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Sample records for bacterial thymidine kinase

  1. Thymidine kinase diversity in bacteria

    DEFF Research Database (Denmark)

    Sandrini, Michael; Clausen, A.R.; Munch-Petersen, B.;

    2006-01-01

    Thymidine kinases (TKs) appear to be almost ubiquitous and are found in nearly all prokaryotes, eukaryotes, and several viruses. They are the key enzymes in thymidine salvage and activation of several anti-cancer and antiviral drugs. We show that bacterial TKs can be subdivided into 2 groups. The...... TKs from Gram-positive bacteria are more closely related to the eukaryotic TK1 enzymes than are TKs from Gram-negative bacteria....

  2. Thymidine kinase diversity in bacteria

    DEFF Research Database (Denmark)

    Sandrini, Michael; Clausen, A.R.; Munch-Petersen, B.; Piskur, Jure

    Thymidine kinases (TKs) appear to be almost ubiquitous and are found in nearly all prokaryotes, eukaryotes, and several viruses. They are the key enzymes in thymidine salvage and activation of several anti-cancer and antiviral drugs. We show that bacterial TKs can be subdivided into 2 groups. The...... TKs from Gram-positive bacteria are more closely related to the eukaryotic TK1 enzymes than are TKs from Gram-negative bacteria....

  3. The use of 14C-FIAU to predict bacterial thymidine kinase presence: Implications for radiolabeled FIAU bacterial imaging

    International Nuclear Information System (INIS)

    Currently available infectious disease imaging techniques cannot differentiate between infection and sterile inflammation or between different types of infections. Recently, radiolabeled FIAU was found to be a substrate for the thymidine kinase (TK) enzyme of multiple pathogenic bacteria, leading to its translational use in the imaging of bacterial infections. Patients with immunodeficiencies, however, are susceptible to a different group of pathogenic bacteria when compared to immunocompetent subjects. In this study, we wanted to predict the usefulness of radiolabeled FIAU in the detection of bacterial infections commonly occurring in patients with immunodeficiencies, in vitro, prior to attempting in vivo imaging with 124I-FIAU-PET. Methods: We obtained representative strains of bacterial pathogens isolated from actual patients with genetic immunodeficiencies. We evaluated the bacterial susceptibility of different strains to the effect of incubation with FIAU, which would implicate the presence of the thymidine kinase (TK) enzyme. We also incubated the bacteria with 14C-FIAU and consequently measured its rate of incorporation in the bacterial DNA using a liquid scintillation counter. Results: Unlike the other bacterial strains, the growth of Pseudomonas aeruginosa was not halted by FIAU at any concentration. All the tested clinical isolates demonstrated different levels of 14C-FIAU uptake, except for P. aeruginosa. Conclusion: Radiolabeled FIAU has been successful in delineating bacterial infections, both in preclinical and pilot translational studies. In patients with immunodeficiencies, Pseudomonas infections are commonly encountered and are usually difficult to differentiate from fungal infections. The use of radiolabeled FIAU for in vivo imaging of those patients, however, would not be useful, considering the apparent lack of TK enzyme in Pseudomonas. One has to keep in mind that not all pathogenic bacteria possess the TK enzyme and as such will not all retain

  4. Cloning of the Koi Herpesvirus Genome as an Infectious Bacterial Artificial Chromosome Demonstrates That Disruption of the Thymidine Kinase Locus Induces Partial Attenuation in Cyprinus carpio koi▿

    Science.gov (United States)

    Costes, B.; Fournier, G.; Michel, B.; Delforge, C.; Raj, V. Stalin; Dewals, B.; Gillet, L.; Drion, P.; Body, A.; Schynts, F.; Lieffrig, F.; Vanderplasschen, A.

    2008-01-01

    Koi herpesvirus (KHV) is the causative agent of a lethal disease in koi and common carp. In the present study, we describe the cloning of the KHV genome as a stable and infectious bacterial artificial chromosome (BAC) clone that can be used to produce KHV recombinant strains. This goal was achieved by the insertion of a loxP-flanked BAC cassette into the thymidine kinase (TK) locus. This insertion led to a BAC plasmid that was stably maintained in bacteria and was able to regenerate virions when permissive cells were transfected with the plasmid. Reconstituted virions free of the BAC cassette but carrying a disrupted TK locus (the FL BAC-excised strain) were produced by the transfection of Cre recombinase-expressing cells with the BAC. Similarly, virions with a wild-type revertant TK sequence (the FL BAC revertant strain) were produced by the cotransfection of cells with the BAC and a DNA fragment encoding the wild-type TK sequence. Reconstituted recombinant viruses were compared to the wild-type parental virus in vitro and in vivo. The FL BAC revertant strain and the FL BAC-excised strain replicated comparably to the parental FL strain. The FL BAC revertant strain induced KHV infection in koi carp that was indistinguishable from that induced by the parental strain, while the FL BAC-excised strain exhibited a partially attenuated phenotype. Finally, the usefulness of the KHV BAC for recombination studies was demonstrated by the production of an ORF16-deleted strain by using prokaryotic recombination technology. The availability of the KHV BAC is an important advance that will allow the study of viral genes involved in KHV pathogenesis, as well as the production of attenuated recombinant candidate vaccines. PMID:18337580

  5. Enzymatic Regulation of Cytosolic Thymidine Kinase 1 and Mitochondrial Thymidine Kinase 2

    DEFF Research Database (Denmark)

    Munch-Petersen, Birgitte

    2010-01-01

    The central enzyme on the de novo pathway for synthesis of DNA precursors, the deoxyribonucleoside triphosphates, is ribonucleotide reductase (RNR). Deoxythymidine triphosphate (dTTP) has a key role in control of RNR activity shifting the specificity from pyrimidine to purine nucleotide reduction....... Apart from the complex de novo synthesis of dTTP through UDP reduction, dTTP is provided through salvage of thymidine catalyzed by the thymidine kinases, the cytosolic and cell cycle regulated TK1 and the mitochondrial and constitutively expressed TK2. The complex enzymatic regulation of TK1 and TK2 and...

  6. Dictyostelium discoideum salvages purine deoxyribonucleosides by highly specific bacterial-like deoxyribonucleoside kinases

    DEFF Research Database (Denmark)

    Sandrini, Michael; Soderbom, F.; Mikkelsen, N.E.;

    2007-01-01

    . discoideum thymidine kinase was similar to the human thymidine kinase 1 and was specific for thymidine with a k(m) of 5.1 mu M. The other two cloned kinases were phylogenetically closer to bacterial deoxyribonucleoside kinases than to the eukaryotic enzymes. D. discoideum deoxyadenosine kinase (DddAK) had a...

  7. Diffenrent affinity of the two forms of human cytosolic thymidine kinase towards pyrimidine analogs

    DEFF Research Database (Denmark)

    Munch-Petersen, B.; Tyrsted, Gerda; Cloos, Lisbeth;

    1995-01-01

    Biokemi, thymidine kinase 1, cytosolic, regulation, ATP activation, azidothymidine, nucleoside analog......Biokemi, thymidine kinase 1, cytosolic, regulation, ATP activation, azidothymidine, nucleoside analog...

  8. Tomato thymidine kinase is subject to inefficient TTP feedback regulation

    DEFF Research Database (Denmark)

    Larsen, Nicolai Balle; Munch-Petersen, Birgitte; Piskur, Jure

    2014-01-01

    A promising suicide gene therapy system to treat gliomas has been reported: the thymidine kinase 1 from tomato (toTK1) combined with the nucleoside analog pro-drug zidovudine (azidothymidine, AZT), which is known to penetrate the blood–brain barrier. Transduction with toTK1 has been found to effi...... reduced TTP-mediated feedback inhibition of the AZT phosphorylation...

  9. Thymidine kinase 1 deficient cells show increased survival rate after UV-induced DNA damage

    DEFF Research Database (Denmark)

    Skovgaard, T; Rasmussen, Lene Juel; Munch-Petersen, Birgitte

    2010-01-01

    enzyme thymidine kinase 1 (TK1) are more resistant to UV-induced DNA damage than TK1 positive cells although they have thymidine triphosphate (dTTP) levels of only half the size of control cells. Our results suggest that higher thymidine levels in the TK- cells caused by defect thymidine salvage to dTTP...

  10. A mathematical model of human thymidine kinase 2 activity

    DEFF Research Database (Denmark)

    Radivoyevitch, Tom; Munch-Petersen, Birgitte; Wang, Liya;

    2011-01-01

    _ The mitochondrial enzyme thymidine kinase 2 (TK2) phosphorylates deoxythymidine (dT) and deoxycytidine (dC) to form dTMP and dCMP, which in cells rapidly become the negative-feedback end-products dTTP and dCTP. TK2 kinetic activity exhibits Hill coefficients of ∼0.5 (apparent negative cooperati......_ The mitochondrial enzyme thymidine kinase 2 (TK2) phosphorylates deoxythymidine (dT) and deoxycytidine (dC) to form dTMP and dCMP, which in cells rapidly become the negative-feedback end-products dTTP and dCTP. TK2 kinetic activity exhibits Hill coefficients of ∼0.5 (apparent negative...

  11. Expression of transferred thymidine kinase genes is controlled by methylation.

    OpenAIRE

    Christy, B.; Scangos, G

    1982-01-01

    Plasmid pTKx-1, containing the herpes simplex virus gene for thymidine kinase (TK) inserted into the BamHI site of plasmid pBR322, was introduced into Ltk- cells by calcium phosphate precipitation in the absence of carrier DNA. Line 101 is a TK+ derivative of Ltk- that contains multiple copies of pTKx-1 in a multimeric structure. A derivative of 101 that retained but no longer expressed the herpes simplex TK genes (termed 101BU1) and derivatives of line 101BU1 that reexpressed the genes (term...

  12. Deoxypyrimidine monophosphate bypass therapy for thymidine kinase 2 deficiency

    OpenAIRE

    Garone, Caterina; Garc??a-D??az, Beatriz; Emmanuele, Valentina; L??pez Garc??a, Luis Carlos; Tadesse, Saba; Akman, Hasan O.; Tanji, Kurenai; Quinzii, Catarina M.; Hirano, Michio

    2014-01-01

    Autosomal recessive mutations in the thymidine kinase 2 gene (TK2) cause mitochondrial DNA depletion, multiple deletions, or both due to loss of TK2 enzyme activity and ensuing unbalanced deoxynucleotide triphosphate (dNTP) pools. To bypass Tk2 deficiency, we administered deoxycytidine and deoxythymidine monophosphates (dCMP+dTMP) to the Tk2 H126N (Tk2 −/− ) knock-in mouse model from postnatal day 4, when mutant mice are phenotypically normal, but biochemically affected. Assessment of 13-day-...

  13. Structures of thymidine kinase 1 of human and mycoplasma origin

    DEFF Research Database (Denmark)

    Welin, Martin; Kosinska, Urszula; Mikkelsen, Nils-Egil;

    2004-01-01

    Cytosolic thymidine kinase, TK1, is a well-known cell cycle regulated enzyme of importance in nucleotide metabolism as well as an activator of antiviral and anticancer drugs as AZT. We have now determined the first structures of the TK1 family, the human and Ureaplasma urealyticum enzymes, in...... complex with the feedback inhibitor dTTP. The TK1s have a tetrameric structure where each subunit contains an alfa/beta-domain that is similar to ATPase domains of members of the RecA structural family and a domain of a new type containing a structural zinc. The zinc ion connects beta-structures at the...... differs fundamentally from the structures of the other deoxyribonucleoside kinases indicating a different evolutionary origin....

  14. Two thymidine kinases and one multisubstrate deoxyribonucleoside kinase salvage DNA precursors in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Anders Ranegaard Clausen, Anders Ranegaard; Girandon, Lenart; Ali, Ashfaq; Knecht, Wolfgang; Rozpedowska, Elzbieta; Sandrini, Michael Paolo; Andreasson, Erik; Munch-Petersen, Birgitte; Piskur, Jure

    2012-01-01

    Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (EC 2.7.1.145) salvage deoxyribonucleosides by transfer of a phosphate group to the 5' of a deoxyribonucleoside. This salvage pathway is well characterized in...... mammals, but in contrast, little is known about how plants salvage deoxyribonucleosides. We show that during salvage, deoxyribonucleosides can be phosphorylated by extracts of Arabidopsis thaliana into corresponding monophosphate compounds with an unexpected preference for purines over pyrimidines....... Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome, we identified two types of genes that could encode enzymes which are involved in the salvage of deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1 (EC 2...

  15. Two thymidine kinases and one multisubstrate deoxyribonucleoside kinase salvage DNA precursors in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Clausen, Anders R.; Girandon, Lenart; Ali, Ashfaq; Knecht, Wolfgang; Rozpedowska, Elzbieta; Sandrini, Michael; Andreasson, Erik; Munch‐Petersen, Birgitte; Piskur, Jure

    2012-01-01

    Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (EC 2.7.1.145) salvage deoxyribonucleosides by transfer of a phosphate group to the 5′ of a deoxyribonucleoside. This salvage pathway is well characterized in...... mammals, but in contrast, little is known about how plants salvage deoxyribonucleosides. We show that during salvage, deoxyribonucleosides can be phosphorylated by extracts of Arabidopsis thaliana into corresponding monophosphate compounds with an unexpected preference for purines over pyrimidines....... Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome, we identified two types of genes that could encode enzymes which are involved in the salvage of deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1 (EC 2...

  16. Mutation hot spots in the canine herpesvirus thymidine kinase gene.

    Science.gov (United States)

    Yamada, Shinya; Matsumoto, Yasunobu; Takashima, Yasuhiro; Otsuka, Haruki

    2005-08-01

    The guanine and cytosine content (GC-content) of alpha-herpesvirus genes are highly variable despite similar genome structures. It is known that drug resistant HSV, which has the genome with a high GC-content (approximately 70%), commonly includes frameshift mutations in homopolymer stretches of guanine (G) and cytosine (C) within the thymidine kinase (TK) gene. However, whether such mutation hotspots exist in the TK gene of canine herpesvirus (CHV) which has a low GC-content was unknown. In this study, we investigated mutations in the TK gene of CHV. CHV was passaged in the presence of iodo-deoxyuridine (IDU), and IDU-resistant clones were isolated. In all IDU-resistant virus clones, mutations in the TK gene were observed. The majority of these mutations were frameshift mutations of an adenine (A) insertion or deletion within either of 2 stretches of eight A's in the TK gene. It was demonstrated that CHV TK mutations frequently occur at a limited number of hot spots within long homopolymer nucleotide stretches. PMID:15965615

  17. Elevated serum thymidine kinase activity in canine splenic hemangiosarcoma*.

    Science.gov (United States)

    Thamm, D H; Kamstock, D A; Sharp, C R; Johnson, S I; Mazzaferro, E; Herold, L V; Barnes, S M; Winkler, K; Selting, K A

    2012-12-01

    Thymidine kinase 1 (TK1) is a soluble biomarker associated with DNA synthesis. This prospective study evaluated serum TK1 activity in dogs presenting with hemoabdomen and a splenic mass. An ELISA using azidothymidine as a substrate was used to evaluate TK1 activity. Sixty-two dogs with hemoabdomen and 15 normal controls were studied. Serum TK1 activity was significantly higher in dogs with hemangiosarcoma (HSA) than in normal dogs (mean ± SEM = 17.0 ± 5.0 and 2.01 ± 0.6, respectively), but not dogs with benign disease (mean ± SEM = 10.0 ± 3.3). Using a cut-off of 6.55 U/L, TK activity demonstrated a sensitivity of 0.52, specificity of 0.93, positive predictive value of 0.94 and negative predictive value of 0.48 for distinguishing HSA versus normal. When interval thresholds of 7.95 U/L were used together, diagnostic utility was increased. Serum TK1 evaluation may help to discriminate between benign disease and HSA in dogs with hemoabdomen and a splenic mass. PMID:22236280

  18. Identification and structural analysis of the thymidine kinase gene of infectious bovine rhinotracheitis virus

    International Nuclear Information System (INIS)

    In an attempt to understand the gene expression of the infectious bovine rhinotracheitis virus (IBRV), the viral thymidine kinase ge (tk), a well regulated viral gene, has been cloned in Escherichia coli HB101 by integrating partially Sau 3A-digested DNA fragments into a cosmid vector, pJB8. Recombinant cosmids were further analyzed by restriction digestions and by Southern blot hybridization. Results showed that this plasmid library comprised all of the IBRV genome with the exception of both termini. The individual recombinant cosmid clones were then transformed to E. coli tdk- mutant strains, Ky895 or C600 tdk- for the selection of the IBRV tk gene. The physical location of the viral DNA inserts of one of the clones, pIBR5, was determined and sequences complementing the tk activity were isolated by subcloning. The E. coli mutant strain mutant strain C600 tdk- harboring pIBRTK partially restores the tk activity by exhibiting a three and half fold increase in the level of the incorporation of [3H]thymidine into bacterial DNA over that of the C600 tdk- mutant. The plasmid, pIBRTK was also used as a probe to examine the expression of IBRV-tk gene in infected MDBK cells. A species of mRNA from infected cells with molecular weight of 2.2 kb was obtained when the total RNA or polyA-enriched RNA were electrophoresized in agarose gels and hybridized with 32P-pIBRTK DNA

  19. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten;

    2010-01-01

    Bacteria and Eukarya share essentially the same family of protein-serine/threonine kinases, also known as the Hanks-type kinases. However, when it comes to protein-tyrosine phosphorylation, bacteria seem to have gone their own way. Bacterial protein-tyrosine kinases (BY-kinases) are bacterial...... enzymes that are unique in exploiting the ATP/GTP-binding Walker motif to catalyze phosphorylation of protein tyrosine residues. Characterized for the first time only a decade ago, BY-kinases have now come to the fore. Important regulatory roles have been linked with these enzymes, via their involvement...... in exopolysaccharide production, virulence, DNA metabolism, stress response and other key functions of the bacterial cell. BY-kinases act through autophosphorylation (mainly in exopolysaccharide production) and phosphorylation of other proteins, which have in most cases been shown to be activated by...

  20. Efficacy of anise oil, dwarf-pine oil and chamomile oil against thymidine-kinase-positive and thymidine-kinase-negative herpesviruses.

    Science.gov (United States)

    Koch, Christine; Reichling, Jürgen; Kehm, Roland; Sharaf, Mona M; Zentgraf, Hanswalter; Schneele, Jürgen; Schnitzler, Paul

    2008-11-01

    The effect of anise oil, dwarf-pine oil and chamomile oil against different thymidine-kinase-positive (aciclovir-sensitive) and thymidine-kinase-negative (aciclovir-resistant) herpes simplex virus type 1 (HSV-1) strains was examined. Clinical HSV-1 isolates containing frameshift mutations in the thymidine kinase (TK) gene, an insertion or a deletion, yield a non-functional thymidine kinase enzyme resulting in phenotypical resistance against aciclovir. The inhibitory activity of three different essential oils against herpes simplex virus isolates was tested in-vitro using a plaque reduction assay. All essential oils exhibited high levels of antiviral activity against aciclovir-sensitive HSV strain KOS and aciclovir-resistant clinical HSV isolates as well as aciclovir-resistant strain Angelotti. At maximum noncytotoxic concentrations of the plant oils, plaque formation was significantly reduced by 96.6-99.9%, when herpesviruses were preincubated with drugs before attachment to host cells. No significant effect on viral infectivity could be achieved by adding these compounds during the replication phase. These results indicate that anise oil, dwarf-pine oil and chamomile oil affected the virus by interrupting adsorption of herpesviruses and in a different manner than aciclovir, which is effective after attachment inside the infected cells. Thus the investigated essential oils are capable of exerting a direct effect on HSV and might be useful in the treatment of drug-resistant viruses. Chamomile oil did not reveal any irritating potential on hen's egg chorioallantoic membrane, demonstrated the highest selectivity index among the oils tested and was highly active against clinically relevant aciclovir-resistant HSV-1 strains. PMID:18957177

  1. Substrate specificity of three viral thymidine kinases (TK): vaccinia virus TK, feline herpesvirus TK, and canine herpesvirus TK.

    Science.gov (United States)

    Solaroli, N; Johansson, M; Balzarini, J; Karlsson, A

    2006-01-01

    In search of novel suicide gene candidates we have cloned and characterized thymidine kinases from three viruses; vaccinia virus TK (VVTK), feline herpesvirus TK (FHV-TK), and canine herpesvirus TK (CHV-TK). Our studies showed that VVTK primarily is a thymidine kinase, with a substrate specificity mainly restricted to dThd and only minor affinity for dCyd. VVTK also is related closely to mammalian thymidine kinase 1 (TK1), with 66% identity and 75% general homology. Although CHV-TK and FHV-TK are sequence related to herpes simplex virus types 1 thymidine kinase (HSV1-TK), with 31% and 35% identity and a general similarity of 54%, the substrate specificity of these enzymes was restricted to dThd and thymidine analogs. PMID:17065088

  2. Genetic determinants of growth phase-dependent and adenovirus 5-responsive expression of the Chinese hamster thymidine kinase gene are contained within thymidine kinase mRNA sequences.

    OpenAIRE

    Lewis, J. A.; Matkovich, D A

    1986-01-01

    We have constructed a chimeric thymidine kinase (TK) minigene, pHe delta 6Ha, which combines the complete coding and 3' noncoding regions of a Chinese hamster TK cDNA with the promoter region and 5' untranslated region of the TK gene of herpes simplex virus type 1. We have transformed rat 4 cells to Tk+ with this gene and analyzed the pattern of TK gene expression in these transformants under various conditions of in vitro cell culture. We find that TK gene expression in these Tk+ transforman...

  3. Comparative active-site mutation study of human and Caenorhabditis elegans thymidine kinase 1

    DEFF Research Database (Denmark)

    Skovgaard, Tine; Uhlin, Ulla; Munch-Petersen, Birgitte

    2012-01-01

    ligands. To improve our understanding of TK1 substrate specificity, we performed a detailed, mutation-based comparative structure-function study of the active sites of two thymidine kinases: HuTK1 and Caenorhabditis elegans TK1 (CeTK1). Specifically, mutations were introduced into the hydrophobic pocket...... surrounding the substrate base. In CeTK1, some of these mutations led to increased activity with deoxycytidine and deoxyguanosine, two unusual substrates for TK1-like kinases. In HuTK1, mutation of T163 to S resulted in a kinase with a 140-fold lower K(m) for the antiviral nucleoside analogue 3'-azido-3...

  4. Serum thymidine kinase in vitamin B12 deficiency

    International Nuclear Information System (INIS)

    In DNA synthesis deoxythymidine kinase (TK) catalyses the conversion of deoxythymidine to deoxythymidine monophosphate (dTMP) via the 'salvage pathway'. Serum deoxythymidine kinase (S=TK) was measured in this study in 75 patients with vitamin B12 deficiency by a new, very sensitive method, using 125I-deoxyuridine as substrate. Elevated S-TK levels were found in those patients who had developed haemolysis and anaemia and the more advanced the disease the higher the S-TK value. Thus there was a highly significant correlation between S-TK, haemoglobin level and lactic dehydrogenase activity. These findings are consistent with the theory that elevated levels of S-TK are due to release from unstable proliferating tissue. (author)

  5. High efficient generation of replication-defective adenoviruses containing thymidine kinase by homogeneous recombination in bacteria

    Institute of Scientific and Technical Information of China (English)

    CONG Tie-chuan; LU Zhe-ming; LI Yong; ZHENG Li; QIN Yong

    2007-01-01

    Background Suicide gene therapy is a widely used molecular treatment for head and neck cancer. In this study, we try to use the method of homogenous recombination in bacteria to clone thymidine kinase gene (tk)-a kind of suicide gene to adenovirus backbone vectors for the construction of replication-defective adenoviruses.Methods pAdTrack-CMV/tk was constructed through subclone of a restriction endonuclease fragment including thymidine kinase gene from plasmid pCMV-tk to another plasmid pAdTrack-CMV, and then co-transfected with supercoiled pAdEasy-1, which was an adenoviral backbone vector except for deletions of E1 and E3, to competent E.coli BJ5183 for homogenous recombination using electroporation procedure. With the same method, pAdTrack-CMV was also co-transformed with pAdEasy-1 for homogenous recombination in BJ5183. Identified with restriction endonuclease Pacl and polymerase chain reaction (PCR), plasmids pAd-GFP/tk and pAd-GFP were successfully constructed. Each of them was digested with Pacl and sequently transfected into human embryo kidney 293 cells (HEK293) using Lipofectamine 2000.Results Comet-like adenovirus-producing foci of Ad-GFP/tk and Ad-GFP were observed after 5 to 7 days of cell culture.After twelve days of packaging, the replication-defective adenoviruses were collected. Identified with PCR, thymidine kinase gene was successfully constructed into Ad-GFP/tk.Conclusion The replication-defective adenoviruses containing thymidine kinase can be constructed more easily by homogenous recombination in bacteria than conventional techniques.

  6. Endothelin-1 activates phospholipase D and thymidine incorporation in fibroblasts overexpressing protein kinase C beta 1.

    OpenAIRE

    Pai, J K; Dobek, E A; Bishop, W R

    1991-01-01

    Endothelins (ETs) are a family of extremely potent vasoconstrictor peptides. In addition, ET-1 acts as a potent mitogen and activates phospholipase C in smooth muscle cells and fibroblasts. We examined the effects of ET-1 on phosphatidylcholine (PC) metabolism and thymidine incorporation in control Rat-6 fibroblasts and in cells that overexpress protein kinase C beta 1 (PKC). PC pools were labeled with [3H]myristic acid, and formation of phosphatidylethanol (PEt), an unambiguous marker of pho...

  7. Structure Guided Development of Novel Thymidine Mimetics targeting Pseudomonas aeruginosa Thymidylate Kinase: from Hit to Lead Generation

    OpenAIRE

    Choi, Jun Yong; Plummer, Mark S.; Starr, Jeremy; Desbonnet, Charlene R.; Soutter, Holly; Chang, Jeanne; Miller, J. Richard; Dillman, Keith; Miller, Alita A.; Roush, William R.

    2012-01-01

    Thymidylate kinase (TMK) is a potential chemotherapeutic target because it is directly involved in the synthesis of an essential component, thymidine triphosphate, in DNA replication. All reported TMK inhibitors are thymidine analogs, which might retard their development as potent therapeutics due to cell permeability and off-target activity against human TMK. A small molecule hit (1, IC50 = 58 μM), which has reasonable inhibition potency against Pseudomonas aeruginosa TMK (PaTMK), was identi...

  8. Studies of the cytosolic thymidine kinase in human cells and comparison to the recombinantly expressed enzyme

    DEFF Research Database (Denmark)

    Kock Jensen, Helle

    Thymidine kinase (TK) is a key enzyme in the salvage pathway of the nucleoside metabolism catalyzing the first phosphorylation step in TTP synthesis. Human cytosolic TK (TKl) is highly cell cycle regulated. TKl is regulated on many different levels of expression and isoforms with altered enzymatic...... identical but further investigations showed some interesting differences. Recombinant TKl is about 10 fold more sensitive towards TTP as inhibitor. Furthermore the effect of removal of ATP from the native TKl on the enzyme kinetics and native molecular weight was not found for recombinant TKl. Native TKl...

  9. Treatment of rat gliomas with recombinant retrovirus harboring Herpes simplex virus thymidine kinase suicide gene

    International Nuclear Information System (INIS)

    The retrovirus vector containing Herpes simplex virus type 1 thymidine kinase (HSVtk) gene was constructed. The vector was transfected into the packaging cell line PG13. It was shown that individual transfected cells differ in the production of recombinant retrovirus and in their susceptibility to be killed by ganciclovir. Recombinant retrovirus with a gibbon envelope was able to transduced the HSVtk gene into rat glioma cells. In vivo studies confirmed the ability of intraperitoneal ganciclovir administration to influence subcutaneous and intracerebral tumors developed after injection of C6 rat glioma cells with subsequent injection of HSVtk retrovirus producing cells. (author)

  10. X rays induce interallelic homologous recombination at the human thymidine kinase gene.

    OpenAIRE

    Benjamin, M B; Little, J B

    1992-01-01

    We have developed a human lymphoblast cell line for the study of interchromosomal homologous recombination at the endogenous thymidine kinase (tk) gene on chromosome 17 (M. B. Benjamin, H. Potter, D. W. Yandell, and J. B. Little, Proc. Natl. Acad. Sci. USA 88:6652-6656, 1991). This cell line (designated 6:86) carries unique heterozygous frameshift mutations in exons 4 and 7 of its endogenous tk alleles and can revert to TK+ by frame-restoring mutations, gene conversion, or reciprocal recombin...

  11. Transfer of herpes simplex virus thymidine kinase synthesized in bacteria by a high-expression plasmid to tissue culture cells by protoplast fusion

    International Nuclear Information System (INIS)

    The introduction of a protein into living tissue culture cells may permit the in vivo study of functions of the protein. The authors have previously described a high-efficiency-expression plasmid, pHETK2, containing the herpes simplex virus type 1 thymidine kinase (TK) gene which, upon temperature induction, causes TK to be synthesized as greater than 4% of the bacterial protein. In this report it is shown that enzymatically active TK was transferred to mouse Ltk- cells by polyethylene glycol-mediated fusion with protoplasts prepared from bacteria containing induced levels of TK. The presence of TK in the Ltk- cells was detected by the incorporation of [3H]thymidine into cell nuclei as measured by autoradiography

  12. Bacterial incorporation of tritiated thymidine and populations of bacteriophagous fauna in the rhizosphere of wheat

    DEFF Research Database (Denmark)

    Christensen, Henrik; Griffiths, Bryan; Christensen, Søren

    1992-01-01

    Bacterial and microfaunal populations, and bacterial productivity measured by tritiated thymidine (3HTdr) incorporation, in the rhizosphere of wheat seedlings were measured. Soil from planted pots was fractionated into rhizosphere and non-rhizosphere (bulk) soil, while unplanted soil was taken from...... pots without plants. Total bacterial counts and biovolume did not differ between fractions but viable (plate) counts were 8 times higher in the rhizosphere compared to bulk and unplanted soil. 3HTdr was incorporated at a constant rate with low variability in bulk or unplanted soil. In rhizosphere soil...... 3HTdr incorporation was lower than in bulk or unplanted soils and showed high variability. The populations of bacterial-feeding protozoa and nematodes indicated that rhizosphere bacterial activity was actually 3–4 times greater in rhizosphere than bulk soil in accordance with the results of the...

  13. Chromatin structure is required to block transcription of the methylated herpes simplex virus thymidine kinase gene

    International Nuclear Information System (INIS)

    Inhibition of herpes simplex virus (HSV) thymidine kinase (TK) gene transcription (pHSV-106, pML-BPV-TK4) by DNA methylation is an indirect effect, which occurs with a latency period of ∼ 8 hr microinjection of the DNA into TK- rat 2 and mouse LTK- cells. The authors have strong evidence that chromatin formation is critical for the transition of the injected DNA from methylation insensitivity to methylation sensitivity. Chromatin was reconstituted in vitro by using methylated and mock-methylated HSV TK DNA and purified chicken histone octamers. After microinjection, the methylated chromatin was always biologically inactive, as tested by autoradiography of the cells after incubation with [3H]thymidine and by RNA dot blot analysis. However, in transformed cell lines, reactivation of the methylated chromatic occurred after treatment with 5-azacytidine. Furthermore, integration of the TK chromatin into the host genome is not required to block expression of the methylated TK gene. Mouse cells that contained the pML-BPV-TK4 chromatin permanently in an episomal state also did not support TK gene expression as long as the TK DNA remained methylated

  14. Detection of bovine herpesvirus 4 glycoprotein B and thymidine kinase DNA by PCR assays in bovine milk

    NARCIS (Netherlands)

    Wellenberg, G.J.; Verstraten, E.; Belak, S.; Verschuren, S.B.E.; Rijsewijk, F.A.M.; Peshev, R.; Oirschot, van J.T.

    2001-01-01

    A polymerase chain reaction (PCR) assay was developed to detect bovine herpesvirus 4 (BHV4) glycoprotein B (gB) DNA, and a nested-PCR assay was modified for the detection of BHV4 thymidine kinase (TK) DNA in bovine milk samples. To identify false-negative PCR results, internal control templates were

  15. Secretion of thymidine kinase to increase the effectivity of suicide gene therapy results in the loss of enzymatic activity

    NARCIS (Netherlands)

    Beerens, A. M. J.; Rots, M. G.; Bermudez, B.; De Vries, E. F. J.; Haisma, H. J.

    2008-01-01

    Low efficiency of gene transfer is one of the major limitations of gene therapy. A solution to this problem may be transmission; by modification of the transgene, the gene product can be secreted and internalized by the surrounding cells. Cancer gene therapy using the herpes simplex thymidine kinase

  16. Plant thymidine kinase 1: a novel efficient suicide gene for malignant glioma therapy

    DEFF Research Database (Denmark)

    Khan, Z.; Knecht, Wolfgang; Willer, Mette;

    2010-01-01

    -type cells. In addition, when neural progenitor cells were used as delivery vectors for toTK1 in intracranial GBM xenografts in nude rats, substantial attenuation of tumor growth was achieved in animals exposed to AZT, and survival of the animals was significantly improved compared with controls. The novel...... for central nervous system (CNS) tumors, several obstacles have been encountered such as inefficient gene transfer to the tumor cells, limited prodrug penetration into the CNS, and inefficient enzymatic activity of the suicide gene. We report here the cloning and successful application of a novel...... thymidine kinase 1 (TK1) from the tomato plant, with favorable characteristics in vitro and in vivo. This enzyme (toTK1) is highly specific for the nucleoside analog prodrug zidovudine (azidothymidine, AZT), which is known to penetrate the blood-brain barrier. An important feature of toTK1 is that it...

  17. Effect of C-terminal of human cytosolic thymidine kinase (TK1) on in vitro stability and enzymatic properties

    DEFF Research Database (Denmark)

    Munch-Petersen, Birgitte; Munch-Petersen, Sune; Berenstein, Dvora;

    2006-01-01

    Thymidine kinase (TK1) is a key enzyme in the salvage pathway of nucleotide metabolism and catalyzes the first rate-limiting step in the synthesis of dTTP, transfer of a gamma-phosphate group from a nucleoside triphosphate to the 5′-hydroxyl group of thymidine, thus forming dTMP. TK1 is cytosolic...... and its activity fluctuates during cell cycle coinciding with the DNA synthesis rate and disappears during mitosis. This fluctuation is important for providing a balanced supply of dTTP for DNA replication. The cell cycle specific activity of TK1 is regulated at the transcriptional level, but...

  18. Cell-cycle-specific interaction of nuclear DNA-binding proteins with a CCAAT sequence from the human thymidine kinase gene

    International Nuclear Information System (INIS)

    Induction of thymidine kinase parallels the onset of DNA synthesis. To investigate the transcriptional regulation of the thymidine kinase gene, the authors have examined whether specific nuclear factors interact in a cell-cycle-dependent manner with sequences upstream of this gene. Two inverted CCAAT boxes near the transcriptional initiation sites were observed to form complexes with nuclear DNA-binding proteins. The nature of the complexes changes dramatically as the cells approach DNA synthesis and correlates well with the previously reported transcriptional increase of the thymidine kinase gene

  19. Glucose transport and apoptosis after gene therapy with HSV thymidine kinase

    International Nuclear Information System (INIS)

    The relation between tumour metabolism and induction of apoptosis by gene therapy was investigated in a rat Morris hepatoma (MH3924A) model expressing the HSV thymidine kinase (HSVtk) gene. In vivo the amount of glucose transporter (GLUT1 and GLUT3 isoforms) expressing cells was determined in tumours of untreated and treated animals using immunohistochemistry. In vitro uptake studies with 2-fluoro-2-deoxy-D-glucose (FDG), 3-O-methylglucose and thymidine (TdR) and a TUNEL (TdT-mediated dUTP nick end labelling) assay for the assessment of apoptosis were done immediately and 24 h after treatment of the recombinant cells with different doses of ganciclovir (GCV). Immunohistochemistry revealed a significant increase in GLUT1 in treated tumours which showed enhanced transport activity for FDG. In vitro the FDG and 3-O-methylglucose uptake increased to 186% when compared with that of the non-treated cells immediately after incubation with GCV. However, 24 h later the FDG uptake had declined to its normal level, whereas the accumulation of 3-O-methylglucose remained elevated. The uptake of TdR, which was determined simultaneously, decreased in the acid-insoluble fraction of the cells to 27% and 11%, respectively, immediately and 24 h after therapy, while in the acid-soluble fraction it increased to 229% and to 167%, respectively. Employing the TUNEL technique, 25% of cells were found to be apoptotic 24 h after the termination of GCV treatment. Inhibition of glucose transport by cytochalasin B or competition with deoxyglucose resulted in a 78% (cytochalasin B) and 88% (deoxyglucose) decrease in FDG uptake in the recombinant hepatoma cells and in an increase in the apoptotic cell fraction. It is concluded that inhibition of enhanced glucose transport in GCV-treated cells increased apoptosis. Therefore, enhanced glucose transport seems to represent a stress reaction of tumour cells dedicated for the prevention of cell death. (orig.)

  20. Construction and identification of recombinant vectors carrying herpes simplex virus thymidine kinase and cytokine genes expressed in gastric carcinoma cell line SGC7901

    OpenAIRE

    Zhang, Jian-Hua; Wan, Ming-Xi; Yuan, Jia-Ying; Pan, Bo-Rong

    2004-01-01

    AIM: To construct and identify the recombinant vectors carrying herpes simplex virus thymidine kinase (HSV-TK) and tumor necrosis factor alpha (TNF-α) or interleukin-2 (IL-2) genes expressed in gastric carcinoma cell line SGC7901.

  1. Expression of the Apoptosis Inhibitor Survivin and its correlation with Thymidine Kinase and Axillary Lymph Node Metastasis in Breast Cancer

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    1 Introduction Many molecular factors have been demonstrated to interfere with cellular proliferation and programmed cell death.One of these factors is a recently discovered member of the “inhibitor of apoptosis protein(IAP)” called survivin. Survivin is abundantly expressed in most solid and hematologic malignancies, but undetectable in normal adult tissues. Interference with survivin function induces pleiotropic cell-division defects and apoptosis. Cytosolic thymidine kinase (TK) is a marker for prolifera...

  2. Cloning of Thymidine Kinase Gene of Duck Plague Virus Using Degenerate PCR

    Institute of Scientific and Technical Information of China (English)

    HAN Xian-jie; WANG Jun-wei

    2005-01-01

    The DNA of duck plague virus (DPV) thymidine kinase (TK) gene was cloned and sequenced from a vaccine virus in the study. Degenerate oligonucleotide primers for the consensus site of herpesvirus UL24, TK, and glycoprotein H(gH) gene were used in the polymerase chain reaction (PCR) to amplify DNA product with 3 741-base-pairs (bp) in size. DNA sequence analysis revealed a 1 077-base-pairs (bp) open reading frame (ORF) encoding a 358 amino acid polypeptide homologous to herpesvirus TK proteins. The predicted TK protein shared 31.2, 41.3, 35.7, 37.4, and 28.4% identity with herpes simplex virus typel, equine herpesvirus type 4, Marek's disease virus 2, herpesvirus turkey, and infectious laryngotracheitis virus, respectively. Comparison of the amino acid sequences of other herpesvirus TK proteins showed that these proteins were not conserved on the whole, otherwise the portion of the TK proteins corresponding to the nucleotide binding domain and the nucleoside binding site were highly conserved among herpesvirus. Comparison with the amino acid sequences of the conserved nucleotide and nucleoside binding domains of other eleven herpesvirus TK proteins to the predicted DPV peptide confirmed its identity as the DPV TK protein.

  3. Loss of arylformamidase with reduced thymidine kinase expression leads to impaired glucose tolerance

    Directory of Open Access Journals (Sweden)

    Alison J. Hugill

    2015-11-01

    Full Text Available Tryptophan metabolites have been linked in observational studies with type 2 diabetes, cognitive disorders, inflammation and immune system regulation. A rate-limiting enzyme in tryptophan conversion is arylformamidase (Afmid, and a double knockout of this gene and thymidine kinase (Tk has been reported to cause renal failure and abnormal immune system regulation. In order to further investigate possible links between abnormal tryptophan catabolism and diabetes and to examine the effect of single Afmid knockout, we have carried out metabolic phenotyping of an exon 2 Afmid gene knockout. These mice exhibit impaired glucose tolerance, although their insulin sensitivity is unchanged in comparison to wild-type animals. This phenotype results from a defect in glucose stimulated insulin secretion and these mice show reduced islet mass with age. No evidence of a renal phenotype was found, suggesting that this published phenotype resulted from loss of Tk expression in the double knockout. However, despite specifically removing only exon 2 of Afmid in our experiments we also observed some reduction of Tk expression, possibly due to a regulatory element in this region. In summary, our findings support a link between abnormal tryptophan metabolism and diabetes and highlight beta cell function for further mechanistic analysis.

