; Results we demonstrate that both the 5' and 3' termini of the IME08 genome starts with base G or A. The presence of a consensus sequence TTGGA|G around the breakpoint of the high frequency read sequences suggests that the terminase cuts the branched pre-genome in a sequence-preferred manner. Our analysis also shows that terminal cleavage is asymmetric, with one end cut at a consensus sequence, and the other end generated randomly. The sequence-preferred cleavage may produce sticky-ends, but with each end being packaged with different efficiencies; Conclusions this study illustrates how high throughput sequencing can be used to probe replication and packaging mechanisms in bacteriophages and/or viruses.

  18. Study of the transfer RNAs coded by T2, T4, and T6 bacteriophages

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    Desai, S.M.; Weiss, S.B.

    1977-07-25

    T2,T4, and T6 bacteriophage tRNAs coding for arginine, leucine, proline, isoleucine, and glycine were isolated under conditions of short term and long term infection of Escherchia coli B cells. The corresponding phage tRNA species were examined for sequence homology by RNA.DNA hybridization analysis and by their relative behavior on reversed phase chromatography. The results indicate that all three T-even phages code for similar tRNA species; however, some tRNA species are homologous, others are not, and not all of the same tRNA species are coded by each bacteriophage. Reversed phase chromatography showed the presence of isoacceptor tRNAs for each phage aminoacyl-tRNA species. Pulse-chase experiments for (/sup 32/P)tRNA/sup Gly/ suggest that the multiple isoacceptor species observed derive from the intracellular modification of a single tRNA/sup Gly/ gene product.

  19. Genomes of the T4-related bacteriophages as windows on microbial genome evolution

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    Miller Eric S

    2010-10-01

    Full Text Available Abstract The T4-related bacteriophages are a group of bacterial viruses that share morphological similarities and genetic homologies with the well-studied Escherichia coli phage T4, but that diverge from T4 and each other by a number of genetically determined characteristics including the bacterial hosts they infect, the sizes of their linear double-stranded (ds DNA genomes and the predicted compositions of their proteomes. The genomes of about 40 of these phages have been sequenced and annotated over the last several years and are compared here in the context of the factors that have determined their diversity and the diversity of other microbial genomes in evolution. The genomes of the T4 relatives analyzed so far range in size between ~160,000 and ~250,000 base pairs (bp and are mosaics of one another, consisting of clusters of homology between them that are interspersed with segments that vary considerably in genetic composition between the different phage lineages. Based on the known biological and biochemical properties of phage T4 and the proteins encoded by the T4 genome, the T4 relatives reviewed here are predicted to share a genetic core, or "Core Genome" that determines the structural design of their dsDNA chromosomes, their distinctive morphology and the process of their assembly into infectious agents (phage morphogenesis. The Core Genome appears to be the most ancient genetic component of this phage group and constitutes a mere 12-15% of the total protein encoding potential of the typical T4-related phage genome. The high degree of genetic heterogeneity that exists outside of this shared core suggests that horizontal DNA transfer involving many genetic sources has played a major role in diversification of the T4-related phages and their spread to a wide spectrum of bacterial species domains in evolution. We discuss some of the factors and pathways that might have shaped the evolution of these phages and point out several parallels

  20. Expression of a cloned denV gene of bacteriophage T4 in Escherichia coli

    International Nuclear Information System (INIS)

    A 713-base-pair Hae III fragment from bacteriophage T4 encompassing the denV gene with its preceding promoter has been cloned in a pBR322-derived positive-selection vector and introduced into a variety of DNA repair-deficient uvr and rec and uvr,rec Escherichia coli strains. The denV gene was found to be expressed, probably from its own promoter, causing pyrimidine dimer incision-deficient uvrA, uvrB, uvrC strains to be rescued by the denV gene. A uvrD (DNA helicase II) strain was also complemented, but to a lesser extent. A wild-type strain did not seem to be affected at the UV doses tested. Surprisingly, all recA, recB, and recC strains tested also showed an increased UV resistance, perhaps by reinforcement of the intact uvr system in these strains. Complementation of denV- T4 strains and host-cell reactivation of lambda phage was also observed in denV+ E. coli strains. Equilibrium sedimentation showed that DNA repair synthesis occurred in a UV-irradiated uvrA E. coli strain carrying the cloned denV gene. Southern blotting confirmed earlier results that the denV gene is located at 64 kilobases on the T4 map. Phage T2 (denV-) did not hybridize to a denV-specific probe

  1. Expression of a cloned denV gene of bacteriophage T4 in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Valerie, K.; Henderson, E.E.; de Riel, J.K.

    1985-07-01

    A 713-base-pair Hae III fragment from bacteriophage T4 encompassing the denV gene with its preceding promoter has been cloned in a pBR322-derived positive-selection vector and introduced into a variety of DNA repair-deficient uvr and rec and uvr,rec Escherichia coli strains. The denV gene was found to be expressed, probably from its own promoter, causing pyrimidine dimer incision-deficient uvrA, uvrB, uvrC strains to be rescued by the denV gene. A uvrD (DNA helicase II) strain was also complemented, but to a lesser extent. A wild-type strain did not seem to be affected at the UV doses tested. Surprisingly, all recA, recB, and recC strains tested also showed an increased UV resistance, perhaps by reinforcement of the intact uvr system in these strains. Complementation of denV- T4 strains and host-cell reactivation of lambda phage was also observed in denV+ E. coli strains. Equilibrium sedimentation showed that DNA repair synthesis occurred in a UV-irradiated uvrA E. coli strain carrying the cloned denV gene. Southern blotting confirmed earlier results that the denV gene is located at 64 kilobases on the T4 map. Phage T2 (denV-) did not hybridize to a denV-specific probe.

  2. Structural remodeling of bacteriophage T4 and host membranes during infection initiation.

    Science.gov (United States)

    Hu, Bo; Margolin, William; Molineux, Ian J; Liu, Jun

    2015-09-01

    The first stages of productive bacteriophage infections of bacterial host cells require efficient adsorption to the cell surface followed by ejection of phage DNA into the host cytoplasm. To achieve this goal, a phage virion must undergo significant structural remodeling. For phage T4, the most obvious change is the contraction of its tail. Here, we use skinny E. coli minicells as a host, along with cryo-electron tomography and mutant phage virions, to visualize key structural intermediates during initiation of T4 infection. We show for the first time that most long tail fibers are folded back against the tail sheath until irreversible adsorption, a feature compatible with the virion randomly walking across the cell surface to find an optimal site for infection. Our data confirm that tail contraction is triggered by structural changes in the baseplate, as intermediates were found with remodeled baseplates and extended tails. After contraction, the tail tube penetrates the host cell periplasm, pausing while it degrades the peptidoglycan layer. Penetration into the host cytoplasm is accompanied by a dramatic local outward curvature of the cytoplasmic membrane as it fuses with the phage tail tip. The baseplate hub protein gp27 and/or the ejected tape measure protein gp29 likely form the transmembrane channel for viral DNA passage into the cell cytoplasm. Building on the wealth of prior biochemical and structural information, this work provides new molecular insights into the mechanistic pathway of T4 phage infection. PMID:26283379

  3. Atomic force microscopy images of T4 bacteriophages on silicon substrates

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    Kolbe, W.F.; Ogletree, D.F.; Salmeron, M.B.

    1991-08-01

    A new atomic force microscope incorporating microfabricated cantilevers and employing laser beam deflection for force detection has been constructed and is being applied to studied of biological material. In this study, T4 bacteriophage virus particles were deposited from solution onto electronic grade flat silicon wafers and imaged in air with the microscope. Microliter droplets of the solution were deposited and either allowed to dry or removed with blotting paper. The images show both isolated viruses and aggregates of various sizes. The external structure as well as strands believed to be DNA streaming out of the virus could be observed. The construction of the microscope and its performance are also described. 19 refs., 4 figs.

  4. Carbon loss during irradiation of T4 bacteriophages and E. coli bacteria in electron microscopes

    Energy Technology Data Exchange (ETDEWEB)

    Dubochet, J.

    1975-08-01

    The loss of /sup 14/C due to electron irradiation has been measured on labeled T4 bacteriophages and E. coli bacteria under conditions relevant for practical electron microscopy for fixed and scanning beam exposure. During irradiation, the remaining material became less and less sensitive to further carbon loss. Surface migration of molecular fragments and adsorbed molecules is involved in the process of beam damage. In CTEM under normal working conditions, the parameters on which the carbon loss depends cannot all be controlled. There is no perceptible carbon loss when the irradiation is made at liquid helium temperature. The material surrounding the biological object, the way in which the electron dose is given to the specimen, and the vacuum conditions in the specimen chamber influence the process. We conclude that secondary reactions of molecular fragments formed after inelastic scattering events are of importance and depend on the layer of molecules adsorbed onto the surface of the specimen. (auth)

  5. Bacteriophage T4 development in Escherichia coli is growth rate dependent.

    Science.gov (United States)

    Rabinovitch, Avinoam; Fishov, Itzhak; Hadas, Hilla; Einav, Monica; Zaritsky, Arieh

    2002-05-01

    Three independent parameters (eclipse and latent periods, and rate of ripening during the rise period) are essential and sufficient to describe bacteriophage development in its bacterial host. A general model to describe the classical "one-step growth" experiment [Rabinovitch et al. (1999a) J. Bacteriol.181, 1687-1683] allowed their calculations from experimental results obtained with T4 in Escherichia coli B/r under different growth conditions [Hadas et al. (1997) Microbiology143, 179-185]. It is found that all three parameters could be described by their dependence solely on the culture doubling time tau before infection. Their functional dependence on tau, derived by a best-fit analysis, was used to calculate burst size values. The latter agree well with the experimental results. The dependence of the derived parameters on growth conditions can be used to predict phage development under other experimental manipulations.

  6. Functional Analysis of the Bacteriophage T4 Rad50 Homolog (gp46) Coiled-coil Domain.

    Science.gov (United States)

    Barfoot, Tasida; Herdendorf, Timothy J; Behning, Bryanna R; Stohr, Bradley A; Gao, Yang; Kreuzer, Kenneth N; Nelson, Scott W

    2015-09-25

    Rad50 and Mre11 form a complex involved in the detection and processing of DNA double strand breaks. Rad50 contains an anti-parallel coiled-coil with two absolutely conserved cysteine residues at its apex. These cysteine residues serve as a dimerization domain and bind a Zn(2+) cation in a tetrathiolate coordination complex known as the zinc-hook. Mutation of the zinc-hook in bacteriophage T4 is lethal, indicating the ability to bind Zn(2+) is critical for the functioning of the MR complex. In vitro, we found that complex formation between Rad50 and a peptide corresponding to the C-terminal domain of Mre11 enhances the ATPase activity of Rad50, supporting the hypothesis that the coiled-coil is a major conduit for communication between Mre11 and Rad50. We constructed mutations to perturb this domain in the bacteriophage T4 Rad50 homolog. Deletion of the Rad50 coiled-coil and zinc-hook eliminates Mre11 binding and ATPase activation but does not affect its basal activity. Mutation of the zinc-hook or disruption of the coiled-coil does not affect Mre11 or DNA binding, but their activation of Rad50 ATPase activity is abolished. Although these mutants excise a single nucleotide at a normal rate, they lack processivity and have reduced repetitive exonuclease rates. Restricting the mobility of the coiled-coil eliminates ATPase activation and repetitive exonuclease activity, but the ability to support single nucleotide excision is retained. These results suggest that the coiled-coiled domain adopts at least two conformations throughout the ATPase/nuclease cycle, with one conformation supporting enhanced ATPase activity and processivity and the other supporting nucleotide excision.

  7. A bacteriophage T4 in vitro system to clone long DNA molecules. Final report, June 1, 1990--January 31, 1996

    Energy Technology Data Exchange (ETDEWEB)

    Rao, V.B.

    1997-09-01

    A summary is presented of the following objectives: development of a bacteriophage T4 in vitro system, and techniques to clone long segments of foreign DNA; development of a giant prohead DNA packaging system that could potentially be used to clone even a megabase size DNA; and development of techniques to rapidly map the cloned DNA inserts.

  8. Crystallization of the carboxy-terminal region of the bacteriophage T4 proximal long tail fibre protein gp34

    International Nuclear Information System (INIS)

    The crystallization of three C-terminal fragments of the bacteriophage T4 protein gp34 is reported. Diffraction data have been obtained for three native crystal forms and two selenomethionine derivatives, one of which contained high-quality anomalous signal

  9. Restriction of a bacteriophage of Streptomyces albus G involving endonuclease SalI.

    Science.gov (United States)

    Chater, K F; Wilde, L C

    1976-11-01

    The bacteriophage Pa16, isolated from soil on Streptomyces albus G, was restricted when transferred from an alternative host back to S. albus G. Extracted unmodified Pa16 deoxyribonucleic acid was cleaved at a single site by a cell-free extract of S. albus G. Fractions cleaving Pal6 deoxyribonucleic acid contained the endonuclease SalI first described by J. Arrand, P. Myers, and R. J. Roberts (unpublished data). A mutant of S. albus G was isolated which was defective in both restriction and modification of Pal6. This mutant lacked SalI activity. It is concluded that SalI is the agent of restriction of Pal6 by S. albus G.

  10. T4-related bacteriophage LIMEstone isolates for the control of soft rot on potato caused by 'Dickeya solani'.

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    Evelien M Adriaenssens

    Full Text Available The bacterium 'Dickeya solani', an aggressive biovar 3 variant of Dickeya dianthicola, causes rotting and blackleg in potato. To control this pathogen using bacteriophage therapy, we isolated and characterized two closely related and specific bacteriophages, vB_DsoM_LIMEstone1 and vB_DsoM_LIMEstone2. The LIMEstone phages have a T4-related genome organization and share DNA similarity with Salmonella phage ViI. Microbiological and molecular characterization of the phages deemed them suitable and promising for use in phage therapy. The phages reduced disease incidence and severity on potato tubers in laboratory assays. In addition, in a field trial of potato tubers, when infected with 'Dickeya solani', the experimental phage treatment resulted in a higher yield. These results form the basis for the development of a bacteriophage-based biocontrol of potato plants and tubers as an alternative for the use of antibiotics.

  11. The Effects of T4 and A3/R Bacteriophages on Differentiation of Human Myeloid Dendritic Cells.

    Science.gov (United States)

    Bocian, Katarzyna; Borysowski, Jan; Zarzycki, Michał; Pacek, Magdalena; Weber-Dąbrowska, Beata; Machcińska, Maja; Korczak-Kowalska, Grażyna; Górski, Andrzej

    2016-01-01

    Bacteriophages (phages) are viruses of bacteria. Here we evaluated the effects of T4 and A3/R bacteriophages, as well as phage-generated bacterial lysates, on differentiation of human myeloid dendritic cells (DCs) from monocytes. Neither of the phages significantly reduced the expression of markers associated with differentiation of DCs and their role in the activation of T cells (CD40, CD80, CD83, CD86, CD1c, CD11c, MHC II, PD-L1, PD-L2, TLR2, TLR4, and CCR7) and phagocytosis receptors (CD64 and DEC-205). By contrast, bacterial lysate of T4 phage significantly decreased the percentages of DEC-205- and CD1c-positive cells. The percentage of DEC-205-positive cells was also significantly reduced in DCs differentiated in the presence of lysate of A3/R phage. Thus while bacteriophages do not substantially affect differentiation of DCs, some products of phage-induced lysis of bacterial cells may influence the differentiation and potentially also some functions of DCs. Our results have important implications for phage therapy of bacterial infections because during infections monocytes recruited to the site of inflammation are an important source of inflammatory DCs. PMID:27582733

  12. The Effects of T4 and A3/R Bacteriophages on Differentiation of Human Myeloid Dendritic Cells.

    Science.gov (United States)

    Bocian, Katarzyna; Borysowski, Jan; Zarzycki, Michał; Pacek, Magdalena; Weber-Dąbrowska, Beata; Machcińska, Maja; Korczak-Kowalska, Grażyna; Górski, Andrzej

    2016-01-01

    Bacteriophages (phages) are viruses of bacteria. Here we evaluated the effects of T4 and A3/R bacteriophages, as well as phage-generated bacterial lysates, on differentiation of human myeloid dendritic cells (DCs) from monocytes. Neither of the phages significantly reduced the expression of markers associated with differentiation of DCs and their role in the activation of T cells (CD40, CD80, CD83, CD86, CD1c, CD11c, MHC II, PD-L1, PD-L2, TLR2, TLR4, and CCR7) and phagocytosis receptors (CD64 and DEC-205). By contrast, bacterial lysate of T4 phage significantly decreased the percentages of DEC-205- and CD1c-positive cells. The percentage of DEC-205-positive cells was also significantly reduced in DCs differentiated in the presence of lysate of A3/R phage. Thus while bacteriophages do not substantially affect differentiation of DCs, some products of phage-induced lysis of bacterial cells may influence the differentiation and potentially also some functions of DCs. Our results have important implications for phage therapy of bacterial infections because during infections monocytes recruited to the site of inflammation are an important source of inflammatory DCs.

  13. The effects of T4 and A3R bacteriophages on differentiation of human myeloid dendritic cells

    Directory of Open Access Journals (Sweden)

    Katarzyna Bocian

    2016-08-01

    Full Text Available Bacteriophages (phages are viruses of bacteria. Here we evaluated the effects of T4 and A3R bacteriophages, as well as phage-generated bacterial lysates, on differentiation of human myeloid dendritic cells (DCs from monocytes. Neither of the phages significantly reduced the expression of markers associated with differentiation of DCs and their role in the activation of T cells (CD40, CD80, CD83, CD86, CD1c, CD11c, MHC II, PD-L1, PD-L2, TLR2, TLR4, and CCR7 and phagocytosis receptors (CD64 and DEC-205. By contrast, bacterial lysate of T4 phage significantly decreased the percentages of DEC-205- and CD1c-positive cells. The percentage of DEC-205-positive cells was also significantly reduced in DCs differentiated in the presence of lysate of A3R phage. Thus while bacteriophages do not substantially affect differentiation of DCs, some products of phage-induced lysis of bacterial cells may influence the differentiation and potentially also some functions of DCs. Our results have important implications for phage therapy of bacterial infections because during infections monocytes recruited to the site of inflammation are an important source of inflammatory DCs.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Genetic effects of cosmic radiation on bacteriophage T4Br/+/ /On materials of biological experiment Soyuz-Apollo/

    Energy Technology Data Exchange (ETDEWEB)

    Iurov, S.S. (Academy of Sciences, Institute of Biological Physics, Pushchino, USSR); Akoev, I.G. (Ministerstvo Zdravookhraneniia SSSR, Institut Mediko-Biologicheskikh Problem, Moscow, USSR)

    1979-01-01

    During the experiment Spore-ring Forming Fungi Biorhythm of the Apollo-Soyuz test project the Rhythm-1 apparatus contained a dried film culture of bacteriophage T4Br(+), growing cultures of Actinomyces and plastic nuclear particle detectors. The following were studied: the frequency of induction of r mutations in the bacteriophage film per 20,000 surviving particles, the spectrum of mutant types obtained (rI, rII, rIII), and the possible molecular mechanisms for the occurrence of rII mutants with due regard to the registered tracks of heavy nuclear particles. The studies showed that the local radiation due to heavy nuclear particle tracks plays a major role in space radiation damage.

  16. Vaccination against Very Virulent Infectious Bursal Disease Virus Using Recombinant T4 Bacteriophage Displaying Viral Protein VP2

    Institute of Scientific and Technical Information of China (English)

    Yong-Chang CAO; Quan-Cheng SHI; Jing-Yun MA; Qing-Mei XIE; Ying-Zuo BI

    2005-01-01

    In order to develop a desirable inexpensive, effective and safe vaccine against the very virulent infectious bursal disease virus (vvIBDV), we tried to take advantage of the emerging T4 bacteriophage surface protein display system. The major immunogen protein VP2 from the vvIBDV strain HK46 was fused to the nonessential T4 phage surface capsid protein, a small outer capsid (SOC) protein, resulting in the 49 kDa SOC-VP2 fusion protein, which was verified by sodium dodecylsulfate polyacrylamide gel electrophoresis and Western blot. Immunoelectromicroscopy showed that the recombinant VP2 protein was successfully displayed on the surface of the T4 phage. The recombinant VP2 protein is antigenic and showed reactivities to various monoclonal antibodies (mAbs) against IBDV, whereas the wild-type phage T4 could not react to any mAb. In addition, the recombinant VP2 protein is immunogenic and elicited specific antibodies in immunized specific pathogen free (SPF) chickens. More significantly, immunization of SPF chickens with the recombinant T4-VP2 phage protected them from infection by the vvIBDV strain HK46. When challenged with the vvIBDV strain HK46 at a dose of 100 of 50% lethal dose (LD50) per chicken 4 weeks after the booster was given, the group vaccinated with the T4-VP2 recombinant phage showed no clinical signs of disease or death, wh ereas the unvaccinated group and the group vaccinated with the wild-type T4phage exhibited 100% clinical signs of disease and bursal damages, and 30%-40% mortality. Collectively,the data herein showed that the T4-displayed VP2 protein might be an inexpensive, effective and safe vaccine candidate against vvIBDV.

  17. Post-transcriptional control by bacteriophage T4: mRNA decay and inhibition of translation initiation

    Directory of Open Access Journals (Sweden)

    Miller Eric S

    2010-12-01

    Full Text Available Abstract Over 50 years of biological research with bacteriophage T4 includes notable discoveries in post-transcriptional control, including the genetic code, mRNA, and tRNA; the very foundations of molecular biology. In this review we compile the past 10 - 15 year literature on RNA-protein interactions with T4 and some of its related phages, with particular focus on advances in mRNA decay and processing, and on translational repression. Binding of T4 proteins RegB, RegA, gp32 and gp43 to their cognate target RNAs has been characterized. For several of these, further study is needed for an atomic-level perspective, where resolved structures of RNA-protein complexes are awaiting investigation. Other features of post-transcriptional control are also summarized. These include: RNA structure at translation initiation regions that either inhibit or promote translation initiation; programmed translational bypassing, where T4 orchestrates ribosome bypass of a 50 nucleotide mRNA sequence; phage exclusion systems that involve T4-mediated activation of a latent endoribonuclease (PrrC and cofactor-assisted activation of EF-Tu proteolysis (Gol-Lit; and potentially important findings on ADP-ribosylation (by Alt and Mod enzymes of ribosome-associated proteins that might broadly impact protein synthesis in the infected cell. Many of these problems can continue to be addressed with T4, whereas the growing database of T4-related phage genome sequences provides new resources and potentially new phage-host systems to extend the work into a broader biological, evolutionary context.

  18. Structure of the Three N-Terminal Immunoglobulin Domains of the Highly Immunogenic Outer Capsid Protein from a T4-Like Bacteriophage

    Energy Technology Data Exchange (ETDEWEB)

    Fokine, Andrei; Islam, Mohammad Z.; Zhang, Zhihong; Bowman, Valorie D.; Rao, Venigalla B.; Rossmann, Michael G. (CUA); (Purdue)

    2011-09-16

    The head of bacteriophage T4 is decorated with 155 copies of the highly antigenic outer capsid protein (Hoc). One Hoc molecule binds near the center of each hexameric capsomer. Hoc is dispensable for capsid assembly and has been used to display pathogenic antigens on the surface of T4. Here we report the crystal structure of a protein containing the first three of four domains of Hoc from bacteriophage RB49, a close relative of T4. The structure shows an approximately linear arrangement of the protein domains. Each of these domains has an immunoglobulin-like fold, frequently found in cell attachment molecules. In addition, we report biochemical data suggesting that Hoc can bind to Escherichia coli, supporting the hypothesis that Hoc could attach the phage capsids to bacterial surfaces and perhaps also to other organisms. The capacity for such reversible adhesion probably provides survival advantages to the bacteriophage.

  19. Interaction of bacteriophage T4 and T7 single-stranded DNA-binding proteins with DNA

    Science.gov (United States)

    Shokri, Leila; Rouzina, Ioulia; Williams, Mark C.

    2009-06-01

    Bacteriophages T4 and T7 are well-studied model replication systems, which have allowed researchers to determine the roles of many proteins central to DNA replication, recombination and repair. Here we summarize and discuss the results from two recently developed single-molecule methods to determine the salt-dependent DNA-binding kinetics and thermodynamics of the single-stranded DNA (ssDNA)-binding proteins (SSBs) from these systems. We use these methods to characterize both the equilibrium double-stranded DNA (dsDNA) and ssDNA binding of the SSBs T4 gene 32 protein (gp32) and T7 gene 2.5 protein (gp2.5). Despite the overall two-orders-of-magnitude weaker binding of gp2.5 to both forms of DNA, we find that both proteins exhibit four-orders-of-magnitude preferential binding to ssDNA relative to dsDNA. This strong preferential ssDNA binding as well as the weak dsDNA binding is essential for the ability of both proteins to search dsDNA in one dimension to find available ssDNA-binding sites at the replication fork.

  20. Mutants of bacteriophage T4 deficient in the ability to induce nuclear disruption: shutoff of host DNA and protein synthesis gene dosage experiments, identification of a restrictive host, and possible biological significance.

    Science.gov (United States)

    Snustad, D P; Bursch, C J; Parson, K A; Hefeneider, S H

    1976-04-01

    The shutoff of host DNA synthesis is delayed until about 8 to 10 min after infection when Escherichia coli B/5 cells were infected with bacteriophage T4 mutants deficient in the ability to induce nuclear disruption (ndd mutants). The host DNA synthesized after infection with ndd mutants is stable in the absence of T4 endonucleases II and IV, but is unstable in the presence of these nucleases. Host protein synthesis, as indicated by the inducibility of beta-galactosidase and sodium dodecyl sulfate-polyacrylamide gel patterns of isoptopically labeled proteins synthesize after infection, is shut off normally in ndd-infected cells, even in the absence of host DNA degradation. The Cal Tech wild-type strain of E. coli CT447 was found to restrict growth of the ndd mutants. Since T4D+ also has a very low efficiency of plating on CT447, we have isolated a nitrosoguanidine-induced derivative of CT447 which yields a high T4D+ efficiency of plating while still restricting the ndd mutants. Using this derivative, CT447 T4 plq+ (for T4 plaque+), we have shown that hos DNA degradation and shutoff of host DNA synthesis occur after infection with either ndd98 X 5 (shutoff delayed) or T4D+ (shutoff normal) with approximately the same kinetics as in E. coli strain B/5. Nuclear disruption occurs after infection of CT447 with ndd+ phage, but not after infection with ndd- phage. The rate of DNA synthesis after infection of CT447 T4 plq+ with ndd98 X 5 is about 75% of the rate observed after infection with T4D+ while the burst size of ndd98 X 5 is only 3.5% of that of T4D+. The results of gene dosage experiments using the ndd restrictive host C5447 suggest that the ndd gene product is required in stoichiometric amounts. The observation by thin-section electron microscopy of two distinct pools of DNA, one apparently phage DNA and the other host DNA, in cells infected with nuclear disruption may be a compartmentalization mechanism which separates the pathways of host DNA degradation and

  1. T4噬菌体的组装、感染及细胞裂解%Assembly, Infection and Cell Lysis of Bacteriophage T4

    Institute of Scientific and Technical Information of China (English)

    颜晨; 逯凯; 任慧英

    2011-01-01

    To further develop and utilize T4 phage display and phage therapy, and open the broader prospect for the progress in study of T4 phage, the important components of T4 phage particles was analyzed by molecular biology techniques, such as the head, the tail and the basement. The protein composition of these structure were confirmed. DNA packing which was an important part in the assembly of T4 phage particle was described in detail. Not only the phenomenon of infection and lysis, but also the superinfection exclusion and lysis inhibition in T4 life cycles introduced with molecular mechanism and impact factor. The assembly and infection mechanism of T4 phage would be known much better by these research.%为深入开发利用T4噬菌体展示系统及T4噬菌体的临床细菌病治疗,应用现代各种分子生物学手段,在蛋白分子和组成结构上对T4噬菌体颗粒的各个重要组成部分进行分析,例如头部、尾部和尾板,描述了其蛋白构成和各种影响因子,并着重介绍了在噬菌体装配中最为重要的DNA的包装.同时针对噬菌体T4的感染与超感染免疫性和感染所导致的细胞裂解和裂解抑制等方面,介绍了这些现象发生的分子机制和影响因素.以期对T4噬菌体的分子装配和侵染机制有更为深刻的了解.

  2. Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Siyang; Gao, Song; Kondabagil, Kiran; Xiang, Ye; Rossmann, Michael G.; Rao, Venigalla B. (CUA); (Purdue)

    2012-04-04

    Tailed DNA bacteriophages assemble empty procapsids that are subsequently filled with the viral genome by means of a DNA packaging machine situated at a special fivefold vertex. The packaging machine consists of a 'small terminase' and a 'large terminase' component. One of the functions of the small terminase is to initiate packaging of the viral genome, whereas the large terminase is responsible for the ATP-powered translocation of DNA. The small terminase subunit has three domains, an N-terminal DNA-binding domain, a central oligomerization domain, and a C-terminal domain for interacting with the large terminase. Here we report structures of the central domain in two different oligomerization states for a small terminase from the T4 family of phages. In addition, we report biochemical studies that establish the function for each of the small terminase domains. On the basis of the structural and biochemical information, we propose a model for DNA packaging initiation.

  3. Overexpression, purification, and partial characterization of ADP-ribosyltransferases modA and modB of bacteriophage T4.

    Science.gov (United States)

    Tiemann, B; Depping, R; Rüger, W

    1999-01-01

    There is increasing experimental evidence that ADP-ribosylation of host proteins is an important means to regulate gene expression of bacteriophage T4. Surprisingly, this phage codes for three different ADP-ribosyltransferases, gene products Alt, ModA, and ModB, modifying partially overlapping sets of host proteins. While gene product Alt already has been isolated as a recombinant protein and its action on host RNA polymerases and transcription regulation have been studied, the nucleotide sequences of the two mod genes was published only recently. Their mode of action in the course of the infection cycle and the consequences of the ADP-ribosylations catalyzed by these enzymes remain to be investigated. Here we describe the cloning of the genes, the overexpression, purification, and partial characterization of ADP-ribosyltransferases ModA and ModB. Both proteins seem to act independently, and the ADP-ribosyl moieties are transferred to different sets of host proteins. While gene product ModA, similarly to the Alt protein, acts also on the alpha-subunit of host RNA polymerase, the ModB activity serves another set of proteins, one of which was identified as the S1 protein associated with the 30S subunit of the E. coli ribosomes.

  4. Phage T4 endonuclease V stimulates DNA repair replication in isolated nuclei from ultraviolet-irradiated human cells, including xeroderma pigmentosum fibroblasts

    International Nuclear Information System (INIS)

    The repair mode of DNA replication has been demonstrated in isolated nuclei from uv-irradiated human cells. Nuclei are incubated in a mixture containing [3H]thymidine triphosphate and bromodeoxyuridine triphosphate in a 1:5 ratio. The 3H at the density of parental DNA in alkaline CsCl density gradients is then a measure of repair. In nuclei prepared from WI38 cells 30 min after irradiation, repair replication is uv-dependent and proceeds at approximately the in vivo rate for 5 min. Repair replication is reduced in irradiated nuclei or in nuclei prepared immediately after irradiation. It is Mg2+-dependent and stimulated by added ATP and deoxyribonucleoside triphosphates. No repair replication is observed in nuclei from xeroderma pigmentosum (complementation group A) cells. However, upon addition of coliphage T4 endonuclease V, which specifically nicks DNA containing pyrimidine dimers, repair replication is observed in nuclei from irradiated xeroderma pigmentosum cells and is stimulated in WI38 nuclei. The reaction then persists for an hour and is dependent upon added ATP and deoxyribonucleoside triphosphates. The repair label is in stretches of roughly 35 nucleotides, as it is in intact cells. Added pancreatic DNase does not promote uv-dependent repair synthesis. Our results support the view that xeroderma pigmentosum (group A) cells are defective in the incision step of the DNA excision repair pathway, and demonstrate the utility of this system for probing DNA repair mechanisms

  5. ModA and ModB, two ADP-ribosyltransferases encoded by bacteriophage T4: catalytic properties and mutation analysis.

    Science.gov (United States)

    Tiemann, Bernd; Depping, Reinhard; Gineikiene, Egle; Kaliniene, Laura; Nivinskas, Rimas; Rüger, Wolfgang

    2004-11-01

    Bacteriophage T4 encodes three ADP-ribosyltransferases, Alt, ModA, and ModB. These enzymes participate in the regulation of the T4 replication cycle by ADP-ribosylating a defined set of host proteins. In order to obtain a better understanding of the phage-host interactions and their consequences for regulating the T4 replication cycle, we studied cloning, overexpression, and characterization of purified ModA and ModB enzymes. Site-directed mutagenesis confirmed that amino acids, as deduced from secondary structure alignments, are indeed decisive for the activity of the enzymes, implying that the transfer reaction follows the Sn1-type reaction scheme proposed for this class of enzymes. In vitro transcription assays performed with Alt- and ModA-modified RNA polymerases demonstrated that the Alt-ribosylated polymerase enhances transcription from T4 early promoters on a T4 DNA template, whereas the transcriptional activity of ModA-modified polymerase, without the participation of T4-encoded auxiliary proteins for middle mode or late transcription, is reduced. The results presented here support the conclusion that ADP-ribosylation of RNA polymerase and of other host proteins allows initial phage-directed mRNA synthesis reactions to escape from host control. In contrast, subsequent modification of the other cellular target proteins limits transcription from phage early genes and participates in redirecting transcription to phage middle and late genes.

  6. Ergothioneine, histidine, and two naturally occurring histidine dipeptides as radioprotectors against gamma-irradiation inactivation of bacteriophages T4 and P22

    Energy Technology Data Exchange (ETDEWEB)

    Hartman, P.E.; Hartman, Z.; Citardi, M.J.

    1988-05-01

    Bacteriophages P22, T4+, and T4os (osmotic shock-resistant mutant with altered capsids) were diluted in 0.85% NaCl and exposed to gamma irradiation (2.79 Gy/min) at room temperature (24 degrees C). T4+ was more sensitive to inactivation than was P22, and the T4os mutant was even more sensitive than T4+. Catalase exhibited a strong protective effect and superoxide dismutase a weaker protection, indicating that H/sub 2/O/sub 2/ or some product derived therefrom was predominant in causing inactivation of plaque formation. Low but significant (0.1-0.3 mM) reduced glutathione (GSH) enhanced phage inactivation, but a higher (1 mM) GSH concentration protected. A similar effect was found for the polyamine, spermidine. In contrast, 0.1 mM L-ergothioneine (2-thiol-L-histidine betaine) exhibited strong protection and 1 mM afforded essentially complete protection. L-Ergothioneine is present in millimolar concentrations in some fungi and is conserved up to millimolar concentrations in critical tissues when consumed by man. L-Histidine and two histidine-containing dipeptides, carnosine and anserine, protected at a concentration of 1 mM, a level at which they are present in striated muscles of various animals.

  7. Divergence of the mRNA targets for the Ssb proteins of bacteriophages T4 and RB69

    Directory of Open Access Journals (Sweden)

    Petrov Vasiliy M

    2004-09-01

    Full Text Available Abstract The single-strand binding (Ssb protein of phage T4 (T4 gp32, product of gene 32 is a mRNA-specific autogenous translational repressor, in addition to being a sequence-independent ssDNA-binding protein that participates in phage DNA replication, repair and recombination. It is not clear how this physiologically essential protein distinguishes between specific RNA and nonspecific nucleic acid targets. Here, we present phylogenetic evidence suggesting that ssDNA and specific RNA bind the same gp32 domain and that plasticity of this domain underlies its ability to configure certain RNA structures for specific binding. We have cloned and characterized gene 32 of phage RB69, a relative of T4 We observed that RB69 gp32 and T4 gp32 have nearly identical ssDNA binding domains, but diverge in their C-terminal domains. In T4 gp32, it is known that the C-terminal domain interacts with the ssDNA-binding domain and with other phage-induced proteins. In translation assays, we show that RB69 gp32 is, like T4 gp32, an autogenous translational repressor. We also show that the natural mRNA targets (translational operators for the 2 proteins are diverged in sequence from each other and yet can be repressed by either gp32. Results of chemical and RNase sensitivity assays indicate that the gp32 mRNA targets from the 2 related phages have similar structures, but differ in their patterns of contact with the 2 repressors. These and other observations suggest that a range of gp32-RNA binding specificities may evolve in nature due to plasticity of the protein-nucleic acid interaction and its response to modulation by the C-terminal domain of this translational repressor.

  8. Interaction of Escherichia coli B and B/4 and Bacteriophage T4D with Berea Sandstone Rock in Relation to Enhanced Oil Recovery.

    Science.gov (United States)

    Chang, P L; Yen, T F

    1984-03-01

    Much research and development is needed to recover oil reserves presently unattainable, and microbially enhanced oil recovery is a technology that may be used for this purpose. To address the problem of bacterial contamination in an oil field injection well region, we connected each end of a Teflon-sleeved Berea sandstone rock to a flask containing nutrient medium. By inoculating one flask with Escherichia coli B, we could observe bacterial growth in the uninoculated flask resulting from the transport and establishment of cells across the rock. Differences in bacterial populations occurred depending on whether bacteriophage T4D was first adsorbed to the rock. The results of these experiments indicate that the inhibition of bacterial establishment within a rock matrix is possible via lytic interaction. Some nonlytic effects are also implied by experiments with B/4 cells, which are T4D-resistant mutants of E. coli B. A 10 to 40% retention of T4 by the rock occurred when it was loaded with 10 to 10 PFU. We also describe a lysogenic system for possible use in microbially enhanced oil recovery techniques.

  9. An elasticity theory for self-assembled protein lattices with application to the martensitic phase transition in bacteriophage T4 tail sheath

    CERN Document Server

    Falk, W M; Falk, Wayne M.; James, Richard D.

    2005-01-01

    We propose an elasticity theory for one and two dimensional arrays of globular proteins for which the free energy is affected by relative position and relative rotation between neighboring molecules. The kinematics of such assemblies is described, the conditions of compatibility are found, a form of the free energy is given, and formulas for applied forces and moments are developed. It is shown that fully relaxed states of sheets consist of helically deformed sheets which themselves are composed of helical chains of molecules in rational directions. We apply the theory to the fascinating contractile deformation that occurs in the tail sheath of the virus bacteriophage T4, which aids its invasion of its bacterial host. Using electron density maps of extended and contracted sheath, we approximate the domains of each molecule by ellipsoids and then evaluate our formulas for the position and orientation of each molecule. We show that, with the resulting kinematic description, the configurations of extended and co...

  10. Studies of viral DNA packaging motors with optical tweezers: a comparison of motor function in bacteriophages φ29, λ, and T4

    Science.gov (United States)

    Smith, Douglas E.; Fuller, Derek N.; Raymer, Dorian M.; Rickgauer, Peter; Grimes, Shelley; Jardine, Paul J.; Anderson, Dwight L.; Catalano, Carlos E.; Kottadiel, Vishal; Rao, Venigalla B.

    2007-09-01

    A key step in the assembly of many viruses is the packaging of double-stranded DNA into a viral procapsid (an empty protein shell) by the action of an ATP-powered portal motor complex. We have developed methods to measure the packaging of single DNA molecules into single viral proheads in real time using optical tweezers. We can measure DNA binding and initiation of translocation, the DNA translocation dynamics, and the filling of the capsid against resisting forces. In addition to studying bacteriophage φ29, we have recently extended these methods to study the E. coli bacteriophages λ and T4, two important model systems in molecular biology. The three systems have different capsid sizes/shapes, genome lengths, and biochemical and structural differences in their packaging motors. Here, we compare and contrast these three systems. We find that all three motors translocate DNA processively and generate very large forces, each exceeding 50 piconewtons, ~20x higher force than generated by the skeletal muscle myosin 2 motor. This high force generation is required to overcome the forces resisting the confinement of the stiff, highly charged DNA at high density within the viral capsids. However, there are also striking differences between the three motors: they exhibit different DNA translocation rates, degrees of static and dynamic disorder, responses to load, and pausing and slipping dynamics.

  11. Coordination and processing of DNA ends during double-strand break repair: the role of the bacteriophage T4 Mre11/Rad50 (MR) complex.

    Science.gov (United States)

    Almond, Joshua R; Stohr, Bradley A; Panigrahi, Anil K; Albrecht, Dustin W; Nelson, Scott W; Kreuzer, Kenneth N

    2013-11-01

    The in vivo functions of the bacteriophage T4 Mre11/Rad50 (MR) complex (gp46/47) in double-strand-end processing, double-strand break repair, and recombination-dependent replication were investigated. The complex is essential for T4 growth, but we wanted to investigate the in vivo function during productive infections. We therefore generated a suppressed triple amber mutant in the Rad50 subunit to substantially reduce the level of complex and thereby reduce phage growth. Growth-limiting amounts of the complex caused a concordant decrease in phage genomic recombination-dependent replication. However, the efficiencies of double-strand break repair and of plasmid-based recombination-dependent replication remained relatively normal. Genetic analyses of linked markers indicated that double-strand ends were less protected from nuclease erosion in the depleted infection and also that end coordination during repair was compromised. We discuss models for why phage genomic recombination-dependent replication is more dependent on Mre11/Rad50 levels when compared to plasmid recombination-dependent replication. We also tested the importance of the conserved histidine residue in nuclease motif I of the T4 Mre11 protein. Substitution with multiple different amino acids (including serine) failed to support phage growth, completely blocked plasmid recombination-dependent replication, and led to the stabilization of double-strand ends. We also constructed and expressed an Mre11 mutant protein with the conserved histidine changed to serine. The mutant protein was found to be completely defective for nuclease activities, but retained the ability to bind the Rad50 subunit and double-stranded DNA. These results indicate that the nuclease activity of Mre11 is critical for phage growth and recombination-dependent replication during T4 infections.

  12. Optical tweezers studies of viral DNA packaging: Motor function and DNA confinement in Bacteriophages phi29, lambda, and T4

    Science.gov (United States)

    Smith, Douglas

    2007-03-01

    In the assembly of many viruses a powerful molecular motor translocates the genome into a pre-assembled capsid. We use optical tweezers to directly measure translocation of a single DNA molecule into the viral capsid. Improved techniques allow us to measure initiation and early stages of packaging. With phi29 the DNA terminal protein was found to cause large variations in the starting point of packaging. Removal of this protein results in terminal initiation, permitting more accurate assessment of motor function and DNA confinement forces. We investigated the role of electrostatic repulsion by varying ionic screening of the DNA. The observed trends are in accord with those theoretically expected considering counter-ion competition; however the forces are larger than expected in comparison with recent theories and DNA ejection measurements. We have recently succeeded in extending our methods to study two other phages: lambda and T4. These systems have unique structural and functional features, presenting an opportunity for comparative studies in this family of molecular motors. Initial measurements show that lambda and T4 translocate DNA several times faster than the phi29 motor, but are more sensitive to applied load.

  13. Fragmentation of bacteriophage S13 replicative from DNA by restriction endonucleases from Hemophilus influenzae and Hemophilus aegyptius.

    NARCIS (Netherlands)

    F.G. Grosveld (Frank); K.M. Ojamaa; J.H. Spencer

    1976-01-01

    textabstractThe restriction enzymes Hind from Hemophilus influenzae and HaeIII from Hemophilus aegyptius cleave bacteriophage S13 replicative form (RF) DNA into 13 and 10 specific fragments, respectively. The sizes of these fragments were estimated by gel electrophoresis, electron microscopy, and py

  14. CRISPR/Cas9-Mediated Phage Resistance Is Not Impeded by the DNA Modifications of Phage T4

    OpenAIRE

    Yaung, Stephanie J.; Kevin M Esvelt; Church, George M

    2014-01-01

    Bacteria rely on two known DNA-level defenses against their bacteriophage predators: restriction-modification and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems. Certain phages have evolved countermeasures that are known to block endonucleases. For example, phage T4 not only adds hydroxymethyl groups to all of its cytosines, but also glucosylates them, a strategy that defeats almost all restriction enzymes. We sought to determine whether the...

  15. Comparative study of the resistance of bacteriophageT4,ΦX174D,MS2 and f2 to gamma radiation%T4、ΦX174D、MS2和f2噬菌体对γ射线抵抗力的比较研究

    Institute of Scientific and Technical Information of China (English)

    陈昭斌; 黄红英; 张朝武

    2008-01-01

    目的 为筛选出辐照灭菌中评价对病毒灭活效果的指示噬菌体,比较研究了噬菌体T4、φX174D、MS2和f2,以及两种细菌参考株大肠杆菌8099和枯草杆菌黑色变种芽孢ATCC 9372对电离辐射γ射线的抵抗力.方法 采用钴源60Co-γ射线辐射源对噬菌体和细菌进行辐照处理;灭活方法采用悬液灭活试验;噬菌体的检测采用双层琼脂法.结果 方差分析表明,不同剂量的γ射线组间(P<0.01)和不同的噬菌体组间(P<0.05),差异均有统计学意义.LIV(灭活对数值)和KL(杀灭对数值)为:(1)T4、ΦX174、f2和MS2 4种噬菌体经100 Gy的γ射线辐照可达到消毒水平(LIV≥4.00 log10);在此剂量下4种噬菌体的LIV分别为:6.31,6.92,5.74和4.46 log10;(2)在前述剂量下,大肠杆菌8099的KL>7.97 log10;(3)在前述剂量下,枯草杆菌黑色变种芽孢ATCC9372的KL为1.61 log10.结论 6种微生物对γ射线的抵抗力由强到弱为:枯草杆菌黑色变种芽孢>噬菌体MS2>噬菌体f2>噬菌体T4>噬菌体φX174>大肠杆菌.%Objective To screen the suitable bacteriophage as virus indictor in irradiation sterilization.Methods Suspensions of bacteriophage T4,φX174D,MS2,and f2,Escherichia coli 8099,and Bacillus subtilis var. niger.sp.ATCC9372 were irradiated with60 Co-γ ray.The mean log10 inactivation value levels of the bacteriophage T4,ΦX174,f2,and MS2 were 6.31,6.92,5.74,and 4.46 log10 respectively,var.niger.sp.ATCC9372 was 1.61 log10.Conclusion The order of resistance of the above six microorganisms to gamma-radiation from the biggest to the smallest iS as follows:Bacillus subtilis var.niger.sp.>bacteriophage MS2>bacteriophage f2>bacteriophage T4bacteriophage φX 174D>E. coli.

  16. Enhancement of DNA repair using topical T4 endonuclease V does not inhibit melanoma formation in Cdk4(R24C/R24C)/Tyr-Nras(Q61K) mice following neonatal UVR.

    Science.gov (United States)

    Hacker, Elke; Muller, H Konrad; Hayward, Nicholas; Fahey, Paul; Walker, Graeme

    2010-02-01

    To further investigate the use of DNA repair-enhancing agents for skin cancer prevention, we treated Cdk4(R24C/R24C)/Nras(Q61K) mice topically with the T4 endonuclease V DNA repair enzyme (known as Dimericine) immediately prior to neonatal ultraviolet radiation (UVR) exposure, which has a powerful effect in exacerbating melanoma development in the mouse model. Dimericine has been shown to reduce the incidence of basal-cell and squamous cell carcinoma. Unexpectedly, we saw no difference in penetrance or age of onset of melanoma after neonatal UVR between Dimericine-treated and control animals, although the drug reduced DNA damage and cellular proliferation in the skin. Interestingly, epidermal melanocytes removed cyclobutane pyrimidine dimers (CPDs) more efficiently than surrounding keratinocytes. Our study indicates that neonatal UVR-initiated melanomas may be driven by mechanisms other than solely that of a large CPD load and/or their inefficient repair. This is further suggestive of different mechanisms by which UVR may enhance the transformation of keratinocytes and melanocytes. PMID:19788533

  17. Enhancement of DNA repair using topical T4 endonuclease V does not inhibit melanoma formation in Cdk4(R24C/R24C)/Tyr-Nras(Q61K) mice following neonatal UVR.

    Science.gov (United States)

    Hacker, Elke; Muller, H Konrad; Hayward, Nicholas; Fahey, Paul; Walker, Graeme

    2010-02-01

    To further investigate the use of DNA repair-enhancing agents for skin cancer prevention, we treated Cdk4(R24C/R24C)/Nras(Q61K) mice topically with the T4 endonuclease V DNA repair enzyme (known as Dimericine) immediately prior to neonatal ultraviolet radiation (UVR) exposure, which has a powerful effect in exacerbating melanoma development in the mouse model. Dimericine has been shown to reduce the incidence of basal-cell and squamous cell carcinoma. Unexpectedly, we saw no difference in penetrance or age of onset of melanoma after neonatal UVR between Dimericine-treated and control animals, although the drug reduced DNA damage and cellular proliferation in the skin. Interestingly, epidermal melanocytes removed cyclobutane pyrimidine dimers (CPDs) more efficiently than surrounding keratinocytes. Our study indicates that neonatal UVR-initiated melanomas may be driven by mechanisms other than solely that of a large CPD load and/or their inefficient repair. This is further suggestive of different mechanisms by which UVR may enhance the transformation of keratinocytes and melanocytes.

  18. In vitro binding of anthrax protective antigen on bacteriophage T4 capsid surface through Hoc-capsid interactions: A strategy for efficient display of large full-length proteins

    International Nuclear Information System (INIS)

    An in vitro binding system is described to display large full-length proteins on bacteriophage T4 capsid surface at high density. The phage T4 icosahedral capsid features 155 copies of a nonessential highly antigenic outer capsid protein, Hoc, at the center of each major capsid protein hexon. Gene fusions were engineered to express the 83-kDa protective antigen (PA) from Bacillus anthracis fused to the N-terminus of Hoc and the 130-kDa PA-Hoc protein was expressed in Escherichia coli and purified. The purified PA-Hoc was assembled in vitro on hoc - phage particles. Binding was specific, stable, and of high affinity. This defined in vitro system allowed manipulation of the copy number of displayed PA and imposed no significant limitation on the size of the displayed antigen. In contrast to in vivo display systems, the in vitro approach allows all the capsid binding sites to be occupied by the 130-kDa PA-Hoc fusion protein. The PA-T4 particles were immunogenic in mice in the absence of an adjuvant, eliciting strong PA-specific antibodies and anthrax lethal toxin neutralizing antibodies. The in vitro display on phage T4 offers a novel platform for potential construction of customized vaccines against anthrax and other infectious diseases

  19. Radiation-induced damage in T4 bacteriophage: the effect of superoxid radicals and molecular oxygen. Progress report, December 1, 1977--November 30, 1978. [. gamma. -irradiated in phosphate buffer suspensions

    Energy Technology Data Exchange (ETDEWEB)

    Samuni, A; Chevion, M; Halpern, Y S; Ilan, Y A; Czapski, G

    1978-01-01

    The sensitivity of T4 bacteriophage towards ..gamma.. irradiation has been studied in phosphate buffer suspensions. The spectrum of the water radicals was controlled by a careful choice of the appropriate saturating gas and the addition of radical scavengers. Thus, it was possible to distinguish between the effects of molecular oxygen and the superoxide radicals formed through its reactions. About 90 percent of the damage was caused by the water radicals formed in the bulk suspensions. These probably affected the phage proteins; only the remainder of the damage involved the viral DNA. The oxygen enhancement ratio observed was not connected in any way with the formation of the superoxide radicals. The results confirmed that the OH radicals are the reactive species, while e/sup -//sub aq/ as well as the superoxide radical do not contribute to the radiodamage.

  20. Control of Escherichia coli O157 on beef at 37, 22 and 4 °C by T5-, T1-, T4-and O1-like bacteriophages.

    Science.gov (United States)

    Liu, H; Niu, Y D; Meng, R; Wang, J; Li, J; Johnson, R P; McAllister, T A; Stanford, K

    2015-10-01

    Efficacy of four bacteriophages (phages) and a cocktail for biocontrol of Escherichia coli O157 was assessed on beef samples stored at 4, 22 and 37 °C. Samples (3 × 3 × 1 cm) were contaminated with E. coli O157 (10(4) CFU/cm(2)) and treated with single phages: T5-like (T5), T1-like (T1), T4-like (T4) and O1-like (O1), or a cocktail at two titers: multiplicity of infection (MOI) = 1000 and MOI = 10. In contrast to previous studies, use of virucidal solution prevented over-estimation of phage efficacy. Irrespective of temperature and MOIs, T5 was most (P supply chain includes hours of storage or transport at temperatures near 4 °C, this study demonstrates phages could significantly reduce E. coli O157 during this period. PMID:26187829

  1. Repairing DNA damage in xeroderma pigmentosum: T4N5 lotion and gene therapy.

    Science.gov (United States)

    Zahid, Sarwar; Brownell, Isaac

    2008-04-01

    Patients with xeroderma pigmentosum (XP) have defective DNA repair and are at a high risk for cutaneous malignancies. Standard treatments for XP are limited in scope and effectiveness. Understanding the molecular etiology of XP has led to the development of novel therapeutic approaches, including enzyme and gene therapies. One new topical treatment utilizing bacteriophage T4 endonuclease 5 (T4N5) in a liposomal lotion is currently in clinical trials and has received a Fast Track designation from the FDA. Gene therapy for XP, while making leaps in preclinical studies, has been slower to develop due to tactical hurdles, but seems to have much potential for future treatment. If these treatments prove effective in lowering the risk of cancer in patients with XP, they may also be found useful in reducing skin cancers in other at-risk patient populations.

  2. CRISPR/Cas9-mediated phage resistance is not impeded by the DNA modifications of phage T4.

    Directory of Open Access Journals (Sweden)

    Stephanie J Yaung

    Full Text Available Bacteria rely on two known DNA-level defenses against their bacteriophage predators: restriction-modification and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-CRISPR-associated (Cas systems. Certain phages have evolved countermeasures that are known to block endonucleases. For example, phage T4 not only adds hydroxymethyl groups to all of its cytosines, but also glucosylates them, a strategy that defeats almost all restriction enzymes. We sought to determine whether these DNA modifications can similarly impede CRISPR-based defenses. In a bioinformatics search, we found naturally occurring CRISPR spacers that potentially target phages known to modify their DNA. Experimentally, we show that the Cas9 nuclease from the Type II CRISPR system of Streptococcus pyogenes can overcome a variety of DNA modifications in Escherichia coli. The levels of Cas9-mediated phage resistance to bacteriophage T4 and the mutant phage T4 gt, which contains hydroxymethylated but not glucosylated cytosines, were comparable to phages with unmodified cytosines, T7 and the T4-like phage RB49. Our results demonstrate that Cas9 is not impeded by N6-methyladenine, 5-methylcytosine, 5-hydroxymethylated cytosine, or glucosylated 5-hydroxymethylated cytosine.

  3. Various Culture Media Effect on T4 Phage Lysis and Production

    Directory of Open Access Journals (Sweden)

    Muhammad Kamran Taj

    2013-11-01

    Full Text Available Studies on bacteriophage growth and its development played a vital role in the history of molecular biology which in turn helped in clarification of many points. Most of the previous studies on bacteriophage development and growth have been performed under optimal conditions for the host cell. While On the other hand these conditions may not be optimal for the T4 bacteriophage. As a matter of fact in nature E. coli faces many unfavorable growth conditions, good example are those conditions prevailing in the human gut in which E.coli manages to survive well. This study characterizes the effects and influences of well-defined physiological conditions on T4 bacteriophage growth and development. In addition to this, T4 bacteriophage interactions with its bacterial host have also been demonstrated.In our present study we observed that the maximum growth and lysis of T4 bacteriophage was on luria-bertani (LB and nutrient media (NM. Moreover the T4 bacteriophage production and lysis was also good in luria-bertani plus glucose (LB+G media but when compared with its production in luria-bertani (LB and nutrient media it was found to be less than these medium. Our study results also showed that in minimal media (MM rate of growth and lysis activity of T4 bacteriophage was lowest as compared to other mentioned medium.

  4. 口蹄疫病毒全衣壳前体(P1)重组T4噬菌体的构建和鉴定%Construction and identification of foot-and-mouth disease virus entire capsid protein precursor (P1) gene recombinant T4 bacteriophage

    Institute of Scientific and Technical Information of China (English)

    田纯见; 毕英佐; 曹永长

    2007-01-01

    以含完整的口蹄疫病毒全衣壳前体基因(P1)的质粒pP1-T为模板进行PCR扩增获得P1基因片段(约2 200 bp),构建整合质粒pR-P1经引物对Pr.78/P1-A2进行PCR扩增筛选插入方向正确的重组质粒,EcoRⅠ酶切和T7启动子测序正确.以pR-P1质粒转化E.coli E2,感染噬菌体ψT4-Z1进行同源重组,经溶菌酶依赖性筛选获得整合成功的噬菌体ψT4-P1.SDS-PAGE检测重组噬菌体出现约98 000预计大小的条带;Western bloting检测表明,重组噬菌体ψT4-P1与口蹄疫阳性血清可发生免疫反应.

  5. Cloning, expression and stable maintenance of a bacteriophage DNA repair gene in E. coli

    International Nuclear Information System (INIS)

    Efforts to clone the bacteriophage T4 DNA repair gene, denV, have met with repeated problems of gene instability. Therefore, oligonucleotide site-directed mutagenesis was used to create new DNA restriction sites closely flanking the structural gene, facilitating the removal of adjacent deleterious T4 sequences. The sequence which encodes the DNA repair enzyme, endonuclease V, was then reconstructed behind the hybrid λ promoter O/sub L/P/sub R/ and its associated ribosome-binding site in a plasmid vector for expression in E. coli. Transformants harboring the expression plasmid bearing the correct reconstruction of denV were screened and selected by colony hybridization to a synthetic bridge oligonucleotide and by restriction analyses. The denV structural gene was found inserted at an equal frequency in both orientations relative to the promoter. These plasmids were transformed into an E. coli strain which is defective for UV excision recombination and repair (uvrA-, recA-). The defective hosts were assayed for survival after UV irradiation, and expression of the denV gene was found to enhance UV survival to levels indicative of full complementation of the uvrA- deficiency. Correspondingly, Western blot analyses of cellular protein extracts from the defective host harboring the denV+ plasmid showed a protein which was immunoreactive with an anti-endonuclease V antibody. Additionally, in infections using a UV-irradiated T4 mutant (T4 denV1, defective for active endonuclease V), these cells fully complemented the mutant phage to wild type survival levels

  6. Nanoscale bacteriophage biosensors beyond phage display

    Directory of Open Access Journals (Sweden)

    Lee JW

    2013-10-01

    Full Text Available Jong-Wook Lee,1 Jangwon Song,1,2 Mintai P Hwang,1 Kwan Hyi Lee1,2 1Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, Korea; 2Department of Biomedical Engineering, University of Science and Technology, Seoul, Korea Abstract: Bacteriophages are traditionally used for the development of phage display technology. Recently, their nanosized dimensions and ease with which genetic modifications can be made to their structure and function have put them in the spotlight towards their use in a variety of biosensors. In particular, the expression of any protein or peptide on the extraluminal surface of bacteriophages is possible by genetically engineering the genome. In addition, the relatively short replication time of bacteriophages offers researchers the ability to generate mass quantities of any given bacteriophage-based biosensor. Coupled with the emergence of various biomarkers in the clinic as a means to determine pathophysiological states, the development of current and novel technologies for their detection and quantification is imperative. In this review, we categorize bacteriophages by their morphology into M13-based filamentous bacteriophages and T4- or T7-based icosahedral bacteriophages, and examine how such advantages are utilized across a variety of biosensors. In essence, we take a comprehensive approach towards recent trends in bacteriophage-based biosensor applications and discuss their outlook with regards to the field of biotechnology. Keywords: biosensing, M13 bacteriophage, T4 bacteriophage, bacterial detection, Escherichia coli, SPR sensor

  7. Applications of repaired endonucleases

    International Nuclear Information System (INIS)

    The possibilities of using antimutagenously various endonucleases are discussed. Since mutageniety of excision repair is considerably lower than the repair in the replication moment or after thereof the intensification of the excision repair of premutation disorders can suppress mutagenesis. Experimental aproaches of using repair endonucleases for sounding premutation changes are described. The optimal object has been chosen for endonuclear sounding of premutation DNA disorders following an ionizing radiation action

  8. Thermodynamic Treatment of Bacteriophage T4B Adsorption Kinetics

    Science.gov (United States)

    Gamow, Rustem I.

    1969-01-01

    The activation energy was measured for the reaction of inactive phage becoming active in the presence and in the absence of l-tryptophan and was found to be about 50% lower in the former case. PMID:4897123

  9. LPS-Activated Monocytes Are Unresponsive to T4 Phage and T4-Generated Escherichia coli Lysate.

    Science.gov (United States)

    Bocian, Katarzyna; Borysowski, Jan; Zarzycki, Michał; Wierzbicki, Piotr; Kłosowska, Danuta; Weber-Dąbrowska, Beata; Korczak-Kowalska, Grażyna; Górski, Andrzej

    2016-01-01

    A growing body of data shows that bacteriophages can interact with different kinds of immune cells. The objective of this study was to investigate whether T4 bacteriophage and T4-generated Escherichia coli lysate affect functions of monocytes, the key population of immune cells involved in antibacterial immunity. To that end, we evaluated how T4 and E. coli lysate influence the expression of main costimulatory molecules including CD40, CD80 and CD86, TLR2, TLR4 on monocytes, as well as the production of IL-6 and IL-12 in cultures of peripheral blood mononuclear cells (PBMCs). Separate experiments were performed on unactivated and LPS-activated PBMCs cultures. Both studied preparations significantly increased the percentage of CD14(+)CD16(-)CD40(+) and CD14(+)CD16(-)CD80(+) monocytes in unactivated PBMCs cultures, as well as the concentration of IL-6 and IL-12 in culture supernates. However, neither purified T4 nor E. coli lysate had any significant effect on monocytes in LPS-activated PBMCs cultures. We conclude that LPS-activated monocytes are unresponsive to phages and products of phage-induced lysis of bacteria. This study is highly relevant to phage therapy because it suggests that in patients with infections caused by Gram-negative bacteria the administration of phage preparations to patients and lysis of bacteria by phages are not likely to overly stimulate monocytes. PMID:27630621

  10. Genome and Proteome of Campylobacter jejuni Bacteriophage NCTC 12673▿†

    Science.gov (United States)

    Kropinski, Andrew M.; Arutyunov, Denis; Foss, Mary; Cunningham, Anna; Ding, Wen; Singh, Amit; Pavlov, Andrey R.; Henry, Matthew; Evoy, Stephane; Kelly, John; Szymanski, Christine M.

    2011-01-01

    Campylobacter jejuni continues to be the leading cause of bacterial food-borne illness worldwide, so improvements to current methods used for bacterial detection and disease prevention are needed. We describe here the genome and proteome of C. jejuni bacteriophage NCTC 12673 and the exploitation of its receptor-binding protein for specific bacterial detection. Remarkably, the 135-kb Myoviridae genome of NCTC 12673 differs greatly from any other proteobacterial phage genome described (including C. jejuni phages CP220 and CPt10) and instead shows closest homology to the cyanobacterial T4-related myophages. The phage genome contains 172 putative open reading frames, including 12 homing endonucleases, no visible means of packaging, and a putative trans-splicing intein. The phage DNA appears to be strongly associated with a protein that interfered with PCR amplification and estimation of the phage genome mass by pulsed-field gel electrophoresis. Identification and analyses of the receptor-binding protein (Gp48) revealed features common to the Salmonella enterica P22 phage tailspike protein, including the ability to specifically recognize a host organism. Bacteriophage receptor-binding proteins may offer promising alternatives for use in pathogen detection platforms. PMID:21965409

  11. Studies on the repair of damaged DNA in bacteriophage, bacterial and mammalian systems. Comprehensive report, 1 February 1981-15 September 1983

    International Nuclear Information System (INIS)

    We have explored the molecular mechanism of the repair of DNA at a number of different levels of biological organization, by investigating bacteriophage, bacterial, yeast and mammalian (including human) cells. We have demonstrated that uv endonuclease of phage T4 not only possesses pyrimidine dimer (PD)-DNA glycosylase activity but also apyrimidinic (AP) endonuclease activity. The demonstration of both activities provided an explanation for the specific endonucleosytic cleavage of DNA at sites of pyrimidine dimers catalyzed by this small protein. A new apurinic/apyrimidinic (AP) endonuclease, specific for sites of of base loss in single stranded DNA has been isolated from E. celi and presumably recognizes these lesions in single stranded regions of duplex DNA. We have partially purified this enzyme and have carried out a preliminary characterization of the activity. We treated xeroderma pigmentosum and normal cells with sodium butyrate in the hope of restoring normal levels of excision repair to the former. Although this result was not obtained, we established that all cells treated with sodium butyrate show enhanced levels of repair synthesis, thus providing a means for increasing the sensitivity of this commonly used technique for measuring DNA repair in mammalian cells in culture

  12. Campylobacter bacteriophages and bacteriophage therapy.

    Science.gov (United States)

    Connerton, P L; Timms, A R; Connerton, I F

    2011-08-01

    Members of the genus Campylobacter are frequently responsible for human enteric disease with occasionally very serious outcomes. Much of this disease burden is thought to arise from consumption of contaminated poultry products. More than 80% of poultry in the UK harbour Campylobacter as a part of their intestinal flora. To address this unacceptably high prevalence, various interventions have been suggested and evaluated. Among these is the novel approach of using Campylobacter-specific bacteriophages, which are natural predators of the pathogen. To optimize their use as therapeutic agents, it is important to have a comprehensive understanding of the bacteriophages that infect Campylobacter, and how they can affect their host bacteria. This review will focus on many aspects of Campylobacter-specific bacteriophages including: their first isolation in the 1960s, their use in bacteriophage typing schemes, their isolation from the different biological sources and genomic characterization. As well as their use as therapeutic agents to reduce Campylobacter in poultry their future potential, including their use in bio-sanitization of food, will be explored. The evolutionary consequences of naturally occurring bacteriophage infection that have come to light through investigations of bacteriophages in the poultry ecosystem will also be discussed.

  13. Nanoscale bacteriophage biosensors beyond phage display.

    Science.gov (United States)

    Lee, Jong-Wook; Song, Jangwon; Hwang, Mintai P; Lee, Kwan Hyi

    2013-01-01

    Bacteriophages are traditionally used for the development of phage display technology. Recently, their nanosized dimensions and ease with which genetic modifications can be made to their structure and function have put them in the spotlight towards their use in a variety of biosensors. In particular, the expression of any protein or peptide on the extraluminal surface of bacteriophages is possible by genetically engineering the genome. In addition, the relatively short replication time of bacteriophages offers researchers the ability to generate mass quantities of any given bacteriophage-based biosensor. Coupled with the emergence of various biomarkers in the clinic as a means to determine pathophysiological states, the development of current and novel technologies for their detection and quantification is imperative. In this review, we categorize bacteriophages by their morphology into M13-based filamentous bacteriophages and T4- or T7-based icosahedral bacteriophages, and examine how such advantages are utilized across a variety of biosensors. In essence, we take a comprehensive approach towards recent trends in bacteriophage-based biosensor applications and discuss their outlook with regards to the field of biotechnology.