  4. Identification of a Bohle iridovirus thymidine kinase gene and demonstration of activity using vaccinia virus.

    Science.gov (United States)

    Coupar, B E H; Goldie, S G; Hyatt, A D; Pallister, J A

    2005-09-01

    In recent years interest in the family Iridoviridae has been renewed by the identification of a number of viruses, particularly from the genus Ranavirus, associated with disease in a range of poikilotherms. Ranaviruses have been isolated from amphibian, piscine and reptilian species. Here we describe an open reading frame (ORF) identified in the genome of Bohle iridovirus (BIV) which contains a nucleotide binding motif conserved within the thymidine kinase (TK) genes of iridoviruses from other genera (lymphocystis disease virus, LCDV, type species of the genus Lymphocystivirus; Chilo iridescent virus, CIV, type species of the genus Iridovirus). The ability of this putative gene to express a functional TK was confirmed by rescue of a TK negative mutant vaccinia virus in the presence of selective media, when expression was controlled by a vaccinia virus promoter. The sequence of the BIV TK was compared with the homologous sequences from epizootic haematopoietic necrosis virus (EHNV), a virus associated with disease in fish, from Wamena iridovirus (WIV) associated with systemic disease in green pythons, and from frog virus 3 (FV3) the ranavirus type species. Comparisons between these sequences and those available from other ranaviruses, other iridoviruses, other DNA viruses and cellular TKs are presented. PMID:15883656

  5. A rapid method for identifying the thymidine kinase genes of avipoxviruses.

    Science.gov (United States)

    Schnitzlein, W M; Ghildyal, N; Tripathy, D N

    1988-08-01

    The thymidine kinase (TK) genes of poxviruses can be rapidly located without using TK- mutants or having to restriction map and clone the viral genomes. Identification of the TK gene is based on in situ gel hybridization with an end-labelled degenerate oligonucleotide probe, representing a consensus sequence near the 3' end of the gene. Restriction fragments of the viral DNAs are electrophoresed in agarose gels and annealed with the probe. Using this method, the TK genes of fowl pox (FPV) and quail pox (QPV) viruses were initially localized to HindIII fragments of approximately 3.8 and 6.7 kb, respectively. After inserting these fragments into pUC 19, recombinant plasmids containing the TK genes were screened by a modified in situ gel annealing procedure. Restriction mapping of the two cloned fragments and subsequent hybridization analysis more precisely placed at least the 3' portion of the FPV and QPV TK genes within a 1.4 kb ClaI-XbaI and 1.7 kb ClaI-PstI fragment, respectively. The site of the FPV TK gene was verified by comparison to the mapped position of the similar gene in an Australian FPV. The location of the QPV TK gene was confirmed by hybridization with the FPV TK gene, despite the apparent divergency of these two genes. PMID:2846602

  6. Ganciclovir uptake in human mammary carcinoma cells expressing herpes simplex virus thymidine kinase

    International Nuclear Information System (INIS)

    Assessment of suicide enzyme activity would have considerable impact on the planning and the individualization of suicide gene therapy of malignant tumors. This may be done by determining the pharmacokinetics of specific substrates. We generated ganciclovir (GCV)-sensitive human mammary carcinoma cell lines after transfection with a retroviral vector bearing the herpes simplex virus thymidine kinase (HSV-tk) gene. Thereafter, uptake measurements and HPLC analyses were performed up to 48 h in an HSV-tk-expressing cell line and in a wild-type cell line using tritiated GCV. HSV-tk-expressing cells showed higher GCV uptake and phosphorylation than control cells, whereas in wild-type MCF7 cells no phosphorylated GCV was detected. In bystander experiments the total GCV uptake was related to the amount of HSV-tk-expressing cells. Furthermore, the uptake of GCV correlated closely with the growth inhibition (r=0.92). Therefore, the accumulation of specific substrates may serve as an indicator of the HSV-tk activity and of therapy outcome. Inhibition and competition experiments demonstrated slow transport of GCV by the nucleoside carriers. The slow uptake and low affinity to HSV-tk indicate that GCV is not an ideal substrate for the nucleoside transport systems or for HSV-tk. This may be the limiting factor for therapy success, necessitating the search for better substrates of HSV-tk

  7. Herpes simplex virus thymidine kinase and ganciclovir suicide gene therapy for human pancreatic cancer

    Institute of Scientific and Technical Information of China (English)

    Jing Wang; Xiao-Xuan Lu; Dao-Zhen Chen; Shu-Feng Li; Li-Shan Zhang

    2004-01-01

    AIM: To investigate the in vitro effects of suicide gene therepy system of herpes simplex virus thymidine kinase gene (HSV-TK) in combination with the treatment of nucleotide analog-ganciclovir (GCV) on human pancreatic cancer, and to provide a novel clinical therapeutic method for human pancreatic cancer.METHODS: We used a replication defective recombinant retrovirus vector GINaTK (bearing HSV-TK gene) to make packaging cell PA317 produce progeny virions. We then transferred the HSV-TK gene to target cells SW1990 using these progeny virions, and treated these gene-modified tumor cells with GCV to study the sensitivity of the cells to GCV and their bystander effects by routine MTT-method.RESULTS: Packaging cell PA317/TK was successfully constructed, and we acquired SW1990/TK through virus progeny infection. These gene-modified pancreatic cancer cells were sensitive to the treatment of GCV compared with unmodified tumor cells (t=4.15, n=10, P<0.0025). We also observed a remarkable bystander effect by mixing two kinds of cells at different ratio.CONCLUSION: Our data demonstrate that HSV-TK/GCV suicide gene therapy system is effective for treating experimental human pancreatic cancer, which is largely resistant to the common therapies, so the suicide gene therapy system may be a potential treatment approach for pancreatic cancer.

  8. Bacterial production determined by [3H]thymidine incorporation in field rhizospheres as evaluated by comparison to rhizodeposition

    DEFF Research Database (Denmark)

    Christensen, Henrik; Rønn, Regin; Ekelund, Flemming; Christensen, Søren

    1995-01-01

    .2–15 × 104 cells g-1 soil) as well as elevated thymidine incorporation (9.7–12 pmol g-1 soil) in rhizosphere soil compared to bulk soil. Rhizodeposition, as determined by several pulse labellings with 14CO2, was estimated to be 412 µg C g-1 dry wt soil in the 0–15 cm layer. Bacterial production, as......In a sandy loam soil cropped to barley bacterial production in the rhizosphere was compared to the results of a parallel investigation on rhizodeposition. Bacterial production was stimulated in the rhizosphere as revealed by an increased biomass of bacteria (643–883 µg C g-1 soil) and protozoa (7...... determined by incorporation of 3H-labelled thymidine converted to bacterial C, revealed a plant-induced formation of 1348 µg bacterial C g-1 soil in the 0–15 cm layer. This is probably the first estimate for bacterial production based on thymidine incorporation which has been compared to an estimate of C...

  9. New Variants of Tomato Thymidine Kinase 1 Selected for Increased Sensitivity of E. coli KY895 towards Azidothymidine

    Energy Technology Data Exchange (ETDEWEB)

    Slot Christiansen, Louise [Department of Biology, Lund University, Lund 22362 (Sweden); Lund Protein Production Platform, Lund University, Lund 22362 (Sweden); Egeblad, Louise [Lund Protein Production Platform, Lund University, Lund 22362 (Sweden); Munch-Petersen, Birgitte [Department of Science, Systems and Models, Roskilde University, Roskilde 4000 (Denmark); Piškur, Jure [Department of Biology, Lund University, Lund 22362 (Sweden); Knecht, Wolfgang, E-mail: Louise.Slot_Christiansen@biol.lu.se [Department of Biology, Lund University, Lund 22362 (Sweden); Lund Protein Production Platform, Lund University, Lund 22362 (Sweden)

    2015-06-08

    Nucleoside analogues (NA) are prodrugs that are phosphorylated by deoxyribonucleoside kinases (dNKs) as the first step towards a compound toxic to the cell. During the last 20 years, research around dNKs has gone into new organisms other than mammals and viruses. Newly discovered dNKs have been tested as enzymes for suicide gene therapy. The tomato thymidine kinase 1 (ToTK1) is a dNK that has been selected for its in vitro kinetic properties and then successfully been tested in vivo for the treatment of malignant glioma. We present the selection of two improved variants of ToTK1 generated by random protein engineering for suicide gene therapy with the NA azidothymidine (AZT). We describe their selection, recombinant production and a subsequent kinetic and biochemical characterization. Their improved performance in killing of E. coli KY895 is accompanied by an increase in specificity for the NA AZT over the natural substrate thymidine as well as a decrease in inhibition by dTTP, the end product of the nucleoside salvage pathway for thymidine. The understanding of the enzymatic properties improving the variants efficacy is instrumental to further develop dNKs for use in suicide gene therapy.

  10. New Variants of Tomato Thymidine Kinase 1 Selected for Increased Sensitivity of E. coli KY895 towards Azidothymidine

    International Nuclear Information System (INIS)

    Nucleoside analogues (NA) are prodrugs that are phosphorylated by deoxyribonucleoside kinases (dNKs) as the first step towards a compound toxic to the cell. During the last 20 years, research around dNKs has gone into new organisms other than mammals and viruses. Newly discovered dNKs have been tested as enzymes for suicide gene therapy. The tomato thymidine kinase 1 (ToTK1) is a dNK that has been selected for its in vitro kinetic properties and then successfully been tested in vivo for the treatment of malignant glioma. We present the selection of two improved variants of ToTK1 generated by random protein engineering for suicide gene therapy with the NA azidothymidine (AZT). We describe their selection, recombinant production and a subsequent kinetic and biochemical characterization. Their improved performance in killing of E. coli KY895 is accompanied by an increase in specificity for the NA AZT over the natural substrate thymidine as well as a decrease in inhibition by dTTP, the end product of the nucleoside salvage pathway for thymidine. The understanding of the enzymatic properties improving the variants efficacy is instrumental to further develop dNKs for use in suicide gene therapy

  11. Spatial and temporal variations in bacterial macromolecule labeling with [methyl-3H]thymidine in a hypertrophic lake

    International Nuclear Information System (INIS)

    The incorporation of [methyl-3H]thymidine into three macromolecular fractions, designated as DNA, RNA, and protein, by bacteria from Hartbeespoort Dam, South Africa, was measured over 1 year by acid-base hydrolysis procedures. Samples were collected at 10 m, which was at least 5 m beneath the euphotic zone. On four occasions, samples were concurrently collected at the surface. Approximately 80% of the label was incorporated into bacterial DNA in surface samples. At 10 m, total incorporation of label into bacterial macromolecules was correlated to bacterial utilization of glucose. The labeling of DNA, which ranged between 0 and 78% of total macromolecule incorporation, was inversely related to glucose uptake, total thymidine incorporation, and euphotic zone algal production. With decreased DNA labeling, increasing proportions of label were found in the RNA fraction and proteins. Enzymatic digestion followed by chromatographic separation of macromolecule fragments indicated that DNA and proteins were labeled while RNA was not. The RNA fraction may represent labeled lipids or other macromolecules or both. The data demonstrated a close coupling between phytoplankton production and heterotrophic bacterial activity in this hypertrophic lake but also confirmed the need for the routine extraction and purification of DNA during [methyl-3H]thymidine studies of aquatic bacterial production

  12. X-rays induce interallelic homologous recombination at the human thymidine kinase gene

    International Nuclear Information System (INIS)

    The authors have developed a human lymphoblast cell line for the study of interchromosomal homologous recombination at the endogenous thymidine kinase (tk) gene on chromosome 17. This cell line (designated 6:86) carries unique heterozygous frameshift mutations in exons 4 and 7 of its endogenous tk alleles and can revert to TK+ by frame-restoring mutations, gene conversion, or reciprocal recombination. Line 6:86 reverts spontaneously to TK+ at a frequency of 10-7 to 10-8, and exposures to X-irradiation or the frameshift mutagen ICR-191 induce increased reversion frequencies in a dose-dependent manner. Another cell line (designated 4:2) carries a homozygous exon 7 frameshift and is not expected to revert through mechanisms other than frame-restoring mutation. Line 4:2 reverts to TK+ at a lower spontaneous frequency than does 6:86 but can be induced with similar kinetics by ICR-191. In contrast to line 6:86, however, X rays did not induce detectable reversion of line 4:2. We have characterized a number of 6:86-derived revertants by means of restriction fragment length polymorphism analysis at tk and linked loci, single-strand conformation polymorphisms, and direct transcript sequencing. For X rays, most revertants retain both original mutations in the genomic DNA, and a subset of these frameshift-retaining revertants produce frameshift-free message, indicating that reversion is the result of reciprocal recombination within the tk gene. Frame-restoring point mutations, restoration of original sequences, and phenocopy reversion by acquisition of aminopterin resistance were also found among X-ray-induced revertants, whereas the ICR-191-induced revertants examined show only loss of the exon 7 frameshift

  13. Utility of serum thymidine kinase activity measurements for cases of bovine leukosis with difficult clinical diagnoses.

    Science.gov (United States)

    Tawfeeq, Mohammad Monir; Miura, Saori; Horiuchi, Noriyuki; Kobayashi, Yoshiyasu; Furuoka, Hidefumi; Inokuma, Hisashi

    2013-01-01

    This study evaluated the clinical usefulness of serum thymidine kinase (TK) activity for diagnosing bovine leukosis cases for which clinical diagnosis was difficult ('BL with difficult diagnosis'). Median TK activity values in 24 'BL with difficult diagnosis' and 36 cattle for which BL was clinically confirmed by cytology findings of enlarged superficial lymph nodes ('clinically confirmed BL') were 36.8 and 39.4 U/l, respectively (no significant difference). The percentage with positive TK activity (> 5.4 U/l) was also similar in both groups (83.3% for 'BL with difficult diagnosis' and 97.2% for 'clinically confirmed BL'). TK activity was significantly higher in cows with 'BL with difficult diagnosis' compared to those with other tumors (N = 13) and those with inflammatory diseases (N = 14). Maximum TK activity in cows with other tumors and inflammatory diseases was not high (cows with other tumors and those with inflammatory diseases were 1.8 and 1.4 IU/l, respectively. Positive TK activity was found in a significantly higher percentage of cows with 'BL with difficult diagnosis' (83.3%) relative to the percentages of cows with other tumors (15.3%) and inflammatory diseases (21.4%). Thus, TK activity is an appropriate marker for detecting BL onset in cows with 'BL with difficult diagnosis' as well as 'clinically confirmed BL' group. While the specificity of TK activity required for BL diagnosis is not clear, simultaneous evaluation of serum lactate dehydrogenase activity may assist in the differential diagnoses of other tumors and inflammatory diseases from BL. PMID:23628971

  14. Discovery of inhibitors of bacterial histidine kinases

    NARCIS (Netherlands)

    Velikova, N.R.

    2014-01-01

    Discovery of Inhibitors of Bacterial Histidine Kinases

    Summary

    The thesis is on novel antibacterial drug discovery (http://youtu.be/NRMWOGgeysM). Using structure-based and fragment-based dru

  15. Isolation and characterization of Dictyostelium thymidine kinase 1 as a calmodulin-binding protein.

    Science.gov (United States)

    O'Day, Danton H; Chatterjee-Chakraborty, Munmun; Wagler, Stephanie; Myre, Michael A

    2005-06-17

    Probing of a cDNA expression library from multicellular development of Dictyostelium discoideum using a recombinant radiolabelled calmodulin probe (35S-VU1-CaM) led to the isolation of a cDNA encoding a putative CaM-binding protein (CaMBP). The cDNA contained an open reading frame of 951 bp encoding a 227aa polypeptide (25.5 kDa). Sequence comparisons led to highly significant matches with cytosolic thymidine kinases (TK1; EC 2.7.1.21) from a diverse number of species including humans (7e-56; 59% Identities; 75% Positives) indicating that the encoded protein is D. discoideum TK1 (DdTK1; ThyB). DdTK1 has not been previously characterized in this organism. In keeping with its sequence similarity with DdTK1, antibodies against humanTK1 recognize DdTK1, which is expressed during growth but decreases in amount after starvation. A CaM-binding domain (CaMBD; 20GKTTELIRRIKRFNFANKKC30) was identified and wild type DdTK1 plus two constructs (DdTK deltaC36, DdTK deltaC75) possessing the domain were shown to bind CaM in vitro but only in the presence of calcium while a construct (DdTK deltaN72) lacking the region failed to bind to CaM. Thus, DdTK1 is a Ca2+-dependent CaMBP. Sequence alignments against TK1 from vertebrates to viruses show that CaM-binding region is highly conserved. The identified CaMBD overlaps the ATP-binding (P-loop) domain suggesting CaM might affect the activity of this kinase. Recombinant DdTK is enzymatically active and showed stimulation by CaM (113+/-0.5%) an in vitro enhancement that was prevented by co-addition of the CaM antagonists W7 (91.2+/-0.8%) and W13 (96.6+/-0.6%). The discovery that TK1 from D. discoideum, and possibly other species including humans and a large number of human viruses, is a Ca2+-dependent CaMBP opens up new avenues for research on this medically relevant protein. PMID:15883042

  16. New Class of Competitive Inhibitor of Bacterial Histidine Kinases

    OpenAIRE

    Gilmour, Raymond; Foster, J. Estelle; Sheng, Qin; McClain, Jonathan R.; Riley, Anna; Sun, Pei-Ming; Ng, Wai-Leung; Yan, Dalai; Nicas, Thalia I.; Henry, Kenneth; Winkler, Malcolm E.

    2005-01-01

    Bacterial histidine kinases have been proposed as targets for the discovery of new antibiotics, yet few specific inhibitors of bacterial histidine kinases have been reported. We report here a novel thienopyridine (TEP) compound that inhibits bacterial histidine kinases competitively with respect to ATP but does not comparably inhibit mammalian serine/threonine kinases. Although it partitions into membranes and does not inhibit the growth of bacterial or mammalian cells, TEP could serve as a s...

  17. Construction of Poxviruses as Cloning Vectors: Insertion of the Thymidine Kinase Gene from Herpes Simplex Virus into the DNA of Infectious Vaccinia Virus

    Science.gov (United States)

    Panicali, Dennis; Paoletti, Enzo

    1982-08-01

    We have constructed recombinant vaccinia viruses containing the thymidine kinase gene from herpes simplex virus. The gene was inserted into the genome of a variant of vaccinia virus that had undergone spontaneous deletion as well as into the 120-megadalton genome of the large prototypic vaccinia variant. This was accomplished via in vivo recombination by contransfection of eukaryotic tissue culture cells with cloned BamHI-digested thymidine kinase gene from herpes simplex virus containing flanking vaccinia virus DNA sequences and infectious rescuing vaccinia virus. Pure populations of the recombinant viruses were obtained by replica filter techniques or by growth of the recombinant virus in biochemically selective medium. The herpes simplex virus thymidine kinase gene, as an insert in vaccinia virus, is transcribed in vivo and in vitro, and the fidelity of in vivo transcription into a functional gene product was detected by the phosphorylation of 5-[125I]iodo-2'-deoxycytidine.

  18. Locating a nucleotide-binding site in the thymidine kinase of vaccinia virus and of herpes simplex virus by scoring triply aligned protein sequences.

    OpenAIRE

    Gentry, G A

    1985-01-01

    Computer techniques were used to locate related segments of amino acid sequences in the thymidine kinases of vaccinia virus and of herpes simplex virus type 1 and in porcine adenylate kinase. As determined by a procedure that evaluates triply aligned sequences, the probability that the similarities among the segments described here arose by chance was no greater than 0.001. Because the sequence in porcine adenylate kinase is a nucleotide phosphate-binding site it is concluded that the segment...

  19. An Oncolytic Adenovirus Expressing Herpes Simplex Virus-Thymidine Kinase for Targeting Cancer Therapy: An in vitro Evaluation

    Institute of Scientific and Technical Information of China (English)

    Fei-qun Zheng; Yin Xu; Yi-de Qin; Ren-jie Yang; Jun Han

    2009-01-01

    Objective: Oncolytic adenovirus, also called conditionally replicating adenovirus (CRAD), has been developed for the treatment of cancer. However, there is a tremendous need to enhance their antitumor efficacy. Here we wish to evaluate whether a strategy that combines the herpes simplex virus-thymidine kinase with oncolytic effects offers a therapeutic advantage.Methods: A novel adenovirus Ad-ETK containing a sequentially positioned promoter of human telomerase reverse transcriptase (hTERT), the coding sequence of E1A gene, an internal ribosome entry site sequence (IRES) and the coding sequence of herpes simplex virus-thymidine kinase (HSV-TK) was constructed. Infection of various cells with Ad-ETK followed by RT-PCR confirmed the expression of E1A and HSV-TK. The oncolytic ability and synergism between oncolytic effects and HSV-TK system was measured. The infection efficiency was determined by flow cytometry.Results: Ad-ETK deliverys E1A and HSV-TK gene, which selectively replicates in hTERT-positive tumor cells, and the progeny virus can reach up to 150 IU/cell. Our in vitro study showed that Ad-ETK plus ganciclovir (GCV) induced an obvious cell death.Conclusion: An oncolytic adenovirus plus the HSV-TK/GCV suicide gene system resulted in a significant improvement in treatment efficacy and it may offer important considerations in the development and preclinical assessments of oncolytic virotherapy.

  20. Nucleoside analogues are activated by bacterial deoxyribonucleoside kinases in a species-specific manner

    DEFF Research Database (Denmark)

    Sandrini, Michael; Clausen, Anders; On, Stephen L. W.;

    2007-01-01

    bactericidal activity against several clinical bacterial isolates and type strains. We identified and subcloned the genes coding for putative deoxyribonucleoside kinases in Escherichia coli, Pasteurella multocida, Salmonella enterica, Yersinia enterocolitica, Bacillus cereus, Clostridium perfringens and....... The tested Gram-negative bacteria were susceptible to 3"-azido-3"-deoxythymidine (AZT) in the concentration range 0.032-31.6 µM except for a single E. coli isolate and two Pseudomonas aeruginosa isolates which were resistant to the tested AZT concentrations. Purified recombinant S. enterica thymidine...... deoxyadenosine kinase had a Km for gemcitabine of 33.5 µM and kcat/Km of 5.1 × 10^3 s-1 M-1 and activates gemcitabine in vivo. S. enterica and B. cereus are now amongst the first bacteria with a completely characterized set of dNK enzymes. Bacterial dNKs efficiently activate nucleoside analogues in a species...

  1. Killing effect of coexpressing cytosine deaminase and thymidine kinase on rat vascular smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    曹慧青; 孟宪敏; 刘冬青; 赵秀文; 丁金凤

    2004-01-01

    Background Vascular smooth muscle cell (VSMC) proliferation following arterial injury plays a critical role in a variety of vascular proliferative disorders, such as atherosclerosis and restenosis after balloon angioplasty. Herpes simplex virus-thymidine kinase (HSV-TK)/ganciclovir (GCV) and E.coli cytosine deaminase (CD)/5-fluorocytosine (5-Fc) suicide gene systems have been successfully employed in cardiovascular gene therapy, respectively. We reasoned that coexpression of both HSV-TK with CD suicide genes would lead to increased cell killing. To test this imagine, the adenoviral vectors expressing TK and/or CD genes were developed and tested on vascular smooth muscle cells. Methods Adenoviral vectors, including Ad-EF1α-CD-cytomegolovirus (CMV)-TK coexpressing both CD and TK double suicide genes, Ad-EF1α-CD and Ad-CMV-TK expressing CD and TK respectively, and control vector Ad-CMV-LacZ, were constructed and prepared with homologous recombination in RecA+E.coli cells. Integration and expression of CD and/or TK gene were identified by PCR and Western blot. Primary cultured VSMCs were infected at a multiplicity of infection (MOI) of 20 with exposure to their matching prodrugs 5-Fc and GCV. Cell mortality was measured by methyl thiazolyl tetrazolium (MTT) assays. Flow cytometry analysis was used to detect cell death. Apoptotic cells were analyzed using Hoechst 33342 fluorescence dye as a DNA probe. Genomic DNA cleavage of apoptotic VSMCs was tested by agarose gel electrophoresis. Results Recombinant adenovirus expressing CD and/or TK suicide genes were successfully constructed. Both single and double suicide genes could be integrated into adenoviral genome and expressed. Cytotoxic effects of Ad-EF1α-CD-CMV-TK double suicide genes combined with 5-Fc and GCV were higher than those of Ad-CMV-TK and Ad-EF1α-CD single gene groups. The rate of cell survival was only (9±3)% in the Ad-EF1α-CD-CMV-TK group, but (37±3)% in the Ad-CMV-TK and (46±4)% in the Ad-EF1

  2. Synthesis and evaluation of thymidine kinase 1-targeting carboranyl pyrimidine nucleoside analogs for boron neutron capture therapy of cancer.

    Science.gov (United States)

    Agarwal, Hitesh K; Khalil, Ahmed; Ishita, Keisuke; Yang, Weilian; Nakkula, Robin J; Wu, Lai-Chu; Ali, Tehane; Tiwari, Rohit; Byun, Youngjoo; Barth, Rolf F; Tjarks, Werner

    2015-07-15

    A library of sixteen 2nd generation amino- and amido-substituted carboranyl pyrimidine nucleoside analogs, designed as substrates and inhibitors of thymidine kinase 1 (TK1) for potential use in boron neutron capture therapy (BNCT) of cancer, was synthesized and evaluated in enzyme kinetic-, enzyme inhibition-, metabolomic-, and biodistribution studies. One of these 2nd generation carboranyl pyrimidine nucleoside analogs (YB18A [3]), having an amino group directly attached to a meta-carborane cage tethered via ethylene spacer to the 3-position of thymidine, was approximately 3-4 times superior as a substrate and inhibitor of hTK1 than N5-2OH (2), a 1st generation carboranyl pyrimidine nucleoside analog. Both 2 and 3 appeared to be 5'-monophosphorylated in TK1(+) RG2 cells, both in vitro and in vivo. Biodistribution studies in rats bearing intracerebral RG2 glioma resulted in selective tumor uptake of 3 with an intratumoral concentration that was approximately 4 times higher than that of 2. The obtained results significantly advance the understanding of the binding interactions between TK1 and carboranyl pyrimidine nucleoside analogs and will profoundly impact future design strategies for these agents. PMID:26087030

  3. Effect of valine 106 on structure-function relation of cytosolic human thymidine kinase - Kinetic properties and oligomerization pattern of nine substitution mutants of V106

    DEFF Research Database (Denmark)

    Frederiksen, Hanne; Berenstein, Dvora; Munch-Petersen, Birgitte

    2004-01-01

    enzymatic properties of the cell-cycle-regulated human cytosolic thymidine kinase 1 (TK1) is investigated. On the basis of the previously observed profound differences between recombinant TK1 with Val106 (V106WT) and Met106 (V106M) in catalytic activity and oligomerization pattern, we designed and...

  4. Measurement of bacterial growth rates in subsurface sediments using the incorporation of tritiated thymidine into DNA.

    Science.gov (United States)

    Thorn, P M; Ventullo, R M

    1988-07-01

    Microbial growth rates in subsurface sediment from three sites were measured using incorporation of tritiated thymidine into DNA. Sampling sites included Lula, Oklahoma, Traverse City, Michigan, and Summit Lake, Wisconsin. Application of the thymidine method to subsurface sediments required (1) thymidine concentrations greater than 125 nM, (2) incubation periods of less than 4 hours, (3) addition of SDS and EDTA for optimum macromolecular extraction, and (4) DNA purification, in order to accurately measure the rate of thymidine incorporation into DNA. Macromolecule extraction recoveries, as well as the percentage of tritium label incorporated into the DNA fraction, were variable and largely dependent upon sediment composition. In general, sandy sediments yielded higher extraction recoveries and demonstrated a larger percentage of label incorporated into DNA than sediments that contained a high silt-clay component. Reported results also indicate that the acid-base hydrolysis procedure routinely used for macromolecular fractionation in water samples may not be routinely applicable to the modified sediment procedure where addition of SDS and EDTA are required for macromolecule extraction. Growth rates exhibited by subsurface communities are relatively slow, ranging from 5.1 to 10.2×10(5) cells g(-1) day(-1). These rates are 2-1,000-fold lower than growth rates measured in surface sediments. These data lend support to the supposition that subsurface microbial communities are nutritionally stressed. PMID:24201529

  5. Overexpression of interleukin 2 receptor, thymidine kinase and immunoglobulin-associated alpha-1 messenger RNA in a clinical case of enzootic bovine leukosis.

    Science.gov (United States)

    Tawfeeq, Mohammad Monir; Tagawa, Michihito; Itoh, Yuuki; Sugimoto, Kazuya; Kobayashi, Yoshiyasu; Inokuma, Hisashi

    2012-09-01

    A 49-month-old Holstein cow with anorexia, tachypnea, enlarged peripheral lymph nodes, and difficulty standing up was suspected of bovine leukosis. Hematological examination revealed lymphocytosis with the presence of neoplastic cells. Increased total lactate dehydrogenase (LDH) activity, isozymes of LDH-2 and LDH-3 activities and thymidine kinase activity were observed. Cytological findings of fine needle aspiration of subiliac lymph nodes indicated lymphosarcoma. Histopathology and antibody analysis confirmed the diagnosis of enzootic bovine leukosis, a B-cell bovine lymphoma caused by bovine leukemia virus. Gene expressions known as biomarkers of hematopoietic neoplasia in human were also examined in the present case. Increased messenger RNA expression of interleukin 2 receptor, thymidine kinase, and immunoglobulin-associated alpha-1 was observed in the case animal. PMID:23037779

  6. Genotypic Characterization of UL23 Thymidine Kinase and UL30 DNA Polymerase of Clinical Isolates of Herpes Simplex Virus: Natural Polymorphism and Mutations Associated with Resistance to Antivirals▿

    OpenAIRE

    Burrel, Sonia; Deback, Claire; Agut, Henri; Boutolleau, David

    2010-01-01

    The molecular mechanisms of herpes simplex virus (HSV) resistance to antiviral drugs interfering with viral DNA synthesis reported so far rely on the presence of mutations within UL23 (thymidine kinase [TK]) and UL30 (DNA polymerase) genes. The interpretation of genotypic antiviral resistance assay results requires the clear distinction between resistance mutations and natural interstrain sequence variations. The objectives of this work were to describe extensively the natural polymorphism of...

  7. Antitumor effects and radiosensitization of cytosine deaminase and thymidine kinase fusion suicide gene on colorectal carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    De-Hua Wu; Li Liu; Long-Hua Chen

    2005-01-01

    AIM: To investigate the killing effect and radiosensitization of double suicide gene mediated by adenovirus on colorectal carcinoma cells.METHODS: Colorectal carcinoma cell line SW480 was transfected with adenovirus expression vector containing cytosine deaminase (CD) and thymidine kinase (Tk) fusion gene. The expression of CD-TK fusion gene was detected by reverse transcriptase-polymerase chain reaction. The toxic effect of ganciclovir (GCV) and 5-fiuorocytosine (5FC) on infected cells was determined by MTT assay. The radiosensitization of double suicide gene was evaluated by clonogenic assay.RESULTS: After prodrugs were used, the survival rate of colorectal carcinoma cells was markedly decreased. When GCV and 5-FC were used in combination, the cytotoxicity and bystandereffect were markedly superior to a single prodrug (x2 = 30.371, P<0.01). Both GCV and 5-FC could sensitize colorectal carcinoma cells to the toxic effect of radiation, and greater radiosensitization was achieved when both prodrug were used in combination. CONCLUSION: CD-TK double suicide gene can kill and radiosensitize colorectal carcinoma cells.

  8. Serum thymidine kinase, a possible marker for monitoring the effect of bone marrow transplant treatment in early recovery phase

    International Nuclear Information System (INIS)

    We measured serum thymidine kinase (TK) activity with a radioenzyme assay system employing [I-125]-iododeoxyuridine as the tracer on serial specimens from five bone marrow transplant (BMT) patients before and after transplantation. The serum level of TK activity in the 4 patients with effective BMT treatment ranged from 3.0 to 16.9 U/L (mean, 7.80 U/L) before transplantation and from 27.3 to 236.1 U/L (mean, 82.95 U/L) after the BMT treatment. Mean serum TK activity increased 13.17-fold (range, 1.68 to 29.14-fold). In contrast, the activity in the patient with ineffective BMT treatment was not significantly different during, before, or after BMT treatment. In addition, serum TK activity in BMT patients was well correlated with the change in the number of leukocytes before and after BMT treatment (r= + 0.709 (p<0.01), y=0.012x + 0.87)

  9. Computational modeling and functional analysis of Herpes simplex virus type-1 thymidine kinase and Escherichia coli cytosine deaminase fusion protein

    International Nuclear Information System (INIS)

    Herpes simplex virus type-1 thymidine kinase (HSV-1TK) and Escherichia coli cytosine deaminase (CD) fusion protein was designed using InsightII software. The structural rationality of the fusion proteins incorporating a series of flexible linker peptide was analyzed, and a suitable linker peptide was chosen for further investigated. The recombinant plasmid containing the coding regions of HSV-1TK and CD cDNA connected by this linker peptide coding sequence was generated and subsequently transfected into the human embryonic kidney 293 cells (HEK293). The Western blotting indicated that the recombinant fusion protein existed as a dimer with a molecular weight of approximately 90 kDa. The toxicity of the prodrug on the recombinant plasmid-transfected human lung cancer cell line NCIH460 was evaluated, which showed that TKglyCD-expressing cells conferred upon cells prodrug sensitivities equivalent to that observed for each enzyme independently. Most noteworthy, cytotoxicity could be enhanced by concurrently treating TKglyCD-expressing cells with prodrugs GCV and 5-FC. The results indicate that we have successfully constructed a HSV-1TKglyCD fusion gene which might have a potential application for cancer gene therapy

  10. An Adenovirus Vector Containing the Suicide Gene Thymidine Kinase for a Broad Application in Cancer Gene Therapy

    Directory of Open Access Journals (Sweden)

    Magalhães GS

    2002-01-01

    Full Text Available Treatment of cancer using gene therapy is based on adding a property to the cell leading to its elimination. One possibility is the use of suicide genes that code for enzymes that transform a pro-drug into a cytotoxic product. The most extensively used is the herpes simplex virus thymidine kinase (TK gene, followed by administration of the antiviral drug ganciclovir (GCV. The choice of the promoter to drive the transcription of a transgene is one of the determinants of a given transfer vector usefulness, as different promoters show different efficiencies depending on the target cell type. In the experiments presented here, we report the construction of a recombinant adenovirus carrying TK gene (Ad-TK driven by three strong promoters (P CMV IE, SV40 and EN1 and its effectiveness in two cell types. Human HeLa and mouse CCR2 tumor cells were transduced with Ad-TK and efficiently killed after addition of GCV. We could detect two sizes of transcripts of TK gene, one derived from the close together P CMV IE/SV40 promoters and the other from the 1.5 Kb downstream EN1 promoter. The relative amounts of these transcripts were different in each cell type thus indicating a higher flexibility of this system.

  11. Cloning of the koi herpesvirus (KHV gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis

    Directory of Open Access Journals (Sweden)

    Gilad Oren

    2005-03-01

    Full Text Available Abstract Background Outbreaks with mass mortality among common carp Cyprinus carpio carpio and koi Cyprinus carpio koi have occurred worldwide since 1998. The herpes-like virus isolated from diseased fish is different from Herpesvirus cyprini and channel catfish virus and was accordingly designated koi herpesvirus (KHV. Diagnosis of KHV infection based on viral isolation and current PCR assays has a limited sensitivity and therefore new tools for the diagnosis of KHV infections are necessary. Results A robust and sensitive PCR assay based on a defined gene sequence of KHV was developed to improve the diagnosis of KHV infection. From a KHV genomic library, a hypothetical thymidine kinase gene (TK was identified, subcloned and expressed as a recombinant protein. Preliminary characterization of the recombinant TK showed that it has a kinase activity using dTTP but not dCTP as a substrate. A PCR assay based on primers selected from the defined DNA sequence of the TK gene was developed and resulted in a 409 bp amplified fragment. The TK based PCR assay did not amplify the DNAs of other fish herpesviruses such as Herpesvirus cyprini (CHV and the channel catfish virus (CCV. The TK based PCR assay was specific for the detection of KHV and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions. The TK based PCR was compared to previously described PCR assays and to viral culture in diseased fish and was shown to be the most sensitive method of diagnosis of KHV infection. Conclusion The TK based PCR assay developed in this work was shown to be specific for the detection of KHV. The TK based PCR assay was more sensitive for the detection of KHV than previously described PCR assays; it was as sensitive as virus isolation which is the golden standard method for KHV diagnosis and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions.

  12. Deoxyribonucleoside kinases in two aquatic bacteria with high specificity for thymidine and deoxyadenosine

    DEFF Research Database (Denmark)

    Tinta, Tinkara; Christiansen, Louise Slot; Konrad, Anke;

    2012-01-01

    Deoxyribonucleoside kinases (dNKs) are essential in the mammalian cell but their 'importance' in bacteria, especially aquatic ones, is less clear. We studied two aquatic bacteria, Gram-negative Flavobacterium psychrophilum JIP02/86 and Polaribacter sp. MED152, for their ability to salvage......, these two bacteria are missing this activity. When tens of available aquatic bacteria genomes were examined for the presence of dNKs, a majority had at least a TK1-like gene, but several lacked any dNKs. Apparently, among aquatic bacteria, the role of the dN salvage varies....

  13. Thymidine kinase 2 deficiency-induced mitochondrial DNA depletion causes abnormal development of adipose tissues and adipokine levels in mice.

    Directory of Open Access Journals (Sweden)

    Joan Villarroya

    Full Text Available Mammal adipose tissues require mitochondrial activity for proper development and differentiation. The components of the mitochondrial respiratory chain/oxidative phosphorylation system (OXPHOS are encoded by both mitochondrial and nuclear genomes. The maintenance of mitochondrial DNA (mtDNA is a key element for a functional mitochondrial oxidative activity in mammalian cells. To ascertain the role of mtDNA levels in adipose tissue, we have analyzed the alterations in white (WAT and brown (BAT adipose tissues in thymidine kinase 2 (Tk2 H126N knockin mice, a model of TK2 deficiency-induced mtDNA depletion. We observed respectively severe and moderate mtDNA depletion in TK2-deficient BAT and WAT, showing both tissues moderate hypotrophy and reduced fat accumulation. Electron microscopy revealed altered mitochondrial morphology in brown but not in white adipocytes from TK2-deficient mice. Although significant reduction in mtDNA-encoded transcripts was observed both in WAT and BAT, protein levels from distinct OXPHOS complexes were significantly reduced only in TK2-deficient BAT. Accordingly, the activity of cytochrome c oxidase was significantly lowered only in BAT from TK2-deficient mice. The analysis of transcripts encoding up to fourteen components of specific adipose tissue functions revealed that, in both TK2-deficient WAT and BAT, there was a consistent reduction of thermogenesis related gene expression and a severe reduction in leptin mRNA. Reduced levels of resistin mRNA were found in BAT from TK2-deficient mice. Analysis of serum indicated a dramatic reduction in circulating levels of leptin and resistin. In summary, our present study establishes that mtDNA depletion leads to a moderate impairment in mitochondrial respiratory function, especially in BAT, causes substantial alterations in WAT and BAT development, and has a profound impact in the endocrine properties of adipose tissues.

  14. Functional Coexpression of HSV-1 Thymidine Kinase and Green Fluorescent Protein: Implications for Noninvasive Imaging of Transgene Expression

    Directory of Open Access Journals (Sweden)

    Andreas Jacobs

    1999-06-01

    Full Text Available Current gene therapy technology is limited by the paucity of methodology for determining the location and magnitude of therapeutic transgene expression in vivo. We describe and validate a paradigm for monitoring therapeutic transgene expression by noninvasive imaging of the herpes simplex virus type 1 thymidine kinase (HSV-1-tk marker gene expression. To test proportional coexpression of therapeutic and marker genes, a model fusion gene comprising green fluorescent protein (gfp and HSV-1-tk genes was generated (tkgfp gene and assessed for the functional coexpression of the gene product, TKGFP fusion protein, in rat 9L gliosarcoma, RG2 glioma, and W256 carcinoma cells. Analysis of the TKGFP protein demonstrated that it can serve as a therapeutic gene by rendering tkgfp transduced cells sensitive to ganciclovir or as a screening marker useful for identifying transduced cells by fluorescence microscopy or fluorescence-activated cell sorting (FACS. TK and GFP activities in the TKGFP fusion protein were similar to corresponding wild-type proteins and accumulation of the HSV-1-tk-specific radiolabeled substrate, 2′-fluoro-2′-deoxy-1β-D-arabino-furanosyl-5-iodo-uracil (FIAU, in stability transduced clones correlated with gfp-fluorescence intensity over a wide range of expression levels. The tkgfp fusion gene itself may be useful in developing novel cancer gene therapy approaches. Valuable information about the efficiency of gene transfer and expression could be obtained by non-invasive imaging of tkgfp expression with FIAU and clinical imaging devices (gamma camera, positron-emission tomography [PET], single photon emission computed tomography [SPECT], and/or direct visualization of gfp expression in situ by fluorescence microscopy or endoscopy.

  15. Combination effect of oncolytic adenovirus therapy and herpes simplex virus thymidine kinase/ganciclovir in hepatic carcinoma animal models

    Institute of Scientific and Technical Information of China (English)

    Fei-qun ZHENG; Yin XU; Ren-jie YANG; Bin WU; Xiao-hua TAN; Yi-de QIN; Qun-wei ZHANG

    2009-01-01

    Aim: Oncolytic adenovirus, also called conditionally replicating adenovirus (CRAD), can selectively propagate in tumor cells and cause cell lysis. The released viral progeny can infect neighboring cancer cells, initiating a cascade that can lead to the ultimate destruction of the tumor. Suicide gene therapy using herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) offers a potential treatment strategy for cancer and is undergoing preclinical trials for a variety of tumors.We hypothesized that HSV-TK gene therapy combined with oncolytic adenoviral therapy would have an enhanced effect compared with the individual effects of the therapies and is a potential novel therapeutic strategy to treat liver cancer. Methods: To address our hypothesis, a novel CRAD was created, which consisted of a telomerase-dependent oncolytic adenovirus engineered to express E1A and HSV-TK genes (Ad-ETK). The combined effect of Ad-ETK and GCV was assessed both in vitro and in vivo in nude mice bearing HepG2 cell-derived tumors. Expression of the therapeutic genes by the transduced tumor cells was analyzed by RT-PCR and Western blotting.Results: We confirmed that Ad-ETK had antitumorigenic effects on human hepatocellular carcinoma (HCC) both in vitro and in vivo, and the TK/GCV system enhanced oncolytic adenoviral therapy. We confirmed that both E1A and HSV-TK genes were expressed in vivo.Conclusion: The Ad-ETK construct should provide a relatively safe and selective approach to killing cancer cells and should be investigated as an adjuvant therapy for hepatocellular carcinoma.

  16. In vitro evaluation of herpes simplex virus type 1 thymidine kinase reporter system in dynamic studies of transcriptional gene regulation

    International Nuclear Information System (INIS)

    The herpes simplex virus type 1 thymidine kinase (HSV1-TK) reporter system is being used to directly and indirectly monitor therapeutic gene expression, immune cell trafficking and protein-protein interactions in various living animals. However, the issues of HSV1-TK enzyme stability in living cells and whether this reporter system is optimal for dynamic studies of gene expression events in genetic imaging have not be addressed. The purpose of the present study was to evaluate the application of this reporter system in dynamic studies of transcriptional gene regulation. To achieve this purpose, we established two tetracycline-inducible murine sarcoma cell lines, tetracycline-turn-off HSV1-tk-expressing cell line (NG4TL4/tet-off-HSV1-tk) and tetracycline-turn-off Luc-expressing cell line (NG4TL4/tet-off-Luc), to create an artificially regulated gene expression model in vitro. The dynamic transcriptional events mediating a series of doxycycline (Dox) inductions were monitored by HSV1-TK or by the firefly luciferase reporter gene using HSV1-TK enzyme activity assay and luciferase assay, respectively. The results of dynamic gene expression studies showed that the luciferase gene is an optimal reporter gene for monitoring short-timescale, dynamic transcriptional events mediating a series of Dox inductions, whereas the HSV1-tk is not optimal to achieve this purpose. Furthermore, the enzyme half-life of HSV1-TK in NG4TL4 cells is about 35 h after cycloheximide-induced protein inhibition. On the other hand, the results of an efflux assay of [131I] FIAU and [3H] GCV revealed that the molecular probe phosphorylated by HSV1-TK can be trapped long term within HSV1-TK stably transformed cells. Therefore, a long half-life radionuclide is not suitable for dynamic gene expression studies. Based on these results, we suggest that the HSV1-TK reporter system is not optimal for monitoring short-timescale dynamic processes such as kinetic gene expression controlled by inducible

  17. Construction of the recombinant vector carrying herpes simplex virus thymidine kinase and cytokine genes expressed in cell line Tca8113

    Institute of Scientific and Technical Information of China (English)

    JI Guang-hui; ZOU Jing-zhi; QU Le; YUE Ying; KUAI Jian-ke

    2004-01-01

    Objective: To construct expression vector containing fusion genes of herpes simplex virus thymidine kinase(Hsv-tk), Interleukin-2(IL-2) with internal ribosome entry sites(IRES), and to assess their expression in cell lineTca8113. Methods: IL-2 cDNA was obtained by reverse transcription. Hsv-tk, IL-2 and IRES genes were amplified by PCR. The purified amplification products were inserted into pGEM-T-Easy, and transformed into E. coli JM109. The purified recombinant plasmids were identified by restriction endonucleases. The recombinant plasmids were digested and pEGFPN3 were linearized, DNA fragments of Hsv-tk, IRES and IL-2 were ligated into linearized pEGFP-N3, and then transferred into E. coli JM109. The recombinant tk-IL-2 genes were cloned separately and introduced into the expression vector pEGFPN3 containing GFP. The recombinant vectors were identified by their restriction sites through PCR. The plasmids pEGFP-TI was also transfected into Tca8113 cells by calcium phosphate method for the expression of fusion proteins. Fusion genes expressing vector PL(TI)SN was generated by the fusion of HSV-tk, IRES and IL-2 with the use of DNA recombination technology. The recombinant retroviruses were transferred into Tca8113 cells by lipofectamine. The positive clones were obtained after G418 selection and named Tca/TI respectively. Results: The pEGFP-TI pasmid was identified respectively by restriction endonucleases, and their fragment sizes were 1 120 bp and 450 bp. The pEGFP-TI pasmid as templates were amplified respectively by PCR, and their PCR products were 1 120 bp and 450 bp. The pEGFP-TI vectors were used to transfect Tca8113 cell, and the cells with fluorescence accounted for 60 % of the total amount. Conclusion: pFGFP- tk- IRES- IL-2 expressing vector is easy to assess the expression of tk-IRES-IL-2-GFP fusion protein localization in transfected cells. The successful construction of expressing vector containing fusion genes of Hsv-tk, IRES and IL-2 may be

  18. A thymidine kinase-negative bovine herpesvirus 5 is highly attenuated for rabbits, but is neuroinvasive and establishes latent infection

    Directory of Open Access Journals (Sweden)

    Sara Campos da Silva

    2011-05-01

    Full Text Available Mutant viral strains deleted in non-essential genes represent useful tools to study the function of specific gene products in the biology of the virus. We herein describe an investigation on the phenotype of a bovine herpesvirus 5 (BoHV-5 recombinant deleted in the gene encoding the enzyme thymidine kinase (TK in rabbits, with special emphasis to neuroinvasiveness and the ability to establish and reactivate latent infection. Rabbits inoculated with the parental virus (SV-507/99 (n=18 at a low titer (10(5.5TCID50 shed virus in nasal secretions in titers up to 10(4.5TCID50 for up to 12 days (average: 9.8 days [5-12] and 5/ 16 developed neurological disease and were euthanized in extremis. Rabbits inoculated with the recombinant BoHV-5TKΔ at a high dose (10(7.1TCID50 also shed virus in nasal secretions, yet to lower titers (maximum: 10(2.3TCID50 and for a shorter period (average: 6.6 days [2-11] and remained healthy. PCR examination of brain sections of inoculated rabbits at day 6 post-infection (pi revealed a widespread distribution of the parental virus, whereas DNA of the recombinant BoHV-5TKΔ-was detected only in the trigeminal ganglia [TG] and olfactory bulbs [OB]. Nevertheless, during latent infection (52pi, DNA of the recombinant virus was detected in the TGs, OBs and also in other areas of the brain, demonstrating the ability of the virus to invade the brain. Dexamethasone (Dx administration at day 65 pi was followed by virus reactivation and shedding by 5/8 rabbits inoculated with the parental strain (mean duration of 4.2 days [1 - 9] and by none of seven rabbits inoculated with the recombinant virus. Again, PCR examination at day 30 post-Dx treatment revealed the presence of latent DNA in the TGs, OBs and in other areas of the brain of both groups. Taken together, these results confirm that the recombinant BoHV-5TKΔ is highly attenuated for rabbits. It shows a reduced ability to replicate in the nose but retains the ability to invade

  19. In vitro evaluation of herpes simplex virus type 1 thymidine kinase reporter system in dynamic studies of transcriptional gene regulation

    Energy Technology Data Exchange (ETDEWEB)

    Hsieh, C.-H. [Department of Medical Radiation Technology and Institute of Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); Liu, R.-S. [Department of Medical Radiation Technology and Institute of Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); National Yang-Ming University Medical School and National PET/Cyclotron Center, Taipei Veterans General Hospital, Taipei 112, Taiwan (China); Wang, H.-E. [Department of Medical Radiation Technology and Institute of Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); Hwang, J.-J. [Department of Medical Radiation Technology and Institute of Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); Deng, W.-P. [Institute of Biomedical Material, Taipei Medical University, Taipei 112, Taiwan (China); Chen, J.-C. [Department of Medical Radiation Technology and Institute of Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); Chen, F.-D. [Department of Medical Radiation Technology and Institute of Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China) and Institute of Radiological Sciences, Central Taiwan University of Science and Technology Taichung 112, Taiwan (China)]. E-mail: d49220009@ym.edu.tw

    2006-07-15

    The herpes simplex virus type 1 thymidine kinase (HSV1-TK) reporter system is being used to directly and indirectly monitor therapeutic gene expression, immune cell trafficking and protein-protein interactions in various living animals. However, the issues of HSV1-TK enzyme stability in living cells and whether this reporter system is optimal for dynamic studies of gene expression events in genetic imaging have not be addressed. The purpose of the present study was to evaluate the application of this reporter system in dynamic studies of transcriptional gene regulation. To achieve this purpose, we established two tetracycline-inducible murine sarcoma cell lines, tetracycline-turn-off HSV1-tk-expressing cell line (NG4TL4/tet-off-HSV1-tk) and tetracycline-turn-off Luc-expressing cell line (NG4TL4/tet-off-Luc), to create an artificially regulated gene expression model in vitro. The dynamic transcriptional events mediating a series of doxycycline (Dox) inductions were monitored by HSV1-TK or by the firefly luciferase reporter gene using HSV1-TK enzyme activity assay and luciferase assay, respectively. The results of dynamic gene expression studies showed that the luciferase gene is an optimal reporter gene for monitoring short-timescale, dynamic transcriptional events mediating a series of Dox inductions, whereas the HSV1-tk is not optimal to achieve this purpose. Furthermore, the enzyme half-life of HSV1-TK in NG4TL4 cells is about 35 h after cycloheximide-induced protein inhibition. On the other hand, the results of an efflux assay of [{sup 131}I] FIAU and [{sup 3}H] GCV revealed that the molecular probe phosphorylated by HSV1-TK can be trapped long term within HSV1-TK stably transformed cells. Therefore, a long half-life radionuclide is not suitable for dynamic gene expression studies. Based on these results, we suggest that the HSV1-TK reporter system is not optimal for monitoring short-timescale dynamic processes such as kinetic gene expression controlled by

  20. Targeted impairment of thymidine kinase 2 expression in cells induces mitochondrial DNA depletion and reveals molecular mechanisms of compensation of mitochondrial respiratory activity

    International Nuclear Information System (INIS)

    Highlights: → We impaired TK2 expression in Ost TK1- cells via siRNA-mediated interference (TK2-). → TK2 impairment caused severe mitochondrial DNA (mtDNA) depletion in quiescent cells. → Despite mtDNA depletion, TK2- cells show high cytochrome oxidase activity. → Depletion of mtDNA occurs without imbalance in the mitochondrial dNTP pool. → Nuclear-encoded ENT1, DNA-pol γ, TFAM and TP gene expression is lowered in TK2- cells. -- Abstract: The mitochondrial DNA (mtDNA) depletion syndrome comprises a clinically heterogeneous group of diseases characterized by reductions of the mtDNA abundance, without associated point mutations or rearrangements. We have developed the first in vitro model to study of mtDNA depletion due to reduced mitochondrial thymidine kinase 2 gene (TK2) expression in order to understand the molecular mechanisms involved in mtDNA depletion syndrome due to TK2 mutations. Small interfering RNA targeting TK2 mRNA was used to decrease TK2 expression in Ost TK1- cells, a cell line devoid of endogenous thymidine kinase 1 (TK1). Stable TK2-deficient cell lines showed a reduction of TK2 levels close to 80%. In quiescent conditions, TK2-deficient cells showed severe mtDNA depletion, also close to 80% the control levels. However, TK2-deficient clones showed increased cytochrome c oxidase activity, higher cytochrome c oxidase subunit I transcript levels and higher subunit II protein expression respect to control cells. No alterations of the deoxynucleotide pools were found, whereas a reduction in the expression of genes involved in nucleoside/nucleotide homeostasis (human equilibrative nucleoside transporter 1, thymidine phosphorylase) and mtDNA maintenance (DNA-polymerase γ, mitochondrial transcription factor A) was observed. Our findings highlight the importance of cellular compensatory mechanisms that enhance the expression of respiratory components to ensure respiratory activity despite profound depletion in mtDNA levels.

  1. Effect of p53 activation on cell growth, thymidine kinase-1 activity, and 3'-deoxy-3'fluorothymidine uptake

    Energy Technology Data Exchange (ETDEWEB)

    Schwartz, Jeffrey L. E-mail: jschwart@u.washington.edu; Tamura, Yasuko; Jordan, Robert; Grierson, John R.; Krohn, Kenneth A

    2004-05-01

    The use of thymidine (TdR) and thymidine analogs such as 3'-deoxy-3'-fluorothymidine (FLT) as positron emission tomography (PET)-based tracers of tumor proliferation rate is based on the hypothesis that measurement of uptake of these nucleosides, a function primarily of thymidine kinase-1 (TK{sub 1}) activity, provides an accurate measure of cell proliferation in tumors. Tumor growth is influenced by many factors including the oxygen concentration within tumors and whether tumor cells have been exposed to cytotoxic therapies. The p53 gene plays an important role in regulating growth under both of these conditions. The goal of this study was to investigate the influence of p53 activation on cell growth, TK{sub 1} activity, and FLT uptake. To accomplish this, TK{sub 1} activity, S phase fraction, and the uptake of FLT were determined in plateau-phase and exponentially growing cultures of an isogenic pair of human tumor cell lines in which p53 expression was normal or inactivated by human papilloma virus type 16 E6 expression. Ionizing radiation exposure was used to stimulate p53 activity and to induce alterations in cell cycle progression. We found that exposure of cells to ionizing radiation induced dose-dependent changes in cell cycle progression in both cell lines. The relationship between S phase percentage, TK{sub 1} activity, and FLT uptake were essentially unchanged in the p53-normal cell line. In contrast, TK{sub 1} activity and FLT uptake remained high in the p53-deficient variant even when S phase percentage was low due to a p53-dependent G2 arrest. We conclude that a functional p53 response is required to maintain the normal relationship between TK1 activity and S phase percentage following radiation exposure.

  2. Evidence for an involvement of thymidine kinase in the excision repair of ultraviolet-irradiated herpes simplex virus in human cells

    International Nuclear Information System (INIS)

    A wild-type strain of herpes simplex virus type 1 (HSV-1:KOS) encoding a functional thymidine kinase (tk+) and a tk- mutant strain (HSV-1:PTK3B) were used to study the role of the viral tk in the repair of UV-irradiated HSV-1 in human cells. UV survival of HSV-1:PTK3B was substantially reduced compared with that of HSV-1:KOS when infecting normal human cells. In contrast, the UV survival of HSV-1:PTK3B was similar to that of HSV-1:KOS when infecting excision repair-deficient cells from a xeroderma pigmentosum patient from complementation group A. These results suggest that the repair of UV-irradiated HSV-1 in human cells depends, in part at least, on expression of the viral tk and that the repair process influenced by tk activity is excision repair or a process dependent on excision repair

  3. In vivo PET imaging with {sup 18}F-FHBG of hepatoma cancer gene therapy using herpes simplex virus thymidine kinase and ganciclovir

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    Lee, TaeSup; Kim, JunYoup; Moon, ByungSeok; Kang, JooHyun; Song, Inho; Kwon, HeeChung; Kim, KyungMin; Cheon, GiJeong; Choi, ChangWoon; Lim, SangMoo [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2007-07-01

    Monitoring gene expression in vivo to evaluate the gene therapy efficacy is a critical issue for scientists and physicians. Non-invasive nuclear imaging can offer information regarding the level of gene expression and its location when an appropriate reporter gene is constructed in the therapeutic gene therapy. Herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) is the most common reporter gene and is used in cancer gene therapy by activating relatively nontoxic compounds, such as acyclovir or ganciclovir (GCV), to induce cell death. In this study, we investigate the feasibility of monitoring cancer gene therapy using retroviral vector transduced HSV1-tk and GCV, in vitro cellular uptake and in vivo animal studies, including biodistribution and small animal positron emission tomography (PET) imaging, were performed in HSV1-tk and luciferase (Luc)-transduced MCA-TK/Luc and enhanced green fluorescent protein (eGFP)-transduced MCA-eGFP hepatoma cell lines.

  4. In vivo PET imaging with 18F-FHBG of hepatoma cancer gene therapy using herpes simplex virus thymidine kinase and ganciclovir

    International Nuclear Information System (INIS)

    Monitoring gene expression in vivo to evaluate the gene therapy efficacy is a critical issue for scientists and physicians. Non-invasive nuclear imaging can offer information regarding the level of gene expression and its location when an appropriate reporter gene is constructed in the therapeutic gene therapy. Herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) is the most common reporter gene and is used in cancer gene therapy by activating relatively nontoxic compounds, such as acyclovir or ganciclovir (GCV), to induce cell death. In this study, we investigate the feasibility of monitoring cancer gene therapy using retroviral vector transduced HSV1-tk and GCV, in vitro cellular uptake and in vivo animal studies, including biodistribution and small animal positron emission tomography (PET) imaging, were performed in HSV1-tk and luciferase (Luc)-transduced MCA-TK/Luc and enhanced green fluorescent protein (eGFP)-transduced MCA-eGFP hepatoma cell lines

  5. Intravenous Administration Is an Effective and Safe Route for Cancer Gene Therapy Using the Bifidobacterium-Mediated Recombinant HSV-1 Thymidine Kinase and Ganciclovir

    Directory of Open Access Journals (Sweden)

    Huicong Zhou

    2016-06-01

    Full Text Available The herpes simplex virus thymidine kinase/ganciclovir (HSV TK/GCV system is one of the best studied cancer suicide gene therapy systems. Our previous study showed that caspase 3 expression was upregulated and bladder tumor growth was significantly reduced in rats treated with a combination of Bifidobacterium (BF and HSV TK/GCV (BF-rTK/GCV. However, it was raised whether the BF-mediated recombinant thymidine kinase combined with ganciclovir (BF-rTK/GCV was safe to administer via venous for cancer gene therapy. To answer this question, the antitumor effects of BF-rTK/GCV were mainly evaluated in a xenograft nude mouse model bearing MKN-45 gastric tumor cells. The immune response, including analysis of cytokine profiles, was analyzed to evaluate the safety of intramuscular and intravenous injection of BF-rTK in BALB/c mice. The results suggested that gastric tumor growth was significantly inhibited in vivo by BF-rTK/GCV. However, the BF-rTK/GCV had no effect on mouse body weight, indicating that the treatment was safe for the host. The results of cytokine profile analysis indicated that intravenous injection of a low dose of BF-rTK resulted in a weaker cytokine response than that obtained with intramuscular injection. Furthermore, immunohistochemical analysis showed that intravenous administration did not affect the expression of immune-associated TLR2 and TLR4. Finally, the BF-rTK/GCV inhibited vascular endothelial growth factor (VEGF expression in mouse model, which is helpful for inhibiting of tumor angiogenesis. That meant intravenous administration of BF-rTK/GCV was an effective and safe way for cancer gene therapy.

  6. Identification of a differentially expressed thymidine kinase gene related to tapping panel dryness syndrome in the rubber tree (Hevea brasiliensis Muell. Arg. by random amplified polymorphic DNA screening

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    Arjunan Thulaseedharan

    2010-01-01

    Full Text Available Tapping panel dryness (TPD syndrome is one of the latex yield affecting factors in the rubber tree (Hevea brasiliensis Mull. Arg.. Therefore, identification of a DNA marker will be highly useful for screening progenies in breeding programs. The major goal of this study was to detect genetic variations and/or identification of gene fragments among 37 Hevea clones by the random amplified polymorphic DNA “fingerprinting” technique. Different levels of DNA polymorphism were detected with various primers and a distinct polymorphic band (2.0 kb was obtained with OPA-17 primer. It was cloned into a plasmid vector for further sequence characterization and the nucleotide sequence shows homology with a novel putative plant thymidine kinase (TK gene, designated as HbTK (Hevea brasiliensis thymidine kinase; GenBank accession number AY130829. The protein HbTK has 67%, 65%, 64%, and 63% similarity to TK genes of Medicago, Oryza, Arabidopsis, and Lyco- persicon, respectively, and it was highly conserved in all species analyzed. The predicted amino acid sequence contained conserved domains of TK proteins in the C-terminal half. Southern blot analysis indicated that HbTK is one of the members of a small gene family. Northern blot results revealed that the expression of the HbTK gene was up-regulated in mature bark tissues of the healthy tree while it was down-regulated in the TPD-affected one. These results suggest that this gene may play important roles in maintaining active nucleotide metabolism during cell division at the tapped site of bark tissues in the healthy tree under stress (tapping conditions for normal latex biosynthesis.

  7. Activation of caspase-3 noninvolved in the bystander effect of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system.

    Science.gov (United States)

    Zhang, Zhihong; Lin, Juqiang; Chu, Jun; Ma, Yan; Zeng, Shaoqun; Luo, Qingming

    2008-01-01

    Use of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system is one of the promising approaches in the rapidly growing area of gene therapy. The "bystander effect," a phenomenon in which HSV-tk+ cells exposed to GCV are toxic to adjacent HSV-tk- cells, was reported to play an important role in suicide gene therapy. However, the mechanism by which HSV-tk/GCV induces the bystander effect is poorly understood. We monitored the activation of caspase-3 in living cells induced by the HSV-tk/GCV system using a genetically encoded fluorescence resonance energy transfer (FRET) probe CD3, , a caspase-3 recognition site fused with a cyan fluorescent protien (CFP) and a red fluorescent protein (DsRed) which we reported and named in a previous paper. Fluorescence protein (FP)-based multicolor cellular labeling, combined with the multichannel fluorescence imaging and FRET imaging techniques, provides a novel and improved approach to directly determine whether the activation of caspase-3 involved in the HSV-tk/GCV system induces cell apoptosis in tk gene-expressing cells and their neighboring cells. FRET ratio images of CD3, and fluorescence images of the fusion protein of thymidine kinase linked with green fluorescent protein (TK-GFP), indicated that HSV-tk/GCV system-induced apoptosis in human adenoid cystic carcinoma (ACC-M) cells was via a caspase-3 pathway, and the activation of caspase-3 was not involved in the bystander effect of HSV-tk/GCV system. PMID:18601533

  8. Thymidine kinase deficient human cells have increased UV sensitivity in their capacity to support herpes simplex virus but normal UV sensitivity for colony formation

    International Nuclear Information System (INIS)

    A thymidine kinase deficient (tk-) and two thymidine kinase proficient (tk+) human cell lines were compared for UV sensitivity using colony-forming ability as well as their capacity to support the plaque formation of herpes simplex type 1 (HSV-1).The tk- line (143 cells) was a derivative of one of the tk+ lines (R970-5), whereas the other tk+ line (AC4 cells) was a derivative of the 143 cells obtained by transfection with purified sheared HSV-2 DNA encoding the viral tk gene. 143, R970-5 and AC4 cells showed a similar UV sensitivity for colony-forming ability. In contrast, the capacity to support HSV-1 plaque formation immediately (within 1 h) afte UV-irradiation was reduced to a greater extent in the 143 cells compared to the R970-5 and AC4 cells. Capacity curves for plaque formation of the HSV-1: KOS wild-type (tk+) strain were similar to those for the HSV-1: PTK3B mutant (tk-) strain were similar to those for the HSV-1: PTK3B mutant (tk-) strain in the 3 cell strains, indicating that the viral tk gene does not influence the ability of HSV-1 to form plaques in UV-irradiated compared to unirradiated human cells. Cellular capacity for HSV-1 plaque formation was found to recover in both tk- and tk+ cells for cultures infected 24 h after UV-irradiation. These results suggest that repair of UV-damaged DNA takes place to a similar extent in both tk- and tk+ human cells, but the kinetics of repair are initially slower in tk-compared to tk+ human cells. (author). 33 refs.; 3 figs.; 1 tab

  9. Targeted impairment of thymidine kinase 2 expression in cells induces mitochondrial DNA depletion and reveals molecular mechanisms of compensation of mitochondrial respiratory activity

    Energy Technology Data Exchange (ETDEWEB)

    Villarroya, Joan, E-mail: joanvillarroya@gmail.com [Institut de Recerca, Hospital Universitari de la Vall d' Hebron, Barcelona (Spain); Institut de Recerca l' Hospital de la Santa Creu i Sant Pau, Barcelona (Spain); Lara, Mari-Carmen [Institut de Recerca, Hospital Universitari de la Vall d' Hebron, Barcelona (Spain); Department of Neurology, Columbia University Medical Center, New York, NY (United States); Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER), ISCIII (Spain); Dorado, Beatriz [Department of Neurology, Columbia University Medical Center, New York, NY (United States); Garrido, Marta [Unitat de Biologia Cel.lular i Molecular, IMIM-Hospital del Mar, Barcelona (Spain); Garcia-Arumi, Elena [Institut de Recerca, Hospital Universitari de la Vall d' Hebron, Barcelona (Spain); Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER), ISCIII (Spain); Meseguer, Anna [Institut de Recerca, Hospital Universitari de la Vall d' Hebron, Barcelona (Spain); Hirano, Michio [Department of Neurology, Columbia University Medical Center, New York, NY (United States); Vila, Maya R. [Institut de Recerca, Hospital Universitari de la Vall d' Hebron, Barcelona (Spain)

    2011-04-08

    Highlights: {yields} We impaired TK2 expression in Ost TK1{sup -} cells via siRNA-mediated interference (TK2{sup -}). {yields} TK2 impairment caused severe mitochondrial DNA (mtDNA) depletion in quiescent cells. {yields} Despite mtDNA depletion, TK2{sup -} cells show high cytochrome oxidase activity. {yields} Depletion of mtDNA occurs without imbalance in the mitochondrial dNTP pool. {yields} Nuclear-encoded ENT1, DNA-pol {gamma}, TFAM and TP gene expression is lowered in TK2{sup -} cells. -- Abstract: The mitochondrial DNA (mtDNA) depletion syndrome comprises a clinically heterogeneous group of diseases characterized by reductions of the mtDNA abundance, without associated point mutations or rearrangements. We have developed the first in vitro model to study of mtDNA depletion due to reduced mitochondrial thymidine kinase 2 gene (TK2) expression in order to understand the molecular mechanisms involved in mtDNA depletion syndrome due to TK2 mutations. Small interfering RNA targeting TK2 mRNA was used to decrease TK2 expression in Ost TK1{sup -} cells, a cell line devoid of endogenous thymidine kinase 1 (TK1). Stable TK2-deficient cell lines showed a reduction of TK2 levels close to 80%. In quiescent conditions, TK2-deficient cells showed severe mtDNA depletion, also close to 80% the control levels. However, TK2-deficient clones showed increased cytochrome c oxidase activity, higher cytochrome c oxidase subunit I transcript levels and higher subunit II protein expression respect to control cells. No alterations of the deoxynucleotide pools were found, whereas a reduction in the expression of genes involved in nucleoside/nucleotide homeostasis (human equilibrative nucleoside transporter 1, thymidine phosphorylase) and mtDNA maintenance (DNA-polymerase {gamma}, mitochondrial transcription factor A) was observed. Our findings highlight the importance of cellular compensatory mechanisms that enhance the expression of respiratory components to ensure respiratory activity

  10. Nucleotide sequence of glycoprotein genes B, C, D, G, H and I, the thymidine kinase and protein kinase genes and gene homologue UL24 of an Australian isolate of canine herpesvirus.

    Science.gov (United States)

    Reubel, Gerhard Herbert; Pekin, Jenny; Webb-Wagg, Kyleen; Hardy, Christopher Miles

    2002-10-01

    We report the complete nucleotide (nt) sequence of nine genes of an Australian isolate of canine herpesvirus (CHV). Four of them are located in the unique short (US) region: glycoprotein (g) genes gG, gD and gI, and the protein kinase gene. Five are in the unique long (UL) region: the thymidine kinase gene, gB, gC, gH, and gene homologue UL24. Partial sequence was determined for four genes, two in the UL region (UL21 and virion protein) and two in the US region (US2 and gE). A repeat sequence of 382 nt with unknown function was identified in the 615 nt intergenic region between gH and UL21. A total of 16.93 kb was sequenced and compared with sequences from CHV isolates from the USA, France, Japan and Australia. Only minor nt and/or amino acid (aa) differences were observed. PMID:12416682

  11. [{sup 11}C]FMAU and [{sup 18}F]FHPG as PET tracers for herpes simplex virus thymidine kinase enzyme activity and human cytomegalovirus infections

    Energy Technology Data Exchange (ETDEWEB)

    Vries, Erik F.J. de E-mail: e.f.j.de.vries@pet.azg.nl; Waarde, Aren van; Harmsen, Marco C.; Mulder, Nanno H.; Vaalburg, Willem; Hospers, Geke A.P

    2000-02-01

    [{sup 11}C]-2'-Fluoro-5-methyl-1-{beta}-D-arabinofuranosyluracil ([{sup 11}C]FMAU) and [{sup 18}F]-9-[(3-fluoro-1-hydroxy-2-propoxy)methyl]guanine ([{sup 18}F]FHPG), radiolabeled representatives of two classes of antiviral agents, were evaluated as tracers for measuring herpes simplex virus thymidine kinase (HSV-tk) enzyme activity after gene transfer and as tracers for localization of active human cytomegalovirus (HCMV) infections. In vitro accumulation experiments revealed that both [{sup 11}C]FMAU and [{sup 18}F]FHPG accumulated significantly more in HSV-tk expressing cells than they did in control cells. [{sup 18}F]FHPG uptake in HSV-tk expressing cells, however, was found to depend strongly on the cell line used, which might be due to cell type dependent membrane transport or cell type dependent substrate specific susceptibility of the enzyme. In vitro, both tracers exhibited a good selectivity for accumulation in HCMV-infected human umbilical vein endothelial cells over uninfected cells. In contrast to [{sup 18}F]FHPG, [{sup 11}C]FMAU uptake in control cells was relatively high due to phosphorylation of the tracer by host kinases. Therefore, [{sup 18}F]FHPG appears to be the more selective tracer not only to predict HSV-tk gene therapy outcome, but also to localize active HCMV infections with PET.

  12. Bacterial production and growth rate estimation from [3H]thymidine incorporation for attached and free-living bacteria in aquatic systems

    International Nuclear Information System (INIS)

    Production and specific growth rates of attached and free-living bacteria were estimated in an oligotrophic marine system, La Salvaje Beach, Vizcaya, Spain, and in a freshwater system having a higher nutrient concentration, Butron River, Vizcaya, Spain. Production was calculated from [methyl-3H]thymidine incorporation by estimating specific conversion factors (cells or micrograms of C produced per mole of thymidine incorporated) for attached and free-living bacteria, respectively, in each system. Conversion factors were not statistically different between attached and free-living bacteria: 6.812 x 1011 and 8.678 x 1011 μg of C mol-1 for free-living and attached bacteria in the freshwater system, and 1.276 x 1011 and 1.354 x 1011 μg of C mol-1 for free-living and attached bacteria in the marine system. Therefore, use of a unique conversion factor for the mixed bacterial population is well founded. However, conversion factors were higher in the freshwater system than in the marine system. This could be due to the different tropic conditions of the two systems. Free-living bacteria contributed the most to production in the two systems (85% in the marine system and 67% in the freshwater system) because of their greater contribution to total biomass. Specific growth rates calculated from production data and biomass data were similar for attached and free-living bacteria

  13. Selective enhancement of radiation response of herpes simplex virus thymidine kinase transduced 9L gliosarcoma cells in vitro and in vivo by antiviral agents

    International Nuclear Information System (INIS)

    Purpose: To demonstrate in a well-characterized tumor model that the radiosensitivity of tumor cells transduced with a herpes simplex virus thymidine kinase gene (HS-tk) would be selectively enhanced by antiviral agents. Methods and Materials: Rat 9L gliosarcoma cells transduced with a retroviral vector containing an HS-tk gene, 9L-tk cells were exposed to various doses of irradiation under either in vitro or in vivo conditions. The radiation sensitizing potential of two antiviral drugs, bromovinyl deoxyuridine (BVdU) and dihydroxymethyl ethyl methyl guanine (acyclovir), was evaluated in vitro. The radiosensitizing ability of BVdU was also evaluated with a 9L-tk tumor growing in the rat brain. Tumors growing in the right hemisphere of rat brains were irradiated stereotactically with single-dose irradiation. Results: The radiation response of 9L-tk cells was selectively enhanced by antiviral agents relative to nontransduced cells. In the cell culture, when a 24-h drug exposure (20 μg/ml) preceded radiation, the sensitizer enhancement ratio (SER) for BVdU and acyclovir was 1.4 ± 0.1 and 1.3 ± 0.1, respectively. Exposure of cells to 10 μg/ml acyclovir for two 24-h periods both pre- and postirradiation resulted in a SER of 1.6 ± 0.1. In vivo, a significant increase in median survival time of rats with 9L-tk tumors was found when BVdU was administered prior to single-dose irradiation relative to the survival time of similar rats receiving radiation alone. Conclusion: An antiviral agent can enhance cell killing by radiation with selective action in cells transduced with the herpes simplex virus thymidine kinase gene. The results suggest that the three-pronged therapy of HS-tk gene transduction, systemically administered antiviral drug, and stereotactically targeted radiation therapy will improve the effectiveness of radiation therapy for the treatment of radioresistant tumors

  14. Selective enhancement of radiation response of herpes simplex virus thymidine kinase transduced 9L gliosarcoma cells in vitro and in vivo by antiviral agents

    International Nuclear Information System (INIS)

    The purpose of this investigation was to demonstrate in a well-characterized tumor model that the radiosensitivity of tumor cells transduced with a herpes simplex virus thymidine kinase gene (HS-tk) would be selectively enhanced by antiviral agents. Rat 9L gliosarcoma cells transduced with a retroviral vector containing an HS-tk gene, 9L-tk cells were exposed to various doses or irradiation under either in vitro or in vivo conditions. The radiation sensitizing potential of two antiviral drugs, bromovinyl deoxyuridine (BVdU) and dihydroxymethyl ethyl methyl guanine (acyclovir), was evaluated in vitro. The radiosensitizing ability of BVdU was also evaluated with a 9L-tk tumor growing in the rat brain. Tumors growing in the right hemisphere of rat brains were irradiated stereotactically with single-dose irradiation. The radiation response of 9L-tk cells was selectively enhanced by antiviral agents relative to nontransduced cells. In the cell culture, when a 24-h drug exposure (20 μg/ml) preceded radiation, the sensitizer enhancement ratio (SER) for BVdU and acyclovir was 1.4 ± 0.1 and 1.3 ± 0.1, respectively. Exposure of cells to 10 μg/ml acyclovir for two 24-h periods both pre- and postirradiation resulted in a SER of 1.6 ± 0.1. In vivo, a significant increase in median survival time of rats with 9L-tk tumors was found when BVdU was administered prior to single-dose irradiation relative to the survival time of similar rats receiving radiation alone. An antiviral agent can enhance cell killing by radiation with selective action in cells transduced with the herpes simplex virus thymidine kinase gene. The results suggest that the three-pronged therapy of HS-tk gene transduction, systemically administered antiviral drug, and stereotactically targeted radiation therapy will improve the effectiveness of radiation therapy for the treatment of radioresistant tumors. 25 refs., 6 figs

  15. Amino acid substitutions in the thymidine kinase gene of induced acyclovir-resistant herpes simplex virus type 1

    Science.gov (United States)

    Hussin, Ainulkhir; Nor, Norefrina Shafinaz Md; Ibrahim, Nazlina

    2013-11-01

    Acyclovir (ACV) is an antiviral drug of choice in healthcare setting to treat infections caused by herpes viruses, including, but not limited to genital herpes, cold sores, shingles and chicken pox. Acyclovir resistance has emerged significantly due to extensive use and misuse of this antiviral in human, especially in immunocompromised patients. However, it remains unclear about the amino acid substitutions in thymidine (TK) gene, which specifically confer the resistance-associated mutation in herpes simplex virus. Hence, acyclovir-resistant HSV-1 was selected at high concentration (2.0 - 4.5 μg/mL), and the TK-gene was subjected to sequencing and genotypic characterization. Genotypic sequences comparison was done using HSV-1 17 (GenBank Accesion no. X14112) for resistance-associated mutation determination whereas HSV-1 KOS, HSV-1 473/08 and HSV clinical isolates sequences were used for polymorphism-associated mutation. The result showed that amino acid substitutions at the non-conserved region (UKM-1: Gln34Lys, UKM-2: Arg32Ser & UKM-5: Arg32Cys) and ATP-binding site (UKM-3: Tyr53End & UKM-4: Ile54Leu) of the TK-gene. These discoveries play an important role to extend another dimension to the evolution of acyclovir-resistant HSV-1 and suggest that selection at high ACV concentration induced ACV-resistant HSV-1 evolution. These findings also expand the knowledge on the type of mutations among acyclovir-resistant HSV-1. In conclusion, HSV-1 showed multiple strategies to exhibit acyclovir resistance, including amino acid substitutions in the TK gene.

  16. Correlations of 18F-fluorothymidine uptake with pathological tumour size, Ki-67 and thymidine kinase 1 expressions in primary and metastatic lymph node colorectal cancer foci

    International Nuclear Information System (INIS)

    To examine correlations of 18F-fluorothymidine (FLT) uptake with pathological tumour size and immunohistochemical Ki-67, and thymidine kinase 1 (TK-1) expressions in primary and metastatic node colorectal cancer foci. Thirty primary cancers (PCs) and 37 metastatic nodes (MNs) were included. FLT uptake was assessed by visual scores (non-visible: 0-1 and visible: 2-4), standardized uptake value (SUV), and correlated with size, Ki-67, and TK-1. SUV was measured in visible lesions. FLT heterogeneity was assessed by visual scores (no heterogeneous uptake: 0 and heterogeneous uptake: 1-4). Forty-two lesions were visible. The visible group showed significantly higher values than the non-visible group in size, Ki-67, and TK-1 (each p 0.05). Heterogeneous FLT uptake was noted in 73 % (22/30) of PCs. FLT uptake correlated with size. Heterogeneous FLT distribution in colorectal cancers may be one of the causes of weak or lack of FLT uptake/Ki-67 or TK-1 correlation. (orig.)

  17. Tanshinone IIA increases the bystander effect of herpes simplex virus thymidine kinase/ganciclovir gene therapy via enhanced gap junctional intercellular communication.

    Directory of Open Access Journals (Sweden)

    Jianyong Xiao

    Full Text Available The bystander effect is an intriguing phenomenon by which adjacent cells become sensitized to drug treatment during gene therapy with herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV. This effect is reported to be mediated by gap junctional intercellular communication (GJIC, and therefore, we postulated that upregulation of genes that facilitate GJIC may enhance the HSV-tk/GCV bystander effect. Previous findings have shown Tanshinone IIA (Tan IIA, a chemical substance derived from a Chinese medicine herb, promotes the upregulation of the connexins Cx26 and Cx43 in B16 cells. Because gap junctions are formed by connexins, we hypothesized that Tan IIA might increase GJIC. Our results show that Tan IIA increased GJIC in B16 melanoma cells, leading to more efficient GCV-induced bystander killing in cells stably expressing HSV-tk. Additionally, in vivo experiments demonstrated that tumors in mice with 10% HSV-tk positive B16 cells and 90% wild-type B16 cells became smaller following treatment with the combination of GCV and Tan IIA as compared to GCV or Tan IIA alone. These data demonstrate that Tan IIA can augment the bystander effect of HSV-tk/GCV system through increased gap junction coupling, which adds strength to the promising strategy that develops connexins inducer to potentiate the effects of suicide gene therapy.

  18. Antitumor effects of non-replicative herpes simplex vectors expressing antiangiogenic proteins and thymidine kinase on Lewis lung carcinoma establishment and growth.

    Science.gov (United States)

    Berto, E; Bozac, A; Volpi, I; Lanzoni, I; Vasquez, F; Melara, N; Manservigi, R; Marconi, P

    2007-09-01

    There is growing evidence that combinations of antiangiogenic proteins with other antineoplastic treatments such as chemo- or radiotherapy and suicide genes-mediated tumor cytotoxicity lead to synergistic effects. In the present work, we tested the activity of two non-replicative herpes simplex virus (HSV)-1-based vectors, encoding human endostatin::angiostatin or endostatin::kringle5 fusion proteins in combination with HSV-1 thymidine kinase (TK) molecule, on endothelial cells (ECs) and Lewis lung carcinoma (LLC) cells. We observed a significant reduction of the in vitro growth, migration and tube formation by primary ECs upon direct infection with the two recombinant vectors or cultivation with conditioned media obtained from the vector-infected LLC cells. Moreover, direct cytotoxic effect of HSV-1 TK on both LLC and ECs was demonstrated. We then tested the vectors in vivo in two experimental settings, that is, LLC tumor growth or establishment, in C57BL/6 mice. The treatment of pre-established subcutaneous tumors with the recombinant vectors with ganciclovir (GCV) induced a significant reduction of tumor growth rate, while the in vitro infection of LLC cells with the antiangiogenic vectors before their implantation in mice flanks, either in presence or absence of GCV, completely abolished the tumor establishment. PMID:17557110

  19. Prognostic utility of gene therapy with herpes simplex virus thymidine kinase for patients with high-grade malignant gliomas: a systematic review and meta analysis.

    Science.gov (United States)

    Zhao, Fei; Tian, Jinhui; An, Lifeng; Yang, Kehu

    2014-06-01

    The aim of this study was to assess the effectiveness of adding viral vector-mediated gene therapy with herpes simplex virus thymidine kinase (HSV-tk) to standard treatment, in comparison with standard treatment alone to treat patients with high-grade gliomas (HGGs). A literature search of the databases PubMed, Embase, the Cochrane Library, Web of Science, and Chinese biomedicine was performed to identify eligible studies. Three randomized controlled trials (involving a total of 532 patients) were included in this systematic review. A meta-analysis of included studies demonstrated a significant increase in median survival time (MST) in patients who were treated with HSV-tk gene therapy (mean deviation 0.59, 95% CI: 0.41-0.76, p standard therapy was superior to gene therapy [odds ratio (OR) = 1.31, p = 0.09]; yet differences in HR and OR between experimental groups and control groups had no statistical significance (p > 0.05). Based on the best available evidence, it appears that adding gene therapy with HSV-tk has some effect in treating HGG patients, especially with respect to MST. However, neither the pooled analysis of OS, nor the combined analysis of tumor progress indicates any significant advantage to adding gene therapy compared with standard treatment alone. More prospective studies are needed to draw solid conclusions about whether gene therapy has significant prognostic advantage. PMID:24756350

  20. Transduction and selection of human T cells with novel CD34/thymidine kinase chimeric suicide genes for the treatment of graft-versus-host disease.

    Science.gov (United States)

    Rettig, Michael P; Ritchey, Julie K; Meyerrose, Todd E; Haug, Jeffrey S; DiPersio, John F

    2003-07-01

    Clinical trials evaluating the herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV) suicide gene therapy system for the control of graft-versus-host disease (GVHD) have been limited by low transduction efficiencies and inefficient selection procedures. In this study, we designed and evaluated a novel chimeric suicide gene consisting of the extracellular and transmembrane domains of human CD34 and full-length HSV-tk (DeltaCD34-tk). High-efficiency transfer of DeltaCD34-tk to primary human T cells was accomplished after a single exposure to VSV-G-pseudotyped, Moloney murine leukemia virus-based retrovirus 48 h after activation of human PBMCs with anti-CD3 and anti-CD28 antibodies immobilized on magnetic beads. Using an optimized 5-day transduction and selection procedure, transduction efficiencies averaged 71%, with isolation purities greater than 95% and yields exceeding 90%. The immunoselected T cells were selectively eliminated by GCV (IC(50) approximately 3 nM), maintained a normal subset composition, exhibited a polyclonal TCR Vbeta family repertoire, and contained 5 or 6 vector copies per transduced cell when optimally transduced. No increase in GCV sensitivity was observed upon incorporation of highly active mutant HSV-tk enzymes into the DeltaCD34-tk suicide gene. T cells modified with the DeltaCD34-tk gene using the optimized protocol should improve the overall efficacy of the HSV-tk/GCV suicide gene therapy method of GVHD control. PMID:12842426

  1. Evaluation of 3'-deoxy-3'-[18F]-fluorothymidine (18F-FLT) kinetics correlated with thymidine kinase-1 expression and cell proliferation in newly diagnosed gliomas

    International Nuclear Information System (INIS)

    The thymidine analog 3'-deoxy-3'-[18F]fluorothymidine (18F-FLT) has been developed as a positron emission tomography (PET) tracer to assess the proliferation activity of tumors in vivo. The present study investigated the relationship between the kinetic parameters of 18F-FLT in vivo and thymidine kinase-1 (TK-1) expression and cell proliferation rate in vitro, and blood-brain barrier (BBB) breakdown in human brain gliomas. A total of 21 patients with newly diagnosed gliomas were examined by 18F-FLT PET kinetic analysis. Maximum standardized uptake value (SUVmax) and tumor-to-normal (T/N) ratio of 18F-FLT in the tumor and 18F-FLT kinetic parameters in the corresponding contralateral region were determined. The expression levels of TK-1 protein and mRNA were determined by immunohistochemistry (IHC) and real-time polymerase chain reaction (PCR), respectively, using surgical specimens. The cell proliferation rate of the tumor was determined in terms of the Ki-67 labeling index. BBB breakdown was evaluated on MR images with contrast enhancement. 18F-FLT SUVmax and T/N ratio were significantly correlated with the influx rate constant (K1; P = 0.001 and P 3). IHC and real-time PCR studies demonstrated a significant correlation between K1 and TK-1 mRNA expression (P = 0.001), but not between k3 and TK-1 protein and mRNA expression. Linear regression analysis revealed a significant correlation between K1 and the Ki-67 index (P = 0.003), but not between k3 and the Ki-67 index. TK-1 mRNA expression was significantly correlated with the Ki-67 index (P = 0.009). 18F-FLT SUVmax and T/N ratio were significantly correlated with BBB breakdown evaluated by contrast enhancement in MR images (P = 0.003 and P = 0.011, respectively). These results indicate that 18F-FLT uptake in the tumor is significantly related to transport through the disrupted BBB, but not through phosphorylation activity. Although the tissue TK-1 expression reflects tumor proliferation activity, the phosphorylation

  2. Growth properties and vaccine efficacy of recombinant pseudorabies virus defective in glycoprotein E and thymidine kinase genes.