  14. Chlamydia bacteriophages.

    Science.gov (United States)

    Śliwa-Dominiak, Joanna; Suszyńska, Ewa; Pawlikowska, Małgorzata; Deptuła, Wiesław

    2013-11-01

    Phages are called "good viruses" due to their ability to infect and kill pathogenic bacteria. Chlamydia are small, Gram-negative (G-) microbes that can be dangerous to human and animals. In humans, these bacteria are etiological agents of diseases such as psittacosis or respiratory tract diseases, while in animals, the infection may result in enteritis in cattle and chronic bowel diseases, as well as miscarriages in sheep. The first-known representative of chlamydiaphages was Chp1. It was discovered in Chlamydia psittaci isolates. Since then, four more species of chlamydiaphages have been identified [Chp2, Chp3, φCPG1 φCPAR39 (φCpn1) and Chp4]. All of them were shown to infect Chlamydia species. This paper described all known chlamydiaphages. They were characterised in terms of origin, host range, and their molecular structure. The review concerns the characterisation of bacteriophages that infects pathogenic and dangerous bacteria with unusual, intracellular life cycles that are pathogenic. In the era of antibiotic resistance, it is difficult to cure chlamydophilosis. Those bacteriophages can be an alternative to antibiotics, but before this happens, we need to get to know chlamydiaphages better. PMID:23903989

  15. Template reporter bacteriophage platform and multiple bacterial detection assays based thereon

    Science.gov (United States)

    Goodridge, Lawrence (Inventor)

    2007-01-01

    The invention is a method for the development of assays for the simultaneous detection of multiple bacteria. A bacteria of interest is selected. A host bacteria containing plasmid DNA from a T even bacteriophage that infects the bacteria of interest is infected with T4 reporter bacteriophage. After infection, the progeny bacteriophage are plating onto the bacteria of interest. The invention also includes single-tube, fast and sensitive assays which utilize the novel method.

  16. The nucleotide sequence of two restriction fragments located in the gene AB region of bacteriophage S13.

    NARCIS (Netherlands)

    F.G. Grosveld (Frank); J.H. Spencer

    1977-01-01

    textabstractThe nucleotide sequence of a double stranded DNA fragment from the gene AB region of bacteriophage S13 DNA has been determined. The fragment was isolated as two adjacent shorter fragments by cleavage of S13 replicative form (RF) DNA with restriction endonuclease III from Hemophilus aegyp

  17. Bacteriophages Infecting Propionibacterium acnes

    Directory of Open Access Journals (Sweden)

    Holger Brüggemann

    2013-01-01

    Full Text Available Viruses specifically infecting bacteria, or bacteriophages, are the most common biological entity in the biosphere. As such, they greatly influence bacteria, both in terms of enhancing their virulence and in terms of killing them. Since the first identification of bacteriophages in the beginning of the 20th century, researchers have been fascinated by these microorganisms and their ability to eradicate bacteria. In this review, we will cover the history of the Propionibacterium acnes bacteriophage research and point out how bacteriophage research has been an important part of the research on P. acnes itself. We will further discuss recent findings from phage genome sequencing and the identification of phage sequence signatures in clustered regularly interspaced short palindromic repeats (CRISPRs. Finally, the potential to use P. acnes bacteriophages as a therapeutic strategy to combat P. acnes-associated diseases will be discussed.

  18. Bacteriophages infecting Propionibacterium acnes.

    Science.gov (United States)

    Brüggemann, Holger; Lood, Rolf

    2013-01-01

    Viruses specifically infecting bacteria, or bacteriophages, are the most common biological entity in the biosphere. As such, they greatly influence bacteria, both in terms of enhancing their virulence and in terms of killing them. Since the first identification of bacteriophages in the beginning of the 20th century, researchers have been fascinated by these microorganisms and their ability to eradicate bacteria. In this review, we will cover the history of the Propionibacterium acnes bacteriophage research and point out how bacteriophage research has been an important part of the research on P. acnes itself. We will further discuss recent findings from phage genome sequencing and the identification of phage sequence signatures in clustered regularly interspaced short palindromic repeats (CRISPRs). Finally, the potential to use P. acnes bacteriophages as a therapeutic strategy to combat P. acnes-associated diseases will be discussed.

  19. Conserved Endonuclease Function of Hantavirus L Polymerase.

    Science.gov (United States)

    Rothenberger, Sylvia; Torriani, Giulia; Johansson, Maria U; Kunz, Stefan; Engler, Olivier

    2016-01-01

    Hantaviruses are important emerging pathogens belonging to the Bunyaviridae family. Like other segmented negative strand RNA viruses, the RNA-dependent RNA polymerase (RdRp) also known as L protein of hantaviruses lacks an intrinsic "capping activity". Hantaviruses therefore employ a "cap snatching" strategy acquiring short 5' RNA sequences bearing 5'cap structures by endonucleolytic cleavage from host cell transcripts. The viral endonuclease activity implicated in cap snatching of hantaviruses has been mapped to the N-terminal domain of the L protein. Using a combination of molecular modeling and structure-function analysis we confirm and extend these findings providing evidence for high conservation of the L endonuclease between Old and New World hantaviruses. Recombinant hantavirus L endonuclease showed catalytic activity and a defined cation preference shared by other viral endonucleases. Based on the previously reported remarkably high activity of hantavirus L endonuclease, we established a cell-based assay for the hantavirus endonuclase function. The robustness of the assay and its high-throughput compatible format makes it suitable for small molecule drug screens to identify novel inhibitors of hantavirus endonuclease. Based on the high degree of similarity to RdRp endonucleases, some candidate inhibitors may be broadly active against hantaviruses and other emerging human pathogenic Bunyaviruses. PMID:27144576

  20. Conserved Endonuclease Function of Hantavirus L Polymerase

    Directory of Open Access Journals (Sweden)

    Sylvia Rothenberger

    2016-05-01

    Full Text Available Hantaviruses are important emerging pathogens belonging to the Bunyaviridae family. Like other segmented negative strand RNA viruses, the RNA-dependent RNA polymerase (RdRp also known as L protein of hantaviruses lacks an intrinsic “capping activity”. Hantaviruses therefore employ a “cap snatching” strategy acquiring short 5′ RNA sequences bearing 5′cap structures by endonucleolytic cleavage from host cell transcripts. The viral endonuclease activity implicated in cap snatching of hantaviruses has been mapped to the N-terminal domain of the L protein. Using a combination of molecular modeling and structure–function analysis we confirm and extend these findings providing evidence for high conservation of the L endonuclease between Old and New World hantaviruses. Recombinant hantavirus L endonuclease showed catalytic activity and a defined cation preference shared by other viral endonucleases. Based on the previously reported remarkably high activity of hantavirus L endonuclease, we established a cell-based assay for the hantavirus endonuclase function. The robustness of the assay and its high-throughput compatible format makes it suitable for small molecule drug screens to identify novel inhibitors of hantavirus endonuclease. Based on the high degree of similarity to RdRp endonucleases, some candidate inhibitors may be broadly active against hantaviruses and other emerging human pathogenic Bunyaviruses.

  1. Direct observation of T4 lysozyme hinge-bending motion by fluorescence correlation spectroscopy.

    Science.gov (United States)

    Yirdaw, Robel B; McHaourab, Hassane S

    2012-10-01

    Bacteriophage T4 Lysozyme (T4L) catalyzes the hydrolysis of the peptidoglycan layer of the bacterial cell wall late in the infection cycle. It has long been postulated that equilibrium dynamics enable substrate access to the active site located at the interface between the N- and C-terminal domains. Crystal structures of WT-T4L and point mutants captured a range of conformations that differ by the hinge-bending angle between the two domains. Evidence of equilibrium between open and closed conformations in solution was gleaned from distance measurements between the two domains but the nature of the equilibrium and the timescale of the underlying motion have not been investigated. Here, we used fluorescence fluctuation spectroscopy to directly detect T4L equilibrium conformational fluctuations in solution. For this purpose, Tetramethylrhodamine probes were introduced at pairs of cysteines in regions of the molecule that undergo relative displacement upon transition from open to closed conformations. Correlation analysis of Tetramethylrhodamine intensity fluctuations reveals hinge-bending motion that changes the relative distance and orientation of the N- and C-terminal domains with ≅ 15 μs relaxation time. That this motion involves interconversion between open and closed conformations was further confirmed by the dampening of its amplitude upon covalent substrate trapping. In contrast to the prevalent two-state model of T4L equilibrium, molecular brightness and number of particles obtained from cumulant analysis suggest that T4L populates multiple intermediate states, consistent with the wide range of hinge-bending angles trapped in the crystal structure of T4L mutants.

  2. Endonuclease V cleaves at inosines in RNA.

    Science.gov (United States)

    Vik, Erik Sebastian; Nawaz, Meh Sameen; Strøm Andersen, Pernille; Fladeby, Cathrine; Bjørås, Magnar; Dalhus, Bjørn; Alseth, Ingrun

    2013-01-01

    Endonuclease V orthologues are highly conserved proteins found in all kingdoms of life. While the prokaryotic enzymes are DNA repair proteins for removal of deaminated adenosine (inosine) from the genome, no clear role for the eukaryotic counterparts has hitherto been described. Here we report that human endonuclease V (ENDOV) and also Escherichia coli endonuclease V are highly active ribonucleases specific for inosine in RNA. Inosines are normal residues in certain RNAs introduced by specific deaminases. Adenosine-to-inosine editing is essential for proper function of these transcripts and defects are linked to various human disease. Here we show that human ENDOV cleaves an RNA substrate containing inosine in a position corresponding to a biologically important site for deamination in the Gabra-3 transcript of the GABA(A) neurotransmitter. Further, human ENDOV specifically incises transfer RNAs with inosine in the wobble position. This previously unknown RNA incision activity may suggest a role for endonuclease V in normal RNA metabolism. PMID:23912683

  3. Massively parallel characterization of restriction endonucleases

    OpenAIRE

    Kamps-Hughes, Nick; Quimby, Aine; Zhu, Zhenyu; Johnson, Eric A.

    2013-01-01

    Restriction endonucleases are highly specific in recognizing the particular DNA sequence they act on. However, their activity is affected by sequence context, enzyme concentration and buffer composition. Changes in these factors may lead to either ineffective cleavage at the cognate restriction site or relaxed specificity allowing cleavage of degenerate ‘star’ sites. Additionally, uncharacterized restriction endonucleases and engineered variants present novel activities. Traditionally, restri...

  4. Characterization of FP22, a large streptomycete bacteriophage with DNA insensitive to cleavage by many restriction enzymes.

    Science.gov (United States)

    Hahn, D R; McHenney, M A; Baltz, R H

    1990-12-01

    Bacteriophage FP22 has a very broad host range within streptomycetes and appeared to form lysogens of Streptomyces ambofaciens ATCC 15154. FP22 shared strong cross-immunity and antibody cross-reactivity with bacteriophage P23, but not with seven other streptomycete bacteriophages. FP22 particles had a head diameter of 71 nm and a tail length of 307 nm. The FP22 genome was 131 kb, which is the largest bacteriophage genome reported for streptomycetes. The G + C content of the genome was 46 mol% and restriction mapping indicated that FP22 DNA had discrete ends. NaCl- and pyrophosphate-resistant deletion mutants were readily isolated and the extent of the deletions defined at least 23 kb of dispensable DNA in two regions of the genome. The DNA was not cleaved by most restriction endonucleases (or isoschizomers) which have been identified in the streptomycetes, including the tetranucleotide cutter MboI (GATC).

  5. Bacteriophages and Biofilms

    Directory of Open Access Journals (Sweden)

    David R. Harper

    2014-06-01

    Full Text Available Biofilms are an extremely common adaptation, allowing bacteria to colonize hostile environments. They present unique problems for antibiotics and biocides, both due to the nature of the extracellular matrix and to the presence within the biofilm of metabolically inactive persister cells. Such chemicals can be highly effective against planktonic bacterial cells, while being essentially ineffective against biofilms. By contrast, bacteriophages seem to have a greater ability to target this common form of bacterial growth. The high numbers of bacteria present within biofilms actually facilitate the action of bacteriophages by allowing rapid and efficient infection of the host and consequent amplification of the bacteriophage. Bacteriophages also have a number of properties that make biofilms susceptible to their action. They are known to produce (or to be able to induce enzymes that degrade the extracellular matrix. They are also able to infect persister cells, remaining dormant within them, but re-activating when they become metabolically active. Some cultured biofilms also seem better able to support the replication of bacteriophages than comparable planktonic systems. It is perhaps unsurprising that bacteriophages, as the natural predators of bacteria, have the ability to target this common form of bacterial life.

  6. Bacteriophages and cancer.

    Science.gov (United States)

    Budynek, Paulina; Dabrowska, Krystyna; Skaradziński, Grzegorz; Górski, Andrzej

    2010-05-01

    Bacteriophages can be used effectively to cure bacterial infections. They are known to be active against bacteria but inactive against eukaryotic cells. Nevertheless, novel observations suggest that phages are not neutral for higher organisms. They can affect physiological and immunological processes which may be crucial to their expected positive effects in therapies. Bacteriophages are a very differentiated group of viruses and at least some of them can influence cancer processes. Phages may also affect the immunological system. In general, they activate the immunological response, for example cytokine secretion. They can also switch the tumor microenvironment to one advantageous for anticancer treatment. On the other hand, bacteriophages are used as a platform for foreign peptides that may induce anticancer effects. As bacterial debris can interfere with bacteriophage activity, phage purification is significant for the final effect of a phage preparation. In this review, results of the influence of bacteriophages on cancer processes are presented which have implications for the perspective application of phage therapy in patients with cancer and the general understanding of the role of bacteriophages in the human organism.

  7. Restriction of bacteriophage plaque formation in Streptomyces spp.

    Science.gov (United States)

    Cox, K L; Baltz, R H

    1984-08-01

    Several Streptomyces species that produce restriction endonucleases were characterized for their ability to propagate 10 different broad host range bacteriophages. Each species displayed a different pattern of plaque formation. A restrictionless mutant of S. albus G allowed plaque formation by all 10 phages, whereas the wild-type strain showed plaques with only 2 phages. DNA isolated from three of the phages was analyzed for the presence of restriction sites for Streptomyces species-encoded enzymes, and a very strong correlation was established between the failure to form plaques on Streptomyces species that produced particular restriction enzymes and the presence of the corresponding restriction sites in the phage DNA. Also, the phages that lacked restriction sites in their DNA generally formed plaques on the corresponding restriction endonuclease-producing hosts at high efficiency. The DNAs from the three phages analyzed also generally contained either many or no restriction sites for the Streptomyces species-produced enzymes, suggesting a strong evolutionary trend to either eliminate all or tolerate many restriction sites. The data indicate that restriction plays a major role in host range determination for Streptomyces phages. Analysis of bacteriophage host ranges of many other uncharacterized Streptomyces hosts has identified four relatively nonrestricting hosts, at least two of which may be suitable hosts for gene cloning. The data also suggest that several restriction systems remain to be identified in the genus Streptomyces.

  8. Bacteriophage therapy against Enterobacteriaceae

    Institute of Scientific and Technical Information of China (English)

    Youqiang; Xu; Yong; Liu; Yang; Liu; Jiangsen; Pei; Su; Yao; Chi; Cheng

    2015-01-01

    The Enterobacteriaceae are a class of gram-negative facultative anaerobic rods, which can cause a variety of diseases, such as bacteremia, septic arthritis, endocarditis, osteomyelitis, lower respiratory tract infections, skin and soft-tissue infections, urinary tract infections, intra-abdominal infections and ophthalmic infections, in humans, poultry, animals and fish. Disease caused by Enterobacteriaceae cause the deaths of millions of people every year, resulting in enormous economic loss. Drug treatment is a useful and efficient way to control Enterobacteriaceae infections. However, with the abuse of antibiotics, drug resistance has been found in growing number of Enterobacteriaceae infections and, as such, there is an urgent need to find new methods of control. Bacteriophage therapy is an efficient alternative to antibiotics as it employs a different antibacterial mechanism. This paper summarizes the history of bacteriophage therapy, its bacteriallytic mechanisms, and the studies that have focused on Enterobacteriaceae and bacteriophage therapy.

  9. Hyperexpansion of RNA Bacteriophage Diversity

    Science.gov (United States)

    Krishnamurthy, Siddharth R.; Janowski, Andrew B.; Zhao, Guoyan; Barouch, Dan; Wang, David

    2016-01-01

    Bacteriophage modulation of microbial populations impacts critical processes in ocean, soil, and animal ecosystems. However, the role of bacteriophages with RNA genomes (RNA bacteriophages) in these processes is poorly understood, in part because of the limited number of known RNA bacteriophage species. Here, we identify partial genome sequences of 122 RNA bacteriophage phylotypes that are highly divergent from each other and from previously described RNA bacteriophages. These novel RNA bacteriophage sequences were present in samples collected from a range of ecological niches worldwide, including invertebrates and extreme microbial sediment, demonstrating that they are more widely distributed than previously recognized. Genomic analyses of these novel bacteriophages yielded multiple novel genome organizations. Furthermore, one RNA bacteriophage was detected in the transcriptome of a pure culture of Streptomyces avermitilis, suggesting for the first time that the known tropism of RNA bacteriophages may include gram-positive bacteria. Finally, reverse transcription PCR (RT-PCR)-based screening for two specific RNA bacteriophages in stool samples from a longitudinal cohort of macaques suggested that they are generally acutely present rather than persistent. PMID:27010970

  10. Hyperexpansion of RNA Bacteriophage Diversity.

    Directory of Open Access Journals (Sweden)

    Siddharth R Krishnamurthy

    2016-03-01

    Full Text Available Bacteriophage modulation of microbial populations impacts critical processes in ocean, soil, and animal ecosystems. However, the role of bacteriophages with RNA genomes (RNA bacteriophages in these processes is poorly understood, in part because of the limited number of known RNA bacteriophage species. Here, we identify partial genome sequences of 122 RNA bacteriophage phylotypes that are highly divergent from each other and from previously described RNA bacteriophages. These novel RNA bacteriophage sequences were present in samples collected from a range of ecological niches worldwide, including invertebrates and extreme microbial sediment, demonstrating that they are more widely distributed than previously recognized. Genomic analyses of these novel bacteriophages yielded multiple novel genome organizations. Furthermore, one RNA bacteriophage was detected in the transcriptome of a pure culture of Streptomyces avermitilis, suggesting for the first time that the known tropism of RNA bacteriophages may include gram-positive bacteria. Finally, reverse transcription PCR (RT-PCR-based screening for two specific RNA bacteriophages in stool samples from a longitudinal cohort of macaques suggested that they are generally acutely present rather than persistent.

  11. Hyperexpansion of RNA Bacteriophage Diversity.

    Science.gov (United States)

    Krishnamurthy, Siddharth R; Janowski, Andrew B; Zhao, Guoyan; Barouch, Dan; Wang, David

    2016-03-01

    Bacteriophage modulation of microbial populations impacts critical processes in ocean, soil, and animal ecosystems. However, the role of bacteriophages with RNA genomes (RNA bacteriophages) in these processes is poorly understood, in part because of the limited number of known RNA bacteriophage species. Here, we identify partial genome sequences of 122 RNA bacteriophage phylotypes that are highly divergent from each other and from previously described RNA bacteriophages. These novel RNA bacteriophage sequences were present in samples collected from a range of ecological niches worldwide, including invertebrates and extreme microbial sediment, demonstrating that they are more widely distributed than previously recognized. Genomic analyses of these novel bacteriophages yielded multiple novel genome organizations. Furthermore, one RNA bacteriophage was detected in the transcriptome of a pure culture of Streptomyces avermitilis, suggesting for the first time that the known tropism of RNA bacteriophages may include gram-positive bacteria. Finally, reverse transcription PCR (RT-PCR)-based screening for two specific RNA bacteriophages in stool samples from a longitudinal cohort of macaques suggested that they are generally acutely present rather than persistent.

  12. Crystal Structure of the Phage T4 Recombinase UvsX and Its Functional Interaction with the T4 SF2 Helicase UvsW

    Energy Technology Data Exchange (ETDEWEB)

    Gajewski, Stefan; Webb, Michael R.; Galkin, Vitold; Egelman, Edward H.; Kreuzer, Kenneth N.; White, Stephen W. (Duke); (UV); (SJCH)

    2012-07-11

    Bacteriophage T4 provides an important model system for studying the mechanism of homologous recombination. We have determined the crystal structure of the T4 UvsX recombinase, and the overall architecture and fold closely resemble those of RecA, including a highly conserved ATP binding site. Based on this new structure, we reanalyzed electron microscopy reconstructions of UvsX-DNA filaments and docked the UvsX crystal structure into two different filament forms: a compressed filament generated in the presence of ADP and an elongated filament generated in the presence of ATP and aluminum fluoride. In these reconstructions, the ATP binding site sits at the protomer interface, as in the RecA filament crystal structure. However, the environment of the ATP binding site is altered in the two filament reconstructions, suggesting that nucleotide cannot be as easily accommodated at the protomer interface of the compressed filament. Finally, we show that the phage helicase UvsW completes the UvsX-promoted strand-exchange reaction, allowing the generation of a simple nicked circular product rather than complex networks of partially exchanged substrates.

  13. Synthesis and magnetic property of T4 virus-supported gold-coated iron ternary nanocomposite

    Energy Technology Data Exchange (ETDEWEB)

    Xu Ziming; Sun Hongjing; Gao Faming, E-mail: fmgao@ysu.edu.cn; Hou Li; Li Na [Yanshan University, Key Laboratory of Applied Chemistry (China)

    2012-12-15

    Herein, we present a novel method based on the use of the symmetrical T4 bacteriophage capsid as a scaffold for preparing the gold-coated iron ternary core/shell nanostructure. Results showed that the thick gold shell was obtained to effectively protect Fe core from oxidation. Magnetic measurements showed that the nanocomposites were superparamagnetic at room temperature with a blocking temperature of about 35 K. At 3 K, its coercivity of 1142.86 Oe was larger than the existing experimental values. The magnetic property of Au/T4 was also tested, demonstrating the source of the magnetic sample arising from the Fe core only. The absorption spectrum of the Fe-Au/T4 complex was measured and compared with gold/virus. Different thickness gold shells were controlled in the synthesis by tuning the Au salt addition. On the basis of results and discussion, we further speculated the general growing mechanism of the template-supported Fe-Au process.

  14. Problem-Solving Test: RNA and Protein Synthesis in Bacteriophage-Infected "E. coli" Cells

    Science.gov (United States)

    Szeberenyi, Jozsef

    2008-01-01

    The classic experiment presented in this problem-solving test was designed to identify the template molecules of translation by analyzing the synthesis of phage proteins in "Escherichia coli" cells infected with bacteriophage T4. The work described in this test led to one of the most seminal discoveries of early molecular biology: it dealt a…

  15. Stability and in vitro DNA packaging of bacteriophages: effects of dextrans, sugars, and polyols

    Energy Technology Data Exchange (ETDEWEB)

    Serwer, P. (The Univ. of Texas Health Science Center, San Antonio); Masker, W.E.; Allen, J.L.

    1983-02-01

    Attempts were made to increase the efficiency of infectious particle formation during the in vitro assembly of bacteriophage T7 from procapsids and DNA. It was found that dextrans and some smaller, related compounds (sucrose and sorbitol) increase this efficiency by a factor of 8 to 50. Dextrans also inhibited elevated temperature-induced emptying of DNA from bacteriophages T7, P22, and T4, suggesting that the stimulation of assembly is caused, at least in part, by the stabilization of packaged DNA in capsids. The data indicated that the sugars and polyols can slow DNA emptying from bacteriophages at elevated temperature whether they permeate the bacteriophage capsid or not. In contrast, the data suggested that permeation of some particle, probably a capsid, results in inhibition of in vitro T7 assembly.

  16. Chlamydial plasmids and bacteriophages.

    Science.gov (United States)

    Pawlikowska-Warych, Małgorzata; Śliwa-Dominiak, Joanna; Deptuła, Wiesław

    2015-01-01

    Chlamydia are absolute pathogens of humans and animals; despite being rather well recognised, they are still open for discovery. One such discovery is the occurrence of extrachromosomal carriers of genetic information. In prokaryotes, such carriers include plasmids and bacteriophages, which are present only among some Chlamydia species. Plasmids were found exclusively in Chlamydia (C.) trachomatis, C. psittaci, C. pneumoniae, C. suis, C. felis, C. muridarum and C. caviae. In prokaryotic organisms, plasmids usually code for genes that facilitate survival of the bacteria in the environment (although they are not essential). In chlamydia, their role has not been definitely recognised, apart from the fact that they participate in the synthesis of glycogen and encode proteins responsible for their virulence. Furthermore, in C. suis it was evidenced that the plasmid is integrated in a genomic island and contains the tetracycline-resistance gene. Bacteriophages specific for chlamydia (chlamydiaphages) were detected only in six species: C. psittaci, C. abortus, C. felis, C. caviae C. pecorum and C. pneumoniae. These chlamydiaphages cause inhibition of the developmental cycle, and delay transformation of reticulate bodies (RBs) into elementary bodies (EBs), thus reducing the possibility of infecting other cells in time. Plasmids and bacteriophages can be used in the diagnostics of chlamydioses; although especially in the case of plasmids, they are already used for detection of chlamydial infections. In addition, bacteriophages could be used as therapeutic agents to replace antibiotics, potentially addressing the problem of increasing antibiotic-resistance among chlamydia.

  17. Resolution of branched DNA substrates by T7 endonuclease I and its inhibition.

    Science.gov (United States)

    Lu, M; Guo, Q; Studier, F W; Kallenbach, N R

    1991-02-01

    Endonuclease I is a multipurpose enzyme implicated in the breakdown of host DNA, packaging of phage DNA, and recombination during the lytic cycle of bacteriophage T7. We investigate here some aspects of the substrate requirements for its activity in resolving branched intermediates similar to Holliday junctions (Holliday, R. (1964) Genet. Res. 5, 282-304) that arise in recombination. The enzyme is able to resolve branched substrates containing very short duplex arms: 4 base pairs suffice. It cleaves 5' to the branch, with a distinct preference for the non-crossover strands in Holliday-like model junctions. Ligands that interact strongly with the branch site can inhibit the enzyme, with KI values in the 10-50 microM range. PMID:1990002

  18. Isolation and characterization of bacteriophages specific to hydrogen-sulfide-producing bacteria.

    Science.gov (United States)

    Gong, Chao; Heringa, Spencer; Singh, Randhir; Kim, Jinkyung; Jiang, Xiuping

    2013-01-01

    The objectives of this study were to isolate and characterize bacteriophages specific to hydrogen-sulfide-producing bacteria (SPB) from raw animal materials, and to develop a SPB-specific bacteriophage cocktail for rendering application. Meat, chicken offal, and feather samples collected from local supermarkets and rendering processing plants were used to isolate SPB (n = 142). Bacteriophages (n = 52) specific to SPB were isolated and purified from the above samples using 18 of those isolated SPB strains as hosts. The host ranges of bacteriophages against 5 selected SPB strains (Escherichia coli, Citrobacter freundii, and Hafnia alvei) were determined. Electron microscopy observation of 9 phages selected for the phage cocktail revealed that 6 phages belonged to the family of Siphoviridae and 3 belonged to the Myoviridae family. Restriction enzyme digestion analysis with endonuclease DraI detected 6 distinguished patterns among the 9 phages. Phage treatment prevented the growth of SPB for up to 10 h with multiplicity of infection ratios of 1, 10, 100, and 1000 in tryptic soy broth at 30 °C, and extended the lag phase of SPB growth for 2 h at 22 °C with multiplicities of infection of 10, 100, and 1000. These results suggest that the selected bacteriophage cocktail has a high potential for phage application to control SPB in raw animal materials destined for the rendering process.

  19. The phage T4 restriction endoribonuclease RegB: a cyclizing enzyme that requires two histidines to be fully active

    OpenAIRE

    Saïda, Fakhri; Uzan, Marc; Bontems, François

    2003-01-01

    The regB gene, from the bacteriophage T4, codes for an endoribonuclease that controls the expression of a number of phage early genes. The RegB protein cleaves its mRNA substrates with an almost absolute specificity in the middle of the tertranucleotide GGAG, making it a unique well-defined restriction endoribonuclease. This striking protein has no homology to any known RNase and its catalytic mechanism has never been investigated. Here, we show, using 31P nuclear magnetic resonance (NMR), th...

  20. Expression of the denV gene of coliphage T4 in UV-sensitive rad mutants of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    A plasmid containing the denV gene from bacteriophage T4, under the control of the yeast alcohol dehydrogenase I (ADC1) promoter, conferred a substantial increase in UV resistance in the UV-sensitive Saccharomyces cerevisiae mutants rad1-2 and rad3-2. The UV resistance of the denV+ yeast cells was cell cycle dependent and correlated well with the level of the denV gene product as measured by immunoblotting and by a photoreversal assay for pyrimidine dimer-DNA glycosylase activity

  1. T4 thyrotoxicosis: an independent disease or the effect of an alteration in the peripheral metabolism of T4

    International Nuclear Information System (INIS)

    Six cases of T4 thyrotoxicosis were observed in 250 patients with hyperthyroidism. In the 6 episodes, the thyrotoxicosis was associated with severe systemic illness or with the admnistration of propanolol, which blocked the peripheral convertion of T4 to T3. These data indicate that T4 thyrotoxicosis reflects an alteration in the peripheral metabolism of T4 produced by systemic illness or drugs. (M.A.C.)

  2. Structure determination and biochemical characterization of a putative HNH endonuclease from Geobacter metallireducens GS-15.

    Directory of Open Access Journals (Sweden)

    Shuang-yong Xu

    Full Text Available The crystal structure of a putative HNH endonuclease, Gmet_0936 protein from Geobacter metallireducens GS-15, has been determined at 2.6 Å resolution using single-wavelength anomalous dispersion method. The structure contains a two-stranded anti-parallel β-sheet that are surrounded by two helices on each face, and reveals a Zn ion bound in each monomer, coordinated by residues Cys38, Cys41, Cys73, and Cys76, which likely plays an important structural role in stabilizing the overall conformation. Structural homologs of Gmet_0936 include Hpy99I endonuclease, phage T4 endonuclease VII, and other HNH endonucleases, with these enzymes sharing 15-20% amino acid sequence identity. An overlay of Gmet_0936 and Hpy99I structures shows that most of the secondary structure elements, catalytic residues as well as the zinc binding site (zinc ribbon are conserved. However, Gmet_0936 lacks the N-terminal domain of Hpy99I, which mediates DNA binding as well as dimerization. Purified Gmet_0936 forms dimers in solution and a dimer of the protein is observed in the crystal, but with a different mode of dimerization as compared to Hpy99I. Gmet_0936 and its N77H variant show a weak DNA binding activity in a DNA mobility shift assay and a weak Mn²⁺-dependent nicking activity on supercoiled plasmids in low pH buffers. The preferred substrate appears to be acid and heat-treated DNA with AP sites, suggesting Gmet_0936 may be a DNA repair enzyme.

  3. Effect of HZE particles and space hadrons on bacteriophages

    Energy Technology Data Exchange (ETDEWEB)

    Iurov, S.S.; Akoev, I.G.; Leonteva, G.A.

    1983-01-01

    The effects of particle radiation of the type encountered in space flight on bacteriophages are investigated. Survival and mutagenesis were followed in dry film cultures or liquid suspensions of T4Br(+) bacteriophage exposed to high-energy (HZE) particles during orbital flight, to alpha particles and accelerator-generated hardrons in the laboratory, and to high-energy cosmic rays at mountain altitudes. The HZE particles and high-energy hadrons are found to have a greater relative biological efficiency than standard gamma radiation, while exhibiting a highly inhomogeneous spatial structure in the observed biological and genetic effects. In addition, the genetic lesions observed are specific to the type of radiation exposure, consisting primarily of deletions and multiple lesions of low revertability, with mode of action depending on the linear energy transfer. 18 references.

  4. Effect of HZE particles and space hadrons on bacteriophages

    Science.gov (United States)

    Yurov, S. S.; Akoev, I. G.; Leont'eva, G. A.

    The effect of high energy (HZE) particles and high energy hadrons on T4Br+ bacteriophage was analyzed. The experiments were done in orbital flight, on high mountains, on an accelerator, and with an alpha particle source. We studied the survival rate of the bacteriophage, the mutation frequency, the mutation spectrum and the revertability under the action of chemical mutagens with a known mechanism of action on DNA. It was found that the biological efficiency of HZE particles and high energy hadrons is greater than that of γ radiation. The spectra of mutations produced by these mutations and the mechanisms of their action are also different. These effects were local, because of the mode of interaction of the radiant energy with biological objects, and depended on the linear energy transfer (LET). The modes have now been experimentally defined.

  5. Evidence that the heterogeneity of a T4 population is the result of heritable traits.

    Directory of Open Access Journals (Sweden)

    Zachary J Storms

    Full Text Available Many bacteriophage populations display heterogeneity in their adsorption characteristics; a portion of the phage population remains free in solution throughout adsorption experiments (residual fraction. This residual fraction generally constitutes a minority of phages that exhibit significantly slower adsorption kinetics than the main phage stock (main fraction. While this phenomenon is likely the result of evolutionary driving forces, the present study demonstrates that the residual fraction is not always the result of phenotypic variations within a single genotype, as is generally thought. Experiments with phage T4 showed that two subgroups with distinct adsorption traits that were passed on to their progeny could be isolated from the original phage stock. Sequencing of genes involved in adsorption revealed two point mutations in gene 37 of residual fraction isolates, which resulted in modifications to the long tail-fiber, the organelle of attachment and host cell recognition. Adsorption studies consistently showed that T4 phage stocks amplified from residual fraction isolates had significantly lower adsorption efficiencies than those amplified from main fractions. The conducted experiments provide convincing evidence that the observed heterogeneity in T4 adsorption behavior is the result of conserved mutations to the phage genome and is not exclusively the result of phenotypic variations within the population. While it is believed high mutation rates exist to hasten phage adaptation, this study shows that this bet hedging strategy can also, in the short term, inadvertently handicap the phage's adsorption capabilities to a given host under normal infection conditions, resulting in the residual fraction observed in adsorption experiments.

  6. Some aspects of the mechanism of bacteriophage function. Final progress report. [Mechanisms of inactivation of bacteriophages by ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Freifelder, D.

    1977-06-12

    Data are summarized from a ten-year study on the radiobiology of phages. The results showed that: phages are inactivated principally by damage to DNA; DNA damage is of two types, base damage and double-strand breakage; double-strand breakage may be lethal because of interruption within a gene, however in phage systems the damage is more fundamental in that only a single DNA fragment is injected into the host; E. coli phage T4 is relatively resistant to inactivation by x-rays; and the rate of production of strand breaks and base damage is nearly the same in bacteriophage and bacteria.

  7. Bacteriophages of methanotrophic bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Tyutikow, F.M. (All-Union Research Inst. for Genetics and Selection of Industrial Microorganisms, Moscow, USSR); Bespalova, I.A.; Rebentish, B.A.; Aleksandrushkina, N.N.; Krivisky, A.S.

    1980-10-01

    Bacteriophages of methanotrophic bacteria have been found in 16 out of 88 studied samples (underground waters, pond water, soil, gas and oil installation waters, fermentor cultural fluids, bacterial paste, and rumen of cattle) taken in different geographic zones of the Soviet Union. Altogether, 23 phage strains were isolated. By fine structure, the phages were divided into two types (with very short or long noncontractile tails); by host range and serological properties, they fell into three types. All phages had guanine- and cytosine-rich double-stranded deoxyribonucleic acid consisting of common nitrogen bases. By all of the above-mentioned properties, all phages within each of the groups were completely identical to one another, but differed from phages of other groups.

  8. Genetically modified bacteriophages.

    Science.gov (United States)

    Sagona, Antonia P; Grigonyte, Aurelija M; MacDonald, Paul R; Jaramillo, Alfonso

    2016-04-18

    Phages or bacteriophages, viruses that infect and replicate inside bacteria, are the most abundant microorganisms on earth. The realization that antibiotic resistance poses a substantial risk to the world's health and global economy is revitalizing phage therapy as a potential solution. The increasing ease by which phage genomes can be modified, owing to the influx of new technologies, has led to an expansion of their natural capabilities, and a reduced dependence on phage isolation from environmental sources. This review will discuss the way synthetic biology has accelerated the construction of genetically modified phages and will describe the wide range of their applications. It will further provide insight into the societal and economic benefits that derive from the use of recombinant phages in various sectors, from health to biodetection, biocontrol and the food industry.

  9. Genetically modified bacteriophages.

    Science.gov (United States)

    Sagona, Antonia P; Grigonyte, Aurelija M; MacDonald, Paul R; Jaramillo, Alfonso

    2016-04-18

    Phages or bacteriophages, viruses that infect and replicate inside bacteria, are the most abundant microorganisms on earth. The realization that antibiotic resistance poses a substantial risk to the world's health and global economy is revitalizing phage therapy as a potential solution. The increasing ease by which phage genomes can be modified, owing to the influx of new technologies, has led to an expansion of their natural capabilities, and a reduced dependence on phage isolation from environmental sources. This review will discuss the way synthetic biology has accelerated the construction of genetically modified phages and will describe the wide range of their applications. It will further provide insight into the societal and economic benefits that derive from the use of recombinant phages in various sectors, from health to biodetection, biocontrol and the food industry. PMID:26906932

  10. Brucella T4SS: the VIP pass inside host cells.

    Science.gov (United States)

    Lacerda, Thais Lourdes Santos; Salcedo, Suzana Pinto; Gorvel, Jean-Pierre

    2013-02-01

    For many Gram-negative bacteria, like Brucella, the type IV secretion system (T4SS) has a critical role in bacterial virulence. In Brucella, the VirB T4SS permits the injection of bacterial effectors inside host cells, leading to subversion of signaling pathways and favoring bacterial growth and pathogenesis. The virB operon promoter is tightly regulated by a combination of transcriptional activators and repressors that are expressed according to the environmental conditions encountered by Brucella. Recent advances have shed light on the Brucella T4SS regulatory mechanisms and also its substrates. Characterization of the targets and functions of these translocated effectors is underway and will help understand the role of the T4SS in the establishment of a replication niche inside host cells.

  11. [Inactivation of T4 phage in water environment using proteinase].

    Science.gov (United States)

    Lü, Wen-zhou; Yang, Qing-xiang; Zhang, Yu; Yang, Min; Zhu, Chun-fang

    2004-09-01

    The inactivation effectiveness of proteinase to viruses was investigated by using T4 phage as a model virus. The results showed that the inactivation effectiveness of proteinase to T4 phage was obvious. In the optimum conditions and 67.5 u/mL concentration, the inactivation rate of proteinase K to T4 phage in sterilized water and in sewage achieved 99.4% and 49.4% respectively in an hour, and achieved >99.9% and 81.1% in three hours. The inactivation rate of the industrial proteinase 1398 to T4 phage in sterilized water achieved 74.4% in an hour. The effects of pH and temperature on the inactivation effectiveness was not evident.

  12. MmoSTI restriction endonuclease, isolated from Morganella morganii infecting a tropical moth, Actias selene, cleaving 5'-|CCNGG-3' sequences.

    Science.gov (United States)

    Skowron, Marta A; Zebrowska, Joanna; Wegrzyn, Grzegorz; Skowron, Piotr M

    2016-02-01

    A type II restriction endonuclease, MmoSTI, from the pathogenic bacterium Morganella morganii infecting a tropical moth, Actias selene, has been detected and biochemically characterized, as a potential etiological differentiation factor. The described REase recognizes interrupted palindromes, i.e., 5'-CCNGG-3' sequences and cleaves DNA leaving 5-nucleotide (nt) long, single-stranded (ss), 5'-cohesive ends, which was determined by three complementary methods: (i) cleavage of custom and standard DNA substrates, (ii) run-off sequencing of cleavage products, and (iii) shotgun cloning and sequencing of bacteriophage lambda (λ) DNA digested with MmoSTI. MmoSTI, the first 5'-CCNGG-3' REase characterized from M. morganii, is a neoschizomer of ScrFI, which cleaves DNA leaving 1-nt long, ss, 5'-cohesive ends. It is a high-frequency cutter and can be isolated from easily cultured bacteria, thus it can potentially serve as a tool for DNA manipulations.

  13. Design and optimization of radioimmunoassay for T4

    International Nuclear Information System (INIS)

    Design and optimization of radioimmunoassay for T4. The results of a radioimmunoassay are influenced by the equipment, the laboratory personnel, the quality of the reagents used and especially by the assay design. A good essay design should yield analytical results with high precision and sensitivity within the desired concentration range and can be performed rapidly and easily at low cost. In order to optimize the assay design for T4 the following parameters were studied: amount and dilution of reagents, incubation temperature and length of time prior to separation, reagent volume and sequence of reagent addition. Optimal results for T4 assay were achieved using 25 ul of T4 standard (or samples), 100 ul of 125I labelled T4 (30,000 cpm) and 100 ul of T4 antiserum (1:200 dilution), incubated at room temperature for 1 hour or at 370C for 45 minutes. The following results were obtained under these conditions: a non specific binding value of (6,8+-0,7)%, a maximum binding (B/T) of (67,8+-2,2)%, a value of (5,0+-0,7)ug/dl for ED50. Results for low medium, and high quality control samples were (1,8+-0,1)ug/dl, (5,6+-0,3)ug/dl, and (14,9+-0,6)ug/dl respectively. (author). 12 figs, 6 refs

  14. Yeast structural gene (APN1) for the major apurinic endonuclease: homology to Escherichia coli endonuclease IV.

    OpenAIRE

    Popoff, S C; Spira, A I; Johnson, A. W.; Demple, B

    1990-01-01

    DNA damage generated by oxygen radicals includes base-free apurinic/apyrimidinic (AP) sites and strand breaks that bear deoxyribose fragments. The yeast Saccharomyces cerevisiae repairs such DNA lesions by using a single major enzyme. We have cloned the yeast structural gene (APN1) encoding this AP endonuclease/3'-repair diesterase by immunological screening of a yeast genomic DNA expression library in lambda gt11. Gene disruption experiments confirm that the Apn1 protein accounts for greater...

  15. Recombinant expression and purification of T4 phage Hoc, Soc, gp23, gp24 proteins in native conformations with stability studies.

    Directory of Open Access Journals (Sweden)

    Paulina Miernikiewicz

    Full Text Available Understanding the biological activity of bacteriophage particles is essential for rational design of bacteriophages with defined pharmacokinetic parameters and to identify the mechanisms of immunobiological activities demonstrated for some bacteriophages. This work requires highly purified preparations of the individual phage structural proteins, possessing native conformation that is essential for their reactivity, and free of incompatible biologically active substances such as bacterial lipopolysaccharide (LPS. In this study we describe expression in E. coli and purification of four proteins forming the surface of the bacteriophage T4 head: gp23, gp24, gphoc and gpsoc. We optimized protein expression using a set of chaperones for effective production of soluble proteins in their native conformations. The assistance of chaperones was critical for production of soluble gp23 (chaperone gp31 of T4 phage and of gpsoc (chaperone TF of E. coli. Phage head proteins were purified in native conditions by affinity chromatography and size-exclusion chromatography. Two-step LPS removal allowed immunological purity grade with the average endotoxin activity less than 1 unit per ml of protein preparation. The secondary structure and stability of the proteins were studied using circular dichroism (CD spectrometry, which confirmed that highly purified proteins preserve their native conformations. In increasing concentration of a denaturant (guanidine hydrochloride, protein stability was proved to increase as follows: gpsoc, gp23, gphoc. The denaturation profile of gp24 protein showed independent domain unfolding with the most stable larger domain. The native purified recombinant phage proteins obtained in this work were shown to be suitable for immunological experiments in vivo and in vitro.

  16. The Caulobacter crescentus transducing phage Cr30 is a unique member of the T4-like family of myophages.

    Science.gov (United States)

    Ely, Bert; Gibbs, Whitney; Diez, Simon; Ash, Kurt

    2015-06-01

    Bacteriophage Cr30 has proven useful for the transduction of Caulobacter crescentus. Nucleotide sequencing of Cr30 DNA revealed that the Cr30 genome consists of 155,997 bp of DNA that codes for 287 proteins and five tRNAs. In contrast to the 67 % GC content of the host genome, the GC content of the Cr30 genome is only 38 %. This lower GC content causes both the codon usage pattern and the amino acid composition of the Cr30 proteins to be quite different from those of the host bacteria. As a consequence, the Cr30 mRNAs probably are translated at a rate that is slower than the normal rate for host mRNAs. A phylogenetic comparison of the genome indicates that Cr30 is a member of the T4-like family that is most closely related to a new group of T-like phages exemplified by фM12.

  17. Developing a programmed restriction endonuclease for highly specific DNA cleavage

    OpenAIRE

    Eisenschmidt, Kristin; Lanio, Thomas; Simoncsits, András; Jeltsch, Albert; Pingoud, Vera; Wende, Wolfgang; Pingoud, Alfred

    2005-01-01

    Specific cleavage of large DNA molecules at few sites, necessary for the analysis of genomic DNA or for targeting individual genes in complex genomes, requires endonucleases of extremely high specificity. Restriction endonucleases (REase) that recognize DNA sequences of 4–8 bp are not sufficiently specific for this purpose. In principle, the specificity of REases can be extended by fusion to sequence recognition modules, e.g. specific DNA-binding domains or triple-helix forming oligonucleotid...

  18. Virulence reduction in Bacteriophage resistant bacteria

    Directory of Open Access Journals (Sweden)

    Marcela eLeón

    2015-04-01

    Full Text Available Bacteriophages can influence the abundance, diversity and evolution of bacterial communities. Several bacteriophages have been reported to add virulence factors to their host and to increase bacterial virulence. However, lytic bacteriophages can also exert a selective pressure allowing the proliferation of strains with reduced virulence. This reduction can be explained because bacteriophages use structures present on the bacterial surface as receptors, which can be virulence factors in different bacterial species. Therefore, strains with modifications in these receptors will be resistant to bacteriophage infection and may also exhibit reduced virulence. This mini-review summarizes the reports on bacteriophage-resistant strains with reductions in virulence, and it discusses the potential consequences in phage therapy and in the use of bacteriophages to select attenuated strains for vaccines.

  19. Characterization of a novel Morganella morganii bacteriophage FSP1 isolated from river water.

    Science.gov (United States)

    Yamaki, Shogo; Omachi, Takuo; Kawai, Yuji; Yamazaki, Koji

    2014-10-01

    Morganella morganii has been identified as a causative agent of opportunistic infections and histamine poisoning. Bacteriophage is a virus and has recently been considered an alternative agent to antibiotics for the control of bacteria that have developed antibiotic resistance. In this study, a novel M. morganii bacteriophage isolated from river water was characterized. The isolated phage, termed FSP1, was purified by polyethylene glycol precipitation followed by cesium chloride density-gradient centrifugation. FSP1 has infectivity against only M. morganii and was identified as a Myoviridae bacteriophage through morphological analysis with transmission electron microscopy. According to the one-step growth curve, the FSP1 latent period, eclipse period, and burst size were 30, 20 min, and 42 PFU infected cell(-1) , respectively. The genome size of FSP1 was estimated to be c. 45.6-49.4 kb by restriction endonuclease analyses. Moreover, challenge testing against M. morganii in vitro revealed that FSP1 had high lytic activity and that the viable cell count of M. morganii was reduced by 6.12 log CFU mL(-1) after inoculation with FSP1 at a multiplicity of infection (MOI) = 10. These results suggested that FSP1 could be used as a biocontrol agent against M. morganii for treatment of infectious disease treatment or food decontamination.

  20. RecA-dependent programmable endonuclease Ref cleaves DNA in two distinct steps.

    Science.gov (United States)

    Ronayne, Erin A; Cox, Michael M

    2014-04-01

    The bacteriophage P1 recombination enhancement function (Ref) protein is a RecA-dependent programmable endonuclease. Ref targets displacement loops formed when an oligonucleotide is bound by a RecA filament and invades homologous double-stranded DNA sequences. Mechanistic details of this reaction have been explored, revealing that (i) Ref is nickase, cleaving the two target strands of a displacement loop sequentially, (ii) the two strands are cleaved in a prescribed order, with the paired strand cut first and (iii) the two cleavage events have different requirements. Cutting the paired strand is rapid, does not require RecA-mediated ATP hydrolysis and is promoted even by Ref active site variant H153A. The displaced strand is cleaved much more slowly, requires RecA-mediated ATP hydrolysis and does not occur with Ref H153A. The two cleavage events are also affected differently by solution conditions. We postulate that the second cleavage (displaced strand) is limited by some activity of RecA protein.

  1. P1 Ref Endonuclease: A Molecular Mechanism for Phage-Enhanced Antibiotic Lethality.

    Science.gov (United States)

    Ronayne, Erin A; Wan, Y C Serena; Boudreau, Beth A; Landick, Robert; Cox, Michael M

    2016-01-01

    Ref is an HNH superfamily endonuclease that only cleaves DNA to which RecA protein is bound. The enigmatic physiological function of this unusual enzyme is defined here. Lysogenization by bacteriophage P1 renders E. coli more sensitive to the DNA-damaging antibiotic ciprofloxacin, an example of a phenomenon termed phage-antibiotic synergy (PAS). The complementary effect of phage P1 is uniquely traced to the P1-encoded gene ref. Ref is a P1 function that amplifies the lytic cycle under conditions when the bacterial SOS response is induced due to DNA damage. The effect of Ref is multifaceted. DNA binding by Ref interferes with normal DNA metabolism, and the nuclease activity of Ref enhances genome degradation. Ref also inhibits cell division independently of the SOS response. Ref gene expression is toxic to E. coli in the absence of other P1 functions, both alone and in combination with antibiotics. The RecA proteins of human pathogens Neisseria gonorrhoeae and Staphylococcus aureus serve as cofactors for Ref-mediated DNA cleavage. Ref is especially toxic during the bacterial SOS response and the limited growth of stationary phase cultures, targeting aspects of bacterial physiology that are closely associated with the development of bacterial pathogen persistence.

  2. P1 Ref Endonuclease: A Molecular Mechanism for Phage-Enhanced Antibiotic Lethality.

    Science.gov (United States)

    Ronayne, Erin A; Wan, Y C Serena; Boudreau, Beth A; Landick, Robert; Cox, Michael M

    2016-01-01

    Ref is an HNH superfamily endonuclease that only cleaves DNA to which RecA protein is bound. The enigmatic physiological function of this unusual enzyme is defined here. Lysogenization by bacteriophage P1 renders E. coli more sensitive to the DNA-damaging antibiotic ciprofloxacin, an example of a phenomenon termed phage-antibiotic synergy (PAS). The complementary effect of phage P1 is uniquely traced to the P1-encoded gene ref. Ref is a P1 function that amplifies the lytic cycle under conditions when the bacterial SOS response is induced due to DNA damage. The effect of Ref is multifaceted. DNA binding by Ref interferes with normal DNA metabolism, and the nuclease activity of Ref enhances genome degradation. Ref also inhibits cell division independently of the SOS response. Ref gene expression is toxic to E. coli in the absence of other P1 functions, both alone and in combination with antibiotics. The RecA proteins of human pathogens Neisseria gonorrhoeae and Staphylococcus aureus serve as cofactors for Ref-mediated DNA cleavage. Ref is especially toxic during the bacterial SOS response and the limited growth of stationary phase cultures, targeting aspects of bacterial physiology that are closely associated with the development of bacterial pathogen persistence. PMID:26765929

  3. P1 Ref Endonuclease: A Molecular Mechanism for Phage-Enhanced Antibiotic Lethality.

    Directory of Open Access Journals (Sweden)

    Erin A Ronayne

    2016-01-01

    Full Text Available Ref is an HNH superfamily endonuclease that only cleaves DNA to which RecA protein is bound. The enigmatic physiological function of this unusual enzyme is defined here. Lysogenization by bacteriophage P1 renders E. coli more sensitive to the DNA-damaging antibiotic ciprofloxacin, an example of a phenomenon termed phage-antibiotic synergy (PAS. The complementary effect of phage P1 is uniquely traced to the P1-encoded gene ref. Ref is a P1 function that amplifies the lytic cycle under conditions when the bacterial SOS response is induced due to DNA damage. The effect of Ref is multifaceted. DNA binding by Ref interferes with normal DNA metabolism, and the nuclease activity of Ref enhances genome degradation. Ref also inhibits cell division independently of the SOS response. Ref gene expression is toxic to E. coli in the absence of other P1 functions, both alone and in combination with antibiotics. The RecA proteins of human pathogens Neisseria gonorrhoeae and Staphylococcus aureus serve as cofactors for Ref-mediated DNA cleavage. Ref is especially toxic during the bacterial SOS response and the limited growth of stationary phase cultures, targeting aspects of bacterial physiology that are closely associated with the development of bacterial pathogen persistence.

  4. Restriction endonucleases digesting DNA in PCR buffer

    Institute of Scientific and Technical Information of China (English)

    LIU Xue-dong; ZHENG Dong; ZHOU Yan-na; MAO Wei-wei; MA Jian-zhang

    2005-01-01

    Six commonly used restriction endonucleases (Res) (Acc I, Ban II, EcoR I, Hind III, Sac I, Sca I) were tested for their ability to directly digest DNA completely in the Polymerase Chain Reaction (PCR) buffers. The results showed that: with the requirement for additional magnesium supplemented as activator, Res, except EcoR I appeared star activity, completely digested unmethylated lambda DNA after overnight incubation in PCR buffer and functioned as equally well as in recommended Restriction Enzyme Buffer provided with each enzyme; all Res tested completely digested PCR products in PCR buffer, it implied digestion of PCR products may often be performed directly in the PCR tube without the requirement for any precipitation or purification steps; and the concentration of MgCl2 from 2.5 mmol·L-1 to 10 mmol·L-1 did not significantly affect activity of Res in PCR buffer. This simplified method for RE digestion of PCR products could have applications in restriction fragment length polymorphism (RFLP) analysis and single-stranded conformational polymorphism (SSCP) analysis of large PCR products. However, usage of this procedure for cloning applications needs further data.

  5. Bacteriophages: back to the future

    Science.gov (United States)

    A Listeria monocytogenes-specific bacteriophage cocktail (ListShield™) was evaluated for its activity against a nalidixic acid-resistant L. monocytogenes (Lm-NalR) isolate on fresh-cut spinach stored under modified atmosphere packaging (MAP) at various temperatures. Pieces (~2x2 cm2) of fresh spinac...

  6. Bacteriophage endolysins as novel antimicrobials

    Science.gov (United States)

    Endolysins are enzymes used by bacteriophages at the end of their replication cycle to degrade the peptidoglycan of the bacterial host from within, resulting in cell lysis and release of progeny virions. Due to the absence of an outer membrane in the Gram-positive bacterial cell wall, endolysins can...

  7. Complete Genome Sequences of Five Bacteriophages That Infect Rhodobacter capsulatus.

    Science.gov (United States)

    Bollivar, David W; Bernardoni, Brooke; Bockman, Matthew R; Miller, Brenda M; Russell, Daniel A; Delesalle, Veronique A; Krukonis, Gregory P; Hatfull, Graham F; Cross, Madeline R; Szewczyk, Marlena M; Eppurath, Atul

    2016-05-26

    Five bacteriophages that infect the Rhodobacter capsulatus strain YW1 were isolated from stream water near Bloomington, Illinois, USA. Two distinct genome types are represented in the newly isolated bacteriophages. These genomes are different from other bacteriophage genomes previously described.

  8. Oral Application of T4 Phage Induces Weak Antibody Production in the Gut and in the Blood

    Directory of Open Access Journals (Sweden)

    Joanna Majewska

    2015-08-01

    Full Text Available A specific humoral response to bacteriophages may follow phage application for medical purposes, and it may further determine the success or failure of the approach itself. We present a long-term study of antibody induction in mice by T4 phage applied per os: 100 days of phage treatment followed by 112 days without the phage, and subsequent second application of phage up to day 240. Serum and gut antibodies (IgM, IgG, secretory IgA were analyzed in relation to microbiological status of the animals. T4 phage applied orally induced anti-phage antibodies when the exposure was long enough (IgG day 36, IgA day 79; the effect was related to high dosage. Termination of phage treatment resulted in a decrease of IgA again to insignificant levels. Second administration of phage induces secretory IgA sooner than that induced by the first administrations. Increased IgA level antagonized gut transit of active phage. Phage resistant E. coli dominated gut flora very late, on day 92. Thus, the immunological response emerges as a major factor determining phage survival in the gut. Phage proteins Hoc and gp12 were identified as highly immunogenic. A low response to exemplary foreign antigens (from Ebola virus presented on Hoc was observed, which suggests that phage platforms can be used in oral vaccine design.

  9. Evaluation of virus removal in MBR using coliphages T4

    Institute of Scientific and Technical Information of China (English)

    ZHENG Xiang; Lü Wenzhou; YANG Min; LIU Junxin

    2005-01-01

    A membrane bioreactor (MBR) with gravity drain was tested for domestic wastewater for 65 days. Results showed that the effluent quality was excellent, and met with the reuse water standard of China (GB/T 18920-2002). Virus removal in the membrane separation process was investigated by employing coliphages T4 as a tracer. Two microfiltration membrane modules, with pore sizes of 0.22 and 0.1 μm, were used to investigate their effects on virus rejection at the transmembrane pressure of 8.5 kPa. It was found that 0.1 μm membrane had complete rejection of virus, and 0.22 μm membrane had significant rejection of virus. In the long- term operation of this MBR, no significant difference was observed between both pore sizes because the virus concentrations of the effluent in both cases were in the same order. Effluent virus concentration at steady state of MBR running was less than 2 PFU/mL. The removal ratios of coliphage T4 in MF processes were more than 105.5. The membrane surface deposits played an important role in the rejection of virus. The formation of cake clay on the membrane surface was the main cause of high rejection of coliphage T4 with MF of 0.22 μm.

  10. The actions of restriction endonucleases on lampbrush chromosomes.

    Science.gov (United States)

    Gould, D C; Callan, H G; Thomas, C A

    1976-07-01

    Lampbrush chromosomes from oocytes of Notophthalmus viridescens were dispersed in media containing restriction endonucleases isolated from Haemophilus and E. coli. These endonucleases cleave duplex DNAs at specific palindromic sequences of nucleotides, and several sensitive sites occur per micron of DNA. The overwhelming majority of the lateral loops of lampbrush chromosomes are extensively fragmented by these endonucleases, but an occasional pair of loops is refractory. A notable example of loops showing this refractory property are the giant loops on chromosome II in the presence of Hae. These loops, whose DNA-containing axes are several hundred micra long, are sensitive to other nucleases such as EcoB, endonuclease I and pancreatic DNase I; their refractory behavior towards Hae therefore indicates that the sequence sensitive to this particular endonuclease is systematically absent. This anomalous property can be comprehended if it be assumed that the axial DNA of the giant loops consists of tandem repeats of a sequence which happens not to include the sensitive site. PMID:987047

  11. UV-induced endonuclease III-sensitive sites at the mating type loci in Saccharomyces cerevisiae are repaired by nucleotide excision repair: RAD7 and RAD16 are not required for their removal from HML alpha.

    Science.gov (United States)

    Reed, S H; Boiteux, S; Waters, R

    1996-03-01

    Ultraviolet irradiation of DNA induces cyclobutane pyrimidine dimers (CPDs) 6-4'-(pyrimidine 2'-one) pyrimidines and pyrimidine hydrates. The dimer is the major photoproduct, and is specifically recognized by endonuclease V of phage T4. Pyrimidine hydrates represent a small fraction of the total photoproducts, and are substrates for endonuclease III of Escherichia coli. We used these enzymes to follow the fate of their substrates in the mating type loci of Saccharomyces cerevisiae. In a RAD strain, CPSs in the transcriptionally active MAT alpha locus are preferentially repaired relative to the inactive HML alpha locus, whilst repair of endonuclease III-sensitive sites is not preferential. The rad1, 2, 3 and 4 mutants, which lack factors that are essential for the incision step of nucleotide excision repair (NER), repair neither CPDs nor endonuclease III-sensitive sites, clearly showing that these lesions are repaired by by NER pathway. Previously it had been shown that the products of the RAD7 and RAD16 genes are required for the NER of CPDs from the HML alpha locus. We show that, in the same locus, these gene products are not needed for removal of endonuclease III-sensitive sites by the same mechanism. This indicates that the components required for NER differ depending on either the type of lesion encountered or on the specific location of the lesion within the genome.

  12. Removal of endotoxins from bacteriophage preparations by extraction with organic solvents.

    Directory of Open Access Journals (Sweden)

    Bożena Szermer-Olearnik

    Full Text Available Lipopolysaccharide (LPS, endotoxin, pyrogen constitutes a very troubling contaminant of crude phage lysates produced in Gram-negative bacteria. Toxicity of LPS depends on the strong innate immunity response including the cytokines. Therefore, its removal is important for bacteriophage applications. In this paper, we present a procedure for extractive removal of endotoxin from bacteriophage preparations with water immiscible solvents (1-octanol or 1-butanol. During extraction most of the phage lytic activity is retained in the aqueous phase, while endotoxin accumulates in the organic solvent. The levels of endotoxin (expressed as endotoxin units, EU in the aqueous bacteriophage-containing fraction determined by limulus amebocyte lysate or EndoLISA assay were exceptionally low. While the initial endotoxin levels in the crude phage lysates ranged between 10(3 and 10(5 EU/ml the average level after organic extraction remaining in the aqueous fraction was 5.3 EU/ml. These values when related to phage titers decreased from 10(3-10(5 EU/10(9 PFU (plaque forming units down to an average of 2.8 EU/10(9 PFU. The purification procedure is scalable, efficient and applicable to all the bacteriophages tested: T4, HAP1 (E. coli and F8 (P. aeruginosa.

  13. Sewage bacteriophage inactivation by cationic porphyrins: influence of light parameters.

    Science.gov (United States)

    Costa, Liliana; Carvalho, Carla M B; Faustino, Maria A F; Neves, Maria G P M S; Tomé, João P C; Tomé, Augusto C; Cavaleiro, José A S; Cunha, Angela; Almeida, Adelaide

    2010-08-01

    Photodynamic therapy has been used to inactivate microorganisms through the use of targeted photosensitizers. Although the photoinactivation of microorganisms has already been studied under different conditions, a systematic evaluation of irradiation characteristics is still limited. The goal of this study was to test how the light dose, fluence rate and irradiation source affect the viral photoinactivation of a T4-like sewage bacteriophage. The experiments were carried out using white PAR light delivered by fluorescent PAR lamps (40 W m(-2)), sun light (600 W m(-2)) and an halogen lamp (40-1690 W m(-2)). Phage suspensions and two cationic photosensitizers (Tetra-Py(+)-Me, Tri-Py(+)-Me-PF) at concentrations of 0.5, 1.0 and 5.0 microM were used. The results showed that the efficacy of the bacteriophage photoinactivation is correlated not only with the sensitizer and its concentration but also with the light source, energy dose and fluence rate applied. Both photosensitizers at 5.0 microM were able to inactivate the T4-like phage to the limit of detection for each light source and fluence rate. However, depending of the light parameters, different irradiation times are required. The efficiency of photoinactivation is dependent on the spectral emission distribution of the light sources used. Considering the same light source and a fixed light dose applied at different fluence rates, phage inactivation was significantly higher when low fluence rates were used. In this way, the light source, fluence rate and total light dose play an important role in the effectiveness of the antimicrobial photodynamic therapy and should always be considered when establishing an optimal antimicrobial protocol. PMID:20563346

  14. T4 genes in the marine ecosystem: studies of the T4-like cyanophages and their role in marine ecology

    Directory of Open Access Journals (Sweden)

    Millard Andrew D

    2010-10-01

    Full Text Available Abstract From genomic sequencing it has become apparent that the marine cyanomyoviruses capable of infecting strains of unicellular cyanobacteria assigned to the genera Synechococcus and Prochlorococcus are not only morphologically similar to T4, but are also genetically related, typically sharing some 40-48 genes. The large majority of these common genes are the same in all marine cyanomyoviruses so far characterized. Given the fundamental physiological differences between marine unicellular cyanobacteria and heterotrophic hosts of T4-like phages it is not surprising that the study of cyanomyoviruses has revealed novel and fascinating facets of the phage-host relationship. One of the most interesting features of the marine cyanomyoviruses is their possession of a number of genes that are clearly of host origin such as those involved in photosynthesis, like the psbA gene that encodes a core component of the photosystem II reaction centre. Other host-derived genes encode enzymes involved in carbon metabolism, phosphate acquisition and ppGpp metabolism. The impact of these host-derived genes on phage fitness has still largely to be assessed and represents one of the most important topics in the study of this group of T4-like phages in the laboratory. However, these phages are also of considerable environmental significance by virtue of their impact on key contributors to oceanic primary production and the true extent and nature of this impact has still to be accurately assessed.

  15. Probing the folded state and mechanical unfolding pathways of T4 lysozyme using all-atom and coarse-grained molecular simulation

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Wenjun, E-mail: wjzheng@buffalo.edu; Glenn, Paul [Department of Physics, University at Buffalo, Buffalo, New York 14260 (United States)

    2015-01-21

    The Bacteriophage T4 Lysozyme (T4L) is a prototype modular protein comprised of an N-terminal and a C-domain domain, which was extensively studied to understand the folding/unfolding mechanism of modular proteins. To offer detailed structural and dynamic insights to the folded-state stability and the mechanical unfolding behaviors of T4L, we have performed extensive equilibrium and steered molecular dynamics simulations of both the wild-type (WT) and a circular permutation (CP) variant of T4L using all-atom and coarse-grained force fields. Our all-atom and coarse-grained simulations of the folded state have consistently found greater stability of the C-domain than the N-domain in isolation, which is in agreement with past thermostatic studies of T4L. While the all-atom simulation cannot fully explain the mechanical unfolding behaviors of the WT and the CP variant observed in an optical tweezers study, the coarse-grained simulations based on the Go model or a modified elastic network model (mENM) are in qualitative agreement with the experimental finding of greater unfolding cooperativity in the WT than the CP variant. Interestingly, the two coarse-grained models predict different structural mechanisms for the observed change in cooperativity between the WT and the CP variant—while the Go model predicts minor modification of the unfolding pathways by circular permutation (i.e., preserving the general order that the N-domain unfolds before the C-domain), the mENM predicts a dramatic change in unfolding pathways (e.g., different order of N/C-domain unfolding in the WT and the CP variant). Based on our simulations, we have analyzed the limitations of and the key differences between these models and offered testable predictions for future experiments to resolve the structural mechanism for cooperative folding/unfolding of T4L.