    Science.gov (United States)

    Wu, Ching-Ying; Liao, Chih-Ming; Chi, Jiun-Ni; Chien, Maw-Sheng; Huang, Chienjin

    2016-07-10

    Pseudorabies virus (PRV) is an alphaherpesvirus that causes pseudorabies (PR), an economically important viral disease of pigs. Marker vaccines were widely used in PR prevention and eradication programs. The purpose of this study was to construct a novel recombinant virus with deletions at defined regions in the glycoprotein E (gE) and thymine kinase (TK) genes by homologous recombination. This study also evaluated the safety and efficacy of the virus for a live attenuated marker vaccine. No significant difference was observed in virus replication between gE gene-deleted (gE(-)), gE/TK double gene-deleted (gE(-)TK(-)), and wild-type PRV by growth curve analysis. However, gE(-)TK(-) PRV was completely attenuated in mice. To evaluate the immunogenicity of gE(-)TK(-) PRV, four 12-week-old specific-pathogen-free pigs per group were immunized intramuscularly with viral titers of 1×10(4), 1×10(5), or 1×10(6) TCID50, followed by intranasal challenge infection with virulent PRV (1×10(8) TCID50) at 3 weeks post vaccination. The gE(-)TK(-) PRV-vaccinated pigs displayed no general adverse effects after immunization and had protective immune responses after PRV challenge. Thus, gE(-)TK(-) PRV was safe and efficacious and might be a potential candidate for a live attenuated marker vaccine against PRV. PMID:27164258

  3. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

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    Lei eShi

    2014-09-01

    Full Text Available Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.

  4. Human telomerase reverse-transcriptase promoter-controlled and herpes simplex virus thymidine kinase-armed adenoviruses for renal cell carcinoma treatment

    Directory of Open Access Journals (Sweden)

    Tian DW

    2013-04-01

    Full Text Available Dawei Tian,1–4 Yan Sun,3 Yang Yang,2,3 Mingde Lei,3 Na Ding,3 Ruifa Han2,31Tianjin Medical University, Tianjin, People's Republic of China; 2Department of Urinary Surgery, 3Tianjin Institute of Urology, Second Hospital of Tianjin Medical University, Tianjin, People's Republic of China; 4Tianjin Nankai Hospital, Tianjin, People's Republic of ChinaAbstract: New treatment strategies are required for renal cell carcinoma (RCC due to its relative insensitivity to conventional radio- and chemotherapies. The promising strategy of tumor inhibition using human telomerase reverse transcriptase (hTERT-controlled herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV in the hTERT promoter-driven HSV-TK/GCV suicide gene system was investigated. Tumor volume, weight, relative proliferation rate, and cell-apoptosis levels were examined in mice injected with adenovirus (Ad-hTERT-HSV-TK and GCV. Increased cell death occurred following treatment with Ads carrying hTERT-HSV-TK/GCV or cytomegalovirus promoter-controlled (CMV-HSV-TK/GCV for human RCC 786-0 and fibroblast MRC-5 cells. In mice, Ad-hTERT-HSV-TK/GCV more specifically inhibited tumor and RCC xenograft growth than Ad-CMV-HSV-TK/GCV (P < 0.05. Furthermore, Ad-hTERT-HSV-TK/GCV did not significantly damage normal fibroblasts or organ systems (heart, lung, liver, brain, kidney, and spleen. Thus, Ad-hTERT-HSV-TK/GCV is an effective RCC inhibitor in human cells in vitro and in vivo mouse models, indicating potential usefulness in RCC-targeted gene therapy.Keywords: hTERT promoter, HSV-TK/GCV, renal cell carcinoma, adenovirus

  5. Correlations of {sup 18}F-fluorothymidine uptake with pathological tumour size, Ki-67 and thymidine kinase 1 expressions in primary and metastatic lymph node colorectal cancer foci

    Energy Technology Data Exchange (ETDEWEB)

    Nakajo, Masatoyo; Nakajo, Masayuki [Kagoshima University, Department of Radiology, Graduate School of Medical and Dental Sciences, Kagoshima (Japan); Nanpuh Hospital, Department of Radiology, Kagoshima (Japan); Kajiya, Yoriko; Tani, Atsushi [Nanpuh Hospital, Department of Radiology, Kagoshima (Japan); Goto, Yuko; Higashi, Michiyo [Kagoshima University, Department of Human Pathology, Graduate School of Medical and Dental Sciences, Kagoshima, 890-8544 (Japan); Jinguji, Megumi; Fukukura, Yoshihiko [Kagoshima University, Department of Radiology, Graduate School of Medical and Dental Sciences, Kagoshima (Japan); Tanaka, Sadao [Nanpuh Hospital, Department of Pathology, Kagoshima (Japan)

    2014-12-15

    To examine correlations of {sup 18}F-fluorothymidine (FLT) uptake with pathological tumour size and immunohistochemical Ki-67, and thymidine kinase 1 (TK-1) expressions in primary and metastatic node colorectal cancer foci. Thirty primary cancers (PCs) and 37 metastatic nodes (MNs) were included. FLT uptake was assessed by visual scores (non-visible: 0-1 and visible: 2-4), standardized uptake value (SUV), and correlated with size, Ki-67, and TK-1. SUV was measured in visible lesions. FLT heterogeneity was assessed by visual scores (no heterogeneous uptake: 0 and heterogeneous uptake: 1-4). Forty-two lesions were visible. The visible group showed significantly higher values than the non-visible group in size, Ki-67, and TK-1 (each p < 0.05). Size correlated significantly with visual score (PC; ρ = 0.74 and MN; ρ = 0.63), SUVmax (PC; ρ = 0.49, and MN; ρ = 0.76), and SUVmean (PC; ρ = 0.40 and MN; ρ = 0.76) (each p < 0.05). Visual score correlated significantly with size (ρ = 0.86), Ki-67max (ρ = 0.35), Ki-67mean (ρ = 0.38), TK-1max (ρ = 0.35) and TK-1mean (ρ = 0.25) (each p < 0.05). No significant correlations were found between FLT uptake and Ki-67 or TK-1 in 42 visible lesions (each p > 0.05). Heterogeneous FLT uptake was noted in 73 % (22/30) of PCs. FLT uptake correlated with size. Heterogeneous FLT distribution in colorectal cancers may be one of the causes of weak or lack of FLT uptake/Ki-67 or TK-1 correlation. (orig.)

  6. Cooperative Therapeutic Effects of Herpes Simplex Virus Thymidine Kinase Gene/Ganciclovir System and Chemotherapeutic Agents on Prostate Cancer in vitro

    Institute of Scientific and Technical Information of China (English)

    XING Yifei; XIAO Yajun; LU Gongcheng; ZENG Fuqing; ZHAO Jun; XIONG Ping; FENG Wei

    2006-01-01

    The killing effects of herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) approach by the addition of several commonly clinical chemotherapeutic agents on hormone refractory prostate cancer (HRPC) cells PC-3m were investigated. After transferring of the HSV-tk gene into PC-3m cells, mRNA and protein expression of HSV-tk was detected by reverse-transcript polymerase chain reaction (RT-PCR) and strept avidin-biotin complex (SABC) immunohistochemical method. The killing effect of GCV, cisplatin (CDDP), etoposide (VP-16), vincristine (VCR), methotrexate (MTX), 5-fluorouracil (5-Fu), and suramin on PC-3m cells was evaluated by morphological assessment analysis, trypan blue exclusion assay and MTT assay respectively. Additionally, the cooperative effect of HSV-tk/GCV system combined with the above agents on the target cancer cells was determined by MTT. Furthermore, apoptosis and necrosis induced by GCV plus 5-Fu or suramin was analyzed by flow cytometry (FCM). The results showed that that there was HSV-tk mRNA and protein expression in pDR2-tk plasmid transduced PC-3m cell. Combination of GCV with VP-16, VCR, 5-Fu or suramin led to an enhanced cellular killing effect, but with CDDP resulted in a reduced one and with MTX in an approximate one. FCM revealed that synergistic use of GCV and 5-fu or suramin resulted in a rather large proportion of apoptosis and necrosis with the apoptosis index being 36.38 % and 35.51%, and the proportion of necrosis being 33.05 % and 28.87 %, respectively. In conclusion, HSV-tk/CGV approach by addition of certain clinical available chemotherapeutic drugs brings on statistically significant enhanced cell killing over single-agent treatment.Our results highlight the potential for such new combination therapies for future treatments of HRPC.

  7. Imaging of Herpes Simplex Virus Type 1 Thymidine Kinase Gene Expression with Radiolabeled 5-(2-iodovinyl)-2'-deoxyuridine (IVDU) in Liver by Hydrodynamic-based Procedure

    International Nuclear Information System (INIS)

    Hydrodynamic-based procedure is a simple and effective gene delivery method to lead a high gene expression in liver tissue. Non-invasive imaging reporter gene system has been used widely with herpes simplex virus type 1 thymidine kinase (HSV1-tk) and its various substrates. In the present study, we investigated to image the expression of HSV1-tk gene with 5-(2-iodovinyl)-2'-deoxyuridine (IVDU) in mouse liver by the hydrodynamicbased procedure. HSV1-tk or enhanced green fluorescence protein (EGFP) encoded plasmid DNA was transferred into the mouse liver by hydrodynamic injection. At 24 h post-injection, RT-PCR, biodistribution, fluorescence imaging, nuclear imaging and digital wholebody autoradiography (DWBA) were performed to confirm transferred gene expression. In RT-PCR assay using mRNA from the mouse liver, specific bands of HSV1-tk and EGFP gene were observed in HSV1-tk and EGFP expressing plasmid injected mouse, respectively. Higher uptake of radiolabeled IVDU was exhibited in liver of HSV1-tk gene transferred mouse by biodistribution study. In fluorescence imaging, the liver showed specific fluorescence signal in EGFP gene transferred mouse. Gamma-camera image and DWBA results showed that radiolabeled IVDU was accumulated in the liver of HSV1-tk gene transferred mouse. In this study, hydrodynamic-based procedure was effective in liver-specific gene delivery and it could be quantified with molecular imaging methods. Therefore, co-expression of HSV1-tk reporter gene and target gene by hydrodynamic-based procedure is expected to be a useful method for the evaluation of the target gene expression level with radiolabeled IVDU

  8. Construction and identification of recombinant vectors carrying herpes simplex virus thymidine kinase and cytokine genes expressed in gastric carcinoma cell line SGC7901

    Institute of Scientific and Technical Information of China (English)

    Jian-Hua Zhang; Ming-Xi Wan; Jia-Ying Yuan; Bo-Rong Pan

    2004-01-01

    AIM: To construct and identify the recombinant vectors carrying herpes simplex virus thymidine kinase (HSVoTK) and tumor necrosis factor alpha (TNF-α) or interleukin-2 (IL-2)genes expressed in gastric carcinoma cell line SGC7901.METHODS: The fragments of HSV-TK, internal ribosome entry sites (IRES) and TNF-α or TL-2 genes were inserted in a TK-IRES-TNF-α or TK-IRES-IL-2 order into pEGFP-N3 and pLXSN to generate the therapeutic vectors pEGFP-TT,pEGFP-TI, pL(TT)SN and pL(TI)SN respectively, which were structurally confirmed by the digestion analysis of restriction endonuclease. The former two plasmids were used for the transient expression of recombinant proteins in the target cells while pL(TT)SN and pL(TI)SN were transfected into SGC7901 cells by lipofectamine for the stable expression of objective genes through G418 selection. The protein products expressed transiently and stably in SGC7901 cells by the constructed vectors were confirmed by fluorescent microscopy and Western blot respectively.RESULTS: The inserted fragments in all constructed plasmids were structurally confirmed to be consistent with that of the published data. In the transient expression, both pEGFP-TT and pEGFP-TI were shown expressed in nearly 50% of the transfected SGC7901 cells. Similarly, the G418 selected vectors PL(-TT)SN and PL(TI)SN were confirmed to be successful in the stable expression of the objective proteins in the target cells.CONCLUSION: The constructed recombinant vectors in the present study that can express the suicide gene TK in combination with cytokines genes may serve as the potential tools to perform more effective investigations in future for the gene therapy of gastric carcinoma.

  9. Targeting of herpes simplex virus 1 thymidine kinase gene sequences into the OCT4 locus of human induced pluripotent stem cells.

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    Wu Ou

    Full Text Available The in vitro differentiation of human induced pluripotent stem cells (hiPSC to generate specific types of cells is inefficient, and the remaining undifferentiated cells may form teratomas. This raises safety concerns for clinical applications of hiPSC-derived cellular products. To improve the safety of hiPSC, we attempted to site-specifically insert a herpes simplex virus 1 thymidine kinase (HSV1-TK suicide gene at the endogenous OCT4 (POU5F1 locus of hiPSC. Since the endogenous OCT4 promoter is active in undifferentiated cells only, we speculated that the HSV1-TK suicide gene will be transcribed in undifferentiated cells only and that the remaining undifferentiated cells can be depleted by treating them with the prodrug ganciclovir (GCV prior to transplantation. To insert the HSV1-TK gene at the OCT4 locus, we cotransfected hiPSC with a pair of plasmids encoding an OCT4-specific zinc finger nuclease (ZFN and a donor plasmid harboring a promoter-less transgene cassette consisting of HSV1-TK and puromycin resistance gene sequences, flanked by OCT4 gene sequences. Puromycin resistant clones were established and characterized regarding their sensitivity to GCV and the site of integration of the HSV1-TK/puromycin resistance gene cassette. Of the nine puromycin-resistant iPSC clones analyzed, three contained the HSV1-TK transgene at the OCT4 locus, but they were not sensitive to GCV. The other six clones were GCV-sensitive, but the TK gene was located at off-target sites. These TK-expressing hiPSC clones remained GCV sensitive for up to 90 days, indicating that TK transgene expression was stable. Possible reasons for our failed attempt to selectively target the OCT4 locus are discussed.

  10. A human osteosarcoma cell line expressing herpes simplex type-1 thymidine kinase: studies with radiolabeled (E)-5-(2-iodovinyl)-2'-fluoro-2'-deoxyuridine

    International Nuclear Information System (INIS)

    Introduction: (E)-5-(2-Iodovinyl)-2'-fluoro-2'-deoxyuridine (IVFRU) is a pyrimidine nucleoside analogue that accumulates selectively in murine cells expressing herpes simplex type-1 thymidine kinase (HSV-1 TK). The uptake of [125I]IVFRU in human 143B osteosarcoma cells transduced with a retroviral vector bearing the HSV-1 TK gene (143B-LTK cells) is now reported. Methods: HSV-1 TK gene expression in 143B-LTK cells was confirmed by Western blotting and reverse transcriptase (RT)-PCR. Cell and subcellular uptake of [125I]IVFRU was determined in cell culture, and whole body biodistribution after intravenous injection of [125I]IVFRU was determined using nude mice bearing implanted 143B or 143B-LTK tumors. Results: Although IVFRU was less toxic to the human cell line expressing HSV-1 TK (143B-LTK) than ganciclovir, both IVFRU and ganciclovir were not toxic to the cell line not expressing HSV-1 TK (143B). When cells were exposed to [125I]IVFRU in vitro, only the 143B-LTK cells accumulated radioactivity. The acid-soluble fraction from 143B-LTK cell lysates contained 8-fold greater activity than the acid-insoluble fraction after an 8-h exposure to [125I]IVFRU. Biodistribution of [125I]IVFRU in nude mice bearing subcutaneous 143B and 143B-LTK tumors revealed widespread distribution of the nucleoside in vivo but with specific localization in 143B-LTK tumors. Conclusion: The underlying biochemical process of metabolic entrapment of IVFRU in human osteosarcoma cells expressing HSV-1 TK is responsible for selective localization in these cells. The differences in subcellular distribution into the nucleic acid fraction, and in cytotoxicity, reflect the importance of cell type and lineage as determinants of the performance of gene imaging radiopharmaceuticals

  11. Adenovirus-Mediated Herpes Simplex Virus Thymidine Kinase Gene Transfer Driver by KDR Promoter in Treatment of Experimental Human HepatocelLular Carcinoma in Nude Mice

    Institute of Scientific and Technical Information of China (English)

    LI Bao-jin; ZHANG Chao; YI Yuan-xue; HAO Ying; LIU Xiao-ping; OU Qing-jia

    2007-01-01

    Objective: To investigate the therapeutic efficacy of adenovirus-mediated herpes simplex virus thymidine kinase (HSV-tk) gene transfer under the driving of KDR promoter (AdKDR-tk) in combination of ganciclovir (GCV) against human hepatocellular carcinoma in nude mice. Methods: HepG2 cell line was implanted subcutaneously into 32 nude mice, which were subsequently divided into 4 groups (n=8 each group): Ganciclovir group (Ⅰ), Ad group (Ⅱ), AdCMV-tk/GCV group (under the driving of CMV promoter) (Ⅲ) and AdKDR-tk/GCV group (Ⅳ). Then intratumoral injection of recombinant adenovirus or Ad was performed in all nude mice, and repeated 24 h later. For the following 10 d GCV was given at a dose of 100 mg/(kg·d), ip. All the treated animals were killed to evaluate the tumor weight and the histopathological changes and the microvessel density of tumors after the treatment was determined. Results: Compared with group Ⅰ, the tumor inhibitory rate was 12.3% in group Ⅲ and 24.5% in group Ⅳ; the inhibition rates were significantly different between group Ⅲ and Ⅳ (P<0.05). The mean MVDs in group Ⅰ, Ⅱ, Ⅲand Ⅳ were 37.4±8.6, 30.6±7.8, 27.6±7.1, and 10.7±4.1 (microvessels/mm2), respectively. Significant differences were found between group Ⅲ and Ⅱ (P<0.05), Ⅳ and Ⅱ (P<0.01), and Ⅳ and Ⅲ (P<0.01). Conclusion: Intratumoral injection of AdKDR-tk results in marked inhibition of HCC growth through inhibition angiogenesis in nude mice. It may be a new treatment approach for human HCC.

  12. Protein tyrosine kinase inhibition and cell proliferation: is the [3H]-thymidine uptake assay representative of the T-lymphocyte proliferation rate?

    Science.gov (United States)

    Spinozzi, F; Pagliacci, M C; Agea, E; Migliorati, G; Riccardi, C; Bertotto, A; Nicoletti, I

    1995-01-01

    T-cell growth is controlled to a large degree by extracellular signals that bind to specific receptors on the surface of cells. A number of these receptors have intrinsic protein tyrosine kinase (PTK) activity. Their action on second messenger generation, and thus on cell proliferation, has been indirectly demonstrated by the decrease in [3H]-thymidine (TdR) uptake that follows co-stimulation of T-cells with mitogens and PTK inhibitors such as genistein (GEN). In this paper we report that the [3H]-TdR uptake assay is not a valid and reliable tool for investigating the proliferative activity of certain T-cell lines. In fact, a concomitant assessment of both [3H]-TdR uptake and cell cycle progression demonstrated that GEN is able to block G2/M progression of Jurkat T-lymphocytes even at doses (5 micrograms/ml) that do not influence [3H]-TdR uptake. Pretreatment with sodium o-vanadate (100 nM) could not reverse the GEN-related cell cycle perturbation, but was able to restore optimal [3H]-TdR uptake. Finally, GEN treatment was able to induce concentration-dependent apoptotic cell death of Jurkat T-cells. The control of cell activation, proliferation and programmed cell death is undoubtedly influenced by receptor-associated PTKs. The final effect on cell survival is almost entirely dependent on the activation state of the cell. The [3H]-TdR uptake assay seems to be inadequate for a correct interpretation of the expected results. PMID:7655707

  13. Short-term variability in bacterial abundance, cell properties, and incorporation of leucine and thymidine in subarctic sea ice

    DEFF Research Database (Denmark)

    Kaartokallio, H.; Søgaard, D.H.; Norman, L.;

    2013-01-01

    cell population properties (by flow cytometry) in subarctic sea ice in SW Greenland. Short-term temporal variability was moderate, and steep environmental gradients, typical for sea ice, were the main drivers of the variability in bacterial cell properties and activity. Low nucleic acid (LNA) bacteria...... brightly fluorescing intracellular inclusions after Nile Blue A staining. High Leu saturating concentrations coupled with the occurrence of PHA-producing organisms further highlight the similarity of sea ice internal habitats to biofilm-like systems rather than to open-water systems....

  14. Optimized thymidylate kinase assay, based on enzymatically synthesized 5-[125I]iododeoxyuridine monophosphate and its application to an immunological study of herpes simplex virus thymidine-thymidylate kinases

    International Nuclear Information System (INIS)

    The biological synthesis and purification of 5-[125I]iododeoxyuridine monophosphate (IdUMP) are described. The specificity of IdUMP as substrate in the thymidylate monophosphate kinase (TMPK) assay is demonstrated, and a 100-fold gain in sensitivity as compared to the conventional TMPK assay is shown. TMPK measurements of isozymes derived from herpes simplex virus (HSV)-infected cells, uninfected cells, and tumor biopsies were performed. The results showed a significant difference in dependence of phosphate donor concentration present for TMPK activity from HSV-infected cells compared to the corresponding activity from uninfected cells, while only a minor difference in pH optima was observed for these enzyme activities. The increased sensitivity made it possible to detect and quantify HSV TMPK-blocking antibodies (ab) present in human sera. Sera from HSV ab-positive individuals were found to block the two HSV TMPKs to varying degrees and with different specificities. The immunological relationship between the TMPK and thymidine kinase (TK) induced by HSV-1 and HSV-2, respectively, was studied by comparing the capacities of different sera to block the two enzymatic activities. The results showed that the capacity to block HSV-1 TK and TMPK was proportional for all of the sera studied, while sera that preferentially blocked only the HSV-2 TMPK or HSV-2 TK were found. It was concluded that the HSV-2 TMPK and TK activities are less related than the corresponding activities for HSV-1 and that the HSV-2 enzyme activities are mediated by different catalytic sites

  15. Modulation of the cytotoxicity of 3'-azido-3'-deoxythymidine and methotrexate after transduction of folate receptor cDNA into human cervical carcinoma: identification of a correlation between folate receptor expression and thymidine kinase activity.

    Science.gov (United States)

    Sun, X L; Jayaram, H N; Gharehbaghi, K; Li, Q J; Xiao, X; Antony, A C

    1999-02-15

    Cervical carcinoma is an AIDS-defining illness. The expression of folate receptors (FRs) in cervical carcinoma (HeLa-IU1) cells was modulated by stable transduction of FR cDNA encapsidated in recombinant adeno-associated virus-2 in the sense and antisense orientation (sense and antisense cells, respectively). Although sense cells proliferated slower than antisense or untransduced cells in vivo and in vitro in 2% (but not 10%) FCS, [methyl-3H]thymidine incorporation into DNA was significantly increased in sense cells in 10% serum; therefore, the basis for this discrepancy was investigated. The activity of thymidine kinase (TK) was subsequently directly correlated with the extent of FR expression in single cell-derived clones of transduced cells. This elevated TK activity was not a result of recruitment of the salvage pathway based on the presence of adequate dTTP pools, normal thymidylate synthase (TS) activity, persistence of increased thymidine incorporation despite the exogenous provision of excess 5,10-methylene-tetrahydrofolate, and documentation of adequate folates in sense cells. The increase in TK activity conferred significant biological properties to sense cells (but not antisense or untransduced cells) as demonstrated by augmented phosphorylation of 3'-azido-3'-deoxythymidine (AZT) and concomitantly greater sensitivity to the cytotoxic effects of AZT. Conversely, sense cells were highly resistant to methotrexate, but this was reversed by the addition of AZT. The direct correlation of FR expression and TK activity indicates a previously unrecognized consequence of FR overexpression. PMID:10029088

  16. Cytotoxicity and cellular uptake of pyrimidine nucleosides for imaging herpes simplex type-1 thymidine kinase (HSV-1 TK) expression in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Morin, Kevin W.; Duan Weili; Xu Lihua; Zhou Aihua; Moharram, Sameh; Knaus, Edward E.; McEwan, Alexander J.B.; Wiebe, Leonard I. E-mail: leonard.wiebe@ualberta.ca

    2004-07-01

    In vivo transfer of the herpes simplex virus type-1 thymidine kinase (HSV-1 TK) gene, with subsequent administration of antiviral drugs such as ganciclovir, has emerged as a promising gene therapy protocol for treating proliferative disorders. The in vitro cytotoxicities (IC{sub 50}) for two series of 5-iodo- and (E)-5-(2-iodovinyl)-substituted 2'-deoxy- and 2'-deoxy-2'-fluoro-pyrimidine nucleosides ranged from millimolar to low nanomolar concentrations in mammalian tumor cell lines (KBALB; R-970-5; 143B; EMT-6) and their counterparts engineered to express HSV-1 TK (KBALB-STK; 143B-LTK). Their HSV-1 TK selectivity indices ranged from one (nonselective) to one million (highly selective) based on cytotoxicity, with FIRU being the least toxic to all cell lines, and FIAU being most toxic. HSV-1 TK selectivity, based on uptake, ranged from 10 to 140, with IVDU being most selective for HSV-1 TK expressing cells, followed by IVFRU, FIRU, FIAU, IVFAU and finally IUDR. Phosphorylation of [{sup 125}I]FIAU led to incorporation of the radiolabel into nucleic acids, whereas IVFRU and FIRU radioactivity was trapped primarily in the nucleotide pool. These data indicate that cytotoxicity does not depend on initial metabolic trapping (e.g., phosphorylation), but on elaboration of the mononucleotides to more cytotoxic anabolites. Lipophilicities and nucleoside transport rates of the six nucleosides tested were within narrow ranges. This supports the premise that cellular biochemistry, and not cellular bioavailability, is responsible for the observed broad range of cytotoxicity and trapping. In vivo biodistribution studies with 5-[{sup 125}I]iodo-2'-fluoro-2'-deoxyribouridine (FIRU), 5-[{sup 125}I]iodo-2'-fluoro-2'-deoxyarabinouridine (FIAU) and (E)-5-(2-[{sup 125}I]iodovinyl)-2'-fluoro-2'-deoxyuridine (IVFRU) demonstrate selective accumulation of all three radiotracers in HSV-1 TK-expressing KBABK-STK tumors, compared to their very low

  17. Preliminary study of MR diffusion weighted imaging in nude mice models of hepatic Bel7402 tumors after adenovirus-mediated cytosine diaminase-thymidine kinase gene therapy

    International Nuclear Information System (INIS)

    Objective: To study the characteristics of DWI in nude mice models of hepatic Bel7402 tumors after treatment with adenovirus-mediated cytosine diaminase-thymidine kinase (Ad. CD-TK) double suicide gene therapy, and then to identify whether DWI can be used for assessing curative effect of postoperative tumors. Methods: Thirty nude mice models of hepatic Bel7402 tumors were successfully created using cell suspension method, after the tumor grew to more than 1 cm in diameter, 20 tumor models were treated by intratumoral administration of Ad. CD-TK for 3 days plus intraperitonea (i.p.) treatment with 5-Fc and GCV for the duration of the study.Then they were randomly divided into three groups during 5-Fc and GCV treatment. The remaining 10 tumor models were used as controls. MR scanning were performed in 10th day before and after tumor implantation in all models by using EPI-SE series and SENSE technology for treatment group. Tumor volumes and ADC values were calculated pretreatment and posttreatment. Cell apoptosis were determined by using TUNEL method. Analyze the change of ADC and apoptosis index (AI) in different times, t test was used for comparison the difference of AI and ADC values respectively. Results: After 10 days,the tumor volumes of the treatment groups and controls were respectively (724.16 ±57.45) mm3, (754.57 ± 66.84) mm3, with no significant difference (t=0.488, P >0.05). The ADC values of the treatment groups were (0.98 ±0.11) × 10-3 mm2/s,the ones of the control groups were (0.68 ±0.04) × 10-3 mm2/s; AI of the treatment groups were (23.25 ±6.57)%, the ones of the control groups were (2.57 ± 0.58)%. There were difference in both groups (t=4.473, 5.874; P<0.01). Conclusion: DWI can be effectively to monitor the early pathological changes of hepatic Bel7402 tumors after Ad. CD-TK double suicide gene therapy, and provide experimental evidences for clinical application. (authors)

  18. Non-invasive in vivo imaging with radiolabelled FIAU for monitoring cancer gene therapy using herpes simplex virus type 1 thymidine kinase and ganciclovir

    International Nuclear Information System (INIS)

    An experimental cancer gene therapy model was employed to develop a non-invasive imaging procedure using radiolabelled 2'-fluoro-2'-deoxy-5-iodo-1-β-d-arabinofuranosyluracil (FIAU) as an enzyme substrate for monitoring retroviral vector-mediated herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) transgene expression. Iodine-131 labelled FIAU was prepared by a no-carrier-added (n.c.a.) synthesis process and lyophilised to give ''hot kits''. The labelling yield was over 95%, with a radiochemical purity of more than 98%. The stability of [131I]FIAU in the form of lyophilised powder (the hot kit) was much better than that in the normal saline solution. The shelf life of the final [131I]FIAU hot kit product is as long as 4 weeks. Cellular uptake of [131I]FIAU after different periods of storage was investigated in vitro with HSV1-tk-retroviral vector transduced NG4TL4-STK and parental non-transduced NG4TL4 murine sarcoma cell lines over an 8-h incubation period. The NG4TL4-STK cells accumulated more radioactivity than NG4TL4 cells in all conditions, and accumulation increased with time up to 8 h. The kinetic profile of the cellular uptake of n.c.a. [131I]FIAU formulated from the lyophilised hot kit or from the stock solution was qualitatively similar. For animal model cancer gene therapy studies, FVB/N mice were inoculated subcutaneously with the HSV1-tk(+) and tk(-) sarcoma cells into the flank to produce tumours. Biodistribution studies showed that tumour/blood ratios were 2, 3.5, 8.2 and 386.8 at 1, 4, 8 and 24 h post injection, respectively, for the HSV1-tk(+) tumours, and 0.5, 0.5, 0.7 and 5.4, respectively, for the HSV1-tk(-) tumours. Radiotracer clearance from blood was completed in 24 h and was bi-exponential. A significant difference in radioactivity accumulation was revealed among the HSV1-tk(+) tumours, the tk(-) tumours and other tissues. At 24 h p.i., higher activity retention was observed in HSV1-tk(+) tumours (9.67%±3.89%ID/g) than in HSV1-tk

  19. Do there exist synergistic antitumor effects by coexpression of herpes simplex virus thymidine kinase with cytokine genes on human gastric cancer cell line SGC7901?

    Institute of Scientific and Technical Information of China (English)

    Jian-Hua Zhang; Ming-Xi Wan; Jia-Ying Yuan; Bo-Rong Pan

    2004-01-01

    AIM: To evaluate the synergistic antitumor effects of herpes simplex virus thymidine kinase (HSV-TK) together with tumor necrosis factor alpha (TNF-α) or interleukin-2 (IL-2) gene expression on gastric cancer cell line SGC7901.METHODS: Recombinant vectors pL(TT)SN and pL(TI)SN,which express TK-IRES-TNF-α and TK-IRES-IL-2 genes separately, as well as the control plasmids pL(TK)SN and pLXSN were employed to transfect PA317 cells respectively to generate the viruses that can stably express the objective genes through G418 selection. The gastric cancer cells were then transfected by the retroviral serum from the package cells and maintained in culture to determine the cell growth and apoptosis. The cytotoxic effects of HSV-TK together with TNF-α or IL-2 gene expression on the transfected cancer cells were evaluated by the cell viability and bystander effects in the presence of GCV supplemented in the cultural medium.RESULTS: Expression of recombinant proteins including TNF-α and IL-2 by stable transfectants was confirmed by Western blotting. The percentage of cell apoptosis in the SGC/0, SGC/TK-TNF-α, SGC/TK-IL-2 and SGC/TK clone was 2.3%, 12.3%, 11.1% and 10.9% respectively at 24 h posttransfection. Cell growth status among all the experimental groups as judged by cell absorbance (,4) at 570nm did not exhibit any significant difference (P>0.05); although it was noted to be slightly lower in the SGC/-TT group. Cell survival rate in SGC/TI, SGC/TT and SGC/TK group was significantly decreased in a dose-dependent manner of GCV compared with that of the SGC/0 group (P<0.05-0.01). Among all studied cells, the SGC/TTm was shown most sensitive to GCV rate of SGC/0 cells was not affected by the presence of GCV bystander effect induced by SGC/TI, SGC/TT- and SGC/TK cells was confirmed by the fact that 20% of these stable transfectants could kill 50% of the co-cultured cells, in which the most prominent bystander effect was found in the circumstance of SGC/TTF presence

  20. Non-invasive in vivo imaging with radiolabelled FIAU for monitoring cancer gene therapy using herpes simplex virus type 1 thymidine kinase and ganciclovir

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Win-Ping; Lai, Wen-Fu [Graduate Institute of Biomedical Materials, Taipei Medical University, Taipei (Taiwan); Yang, Wen K.; Yang, Den-Mei [Institute of Biological Science, Academic Sinica, Taipei (Taiwan); Liu, Ren-Shyan [Department of Nuclear Medicine and National PET Cyclotron Center, Veterans General Hospital, Taipei (Taiwan); Hwang, Jeng-Jong; Wang, Hsin-Ell [Institute of Radiological Science, National Yang-Ming University, 155, Sec. 2, Lih-Nong Street, 112, Pei-tou, Taipei (Taiwan); Fu, Ying-Kai [Institute of Nuclear Energy, Atomic Energy Council, Taoyuan (Taiwan)

    2004-01-01

    An experimental cancer gene therapy model was employed to develop a non-invasive imaging procedure using radiolabelled 2'-fluoro-2'-deoxy-5-iodo-1-{beta}-d-arabinofuranosyluracil (FIAU) as an enzyme substrate for monitoring retroviral vector-mediated herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) transgene expression. Iodine-131 labelled FIAU was prepared by a no-carrier-added (n.c.a.) synthesis process and lyophilised to give ''hot kits''. The labelling yield was over 95%, with a radiochemical purity of more than 98%. The stability of [{sup 131}I]FIAU in the form of lyophilised powder (the hot kit) was much better than that in the normal saline solution. The shelf life of the final [{sup 131}I]FIAU hot kit product is as long as 4 weeks. Cellular uptake of [{sup 131}I]FIAU after different periods of storage was investigated in vitro with HSV1-tk-retroviral vector transduced NG4TL4-STK and parental non-transduced NG4TL4 murine sarcoma cell lines over an 8-h incubation period. The NG4TL4-STK cells accumulated more radioactivity than NG4TL4 cells in all conditions, and accumulation increased with time up to 8 h. The kinetic profile of the cellular uptake of n.c.a. [{sup 131}I]FIAU formulated from the lyophilised hot kit or from the stock solution was qualitatively similar. For animal model cancer gene therapy studies, FVB/N mice were inoculated subcutaneously with the HSV1-tk(+) and tk(-) sarcoma cells into the flank to produce tumours. Biodistribution studies showed that tumour/blood ratios were 2, 3.5, 8.2 and 386.8 at 1, 4, 8 and 24 h post injection, respectively, for the HSV1-tk(+) tumours, and 0.5, 0.5, 0.7 and 5.4, respectively, for the HSV1-tk(-) tumours. Radiotracer clearance from blood was completed in 24 h and was bi-exponential. A significant difference in radioactivity accumulation was revealed among the HSV1-tk(+) tumours, the tk(-) tumours and other tissues. At 24 h p.i., higher activity retention was observed

  1. In vivo evaluation of the uptake of [(123)I]FIAU, [(123)I]IVFRU and [(123)I]IVFAU by normal mouse brain: potential for noninvasive assessment of HSV-1 thymidine kinase gene expression in gliomas.

    Science.gov (United States)

    Li, H-F; Winkeler, A; Moharram, S; Knaus, E E; Dittmar, K; Stöckle, M; Heiss, W D; Wiebe, L I; Jacobs, A H; Jacob, A J

    2008-01-01

    Radioiodinated 5-iodo-1-(2-fluoro-2-deoxy-beta-D-arabinofuranosyl)uracil (F *IAU) is most commonly used for noninvasive assessment of herpes simplex virus type 1 thymidine kinase (HSV-1-tk) gene expression. However, it does not permeate the intact blood-brain barrier (BBB) because of its moderate lipophilicity. In this work, three iodo-nucleosides, FIAU, IVFRU, and IVFAU, were radiolabeled with iodine-123 and tested for permeation of the BBB in mice and for potential measurement of HSV-1-tk gene expression in gliomas. The results demonstrate that brain uptake and retention of these nucleosides is not directly related to their lipophilicity. The low brain uptake of IVFAU, in conjunction with its higher and constant brain/blood ratio, may reflect greater stability against hydrolysis of the N-glycosidic bond. In vivo PET evaluations of [(124)I]IVFRU and [(124)I]IVFAU in tumor-bearing mice are warranted. PMID:18188770

  2. Bacterial assay to study plant sensor histidine kinases

    Czech Academy of Sciences Publication Activity Database

    Spíchal, Lukáš

    New York City : Humana Press, 2011 - (Dissmeyer, N.; Schnittger, A.), s. 139-147 ISBN 978-1-61779-263-2. - (Methods in Molecular Biology. 779) R&D Projects: GA ČR GA301/08/1649; GA MŠk(CZ) LC06034 Institutional research plan: CEZ:AV0Z50380511 Keywords : Cytokinins * histidine kinases * CRE1/AHK4 Subject RIV: EF - Botanics http://www.ncbi.nlm.nih.gov/pubmed/21837564

  3. A novel quantitative kinase assay using bacterial surface display and flow cytometry.

    Directory of Open Access Journals (Sweden)

    Sónia Troeira Henriques

    Full Text Available The inhibition of tyrosine kinases is a successful approach for the treatment of cancers and the discovery of kinase inhibitor drugs is the focus of numerous academic and pharmaceutical laboratories. With this goal in mind, several strategies have been developed to measure kinase activity and to screen novel tyrosine kinase inhibitors. Nevertheless, a general non-radioactive and inexpensive approach, easy to implement and adapt to a range of applications, is still missing. Herein, using Bcr-Abl tyrosine kinase, an oncogenic target and a model protein for cancer studies, we describe a novel cost-effective high-throughput screening kinase assay. In this approach, named the BacKin assay, substrates displayed on a Bacterial cell surface are incubated with Kinase and their phosphorylation is examined and quantified by flow cytometry. This approach has several advantages over existing approaches, as using bacteria (i.e. Escherichia coli to display peptide substrates provides a self renewing solid support that does not require laborious chemical strategies. Here we show that the BacKin approach can be used for kinetic and mechanistic studies, as well as a platform to characterize and identify small-molecule or peptide-based kinase inhibitors with potential applications in drug development.

  4. Uptake of positron emission tomography tracers in experimental bacterial infections: a comparative biodistribution study of radiolabeled FDG, thymidine, l-methionine, {sup 67}Ga-citrate, and {sup 125}I-HSA

    Energy Technology Data Exchange (ETDEWEB)

    Sugawara, Y.; Gutowski, T.D.; Fisher, S.J.; Brown, R.S. [Michigan Univ., Ann Arbor (United States). Medical Center; Wahl, R.L. [Michigan Univ., Ann Arbor (United States). Medical Center]|[Department of Radiology, University of Michigan Medical Center, Ann Arbor (United States)

    1999-04-29

    The purpose of this study was to evaluate the localization of positron emission tomography (PET) tracers [2-deoxy-2-fluoro-d-glucose (FDG), thymidine, and l-methionine] in sites of bacterial infection, and to contrast this with that of other tracers. The left calf muscles of rats were infected with a suspension of Escherichia coli and the biodistribution of {sup 18}F- or {sup 3}H-FDG, {sup 3}H-thymidine, l-{sup 11}C- or {sup 3}H-methionine, gallium-67 citrate ({sup 67}Ga-citrate) and iodine-125 human serum albumin ({sup 125}I-HSA) was determined in these animals. {sup 3}H-FDG uptake in the infectious foci was evaluated by autoradiography of histological sections. Although {sup 18}F-FDG, {sup 67}Ga-citrate, and {sup 125}I-HSA showed comparatively high uptake in the infected muscle [the percentage activity of injected dose (ID) per gram of tissue normalized for rat weight in kilogram (%ID/g) x kg at 2 h postinjection was as follows: {sup 18}F-FDG, 0.184{+-}0.026 to 0.218{+-}0.046; {sup 67}Ga-citrate, 0.221{+-}0.016; {sup 125}I-HSA, 0.198{+-}0.019], the infected muscle to blood ratio was much higher for {sup 18}F-FDG than for {sup 67}Ga-citrate or {sup 125}I-HSA ({sup 18}F-FDG, 10.31{+-}0.76 to 14.89{+-}2.26; {sup 67}Ga-citrate, 1.24{+-}0.67; {sup 125}I-HSA, 0.20{+-}0.02). The draining reactive lymph nodes also showed higher accumulation of {sup 18}F-FDG than of {sup 67}Ga-citrate or {sup 125}I-HSA. The uptake of {sup 3}H-thymidine and l-{sup 11}C- or {sup 3}H-methionine in the infected muscle was lower than that of {sup 18}F- or {sup 3}H-FDG at 2 h postinjection, {sup 3}H-thymidine = 0.039{+-}0.005 and L-{sup 3}H-methionine = 0.063{+-}0.007 (%ID/g) x kg. Autoradiographs showed that the highest {sup 3}H-FDG uptake was seen in the area of inflammatory cell infiltration surrounding the necrotic region. In conclusion, {sup 18}F-FDG, which rapidly accumulates in sites of bacterial infection and in reactive lymph nodes with a high target to background ratio, appears to be

  5. Uptake of positron emission tomography tracers in experimental bacterial infections: a comparative biodistribution study of radiolabeled FDG, thymidine, l-methionine, 67Ga-citrate, and 125I-HSA

    International Nuclear Information System (INIS)

    The purpose of this study was to evaluate the localization of positron emission tomography (PET) tracers [2-deoxy-2-fluoro-d-glucose (FDG), thymidine, and l-methionine] in sites of bacterial infection, and to contrast this with that of other tracers. The left calf muscles of rats were infected with a suspension of Escherichia coli and the biodistribution of 18F- or 3H-FDG, 3H-thymidine, l-11C- or 3H-methionine, gallium-67 citrate (67Ga-citrate) and iodine-125 human serum albumin (125I-HSA) was determined in these animals. 3H-FDG uptake in the infectious foci was evaluated by autoradiography of histological sections. Although 18F-FDG, 67Ga-citrate, and 125I-HSA showed comparatively high uptake in the infected muscle [the percentage activity of injected dose (ID) per gram of tissue normalized for rat weight in kilogram (%ID/g) x kg at 2 h postinjection was as follows: 18F-FDG, 0.184±0.026 to 0.218±0.046; 67Ga-citrate, 0.221±0.016; 125I-HSA, 0.198±0.019], the infected muscle to blood ratio was much higher for 18F-FDG than for 67Ga-citrate or 125I-HSA (18F-FDG, 10.31±0.76 to 14.89±2.26; 67Ga-citrate, 1.24±0.67; 125I-HSA, 0.20±0.02). The draining reactive lymph nodes also showed higher accumulation of 18F-FDG than of 67Ga-citrate or 125I-HSA. The uptake of 3H-thymidine and l-11C- or 3H-methionine in the infected muscle was lower than that of 18F- or 3H-FDG at 2 h postinjection, 3H-thymidine = 0.039±0.005 and L-3H-methionine = 0.063±0.007 (%ID/g) x kg. Autoradiographs showed that the highest 3H-FDG uptake was seen in the area of inflammatory cell infiltration surrounding the necrotic region. In conclusion, 18F-FDG, which rapidly accumulates in sites of bacterial infection and in reactive lymph nodes with a high target to background ratio, appears to be a promising infection detection agent. (orig.)

  6. Effect of valine 106 on structure-function relation of cytosolic human thymidine kinase - Kinetic properties and oligomerization pattern of nine substitution mutants of V106

    DEFF Research Database (Denmark)

    Frederiksen, Hanne; Berenstein, Dvora; Munch-Petersen, Birgitte

    2004-01-01

    characterized nine mutants of amino acid 106 differing in size, conformation and polarity. According to their oligomerization pattern and thymidine kinetics, the TK1 mutants can be divided into two groups. Group I (V106A, V106I and V106T) behaves like V106WT, in that pre-assay exposure to ATP induces reversible...... transition from a dimer with low catalytic activity to a tetramer with high catalytic activity. Group II (V106G, V106H, V106K, V106L and V106Q) behaves like V106M in that they are permanently high activity tetramers, irrespective of ATP exposure. We conclude that size and conformation of amino acid 106 are...

  7. Thymidine Phosphorylase Inhibitors

    Czech Academy of Sciences Publication Activity Database

    Nencka, Radim

    Karachi : Bentham Science Publishers, 2011 - (Atta-ur-Rahman, F.; Choudhary, M.), s. 116-147 ISBN 978-1-60805-162-5 R&D Projects: GA MŠk 1M0508; GA AV ČR 1QS400550501 Institutional research plan: CEZ:AV0Z40550506 Keywords : thymidine phosphorylase inhibitors * angiogenesis * cancer chemotherapy Subject RIV: CC - Organic Chemistry

  8. Expression of thymidine kinase mediated by a novel non-viral delivery system under the control of vascular endothelial growth factor receptor 2 promoter selectively kills human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Ying Wang; Hui-Xiong Xu; Ming-De Lu; Qing Tang

    2008-01-01

    AIM: To investigate the killing efficiency of a recombinant plasmid containing a thymidine kinase (TK) domain insert driven by the vascular endothelial growth factor receptor 2 (VEGFR2) promoter (KDR) on vascular endothelial cells.METHODS: The KDR-TK fragment was extracted from pBluescript 11 KDR-TK plasmid by enzymatic digestion with XhoI and Sa/I. The enhanced green fluorescence protein (EGFP) carrier was extracted from pEGFP by the same procedure. The KDR-TK was inserted into the pEGFP carrier to construct pEGFP-KDR-TK. Using ultrasound irradiation and microbubble,pEGFP-KDR-TK was transferred into human umbilical vein endothelial cells (HUVECs). The transient infection rate was estimated by green fluorescent protein (GFP)expression. Transfected HUVECs, non-transfected HUVECs, and HepG2 cells were cultured in the presence of different concentrations of ganciclovir (GCV), and the killing efficacy of HSV-TK/GCV was analyzed by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTr) assay.RESULTS: The recombinant pEGFP-KDR-TK was successfully constructed by inserting the KDR-TK fragment into the pEGFP carrier. Transfected HUVECs showed cytoplasmic green fluorescence, and the transient transfection rate was about 20.3%. Pools of G418-resistant cells exhibited a higher sensitivity to the prodrug/GCV compared to non-transfected HUVECs or non-transfected HepG2 cells, respectively.CONCLUSION: KDR promoter and the suicide gene/prodrug system mediated by diagnostic ultrasound combined with microbubble can significantly kill HUVECs.Such therapy may present a novel and attractive approach to target gene therapy on tumor vessels.

  9. Imaging herpes viral thymidine kinase-1 reporter gene expression with a new 18F-labeled probe: 2'-fluoro-2'-deoxy-5-[18F]fluoroethyl-1-β-D-arabinofuranosyl uracil

    International Nuclear Information System (INIS)

    The preparation and radiolabeling of 2'-fluoro-2'-deoxy-1-β-D-arabinofuranosyl-5-(2-fluoroethyl)-uracil (FFEAU) with 18F and its evaluation as a probe for imaging herpes simplex virus 1 thymidine kinase (HSV1-tk) gene expression are described. 2'-Fluoro-2'-deoxy-3',5'-di-O-benzoyl-1-β-D-arabinofuranosyl-3-N-benzoyl-5- (2-[18F]fluoroethyl)-uracil 12 was prepared by nucleophilic substitution of the corresponding tosyl 8 or trifluoroethanesulfonyl 9 derivative with n-Bu4N[18F]F. Base hydrolysis was used to remove the benzoyl protecting groups, followed by HPLC purification, to afford [18F]FFEAU 13. The trifluoroethanesulfonyl substrate 9 appears to be the better labeling precursor. Carrier n-Bu4NF was added to the labeling reaction, which resulted in specific activities of 40-70 Ci/mmol (estimated). Radiochemical purity averaged 94±4%. Although [18F]FFEAU was obtained in low radiochemical yield with 9 and further optimization of the radiosynthesis will be required, sufficient product was available for a series of in vitro and in vivo studies. [18F]FFEAU was directly compared with [3H]TdR in a series of in vitro accumulation studies involving a HSV1-tk stably transduced cell line, RG2TK+ and a nontransduced, wild-type RG2 cells. The initial in vitro and in vivo imaging studies are promising; FFEAU has in vitro accumulation and sensitivity characteristics similar to that previously reported for FIAU, but greater selectivity than FIAU due to lower uptake and retention in nontransduced cells and tissues. The animal imaging experiment showed low levels of radioactivity in the lungs, with little or no radioactivity seen in the heart, liver, spleen and intestines.

  10. Expression and characterization of the integral membrane domain of bacterial histidine kinase SCO3062 for structural studies

    International Nuclear Information System (INIS)

    Bacterial histidine kinases play an important role in the response to external stimuli. Structural studies of the histidine kinase transmembrane domain are challenging due to difficulties in protein expression and sample preparation. After carrying out expression screening of a series of histidine kinases, we investigated sample preparation methods for obtaining high quality samples of the periplasmic and transmembrane domain (PTD) of the bacterial histidine kinase SCO3062. Various sample conditions were tested for their ability to give homogeneous NMR spectra of the SCO3062 PTD with well-resolved resonances. Circular dichroism and 3D 15N-edited NOESY spectrum results demonstrate that the SCO3062 PTD is predominantly α-helical. This method should be applicable to the NMR analysis of other transmembrane proteins

  11. Expression and characterization of the integral membrane domain of bacterial histidine kinase SCO3062 for structural studies.

    Science.gov (United States)

    Yeo, Kwon Joo; Kwak, Su-Nam; Kim, Hyun Jung; Cheong, Chaejoon; Kim, Myung Hee; Jeon, Young Ho

    2008-11-14

    Bacterial histidine kinases play an important role in the response to external stimuli. Structural studies of the histidine kinase transmembrane domain are challenging due to difficulties in protein expression and sample preparation. After carrying out expression screening of a series of histidine kinases, we investigated sample preparation methods for obtaining high quality samples of the periplasmic and transmembrane domain (PTD) of the bacterial histidine kinase SCO3062. Various sample conditions were tested for their ability to give homogeneous NMR spectra of the SCO3062 PTD with well-resolved resonances. Circular dichroism and 3D (15)N-edited NOESY spectrum results demonstrate that the SCO3062 PTD is predominantly alpha-helical. This method should be applicable to the NMR analysis of other transmembrane proteins. PMID:18789898

  12. Reconstitution of an efficient thymidine salvage pathway in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Vernis, L.; Piskur, Jure; Diffley, J.F.X.

    2003-01-01

    The budding yeast Saccharomyces cerevisiae is unable to incorporate exogenous nucleosides into DNA. We have made a number of improvements to existing strategies to reconstitute an efficient thymidine salvage pathway in yeast. We have constructed strains that express both a nucleoside kinase as well...

  13. New Kinase Regulation Mechanism Found in HipBA: a Bacterial Persistence Switch

    Energy Technology Data Exchange (ETDEWEB)

    Evdokimov, A.; Voznesensky, I; Fennell, K; Anderson, M; Smith, J; Fisher, D

    2009-01-01

    Bacterial persistence is the ability of individual cells to randomly enter a period of dormancy during which the cells are protected against antibiotics. In Escherichia coli, persistence is regulated by the activity of a protein kinase HipA and its DNA-binding partner HipB, which is a strong inhibitor of both HipA activity and hip operon transcription. The crystal structure of the HipBA complex was solved by application of the SAD technique to a mercury derivative. In this article, the fortuitous and interesting effect of mercury soaks on the native HipBA crystals is discussed as well as the intriguing tryptophan-binding pocket found on the HipA surface. A HipA-regulation model is also proposed that is consistent with the available structural and biochemical data.

  14. New kinase regulation mechanism found in HipBA: a bacterial persistence switch.

    Science.gov (United States)

    Evdokimov, Artem; Voznesensky, Igor; Fennell, Kimberly; Anderson, Marie; Smith, James F; Fisher, Douglas A

    2009-08-01

    Bacterial persistence is the ability of individual cells to randomly enter a period of dormancy during which the cells are protected against antibiotics. In Escherichia coli, persistence is regulated by the activity of a protein kinase HipA and its DNA-binding partner HipB, which is a strong inhibitor of both HipA activity and hip operon transcription. The crystal structure of the HipBA complex was solved by application of the SAD technique to a mercury derivative. In this article, the fortuitous and interesting effect of mercury soaks on the native HipBA crystals is discussed as well as the intriguing tryptophan-binding pocket found on the HipA surface. A HipA-regulation model is also proposed that is consistent with the available structural and biochemical data. PMID:19622872

  15. Evaluation of localized bacterial infection using radioisotope-labeled nucleosides imaging modality

    International Nuclear Information System (INIS)

    Conventional diagnostic methods for infections are difficult to distinguish localized bacterial infections from sites of sterile inflammation. For this reason, the importance of developing methods to image bacterial infections is widely recognized. In this study to acquire bacterial infection imaging with radiolabeled nucleosides, in vitro bacterial thymidine kinase (tk) activities of Salmonella typhimurium with [18F]FLT and [125I]IVDU were measured and localized infections model in BALB/c mice was imaged with [18F]FLT or [125I]FIAU

  16. Loss of the DNA Damage Repair Kinase ATM Impairs Inflammasome-Dependent Anti-Bacterial Innate Immunity.

    Science.gov (United States)

    Erttmann, Saskia F; Härtlova, Anetta; Sloniecka, Marta; Raffi, Faizal A M; Hosseinzadeh, Ava; Edgren, Tomas; Rofougaran, Reza; Resch, Ulrike; Fällman, Maria; Ek, Torben; Gekara, Nelson O

    2016-07-19

    The ATM kinase is a central component of the DNA damage repair machinery and redox balance. ATM dysfunction results in the multisystem disease ataxia-telangiectasia (AT). A major cause of mortality in AT is respiratory bacterial infections. Whether ATM deficiency causes innate immune defects that might contribute to bacterial infections is not known. Here we have shown that loss of ATM impairs inflammasome-dependent anti-bacterial innate immunity. Cells from AT patients or Atm(-/-) mice exhibited diminished interleukin-1β (IL-1β) production in response to bacteria. In vivo, Atm(-/-) mice were more susceptible to pulmonary S. pneumoniae infection in a manner consistent with inflammasome defects. Our data indicate that such defects were due to oxidative inhibition of inflammasome complex assembly. This study reveals an unanticipated function of reactive oxygen species (ROS) in negative regulation of inflammasomes and proposes a theory for the notable susceptibility of AT patients to pulmonary bacterial infection. PMID:27421701

  17. Efficacy and safety of dendrimer nanoparticles with coexpression of tumor necrosis factor-α and herpes simplex virus thymidine kinase in gene radiotherapy of the human uveal melanoma OCM-1 cell line

    Directory of Open Access Journals (Sweden)

    Wang YC

    2013-10-01

    Full Text Available Yingchih Wang,1,* Li Mo,2,* Wenbin Wei,1 Xuehui Shi1 1Beijing Tongren Eye Center, Capital Medical University, Beijing, People's Republic of China; 2Department of Ophthalmology, The 180th Hospital of PLA, Quanzhou, People's Republic of China *These authors contributed equally to this work Abstract: Human uveal melanoma is the most common primary intraocular tumor, and brachytherapy is one of the most common and effective treatment strategies. In order to find a safer and more effective way to increase the radio sensitivity of the tumor, we tried to use the dendrimer nanoparticle performing coexpression gene radiotherapy. In this study, we constructed recombinant DNA plasmids (early growth response-1 tumor necrosis factor-α [pEgr1-TNFα], pEgr1 thymidine kinase [TK], and pEgr1-TNFα -TK according to the Egr1 promoter sequence. The sequences of human TNFα and herpes simplex virus (HSV TK that were published by GenBank. Agarose gel electrophoresis and DNA sequencing had proven that we constructed the double-gene recombined plasmids pEgr1-TNF-TK correctly, as well as the plasmids pEgr1-TNFα and pEgr1-TK. The dendrimer nanoparticles combined with plasmid DNA as dendriplexes were verified with agarose gel electrophoresis and observed by transmission electron microscopy (TEM and scanning electron microscopy to define size and shape. Zeta potential was measured using a Zetasizer analyzer. Optimal size and neutral zeta-potential characteristics of dendriplexes were achieved for the transfection studies. DNase I examination proved that the dendriplexes could protect plasmid DNA for at least 6 hours. The recombinant plasmids were transfected with dendrimer nanoparticles into the human choroidal melanoma OCM-1 cell line, followed by exposure to iodine-125 (125I after transfection. After transfection with dendrimer nanoparticles and the irradiation of 125I, the gene expressions of TNFα and HSV1-TK were significantly increased at the protein level by

  18. The experimental study of reporter probe 131I-FIAU in neonatal cardiac myocytes after transfer of herpes simplex virus type 1 thymidine kinase reporter gene by different vectors

    International Nuclear Information System (INIS)

    Objective: Reporter gene imaging is a promising approach for noninvasive monitoring of cardiac gene therapy. In the present study, the recombinant plasmid and adenoviral vector carrying reporter gene. herpes simplex virus type 1 thymidine kinase (HSV1-tk), were constructed and transferred into nee-natal cardiac myocytes, and a series of in vitro studies were carried out on the cells transferred to evaluate the uptake of radiolabeled reporter probe and to compare both vectors for cardiac reporter gene imaging. Methods: Neonatal cardiac myocytes were obtained from rat heart by single collagenase digestion. HSVI-tk. chosen as the reporter gene.was inserted into adenovirus vector (Ad5-tk) and plasmid (pDC316-tk), thus it could be transferred into neonatal cardiac myocytes. Recombinant adenovirus containing enhanced green fluorescent protein (Ad5-EGFP) was used as control. Recombinant plasmid was coated with lipofectamine TM 2000 (pDC316-tk/lipoplex). The specific reporter probe of HSV1-tk, 2'-fluoro-2'-deoxy-l-β-D-arabinofuranosyl-uracil (FAU), was labeled with 131I by solid phase oxidation with lodogen. Product wag purified on a reverse. phase Sep-Pak C18 column and the radiochemical purity wag then assessed. The accumulation of it in the transferred cardiac myocytes wag detected as uptake rate. Furthermore, mRNA expression of HSV1-tk was detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), while its protein expression wag located by immunocytochemistry. Results: FAU could be labeled with 131I and the labeling efficiency was (53.82 ±2.05)%. The radiochemical purity was (94.85 ± 1.76)% after purification, and it kept stable in vitro for at least 24h. Time-dependent increase of the ac- cumulation of 131I-FIAU was observed in both Ad5-tk group and pDC316-tk/lipoplex group. and the highest uptake rate occurred at 5h, with peak values of (12.55 ± 0.37)% and (2.09 ± 0.34)% respectively. However, it also indicated that greater

  19. Estimations of the DNA Synthesis Rate of Bone Marrow Cells after Administration of Labelled Thymidine In Vitro

    International Nuclear Information System (INIS)

    Bone marrow cells are incubated with labelled thymidine under varying in vitro conditions. The incorporation rate of labelled thymidine into DNA is influenced by the condition and duration of. the in vitro incubation. Similar influences operate on the pool size of labelled thymidine phosphates. Up to concentrations of 10-6 M thymidine in the incubation medium there is a linear relation of thymidine concentration and thymidine incorporation into DNA. Concentrations of thymidine exceeding 10-6 M lead to increasing inhibition of the thymidine kinase. The endogenous formation of thymidylate cannot be inhibited entirely by exogenous thymidine supply. Consequently, determinations of the DNA synthesis rate from the incorporated amount of labelled thymidine have to be corrected for the respective endogenous thymidylate contribution. A better procedure is to block the formation of endogenous thymidylate by means of amethopterin. Standard conditions are described, under which an undisturbed synthesis of DNA thymine from exogenous thymidine only takes place. Determinations can be performed by means of autoradiographic or biochemical techniques. By application of the semi-automatic grain counting technique, after sufficient autoradiographic standardization, evaluations of DNA synthesis rates and DNA synthesis times of different cell types in the bone marrow become practicable. (author)

  20. Thymidine incorporation by the microbial community of standing dead Spartina alterniflora

    International Nuclear Information System (INIS)

    Thymidine incorporation by the microbial community on standing dead leaves of Spartina alterniflora did not obey many of the assumptions inherent in the use of the technique in planktonic systems. Incorporation rates of [methyl-3H]thymidine were nonsaturable over a wide concentration range (101 to 105 nM). Owing to metabolism by both fungi and bacteria, a major fraction of the radiolabel (mean, 48%) appeared in protein. Extraction of the radiolabeled macromolecules were inefficient, averaging 8.8%. Based on an empirically derived conversion factor, 4 x 1018 cells mol of thymidine-1, doubling times ranged from 4 to 69 h for the epiphytic bacterial assemblage

  1. Tyrosine protein kinase inhibitors block invasin-promoted bacterial uptake by epithelial cells.

    OpenAIRE

    Rosenshine, I.; Duronio, V; Finlay, B B

    1992-01-01

    The ability to enter into (invade) mammalian cells is an essential virulence determinant of many pathogenic bacteria and intracellular parasites. These organisms are internalized by host cells upon attachment to their surface. However, the mechanisms used by intracellular parasites to induce internalization into host cells have not been defined. We found that the protein kinase inhibitor staurosporine blocks invasion by some pathogenic bacteria, including Yersinia enterocolitica and Yersinia ...

  2. Assessment of Thymidine Phosphorylase Function: Measurement of Plasma Thymidine (and Deoxyuridine) and Thymidine Phosphorylase Activity

    Science.gov (United States)

    Martí, Ramon; López, Luis C.; Hirano, Michio

    2016-01-01

    We describe detailed methods to measure thymidine (dThd) and deoxyuridine (dUrd) concentrations and thymidine phosphorylase (TP) activity in biological samples. These protocols allow the detection of TP dysfunction in patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Since the identification of mutations in TϒMP, the gene encoding TP, as the cause of MNGIE (Nishino et al. Science 283:689–692, 1999), the assessment of TP dysfunction has become the best screening method to rule out or confirm MNGIE in patients. TϒMP sequencing, to find the causative mutations, is only needed when TP dysfunction is detected. dThd and dUrd are measured by resolving these compounds with high-performance liquid chromatography (HPLC) followed by the spectrophotometric monitoring of the eluate absorbance at 267 nm (HPLC-UV). TP activity can be measured by an endpoint determination of the thymine formed after 1 h incubation of the buffy coat homogenate in the presence of a large excess of its substrate dThd, either spectrophotometrically or by HPLC-UV. PMID:22215544

  3. The effect of low doses on the metabolism of thymidine in bone marrow cells of NMRI mice; an in vivo - in vitro study using labeled nucleic acid precursors

    International Nuclear Information System (INIS)

    Low-dose whole-body exposure of mice to less than 0.01 Gy causes a temporary inhibition of incorporation of thymidine into DNA of bone-marrow cells in vitro. This inhibition has its maximum at 4 hours after irradiation. The low-dose effect is due to the temporary inhibition of cellular thymidine kinase. This decrease in thymidine kinase activity involves only 35% of the entire cellular enzyme activity and, analogous to the depression of thymidine incorporation into the cells, is only seen in the presence of a physiological concentration of NaHCO3. The inhibition of TdR-kinase at very low doses should not be viewed as an expression of cell damage but might be part of a cellular protection mechanism different from known repair systems. (orig.)

  4. The pyrimidine nucleotide carrier PNC1 and mitochondrial trafficking of thymidine phosphates in cultured human cells

    Energy Technology Data Exchange (ETDEWEB)

    Franzolin, Elisa; Miazzi, Cristina; Frangini, Miriam; Palumbo, Elisa; Rampazzo, Chiara [Department of Biology, University of Padova, Via Ugo Bassi 58B, I-35131 Padova (Italy); Bianchi, Vera, E-mail: vbianchi@bio.unipd.it [Department of Biology, University of Padova, Via Ugo Bassi 58B, I-35131 Padova (Italy)

    2012-10-15

    In cycling cells cytosolic de novo synthesis of deoxynucleotides is the main source of precursors for mitochondrial (mt) DNA synthesis. The transfer of deoxynucleotides across the inner mt membrane requires protein carriers. PNC1, a SLC25 family member, exchanges pyrimidine nucleoside triphosphates in liposomes and its downregulation decreases mtUTP concentration in cultured cells. By an isotope-flow protocol we confirmed transport of uridine nucleotides by PNC1 in intact cultured cells and investigated PNC1 involvement in the mt trafficking of thymidine phosphates. Key features of our approach were the manipulation of PNC1 expression by RNA interference or inducible overexpression, the employment of cells proficient or deficient for cytosolic thymidine kinase (TK1) to distinguish the direction of flow of thymidine nucleotides across the mt membrane during short pulses with [{sup 3}H]-thymidine, the determination of mtdTTP specific radioactivity to quantitate the rate of mtdTTP export to the cytoplasm. Downregulation of PNC1 in TK1{sup -} cells increased labeled dTTP in mitochondria due to a reduced rate of export. Overexpression of PNC1 in TK1{sup +} cells increased mtdTTP pool size and radioactivity, suggesting an involvement in the import of thymidine phosphates. Thus PNC1 is a component of the network regulating the mtdTTP pool in human cells. -- Highlights: Black-Right-Pointing-Pointer Thymidine phosphates exchange between mitochondria and cytosol in mammalian cells. Black-Right-Pointing-Pointer siRNA-downregulation of PNC1 delays mitochondrial dTTP export in TK1{sup -} cells. Black-Right-Pointing-Pointer PNC1 overexpression accumulates dTTP in mitochondria of TK1{sup +} cells. Black-Right-Pointing-Pointer PNC1 exchanges thymidine nucleotides across the mitochondrial inner membrane. Black-Right-Pointing-Pointer PNC1 participates in the regulation of the mtdTTP pool supporting mtDNA synthesis.

  5. The pyrimidine nucleotide carrier PNC1 and mitochondrial trafficking of thymidine phosphates in cultured human cells

    International Nuclear Information System (INIS)

    In cycling cells cytosolic de novo synthesis of deoxynucleotides is the main source of precursors for mitochondrial (mt) DNA synthesis. The transfer of deoxynucleotides across the inner mt membrane requires protein carriers. PNC1, a SLC25 family member, exchanges pyrimidine nucleoside triphosphates in liposomes and its downregulation decreases mtUTP concentration in cultured cells. By an isotope-flow protocol we confirmed transport of uridine nucleotides by PNC1 in intact cultured cells and investigated PNC1 involvement in the mt trafficking of thymidine phosphates. Key features of our approach were the manipulation of PNC1 expression by RNA interference or inducible overexpression, the employment of cells proficient or deficient for cytosolic thymidine kinase (TK1) to distinguish the direction of flow of thymidine nucleotides across the mt membrane during short pulses with [3H]-thymidine, the determination of mtdTTP specific radioactivity to quantitate the rate of mtdTTP export to the cytoplasm. Downregulation of PNC1 in TK1− cells increased labeled dTTP in mitochondria due to a reduced rate of export. Overexpression of PNC1 in TK1+ cells increased mtdTTP pool size and radioactivity, suggesting an involvement in the import of thymidine phosphates. Thus PNC1 is a component of the network regulating the mtdTTP pool in human cells. -- Highlights: ► Thymidine phosphates exchange between mitochondria and cytosol in mammalian cells. siRNA-downregulation of PNC1 delays mitochondrial dTTP export in TK1− cells. ► PNC1 overexpression accumulates dTTP in mitochondria of TK1+ cells. ► PNC1 exchanges thymidine nucleotides across the mitochondrial inner membrane. ► PNC1 participates in the regulation of the mtdTTP pool supporting mtDNA synthesis.

  6. The experimental research on the osteosarcoma U-2 cells transfected by thymidine kinase gene in vitro%TK基因转染骨肉瘤U-2细胞体外实验研究

    Institute of Scientific and Technical Information of China (English)

    李晋惠; 吕智; 马卓; 冯毅; 王军; 兰彦平; 张桦栋

    2012-01-01

    Objective To study the killing effects of the liposome-me dialed thyrnidine kinase (TK)/ gandclovir (GCV) system on human osteosarcoma cells and its bystander effects. Methods The recombinant piasmid plRES-EGFP-tk was constructed, and then was prepared and transformed into Escherichia coli BL21 competent cells. And the sequencing was identified. The recombinant piasmid deoxyribonucleic acid (DNA) was extracted and purified, and the human osteosarcoma cell line U-2 osteosarcoma (OS) was cultured and transfected. The ceil growth state was analyzed by flow cytomctry and observed under inverted fluorescence microscope. After transfection, the supernatant of culture medium was added to the TK. (-) U-2 OS, and then its bystander effects were observed. Results The recombinant piasmid pIRES-EGFP-tk containing complementary deoxyribonucleic acid (cDNA) fragments with TK gene was successfully constructed. By such method as polymerase chain reaction (PCR) and so on. The size and position of plasmids were identified, and the results were consistent with the experimental design. Under fluorescence microscope, U-2 OS (TK-) was observed to show green fluorescence. The supernatant of culture medium of U-2 OS TK/GCV system had a significant effect of inducing apoptosis. However, when the supernatant was filtrated with 0.2μrn filter membrane, its effects disappeared. Conclusions The recombinant piasmid pIRES-EGFP-tk is successfully constructed. The experiment in vitro demonstrated that the human osteosarcoma cell line U-2 OS is sensitive to the liposome-mediated TK/GCV system and its bystander effects are significant. It is expected to become a new gene therapy approach for the osteosarcoma.%目的 研究脂质体介导的TK/GCV系统对人骨肉瘤细胞的杀伤作用及其产生的旁观者效应.方法 构建重组质粒pIRES-EGFP-tk,制备和转化感受态大肠杆菌BL21以及鉴定测序,提取、纯化重组质粒DNA,培养和转染人骨肉瘤细胞株U-2\tOS,流式细

  7. Thymidine kinase 1 regulatory fine-tuning through tetramer formation

    DEFF Research Database (Denmark)

    Mutahir, Zeeshan; Clausen, Anders R.; Andersson, Karl-Magnus; Wisen, Sofia M; Munch-Petersen, Birgitte; Piskur, Jure

    2013-01-01

    concentration-dependent transition of TK1 from a dimer with low catalytic efficiency to a tetramer with high catalytic efficiency. This regulatory fine-tuning serves as an additional control to provide a balanced pool of nucleic acid precursors in the cell. We subcloned and over-expressed 10 different TK1s...

  8. Overexpression of Rice Wall-Associated Kinase 25 (OsWAK25) Alters Resistance to Bacterial and Fungal Pathogens.

    Science.gov (United States)

    Harkenrider, Mitch; Sharma, Rita; De Vleesschauwer, David; Tsao, Li; Zhang, Xuting; Chern, Mawsheng; Canlas, Patrick; Zuo, Shimin; Ronald, Pamela C

    2016-01-01

    Wall-associated kinases comprise a sub-family of receptor-like kinases that function in plant growth and stress responses. Previous studies have shown that the rice wall-associated kinase, OsWAK25, interacts with a diverse set of proteins associated with both biotic and abiotic stress responses. Here, we show that wounding and BTH treatments induce OsWAK25 transcript expression in rice. We generated OsWAK25 overexpression lines and show that these lines exhibit a lesion mimic phenotype and enhanced expression of rice NH1 (NPR1 homolog 1), OsPAL2, PBZ1 and PR10. Furthermore, these lines show resistance to the hemibiotrophic pathogens, Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe oryzae, yet display increased susceptibility to necrotrophic fungal pathogens, Rhizoctonia solani and Cochliobolus miyabeanus. PMID:26795719

  9. Bacterial abundance and production in the central and eastern Arabian Sea

    Digital Repository Service at National Institute of Oceanography (India)

    Ramaiah, N.; Raghukumar, S.; Gauns, M.

    Seasonal and spatial variations in bacterial and picoplankton abundances and bacterial production (thymidine incorporation rates) were determined in the water column up to 150 m in several stations in the central and eastern Arabian Sea. Higher...

  10. A Serine-Threonine Kinase (StkP Regulates Expression of the Pneumococcal Pilus and Modulates Bacterial Adherence to Human Epithelial and Endothelial Cells In Vitro.

    Directory of Open Access Journals (Sweden)

    Jenny A Herbert

    Full Text Available The pneumococcal serine threonine protein kinase (StkP acts as a global regulator in the pneumococcus. Bacterial mutants deficient in StkP are less virulent in animal models of infection. The gene for this regulator is located adjacent to the gene for its cognate phosphatase in the pneumococcal genome. The phosphatase dephosphorylates proteins phosphorylated by StkP and has been shown to regulate a number of key pneumococcal virulence factors and to modulate adherence to eukaryotic cells. The role of StkP in adherence of pneumococci to human cells has not previously been reported. In this study we show StkP represses the pneumococcal pilus, a virulence factor known to be important for bacterial adhesion. In a serotype 4 strain regulation of the pilus by StkP modulates adherence to human brain microvascular endothelial cells (HBMEC and human lung epithelial cells. This suggests that the pneumococcal pilus may play a role in adherence during infections such as meningitis and pneumonia. We show that regulation of the pilus occurs at the population level as StkP alters the number of pili-positive cells within a single culture. As far as we are aware this is the first gene identified outside of the pilus islet that regulates the biphasic expression of the pilus. These findings suggest StkPs role in cell division may be linked to regulation of expression of a cell surface adhesin.

  11. Rates of [3H]thymidine incorporation into thymidine triphosphate vs nuclear matrix DNA in vivo

    International Nuclear Information System (INIS)

    Previous studies in vitro have indicated the possible existence of more than one thymidine pool accessible to DNA replication forks. To investigate this possibility in vivo, the incorporation of [3H]thymidine (3H-Thd) into matrix DNA and soluble thymidine triphosphate (TTP) was examined in regenerating rat liver. Nuclear matrix-attached DNA, although only 2% of total nuclear DNA, represents the most newly replicated DNA in eukaryotic cells, thereby providing a very sensitive means of examining precursor incorporation kinetics. Partially hepatectomized rats were injected with 200 μC 3H-Thd via the hepatic portal vein. After various pulse times, matrix DNA and/or soluble TTP were extracted and their specific activities determined. After only 1 min, the labeling of matrix DNA had already reached a maximum. Matrix DNA contained 27% of the label at 2 min, 11% at 5 min, and only 5% at 10 min with respect to total DNA. Contrary to the kinetics found for matrix DNA, 3H-Thd incorporation into TTP was still increasing at early pulse times and had not peaked even after a 10 min pulse. Five min after injection, TTP specific activity was 3050 dpm/nmole and was 7030 dpm/nmole after 10 min. Since the peak specific activity for [3H]-Thd incorporation into matrix DNA precedes that for [3H]-Thd into TTP, there may be channeling of exogenous thymidine directly to the site of DNA synthesis bypassing existing nucleotide pools

  12. A fluorescent surrogate of thymidine in duplex DNA.

    Science.gov (United States)

    Mata, Guillaume; Schmidt, Olivia P; Luedtke, Nathan W

    2016-03-17

    is a new fluorescent thymidine mimic composed of 2'-deoxyuridine fused to dimethylaniline. exhibits the same pKa and base pairing characteristics as native thymidine residues, and its fluorescence properties are highly sensitive to nucleobase ionization, base pairing and metal binding. PMID:26954231

  13. Consequences of Accounting for Isotopic Dilution in Thymidine Incorporation Assays

    Science.gov (United States)

    Chrzanowski, Thomas H.

    1988-01-01

    Rates of thymidine incorporation into DNA were corrected for isotope dilution by internal nucleotide pools and were compared with rates obtained from uncorrected data. Differences as large as 109% were observed between corrected and uncorrected estimates of thymidine incorporation. The degree of underestimation varied seasonally and, to a lesser extent, spatially. PMID:16347698

  14. Consequences of Accounting for Isotopic Dilution in Thymidine Incorporation Assays

    OpenAIRE

    Chrzanowski, Thomas H.

    1988-01-01

    Rates of thymidine incorporation into DNA were corrected for isotope dilution by internal nucleotide pools and were compared with rates obtained from uncorrected data. Differences as large as 109% were observed between corrected and uncorrected estimates of thymidine incorporation. The degree of underestimation varied seasonally and, to a lesser extent, spatially.

  15. TaCPK2-A, a calcium-dependent protein kinase gene that is required for wheat powdery mildew resistance enhances bacterial blight resistance in transgenic rice.

    Science.gov (United States)

    Geng, Shuaifeng; Li, Aili; Tang, Lichuan; Yin, Lingjie; Wu, Liang; Lei, Cailin; Guo, Xiuping; Zhang, Xin; Jiang, Guanghuai; Zhai, Wenxue; Wei, Yuming; Zheng, Youliang; Lan, Xiujin; Mao, Long

    2013-08-01

    Calcium-dependent protein kinases (CPKs) are important Ca2+ signalling components involved in complex immune and stress signalling networks; but the knowledge of CPK gene functions in the hexaploid wheat is limited. Previously, TaCPK2 was shown to be inducible by powdery mildew (Blumeria graminis tritici, Bgt) infection in wheat. Here, its functions in disease resistance are characterized further. This study shows the presence of defence-response and cold-response cis-elements on the promoters of the A subgenome homoeologue (TaCPK2-A) and D subgenome homoeologue (TaCPK2-D), respectively. Their expression patterns were then confirmed by quantitative real-time PCR (qRT-PCR) using genome-specific primers, where TaCPK2-A was induced by Bgt treatment while TaCPK2-D mainly responded to cold treatment. Downregulation of TaCPK2-A by virus-induced gene silencing (VIGS) causes loss of resistance to Bgt in resistant wheat lines, indicating that TaCPK2-A is required for powdery mildew resistance. Furthermore, overexpression of TaCPK2-A in rice enhanced bacterial blight (Xanthomonas oryzae pv. oryzae, Xoo) resistance. qRT-PCR analysis showed that overexpression of TaCPK2-A in rice promoted the expression of OsWRKY45-1, a transcription factor involved in both fungal and bacterial resistance by regulating jasmonic acid and salicylic acid signalling genes. The opposite effect was found in wheat TaCPK2-A VIGS plants, where the homologue of OsWRKY45-1 was significantly repressed. These data suggest that modulation of WRKY45-1 and associated defence-response genes by CPK2 genes may be the common mechanism for multiple disease resistance in grass species, which may have undergone subfunctionalization in promoters before the formation of hexaploid wheat. PMID:23918959

  16. In Vitro Metabolism of H3 Thymidine

    International Nuclear Information System (INIS)

    The in vitro metabolism and fate of the DNA precursor H3 thymidine, H3TDR, (1.9 c/mMole) was studied in human leukaemic blood and normal dog marrow. New DNA synthesis was estimated by determining the labelling indices and grain counts of cell autoradiograms made from the mixtures at 1 h. Cell labelling could readily be depressed by adding minute amounts of unlabelled TDR to H3TDR initially, demonstrating the smallness of the TDR pool. When TDR was added after 20 min to the incubation mixture, no depression of labelling occurred.. This 20 min period defines the H3TDR incorporation period. Supernatants at 1 h still contained considerable H3 activity yet failed to label fresh added cells. These ''used'' supernatants contained H3TDR as the major H3 compound, and only small amounts of H3 thymine; H3 water was not formed. Speculations on the mechanisms involved are presented. (author)

  17. Identification and characterization of transforming growth factor β-activated kinase 1 from Litopenaeus vannamei involved in anti-bacterial host defense.

    Science.gov (United States)

    Wang, Sheng; Li, Haoyang; L, Kai; Qian, Zhe; Weng, Shaoping; He, Jianguo; Li, Chaozheng

    2016-05-01

    LvTAK1, a member of transforming growth factor β-activated kinase 1 (TAK1) families, has been identified from Litopenaeus vannamei in this study. The full length of LvTAK1 is 2670 bp, including a 2277 bp open reading frame (ORF) that encoded a putative protein of 758 amino acids with a calculated molecular weight of ∼83.4 kDa LvTAK1 expression was most abundant in muscles and was up-regulated in gills after LPS, Vibrio parahaemolyticus, Staphhylococcu saureus, Poly (I:C) and WSSV challenge. Both in vivo and in vitro experiments indicated that LvTAK1 could activate the expression of several antimicrobial peptide genes (AMPs). In addition, the dsRNA-mediated knockdown of LvTAK1 enhanced the susceptibility of shrimps to Vibrio parahaemolyticus, a kind of Gram-negative bacteria. These results suggested LvTAK1 played important roles in anti-bacterial infection. CoIP and subcellular localization assay demonstrated that LvTAK1 could interact with its binding protein LvTAB2, a key component of IMD pathway. Moreover, over-expression of LvTAK1 in Drosophila S2 cell could strongly induce the promoter activity of Diptericin (Dpt), a typical AMP which is used to read out of the activation of IMD pathway. These findings suggested that LvTAK1 could function as a component of IMD pathway. Interestingly, with the over-expression of LvTAK1 in S2 cell, the promoter activity of Metchnikowin (Mtk), a main target gene of Toll/Dif pathway, was up-regulated over 30 times, suggesting that LvTAK1 may also take part in signal transduction of the Toll pathway. In conclusion, we provided some evidences that the involvement of LvTAK1 in the regulation of both Toll and IMD pathways, as well as innate immune against bacterial infection in shrimp. PMID:27033469

  18. DNA immunization with a herpes simplex virus 2 bacterial artificial chromosome

    International Nuclear Information System (INIS)

    Construction of a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) is described. BAC vector sequences were inserted into the thymidine kinase gene of HSV-2 by homologous recombination. DNA from cells infected with the resulting recombinant virus was transformed into E. coli, and colonies containing the HSV-2 BAC (HSV2-BAC) were isolated and analyzed for the expected genotype. HSV2-BAC DNA was infectious when transfected back into mammalian cells and the resulting virus was thymidine kinase negative. When used to immunize mice, the HSV2-BAC DNA elicited a strong HSV-2 specific antibody response that was equal to or greater than live virus immunization. Further, HSV2-BAC immunization was protective when animals were challenged with a lethal dose of virus. The utility of the HSV2-BAC for construction of recombinant virus genomes was demonstrated by elimination of the HSV-2 glycoprotein D (gD) gene. A recombinant HSV-2 BAC with the gD gene deleted was isolated and shown to be incapable of producing infectious virus following transfection unless an HSV gD gene was expressed in a complementing cell line. Immunization of mice with the HSV2 gD-BAC also elicited an HSV-2 specific antibody response and was protective. The results demonstrate the feasibility of DNA immunization with HSV-2 bacterial artificial chromosomes for replicating and nonreplicating candidate HSV-2 vaccines, as well as the utility of BAC technology for construction and maintenance of novel HSV-2 vaccines. The results further suggest that such technology will be a powerful tool for dissecting the immune response to HSV-2

  19. Reduction of bacterial growth by a vesicular-arbuscular mycorrhizal fungus in the rhizosphere of cucumber (Cucumis sativus L.)

    DEFF Research Database (Denmark)

    Christensen, H.; Jakobsen, I.

    1993-01-01

    incorporation of [H-3]-thymidine was around one order of magnitude lower compared to other rhizosphere measurements, probably because pseudomonads that did not incorporate [H-3]-thymidine dominated the bacterial population. The VAM probably decreased the amount of plant root-derived organic matter available for...

  20. Deoxyribonucleoside kinases: two enzyme families catalyze the same reaction

    DEFF Research Database (Denmark)

    Sandrini, Michael; Piskur, Jure

    2005-01-01

    Mammals have four deoxyribonucleoside kinases, the cytoplasmic (TK1) and mitochondrial (TK2) thymidine kinases, and the deoxycytidine (dCK) and deoxyguanosine (dGK) kinases, which salvage the precursors for nucleic acids synthesis. In addition to the native deoxyribonucleoside substrates, the kin...... kinases can phosphorylate and thereby activate a variety of anti-cancer and antiviral prodrugs. Recently, the crystal structure of human TK1 has been solved and has revealed that enzymes with fundamentally different origins and folds catalyze similar, crucial cellular reactions....

  1. 腺病毒为载体的单纯疱疹病毒胸苷激酶/更昔洛韦系统联合光动力治疗口腔恶性肿瘤的初步观察%The initial observation of adenovirus vector-mediated herpes simplex virus-thymidine kinase gene/ganciclovir system and photodynamic therapy for oral malignant tumor treatments

    Institute of Scientific and Technical Information of China (English)

    陈世璋; 张燕升; 范忠; 李维弟; 郭莹; 王潇; 陈剑飞; 李宁

    2011-01-01

    Objective To evaluate the method of adenovirus vector-mediated herpes simplex virus-thymidine kinase gene (ADV-TK)/ganciclovir(GCV) system and photodynamic therapy (PUT) for treating the oral malignant tumor. Methods Ten patients who were suffering from oral malignant tumor of the different positions were selected, and injected with the hematoporphyrin derivative (HPD), irradiated by picking the corresponding laser frequency, and injected the ADV-TK gene into the tumor body and periphery. By the imaging and the hemodynamics analysis, the clinical efficacy after infusing vein with GCV was assessed. Results After the combination method treatments, patients' tumors appeared clear shrinkage or complete extinction. There was obvious fall of the blood flow volume in the tumor body. Imaging results showed significantly differences. So it was a perfect and effective treatment Conclusion There are many advantages to apply the ADV-TK/GCV system and PDT treatment on the oral malignant tumor, minimal side effects and greater clinical security. It is a safe and credible therapy which can be offered for curing the oral malignant tumor systematically.%目的 应用腺病毒为载体的单纯疱疹病毒胸苷激酶(ADV-TK)/更昔洛韦(GCV)系统联合光动力疗法对口腔恶性肿瘤复发患者进行治疗,探讨此法治疗口腔恶性肿瘤的临床效果.方法 选取10名口腔不同部位的恶性肿瘤复发患者,用光敏剂——血卟啉衍生物(HPD)静脉给药,选择相应激光输出功率定时照射,以ADV-TK基因行瘤体及周边注射,经GCV静脉输入治疗后,通过影像学及瘤体血流动力学分析进行临床疗效评估.结果 经联合法治疗后,患者肿瘤明显缩小(甚至完全消失),瘤体内血流量降低,影像结果对比改变明显,治疗效果理想.结论 将ADV-TK/GCV系统联合光动力治疗口腔恶性肿瘤具有显著的抗肿瘤效应,具备副作用轻微、临床安全性高的特点,为系统性治疗相关肿瘤

  2. Cloning and molecular characterization of the murine macrophage "68-kDa" protein kinase C substrate and its regulation by bacterial lipopolysaccharide.

    OpenAIRE

    Seykora, J T; Ravetch, J V; Aderem, A

    1991-01-01

    We have isolated and characterized a cDNA clone encoding the murine macrophage 68-kDa protein kinase C substrate, which is homologous to the 80- to 87-kDa protein identified by the acronym MARCKS (myristoylated alanine-rich C kinase substrate). The murine MARCKS cDNA clone encodes an acidic protein of 309 amino acids with a calculated molecular weight of 29,661. Transfection of the murine MARCKS gene into TK-L fibroblasts produced a myristoylated protein kinase C substrate that migrated on SD...