  16. Bacteriophage Procurement for Therapeutic Purposes.

    Science.gov (United States)

    Weber-Dąbrowska, Beata; Jończyk-Matysiak, Ewa; Żaczek, Maciej; Łobocka, Małgorzata; Łusiak-Szelachowska, Marzanna; Górski, Andrzej

    2016-01-01

    Bacteriophages (phages), discovered 100 years ago, are able to infect and destroy only bacterial cells. In the current crisis of antibiotic efficacy, phage therapy is considered as a supplementary or even alternative therapeutic approach. Evolution of multidrug-resistant and pandrug-resistant bacterial strains poses a real threat, so it is extremely important to have the possibility to isolate new phages for therapeutic purposes. Our phage laboratory and therapy center has extensive experience with phage isolation, characterization, and therapeutic application. In this article we present current progress in bacteriophages isolation and use for therapeutic purposes, our experience in this field and its practical implications for phage therapy. We attempt to summarize the state of the art: properties of phages, the methods for their isolation, criteria of phage selection for therapeutic purposes and limitations of their use. Perspectives for the use of genetically engineered phages to specifically target bacterial virulence-associated genes are also briefly presented. PMID:27570518

  17. Bacteriophage biocontrol of foodborne pathogens.

    Science.gov (United States)

    Kazi, Mustafa; Annapure, Uday S

    2016-03-01

    Bacteriophages are viruses that only infect bacterial cells. Phages are categorized based on the type of their life cycle, the lytic cycle cause lysis of the bacterium with the release of multiple phage particles where as in lysogenic phase the phage DNA is incorporated into the bacterial genome. Lysogeny does not result in lysis of the host. Lytic phages have several potential applications in the food industry as biocontrol agents, biopreservatives and as tools for detecting pathogens. They have also been proposed as alternatives to antibiotics in animal health. Two unique features of phage relevant for food safety are that they are harmless to mammalian cells and high host specificity, keeping the natural microbiota undisturbed. However, the recent approval of bacteriophages as food additives has opened the discussion about 'edible viruses'. This article reviews in detail the application of phages for the control of foodborne pathogens in a process known as "biocontrol". PMID:27570260

  18. Immobilization of Active Bacteriophages on Polyhydroxyalkanoate Surfaces.

    Science.gov (United States)

    Wang, Chanchan; Sauvageau, Dominic; Elias, Anastasia

    2016-01-20

    A rapid, efficient technique for the attachment of bacteriophages (phages) onto polyhydroxyalkanoate (PHA) surfaces has been developed and compared to three reported methods for phage immobilization. Polymer surfaces were modified to facilitate phage attachment using (1) plasma treatment alone, (2) plasma treatment followed by activation by 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS), (3) plasma-initiated acrylic acid grafting, or (4) plasma-initiated acrylic acid grafting with activation by EDC and sulfo-NHS. The impact of each method on the surface chemistry of PHA was investigated using contact angle analysis and X-ray photoelectron spectroscopy. Each of the four treatments was shown to result in both increased hydrophilicity and in the modification of the surface functional groups. Modified surfaces were immersed in suspensions of phage T4 for immobilization. The highest level of phage binding was observed for the surfaces modified by plasma treatment alone. The change in chemical bond states observed for surfaces that underwent plasma treatment is suspected to be the cause of the increased binding of active phages. Plasma-treated surfaces were further analyzed through phage-staining and fluorescence microscopy to assess the surface density of immobilized phages and their capacity to capture hosts. The infective capability of attached phages was confirmed by exposing the phage-immobilized surfaces to the host bacteria Escherichia coli in both plaque and infection dynamic assays. Plasma-treated surfaces with immobilized phages displayed higher infectivity than surfaces treated with other methods; in fact, the equivalent initial multiplicity of infection was 2 orders of magnitude greater than with other methods. Control samples - prepared by immersing polymer surfaces in phage suspensions (without prior plasma treatment) - did not show any bacterial growth inhibition, suggesting they did not bind

  19. Transduction of plasmid DNA in Streptomyces spp. and related genera by bacteriophage FP43.

    Science.gov (United States)

    McHenney, M A; Baltz, R H

    1988-05-01

    A segment (hft) of bacteriophage FP43 DNA cloned into plasmid pIJ702 mediated high-frequency transduction of the resulting plasmid (pRHB101) by FP43 in Streptomyces griseofuscus. The transducing particles contained linear concatemers of plasmid DNA. Lysates of FP43 prepared on S. griseofuscus containing pRHB101 also transduced many other Streptomyces species, including several that restrict plaque formation by FP43 and at least two that produce restriction endonucleases that cut pRHB101 DNA. Transduction efficiencies in different species were influenced by the addition of anti-FP43 antiserum to the transduction plates, the temperature for cell growth before transduction, the multiplicity of infection, and the host on which the transducing lysate was prepared. FP43 lysates prepared on S. griseofuscus(pRHB101) also transduced species of Streptoverticillium, Chainia, and Saccharopolyspora.

  20. Surface plasmon resonance detection of E. coli and methicillin-resistant S. aureus using bacteriophages.

    Science.gov (United States)

    Tawil, Nancy; Sacher, Edward; Mandeville, Rosemonde; Meunier, Michel

    2012-01-01

    Early diagnosis and appropriate treatment of Escherichia coli (E. coli) O157:H7 and methicillin-resistant Staphylococcus aureus (MRSA) are key elements in preventing resultant life-threatening illnesses, such as hemorrhagic colitis, hemolytic uremic syndrome, and septicemia. In this report, we describe the use of surface plasmon resonance (SPR) for the biodetection of pathogenic bacteria, using bacteriophages as the recognition elements. T4 bacteriophages were used to detect E. coli, while a novel, highly specific phage was used to detect MRSA. We found that the system permits label-free, real-time, specific, rapid and cost-effective detection of pathogens, for concentrations of 10(3) colony forming units/milliliter, in less than 20 min. This system promises to become a diagnostic tool for bacteria that cause major public concern for food safety, bioterrorism, and nosocomial infections.

  1. Effect of thyroxine on cellular oxygen-consumption and glucose uptake: evidence of an effect of total T4 and not "free T4"

    DEFF Research Database (Denmark)

    Kvetny, J; Matzen, L E

    1990-01-01

    in human mononuclear blood cells. Cells were incubated in protein free medium and in human serum totally depleted of thyroid hormones by resin treatment and fixed amounts of T4 (total T4 = 0-50-100-5000 nmol/l; free T4 = 0-5-11-5600 pmol/l) were added. Thyroxine stimulated glucose uptake and oxygen...

  2. I-TevI, the endonuclease encoded by the mobile td intron, recognizes binding and cleavage domains on its DNA target.

    OpenAIRE

    Bell-Pedersen, D; Quirk, S M; Bryk, M; Belfort, M

    1991-01-01

    Mobility of the phage T4 td intron depends on activity of an intron-encoded endonuclease (I-TevI), which cleaves a homologous intronless (delta In) target gene. The double-strand break initiates a recombination event that leads to intron transfer. We found previously that I-TevI cleaves td delta In target DNA 23-26 nucleotides upstream of the intron insertion site. DNase I-footprinting experiments and gel-shift assays indicate that I-TevI makes primary contacts around the intron insertion sit...

  3. Use of Bacteriophages to control bacterial pathogens

    Science.gov (United States)

    Lytic bacteriophages can provide a natural method and an effective alternative to antibiotics to reduce bacterial pathogens in animals, foods, and other environments. Bacteriophages (phages) are viruses which infect bacterial cells and eventually kill them through lysis, and represent the most abun...

  4. Programming Bacteriophages by Swapping Their Specificity Determinants.

    Science.gov (United States)

    Goren, Moran G; Yosef, Ido; Qimron, Udi

    2015-12-01

    Bacteriophages, bacteria's natural enemies, may serve as potent antibacterial agents. Their specificity for certain bacterial sub-species limits their effectiveness, but allows selective targeting of bacteria. Lu and colleagues present a platform for such targeting through alteration of bacteriophages' host specificity by swapping specificity domains in their host-recognition ligand.

  5. Single molecule studies of DNA packaging by bacteriophages

    Science.gov (United States)

    Fuller, Derek Nathan

    The DNA packaging dynamics of bacteriophages φ29, gamma, and T4 were studied at the single molecule level using a dual trap optical tweezers. Also, a method for producing long DNA molecules by PCR for optical tweezers studies of protein DNA interactions is presented and thoroughly characterized. This DNA preparation technique provided DNA samples for the φ29 and T4 studies. In the studies of φ29, the role of charge was investigated by varying the ionic conditions of the packaging buffer. Ionic conditions in which the DNA charge was highly screened due to divalent and trivalent cations showed the lowest resistance to packaging of the DNA to high density. This confirmed the importance of counterions in shielding the DNA interstrand repulsion when packaged to high density. While the ionic nature of the packaging buffer had a strong effect on packaging velocities, there was no clear trend between the counterion-screened charge of the DNA and the maximum packaging velocity. The packaging studies of lambda and T4 served as systems for comparative studies with φ29. Each system showed similarities to the φ29 system and unique differences. Both the lambda and T4 packaging motors were capable of generating forces in excess of 50 pN and showed remarkably high processivity, similar to φ29. However, dynamic structural transitions were observed with lambda that are not observed with φ29. The packaging of the lambda genome showed capsid expansion at approximately 30 percent of the genome packaged and capsid rupture at 90 percent of the genome packaged in the absence of capsid stabilizing protein gpD. Unique to the T4 packaging motor, packaging dynamics showed a remarkable amount of variability in velocities. This variability was seen both within individual packaging phages and from one phage to the next. This is possibly due to different conformational states of the packaging machinery. Additionally, lambda and T4 had average packaging velocities under minimal load of 600

  6. Visualizing phosphodiester-bond hydrolysis by an endonuclease

    DEFF Research Database (Denmark)

    Molina, Rafael; Stella, Stefano; Redondo, Pilar;

    2015-01-01

    The enzymatic hydrolysis of DNA phosphodiester bonds has been widely studied, but the chemical reaction has not yet been observed. Here we follow the generation of a DNA double-strand break (DSB) by the Desulfurococcus mobilis homing endonuclease I-DmoI, trapping sequential stages of a two-metal-...

  7. Diversity of Endonuclease V: From DNA Repair to RNA Editing

    Directory of Open Access Journals (Sweden)

    Isao Kuraoka

    2015-09-01

    Full Text Available Deamination of adenine occurs in DNA, RNA, and their precursors via a hydrolytic reaction and a nitrosative reaction. The generated deaminated products are potentially mutagenic because of their structural similarity to natural bases, which in turn leads to erroneous nucleotide pairing and subsequent disruption of cellular metabolism. Incorporation of deaminated precursors into the nucleic acid strand occurs during nucleotide synthesis by DNA and RNA polymerases or base modification by DNA- and/or RNA-editing enzymes during cellular functions. In such cases, removal of deaminated products from DNA and RNA by a nuclease might be required depending on the cellular function. One such enzyme, endonuclease V, recognizes deoxyinosine and cleaves 3' end of the damaged base in double-stranded DNA through an alternative excision repair mechanism in Escherichia coli, whereas in Homo sapiens, it recognizes and cleaves inosine in single-stranded RNA. However, to explore the role of endonuclease V in vivo, a detailed analysis of cell biology is required. Based on recent reports and developments on endonuclease V, we discuss the potential functions of endonuclease V in DNA repair and RNA metabolism.

  8. Characterization of bacteriophages infecting Streptomyces erythreus and properties of phage-resistant mutants.

    Science.gov (United States)

    Donadio, S; Paladino, R; Costanzi, I; Sparapani, P; Schreil, W; Iaccarino, M

    1986-06-01

    Three bacteriophages infecting Streptomyces erythreus, called G3, G4 and G5, were isolated and characterized. They contain double-stranded linear DNA molecules with cohesive ends. The restriction map of G3 DNA (48 kilobases long) for four restriction endonucleases and that of G4 DNA (43 kilobases long) for seven restriction endonucleases are reported. Restriction analysis and hybridization experiments showed that G3 and G4 share little DNA homology, while G4 and G5 are apparently identical except for an additional EcoRI site present in G5. The region containing this EcoRI site has been mapped on G4 DNA. Microbiological and serological data showed that G5 is very similar to G4. G3- and G4-resistant mutants of S. erythreus PS1 were isolated. The screening of phage-resistant mutants showed a high frequency of strains with increased erythromycin production. The mechanism of phage resistance of strain PS3 (G3 resistant) and of strain PS16 (G4 resistant) was examined. The DNA of the resistant strains contains no phage DNA, ruling out lysogeny as a cause of phage resistance. Transfection of strains PS1, PS3, and PS16 with DNA of the three phages showed the same efficiency, indicating that resistance is at the level of the bacterial wall.

  9. Magnetic diagnostics on the Lockheed Martin T4 Experiment

    Science.gov (United States)

    Rhoads, John

    2015-11-01

    The Lockheed Martin T4 Experiment is a magnetically encapsulated linear ring cusp confinement device designed to study the physics relevant to the Compact Fusion Reactor program. As part of the diagnostics suite, an invasive three-axis magnetic probe and several flux loops have been constructed and installed. The probe was designed to reduce electrostatic pick-up by differentially amplifying two counter-wound coils for each axis. The flux loops are designed to detect plasma diamagnetism after accounting for the flux due to the background magnetic field. This mandates that the temporal evolution of the background field must be properly taken into account in order to discern the plasma response. To this end, both hardware and software techniques have been employed. Diagnostic designs and preliminary measurements will be presented.

  10. Pathogenic assays of acanthamoeba belonging to the t4 genotype.

    Directory of Open Access Journals (Sweden)

    Hamed Mirjalali

    2013-12-01

    Full Text Available Acanthamoeba genus is introduced as opportunistic and cosmopolitan parasite. Monkey and wistar rat are appropriate models for experimental study on Acanthamoeba infection. In this study Acanthamoeba spp. were isolated from hot spring (HS, windows dust (WD and a corneal sample of keratitis patient (KP and their pathogenicity surveyed by in vitro and in vivo tests.Isolates of Acanthamoeba were cultivated axenically for 12 months in PYG medium. Overall, 30 wistar rats, in 6 equal groups were used for developing experimental Acanthamoeba keratitis (AK and Granulomatous Amoebic Encephalitis (GAE. The Keratitis and Granulomatous Encephalitis experiments were performed by intrastromal and intranasal inoculation of Acanthamoeba cysts, respectively. Pathogenicity of the three isolates was also evaluated by in vitro test using osmotolerance and temperature tolerance assays. Identification of genotypes were performed by PCR technique and sequencing.None of the isolates could perform AK and GAE in wistar rats, although all isolates were described as T4 genotype. Isolates obtained from KP and WD could grow only in 30 °C, but not in 37 °C and 40 °C. On the other hand, HS isolate grew in 30 °C and 37 °C but not in 40 °C. Moreover, all of isolate grew in 0.5 M mannitol but not in 1 M and 1.5 M.T4 isolates with a long-term axenic culture and different factors related to host and parasite may play role in pathogenicity of these free-living amoebae.

  11. Two bacteriophages of Clostridium difficile.

    OpenAIRE

    Mahony, D E; Bell, P D; Easterbrook, K. B.

    1985-01-01

    Two temperate bacteriophages of differing morphology and host range were isolated by screening 94 isolates of Clostridium difficile. Phage 41 had a 300-nm flexible tail, whereas phage 56 had a shorter tail with a contractile sheath. Electron microscopy of phage 56 lysates exposed to elevated magnesium concentrations showed small virus-like particles which were 21 nm in diameter. The addition of MgCl2 to semisolid agar overlays enhanced both the titer and plaque size of phage 56. Phage 56 was ...

  12. Isolation and Characterization of a Virulent Bacteriophage φPA-HF17 of Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Fang Han

    2014-09-01

    Full Text Available Pseudomonas aeruginosa, an important causative agent of nosocomial infection, is found throughout the hospital environment in moist reservoirs, and multidrug-resistant strains of P. aeruginosa have been increasingly reported worldwide. Bacteriophages are often considered potential therapeutic candidates in treating infectious diseases. In this study, a novel virulent bacteriophage φPA-HF17, specific infecting clinical isolates of P. aeruginosa, was isolated and characterized from environmental sewage. Transmission electron microscopy showed that phage φPA-HF17 had an icosahedral head with a very short tail, and exhibited characteristics typical of a podovirus. Restriction analysis indicated that phage φPA-HF17 was a double-stranded DNA virus, which might be digested by some restriction endonucleases. Phage φPA-HF17 was highly infectious with a rapid adsorption (>90% adsorbed in 4 min. In a one-step growth experiment, phage φPA-HF17 was shown having a latent period of 10 minute, with corresponding burst sizes of 200 PFU/cell. Furthermore, when φPA-HF17 alone was incubated at different pHs and different temperatures, the phage was stable over a wide pH range (4 to 10 and at extreme temperature (50°C. These results suggest that φPA-HF17 may be candidate therapeutic phage to control P. aeruginosa infection.

  13. Inactivation of E. coli, B. subtilis spores, and MS2, T4, and T7 phage using UV/H{sub 2}O{sub 2} advanced oxidation

    Energy Technology Data Exchange (ETDEWEB)

    Mamane, Hadas [School of Mechanical Engineering and Porter School of Environmental Studies, Tel-Aviv University, Tel-Aviv 69978 (Israel)]. E-mail: hadasmg@post.tau.ac.il; Shemer, Hilla [Department of Civil and Environmental Engineering, Duke University, Durham, NC 27708-0287 (United States); Linden, Karl G. [Department of Civil and Environmental Engineering, Duke University, Durham, NC 27708-0287 (United States)

    2007-07-31

    The goal of this study was to evaluate the potential of an advanced oxidation process (AOP) for microbiocidal and virucidal inactivation. The viruses chosen for this study were bacteriophage MS2, T4, and T7. In addition, Bacillus subtilis spores and Escherichia coli were studied. By using H{sub 2}O{sub 2} in the presence of filtered ultraviolet (UV) irradiation (UV/H{sub 2}O{sub 2}) to generate wavelengths above 295 nm, the direct UV photolysis disinfection mechanism was minimized, while disinfection by H{sub 2}O{sub 2} was also negligible. Virus T4 and E. coli in phosphate buffered saline (PBS) were sensitive to >295 nm filtered UV irradiation (without H{sub 2}O{sub 2}), while MS2 was very resistant. Addition of H{sub 2}O{sub 2} at 25 mg/l in the presence of filtered UV irradiation over a 15 min reaction time did not result in any additional disinfection of virus T4, while an additional one log inactivation for T7 and 2.5 logs for MS2 were obtained. With E. coli, only a slight additional effect was observed when H{sub 2}O{sub 2} was added. B. subtilis spores did not show any inactivation at any of the conditions used in this study. The OH radical exposure (CT value) was calculated to present the relationship between the hydroxyl radical dose and microbial inactivation.

  14. Inactivation of E. coli, B. subtilis spores, and MS2, T4, and T7 phage using UV/H2O2 advanced oxidation

    International Nuclear Information System (INIS)

    The goal of this study was to evaluate the potential of an advanced oxidation process (AOP) for microbiocidal and virucidal inactivation. The viruses chosen for this study were bacteriophage MS2, T4, and T7. In addition, Bacillus subtilis spores and Escherichia coli were studied. By using H2O2 in the presence of filtered ultraviolet (UV) irradiation (UV/H2O2) to generate wavelengths above 295 nm, the direct UV photolysis disinfection mechanism was minimized, while disinfection by H2O2 was also negligible. Virus T4 and E. coli in phosphate buffered saline (PBS) were sensitive to >295 nm filtered UV irradiation (without H2O2), while MS2 was very resistant. Addition of H2O2 at 25 mg/l in the presence of filtered UV irradiation over a 15 min reaction time did not result in any additional disinfection of virus T4, while an additional one log inactivation for T7 and 2.5 logs for MS2 were obtained. With E. coli, only a slight additional effect was observed when H2O2 was added. B. subtilis spores did not show any inactivation at any of the conditions used in this study. The OH radical exposure (CT value) was calculated to present the relationship between the hydroxyl radical dose and microbial inactivation

  15. Propagating the missing bacteriophages: a large bacteriophage in a new class

    Directory of Open Access Journals (Sweden)

    Hardies Stephen C

    2007-02-01

    Full Text Available Abstract The number of successful propagations/isolations of soil-borne bacteriophages is small in comparison to the number of bacteriophages observed by microscopy (great plaque count anomaly. As one resolution of the great plaque count anomaly, we use propagation in ultra-dilute agarose gels to isolate a Bacillus thuringiensis bacteriophage with a large head (95 nm in diameter, tail (486 × 26 nm, corkscrew-like tail fibers (187 × 10 nm and genome (221 Kb that cannot be detected by the usual procedures of microbiology. This new bacteriophage, called 0305φ8-36 (first number is month/year of isolation; remaining two numbers identify the host and bacteriophage, has a high dependence of plaque size on the concentration of a supporting agarose gel. Bacteriophage 0305φ8-36 does not propagate in the traditional gels used for bacteriophage plaque formation and also does not produce visible lysis of liquid cultures. Bacteriophage 0305φ8-36 aggregates and, during de novo isolation from the environment, is likely to be invisible to procedures of physical detection that use either filtration or centrifugal pelleting to remove bacteria. Bacteriophage 0305φ8-36 is in a new genomic class, based on genes for both structural components and DNA packaging ATPase. Thus, knowledge of environmental virus diversity is expanded with prospect of greater future expansion.

  16. Immunocompatibility of Bacteriophages as Nanomedicines

    Directory of Open Access Journals (Sweden)

    Tranum Kaur

    2012-01-01

    Full Text Available Bacteriophage-based medical research provides the opportunity to develop targeted nanomedicines with heightened efficiency and safety profiles. Filamentous phages also can and have been formulated as targeted drug-delivery nanomedicines, and phage may also serve as promising alternatives/complements to antibiotics. Over the past decade the use of phage for both the prophylaxis and the treatment of bacterial infection, has gained special significance in view of a dramatic rise in the prevalence of antibiotic resistance bacterial strains. Two potential medical applications of phages are the treatment of bacterial infections and their use as immunizing agents in diagnosis and monitoring patients with immunodeficiencies. Recently, phages have been employed as gene-delivery vectors (phage nanomedicine, for nearly half a century as tools in genetic research, for about two decades as tools for the discovery of specific target-binding proteins and peptides, and for almost a decade as tools for vaccine development. As phage applications to human therapeutic development grow at an exponential rate, it will become essential to evaluate host immune responses to initial and repetitive challenges by therapeutic phage in order to develop phage therapies that offer suitable utility. This paper examines and discusses phage nanomedicine applications and the immunomodulatory effects of bacteriophage exposure and treatment modalities.

  17. Interrupted thymidylate synthase gene of bacteriophages T2 and T6 and other potential self-splicing introns in the T-even bacteriophages

    Energy Technology Data Exchange (ETDEWEB)

    Chu, F.K.; Maley, F.; Martinez, J.; Maley, G.F.

    1987-09-01

    Southern hybridization analyses of procaryotic DNA from Escherchia coli, lambda bacteriophage, and T1 to T7 phages were carried out. The hybridization probes used consisted of DNA restriction fragments derived from the T4 phage intron-containing thymidylate synthase gene (td) and short synthetic oligodeoxynucleotides defining specific exon and intron regions of the gene. It was shown that intact as well as restricted DNA from the T-even phages hybridized not only to both T4 phage td intron- and exon-specific probes but also to probes defining the td 5' (exon I-intron) and 3' (intron-exon II) presplice junctions. These data strongly suggest that, analogous to the T4 phage, only the T2 and T6 phages among the procaryotes tested contain interrupted td genes. The td intervening sequence in each phage is roughly 1 kilobase pair (kb) in size and interrupts the td gene at a site analogous to that in the T4 phage. This was confirmed by data from Northern (RNA) hybridization analysis of td-specific in vitro transcripts of these phage DNAs. (..cap alpha..-/sup 32/P)GTP in vitro labeling of total RNA from T4 phage-infected cells produced five species of labeled RNAs that were 1, 0.9, 0.83, 0.75, and 0.6 kb in size. Only the 1-, 0.9-, and 0.75-kb species were labeled in RNA from T2- or T6-infected cells. The commonly present 1-kb RNA is the excised td intron, which exists in both linear and circular forms in the respective T-even-phage-infected cells, while the 0.6-kb RNA unique to T4 may be the excised intron derived from the ribonucleotide reductase small subunit gene (nrdB) of the phage. The remaining labeled RNA species are likely candidates for other self-splicing introns.

  18. Interrupted thymidylate synthase gene of bacteriophages T2 and T6 and other potential self-splicing introns in the T-even bacteriophages

    International Nuclear Information System (INIS)

    Southern hybridization analyses of procaryotic DNA from Escherchia coli, λ bacteriophage, and T1 to T7 phages were carried out. The hybridization probes used consisted of DNA restriction fragments derived from the T4 phage intron-containing thymidylate synthase gene (td) and short synthetic oligodeoxynucleotides defining specific exon and intron regions of the gene. It was shown that intact as well as restricted DNA from the T-even phages hybridized not only to both T4 phage td intron- and exon-specific probes but also to probes defining the td 5' (exon I-intron) and 3' (intron-exon II) presplice junctions. These data strongly suggest that, analogous to the T4 phage, only the T2 and T6 phages among the procaryotes tested contain interrupted td genes. The td intervening sequence in each phage is roughly 1 kilobase pair (kb) in size and interrupts the td gene at a site analogous to that in the T4 phage. This was confirmed by data from Northern (RNA) hybridization analysis of td-specific in vitro transcripts of these phage DNAs. [α-32P]GTP in vitro labeling of total RNA from T4 phage-infected cells produced five species of labeled RNAs that were 1, 0.9, 0.83, 0.75, and 0.6 kb in size. Only the 1-, 0.9-, and 0.75-kb species were labeled in RNA from T2- or T6-infected cells. The commonly present 1-kb RNA is the excised td intron, which exists in both linear and circular forms in the respective T-even-phage-infected cells, while the 0.6-kb RNA unique to T4 may be the excised intron derived from the ribonucleotide reductase small subunit gene (nrdB) of the phage. The remaining labeled RNA species are likely candidates for other self-splicing introns

  19. Partial characterization of Acanthamoeba castellanii (T4 genotype) DNase activity.

    Science.gov (United States)

    Iqbal, Junaid; Panjwani, Shamvil; Siddiqui, Ruqaiyyah; Khan, Naveed Ahmed

    2015-02-01

    The deoxyribonuclease (DNase) activities of Acanthamoeba castellanii belonging to the T4 genotype were investigated. Using zymographic assays, the DNase activities had approximate molecular masses of 25 and 35 kDa. A. castellanii DNases exhibited activity at wide-ranging temperature of up to 60 °C and at pH ranging from 4 to 9. The DNases activities were unaffected by proteinase-K treatment, divalent cations such as Ca(++), Cu(++), Mg(++), and Zn(++), or divalent cation chelating agent ethylenediaminetetraacetic acid (EDTA) or sodium dodecyl sulfate (SDS). The non-reliance on divalent cations and homology data suggests that A. castellanii DNases belong to the class of eukaryotic lysosomal DNase II but exhibit robust properties. The DNases activity in A. castellanii interfered with the genomic DNA extraction. Extraction methods involving EDTA, SDS, and proteinase-K resulted in low yield of genomic DNA. On the other hand, these methods resulted in high yield of genomic DNA from human cells suggesting the robust nature of A. castellanii DNases that are unaffected by reagents normally used in blocking eukaryotic DNases. In contrast, the use of chaotropic agent such as guanidine thiocyanate improved the yield of genomic DNA from A. castellanii cells significantly. Further purification and characterization of Acanthamoeba DNases is needed to study their non-classic distinct properties and to determine their role in the biology, cellular differentiation, cell cycle progression, and arrest of Acanthamoeba.

  20. Purification and characterization of the x-ray endonuclease of Escherichia coli

    International Nuclear Information System (INIS)

    This work concerns the purification and characterization of the x-ray endonuclease of E. coli. The x-ray endonuclease was purified by chromatography on DNA-agarose, Sephadex gel filtration, hydroxylapatite chromatography, and phosphocellulose chromatography. Parallel assays on modified DNA and oligonucleotide substances established that the x-ray endonuclease was active on DNA contain in apurinic and apyrimidinic sites, thymine glycol and urea residues, and undefined lesions produced by UV and X radiation. Characterization of the x-ray endonuclease by gel filtration gave a molecular weight of about 25,000 dalton while SDS-polyacrylamide gel electrophoresis of the most purified preparations showed a single band corresponding to a molecular weight of about 13,000 daltons. Analysis of DNA substrates following x-ray endonuclease treatment showed that the x-ray endonuclease nicked at the 3' side of a base lesion to yield 3'OH and 5'PO termini. Analysis of the acid/alcohol soluble products of the digestion of specifically modified synthetic poly dT:dA by the x-ray endonuclease showed this enzyme to have DNA glycosylase activities that released both thymine glycol and urea residues from DNA. Inhibitor studies showed the thymine-glycol endonuclease activity was inhibited by NEM while the AP endonuclease was not. NEM was also shown to inhibit endonuclease activity on UV-irradiated DNA, X-irradiated DNA, and urea-containing DNA

  1. Identification of leptospiral isolates by bacterial restriction endonuclease analysis (Brenda

    Directory of Open Access Journals (Sweden)

    Venkatesha M

    2001-01-01

    Full Text Available DNA samples from 19 reference serovars belonging to 19 different serogroups of Leptospira interrogans and two serovars belonging to Leptospira biflexa were examined by bacterial restriction endonuclease analysis using EcoR I and Hae III enzymes. All the serovars gave unique restriction patterns that differed from each other. DNA from 10 local isolates digested with these enzymes produced patterns which on comparison with the standard patterns produced by reference strains could be identified to serovar level.

  2. Computational redesign of endonuclease DNA binding and cleavage specificity

    Science.gov (United States)

    Ashworth, Justin; Havranek, James J.; Duarte, Carlos M.; Sussman, Django; Monnat, Raymond J.; Stoddard, Barry L.; Baker, David

    2006-06-01

    The reprogramming of DNA-binding specificity is an important challenge for computational protein design that tests current understanding of protein-DNA recognition, and has considerable practical relevance for biotechnology and medicine. Here we describe the computational redesign of the cleavage specificity of the intron-encoded homing endonuclease I-MsoI using a physically realistic atomic-level forcefield. Using an in silico screen, we identified single base-pair substitutions predicted to disrupt binding by the wild-type enzyme, and then optimized the identities and conformations of clusters of amino acids around each of these unfavourable substitutions using Monte Carlo sampling. A redesigned enzyme that was predicted to display altered target site specificity, while maintaining wild-type binding affinity, was experimentally characterized. The redesigned enzyme binds and cleaves the redesigned recognition site ~10,000 times more effectively than does the wild-type enzyme, with a level of target discrimination comparable to the original endonuclease. Determination of the structure of the redesigned nuclease-recognition site complex by X-ray crystallography confirms the accuracy of the computationally predicted interface. These results suggest that computational protein design methods can have an important role in the creation of novel highly specific endonucleases for gene therapy and other applications.

  3. Human repair endonuclease incises DNA at cytosine photoproducts

    International Nuclear Information System (INIS)

    The nature of DNA damage by uvB and uvC irradiation was investigated using a defined sequence of human DNA. A UV-irradiated, 3'-end-labeled, 92 base pair sequence from the human alphoid segment was incubated with a purified human lymphoblast endonuclease that incises DNA at non-dimer photoproducts. Analysis by polyacrylamide gel electrophoresis identified all sites of endonucleolytic incision as cytosines. These were found in regions of the DNA sequence lacking adjacent pyrimidines and therefore are neither cyclobutane pyrimidine dimers nor 6-4'-pyrimidines. Incision at cytosine photoproducts was not detected at loci corresponding to alkali-labile sites in either control or irradiated substrates. This demonstrates that the bands detected after the enzymic reactions were not the result of DNA strand breaks, base loss sites or ring-opened cytosines. The optimal wavelengths for formation of cytosine photoproducts are 270-295 nm, similar to those associated with maximal tumor yields in animal ultraviolet carcinogenesis studies. Irradiation by monochromatic 254 nm light resulted in reduced cytosine photoproduct formation. This human UV endonuclease has an apparently identical substrate specificity to E. coli endonuclease III. Both the human and bacterial enzymes incise cytosine moieties in UV irradiated DNA and modified thymines in oxidized DNA

  4. Novel DNA packaging recognition in the unusual bacteriophage N15

    International Nuclear Information System (INIS)

    Phage lambda's cosB packaging recognition site is tripartite, consisting of 3 TerS binding sites, called R sequences. TerS binding to the critical R3 site positions the TerL endonuclease for nicking cosN to generate cohesive ends. The N15 cos (cosN15) is closely related to cosλ, but whereas the cosBN15 subsite has R3, it lacks the R2 and R1 sites and the IHF binding site of cosBλ. A bioinformatic study of N15-like phages indicates that cosBN15 also has an accessory, remote rR2 site, which is proposed to increase packaging efficiency, like R2 and R1 of lambda. N15 plus five prophages all have the rR2 sequence, which is located in the TerS-encoding 1 gene, approximately 200 bp distal to R3. An additional set of four highly related prophages, exemplified by Monarch, has R3 sequence, but also has R2 and R1 sequences characteristic of cosB–λ. The DNA binding domain of TerS-N15 is a dimer. - Highlights: • There are two classes of DNA packaging signals in N15-related phages. • Phage N15's TerS binding site: a critical site and a possible remote accessory site. • Viral DNA recognition signals by the λ-like bacteriophages: the odd case of N15

  5. Novel DNA packaging recognition in the unusual bacteriophage N15

    Energy Technology Data Exchange (ETDEWEB)

    Feiss, Michael [Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242 (United States); Geyer, Henriette, E-mail: henriettegeyer@gmail.com [Division of Viral Infections, Robert Koch Institute, Berlin (Germany); Division of Viral Infections, Robert Koch Institute, Berlin (Germany); Klingberg, Franco, E-mail: franco.klingberg@thermofisher.com [Flow Cytometry, Imaging & Microscopy, Thermo Fisher Scientific, Frankfurter Strasse 129B 64293 Darmstadt (Germany); Flow Cytometry, Imaging & Microscopy, Thermo Fisher Scientific, Frankfurter Strasse 129B 64293 Darmstadt (Germany); Moreno, Norma, E-mail: nmoreno@islander.tamucc.edu [Texas A& M University – Corpus Christi, 6300 Ocean Drive, Corpus Christi, TX 78412, United States. (United States); Texas A& M University – Corpus Christi, 6300 Ocean Drive, Corpus Christi, TX 78412, United States. (United States); Forystek, Amanda, E-mail: eamanda-forystek@uiowa.edu [Flow Cytometry, Imaging & Microscopy, Thermo Fisher Scientific, Frankfurter Strasse 129B 64293 Darmstadt (Germany); Room # 2911 JPP, Dept. of Psychiatry, The University of Iowa, 200 Hawkins Drive, Iowa City, Iowa, 52242 (United States); Maluf, Nasib Karl, E-mail: fKarl.Maluf@ap-lab.com [Flow Cytometry, Imaging & Microscopy, Thermo Fisher Scientific, Frankfurter Strasse 129B 64293 Darmstadt (Germany); Alliance Protein Laboratories, Inc. 6042 Cornerstone Court West, Suite ASan Diego, CA 92121, USA. (United States); Sippy, Jean [Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242 (United States)

    2015-08-15

    Phage lambda's cosB packaging recognition site is tripartite, consisting of 3 TerS binding sites, called R sequences. TerS binding to the critical R3 site positions the TerL endonuclease for nicking cosN to generate cohesive ends. The N15 cos (cos{sup N15}) is closely related to cos{sup λ}, but whereas the cosB{sup N15} subsite has R3, it lacks the R2 and R1 sites and the IHF binding site of cosB{sup λ}. A bioinformatic study of N15-like phages indicates that cosB{sup N15} also has an accessory, remote rR2 site, which is proposed to increase packaging efficiency, like R2 and R1 of lambda. N15 plus five prophages all have the rR2 sequence, which is located in the TerS-encoding 1 gene, approximately 200 bp distal to R3. An additional set of four highly related prophages, exemplified by Monarch, has R3 sequence, but also has R2 and R1 sequences characteristic of cosB–λ. The DNA binding domain of TerS-N15 is a dimer. - Highlights: • There are two classes of DNA packaging signals in N15-related phages. • Phage N15's TerS binding site: a critical site and a possible remote accessory site. • Viral DNA recognition signals by the λ-like bacteriophages: the odd case of N15.

  6. [Friedrich Mauz: T4 assessor and military psychiatrist].

    Science.gov (United States)

    Silberzahn-Jandt, G; Schmuhl, H-W

    2012-03-01

    Friedrich Mauz is one of the medical perpetrators of the second tier whose biography is difficult to comprehend. Autobiographies from three different political systems exist - Weimar Republic, the Third Reich, and postwar Germany in which he constantly reinvented himself. While after 1933 he suddenly emphasized his participation in the civil war turmoil during the early period of the Weimar Republic and his patriotism, he then depicted himself after 1945 as an apolitical person characterized by Württemberg pietism who inwardly rejected the Nazi State but had found himself prepared to accept "all sorts of humiliating concessions." He claimed that he had always remained true to his scientific code of conduct and had distanced himself from psychiatric genetics. In point of fact, Mauz was among those exonerated in the denazification trial in 1946 and was able to pursue his career in the Federal Republic of Germany. However, if the sources are read against the grain, a different picture emerges. Mauz's career stalled in the 1930s, not because he had been politically offensive, but because his scientific work was flimsy and considered lacking originality, particularly since he had chosen constitution research and psychotherapy as his main fields of interest, which were overshadowed by research in genetic psychiatry in the 1930s. Mauz tendered his services to the Nazi policy of genetic health, served as a medical assessor in proceedings based on the "Law for the Prevention of Genetically Diseased Offspring," permitted himself to be recruited for the T4 program as a medical expert, even participated in the deliberations on a future "Law on Euthanasia," and as a consulting psychiatrist for the German Armed Forces contributed to military medicine. PMID:22399061

  7. [Friedrich Mauz: T4 assessor and military psychiatrist].

    Science.gov (United States)

    Silberzahn-Jandt, G; Schmuhl, H-W

    2012-03-01

    Friedrich Mauz is one of the medical perpetrators of the second tier whose biography is difficult to comprehend. Autobiographies from three different political systems exist - Weimar Republic, the Third Reich, and postwar Germany in which he constantly reinvented himself. While after 1933 he suddenly emphasized his participation in the civil war turmoil during the early period of the Weimar Republic and his patriotism, he then depicted himself after 1945 as an apolitical person characterized by Württemberg pietism who inwardly rejected the Nazi State but had found himself prepared to accept "all sorts of humiliating concessions." He claimed that he had always remained true to his scientific code of conduct and had distanced himself from psychiatric genetics. In point of fact, Mauz was among those exonerated in the denazification trial in 1946 and was able to pursue his career in the Federal Republic of Germany. However, if the sources are read against the grain, a different picture emerges. Mauz's career stalled in the 1930s, not because he had been politically offensive, but because his scientific work was flimsy and considered lacking originality, particularly since he had chosen constitution research and psychotherapy as his main fields of interest, which were overshadowed by research in genetic psychiatry in the 1930s. Mauz tendered his services to the Nazi policy of genetic health, served as a medical assessor in proceedings based on the "Law for the Prevention of Genetically Diseased Offspring," permitted himself to be recruited for the T4 program as a medical expert, even participated in the deliberations on a future "Law on Euthanasia," and as a consulting psychiatrist for the German Armed Forces contributed to military medicine.

  8. Bacteriophages of Leuconostoc, Oenococcus, and Weissella

    DEFF Research Database (Denmark)

    Kot, Witold; Neve, Horst; Heller, Knut J;

    2014-01-01

    can be classified as either Ln. mesenteroides or Ln. pseudomesenteroides. They are important flavor producers in dairy fermentations and they initiate nearly all vegetable fermentations. Therefore, bacteriophages attacking Leuconostoc strains may negatively influence the production process....... Bacteriophages attacking Leuconostoc strains were first reported in 1946. Since then, the majority of described Leuconostoc phages was isolated from either dairy products or fermented vegetable products. Both lytic and temperate phages of Leuconostoc were reported. Most of Leuconostoc phages examined using...

  9. Characterization and purification of bacteriophages using chromatofocusing.

    Science.gov (United States)

    Brorson, Kurt; Shen, Hong; Lute, Scott; Pérez, Jessica Soto; Frey, Douglas D

    2008-10-17

    The technique of chromatofocusing was applied to the characterization and purification of three bacteriophages that are routinely used for testing virus filters: phiX174, PR772, and PP7. Chemically well-defined eluent buffers were used, instead of the more commonly used chromatofocusing polyampholyte buffers. Chromatographic column packings were selected to minimize band broadening by confining bacteriophage adsorption solely to the exterior particle surface. Under the conditions used it was determined that bacteriophages could be made to focus into narrow bands in a retained pH gradient with recoveries of live phage that ranged from 15 to nearly 100% as determined by a plaque-forming assay. Retention times and apparent isoelectric point data were obtained for samples consisting either of purified bacteriophage, or samples consisting of crude preparations of bacteriophages containing host cell impurities. Isoelectric point estimates were obtained using modified, previously described models. The results obtained suggest that chromatofocusing is a simple and rapid method for obtaining approximate isoelectric points for bacteriophages and probably other types of viruses. It is also likely a useful method for purifying these materials.

  10. Establishment of an Enzyme Linked Immunosorbent Assay for Total Thyroxine (T4

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A sensitive and specific ELISA for total thyroxine (T4) is established. The anti-T4 antibody is coatedon the microtiter plate, the T4 antigen is conjugated to the biotin. The label is horseradish peroxidase(HRP)

  11. Pathogen detection using engineered bacteriophages.

    Science.gov (United States)

    Smartt, Abby E; Xu, Tingting; Jegier, Patricia; Carswell, Jessica J; Blount, Samuel A; Sayler, Gary S; Ripp, Steven

    2012-04-01

    Bacteriophages, or phages, are bacterial viruses that can infect a broad or narrow range of host organisms. Knowing the host range of a phage allows it to be exploited in targeting various pathogens. Applying phages for the identification of microorganisms related to food and waterborne pathogens and pathogens of clinical significance to humans and animals has a long history, and there has to some extent been a recent revival in these applications as phages have become more extensively integrated into novel detection, identification, and monitoring technologies. Biotechnological and genetic engineering strategies applied to phages are responsible for some of these new methods, but even natural unmodified phages are widely applicable when paired with appropriate innovative detector platforms. This review highlights the use of phages as pathogen detector interfaces to provide the reader with an up-to-date inventory of phage-based biodetection strategies.

  12. Host receptors for bacteriophage adsorption.

    Science.gov (United States)

    Bertozzi Silva, Juliano; Storms, Zachary; Sauvageau, Dominic

    2016-02-01

    The adsorption of bacteriophages (phages) onto host cells is, in all but a few rare cases, a sine qua non condition for the onset of the infection process. Understanding the mechanisms involved and the factors affecting it is, thus, crucial for the investigation of host-phage interactions. This review provides a survey of the phage host receptors involved in recognition and adsorption and their interactions during attachment. Comprehension of the whole infection process, starting with the adsorption step, can enable and accelerate our understanding of phage ecology and the development of phage-based technologies. To assist in this effort, we have established an open-access resource--the Phage Receptor Database (PhReD)--to serve as a repository for information on known and newly identified phage receptors. PMID:26755501

  13. 子代T4的PCR和real time PCR测定%Quantification of progeny T4 by PCR and real-time PCR

    Institute of Scientific and Technical Information of China (English)

    刘邻渭; 杨公明

    2007-01-01

    目的:为建立通过测定转换样品T4而检验样品大肠杆菌的新方法,探讨快速测定转换样品中T4的PCR技术.方法:以T4DNA纯化样品水稀释液和转换样品沸水浴处理产物分别为模板样液,经过两对引物PCR及real time PCR的试用和比较,优选定量检测转换样品中T4的适宜方法和条件.结果:源自T4 ligase的一对引物具有较高的灵敏度和特异性;针对纯化T4DNA配制的模板样液,优化的PCR和real time PCR至少可分别精确检出39.25和3.925 pg/ml的T4DNA;针对沸水浴处理转换样品制备的模板样液,PCR和real time PCR可清楚区别不同浓度的转换样品,PCR的检出极限为500 PFU/ml T4的转换样品,real time PCR的检测极限为35 PFU/ml T4的转换样品.结论:采用PCR和real time PCR可快速定量测定转换样品中的T4含量,real time PCR比PCR的灵敏度高约一个数量级.

  14. Neisseria gonorrhoeae FA1090 Carries Genes Encoding Two Classes of Vsr Endonucleases

    OpenAIRE

    Kwiatek, Agnieszka; Łuczkiewicz, Maciej; Bandyra, Katarzyna; Stein, Daniel C.; Piekarowicz, Andrzej

    2010-01-01

    A very short patch repair system prevents mutations resulting from deamination of 5-methylcytosine to thymine. The Vsr endonuclease is the key enzyme of this system, providing sequence specificity. We identified two genes encoding Vsr endonucleases V.NgoAXIII and V.NgoAXIV from Neisseria gonorrhoeae FA1090 based on DNA sequence similarity to genes encoding Vsr endonucleases from other bacteria. After expression of the gonococcal genes in Escherichia coli, the proteins were biochemically chara...

  15. Class I self-splicing introns are found in the T-even bacteriophage family

    International Nuclear Information System (INIS)

    The thymidylate synthase gene (td) and ribonucleotide reductase B2 subunit gene (nrdB) EMBO both of bacteriophage T4 in origin, are procaryotic intron-containing protein-encoding genes. To screen for other procaryotic introns, southern hybridization analysis of several procaryotic genomes was carried out, using T4 phage td DNA restriction fragments and synthetic oligodeoxynucleotides defining strategic td exon and intron regions. Furthermore, the labeling pattern of total RNA with [α-32P]GTP, a typical reaction of self-splicing RNAs (class I), was examined. Experimental data implicate multiple self-splicing introns only in the T-even phages: five (1, 0.9, 0.83, 0.75 and 0.6 kb) in T4 and three (1, 0.9 and 0.75 kb) each in T2 and T6 phages. Northern hybridization analysis of total RNA extracted from T-even phage-infected cells confirms that the 1 kb RNA from each phage is in fact the excised intron segment from the precursor RNA transcribed from an intron-containing td gene in each case. This RNA cyclizes to form a contiguous circular molecule. The 0.6 kb RNA is most likely the T4 phage nrdB intron which seems to be absent from the corresponding gene in T2 and T6. The remaining RNA species are candidates for other self-splicing introns in these phages

  16. Complete Genome Sequence of Bacillus thuringiensis Bacteriophage BMBtp2

    OpenAIRE

    Dong, Zhaoxia; Peng, Donghai; Wang, Yueying; Zhu, Lei; Ruan, Lifang; Sun, Ming

    2013-01-01

    Bacillus thuringiensis is an insect pathogen which has been widely used for biocontrol. During B. thuringiensis fermentation, lysogenic bacteriophages cause severe losses of yield. Here, we announce the complete genome sequence of a bacteriophage, BMBtp2, which is induced from B. thuringiensis strain YBT-1765, which may be helpful to clarify the mechanism involved in bacteriophage contamination.

  17. 21 CFR 866.2050 - Staphylococcal typing bacteriophage.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Staphylococcal typing bacteriophage. 866.2050 Section 866.2050 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... Staphylococcal typing bacteriophage. (a) Identification. A staphylococcal typing bacteriophage is a...

  18. Adenosine Triphosphate Stimulates Aquifex aeolicus MutL Endonuclease Activity

    OpenAIRE

    Jerome Mauris; Thomas C Evans

    2009-01-01

    BACKGROUND: Human PMS2 (hPMS2) homologues act to nick 5' and 3' to misincorporated nucleotides during mismatch repair in organisms that lack MutH. Mn(++) was previously found to stimulate the endonuclease activity of these homologues. ATP was required for the nicking activity of hPMS2 and yPMS1, but was reported to inhibit bacterial MutL proteins from Thermus thermophilus and Aquifex aeolicus that displayed homology to hPMS2. Mutational analysis has identified the DQHA(X)(2)E(X)(4)E motif pre...

  19. Adenosine triphosphate stimulates Aquifex aeolicus MutL endonuclease activity.

    Directory of Open Access Journals (Sweden)

    Jerome Mauris

    Full Text Available BACKGROUND: Human PMS2 (hPMS2 homologues act to nick 5' and 3' to misincorporated nucleotides during mismatch repair in organisms that lack MutH. Mn(++ was previously found to stimulate the endonuclease activity of these homologues. ATP was required for the nicking activity of hPMS2 and yPMS1, but was reported to inhibit bacterial MutL proteins from Thermus thermophilus and Aquifex aeolicus that displayed homology to hPMS2. Mutational analysis has identified the DQHA(X(2E(X(4E motif present in the C-terminus of PMS2 homologues as important for endonuclease activity. METHODOLOGIES/PRINCIPAL FINDINGS: We examined the effect ATP had on the Mn(++ induced nicking of supercoiled pBR322 by full-length and mutant A. aeolicus MutL (Aae MutL proteins. Assays were single time point, enzyme titration experiments or reaction time courses. The maximum velocity for MutL nicking was determined to be 1.6+/-0.08x10(-5 s(-1 and 4.2+/-0.3x10(-5 s(-1 in the absence and presence of ATP, respectively. AMPPNP stimulated the nicking activity to a similar extent as ATP. A truncated Aae MutL protein composed of only the C-terminal 123 amino acid residues was found to nick supercoiled DNA. Furthermore, mutations in the conserved C-terminal DQHA(X(2E(X(4E and CPHGRP motifs were shown to abolish Aae MutL endonuclease activity. CONCLUSIONS: ATP stimulated the Mn(++ induced endonuclease activity of Aae MutL. Experiments utilizing AMPPNP implied that the stimulation did not require ATP hydrolysis. A mutation in the DQHA(X(2E(X(4E motif of Aae MutL further supported the role of this region in endonclease activity. For the first time, to our knowledge, we demonstrate that changing the histidine residue in the conserved CPHGRP motif abolishes endonucleolytic activity of a hPMS2 homologue. Finally, the C-terminal 123 amino acid residues of Aae MutL were sufficient to display Mn(++ induced nicking activity.

  20. Nucleosomes Inhibit Cas9 Endonuclease Activity in Vitro.

    Science.gov (United States)

    Hinz, John M; Laughery, Marian F; Wyrick, John J

    2015-12-01

    During Cas9 genome editing in eukaryotic cells, the bacterial Cas9 enzyme cleaves DNA targets within chromatin. To understand how chromatin affects Cas9 targeting, we characterized Cas9 activity on nucleosome substrates in vitro. We find that Cas9 endonuclease activity is strongly inhibited when its target site is located within the nucleosome core. In contrast, the nucleosome structure does not affect Cas9 activity at a target site within the adjacent linker DNA. Analysis of target sites that partially overlap with the nucleosome edge indicates that the accessibility of the protospacer-adjacent motif (PAM) is the critical determinant of Cas9 activity on a nucleosome.

  1. Bacteriophages with the Ability to Degrade Uropathogenic Escherichia Coli Biofilms

    Directory of Open Access Journals (Sweden)

    Amee Manges

    2012-04-01

    Full Text Available Escherichia coli-associated urinary tract infections (UTIs are among the most common bacterial infections in humans. UTIs are usually managed with antibiotic therapy, but over the years, antibiotic-resistant strains of uropathogenic E. coli (UPEC have emerged. The formation of biofilms further complicates the treatment of these infections by making them resistant to killing by the host immune system as well as by antibiotics. This has encouraged research into therapy using bacteriophages (phages as a supplement or substitute for antibiotics. In this study we characterized 253 UPEC in terms of their biofilm-forming capabilities, serotype, and antimicrobial resistance. Three phages were then isolated (vB_EcoP_ACG-C91, vB_EcoM_ACG-C40 and vB_EcoS_ACG-M12 which were able to lyse 80.5% of a subset (42 of the UPEC strains able to form biofilms. Correlation was established between phage sensitivity and specific serotypes of the UPEC strains. The phages’ genome sequences were determined and resulted in classification of vB_EcoP_ACG-C91 as a SP6likevirus, vB_EcoM_ACG-C40 as a T4likevirus and vB_EcoS_ACG-M12 as T1likevirus. We assessed the ability of the three phages to eradicate the established biofilm of one of the UPEC strains used in the study. All phages significantly reduced the biofilm within 2–12 h of incubation.

  2. Structural stability and endonuclease activity of a PI-SceI GFP-fusion protein

    Directory of Open Access Journals (Sweden)

    Alireza G. Senejani, J. Peter Gogarten

    2007-01-01

    Full Text Available Homing endonucleases are site-specific and rare cutting endonucleases often encoded by intron or intein containing genes. They lead to the rapid spread of the genetic element that hosts them by a process termed 'homing'; and ultimately the allele containing the element will be fixed in the population. PI-SceI, an endonuclease encoded as a protein insert or intein within the yeast V-ATPase catalytic subunit encoding gene (vma1, is among the best characterized homing endonucleases. The structures of the Sce VMA1 intein and of the intein bound to its target site are known. Extensive biochemical studies performed on the PI-SceI enzyme provide information useful to recognize critical amino acids involved in self-splicing and endonuclease functions of the protein. Here we describe an insertion of the Green Fluorescence Protein (GFP into a loop which is located between the endonuclease and splicing domains of the Sce VMA1 intein. The GFP is functional and the additional GFP domain does not prevent intein excision and endonuclease activity. However, the endonuclease activity of the newly engineered protein was different from the wild-type protein in that it required the presence of Mn2+ and not Mg2+ metal cations for activity.

  3. Crystal structure of an avian influenza polymerase PA[subscript N] reveals an endonuclease active site

    Energy Technology Data Exchange (ETDEWEB)

    Yuan, Puwei; Bartlam, Mark; Lou, Zhiyong; Chen, Shoudeng; Zhou, Jie; He, Xiaojing; Lv, Zongyang; Ge, Ruowen; Li, Xuemei; Deng, Tao; Fodor, Ervin; Rao, Zihe; Liu, Yingfang; (NU Sinapore); (Nankai); (Oxford); (Chinese Aca. Sci.); (Tsinghua)

    2009-11-10

    The heterotrimeric influenza virus polymerase, containing the PA, PB1 and PB2 proteins, catalyses viral RNA replication and transcription in the nucleus of infected cells. PB1 holds the polymerase active site and reportedly harbours endonuclease activity, whereas PB2 is responsible for cap binding. The PA amino terminus is understood to be the major functional part of the PA protein and has been implicated in several roles, including endonuclease and protease activities as well as viral RNA/complementary RNA promoter binding. Here we report the 2.2 angstrom (A) crystal structure of the N-terminal 197 residues of PA, termed PA(N), from an avian influenza H5N1 virus. The PA(N) structure has an alpha/beta architecture and reveals a bound magnesium ion coordinated by a motif similar to the (P)DX(N)(D/E)XK motif characteristic of many endonucleases. Structural comparisons and mutagenesis analysis of the motif identified in PA(N) provide further evidence that PA(N) holds an endonuclease active site. Furthermore, functional analysis with in vivo ribonucleoprotein reconstitution and direct in vitro endonuclease assays strongly suggest that PA(N) holds the endonuclease active site and has critical roles in endonuclease activity of the influenza virus polymerase, rather than PB1. The high conservation of this endonuclease active site among influenza strains indicates that PA(N) is an important target for the design of new anti-influenza therapeutics.

  4. Structural and biochemical basis for development of influenza virus inhibitors targeting the PA endonuclease.

    Directory of Open Access Journals (Sweden)

    Rebecca M DuBois

    Full Text Available Emerging influenza viruses are a serious threat to human health because of their pandemic potential. A promising target for the development of novel anti-influenza therapeutics is the PA protein, whose endonuclease activity is essential for viral replication. Translation of viral mRNAs by the host ribosome requires mRNA capping for recognition and binding, and the necessary mRNA caps are cleaved or "snatched" from host pre-mRNAs by the PA endonuclease. The structure-based development of inhibitors that target PA endonuclease is now possible with the recent crystal structure of the PA catalytic domain. In this study, we sought to understand the molecular mechanism of inhibition by several compounds that are known or predicted to block endonuclease-dependent polymerase activity. Using an in vitro endonuclease activity assay, we show that these compounds block the enzymatic activity of the isolated PA endonuclease domain. Using X-ray crystallography, we show how these inhibitors coordinate the two-metal endonuclease active site and engage the active site residues. Two structures also reveal an induced-fit mode of inhibitor binding. The structures allow a molecular understanding of the structure-activity relationship of several known influenza inhibitors and the mechanism of drug resistance by a PA mutation. Taken together, our data reveal new strategies for structure-based design and optimization of PA endonuclease inhibitors.

  5. Bacteriophage-Based Pathogen Detection

    Science.gov (United States)

    Ripp, Steven

    Considered the most abundant organism on Earth, at a population approaching 1031, bacteriophage, or phage for short, mediate interactions with myriad bacterial hosts that has for decades been exploited in phage typing schemes for signature identification of clinical, food-borne, and water-borne pathogens. With over 5,000 phage being morphologically characterized and grouped as to susceptible host, there exists an enormous cache of bacterial-specific sensors that has more recently been incorporated into novel bio-recognition assays with heightened sensitivity, specificity, and speed. These assays take many forms, ranging from straightforward visualization of labeled phage as they attach to their specific bacterial hosts to reporter phage that genetically deposit trackable signals within their bacterial hosts to the detection of progeny phage or other uniquely identifiable elements released from infected host cells. A comprehensive review of these and other phage-based detection assays, as directed towards the detection and monitoring of bacterial pathogens, will be provided in this chapter.

  6. Photodynamic Inactivation of Mammalian Viruses and Bacteriophages

    Directory of Open Access Journals (Sweden)

    Liliana Costa

    2012-06-01

    Full Text Available Photodynamic inactivation (PDI has been used to inactivate microorganisms through the use of photosensitizers. The inactivation of mammalian viruses and bacteriophages by photosensitization has been applied with success since the first decades of the last century. Due to the fact that mammalian viruses are known to pose a threat to public health and that bacteriophages are frequently used as models of mammalian viruses, it is important to know and understand the mechanisms and photodynamic procedures involved in their photoinactivation. The aim of this review is to (i summarize the main approaches developed until now for the photodynamic inactivation of bacteriophages and mammalian viruses and, (ii discuss and compare the present state of the art of mammalian viruses PDI with phage photoinactivation, with special focus on the most relevant mechanisms, molecular targets and factors affecting the viral inactivation process.

  7. Photodynamic inactivation of mammalian viruses and bacteriophages.

    Science.gov (United States)

    Costa, Liliana; Faustino, Maria Amparo F; Neves, Maria Graça P M S; Cunha, Angela; Almeida, Adelaide

    2012-07-01

    Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers. The inactivation of mammalian viruses and bacteriophages by photosensitization has been applied with success since the first decades of the last century. Due to the fact that mammalian viruses are known to pose a threat to public health and that bacteriophages are frequently used as models of mammalian viruses, it is important to know and understand the mechanisms and photodynamic procedures involved in their photoinactivation. The aim of this review is to (i) summarize the main approaches developed until now for the photodynamic inactivation of bacteriophages and mammalian viruses and, (ii) discuss and compare the present state of the art of mammalian viruses PDI with phage photoinactivation, with special focus on the most relevant mechanisms, molecular targets and factors affecting the viral inactivation process.

  8. Fragment-Based Identification of Influenza Endonuclease Inhibitors.

    Science.gov (United States)

    Credille, Cy V; Chen, Yao; Cohen, Seth M

    2016-07-14

    The influenza virus is responsible for millions of cases of severe illness annually. Yearly variance in the effectiveness of vaccination, coupled with emerging drug resistance, necessitates the development of new drugs to treat influenza infections. One attractive target is the RNA-dependent RNA polymerase PA subunit. Herein we report the development of inhibitors of influenza PA endonuclease derived from lead compounds identified from a metal-binding pharmacophore (MBP) library screen. Pyromeconic acid and derivatives thereof were found to be potent inhibitors of endonuclease. Guided by modeling and previously reported structural data, several sublibraries of molecules were elaborated from the MBP hits. Structure-activity relationships were established, and more potent molecules were designed and synthesized using fragment growth and fragment merging strategies. This approach ultimately resulted in the development of a lead compound with an IC50 value of 14 nM, which displayed an EC50 value of 2.1 μM against H1N1 influenza virus in MDCK cells. PMID:27291165

  9. Antiviral effect of cationic compounds on bacteriophages

    Directory of Open Access Journals (Sweden)

    Mai Huong eChatain-Ly

    2013-03-01

    Full Text Available The antiviral activity of several cationic compounds - cetytrimethylammonium (CTAB, chitosan, nisin and lysozyme - was investigated on the bacteriophage c2 (DNA head and non-contractile tail infecting Lactococcus strains and the bacteriophage MS2 (F-specific RNA infecting E.coli. Firstly, these activities were evaluated in a phosphate buffer pH 7- 10 mM. The CTAB had a virucidal effect on the Lactococcus bacteriophages, but not on the MS2. After 1 min of contact with 0.125 mM CTAB, the c2 population was reduced from 6 log(pfu/mL to 1,5 log(pfu/mL and completely deactivated at 1 mM. On the contrary, chitosan inhibited the MS2 more than it did the bacteriophages c2. No antiviral effect was observed for the nisin or the lysozyme on bacteriophages after 1 min of treatment. A 1 and 2.5 log reduction was respectively observed for nisin and lysozyme when the treatment time increased (5 or 10 min. These results showed that the antiviral effect depended both on the virus and structure of the antimicrobial compounds. The antiviral activity of these compounds was also evaluated in different physico-chemical conditions and in complex matrices. The antiviral activity of CTAB was impaired in acid pH and with an increase of the ionic strength. These results might be explained by the electrostatic interactions between cationic compounds and negatively charged particles such as bacteriophages or other compounds in a matrix. Milk proved to be protective suggesting the components of food could interfere with antimicrobial compounds.

  10. Genomic Sequencing and Biological Characteristics of a Novel Escherichia Coli Bacteriophage 9g, a Putative Representative of a New Siphoviridae Genus

    Directory of Open Access Journals (Sweden)

    Eugene E. Kulikov

    2014-12-01

    Full Text Available Bacteriophage 9g was isolated from horse feces using Escherichia coli C600 as a host strain. Phage 9g has a slightly elongated capsid 62 × 76 nm in diameter and a non-contractile tail about 185 nm long. The complete genome sequence of this bacteriophage consists of 56,703 bp encoding 70 predicted open reading frames. The closest relative of phage 9g is phage PhiJL001 infecting marine alpha-proteobacterium associated with Ircinia strobilina sponge, sharing with phage 9g 51% of amino acid identity in the main capsid protein sequence. The DNA of 9g is resistant to most restriction endonucleases tested, indicating the presence of hypermodified bases. The gene cluster encoding a biosynthesis pathway similar to biosynthesis of the unusual nucleoside queuosine was detected in the phage 9g genome. The genomic map organization is somewhat similar to the typical temperate phage gene layout but no integrase gene was detected. Phage 9g efficiently forms stable associations with its host that continues to produce the phage over multiple passages, but the phage can be easily eliminated via viricide treatment indicating that no true lysogens are formed. Since the sequence, genomic organization and biological properties of bacteriophage 9g are clearly distinct from other known Enterobacteriaceae phages, we propose to consider it as the representative of a novel genus of the Siphoviridae family.

  11. Inteins, introns, and homing endonucleases: recent revelations about the life cycle of parasitic genetic elements

    Directory of Open Access Journals (Sweden)

    Hilario Elena

    2006-11-01

    Full Text Available Abstract Self splicing introns and inteins that rely on a homing endonuclease for propagation are parasitic genetic elements. Their life-cycle and evolutionary fate has been described through the homing cycle. According to this model the homing endonuclease is selected for function only during the spreading phase of the parasite. This phase ends when the parasitic element is fixed in the population. Upon fixation the homing endonuclease is no longer under selection, and its activity is lost through random processes. Recent analyses of these parasitic elements with functional homing endonucleases suggest that this model in its most simple form is not always applicable. Apparently, functioning homing endonuclease can persist over long evolutionary times in populations and species that are thought to be asexual or nearly asexual. Here we review these recent findings and discuss their implications. Reasons for the long-term persistence of a functional homing endonuclease include: More recombination (sexual and as a result of gene transfer than previously assumed for these organisms; complex population structures that prevent the element from being fixed; a balance between active spreading of the homing endonuclease and a decrease in fitness caused by the parasite in the host organism; or a function of the homing endonuclease that increases the fitness of the host organism and results in purifying selection for the homing endonuclease activity, even after fixation in a local population. In the future, more detailed studies of the population dynamics of the activity and regulation of homing endonucleases are needed to decide between these possibilities, and to determine their relative contributions to the long term survival of parasitic genes within a population. Two outstanding publications on the amoeba Naegleria group I intron (Wikmark et al. BMC Evol Biol 2006, 6:39 and the PRP8 inteins in ascomycetes (Butler et al.BMC Evol Biol 2006, 6:42 provide

  12. H. pylori-Induced DNA Strand Breaks Are Introduced by Nucleotide Excision Repair Endonucleases and Promote NF-κB Target Gene Expression

    Directory of Open Access Journals (Sweden)

    Mara L. Hartung

    2015-10-01

    Full Text Available The human bacterial pathogen Helicobacter pylori exhibits genotoxic properties that promote gastric carcinogenesis. H. pylori introduces DNA double strand breaks (DSBs in epithelial cells that trigger host cell DNA repair efforts. Here, we show that H. pylori-induced DSBs are repaired via error-prone, potentially mutagenic non-homologous end-joining. A genome-wide screen for factors contributing to DSB induction revealed a critical role for the H. pylori type IV secretion system (T4SS. Inhibition of transcription, as well as NF-κB/RelA-specific RNAi, abrogates DSB formation. DSB induction further requires β1-integrin signaling. DSBs are introduced by the nucleotide excision repair endonucleases XPF and XPG, which, together with RelA, are recruited to chromatin in a highly coordinated, T4SS-dependent manner. Interestingly, XPF/XPG-mediated DNA DSBs promote NF-κB target gene transactivation and host cell survival. In summary, H. pylori induces XPF/XPG-mediated DNA damage through activation of the T4SS/β1-integrin signaling axis, which promotes NF-κB target gene expression and host cell survival.