  3. The phtC-phtD Locus Equips Legionella pneumophila for Thymidine Salvage and Replication in Macrophages

    Science.gov (United States)

    Fonseca, Maris V.; Sauer, John-Demian; Crepin, Sebastien; Byrne, Brenda

    2014-01-01

    The phagosomal transporter (Pht) family of the major facilitator superfamily (MFS) is encoded by phylogenetically related intracellular gammaproteobacteria, including the opportunistic pathogen Legionella pneumophila. The location of the pht genes between the putative thymidine kinase (tdk) and phosphopentomutase (deoB) genes suggested that the phtC and phtD loci contribute to thymidine salvage in L. pneumophila. Indeed, a phtC+ allele in trans restored pyrimidine uptake to an Escherichia coli mutant that lacked all known nucleoside transporters, whereas a phtD+ allele did not. The results of phenotypic analyses of L. pneumophila strains lacking phtC or phtD strongly indicate that L. pneumophila requires PhtC and PhtD function under conditions where sustained dTMP synthesis is compromised. First, in broth cultures that mimicked thymidine limitation or starvation, L. pneumophila exhibited a marked requirement for PhtC function. Conversely, mutation of phtD conferred a survival advantage. Second, in medium that lacked thymidine, multicopy phtC+ or phtD+ alleles enhanced the survival of L. pneumophila thymidylate synthase (thyA)-deficient strains, which cannot synthesize dTMP endogenously. Third, under conditions in which transport of the pyrimidine nucleoside analog 5-fluorodeoxyuridine (FUdR) would inhibit growth, PhtC and PhtD conferred a growth advantage to L. pneumophila thyA+ strains. Finally, when cultured in macrophages, L. pneumophila required the phtC-phtD locus to replicate. Accordingly, we propose that PhtC and PhtD contribute to protect L. pneumophila from dTMP starvation during its intracellular life cycle. PMID:24478086

  4. Photoreversal-dependent release of thymidine and thymidine monophosphate from pyrimidine dimer-containing DNA excision fragments isolated from ultraviolet-damaged human fibroblasts

    International Nuclear Information System (INIS)

    To elucidate the enzymatic excision-repair process operative on cyclobutane-type pyrimidine photodimers in human dermal fibroblasts, we have examined excised dimer-containing material recovered in the trichloroacetic acid soluble fraction from far-ultraviolet-irradiated (254 nm, 40 J m-2) and incubated (24 h) cell cultures. The excised DNA photoproducts were found in oligonucleotide fragments with an estimated mean chain length of approximately 3.7 bases. Exposure of these isolated excision fragments, labeled with [3H]thymidine (dT), to a secondary, dimer-photoreversing fluence of far-UV (5.5 kJ m-2) resulted in the release of free dT and thymidine monophosphate (TMP). Photorelease of these two radioactive species was measured by high-performance liquid chromatography, with TMP being detected as the increase in dT following bacterial alkaline phosphatase treatment. These data imply that the photoliberated dT and TMP moieties were attached to the excision fragments solely by the cyclobutane ring of the dimer. No evidence was obtained for the photoliberation of free thymine, thus corroborating a conclusion reached by others that the excision of dimers in human cells is not initiated by scission of an intradimer N-glycosyl bond. The sum of the tritium label recovered in dT plus TMP corresponded to approximately 40% of that disappearing from thymine-containing dimers on photoreversal, suggesting that in about 80% of the isolated excision fragments the dimer is located at one end of the oligonucleotide and contains a break in its internal phosphodiester bond

  5. Autoradiographic Study of Incorporation of Tritiated Thymidine in the Rat

    International Nuclear Information System (INIS)

    The authors used tritiated thymidine to evaluate desoxyribonucleic acid synthesis in various rat organs. They show by autoradiographs that this synthesis takes place mainly in tissue having intensive mitotic activity (bone marrow, seminiferous tubules of the testis, mucous membrane of the intestine, oesophagus and tongue). The authors also studied the regeneration of the convoluted renal tubules during the months following local irradiation of the kidney at various doses. (author)

  6. Photoaddition of p-aminobenzoil acid to thymine and thymidine

    International Nuclear Information System (INIS)

    Several studies in the literature have shown that DNA is damaged after UV irradiation in the presence of the sunscreen agent p-aminobenzoic acid (PABA), both in vivo and in vitro. One type of damage has been shown to be the result of increased yields of pyrimidime cyclobutane dimer formation. However, it has been suggested that other types of lesions are produced as well. We have studied the photochemistry of the thymine-PABA and thymidine-PABA systems and report here the isolation and characterization of thymine-PABA and thymidine-PABA photoadducts. These products have been identified, respectively as 5-(2-amino-5-carboxyphenyl)-5,6-dihydrothymine and isomeric forms of 5-(2-amino-5-carboxyphenyl)-5,6-dihydrothymidine. The quantum yields for the formation of these ducts in deaerated aqueous solutions at pH 7.0 have been determined to be 9.5 x 10-4 and 4.3 x 10-3 for the thymine and thymidine based adducts respectively. A pH profile for the thymine-PABA system indicated a maximum quantum yield for adduct formation at pH 6.5, although it could be detected over the whole pH range studied (pH 3.5-11.0). (Author)

  7. Bacterial secondary production on vascular plant detritus: relationships to detritus composition and degradation rate.

    OpenAIRE

    Moran, M.A. (María Asunción); Hodson, R E

    1989-01-01

    Bacterial production at the expense of vascular plant detritus was measured for three emergent plant species (Juncus effusus, Panicum hemitomon, and Typha latifolia) degrading in the littoral zone of a thermally impacted lake. Bacterial secondary production, measured as tritiated thymidine incorporation into DNA, ranged from 0.01 to 0.81 microgram of bacterial C mg of detritus-1 day-1. The three plant species differed with respect to the amount of bacterial productivity they supported per mil...

  8. Responses of Baltic Sea Ice and Open-Water Natural Bacterial Communities to Salinity Change

    OpenAIRE

    Kaartokallio, Hermanni; Laamanen, Maria; Sivonen, Kaarina

    2005-01-01

    To investigate the responses of Baltic Sea wintertime bacterial communities to changing salinity (5 to 26 practical salinity units), an experimental study was conducted. Bacterial communities of Baltic seawater and sea ice from a coastal site in southwest Finland were used in two batch culture experiments run for 17 or 18 days at 0°C. Bacterial abundance, cell volume, and leucine and thymidine incorporation were measured during the experiments. The bacterial community structure was assessed u...

  9. Nucleoside phosphonic acids in thymidine phosphorylase inhibition: Structure - activity relationship

    Czech Academy of Sciences Publication Activity Database

    Panova, Natalya; Kóšiová, Ivana; Petrová, Magdalena; Vaněk, Václav; Liboska, Radek; Kovačková, Soňa; Kočalka, Petr; Králíková, Šárka; Točík, Zdeněk; Páv, Ondřej; Pačes, Ondřej; Rejman, Dominik; Rosenberg, Ivan

    -, č. 52 (2008), s. 665-666. ISSN 0261-3166. [Joint Symposium of the International Roundtable on Nucleosides, Nucleotides and Nucleic Acids /18./ and the International Symposium on Nucleic Acid Chemistry /35./. Kyoto, 08.09.2008-12.09.2008] R&D Projects: GA MŠk(CZ) LC06061; GA MŠk(CZ) LC06077; GA MŠk LC512 Institutional research plan: CEZ:AV0Z40550506 Keywords : thymidine phosphorylase * inhibitors * phosphonic acids Subject RIV: CC - Organic Chemistry

  10. Stimulation of bacterial DNA synthesis by algal exudates in attached algal-bacterial consortia

    International Nuclear Information System (INIS)

    Algal-bacterial consortia attached to polystyrene surfaces were prepared in the laboratory by using the marine diatom Amphora coffeaeformis and the marine bacterium Vibrio proteolytica (the approved name of this bacterium is Vibrio proteolyticus. The organisms were attached to the surfaces at cell densities of approximately 5 x 104 cells cm-2 (diatoms) and 5 x 106 cells cm-2 (bacteria). The algal-bacterial consortia consistently exhibited higher rates of [3H]thymidine incorporation than did biofilms composed solely of bacteria. The rates of [3H]thymidine incorporation by the algal-bacterial consortia were fourfold greater than the rates of incorporation by monobacterial biofilms 16 h after biofilm formation and were 16-fold greater 70 h after biofilm formation. Extracellular material released from the attached Amphora cells supported rates of bacterial activity (0.8 x 10-21 mol to 17.9 x 10-21 mol of [3H]thymidine incorporated cell -1 h-1) and growth (doubling time, 29.5 to 1.4 days) comparable to values reported for a wide variety of marine and freshwater ecosystems. In the presence of sessile diatom populations, DNA synthesis by attached V. proteolytica cells was light dependent and increased with increasing algal abundance. The metabolic activity of diatoms thus appears to be the rate-limiting process in biofilm development on illuminated surfaces under conditions of low bulk-water dissolved organic carbon

  11. Thymidine phosphorylase influences [{sup 18}F]fluorothymidine uptake in cancer cells and patients with non-small cell lung cancer

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seung Jin [Gachon University, College of Pharmacy, Incheon (Korea, Republic of); Yeo, Jeong Seok [Dongguk University Ilsan Hospital, Department of Nuclear Medicine, Goyang-si (Korea, Republic of); Lee, Haeng Jung; Lee, Eun Jung; Kim, Seog Young [Asan Medical Center, Institute for Innovative Cancer Research, Seoul (Korea, Republic of); Jang, Se Jin [University of Ulsan College of Medicine, Department of Pathology, Asan Medical Center, Seoul (Korea, Republic of); Lee, Jong Jin; Ryu, Jin-Sook; Moon, Dae Hyuk [University of Ulsan College of Medicine, Department of Nuclear Medicine, Asan Medical Center, Seoul (Korea, Republic of)

    2014-07-15

    Thymidine phosphorylase (TP), a key enzyme in the pyrimidine nucleoside salvage pathway, catalyses the reversible phosphorylation of thymidine, thereby generating thymine and 2-deoxy-D-ribose-1-phosphate. By regulating the levels of endogenous thymidine, TP may influence [{sup 18}F]fluorothymidine ([{sup 18}F]FLT) uptake. We investigated the effect of TP activity on [{sup 18}F]FLT uptake by tumours. Uptake of [{sup 3}H]FLT and [{sup 3}H]thymidine ([{sup 3}H]Thd) and the activities of TP, thymidine kinase 1 (TK1), and equilibrative nucleoside transporter 1 (ENT1) were determined in exponentially growing A431, A549, HT29, HOP92, ACHN, and SKOV3 cells in the presence or absence of tipiracil hydrochloride, a TP inhibitor. Eighty-five non-small cell lung cancer tissues from a patient cohort that was previously studied with [{sup 18}F]FLT positron emission tomography (PET) were retrieved and subjected to immunohistochemical analysis of TP expression. Factors that affected the maximum standardised uptake value (SUVmax) of [{sup 18}F]FLT-PET were identified by multiple linear regression analysis. A431 cells had the highest TP activity; A549 and HT29 cells had moderate TP activity; and ACHN, SKOV3, and HOP92 cells had little detectable TP activity. Cell lines with high TP activity took up more [{sup 3}H]FLT than [{sup 3}H]Thd, whereas cells with little TP activity took up more [{sup 3}H]Thd than [{sup 3}H]FLT. In cells with high TP activity, TP inhibition decreased [{sup 3}H]FLT uptake and increased [{sup 3}H]Thd uptake. However, TP inhibition had no effect on ACHN, SKOV3, and HOP92 cells. TP inhibition did not change TK1 or ENT1 activity, but did increase the intracellular level of thymidine. The SUVmax of [{sup 18}F]FLT was affected by three independent factors: Ki-67 expression (P < 0.001), immunohistochemical TP score (P < 0.001), and tumour size (P = 0.015). TP activity influences [{sup 18}F]FLT uptake, and may explain preferential uptake of [{sup 18}F]FLT over [{sup 3

  12. Primary and Bacterial Secondary Production in a Southwestern Reservoir

    OpenAIRE

    Chrzanowski, Thomas H.; Hubbard, James G

    1988-01-01

    Rates of primary and bacterial secondary production in Lake Arlington, Texas, were determined. The lake is a warm (annual temperature range, 7 to 32°C), shallow, monomictic reservoir with limited macrophyte development in the littoral zone. Samples were collected from six depths within the photic zone from a site located over the deepest portion of the lake. Primary production and bacterial production were calculated from NaH14CO3 and [methyl-3H]thymidine incorporation, respectively. Peak ins...

  13. Construction of an infectious clone of canine herpesvirus genome as a bacterial artificial chromosome.

    Science.gov (United States)

    Arii, Jun; Hushur, Orkash; Kato, Kentaro; Kawaguchi, Yasushi; Tohya, Yukinobu; Akashi, Hiroomi

    2006-04-01

    Canine herpesvirus (CHV) is an attractive candidate not only for use as a recombinant vaccine to protect dogs from a variety of canine pathogens but also as a viral vector for gene therapy in domestic animals. However, developments in this area have been impeded by the complicated techniques used for eukaryotic homologous recombination. To overcome these problems, we used bacterial artificial chromosomes (BACs) to generate infectious BACs. Our findings may be summarized as follows: (i) the CHV genome (pCHV/BAC), in which a BAC flanked by loxP sites was inserted into the thymidine kinase gene, was maintained in Escherichia coli; (ii) transfection of pCHV/BAC into A-72 cells resulted in the production of infectious virus; (iii) the BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus CHV/BAC by co-infection with CHV/BAC and a recombinant adenovirus that expressed the Cre recombinase; and (iv) a recombinant virus in which the glycoprotein C gene was deleted was generated by lambda recombination followed by Flp recombination, which resulted in a reduction in viral titer compared with that of the wild-type virus. The infectious clone pCHV/BAC is useful for the modification of the CHV genome using bacterial genetics, and CHV/BAC should have multiple applications in the rapid generation of genetically engineered CHV recombinants and the development of CHV vectors for vaccination and gene therapy in domestic animals. PMID:16515874

  14. Characterization of three mitogen-activated protein kinases (MAPK) genes reveals involvement of ERK and JNK, not p38 in defense against bacterial infection in Yesso scallop Patinopecten yessoensis.

    Science.gov (United States)

    Sun, Yan; Zhang, Lingling; Zhang, Meiwei; Li, Ruojiao; Li, Yangping; Hu, Xiaoli; Wang, Shi; Bao, Zhenmin

    2016-07-01

    Mitogen-activated protein kinases (MAPKs) are protein Ser/Thr kinases that play a vital role in innate immune responses by converting extracellular stimuli into a wide range of cellular responses. Although MAPKs have been extensively studied in various vertebrates and invertebrates, our current understanding of MAPK signaling cascade in scallop is in its infancy. In this study, three MAPK genes (PyERK, PyJNK, and Pyp38) were identified from Yesso scallop Patinopecten yessoensis. The open reading frame of PyERK, PyJNK, and Pyp38 was 1104, 1227, and 1104 bp, encoding 367, 408, and 367 amino acids, respectively. Conservation in some splicing sites was revealed across the three PyMAPKs, suggesting the common descent of MAPKs genes. The expression profiles of PyMAPKs over the course of ten different developmental stages showed that they had different expression patterns. In adult scallops, PyMAPKs were primarily expressed in muscles, hemocytes, gill, and mantle. To gain insights into their role in innate immunity, we investigated their expression profiles after infection with Gram-positive bacteria (Micrococcus luteus) and Gram-negative bacteria (Vibrio anguillarum). Significant difference in gene expression was only found in PyERK and PyJNK, but not Pyp38, suggesting Pyp38 may not participate in immune response to bacterial infection. Besides, PyERK and PyJNK exhibited more drastic change against the invasion of V. anguillarum than M. luteus, suggesting they could be more sensitive to Gram-negative bacteria than Gram-positive bacteria. This study provides valuable resource for elucidating the role of MAPK signal pathway in bivalve innate immune response. PMID:27155450

  15. Interleukin 1 increases thymidine labeling index of normal tissues of mic but not the tumor

    International Nuclear Information System (INIS)

    This study was conducted to investigate the action of human recombinant interleukin 1 as a radioprotector for different mouse normal cells other than bone marrow cells. Semi-continuous injections of tritiated thymidine were administered every 6 h, over 24 h to determine thymidine labeling index. Mice were injected with recombinant human interleukin 1 24 h prior to tritiated thymidine and were compared to control animals that did not receive interleukin 1. Mice were killed 1 h after the last thymidine injection. The 24 h thymidine labeling index for normal tissues and RIF-1 tumor was determined. Labeling indices were also determined 1-14 days after a series of fractionated irradiations with or without pretreatment with a single dose of interleukin 1 administered 24 h prior to the first radiation. The thymidine labeling index of normal tissues was higher following the injection of recombinant human interleukin 1 24 h before radiolabeling. This was found in all normal tissues tested. The thymidine labeling index of RIF-1 fibrosarcoma was not affected by interleukin 1 injection. A single interleukin 1 injection 24 h before the first radiation fraction also increased the thymidine labeling indices of normal tissues after localized fractionated irradiation. The thymidine labeling index of RIF-1 tumor was not increased by interleukin 1 administration except after relatively high radiation doses (20 Gy in five fractions). The ability of interleukin 1 to enhance the thymidine labeling index declined after the first day following the completion of fractionated irradiation. Recombinant human interleukin 1 increased the 24 h thymidine labeling index in normal tissues in mice, but not in RIF-1 tumor. Fractionated irradiation could maintain the effect of a single dose of interleukin 1, administered 24 h prior to the first fraction, up to 24 h after the end of radiation. 25 refs., 3 figs., 1 tab

  16. Imaging of Viral Thymidine Kinase Gene Expression by Replicating Oncolytic Adenovirus and Prediction of Therapeutic Efficacy

    OpenAIRE

    Kim, Eun-Jung; Yoo, Ji Young; Choi, Young-Hwan; Ahn, Keun-Jae; Lee, Jong-Doo; Yun, Chae-Ok; Yun, Mijin

    2008-01-01

    Purpose We have used a genetically attenuated adenoviral vector which expresses HSVtk to assess the possible additive role of suicidal gene therapy for enhanced oncolytic effect of the virus. Expression of TK was measured using a radiotracer-based molecular counting and imaging system. Materials and Methods Replication-competent recombinant adenoviral vector (Ad-ΔE1B19/55) was used in this study, whereas replication-incompetent adenovirus (Ad-ΔE1A) was generated as a control. Both Ad-ΔE1B19/5...

  17. Characterization of Oligomeric and Kinetic Properties of Tomato Thymidine Kinase 1

    DEFF Research Database (Denmark)

    Mutahir, Zeeshan; Larsen, Nicolai Balle; Andersson, Karl-Magnus;

    2011-01-01

    AZT can easily penetrate the blood–brain barrier and toTK1 can efficiently phosphorylate AZT and also AZT-monophosphate. In a pursuit to further understand the properties of toTK1, we examined the oligomerization properties of recombinant toTK1 and its effect on enzyme kinetics. Previously, it has...

  18. The development of herpes simplex virus thymidine kinase suicide gene imaging

    International Nuclear Information System (INIS)

    Suicide gene treatment of tumor catches more and more attention in recent years. Cells transferred with suicide gene from virus or bacteria will express specific enzymes and transform innocuous prodrugs into highly toxic chemotherapeutic drugs. As a result, the cells will be killed. Radionuclide labeled probe can display the biologic characteristics of suicide gene in vivo. This article reviews the development of HSV-tk gene imaging. (authors)

  19. Serum thymidine kinase--a marker of bone marrow toxicity during treatment with zidovudine

    DEFF Research Database (Denmark)

    Pedersen, C; Ingeberg, S; Teglbjaerg, L S

    1989-01-01

    0.01) in the zidovudine group, whereas it remained stable in the placebo (control) group. On the basis of this observation, the value of S-TK measurements as a predictor of bone marrow toxicity during zidovudine therapy was investigated in 42 patients with AIDS or ARC who received zidovudine as part...... of their usual treatment. There was a significant association between S-TK, haemoglobin and neutrophil counts measured after the first 4 weeks of therapy and the risk of developing bone marrow toxicity during the following 6 months. Combined, measurements of S-TK and neutrophil counts seem to be well...... suited for the identification of patients who have a high probability for developing bone marrow toxicity during zidovudine treatment....

  20. In vitro thymidine labelling of human pulmonary neoplasms

    International Nuclear Information System (INIS)

    The in vitro thymidine labelling indices (TLI) of 58 human lung tumours were assessed using autoradiography. The labelling technique involved incubation of 1 mm3 tumour fragments with 3H-thymidine (5μCi ml-1) under conditions of hyperbaric oxygenation at a pressure of 3 atmospheres. Only a rim of labelling was achieved along the edges of fragments and the depth of this rim varied from tumour to tumour. A technique for counting TLIs was therefore devised to take this into account. In general, those tumours showing low TLI values of < 5.0% showed a greater depth of labelling. The common malignant tumours of the bronchus showed a wide range of values (2.2-30.4%) though the adenocarcinomata had a lower average value than the other groups. With the squamous carcinomata a relationship with differentiation was shown. The mean value for small cell carcinomata (16.9%) - a highly aggressive tumour - was no higher than for the other groups. The low grade malignant tumours showed TLIs of < 3.0% and these values correlate with their less aggressive clinical behaviour. Labelling of stromal cells and inflammatory cells varied greatly from tumour to tumour; however, no correlation was found with the TLIs of tumour cells. (author)

  1. A stable isotope method for measurement of thymidine incorporation into DNA

    International Nuclear Information System (INIS)

    A method has been developed for the measurement of DNA synthesis in vivo using the incorporation of multilabeled, nonradioactive thymidine. Simultaneous intraperitoneal injection of hexalabeled thymidine and tritiated thymidine into a normal adult rat resulted in the incorporation of both labeled nucleosides into the DNA cells undergoing replication. The DNA of several tissues and organs was analysed, including liver, thymus, spleen, bone marrow, and small intestine. Following extraction with hot trichloroacetic acid, acid hydrolysis, and thin-layer chromatography of the hydrolysates, the isotopic compositions of the thymine products were determined by field ionization mass spectrometry and by scintillation counting. The relative incorporation of radioactive and stable isotope-labeled thymidine was similar in all tissues, and corresponded to the ratio of the two labeled nucleosides in the injected material. These results indicate the feasibility of utilizing thymidine multilabeled with stable isotopes for measurement of cellular proliferation rates in conjunction with cancer therapy. (author)

  2. Bacterial gastroenteritis

    Science.gov (United States)

    Infectious diarrhea - bacterial gastroenteritis; Acute gastroenteritis; Gastroenteritis - bacterial ... Bacterial gastroenteritis can affect 1 person or a group of people who all ate the same food. It is ...

  3. Uptake, excretion, and radiation hazards of tritiated thymidine in humans

    International Nuclear Information System (INIS)

    Nine patients with malignant disease were given iv injections of tritiated thymidine, 0.2 mCi/kg, for tumor cell kinetics studies. Serial plasma, urine, saliva, and air vapor samples were collected variously for up to 79 days, and tritium activity was measured. The initial half-life of plasma activity was rapid. After 1 day, the activity decayed with a half-life of 10.8 days, indicating equilibration of activity with the total body water. Urine activity was over 100 times the plasma activity within 1 hr, with equilibration approaching the plasma activity after 2 days, and then decayed at a similar rate. Saliva and air vapor activity increased to plasma levels and then decayed at the same rate as did plasma activity. In the first 24 hr, approximately one-third of the total injected activity was excreted in the urine. During the first 12 days there were 54.2% urinary and 10.6% insensible losses. Maximum losses determined by extrapolation of observed data were 68% urinary and 19.5% insensible losses, or a total of 87.5%. Approximately 7% of the injected activity may represent material initially incorporated into DNA but later metabolized and excreted. The radiation dose from total body water is estimated at 0.69 rad. The estimated dose absorbed by cell nuclei from incorporated material is a maximum of 20.5 rads. These radiation doses would not seem to contraindicate injection of 0.2 mCi tritiated thymidine per kg to patients in this clinical and experimental setting. Measurements of activity in personnel and room air indicate that the use of such doses is not hazardous if appropriate precautions are followed

  4. Host Signal Transduction and Protein Kinases Implicated in Legionella Infection

    OpenAIRE

    Hempstead, Andrew D.; Isberg, Ralph R.

    2013-01-01

    Modulation of the phosphorylation status of proteins by both kinases and phosphatases plays an important role in cellular signal transduction. Challenge of host cells by Legionella pneumophila manipulates the phosphorylation state of multiple host factors. These changes play roles in bacterial uptake, vacuole modification, cellular survival, and the immune response. In addition to modification by host cell kinases in response to the bacterium, L. pneumophila translocates bacterial kinases int...

  5. Liquid scintillation counting of 3H-thymidine incorporated into rat lens DNA

    International Nuclear Information System (INIS)

    DNA synthesis in the lens has previously been localized by autoradiography following incorporation of 3H-thymidine. For the quantification of DNA synthesis in the lens, pooling of lenses and extraction of the DNA for liquid scintillation counting, has formerly been adapted. In the present investigation a method has been developed for the extraction of the unincorporated tracer from whole lenses after short time incubation in a medium containing 3H-thymidine. The 3H-thymidine incorporated into individual lenses was then detected by liquid scintillation counting after dissolution of the lenses. The sources of the variation in the method are evaluated. (author)

  6. Transcriptional profiling of thymidine-producing strain recombineered from Escherichia coli BL21

    Directory of Open Access Journals (Sweden)

    Jin-Sook Kim

    2015-12-01

    Full Text Available DNA microarrays were used to compare the expression profiles of a thymidine overproducing strain (BLT013 and its isogenic parent, Escherichia coli BL21(DE3, when each was grown under well-defined thymidine production conditions with glycerol as carbon source. Here we describe the experimental procedures and methods in detail to reproduce the results and provide resource to be applied to similar engineering approach (available at Gene Expression Omnibus database under GSE69963. Taken together, the microarray data provide a basis for new testable hypotheses regarding enhancement of thymidine productivity and attaining a more complete understanding of nucleotide metabolism in bacteria.

  7. The Legionella Kinase LegK2 Targets the ARP2/3 Complex To Inhibit Actin Nucleation on Phagosomes and Allow Bacterial Evasion of the Late Endocytic Pathway

    Science.gov (United States)

    Michard, Céline; Sperandio, Daniel; Baïlo, Nathalie; Pizarro-Cerdá, Javier; LeClaire, Lawrence; Chadeau-Argaud, Elise; Pombo-Grégoire, Isabel; Hervet, Eva; Vianney, Anne; Gilbert, Christophe; Faure, Mathias; Cossart, Pascale

    2015-01-01

    ABSTRACT Legionella pneumophila, the etiological agent of legionellosis, replicates within phagocytic cells. Crucial to biogenesis of the replicative vacuole is the Dot/Icm type 4 secretion system, which translocates a large number of effectors into the host cell cytosol. Among them is LegK2, a protein kinase that plays a key role in Legionella infection. Here, we identified the actin nucleator ARP2/3 complex as a target of LegK2. LegK2 phosphorylates the ARPC1B and ARP3 subunits of the ARP2/3 complex. LegK2-dependent ARP2/3 phosphorylation triggers global actin cytoskeleton remodeling in cells, and it impairs actin tail formation by Listeria monocytogenes, a well-known ARP2/3-dependent process. During infection, LegK2 is addressed to the Legionella-containing vacuole surface and inhibits actin polymerization on the phagosome, as revealed by legK2 gene inactivation. Consequently, LegK2 prevents late endosome/lysosome association with the phagosome and finally contributes to remodeling of the bacterium-containing phagosome into a replicative niche. The inhibition of actin polymerization by LegK2 and its effect on endosome trafficking are ARP2/3 dependent since it can be phenocopied by a specific chemical inhibitor of the ARP2/3 complex. Thus, LegK2-ARP2/3 interplay highlights an original mechanism of bacterial virulence with an unexpected role in local actin remodeling that allows bacteria to control vesicle trafficking in order to escape host defenses. PMID:25944859

  8. Biological indicators for radiation exposure. Thymidine concentration in human serum as 'biological dosemeter'?

    International Nuclear Information System (INIS)

    The in vivo test of blood from partial and whole-body irradiated patients revealed no strict differences in radiosensitivity in correlation to the dose, only tendencies for radiation-effects. The serum thymidine concentration appeared to be also dependent on non-investigated factors, such as diseases and previous therapies. Therefore, the suitability of thymidine concentration in blood as a 'biochemical dosimeter' could not be demonstrated. (orig.)

  9. Vorinostat synergises with capecitabine through upregulation of thymidine phosphorylase

    Science.gov (United States)

    Di Gennaro, E; Piro, G; Chianese, M I; Franco, R; Cintio, A Di; Moccia, T; Luciano, A; de Ruggiero, I; Bruzzese, F; Avallone, A; Arra, C; Budillon, A

    2010-01-01

    Background: Potentiation of anticancer activity of capecitabine is required to improve its therapeutic index. In colorectal cancer (CRC) cells, we evaluated whether the histone deacetylase-inhibitor vorinostat may induce synergistic antitumour effects in combination with capecitabine by modulating the expression of thymidine phosphorylase (TP), a key enzyme in the conversion of capecitabine to 5-florouracil (5-FU), and thymidylate synthase (TS), the target of 5-FU. Methods: Expression of TP and TS was measured by real-time PCR, western blotting and immunohistochemistry. Knockdown of TP was performed by specific small interfering RNA. Antitumour activity of vorinostat was assessed in vitro in combination with the capecitabine active metabolite deoxy-5-fluorouridine (5′-DFUR) according to the Chou and Talay method and by evaluating apoptosis as well as in xenografts-bearing nude mice in combination with capecitabine. Results: Vorinostat induced both in vitro and in vivo upregulation of TP as well as downregulation of TS in cancer cells, but not in ex vivo treated peripheral blood lymphocytes. Combined treatment with vorinostat and 5′-DFUR resulted in a synergistic antiproliferative effect and increased apoptotic cell death in vitro. This latter effect was impaired in cells where TP was knocked. In vivo, vorinostat plus capecitabine potently inhibited tumour growth, increased apoptosis and prolonged survival compared with control or single-agent treatments. Conclusions: Overall, this study suggests that the combination of vorinostat and capecitabine is an innovative antitumour strategy and warrants further clinical evaluation for the treatment of CRC. PMID:21045833

  10. Casein kinases

    DEFF Research Database (Denmark)

    Issinger, O G

    1993-01-01

    subunits are highly conserved during evolution. The relationship between CK-2 alpha from humans and plants is still 73%. Similar relationships are reported for the beta-subunit. Chromosomal assignment of CK-2 alpha shows two gene loci, one of which is a pseudogene. They are located on different chromosomes...... genetic changes are necessarily involved; the observed changes may be entirely due to a signal transduction pathway where CK-2 could be phosphorylated by another kinase(s). CK-2 cDNAs from various organisms have been isolated and characterized. From the deduced amino acid sequence it turns out that CK-2...

  11. A technique for improved resolution of [3H]thymidine autoradiography in the avian embryo: a preliminary report

    International Nuclear Information System (INIS)

    A technique is described for producing pulse-like effects in [3H]thymidine autoradiography in chick embryos. This procedure involves combining thymidine with reserpine, which temporarily inhibits thymidine incorporation. The concept is to introduce thymidine, allow time for its incorporation, then introduce reserpine, so that subsequent uptake is inhibited, and a relatively discrete label will appear in cells generated at the time of thymidine administration. The best results occurred when thymidine was administered 12 h prior to reserpine, or when the two were introduced simultaneously. A dose of 0.004 mg of reserpine produced the suppression, but had no deleterious effects, in terms of embryonic survival or in long-lasting changes in cell numbers, as reflected by cell counts in the trochlear nucleus, a population which was undergoing proliferation at the time of reserpine injection. Thus, this technique appears to hold considerable promise for improving the precision of the autoradiography procedure in avian embryos. (Auth.)

  12. Role of the chronic bacterial infection in urinary bladder carcinogenesis

    International Nuclear Information System (INIS)

    The purpose of this thesis was to determine whether or not bacterial infection of the urinary bladder had a role in urinary bladder carcinogenesis. To investigate this proposition, four separate studies were conducted. The first study developed an experimental animal model where bacterial infection of the urinary bladder could be introduced and maintained for a period in excess of one year. The method of infection, inoculation of bacteria (Escherichia coli type 04) subserosally into the vesical wall, successfully caused persistent infection in the majority of animals. In the second study the temporal effects of bacterial infection on the induction of urothelial ornithine decarboxylase (ODC) and 3H-thymidine uptake and DNA synthesis were examined. Bacterial infection of the urinary bladder induced urothelial ODC with a peak in enzyme activity 6 hr after infection.3H-Thymidine uptake and DNA synthesis peaked 48 hr after infection and coincided with the urothelial hyperplasia that occurred in response to the infection. In the third study the specific bladder carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) was given to rats concurrent with the urinary bacterial infection. In the fourth study rats were administered sodium nitrate and either dibutylamine or piperazine in the drinking water. The infected group developed bladder tumors while none were detected in the non-infected rats. From these studies it may be concluded that bacterial infection may have a significant role in the process of urinary bladder carcinogenesis

  13. Recovery of infectious virus from full-length cowpox virus (CPXV DNA cloned as a bacterial artificial chromosome (BAC

    Directory of Open Access Journals (Sweden)

    Roth Swaantje J

    2011-01-01

    Full Text Available Abstract Transmission from pet rats and cats to humans as well as severe infection in felids and other animal species have recently drawn increasing attention to cowpox virus (CPXV. We report the cloning of the entire genome of cowpox virus strain Brighton Red (BR as a bacterial artificial chromosome (BAC in Escherichia coli and the recovery of infectious virus from cloned DNA. Generation of a full-length CPXV DNA clone was achieved by first introducing a mini-F vector, which allows maintenance of large circular DNA in E. coli, into the thymidine kinase locus of CPXV by homologous recombination. Circular replication intermediates were then electroporated into E. coli DH10B cells. Upon successful establishment of the infectious BR clone, we modified the full-length clone such that recombination-mediated excision of bacterial sequences can occur upon transfection in eukaryotic cells. This self-excision of the bacterial replicon is made possible by a sequence duplication within mini-F sequences and allows recovery of recombinant virus progeny without remaining marker or vector sequences. The in vitro growth properties of viruses derived from both BAC clones were determined and found to be virtually indistinguishable from those of parental, wild-type BR. Finally, the complete genomic sequence of the infectious clone was determined and the cloned viral genome was shown to be identical to that of the parental virus. In summary, the generated infectious clone will greatly facilitate studies on individual genes and pathogenesis of CPXV. Moreover, the vector potential of CPXV can now be more systematically explored using this newly generated tool.

  14. Comparison of thymidine phosphorylase expression and prognostic factors in gallbladder and bile duct cancer

    Directory of Open Access Journals (Sweden)

    You Young

    2010-10-01

    Full Text Available Abstract Background Biliary tract cancers have limitations in information about different location-related pathogenesis and clinico-pathological characteristics. The goal of this study was to investigate anatomical site-related similarities and differences in biliary tract cancers and to assess the expression and clinical significance of functional proteins such as p53, cyclin D1, survivin, thymidine phosphorylase, and ERCC1. Methods One hundred and sixty-one patients with biliary tract adenocarcinomas, who underwent curative or palliative surgery in a single institution between October 1994 and December 2003 were evaluated, retrospectively. The level of protein expression of p53, cyclin D1, survivin, thymidine phosphorylase, and ERCC1 was assessed by immunohistochemistry. Results With respect to clinico-pathological characteristics, gallbladder cancer was more frequent in women, and bile duct cancer was more common in men. Perineural invasion was more common in bile duct cancer. Recurrence as a distant metastasis was more common in gallbladder cancer. Immunohistochemical analysis revealed that thymidine phosphorylase expression was significantly higher in gallbladder cancer than in bile duct cancer. Positive thymidine phosphorylase and p53 staining were associated with an advanced stage. Differentiation, vascular invasion, perineural invasion, lymphatic invasion, lymph node metastasis, and TNM stage independently predicted poor prognosis in biliary tract cancer. These correlations were seen more clearly in gallbladder cancer. The immunohistochemical staining patterns of p53, cyclin D1, survivin, thymidine phosphorylase, and ERCC1 showed no prognostic significance in biliary tract cancers. Conclusions We concluded that gallbladder and bile duct cancers are considered to be separate diseases with different clinico-pathological characteristics and prognostic factors. In addition, we hypothesize that high expression of thymidine phosphorylase by

  15. Detection of apoptotic cells by selective precipitation of [3H]thymidine-labelled DNA.

    OpenAIRE

    Patki, A H; Lederman, M M

    1996-01-01

    Apoptosis is characterized by systematic fragmentation of high-molecular-weight DNA into oligonucleosome fragments. A sensitive method for detection of apoptotic cells involving [3H]thymidine-labelled DNA is presented. Cells from mid-log-phase cultures were labelled with [3H]thymidine for 15 to 18 h and then exposed to gamma irradiation to induce apoptosis. A modified Hirt method was used to separate low-molecular-weight DNA from high-molecular-weight DNA. The percentage of fragmented DNA and...

  16. Tritiated thymidine uptake in chondrocytes of chickens afflicted with tibial dyschondroplasia

    International Nuclear Information System (INIS)

    3H-Thymidine was localized in sections of growth-plate cartilage and associated tibial dyschondroplastic lesion by autoradiography. One hour after 3H-thymidine was injected, radioactivity was found in the proliferating zone; after 48 hr it was also in the hypertrophic zone, and by 96 hr it was present in cells that were 4 to 5 mm into the lesion. This indicates that the lesion develops from the growth plate itself. The life span of the cells in the growth plate appears to be about 48 hr

  17. The Reactivity of Thymine and Thymidine 5,6-Epoxides with Organometallic Reagents - A Route to Thymidine (6-4) Photoproduct Analogues.

    Science.gov (United States)

    Wrigstedt, Pauli; Kavakka, Jari; Heikkinen, Sami; Nieger, Martin; Räisänen, Minna; Repo, Timo

    2016-05-01

    This report describes an efficient procedure for the generation and isolation of various thymine and thymidine 5,6-epoxides from the corresponding trans-5,6-bromohydrins by reaction with triethylamine. The quantitative isolation of the epoxides, accomplished by solvent precipitation of triethylamine hydrobromide, enabled their regiospecific ring-opening at C6 position by organometallic nucleophiles. The reaction was amenable to a broad range of alkyl, aryl, alkenyl, and alkynyl organomagnesium, -zinc, -aluminum, or -boron reagents, although the reactivity was strongly affected by the electronic effects of N3 protecting group. Additionally, the reaction featured excellent cis-diastereoselectivity providing access to C6-carbon-functionalized dihydrothymidine cis-alcohols, which are synthetic derivatives of UV-induced DNA lesions, namely, thymidine (6-4) photoproducts. PMID:27080560

  18. The significance of 3H-thymidine degradation in cell culture experiments with special reference to rheumatoid arthritis

    International Nuclear Information System (INIS)

    The degradation of 3H-thymidine under various cell culture conditions was analysed. It was found that a half of 3H-thymidine was degraded to 3H-thymine during 24 hours in PHA stimulation of blood lymphocytes. A control culture in which PHA was not added also caused 3H-thymidine degradation. 3H-thymidine degradation was prevented by adding 5-nitrouracil to the incubation medium at a concentration of 0.577 mg/ml. At the same time 5-NU increased 3H-thymidine incorporation into lymphocytes by 47%. 5-NU also eliminated the inhibitory effect of rheumatoid arthritis synovial tissue eluate on PHA stimulation. In addition 5-NU and nonradioactive thymine increased the 3H-thymidine labelling index of fresh rheumatoid arthritis synovial membrane biopsies, and also more of the isotope was accumulated in the individual cells of the membrane. These studies demonstrate that 3H-thymidine degradation is an important phenomenon in cell cultures and that it can be prevented effectively by using 5-nitrouracil with 3H-thymidine. (author)

  19. Kinetics of staphylococcal opsonization, attachment, ingestion and killing by human polymorphonuclear leukocytes: a quantitative assay using [3H]thymidine labelled bacteria

    International Nuclear Information System (INIS)

    A method has been developed for studying quantitatively the separate processes of bacterial opsonization, phagocytosis and killing by hyman polymorphonuclear leukocytes using [3H]thymidine labelled staphylococcus aureus. Phagocytosis is determined by assaying for leukocytes-associated radioactivity after different centrifugation and washing the leukoctes. Opsonization is studied by incubating bacteria with an opsonic source for varying durations and then adding leukocytes. By treatment of samples with the muralytic enzyme, lysostaphin, the attachment and ingestion phases of phagocytosis can be separated. Sampling for colony forming units after disruption of the leukocytes permits the measurement of gacterial killing. Using this method, differences in the kinetics of staphylococcal opsonization by normal and C2 deficient sera were defined, opsonic influences on the attachment and ingestion phases of phagocytosis were delineated, and the influences of different opsonins and leukocyte populations on killing were determined

  20. Analysis of the inhibitory effects of VP-16-213 (etoposide) and podophyllotoxin on thymidine transport and metabolism in Ehrlich ascites tumor cells in vitro

    International Nuclear Information System (INIS)

    Uptake of 3H after exposure of cells to [3H]-thymidine is characterized by a rapid initial velocity that approximates membrane transport followed by a slower rate of uptake that parallels the accumulation of phosphorylated derivatives of thymidine, primarily thymidine triphosphate, within the cell. The high rate of thymidine transport relative to thymidine metabolism to the triphosphate within the cell decreases as the extracellular nucleoside concentration is reduced due to a much greater decrease in membrane transport than the subsequent metabolic step. Hence, as extracellular thymidine is decreased, transport becomes increasingly rate limiting to metabolism within the cell. VP-16-213 (etoposide) or podophyllotoxin inhibits the initial uptake rate for thymidine and, as a consequence, inhibits the intracellular formation of thymidine triphosphate. When extracellular thymidine is high, inhibitory effects on transport are transient, and the net rate of thymidine triphosphate accumulation within drug-treated cells rapidly approaches a velocity comparable to that of control cells, indicating no direct VP-16-213 or podophyllotoxin effect on nucleoside and nucleotide phosphorylation. When extracellular thymidine is reduced so that transport is rate limiting to metabolism, the duration of the inhibitory effects of VP-16-213 on thymidine triphosphate formation is prolonged. A secondary effect of VP-16-213 becomes manifest beyond 10 min of incubation with [3H]thymidine with the virtual complete cessation of thymidine incorporation into the acid precipitate without any change in the thymidine triphosphate level. This late effect is not observed with podophyllotoxin and indicates a direct effect of VP-16-213 on DNA synthesis that is distinct from the earlier inhibitory effect on thymidine phosphorylation, which is secondary to membrane transport

  1. Pyrimidine acyclic nucleoside phosphonates and their phosphorylated analogs as potential multisubstrate inhibitors of thymidine phosphorylase

    Czech Academy of Sciences Publication Activity Database

    Pomeisl, Karel; Votruba, Ivan; Holý, Antonín; Pohl, Radek; Blažek, Jiří; Krečmerová, Marcela

    Ottawa : -, 2012. -. [Ottawa 2012 International Symposium On Biochemistry and Biophysics. 24.10.2012-25.10.2012, Ottawa] R&D Projects: GA MPO FR-TI4/625 Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * thymidine phosphorylase * phosphorylation * pyrimidines Subject RIV: CC - Organic Chemistry

  2. Discovery of 5-substituted-6-chlorouracils as novel thymidine phosphorylase inhibitors

    Czech Academy of Sciences Publication Activity Database

    Nencka, Radim; Votruba, Ivan; Hřebabecký, Hubert; Tloušťová, Eva; Masojídková, Milena; Holý, Antonín

    Bern : DCB University of Bern, 2006. s. 224. [IRT-International Roundtable on Nucleosides, Nucleotides and Nucleic Acids /17./. 03.09.2006-07.09.2006, Bern] R&D Projects: GA MŠk(CZ) 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : thymidine phosphorylase * 5-substituted-6-chlorouracils * angiogenesis Subject RIV: CC - Organic Chemistry

  3. Discovery of 5-substituted-6-chlorouracils as efficient inhibitors of human thymidine phosphorylase

    Czech Academy of Sciences Publication Activity Database

    Nencka, Radim; Votruba, Ivan; Hřebabecký, Hubert; Jansa, Petr; Tloušťová, Eva; Horská, Květoslava; Masojídková, Milena; Holý, Antonín

    2007-01-01

    Roč. 50, č. 24 (2007), s. 6016-6023. ISSN 0022-2623 R&D Projects: GA MŠk 1M0508; GA AV ČR 1QS400550501 Institutional research plan: CEZ:AV0Z40550506 Keywords : thymidine phosphorylase inhibitors Subject RIV: CC - Organic Chemistry Impact factor: 4.895, year: 2007

  4. Benthic bacterial biomass and production in the Hudson River estuary

    International Nuclear Information System (INIS)

    Bacterial biomass, production, and turnover were determined for two freshwater march sites and a site in the main river channel along the tidally influenced Hudson River. The incorporation of [methyl-3H]thymidine into DNA was used to estimate the growth rate of surface and anaerobic bacteria. Bacterial production at marsh sites was similar to, and in some cases considerably higher than, production estimates reported for other aquatic wetland and marine sediment habitats. Production averaged 1.8-2.8 mg C·m-2· hour-1 in marsh sediments. Anaerobic bacteria in marsh sediment incorporated significant amounts of [methyl-3H]thymidine into DNA. Despite differences in dominant vegatation and tidal regime, bacterial biomass was similar (1 x 103 ± 0.08 mg C·m-2) in Trapa, Typha, and Nuphar aquatic macrophyte communities. Bacterial abundance and productivity were lower in sandy sediments associated with Scirpus communities along the Hudson River (0.2 x 103 ± 0.05 mg C·m-2 and 0.3 ± 0.23 mg C · m-2· hour-1, respectively)

  5. [Effect of x-rays on the intracellular transport of thymidine in human lymphocytes stimulated by phytohemagglutinins].

    Science.gov (United States)

    Catena, C; Mattoni, A

    1984-08-31

    The effect of X-irradiation on thymidine transport in human lymphocytes PHA stimulated was investigated. Mediated transport sistem, the predominant mechanism at low extracellular concentration of thymidine (less than 10(-7) M) in the medium is highly radiosensitive. The transport sistem was damaged considerably by high doses of X-rays (at least until 10 Krad); the decrease of thymidine uptake was a function of the time of incubation after irradiation. It suggest that repair mechanisms are not involved at high doses of X-rays within 120 minutes of incubation. PMID:6497982

  6. A phase II trial of thymidine and carboplatin for recurrent malignant glioma: a North American Brain Tumor Consortium Study.

    OpenAIRE

    Robins, H. Ian; Chang, Susan M.; Prados, Michael D.; Yung, W.K. Alfred; Hess, Kenneth; Schiff, David; Greenberg, Harry; Fink, Karen; Nicolas, Kelly; Kuhn, John G.; Cloughesy, Timothy; Junck, Larry; Mehta, Minesh

    2002-01-01

    This phase II study in recurrent high-grade glioma evaluated the response rate, toxicities, and time to treatment failure of high-dose carboplatin modulated by a 24-h infusion of thymidine (75 g/m(2)). The trial was based on preclinical data and a prior phase I study ( J. Clin. Oncol. 17, 2922-2931, 1999); a phase II recurrent high-grade glioma study was initiated in July of 1998. Thymidine was given over 24 h; carboplatin was given over 20 min at hour 20 of the thymidine infusion. The starti...

  7. Determination of 3H-thymidine incorporation into ovarian cancer cells and its validity with regard to cytostatic therapy

    International Nuclear Information System (INIS)

    The incorporation of 3H-thymidine in ovarian cancer cells determined by autoradiography represents a method of additional tumor characterization. The in vitro tests allowed roughly hints about the efficiency of an intended therapy in 49 patients suffering from ovarian cancer. High risk patients ought to be excluded from chemotherapy, if the incorporation of thymidine is low. For this decision the results of oncobiograms and clinical parameters were evaluated. The 3H-thymidine incorporation enables to select an individual kind of therapy for any patient with regard to success and duration of treatment. (author)

  8. In vivo evaluation of the 3-carboranyl thymidine analogue (3-CTA), N5-2OH, for neutron capture therapy

    International Nuclear Information System (INIS)

    The purpose of the present study was to evaluate a 3 CTA, designated N5-2OH, as a boron delivery agent for NCT. Target validation was established using the thymidine kinase 1 (+) wild type L929 cell line and its TK1(-) counterpart, which were implanted subcutaneously into NIH nu/nu mice. 10B-enriched N5-2OH, solubilized in DMSO (50μg 10B in 15μl), was administered by 2 intratumoral (i.t.) injections at 2 h intervals. Two hours later the animals were irradiated at the MITR-II Research Reactor, following which tumor volumes were determined over a period of 30 days. Mice bearing TK1(+) wild type tumors, which had received N5-2OH, had a 15 fold inhibition in tumor growth compared to TK1(-) controls (247 versus 3,603 mm3). Based on these data, biodistribution and therapy studies were initiated in F98 glioma bearing rats. Animals received 500μg of N5-2OH, administered intracerebrally (i.c.) by convection enhanced delivery (CED) using ALZET pumps (8μl/h for 24 h). The tumor boron concentration was 17.3μg/g compared to undetectable amounts in normal brain and blood. BNCT was carried out 14 d following i.c. implantation of 103 F98 glioma cells and 24 h following CED of N5-2OH (500μg/200μl). The mean survival time (MST) of these animals was 38 d compared to 31 d and 25 d, respectively, for irradiated and untreated controls. Studies are planned to optimize the delivery and formulation of N5-2OH and additional therapy studies will be carried out using N5-2OH in combination with BPA and BSH. (author)

  9. A General Approach to the Non-Invasive Imaging of Transgenes Using Cis-Linked Herpes Simplex Virus Thymidine Kinase

    Directory of Open Access Journals (Sweden)

    Juri G. Tjuvajev

    1999-10-01

    Full Text Available Non-invasive imaging of gene expression opens new prospects for the study of transgenic animals and the implementation of genetically based therapies in patients. We have sought to establish a general paradigm to enable whole body non-invasive imaging of any transgene. We show that the expression and imaging of HSV1-tk (a marker gene can be used to monitor the expression of the LacZ gene (a second gene under the transcriptional control of a single promoter within a bicistronic unit that includes a type II internal ribosomal entry site. In cells bearing a single copy of the vector, the expression of the two genes is proportional and constant, both in vitro and in vivo. We demonstrate that non-invasive imaging of HSV1-tk gene accurately reflects the topology and activity of the other cis-linked transgene.

  10. G1/S-regulated E2F-containing protein complexes bind to the mouse thymidine kinase gene promoter

    DEFF Research Database (Denmark)

    Dou, Q P; Zhao, S; Levin, A H;

    1994-01-01

    complexes were also investigated. Studies using specific antibodies revealed that p107, a retinoblastoma-like protein, was present in both E2F-G0/G1 and E2F.S, whereas cyclin E.cyclin A.cdk2 were only present in E2F.S complex(es). These data suggest that removal of the p107-containing E2F.G0/G1 complex, a...

  11. Fusion of herpes simplex virus thymidine kinase to VP22 does not result in intercellular trafficking of the protein

    NARCIS (Netherlands)

    Beerens, A. M. J.; Rots, M. G.; de Vries, E. F. J.; Haisma, H. J.

    2007-01-01

    Suicide gene therapy is a promising approach for the treatment of cancer. Current protocols, however, suffer from low efficiency. We tried to alleviate this problem by developing a transgene that will spread from the initially transduced cell to the surrounding cells (transmission). We used herpes s

  12. Predicting Response of Ovarian Cancer to Paclitaxel Treatment: Mathematical Modelling Based on Trend of CA125, TPS and Thymidine Kinase

    Czech Academy of Sciences Publication Activity Database

    Pecen, Ladislav; Kalábová, R.; Šimíčková, M.; Vondráček, J.; Valík, D.

    2001-01-01

    Roč. 47, 6 Suppl. Part 2 (2001), s. 126-126. ISSN 0009-9147. [Annual Meeting /53./. 29.07.2001-02.08.2001, Chicago] R&D Projects: GA MZd NC6405 Institutional research plan: AV0Z1030915 Subject RIV: BA - General Mathematics

  13. A Method for Quantification of Nucleotides and Nucleotide Analogues in Thymidine Kinase Assays Utilizing Lanthanum Phosphate Co-Precipitation

    OpenAIRE

    Gammon, ST; Bernstein, M; Schuster, DP; Piwnica-Worms, D

    2007-01-01

    Current methodologies for quantifying radiolabeled nucleoside monophosphates and nucleoside analogues result in high retention of unphosphorylated guanosine nucleosides in the case of lanthanum chloride precipitation or inconsistent retention of nucleotides in the case of DEAE cellulose filter papers. This study describes an innovative method for quantifying TK activity that is compatible with both purine and pyrimidine nucleoside analogues by utilizing lanthanum phosphate co-precipitation at...

  14. Fluoride stimulates [3H]thymidine incorporation and alkaline phosphatase production by human osteoblasts

    International Nuclear Information System (INIS)

    The effect of sodium fluoride on alkaline phosphatase (ALP) release and [3H]thymidine uptake by human osteoblasts in culture was investigated. Sodium fluoride stimulated both ALP release and [3H]thymidine uptake at concentrations of sodium fluoride greater than 250 mumol/L. This stimulation was similar in magnitude to that induced by 1,25-dihydroxycholecalciferol. The fluoride-induced increase in ALP was inhibited by verapamil, a calcium channel blocker. We conclude that sodium fluoride stimulates osteoblasts to proliferate and to release ALP. This stimulation by fluoride is dependent on calcium influx. Fluoride-induced stimulation of human osteoblasts may be relevant to its effect in enhancing bone formation in patients with osteoporosis

  15. The protein that binds to DNA base J in trypanosomatids has features of a thymidine hydroxylase.

    Science.gov (United States)

    Yu, Zhong; Genest, Paul-André; ter Riet, Bas; Sweeney, Kate; DiPaolo, Courtney; Kieft, Rudo; Christodoulou, Evangelos; Perrakis, Anastassis; Simmons, Jana M; Hausinger, Robert P; van Luenen, Henri G A M; Rigden, Daniel J; Sabatini, Robert; Borst, Piet

    2007-01-01

    Trypanosomatids contain an unusual DNA base J (beta-d-glucosylhydroxymethyluracil), which replaces a fraction of thymine in telomeric and other DNA repeats. To determine the function of base J, we have searched for enzymes that catalyze J biosynthesis. We present evidence that a protein that binds to J in DNA, the J-binding protein 1 (JBP1), may also catalyze the first step in J biosynthesis, the conversion of thymine in DNA into hydroxymethyluracil. We show that JBP1 belongs to the family of Fe(2+) and 2-oxoglutarate-dependent dioxygenases and that replacement of conserved residues putatively involved in Fe(2+) and 2-oxoglutarate-binding inactivates the ability of JBP1 to contribute to J synthesis without affecting its ability to bind to J-DNA. We propose that JBP1 is a thymidine hydroxylase responsible for the local amplification of J inserted by JBP2, another putative thymidine hydroxylase. PMID:17389644

  16. Estrogen-progestin pharmacodynamics of the postmenopausal endometrium studied by thymidine labeling

    Energy Technology Data Exchange (ETDEWEB)

    Friedrich, E.R.; Meyer, J.S.

    1982-06-01

    Five postmenopausal women were treated with conjugated equine estrogens, 1.25 mg tablets for 25 days, and medroxyprogesterone acetate, 10 mg tablets, in combination with the last 10 estrogen doses. Twenty-five endometrial biopsy specimens were incubated in vitro with tritiated thymidine and radioautographic slides were prepared. Within five days of estrogen treatment the thymidine labeling index (TLI) in both glands and stromal cells increased from a very low resting state to relatively high levels of DNA synthesis and cell proliferation. Within five days after addition of progestin, epithelial TLIs decreased to low levels and returned to minimal baseline levels four days after the last steroid dose. Analysis of variances indicated significant changes in epithelial cells (P less than 0.0001) confirming that the proliferative effect of estrogens was suppressed during the progestin phase. Stromal TLI changes were not significant (P . 0.46).

  17. The Use of Tritium-Labelled Thymidine in Studies on the Synthesis of Deoxyribonucleic Acids

    International Nuclear Information System (INIS)

    In the course of studies on the biosynthesis of DNA some experiments were performed using Ehrlich ascites cells, examining the uptake and incorporation of H3-thymidine. After in-vitro incubation of the cells with this compound, autoradiographs of the cells were made and DNA was also isolated and assayed for H3 activity using a flow-counter. A comparison of the two methods of assay showed a marked discrepancy; the H3 activity per cell, calculated from the autoradiographs always appeared to be greater than the activity of the isolated DNA. Subsequent flow-counter assay of the activity of washed, homogenized whole cells gave figures which agreed with the autoradiographic assay. It thus appeared that, in this system, the use of autoradiography as a measure for DNA synthesis was open to criticism. Analysis of the bound, non-DNA activity has been made and similar studies of total cellular H3 activity and the activity of isolated DNA have been undertaken on other cell types; these have also shown similar effects. From this information it has been possible to divide the synthetic process into various stages: (1) The initial incorporation of thymidine into the cell; (2) Subsequent phosphorylation in at least two steps; (3) Polymerization of the phosphorylated thymidine into DNA. Thus, although the assumption that the incorporation of thymidine into the cell gives a measure of DNA synthesis is an oversimplification, it would seem that considerable information about the preliminary stages in the process can be obtained by use of this tracer. (author)

  18. Variability of the thymidine labeling index in squamous cell carcinoma of the head and neck

    International Nuclear Information System (INIS)

    Tritiated thymidine (3HTdR) labeling is the standard technique for determining the kinetic activity of tumors. This method has been used to label multiple sections of tumor specimens obtained from seven patients with advanced squamous cell carcinoma of the head and neck. Considerable variability was observed in the labeling index in different sites from the same specimen. To reduce the large sampling error due to heterogeneity, we recommend that an average value be determined from multiple sections when employing this technique

  19. Noninvasive measurement of liver regeneration with positron emission tomography and [2-11C]thymidine

    International Nuclear Information System (INIS)

    The feasibility of liver regeneration determination with [2-11C]thymidine and positron emission tomography was investigated in partially hepatectomized rats. Serial tomographic scans were performed over a 120-minute period after injection of [2-11C]thymidine together with tritium-labeled thymidine. Within 10 minutes after injection, positron emission tomography scans showed a twofold higher hepatic uptake in regenerating than in nonregenerating livers. Time-activity curves over the liver area indicated that the maximal uptake was followed by a faster decrease of 11C radioactivity in controls than in regenerating animals, so that total 11C activity remaining in the liver at 120 minutes accounted for 68% of maximum in regenerating and only 38% in controls. Tissue distribution studies performed at 120 minutes showed that total 11C radioactivity, expressed in percent injected dose per gram, was six times higher in regenerating livers than in controls (0.62% ± 0.07% in regenerating livers and 0.10% ± 0.03% in nonregenerating livers; P less than 0.001) and correlated with 3H radioactivity measured in the nuclear fraction (r = 0.92; P less than 0.001). When the hepatic uptake was expressed in percent of dose per organ, the difference between both groups increased (2.31% ± 0.23% in regenerating livers and 0.29% ± 0.02% in nonregenerating livers; P less than 0.001) because of higher weight of regenerating livers than of nonregenerating livers (3.83 ± 0.11 g in regenerating livers and 2.96 ± 0.16 g in nonregenerating livers; P less than 0.001). In other organs examined, no difference in 11C radioactivity was found between the two groups of rats. These results indicated the potential usefulness of [2-11C]thymidine and positron emission tomography for noninvasive measurement of liver regeneration

  20. Design and synthesis of 5,6-disubstituted uracil derivatives as potent inhibitors of thymidine phosphorylase

    Czech Academy of Sciences Publication Activity Database

    Nencka, Radim; Votruba, Ivan; Hřebabecký, Hubert; Tloušťová, Eva; Horská, Květoslava; Masojídková, Milena; Holý, Antonín

    Praha : ÚOCHB AV ČR, 2005 - (Hocek, M.), s. 441-442 ISBN 80-86241-25-4. - (Collection Symposium Series. 7). [Chemistry of Nucleic Acid Components /13./. Špindlerův Mlýn (CZ), 03.09.2005-09.09.2005] R&D Projects: GA ČR(CZ) GA203/03/0089 Institutional research plan: CEZ:AV0Z40550506 Keywords : thymidine phosphorylase inhibitor * uracil * angiogenesis Subject RIV: CC - Organic Chemistry

  1. Increased tritiated thymidine-labeling index of bone marrow myeloblasts following BCG administration in man

    International Nuclear Information System (INIS)

    The in vitro (3H)thymidine-labeling index of bone marrow myeloblasts was determined in eight hematologically normal individuals before and after the administration of Bacillus Calmette-Guerin twice weekly for 2 weeks. The mean labeling indexes of meyloblasts were 30.5 and 45.2% (P<0.004). Further characterization of this stimulation of proliferation of bone marrow myeloblasts could provide some rationale for combining cytotoxic drugs and immunotherapy in cancer patients. (orig./AJ)

  2. Lymphocyte transformation (by [3H]thymidine) in mice infected with Trichinella spiralis

    International Nuclear Information System (INIS)

    The blastogenic response of infected and normal mice (to stimulation by specific antigen and nonspecific antigens) was followed by the uptake of [3H]thymidine. It was established that there was a minimum number of cells (1 x 106) per culture for these responses to be observed and that for specific stimulation by T. spiralis soluble antigen 10μg per culture gave optimal results. (author)

  3. Serine/threonine/tyrosine phosphorylation regulates DNA binding of bacterial transcriptional regulators

    DEFF Research Database (Denmark)

    Kalantari, Aida; Derouiche, Abderahmane; Shi, Lei; Mijakovic, Ivan

    2015-01-01

    Reversible phosphorylation of bacterial transcriptional regulators (TRs) belonging to the family of two-component systems (TCSs) is a well-established mechanism for regulating gene expression. Recent evidence points to the fact that reversible phosphorylation of bacterial TRs on other types of....... Here, we present an overview of different classes of bacterial TR phosphorylated and regulated by serine/threonine and tyrosine kinases. Particular attention is given to examples when serine/threonine and tyrosine kinases interact with TCSs, phosphorylating either the histidine kinases or the response...... regulators. We argue that these promiscuous kinases connect several signal transduction pathways and serve the role of signal integration....

  4. The tomato leucine-rich repeat receptor-like kinases SlSERK3A and SlSERK3B have overlapping functions in bacterial and nematode innate immunity.

    Directory of Open Access Journals (Sweden)

    Hsuan-Chieh Peng

    Full Text Available The Somatic Embryogenesis Receptor Kinase 3 (SERK3/Brassinosteroid (BR Insensitive 1-Associated Kinase 1 (BAK1 is required for pattern-triggered immunity (PTI in Arabidopsis thaliana and Nicotiana benthamiana. Tomato (Solanum lycopersicum has three SlSERK members. Two of them exhibit particularly high levels of sequence similarity to AtSERK3 and, therefore, were named SlSERK3A and SlSERK3B. To characterize a role for SlSERK3A and SlSERK3B in defense, we suppressed each gene individually or co-silenced both using virus-induced gene silencing (VIGS in the tomato cv. Moneymaker. Co-silencing SlSERK3A and SlSERK3B resulted in spontaneous necrotic lesions and reduced sensitivity to exogenous BR treatment. Silencing either SlSERK3A or SlSERK3B resulted in enhanced susceptibility to root knot-nematode and to non-pathogenic Pseudomonas syringae pv. tomato (Pst DC3000 hrcC indicating that both SlSERK3s are positive regulators of defense. Interestingly, silencing SlSERK3B, but not SlSERK3A, resulted in enhanced susceptibility to the pathogenic strain Pst DC3000 indicating distinct roles for these two SlSERK3 paralogs. SlSERK3A and SlSERK3B are active kinases, localized to the plasma membrane, and interact in vivo with the Flagellin Sensing 2 receptor in a flg22-dependent manner. Complementation of the Atserk3/bak1-4 mutant with either SlSERK3A or SlSERK3B partially rescued the mutant phenotype. Thus, SlSERK3A and SlSERK3B are likely to constitute tomato orthologs of BAK1.

  5. Effects of four aromatic organic pollutants on microbial glucose metabolism and thymidine incorporation in marine sediments

    International Nuclear Information System (INIS)

    The metabolism of D-(U-14C)glucose and the incorporation of (methyl-3H)thymidine by aerobic and anaerobic marine sediment microbes exposed to 1 to 1000 ppm anthracene, naphthalene, p,p'-dichlorodiphenyltrichloroethane, and pentachlorophenol were examined. Cell-specific rates of (14C)glucose metabolism averaged 1.7 x 10-21 and 0.5 x 10-21 mol/min per cell for aerobic and anaerobic sediment slurries, respectively; (3H)thymidine incorporation rates averaged 43 x 10-24 and 9 x 10-24 mol/min per cell for aerobic and anaerobic slurries, respectively. Aerobic sediments exposed to three of the organic pollutants for 2 to 7 days showed recovery of both activities. Anaerobic sediments showed little recovery after 2 days of pre-exposure to the pollutants. The authors conclude that (i) anaerobic sediments are more sensitive than aerobic sediments to pollutant additions; (ii) (3H)thymidine incorporation is more sensitive to pollutant additions than is (14C)glucose metabolism; and (iii) the toxicity of the pollutants increased in the following order: anthracene, p,p'-dichlorodiphenyltrichloroethane, naphthalene, and pentachlorophenol

  6. Post-mortem 3H-thymidine incorporation in human epidermis and oral mucosa

    International Nuclear Information System (INIS)

    Using the 3H-thymidine labelling method, the authors studied post-mortem incorporation activity in the epidermis and oral mucosa of corpses which were stored with their clothes on under conditions of normal room temperature (+200) and of cooling (+40C). Samples were taken in the form of skin punches at 2 h or 4 h intervals, respec.. Using histo-autoradiograms, the incorporation of 3H-thymidine in dependence from the time interval between the points of time of death and sampling were determined in situe and given as the ratio of labelled cells of the germinative layer per 100 μm length of basement membrane. A linear drop of post-mortem thymidine incorporation rates in epidermis and oral mucosa was found in human corpse skin correlating with increasing temporal distance from the point of time of death. Incorporation rates in the oral mucosa were markedly higher (by a factor of 3 to 5) than those of the epidermis which agrees well with in vivo conditions. No labelling of cell nuclei, i.e. no synthetic activity of the germinative layer, could be detected in the epidermis 35-40 h after individual death at the latest (in the oral mucosa after 45-50 h). However, clear incorporation activities could be observed in the germinative layer of epidermis and oral mucosa after more than 4 d in the case of storage at +40C. (orig./MG)

  7. Fructose Degradation in the Haloarchaeon Haloferax volcanii Involves a Bacterial Type Phosphoenolpyruvate-Dependent Phosphotransferase System, Fructose-1-Phosphate Kinase, and Class II Fructose-1,6-Bisphosphate Aldolase

    OpenAIRE

    Pickl, Andreas; Johnsen, Ulrike; Schönheit, Peter

    2012-01-01

    The halophilic archaeon Haloferax volcanii utilizes fructose as a sole carbon and energy source. Genes and enzymes involved in fructose uptake and degradation were identified by transcriptional analyses, deletion mutant experiments, and enzyme characterization. During growth on fructose, the gene cluster HVO_1495 to HVO_1499, encoding homologs of the five bacterial phosphotransferase system (PTS) components enzyme IIB (EIIB), enzyme I (EI), histidine protein (HPr), EIIA, and EIIC, was highly ...

  8. Effect of morphine on 3H-thymidine incorporation in the subependyma of the rat: an autoradiographic study

    International Nuclear Information System (INIS)

    Following morphine treatment, an autoradiographic study investigated the uptake of 3H-thymidine by the subependymal cells in the rat brain. 3H-thymidine was administered subcutaneously to adult, male Sprague-Dawley rats 30 minutes after saline or morphine (19 mg/kg) injection. The animals were sacrified 1 hour after 3H-thymidine administration. In some experiments the opioid antagonist, naloxone, was given alone 45 minutes before 3H-thymidine or 125 minutes before morphine treatment. Three areas of the subependyma were evaluated in terms of the percentage labeled cells and number of grains per nucleus, and a dorsal-to-ventral gradiant was described. Morphine treatment significantly increased the number of 3H-thymidine labeled subependymal cells and number of grains/nucleus within labeled cells. Examination of the distribution of grains/nucleus showed that morphine-treated animals had significantly more cells labeled with 30 or more grains than did saline-injected controls. Prior administration of naloxone blocked the increased 3H-thymidine uptake in morphine-treated animals but had no significant influence on cell proliferation when administered alone. The data are discussed in terms of morphine's possible dual influence on mechanisms which enhance cell transition from G to S phase and/or which accelerate DNA synthesis once these cells have entered the S phase of cell replication

  9. Effect of morphine on /sup 3/H-thymidine incorporation in the subependyma of the rat: an autoradiographic study

    Energy Technology Data Exchange (ETDEWEB)

    Miller, C.R.; O' Steen, W.K.; Deadwyler, S.A.

    1982-06-20

    Following morphine treatment, an autoradiographic study investigated the uptake of /sup 3/H-thymidine by the subependymal cells in the rat brain. /sup 3/H-thymidine was administered subcutaneously to adult, male Sprague-Dawley rats 30 minutes after saline or morphine (19 mg/kg) injection. The animals were sacrified 1 hour after /sup 3/H-thymidine administration. In some experiments the opioid antagonist, naloxone, was given alone 45 minutes before /sup 3/H-thymidine or 125 minutes before morphine treatment. Three areas of the subependyma were evaluated in terms of the percentage labeled cells and number of grains per nucleus, and a dorsal-to-ventral gradiant was described. Morphine treatment significantly increased the number of /sup 3/H-thymidine labeled subependymal cells and number of grains/nucleus within labeled cells. Examination of the distribution of grains/nucleus showed that morphine-treated animals had significantly more cells labeled with 30 or more grains than did saline-injected controls. Prior administration of naloxone blocked the increased /sup 3/H-thymidine uptake in morphine-treated animals but had no significant influence on cell proliferation when administered alone. The data are discussed in terms of morphine's possible dual influence on mechanisms which enhance cell transition from G to S phase and/or which accelerate DNA synthesis once these cells have entered the S phase of cell replication.

  10. The study of the effects of mechanical vibration at infrasound frequency on [(3)H]-thymidine incorporation into DNA of E. coli K-12.

    Science.gov (United States)

    Martirosyan, Varsik; Baghdasaryan, Naira; Ayrapetyan, Sinerik

    2013-03-01

    The aim of the present work was to investigate the frequency-dependent effects of mechanical vibration at infrasound frequency (MV at IS frequency or MV) on E. coli K-12 growth by investigating the cell proliferation, using radioactive [(3)H]-thymidine assay. The frequency-dependent effects of MV were shown that it could either stimulate or inhibit the growth of microbes. However, the mechanism through which the MV effects affect the bacterial cells is not clear yet. It was suggested that the aqua medium can serve as a target through which the biological effect of MV on microbes could be realized. To check this hypothesis the frequency-dependent effect (2, 4, 6, 8, 10 Hz) of MV on the bacterial growth in cases of exposure the preliminary treated microbes-free medium and microbes containing medium were studied. It has been shown that MV at 4, 8, and 10 Hz frequency has inhibition effects, while at 2 and 6 Hz has stimulation effects on cell proliferation. PMID:23046076

  11. Bacterial Vaginosis

    Science.gov (United States)

    ... 586. Related Content STDs during Pregnancy Fact Sheet Pregnancy and HIV, Viral Hepatitis, and STD Prevention Pelvic Inflammatory Disease ( ... Bacterial Vaginosis (BV) Chlamydia Gonorrhea Genital Herpes Hepatitis HIV/AIDS & STDs Human Papillomavirus ... STDs See Also Pregnancy Reproductive ...

  12. Bacterial Meningitis

    Science.gov (United States)

    ... Schedules Preteen & Teen Vaccines Meningococcal Disease Sepsis Bacterial Meningitis Recommend on Facebook Tweet Share Compartir On this ... serious disease. Laboratory Methods for the Diagnosis of Meningitis This manual summarizes laboratory methods used to isolate, ...

  13. Prostatitis - bacterial

    Science.gov (United States)

    Any bacteria that can cause a urinary tract infection can cause acute bacterial prostatitis. Infections spread through sexual contact can cause prostatitis. These include chlamydia and gonorrhea . Sexually transmitted ...

  14. Bacterial Conjunctivitis

    OpenAIRE

    Köhle, Ülkü; Kükner, Şahap

    2003-01-01

    Conjunctivitis is an infection of the conjunctiva, generally characterized by irritation, itching, foreign body sensation, tearing and discharge. Bacterial conjunctivitis may be distinguished from other types of conjunctivitis by the presence of yellow–white mucopurulent discharge. It is the most common form of ocular infection all around the world. Staphylococcus species are the most common bacterial pathogenes, followed by Streptococcus pneumoniae and Haemophilus i...

  15. Dual-label radioisotope method for simultaneously measuring bacterial production and metabolism in natural waters

    International Nuclear Information System (INIS)

    Bacterial production and amino acid metabolism in aquatic systems can be estimated by simultaneous incubation of water samples with both tritiated methyl-thymidine and 14C-labeled amino acids. This dual-label method not only saves time, labor, and materials, but also allows determination of these two parameters in the same microbial subcommunity. Both organic carbon incorporation and respiration can be estimated. The method is particularly suitable for large-scale field programs and has been used successfully with eutrophic estuarine samples as well as with oligotrophic oceanic water. In the mesohaline portion of Chesapeake Bay, thymidine incorporation ranged seasonally from 2 to 635 pmol liter-1 h-1 and amino acid turnover rates ranged from 0.01 to 28.4% h-1. Comparison of thymidine incorporation with amino acid turnover measurements made at a deep, midbay station in 1985 suggested a close coupling between bacterial production and amino acid metabolism during most of the year. However, production-specific amino acid turnover rates increased dramatically in deep bay waters during the spring phytoplankton bloom, indicating transient decoupling of bacterial production from metabolism. Ecological features such as this are readily detectable with the dual-label method

  16. Diversification of Lrk/Tak Kinase Gene Clusters is Associated with Subfunctionalization and Cultivar-specific Transcript Accumulation in Barley

    Science.gov (United States)

    Lrk (Lr10 receptor-like kinase) and Tak (Triticum aestivum kinase) belong to the receptor-like kinase (RLK) super-gene family in higher plants. Three Lrk/Tak gene regions spanning greater than 600 kb were identified via a genome-wide survey of barley gene-rich BAC (Bacterial Artificial Chromosome) ...

  17. CK (Creatine Kinase) Test

    Science.gov (United States)

    ... be limited. Home Visit Global Sites Search Help? Creatine Kinase Share this page: Was this page helpful? Also known as: CK; Total CK; Creatine Phosphokinase; CPK Formal name: Creatine Kinase Related tests: ...

  18. Bacterial carbonatogenesis

    International Nuclear Information System (INIS)

    Several series of experiments in the laboratory as well as in natural conditions teach that the production of carbonate particles by heterotrophic bacteria follows different ways. The 'passive' carbonatogenesis is generated by modifications of the medium that lead to the accumulation of carbonate and bicarbonate ions and to the precipitation of solid particles. The 'active' carbonatogenesis is independent of the metabolic pathways. The carbonate particles are produced by ionic exchanges through the cell membrane following still poorly known mechanisms. Carbonatogenesis appears to be the response of heterotrophic bacterial communities to an enrichment of the milieu in organic matter. The active carbonatogenesis seems to start first. It is followed by the passive one which induces the growth of initially produced particles. The yield of heterotrophic bacterial carbonatogenesis and the amounts of solid carbonates production by bacteria are potentially very high as compared to autotrophic or chemical sedimentation from marine, paralic or continental waters. Furthermore, the bacterial processes are environmentally very ubiquitous; they just require organic matter enrichment. Thus, apart from purely evaporite and autotrophic ones, all Ca and/or Mg carbonates must be considered as from heterotrophic bacterial origin. By the way, the carbon of carbonates comes from primary organic matter. Such considerations ask questions about some interpretations from isotopic data on carbonates. Finally, bacterial heterotrophic carbonatogenesis appears as a fundamental phase in the relationships between atmosphere and lithosphere and in the geo-biological evolution of Earth. (author)

  19. Influence of the products of radiolysis of uracil and thymidine on the biosynthesis of DNA of Ehrlich's ascites cells

    Energy Technology Data Exchange (ETDEWEB)

    Polverelli, M.; Teoule, R.

    1972-09-01

    From ninth annual meeting of the European society for Radiation Biology; Rome, Italy (26 8ep 1972). The effect of gamma radiolysis products in aqueous solution of uracil and thymidine on the normal replicative biosynthesis of DNA of Ehrlich's ascites cells incubated in vitro in the presence of radioactive precursors of DNA (/sup 14/C-methyl thymidine or (/sup 14/C/sub 2/-d esoxyrytidine) was studied. The inhibiting effect of the thymidine radiolysis products, desoxy-2' - BETA -D-ribofuranosyl-1-hydroxy-5methyl-5-barbituric acid and hydroxy-6-dihydro-5, 6-thymidine, and the uracil radiolysis products, alloxanne and parabanic acid, was shown. (JSR)

  20. Tritium distribution in newborn mice after providing mother mice with drinking water containing tritiated thymidine

    International Nuclear Information System (INIS)

    Throughout gestation pregnant mice received drinking water which contained [methyl-3H]thymidine (18.5 kBq/ml). The newborn mice were divided into two groups. One group was nursed by their own mothers, which were further supplied with tritiated thymidine until 4 weeks after delivery (Experiment I). The other group was nursed by ''nonradioactive mothers'' which were given no tritiated thymidine (Experiment II). Tritium incorporation into the small molecular components of the acid-soluble fraction, lipid, RNA, DNA, and protein was analyzed for the newborn mice at various ages. In Experiment II, total radioactivity per gram tissue decreased initially after birth with a half life of 2.5-2.9 days in spleen, liver, intestine, stomach, thymus, lung, kidney, heart, and brain. At about 2 weeks after birth, a slower component of tritium elimination due mainly to the DNA-bound tritium appeared. Specific activity of DNA at birth was organ specific, highest in heart and lowest in thymus. Cumulative absorbed dose in various organs was estimated for the first 4 weeks after birth based upon an assumption that total and DNA-bound tritium are uniformly distributed. The result showed that organ specificity of dose accumulation is obvious for DNA-bound tritium, highest in spleen (1.15 mGy) and lowest in brain (0.13 mGy). It was also shown that the tritium supply from mother's milk is of minor importance for dose accumulation of DNA-bound tritium in the cell nuclei of organs of suckling mice

  1. Tritium distribution in newborn mice after providing mother mice with drinking water containing tritiated thymidine

    International Nuclear Information System (INIS)

    Throughout gestation pregnant mice received drinking water which contained [methyl-3H]thymidine (18.5 kBq/ml). The newborn mice were divided into two groups. One group was nursed by their own mothers, which were further supplied with tritiated thymidine until 4 weeks after delivery (Experiment I). The other group was nursed by nonradioactive mothers which were given no tritiated thymidine (Experiment II). Tritium incorporation into the small molecular components of the acid-soluble fraction, lipid, RNA, DNA, and protein was analyzed for the newborn mice at various ages. In Experiment II, total radioactivity per gram tissue decreased initially after birth with a half life of 2.5 to 2.9 days in spleen, liver, intestine, stomach, thymus, lung, kidney, heart, and brain. At about 2 weeks after birth, a slower component of tritium elimination due mainly to the DNA-bound tritium appeared. Specific activity of DNA at birth was organ specific, highest in heart and lowest in thymus. Cumulative absorbed dose in various organs was estimated for the first 4 weeks after birth based upon an assumption that total and DNA-bound tritium are uniformly distributed. The result showed that organ specificity of dose accumulation is obvious for DNA-bound tritium, highest in spleen (1.15 mGy) and lowest in brain (0.13 mGy). It was also shown that the tritium supply from mother's milk is of minor importance for dose accumulation of DNA-bound tritium in the cell nuclei of organs of suckling mice

  2. Comparative Study between topical applications liposomally entrapped DNA repair enzymes and thymidine dinucleotide as radioprotectors

    International Nuclear Information System (INIS)

    The delivery of active agents to the skin by liposome carriers received great interest during the last three decades. This is based on their potential to enclose various types of biological materials and to deliver them to diverse cell types. Recent work suggests that liposomes as vehicles for topical drug delivery may be superior to conventional preparations. Also, topical application of DNA repair enzymes to irradiated skin increases the rate of repair of DNA potentially damaged cells. Moreover, thymidine dinucleotide is a new skin photo-protective agent against non-ionizing radiation through induction of DNA repair. Gamma irradiation can produce DNA damage in human skin. DNA mutations have an important role in the development of skin cancer and precancerous skin lesions. Albino rats were irradiated with Cobalt-60 gamma radiation with different doses (0.5, 1.5, 3 Gy), and were treated by either thymidine dinucleotide or liposomally entrapped DNA repair enzymes topically 24 hours before irradiation. Evaluation was done histopathologically by H and E stain. Computerized image analyzer using Masson's trichrome stain was also done. Gamma radiation produced epidermal thinning and dermal inflammatory cells together with collagen fragmentation and clumping in a dose-dependent manner. Comparing between both thymidine dinucleotide and liposomally entrapped DNA repair enzymes pretreated and irradiated rats. Low dose irradiation (0.5 Gy) together with previous drugs showed preservation of epidermis with no inflammatory cells and also it maintained the normal architecture of collagen bundles. However, they were ineffective with higher doses. In conclusion our results may suggest that the effects of gamma radiation on the skin at low dose could be minimized by the use of these drugs before exposure

  3. The endozepine ODN stimulates [3H]thymidine incorporation in cultured rat astrocytes

    International Nuclear Information System (INIS)

    High concentrations of diazepam-binding inhibitor (DBI) mRNA have been detected in astrocytoma, suggesting that DBI-derived peptides may play a role in glial cell proliferation. In the present study, we have investigated the effect of a processing product of DBI, the octadecaneuropeptide ODN, on DNA synthesis in cultured rat astrocytes. At very low concentrations (10-14 to 10-11 M), ODN caused a dose-dependent increase of [3H]thymidine incorporation. At higher doses (10-10 to 10-5 M), the effect of ODN gradually declined. The central-type benzodiazepine receptor antagonist flumazenil (10-6 M) completely suppressed the stimulatory action of ODN whereas the peripheral-type benzodiazepine receptor ligand, PK11195 (10-6 M) had no effect. The ODN-induced stimulation of [3H]thymidine incorporation was mimicked by methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM). The GABAA receptor antagonist bicuculline (10-4 M) suppressed the effect of both ODN and DMCM on DNA synthesis. Exposure of cultured astrocytes to the specific GABAA agonist 3APS (10-10 to 10-4 M) also induced a dose-related increase of [3H]thymidine incorporation. The present study indicates that ODN, acting through central-type benzodiazepine receptors associated with the GABAA receptor complex, stimulates DNA synthesis in rat glial cells. These data provide evidence for an autocrine role of endozepines in the control of glial cell proliferation. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  4. Variability of the thymidine labeling index in squamous cell carcinoma of the head and neck

    Energy Technology Data Exchange (ETDEWEB)

    Greenberg, B.; Woo, L.; Blatchford, S.; Aguirre, M.; Garewal, H.

    1988-06-01

    Tritiated thymidine (/sup 3/HTdR) labeling is the standard technique for determining the kinetic activity of tumors. This method has been used to label multiple sections of tumor specimens obtained from seven patients with advanced squamous cell carcinoma of the head and neck. Considerable variability was observed in the labeling index in different sites from the same specimen. To reduce the large sampling error due to heterogeneity, we recommend that an average value be determined from multiple sections when employing this technique.

  5. Multisubstrate inhibitors of thymidine phosphorylase derived from 8-aza-7,9-dideazaxanthine

    Czech Academy of Sciences Publication Activity Database

    Mařák, David; Otmar, Miroslav; Votruba, Ivan; Dračínský, Martin; Holý, Antonín

    Marburg : University of Marburg, 2006. s. 111. ISBN 3-89703-685-1. [Joint Meeting of the Czech, German and Hungarian Pharmaceutical Societies. 04.10.2006-07.10.2006, Marburg] R&D Projects: GA MŠk(CZ) 1M0508 Grant ostatní: Descartes Prize(XE) HPAW-2002-100096 Institutional research plan: CEZ:AV0Z40550506 Keywords : thymidine phosphorylase * 8-Aza-7,9-dideazaxanthine * pyrrolo[3,4-d]pyrimidine Subject RIV: CC - Organic Chemistry

  6. 3H-thymidine labelling pattern of preleptotene chromosone condensation stages in the foetal sheep ovary

    International Nuclear Information System (INIS)

    A sequence of morphological events from premeiotic interphase to leptotene has been described in the ovaries of 64-day old sheep foetuses. The nuclear changes throughout the condensation and decondensation stages showed a sequential pattern. After a flash of tritiated thymidine, the labelling pattern of these figures demonstrated that those stages followed premeiotic DNA synthesis, i.e. belonged to meiotic prophase. However, with our methodology, this sequence of morphological events could not be confirmed due to a high asynchronism in the oogenetic processes

  7. Effects of thymidine phosphorylase on tumor aggressiveness and 5-fluorouracil sensitivity in cholangiocarcinoma

    Institute of Scientific and Technical Information of China (English)

    Jongkonnee; Thanasai; Temduang; Limpaiboon; Patcharee; Jearanaikoon; Banchob; Sripa; Chawalit; Pairojkul; Srisurang; Tantimavanich; Masanao; Miwa

    2010-01-01

    AIM: To evaluate the role of thymidine phosphorylase (TP) in cholangiocarcinoma using small interfering RNA (siRNA). METHODS: A human cholangiocarcinoma-derived cell line KKU-M139, which has a naturally high level of endogenous TP, had TP expression transiently knocked down using siRNA. Cell growth, migration, in vitro angiogenesis, apoptosis, and cytotoxicity were assayed in TP knockdown and wild-type cell lines. RESULTS: TP mRNA and protein expression were decreased by 87.1% ± 0.49% and 72.5% ± 3.2%, resp...