  13. Do we need still more trials on T-4 and T-3 combination therapy in hypothyroidism?

    NARCIS (Netherlands)

    W. Wiersinga

    2009-01-01

    Approximately 10% of hypothyroid patients are dissatisfied with the outcome of levothyroxine replacement. It is unlikely that slight over- or under-treatment with thyroxine (T-4) explains remaining complaints Meta-analysis of randomized clinical trials shows no advantage of T-4/tri-iodothyronine (T-

  14. A stochastic model for bacteriophage therapies

    CERN Document Server

    Bardina, Xavier; Rovira, Carles; Tindel, Samy

    2011-01-01

    In this article, we analyze a system modeling bacteriophage treatments for infections in a noisy context. In the small noise regime, we show that after a reasonable amount of time the system is close to a sane equilibrium (which is a relevant biologic information) with high probability. Mathematically speaking, our study hinges on concentration techniques for delayed stochastic differential equations.

  15. Bacteriophages as surface and ground water tracers

    Directory of Open Access Journals (Sweden)

    P. Rossi

    1998-01-01

    Full Text Available Bacteriophages are increasingly used as tracers for quantitative analysis in both hydrology and hydrogeology. The biological particles are neither toxic nor pathogenic for other living organisms as they penetrate only a specific bacterial host. They have many advantages over classical fluorescent tracers and offer the additional possibility of multi-point injection for tracer tests. Several years of research make them suitable for quantitative transport analysis and flow boundary delineation in both surface and ground waters, including karst, fractured and porous media aquifers. This article presents the effective application of bacteriophages based on their use in differing Swiss hydrological environments and compares their behaviour to conventional coloured dye or salt-type tracers. In surface water and karst aquifers, bacteriophages travel at about the same speed as the typically referenced fluorescent tracers (uranine, sulphurhodamine G extra. In aquifers of interstitial porosity, however, they appear to migrate more rapidly than fluorescent tracers, albeit with a significant reduction in their numbers within the porous media. This faster travel time implies that a modified rationale is needed for defining some ground water protection area boundaries. Further developments of other bacteriophages and their documentation as tracer methods should result in an accurate and efficient tracer tool that will be a proven alternative to conventional fluorescent dyes.

  16. ADSORPTION OF BACTERIOPHAGES ON CLAY MINERALS

    Science.gov (United States)

    Theability to predict the fate of microorganisms in soil is dependent on an understanding of the process of their sorption on soil and subsurface materials. Presently, we have focused on studying the thermodynamics of sorption of bacteriophages (T-2, MS-2, and

  17. Comparative genomics of Shiga toxin encoding bacteriophages

    Directory of Open Access Journals (Sweden)

    Smith Darren L

    2012-07-01

    Full Text Available Abstract Background Stx bacteriophages are responsible for driving the dissemination of Stx toxin genes (stx across their bacterial host range. Lysogens carrying Stx phages can cause severe, life-threatening disease and Stx toxin is an integral virulence factor. The Stx-bacteriophage vB_EcoP-24B, commonly referred to as Ф24B, is capable of multiply infecting a single bacterial host cell at a high frequency, with secondary infection increasing the rate at which subsequent bacteriophage infections can occur. This is biologically unusual, therefore determining the genomic content and context of Ф24B compared to other lambdoid Stx phages is important to understanding the factors controlling this phenomenon and determining whether they occur in other Stx phages. Results The genome of the Stx2 encoding phage, Ф24B was sequenced and annotated. The genomic organisation and general features are similar to other sequenced Stx bacteriophages induced from Enterohaemorrhagic Escherichia coli (EHEC, however Ф24B possesses significant regions of heterogeneity, with implications for phage biology and behaviour. The Ф24B genome was compared to other sequenced Stx phages and the archetypal lambdoid phage, lambda, using the Circos genome comparison tool and a PCR-based multi-loci comparison system. Conclusions The data support the hypothesis that Stx phages are mosaic, and recombination events between the host, phages and their remnants within the same infected bacterial cell will continue to drive the evolution of Stx phage variants and the subsequent dissemination of shigatoxigenic potential.

  18. Bacteriophages as surface and ground water tracers

    Science.gov (United States)

    Rossi, P.; Dörfliger, N.; Kennedy, K.; Müller, I.; Aragno, M.

    Bacteriophages are increasingly used as tracers for quantitative analysis in both hydrology and hydrogeology. The biological particles are neither toxic nor pathogenic for other living organisms as they penetrate only a specific bacterial host. They have many advantages over classical fluorescent tracers and offer the additional possibility of multi-point injection for tracer tests. Several years of research make them suitable for quantitative transport analysis and flow boundary delineation in both surface and ground waters, including karst, fractured and porous media aquifers. This article presents the effective application of bacteriophages based on their use in differing Swiss hydrological environments and compares their behaviour to conventional coloured dye or salt-type tracers. In surface water and karst aquifers, bacteriophages travel at about the same speed as the typically referenced fluorescent tracers (uranine, sulphurhodamine G extra). In aquifers of interstitial porosity, however, they appear to migrate more rapidly than fluorescent tracers, albeit with a significant reduction in their numbers within the porous media. This faster travel time implies that a modified rationale is needed for defining some ground water protection area boundaries. Further developments of other bacteriophages and their documentation as tracer methods should result in an accurate and efficient tracer tool that will be a proven alternative to conventional fluorescent dyes.

  19. An Undergraduate Laboratory Activity Demonstrating Bacteriophage Specificity

    Directory of Open Access Journals (Sweden)

    Mary E. Allen

    2013-02-01

    Full Text Available Bacteriophage are among the most diverse and numerous microbes inhabiting our planet. Yet many laboratory activities fail to engage students in meaningful exploration of their diversity, unique characteristics, and abundance. In this curriculum activity students use a standard plaque assay to enumerate bacteriophage particles from a natural sample and use the scientific method to address questions about host specificity and diversity. A raw primary sewage sample is enriched for bacteriophage using hosts in the family Enterobacteriaceae. Students hypothesize about host specificity and use quantitative data (serial dilution and plaque assay to test their hypotheses. Combined class data also help them answer questions about phage diversity. The exercise was field tested with a class of 47 students using pre- and posttests. For all learning outcomes posttest scores were higher than pretest scores at or below p = 0.01. Average individualized learning gain (G was also calculated for each learning outcome. Students’ use of scientific language in reference to bacteriophage and host interaction significantly improved (p = 0.002; G = 0.50. Improved means of expression helped students construct better hypotheses on phage host specificity (G = 0.31, p = 0.01 and to explain the plaque assay method (G = 0.33, p = 0.002. At the end of the exercise students also demonstrated improved knowledge and understanding of phage specificity as related to phage therapy in humans (p < 0.001; G = 51.

  20. pp-GalNAc-T4表达载体的构建

    Institute of Scientific and Technical Information of China (English)

    李喆; 郭向红; 吴士良

    2011-01-01

    目的 构建pp - GalNAc - T4的正义表达载体.方法 采用质粒转染法构建真核表达载体.结果 构建了pp -GalNAc-T4的正义表达载体,并得到了鉴定.结论 构建了pp - GalNAc - T4的正义表达载体,研究发现了在pp - GalNAc - T4c DNA转染NB4细胞株后,在mRNA水平及蛋白水平能稳定上调pp-GalNAc-T4的表达,可以用于以后的研究.

  1. A ligation-independent cloning method using nicking DNA endonuclease.

    Science.gov (United States)

    Yang, Jie; Zhang, Zhihong; Zhang, Xin A; Luo, Qingming

    2010-11-01

    Using nicking DNA endonuclease (NiDE), we developed a novel technique to clone DNA fragments into plasmids. We created a NiDE cassette consisting of two inverted NiDE substrate sites sandwiching an asymmetric four-base sequence, and NiDE cleavage resulted in 14-base single-stranded termini at both ends of the vector and insert. This method can therefore be used as a ligation-independent cloning strategy to generate recombinant constructs rapidly. In addition, we designed and constructed a simple and specific vector from an Escherichia coli plasmid back-bone to complement this cloning method. By cloning cDNAs into this modified vector, we confirmed the predicted feasibility and applicability of this cloning method. PMID:21091446

  2. Mitochondrial endonuclease G mediates breakdown of paternal mitochondria upon fertilization.

    Science.gov (United States)

    Zhou, Qinghua; Li, Haimin; Li, Hanzeng; Nakagawa, Akihisa; Lin, Jason L J; Lee, Eui-Seung; Harry, Brian L; Skeen-Gaar, Riley Robert; Suehiro, Yuji; William, Donna; Mitani, Shohei; Yuan, Hanna S; Kang, Byung-Ho; Xue, Ding

    2016-07-22

    Mitochondria are inherited maternally in most animals, but the mechanisms of selective paternal mitochondrial elimination (PME) are unknown. While examining fertilization in Caenorhabditis elegans, we observed that paternal mitochondria rapidly lose their inner membrane integrity. CPS-6, a mitochondrial endonuclease G, serves as a paternal mitochondrial factor that is critical for PME. We found that CPS-6 relocates from the intermembrane space of paternal mitochondria to the matrix after fertilization to degrade mitochondrial DNA. It acts with maternal autophagy and proteasome machineries to promote PME. Loss of cps-6 delays breakdown of mitochondrial inner membranes, autophagosome enclosure of paternal mitochondria, and PME. Delayed removal of paternal mitochondria causes increased embryonic lethality, demonstrating that PME is important for normal animal development. Thus, CPS-6 functions as a paternal mitochondrial degradation factor during animal development.

  3. Human RECQL5beta stimulates flap endonuclease 1

    DEFF Research Database (Denmark)

    Speina, Elzbieta; Dawut, Lale; Hedayati, Mohammad;

    2010-01-01

    devoid of RECQL1 and RECQL5 display increased chromosomal instability. Here, we report the physical and functional interaction of the large isomer of RECQL5, RECQL5beta, with the human flap endonuclease 1, FEN1, which plays a critical role in DNA replication, recombination and repair. RECQL5beta...... dramatically stimulates the rate of FEN1 cleavage of flap DNA substrates. Moreover, we show that RECQL5beta and FEN1 interact physically and co-localize in the nucleus in response to DNA damage. Our findings, together with the previous literature on WRN, BLM and RECQL4's stimulation of FEN1, suggests...... that the ability of RecQ helicases to stimulate FEN1 may be a general feature of this class of enzymes. This could indicate a common role for the RecQ helicases in the processing of oxidative DNA damage....

  4. STUDIES ON THE PURIFICATION OF BACTERIOPHAGE.

    Science.gov (United States)

    Kalmanson, G; Bronfenbrenner, J

    1939-11-20

    A simple method of concentrating and purifying bacteriophage has been described. The procedure consisted essentially in collecting the active agent on a reinforced collodion membrane of a porosity that would just retain all the active agent and permit extraneous material to pass through. Advantage was taken of the fact that B. coli will proliferate and regenerate bacteriophage in a completely diffusible synthetic medium with ammonia as the only source of nitrogen, which permitted the purification of the bacteriophage by copious washing. The material thus obtained was concentrated by suction and after thorough washing possessed all the activity of the original filtrate. It was labile, losing its activity in a few days on standing, and was quickly and completely inactivated upon drying. This material contained approximately 15 per cent of nitrogen and with 2 or 3 mg. samples of inactive dry residue it was possible to obtain positive protein color tests. The concentrated and purified bacteriophage has about 10(-14) mg. of nitrogen, or 6 x 10(-17) gm. of protein per unit of lytic activity. Assuming that each unit of activity represents a molecule, the calculated maximum average molecular weight would be approximately 36,000,000, and on the assumption of a spherical shape of particles and a density of 1.3, the calculated radius would be about 22 millimicra. By measurement of the diffusion rate, the average radius of particle of the fraction of the purified bacteriophage which diffuses most readily through a porous plate was found to be of the order of magnitude of 9 millimicra, or of a calculated molecular weight of 2,250,000. Furthermore, when this purified bacteriophage was fractionated by forcing it through a thin collodion membrane, which permits the passage of only the smaller particles, it was possible to demonstrate in the ultrafiltrate active particles of about 2 millimicra in radius, and of a calculated molecular weight of 25,000. It was of interest to apply

  5. The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection.

    Directory of Open Access Journals (Sweden)

    Qingwen Jin

    Full Text Available Insertion of T4 lysozyme (T4L into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed.We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects.Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1 infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5.Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

  6. Biogeography of bacteriophages at four hydrothermal vent sites in the Antarctic based on g23 sequence diversity.

    Science.gov (United States)

    Millard, Andrew D; Pearce, David; Zwirglmaier, Katrin

    2016-04-01

    In this study, which was carried out within the ChEsSO consortium project (Chemosynthetically driven ecosystems south of the Polar Front), we sampled two hydrothermal vent sites on the East Scotia Ridge, Scotia Sea, one in the Kemp Caldera, South Sandwich Arc and one in the Bransfield Strait, north-west of the Antarctic Peninsula, which exhibit strong differences in their chemical characteristics. We compared a subset of their bacteriophage population by Sanger- and 454-sequencing of g23, which codes for the major capsid protein of T4likeviruses. We found that the sites differ vastly in their bacteriophage diversity, which reflects the differences in the chemical conditions and therefore putatively the differences in microbial hosts living at these sites. Comparing phage diversity in the vent samples to other aquatic samples, the vent samples formed a distinct separate cluster, which also included the non-vent control samples that were taken several hundred meters above the vent chimneys. This indicates that the influence of the vents on the microbial population and therefore also the bacteriophage population extends much further than anticipated.

  7. Biogeography of bacteriophages at four hydrothermal vent sites in the Antarctic based on g23 sequence diversity.

    Science.gov (United States)

    Millard, Andrew D; Pearce, David; Zwirglmaier, Katrin

    2016-04-01

    In this study, which was carried out within the ChEsSO consortium project (Chemosynthetically driven ecosystems south of the Polar Front), we sampled two hydrothermal vent sites on the East Scotia Ridge, Scotia Sea, one in the Kemp Caldera, South Sandwich Arc and one in the Bransfield Strait, north-west of the Antarctic Peninsula, which exhibit strong differences in their chemical characteristics. We compared a subset of their bacteriophage population by Sanger- and 454-sequencing of g23, which codes for the major capsid protein of T4likeviruses. We found that the sites differ vastly in their bacteriophage diversity, which reflects the differences in the chemical conditions and therefore putatively the differences in microbial hosts living at these sites. Comparing phage diversity in the vent samples to other aquatic samples, the vent samples formed a distinct separate cluster, which also included the non-vent control samples that were taken several hundred meters above the vent chimneys. This indicates that the influence of the vents on the microbial population and therefore also the bacteriophage population extends much further than anticipated. PMID:26903011

  8. Effects of 18 months of L-T4 replacement in women with subclinical hypothyroidism.

    LENUS (Irish Health Repository)

    Adrees, M

    2012-02-01

    CONTEXT: Some of the cardiovascular and renal abnormalities seen in overt hypothyroidism have also been reported in subclinical hypothyroidism (SCH). Short-term L-T4 replacement in SCH improves cardiovascular risk markers and reduces carotid intima-media thickness (CIMT), a surrogate marker of atherosclerosis. The haemodynamic and renal effects of L-T4 replacement in SCH are poorly understood. OBJECTIVES: To compare cardiovascular risk factors and renal variables in women with SCH and normal women. To study the effects of L-T4 replacement in SCH subjects on these variables and on structural and functional changes in common carotid and brachial arteries. DESIGN: Fifty-six women with SCH before and after L-T4 replacement for 18 months and 56 normal women of similar age distribution were studied. Blood Pressure (BP), plasma lipids and homocysteine were measured and renal function evaluated [estimation of glomerular filtration rate (eGFR) using standard equations and measurement of serum Cystatin-C] in women with SCH before and after 18 months of l-T4, and in healthy women. CIMT and endothelial function (using brachial artery ultrasound) were studied before and after L-T4 in a subgroup of women with SCH. RESULTS: Systolic and diastolic BP, total cholesterol, triglyceride, LDL-cholesterol, lipoprotein(a) and homocysteine were greater in SCH (P < 0.05), and following L-T4 replacement decreased (P < 0.05) to levels that no longer differed from normal subjects. Estimated GFR was reduced and serum Cystatin-C increased (P < 0.05) in SCH. These variables also normalized following L-T4. Following L-T4 replacement the carotid artery baseline diameter increased by 7.1% and CIMT decreased by a mean value of 13%, while brachial artery diameter increased basally by 12.5% and following endothelium-dependent vasodilatation by 17.5% (P < 0.05). However, the increment following reactive hyperaemia did not differ before or following L-T4 replacement. CONCLUSION: Normalization of

  9. Oxygen effect in bacteriophages irradiated in different media. 1. Irradiation in salt solution

    Energy Technology Data Exchange (ETDEWEB)

    Korystov, Yu.N.; Veksler, F.B. (AN SSSR, Pushchino-na-Oke. Inst. Biologicheskoj Fiziki)

    1983-09-14

    The oxygen effect (OE) on bacteriophage T4 in a salt solution was studied. It is shown that the sign and magnitude of OE depend on the conditions of the postirradiation incubation of the phage in irradiated medium. The direct OE is due to postirradiation lesion of the phage by hydrogen peroxide which is formed in greater amounts after irradiation in oxygen than in anoxia. The addition of catalase is shown to eliminate the postirradiation inactivation of the phage. In this case an opposite OE is observed. The mechanism of this effect is a scavenge of hydrogen atoms which damage the phage by oxygen. In the presence of catalase the OE depends also on pH of the solution. It is suggested that the hydroxyl radical arising from the reaction of H/sub 2/O/sub 2/ with Fe/sup 2 +/ is responsible for the damaging effect of H/sub 2/O/sub 2/.

  10. Evolution and the complexity of bacteriophages

    Directory of Open Access Journals (Sweden)

    Serwer Philip

    2007-03-01

    Full Text Available Abstract Background The genomes of both long-genome (> 200 Kb bacteriophages and long-genome eukaryotic viruses have cellular gene homologs whose selective advantage is not explained. These homologs add genomic and possibly biochemical complexity. Understanding their significance requires a definition of complexity that is more biochemically oriented than past empirically based definitions. Hypothesis Initially, I propose two biochemistry-oriented definitions of complexity: either decreased randomness or increased encoded information that does not serve immediate needs. Then, I make the assumption that these two definitions are equivalent. This assumption and recent data lead to the following four-part hypothesis that explains the presence of cellular gene homologs in long bacteriophage genomes and also provides a pathway for complexity increases in prokaryotic cells: (1 Prokaryotes underwent evolutionary increases in biochemical complexity after the eukaryote/prokaryote splits. (2 Some of the complexity increases occurred via multi-step, weak selection that was both protected from strong selection and accelerated by embedding evolving cellular genes in the genomes of bacteriophages and, presumably, also archaeal viruses (first tier selection. (3 The mechanisms for retaining cellular genes in viral genomes evolved under additional, longer-term selection that was stronger (second tier selection. (4 The second tier selection was based on increased access by prokaryotic cells to improved biochemical systems. This access was achieved when DNA transfer moved to prokaryotic cells both the more evolved genes and their more competitive and complex biochemical systems. Testing the hypothesis I propose testing this hypothesis by controlled evolution in microbial communities to (1 determine the effects of deleting individual cellular gene homologs on the growth and evolution of long genome bacteriophages and hosts, (2 find the environmental conditions that

  11. Cloning and characterization of a wheat homologue of apurinic/apyrimidinic endonuclease Ape1L.

    Directory of Open Access Journals (Sweden)

    Botagoz Joldybayeva

    Full Text Available BACKGROUND: Apurinic/apyrimidinic (AP endonucleases are key DNA repair enzymes involved in the base excision repair (BER pathway. In BER, an AP endonuclease cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases and/or oxidative damage. A Triticum aestivum cDNA encoding for a putative homologue of ExoIII family AP endonucleases which includes E. coli Xth, human APE1 and Arabidopsis thaliana AtApe1L has been isolated and its protein product purified and characterized. METHODOLOGY/PRINCIPAL FINDINGS: We report that the putative wheat AP endonuclease, referred here as TaApe1L, contains AP endonuclease, 3'-repair phosphodiesterase, 3'-phosphatase and 3' → 5' exonuclease activities. Surprisingly, in contrast to bacterial and human AP endonucleases, addition of Mg(2+ and Ca(2+ (5-10 mM to the reaction mixture inhibited TaApe1L whereas the presence of Mn(2+, Co(2+ and Fe(2+ cations (0.1-1.0 mM strongly stimulated all its DNA repair activities. Optimization of the reaction conditions revealed that the wheat enzyme requires low divalent cation concentration (0.1 mM, mildly acidic pH (6-7, low ionic strength (20 mM KCl and has a temperature optimum at around 20 °C. The steady-state kinetic parameters of enzymatic reactions indicate that TaApe1L removes 3'-blocking sugar-phosphate and 3'-phosphate groups with good efficiency (kcat/KM = 630 and 485 μM(-1 · min(-1, respectively but possesses a very weak AP endonuclease activity as compared to the human homologue, APE1. CONCLUSIONS/SIGNIFICANCE: Taken together, these data establish the DNA substrate specificity of the wheat AP endonuclease and suggest its possible role in the repair of DNA damage generated by endogenous and environmental factors.

  12. Characterization of Borrelia burgdorferi isolates by restriction endonuclease analysis and DNA hybridization.

    OpenAIRE

    LeFebvre, R B; Perng, G C; Johnson, R C

    1989-01-01

    Genomes of several Borrelia burgdorferi isolates from North America and Europe were characterized by restriction endonuclease analysis and DNA hybridization using labeled B. burgdorferi whole-cell DNA (strain ATCC 35210). Several different restriction and homology patterns were observed among these isolates, indicating genotypic heterogeneity within this genus and species. It was concluded from this study that restriction endonuclease analysis of B. burgdorferi whole-cell DNA may be a reliabl...

  13. Analyzing the forces binding a restriction endonuclease to DNA using a synthetic nanopore

    OpenAIRE

    Dorvel, B.; Sigalov, G.; Zhao, Q.; Comer, J.; Dimitrov, V; Mirsaidov, U.; Aksimentiev, A.; Timp, G.

    2009-01-01

    Restriction endonucleases are used prevalently in recombinant DNA technology because they bind so stably to a specific target sequence and, in the presence of cofactors, cleave double-helical DNA specifically at a target sequence at a high rate. Using synthetic nanopores along with molecular dynamics (MD), we have analyzed with atomic resolution how a prototypical restriction endonuclease, EcoRI, binds to the DNA target sequence—GAATTC—in the absence of a Mg2+ ion cofactor. We have previously...

  14. Cleavage and protection of locked nucleic acid-modified DNA by restriction endonucleases

    DEFF Research Database (Denmark)

    Crouzier, Lucile; Dubois, Camille; Wengel, Jesper;

    2012-01-01

    Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far. We herein for the first time report cleavage by restriction endonuclease of LNA-modified DNA oligonucleotides. The experiments revealed that RsaI is an efficient enzyme capable of recognizing and cleaving...... LNA-modified DNA oligonucleotides. Furthermore, introduction of LNA nucleotides protects against cleavage by the restriction endonucleases PvuII, PstI, SacI, KpnI and EcoRI....

  15. T4-induced antipolarity: temporal heterogeneity in response of early transcription units.

    OpenAIRE

    Thermes, C; Brody, E

    1984-01-01

    When T4 infects Escherichia coli in the absence of protein synthesis, rho-mediated termination takes place on early polycistronic transcription units. During the early period of development, the appearance of delayed early transcripts becomes insensitive to the inhibition of protein synthesis. In the absence of the T4 gene product mot, an inducer for the middle mode of transcription, only the early polycistronic messengers are synthesized. In mot- -infected cells, the synthesis of the distal ...

  16. A novel DNA joining activity catalyzed by T4 DNA ligase.

    OpenAIRE

    Western, L M; Rose, S. J.

    1991-01-01

    The use of T4 and E. coli DNA ligases in genetic engineering technology is usually associated with nick-closing activity in double stranded DNA or ligation of 'sticky-ends' to produce recombinant DNA molecules. We describe in this communication the ability of T4 DNA ligase to catalyze intramolecular loop formation between annealed oligodeoxyribonucleotides wherein Watson-Crick base pairing is absent on one side of the ligation site. Enzyme concentration, loop size, substrate specificity, and ...

  17. Bacteriophages and bacteriophage-derived endolysins as potential therapeutics to combat Gram-positive spore forming bacteria.

    Science.gov (United States)

    Nakonieczna, A; Cooper, C J; Gryko, R

    2015-09-01

    Since their discovery in 1915, bacteriophages have been routinely used within Eastern Europe to treat a variety of bacterial infections. Although initially ignored by the West due to the success of antibiotics, increasing levels and diversity of antibiotic resistance is driving a renaissance for bacteriophage-derived therapy, which is in part due to the highly specific nature of bacteriophages as well as their relative abundance. This review focuses on the bacteriophages and derived lysins of relevant Gram-positive spore formers within the Bacillus cereus group and Clostridium genus that could have applications within the medical, food and environmental sectors.

  18. Application of bacteriophages in sensor development.

    Science.gov (United States)

    Peltomaa, Riikka; López-Perolio, Irene; Benito-Peña, Elena; Barderas, Rodrigo; Moreno-Bondi, María Cruz

    2016-03-01

    Bacteriophage-based bioassays are a promising alternative to traditional antibody-based immunoassays. Bacteriophages, shortened to phages, can be easily conjugated or genetically engineered. Phages are robust, ubiquitous in nature, and harmless to humans. Notably, phages do not usually require inoculation and killing of animals; and thus, the production of phages is simple and economical. In recent years, phage-based biosensors have been developed featuring excellent robustness, sensitivity, and selectivity in combination with the ease of integration into transduction devices. This review provides a critical overview of phage-based bioassays and biosensors developed in the last few years using different interrogation methods such as colorimetric, enzymatic, fluorescence, surface plasmon resonance, quartz crystal microbalance, magnetoelastic, Raman, or electrochemical techniques.

  19. Detection of bacteria with bioluminescent reporter bacteriophage.

    Science.gov (United States)

    Klumpp, Jochen; Loessner, Martin J

    2014-01-01

    Bacteriophages are viruses that exclusively infect bacteria. They are ideally suited for the development of highly specific diagnostic assay systems. Bioluminescent reporter bacteriophages are designed and constructed by integration of a luciferase gene in the virus genome. Relying on the host specificity of the phage, the system enables rapid, sensitive, and specific detection of bacterial pathogens. A bioluminescent reporter phage assay is superior to any other molecular detection method, because gene expression and light emission are dependent on an active metabolism of the bacterial cell, and only viable cells will yield a signal. In this chapter we introduce the concept of creating reporter phages, discuss their advantages and disadvantages, and illustrate the advances made in developing such systems for different Gram-negative and Gram-positive pathogens. The application of bioluminescent reporter phages for the detection of foodborne pathogens is emphasized.

  20. Activity-based in vitro selection of T4 DNA ligase

    International Nuclear Information System (INIS)

    Recent in vitro methodologies for selection and directed evolution of proteins have concentrated not only on proteins with affinity such as single-chain antibody but also on enzymes. We developed a display technology for selection of T4 DNA ligase on ribosome because an in vitro selection method for DNA ligase had never been developed. The 3' end of mRNA encoding the gene of active or inactive T4 DNA ligase-spacer peptide fusion protein was hybridized to dsDNA fragments with cohesive ends, the substrate of T4 DNA ligase. After in vitro translation of the mRNA-dsDNA complex in a rabbit reticulocyte system, a mRNA-dsDNA-ribosome-ligase complex was produced. T4 DNA ligase enzyme displayed on a ribosome, through addition of a spacer peptide, is able to react with dsDNA in the complex. The complex expressing active ligase was biotinylated by ligation with another biotinylated dsDNA probe and selected with streptavidin-coated magnetic beads. We effectively selected active T4 DNA ligase from a small amount of protein. The gene of the active T4 DNA ligase was enriched 40 times from a mixture of active and inactive genes using this selection strategy. This ribosomal display strategy may have high potential to be useful for selection of other enzymes associated with DNA

  1. Bacteriophages as recognition and identification agents

    Energy Technology Data Exchange (ETDEWEB)

    Teodorescu, M.C.; Gaspar, A.

    1987-04-23

    Bacteriophages are employed as agents for recognition and identification of molecules and cellular materials, using their ability to recognize their bacterial host, by coating them with antibodies or by selecting them to perform in a manner analogous to antibodies. Visibility for identification is effected by incorporating a fluorescent agent, a radioisotope, a metal, an enzyme, or other staining material. The method of this invention may be utilized in selected clinical procedures, and is adaptable to use in an assay kit.

  2. Going viral: designing bioactive surfaces with bacteriophage.

    Science.gov (United States)

    Hosseinidoust, Zeinab; Olsson, Adam L J; Tufenkji, Nathalie

    2014-12-01

    Bacteriophage-functionalized bioactive surfaces are functional materials that can be used as antimicrobial surfaces in medical applications (e.g., indwelling medical devices or wound dressings) or as biosensors for bacterial capture and detection. Despite offering immense potential, designing efficient phage-functionalized bioactive surfaces is hampered by a number of challenges. This review offers an overview of the current state of knowledge in this field and presents a critical perspective of the technological promises and challenges.

  3. A new look at bacteriophage phylogenomics

    OpenAIRE

    Nóbrega, Franklin; Pinto, Graça; Azeredo, Joana; Kluskens, Leon

    2012-01-01

    Bacteriophages or phages are viruses that only infect bacteria. The International Committee on Taxonomy of Viruses classified these viruses in accordance with the morphology of their free virion particles and type and size of their genome. This system fails on the classification of several phages, which have their genome already sequenced. It also requires a morphological analysis by transmission electron microscopy, which is very expensive and time consuming [1]. In 2002 Rohwe...

  4. Genomic impact of CRISPR immunization against bacteriophages.

    Science.gov (United States)

    Barrangou, Rodolphe; Coûté-Monvoisin, Anne-Claire; Stahl, Buffy; Chavichvily, Isabelle; Damange, Florian; Romero, Dennis A; Boyaval, Patrick; Fremaux, Christophe; Horvath, Philippe

    2013-12-01

    CRISPR (clustered regularly interspaced short palindromic repeats) together with CAS (RISPR-associated) genes form the CRISPR-Cas immune system, which provides sequence-specific adaptive immunity against foreign genetic elements in bacteria and archaea. Immunity is acquired by the integration of short stretches of invasive DNA as novel 'spacers' into CRISPR loci. Subsequently, these immune markers are transcribed and generate small non-coding interfering RNAs that specifically guide nucleases for sequence-specific cleavage of complementary sequences. Among the four CRISPR-Cas systems present in Streptococcus thermophilus, CRISPR1 and CRISPR3 have the ability to readily acquire new spacers following bacteriophage or plasmid exposure. In order to investigate the impact of building CRISPR-encoded immunity on the host chromosome, we determined the genome sequence of a BIM (bacteriophage-insensitive mutant) derived from the DGCC7710 model organism, after four consecutive rounds of bacteriophage challenge. As expected, active CRISPR loci evolved via polarized addition of several novel spacers following exposure to bacteriophages. Although analysis of the draft genome sequence revealed a variety of SNPs (single nucleotide polymorphisms) and INDELs (insertions/deletions), most of the in silico differences were not validated by Sanger re-sequencing. In addition, two SNPs and two small INDELs were identified and tracked in the intermediate variants. Overall, building CRISPR-encoded immunity does not significantly affect the genome, which allows the maintenance of important functional properties in isogenic CRISPR mutants. This is critical for the development and formulation of sustainable and robust next-generation starter cultures with increased industrial lifespans.

  5. Isolation of Arthrobacter Bacteriophage from Soil †

    OpenAIRE

    Germida, James J.; Casida, L. E.

    1981-01-01

    Soil was percolated with water and various nutrient solutions, and then the percolates were analyzed for bacteriophages which produced plaques on various Arthrobacter strains. The water percolates did not contain detectable phage. In contrast, phages for A. globiformis strains ATCC 8010 and 4336, and for several recent Arthrobacter species soil isolates, were easily detected in nutrient broth, soil extract, and cation-complete medium percolates. These percolates did not contain phage that pro...

  6. Use of bacteriophages to control biofilms

    OpenAIRE

    Sillankorva, Sanna

    2009-01-01

    Tese de doutoramento em Engenharia Química e Biológica After several years of abandonment, the use of bacteriophages (phages) for killing bacteria has withdrawn recent attention and reappraisal. This has led to a vast phage research, in varied fields, with impressive outcomes and currently several studies are ongoing with animals, horticulture and agriculture products, and even with humans. Despite this enthusiasm, there is a lack of research conserning phage utilization to red...

  7. A putative Type IIS restriction endonuclease GeoICI from Geobacillus sp.--A robust, thermostable alternative to mezophilic prototype BbvI.

    Science.gov (United States)

    Zebrowska, Joanna; Zolnierkiewicz, Olga; Skowron, Marta A; Zylicz-Stachula, Agnieszka; Jezewska-Frackowiak, Joanna; Skowron, Piotr M

    2016-03-01

    Screening of extreme environments in search for novel microorganisms may lead to the discovery of robust enzymes with either new substrate specificities or thermostable equivalents of those already found in mesophiles, better suited for biotechnology applications. Isolates from Iceland geysers' biofilms, exposed to a broad range of temperatures, from ambient to close to water boiling point, were analysed for the presence of DNA-interacting proteins, including restriction endonucleases (REases). GeoICI, a member of atypical Type IIS REases, is the most thermostable isoschizomer of the prototype BbvI, recognizing/cleaving 5'-GCAGC(N8/12)-3'DNA sequences. As opposed to the unstable prototype, which cleaves DNA at 30°C, GeoICI is highly active at elevated temperatures, up to 73°C and over a very wide salt concentration range. Recognition/cleavage sites were determined by: (i) digestion of plasmid and bacteriophage lambda DNA (Λ); (ii) cleavage of custom PCR substrates, (iii) run-off sequencing of GeoICI cleavage products and (iv) shotgun cloning and sequencing of Λ DNA fragmented with GeoICI. Geobacillus sp. genomic DNA was PCR-screened for the presence of other specialized REases-MTases and as a result, another putative REase- MTase, GeoICII, related to the Thermus sp. family of bifunctional REases-methyltransferases (MTases) was detected. PMID:26949085

  8. A putative Type IIS restriction endonuclease GeoICI from Geobacillus sp. – A robust, thermostable alternative to mezophilic prototype BbvI

    Indian Academy of Sciences (India)

    Joanna Zebrowska; Olga Zołnierkiewicz; Marta A Skowron; Agnieszka Zylicz-Stachula; Joanna Jezewska-Frackowiak; Piotr M Skowron

    2016-03-01

    Screening of extreme environments in search for novel microorganisms may lead to the discovery of robust enzymes with either new substrate specificities or thermostable equivalents of those already found in mesophiles, better suited for biotechnology applications. Isolates from Iceland geysers’ biofilms, exposed to a broad range of temperatures, from ambient to close to water boiling point, were analysed for the presence of DNA-interacting proteins, including restriction endonucleases (REases). GeoICI, a member of atypical Type IIS REases, is the most thermostable isoschizomer of the prototype BbvI, recognizing/cleaving 5′-GCAGC(N8/12)-3′ DNA sequences. As opposed to the unstable prototype, which cleaves DNA at 30°C, GeoICI is highly active at elevated temperatures, up to 73°C and over a very wide salt concentration range. Recognition/cleavage sites were determined by: (i) digestion of plasmid and bacteriophage lambda DNA (λ); (ii) cleavage of custom PCR substrates, (iii) run-off sequencing of GeoICI cleavage products and (iv) shotgun cloning and sequencing of λ DNA fragmented with GeoICI. Geobacillus sp. genomic DNA was PCR-screened for the presence of other specialized REases-MTases and as a result, another putative REase-MTase, GeoICII, related to the Thermus sp. family of bifunctional REases-methyltransferases (MTases) was detected.

  9. Genetically modified bacteriophages in applied microbiology.

    Science.gov (United States)

    Bárdy, P; Pantůček, R; Benešík, M; Doškař, J

    2016-09-01

    Bacteriophages represent a simple viral model of basic research with many possibilities for practical application. Due to their ability to infect and kill bacteria, their potential in the treatment of bacterial infection has been examined since their discovery. With advances in molecular biology and gene engineering, the phage application spectrum has been expanded to various medical and biotechnological fields. The construction of bacteriophages with an extended host range or longer viability in the mammalian bloodstream enhances their potential as an alternative to conventional antibiotic treatment. Insertion of active depolymerase genes to their genomes can enforce the biofilm disposal. They can also be engineered to transfer various compounds to the eukaryotic organisms and the bacterial culture, applicable for the vaccine, drug or gene delivery. Phage recombinant lytic enzymes can be applied as enzybiotics in medicine as well as in biotechnology for pathogen detection or programmed cell death in bacterial expression strains. Besides, modified bacteriophages with high specificity can be applied as bioprobes in detection tools to estimate the presence of pathogens in food industry, or utilized in the control of food-borne pathogens as part of the constructed phage-based biosorbents.

  10. Genetically modified bacteriophages in applied microbiology.

    Science.gov (United States)

    Bárdy, P; Pantůček, R; Benešík, M; Doškař, J

    2016-09-01

    Bacteriophages represent a simple viral model of basic research with many possibilities for practical application. Due to their ability to infect and kill bacteria, their potential in the treatment of bacterial infection has been examined since their discovery. With advances in molecular biology and gene engineering, the phage application spectrum has been expanded to various medical and biotechnological fields. The construction of bacteriophages with an extended host range or longer viability in the mammalian bloodstream enhances their potential as an alternative to conventional antibiotic treatment. Insertion of active depolymerase genes to their genomes can enforce the biofilm disposal. They can also be engineered to transfer various compounds to the eukaryotic organisms and the bacterial culture, applicable for the vaccine, drug or gene delivery. Phage recombinant lytic enzymes can be applied as enzybiotics in medicine as well as in biotechnology for pathogen detection or programmed cell death in bacterial expression strains. Besides, modified bacteriophages with high specificity can be applied as bioprobes in detection tools to estimate the presence of pathogens in food industry, or utilized in the control of food-borne pathogens as part of the constructed phage-based biosorbents. PMID:27321680

  11. Call for a dedicated European legal framework for bacteriophage therapy.

    Science.gov (United States)

    Verbeken, Gilbert; Pirnay, Jean-Paul; Lavigne, Rob; Jennes, Serge; De Vos, Daniel; Casteels, Minne; Huys, Isabelle

    2014-04-01

    The worldwide emergence of antibiotic resistances and the drying up of the antibiotic pipeline have spurred a search for alternative or complementary antibacterial therapies. Bacteriophages are bacterial viruses that have been used for almost a century to combat bacterial infections, particularly in Poland and the former Soviet Union. The antibiotic crisis has triggered a renewed clinical and agricultural interest in bacteriophages. This, combined with new scientific insights, has pushed bacteriophages to the forefront of the search for new approaches to fighting bacterial infections. But before bacteriophage therapy can be introduced into clinical practice in the European Union, several challenges must be overcome. One of these is the conceptualization and classification of bacteriophage therapy itself and the extent to which it constitutes a human medicinal product regulated under the European Human Code for Medicines (Directive 2001/83/EC). Can therapeutic products containing natural bacteriophages be categorized under the current European regulatory framework, or should this framework be adapted? Various actors in the field have discussed the need for an adapted (or entirely new) regulatory framework for the reintroduction of bacteriophage therapy in Europe. This led to the identification of several characteristics specific to natural bacteriophages that should be taken into consideration by regulators when evaluating bacteriophage therapy. One important consideration is whether bacteriophage therapy development occurs on an industrial scale or a hospital-based, patient-specific scale. More suitable regulatory standards may create opportunities to improve insights into this promising therapeutic approach. In light of this, we argue for the creation of a new, dedicated European regulatory framework for bacteriophage therapy. PMID:24500660

  12. Isolation and characterization of bacteriophages of Salmonella enterica serovar Pullorum.

    Science.gov (United States)

    Bao, H; Zhang, H; Wang, R

    2011-10-01

    In this study, 2 bacteriophages of Salmonella Pullorum were isolated using an enrichment protocol and the double agar layer method. They were named PSPu-95 and PSPu-4-116, respectively, against clinical isolates of Salmonella Pullorum SPu-95 and SPu-116. The host ranges of the 2 bacteriophages were determined by performing spot tests with 20 bacteria strains. Both bacteriophages had wide host ranges. Bacteriophage PSPu-95 had a lytic effect on 17 of the 20 isolates (85%), and PSPu-4-116 produced a lytic effect on 14 isolates (70%) and was the only bacteriophage that produced a clear plaque on enterotoxigenic Escherichia coli K88. Transmission electron microscopy revealed the bacteriophages belonged to the order Caudovirales. Bacteriophage PSPu-95 was a member of the family Siphoviridae, but bacteriophage PSPu-4-116 belonged to the family Myoviridae. Both had a double-stranded DNA, which was digested with HindIII or EcoRI, that was estimated to be 58.3 kbp (PSPu-95) and 45.2 kbp (PSPu-4-116) by 1% agar electrophoresis. One-step growth kinetics showed that the latent periods were all less than 20 min, and the burst size was 77.5 pfu/cell for PSPu-95 and 86 pfu/cell for PSPu-4-116. The bacteriophages were able to survive in a pH range between 4 and 10, and they were able to survive in a treatment of 70°C for 60 min. The characterizations of these 2 bacteriophages were helpful in establishing a basis for adopting the most effective bacteriophage to control bacteria in the poultry industry.

  13. Bacteriophage P70: unique morphology and unrelatedness to other Listeria bacteriophages.

    Science.gov (United States)

    Schmuki, Martina M; Erne, Doris; Loessner, Martin J; Klumpp, Jochen

    2012-12-01

    Listeria monocytogenes is an important food-borne pathogen, and its bacteriophages find many uses in detection and biocontrol of its host. The novel broad-host-range virulent phage P70 has a unique morphology with an elongated capsid. Its genome sequence was determined by a hybrid sequencing strategy employing Sanger and PacBio techniques. The P70 genome contains 67,170 bp and 119 open reading frames (ORFs). Our analyses suggest that P70 represents an archetype of virus unrelated to other known Listeria bacteriophages.

  14. Alteration of the Specificity of PstⅠRestriction Endonuclease

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The influence of factors on the substrate-specificity of Pst Ⅰ restriction endonuclease has been studied with the method of electrophoresis. The results show that, the specificity of Pst Ⅰ almost can not be influenced by the single alteration of the concentration of Tris*HCl, Mg2+ or Na+ in the reaction system, but it can be altered by the reduction of any two of them. The specificity can not be altered by the single alteration of pH or the replacement of Mg2+ with Mn2+. The addition of glycerol or dimethylsulphoxide (DMSO) to the reaction system results in the relaxation of the substrate-specificity of Pst Ⅰ , but dimethylmethylformide, glycol and ethyl alcohol can not bring about the alteration of Pst Ⅰ specificity. Through the method of cloning and sequencing, the nucleotides of No.1 and 6 in the recognition sequence of Pst Ⅰ have changed (1C→A or 6G→T). Used with the enzyme analysis of an artificially synthetic DNA segment containing a special sequence, the nucleotides of No.1 and 6 have both changed (1C→A and 6G→T). The recognition sequence of Pst Ⅰ is speculated to be changed from CTGCA↓G to TGCA↓.

  15. Effects of Dimerization of Serratia marcescens Endonuclease on Water Dynamics.

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Chuanying; Beck, Brian W.; Krause, Kurt; Weksberg, Tiffany E.; Pettitt, Bernard M.

    2007-02-15

    The research described in this product was performed in part in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. The dynamics and structure of Serratia marcescens endonuclease and its neighboring solvent are investigated by molecular dynamics (MD). Comparisons are made with structural and biochemical experiments. The dimer form is physiologic and functions more processively than the monomer. We previously found a channel formed by connected clusters of waters from the active site to the dimer interface. Here, we show that dimerization clearly changes correlations in the water structure and dynamics in the active site not seen in the monomer. Our results indicate that water at the active sites of the dimer is less affected compared with bulk solvent than in the monomer where it has much slower characteristic relaxation times. Given that water is a required participant in the reaction, this gives a clear advantage to dimerization in the absence of an apparent ability to use both active sites simultaneously.

  16. Experience of the Eliava Institute in bacteriophage therapy

    Institute of Scientific and Technical Information of China (English)

    Mzia; Kutateladze

    2015-01-01

    <正>The rapid propagation of multidrug resistant bacterial strains is leading to renewed interest in bacteriophage therapy.With challenges in the treatment of bacterial infections,it is essential for people worldwide to understand how alternative approaches,such as bacteriophages,could be used to combat antibiotic resistant bacteria.The Eliava Institute

  17. Sequence and comparative analysis of Leuconostoc dairy bacteriophages

    DEFF Research Database (Denmark)

    Kot, Witold; Hansen, Lars Henrik; Neve, Horst;

    2014-01-01

    Bacteriophages attacking Leuconostoc species may significantly influence the quality of the final product. There is however limited knowledge of this group of phages in the literature. We have determined the complete genome sequences of nine Leuconostoc bacteriophages virulent to either Leuconostoc...

  18. Bacteriophages: The viruses for all seasons of molecular biology

    Directory of Open Access Journals (Sweden)

    Karam Jim D

    2005-03-01

    Full Text Available Abstract Bacteriophage research continues to break new ground in our understanding of the basic molecular mechanisms of gene action and biological structure. The abundance of bacteriophages in nature and the diversity of their genomes are two reasons why phage research brims with excitement. The pages of Virology Journal will reflect the excitement of the "New Phage Biology."

  19. POSSIBILITES OF BACTERIOPHAGES APPLICATION IN SURGERY AND TRANSPLANTATION

    Directory of Open Access Journals (Sweden)

    N.I. Gabrielyan

    2012-01-01

    Full Text Available The review of the modern data about bacteriophages and to their application to surgery is presented. Interest to bacteriophages is closely connected with an urgency of a problem of postoperative infectious complications and to resistance increase nosocomial species microbes to antibiotics. Successful demonstrative application of bacteriophages on experimental models for a reduction of is conditional-pathogenic microbes in biofilms, for treatment septicemia at the animals, caused resistance species P. aeruginosa, Klebsiella spp., Staphylococcus and other microbes is described. Positive results on application of bacteriophages in surgery are received at treatment of the infected wounds, peritonitis, infectious complications after liver and kidney transplantation. New mechanisms of action of bacteriophages, including their influence on transplantology immunity are resulted. Use of phages as alternatives of treatment and preventive maintenance of a superinfection at imunocomprometive patients is perspective. 

  20. The effects of bacteriophage and nanoparticles on microbial processes

    Science.gov (United States)

    Moody, Austin L.

    There are approximately 1031 tailed phages in the biosphere, making them the most abundant organism. Bacteriophages are viruses that infect bacteria. Due to the large diversity and abundance, no two bacteriophages that have been isolated are genetically the same. Phage products have potential in disease therapy to solve bacteria-related problems, such as infections resulting from resistant strains of Staphylococcus aureus. A bacteriophage capable of infecting methicillin-resistant S. aureus (MRSA) was isolated from bovine hair. The bacteriophage, named JB phage, was characterized using purification, amplification, cesium chloride banding, scanning electron microscopy, and transmission electron microscopy. JB phage and nanoparticles were used in various in vitro and in vivo models to test their effects on microbial processes. Scanning and transmission electron microscopy studies revealed strong interactions between JB phage and nanoparticles, which resulted in increased bacteriophage infectivity. JB phage and nanoparticle cocktails were used as a therapeutic to treat skin and systemic infections in mice caused by MRSA.

  1. [THE IDENTIFICATION AND DIFFERENTIATION OF BACTERIOPHAGES OF HUMAN PATHOGENIC VIBRIO].

    Science.gov (United States)

    Gaevskaia, N E; Kudriakova, T A; Makedonova, L D; Kachkina, G V

    2015-04-01

    The issue of identification and differentiation of large group of bacteriophages of human pathogenic vibrio is still unresolved. In research and practical applied purposes it is important to consider characteristics of bacteriophages for establishing similarity and differences between them. The actual study was carried out to analyze specimens of DNA-containing bacteriophages of pathogenic vibrio. The overwhelming majority of them characterized by complicated type of symmetry--phages with double-helical DNA and also phages with mono-helical DNA structure discovered recently in vibrio. For the first time, the general framework of identification and differentiation of bacteriophages of pathogenic vibrio was developed. This achievement increases possibility to establish species assignment of phages and to compare with phages registered in the database. "The collection of bacteriophages and test-strains of human pathogenic vibrio" (No2010620549 of 24.09.210).

  2. Analysis of T4SS-induced signaling by H. pylori using quantitative phosphoproteomics

    Directory of Open Access Journals (Sweden)

    Frithjof eGlowinski

    2014-07-01

    Full Text Available Helicobacter pylori is a Gram-negative bacterial pathogen colonizing the human stomach. Infection with H. pylori causes chronic inflammation of the gastric mucosa and may lead to peptic ulceration and/or gastric cancer. A major virulence determinant of H. pylori is the type IV secretion system (T4SS, which is used to inject the virulence factor CagA into the host cell, triggering a wide range of cellular signaling events. Here, we used a phosphoproteomic approach to investigate tyrosine signaling in response to host-pathogen interaction, using stable isotope labeling in cell culture (SILAC of AGS cells to obtain a differential picture between multiple infection conditions. Cells were infected with wild type H. pylori P12, a P12ΔCagA deletion mutant, and a P12ΔT4SS deletion mutant to compare signaling changes over time and in the absence of CagA or the T4SS. Tryptic peptides were enriched for tyrosine (Tyr phosphopeptides and analysed by nano-LC-Orbitrap MS. In total, 58 different phosphosites were found to be regulated following infection. The majority of phosphosites identified were kinases of the MAPK familiy. CagA and the T4SS were found to be key regulators of Tyr phosphosites. Our findings indicate that CagA primarily induces activation of ERK1 and integrin linked factors, whereas the T4SS primarily modulates JNK and p38 activation.

  3. Attenuated recombinant vaccinia virus expressing oncofetal antigen (tumor-associated antigen) 5T4 induces active therapy of established tumors.

    Science.gov (United States)

    Mulryan, Kate; Ryan, Matthew G; Myers, Kevin A; Shaw, David; Wang, Who; Kingsman, Susan M; Stern, Peter L; Carroll, Miles W

    2002-10-01

    The human oncofetal antigen 5T4 (h5T4) is a transmembrane glycoprotein overexpressed by a wide spectrum of cancers, including colorectal, ovarian, and gastric, but with a limited normal tissue expression. Such properties make 5T4 an excellent putative target for cancer immunotherapy. The murine homologue of 5T4 (m5T4) has been cloned and characterized, which allows for the evaluation of immune intervention strategies in "self-antigen" in vivo tumor models. We have constructed recombinant vaccinia viruses based on the highly attenuated and modified vaccinia virus ankara (MVA strain), expressing h5T4 (MVA-h5T4), m5T4 (MVA-m5T4), and Escherichia coli LacZ (MVA-LacZ). Immunization of BALB/c and C57BL/6 mice with MVA-h5T4 and MVA-m5T4 constructs induced antibody responses to human and mouse 5T4, respectively. C57BL/6 and BALB/c mice vaccinated with MVA-h5T4 were challenged with syngeneic tumor line transfectants, B16 melanoma, and CT26 colorectal cells that express h5T4. MVA-h5T4-vaccinated mice showed significant tumor retardation compared with mice vaccinated with MVA-LacZ or PBS. In active treatment studies, inoculation with MVA-h5T4 was able to treat established CT26-h5T4 lung tumor and to a lesser extent B16.h5T4 s.c. tumors. Additionally, when C57BL/6 mice vaccinated with MVA-m5T4 were challenged with B16 cells expressing m5T4, resulting growth of the tumors was significantly retarded compared with control animals. Furthermore, mice vaccinated with MVA-m5T4 showed no signs of autoimmune toxicity. These data support the use of MVA-5T4 for tumor immunotherapy. PMID:12481437

  4. Variation in serum T3, T4, TB G in children and adolescents zaire normal

    International Nuclear Information System (INIS)

    Variation of serum T4, T3 and TBG concentration in normal zairian children and adolescent. Serum T4, T3 and TBG radioimmunoassays were carried out in 155 healthy zairian children and adolescent. Their age ranged from 0 to 21 years. They were clinically euthyroid and not taking any drug which could interfer with RIA measurement. Serum T4 level remaind inchanged in all age groups. Serum T3 showed a progressive decrease in 0 to 10 years old groups of children. There after, these decrease became significantelly greater in 16 to 21 years old groups. Serum TBG level was essentially equal in 0 to 10 years old groups. There after it showed a significantelly decrease in 11 to 21 years old groups.

  5. Comparison of Immunoassay methods for T3, T4 and TSH

    International Nuclear Information System (INIS)

    Measurements of T3, T4 and TSH have been considered very important in the diagnosis and monitoring of thyroid diseases both overt and subclinical. These subclinical diseases are actively seeking for years, both in healthy patients and hospitalized for other illnesses; and in the population over 35 years, especially women, in health checkups. The active search for these diseases requires the use of rapid and reliable techniques; that can be developed massively, with good level of detectability and comparable. The overall objective is to present the evaluation of different immunoassay techniques with respect to the RIA and IRMA: ELISA, chemiluminescence, Amplified Chemiluminescence, electrochemiluminescence Immunofluorescence. Compare including automatic methods and analyze the cost and feasibility of them for laboratory immunoassay. ELISA colorimetric technique for dosing was comparable to RIA T4, not for T3. Chemiluminescence (AMERLITE) compared to dosing RIA and IRMA T4 to TSH proved to be valid for both. Amplified Chemiluminescence (Immulite) compared to IRMA for TSH was no significant difference. Electrochemiluminescence (Elecsys 2010) compared to T3 and T4 RIA and IRMA for TSH, no significant differences for T4 and TSH; but no variation to T3. Immunofluorescence (AIA-600) used to compare with RIA for T3 and T4, and TSH IRMA, no significant differences for the measured analytes. Benchmarking of automatic methods suggests that the most thrifty of trials is Immunofluorescence the AIA-600, regarding calibration and control, programming time, randomization and the ability to save the value of the fluorescence deferred calculations for tests without valid at the time of realizing calibration. Analyzing the cost and feasibility of these methods for laboratory immunoassay, we must consider that their characteristics electrochemiluminescence is the fastest, but its price is prohibitive for our health systems. The AIA-600 appears to be the method of choice for its

  6. M13 Bacteriophage Based Protein Sensors

    Science.gov (United States)

    Lee, Ju Hun

    Despite significant progress in biotechnology and biosensing, early detection and disease diagnosis remains a critical issue for improving patient survival rates and well-being. Many of the typical detection schemes currently used possess issues such as low sensitivity and accuracy and are also time consuming to run and expensive. In addition, multiplexed detection remains difficult to achieve. Therefore, developing advanced approaches for reliable, simple, quantitative analysis of multiple markers in solution that also are highly sensitive are still in demand. In recent years, much of the research has primarily focused on improving two key components of biosensors: the bio-recognition agent (bio-receptor) and the transducer. Particular bio-receptors that have been used include antibodies, aptamers, molecular imprinted polymers, and small affinity peptides. In terms of transducing agents, nanomaterials have been considered as attractive candidates due to their inherent nanoscale size, durability and unique chemical and physical properties. The key focus of this thesis is the design of a protein detection and identification system that is based on chemically engineered M13 bacteriophage coupled with nanomaterials. The first chapter provides an introduction of biosensors and M13 bacteriophage in general, where the advantages of each are provided. In chapter 2, an efficient and enzyme-free sensor is demonstrated from modified M13 bacteriophage to generate highly sensitive colorimetric signals from gold nanocrystals. In chapter 3, DNA conjugated M13 were used to enable facile and rapid detection of antigens in solution that also provides modalities for identification. Lastly, high DNA loadings per phage was achieved via hydrozone chemistry and these were applied in conjunction with Raman active DNA-gold/silver core/shell nanoparticles toward highly sensitive SERS sensing.

  7. Ecological study of bacteriophages of Vibrio natriegens

    Energy Technology Data Exchange (ETDEWEB)

    Zachary, A.

    1978-03-01

    Effects of temperature and anaerobic conditions on the replication of two bacteriophages, nt-1 and nt-6, of the estuarine bacterium Vibrio natriegens were studied. Reduction in temperature resulted in longer latent periods and reduced burst sizes for both phages. Replication under anaerobic conditions resulted in longer latent periods; however, phage nt-6 had a reduced burst size, whereas phage nt-1 had an increased burst size, resulting in a rate of phage production nearly equal to that observed under aerobic conditions. Therefore the distribution of the phages in marsh areas could be influenced by temperature and anaerobiosis.

  8. Bacteriophage biosensors for antibiotic-resistant bacteria.

    Science.gov (United States)

    Sorokulova, Irina; Olsen, Eric; Vodyanoy, Vitaly

    2014-03-01

    An increasing number of disease-causing bacteria are resistant to one or more anti-bacterial drugs utilized for therapy. Early and speedy detection of these pathogens is therefore very important. Traditional pathogen detection techniques, that include microbiological and biochemical assays are long and labor-intensive, while antibody or DNA-based methods require substantial sample preparation and purification. Biosensors based on bacteriophages have demonstrated remarkable potential to surmount these restrictions and to offer rapid, efficient and sensitive detection technique for antibiotic-resistant bacteria.

  9. Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

    Energy Technology Data Exchange (ETDEWEB)

    Kennedy, Edward M.; Cullen, Bryan R., E-mail: bryan.cullen@duke.edu

    2015-05-15

    CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

  10. Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

    International Nuclear Information System (INIS)

    CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

  11. Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment.

    Science.gov (United States)

    Kennedy, Edward M; Cullen, Bryan R

    2015-05-01

    CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

  12. HEPATIC ENZYME INDUCERS INCREASE THYROXINE (T4) CATABOLISM IN HUMAN AND RAT HEPATOCYTES

    Science.gov (United States)

    Nuclear receptor agonists such as 3-methylcholantrene (3-MC), phenobarbital (PB), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153), and, pregnenolone-16a-carbonitrile (PCN) decrease serum thyroxine (T4) concentrations in rats. It appears that this decrease occurs through the induction...

  13. THYROXINE (T4) CATABOLISM IN HUMAN AND RAT HEPATOCYTES INCREASES FOLLOWING EXPOSURE TO HEPATIC ENZYME INDUCERS

    Science.gov (United States)

    Nuclear receptor agonists phenobarbital (PB), 3-methylcholanthrene (3MC), pregnenolone-16a-carbonitrile (PCN), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153), and 2,2' ,4,4'-tetrabromodiphenyl ether (BDE 47) decrease serum thyroxine (T4) in rats. This decrease is thought to occur th...

  14. Congenital hypothyroidism - precocious diagnostic for radioimmunoassay of the neonatal T4 and TSH

    International Nuclear Information System (INIS)

    The authors studied the results of the neonatal T4 and TSH in 31,572 children that were screened for congenital hjypothyroidism. Precocious diagnosis were made in 8 cases of congenital hypothyroidism and in 2 cases of TBG deficiency. The frequence of congenital hypothyroidism found in this study was 1/3,946. (author)

  15. PCB-153 AND BDE-47 INCREASE THYROXINE T4) CATABOLISM IN RAT AND HUMAN HEPATOCYTES

    Science.gov (United States)

    Previous studies demonstrate that in vivo exposure to 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153) and 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) decrease serum thyroxine (T4) levels in rats. This decrease is thought to occur through the induction of hepatic metabolizing enzymes ...

  16. Effects of hepatic enzyme inducers on thyroxine (T4) catabolism in primary rat hepatocytes

    Science.gov (United States)

    Nuclear receptor agonists such as phenobarbital (PB), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153), and 3-methylcholantrene (3-MC) decrease circulating thyroxine (T4) concentrations in rats. It is suspected that this decrease occurs through the induction of hepatic metabolizing en...

  17. Modular architecture of the T4 phage superfamily: A conserved core genome and a plastic periphery

    International Nuclear Information System (INIS)

    Among the most numerous objects in the biosphere, phages show enormous diversity in morphology and genetic content. We have sequenced 7 T4-like phages and compared their genome architecture. All seven phages share a core genome with T4 that is interrupted by several hyperplastic regions (HPRs) where most of their divergence occurs. The core primarily includes homologues of essential T4 genes, such as the virion structure and DNA replication genes. In contrast, the HPRs contain mostly novel genes of unknown function and origin. A few of the HPR genes that can be assigned putative functions, such as a series of novel Internal Proteins, are implicated in phage adaptation to the host. Thus, the T4-like genome appears to be partitioned into discrete segments that fulfil different functions and behave differently in evolution. Such partitioning may be critical for these large and complex phages to maintain their flexibility, while simultaneously allowing them to conserve their highly successful virion design and mode of replication

  18. The identification and optimization of a N-hydroxy urea series of flap endonuclease 1 inhibitors.

    Science.gov (United States)

    Tumey, L Nathan; Bom, David; Huck, Bayard; Gleason, Elizabeth; Wang, Jianmin; Silver, Daniel; Brunden, Kurt; Boozer, Sherry; Rundlett, Stephen; Sherf, Bruce; Murphy, Steven; Dent, Tom; Leventhal, Christina; Bailey, Andrew; Harrington, John; Bennani, Youssef L

    2005-01-17

    Flap endonuclease-1 (FEN1) is a key enzyme involved in base excision repair (BER), a primary pathway utilized by mammalian cells to repair DNA damage. Sensitization to DNA damaging agents is a potential method for the improvement of the therapeutic window of traditional chemotherapeutics. In this paper, we describe the identification and SAR of a series of low nanomolar FEN1 inhibitors. Over 1000-fold specificity was achieved against a related endonuclease, xeroderma pigmentosum G (XPG). Two compounds from this series significantly potentiate the action of methyl methanesulfonate (MMS) and temozolamide in a bladder cancer cell line (T24). To our knowledge, these are the most potent endonuclease inhibitors reported to date. PMID:15603939

  19. Endonuclease-rolling circle amplification-based method for sensitive analysis of DNA-binding protein

    Institute of Scientific and Technical Information of China (English)

    Min Li Li; Dong Rui Zhou; Hong Zhao; Jin Ke Wang; Zu Hong Lu

    2009-01-01

    A sensitive approach for the qualitative detection of DNA-binding protein on the microarray was developed. DNA complexes in which a partial duplex region is formed from a biotin-primer and a circle single strand DNA (ssDNA) were spotted on a microarray. The endonuclease recognition site (ERS) and the DNA-binding sites (DBS) were arranged side by side within the duplex region. The working principle of the detection system is described as follows: when the DNA-binding protein capture the DBS, the endonuclease could not attach to the ERS, and the immobilized primer in the DNA complex could be extended along the circle ssDNA by rolling circle amplification (RCA). When no protein protects the DBS, the ERS could be attacked by the endonuclease and subsequently no rolling circle amplification occurs. Thereby we can detect the sequence specific DNA-binding activity with high-sensitivity due to the signal amplification of RCA.

  20. N-acylhydrazone inhibitors of influenza virus PA endonuclease with versatile metal binding modes

    Science.gov (United States)

    Carcelli, Mauro; Rogolino, Dominga; Gatti, Anna; de Luca, Laura; Sechi, Mario; Kumar, Gyanendra; White, Stephen W.; Stevaert, Annelies; Naesens, Lieve

    2016-08-01

    Influenza virus PA endonuclease has recently emerged as an attractive target for the development of novel antiviral therapeutics. This is an enzyme with divalent metal ion(s) (Mg2+ or Mn2+) in its catalytic site: chelation of these metal cofactors is an attractive strategy to inhibit enzymatic activity. Here we report the activity of a series of N-acylhydrazones in an enzymatic assay with PA-Nter endonuclease, as well as in cell-based influenza vRNP reconstitution and virus yield assays. Several N-acylhydrazones were found to have promising anti-influenza activity in the low micromolar concentration range and good selectivity. Computational docking studies are carried on to investigate the key features that determine inhibition of the endonuclease enzyme by N-acylhydrazones. Moreover, we here describe the crystal structure of PA-Nter in complex with one of the most active inhibitors, revealing its interactions within the protein’s active site.

  1. RNA-dependent DNA endonuclease Cas9 of the CRISPR system: Holy Grail of genome editing?

    Science.gov (United States)

    Gasiunas, Giedrius; Siksnys, Virginijus

    2013-11-01

    Tailor-made nucleases for precise genome modification, such as zinc finger or TALE nucleases, currently represent the state-of-the-art for genome editing. These nucleases combine a programmable protein module which guides the enzyme to the target site with a nuclease domain which cuts DNA at the addressed site. Reprogramming of these nucleases to cut genomes at specific locations requires major protein engineering efforts. RNA-guided DNA endonuclease Cas9 of the type II (clustered regularly interspaced short palindromic repeat) CRISPR-Cas system uses CRISPR RNA (crRNA) as a guide to locate the DNA target and the Cas9 protein to cut DNA. Easy programmability of the Cas9 endonuclease using customizable RNAs brings unprecedented flexibility and versatility for targeted genome modification. We highlight the potential of the Cas9 RNA-guided DNA endonuclease as a novel tool for genome surgery, and discuss possible constraints and future prospects.

  2. Effects of environmental and clinical interferents on the host capture efficiency of immobilized bacteriophages.

    Science.gov (United States)

    Dixon, Daniel V; Hosseinidoust, Zeinab; Tufenkji, Nathalie

    2014-03-25

    Bacteriophage-functionalized surfaces are a new class of advanced functional material and have been demonstrated to be applicable for use as antimicrobial surfaces in medical applications (e.g., indwelling medical devices or wound dressings) or as biosensors for bacterial capture and detection. However, the complex composition of many real life samples (e.g., blood, natural waters, etc.) can potentially interfere with the interaction of phage and its bacterial host, leading to a decline in the efficiency of the phage-functionalized surface. In this study, the bacterial capture efficiency of two model phage-functionalized surfaces was assessed in the presence of potential environmental and biomedical interferents. The two phage-bacteria systems used in this study are PRD1 with Salmonella Typhimurium and T4 with Escherichia coli. The potential interferents tested included humic and fulvic acids, natural groundwater, colloidal latex microspheres, host extracellular polymeric substances (EPS), albumin, fibrinogen, and human serum. EPS and human serum decreased the host capture efficiency for immobilized PRD1 and T4, and also impaired the infectivity of the nonimmobilized (planktonic) phage. Interestingly, humic and fulvic acids reduced the capture efficiency of T4-functionalized surfaces, even though they did not lead to inactivation of the suspended virions. Neither humic nor fulvic acids affected the capture efficiency of PRD1. These findings demonstrate the inadequacy of traditional phage selection methods (i.e., infectivity of suspended phage toward its host in clean buffer) for designing advanced functional materials and further highlight the importance of taking into account the environmental conditions in which the immobilized phage is expected to function.

  3. 2012 ETA Guidelines: The Use of L-T4 + L-T3 in the Treatment of Hypothyroidism

    DEFF Research Database (Denmark)

    Nygaard, Birte

    2012-01-01

    Background: Data suggest symptoms of hypothyroidism persist in 5–10% of levothyroxine (L-T4)-treated hypothyroid patients with normal serum thyrotrophin (TSH). The use of L-T4 + liothyronine (L-T3) combination therapy in such patients is controversial. The ETA nominated a task force to review....... There is insufficient evidence that L-T4 + L-T3 combination therapy is better than L-T4 monotherapy, and it is recommended that L-T4 monotherapy remains the standard treatment of hypothyroidism. L-T4 + L-T3 combination therapy might be considered as an experimental approach in compliant L-T4-treated hypothyroid...

  4. Comparative effect of Citrus sinensis and carbimazole on serum T 4 , T 3 and TSH levels

    Directory of Open Access Journals (Sweden)

    Okon Uduak Uduak

    2014-01-01

    Full Text Available Background: There are previous independent reports on the anti-thyroid property of Citrus sinensis. This isoflavones and phenolic acid-rich natural agent is widely consumed as dietary supplement, thus the need to investigate its comparative effect with a standard anti-thyroid drug on T 4 , T 3 and thyroid stimulating hormone (TSH levels. Objective: To compare the effect of Citrus sinensis and carbimazole (CARB on blood levels of thyroid hormones (T 4 and T 3 and TSH. Materials and Methods: Male wistar albino rats weighing 100-150 g were employed in this research. The rats were randomly assigned to four groups of seven rats per group. Group I served as control and were administered distilled water while groups II-IV were administered with 1500 mg/kg of Citrus sinensis (fresh orange juice; FOJ, 0.1 μg/g of levothyroxine (LVT and 0.01 mg/g of CARB, respectively, per oral once daily for 28 days. The animals were sacrificed under chloroform anaesthesia and blood sample collected by cardiac puncture and processed by standard method to obtain serum. TSH, T 4 and T 3 were assayed with the serum using ARIA II automated radioimmunoassay instrument. Results: The results showed that TSH level was significantly (P < 0.05 decreased in LVT treated group compared with the FOJ group. T 4 was significantly (P < 0.05 decreased in the FOJ and CARB groups compared with the control and LVT groups. LVT significantly increased T 4 when compared with FOJ group. T 3 was significantly (P < 0.05 decreased in the CARB group compared with the control. Conclusion: These findings suggest that FOJ alters thyroid hormones metabolism to reduce their serum levels with a compensatory elevations of TSH level in a direction similar to CARB.

  5. Bacteriophages and Their Role in Food Safety

    Directory of Open Access Journals (Sweden)

    Sanna M. Sillankorva

    2012-01-01

    Full Text Available The interest for natural antimicrobial compounds has increased due to alterations in consumer positions towards the use of chemical preservatives in foodstuff and food processing surfaces. Bacteriophages fit in the class of natural antimicrobial and their effectiveness in controlling bacterial pathogens in agro-food industry has led to the development of different phage products already approved by USFDA and USDA. The majority of these products are to be used in farm animals or animal products such as carcasses, meats and also in agricultural and horticultural products. Treatment with specific phages in the food industry can prevent the decay of products and the spread of bacterial diseases and ultimately promote safe environments in animal and plant food production, processing, and handling. This is an overview of recent work carried out with phages as tools to promote food safety, starting with a general introduction describing the prevalence of foodborne pathogens and bacteriophages and a more detailed discussion on the use of phage therapy to prevent and treat experimentally induced infections of animals against the most common foodborne pathogens, the use of phages as biocontrol agents in foods, and also their use as biosanitizers of food contact surfaces.

  6. Bacteriophage recombination systems and biotechnical applications.

    Science.gov (United States)

    Nafissi, Nafiseh; Slavcev, Roderick

    2014-04-01

    Bacteriophage recombination systems have been widely used in biotechnology for modifying prokaryotic species, for creating transgenic animals and plants, and more recently, for human cell gene manipulation. In contrast to homologous recombination, which benefits from the endogenous recombination machinery of the cell, site-specific recombination requires an exogenous source of recombinase in mammalian cells. The mechanism of bacteriophage evolution and their coexistence with bacterial cells has become a point of interest ever since bacterial viruses' life cycles were first explored. Phage recombinases have already been exploited as valuable genetic tools and new phage enzymes, and their potential application to genetic engineering and genome manipulation, vectorology, and generation of new transgene delivery vectors, and cell therapy are attractive areas of research that continue to be investigated. The significance and role of phage recombination systems in biotechnology is reviewed in this paper, with specific focus on homologous and site-specific recombination conferred by the coli phages, λ, and N15, the integrase from the Streptomyces phage, ΦC31, the recombination system of phage P1, and the recently characterized recombination functions of Yersinia phage, PY54. Key steps of the molecular mechanisms involving phage recombination functions and their application to molecular engineering, our novel exploitations of the PY54-derived recombination system, and its application to the development of new DNA vectors are discussed.

  7. Understanding Bacteriophage Specificity in Natural Microbial Communities

    Directory of Open Access Journals (Sweden)

    Britt Koskella

    2013-03-01

    Full Text Available Studying the coevolutionary dynamics between bacteria and the bacteriophage viruses that infect them is critical to understanding both microbial diversity and ecosystem functioning. Phages can play a key role in shaping bacterial population dynamics and can significantly alter both intra- and inter-specific competition among bacterial hosts. Predicting how phages might influence community stability and apparent competition, however, requires an understanding of how bacteria-phage interaction networks evolve as a function of host diversity and community dynamics. Here, we first review the progress that has been made in understanding phage specificity, including the use of experimental evolution, we then introduce a new dataset on natural bacteriophages collected from the phyllosphere of horse chestnut trees, and finally we highlight that bacterial sensitivity to phage is rarely a binary trait and that this variation should be taken into account and reported. We emphasize that there is currently insufficient evidence to make broad generalizations about phage host range in natural populations, the limits of phage adaptation to novel hosts, or the implications of phage specificity in shaping microbial communities. However, the combination of experimental and genomic approaches with the study of natural communities will allow new insight to the evolution and impact of phage specificity within complex bacterial communities.

  8. Bacteriophage recombination systems and biotechnical applications.

    Science.gov (United States)

    Nafissi, Nafiseh; Slavcev, Roderick

    2014-04-01

    Bacteriophage recombination systems have been widely used in biotechnology for modifying prokaryotic species, for creating transgenic animals and plants, and more recently, for human cell gene manipulation. In contrast to homologous recombination, which benefits from the endogenous recombination machinery of the cell, site-specific recombination requires an exogenous source of recombinase in mammalian cells. The mechanism of bacteriophage evolution and their coexistence with bacterial cells has become a point of interest ever since bacterial viruses' life cycles were first explored. Phage recombinases have already been exploited as valuable genetic tools and new phage enzymes, and their potential application to genetic engineering and genome manipulation, vectorology, and generation of new transgene delivery vectors, and cell therapy are attractive areas of research that continue to be investigated. The significance and role of phage recombination systems in biotechnology is reviewed in this paper, with specific focus on homologous and site-specific recombination conferred by the coli phages, λ, and N15, the integrase from the Streptomyces phage, ΦC31, the recombination system of phage P1, and the recently characterized recombination functions of Yersinia phage, PY54. Key steps of the molecular mechanisms involving phage recombination functions and their application to molecular engineering, our novel exploitations of the PY54-derived recombination system, and its application to the development of new DNA vectors are discussed. PMID:24442504

  9. Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering

    OpenAIRE

    Aouida, Mustapha; Eid, Ayman; Ali, Zahir; Cradick, Thomas; Lee, Ciaran; Deshmukh, Harshavardhan; Atef, Ahmed; AbuSamra, Dina; Gadhoum, Samah Zeineb; Merzaban, Jasmeen; Bao, Gang; Mahfouz, Magdy

    2015-01-01

    The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spac...

  10. The effects of addition of mononucleotides on Sma nuc endonuclease activity.

    Science.gov (United States)

    Romanova, Julia; Filimonova, Maria

    2012-01-01

    Examination of the effects of mononucleotides on Sma nuc endonuclease originated from Gram negative bacterium Serratia marcescens displayed that any mononucleotide produced by Sma nuc during hydrolysis of DNA or RNA may regulate the enzyme activity affecting the RNase activity without pronounced influence on the activity towards DNA. The type of carbohydrate residue in mononucleotides does not affect the regulation. In contrast, the effects depend on the type of bases in nucleotides. AMP or dAMP was classified as a competitive inhibitor of partial type. GMP, UMP, and CMP were found to be uncompetitive inhibitors that suggest a specific site(s) for the nucleotide(s) binding in Sma nuc endonuclease.

  11. Combined use of Bacteriophage K and a novel Bacteriophage to reduce Staphylococcus aureus biofilm formation

    DEFF Research Database (Denmark)

    Alves, D.R.; Gaudion, A.; Bean, J.E.;

    2014-01-01

    Biofilms are major causes of impairment of wound healing and patient morbidity. One of the most common and aggressive wound pathogens is Staphylococcus aureus, displaying a large repertoire of virulence factors and commonly reduced susceptibility to antibiotics, such as the spread of methicillin-......-resistant S. aureus (MRSA). Bacteriophages are obligate parasites of bacteria. They multiply intracellularly and lyse their bacterial host, releasing their progeny. We isolated a novel phage, DRA88, which has a ...

  12. Bacteria vs. Bacteriophages: Parallel Evolution of Immune Arsenals.

    Science.gov (United States)

    Shabbir, Muhammad A B; Hao, Haihong; Shabbir, Muhammad Z; Wu, Qin; Sattar, Adeel; Yuan, Zonghui

    2016-01-01

    Bacteriophages are the most common entities on earth and represent a constant challenge to bacterial populations. To fend off bacteriophage infection, bacteria evolved immune systems to avert phage adsorption and block invader DNA entry. They developed restriction-modification systems and mechanisms to abort infection and interfere with virion assembly, as well as newly recognized clustered regularly interspaced short palindromic repeats (CRISPR). In response to bacterial immune systems, bacteriophages synchronously evolved resistance mechanisms, such as the anti-CRISPR systems to counterattack bacterial CRISPR-cas systems, in a continuing evolutionary arms race between virus and host. In turn, it is fundamental to the survival of the bacterial cell to evolve a system to combat bacteriophage immune strategies.

  13. Bacteria vs. bacteriophages: parallel evolution of immune arsenals

    Directory of Open Access Journals (Sweden)

    Muhammad Abu Bakr Shabbir

    2016-08-01

    Full Text Available Bacteriophages are the most common entities on earth and represent a constant challenge to bacterial populations. To fend off bacteriophage infection, bacteria evolved immune systems to avert phage adsorption and block invader DNA entry. They developed restriction-modification systems and mechanisms to abort infection and interfere with virion assembly, as well as newly recognized clustered regularly interspaced short palindromic repeats (CRISPR. In response to bacterial immune systems, bacteriophages synchronously evolved resistance mechanisms, such as the anti-CRISPR systems to counterattack bacterial CRISPR-cas systems, in a continuing evolutionary arms race between virus and host. In turn, it is fundamental to the survival of the bacterial cell to evolve a system to combat bacteriophage immune strategies.

  14. Antimicrobial bacteriophage-derived proteins and therapeutic applications

    Science.gov (United States)

    Antibiotics have the remarkable power to control bacterial infections. Unfortunately, widespread use, whether regarded as prudent or not, has favored the emergence and persistence of antibiotic resistant strains of human pathogenic bacteria, resulting in a global health threat. Bacteriophages (pha...

  15. Relationships among different strains of T7 and among T7-related bacteriophages

    Energy Technology Data Exchange (ETDEWEB)

    Studier, F.W.

    1979-05-01

    DNAs of the related bacteriophages T7, T3, PHI II, H, PHI I, and W31 have been cut with the restriction endonuclease HpaI and the DNA fragments analyzed by gel electrophoresis. A characteristic pattern of fragments was produced from each DNA, only PHI II and H DNA giving patterns that were obviously related to each other. Thus HpaI restriction patterns can be used to provide rapid and positive identification of these closely related phages. Restriction analysis, together with plating behavior and biochemical characteristics, has revealed a remarkable diversity among strains of the same phage from different laboratories. Of T7 strains received from 19 sources only 8 corresponded to the original wild-type T7, the others being different pure deletion mutants of T7, mixtures of wild-type and different mutants, one host-range mutant, and two strains that were actually T3. Divergence in different lines of other T7-related phages was also observed, as were same additional cases of mistaken identity. Deletions in the gene 0.7 region seem to be a common type of change in all of the T7-related phages. Some misconceptions that have arisen because of strain differences or cases of mistaken identity can now be corrected. Some of the best previous measurements of the molecular weight of T7 DNA were apparently made on DNA from deletion mutants, leading to a commonly accepted value for the molecular weight of T7 DNA that is too low. The T7-related phages are generally thought to be female specific, but in fact authentic T3 plates equally well on isogenic male and female strains of Escherichia coli.

  16. Bacteriophages, revitalized after 100 years in the shadow of antibiotics

    Institute of Scientific and Technical Information of China (English)

    Hongping; Wei

    2015-01-01

    <正>The year 2015 marks 100 years since Dr.Frederick Twort discovered the"filterable lytic factor",which was later independently discovered and named "bacteriophage" by Dr.Felix d’Herelle.On this memorable centennial,it is exciting to see a special issue published by Virologica Sinica on Phages and Therapy.In this issue,readers will not only fi nd that bacteriophage research is a

  17. Bacteriophage-based nanoprobes for rapid bacteria separation

    Science.gov (United States)

    Chen, Juhong; Duncan, Bradley; Wang, Ziyuan; Wang, Li-Sheng; Rotello, Vincent M.; Nugen, Sam R.

    2015-10-01

    The lack of practical methods for bacterial separation remains a hindrance for the low-cost and successful development of rapid detection methods from complex samples. Antibody-tagged magnetic particles are commonly used to pull analytes from a liquid sample. While this method is well-established, improvements in capture efficiencies would result in an increase of the overall detection assay performance. Bacteriophages represent a low-cost and more consistent biorecognition element as compared to antibodies. We have developed nanoscale bacteriophage-tagged magnetic probes, where T7 bacteriophages were bound to magnetic nanoparticles. The nanoprobe allowed the specific recognition and attachment to E. coli cells. The phage magnetic nanprobes were directly compared to antibody-conjugated magnetic nanoprobes. The capture efficiencies of bacteriophages and antibodies on nanoparticles for the separation of E. coli K12 at varying concentrations were determined. The results indicated a similar bacteria capture efficiency between the two nanoprobes.The lack of practical methods for bacterial separation remains a hindrance for the low-cost and successful development of rapid detection methods from complex samples. Antibody-tagged magnetic particles are commonly used to pull analytes from a liquid sample. While this method is well-established, improvements in capture efficiencies would result in an increase of the overall detection assay performance. Bacteriophages represent a low-cost and more consistent biorecognition element as compared to antibodies. We have developed nanoscale bacteriophage-tagged magnetic probes, where T7 bacteriophages were bound to magnetic nanoparticles. The nanoprobe allowed the specific recognition and attachment to E. coli cells. The phage magnetic nanprobes were directly compared to antibody-conjugated magnetic nanoprobes. The capture efficiencies of bacteriophages and antibodies on nanoparticles for the separation of E. coli K12 at varying

  18. Targeting Antibacterial Agents by Using Drug-Carrying Filamentous Bacteriophages

    OpenAIRE

    Yacoby, Iftach; Shamis, Marina; Bar, Hagit; Shabat, Doron; Benhar, Itai

    2006-01-01

    Bacteriophages have been used for more than a century for (unconventional) therapy of bacterial infections, for half a century as tools in genetic research, for 2 decades as tools for discovery of specific target-binding proteins, and for nearly a decade as tools for vaccination or as gene delivery vehicles. Here we present a novel application of filamentous bacteriophages (phages) as targeted drug carriers for the eradication of (pathogenic) bacteria. The phages are genetically modified to d...

  19. Alternative bacteriophage life cycles: the carrier state of Campylobacter jejuni

    OpenAIRE

    Siringan, Patcharin; Connerton, Phillippa L.; Cummmings, Nicola J.; Connerton, Ian F.

    2014-01-01

    Members of the genus Campylobacter are frequently responsible for human enteric disease, often through consumption of contaminated poultry products. Bacteriophages are viruses that have the potential to control pathogenic bacteria, but understanding their complex life cycles is key to their successful exploitation. Treatment of Campylobacter jejuni biofilms with bacteriophages led to the discovery that phages had established a relationship with their hosts typical of the carrier state life cy...

  20. Engineered bacteriophage targeting gene networks as adjuvants for antibiotic therapy

    OpenAIRE

    Lu, Timothy K.; Collins, James J.

    2009-01-01

    Antimicrobial drug development is increasingly lagging behind the evolution of antibiotic resistance, and as a result, there is a pressing need for new antibacterial therapies that can be readily designed and implemented. In this work, we engineered bacteriophage to overexpress proteins and attack gene networks that are not directly targeted by antibiotics. We show that suppressing the SOS network in Escherichia coli with engineered bacteriophage enhances killing by quinolones by several orde...

  1. Radiation-induced osteochondroma of the T4 vertebra causing spinal cord compression

    Energy Technology Data Exchange (ETDEWEB)

    Gorospe, Luis; Madrid-Muniz, Carmen; Royo, Aranzazu; Garcia-Raya, Pilar [Department of Radiology, La Paz University Hospital, Madrid (Spain); Alvarez-Ruiz, Fernando [Department of Neurosurgery, La Paz University Hospital, Madrid (Spain); Lopez-Barea, Fernando [Department of Pathology, La Paz University Hospital, Madrid (Spain)

    2002-04-01

    A case of a radiation-induced osteochondroma arising from the vertebral body of T4 in an 18-year-old man is reported. The patient presented with a history of progressive left lower extremity weakness. At 7 years of age, he had undergone resection of a cerebellar medulloblastoma and received adjunctive craniospinal irradiation and systemic chemotherapy. Both CT and MR imaging revealed an extradural mass contiguous with the posteroinferior endplate of the T4 vertebral body. This case indicates that radiation-induced osteochondroma should be considered in the differential diagnosis of patients with symptoms of myelopathy or nerve root compression and a history of radiation therapy involving the spine in childhood. (orig.)

  2. Removal of particle-associated bacteriophages by dual-media filtration at different filter cycle stages and impacts on subsequent UV disinfection

    Energy Technology Data Exchange (ETDEWEB)

    Templeton, M.R.; Andrews, R.C.; Hofmann, R. [University of Toronto, Toronto, ON (Canada). Dept. of Civil Engineering

    2007-06-15

    This bench-scale study investigated the passage of particle-associated bacteriophage through a dual-media (anthracite-sand) filter over a complete filter cycle and the effect on subsequent ultraviolet (UV) disinfection. Two model viruses, bacteriophages MS2 and T4, were considered. The water matrix was de-chlorinated tap water with either kaolin or Aldrich humic acid (AHA) added and coagulated with alum to form floc before filtration. Filter effluent samples were collected for phage enumeration during three filter cycle stages: (I) filter ripening; (ii) stable operation; and (iii) end of filter cycle. Influent and filter effluent samples were subsequently exposed to UV light (254 nm) at 40 mJ/cm{sup 2} using a low pressure UV collimated beam. The study found statistically significant differences ({alpha} = 0.05) in the quantity of particle-associated phage present in the filter effluent during the three stages of filtration. There was reduced UV disinfection efficiency due to the presence of particle- associated phage in the filter effluent in trials with bacteriophage MS2 and humic acid floc. Unfiltered influent water samples also resulted in reduced UV inactivation of phage relative to particle-free control conditions for both phages. Trends in filter effluent turbidity corresponded with breakthrough of particle-associated phage in the filter effluent. The results therefore suggest that maintenance of optimum filtration conditions upstream of UV disinfection is a critical barrier to particle-associated viruses.

  3. Prognostic factors of T4 gastric cancer patients undergoing potentially curative resection

    Institute of Scientific and Technical Information of China (English)

    Naoto Fukuda; Yasuyuki Sugiyama; Joji Wada

    2011-01-01

    AIM: To investigate the prognostic factors of T4 gas-tric cancer patients without distant metastasis who could undergo potentially curative resection. METHODS: We retrospectively analyzed the clinical data of 71 consecutive patients diagnosed with T4 gas-tric cancer and who underwent curative gastrectomy at our institutions. The clinicopathological factors that could be associated with overall survival were evalu-ated. The cumulative survival was determined by the Kaplan-Meier method, and univariate comparisons be-tween the groups were performed using the log-rank test. Multivariate analysis was performed using the Cox proportional hazard model and a step-wise procedure.RESULTS: The study patients comprised 53 men (74.6%) and 18 women (25.4%) aged 39-89 years (mean, 68.9 years). Nineteen patients (26.8%) had postoperative morbidity: pancreatic fistula developed in 6 patients (8.5%) and was the most frequent compli-cation, followed by anastomosis stricture in 5 patients (7.0%). During the follow-up period, 28 patients (39.4%) died because of gastric cancer recurrence, and 3 (4.2%) died because of another disease or accident. For all patients, the estimated overall survival was 34.1% at 5 years. Univariate analyses identified the following statis-tically significant prognostic factors in T4 gastric cancer patients who underwent potentially curative resection: peritoneal washing cytology (P < 0.01), number of met-astatic lymph nodes (P < 0.05), and venous invasion (P < 0.05). In multivariate analyses, only peritoneal wash-ing cytology was identified as an independent prognos-tic factor (HR = 3.62, 95% CI = 1.37-9.57) for long-term survival. CONCLUSION: Positive peritoneal washing cytology was the only independent poor prognostic factor for T4 gastric cancer patients who could be treated with potentially curative resection.

  4. Pulsating strings from two-dimensional CFT on (T4N/S(N

    Directory of Open Access Journals (Sweden)

    Carlos Cardona

    2015-04-01

    Full Text Available We propose a state from the two-dimensional conformal field theory on the orbifold (T4N/S(N as a dual description for a pulsating string moving in AdS3. We show that, up to first order in the deforming parameter, the energy in both descriptions has the same dependence on the mode number, but with a non-trivial function of the coupling.

  5. Interleukin 2-dependent release of interleukin 3 activity by T4+ human T-cell clones.

    OpenAIRE

    Ythier, A A; Abbud-Filho, M; Williams, J.M.; Loertscher, R; Schuster, M W; Nowill, A; Hansen, J A; Maltezos, D; Strom, T.B.

    1985-01-01

    We have isolated and studied two alloreactive, T4+, human lymphocyte clones that release interleukin 2 (IL-2) and interleukin 3 (IL-3) bioactivities upon stimulation with IL-2, alloantigen, or Sepharose-conjugated antibodies directed against the T3 protein. Anti-IL-2 receptor monoclonal antibodies block IL-2-, alloantigen-, or anti-T3-stimulated IL-3 release. Hence, release of IL-3 activity in each circumstance is rigorously dependent upon activation of the IL-2 receptor. Even low, nonmitogen...

  6. Change points in the series of T4 counts prior to AIDS.

    Science.gov (United States)

    Kiuchi, A S; Hartigan, J A; Holford, T R; Rubinstein, P; Stevens, C E

    1995-03-01

    The absolute number of T4 cells has been established as an important clinical marker of disease progression to acquired immunodeficiency syndrome (AIDS) in persons infected with human immunodeficiency virus (HIV). Series of T4 counts are analyzed from the 131 homosexual men who entered the New York Blood Center Study in 1984, mostly seropositive for HIV, and who developed AIDS as participants by 1990. These series exhibit a gradual decline of the log(T4) count followed by a more rapid decline close to the time of the development of AIDS. Empirical Bayes and hierarchical Bayes change point models are proposed to estimate the distribution of the time before AIDS when this rapid decline begins. Results using the EM Algorithm and Markov chain Monte Carlo indicate that the mean change point occurs approximately 1 year before diagnosis with a standard deviation of 9 months. Detection of a change point may indicate that an AIDS diagnosis is increasingly likely for an individual HIV-positive but AIDS-free. PMID:7766779

  7. T3/T4 thoracic sympathictomy and compensatory sweating in treatment of palmar hyperhidrosis

    Institute of Scientific and Technical Information of China (English)

    YANG Jie; TAN Jia-ju; YE Guo-lin; GU Wei-quan; WANG Jun; LIU Yan-guo

    2007-01-01

    Background Compensatory sweating (CS) is one of the most common postoperative complications after thoracic sympathectomy, sympathicotomy or endoscopic sympathetic block (ESB) for palmar hyperhidrosis. This study was conducted to examine the relevance between CS and the sympathetic segment being transected in the surgical treatment of palmar hyperhidrosis, and thus to detect the potential mechanism of the occurrence of CS.Methods Between October 2004 and June 2006, 163 patients with primary hyperhidrosis were randomly divided into two groups, T3 sympathicotomy (78 patients) and T4 sympathicotomy(85), who were operated upon under general anesthesia via single lumen intubation and intercostal video-mediastinoscopy (VM).Results No morbidity or mortality occurred. Palmar hyperhidrosis was cured in all patients. Follow-up(mean (13.8±6.2)months) showed no recurrence of palmar hyperhidrosis. The difference of rates of mild CS in groups T3 and T4 was of no statistical significance. The rate of moderate CS was significantly lower in group T4 than in group T3. No severe CS occurred.Conclusion The rates of occurrence and severity of CS are lowered with the lower sympathetic chain being transected.

  8. Key Players in I-DmoI Endonuclease Catalysis Revealed from Structure and Dynamics

    DEFF Research Database (Denmark)

    Molina, Rafael; Besker, Neva; Marcaida, Maria Jose;

    2016-01-01

    Homing endonucleases, such as I-DmoI, specifically recognize and cleave long DNA target sequences (∼20 bp) and are potentially powerful tools for genome manipulation. However, inefficient and off-target DNA cleavage seriously limits specific editing in complex genomes. One approach to overcome th...

  9. Molecular Recognition of DNA Damage Sites by Apurinic/Apyrimidinic Endonucleases

    Energy Technology Data Exchange (ETDEWEB)

    Braun, W. A.

    2005-07-28

    The DNA repair/redox factor AP endonuclease 1 (APE1) is a multifunctional protein which is known to to be essential for DNA repair activity in human cells. Structural/functional analyses of the APE activity is thus been an important research field to assess cellular defense mechanisms against ionizing radiation.

  10. Arthrobacter luteus restriction endonuclease cleavage map of X174 RF DNA

    NARCIS (Netherlands)

    Vereijken, J.M.; Mansfeld, A.D.M. van; Baas, P.D.; Jansz, H.S.

    1975-01-01

    Cleavage of X174 RF DNA with the restriction endonuclease from Arthrobacter luteus (Alu I) produces 23 fragments of approximately 24–1100 base pairs in length. The order of most of these fragments has been established by digestion of Haemophilus influenzae Rd (Hind II) and Haemophilus aegyptius (Hae

  11. The identification and optimization of 2,4-diketobutyric acids as flap endonuclease 1 inhibitors.

    Science.gov (United States)

    Tumey, L Nathan; Huck, Bayard; Gleason, Elizabeth; Wang, Jianmin; Silver, Daniel; Brunden, Kurt; Boozer, Sherry; Rundlett, Stephen; Sherf, Bruce; Murphy, Steven; Bailey, Andrew; Dent, Tom; Leventhal, Christina; Harrington, John; Bennani, Youssef L

    2004-10-01

    There have been several recent reports of chemopotentiation via inhibition of DNA repair processes. Flap endonuclease 1 (FEN1) is a key enzyme involved in base excision repair (BER), a primary pathway utilized by mammalian cells to repair DNA damage. In this report, we describe the identification and SAR of a series of 2,4-diketobutyric acid FEN1 inhibitors. PMID:15341951

  12. Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering

    KAUST Repository

    Aouida, Mustapha

    2015-07-30

    The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15–39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.

  13. Endonuclease modified comet assay for oxidative DNA damage induced by detection of genetic toxiants

    Institute of Scientific and Technical Information of China (English)

    赵健

    2014-01-01

    Objective The aim of this study was to investigate the use of the lesion-specific endonucleases-modifiedcomet assay for analysis of DNA,oxidation in cell lines.Methods DNA breaks and oxidative damage were evaluated by normal alkaline and formamidopyrimidine-DNAglycosylase(FPG)modified comet assays.Cytotoxicity was assessed by MTT method.The human bronchial epi-

  14. Effect of bacteriophage infection in combination with tobramycin on the emergence of resistance in Escherichia coli and Pseudomonas aeruginosa biofilms.

    Science.gov (United States)

    Coulter, Lindsey B; McLean, Robert J C; Rohde, Rodney E; Aron, Gary M

    2014-10-03

    Bacteriophage infection and antibiotics used individually to reduce biofilm mass often result in the emergence of significant levels of phage and antibiotic resistant cells. In contrast, combination therapy in Escherichia coli biofilms employing T4 phage and tobramycin resulted in greater than 99% and 39% reduction in antibiotic and phage resistant cells, respectively. In P. aeruginosa biofilms, combination therapy resulted in a 60% and 99% reduction in antibiotic and PB-1 phage resistant cells, respectively. Although the combined treatment resulted in greater reduction of E. coli CFUs compared to the use of antibiotic alone, infection of P. aeruginosa biofilms with PB-1 in the presence of tobramycin was only as effective in the reduction of CFUs as the use of antibiotic alone. The study demonstrated phage infection in combination with tobramycin can significantly reduce the emergence of antibiotic and phage resistant cells in both E. coli and P. aeruginosa biofilms, however, a reduction in biomass was dependent on the phage-host system.

  15. Bacteriophages as Bactericides in Plant Protection

    Directory of Open Access Journals (Sweden)

    Aleksa Obradović

    2009-01-01

    Full Text Available Control of plant pathogenic bacteria is a serious problem in production of many agricultural crops. High multiplication rate, adaptability and life inside plant tissue make bacteria unsuitable and inaccessible for most of control measures. Consequently, the list of bactericides available for plant protection is very short. Lately, biological control measures have been intensively studied as a potential solution of the problem. Investigation of bacteriophages,viruses that attack bacteria, is a fast-expanding area of research in plant protection. Several experiments have shown that they can be used as a very efficient tool for control of plant pathogenic bacteria. The fact that they are widespread natural bacterial enemies, simple for cultivation and management, host-specific, suitable for integration with other control practices, human and environment friendly, provide a great advantage for the application of phages over other bactericides.

  16. Bacteriophage T7 DNA polymerase — Sequenase

    Directory of Open Access Journals (Sweden)

    Bin eZhu

    2014-04-01

    Full Text Available An ideal DNA polymerase for chain-terminating DNA sequencing should possess the following features: 1 incorporate dideoxy- and other modified nucleotides at an efficiency similar to that of the cognate deoxynucleotides; 2 high processivity; 3 high fidelity in the absence of proofreading/exonuclease activity; and 4 production of clear and uniform signals for detection. The DNA polymerase encoded by bacteriophage T7 is naturally endowed with or can be engineered to have all these characteristics. The chemically or genetically modified enzyme (Sequenase expedited significantly the development of DNA sequencing technology. This article reviews the history of studies on T7 DNA polymerase with emphasis on the serial key steps leading to its use in DNA sequencing. Lessons from the study and development of T7 DNA polymerase have and will continue to enlighten the characterization of novel DNA polymerases from newly discovered microbes and their modification for use in biotechnology.

  17. Bacteriophage endolysins: applications for food safety.

    Science.gov (United States)

    Schmelcher, Mathias; Loessner, Martin J

    2016-02-01

    Bacteriophage endolysins (peptidoglycan hydrolases) have emerged as a new class of antimicrobial agents useful for controlling bacterial infection or other unwanted contaminations in various fields, particularly in the light of the worldwide increasing frequency of drug-resistant pathogens. This review summarizes and discusses recent developments regarding the use of endolysins for food safety. Besides the use of native and engineered endolysins for controlling bacterial contamination at different points within the food production chain, this also includes the application of high-affinity endolysin-derived cell wall binding domains for rapid detection of pathogenic bacteria. Novel approaches to extend the lytic action of endolysins towards Gram-negative cells will also be highlighted.

  18. Why Be Temperate: Lessons from Bacteriophage λ.

    Science.gov (United States)

    Gandon, Sylvain

    2016-05-01

    Many pathogens have evolved the ability to induce latent infections of their hosts. The bacteriophage λ is a classical model for exploring the regulation and the evolution of latency. Here, I review recent experimental studies on phage λ that identify specific conditions promoting the evolution of lysogenic life cycles. In addition, I present specific adaptations of phage λ that allow this virus to react plastically to variations in the environment and to reactivate its lytic life cycle. All of these different examples are discussed in the light of evolutionary epidemiology theory to disentangle the different evolutionary forces acting on temperate phages. Understanding phage λ adaptations yield important insights into the evolution of latency in other microbes, including several life-threatening human pathogens. PMID:26946976

  19. Surgical treatment of Ⅲb-T_4 lung cancer invading left atrium and great vessels

    Institute of Scientific and Technical Information of China (English)

    WANG Xiao-xin; LIU Tong-lin; YIN Xing-ru

    2010-01-01

    Background Ⅲb-T_4 non-small cell lung cancer (NSCLC) is commonly considered a contraindication to surgery, although chemo-radiotherapy also achieves a poor survival rate. We reviewed our experience with T_4 NSCLC patients who underwent surgery to explore the indications and prognostic factors of surgical treatment of lung cancer invading the left atrium and great vessels.Methods We investigated a cohort of 105 patients, 79 men and 26 women, who underwent surgery from May 1996 to July 2008. Their pathological staging was T_4N_(0-2)M_0. The median age was 59 years, ranging from 36 to 75 years. Patients were grouped based on invading sites: tumors invading the left atrium (LA group), tumors invading the superior vena cava (SVC group), and tumors invading the intrapericardial pulmonary artery (PA group). Patients were further characterized based upon the type of operation, complete resection and incomplete resection groups, and on the lymph node pathological status, N_0, N_1 and N_2 groups. We calculated the overall five-year survival rate.Results All patients received resection of primary lesions, with partial resection of the left atrium in the LA group (n=25), angioplasty of superior vena cava in the SVC group (n=23) and intrapericardial ligation of the pulmonary artery in the PA group (n=57). Complete resection was possible in 77 patients (73.3%). The overall survival rate of the 105 patients was 41.0% at 5 years; 36.0% for the LA group, 34.8% for the SVC group and 45.6% for the PA group. Pathological N status significantly influenced the overall 5-year survival rate; 61.5% for No, 51.1% for the N_1 and 11.8% for the N_2 groups (N_2 group versus No group, P <0.0001, N_2 versus N_1 group, P <0.0001). Surgical resection also influenced survival; 49.4% for the complete resection group and 17.9% for the incomplete resection group (P <0.0001). Cox regression analysis demonstrated that pathological N status was a significant independent predictor of prognosis

  20. Bacteriophages of Leuconostoc, Oenococcus and Weissella

    Directory of Open Access Journals (Sweden)

    Witold P. Kot

    2014-04-01

    Full Text Available Leuconostoc (Ln., Weissella and Oenococcus form a group of related genera of lactic acid bacteria, which once all shared the name Leuconostoc. They are associated with plants, fermented vegetable products, raw milk, dairy products, meat and fish. Most of industrially relevant Leuconostoc strains can be classified as either Ln. mesenteroides or Ln. pseudomesenteroides. They are important flavor producers in dairy fermentations and they initiate nearly all vegetable fermentations. Therefore bacteriophages attacking Leuconostoc strains may negatively influence the production process. Bacteriophages attacking Leuconostoc strains were first reported in 1946. Since then, the majority of described Leuconostoc phages was isolated from either dairy products or fermented vegetable products. Both lytic and temperate phages of Leuconostoc were reported. Most of Leuconostoc phages examined using electron microscopy belong to the Siphoviridae family and differ in morphological details. Hybridization and comparative genomic studies of Leuconostoc phages suggest that they can be divided into several groups, however overall diversity of Leuconostoc phages is much lower as compared to e.g. lactococcal phages. Several fully sequenced genomes of Leuconostoc phages have been deposited in public databases. Lytic phages of Leuconostoc can be divided into two host species-specific groups with similarly organized genomes that shared very low nucleotide similarity. Phages of dairy Leuconostoc have rather limited host-ranges. The receptor binding proteins of two lytic Ln. pseudomesenteroides phages have been identified. Molecular tools for detection of dairy Leuconostoc phages have been developed. The rather limited data on phages of Oenococcus and Weissella show that i lysogeny seems to be abundant in Oenococcus strains, and ii several phages infecting Weissella cibaria are also able to productively infect strains of other Weissella species and even strains of the genus

  1. Montmorillonite-induced Bacteriophage φ6 Disassembly

    Science.gov (United States)

    Trusiak, A.; Gottlieb, P.; Katz, A.; Alimova, A.; Steiner, J. C.; Block, K. A.

    2012-12-01

    It is estimated that there are 1031 virus particles on Earth making viruses an order of magnitude more prevalent in number than prokaryotes with the vast majority of viruses being bacteriophages. Clays are a major component of soils and aquatic sediments and can react with RNA, proteins and bacterial biofilms. The clays in soils serve as an important moderator between phage and their host bacteria, helping to preserve the evolutionary balance. Studies on the effects of clays on viral infectivity have given somewhat contradictory results; possibly a consequence of clay-virus interactions being dependent on the unique structure of particular viruses. In this work, the interaction between montmorillonite and the bacteriophage φ6 is investigated. φ6 is a member of the cystovirus family that infects Pseudomonas syringe, a common plant pathogen. As a member of the cystovirus family with an enveloped structure, φ6 serves as a model for reoviruses, a human pathogen. Experiments were conducted with φ6 suspended in dilute, purified homoionic commercial-grade montmorillonite over a range of virus:clay ratios. At a 1:100000 virus:clay ratio, the clay reduced viral infectivity by 99%. The minimum clay to virus ratio which results in a measurable reduction of P. syringae infection is 1:1. Electron microscopy demonstrates that mixed suspensions of smectite and virus co-aggregate to form flocs encompassing virions within the smectite. Both free viral particles as well as those imbedded in the flocs are seen in the micrographs to be missing the envelope- leaving only the nucleocapsid (NC) intact; indicating that smectite inactivates the virus by envelope disassembly. These results have strong implications in the evolution of both the φ6 virus and its P. syringae host cells. TEM of aggregate showing several disassembled NCs.

  2. A Hypothesis for Bacteriophage DNA Packaging Motors

    Directory of Open Access Journals (Sweden)

    Philip Serwer

    2010-08-01

    Full Text Available The hypothesis is presented that bacteriophage DNA packaging motors have a cycle comprised of bind/release thermal ratcheting with release-associated DNA pushing via ATP-dependent protein folding. The proposed protein folding occurs in crystallographically observed peptide segments that project into an axial channel of a protein 12-mer (connector that serves, together with a coaxial ATPase multimer, as the entry portal. The proposed cycle begins when reverse thermal motion causes the connector’s peptide segments to signal the ATPase multimer to bind both ATP and the DNA molecule, thereby producing a dwell phase recently demonstrated by single-molecule procedures. The connector-associated peptide segments activate by transfer of energy from ATP during the dwell. The proposed function of connector/ATPase symmetry mismatches is to reduce thermal noise-induced signaling errors. After a dwell, ATP is cleaved and the DNA molecule released. The activated peptide segments push the released DNA molecule, thereby producing a burst phase recently shown to consist of four mini-bursts. The constraint of four mini-bursts is met by proposing that each mini-burst occurs via pushing by three of the 12 subunits of the connector. If all four mini-bursts occur, the cycle repeats. If the mini-bursts are not completed, a second cycle is superimposed on the first cycle. The existence of the second cycle is based on data recently obtained with bacteriophage T3. When both cycles stall, energy is diverted to expose the DNA molecule to maturation cleavage.

  3. Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4.

    Science.gov (United States)

    Shaw, D M; Embleton, M J; Westwater, C; Ryan, M G; Myers, K A; Kingsman, S M; Carroll, M W; Stern, P L

    2000-12-15

    The oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell surface. It is defined by a monoclonal antibody, mAb5T4, that recognises a conformational extracellular epitope in the molecule. Overexpression of 5T4 antigen by tumours of several types has been linked with disease progression and poor clinical outcome. Its restricted expression in non-malignant tissue makes 5T4 antigen a suitable target for the development of antibody directed therapies. The use of murine monoclonal antibodies for targeted therapy allows the tumour specific delivery of therapeutic agents. However, their use has several drawbacks, including a strong human anti-mouse immune (HAMA) response and limited tumour penetration due to the size of the molecules. The use of antibody fragments leads to improved targeting, pharmacokinetics and a reduced HAMA. A single chain antibody (scFv) comprising the variable regions of the mAb5T4 heavy and light chains has been expressed in Escherichia coli. The addition of a eukaryotic leader sequence allowed production in mammalian cells. The two 5T4 single chain antibodies, scFv5T4WT19 and LscFv5T4, described the same pattern of 5T4 antigen expression as mAb5T4 in normal human placenta and by FACS. Construction of a 5T4 extracellular domain-IgGFc fusion protein and its expression in COS-7 cells allowed the relative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay. MAb5T4 has a high affinity, K(D)=1.8x10(-11) M, as did both single chain antibodies, scFv5T4WT19 K(D)=2.3x10(-9) M and LscFv5T4 K(D)=7.9x10(-10) M. The small size of this 5T4 specific scFv should allow construction of fusion proteins with a range of biological response modifiers to be prepared whilst retaining the improved pharmacokinetic properties of scFvs. PMID:11113573

  4. Single-stranded endonuclease activity in the excretory--secretory products of Trichinella spiralis and Trichinella pseudospiralis

    OpenAIRE

    Mak, C.; Chung, YYY; Ko, RCC

    2000-01-01

    A novel acidic extracellular single-stranded endonuclease was demonstrated for the first time in the excretory-secretory (E-S) products of 2 species of Trichinella. Unlike the double-stranded endonuclease reported earlier, the single-stranded molecule is divalent cation independent and is detected in both T. spiralis and T. pseudospiralis E-S products. It hydrolysed single-stranded DNA and RNA at comparable rates. The single-stranded endonuclease was sensitive to inhibition by Zn2+ and to hig...

  5. Intensity-modulated radiation therapy for T4 nasopharyngeal carcinoma. Treatment results and locoregional

    Energy Technology Data Exchange (ETDEWEB)

    Chen, J.L.Y.; Tsai, C.L.; Chen, W.Y.; Wang, C.W. [National Taiwan Univ. Hospital, Taipei (China). Div. of Radiation Oncology; Huang, Y.S.; Chen, Y.F. [National Taiwan Univ. Hospital, Taipei (China). Dept. of Medical Imaging; Kuo, S.H. [National Taiwan Univ. Hospital, Taipei (China). Div. of Radiation Oncology; National Taiwan Univ. College of Medicine, Taipei (China). Graduate Inst. of Clinical Medicine; Hong, R.L. [National Taiwan Univ. Hospital, Taipei (China). Div. of Medical Oncology; Ko, J.Y.; Lou, P.J. [National Taiwan Univ. Hospital, Taipei (China). Dept. of Otolaryngology

    2013-12-15

    Purpose: The purpose of this work was to examine outcomes in patients with T4 nasopharyngeal carcinoma (NPC) treated with intensity-modulated radiation therapy (IMRT). Methods and materials: Between 2007 and 2010, 154 patients with nonmetastatic T4 NPC were treated with IMRT to a total dose of 70 Gy in 33-35 fractions. In addition, 97 % of patients received concurrent platinum-based chemotherapy. The median follow-up time was 52.8 months. Results: The rates of 5-year actuarial locoregional control, distant metastasis-free survival, progression free-survival, and overall survival (OS) were 81.2, 72.2, 61.9, and 78.1 %, respectively. A total of 27 patients had locoregional recurrence: 85.2 % in-field failures, 11.1 % marginal failures, and 3.7 % out-of-field failures. Fourteen patients with locoregional recurrence received aggressive treatments, including nasopharyngectomy, neck dissection, or re-irradiation, and the 5-year OS rate tended to be better (61.9 %) compared to those receiving conservative treatment (32.0 %, p = 0.051). In patients treated with 1 course of radiotherapy, grade {>=} 3 toxicities of ototoxicity, neck fibrosis, xerostomia, epistaxis, and radiographic temporal lobe necrosis occurred in 18.2, 9.8, 6.3, 2.1, and 5.6 % of patients, respectively. Increased ototoxicity, osteonecrosis, severe nasal bleeding, and temporal necrosis were observed in patients treated by re-irradiation. Conclusion: IMRT offers good locoregional control in patients with T4 NPC. For patients with locoregional recurrence after definitive radiotherapy, aggressive local treatment may be considered for a better outcome. (orig.)

  6. Measurement of thyroid hormones (thyroxine, T4; triiodothyronine, T3) in captive nondomestic felids.

    Science.gov (United States)

    Rodini, Débora Cattaruzzi; Felippe, Erika Cristiane Gutierrez; Oliveira, Cláudio Alvarenga

    2007-03-01

    The aim of this research was to obtain basic values for the evaluation of thyroid function in nondomestic felids. Serum thyroid hormone concentrations (thyroxine, T4; triiodothyronine, T3) were measured by radioimmunoassay in 145 cats, representing nine species of captive nondomestic felids: jaguar (Panthera onca), n = 49; puma (Puma concolor), n = 10; ocelot (Leopardus pardalis), n = 22; oncilla (Leopardus tigrinus), n = 12; geoffroy (Oncifelis geoffroyi), n = 4; jaguarundi (Herpailurus yaguarondi), n = 8; margay (Leopardus wiedii), n = 7; lion (Panthera leo), n = 26; and tiger (Panthera tigris), n = 7. For each species, mean +/- SEM of T3 and T4, respectively, were as follows: jaguar, 0.56 +/- 0.03 and 9.7 +/- 0.8 ng/ml; puma, 0.67 +/- 0.04 and 11.2 +/- 1.2 ng/ml; ocelot, 0.48 +/- 0.03 and 13.8 +/- 1.5 ng/ml; oncilla, 0.43 +/- 0.03 and 10.0 +/- 1.6 ng/ml; geoffroy, 0.44 +/- 0.04 and 8.0 +/- 0.16 ng/ml; jaguarundi, 0.7 +/- 0.03 and 5.0 +/- 1.0 ng/ml; margay, 0.48 +/- 0.04 and 12.2 +/- 2.3 ng/ml; lion, 0.43 +/- 0.02 and 5.7 +/- 2.6 ng/ml; and tiger, 0.66 +/- 0.03 and 12.6 +/- 0.9 ng/ml. Within species, T3 and T4 concentrations did not differ (P > 0.05) between males and females. PMID:17469287

  7. The isolation and characterization of Campylobacter jejuni bacteriophages from free range and indoor poultry.

    Science.gov (United States)

    Owens, Jane; Barton, Mary D; Heuzenroeder, Michael W

    2013-02-22

    Six hundred and sixty one samples - primarily fresh chicken faeces - were processed to isolate wild type Campylobacter jejuni bacteriophages, via overlay agar methods using C. jejuni NCTC 12662. The aims of this study were to isolate and purify bacteriophages and then test for their ability to lyse field strains of C. jejuni in vitro. Of all samples processed, 130 were positive for bacteriophages. A distinct difference was observed between samples from different poultry enterprises. No bacteriophages could be isolated from indoor broilers. The majority of bacteriophages were isolated from free range poultry - both broilers and egg layers. Bacteriophages were purified and then selected for characterization based on their ability to produce clear lysis on plaque assay, as opposed to turbid plaques. Two hundred and forty one C. jejuni field isolates were tested for sensitivity to the bacteriophages. Lysis was graded subjectively and any minimal lysis was excluded. Using this system, 59.0% of the C. jejuni isolates showed significant sensitivity to at least one bacteriophage. The sensitivity to individual bacteriophages ranged from 10.0% to 32.5% of the C. jejuni isolates. Five bacteriophages were examined by electron microscopy and determined to belong to the Myoviridae family. The physical size, predicted genetic composition and genome size of the bacteriophages correlated well with other reported Campylobacter bacteriophages. The reasons for the observed difference between indoor broilers and free range poultry is unknown, but are postulated to be due to differences in the Campylobacter population in birds under different rearing conditions.

  8. On symmetries of N=(4,4) sigma models on T^4

    CERN Document Server

    Volpato, Roberto

    2014-01-01

    Motivated by an analogous result for K3 models, we classify all groups of symmetries of non-linear sigma models on a torus T^4 that preserve the N=(4,4) superconformal algebra. The resulting symmetry groups are isomorphic to certain subgroups of the Weyl group of E8, that plays a role similar to the Conway group for the case of K3 models. Our analysis heavily relies on the triality automorphism of the T-duality group SO(4,4,Z). As a byproduct of our results, we discover new explicit descriptions of K3 models as asymmetric orbifolds of torus CFTs.

  9. Pathology of advanced buccal mucosa cancer involving masticator space (T4b

    Directory of Open Access Journals (Sweden)

    N P Trivedi

    2015-01-01

    Full Text Available BACKGROUND: Buccal mucosa cancer involving masticator space is classified as very advanced local disease (T4b. The local recurrence rate is very high due to poor understanding of the extent of tumor spread in masticator space and technically difficult surgical clearance. The objective of this study is to understand the extent of tumor spread in masticator space to form basis for appropriate surgical resection. MATERIALS AND METHODS: All consecutive patients with T4b-buccal cancer underwent compartment resection, with complete anatomical removal of involved soft-tissue structures. Specimens were systematically studied to understand the extent of invasion of various structures. The findings of clinical history, imaging and pathologic evaluation were compared and the results were evaluated. RESULTS: A total of 45 patients with advanced buccal cancer (T4b were included in this study. The skin, mandible and lymph nodes were involved in 30, 24 and 17 cases respectively. The pterygoid muscles were involved in 34 cases (medial-pterygoid in 12 and both pterygoids in 22 cases and masseter-muscle in 32 cases. Average distance for soft-tissue margins after compartment surgery was 2 cm and the margins were positive in 3 cases. The group with involvement of medial pterygoid muscle had safest margin with compartment surgery while it was also possible to achieve negative margins for group involving lateral pterygoid muscle and plates.The involvement of pterygomaxillary fissure was area of concern and margin was positive in 2 cases with one patient developing local recurrence with intracranial extension. At 21 months median follow-up (13-35 months, 38 patients were alive without disease while two developed local recurrence at the skull base.CONCLUSIONS: T4b buccal cancers have significant soft-tissue involvement in the masticator space. En bloc removal of all soft-tissues in masticator space is advocated to remove tumor contained within space. The compartment

  10. Constructive Tensorial Group Field Theory II: The $U(1)-T^4_4$ Model

    CERN Document Server

    Lahoche, Vincent

    2015-01-01

    In this paper we continue our program of non-pertubative constructions of tensorial group field theories (TGFT). We prove analyticity and Borel summability in a suitable domain of the coupling constant of the simplest super-renormalizable TGFT which contains some ultraviolet divergencies, namely the color-symmetric quartic melonic rank-four model with Abelian $U(1)$ gauge invariance, nicknamed $U(1)-T^4_4$. We use a multiscale loop vertex expansion. It is an extension of the loop vertex expansion (the basic constructive technique for non-local theories) which is required for theories that involve non-trivial renormalization.

  11. Conserved structural chemistry for incision activity in structurally non-homologous apurinic/apyrimidinic endonuclease APE1 and endonuclease IV DNA repair enzymes.

    Energy Technology Data Exchange (ETDEWEB)

    Tsutakawa, Susan E.; Shin, David S.; Mol, Clifford D.; Izum, Tadahide; Arvai, Andrew S.; Mantha, Anil K.; Szczesny, Bartosz; Ivanov, Ivaylo N.; Hosfield, David J.; Maiti, Buddhadev; Pique, Mike E.; Frankel, Kenneth A.; Hitomi, Kenichi; Cunningham, Richard P.; Mitra, Sankar; Tainer, John A.

    2013-03-22

    Non-coding apurinic/apyrimidinic (AP) sites in DNA form spontaneously and as DNA base excision repair intermediates are the most common toxic and mutagenic in vivo DNA lesion. For repair, AP sites must be processed by 5' AP endonucleases in initial stages of base repair. Human APE1 and bacterial Nfo represent the two conserved 5' AP endonuclease families in the biosphere; they both recognize AP sites and incise the phosphodiester backbone 5' to the lesion, yet they lack similar structures and metal ion requirements. Here, we determined and analyzed crystal structures of a 2.4 ? resolution APE1-DNA product complex with Mg(2+) and a 0.92 Nfo with three metal ions. Structural and biochemical comparisons of these two evolutionarily distinct enzymes characterize key APE1 catalytic residues that are potentially functionally similar to Nfo active site components, as further tested and supported by computational analyses. We observe a magnesium-water cluster in the APE1 active site, with only Glu-96 forming the direct protein coordination to the Mg(2+). Despite differences in structure and metal requirements of APE1 and Nfo, comparison of their active site structures surprisingly reveals strong geometric conservation of the catalytic reaction, with APE1 catalytic side chains positioned analogously to Nfo metal positions, suggesting surprising functional equivalence between Nfo metal ions and APE1 residues. The finding that APE1 residues are positioned to substitute for Nfo metal ions is supported by the impact of mutations on activity. Collectively, the results illuminate the activities of residues, metal ions, and active site features for abasic site endonucleases.

  12. Bacteriophage therapy for safeguarding animal and human health: a review.

    Science.gov (United States)

    Tiwari, Ruchi; Dhama, Kuldeep; Kumar, Amit; Rahal, Anu; Kapoor, Sanjay

    2014-02-01

    Since the discovery of bacteriophages at the beginning of the 19th century their contribution to bacterial evolution and ecology and use in a variety of applications in biotechnology and medicine has been recognized and understood. Bacteriophages are natural bacterial killers, proven as best biocontrol agents due to their ability to lyse host bacterial cells specifically thereby helping in disease prevention and control. The requirement of such therapeutic approach is straight away required in view of the global emergence of Multidrug Resistant (MDR) strains of bacteria and rapidly developing resistance to antibiotics in both animals and humans along with increasing food safety concerns including of residual antibiotic toxicities. Phage typing is a popular tool to differentiate bacterial isolates and to identify and characterize outbreak-associated strains of Salmonella, Campylobacter, Escherichia and Listeria. Numerous methods viz. plaque morphology, ultracentrifugation in the density gradient of CsCl2, and random amplified polymorphic DNA (RAPD) have been found to be effective in detection of various phages. Bacteriophages have been isolated and recovered from samples of animal waste products of different livestock farms. High titer cocktails of broad spectrum lytic bacteriophages are usually used for clinical trial for assessing their therapeutic efficacy against antibiotic unresponsive infections in different animals. Bacteriophage therapy also helps to fight various bacterial infections of poultry viz. colibacillosis, salmonellosis and listeriosis. Moreover, the utility of phages concerning biosafety has raised the importance to explore and popularize the therapeutic dimension of this promising novel therapy which forms the topic of discussion of the present review.

  13. Bacteriophages as an alternative strategy for fighting biofilm development.

    Science.gov (United States)

    Parasion, Sylwia; Kwiatek, Magdalena; Gryko, Romuald; Mizak, Lidia; Malm, Anna

    2014-01-01

    The ability of microbes to form biofilms is an important element of their pathogenicity, and biofilm formation is a serious challenge for today's medicine. Fighting the clinical complications associated with biofilm formation is very difficult and linked to a high risk of failure, especially in a time of increasing bacterial resistance to antibiotics. Bacterial species most commonly isolated from biofilms include coagulase-negative staphylococci, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. The frequent failure of antibiotic therapy led researchers to look for alternative methods and experiment with the use of antibacterial factors with a mechanism of action different from that of antibiotics. Experimental studies with bacteriophages and mixtures thereof, expressing lytic properties against numerous biofilm-forming bacterial species showed that bacteriophages may both prevent biofilm formation and contribute to eradication of biofilm bacteria. A specific role is played here by phage depolymerases, which facilitate the degradation of extracellular polymeric substances (EPS) and thus the permeation of bacteriophages into deeper biofilm layers and lysis of the susceptible bacterial cells. Much hope is placed in genetic modifications of bacteriophages that would allow the equipping bacteriophages with the function of depolymerase synthesis. The use of phage cocktails prevents the development of phage-resistant bacteria.

  14. Alternative bacteriophage life cycles: the carrier state of Campylobacter jejuni.

    Science.gov (United States)

    Siringan, Patcharin; Connerton, Phillippa L; Cummings, Nicola J; Connerton, Ian F

    2014-01-01

    Members of the genus Campylobacter are frequently responsible for human enteric disease, often through consumption of contaminated poultry products. Bacteriophages are viruses that have the potential to control pathogenic bacteria, but understanding their complex life cycles is key to their successful exploitation. Treatment of Campylobacter jejuni biofilms with bacteriophages led to the discovery that phages had established a relationship with their hosts typical of the carrier state life cycle (CSLC), where bacteria and bacteriophages remain associated in equilibrium. Significant phenotypic changes include improved aerotolerance under nutrient-limited conditions that would confer an advantage to survive in extra-intestinal environments, but a lack in motility eliminated their ability to colonize chickens. Under these circumstances, phages can remain associated with a compatible host and continue to produce free virions to prospect for new hosts. Moreover, we demonstrate that CSLC host bacteria can act as expendable vehicles for the delivery of bacteriophages to new host bacteria within pre-colonized chickens. The CSLC represents an important phase in the ecology of Campylobacter bacteriophage. PMID:24671947

  15. Hormonal evaluation of T4 and T3 through radioimmunoassay in younglings of rats with hypothyroidism

    International Nuclear Information System (INIS)

    The onset of fetal thyroid function occurs about 17-18 days after conception in the rat. The maternal hypothyroidism which occurs during gestation provokes alteration in the rat after birth. Due to this alteration, we decided to analyze the hormonal modification in the newborn rats. The hypothyroidism was induced in normal dams, which were being treated for 7 days with MMI (in the concentration of 0,03% in drinking water) before mating. Another dam group which was submitted to an induction of hypothyroidism maintained the treatment with MMI for 13 days during gestation. The hormones were assessed by radioimmunoassay technique. It was seen that the rats which were born from hypothyroid dams suffered alterations on its T4 and T3 hormone levels concerning to 10, 30 and 60 days after birth. There was also modifications on their weight and size. The growth is affected throughout post-natal life by thyroid hormones, which has a facilitator influence on growth hormone economy, as opposed to the inhibitory effects on TSH economy. The administration of MMI bars the fetal thyroid gland function, causing a decrease of both T4 and T3 levels, even after the birth, indicating that the maternal hypothyroidism influences on the post-natal life of the rat. (author)

  16. Experimental infection of T4 Acanthamoeba genotype determines the pathogenic potential.

    Science.gov (United States)

    Alves, Daniella de Sousa Mendes Moreira; Moraes, Aline Silva; Alves, Luciano Moreira; Gurgel-Gonçalves, Rodrigo; Lino Junior, Ruy de Souza; Cuba-Cuba, César Augusto; Vinaud, Marina Clare

    2016-09-01

    T4 is the Acanthamoeba genotype most related to cases of granulomatous amoebic encephalitis (GAE) in immunocompromised patients and of keratitis in contact lens wearers. The determination of the pathogenic potential of Acanthamoeba clinical and environmental isolates using experimental models is extremely important to elucidate the capacity of free-living organisms to establish and cause disease in hosts. The aim of this study was to compare and evaluate the histopathology and culture between two different routes of experimental infection of T4 Acanthamoeba isolated from environmental and clinical source in mice (intracranial and intraperitoneal). Swiss isogenic healthy mice were inoculated with 10(4) trophozoites by intracranial (IC) and intraperitoneal (IP) routes and observed during 21 days. The brains from animals inoculated by the IC route were collected and from the animals of the IP inoculation group, the brains, livers, kidneys, spleens, and lungs were removed. The organs were prepared and appropriately divided to be evaluated with histopathology and culture. There was no significant difference between the inoculation routes in terms of isolates recovery (χ(2) = 0.09; p = 0.76). In the IC group, isolate recovery rate was significantly higher in histopathology than the one achieved by culture (χ(2) = 6.45; p protozoa, improving the knowledge on free-living amoebae isolates. PMID:27164833

  17. Investigation of ferromagnetic filled skutterudite compounds EuT4Sb12 (T = Fe, Ru, Os)

    International Nuclear Information System (INIS)

    The physical properties of single crystals of filled skutterudite compounds EuT4Sb12 (T = Fe, Ru, Os) have been investigated by means of x-ray diffraction, electrical resistivity, specific heat, magnetization, and x-ray absorption spectroscopy measurements. The Eu-based materials crystallize in the LaFe4P12-type structure (space group Im3-bar). A small Eu deficiency is encountered for Eu0.95Fe4Sb12, while the homologous compounds, Eu1.0Ru4Sb12 and Eu1.0Os4Sb12, reveal full occupancy of the Eu site. Eu0.95Fe4Sb12 appears to exhibit either canted ferromagnetic or ferrimagnetic order at TC = 88 K whereas EuRu4Sb12 and EuOs4Sb12 are ferromagnets with Curie temperatures of TC = 4 and 9 K, respectively. X-ray absorption near edge spectroscopy measurements reveal a nearly divalent Eu electronic configuration for EuT4Sb12 (T = Fe, Ru, Os) with no significant change with T atom or temperature

  18. Mini Transsternal Approach to the Anterior High Thoracic Spine (T1–T4 Vertebrae

    Directory of Open Access Journals (Sweden)

    Christian Brogna

    2016-01-01

    Full Text Available Purpose. The anterior high thoracic spine is one of the most complex segments to be accessed surgically due to anatomical constraints and transitional characteristics. We describe in detail the mini transsternal approach to metastatic, infective, traumatic, and degenerative pathologies of T1 to T4 vertebral bodies. We analyse our surgical series, indications, and outcomes. Methods. Over a 5-year period 18 consecutive patients with thoracic myelopathy due to metastatic, infective, traumatic, and degenerative pathologies with T1 to T4 vertebral bodies involvement received a mini transsternal approach with intraoperative monitoring. Frankel scoring system was used to grade the neurological status. Results. Mean follow-up was 40 months. 78% patients improved in Frankel grade after surgery and 22% patients remained unchanged. Average operation time was 210 minutes. There were no intraoperative complications. One patient developed postoperative pneumonia successfully treated with antibiotics. Conclusion. The mini transsternal is a safe approach for infective, metastatic, traumatic, and degenerative lesions affecting the anterior high thoracic spine and the only one allowing an early and direct visualisation of the anterior theca. This approach overcomes the anatomical constraints of this region and provides adequate room for optimal reconstruction and preservation of spinal alignment in the cervicothoracic transition zone with good functional patient outcomes.

  19. Virtual unit delay for digital frequency adaptive T/4 delay phase-locked loop system

    DEFF Research Database (Denmark)

    Yang, Yongheng; Zhou, Keliang; Blaabjerg, Frede

    2016-01-01

    -controller/processor with a fixed sampling rate considering the cost and complexity, where the number of unit delays that have been adopted should be an integer. For instance, in conventional digital control systems, a single-phase T/4 Delay Phase-Locked Loop (PLL) system takes 50 unit delays (i.e., in a 50-Hz system......Digital micro-controllers/processors enable the cost-effective control of grid-connected power converter systems in terms of system monitoring, signal processing (e.g., grid synchronization), control (e.g., grid current and voltage control), etc. Normally, the control is implemented in a micro...... with a sampling frequency of 10-kHz) to create a 90 ◦-lagging voltage in order to achieve the grid synchronization with the orthogonal voltage system. However, in practice, the grid frequency is a time-variant parameter due to various eventualities, and thus rounding the number of the unit delays for the T/4...

  20. Experimental Study of Stationary Shoulder Friction Stir Welded 7N01-T4 Aluminum Alloy

    Science.gov (United States)

    Ji, S. D.; Meng, X. C.; Li, Z. W.; Ma, L.; Gao, S. S.

    2016-03-01

    Stationary shoulder friction stir welding (SSFSW) was successfully used to weld 7N01-T4 aluminum alloy with the thickness of 4 mm. Effects of welding speed on formations, microstructures, and mechanical properties of SSFSW joint were investigated in detail. Under a constant rotational velocity of 2000 rpm, defect-free joints with smooth surface and small flashes are attained using welding speeds of 20 and 30 mm/min. Macrostructure of nugget zone in cross section presents kettle shape. For 7N01-T4 aluminum alloy with low thermal conductivity, decreasing welding speed is beneficial to surface formation of joint. With the increase of welding speed, mechanical properties of joints firstly increase and then decrease. When the welding speed is 30 mm/min, the tensile strength and elongation of joint reach the maximum values of 379 MPa and 7.9%, equivalent to 84.2 and 52% of base material, respectively. Fracture surface morphology exhibits typical ductile fracture. In addition, the minimum hardness value of joint appears in the heat affected zone.

  1. Microstructure and mechanical properties of friction stir welded thin sheets of 2024-T4 aluminum alloy

    Institute of Scientific and Technical Information of China (English)

    LI Lian; TONG Jian-hua; WAN Fa-rong; LONG Yi

    2006-01-01

    Friction stir welding (FSW) is a new and promising welding processing that can produce low-cost and high-quality joints of aluminum alloys. 1 mm thick sheets of 2024-T4 aluminum alloys which are always used as building and decorating materials were welded by FSW. The microstrueture and mechanical properties of friction stir welded 1 mm thick sheets of 2024-T4 aluminum alloy were studied. It was found that the thinner the 2024 aluminum alloy, the larger the FSW technological parameters field. The grains size of weld nugget zone (WNZ) is approximately 10 times smaller than that of the parent material, but the second phase in the material is not refined apparently in the welding. The FS welded joints have about 40% higher yield strength than the parent material,but the elongation of FS welded joints is under about 50% of the parent material. The electron backscattered diffraction (EBSD)results show that there are much more low angle boundaries (LAB) in WNZ than that in parent material, which indicates that FSW causes a number of sub-grain structures in WNZ, and this is also the reason of the increase of yield strength and Vickers hardness of the welded joint.

  2. Sequence variability of Campylobacter temperate bacteriophages

    Directory of Open Access Journals (Sweden)

    Ng Lai-King

    2008-03-01

    Full Text Available Abstract Background Prophages integrated within the chromosomes of Campylobacter jejuni isolates have been demonstrated very recently. Prior work with Campylobacter temperate bacteriophages, as well as evidence from prophages in other enteric bacteria, suggests these prophages might have a role in the biology and virulence of the organism. However, very little is known about the genetic variability of Campylobacter prophages which, if present, could lead to differential phenotypes in isolates carrying the phages versus those that do not. As a first step in the characterization of C. jejuni prophages, we investigated the distribution of prophage DNA within a C. jejuni population assessed the DNA and protein sequence variability within a subset of the putative prophages found. Results Southern blotting of C. jejuni DNA using probes from genes within the three putative prophages of the C. jejuni sequenced strain RM 1221 demonstrated the presence of at least one prophage gene in a large proportion (27/35 of isolates tested. Of these, 15 were positive for 5 or more of the 7 Campylobacter Mu-like phage 1 (CMLP 1, also designated Campylobacter jejuni integrated element 1, or CJIE 1 genes tested. Twelve of these putative prophages were chosen for further analysis. DNA sequencing of a 9,000 to 11,000 nucleotide region of each prophage demonstrated a close homology with CMLP 1 in both gene order and nucleotide sequence. Structural and sequence variability, including short insertions, deletions, and allele replacements, were found within the prophage genomes, some of which would alter the protein products of the ORFs involved. No insertions of novel genes were detected within the sequenced regions. The 12 prophages and RM 1221 had a % G+C very similar to C. jejuni sequenced strains, as well as promoter regions characteristic of C. jejuni. None of the putative prophages were successfully induced and propagated, so it is not known if they were functional or

  3. Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase

    OpenAIRE

    Klingenspor Martin; Fromme Tobias

    2007-01-01

    Abstract Background The subcloning of a DNA fragment from an entry vector into a destination vector is a routinely performed task in molecular biology labs. Results We here present a novel benchtop procedure to achieve rapid recombination into any destination vector of choice with the sole requirement of an endonuclease recognition site. The method relies on a specifically designed entry vector and the combined action of type II and type IIs endonucleases with ligase. The formulation leads to...

  4. Yeast redoxyendonuclease, a DNA repair enzyme similar to Escherichia coli endonuclease III

    Energy Technology Data Exchange (ETDEWEB)

    Gossett, J.; Lee, K.; Cunningham, R.P.; Doetsch, P.W.

    1988-04-05

    A DNA repair endonuclease (redoxyendonuclease) was isolated from bakers' yeast (Saccharomyces cerevisiae). The enzyme has been purified by a series of column chromatography steps and cleaves OsO/sub 4/-damaged, double-stranded DNA at sites of thymine glycol and heavily UV-irradiated DNA at sites of cytosine, thymine, and guanine photoproducts. The base specificity and mechanism of phosphodiester bond cleavage for the yeast redoxyendonuclease appear to be identical with those of Escherichia coli endonuclease III when thymine glycol containing, end-labeled DNA fragments of defined sequence are employed as substrates. Yeast redoxyendonuclease has an apparent molecular size of 38,000-42,000 daltons and is active in the absence of divalent metal cations. The identification of such an enzyme in yeast may be of value in the elucidation of the biochemical basis for radiation sensitivity in certain yeast mutants.

  5. Does quantum entanglement in DNA synchronize the catalytic centers of type II restriction endonucleases?

    CERN Document Server

    Kurian, P; Lindesay, J

    2014-01-01

    Several living systems have been examined for their apparent optimization of structure and function for quantum behavior at biological length scales. Orthodox type II endonucleases, the largest class of restriction enzymes, recognize four-to-eight base pair sequences of palindromic DNA, cut both strands symmetrically, and act without an external metabolite such as ATP. While it is known that these enzymes induce strand breaks by attacking phosphodiester bonds, what remains unclear is the mechanism by which cutting occurs in concert at the catalytic centers. Previous studies indicate the primacy of intimate DNA contacts made by the specifically bound enzyme in coordinating the two synchronized cuts. We propose that collective electronic behavior in the DNA helix generates coherent oscillations, quantized through boundary conditions imposed by the endonuclease, that provide the energy required to break two phosphodiester bonds. Such quanta may be preserved in the presence of thermal noise and electromagnetic in...

  6. UVI31+ is a DNA endonuclease that dynamically localizes to chloroplast pyrenoids in C. reinhardtii.

    Directory of Open Access Journals (Sweden)

    Manish Shukla

    Full Text Available UVI31+ is an evolutionarily conserved BolA family protein. In this study we examine the presence, localization and possible functions of this protein in the context of a unicellular alga, Chlamydomonas reinhardtii. UVI31+ in C. reinhardtii exhibits DNA endonuclease activity and is induced upon UV stress. Further, UVI31+ that normally localizes to the cell wall and pyrenoid regions gets redistributed into punctate foci within the whole chloroplast, away from the pyrenoid, upon UV stress. The observed induction upon UV-stress as well as the endonuclease activity suggests plausible role of this protein in DNA repair. We have also observed that UV31+ is induced in C. reinhardtii grown in dark conditions, whereby the protein localization is enhanced in the pyrenoid. Biomolecular interaction between the purified pyrenoids and UVI31+ studied by NMR demonstrates the involvement of the disordered loop domain of the protein in its interaction.

  7. The Effects of Addition of Mononucleotides on Sma nuc Endonuclease Activity

    Directory of Open Access Journals (Sweden)

    Julia Romanova

    2012-01-01

    Full Text Available Examination of the effects of mononucleotides on Sma nuc endonuclease originated from Gram negative bacterium Serratia marcescens displayed that any mononucleotide produced by Sma nuc during hydrolysis of DNA or RNA may regulate the enzyme activity affecting the RNase activity without pronounced influence on the activity towards DNA. The type of carbohydrate residue in mononucleotides does not affect the regulation. In contrast, the effects depend on the type of bases in nucleotides. AMP or dAMP was classified as a competitive inhibitor of partial type. GMP, UMP, and CMP were found to be uncompetitive inhibitors that suggest a specific site(s for the nucleotide(s binding in Sma nuc endonuclease.

  8. Bacteriophage based probes for pathogen detection.

    Science.gov (United States)

    Singh, Amit; Arutyunov, Denis; Szymanski, Christine M; Evoy, Stephane

    2012-08-01

    Rapid and specific detection of pathogenic bacteria is important for the proper treatment, containment and prevention of human, animal and plant diseases. Identifying unique biological probes to achieve a high degree of specificity and minimize false positives has therefore garnered much interest in recent years. Bacteriophages are obligate intracellular parasites that subvert bacterial cell resources for their own multiplication and production of disseminative new virions, which repeat the cycle by binding specifically to the host surface receptors and injecting genetic material into the bacterial cells. The precision of host recognition in phages is imparted by the receptor binding proteins (RBPs) that are often located in the tail-spike or tail fiber protein assemblies of the virions. Phage host recognition specificity has been traditionally exploited for bacterial typing using laborious and time consuming bacterial growth assays. At the same time this feature makes phage virions or RBPs an excellent choice for the development of probes capable of selectively capturing bacteria on solid surfaces with subsequent quick and automatic detection of the binding event. This review focuses on the description of pathogen detection approaches based on immobilized phage virions as well as pure recombinant RBPs. Specific advantages of RBP-based molecular probes are also discussed.

  9. Self-Assembly of Pyridine-Modified Lipoic Acid Derivatives on Gold and Their Interaction with Thyroxine (T4

    Directory of Open Access Journals (Sweden)

    Pierangelo Metrangolo

    2013-02-01

    Full Text Available Pyridyl derivatives of lipoic acid were prepared as ligands for the study of the interaction with thyroxine (T4. Thin self-assembled films of the ligands were prepared in 70% ethanol on gold and their interaction with T4 was studied by titration experiments in an aqueous buffer solution using Surface Plasmon Resonance (SPR. The thickness and refractive index of the ligand layers were calculated from SPR spectra recorded in two media, also allowing for surface coverage and the density of the layers to be estimated. Two ligands, a 4-pyridyl and a bis(2-hydroxyethyl derivative of lipoic acid, were selected to investigate the feasibility for producing molecularly imprinted self-assembled layers on gold for T4. The methodology was to co-assemble T4 and the ligand onto the gold surface, elute the T4 from the layer under alkaline conditions, and study the rebinding of T4 to the layer. Multiple elution/rebinding cycles were conducted in different buffer solutions, and rebinding of T4 could be observed, with a moderate binding affinity that depended greatly on the solvent used. More optimal binding was observed in HBS buffer, and the affinity of the interaction could be slightly increased when the 4-pyridyl and bis(2-hydroxy-ethyl derivatives of lipoic acid were combined in the imprinted layer.

  10. Mitochondrial Targeted Endonuclease III DNA Repair Enzyme Protects against Ventilator Induced Lung Injury in Mice

    OpenAIRE

    Masahiro Hashizume; Marc Mouner; Joshua M. Chouteau; Gorodnya, Olena M.; Ruchko, Mykhaylo V.; Wilson, Glenn L.; Gillespie, Mark N.; Parker, James C.

    2014-01-01

    The mitochondrial targeted DNA repair enzyme, 8-oxoguanine DNA glycosylase 1, was previously reported to protect against mitochondrial DNA (mtDNA) damage and ventilator induced lung injury (VILI). In the present study we determined whether mitochondrial targeted endonuclease III (EndoIII) which cleaves oxidized pyrimidines rather than purines from damaged DNA would also protect the lung. Minimal injury from 1 h ventilation at 40 cmH2O peak inflation pressure (PIP) was reversed by EndoIII pret...

  11. A ribonucleoprotein complex protects the interleukin-6 mRNA from degradation by distinct herpesviral endonucleases.

    Directory of Open Access Journals (Sweden)

    Mandy Muller

    2015-05-01

    Full Text Available During lytic Kaposi's sarcoma-associated herpesvirus (KSHV infection, the viral endonuclease SOX promotes widespread degradation of cytoplasmic messenger RNA (mRNA. However, select mRNAs escape SOX-induced cleavage and remain robustly expressed. Prominent among these is interleukin-6 (IL-6, a growth factor important for survival of KSHV infected B cells. IL-6 escape is notable because it contains a sequence within its 3' untranslated region (UTR that can confer protection when transferred to a SOX-targeted mRNA, and thus overrides the endonuclease targeting mechanism. Here, we pursued how this protective RNA element functions to maintain mRNA stability. Using affinity purification and mass spectrometry, we identified a set of proteins that associate specifically with the protective element. Although multiple proteins contributed to the escape mechanism, depletion of nucleolin (NCL most severely impacted protection. NCL was re-localized out of the nucleolus during lytic KSHV infection, and its presence in the cytoplasm was required for protection. After loading onto the IL-6 3' UTR, NCL differentially bound to the translation initiation factor eIF4H. Disrupting this interaction, or depleting eIF4H, reinstated SOX targeting of the RNA, suggesting that interactions between proteins bound to distant regions of the mRNA are important for escape. Finally, we found that the IL-6 3' UTR was also protected against mRNA degradation by the vhs endonuclease encoded by herpes simplex virus, despite the fact that its mechanism of mRNA targeting is distinct from SOX. These findings highlight how a multitude of RNA-protein interactions can impact endonuclease targeting, and identify new features underlying the regulation of the IL-6 mRNA.

  12. A ribonucleoprotein complex protects the interleukin-6 mRNA from degradation by distinct herpesviral endonucleases.

    Science.gov (United States)

    Muller, Mandy; Hutin, Stephanie; Marigold, Oliver; Li, Kathy H; Burlingame, Al; Glaunsinger, Britt A

    2015-05-01

    During lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection, the viral endonuclease SOX promotes widespread degradation of cytoplasmic messenger RNA (mRNA). However, select mRNAs escape SOX-induced cleavage and remain robustly expressed. Prominent among these is interleukin-6 (IL-6), a growth factor important for survival of KSHV infected B cells. IL-6 escape is notable because it contains a sequence within its 3' untranslated region (UTR) that can confer protection when transferred to a SOX-targeted mRNA, and thus overrides the endonuclease targeting mechanism. Here, we pursued how this protective RNA element functions to maintain mRNA stability. Using affinity purification and mass spectrometry, we identified a set of proteins that associate specifically with the protective element. Although multiple proteins contributed to the escape mechanism, depletion of nucleolin (NCL) most severely impacted protection. NCL was re-localized out of the nucleolus during lytic KSHV infection, and its presence in the cytoplasm was required for protection. After loading onto the IL-6 3' UTR, NCL differentially bound to the translation initiation factor eIF4H. Disrupting this interaction, or depleting eIF4H, reinstated SOX targeting of the RNA, suggesting that interactions between proteins bound to distant regions of the mRNA are important for escape. Finally, we found that the IL-6 3' UTR was also protected against mRNA degradation by the vhs endonuclease encoded by herpes simplex virus, despite the fact that its mechanism of mRNA targeting is distinct from SOX. These findings highlight how a multitude of RNA-protein interactions can impact endonuclease targeting, and identify new features underlying the regulation of the IL-6 mRNA. PMID:25965334

  13. RsaI: a new sequence-specific endonuclease activity from Rhodopseudomonas sphaeroides.

    OpenAIRE

    Lynn, S P; Cohen, L K; Kaplan, S; Gardner, J F

    1980-01-01

    A new type II sequence-specific endonuclease, RsaI, has been identified from Rhodopseudomonas sphaeroides strain 28/5. An RsaI purification scheme that yields enzyme which is free of contaminating exonuclease and phosphatase activities after a single column fractionation has been developed. The enzyme recognized the tetranucleotide sequence 5'-GTAC-3' and cleaved between the T and A, thereby generating flush ends. RsaI should be extremely useful in deoxyribonucleic acid sequencing experiments.

  14. Human papillomavirus DNA from warts for typing by endonuclease restriction patterns: purification by alkaline plasmid methods.

    Science.gov (United States)

    Chinami, M; Tanikawa, E; Hachisuka, H; Sasai, Y; Shingu, M

    1990-01-01

    The alkaline plasmid DNA extraction method of Birnboim and Doly was applied for the isolation of human papillomavirus (HPV) from warts. Tissue from common and plantar warts was digested with proteinase K, and the extrachromosomal circular covalently-closed form of HPV-DNA was rapidly extracted by alkaline sodium dodecyl sulphate and phenol-chloroform treatment. Recovery of HPV-DNA from the tissue was sufficient for determination of endonuclease restriction patterns by agarose gel electrophoresis.

  15. Metal-chelating 2-hydroxyphenyl amide pharmacophore for inhibition of influenza virus endonuclease.

    Science.gov (United States)

    Carcelli, Mauro; Rogolino, Dominga; Bacchi, Alessia; Rispoli, Gabriele; Fisicaro, Emilia; Compari, Carlotta; Sechi, Mario; Stevaert, Annelies; Naesens, Lieve

    2014-01-01

    The influenza virus PA endonuclease is an attractive target for development of novel anti-influenza virus therapeutics. Reported PA inhibitors chelate the divalent metal ion(s) in the enzyme's catalytic site, which is located in the N-terminal part of PA (PA-Nter). In this work, a series of 2-hydroxybenzamide-based compounds have been synthesized and biologically evaluated in order to identify the essential pharmacophoric motif, which could be involved in functional sequestration of the metal ions (probably Mg(2+)) in the catalytic site of PA. By using HL(1), H2L(2), and HL(3) as model ligands with Mg(2+) ions, we isolated and fully characterized a series of complexes and tested them for inhibitory activity toward PA-Nter endonuclease. H2L(2) and the corresponding Mg(2+) complex showed an interesting inhibition of the endonuclease activity. The crystal structures of the uncomplexed HL(1) and H2L(2) and of the isolated magnesium complex [Mg(L(3))2(MeOH)2]·2MeOH were solved by X-ray diffraction analysis. Furthermore, the speciation models for HL(1), H2L(2), and HL(3) with Mg(2+) were obtained, and the formation constants of the complexes were measured. Preliminary docking calculations were conducted to investigate the interactions of the title compounds with essential amino acids in the PA-Nter active site. These findings supported the "two-metal" coordination of divalent ions by a donor triad atoms chemotype as a powerful strategy to develop more potent PA endonuclease inhibitors.

  16. Interplay Between Bacteriophages and Restriction-Modification Systems in Enterococci

    Directory of Open Access Journals (Sweden)

    Pristas Peter

    2014-06-01

    Full Text Available The complete genomes of Enterococcus faecalis bacteriophages were analyzed for tetranucleotide words avoidance. Very similar tetranucleotide composition was found in all tested genomes with strong underrepresentation of palindromic GATC and GGCC words. This avoidance could be explained as a protection mechanism against host restriction-modification systems as a clear correlation was found between avoidance of palindromic words and the specificity of E. faecalis restriction and modification systems. No similar avoidance of tetranucleotide words was observed for non-palindromic words. A weak correlation was observed between avoidance of tetranucleotide palindromes in bacteriophage genomes and the possession of phage encoded DNA methyltransferases confirming the interrelation between bacteriophage genomes composition and restriction and modification systems in enterococci

  17. A quorum-sensing-induced bacteriophage defense mechanism

    DEFF Research Database (Denmark)

    Høyland-Kroghsbo, Nina Molin; Mærkedahl, Rasmus Baadsgaard; Svenningsen, Sine

    2013-01-01

    One of the key determinants of the size, composition, structure, and development of a microbial community is the predation pressure by bacteriophages. Accordingly, bacteria have evolved a battery of antiphage defense strategies. Since maintaining constantly elevated defenses is costly, we...... understanding of the factors that naturally shape microbial communities is required. One of the key factors in this context is the interactions between bacteria and the most abundant biological entities on Earth, namely, the bacteriophages that prey on bacteria. This proof-of-principle study shows that quorum...... sensing plays an important role in determining the susceptibility of E. coli to infection by bacteriophages ¿ and ¿. On the basis of our findings in the classical Escherichia coli-¿ model system, we suggest that quorum sensing may serve as a general strategy to protect bacteria specifically under...

  18. Analysis on virus resistance and fruit quality for T4 generation of transgenic papaya

    Institute of Scientific and Technical Information of China (English)

    WEI Xiangdong; LAN Congyu; LU Zhijing; YE Changming

    2007-01-01

    Molecular biological characterization,fruit characters,and nutrients were analyzed for T4 generation of transgenic papaya.All transgenic papaya plants with the mutated replicase (RP) gene from papaya ringspot virus (PRSV) showed high resistance or immunity against PRSV in the field.The RP transgene can be steadily inherited to,and expressed at RNA level,the progenies.The growth characteristics of transgenic papaya were much better than nontransgenic papaya in the field.The non-transgenic papaya seedlings began to show typical symptoms caused by PRSV after being inoculated with PRSV.They died quickly and never grew to produce fruit.The adult trees developed yellow leaves and produced smaller fruits and were doomed to a slow death after some time,while most oftransgenic papaya plants (about 91.8%) did not show any symptoms caused by PRSV,and produced more,bigger,and high quality fruits.Compared with non-transgenic plants,the fresh fruit length of T4 generation of transgenic papaya increased 2.6%-5%,and the diameter decreased 0.6%-1.5%.The flesh thickness of fresh fruit increased 12%-15%,which made it fitter for eating.Although the fresh fruit quality changed,there was no significant difference between transgenic and non-transgenic papaya.The quality characteristics of dry fruit including the contents of water,lipid,N,protein,reduced sugar,vitamin A,vitamin C,and carotene in the T4 generation of transgenic papaya were all the same as their non-transgenic parents.This means that transgenic plants and non-transgenic plants are substantially equivalent,and the transgene has no effect on dry fruit quality.In this study,we found that vitamin A and vitamin C in red-fleshed papaya were 1.4-1.8 and 1.78-2.07 times more than the yellow-fleshed ones,respectively,while N and protein were only 84.2%-92.1% and 82.1%-98.9% of the yellow-fleshed ones.

  19. Investigation of the salicylaldehyde thiosemicarbazone scaffold for inhibition of influenza virus PA endonuclease.

    Science.gov (United States)

    Rogolino, Dominga; Bacchi, Alessia; De Luca, Laura; Rispoli, Gabriele; Sechi, Mario; Stevaert, Annelies; Naesens, Lieve; Carcelli, Mauro

    2015-10-01

    The influenza virus PA endonuclease is an attractive target for the development of novel anti-influenza virus therapeutics, which are urgently needed because of the emergence of drug-resistant viral strains. Reported PA inhibitors are assumed to chelate the divalent metal ion(s) (Mg²⁺ or Mn²⁺) in the enzyme's catalytic site, which is located in the N-terminal part of PA (PA-Nter). In the present work, a series of salicylaldehyde thiosemicarbazone derivatives have been synthesized and evaluated for their ability to inhibit the PA-Nter catalytic activity. Compounds 1-6 have been evaluated against influenza virus, both in enzymatic assays with influenza virus PA-Nter and in virus yield assays in MDCK cells. In order to establish a structure-activity relationship, the hydrazone analogue of the most active thiosemicarbazone has also been evaluated. Since chelation may represent a mode of action of such class of molecules, we studied the interaction of two of them, one with and one without biological activity versus the PA enzyme, towards Mg²⁺, the ion that is probably involved in the endonuclease activity of the heterotrimeric influenza polymerase complex. The crystal structure of the magnesium complex of the o-vanillin thiosemicarbazone ligand 1 is also described. Moreover, docking studies of PA endonuclease with compounds 1 and 2 were performed, to further analyse the possible mechanism of action of this class of inhibitors. PMID:26323352

  20. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells.

    Science.gov (United States)

    Kim, Daesik; Kim, Jungeun; Hur, Junho K; Been, Kyung Wook; Yoon, Sun-Heui; Kim, Jin-Soo

    2016-08-01

    Programmable clustered regularly interspaced short palindromic repeats (CRISPR) Cpf1 endonucleases are single-RNA-guided (crRNA) enzymes that recognize thymidine-rich protospacer-adjacent motif (PAM) sequences and produce cohesive double-stranded breaks (DSBs). Genome editing with CRISPR-Cpf1 endonucleases could provide an alternative to CRISPR-Cas9 endonucleases, but the determinants of targeting specificity are not well understood. Using mismatched crRNAs we found that Cpf1 could tolerate single or double mismatches in the 3' PAM-distal region, but not in the 5' PAM-proximal region. Genome-wide analysis of cleavage sites in vitro for eight Cpf1 nucleases using Digenome-seq revealed that there were 6 (LbCpf1) and 12 (AsCpf1) cleavage sites per crRNA in the human genome, fewer than are present for Cas9 nucleases (>90). Most Cpf1 off-target cleavage sites did not produce mutations in cells. We found mismatches in either the 3' PAM-distal region or in the PAM sequence of 12 off-target sites that were validated in vivo. Off-target effects were completely abrogated by using preassembled, recombinant Cpf1 ribonucleoproteins.

  1. Real-time quantitative nicking endonuclease-mediated isothermal amplification with small molecular beacons.

    Science.gov (United States)

    Xu, Wentao; Wang, Chenguang; Zhu, Pengyu; Guo, Tianxiao; Xu, Yuancong; Huang, Kunlun; Luo, Yunbo

    2016-04-21

    Techniques of isothermal amplification have recently made great strides, and have generated significant interest in the field of point-of-care detection. Nicking endonuclease-mediated isothermal amplification (NEMA) is an example of simple isothermal technology. In this paper, a real-time quantitative nicking endonuclease-mediated isothermal amplification with small molecular beacons (SMB-NEMA) of improved specificity and sensitivity is described. First, we optimized the prohibition of de novo synthesis by choosing Nt·BstNBI endonuclease. Second, the whole genome was successfully amplified with Nt·BstNBI (6 U), betaine (1 M) and trehalose (60 mM) for the first time. Third, we achieved 10 pg sensitivity for the first time after adding a small molecular beacon that spontaneously undergoes a conformational change when hybridizing to target, and the practical test validated the assay's application. The small molecular beacon has a similar melting temperature to the reaction temperature, but is approximately 10 bp shorter than the length of a traditional molecular beacon. A new threshold regulation was also established for isothermal conditions. Finally, we established a thermodynamic model for designing small molecular beacons. This multistate model is more correct than the traditional algorithm. This theoretical and practical basis will help us to monitor SMB-NEMA in a quantitative way. In summary, our SMB-NEMA method allows the simple, specific and sensitive assessment of isothermal DNA quantification. PMID:27027375

  2. Prognostic Significance of Human Apurinic/Apyrimidinic Endonuclease (APE/Ref-1) Expression in Rectal Cancer Treated With Preoperative Radiochemotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jun-Sang, E-mail: k423j@cnu.ac.kr [Department of Radiation Oncology, College of Medicine, Chungnam National University, Daejeon (Korea, Republic of); Cancer Research Institute, Chungnam National University, Daejeon (Korea, Republic of); Kim, Jin-Man [Cancer Research Institute, Chungnam National University, Daejeon (Korea, Republic of); Department of Pathology, College of Medicine, Chungnam National University, Daejeon (Korea, Republic of); Liang, Zhe Long [Department of Pathology, College of Medicine, Chungnam National University, Daejeon (Korea, Republic of); Jang, Ji Young [Department of Radiation Oncology, College of Medicine, Chosun University, Gwangju (Korea, Republic of); Kim, Sup; Huh, Gil Ja; Kim, Ki-Hwan [Department of Radiation Oncology, College of Medicine, Chungnam National University, Daejeon (Korea, Republic of); Cho, Moon-June [Department of Radiation Oncology, College of Medicine, Chungnam National University, Daejeon (Korea, Republic of); Cancer Research Institute, Chungnam National University, Daejeon (Korea, Republic of)

    2012-01-01

    Purpose: Human apurinic endonuclease/redox factor 1 (APE/Ref-1) mediates repair of radiation-induced DNA lesions and regulates transcription via redox-based activation. We investigated the predictive and prognostic significance of APE/Ref-1 expression in pretreatment biopsy specimens in locally advanced rectal cancer (LARC) (cT3-T4 or N+). Methods and Materials: APE/Ref-1 expression was analyzed by immunohistochemistry in pretreatment biopsy specimens obtained from 83 patients with LARC. Patients received preoperative radiotherapy of 50.4 Gy in 28 fractions, combined with oral capecitabine and leucovorin chemotherapy, followed by curative surgery. The prognostic significance of various clinicopathologic characteristics, including APE/Ref-1 protein expression, was evaluated. Results: APE/Ref-1 was expressed in 97% of patient samples. Exclusive APE/Ref-1 nuclear staining was observed in 49 of 83 samples (59%), and mixed nuclear and cytoplasmic staining was observed in 31 samples (37%). APE/Ref-1 nuclear expression levels were low in 49 patients (59%) and high in 34 patients (41%). The level of APE/Ref-1 nuclear expression was not a prognostic factor for overall and disease-free survival. Cytoplasmic expression of APE/Ref-1 was a borderline-significant predictive factor for pathologic tumor response (p = 0.08) and a significant prognostic factor for disease-free survival, as shown by univariate analysis (p = 0.037). Multivariate analysis confirmed that cytoplasmic localization of APE/Ref-1 is a significant predictor of disease-free survival (hazard ratio, 0.45; p = 0.046). Conclusions: APE/Ref-1 was expressed in a majority of pretreatment biopsy specimens from patients with LARC. The level of APE/Ref-1 nuclear expression was not a significant predictive and prognostic factor; however, cytoplasmic localization of the protein was negatively associated with disease-free survival. These results indicate that cytoplasmic expression of APE/Ref-1 represents an adverse

  3. Bacteriophages as potential treatment option for antibiotic resistant bacteria.

    Science.gov (United States)

    Bragg, Robert; van der Westhuizen, Wouter; Lee, Ji-Yun; Coetsee, Elke; Boucher, Charlotte

    2014-01-01

    The world is facing an ever-increasing problem with antibiotic resistant bacteria and we are rapidly heading for a post-antibiotic era. There is an urgent need to investigate alterative treatment options while there are still a few antibiotics left. Bacteriophages are viruses that specifically target bacteria. Before the development of antibiotics, some efforts were made to use bacteriophages as a treatment option, but most of this research stopped soon after the discovery of antibiotics. There are two different replication options which bacteriophages employ. These are the lytic and lysogenic life cycles. Both these life cycles have potential as treatment options. There are various advantages and disadvantages to the use of bacteriophages as treatment options. The main advantage is the specificity of bacteriophages and treatments can be designed to specifically target pathogenic bacteria while not negatively affecting the normal microbiota. There are various advantages to this. However, the high level of specificity also creates potential problems, the main being the requirement of highly specific diagnostic procedures. Another potential problem with phage therapy includes the development of immunity and limitations with the registration of phage therapy options. The latter is driving research toward the expression of phage genes which break the bacterial cell wall, which could then be used as a treatment option. Various aspects of phage therapy have been investigated in studies undertaken by our research group. We have investigated specificity of phages to various avian pathogenic E. coli isolates. Furthermore, the exciting NanoSAM technology has been employed to investigate bacteriophage replication and aspects of this will be discussed.

  4. Bacteriophages as potential treatment option for antibiotic resistant bacteria.

    Science.gov (United States)

    Bragg, Robert; van der Westhuizen, Wouter; Lee, Ji-Yun; Coetsee, Elke; Boucher, Charlotte

    2014-01-01

    The world is facing an ever-increasing problem with antibiotic resistant bacteria and we are rapidly heading for a post-antibiotic era. There is an urgent need to investigate alterative treatment options while there are still a few antibiotics left. Bacteriophages are viruses that specifically target bacteria. Before the development of antibiotics, some efforts were made to use bacteriophages as a treatment option, but most of this research stopped soon after the discovery of antibiotics. There are two different replication options which bacteriophages employ. These are the lytic and lysogenic life cycles. Both these life cycles have potential as treatment options. There are various advantages and disadvantages to the use of bacteriophages as treatment options. The main advantage is the specificity of bacteriophages and treatments can be designed to specifically target pathogenic bacteria while not negatively affecting the normal microbiota. There are various advantages to this. However, the high level of specificity also creates potential problems, the main being the requirement of highly specific diagnostic procedures. Another potential problem with phage therapy includes the development of immunity and limitations with the registration of phage therapy options. The latter is driving research toward the expression of phage genes which break the bacterial cell wall, which could then be used as a treatment option. Various aspects of phage therapy have been investigated in studies undertaken by our research group. We have investigated specificity of phages to various avian pathogenic E. coli isolates. Furthermore, the exciting NanoSAM technology has been employed to investigate bacteriophage replication and aspects of this will be discussed. PMID:24619620

  5. Acanthamoeba genotype T4 from the UK and Iran and isolation of the T2 genotype from clinical isolates

    OpenAIRE

    A.H. Maghsood; Sissons, J.; Rezaian, M.; Nolder, D; Warhurst, D.; Khan, Naveed Ahmed

    2005-01-01

    The majority of the keratitis-causing Acanthamoeba isolates are genotype T4. In an attempt to determine whether predominance of T4 isolates in Acanthamoeba keratitis is due to greater virulence or greater prevalence of this genotype, Acanthamoeba genotypes were determined for 13 keratitis isolates and 12 environmental isolates from Iran. Among 13 clinical isolates, eight (61.5 %) belonged to T4, two (15.3 %) belonged to T3 and three (23 %) belonged to the T2 genotype. In contrast, the majorit...

  6. Mediator proteins orchestrate enzyme-ssDNA assembly during T4 recombination-dependent DNA replication and repair

    OpenAIRE

    Bleuit, Jill S.; Xu, Hang; Ma, Yujie; Wang, Tongsheng; Liu, Jie; Morrical, Scott W.

    2001-01-01

    Studies of recombination-dependent replication (RDR) in the T4 system have revealed the critical roles played by mediator proteins in the timely and productive loading of specific enzymes onto single-stranded DNA (ssDNA) during phage RDR processes. The T4 recombination mediator protein, uvsY, is necessary for the proper assembly of the T4 presynaptic filament (uvsX recombinase cooperatively bound to ssDNA), leading to the recombination-primed initiation of leading strand DNA synthesis. In the...

  7. 温敏型核雄性不育系番茄T4育性的研究%Studies on Fertility of Thermo-sensitive Genic Male-sterile Tomato,Lycopersicum esculentum Mill.—T4

    Institute of Scientific and Technical Information of China (English)

    王先裕; 于分弟; 梁聪耀; 李良劭; 黄维娜; 杜永臣; 刘政国; 曾东; 潘立凡

    2011-01-01

    以温敏型核雄性不育系番茄T4及对照First为试材,通过调查花粉发芽率、自交结实率、果实种子数以及采用Tricine-SDS-PAGE电泳方法分析叶片及花药蛋白质的变化,研究T4的育性转化,并分析其在可育和不育条件下花粉发芽率、自交结实率、果实种子数、叶片及花药蛋白质的差异.结果表明:28℃/18℃(昼/夜)处理的T4花粉发芽率、自交结实率最高,果实种子数最多,与对照First差异不显著,表明此时T4恢复部分育性;28℃/24℃和28℃/12℃处理的T4花粉发芽率、自交结实率和果实种子数与对照差异均达到极显著水平,表现为不育.不同温度处理的T4花药蛋白质与叶片蛋白质差异均达到显著水平;28℃/24℃和28℃/12℃处理的T4叶片蛋白质与对照差异显著,但28 ℃/18℃处理的差异不明显.%Taking thermo-sensitive genie male-sterile tomato Lycopersicum esculentum Mill. T4 and the control First as experimental materials, this paper investigated pollen germination rate,self-breeding rate and fruit seed number, analyzed the protein changes in leaf and anther by Tricine-SDS-PAGE electrophoresis to comprehend fertile conversion of T4. The paper also analyzed the pollen germination rate, self-breeding rate and fruit seed number, protein differences in leaf and anther under fertility and sterility conditions. The results showed that pollen germination rate, self-breeding rate and fruit seed number of T4 were the highest with 28 ^C/IS V, ( day/night ) treatment. There was no significant difference compared with the control First. It indicated that T4 had restored partial fertility. But, with 28 t/24 °C and 28 t/12 X. Treatments the pollen germination rate, self-breeding rate and fruit seed number of T4 had significant differences with the control First, and T4 expressed as sterile line. There were significant differences in leaf protein and anther protein of T4 under different temperature treatments

  8. Bacteriophages of Soft Rot Enterobacteriaceae-a minireview.

    Science.gov (United States)

    Czajkowski, Robert

    2016-01-01

    Soft rot Enterobacteriaceae (Pectobacterium spp. and Dickeya spp., formerly pectinolytic Erwinia spp.) are ubiquitous necrotrophic bacterial pathogens that infect a large number of different plant species worldwide, including economically important crops. Despite the fact that these bacteria have been studied for more than 50 years, little is known of their corresponding predators: bacteriophages, both lytic and lysogenic. The aim of this minireview is to critically summarize recent ecological, biological and molecular research on bacteriophages infecting Pectobacterium spp. and Dickeya spp. with the main focus on current and future perspectives in that field.

  9. Engineered enzymatically active bacteriophages and methods of uses thereof

    Energy Technology Data Exchange (ETDEWEB)

    Collins, James J (Newton, MA); Kobayashi, Hideki (Yokohama, JP); Kearn, Mads (Ottawa, CA); Araki, Michihiro (Minatoku, JP); Friedland, Ari (Boston, MA); Lu, Timothy Kuan-Ta (Palo Alto, CA)

    2012-05-22

    The present invention provides engineered bacteriophages that express at least one biofilm degrading enzyme on their surface and uses thereof for degrading bacterial biofilms. The invention also provides genetically engineered bacteriophages expressing the biofilm degrading enzymes and proteins necessary for the phage to replicate in different naturally occurring biofilm producing bacteria. The phages of the invention allow a method of biofilm degradation by the use of one or only a few administration of the phage because the system using these phages is self perpetuating, and capable of degrading biofilm even when the concentration of bacteria within the biofilm is low.

  10. Molecular and chemical engineering of bacteriophages for potential medical applications.

    Science.gov (United States)

    Hodyra, Katarzyna; Dąbrowska, Krystyna

    2015-04-01

    Recent progress in molecular engineering has contributed to the great progress of medicine. However, there are still difficult problems constituting a challenge for molecular biology and biotechnology, e.g. new generation of anticancer agents, alternative biosensors or vaccines. As a biotechnological tool, bacteriophages (phages) offer a promising alternative to traditional approaches. They can be applied as anticancer agents, novel platforms in vaccine design, or as target carriers in drug discovery. Phages also offer solutions for modern cell imaging, biosensor construction or food pathogen detection. Here we present a review of bacteriophage research as a dynamically developing field with promising prospects for further development of medicine and biotechnology.

  11. Salmonella and Campylobacter: Antimicrobial resistance and bacteriophage control in poultry.

    Science.gov (United States)

    Grant, Ar'Quette; Hashem, Fawzy; Parveen, Salina

    2016-02-01

    Salmonella and Campylobacter are major causes of foodborne related illness and are traditionally associated with consuming undercooked poultry and/or consuming products that have been cross contaminated with raw poultry. Many of the isolated Salmonella and Campylobacter that can cause disease have displayed antimicrobial resistance phenotypes. Although poultry producers have reduced on-the-farm overuse of antimicrobials, antimicrobial resistant Salmonella and Campylobacter strains still persist. One method of bio-control, that is producing promising results, is the use of lytic bacteriophages. This review will highlight the current emergence and persistence of antimicrobial resistant Salmonella and Campylobacter recovered from poultry as well as bacteriophage research interventions and limitations.

  12. Mechanism of bacteriophage conversion of lipase activity in Staphylococcus aureus.

    OpenAIRE

    Lee, C Y; Iandolo, J J

    1985-01-01

    Staphylococcus aureus PS54 harbors two temperate bacteriophages and manifests no lipase activity on egg yolk agar. Curing of one of the resident prophages (L54a) restores lipase activity. To study the mechanism of bacteriophage conversion, the prophage was cured, and the gene encoding lipase activity was cloned into pBR322 in Escherichia coli on a 2.9-kilobase DNA fragment of the chromosome. The fragment was subcloned into a shuttle vector and subsequently transformed into S. aureus and Bacil...

  13. Environmental augmentation with bacteriophage prevents colibacillosis in broiler chickens.

    Science.gov (United States)

    El-Gohary, F A; Huff, W E; Huff, G R; Rath, N C; Zhou, Z Y; Donoghue, A M

    2014-11-01

    Bacteriophages are viruses that kill bacteria. They are plentiful in nature; are safe, having no known activity to human or animal cells; and are an attractive alternative to antibiotics. The objectives of this research were to establish an experimental model of colibacillosis induced by indirect exposure to Escherichia coli and to determine if bacteriophage could protect the birds from developing colibacillosis. In study 1 there were 6 treatments with 2 replicate pens of 25 birds. The treatments were control warm brooded; control cold stressed; litter inoculated with E. coli, warm brooded; litter inoculated with E. coli, cold stressed; seeder birds (5 per pen) challenged with E. coli, warm brooded; and seeder birds (5 per pen), cold stressed. The study concluded when the birds were 3 wk of age. Body weights at 1, 2, and 3 wk of age were significantly decreased (P ≤ 0.05) by cold stress, decreased at 1 and 2 wk of age by both the litter and seeder bird treatments compared with the control treatment and by the seeder bird treatment at 3 wk of age. Study 2 consisted of 8 treatments with 2 replicate pens of 20 birds per treatment. The treatments were control, warm brooded; control, cold stressed; litter inoculated with E. coli, cold stressed; and seeder birds (5/pen) challenged with E. coli, cold stressed with and without bacteriophage treatment. In the bacteriophage treatments the bacteriophages were sprayed on the litter. The study was concluded at 3 wk of age. Body weights at 1 wk of age were significantly (P ≤ 0.05) decreased from the control treatment by the seeder bird treatment and were significantly (P ≤ 0.05) higher in all the bacteriophage treatments compared with their matched untreated treatments, except in the control cold stressed treatment. Mortality was significantly (P ≤ 0.05) decreased by bacteriophage in the litter challenged treatment. These data suggest that augmentation of the environment with bacteriophage is a practical and efficacious

  14. Production of antisera and development of radioimmunoassay for serum T3, T4, and ferritin

    International Nuclear Information System (INIS)

    In this study twelve local rabbits and sixteen New-zealand rabbits were subjected to immunization against T3 and T4 immunogens. Two local sheep (ovis aris) were immunized against human liver ferritin. The T3 and T4 immunogens were prepared by conjugation of the haptens to carrier proteins (bovine serum albumin ''BSA'' and horse serum protein ''HSP''), using water soluble carboiimide as coupling agent. The local and New-zealand rabbits were immunized against these conjugates emulsified in freund's complete adjuvant (FCA) in the first and second injections, and emulsified in freund's incomplete adjuvant (FIA) in the following injections. The blood samples obtained from rabbits after each injection were tested for antibodies as well as for the effect of immunization on rabbits biochemical and haematological parameters. The blood samples obtained from sheep were tested for anti-ferritin antibodies using crude antiserum, then this antiserum was purified using ammonium sulphate. A part of it was adsorbed physically onto polystyrene beads while the other part was linked chemically to magnitisable particles inorder to develop to IRMAs. The purified antiferritin antibody was diluted 200,000 folds before being coated to polystyrene beads, and different dilutions were tried with coupling to magnetic solid phase. Optimization and validation procedures for the two IRMAs ferritin were performed. The results obtained showed poor response of rabbits to immunization against T3 and T4 immunogen conjugates, where the percent bound (B%) of tracer with the antibody ranged from (0.0-22%) for local rabbits using charcoal seperation technique, and (0.0-2.9%) using second antibody precipitation technique. The B% for the antiserum obtained from New-zealand rabbits ranged from (0.0-18.1) using second antibody precipitation technique. Serum T3, T4 and TSH of the immunized rabbits were measured and found to be not significantly different form the controls (p=0.2211, 0.098, 0.35 respectively

  15. A boy with 9p+ resulting from maternal t(4;9) translocation: a case report

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Increasing cases of 9p syndrome have been reported since the first description in 1970.In the present case, a extra segment of the end of chromosome 9p resulting from a maternally inherited translocation t(4;9)(q31;p24) was described in a liveborn boy with mental retardation and multiple congenital anomalies. The extra part of chromosome 9p includes segment of the chromosome region 4q31→qter of his mother but deletes a small segment 9p24→pter. To our knowledge, this is the first case described in a liveborn child. This paper also includes a review and tabulation of clinical features seen in the 34 reported cases. The new case had most characteristics reported cases besides worried-face, fat and abnormal lower digestion tract, which is a modification and double expansion of the previous summarization on 9p syndrome.

  16. Pollution reduction technology program for class T4(JT8D) engines

    Science.gov (United States)

    Roberts, R.; Fiorentino, A. J.; Diehl, L. A.

    1977-01-01

    The technology required to develop commercial gas turbine engines with reduced exhaust emissions was demonstrated. Can-annular combustor systems for the JT8D engine family (EPA class T4) were investigated. The JT8D turbofan engine is an axial-flow, dual-spool, moderate-bypass-ratio design. It has a two-stage fan, a four-stage low-pressure compressor driven by a three-stage low-pressure turbine, and a seven-stage high-pressure compressor driven by a single-stage high-pressure turbine. A cross section of the JT8D-17 showing the mechanical configuration is given. Key specifications for this engine are listed.

  17. SIMULATION ON TEMPERATURE FIELD OF FRICTION STIR WELDED JOINTS OF 2024-T4 Al

    Institute of Scientific and Technical Information of China (English)

    S.X. Lü; J.C. Yan; W.G. Li; S.Q. Yang

    2005-01-01

    e thermal model of FSW based on the thermal elastic-plastic finite element method, and the transient temperature distribution of FS welded joints of 2024-T4 Al was simulated by using this model, which provides useful information for the investigation of FSW process. Simulation results show that the temperature distribution of the weld gradually decreases toward periphery in a radiate format, whose center is the probe, and the highest temperature in the weld can reach about 400℃. The initial terminal of the weld is a zone, in which the temperature gradient is great, and defects of the welding are easily produced in this zone. Temperature change at the end of the welded joint is as layer variation, the local serious defects are not easy to produce in this zone.

  18. A Strategic Framework for Utilizing Late-Stage (T4) Translation Research to Address Health Inequities.

    Science.gov (United States)

    Lopez-Class, Maria; Peprah, Emmanuel; Zhang, Xinzhi; Kaufmann, Peter G; Engelgau, Michael M

    2016-01-01

    Achieving health equity requires that every person has the opportunity to attain their full health potential and no one is disadvantaged from achieving this potential because of social position or other socially determined circumstances. Inequity experienced by populations of lower socioeconomic status is reflected in differences in health status and mortality rates, as well as in the distribution of disease, disability and illness across these population groups. This article gives an overview of the health inequities literature associated with heart, lung, blood and sleep (HLBS) disorders. We present an ecological framework that provides a theoretical foundation to study late-stage T4 translation research that studies implementation strategies for proven effective interventions to address health inequities. PMID:27440979

  19. Long-Term Atmospheric Corrosion of Aluminum Alloy 2024-T4 in a Coastal Environment

    Science.gov (United States)

    Zhang, Teng; He, Yuting; Cui, Ronghong; An, Tao

    2015-07-01

    Aluminum alloy 2024-T4 specimens were exposed to atmosphere for 7, 12, and 20 years, respectively, to study long-term corrosion in a coastal environment. One-directional corrosion region and cross-directional corrosion region were defined according to corrosion characters. The statistical regularities, surface appearance, corrosion products, and cross-sectional morphology of both regions were investigated. It was found that the minimum remaining thicknesses of each region can be described by a normal distribution and linearly decrease as the exposure time is increased from 7 to 20 years. The corrosion pits, chlorine ions, and interlinked inner pits are promoting exfoliation, and the alloy's corrosion susceptibility along the long transverse direction is strongly location dependent due to the restrictions imposed by the side material.

  20. Isolation of Acanthamoeba Genotype T4 from a Non-Contact Lens Wearer from the Philippines

    Science.gov (United States)

    Buerano, Corazon C.; Trinidad, Abigail D.; Fajardo, Lindsay Sydney N.; Cua, Irwin Y.; Baclig, Michael O.; Natividad, Filipinas F.

    2014-01-01

    We report the case of a 76-year old Filipino male who presented with pain, redness, and blurring of vision of the right eye. Corneal scraping was done and sent to the St. Luke’s Research and Biotechnology Group for detection and identification of the infectious agent. Morphological detection was performed by allowing the organism from the scraping to grow in 1.5% non-nutrient agar plate with heat-killed E. coli. Trophozoites with acanthopodia and double-walled cysts characteristic of Acanthamoeba were observed within the first and second week of observations, respectively. Molecular identification of the amoebae at the genus level based on the presence of Acanthamoeba-specific amplimer S1, ASA.S1 confirmed the morphological identification. Genotyping through sequence revealed that the organism belonged to T4, which is the genotype commonly present in the eye of keratitis patients. PMID:25589879

  1. Study of residual stresses in tailor rolled blanked Al5J32-T4 sheets

    Institute of Scientific and Technical Information of China (English)

    KIM Dongok; KIM Jinpyeong; LEE Yong; KWAK Heeman; RYU Yongmun; HAN Beomsuck

    2006-01-01

    Several automotive parts such as door panels have been manufactured by using load-adapted blanks for crash optimization and weight minimization.Recently, Tailor Rolled Blanks (TRB) has been introduced to remove the disadvantages of a welding process which was used in joining panel components.TRB offers better structural design capabilities due to the seamless transitions on the panels with different thicknesses.In spite of the advantages of the process, TRB leaves internal stresses in the panel.This residual stresses lower the formability of Tailor Rolled Blanked (TRBed) parts and cause cracks near severe curvature during subsequent forming processes.In this research, the residual stresses of TRBed Al5J32-T4 sheets were studied by X-ray stress analysis, and also microstructure was observed along the rolling direction.In addition, heat treatment was done after TRB process in order to compare the residual stresses to that of the TRBed sheets before the heat treatment.

  2. Possible application of RIA-TEST-T4 (URVJT-Kosice) in congenital hypothyroidism screening

    International Nuclear Information System (INIS)

    Using a kit for serum thyroxine level estimation (RIA-TEST-T4) commercially made by the Kosice Institute of Radioecology and Applied Nuclear Techniques, the authors modified a version for thyroxine level determination in dry blood drops. Within the 30 to 60 ng.ml-1 range, i.e.; well within the region necessary for congenital hypothyroidism screening, sensitivity proved to be 36 imp.s-1.ng-1.ml. Accuracy at 20 ng.ml-1 was 13%, at 50 ng.ml-1 - 8%, and at 70 ng.ml-1 - 7.6%. Compared with imported kits the cost of examination of one neonate was relatively low. The method lends itself well to mechanization, and screening evaluation time can be reduced to a few minutes. (author)

  3. CCD photometry of active Centaur 166P/2001 T4 (NEAT)

    Science.gov (United States)

    Shi, J. C.; Ma, Y. H.

    2015-12-01

    To study the secular evolution of the activity of Centaur 166P/2001 T4 (Near Earth Asteroid Tracking, NEAT) and its physical properties, we present the results of optical observations of the Centaur taken on 2009 March 29 with the Keck 10-m telescope located atop Mauna Kea, Hawaii. It was still active at rh = 11.9 au post-perihelion. An upper limit of the nucleus radius of aN < 14.32 km is derived. The colour index is B - R = 1.59 ± 0.05. The Afρ value is measured to be 288 ± 19 cm and the corresponding dust production rate is 252 kg s-1. Finally, a possible mechanism of activity is discussed.

  4. Toluene 4-Monooxygenase and its Complex with Effector Protein T4moD

    Energy Technology Data Exchange (ETDEWEB)

    Bailey, Lucas J.; Fox, Brian G. (UW)

    2012-10-16

    Toluene 4-monooxygenase (T4MO) is a multiprotein diiron enzyme complex that catalyzes the regiospecific oxidation of toluene to p-cresol. Catalytic function requires the presence of a small protein, called the effector protein. Effector protein exerts substantial control on the diiron hydroxylase catalytic cycle through protein-protein interactions. High-resolution crystal structures of the stoichometric hydroxylase and effector protein complex described here reveal how protein-protein interactions and reduction of the diiron center produce an active site configuration poised for reaction with O{sub 2}. Further information from crystal structures of mutated isoforms of the hydroxylase and a peroxo adduct is combined with catalytic results to give a fuller picture of the geometry of the enzyme-substrate complex used for the high fidelity oxidation of hydrocarbon substrates.

  5. The complete AdS_3 x S^3 x T^4 worldsheet S-matrix

    CERN Document Server

    Borsato, Riccardo; Sfondrini, Alessandro; Stefanski, Bogdan

    2014-01-01

    We derive the non-perturbative worldsheet S matrix for fundamental excitations of Type IIB superstring theory on AdS_3 x S^3 x T^4 with Ramond-Ramond flux. To this end, we study the off-shell symmetry algebra of the theory and its representations. We use these to determine the S matrix up to scalar factors and we derive the crossing equations that these scalar factors satisfy. Our treatment automatically includes fundamental massless excitations, removing a long-standing obstacle in using integrability to study the AdS_3/CFT_2 correspondence. The present paper contains a detailed derivation of results first announced in arXiv:1403.4543.

  6. Nueva Área Terminal (NAT del aeropuerto de Madrid-Barajas T-4

    Directory of Open Access Journals (Sweden)

    Lamela, A.

    2006-03-01

    Full Text Available This paper deals with the New Terminal Area of the Barajas airport. Its first part indicates the participants in the design and construction, as well as some general data. In the second part, organised like a data chart, technical data, such as general information, construction aspects, parking area, data about the building itself and others, are expressed in a concise way. The third part analyzes the construction aspects of the NAT Building, related to the power efficiency. The paper ends with a note that the author of the project has considered of interest about the functionality of the T4. Drawings, details and photographs that show the work in their total magnitude are also included.El articulo que se presenta trata de la Nueva Área Terminal (NAT del aeropuerto de Barajas y está diferenciado en una primera parte, donde se indican los créditos del proyecto y de la construcción, así como algunos datos generales. En el punto 2 y organizado como ficha, aparecen de forma escueta los datos técnicos, tales como: información general, sobre la construcción, sobre los aparcamientos, sobre el edificio terminal y otras. En el punto 3 se analizan los aspectos de la edificación NAT, relacionados con la eficiencia energética y finaliza el artículo con una nota sobre la funcionalidad de la T4, que el autor del proyecto ha considerado de interés. Se incluyen planos detalles y fotografías que muestran la obra en su total magnitud.

  7. High-dose radiotherapy alone for patients with T4-stage laryngeal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Mucha-Malecka, A. [Maria Sklodowska-Curie Memorial Institute, Krakow (Poland). Dept. of Radiation Oncology; Skladowski, K. [Maria Sklodowska-Curie Memorial Institute, Gliwice (Poland). Dept. of Radiation Oncology

    2013-08-15

    Background and purpose: The purpose of this retrospective study was to report on the efficacy of radiotherapy alone in patients with T4-stage laryngeal cancer and to establish the prognostic value of (a) the size and location of the extralaryngeal tumor extensions and (b) of emergency tracheostomy. Patients and methods: A group of 114 patients were treated with definitive radiotherapy between 1990 and 1996. The piriform recess was involved in 37 cases (33 %), the base of the tongue and glosso-epiglottic vallecula in 34 cases (30 %), and the hypopharyngeal wall in 10 cases (9 %). In 16 cases (14 %), emergency tracheostomy was performed before radiotherapy. The mean total dose was 68 Gy (range, 60-77.6 Gy). The mean treatment time was 49 days (range, 42-74 days). Results: Actuarial 3-year local control (LC) was noted in 42 % of patients, disease-free survival (DFS) in 35 %, and overall survival (OS) in 40 %. The best prognosis was for the lesion suspected of cartilage infiltration: 56 % 3-year LC. The worst results were noted in the cases with massive infiltrations spreading from the larynx through the hypopharynx: 13 % 3-year LC. Emergency tracheostomy before radiotherapy was significantly connected with the worst treatment results (p = 0.000): 3-year LC in patients with tracheostomy was 0 % vs. 48 % in patients without tracheostomy. Conclusion: Conventional radiotherapy of T4 laryngeal cancer is a method of treatment with limited effectiveness. The efficacy of radiotherapy is dependent on the location and extent of extralaryngeal infiltrations. Emergency tracheostomy is a prognostic factor connected with the worst prognosis. (orig.)

  8. Norovirus and FRNA bacteriophage determined by RT-qPCR and infectious FRNA bacteriophage in wastewater and oysters.

    Science.gov (United States)

    Flannery, John; Keaveney, Sinéad; Rajko-Nenow, Paulina; O'Flaherty, Vincent; Doré, William

    2013-09-15

    Norovirus (NoV), the leading cause of adult non-bacterial gastroenteritis can be commonly detected in wastewater but the extent of NoV removal provided by wastewater treatment plants (WWTPs) is unclear. We monitored a newly commissioned WWTP with UV disinfection on a weekly basis over a six month period for NoV using RT-qPCR and for FRNA bacteriophage GA using both RT-qPCR (total concentration) and a plaque assay (infectious concentration). Mean concentrations of NoV GI and GII in influent wastewater were reduced by 0.25 and 0.41 log10 genome copies 100 ml(-1), respectively by the WWTP. The mean concentration of total FRNA bacteriophage GA was reduced by 0.35 log genome copies 100 ml(-1) compared to a reduction of infectious FRNA bacteriophage GA of 2.13 log PFU 100 ml(-1). A significant difference between concentrations of infectious and total FRNA bacteriophage GA was observed in treated, but not in untreated wastewaters. We conclude that RT-qPCR in isolation underestimates the reduction of infectious virus during wastewater treatment. We further compared the concentrations of infectious virus in combined sewer overflow (CSO) and UV treated effluents using FRNA bacteriophage GA. A greater percentage (98%) of infectious virus is released in CSO discharges than UV treated effluent (44%). Following a CSO discharge, concentrations of NoV GII and infectious FRNA bacteriophage GA in oysters from less than the limit of detection to 3150 genome copies 100 g(-1) and 1050 PFU 100 g(-1) respectively.

  9. Early determination of the congenital hypothyroidism by means of the cuantification of STH and T4 by radioimmunoassay method

    International Nuclear Information System (INIS)

    It was investigated the advantages and disadvantages and conditions of working for the diagnosis of the congenital hypothyroidism by RIA, and its future implementation. It was determined concentration of neonatal STH and neonatal T4 in whole blood of 517 new borns, obtained by capilar punction and recollected in filter paper. 261 samples were found with levels of T4 between normal limits and STH between 0 and 12.5 u U/ml correspond to a normal thyroid function. 29 samples with normal T4 and STH above 30 u U/ml, correspond to a possible primary hypothyroidism. 171 samples with normal T4 and STH between 12.5 and 30 u U/ml were found. (Author)

  10. Novel bacteriophages containing a genome of another bacteriophage within their genomes.

    Directory of Open Access Journals (Sweden)

    Maud M Swanson

    Full Text Available A novel bacteriophage infecting Staphylococus pasteuri was isolated during a screen for phages in Antarctic soils. The phage named SpaA1 is morphologically similar to phages of the family Siphoviridae. The 42,784 bp genome of SpaA1 is a linear, double-stranded DNA molecule with 3' protruding cohesive ends. The SpaA1 genome encompasses 63 predicted protein-coding genes which cluster within three regions of the genome, each of apparently different origin, in a mosaic pattern. In two of these regions, the gene sets resemble those in prophages of Bacillus thuringiensis kurstaki str. T03a001 (genes involved in DNA replication/transcription, cell entry and exit and B. cereus AH676 (additional regulatory and recombination genes, respectively. The third region represents an almost complete genome (except for the short terminal segments of a distinct bacteriophage, MZTP02. Nearly the same gene module was identified in prophages of B. thuringiensis serovar monterrey BGSC 4AJ1 and B. cereus Rock4-2. These findings suggest that MZTP02 can be shuttled between genomes of other bacteriophages and prophages, leading to the formation of chimeric genomes. The presence of a complete phage genome in the genome of other phages apparently has not been described previously and might represent a 'fast track' route of virus evolution and horizontal gene transfer. Another phage (BceA1 nearly identical in sequence to SpaA1, and also including the almost complete MZTP02 genome within its own genome, was isolated from a bacterium of the B. cereus/B. thuringiensis group. Remarkably, both SpaA1 and BceA1 phages can infect B. cereus and B. thuringiensis, but only one of them, SpaA1, can infect S. pasteuri. This finding is best compatible with a scenario in which MZTP02 was originally contained in BceA1 infecting Bacillus spp, the common hosts for these two phages, followed by emergence of SpaA1 infecting S. pasteuri.

  11. The peripheral conversion of thyroxine (T4) into triiodothyronine (T3) and reverse triiodothyronine (rT3)

    International Nuclear Information System (INIS)

    The aim of this study was to delineate several physiological, pathological and pharmacological factors involved in the peripheral conversion of thyroxine (T4), using radioimmunoassay. The determination of normal values of these tests under basal circumstances and after stimulation with thyrotropin-releasing-hormone is presented, and some physiological factors which may modulate the conversion of T4 are discussed. Results are presented of the thyroid function tests in patients with thyroid disease and with acute non-thyroidal diseases. (Auth.)

  12. Effects of bacteriophage traits on plaque formation

    Directory of Open Access Journals (Sweden)

    Kannoly Sherin

    2011-08-01

    Full Text Available Abstract Background The appearance of plaques on a bacterial lawn is one of the enduring imageries in modern day biology. The seeming simplicity of a plaque has invited many hypotheses and models in trying to describe and explain the details of its formation. However, until now, there has been no systematic experimental exploration on how different bacteriophage (phage traits may influence the formation of a plaque. In this study, we constructed a series of isogenic λ phages that differ in their adsorption rate, lysis timing, or morphology so that we can determine the effects if these changes on three plaque properties: size, progeny productivity, and phage concentration within plaques. Results We found that the adsorption rate has a diminishing, but negative impact on all three plaque measurements. Interestingly, there exists a concave relationship between the lysis time and plaque size, resulting in an apparent optimal lysis time that maximizes the plaque size. Although suggestive in appearance, we did not detect a significant effect of lysis time on plaque productivity. Nonetheless, the combined effects of plaque size and productivity resulted in an apparent convex relationship between the lysis time and phage concentration within plaques. Lastly, we found that virion morphology also affected plaque size. We compared our results to the available models on plaque size and productivity. For the models in their current forms, a few of them can capture the qualitative aspects of our results, but not consistently in both plaque properties. Conclusions By using a collection of isogenic phage strains, we were able to investigate the effects of individual phage traits on plaque size, plaque productivity, and average phage concentration in a plaque while holding all other traits constant. The controlled nature of our study allowed us to test several model predictions on plaque size and plaque productivity. It seems that a more realistic theoretical

  13. STUDIES ON THE BACTERIOPHAGE OF D'HERELLE : VII. ON THE PARTICULATE NATURE OF BACTERIOPHAGE.

    Science.gov (United States)

    Bronfenbrenner, J

    1927-04-30

    When filtrates of lysed cultures (bacteriophage) are subjected to prolonged dialysis under osmotic pressure against water, the presence of the lytic agent can be detected outside the membrane only during the first few days. The residue remaining inside the membrane contains the bulk of the original lytic agent, and yet it is no longer capable of diffusing into the outer solution. The interruption of diffusion is shown not to be due to any alteration in the permeability of the membrane. Moreover, the residue fails to diffuse through a fresh membrane of similar permeability, while the dialyzed portion of the phage passes quantitatively through a new membrane. When ultrafiltration under pressure was substituted for dialysis, the residue on the filter could be washed repeatedly with water without giving off into the filtrate any more active agent. However, if broth was substituted for water, a renewed diffusion of the active agent resulted. These results are interpreted as indicating that the colloidal particles present in the lytic filtrates (and apparently endowed with properties of bacteriophage) do not represent autonomous units of the active agent, but merely serve as a vehicle on which the agent is adsorbed. The vary in size within limits wide enough to permit fractionation by means of ultrafiltration. When the coarser particles retained by the ultrafilter are washed with broth, some of the active agent is detached from its coarse vehicle particles. The agent, now more highly dispersed, is capable of passing the filter which held it back previously. Preparation of a simple ultrafilter used in these experiments is given in detail.

  14. More Is Better: Selecting for Broad Host Range Bacteriophages.

    Science.gov (United States)

    Ross, Alexa; Ward, Samantha; Hyman, Paul

    2016-01-01

    Bacteriophages are viruses that infect bacteria. In this perspective, we discuss several aspects of a characteristic feature of bacteriophages, their host range. Each phage has its own particular host range, the range of bacteria that it can infect. While some phages can only infect one or a few bacterial strains, other phages can infect many species or even bacteria from different genera. Different methods for determining host range may give different results, reflecting the multiple mechanisms bacteria have to resist phage infection and reflecting the different steps of infection each method depends on. This makes defining host range difficult. Another difficulty in describing host range arises from the inconsistent use of the words "narrow" and especially "broad" when describing the breadth of the host range. Nearly all bacteriophages have been isolated using a single host strain of bacteria. While this procedure is fairly standard, it may more likely produce narrow rather than broad host range phage. Our results and those of others suggest that using multiple host strains during isolation can more reliably produce broader host range phages. This challenges the common belief that most bacteriophages have a narrow host range. We highlight the implications of this for several areas that are affected by host range including horizontal gene transfer and phage therapy. PMID:27660623

  15. Bacteriophage for prophylaxis and therapy in cattle, poultry, and pigs.

    Science.gov (United States)

    The successful use of virulent (lytic) bacteriophages (phages) in preventing and treating neonatal enterotoxigenic Escherichia coli infections in calves, lambs and pigs has prompted investigation of other applications phage therapy in food animals. While results have been very variable, some indica...

  16. Genome Sequences of Gordonia terrae Bacteriophages Phinally and Vivi2.

    Science.gov (United States)

    Pope, Welkin H; Anderson, Kaitlyn C; Arora, Charu; Bortz, Michael E; Burnet, George; Conover, David H; D'Incau, Gina M; Ghobrial, Jonathan A; Jonas, Audrey L; Migdal, Emily J; Rote, Nicole L; German, Brian A; McDonnell, Jill E; Mezghani, Nadia; Schafer, Claire E; Thompson, Paige K; Ulbrich, Megan C; Yu, Victor J; Furbee, Emily C; Grubb, Sarah R; Warner, Marcie H; Montgomery, Matthew T; Garlena, Rebecca A; Russell, Daniel A; Jacobs-Sera, Deborah; Hatfull, Graham F

    2016-08-18

    Bacteriophages Phinally and Vivi2 were isolated from soil from Pittsburgh, Pennsylvania, USA, using host Gordonia terrae 3612. The Phinally and Vivi2 genomes are 59,265 bp and 59,337 bp, respectively, and share sequence similarity with each other and with GTE6. Fewer than 25% of the 87 to 89 putative genes have predictable functions.

  17. Bacteriophages Limit the Existence Conditions for Conjugative Plasmids

    Science.gov (United States)

    Wood, A. Jamie; Dytham, Calvin; Pitchford, Jonathan W.; Truman, Julie; Spiers, Andrew; Paterson, Steve; Brockhurst, Michael A.

    2015-01-01

    ABSTRACT Bacteriophages are a major cause of bacterial mortality and impose strong selection on natural bacterial populations, yet their effects on the dynamics of conjugative plasmids have rarely been tested. We combined experimental evolution, mathematical modeling, and individual-based simulations to explain how the ecological and population genetics effects of bacteriophages upon bacteria interact to determine the dynamics of conjugative plasmids and their persistence. The ecological effects of bacteriophages on bacteria are predicted to limit the existence conditions for conjugative plasmids, preventing persistence under weak selection for plasmid accessory traits. Experiments showed that phages drove faster extinction of plasmids in environments where the plasmid conferred no benefit, but they also revealed more complex effects of phages on plasmid dynamics under these conditions, specifically, the temporary maintenance of plasmids at fixation followed by rapid loss. We hypothesized that the population genetic effects of bacteriophages, specifically, selection for phage resistance mutations, may have caused this. Further mathematical modeling and individual-based simulations supported our hypothesis, showing that conjugative plasmids may hitchhike with phage resistance mutations in the bacterial chromosome. PMID:26037122

  18. More Is Better: Selecting for Broad Host Range Bacteriophages.

    Science.gov (United States)

    Ross, Alexa; Ward, Samantha; Hyman, Paul

    2016-01-01

    Bacteriophages are viruses that infect bacteria. In this perspective, we discuss several aspects of a characteristic feature of bacteriophages, their host range. Each phage has its own particular host range, the range of bacteria that it can infect. While some phages can only infect one or a few bacterial strains, other phages can infect many species or even bacteria from different genera. Different methods for determining host range may give different results, reflecting the multiple mechanisms bacteria have to resist phage infection and reflecting the different steps of infection each method depends on. This makes defining host range difficult. Another difficulty in describing host range arises from the inconsistent use of the words "narrow" and especially "broad" when describing the breadth of the host range. Nearly all bacteriophages have been isolated using a single host strain of bacteria. While this procedure is fairly standard, it may more likely produce narrow rather than broad host range phage. Our results and those of others suggest that using multiple host strains during isolation can more reliably produce broader host range phages. This challenges the common belief that most bacteriophages have a narrow host range. We highlight the implications of this for several areas that are affected by host range including horizontal gene transfer and phage therapy.

  19. Complete Genome Sequence of Bacillus thuringiensis Bacteriophage Smudge.

    Science.gov (United States)

    Cornell, Jessica L; Breslin, Eileen; Schuhmacher, Zachary; Himelright, Madison; Berluti, Cassandra; Boyd, Charles; Carson, Rachel; Del Gallo, Elle; Giessler, Caris; Gilliam, Benjamin; Heatherly, Catherine; Nevin, Julius; Nguyen, Bryan; Nguyen, Justin; Parada, Jocelyn; Sutterfield, Blake; Tukruni, Muruj; Temple, Louise

    2016-01-01

    Smudge, a bacteriophage enriched from soil using Bacillus thuringiensis DSM-350 as the host, had its complete genome sequenced. Smudge is a myovirus with a genome consisting of 292 genes and was identified as belonging to the C1 cluster of Bacillus phages. PMID:27540049

  20. Complete Genome Sequence of Bacillus thuringiensis Bacteriophage Smudge

    Science.gov (United States)

    Cornell, Jessica L.; Breslin, Eileen; Schuhmacher, Zachary; Himelright, Madison; Berluti, Cassandra; Boyd, Charles; Carson, Rachel; Del Gallo, Elle; Giessler, Caris; Gilliam, Benjamin; Heatherly, Catherine; Nevin, Julius; Nguyen, Bryan; Nguyen, Justin; Parada, Jocelyn; Sutterfield, Blake; Tukruni, Muruj

    2016-01-01

    Smudge, a bacteriophage enriched from soil using Bacillus thuringiensis DSM-350 as the host, had its complete genome sequenced. Smudge is a myovirus with a genome consisting of 292 genes and was identified as belonging to the C1 cluster of Bacillus phages. PMID:27540049

  1. Complete Genome Sequence of Bacillus thuringiensis Bacteriophage Smudge.

    Science.gov (United States)

    Cornell, Jessica L; Breslin, Eileen; Schuhmacher, Zachary; Himelright, Madison; Berluti, Cassandra; Boyd, Charles; Carson, Rachel; Del Gallo, Elle; Giessler, Caris; Gilliam, Benjamin; Heatherly, Catherine; Nevin, Julius; Nguyen, Bryan; Nguyen, Justin; Parada, Jocelyn; Sutterfield, Blake; Tukruni, Muruj; Temple, Louise

    2016-08-18

    Smudge, a bacteriophage enriched from soil using Bacillus thuringiensis DSM-350 as the host, had its complete genome sequenced. Smudge is a myovirus with a genome consisting of 292 genes and was identified as belonging to the C1 cluster of Bacillus phages.

  2. Role of osmotic and hydrostatic pressures in bacteriophage genome ejection

    NARCIS (Netherlands)

    Lemay, S.G.; Panja, D.; Molineux, I.J.

    2013-01-01

    A critical step in the bacteriophage life cycle is genome ejection into host bacteria. The ejection process for double-stranded DNA phages has been studied thoroughly in vitro, where after triggering with the cellular receptor the genome ejects into a buffer. The experimental data have been interpre

  3. More Is Better: Selecting for Broad Host Range Bacteriophages

    Science.gov (United States)

    Ross, Alexa; Ward, Samantha; Hyman, Paul

    2016-01-01

    Bacteriophages are viruses that infect bacteria. In this perspective, we discuss several aspects of a characteristic feature of bacteriophages, their host range. Each phage has its own particular host range, the range of bacteria that it can infect. While some phages can only infect one or a few bacterial strains, other phages can infect many species or even bacteria from different genera. Different methods for determining host range may give different results, reflecting the multiple mechanisms bacteria have to resist phage infection and reflecting the different steps of infection each method depends on. This makes defining host range difficult. Another difficulty in describing host range arises from the inconsistent use of the words “narrow” and especially “broad” when describing the breadth of the host range. Nearly all bacteriophages have been isolated using a single host strain of bacteria. While this procedure is fairly standard, it may more likely produce narrow rather than broad host range phage. Our results and those of others suggest that using multiple host strains during isolation can more reliably produce broader host range phages. This challenges the common belief that most bacteriophages have a narrow host range. We highlight the implications of this for several areas that are affected by host range including horizontal gene transfer and phage therapy. PMID:27660623

  4. Multiple roles of genome-attached bacteriophage terminal proteins

    Energy Technology Data Exchange (ETDEWEB)

    Redrejo-Rodríguez, Modesto; Salas, Margarita, E-mail: msalas@cbm.csic.es

    2014-11-15

    Protein-primed replication constitutes a generalized mechanism to initiate DNA or RNA synthesis in linear genomes, including viruses, gram-positive bacteria, linear plasmids and mobile elements. By this mechanism a specific amino acid primes replication and becomes covalently linked to the genome ends. Despite the fact that TPs lack sequence homology, they share a similar structural arrangement, with the priming residue in the C-terminal half of the protein and an accumulation of positively charged residues at the N-terminal end. In addition, various bacteriophage TPs have been shown to have DNA-binding capacity that targets TPs and their attached genomes to the host nucleoid. Furthermore, a number of bacteriophage TPs from different viral families and with diverse hosts also contain putative nuclear localization signals and localize in the eukaryotic nucleus, which could lead to the transport of the attached DNA. This suggests a possible role of bacteriophage TPs in prokaryote-to-eukaryote horizontal gene transfer. - Highlights: • Protein-primed genome replication constitutes a strategy to initiate DNA or RNA synthesis in linear genomes. • Bacteriophage terminal proteins (TPs) are covalently attached to viral genomes by their primary function priming DNA replication. • TPs are also DNA-binding proteins and target phage genomes to the host nucleoid. • TPs can also localize in the eukaryotic nucleus and may have a role in phage-mediated interkingdom gene transfer.

  5. Effect of gamma irradiation on bacteriophages used as viral indicators.

    Science.gov (United States)

    Jebri, Sihem; Hmaied, Fatma; Jofre, Juan; MariemYahya; Mendez, Javier; Barkallah, Insaf; Hamdi, Moktar

    2013-07-01

    This study aimed to examine the susceptibility of indicator bacteriophages towards γ-radiation to evaluate their appropriateness as viral indicators for water quality control. The effects of γ-radiation on naturally occurring somatic coliphages, F-specific coliphages and Escherichia coli were examined in raw sewage and sewage sludge. As well, the effects of radiation on bacteriophages ΦX174 and MS2, and E. coli all grown in the laboratory and seeded in distilled water, autoclaved raw sewage and a 1% peptone solution were evaluated. The inactivation of E. coli was fairly similar in all matrices. In contrast, inactivation of bacteriophages was significantly greater in distilled water than in the other matrices. These results showed the great influence of the matrix characteristics on virus inactivation. Somatic coliphages in raw sewage and sewage sludge and ΦX174 in autoclaved sewage were inactivated similarly and were far more resistant than F-specific coliphages, MS2 and E. coli. As well, F-specific RNA bacteriophages in raw sewage and sewage sludge and MS2 in autoclaved sewage were inactivated similarly and were more resistant than E. coli. In contrast, MS2 was more susceptible to γ-radiation than E. coli in distilled water. Our results showed that ΦX174 is a suitable indicator for estimating virus inactivation by γ-irradiation and corroborate the use of somatic coliphages to survey the viral quality of treated water and sludges.

  6. Multiple roles of genome-attached bacteriophage terminal proteins

    International Nuclear Information System (INIS)

    Protein-primed replication constitutes a generalized mechanism to initiate DNA or RNA synthesis in linear genomes, including viruses, gram-positive bacteria, linear plasmids and mobile elements. By this mechanism a specific amino acid primes replication and becomes covalently linked to the genome ends. Despite the fact that TPs lack sequence homology, they share a similar structural arrangement, with the priming residue in the C-terminal half of the protein and an accumulation of positively charged residues at the N-terminal end. In addition, various bacteriophage TPs have been shown to have DNA-binding capacity that targets TPs and their attached genomes to the host nucleoid. Furthermore, a number of bacteriophage TPs from different viral families and with diverse hosts also contain putative nuclear localization signals and localize in the eukaryotic nucleus, which could lead to the transport of the attached DNA. This suggests a possible role of bacteriophage TPs in prokaryote-to-eukaryote horizontal gene transfer. - Highlights: • Protein-primed genome replication constitutes a strategy to initiate DNA or RNA synthesis in linear genomes. • Bacteriophage terminal proteins (TPs) are covalently attached to viral genomes by their primary function priming DNA replication. • TPs are also DNA-binding proteins and target phage genomes to the host nucleoid. • TPs can also localize in the eukaryotic nucleus and may have a role in phage-mediated interkingdom gene transfer

  7. Chromosomal duplications and cointegrates generated by the bacteriophage lamdba Red system in Escherichia coli K-12

    Directory of Open Access Journals (Sweden)

    Nadkarni Ashwini

    2004-12-01

    Full Text Available Abstract Background An Escherichia coli strain in which RecBCD has been genetically replaced by the bacteriophage λ Red system engages in efficient recombination between its chromosome and linear double-stranded DNA species sharing sequences with the chromosome. Previous studies of this experimental system have focused on a gene replacement-type event, in which a 3.5 kbp dsDNA consisting of the cat gene and flanking lac operon sequences recombines with the E. coli chromosome to generate a chloramphenicol-resistant Lac- recombinant. The dsDNA was delivered into the cell as part of the chromosome of a non-replicating λ vector, from which it was released by the action of a restriction endonuclease in the infected cell. This study characterizes the genetic requirements and outcomes of a variety of additional Red-promoted homologous recombination events producing Lac+ recombinants. Results A number of observations concerning recombination events between the chromosome and linear DNAs were made: (1 Formation of Lac+ and Lac- recombinants depended upon the same recombination functions. (2 High multiplicity and high chromosome copy number favored Lac+ recombinant formation. (3 The Lac+ recombinants were unstable, segregating Lac- progeny. (4 A tetracycline-resistance marker in a site of the phage chromosome distant from cat was not frequently co-inherited with cat. (5 Recombination between phage sequences in the linear DNA and cryptic prophages in the chromosome was responsible for most of the observed Lac+ recombinants. In addition, observations were made concerning recombination events between the chromosome and circular DNAs: (6 Formation of recombinants depended upon both RecA and, to a lesser extent, Red. (7 The linked tetracycline-resistance marker was frequently co-inherited in this case. Conclusions The Lac+ recombinants arise from events in which homologous recombination between the incoming linear DNA and both lac and cryptic prophage

  8. Two-stage, self-cycling process for the production of bacteriophages

    Directory of Open Access Journals (Sweden)

    Cooper David G

    2010-11-01

    Full Text Available Abstract Background A two-stage, self-cycling process for the production of bacteriophages was developed. The first stage, containing only the uninfected host bacterium, was operated under self-cycling fermentation (SCF conditions. This automated method, using the derivative of the carbon dioxide evolution rate (CER as the control parameter, led to the synchronization of the host bacterium. The second stage, containing both the host and the phage, was operated using self-cycling infection (SCI with CER and CER-derived data as the control parameters. When each infection cycle was terminated, phages were harvested and a new infection cycle was initiated by adding host cells from the SCF (first stage. This was augmented with fresh medium and the small amount of phages left from the previous cycle initiated the next infection cycle. Both stages were operated independently, except for this short period of time when the SCF harvest was added to the SCI to initiate the next cycle. Results It was demonstrated that this mode of operation resulted in stable infection cycles if the growth of the host cells in the SCF was synchronized. The final phage titers obtained were reproducible among cycles and were as good as those obtained in batch productions performed under the same conditions (medium, temperature, initial multiplicity of infection, etc.. Moreover, phages obtained in different cycles showed no important difference in infectivity. Finally, it was shown that cell synchronization of the host cells in the first stage (SCF not only maintained the volumetric productivity (phages per volume but also led to higher specific productivity (phage per cell per hour in the second stage (SCI. Conclusions Production of bacteriophage T4 in the semi-continuous, automated SCF/SCI system was efficient and reproducible from cycle to cycle. Synchronization of the host in the first stage prior to infection led to improvements in the specific productivity of phages in

  9. Investigating hexameric helicases: Single-molecule studies of DnaB and T4 gp41

    Science.gov (United States)

    Saleh, Omar; Ribeck, Noah; Berezney, John

    2011-03-01

    Hexameric, ring-shaped motor proteins serve as replicative helicases in many systems. They function by encircling and translocating along ssDNA, denaturing dsDNA in advance of its motion by sterically occluding the complementary strand to the outside of the ring. We investigate the helicase activity of two such motors using single-molecule measurements with magnetic tweezers. First, we measure the activity of the E. coli helicase DnaB complexed with the tau subunit of the Pol III holoenzyme. Tau is known from bulk measurements to stimulate DnaB activity (Kim et al., Cell, 1996); we investigate the means of this stimulation. Second, we measure helicase activity of the T4 phage helicase gp41 in multiple tethered DNA geometries. Previous work on DnaB showed a dependence of helicase activity on DNA geometry (Ribeck et al., Biophys. J., 2010); here, we test gp41 for similar behavior to see whether it is a common characteristic of hexameric helicases.

  10. Improvement on the Fatigue Performance of 2024-T4 Alloy by Synergistic Coating Technology

    Directory of Open Access Journals (Sweden)

    Xi-Shu Wang

    2014-05-01

    Full Text Available In this paper, rotating bending fatigue tests of 2024-T4 Al alloy with different oxide coatings were carried out. Compared to the uncoated and previously reported oxide coatings of aluminum alloys, the fatigue strength is able to be enhanced by using a novel oxide coating with sealing pore technology. These results indicate that the better the coating surface quality is, the more excellent the fatigue performance under rotating bending fatigue loading is. The improvement on the fatigue performance is mainly because the fatigue crack initiation and the early stage of fatigue crack growth at the coating layer can be delayed after PEO coating with pore sealing. Therefore, it is a so-called synergistic coating technology for various uses, including welding thermal cracks and filling micro-pores. The effects of different oxide coatings on surface hardness, compressive residual stress, morphology and fatigue fracture morphology are discussed. A critical compressive residual stress of about 95–100 MPa is proposed.

  11. Oral T4-like phage cocktail application to healthy adult volunteers from Bangladesh

    Energy Technology Data Exchange (ETDEWEB)

    Sarker, Shafiqul Alam, E-mail: sasarker@icddrb.org [International Centre for Diarrhoeal Diseases Research, Bangladesh (icddr,b), 68 Shaheed Tajuddin Ahmed Sharani, Mohakhali, Dhaka 1212 (Bangladesh); McCallin, Shawna; Barretto, Caroline [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland); Berger, Bernard, E-mail: bernard.berger@rdls.nestle.com [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland); Pittet, Anne-Cecile [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland); Sultana, Shamima, E-mail: shamima@icddrb.org [International Centre for Diarrhoeal Diseases Research, Bangladesh (icddr,b), 68 Shaheed Tajuddin Ahmed Sharani, Mohakhali, Dhaka 1212 (Bangladesh); Krause, Lutz, E-mail: ltz.krause@gmail.com [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland); Huq, Sayeda, E-mail: sayeeda@mail.icddrb.org [International Centre for Diarrhoeal Diseases Research, Bangladesh (icddr,b), 68 Shaheed Tajuddin Ahmed Sharani, Mohakhali, Dhaka 1212 (Bangladesh); Bibiloni, Rodrigo, E-mail: Rodrigo.Bibiloni@agresearch.co.nz [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland); Bruttin, Anne, E-mail: anne.bruttin@rdls.nestle.com [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland); Reuteler, Gloria, E-mail: gloria.reuteler@rdls.nestle.com [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland); Bruessow, Harald, E-mail: harald.bruessow@rdls.nestle.com [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland)

    2012-12-20

    The genomic diversity of 99 T4-like coliphages was investigated by sequencing an equimolar mixture with Illumina technology and screening them against different databases for horizontal gene transfer and undesired genes. A 9-phage cocktail was given to 15 healthy adults from Bangladesh at a dose of 3 Multiplication-Sign 10{sup 9} and 3 Multiplication-Sign 10{sup 7} plaque-forming units and placebo respectively. Phages were detected in 64% of the stool samples when subjects were treated with higher titer phage, compared to 30% and 28% with lower-titer phage and placebo, respectively. No Escherichia coli was present in initial stool samples, and no amplification of phage was observed. One percent of the administered oral phage was recovered from the feces. No adverse events were observed by self-report, clinical examination, or from laboratory tests for liver, kidney, and hematology function. No impact of oral phage was seen on the fecal microbiota composition with respect to bacterial 16S rRNA from stool.

  12. Technical performance evaluation of the MyT4 point of care technology for CD4+ T cell enumeration.

    Directory of Open Access Journals (Sweden)

    Matilu Mwau

    Full Text Available Though absolute CD4+ T cell enumeration is the primary gateway to antiretroviral therapy initiation for HIV-positive patients in all developing countries, patient access to this critical diagnostic test is relatively poor. We technically evaluated the performance of a newly developed point-of-care CD4+ T cell technology, the MyT4, compared with conventional CD4+ T cell testing technologies.Over 250 HIV-positive patients were consecutively enrolled and their blood tested on the MyT4, BD FACSCalibur, and BD FACSCount.Compared with the BD FACSCount, the MyT4 had an r2 of 0.7269 and a mean bias of -23.37 cells/µl. Compared with the BD FACSCalibur, the MyT4 had an r2 of 0.5825 and a mean bias of -46.58 cells/µl. Kenya currently uses a CD4+ T cell test threshold of 350 cells/µl to determine patient eligibility for antiretroviral therapy. At this threshold, the MyT4 had a sensitivity of 95.3% (95% CI: 88.4-98.7% and a specificity of 87.9% (95% CI: 82.3-92.3% compared with the BD FACSCount and sensitivity and specificity of 88.2% (95% CI: 79.4-94.2% and 84.2% (95% CI: 78.2-89.2%, respectively, compared with the BD FACSCalibur. Finally, the MyT4 had a coefficient of variation of 12.80% compared with 14.03% for the BD FACSCalibur.We conclude that the MyT4 performed well at the current 350 cells/µl ART initiation eligibility threshold when used by lower cadres of health care facility staff in rural clinics compared to conventional CD4+ T cell technologies.

  13. Divalent metal ion differentially regulates the sequential nicking reactions of the GIY-YIG homing endonuclease I-BmoI.

    Directory of Open Access Journals (Sweden)

    Benjamin P Kleinstiver

    Full Text Available Homing endonucleases are site-specific DNA endonucleases that function as mobile genetic elements by introducing double-strand breaks or nicks at defined locations. Of the major families of homing endonucleases, the modular GIY-YIG endonucleases are least understood in terms of mechanism. The GIY-YIG homing endonuclease I-BmoI generates a double-strand break by sequential nicking reactions during which the single active site of the GIY-YIG nuclease domain must undergo a substantial reorganization. Here, we show that divalent metal ion plays a significant role in regulating the two independent nicking reactions by I-BmoI. Rate constant determination for each nicking reaction revealed that limiting divalent metal ion has a greater impact on the second strand than the first strand nicking reaction. We also show that substrate mutations within the I-BmoI cleavage site can modulate the first strand nicking reaction over a 314-fold range. Additionally, in-gel DNA footprinting with mutant substrates and modeling of an I-BmoI-substrate complex suggest that amino acid contacts to a critical GC-2 base pair are required to induce a bottom-strand distortion that likely directs conformational changes for reaction progress. Collectively, our data implies mechanistic roles for divalent metal ion and substrate bases, suggesting that divalent metal ion facilitates the re-positioning of the GIY-YIG nuclease domain between sequential nicking reactions.

  14. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

    OpenAIRE

    Sternberg, Samuel H.; Redding, Sy; Jinek, Martin; Greene, Eric C.; Doudna, Jennifer A.

    2014-01-01

    The CRISPR-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA:DNA base-pairing to target foreign DNA in bacteria. Cas9:guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9:RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9:RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). ...

  15. Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation

    OpenAIRE

    Jinek, Martin; Jiang, Fuguo; Taylor, David W.; Sternberg, Samuel H.; Kaya, Emine; MA, ENBO; Anders, Carolin; Hauer, Michael; Zhou, Kaihong; Lin, Steven; Kaplan, Matias; Anthony T Iavarone; Charpentier, Emmanuelle; Nogales, Eva; Doudna, Jennifer A.

    2014-01-01

    Type II CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all C...

  16. Innate and adaptive immunity in bacteria: mechanisms of programmed genetic variation to fight bacteriophages.

    Science.gov (United States)

    Bikard, David; Marraffini, Luciano A

    2012-02-01

    Bacteria are constantly challenged by bacteriophages (viruses that infect bacteria), the most abundant microorganism on earth. Bacteria have evolved a variety of immunity mechanisms to resist bacteriophage infection. In response, bacteriophages can evolve counter-resistance mechanisms and launch a 'virus versus host' evolutionary arms race. In this context, rapid evolution is fundamental for the survival of the bacterial cell. Programmed genetic variation mechanisms at loci involved in immunity against bacteriophages generate diversity at a much faster rate than random point mutation and enable bacteria to quickly adapt and repel infection. Diversity-generating retroelements (DGRs) and phase variation mechanisms enhance the generic (innate) immune response against bacteriophages. On the other hand, the integration of small bacteriophage sequences in CRISPR loci provide bacteria with a virus-specific and sequence-specific adaptive immune response. Therefore, although using different molecular mechanisms, both prokaryotes and higher organisms rely on programmed genetic variation to increase genetic diversity and fight rapidly evolving infectious agents.

  17. Novel Bacteroides host strains for detection of human- and animal-specific bacteriophages in water.

    Science.gov (United States)

    Wicki, Melanie; Auckenthaler, Adrian; Felleisen, Richard; Tanner, Marcel; Baumgartner, Andreas

    2011-03-01

    Bacteriophages active against specific Bacteroides host strains were shown to be suitable for detection of human faecal pollution. However, the practical application of this finding is limited because some specific host strains were restricted to certain geographic regions. In this study, novel Bacteroides host strains were isolated that discriminate human and animal faecal pollution in Switzerland. Two strains specific for bacteriophages present in human faecal contamination and three strains specific for bacteriophages indicating animal faecal contamination were evaluated. Bacteriophages infecting human strains were exclusively found in human wastewater, whereas animal strains detected bacteriophages only in animal waste. The newly isolated host strains could be used to determine the source of surface and spring water faecal contamination in field situations. Applying the newly isolated host Bacteroides thetaiotaomicron ARABA 84 for detection of bacteriophages allowed the detection of human faecal contamination in spring water.

  18. Simultaneous loss of bacteriophage receptor and coaggregation mediator activities in Actinomyces viscosus MG-1.

    OpenAIRE

    Tylenda, C A; Enriquez, E.; Kolenbrander, P. E.; Delisle, A L

    1985-01-01

    Actinomyces bacteriophages were used as tools to study coaggregation between actinomyces and streptococci. Four bacteriophage isolates, phages AV-1, AV-2, AV-3, and 1281, bound to coaggregation group A Actinomyces viscosus and to group E A. naeslundii. No binding to groups B, C, D, or F was observed. Only A. viscosus MG-1 was capable of supporting a productive infection by these phages. Spontaneously occurring bacteriophage-resistant mutants of A. viscosus MG-1 were isolated and were shown to...

  19. Identification of potential influenza virus endonuclease inhibitors through virtual screening based on the 3D-QSAR model.

    Science.gov (United States)

    Kim, J; Lee, C; Chong, Y

    2009-01-01

    Influenza endonucleases have appeared as an attractive target of antiviral therapy for influenza infection. With the purpose of designing a novel antiviral agent with enhanced biological activities against influenza endonuclease, a three-dimensional quantitative structure-activity relationships (3D-QSAR) model was generated based on 34 influenza endonuclease inhibitors. The comparative molecular similarity index analysis (CoMSIA) with a steric, electrostatic and hydrophobic (SEH) model showed the best correlative and predictive capability (q(2) = 0.763, r(2) = 0.969 and F = 174.785), which provided a pharmacophore composed of the electronegative moiety as well as the bulky hydrophobic group. The CoMSIA model was used as a pharmacophore query in the UNITY search of the ChemDiv compound library to give virtual active compounds. The 3D-QSAR model was then used to predict the activity of the selected compounds, which identified three compounds as the most likely inhibitor candidates.

  20. Necrotizing Meningoencephalitis in a Captive Black and White Ruffed Lemur (Varecia variegata variegata) Caused by Acanthamoeba T4 Genotype.

    Science.gov (United States)

    Gaide, N; Pelandakis, M; Robveille, C; Albaric, O; Jouvion, G; Souchon, M; Risler, A; Abadie, J

    2015-11-01

    A mature male, black and white ruffed lemur (Varecia variegata variegata) died in a zoological garden after a 4-day history of lethargy and non-responsive convulsions. Necropsy and histopathological examinations revealed acute necrotizing and haemorrhagic meningoencephalitis with intralesional amoebas confirmed by immunohistochemistry. Acanthamoeba T4 genotype was identified as the causative agent of the brain lesion, based on amplification and sequencing of 18S ribosomal RNA genes. The presence of free-living amoebas in water and mud from the lemur's environment was investigated by morphological and molecular analyses. The two predominant genera, representing 80% of isolated amoebas, were Naegleria spp. and Acanthamoeba spp. All Acanthamoeba isolates belonged to the T4 genotype. To the author's knowledge, this is the first report of a meningoencephalitis due to Acanthamoeba T4 genotype in Lemuridae with concurrent analysis of pathological tissues and environment.

  1. Creating directed double-strand breaks with the Ref protein: a novel RecA-dependent nuclease from bacteriophage P1.

    Science.gov (United States)

    Gruenig, Marielle C; Lu, Duo; Won, Sang Joon; Dulberger, Charles L; Manlick, Angela J; Keck, James L; Cox, Michael M

    2011-03-11

    The bacteriophage P1-encoded Ref protein enhances RecA-dependent recombination in vivo by an unknown mechanism. We demonstrate that Ref is a new type of enzyme; that is, a RecA-dependent nuclease. Ref binds to ss- and dsDNA but does not cleave any DNA substrate until RecA protein and ATP are added to form RecA nucleoprotein filaments. Ref cleaves only where RecA protein is bound. RecA functions as a co-nuclease in the Ref/RecA system. Ref nuclease activity can be limited to the targeted strands of short RecA-containing D-loops. The result is a uniquely programmable endonuclease activity, producing targeted double-strand breaks at any chosen DNA sequence in an oligonucleotide-directed fashion. We present evidence indicating that cleavage occurs in the RecA filament groove. The structure of the Ref protein has been determined to 1.4 Å resolution. The core structure, consisting of residues 77-186, consists of a central 2-stranded β-hairpin that is sandwiched between several α-helical and extended loop elements. The N-terminal 76 amino acid residues are disordered; this flexible region is required for optimal activity. The overall structure of Ref, including several putative active site histidine residues, defines a new subclass of HNH-family nucleases. We propose that enhancement of recombination by Ref reflects the introduction of directed, recombinogenic double-strand breaks.

  2. Creating Directed Double-strand Breaks with the Ref Protein: A Novel Rec A-Dependent Nuclease from Bacteriophage P1

    Energy Technology Data Exchange (ETDEWEB)

    Gruenig, Marielle C.; Lu, Duo; Won, Sang Joon; Dulberger, Charles L.; Manlick, Angela J.; Keck, James L.; Cox, Michael M. (UW)

    2012-03-16

    The bacteriophage P1-encoded Ref protein enhances RecA-dependent recombination in vivo by an unknown mechanism. We demonstrate that Ref is a new type of enzyme; that is, a RecA-dependent nuclease. Ref binds to ss- and dsDNA but does not cleave any DNA substrate until RecA protein and ATP are added to form RecA nucleoprotein filaments. Ref cleaves only where RecA protein is bound. RecA functions as a co-nuclease in the Ref/RecA system. Ref nuclease activity can be limited to the targeted strands of short RecA-containing D-loops. The result is a uniquely programmable endonuclease activity, producing targeted double-strand breaks at any chosen DNA sequence in an oligonucleotide-directed fashion. We present evidence indicating that cleavage occurs in the RecA filament groove. The structure of the Ref protein has been determined to 1.4 {angstrom} resolution. The core structure, consisting of residues 77-186, consists of a central 2-stranded {beta}-hairpin that is sandwiched between several {alpha}-helical and extended loop elements. The N-terminal 76 amino acid residues are disordered; this flexible region is required for optimal activity. The overall structure of Ref, including several putative active site histidine residues, defines a new subclass of HNH-family nucleases. We propose that enhancement of recombination by Ref reflects the introduction of directed, recombinogenic double-strand breaks.

  3. Rapid and accurate detection of bacteriophage activity against Escherichia coli O157:H7 by propidium monoazide real-time PCR.

    Science.gov (United States)

    Liu, Hui; Niu, Yan D; Li, Jinquan; Stanford, Kim; McAllister, Tim A

    2014-01-01

    Conventional methods to determine the efficacy of bacteriophage (phage) for biocontrol of E. coli require several days, due to the need to culture bacteria. Furthermore, cell surface-attached phage particles may lyse bacterial cells during experiments, leading to an overestimation of phage activity. DNA-based real-time quantitative polymerase chain reaction (qPCR) is a fast, sensitive, and highly specific means of enumerating pathogens. However, qPCR may underestimate phage activity due to its inability to distinguish viable from nonviable cells. In this study, we evaluated the suitability of propidium monoazide (PMA), a microbial membrane-impermeable dye that inhibits amplification of extracellular DNA and DNA within dead or membrane-compromised cells as a means of using qPCR to identify only intact E. coli cells that survive phage exposure. Escherichia coli O157:H7 strain R508N and 4 phages (T5-like, T1-like, T4-like, and O1-like) were studied. Results compared PMA-qPCR and direct plating and confirmed that PMA could successfully inhibit amplification of DNA from compromised/damaged cells E. coli O157:H7. Compared to PMA-qPCR, direct plating overestimated (P bacteriophage for biocontrol of E. coli O157:H7.

  4. Stereotactic body radiotherapy for T3 and T4N0M0 non-small cell lung cancer.

    Science.gov (United States)

    Eriguchi, Takahisa; Takeda, Atsuya; Sanuki, Naoko; Nishimura, Shuichi; Takagawa, Yoshiaki; Enomoto, Tatsuji; Saeki, Noriyuki; Yashiro, Kae; Mizuno, Tomikazu; Aoki, Yousuke; Oku, Yohei; Yokosuka, Tetsuya; Shigematsu, Naoyuki

    2016-06-01

    To evaluate the outcomes and feasibility of stereotactic body radiotherapy (SBRT) for cT3 and cT4N0M0 non-small cell lung cancer (NSCLC), 25 patients with localized primary NSCLC diagnosed as cT3 or cT4N0M0, given SBRT between May 2005 and July 2013, were analyzed. All patients had inoperable tumors. The major reasons for tumors being unresectable were insufficient respiratory function for curative resection, advanced age (>80 years old) or technically inoperable due to invasion into critical organs. The median patient age was 79 years (range; 60-86). The median follow-up duration was 25 months (range: 5-100 months). The 2-year overall survival rates for T3 and T4 were 57% and 69%, respectively. The 2-year local control rates for T3 and T4 were 91% and 68%, respectively. As for toxicities, Grade 0-1, Grade 2 and Grade 3 radiation pneumonitis occurred in 23, 1 and 1 patient, respectively. No other acute or symptomatic late toxicities were reported. Thirteen patients who had no local, mediastinal or intrapulmonary progression at one year after SBRT underwent pulmonary function testing. The median variation in pre-SBRT and post-SBRT forced expiratory volume in 1 s (FEV1) values was -0.1 (-0.8-0.8). This variation was not statistically significant (P = 0.56). Forced vital capacity (FVC), vital capacity (VC), %VC and %FEV1 also showed no significant differences. SBRT for cT3 and cT4N0M0 NSCLC was both effective and feasible. Considering the favorable survival and low morbidity rate, SBRT is a potential treatment option for cT3 and cT4N0M0 NSCLC. PMID:26983978

  5. Prevalence and pattern of nodal metastasis in pT4 gingivobuccal cancers and its implications for treatment

    Directory of Open Access Journals (Sweden)

    H Narendra

    2010-01-01

    Full Text Available Context: The pattern of nodal spread in oral cancers is largely predictable and treatment of neck can be tailored with this knowledge. Most studies available on the pattern are from the western world and for early cancers of the tongue and floor of the mouth. Aims: The present study was aimed to evaluate the prevalence and pattern of nodal metastasis in patients with pathologic T4 (pT4 buccal/alveolar cancers. Settings and Design: Medical records of the patients with pT4 primary buccal and alveolar squamous cell carcinomas treated by single-stage resection of primary tumor and neck dissection at Gujarat Cancer and Research Institute (GCRI, Ahmedabad, a regional cancer center in India, during September 2004 to August 2006, were analyzed for nodal involvement. Materials and Methods: The study included 127 patients with pT4 buccal/alveolar cancer. Data pertaining to clinical nodal status, histologic grade, pT and pN status (TNM classification of malignant tumors, UICC, 6th edition, 2002, total number of nodes removed, and those involved by tumor, and levels of nodal involvement were recorded. Statistical analysis was performed using the Chi-square test. Results: Fifty percent of the patients did not have nodal metastasis on final histopathology. Occult metastasis rate was 23%. All of these occurred in levels I to III. Among those with clinically palpable nodes, level V involvement was seen only in 4% of the patients with pT4 buccal cancer and 3% of the patients with alveolar cancer. Conclusions: Elective treatment of the neck in the form of selective neck dissection of levels I to III is needed for T4 cancers of gingivobuccal complex due to a high rate of occult metastasis. Selected patients with clinically involved nodes could be well served by a selective neck dissection incorporating levels I to III or IV.

  6. Intervalos de referencia para concentraciones séricas de T3 y T4. Estudio preliminar

    Directory of Open Access Journals (Sweden)

    Cecilia Miranda Pantoja

    2013-12-01

    Full Text Available Fundamento: la determinación cuantitativa de las hormonas tiroideas T3 y T4 reviste gran importancia en el diagnóstico y la evaluación del hipertiroidismo, en especial del hipertiroidismo aislado causado por T3.Objetivo: establecer los intervalos de referencia de T3 y T4 en el laboratorio de medicina nuclear del Hospital General Universitario Dr. Gustavo Aldereguía Lima de Cienfuegos. Métodos: estudio descriptivo y prospectivo realizado mediante el método de radioinmunoanálisis, competencia que se establece entre la T3 y T4 sin marcar, y la marcada por un número limitado de los sitios de unión del anticuerpo específico. Al hacer reaccionar una cantidad fija de trazador y anticuerpo con diferentes cantidades del ligando sin marcar, la cantidad de trazador unido por el anticuerpo será inversamente proporcional a la concentración del ligando sin marcar. Resultados: los valores obtenidos se describen según una distribución gaussiana (media aritmética = 117, desviación estándar =31 para T4; media aritmética = 2,64, desviación estándar = 0,64 para T3, comprobado mediante un test de Chi cuadrado. Los rangos de valores normales obtenidos fueron de de 55 – 178 nmol/L y 1,4 – 3,9 nmol/L para T4 y T3 respectivamente. Conclusiones: los intervalos de referencia obtenidos resultaron más amplios que los propuestos por el productor, sobre todo en el caso de T4.

  7. Increased thyroidal T4 to T3 conversion in autonomously functioning thyroid adenoma: from euthyroidism to thyrotoxicosis.

    LENUS (Irish Health Repository)

    Solter, M

    2012-01-31

    AIM: The aim was to investigate whether the intrathyroid conversion of T4 to T3 in autonomously functioning thyroid adenoma (AFTA) tissue could influence serum T3 levels and suppression of TSH, especially in patients with borderline thyroid function. PATIENTS AND METHODS: In ten patients with AFTA, thyroidal conversion of T4 to T3 was investigated in nodular and paranodular, TSH-suppressed tissue. All patients had normal serum T4 and suppressed TSH. Serum T3 was normal in six, and borderline or slightly increased in four. AFTA and paranodular tissues were surgically removed and frozen at -70 degrees C, then homogenized in a glass homogenizer, centrifuged at 100,000xg, and particulate fraction collected as a pellet. Analysis mixture consisted of thyroid enzyme suspension in 50 mumol\\/L TRIS buffer with 5 mumol DTT and 200 muL 1.3 mumol T4. Incubation was performed at 37 degrees C and the generation of T3 measured after 5, 10, 20 and 40 minutes respectively. RESULTS: T3 production (pmol\\/mg protein) was significantly higher in AFTA than in paranodular tissues (8.8 1.2\\/Mean +\\/- SE\\/vs. 1.8 +\\/- 0.2; p<0.01), and excessively high (9.8, 14.1, 14.2 and 15.0) in four patients with borderline or slightly supranormal serum T3. A significant correlation was found between serum T3 concentrations and T3 generation (T4 conversion) in AFTA tissues. CONCLUSION: Results suggest that increased thyroidal T4 to T3 conversion in AFTA tissue could be involved in an increased delivery of T3, increased serum T3 and suppressed serum TSH, particularly in patients with the disease evolving from euthyroid to an early hyperthyroid phase.

  8. Methods for generation of reporter phages and immobilization of active bacteriophages on a polymer surface

    Science.gov (United States)

    Applegate, Bruce Michael (Inventor); Perry, Lynda Louise (Inventor); Morgan, Mark Thomas (Inventor); Kothapalli, Aparna (Inventor)

    2012-01-01

    Novel reporter bacteriophages are provided. Provided are compositions and methods that allow bacteriophages that are used for specific detection or killing of E. coli 0157:H7 to be propagated in nonpathogenic E. coli, thereby eliminating the safety and security risks of propagation in E. coli 0157:H7. Provided are compositions and methods for attaching active bacteriophages to the surface of a polymer in order to kill target bacteria with which the phage comes into contact. Provided are modified bacteriophages immobilized to a surface, which capture E. coli 0157:H7 and cause the captured cells to emit light or fluorescence, allowing detection of the bacteria in a sample.

  9. Identification of a novel site specific endonuclease produced by Mycoplasma fermentans: discovery while characterizing DNA binding proteins in T lymphocyte cell lines.

    OpenAIRE

    Halden, N F; Wolf, J B; Leonard, W J

    1989-01-01

    We have discovered a new restriction endonuclease, MfeI, in nuclear extracts from T cells contaminated with Mycoplasma fermentans. This endonuclease was identified while studying proteins binding to the interleukin-2 receptor alpha chain gene promoter. MfeI cuts at the recognition sequence C'AATTG generating EcoRI compatible cohesive ends. Potential applications are discussed.

  10. Intervalos de referencia para concentraciones séricas de T3 y T4. Estudio preliminar

    OpenAIRE

    Cecilia Miranda Pantoja; Carlos Alberto Miranda Pantoja; Daineris Quintana Castellanos

    2013-01-01

    Fundamento: la determinación cuantitativa de las hormonas tiroideas T3 y T4 reviste gran importancia en el diagnóstico y la evaluación del hipertiroidismo, en especial del hipertiroidismo aislado causado por T3.Objetivo: establecer los intervalos de referencia de T3 y T4 en el laboratorio de medicina nuclear del Hospital General Universitario Dr. Gustavo Aldereguía Lima de Cienfuegos. Métodos: estudio descriptivo y prospectivo realizado mediante el método de radioinmunoanálisis, competencia q...

  11. African swine fever virus AP endonuclease is a redox-sensitive enzyme that repairs alkylating and oxidative damage to DNA

    OpenAIRE

    Redrejo-Rodríguez, Modesto; Alexander A Ishchenko; Saparbaev, Murat K.; Salas, María L.; Salas, José

    2009-01-01

    African swine fever virus (ASFV) encodes an AP endonuclease (pE296R) which is essential for virus growth in swine macrophages. We show here that the DNA repair functions of pE296R (AP endonucleolytic, 3′ → 5′ exonuclease, 3′-diesterase and nucleotide incision repair (NIR) activities) and DNA binding are inhibited by reducing agents. Protein pE296R contains one intramolecular disulfide bond, whose disruption by reducing agents might perturb the interaction of the viral AP endonuclease with the...

  12. Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

    Science.gov (United States)

    Verissimo-Villela, Erika; Kitahara-Oliveira, Milene Yoko; dos Reis, Ana Beatriz de Bragança; Albano, Rodolpho Mattos; Da-Cruz, Alda Maria; Bello, Alexandre Ribeiro

    2016-01-01

    During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins. PMID:27223868

  13. Characterization of DNA binding activities of over-expressed kpnI restriction endonuclease and modification methylase

    OpenAIRE

    Chandrashekaran, Siddamadappa; Babu, Padmanabhan; Nagaraja, Valakunja

    1999-01-01

    The genes encoding the KpnI restriction endonuclease and methyltransferase from Klebsiella pneumoniae have been cloned and expressed in Escherchia coli using a two plasmid strategy. The gene for KpnI methylase with its promoter was cloned and expressed in pACYC184. Even though the methylase clone is in a low copy number plasmid pACMK, high level expression of methylase is achieved. A hyper-expressing clone of KpnI endonuclease, pETRK was engineered by cloning the R gene into the T7 expression...

  14. Optical mapping of a rice B AC clone using restriction endonuclease and imaging with fluorescent microscopy at single molecule level

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A method of constructing restriction map by optical mapping and single molecule fluorescent microscopy is described. DNA molecules were aligned and adsorbed on a glass coverslip surface by a mbdified "molecular combing"technique, and then the surface-immobilized DNAs were cleaved in situ with a restriction endonuclease. Individual DNA molecules digested by the endonuclease EcoR I were observable with fluorescent microscopy. Using optical mapping, a physical map of a rice bacterial artificial chromosome clone was constructed. This method will facilitate genomic mapping and tracing the dynamic process in real time at a single molecule level with fluorescence microscopy.

  15. Identification and characterization of inhibitors of human apurinic/apyrimidinic endonuclease APE1.

    Directory of Open Access Journals (Sweden)

    Anton Simeonov

    Full Text Available APE1 is the major nuclease for excising abasic (AP sites and particular 3'-obstructive termini from DNA, and is an integral participant in the base excision repair (BER pathway. BER capacity plays a prominent role in dictating responsiveness to agents that generate oxidative or alkylation DNA damage, as well as certain chain-terminating nucleoside analogs and 5-fluorouracil. We describe within the development of a robust, 1536-well automated screening assay that employs a deoxyoligonucleotide substrate operating in the red-shifted fluorescence spectral region to identify APE1 endonuclease inhibitors. This AP site incision assay was used in a titration-based high-throughput screen of the Library of Pharmacologically Active Compounds (LOPAC(1280, a collection of well-characterized, drug-like molecules representing all major target classes. Prioritized hits were authenticated and characterized via two high-throughput screening assays -- a Thiazole Orange fluorophore-DNA displacement test and an E. coli endonuclease IV counterscreen -- and a conventional, gel-based radiotracer incision assay. The top, validated compounds, i.e. 6-hydroxy-DL-DOPA, Reactive Blue 2 and myricetin, were shown to inhibit AP site cleavage activity of whole cell protein extracts from HEK 293T and HeLa cell lines, and to enhance the cytotoxic and genotoxic potency of the alkylating agent methylmethane sulfonate. The studies herein report on the identification of novel, small molecule APE1-targeted bioactive inhibitor probes, which represent initial chemotypes towards the development of potential pharmaceuticals.

  16. Atypical myxomatosis--virus isolation, experimental infection of rabbits and restriction endonuclease analysis of the isolate.

    Science.gov (United States)

    Psikal, I; Smíd, B; Rodák, L; Valícek, L; Bendová, J

    2003-08-01

    Atypical form of myxomatosis, which caused non-lethal and clinically mild disease in domestic rabbits 1 month after immunization with a commercially available vaccine MXT, is described. The isolated myxoma virus designated as Litovel 2 (Li-2) did not induce systemic disease following subcutaneous and intradermal applications in susceptible experimental rabbits but led to the immune response demonstrated by ELISA. No severe disease was induced in those Li-2 inoculated rabbits by challenge with the virulent strains Lausanne (Lu) or Sanar (SA), while the control animals showed nodular form of myxomatosis with lethal course of the illness. Restriction fragment length polymorphism (RFLP) of genomic DNA with KpnI and BamHI endonucleases was used for genetic characterization of the Li-2 isolate, the vaccine strain MXT and both virulent strains Lu and SA, respectively. In general, RFLP analysis has shown to be informative for inferring genetic relatedness between myxoma viruses. Based on restriction endonuclease DNA fragment size distribution, it was evident that the pathogenic strain SA is genetically related to the reference strain Lu and the isolate Li-2 is more related, but not identical, to the vaccination strain MXT. PMID:14628995

  17. EENdb: a database and knowledge base of ZFNs and TALENs for endonuclease engineering.

    Science.gov (United States)

    Xiao, An; Wu, Yingdan; Yang, Zhipeng; Hu, Yingying; Wang, Weiye; Zhang, Yutian; Kong, Lei; Gao, Ge; Zhu, Zuoyan; Lin, Shuo; Zhang, Bo

    2013-01-01

    We report here the construction of engineered endonuclease database (EENdb) (http://eendb.zfgenetics.org/), a searchable database and knowledge base for customizable engineered endonucleases (EENs), including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). EENs are artificial nucleases designed to target and cleave specific DNA sequences. EENs have been shown to be a very useful genetic tool for targeted genome modification and have shown great potentials in the applications in basic research, clinical therapies and agricultural utilities, and they are specifically essential for reverse genetics research in species where no other gene targeting techniques are available. EENdb contains over 700 records of all the reported ZFNs and TALENs and related information, such as their target sequences, the peptide components [zinc finger protein-/transcription activator-like effector (TALE)-binding domains, FokI variants and linker peptide/framework], the efficiency and specificity of their activities. The database also lists EEN engineering tools and resources as well as information about forms and types of EENs, EEN screening and construction methods, detection methods for targeting efficiency and many other utilities. The aim of EENdb is to represent a central hub for EEN information and an integrated solution for EEN engineering. These studies may help to extract in-depth properties and common rules regarding ZFN or TALEN efficiency through comparison of the known ZFNs or TALENs.

  18. Decisive role of apurinic/apyrimidinic endonuclease/Ref-1 in initiation of cell death.

    Science.gov (United States)

    Cho, Kyoung Joo; Kim, Hyun Jeong; Park, Soo Chul; Kim, Hyun Woo; Kim, Gyung Whan

    2010-11-01

    The apurinic/apyrimidinic endonuclease/redox effector factor-1 (APE/Ref-1) is involved in the base excision repair of apurinic/apyrimidinic sites induced by oxidative DNA damage. APE/Ref-1 was decreased by kainic acid (KA) injury in a time-dependent manner at the level of proteins, not transcripts. We investigated whether alteration of APE/Ref-1 amounts would influence hippocampal cell fate, survival or death, after KA injury. Overexpression of APE/Ref-1 using adenovirus and restoration of APE small peptides significantly reduced KA-induced hippocampal cell death. Both silencing of APE/Ref-1 by siRNA and inhibition of endonuclease by an antibody significantly increased caspase-3 activity and apoptotic cell death triggered from the early time after exposure to KA. These findings suggest that cell death is initiated by reducing APE/Ref-1 protein and inhibiting its repair function in spite of enough protein amounts. In conclusion, APE/Ref-1 may be a regulator of cell death initiation, and APE small peptides could provide molecular mechanism-based therapies for neuroprotection in progressive excitotoxic neuronal damage.

  19. Polymerase-endonuclease amplification reaction (PEAR for large-scale enzymatic production of antisense oligonucleotides.

    Directory of Open Access Journals (Sweden)

    Xiaolong Wang

    Full Text Available Antisense oligonucleotides targeting microRNAs or their mRNA targets prove to be powerful tools for molecular biology research and may eventually emerge as new therapeutic agents. Synthetic oligonucleotides are often contaminated with highly homologous failure sequences. Synthesis of a certain oligonucleotide is difficult to scale up because it requires expensive equipment, hazardous chemicals and a tedious purification process. Here we report a novel thermocyclic reaction, polymerase-endonuclease amplification reaction (PEAR, for the amplification of oligonucleotides. A target oligonucleotide and a tandem repeated antisense probe are subjected to repeated cycles of denaturing, annealing, elongation and cleaving, in which thermostable DNA polymerase elongation and strand slipping generate duplex tandem repeats, and thermostable endonuclease (PspGI cleavage releases monomeric duplex oligonucleotides. Each round of PEAR achieves over 100-fold amplification. The product can be used in one more round of PEAR directly, and the process can be further repeated. In addition to avoiding dangerous materials and improved product purity, this reaction is easy to scale up and amenable to full automation. PEAR has the potential to be a useful tool for large-scale production of antisense oligonucleotide drugs.

  20. Expression, purification and crystallization of two endonuclease III enzymes from Deinococcus radiodurans.

    Science.gov (United States)

    Sarre, Aili; Ökvist, Mats; Klar, Tobias; Moe, Elin; Timmins, Joanna

    2014-12-01

    Endonuclease III is a bifunctional DNA glycosylase that removes a wide range of oxidized bases in DNA. Deinococcus radiodurans is an extreme radiation-resistant and desiccation-resistant bacterium and possesses three genes encoding endonuclease III enzymes in its genome: DR2438 (EndoIII-1), DR0289 (EndoIII-2) and DR0982 (EndoIII-3). Here, EndoIII-1 and an N-terminally truncated form of EndoIII-3 (EndoIII-3Δ76) have been expressed, purified and crystallized, and preliminary X-ray crystallographic analyses have been performed to 2.15 and 1.31 Å resolution, respectively. The EndoIII-1 crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 181.38, b = 38.56, c = 37.09 Å, β = 89.34° and one molecule per asymmetric unit. The EndoIII-3Δ76 crystals also belonged to the monoclinic space group C2, but with unit-cell parameters a = 91.47, b = 40.53, c = 72.47 Å, β = 102.53° and one molecule per asymmetric unit. The EndoIII-1 structure was determined by molecular replacement, while the truncated EndoIII-3Δ76 structure was determined by single-wavelength anomalous dispersion phasing. Refinement of the structures is in progress.

  1. Spatiotemporal Regulation of a Legionella pneumophila T4SS Substrate by the Metaeffector SidJ

    Science.gov (United States)

    Jeong, Kwang Cheol; Sexton, Jessica A.; Vogel, Joseph P.

    2015-01-01

    Modulation of host cell function is vital for intracellular pathogens to survive and replicate within host cells. Most commonly, these pathogens utilize specialized secretion systems to inject substrates (also called effector proteins) that function as toxins within host cells. Since it would be detrimental for an intracellular pathogen to immediately kill its host cell, it is essential that secreted toxins be inactivated or degraded after they have served their purpose. The pathogen Legionella pneumophila represents an ideal system to study interactions between toxins as it survives within host cells for approximately a day and its Dot/Icm type IVB secretion system (T4SS) injects a vast number of toxins. Previously we reported that the Dot/Icm substrates SidE, SdeA, SdeB, and SdeC (known as the SidE family of effectors) are secreted into host cells, where they localize to the cytoplasmic face of the Legionella containing vacuole (LCV) in the early stages of infection. SidJ, another effector that is unrelated to the SidE family, is also encoded in the sdeC-sdeA locus. Interestingly, while over-expression of SidE family proteins in a wild type Legionella strain has no effect, we found that their over-expression in a ∆sidJ mutant completely inhibits intracellular growth of the strain. In addition, we found expression of SidE proteins is toxic in both yeast and mammalian HEK293 cells, but this toxicity can be suppressed by co-expression of SidJ, suggesting that SidJ may modulate the function of SidE family proteins. Finally, we were able to demonstrate both in vivo and in vitro that SidJ acts on SidE proteins to mediate their disappearance from the LCV, thereby preventing lethal intoxication of host cells. Based on these findings, we propose that SidJ acts as a metaeffector to control the activity of other Legionella effectors. PMID:25774515

  2. Spatiotemporal regulation of a Legionella pneumophila T4SS substrate by the metaeffector SidJ.

    Directory of Open Access Journals (Sweden)

    Kwang Cheol Jeong

    2015-03-01

    Full Text Available Modulation of host cell function is vital for intracellular pathogens to survive and replicate within host cells. Most commonly, these pathogens utilize specialized secretion systems to inject substrates (also called effector proteins that function as toxins within host cells. Since it would be detrimental for an intracellular pathogen to immediately kill its host cell, it is essential that secreted toxins be inactivated or degraded after they have served their purpose. The pathogen Legionella pneumophila represents an ideal system to study interactions between toxins as it survives within host cells for approximately a day and its Dot/Icm type IVB secretion system (T4SS injects a vast number of toxins. Previously we reported that the Dot/Icm substrates SidE, SdeA, SdeB, and SdeC (known as the SidE family of effectors are secreted into host cells, where they localize to the cytoplasmic face of the Legionella containing vacuole (LCV in the early stages of infection. SidJ, another effector that is unrelated to the SidE family, is also encoded in the sdeC-sdeA locus. Interestingly, while over-expression of SidE family proteins in a wild type Legionella strain has no effect, we found that their over-expression in a ∆sidJ mutant completely inhibits intracellular growth of the strain. In addition, we found expression of SidE proteins is toxic in both yeast and mammalian HEK293 cells, but this toxicity can be suppressed by co-expression of SidJ, suggesting that SidJ may modulate the function of SidE family proteins. Finally, we were able to demonstrate both in vivo and in vitro that SidJ acts on SidE proteins to mediate their disappearance from the LCV, thereby preventing lethal intoxication of host cells. Based on these findings, we propose that SidJ acts as a metaeffector to control the activity of other Legionella effectors.

  3. Study on the Interaction of T(4-Mop)PS4 with BSA%T(4-Mop) PS4卟啉与牛血清蛋白相互作用的研究

    Institute of Scientific and Technical Information of China (English)

    张红芬; 郭玉晶; 董川

    2012-01-01

    Using meso-terakis(4-methoxyl-3-sulfonatophenyl) porphyrin(T(4-Mop)PS4 ) as an electrochemical probe, a new electroanalytical method for the determination of bovine serum albumin (BSA) was established based on the interaction of BSA with T(4-Mop)PS4. A good linear relationship was obtained between the variation of peak current and concentration of BSA in the range of 2. 0 x 10-6 -1.0 ×l0-5 mol · L-1. The limit of detection ( LOD) was 1. 2 ×10-6 mol · L-1 and the relative standard deviation( RSD, n = 8 ) for 5. 0 × 10-6 mol · L-1 BSA was 2. 0% . The supramolecular complex of T(4-Mop)PS4 with BSA was studied by three different methods, including spectrophotom-etry, fluorometry and linear sweep voltammetric (LSV) methods. The binding ratio and binding constant of T(4-Mop)PS4 with BSA were obtained. The experimental data demonstrated that T(4-Mop) PS4 could form a 1 : 1 stable supramolecular complex with BSA, and hydrophobic interaction is the main reaction between them.%以四-(4-甲氧基-3-磺酸基苯)卟啉(T(4-Mop)PS4)为探针,通过T(4-Mop) PS4与牛血清白蛋白(BSA)的相互作用,建立了测定BSA的电化学分析方法.T(4-Mop)PS4的峰电流变化(AIp)与BSA在2.0×10-6~ 1.0×10-5mol·L-1范围内呈良好的线性关系,检出限为1.2×10-6mol· L-1;对5.0×10-6mol·L-1BSA平行测定8次,其相对标准偏差为2.0%,回收率为95%~104%.组氨酸、缬氨酸、苯丙氨酸、丝氨酸、异白氨酸、谷氨酰胺、苏氨酸等氨基酸对BSA的测定不产生干扰.采用紫外可见光度法、荧光光谱法和线性扫描伏安法( LSV)研究了T(4-Mop) PS4与BSA之间的相互作用,并测定了二者相互作用的结合常数和结合比.研究表明,T(4-Mop)PS4与BSA之间主要以疏水作用力结合,形成了1:1的稳定复合物.

  4. STUDIES ON THE BACTERIOPHAGE OF D'HERELLE : IV. CONCERNING THE ONENESS OF THE BACTERIOPHAGE.

    Science.gov (United States)

    Bronfenbrenner, J J; Korb, C

    1925-11-30

    Lytic filtrates, active against Bacillus dysenterioe Shiga, Bacillus coli, Bacillus pestis cavioe, and staphylococcus respectively, proved to be differently affected by changes in hydrogen ion concentration. Anti-staphylococcus lysin was the least resistant of the four, showing deterioration in 3 hours at 7 degrees C. beyond the zone of hydrogen ion concentration limited by C(H) = 6.3 x 10(-5) and C(H) = 1.6 x 10(-9). Under the same conditions, the zone of resistance of anti-coli filtrate lay between C(H) = 2.7 x 10(-3) and C(H) = 2.5 x 10(-11), and that of anti-Shiga between C(H) = 1-7 x 10(-4) and C(H) = 1-3 x 10(-11). Anti-pestis cavioe filtrate was most resistant of the four, retaining its full activity in the zone from C(H) = 1 x 10(-3) to C(H) = 3.5 x 10(-12). The fact that these differences in individual resistance persisted, notwithstanding the repeated passage of lytic filtrates through cultures of bacteria other than those against which they were primarily active, seems to offer evidence in favor of a multiplicity of bacteriophages.

  5. Insights into Bacteriophage Application in Controlling Vibrio Species.

    Science.gov (United States)

    Letchumanan, Vengadesh; Chan, Kok-Gan; Pusparajah, Priyia; Saokaew, Surasak; Duangjai, Acharaporn; Goh, Bey-Hing; Ab Mutalib, Nurul-Syakima; Lee, Learn-Han

    2016-01-01

    Bacterial infections from various organisms including Vibrio sp. pose a serious hazard to humans in many forms from clinical infection to affecting the yield of agriculture and aquaculture via infection of livestock. Vibrio sp. is one of the main foodborne pathogens causing human infection and is also a common cause of losses in the aquaculture industry. Prophylactic and therapeutic usage of antibiotics has become the mainstay of managing this problem, however, this in turn led to the emergence of multidrug resistant strains of bacteria in the environment; which has raised awareness of the critical need for alternative non-antibiotic based methods of preventing and treating bacterial infections. Bacteriophages - viruses that infect and result in the death of bacteria - are currently of great interest as a highly viable alternative to antibiotics. This article provides an insight into bacteriophage application in controlling Vibrio species as well underlining the advantages and drawbacks of phage therapy. PMID:27486446

  6. Bacteriophages as Weapons Against Bacterial Biofilms in the Food Industry.

    Science.gov (United States)

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

    2016-01-01

    Microbiological contamination in the food industry is often attributed to the presence of biofilms in processing plants. Bacterial biofilms are complex communities of bacteria attached to a surface and surrounded by an extracellular polymeric material. Their extreme resistance to cleaning and disinfecting processes is related to a unique organization, which implies a differential bacterial growth and gene expression inside the biofilm. The impact of biofilms on health, and the economic consequences, has promoted the development of different approaches to control or remove biofilm formation. Recently, successful results in phage therapy have boosted new research in bacteriophages and phage lytic proteins for biofilm eradication. In this regard, this review examines the environmental factors that determine biofilm development in food-processing equipment. In addition, future perspectives for the use of bacteriophage-derived tools as disinfectants are discussed. PMID:27375566

  7. Bacteriophages as Weapons Against Bacterial Biofilms in the Food Industry.

    Science.gov (United States)

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

    2016-01-01

    Microbiological contamination in the food industry is often attributed to the presence of biofilms in processing plants. Bacterial biofilms are complex communities of bacteria attached to a surface and surrounded by an extracellular polymeric material. Their extreme resistance to cleaning and disinfecting processes is related to a unique organization, which implies a differential bacterial growth and gene expression inside the biofilm. The impact of biofilms on health, and the economic consequences, has promoted the development of different approaches to control or remove biofilm formation. Recently, successful results in phage therapy have boosted new research in bacteriophages and phage lytic proteins for biofilm eradication. In this regard, this review examines the environmental factors that determine biofilm development in food-processing equipment. In addition, future perspectives for the use of bacteriophage-derived tools as disinfectants are discussed.

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

    Directory of Open Access Journals (Sweden)

    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.

  9. Bacteriophages and their implications on future biotechnology: a review

    Directory of Open Access Journals (Sweden)

    Haq Irshad

    2012-01-01

    Full Text Available Abstract Recently it has been recognized that bacteriophages, the natural predators of bacteria can be used efficiently in modern biotechnology. They have been proposed as alternatives to antibiotics for many antibiotic resistant bacterial strains. Phages can be used as biocontrol agents in agriculture and petroleum industry. Moreover phages are used as vehicles for vaccines both DNA and protein, for the detection of pathogenic bacterial strain, as display system for many proteins and antibodies. Bacteriophages are diverse group of viruses which are easily manipulated and therefore they have potential uses in biotechnology, research, and therapeutics. The aim of this review article is to enable the wide range of researchers, scientists, and biotechnologist who are putting phages into practice, to accelerate the progress and development in the field of biotechnology.

  10. BACTERIOPHAGE ENDOLYSINS AND THEIR USE IN BIOTECHNOLOGICAL PROCESSES

    Directory of Open Access Journals (Sweden)

    Lenka Tišáková

    2014-02-01

    Full Text Available Bacteriophage endolysins are peptidoglycan hydrolases, produced in the lytic system of bacteriophage in order to lyse host peptidoglycan from within and release virions into the environment. Phages infecting Gram-positive bacteria express endolysin genes with the characteristic modular structure, consisting of at least two functional domains: N-terminal enzymatically active domain (EAD and C-terminal cell wall binding domain (CBD. CBDs specifically recognize ligands and bind to the bacterial cell wall, whereas EAD catalyze lysis of the peptidoglycan bonds. The reveal of endolysin modular structure leads to new opportunities for domain swapping, construction of chimeras and production of specifically engineered recombinant endolysins and their functional domains with the diverse biotechnological applications from without, such as in detection, elimination and biocontrol of pathogens, or as anti-bacterials in experimental therapy.

  11. Bacteriophage exclusion, a new defense system

    Science.gov (United States)

    Barrangou, Rodolphe; van der Oost, John

    2015-01-01

    The ability to withstand viral predation is critical for survival of most microbes. Accordingly, a plethora of phage resistance systems has been identified in bacterial genomes (Labrie et al, 2010), including restriction-modification systems (R-M) (Tock & Dryden, 2005), abortive infection (Abi) (Chopin et al, 2005), Argonaute-based interference (Swarts et al, 2014), as well as clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein (Cas) adaptive immune system (CRISPR-Cas) (Barrangou & Marraffini, 2014; Van der Oost et al, 2014). Predictably, the dark matter of bacterial genomes contains a wealth of genetic gold. A study published in this issue of The EMBO Journal by Goldfarb et al (2015) unveils bacteriophage exclusion (BREX) as a novel, widespread bacteriophage resistance system that provides innate immunity against virulent and temperate phage in bacteria. PMID:25502457

  12. The nucleotide sequence of the bacteriophage T5 ltf gene.

    Science.gov (United States)

    Kaliman, A V; Kulshin, V E; Shlyapnikov, M G; Ksenzenko, V N; Kryukov, V M

    1995-06-01

    The nucleotide sequence of the bacteriophage T5 Bg/II-BamHI fragment (4,835 bp in length) known to carry a gene encoding the LTF protein which forms the phage L-shaped tail fibers was determined. It was shown to contain an open reading frame for 1,396 amino acid residues that corresponds to a protein of 147.8 kDa. The coding region of ltf gene is preceded by a typical Shine-Dalgarno sequence. Downstream from the ltf gene there is a strong transcription terminator. Data bank analysis of the LTF protein sequence reveals 55.1% identity to the hypothetical protein ORF 401 of bacteriophage lambda in a segment of 118 amino acids overlap. PMID:7789514

  13. A method for the detection of bacteriophages from ocean water

    Science.gov (United States)

    Moebus, K.

    1980-03-01

    A method for the isolation of bacteriophages from ocean water is described. It precludes sample storage before starting phage-enrichment cultures and provides for the use of 3 sub-samples enriched with organic nutrients after 1, 2 and 3 days of incubation. The method was used with samples collected from 6 m below the surface at 48 stations between the European continental shelf and the Sargasso Sea. With 213 among 931 bacterial isolates about 250 strains of bacteriophages were detected by two methods of different sensitivity. From 14 samples taken east of the Azores 115 host bacteria have been found versus only 98 from 34 samples collected at westerly stations. The employment of more than one sub-sample per station as well as the use of more sensitive phage-detection procedures was found to be more advantageous the lower the concentration of cultivatable bacteria in a sample.

  14. Insights into bacteriophage application in controlling Vibrio species

    Directory of Open Access Journals (Sweden)

    Vengadesh Letchumanan

    2016-07-01

    Full Text Available Bacterial infections from various organisms including Vibrio sp. pose a serious hazard to humans in many forms from clinical infection to affecting the yield of agriculture and aquaculture via infection of livestock. Vibrio sp. is one of the main foodborne pathogens causing human infection and is also a common cause of losses in the aquaculture industry. Prophylactic and therapeutic usage of antibiotics has become the mainstay of managing this problem, however this in turn led to the emergence of multidrug resistant strains of bacteria in the environment; which has raised awareness of the critical need for alternative non antibiotic based methods of preventing and treating bacterial infections. Bacteriophages - viruses that infect and result in the death of bacteria – are currently of great interest as a highly viable alternative to antibiotics. This article provides an insight into bacteriophage application in controlling Vibrio species as well underlining the advantages and drawbacks of phage therapy.

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

    Science.gov (United States)

    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.

  16. Sequence and comparative analysis of Leuconostoc dairy bacteriophages.

    Science.gov (United States)

    Kot, Witold; Hansen, Lars H; Neve, Horst; Hammer, Karin; Jacobsen, Susanne; Pedersen, Per D; Sørensen, Søren J; Heller, Knut J; Vogensen, Finn K

    2014-04-17

    Bacteriophages attacking Leuconostoc species may significantly influence the quality of the final product. There is however limited knowledge of this group of phages in the literature. We have determined the complete genome sequences of nine Leuconostoc bacteriophages virulent to either Leuconostoc mesenteroides or Leuconostoc pseudomesenteroides strains. The phages have dsDNA genomes with sizes ranging from 25.7 to 28.4 kb. Comparative genomics analysis helped classify the 9 phages into two classes, which correlates with the host species. High percentage of similarity within the classes on both nucleotide and protein levels was observed. Genome comparison also revealed very high conservation of the overall genomic organization between the classes. The genes were organized in functional modules responsible for replication, packaging, head and tail morphogenesis, cell lysis and regulation and modification, respectively. No lysogeny modules were detected. To our knowledge this report provides the first comparative genomic work done on Leuconostoc dairy phages.

  17. Insights into Bacteriophage Application in Controlling Vibrio Species

    Science.gov (United States)

    Letchumanan, Vengadesh; Chan, Kok-Gan; Pusparajah, Priyia; Saokaew, Surasak; Duangjai, Acharaporn; Goh, Bey-Hing; Ab Mutalib, Nurul-Syakima; Lee, Learn-Han

    2016-01-01

    Bacterial infections from various organisms including Vibrio sp. pose a serious hazard to humans in many forms from clinical infection to affecting the yield of agriculture and aquaculture via infection of livestock. Vibrio sp. is one of the main foodborne pathogens causing human infection and is also a common cause of losses in the aquaculture industry. Prophylactic and therapeutic usage of antibiotics has become the mainstay of managing this problem, however, this in turn led to the emergence of multidrug resistant strains of bacteria in the environment; which has raised awareness of the critical need for alternative non-antibiotic based methods of preventing and treating bacterial infections. Bacteriophages – viruses that infect and result in the death of bacteria – are currently of great interest as a highly viable alternative to antibiotics. This article provides an insight into bacteriophage application in controlling Vibrio species as well underlining the advantages and drawbacks of phage therapy. PMID:27486446

  18. Choline-containing bacteriophage receptors in Streptococcus pneumoniae.

    OpenAIRE

    Lopez, R. (Rafael); Garcia, E.; Garcia, P.; Ronda, C; Tomasz, A.

    1982-01-01

    Choline-containing teichoic acid seems to be essential for the adsorption of bacteriophage Dp-1 to pneumococci. This conclusion is based on the following observations: In contrast to pneumococci grown in choline-containing medium, cells grown in medium containing ethanolamine or other submethylated aminoalcohols instead of choline were found to be resistant to infection by Dp-1. Live choline-grown bacteria and heat- or UV-inactivated cells and purified cell walls prepared from these cells wer...

  19. Bacteriophages : an underestimated role in human and animal health ?

    OpenAIRE

    Marianne eDe Paepe; Marion eLeclerc; Tinsley, Colin R.; Marie-Agnès ePetit

    2014-01-01

    Metagenomic approaches applied to viruses have highlighted their prevalence in almost all microbial ecosystems investigated. In all ecosystems, notably those associated with humans or animals, the viral fraction is dominated by bacteriophages. Whether they contribute to dysbiosis, i.e. the departure from microbiota composition in symbiosis at equilibrium and entry into a state favoring human or animal disease is unknown at present. This review summarizes what has been learnt on phages associa...

  20. Isolation of Actinomyces bacteriophage from human dental plaque.

    OpenAIRE

    Tylenda, C A; Calvert, C.; Kolenbrander, P. E.; Tylenda, A

    1985-01-01

    Human dental plaque samples were screened for the presence of bacteriophage for Actinomyces viscosus and Streptococcus sanguis. None of the 336 samples yielded phage for S. sanguis, but 10 contained virulent actinomyces phage. A high host cell specificity was observed in that one phage isolate infected only A. viscosus T14V, eight phage isolates infected only A. viscosus MG-1, and one infected both strains. None was capable of productively infecting various other actinomyces strains that repr...

  1. Genetic analysis of bacteriophages from clinical and environmental samples

    OpenAIRE

    Knapik, Kamila Z.

    2013-01-01

    Bacteriophages, viruses infecting bacteria, are uniformly present in any location where there are high numbers of bacteria, both in the external environment and the human body. Knowledge of their diversity is limited by the difficulty to culture the host species and by the lack of the universal marker gene present in all viruses. Metagenomics is a powerful tool that can be used to analyse viral communities in their natural environments. The aim of this study was to investigate diverse populat...

  2. Identification of mutations conferring 5-azacytidine resistance in bacteriophage

    OpenAIRE

    Arribas, María; Cabanillas, Laura; Lázaro, Ester

    2011-01-01

    RNA virus replication takes place at a very high error rate, and additional increases in this parameter can produce the extinction of virus infectivity. Nevertheless, RNA viruses can adapt to conditions of increased mutagenesis, which demonstrates that selection of beneficial mutations is also possible at higher-than-standard error rates. In this study we have analysed the evolutionary behaviour of bacteriophage Qβ populations when replication proceeds in the presence of the mutagenic nucleos...

  3. P. fluorescens biofilm control using bacteriophage ΦS1

    OpenAIRE

    Sillankorva, Sanna; Oliveira, Rosário; Vieira, M. J.; Sutherland, Ian W.; Azeredo, Joana

    2004-01-01

    Pseudomonas fluorescens biofilms contribute to the spoilage of dairy industry products due to the proteolytic activity of some Pseudomonas fluorescens strains. The eradication of these biofilms is difficult using the traditional chemical biocides due to the low removal action of these agents. Additionally chemical control leaves inactivated cells attached to the surface that tends to provide an ideal environment for further bacterial adhesion and growth. Bacteriophages can be seen as good alt...

  4. Ecology of Anti-Biofilm Agents I: Antibiotics versus Bacteriophages

    OpenAIRE

    Abedon, Stephen T.

    2015-01-01

    Bacteriophages, the viruses that infect bacteria, have for decades been successfully used to combat antibiotic-resistant, chronic bacterial infections, many of which are likely biofilm associated. Antibiotics as anti-biofilm agents can, by contrast, be inefficacious against even genetically sensitive targets. Such deficiencies in usefulness may result from antibiotics, as naturally occurring compounds, not serving their producers, in nature, as stand-alone disruptors of mature biofilms. Anti-...

  5. Bacteriophages infecting Bacteroides as a marker for microbial source tracking.

    Science.gov (United States)

    Jofre, Joan; Blanch, Anicet R; Lucena, Francisco; Muniesa, Maite

    2014-05-15

    Bacteriophages infecting certain strains of Bacteroides are amid the numerous procedures proposed for tracking the source of faecal pollution. These bacteriophages fulfil reasonably well most of the requirements identified as appropriate for a suitable marker of faecal sources. Thus, different host strains are available that detect bacteriophages preferably in water contaminated with faecal wastes corresponding to different animal species. For phages found preferably in human faecal wastes, which are the ones that have been more extensively studied, the amounts of phages found in waters contaminated with human fecal samples is reasonably high; these amounts are invariable through the time; their resistance to natural and anthropogenic stressors is comparable to that of other relatively resistant indicator of faecal pollution such us coliphages; the abundance ratios of somatic coliphages and bacteriophages infecting Bacteroides thetaiotaomicron GA17 are unvarying in recent and aged contamination; and standardised detection methods exist. These methods are easy, cost effective and provide data susceptible of numerical analysis. In contrast, there are some uncertainties regarding their geographical stability, and consequently suitable hosts need to be isolated for different geographical areas. However, a feasible method has been described to isolate suitable hosts in a given geographical area. In summary, phages infecting Bacteroides are a marker of faecal sources that in our opinion merits being included in the "toolbox" for microbial source tracking. However, further research is still needed in order to make clear some uncertainties regarding some of their characteristics and behaviour, to compare their suitability to the one of emerging methods such us targeting Bacteroidetes by qPCR assays; or settling molecular methods for their determination.

  6. Standardization of neonatal T4 by RIA using blood droplets on filter paper for the detection of neonatal hypothyroidism

    International Nuclear Information System (INIS)

    The aim of this work was to assist the national programme for the prevention of endemic goitre by devising a reliable low-cost technique for the detection of neonatal hypothyroidism. RIA determinations of T4 using bulk reagents were standardized by the second antibody method. 4 refs, 4 figs

  7. Preoperative FDG-PET/CT Is an Important Tool in the Management of Patients with Thick (T4) Melanoma.

    Science.gov (United States)

    Arrangoiz, Rodrigo; Papavasiliou, Pavlos; Stransky, Carrie A; Yu, Jian Q; Tianyu, Li; Sigurdson, Elin R; Berger, Adam C; Farma, Jeffrey M

    2012-01-01

    The yield of preoperative PET/CT (PET/CT) for regional and distant metastases for thin/intermediate thickness melanoma is low. Objective of this study is to determine if PET/CT performed for T4 melanomas helps guide management and alter treatment plans. Methods. Retrospective cohort of 216 patients with T4 melanomas treated at two tertiary institutions. Fifty-six patients met our inclusion criteria (T4 lesion, PET/CT and no clinical evidence of metastatic disease). Results. Fifty-six patients (M: 32, F: 24) with median tumor thickness of 6 mm were identified. PET/CT recognized twelve with regional and four patients with metastatic disease. Melanoma-related treatment plan was altered in 11% of the cases based on PET/CT findings. PET/CT was negative 60% of the time, in 35% of the cases; it identified incidental findings that required further evaluation. Conclusion. Patients with T4 lesions, PET/CT changed the treatment plan 18% of the time. Regional findings changed the surgical treatment plan in 11% and the adjuvant plan in 7% of our cases due to the finding of metastatic disease. Additionally 20 patients had incidental findings that required further workup. In this subset of patients, we feel there is a benefit to PET/CT, and further studies should be performed to validate our findings.

  8. Preoperative FDG-PET/CT Is an Important Tool in the Management of Patients with Thick (T4 Melanoma

    Directory of Open Access Journals (Sweden)

    Rodrigo Arrangoiz

    2012-01-01

    Full Text Available The yield of preoperative PET/CT (PET/CT for regional and distant metastases for thin/intermediate thickness melanoma is low. Objective of this study is to determine if PET/CT performed for T4 melanomas helps guide management and alter treatment plans. Methods. Retrospective cohort of 216 patients with T4 melanomas treated at two tertiary institutions. Fifty-six patients met our inclusion criteria (T4 lesion, PET/CT and no clinical evidence of metastatic disease. Results. Fifty-six patients (M: 32, F: 24 with median tumor thickness of 6 mm were identified. PET/CT recognized twelve with regional and four patients with metastatic disease. Melanoma-related treatment plan was altered in 11% of the cases based on PET/CT findings. PET/CT was negative 60% of the time, in 35% of the cases; it identified incidental findings that required further evaluation. Conclusion. Patients with T4 lesions, PET/CT changed the treatment plan 18% of the time. Regional findings changed the surgical treatment plan in 11% and the adjuvant plan in 7% of our cases due to the finding of metastatic disease. Additionally 20 patients had incidental findings that required further workup. In this subset of patients, we feel there is a benefit to PET/CT, and further studies should be performed to validate our findings.

  9. Nestin expression associates with poor prognosis and triple negative phenotype in locally advanced (T4 breast cancer

    Directory of Open Access Journals (Sweden)

    F. Piras

    2011-11-01

    Full Text Available Nestin, an intermediate filament protein, has traditionally been noted for its importance as a neural stem cell marker. However, in recent years, expression of nestin has shown to be associated with general proliferation of progenitor cell populations within neoplasms. There is no reported study addressing nestin expression in T4 breast cancer patients. Thus, the aim of the present study was to investigate, through immunohistochemistry, the expression and distribution of nestin in T4 breast cancer, in order to determine its association with clinical and pathological parameters as well as with patients’ outcome. Nestin was detectable in tumoral cells and in endothelial cells of blood microvessels, and it is significantly expressed in triple-negative and in inflammatory breast cancer (IBC subgroups of T4 breast tumours. The Kaplan-Meier analysis showed that the presence of nestin in tumoral cells significantly predicted poor prognosis at 5-years survival (P=0.02 and with borderline significance at 10-years of survival (P=0.05 in T4 breast cancer patients. On the basis of these observations, we speculate that nestin expression may characterize tumours with an aggressive clinical behavior, suggesting that the presence of nestin in tumoral cells and vessels may be considered an important factor that leads to a poor prognosis. Further studies are awaited to define the biological role of nestin in the etiology of these subgroups of breast cancers.

  10. Evaluation of an indigenous magnetic rack using prolactin IRMA and thyroxine (T4) RIA as model assays

    International Nuclear Information System (INIS)

    The performance quality feature of an indigenous magnetic rack has been evaluated using prolactin IRMA kit of BRIT and T4 RIA kit of RMC as model assays. Similar data obtained while using an imported commercial magnetic rack for the same assays are also reported. Our results confirmed the satisfactory quality and utility of the indigenous rack fabricated. (author)

  11. THYROXINE (T4) CATABOLISM IN HUMAN AND RAT HEPATOCYTES INCREASES FOLLOWING EXPOSURE TO PROTOTYPICAL HEPATIC ENZYME INDUCERS

    Science.gov (United States)

    Nuclear receptor agonists such as phenobarbital (PB), 3-methylcholantrene (3MC), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153), and, pregnenolone-16a-carbonitrile (PCN) decrease serum thyroxine (T4) concentrations in rats. This decrease is thought to occur through the induction of ...

  12. HAZ development and accelerated post-weld natural ageing in ultrasonic spot welding aluminium 6111-T4 automotive sheet

    International Nuclear Information System (INIS)

    By careful hardness measurements, supported by electron microscopy, it has been shown that there is a clearly observable heat-affected zone (HAZ) when ultrasonic spot welding (USW) aluminium automotive alloys like AA6111-T4, the severity of which depends on the welding energy. In contrast, it has been previously reported that because of the low-energy input with USW no HAZ can be detected. Immediately after welding, softening is seen in the weld zone relative to the normal T4 starting condition. However, this is rapidly recovered by natural ageing, which masks the presence of a HAZ, and the weld strength over long natural ageing times significantly exceeds that of the parent T4 material. This behaviour is caused by dissolution of the solute clusters and Guinier–Preston zones present in the T4 sheet due to the high weld temperatures, which were recorded to reach over 400 °C at the weld interface, followed by accelerated post-weld natural ageing. Interestingly, the weld zone was found to naturally age faster, and to a more advanced state, than the parent material. Modelling has been used to demonstrate that a large excess vacancy concentration can potentially be generated by the high-strain-rate dynamic deformation in USW, which can accelerate the natural ageing response seen in the weld.

  13. Cryptic t(4;11) encoding MLL-AF4 due to insertion of 5' MLL sequences in chromosome 4

    NARCIS (Netherlands)

    von Bergh, A; Gargallo, P; De Prijck, B; Vranckx, H; Marschalek, R; Larripa, I; Kluin, P; Schuuring, E; Hagemeijer, A

    2001-01-01

    The t(4;11) translocation is the cytogenetic hallmark of a subset of acute lymphoblastic leukemias characterized by pro-B immunophenotype and a dismal prognosis. This translocation fuses the MLL gene on chromosome band 11q23 and the AF4 gene on 4q21, resulting in the expression of fusion transcripts

  14. Determinants of prognosis in breast cancer patients with tumor involvement of the skin (pT4b).

    NARCIS (Netherlands)

    Wieland, A.W.; Louwman, M.W.; Voogd, A.C.; Beek, M.W. van; Vreugdenhil, G.R.; Roumen, R.M.H.

    2004-01-01

    Determinants of prognosis were studied in patients with breast cancer with histologically proven tumor extension to the skin without clinical evidence of distant metastases (i.e., pT4b N0-3 M0). Data were collected retrospectively on 77 consecutive patients diagnosed in one community teaching hospit

  15. Isolation of Lactic Acid Bacteria Bacteriophages from Dairy Products

    Directory of Open Access Journals (Sweden)

    Elnaz Shokrani

    2013-09-01

    Full Text Available Backgrounds: Lactococcus lactis (L. lactis is one of the most important microorganisms used in dairy industry for production of fermented milk products. Bacteriophages which attack  L. lactis are a serious threat to the dairy industry because of their negative effects on fermentation processes. Methods: Samples of raw milk were examined for the presence of lactococcal bacteriophages. Samples were centrifuged and then filtered through 0.45µm pore size filters. The filtrates were added to early-exponential cultures of Lactococcus lactis subspp. Lactis (PTCC 1336. Overlay method was used to detect the formation of plaques. After isolation and concentration of phages, serial dilutions of phage stock were used to determine titer of phage in concentrated sample. Electron Microscopy was used for observation and characterization of structural details of bacteriophages. Results: Two phages were isolated; one of them had a hexagonal head of 45×30 nm in diameter and a flexible non-contractile tail of 70nm long which belonged to Siphoviridae. The other had a short tail and a hexagonal head of 53×60 nm in diameter which was a member of Podoviridae family. Conclusion: In this study, for the first time, two phages were isolated from milk. This does not reduce the significance of phage control in different stages of the production. The spread of the phages in the production plant can be very harmful.

  16. Temperate bacteriophages collected by outer membrane vesicles in Komagataeibacter intermedius.

    Science.gov (United States)

    Kharina, Alla; Podolich, Olga; Faidiuk, Iuliia; Zaika, Sergiy; Haidak, Andriy; Kukharenko, Olga; Zaets, Iryna; Tovkach, Fedor; Reva, Oleg; Kremenskoy, Maxim; Kozyrovska, Natalia

    2015-04-01

    The acetic acid bacteria have mainly relevance for bacterial cellulose production and fermented bio-products manufacture. The purpose of this study was to identify temperate bacteriophages in a cellulose-producing bacterial strain Komagataeibacter intermedius IMBG180. Prophages from K. intermedius IMBG180 were induced with mitomycin C and nalidixic acid. Transmission electron microscopy analysis exhibited tailed bacteriophages belonging to Myoviridae. A PCR assay targeting the capsid gene of the myoviruses proved phylogenetic position of induced phages. Nalidixic acid was poor inducer of prophages, however, it induced the OMV-like particles release. Size of OMVs depended on an antibiotic applied for phage induction and varied in the range of 30-80 and 120-200 nm. Inside some of them, tails of phages have been visible. Under conditions, inducing prophages, OMVs acted as the collectors of formed phage particles, using outer membrane receptors for phage detection (in this case, outer membrane siderophore receptor), and fulfilled therefore "a cleaning," as well as defensive functions, preventing bacteriophage spread outside population. This is the first description of myoviruses affiliated to K. intermedius, as well as outer membrane vesicles interaction with phages within this host.

  17. Bacteriophages and medical oncology: targeted gene therapy of cancer.

    Science.gov (United States)

    Bakhshinejad, Babak; Karimi, Marzieh; Sadeghizadeh, Majid

    2014-08-01

    Targeted gene therapy of cancer is of paramount importance in medical oncology. Bacteriophages, viruses that specifically infect bacterial cells, offer a variety of potential applications in biomedicine. Their genetic flexibility to go under a variety of surface modifications serves as a basis for phage display methodology. These surface manipulations allow bacteriophages to be exploited for targeted delivery of therapeutic genes. Moreover, the excellent safety profile of these viruses paves the way for their potential use as cancer gene therapy platforms. The merge of phage display and combinatorial technology has led to the emergence of phage libraries turning phage display into a high throughput technology. Random peptide libraries, as one of the most frequently used phage libraries, provide a rich source of clinically useful peptide ligands. Peptides are known as a promising category of pharmaceutical agents in medical oncology that present advantages such as inexpensive synthesis, efficient tissue penetration and the lack of immunogenicity. Phage peptide libraries can be screened, through biopanning, against various targets including cancer cells and tissues that results in obtaining cancer-homing ligands. Cancer-specific peptides isolated from phage libraries show huge promise to be utilized for targeting of various gene therapy vectors towards malignant cells. Beyond doubt, bacteriophages will play a more impressive role in the future of medical oncology.

  18. Bacteriophages and medical oncology: targeted gene therapy of cancer.

    Science.gov (United States)

    Bakhshinejad, Babak; Karimi, Marzieh; Sadeghizadeh, Majid

    2014-08-01

    Targeted gene therapy of cancer is of paramount importance in medical oncology. Bacteriophages, viruses that specifically infect bacterial cells, offer a variety of potential applications in biomedicine. Their genetic flexibility to go under a variety of surface modifications serves as a basis for phage display methodology. These surface manipulations allow bacteriophages to be exploited for targeted delivery of therapeutic genes. Moreover, the excellent safety profile of these viruses paves the way for their potential use as cancer gene therapy platforms. The merge of phage display and combinatorial technology has led to the emergence of phage libraries turning phage display into a high throughput technology. Random peptide libraries, as one of the most frequently used phage libraries, provide a rich source of clinically useful peptide ligands. Peptides are known as a promising category of pharmaceutical agents in medical oncology that present advantages such as inexpensive synthesis, efficient tissue penetration and the lack of immunogenicity. Phage peptide libraries can be screened, through biopanning, against various targets including cancer cells and tissues that results in obtaining cancer-homing ligands. Cancer-specific peptides isolated from phage libraries show huge promise to be utilized for targeting of various gene therapy vectors towards malignant cells. Beyond doubt, bacteriophages will play a more impressive role in the future of medical oncology. PMID:25012686

  19. MetaPhinder—Identifying Bacteriophage Sequences in Metagenomic Data Sets

    Science.gov (United States)

    Villarroel, Julia; Lund, Ole; Voldby Larsen, Mette; Nielsen, Morten

    2016-01-01

    Bacteriophages are the most abundant biological entity on the planet, but at the same time do not account for much of the genetic material isolated from most environments due to their small genome sizes. They also show great genetic diversity and mosaic genomes making it challenging to analyze and understand them. Here we present MetaPhinder, a method to identify assembled genomic fragments (i.e.contigs) of phage origin in metagenomic data sets. The method is based on a comparison to a database of whole genome bacteriophage sequences, integrating hits to multiple genomes to accomodate for the mosaic genome structure of many bacteriophages. The method is demonstrated to out-perform both BLAST methods based on single hits and methods based on k-mer comparisons. MetaPhinder is available as a web service at the Center for Genomic Epidemiology https://cge.cbs.dtu.dk/services/MetaPhinder/, while the source code can be downloaded from https://bitbucket.org/genomicepidemiology/metaphinder or https://github.com/vanessajurtz/MetaPhinder. PMID:27684958

  20. Alternatives to antibiotics: utilization of bacteriophage to treat colibacillosis and prevent foodborne pathogens.

    Science.gov (United States)

    Huff, W E; Huff, G R; Rath, N C; Balog, J M; Donoghue, A M

    2005-04-01

    Bacteriophages are viruses that infect and kill bacteria. Bacteriophage do not infect animal and plant cells, which makes them a potentially safe alternative to antibiotics. We have been conducting research on the efficacy of bacteriophage to prevent and treat colibacillosis in poultry. Bacteriophages that were lytic to a non-motile, serotype 02 isolate of Escherichia coli were isolated from municipal wastewater treatment plants and poultry processing plants. This E. coli isolate is pathogenic to poultry, causing severe respiratory and systemic infections. Two bacteriophage isolates were selected for use in studies designed to determine the efficacy of these bacteriophage to prevent and treat severe colibacillosis in poultry. Colibacillosis was induced by injecting 6 x 10(4) cfu of E. coli into the thoracic air sac when birds were 1 wk of age. Initial studies demonstrated that mortality was significantly reduced from 85 to 35% when the challenge culture was mixed with equal titers of bacteriophage, and the birds were completely protected when the challenge culture was mixed with 10 pfu of bacteriophage. In subsequent studies, we have shown that an aerosol spray of bacteriophage given to birds prior to this E. coli challenge could significantly reduce mortality even when given 3 d prior to the E. coli challenge. Our research on treating colibacillosis in poultry has demonstrated that an intramuscular injection of bacteriophage given 24 or 48 h after the birds were challenged rescued the birds from this severe E. coli infection. We have demonstrated that bacteriophage can be used to prevent and treat colibacillosis in poultry and may provide an effective alternative to antibiotic use in animal production. PMID:15844825

  1. Chromosomal aberrations induced by the restriction endonucleases Alu I and Bam HI: comparison with X-rays

    International Nuclear Information System (INIS)

    Dose-effect relationships for the frequencies of polycentric chromosomes induced by the restriction endonucleases Alu I and Bam HI and by X-rays in Chinese hamster ovary (CHO) cells were analyzed and compared. 1 Gy of X-rays produce the same frequency of polycentric chromosomes as 2 units Alu I and 7.9 units Bam HI. (author)

  2. Crystal Structure of the Homing Endonuclease I-CvuI Provides a New Template for Genome Modification

    DEFF Research Database (Denmark)

    Molina, Rafael; Redondo, Pilar; López-Méndez, Blanca;

    2015-01-01

    Homing endonucleases recognize and generate a DNA double-strand break, which has been used to promote gene targeting. These enzymes recognize long DNA stretches; they are highly sequence-specific enzymes and display a very low frequency of cleavage even in complete genomes. Although a large numbe...

  3. Ultraviolet-endonuclease activity in cell extracts of Saccharomyces cerevisiae mutants defective in excision of pyrimidine dimers

    International Nuclear Information System (INIS)

    Cell-free extracts of ultraviolet-sensitive mutants of Saccharomyces cerevisiae defective in excision of pyrimidine dimers, rad1, rad2, rad3, rad4, rad10, and rad16, as well as the extracts of the wild-type strain RAD+, display ultraviolet-endonuclease activity

  4. A newly discovered Bordetella species carries a transcriptionally active CRISPR-Cas with a small Cas9 endonuclease

    Science.gov (United States)

    The Cas9 endonuclease of the Type II-a clustered regularly interspersed short palindromic repeats (CRISPR), of Streptococcus pyogenes (SpCas9) has been adapted as a widely used tool for genome editing and genome engineering. Herein, we describe a gene encoding a novel Cas9 ortholog (BpsuCas9) and th...

  5. Lucanthone and its derivative hycanthone inhibit apurinic endonuclease-1 (APE1 by direct protein binding.

    Directory of Open Access Journals (Sweden)

    Mamta D Naidu

    Full Text Available Lucanthone and hycanthone are thioxanthenone DNA intercalators used in the 1980s as antitumor agents. Lucanthone is in Phase I clinical trial, whereas hycanthone was pulled out of Phase II trials. Their potential mechanism of action includes DNA intercalation, inhibition of nucleic acid biosyntheses, and inhibition of enzymes like topoisomerases and the dual function base excision repair enzyme apurinic endonuclease 1 (APE1. Lucanthone inhibits the endonuclease activity of APE1, without affecting its redox activity. Our goal was to decipher the precise mechanism of APE1 inhibition as a prerequisite towards development of improved therapeutics that can counteract higher APE1 activity often seen in tumors. The IC(50 values for inhibition of APE1 incision of depurinated plasmid DNA by lucanthone and hycanthone were 5 µM and 80 nM, respectively. The K(D values (affinity constants for APE1, as determined by BIACORE binding studies, were 89 nM for lucanthone/10 nM for hycanthone. APE1 structures reveal a hydrophobic pocket where hydrophobic small molecules like thioxanthenones can bind, and our modeling studies confirmed such docking. Circular dichroism spectra uncovered change in the helical structure of APE1 in the presence of lucanthone/hycanthone, and notably, this effect was decreased (Phe266Ala or Phe266Cys or Trp280Leu or abolished (Phe266Ala/Trp280Ala when hydrophobic site mutants were employed. Reduced inhibition by lucanthone of the diminished endonuclease activity of hydrophobic mutant proteins (as compared to wild type APE1 supports that binding of lucanthone to the hydrophobic pocket dictates APE1 inhibition. The DNA binding capacity of APE1 was marginally inhibited by lucanthone, and not at all by hycanthone, supporting our hypothesis that thioxanthenones inhibit APE1, predominantly, by direct interaction. Finally, lucanthone-induced degradation was drastically reduced in the presence of short and long lived free radical scavengers, e

  6. Pasteurella haemolytica bacteriophage: identification, partial characterization, and relationship of temperate bacteriophages from isolates of Pasteurella haemolytica (biotype A, serotype 1)

    Energy Technology Data Exchange (ETDEWEB)

    Richards, A.B.; Renshaw, H.W.; Sneed, L.W.

    1985-05-01

    Pasteurella haemolytica (biotype A, serotype 1) isolates (n = 15) from the upper respiratory tract of clinically normal cattle, as well as from lung lesions from cases of fatal bovine pasteurellosis, were examined for the presence of bacteriophage after irradiation with UV light. Treatment of all P haemolytica isolates with UV irradiation resulted in lysis of bacteria due to the induction of vegetative development of bacteriophages. The extent of growth inhibition and bacterial lysis in irradiated cultures was UV dose-dependent. Bacterial cultures exposed to UV light for 20 s reached peak culture density between 60 and 70 minutes after irradiation; thereafter, culture density declined rapidly, so that by 120 minutes, it was approximately 60% of the original value. When examined ultrastructurally, lytic cultures from each isolate revealed bacteriophages with an overall length of approximately 200 nm and that appeared to have a head with icosahedral symmetry and a contractile tail. Cell-free filtrate from each noninduced bacterial isolate was inoculated onto the other bacterial isolates in a cross-culture sensitivity assay for the presence of phages lytic for the host bacterial isolates. Zones of lysis (plaques) did not develop when bacterial lawns grown from the different isolates were inoculated with filtrates from the heterologous isolates.

  7. Inactivation of Escherichia coli O157 Bacteriophages by Using a Mixture of Ferrous Sulfate and Tea Extract.

    Science.gov (United States)

    Liu, Hui; Meng, Ruiqiang; Wang, Jiaying; Niu, Yan D; Li, Jinquan; Stanford, Kim; McAllister, Tim A

    2015-12-01

    Bacteriophages (phages) have been used for biocontrol of Escherichia coli O157 and other pathogenic bacteria in many different matrices and foods, but few studies have included inactivation of residual phages in culture medium before plating and enumeration of surviving host bacteria for the assessment of phage efficacy. This oversight may lead to overestimation of phage efficacy. The ability of virucidal solution containing a mixture of ferrous sulfate [iron(II) sulfate, FeSO4] and tea extract [Fe(II)T] to inactivate residual T5-like, T1-like, T4-like, and rV5-like phages was assessed using E. coli O157 as the host. At concentrations of ≥10 mM FeSO4, all phages were not detected after 20 min in a broth culture model. Compared with the virucidal solution-free samples (1 to 96% recovery), Fe(II)T (10 mM FeSO4 plus 15% tea extract) recovered a greater (P < 0.01) number of E. coli O157 from phage-treated broth culture (97 to 100% recovery) and beef samples (52 to 100% recovery). Moreover, with the addition of Fe(II)T, the number of bacteria surviving after exposure to T5-like or T4-like phages was greater (P < 0.01) than that after exposure to T1-like or rV5-like phages. Consequently, use of a virucide for phage inactivation is recommended to improve the accuracy of evaluations of phage efficacy for biocontrol of E. coli O157.

  8. Isolation and characterization of a lytic bacteriophage φKp-lyy15 of Klebsiella pneumoniae

    Institute of Scientific and Technical Information of China (English)

    Yinyin; Lu; Hongyan; Shi; Zhe; Zhang; Fang; Han; Jinghua; Li; Yanbo; Sun

    2015-01-01

    <正>Dear Editor,Bacteriophages(phages)are viruses that specifically infect and kill bacteria.They are ubiquitous throughout all environments that bacteria inhabit.Following their discovery by F.W.Twort in 1915 and F.d’Herele in 1917,bacteriophages were recognized as potential agents to treat bacterial diseases and phage therapy has been used

  9. Detection of bacteriophage-infected cells of Lactococcus lactis using flow cytometry

    DEFF Research Database (Denmark)

    Michelsen, Ole; Cuesta-Dominguez, Álvaro; Albrektsen, Bjarne;

    2007-01-01

    Bacteriophage infection in dairy fermentation constitutes a serious problem worldwide. We have studied bacteriophage infection in Lactococcus lactis by using the flow cytometer. The first effect of the infection of the bacterium is a change from cells in chains toward single cells. We interpret...

  10. Polymer-based delivery systems for support and delivery of bacteriophages

    Science.gov (United States)

    Brown, Alyssa Marie

    One of the most urgent problems in the fields of medicine and agriculture is the decreasing effectiveness of antibiotics. Once a miracle drug, antibiotics have recently become associated with the creation of antibiotic-resistant bacteria. The main limitations of these treatments include lack of both adaptability and specificity. To overcome these shortcomings of current antibiotic treatments, there has been a renewed interest in bacteriophage research. Bacteriophages are naturally-occurring viruses that lyse bacteria. They are highly specific, with each bacteriophage type lysing a narrow range of bacteria strains. Bacteriophages are also ubiquitous biological entities, populating environments where bacterial growth is supported. Just as humans are exposed to bacteria in their daily lives, we are exposed to bacteriophages as well. To use bacteriophages in practical applications, they must be delivered to the site of an infection in a controlled-release system. Two systems were studied to observe their support of bacteriophage lytic activity, as well as investigate the possibility of controlling bacteriophage release rates. First, hydrogels were studied, using crosslinking and blending techniques to achieve a range of release profiles. Second, polyanhydride microparticles were studied, evaluating release rates as a function of monomer chemistries.

  11. [The bacteriophages Yersinia pseudotuberculosis: the detection in strains of different O-serovars and their identification].

    Science.gov (United States)

    Makedonova, L D; Kudriakova, T A; Kachkina, G V; Gaevskaia, N E

    2013-08-01

    The sample included five indicator pseudotuberculosis strains. The application of these strains permitted to isolate out of 161 strains of Y. pseudotuberculosis 9 bacteriophages identical by their morphologic and serologic characteristics but having individual particularities in their lytic activity. The test on sensitivity to bacteriophages can be used in laboratory diagnostic to differentiate the strains of Yersinia pseudotuberculosis.

  12. Biocontrol of Escherichia coli O157:H7 using a bacteriophage cocktail in laboratory media

    Science.gov (United States)

    Bacteriophages are natural enemies of bacteria, and therefore, logical candidates to evaluate as antibacterial agents for the control of foodborne pathogens. The effect of a bacteriophage treatment on the prevention of E. coli O157:H7 growth was investigated in Tryptic Soy Broth (TSB) laboratory med...

  13. Complete Genome Sequence of a Myoviridae Bacteriophage Infecting Salmonella enterica Serovar Typhimurium.

    Science.gov (United States)

    Paradiso, Rubina; Orsini, Massimiliano; Bolletti Censi, Sergio; Borriello, Giorgia; Galiero, Giorgio

    2016-01-01

    The bacteriophage 118970_sal3 was isolated from water buffalo feces in southern Italy, exhibiting lytic activity against Salmonella enterica serovar Typhimurium. This bacteriophage belongs to the Myoviridae family and has a 39,464-bp double-stranded DNA (ds-DNA) genome containing 53 coding sequences (CDSs). PMID:27688333

  14. 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; Borriello, Giorgia

    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

  15. Complete Genome Sequence of a Myoviridae Bacteriophage Infecting Salmonella enterica Serovar Typhimurium

    Science.gov (United States)

    Paradiso, Rubina; Orsini, Massimiliano; Bolletti Censi, Sergio; Galiero, Giorgio

    2016-01-01

    The bacteriophage 118970_sal3 was isolated from water buffalo feces in southern Italy, exhibiting lytic activity against Salmonella enterica serovar Typhimurium. This bacteriophage belongs to the Myoviridae family and has a 39,464-bp double-stranded DNA (ds-DNA) genome containing 53 coding sequences (CDSs). PMID:27688333

  16. 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

  17. [The biopolitical production "impaired" subjects within the scope of "Action T4" - a re-reading of "I accuse"].

    Science.gov (United States)

    Offermann, Stefan

    2013-01-01

    The history of the Nazi "euthanasia programme" named "Aktion T4" has been examined from a biopolitical perspective for some time. However, these studies have not focused on the analytics of biopolitical practices of subjectivization as an essential element of the Foucaultian concept. The use of such a theoretical approach can be used in combination with new and substantial empirical research results. The Heidelberg DFG research programme evaluated the "T4" medical records statistically in order to conclusively determine which features had significant importance for selection by the "T4" experts. The inability to work or having an incurable mental illness were the criteria by which psychiatrists and "T4" experts subjectivized patients as "lives not worth living". Considering these new results and a biopolitical approach as starting point, it is time to reconsider the Nazi "euthanasia" propaganda movie "Ich klage an" ("I Accuse", 1941). When scholars began to study the production process of the movie in the 1980s, they looked at the narrative and the movie characters from an instrumental perspective. In other words, they examined how the "T4" protagonists and filmmakers sought to create a film which affected the viewers' opinion in a specific way intentionally influenced by them. According to that line of thought, the character Hanna was neglected because she was considered to be the morally inoffensive disguise of the intended propaganda massage. However, two works from the 1990s which were gender history-oriented finally focused on Hanna and the way the film narrative turns her into a subject "not worth living". Based on these considerations, this article states its thesis. The criteria of the film to subjectivize Hanna share many basic characteristics with those of the subjectivization process of the "T4" victims. To prove this statement, the analytics of the movie are combined with the results of the DFG project. Through the combination of both types of sources

  18. Comparison of Genomes of Three Xanthomonas oryzae Bacteriophages

    Directory of Open Access Journals (Sweden)

    Chen Hui-Yi

    2007-11-01

    Full Text Available Abstract Background Xp10 and OP1 are phages of Xanthomonas oryzae pv. oryzae (Xoo, the causative agent of bacterial leaf blight in rice plants, which were isolated in 1967 in Taiwan and in 1954 in Japan, respectively. We recently isolated the Xoo phage Xop411. Results The linear Xop411 genome (44,520 bp, 58 ORFs sequenced here is 147 bp longer than that of Xp10 (60 ORFs and 735 bp longer than that of OP1 (59 ORFs. The G+C contents of OP1 (51% and Xop411 and Xp10 (52% each are less than that of the host (65%. The 9-bp 3'-overhangs (5'-GGACAGTCT-3' in Xop411 and Xp10 are absent from OP1. More of the deduced Xop411 proteins share higher degrees of identity with Xp10 than with OP1 proteins, while the right end of the genomes of Xp10 and OP1, containing all predicted promoters, share stronger homology. Xop411, Xp10, and OP1 contain 8, 7, and 6 freestanding HNH endonuclease genes, respectively. These genes can be classified into five groups depending on their possession of the HNH domain (HNN or HNH type and/or AP2 domain in intact or truncated forms. While the HNN-AP2 type endonuclease genes dispersed in the genome, the HNH type endonuclease genes, each with a unique copy, were located within the same genome context. Mass spectrometry and N-terminal sequencing showed nine Xop411 coat proteins, among which three were identified, six were assigned as coat proteins (4 and conserved phage proteins (2 in Xp10. The major coat protein, in which only the N-terminal methionine is removed, appears to exist in oligomeric forms containing 2 to 6 subunits. The three phages exhibit different patterns of domain duplication in the N-terminus of the tail fiber, which are involved in determination of the host range. Many short repeated sequences are present in and around the duplicated domains. Conclusion Geographical separation may have confined lateral gene transfer among the Xoo phages. The HNN-AP2 type endonucleases were more likely to transfer their genes

  19. Removal of MS2, Qβ and GA bacteriophages during drinking water treatment at pilot scale.

    Science.gov (United States)

    Boudaud, Nicolas; Machinal, Claire; David, Fabienne; Fréval-Le Bourdonnec, Armelle; Jossent, Jérôme; Bakanga, Fanny; Arnal, Charlotte; Jaffrezic, Marie Pierre; Oberti, Sandrine; Gantzer, Christophe

    2012-05-15

    The removal of MS2, Qβ and GA, F-specific RNA bacteriophages, potential surrogates for pathogenic waterborne viruses, was investigated during a conventional drinking water treatment at pilot scale by using river water, artificially and independently spiked with these bacteriophages. The objective of this work is to develop a standard system for assessing the effectiveness of drinking water plants with respect to the removal of MS2, Qβ and GA bacteriophages by a conventional pre-treatment process (coagulation-flocculation-settling-sand filtration) followed or not by an ultrafiltration (UF) membrane (complete treatment process). The specific performances of three UF membranes alone were assessed by using (i) pre-treated water and (ii) 0.1 mM sterile phosphate buffer solution (PBS), spiked with bacteriophages. These UF membranes tested in this work were designed for drinking water treatment market and were also selected for research purpose. The hypothesis serving as base for this study was that the interfacial properties for these three bacteriophages, in terms of electrostatic charge and the degree of hydrophobicity, could induce variations in the removal performances achieved by drinking water treatments. The comparison of the results showed a similar behaviour for both MS2 and Qβ surrogates whereas it was particularly atypical for the GA surrogate. The infectious character of MS2 and Qβ bacteriophages was mostly removed after clarification followed by sand filtration processes (more than a 4.8-log reduction) while genomic copies were removed at more than a 4.0-log after the complete treatment process. On the contrary, GA bacteriophage was only slightly removed by clarification followed by sand filtration, with less than 1.7-log and 1.2-log reduction, respectively. After the complete treatment process achieved, GA bacteriophage was removed with less than 2.2-log and 1.6-log reduction, respectively. The effectiveness of the three UF membranes tested in terms of

  20. Atomic Structure and Biochemical Characterization of an RNA Endonuclease in the N Terminus of Andes Virus L Protein.

    Directory of Open Access Journals (Sweden)

    Yaiza Fernández-García

    2016-06-01

    Full Text Available Andes virus (ANDV is a human-pathogenic hantavirus. Hantaviruses presumably initiate their mRNA synthesis by using cap structures derived from host cell mRNAs, a mechanism called cap-snatching. A signature for a cap-snatching endonuclease is present in the N terminus of hantavirus L proteins. In this study, we aimed to solve the atomic structure of the ANDV endonuclease and characterize its biochemical features. However, the wild-type protein was refractory to expression in Escherichia coli, presumably due to toxic enzyme activity. To circumvent this problem, we introduced attenuating mutations in the domain that were previously shown to enhance L protein expression in mammalian cells. Using this approach, 13 mutant proteins encompassing ANDV L protein residues 1-200 were successfully expressed and purified. Protein stability and nuclease activity of the mutants was analyzed and the crystal structure of one mutant was solved to a resolution of 2.4 Å. Shape in solution was determined by small angle X-ray scattering. The ANDV endonuclease showed structural similarities to related enzymes of orthobunya-, arena-, and orthomyxoviruses, but also differences such as elongated shape and positively charged patches surrounding the active site. The enzyme was dependent on manganese, which is bound to the active site, most efficiently cleaved single-stranded RNA substrates, did not cleave DNA, and could be inhibited by known endonuclease inhibitors. The atomic structure in conjunction with stability and activity data for the 13 mutant enzymes facilitated inference of structure-function relationships in the protein. In conclusion, we solved the structure of a hantavirus cap-snatching endonuclease, elucidated its catalytic properties, and present a highly active mutant form, which allows for inhibitor screening.

  1. Atomic Structure and Biochemical Characterization of an RNA Endonuclease in the N Terminus of Andes Virus L Protein.

    Science.gov (United States)

    Fernández-García, Yaiza; Reguera, Juan; Busch, Carola; Witte, Gregor; Sánchez-Ramos, Oliberto; Betzel, Christian; Cusack, Stephen; Günther, Stephan; Reindl, Sophia

    2016-06-01

    Andes virus (ANDV) is a human-pathogenic hantavirus. Hantaviruses presumably initiate their mRNA synthesis by using cap structures derived from host cell mRNAs, a mechanism called cap-snatching. A signature for a cap-snatching endonuclease is present in the N terminus of hantavirus L proteins. In this study, we aimed to solve the atomic structure of the ANDV endonuclease and characterize its biochemical features. However, the wild-type protein was refractory to expression in Escherichia coli, presumably due to toxic enzyme activity. To circumvent this problem, we introduced attenuating mutations in the domain that were previously shown to enhance L protein expression in mammalian cells. Using this approach, 13 mutant proteins encompassing ANDV L protein residues 1-200 were successfully expressed and purified. Protein stability and nuclease activity of the mutants was analyzed and the crystal structure of one mutant was solved to a resolution of 2.4 Å. Shape in solution was determined by small angle X-ray scattering. The ANDV endonuclease showed structural similarities to related enzymes of orthobunya-, arena-, and orthomyxoviruses, but also differences such as elongated shape and positively charged patches surrounding the active site. The enzyme was dependent on manganese, which is bound to the active site, most efficiently cleaved single-stranded RNA substrates, did not cleave DNA, and could be inhibited by known endonuclease inhibitors. The atomic structure in conjunction with stability and activity data for the 13 mutant enzymes facilitated inference of structure-function relationships in the protein. In conclusion, we solved the structure of a hantavirus cap-snatching endonuclease, elucidated its catalytic properties, and present a highly active mutant form, which allows for inhibitor screening. PMID:27300328

  2. Screening and identification of receptor antagonist for shiga toxin from random peptides displayed on filamentous bacteriophages

    Institute of Scientific and Technical Information of China (English)

    韩照中; 苏国富; 黄翠芬

    1999-01-01

    The bacteriophage clones which can bind with shiga toxin B subunit (StxB) and inhibit cytotoxicity of shiga toxin were obtained by using antibody capturing method from a 15-mer random peptide library displayed on the surface of bacteriophage fd. Among them, one peptide encoded by the random DNA region of a selected bacteriophage (A12) was synthesized and tested in vitro and in vivo, where the peptide competed with the receptor of shiga toxin to bind StxB, and inhibited the cytotoxicity and enterotoxicity of shiga toxin. The peptide can also block other apparently unrelated StxB binding bacteriophage (A3), which suggests that there are overlapping StxB interaction sites for those ligands with different sequences. The results provide a demonstration of bacteriophage display to screen peptide ligands for a small and/or unable biotinylated molecule by antibodies-capturing strategy, and take the lead for the development of receptor antagonists for shiga toxin.

  3. Bacteriophages as anti-infective agents: recent developments and regulatory challenges.

    Science.gov (United States)

    Gilmore, Brendan F

    2012-05-01

    The biennial meeting on 'Exploiting Bacteriophages for Bioscience, Biotechnology and Medicine', held in London, UK, on 20 January 2012, and chaired by George Salmond (University of Cambridge, UK) hosted over 50 participants representing 13 countries. The highly multidisciplinary meeting covered a diverse range of topics, reflecting the current expansion of interest in this field, including the use of bacteriophages as the source of biochemical reagents for molecular biology, bacteriophages for the treatment of human and animal diseases, bacteriophage-based diagnostics and therapeutic delivery technologies and necessity for, and regulatory challenges associated with, robust clinical trials of phage-based therapeutics. This report focuses on a number of presentations from the meeting relating to cutting-edge research on bacteriophages as anti-infective agents.

  4. Comparison of Mycoplasma ovipneumoniae isolates using bacterial restriction endonuclease DNA analysis and SDS-PAGE.

    Science.gov (United States)

    Mew, A J; Ionas, G; Clarke, J K; Robinson, A J; Marshall, R B

    1985-12-01

    Sixteen isolates of Mycoplasma ovipneumoniae recovered from the nasal tract or lungs of sheep from different flocks in New Zealand were examined by bacterial restriction endonuclease DNA analysis (BRENDA) using EcoR1 and by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). All isolates gave BRENDA patterns which differed entirely from one another. Following 20 serial passages (corresponding to approximately 67 generations) of an isolate, no change was detected in the BRENDA pattern. When eight isolates were examined by SDS-PAGE most bands were common but, nevertheless, each isolate was unique in the sense that they differed from one another in one or more bands. The marked heterogeneity of patterns observed when strains of M. ovipneumoniae are compared by BRENDA, together with the stability of such patterns over many generations, will enable this approach to be used to study the epidemiology of individual strains of M. ovipneumoniae within a flock.

  5. Primary processing of CRISPR RNA by the endonuclease Cas6 in Staphylococcus epidermidis.

    Science.gov (United States)

    Wakefield, Noelle; Rajan, Rakhi; Sontheimer, Erik J

    2015-10-01

    In many bacteria and archaea, an adaptive immune system (CRISPR-Cas) provides immunity against foreign genetic elements. This system uses CRISPR RNAs (crRNAs) derived from the CRISPR array, along with CRISPR-associated (Cas) proteins, to target foreign nucleic acids. In most CRISPR systems, endonucleolytic processing of crRNA precursors (pre-crRNAs) is essential for the pathway. Here we study the Cas6 endonuclease responsible for crRNA processing in the Type III-A CRISPR-Cas system from Staphylococcus epidermidis RP62a, a model for Type III-A CRISPR-Cas systems, and define substrate requirements for SeCas6 activity. We find that SeCas6 is necessary and sufficient for full-length crRNA biogenesis in vitro, and that it relies on both sequence and stem-loop structure in the 3' half of the CRISPR repeat for recognition and processing.

  6. Sequential and Multistep Substrate Interrogation Provides the Scaffold for Specificity in Human Flap Endonuclease 1

    KAUST Repository

    Sobhy, M.

    2013-06-06

    Human flap endonuclease 1 (FEN1), one of the structure-specific 5\\' nucleases, is integral in replication, repair, and recombination of cellular DNA. The 5\\' nucleases share significant unifying features yet cleave diverse substrates at similar positions relative to 5\\' end junctions. Using single-molecule Förster resonance energy transfer, we find a multistep mechanism that verifies all substrate features before inducing the intermediary-DNA bending step that is believed to unify 5\\' nuclease mechanisms. This is achieved by coordinating threading of the 5\\' flap of a nick junction into the conserved capped-helical gateway, overseeing the active site, and bending by binding at the base of the junction. We propose that this sequential and multistep substrate recognition process allows different 5\\' nucleases to recognize different substrates and restrict the induction of DNA bending to the last common step. Such mechanisms would also ensure the protection ofDNA junctions from nonspecific bending and cleavage. 2013 The Authors.

  7. Distinct facilitated diffusion mechanisms by E. coli Type II restriction endonucleases.

    Science.gov (United States)

    Pollak, Adam J; Chin, Aaron T; Reich, Norbert O

    2014-11-18

    The passive search by proteins for particular DNA sequences involving nonspecific DNA is essential for gene regulation, DNA repair, phage defense, and diverse epigenetic processes. Distinct mechanisms contribute to these searches, and it remains unresolved as to which mechanism or blend of mechanisms best suits a particular protein and, more importantly, its biological role. To address this, we compare the translocation properties of two well-studied bacterial restriction endonucleases (ENases), EcoRI and EcoRV. These dimeric, magnesium-dependent enzymes hydrolyze related sites (EcoRI ENase, 5'-GAATTC-3'; EcoRV ENase, 5'-GATATC-3'), leaving overhangs and blunt DNA segments, respectively. Here, we demonstrate that the extensive sliding by EcoRI ENase, involving sliding up to ∼600 bp prior to dissociating from the DNA, contrasts with a larger reliance on hopping mechanism(s) by EcoRV ENase. The mechanism displayed by EcoRI ENase results in a highly thorough search of DNA, whereas the EcoRV ENase mechanism results in an extended, yet less rigorous, interrogation of DNA sequence space. We describe how these mechanistic distinctions are complemented by other aspects of these endonucleases, such as the 10-fold higher in vivo concentrations of EcoRI ENase compared to that of EcoRV ENase. Further, we hypothesize that the highly diverse enzyme arsenal that bacteria employ against foreign DNA involves seemingly similar enzymes that rely on distinct but complementary search mechanisms. Our comparative approach reveals how different proteins utilize distinct site-locating strategies. PMID:25350874

  8. EXPRESSION AND DELETION ANALYSIS OF EcoRII ENDONUCLEASE AND METHYLASE GENE

    Institute of Scientific and Technical Information of China (English)

    刘金毅; 赵晓娟; 孟雁; 沈洁; 薛越强; 史顺娣; 蔡有余

    2001-01-01

    Objective. To clone complete EcoRII restriction endonuclease gene (ecoRllR) and methyltransferase gene(ecoRllM) in one ector and to analyze the coordinating expression of this whole R-M system.Methods. Unidirectional deletion subclones were constructed with ExolII. ecoRllR/M genes were preliminari-ly located in the cloned fragment according to the enzyme activities of subclones. Exact deletion sites were deter-mined by sequencing, and transcriptional start sites were determined by S1 mapping.Results. The DNA fragment which was cloned into pBluescript SK + contained intact ecoRIlR gene andecoRllM gene, anc two transcriptional start sites of ecoRllR gene were determined. 132bp to 458bp from 3' endof ecoRllR gene ar.e indispensable to enzyme activities and deletion of 202bp from 3' end of ecoRllM gene madeenzyme lose the capability in DNA protection to resist specific cut with EcoRII endonuclease (EcoRII. R). Dele-tion of the coding ar d flanking sequences of one gene did not affect the expression of the other gene, and the recombi-nants only containing ecoRllR gene appeared to be lethal to dcm+ host.Conclusion. scoRllM gene linking closely to ecoRIIR gene is very important for the existence of the R-M sys-tem in process of evolution, but the key to control EcoRlI R-M order may not exist in transcriptional level .``Liu Jmy,Corresponding author.

  9. Inhibition of DNA restrictive endonucleases by aqueous nanoparticle suspension of methanophosphonate fullerene derivatives and its mechanisms

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Aqueous nanoparticle suspension of fullerene and its derivatives are currently attracting much attention. To determine the effects of aqueous nanoparticle suspension of a mono-methanophosphonate fullerene and bis-methanophosphonate fullerene (denoted as n-MMPF and n-BMPF, respectively) on the activities of DNA restrictive endonucleases, plasmid pEGFP-N1 was cleaved at a single but differently restrictive site by EcoR I, BamH I, and isozymes Cfr9 I and Xma I, respectively. Both n-MMPF and n-BMPF inhibited the activity of EcoR I, while n-BMPF exhibited stronger inhibition than n-MMPF. Addition of n-BMPF into reaction mixtures inhibited the activities of all the four enzymes, and IC50 values for EcoR I, BamH I, Cfr9 I and Xma I were 4.3, >30, 11.7 and 8.3 μmol/L, respectively. When EcoR I was completely inhibited by n-BMPF, addition of excess amounts of pEGFP-N1 could not produce the product linear plasmid; however, increase of EcoR I amounts antagonized EcoR I inhibition of n-BMPF. Two scavengers of reactive oxygen species (ROS), mannitol and sodium azide at the concentrations of 2-10 mmol/L, did not reverse inhibition of n-BMPF, implying that this inhibition probably is not correlated to ROS. These results suggested that aqueous nano-fullerenes might act as inhibitors of DNA restrictive endonucleases.

  10. Cyanobacterial ribosomal RNA genes with multiple, endonuclease-encoding group I introns

    Directory of Open Access Journals (Sweden)

    Turner Seán

    2007-09-01

    Full Text Available Abstract Background Group I introns are one of the four major classes of introns as defined by their distinct splicing mechanisms. Because they catalyze their own removal from precursor transcripts, group I introns are referred to as autocatalytic introns. Group I introns are common in fungal and protist nuclear ribosomal RNA genes and in organellar genomes. In contrast, they are rare in all other organisms and genomes, including bacteria. Results Here we report five group I introns, each containing a LAGLIDADG homing endonuclease gene (HEG, in large subunit (LSU rRNA genes of cyanobacteria. Three of the introns are located in the LSU gene of Synechococcus sp. C9, and the other two are in the LSU gene of Synechococcus lividus strain C1. Phylogenetic analyses show that these introns and their HEGs are closely related to introns and HEGs located at homologous insertion sites in organellar and bacterial rDNA genes. We also present a compilation of group I introns with homing endonuclease genes in bacteria. Conclusion We have discovered multiple HEG-containing group I introns in a single bacterial gene. To our knowledge, these are the first cases of multiple group I introns in the same bacterial gene (multiple group I introns have been reported in at least one phage gene and one prophage gene. The HEGs each contain one copy of the LAGLIDADG motif and presumably function as homodimers. Phylogenetic analysis, in conjunction with their patchy taxonomic distribution, suggests that these intron-HEG elements have been transferred horizontally among organelles and bacteria. However, the mode of transfer and the nature of the biological connections among the intron-containing organisms are unknown.

  11. Structural and functional characterization of two unusual endonuclease III enzymes from Deinococcus radiodurans.

    Science.gov (United States)

    Sarre, Aili; Ökvist, Mats; Klar, Tobias; Hall, David R; Smalås, Arne O; McSweeney, Sean; Timmins, Joanna; Moe, Elin

    2015-08-01

    While most bacteria possess a single gene encoding the bifunctional DNA glycosylase Endonuclease III (EndoIII) in their genomes, Deinococcus radiodurans possesses three: DR2438 (DrEndoIII1), DR0289 (DrEndoIII2) and DR0982 (DrEndoIII3). Here we have determined the crystal structures of DrEndoIII1 and an N-terminally truncated form of DrEndoIII3 (DrEndoIII3Δ76). We have also generated a homology model of DrEndoIII2 and measured activity of the three enzymes. All three structures consist of two all α-helical domains, one of which exhibits a [4Fe-4S] cluster and the other a HhH-motif, separated by a DNA binding cleft, similar to previously determined structures of endonuclease III from Escherichia coli and Geobacillus stearothermophilus. However, both DrEndoIII1 and DrEndoIII3 possess an extended HhH motif with extra helical features and an altered electrostatic surface potential. In addition, the DNA binding cleft of DrEndoIII3 seems to be less accessible for DNA interactions, while in DrEndoIII1 it seems to be more open. Analysis of the enzyme activities shows that DrEndoIII2 is most similar to the previously studied enzymes, while DrEndoIII1 seems to be more distant with a weaker activity towards substrate DNA containing either thymine glycol or an abasic site. DrEndoIII3 is the most distantly related enzyme and displays no detectable activity towards these substrates even though the suggested catalytic residues are conserved. Based on a comparative structural analysis, we suggest that the altered surface potential, shape of the substrate-binding pockets and specific amino acid substitutions close to the active site and in the DNA interacting loops may underlie the unexpected differences in activity.

  12. Label-free Gram-negative bacteria detection using bacteriophage-adhesin-coated long-period gratings.

    Science.gov (United States)

    Brzozowska, Ewa; Koba, Marcin; Śmietana, Mateusz; Górska, Sabina; Janik, Monika; Gamian, Andrzej; Bock, Wojtek J

    2016-03-01

    This paper presents a novel application of a highly sensitive sensor based on long-period gratings (LPGs) coated with T4 bacteriophage adhesin for Gram-negative bacteria detection. We show here, that the sensor evidently recognizes Escherichia coli K-12 (PCM2560), whereas in the reference tests - ELISA and BIAcore - the results are questionable. For LPGs sensor the resonant wavelength shift observed for E. coli K-12 was approximately half of that measured for E.coli B (positive control). The BIAcore readings (RU) for E. coli K-12 were at 10% level of the signal obtained for E .coli B. These results confirm the improved sensitivity of the LPGs sensor. Moreover, we also show that application of adhesin may allow for efficient detection of E. coli O111 (PCM418), Klebsiella pneumoniae O1 (PCM1) and Yersinia enterocolitica O1 (PCM1879). The specificity of binding bacteria by the adhesin is discussed and it is determined by a distinct region of lipopolysaccharide receptors and/or by the presence of outer-membrane protein C in an outer membrane of Gram-negative bacteria.

  13. An unconventional approach to impedance microbiology: detection of culture media conductivity variations due to bacteriophage generated lyses of host bacteria.

    Science.gov (United States)

    Mortari, Alessia; Adami, Andrea; Lorenzelli, Leandro

    2015-05-15

    A novel and unconventional approach to impedance microbiology has been under investigation. In our approach, solution conductivity variations are generated from bacteriophage lyses of infected host cells and the consequent release of conductive endoplasmic material. To sensitively detect the lysis, low conductive growth media have been developed. A microchip has been fabricated to perform the analysis. The microchip is made of two bare gold electrodes and PDMS microchamber of 36 nL volume. Escherichia coli and selective phages T4 have been used as case study. Proof-of-principle experiments are here presented and discussed. The method was characterised in a wide range between 10(4) and 10(8) CFU/mL, where linear relation was found between conductivity variation and cell concentration in a log10 vs. log10 plot. The method is suited to integration with sample preparation based on phage-functionalised magnetic beads. It has a potential detection limit below 1 CFU/chamber and a total assay time of less than 1 h.

  14. The lectin domain of UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-T4 directs its glycopeptide specificities

    DEFF Research Database (Denmark)

    Hassan, H; Reis, C A; Bennett, E P;

    2000-01-01

    for differential regulation of O-glycan attachment sites and density. Recently, it has emerged that some GalNAc-transferase isoforms in vitro selectively function with partially GalNAc O-glycosylated acceptor peptides rather than with the corresponding unglycosylated peptides. O-Glycan attachment to selected sites......, most notably two sites in the MUC1 tandem repeat, is entirely dependent on the glycosylation-dependent function of GalNAc-T4. Here we present data that a putative lectin domain found in the C terminus of GalNAc-T4 functions as a GalNAc lectin and confers its glycopeptide specificity. A single amino...... by free GalNAc, while the activity with peptides was unaffected....

  15. Acanthamoeba genotypes T2, T4, and T11 in soil sources from El Hierro island, Canary Islands, Spain.

    Science.gov (United States)

    Reyes-Batlle, María; Zamora-Herrera, Jonadab; Vargas-Mesa, Alejandro; Valerón-Tejera, Marco Antonio; Wagner, Carolina; Martín-Navarro, Carmen Ma; López-Arencibia, Atteneri; Sifaoui, Ines; Martínez-Carretero, Enrique; Valladares, Basilio; Piñero, José E; Lorenzo-Morales, Jacob

    2016-08-01

    The genus Acanthamoeba includes pathogenic strains which are causative agents of keratitis and encephalitis that often may end fatal in humans and other animals. In the present study, forty soil samples were collected in the island of El Hierro, Canary Islands, Spain, and checked for the presence of Acanthamoeba. Samples were cultivated onto 2 % non-nutrient agar plates seeded with a layer of heat killed Escherichia coli. Amplification by PCR and sequencing of the DF3 region of the 18S rDNA of Acanthamoeba was carried out in order to confirm morphological identification of the amoebae. Furthermore, Acanthamoeba spp. was isolated from 47.5 % of soil samples. Moreover, genotypes T2, T4, and T11 were identified in these samples. To the best of our knowledge, this is the first study to establish genotypes T2, T4, and T11 in soil sources from El Hierro island. PMID:27075307

  16. Co-administration of Apelin and T4 Protects Inotropic and Chronotropic Changes Occurring in Hypothyroid Rats

    Energy Technology Data Exchange (ETDEWEB)

    Akhondali, Zahra; Badavi, Mohammad; Dianat, Mahin, E-mail: dianat@ajums.ac.ir; Faraji, Farzaneh [Physiology Research Center and Department of Physiology, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz (Iran, Islamic Republic of)

    2015-09-15

    One of the most important thyroid hormone targets is the cardiovascular system. Hemodynamic changes, such as decreased resting heart rate (HR), myocardial contractility, and cardiac output, and increased diastolic pressure and systemic vascular resistance, have been observed in hypothyroid patients. Moreover, in these patients, ECG changes include sinus bradycardia and low voltage complexes (P waves or QRS complexes). This study aimed at evaluating the prophylactic effect of apelin on HR changes and QRS voltage that occur in propylthiouracil (PTU)-induced hypothyroid rats. In this study, 48 adult male Wistar rats weighing 170-235g were randomly divided into 6 groups: Control group (normal saline ip injection + tap water gavage); P group (PTU 0.05%, in drinking water); A group (apelin 200 µg.kg{sup -1}.day{sup -1}, ip); PA group [co-administration of PTU and apelin]; PT group [co-administration of PTU + T4 (0.2 mg/g per day, gavage)]; and PAT group (co-administration of PTU, apelin and T4). All experiments were performed for 28 consecutive days, and then the animals were anesthetized with an ip injection of ketamine (80 mg/kg) and xylazine (12 mg/kg). Lead II electrocardiogram was recorded to calculate HR and QRS voltage. Heart rate and QRS voltage increased more significantly in the hypothyroid group that consumed both apelin and T4 (201 ± 4 beat/min, 0.71 ± 0.02 mv vs. hypothyroid 145 ± 9 beat/min, 0.563 ± 0.015 mv; respectively). The co-administration of apelin and T4 showed a protective effect on QRS voltage and HR in PTU‑induced hypothyroid rats.

  17. Top-down controls on bacterial community structure: microbial network analysis of bacteria, T4-like viruses and protists

    OpenAIRE

    Chow, Cheryl-Emiliane T.; Kim, Diane Y.; Sachdeva, Rohan; Caron, David A.; Fuhrman, Jed A

    2013-01-01

    Characterizing ecological relationships between viruses, bacteria and protists in the ocean are critical to understanding ecosystem function, yet these relationships are infrequently investigated together. We evaluated these relationships through microbial association network analysis of samples collected approximately monthly from March 2008 to January 2011 in the surface ocean (0–5 m) at the San Pedro Ocean Time series station. Bacterial, T4-like myoviral and protistan communities were desc...

  18. Microstructural Characteristics and Mechanical Properties of Friction Stir Spot Welded 2A12-T4 Aluminum Alloy

    OpenAIRE

    Huijie Liu; Yunqiang Zhao; Xingye Su; Lilong Yu; Juncai Hou

    2013-01-01

    2A12-T4 aluminum alloy was friction stir spot welded, and the microstructural characteristics and mechanical properties of the joints were investigated. A softened microstructural region existed in the joint, and it consisted of stir zone (SZ), thermal mechanically affected zone (TMAZ), and heat affected zone (HAZ). The minimum hardness was located in TMAZ, and the average hardness value in SZ can be improved by appropriately increasing welding heat input. The area of complete bonding region ...

  19. Acanthamoeba castellanii of the T4 genotype is a potential environmental host for Enterobacter aerogenes and Aeromonas hydrophila

    OpenAIRE

    Yousuf, Farzana Abubakar; Siddiqui, Ruqaiyyah; Khan, Naveed Ahmed

    2013-01-01

    Background Acanthamoeba can interact with a wide range of microorganisms such as viruses, algae, yeasts, protists and bacteria including Legionella pneumophila, Pseudomonas aeruginosa, Vibrio cholerae, Helicobacter pylori, Listeria monocytogenes, Mycobacterium spp., and Escherichia coli. In this capacity, Acanthamoeba has been suggested as a vector in the transmission of bacterial pathogens to the susceptible hosts. Methods Here, we used a keratitis isolate of A. castellanii of the T4 genotyp...

  20. Co-administration of Apelin and T4 Protects Inotropic and Chronotropic Changes Occurring in Hypothyroid Rats

    International Nuclear Information System (INIS)

    One of the most important thyroid hormone targets is the cardiovascular system. Hemodynamic changes, such as decreased resting heart rate (HR), myocardial contractility, and cardiac output, and increased diastolic pressure and systemic vascular resistance, have been observed in hypothyroid patients. Moreover, in these patients, ECG changes include sinus bradycardia and low voltage complexes (P waves or QRS complexes). This study aimed at evaluating the prophylactic effect of apelin on HR changes and QRS voltage that occur in propylthiouracil (PTU)-induced hypothyroid rats. In this study, 48 adult male Wistar rats weighing 170-235g were randomly divided into 6 groups: Control group (normal saline ip injection + tap water gavage); P group (PTU 0.05%, in drinking water); A group (apelin 200 µg.kg-1.day-1, ip); PA group [co-administration of PTU and apelin]; PT group [co-administration of PTU + T4 (0.2 mg/g per day, gavage)]; and PAT group (co-administration of PTU, apelin and T4). All experiments were performed for 28 consecutive days, and then the animals were anesthetized with an ip injection of ketamine (80 mg/kg) and xylazine (12 mg/kg). Lead II electrocardiogram was recorded to calculate HR and QRS voltage. Heart rate and QRS voltage increased more significantly in the hypothyroid group that consumed both apelin and T4 (201 ± 4 beat/min, 0.71 ± 0.02 mv vs. hypothyroid 145 ± 9 beat/min, 0.563 ± 0.015 mv; respectively). The co-administration of apelin and T4 showed a protective effect on QRS voltage and HR in PTU‑induced hypothyroid rats