  8. Labeling cells in microtiter plates for determination of [3H]thymidine uptake.

    Science.gov (United States)

    Shevach, E M

    2001-05-01

    A number of protocols in Current Protocols in Immunology use as their end-point the determination of cell proliferation by determining the incorporation of [(3)H]thymidine into cellular DNA. This appendix presents a protocol in which the radioactive label is added during the last 4 to 24 hr of the culture. A semiautomated cell harvesting apparatus is then used to lyse the cells with water and precipitate the labeled DNA on glass fiber filters. The filter pads are then dried and counted by standard liquid scintillation counting techniques in a scintillation counter. PMID:18432656

  9. Kinetics of cell labeling and thymidine replacement after continuous infusion of halogenated pyrimidines in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez, R.; Ritter, M.A.; Fowler, J.F.; Kinsella, T.J. (Univ. of Wisconsin Medical School, Madison, WI (United States))

    1994-04-30

    The authors present experiments on an in vivo human tumor xenograft continuously exposed to a fixed serum concentration of halogenated pyrimidines so as to study the kinetics of cell labeling and thymidine replacement. Human colon tumor (HCT-116) cells were injected subcutaneously into nude mice. After 10 days, most animals (>90%) developed measurable tumor nodules with a volume doubling time of 5 [+-] 1 days. Once the tumors reached a cross-sectional area of 0.25-0.30 cm[sup 2], miniosmotic pumps were implanted to deliver a dose of 100 mg/kg/day of IdUrd (iododeoxyuridine) by continuous infusion. After an IdUrd exposure time of 1-7 days, blood and tumor tissue were collected. The steady state serum IdUrd concentration was 0.95 [+-] 0.1 [mu]M, which is a clinically relevant concentration for a prolonged continuous intravenous infusion. The tumor cell potential doubling time (T[sub pot]) was 25. The percent IdUrd thymidine replacement and the fraction of cells labeled followed exponential saturation kinetics with a halflife of 33 and 27 h, respectively. After 5 days of exposure, the thymidine replacement in tumor cells was 2.0% and the fraction of tumor cells labeled was 94%. Immunohistochemical staining of IdUrd labeled tumor tissues showed an exposure-dependent gradient of cellular labeling that was initially highest in regions close to blood vessels. After 4 days of exposure at 100 mg/kg/day, there was an increase in the fraction of cells in G[sub 0] + G[sub 1] and a decrease in the S phase population, suggesting a block between G[sub 1] and S phase. They conclude that the in vivo kinetics of IdUrd thymidine replacement and fraction of cells labeled after continuous exposure followed exponential saturation kinetics with a halflife of approximately the potential doubling time of the tumor cell population. Some form of prolonged, or briefly interrupted, continuous infusion should be considered for clinical administration. 48 refs., 5 figs.

  10. Structural basis for the changed substrate specificity of Drosophila melanogaster deoxyribonucleoside kinase mutant N64D

    DEFF Research Database (Denmark)

    Welin, M.; Skovgaard, T.; Knecht, Wolfgang;

    2005-01-01

    The Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) double mutant N45D/N64D was identified during a previous directed evolution study. This mutant enzyme had a decreased activity towards the natural substrates and decreased feedback inhibition with dTTP, whereas the activity with 3......'-modified nucleoside analogs like 3'-azidothymidine ( AZT) was nearly unchanged. Here, we identify the mutation N64D as being responsible for these changes. Furthermore, we crystallized the mutant enzyme in the presence of one of its substrates, thymidine, and the feedback inhibitor, dTTP. The introduction...

  11. Bacterial Adhesion & Blocking Bacterial Adhesion

    DEFF Research Database (Denmark)

    Vejborg, Rebecca Munk

    2008-01-01

    parameters, which influence the transition from a planktonic lifestyle to a sessile lifestyle, have been studied. Protein conditioning film formation was found to influence bacterial adhesion and subsequent biofilm formation considerable, and an aqueous extract of fish muscle tissue was shown to...... tract to the microbial flocs in waste water treatment facilities. Microbial biofilms may however also cause a wide range of industrial and medical problems, and have been implicated in a wide range of persistent infectious diseases, including implantassociated microbial infections. Bacterial adhesion is...... the first committing step in biofilm formation, and has therefore been intensely scrutinized. Much however, still remains elusive. Bacterial adhesion is a highly complex process, which is influenced by a variety of factors. In this thesis, a range of physico-chemical, molecular and environmental...

  12. Bacterial lipases

    NARCIS (Netherlands)

    Jaeger, Karl-Erich; Ransac, Stéphane; Dijkstra, Bauke W.; Colson, Charles; Heuvel, Margreet van; Misset, Onno

    1994-01-01

    Many different bacterial species produce lipases which hydrolyze esters of glycerol with preferably long-chain fatty acids. They act at the interface generated by a hydrophobic lipid substrate in a hydrophilic aqueous medium. A characteristic property of lipases is called interfacial activation, mea

  13. Bacterial Ecology

    DEFF Research Database (Denmark)

    Fenchel, Tom

    2011-01-01

    Bacterial ecology is concerned with the interactions between bacteria and their biological and nonbiological environments and with the role of bacteria in biogeochemical element cycling. Many fundamental properties of bacteria are consequences of their small size. Thus, they can efficiently exploit...

  14. Biological indicators of radiation exposure. Experimental study of radiation-induced changes of thymidine level in human serum

    International Nuclear Information System (INIS)

    The study summarizes the results of the search for a reliable quantitative biochemical indicator system for ionizing radiation in man. Thymidine seems to be a promising indicator in the serum. The analysis of thymidine is based on the inhibited incorporation of 125I-UdR into DNA of fibroblast cells in the presence of serum of the irradiated mice. Maximal inhibition of the I-UdR-incorporation occurs 4 hours after irradiation. The data from animal experiments show in the dose range between 0.005 and 1 Gy a dose effect relationship which was multiply reproduced. The main objective of the experiments is to test the transferability of results from mouse to man. What little is known on these effects in humans was learned from investigations in radiotherapy patients. Up to now, 22 radiotherapy patients have been examined and in no case dose dependency for the increase of thymidine concentration in human serum as a function of radiation dose has been observed. Among the tumour patients investigated, three groups were found to be of special interest: one group with a low initial thymidine concentration increasing after irradiation, a second group of patients with a high initial thymidine concentration decreasing after irradiation and a third group with a middle initial thymidine concentration not changing after irradiation. A dependency of the effect from time, as it occurs in the mouse, has not been observed in man until now. Thus the applicability of this biochemical indicator system as evidence of a radiation exposure in man has not been proven. (orig./MG)

  15. Use of Tritiated Thymidine to Study the Origin and Fate of Inflammatory Cells

    International Nuclear Information System (INIS)

    In a series of experiments mice were injected with tritiated thymidine at various times following a challenging injection of either tetanus or diphtheria toxoid and the number and proportion of mononuclear cells synthesizing DNA at the site of injection determined. It was noted that the increase in mononuclear inflammatory cells was not preceded by a similar decrease in cells synthesizing DNA. This indicates that the majority of inflammatory mononuclear cells must migrate into the inflamed area, presumably from the blood vessels. Inflammatory cells labelled with tritiated thymidine were injected into the site of inflammation, and autopsies performed at various times. Labelled cells were found not only in the inflammatory area, but in the spleen, bone marrow and lymph nodes. These experiments indicate that as the inflammation subsides, inflammatory cells pass back into the lymphatic and blood vascular systems and eventually some find their way to the hemopoietic tissues of the body. Experiments will be reported indicating formation of plasma cells by inflammatory mononuclear cells. These findings will be discussed in relation to hemopoiesis. (author)

  16. Anti-proliferative effects of protein kinase C inhibitors in human keratinocytes.

    Science.gov (United States)

    Hegemann, L; Bonnekoh, B; van Rooijen, L A; Mahrle, G

    1992-07-01

    Various lines of evidence indicate that protein kinase C, a key enzyme in transmembraneous signal transduction, is involved in the regulation of keratinocyte proliferation. In the present study we have investigated the effects of various structurally unrelated protein kinase C inhibitors on the proliferation of HaCa T cells, a non-tumorigenic human keratinocyte cell line. All protein kinase C inhibitors dose-dependently inhibited cell proliferation as assessed by the incorporation of radioactively labelled thymidine and amino acids as well as the increase in total protein content in keratinocytes. The potencies of the drugs to inhibit cell proliferation were strongly correlated to their inhibitory potency on purified protein kinase C, displaying a correlation coefficient of 0.97. Methotrexate, an anti-proliferative drug, was found not to inhibit protein kinase C. Therefore, our data provide evidence that protein kinase C is crucially involved in the regulation of keratinocyte proliferation but is not the only target of anti-proliferative drug action. PMID:1390454

  17. Evaluation of [methyl- 14C]4'-thio-thymidine for DNA synthesis imaging in vivo

    International Nuclear Information System (INIS)

    Objective: In order to obtain a thymidine analog that might prove simpler to use for imaging DNA synthesis and follow the same biochemistry of thymidine in vivo, we evaluated [methyl- 11C]4'-thio-thymidine ([methyl- 11C]S-dThd) by using the [14C]-labeled counterpart ([methyl- 14C]S-dThd). Methods: [methyl-14C]S-dThd was synthesized by rapid methylation of 5-trimethyl-stannyl-4' -thio-2' -deoxyuridine via a palladium mediated Stille-coupling reaction with [14C]methyl iodide. Degradation of [methyl- 14C]S-dThd when incubated in human blood was analyzed by HPLC. The in vivo potential of [methyl- 14C]S-dThd was evaluated by distribution study of EMT-6 mammary carcinoma-bearing mice. Gemcitabine, a potent inhibitor of DNA synthesis, was used to modulate cell proliferation. Tissue extraction was also performed to investigate the incorporation of [methyl-14C]S-dThd into DNA. Results: [methyl- 14C]S-dThd was obtained in 31-41% radiochemical yield (calculated from [14C]methyl iodide) at 130, 5 min reaction in N,N-dimethylforamide. After semi-preparative HPLC purification, radiochemical purity of [methyl- 14C]S-dThd was >99% and the specific activity was 2.04 GBq/mmol (according to the specific activity of [14C]methyl iodide). Incubation with human blood demonstrated rapid degradation of [2- 14C]thymidine. In contrast, [methyl- 14C]S-dThd was stable with less than 3% degradation at 60 min. In vivo distribution study showed progressive accumulation of radioactivity in proliferating tissues (spleen, thymus, duodenum and tumor). On the other hand, the washout of radioactivity by the non-proliferating tissues (lung, liver, kidney and muscle) appeared nearly exponential. The tumor uptake of [methyl- 14C]S-dThd was high (8.8%ID/g at 60 min) and selective (Tumor to blood ratio: 12.2 at 60 min). Gemicitabine pretreatment significantly reduced the tumor uptake of [methyl- 14C]S-dThd. Relative blood flow as measured by the uptake 4-[N-Methyl- 14C]iodoantipyrine was similar in the

  18. Kinetic observation of rapid electron transfer between thymine and thymidine anion radicals and caffeic acid: a pulse radiolysis study

    International Nuclear Information System (INIS)

    Rapid electron transfer from thymine or thymidine anion radicals to caffeic acid with rate constant of 1 x 109 M-1s-1 was observed by pulse radiolysis, leading to the formation of anion radicals of caffeic acid which is characterized with absorption maximum at 360nm. Caffeic acid has a higher one-electron reduction potential than the target molecule (thymine or thymidine) and acts as a electrophilic protector which prevent the target anion radical from its irreversible protonation at C6 leading to its 5-yl radical via fast electron transfer. The kinetic demonstrations have provided dynamic evidence of charge transfer protection mechanism. (author)

  19. Detection of DNA damage: effect of thymidine glycol residues on the thermodynamic, substrate and interfacial acoustic properties of oligonucleotide duplexes.

    Science.gov (United States)

    Yang, F; Romanova, E; Kubareva, E; Dolinnaya, N; Gajdos, V; Burenina, O; Fedotova, E; Ellis, J S; Oretskaya, T; Hianik, T; Thompson, M

    2009-01-01

    Thymidine glycol residues in DNA are biologically active oxidative molecular damage sites caused by ionizing radiation and other factors. One or two thymidine glycol residues were incorporated in 19- to 31-mer DNA fragments during automatic oligonucleotide synthesis. These oligonucleotide models were used to estimate the effect of oxidized thymidines on the thermodynamic, substrate and interfacial acoustic properties of DNA. UV-monitoring melting data revealed that modified residues in place of thymidines destabilize the DNA double helix by 8-22 degrees C, depending on the number of lesions, the length of oligonucleotide duplexes and their GC-content. The diminished hybridizing capacity of modified oligonucleotides is presumably due to the loss of aromaticity and elevated hydrophilicity of thymine glycol in comparison to the thymine base. According to circular dichroism (CD) data, the modified DNA duplexes retain B-form geometry, and the thymidine glycol residue introduces only local perturbations limited to the lesion site. The rate of DNA hydrolysis by restriction endonucleases R.MvaI, R.Bst2UI, R.MspR9I and R.Bme1390I is significantly decreased as the thymidine glycol is located in the central position of the double-stranded recognition sequences 5'-CC / WGG-3' (W = A, T) or 5'-CC / NGG-3' (N = A, T, G, C) adjacent to the cleavage site. On the other hand, the catalytic properties of enzymes R.Psp6I and R.BstSCI recognizing the similar sequence are not changed dramatically, since their cleavage site is separated from the point of modification by several base-pairs. Data obtained by gel-electrophoretic analysis of radioactive DNA substrates were confirmed by direct spectrophotometric assay developed by the authors. The effect of thymidine glycol was also observed on DNA hybridization at the surface of a thickness-shear mode acoustic wave device. A 1.9-fold decrease in the rate of duplex formation was noted for oligonucleotides carrying one or two thymidine glycol

  20. Incorporation of 3H-thymidine during early developmental stages of ovoviviparous and oviparous embryos in the brine

    International Nuclear Information System (INIS)

    DNA synthesis in the embryos during various developmental stages in the brood pouch of the Artemia female as well as the encysted eggs produced by oviparity was studied by autoradiography using 3H-thymidine. Nuclei of the embryos through the blastula stage in the brood pouch were labelled, regardless of the existence of the thick shell surrounding the embryos. However, incorporation of 3H-thymidine into nuclei was absent in the stages between gastrula and early nauplii which had developed in the brood pouch or were delivered from encysted dry eggs. (author)

  1. [Bacterial vaginosis].

    Science.gov (United States)

    Romero Herrero, Daniel; Andreu Domingo, Antonia

    2016-07-01

    Bacterial vaginosis (BV) is the main cause of vaginal dysbacteriosis in the women during the reproductive age. It is an entity in which many studies have focused for years and which is still open for discussion topics. This is due to the diversity of microorganisms that cause it and therefore, its difficult treatment. Bacterial vaginosis is probably the result of vaginal colonization by complex bacterial communities, many of them non-cultivable and with interdependent metabolism where anaerobic populations most likely play an important role in its pathogenesis. The main symptoms are an increase of vaginal discharge and the unpleasant smell of it. It can lead to serious consequences for women, such as an increased risk of contracting sexually transmitted infections including human immunodeficiency virus and upper genital tract and pregnancy complications. Gram stain is the gold standard for microbiological diagnosis of BV, but can also be diagnosed using the Amsel clinical criteria. It should not be considered a sexually transmitted disease but it is highly related to sex. Recurrence is the main problem of medical treatment. Apart from BV, there are other dysbacteriosis less characterized like aerobic vaginitis of which further studies are coming slowly but are achieving more attention and consensus among specialists. PMID:27474242

  2. Protein kinase CK2 structure-function relationship

    DEFF Research Database (Denmark)

    Boldyreff, B; Meggio, F; Pinna, L A;

    1994-01-01

    Protein kinase CK2 subunits alpha and beta were expressed either separately or together in a bacterial expression system (pT7-7/BL21(DE3)) and purified to homogeneity. After mixing the subunits, a CK2 holoenzyme (alpha 2 beta 2) was spontaneously reconstituted, which displays identical features as...

  3. Effects of low-energy electrons on DNA constituents: effective cross sections for condensed thymidine

    Science.gov (United States)

    Panajotovic, Radmila

    2009-05-01

    Since the first experiments of low-energy electron scattering from condensed DNA [1] have been performed, the interest in studying low-energy electron-biomolecule interactions has been increasing. Knowledge of effective cross sections for single- and double-strand breaks of DNA and for vibrational and electronic excitation of nucleic bases and nucleosides are opening the door to better understanding of effects of radiation on live tissue and possibly indicating interaction pathways leading to gene mutations and cancer. The strong variation of effective cross sections for DNA single-strand breaks with incident electron energy and the resonant enhancement at 1 eV suggested that considerable damage is inflicted by very low-energy electrons to DNA, and indicates the important role of π* shape resonances in the bond-breaking process. However, the complexity of DNA, even if studied as a short single-strand chain, imposes a need to perform measurements on its isolated constituents, such as nucleic bases and nucleosides. Thymidine is one of the most important nucleosides of DNA and an important component of antiviral compounds. In the condensed phase, thymidine's 2'-deoxyribose ring is in the pentose sugar ring form, which is a true conformation of this nucleoside in DNA. Results from High-Resolution Electron Energy Loss [2] study of monomolecular films of thymidine will be discussed and the presence of resonances in the effective cross sections at incident energy below 5 eV will be commented as a possible indication of the dissociative electron attachment. In addition, results on the resonance structures in the effective cross sections for electronic excitations for the incident electron energy from 1.5 to 12 eV will be discussed as a possible pathway for strand brakes in DNA. [4pt] [1] Boudaiffa B, Cloutier P, Hunting D, Huels M A and Sanche L 2002 Rad. Res. 157 227-234[0pt] [2] Panajotovic R, Martin F, Cloutier P, Hunting, D, and Sanche L, 2006 Rad.Res. 165 452

  4. Muscle phosphorylase kinase deficiency

    DEFF Research Database (Denmark)

    Preisler, N; Orngreen, M C; Echaniz-Laguna, A; Laforet, P; Lonsdorfer-Wolf, E; Doutreleau, S; Geny, B; Akman, H O; Dimauro, S; Vissing, J

    2012-01-01

    To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD).......To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD)....

  5. 5-Bromo (or chloro)-6-azido-5,6-dihydro-2' -deoxyuridine and -thymidine derivatives with potent antiviral activity.

    Science.gov (United States)

    Kumar, Rakesh

    2002-02-11

    Synthesis, antiviral, and cytotoxic activities of 5-bromo (or chloro)-6-azido-5,6-dihydro-2' -deoxyuridine (4,5) and -thymidine (6,7) are reported. Compounds 4 and 5 exhibited a broad spectrum of antiherpes activity against (HSV-1, HSV-2, HCMV, and VZV). PMID:11814776

  6. Design and synthesis of novel 5,6-disubstituted uracil derivatives as potent inhibitors of thymidine phosphorylase

    Czech Academy of Sciences Publication Activity Database

    Nencka, Radim; Votruba, Ivan; Hřebabecký, Hubert; Tloušťová, Eva; Horská, Květoslava; Masojídková, Milena; Holý, Antonín

    2006-01-01

    Roč. 16, č. 5 (2006), s. 1335-1337. ISSN 0960-894X R&D Projects: GA ČR(CZ) GA203/03/0089 Institutional research plan: CEZ:AV0Z40550506 Keywords : thymidine phosphorylase inhibitor * disubstituted uracils * angiogenesis Subject RIV: CC - Organic Chemistry Impact factor: 2.538, year: 2006

  7. Influence of dehydroepiandrosterone on G-6-PD activity and /sup 3/H-thymidine uptake of human lymphocytes in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Ennas, M.G.; Laconi, S.; Dessi, S.; Milia, G.; Murru, M.R.; Manconi, P.E.

    1987-01-01

    Dehydroepiandrosterone (DHEA) was found to inhibit experimental cancer development in mouse and rat lung, colon and mammary gland. Since DHEA is a potent inhibitor of mammalian G-6-PD, the hypothesis that the compound could inhibit cell proliferation through an inhibition of the pentose phosphate pathway has been formulated. We studied the effects of DHEA on the proliferation in vitro of human lymphocytes induced by several mitogens (PHA, ConA and PWM), measuring /sup 3/H-thymidine uptake. DHEA inhibited /sup 3/H-thymidine uptake of mitogen-stimulated cells from both G-6-PD+ and G-6-PD- (mediterranean type deficiency) individuals in a dose-dependent and reversible fashion. The inhibitory effect was found even if DHEA was added to cells in the last hours of culture, simultaneously with the addition of /sup 3/H-thymidine. These data suggest that the inhibition of thymidine uptake induced by DHEA on human lymphocytes probably does not depend on the inhibition of G-6-PD.

  8. Influence of dehydroepiandrosterone on G-6-PD activity and 3H-thymidine uptake of human lymphocytes in vitro

    International Nuclear Information System (INIS)

    Dehydroepiandrosterone (DHEA) was found to inhibit experimental cancer development in mouse and rat lung, colon and mammary gland. Since DHEA is a potent inhibitor of mammalian G-6-PD, the hypothesis that the compound could inhibit cell proliferation through an inhibition of the pentose phosphate pathway has been formulated. We studied the effects of DHEA on the proliferation in vitro of human lymphocytes induced by several mitogens (PHA, ConA and PWM), measuring 3H-thymidine uptake. DHEA inhibited 3H-thymidine uptake of mitogen-stimulated cells from both G-6-PD+ and G-6-PD- (mediterranean type deficiency) individuals in a dose-dependent and reversible fashion. The inhibitory effect was found even if DHEA was added to cells in the last hours of culture, simultaneously with the addition of 3H-thymidine. These data suggest that the inhibition of thymidine uptake induced by DHEA on human lymphocytes probably does not depend on the inhibition of G-6-PD

  9. Kinome profiling of Arabidopsis using arrays of kinase consensus substrates

    Directory of Open Access Journals (Sweden)

    Pieterse Corné MJ

    2007-02-01

    Full Text Available Abstract Background Kinome profiling aims at the parallel analysis of kinase activities in a cell. Novel developed arrays containing consensus substrates for kinases are used to assess those kinase activities. The arrays described in this paper were already used to determine kinase activities in mammalian systems, but since substrates from many organisms are present we decided to test these arrays for the determination of kinase activities in the model plant species Arabidopsis thaliana. Results Kinome profiling using Arabidopsis cell extracts resulted in the labelling of many consensus peptides by kinases from the plant, indicating the usefulness of this kinome profiling tool for plants. Method development showed that fresh and frozen plant material could be used to make cell lysates containing active kinases. Dilution of the plant extract increased the signal to noise ratio and non-radioactive ATP enhances full development of spot intensities. Upon infection of Arabidopsis with an avirulent strain of the bacterial pathogen Pseudomonas syringae pv. tomato, we could detect differential kinase activities by measuring phosphorylation of consensus peptides. Conclusion We show that kinome profiling on arrays with consensus substrates can be used to monitor kinase activities in plants. In a case study we show that upon infection with avirulent P. syringae differential kinase activities can be found. The PepChip can for example be used to purify (unknown kinases that play a role in P. syringae infection. This paper shows that kinome profiling using arrays of consensus peptides is a valuable new tool to study signal-transduction in plants. It complements the available methods for genomics and proteomics research.

  10. Prebiotic phosphorylation of thymidine at 65 C in simulated desert conditions.

    Science.gov (United States)

    Bishop, M. J.; Lohrmann, R.; Orgel, L. E.

    1972-01-01

    The phosphorylation of thymidine is described for a variety of conditions at 65 C to demonstrate that the reaction could readily take place in deserts at the present time. This might be used as an indication that urea-phosphate mixtures could have been important as phosphorylating agents on the primitive earth. Reaction products were identified by comparing their chromatographic and electrophoretic mobilities with those of authentic materials and by enzymatic degradation. The results show that good yields of nucleotides are obtained when nucleosides are heated with urea-phospate mixtures at 65 C. Reactions proceed more rapidly at moderate humidities than in a stream of dry nitrogen. Occasional wetting results in even faster and more extensive reactions. There was no reaction for a mixture of urea and trimetaphosphate.

  11. Synthesis and preliminary biological evaluation of a technetium-99m labeled thymidine analog

    Institute of Scientific and Technical Information of China (English)

    Chun Xiong Lu; Zheng Wu Wang; Quan Fu Jiang; Jie Tang; Cheng Tan; Jian Kang Zhang

    2011-01-01

    The synthesis and labeling of 99mTc-N3-{N'-[2-sulfanyl-ethylamino)acetyl]-2-aminoethyl-sulfanyl-l-hexanamide}thymidine (99mTc-NHT) were studied. In the presence of sodium glucoheptonate (GH) and ethylene diamine tetraacetic acid (EDTA), 99mTc-NHT was obtained by using bisaminoethanethiol (N2S2) as a bifunctional coupling agent. The radiochemical purity of the 99mTc-NHT was over 95%. Biodistribution of 99mTc-NHT was performed in hepatoma HepA tumor-bearing mice. At 2 h p.i., the ratios of tumor-to-muscle, tumor-to-bone and tumor-to-blood were 4.41 ± 0.32, 2.45 ± 0.24 and 1.51 ±0.18, respectively.

  12. Postmortal incorporation of 3H-thymidine into human oral mucosa at +40C ambient temperature

    International Nuclear Information System (INIS)

    The postmortal behaviour of the skin was studied under the conditions of 'normal' autolysis. Skin samples with a diameter of 3 mm were taken every 2 or 4 hours in the period between 1 and 56 h p.m. or between 48 and 100 h p.m. The incorporation of 3H-thymidine, represented by the quotient of labelled germ layer cells per 100μ length of basal membrane, was studied in situ as a function of the time interval between death and sampling, using histoautoradiographs. Postmortal incorporation into the oral mucosa was higher by a factor 3 to 5 than incorporation into the epidermis; similar results were obtained in vivo. Experiments on refrigerated skin showed a strong temperature influence on the decrease of epithelial DNA synthesis activity. At +40C, it continued for more than 4 days in the oral mucosa, showing an undulating course of the 'proliferation activities'. (orig./MG)

  13. Role of sugar in controlling reaction pathways: A study with thymine and thymidine

    International Nuclear Information System (INIS)

    Magnetic field effect in conjunction with laser flash photolysis have been used for studying interactions of 9,10-anthraquinone and 2-methyl 1,4-naphthoquinone (menadione) with a DNA base, thymine (Thy) and its nucleoside, thymidine (dThd). Irrespective of medium Thy has been found to support both electron transfer (ET) and hydrogen abstraction with the quinones while dThd has exhibited a complete reluctance towards ET. This unique behavior of dThd has been attributed to a failure in attaining aromaticity by virtue of keto-enol tautomerism upon addition of a sugar moiety. Electron withdrawing effect of sugar unit is also considered responsible for reduction of ET from dThd. Again both Thy and dThd have exhibited hydrogen abstraction in homogeneous medium, which is normally unexpected. The above behaviors of the bases have been explained on the basis of their chemical structures.

  14. Positron imaging feasibility studies: Short term uptake of thymidine in normal and neoplastic tissues

    International Nuclear Information System (INIS)

    The uptake of labeled thymidine (TdR) as measured by labelling index has provided useful experimental and clinical data regarding tumor cell cycle. This approach is limited by the requirement for repeated tissue sampling, but could potentially be extended to in vivo measurements using C-11 TdR and positron emission tomography (PET). Since TdR is rapidly cleared from the circulation, there is concern that the distribution in vivo might reflect blood flow and not cellular metabolism. Therefore, uptake of H-3 TdR was studied in AKR mice, both normal and with spontaneous lymphoma, and in organs and tumors of dogs with spontaneous tumors. Uptake was compared to relative blood flow as measured by the distribution of C-14 iodantipyrine. Metabolic H-3 water was removed by air drying the samples before oxidizing them for counting. The initial distribution of thymidine in normal mice, measured 20 seconds after injection, correlated with relative perfusion measurements. However, between one and sixty minutes after injection there was no correlation of TdR uptake with perfusion in any of the systems studied. This indicates that the distribution more than one minute after injection is primarily dependent on subsequent redistribution and/or metabolism of TdR, rather than the initial distribution by blood flow. The time activity curve of TdR content in normal mouse organs demonstrated that organs with high rates of proliferation retail all the labeled TdR initially taken up. Organs with low rates of proliferation lost their label in a nearly exponential wash out. These studies provide further evidence of the feasibility of using C-11 TdR for PET and provide the experimental basis for developing kinetic models of TdR metabolism to interpret PET studies

  15. The Effect of Tritiated Thymidine on the Morphogenesis of Lateral Roots

    International Nuclear Information System (INIS)

    Along the axis of a primary root exists a series of successive developmental stages of the same organ — the lateral root, a system ideal for assaying effects of various agents on organogenesis. Primary roots of several species grown in H3-thymidine in various concentrations arid for different periods of time, show a region devoid of lateral roots which corresponds closely to the region of differentiation at time of treatment. Anatomical analysis combined with autoradiography indicates that ''hot'' pericycle cells may be capable of 1 or 2 cell divisions before further development of the lateral root is inhibited. There is evidence that a minimum number of cells must be affected before substitution by adjacent cells is eliminated. This may be a function of the length of the cell division cycle and thus prescribe the duration of treatment for the desired morphological effect. Root primordia already established at time of treatment do riot appear affected if the amount of radioactivity is chosen discriminately. In Zea mays the effect of ''nuclear irradiation'' on differentiation has been compared with the effect of X-rays and gamma irradiation (Co60). In general the external irradiation results in a more diffuse disturbance of both lateral and primary root growth. When duration of exposure and dose of an external source are properly chosen the internal effect of tritiated thymidine can be approached. The system offers opportunity to discriminate between damage due to nuclear irradiation (genetic effects ?) and general irradiation (genetic + physiological ?). Pisum sativum and Cucumis sativus have also been used. Current Work involves autoradiography to determine the amount of chromosomal radiation needed to disturb these developmental processes. (author)

  16. Expression, purification, crystallization and preliminary X-ray structure analysis of Vibrio cholerae uridine phosphorylase in complex with thymidine

    International Nuclear Information System (INIS)

    The expression and purification of uridine phosphorylase from V. cholerae and the crystallization and preliminary X-ray structure analysis of the complex of this enzyme with thymidine are reported. A high-resolution structure of the complex of Vibrio cholerae uridine phosphorylase (VchUPh) with its physiological ligand thymidine is important in order to determine the mechanism of the substrate specificity of the enzyme and for the rational design of pharmacological modulators. Here, the expression and purification of VchUPh and the crystallization of its complex with thymidine are reported. Conditions for crystallization were determined with an automated Cartesian Dispensing System using The Classics, MbClass and MbClass II Suites crystallization kits. Crystals of the VchUPh–thymidine complex (of dimensions ∼200–350 µm) were grown by the sitting-drop vapour-diffusion method in ∼7 d at 291 K. The crystallization solution consisted of 1.5 µl VchUPh (15 mg ml−1), 1 µl 0.1 M thymidine and 1.5 µl reservoir solution [15%(w/v) PEG 4000, 0.2 M MgCl2.6H2O in 0.1 M Tris–HCl pH 8.5]. The crystals diffracted to 2.12 Å resolution and belonged to space group P21 (No. 4), with unit-cell parameters a = 91.80, b = 95.91, c = 91.89 Å, β = 119.96°. The Matthews coefficient was calculated as 2.18 Å3 Da−1; the corresponding solvent content was 43.74%

  17. The partial molar heat capacity, expansion, isentropic, and isothermal compressions of thymidine in aqueous solution at T = 298.15 K

    International Nuclear Information System (INIS)

    Highlights: → Solution densities and sound speeds were measured for aqueous solutions of thymidine. → Partial molar volumetric properties at infinite dilution and T = 298.15 K were derived. → The partial molar isentropic and isothermal compressions are of opposite signs. → The partial molar heat capacity for thymidine at infinite dilution was determined. - Abstract: Solution densities have been determined for aqueous solutions of thymidine at T = (288.15, 298.15, 303.15, and 313.15) K. The partial molar volumes at infinite dilution, V20, obtained from the density data were used to derive the partial molar isobaric expansion at infinite dilution for thymidine at T = 298.15 K, E20{E20=(∂V20/∂T)p}. The partial molar heat capacity at infinite dilution for thymidine, Cp,20, at T = 298.15 K has also been determined. Sound speeds have been measured for aqueous solutions of thymidine at T = 298.15 K. The partial molar isentropic compression at infinite dilution, KS,20, and the partial molar isothermal compression at infinite dilution, KT,20{KT,20=-(∂V20/∂P)T}, have been derived from the sound speed data. The V20, E20, Cp,20, and KS,20 results for thymidine are critically compared with those available from the literature.

  18. Creatine kinase in the serum of patients with acute infections of the central nervous system

    DEFF Research Database (Denmark)

    Peterslund, N A; Heinsvig, E M; Christensen, K D

    1985-01-01

    Serum creatine kinase was assessed in 94 consecutive patients without convulsions admitted to hospital due to suspicion of infection of the central nervous system. No reliable discrimination between patients with aseptic and those with bacterial meningitis was obtained. Patients with bacterial...... bacterial meningitis. The highest serum CK value found in patients with encephalitis was 725 U/l. Reference values for control patients with meningism were 16-269 U/1. In a subset of 9 patients creatine kinase isoenzyme analysis was performed. In all cases only muscle type (MM) isoenzyme was found...

  19. Bacterial hydrodynamics

    CERN Document Server

    Lauga, Eric

    2015-01-01

    Bacteria predate plants and animals by billions of years. Today, they are the world's smallest cells yet they represent the bulk of the world's biomass, and the main reservoir of nutrients for higher organisms. Most bacteria can move on their own, and the majority of motile bacteria are able to swim in viscous fluids using slender helical appendages called flagella. Low-Reynolds-number hydrodynamics is at the heart of the ability of flagella to generate propulsion at the micron scale. In fact, fluid dynamic forces impact many aspects of bacteriology, ranging from the ability of cells to reorient and search their surroundings to their interactions within mechanically and chemically-complex environments. Using hydrodynamics as an organizing framework, we review the biomechanics of bacterial motility and look ahead to future challenges.

  20. Type 1 pilus-mediated bacterial invasion of bladder epithelial cells

    OpenAIRE

    Martinez, Juan J.; Mulvey, Matthew A.; Schilling, Joel D.; Pinkner, Jerome S.; Hultgren, Scott J.

    2000-01-01

    Most strains of uropathogenic Escherichia coli (UPEC) encode filamentous adhesive organelles called type 1 pili. We have determined that the type 1 pilus adhesin, FimH, mediates not only bacterial adherence, but also invasion of human bladder epithelial cells. In contrast, adherence mediated by another pilus adhesin, PapG, did not initiate bacterial internalization. FimH-mediated invasion required localized host actin reorganization, phosphoinositide 3-kinase (PI 3-kinase) activation and host...

  1. Exploring the diversity of protein modifications: special bacterial phosphorylation systems

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Grangeasse, Christophe; Turgay, Kürşad

    2016-01-01

    physiology, and regulatory networks. Investigating these unusual bacterial kinase and phosphatases is not only important to understand their role in bacterial physiology but will help to generally understand the full potential and evolution of protein phosphorylation for signal transduction, protein...... has been most thoroughly investigated. Unlike in eukarya, a large diversity of enzyme families has been shown to phosphorylate and dephosphorylate proteins on various amino acids with different chemical properties in bacteria. In this review, after a brief overview of the known bacterial...... phosphorylation systems, we focus on more recently discovered and less widely known kinases and phosphatases. Namely, we describe in detail tyrosine- and arginine-phosphorylation together with some examples of unusual serine-phosphorylation systems and discuss their potential role and function in bacterial...

  2. Effect of transforming growth factor beta (TGF-β) receptor I kinase inhibitor on prostate cancer bone growth

    OpenAIRE

    Wan, Xinhai; Li, Zhi-gang; Yingling, Jonathan M.; Yang, Jun; Starbuck, Michael W.; Ravoori, Murali K.; Kundra, Vikas; Vazquez, Elba; Navone, Nora M.

    2011-01-01

    Transforming growth factor beta 1 (TGF-β1) has been implicated in the pathogenesis of prostate cancer (PCa) bone metastasis. In this study, we tested the antitumor efficacy of a selective TGF-β receptor I kinase inhibitor, LY2109761, in preclinical models. The effect of LY2109761 on the growth of MDA PCa 2b and PC-3 human PCa cells and primary mouse osteoblasts (PMOs) was assessed in vitro by measuring radiolabeled thymidine incorporation into DNA. In vivo, the right femurs of male SCID mice ...

  3. Multiple host kinases contribute to Akt activation during Salmonella infection.

    Directory of Open Access Journals (Sweden)

    Bernhard Roppenser

    Full Text Available SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche. One important function of SopB is activation of the pro-survival kinase Akt/protein kinase B in the infected host cell. Here, we examine the mechanism of Akt activation by SopB during Salmonella infection. We show that SopB-mediated Akt activation is only partially sensitive to PI3-kinase inhibitors LY294002 and wortmannin in HeLa cells, suggesting that Class I PI3-kinases play only a minor role in this process. However, depletion of PI(3,4 P2/PI(3-5 P3 by expression of the phosphoinositide 3-phosphatase PTEN inhibits Akt activation during Salmonella invasion. Therefore, production of PI(3,4 P2/PI(3-5 P3 appears to be a necessary event for Akt activation by SopB and suggests that non-canonical kinases mediate production of these phosphoinositides during Salmonella infection. We report that Class II PI3-kinase beta isoform, IPMK and other kinases identified from a kinase screen all contribute to Akt activation during Salmonella infection. In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell. Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host.

  4. Pyruvate kinase blood test

    Science.gov (United States)

    ... break down faster than normal, a condition called hemolytic anemia . This test helps diagnose pyruvate kinase deficiency (PKD) . ... Pa: Elsevier Saunders; 2011:chap 32. Gallagher PG. Hemolytic anemias: red cell membrane and metabolic defects In: Goldman ...

  5. Tritiated Thymidine as Tracer in DNA Metabolism and Cell Dynamics of Experimental Myeloid Leukaemia

    International Nuclear Information System (INIS)

    Tritium has been used as an isotopic tracer in a variety of biological problems in Israel. We wish to report, in particular, some findings in which tritiated thymidine (TH3) has been used to follow the cell dynamics in experimental myeloid leukaemia and also to investigate the mechanism of its incorporation into the DNA of these and Ehrlich ascites tumour cells. The leukaemic cells were labelled in-vivo by injecting the TH3 into the jugular vein. The dose was 1 pc/g/rat. The rate of appearance of the labelled cells in the peripheral blood and in the ascitic tumour of the animal, was estimated. In other experiments the rate of the dilution of the label in the nuclei was evaluated and thus it was possible to estimate the cellular doubling time in the myelocyte population. The dynamics of transfused leukaemia cells were investigated by injecting labelled myelocytes into the jugular vein of normal and leukaemic rats. Their rate of disappearance from the blood was measured. Various organs were examined for the labelled cells and it was found that soon after injection the cells were mainly trapped by the lungs, later by the spleen and to a lesser extent by the liver. After 24 h no labelled cells were detectable in any of the organs. Information was thus obtained on the fate of the leukaemic myelocytes in various organs of the normal and leukaemic animals. In in-vitro experiments, TH3 was added to the cell suspension in a concentration of 1 p.c/ml. In the course of the in-vitro labelling it was observed that the number of labelled cells was 40 times higher than the number of mitoses. (The same was found also after administering the TH3 in-vivo.) The rate of incorporation of the TH3 was established. Concentrations between 0.0036 p mole x 103 and 1.8 μmolex 10-3 were tested. It was found that the per cent of cells incorporating the label is constant for the various concentrations of thymidine. The number of grains per nucleus increased with the increase of the

  6. Bacterial activity and bacterioplankton diversity in the eutrophic River Warnow--direct measurement of bacterial growth efficiency and its effect on carbon utilization.

    Science.gov (United States)

    Warkentin, Mareike; Freese, Heike M; Schumann, Rhena

    2011-01-01

    The influence of bacterial activity and diversity on bacterial growth efficiency was investigated in a flatland river. Eutrophic River Warnow drains predominantly agricultural land and is heavily loaded with nutrients, dissolved and particulate organic matter (DOM and POM), especially humic substances. Although the water column bacterial community consists of many inactive or damaged cells, bacterioplankton sustained a high bacterial secondary production of 0.2-14.5 μg C L(-1) h(-1) and a high DNA synthesis (thymidine uptake) of 6.1-15.5 μg C L(-1) h(-1). The direct and short-term measurement of bacterial respiration (by optodes) revealed high respiration rates especially in summer leading to directly estimated bacterial growth efficiencies (BGE) of 2-28%. These values are compared to calculations based only on bacterial production, which considerably overestimated BGEs. From all these data, River Warnow can be characterized as a strongly remineralizing system. River Warnow was dominated among others by Cytophaga/Flavobacteria and Actinobacteria which are typical for organic rich waters because of their ability to degrade high molecular weight compounds. However, community composition did not significantly affect BGE. PMID:20676625

  7. Primary and Bacterial Secondary Production in a Southwestern Reservoir

    Science.gov (United States)

    Chrzanowski, Thomas H.; Hubbard, James G.

    1988-01-01

    Rates of primary and bacterial secondary production in Lake Arlington, Texas, were determined. The lake is a warm (annual temperature range, 7 to 32°C), shallow, monomictic reservoir with limited macrophyte development in the littoral zone. Samples were collected from six depths within the photic zone from a site located over the deepest portion of the lake. Primary production and bacterial production were calculated from NaH14CO3 and [methyl-3H]thymidine incorporation, respectively. Peak instantaneous production ranged between 14.8 and 220.5 μg of C liter−1 h−1. There were two distinct periods of high rates of production. From May through July, production near the metalimnion exceeded 100 μg of C liter−1 h−1. During holomixis, production throughout the water column was in excess of 100 μg of C liter−1 h−1 and above 150 μg of C liter−1 h−1 near the surface. Annual areal primary production was 588 g of C m−2. Bacterial production was markedly seasonal. Growth rates during late fall through spring were typically around 0.002 h−1, and production rates were typically 5 μg of C liter−1 h−1. Growth rates were higher during warmer parts of the year and reached 0.03 h−1 by August. The maximum instantaneous rate of bacterial production was approximately 45 μg of C liter−1 h−1. Annual areal bacterial production was 125 g of C m−2. Temporal and spatial distributions of bacterial numbers and activities coincided with temporal and spatial distributions of primary production. Areal primary and bacterial secondary production were highly correlated (r = 0.77, n = 15, P < 0.002). PMID:16347577

  8. Radioactive DNA labelling with 3H-thymidine: effects of internal irradiation on cell growth, chromatin structure and gene activity

    International Nuclear Information System (INIS)

    The growth of jungarian hamster fibroblasts 4/21 is inhibited by 3H-thymidine present in a culture medium in concentrations from 18.5 to 740 KBq/ml. As judged from the gradient elution of DNA from isolated nuclei (the nucleoprotein-celite chromatography), DNA fragmentation increases together with the increase in 3H-thynidine concentration and the decrease in the cell growth rate. DNA fragmentation does not activate the family of heat shock genes (hsp70). On the contrary, the hsp70 gene transcription is somewhat inhibited in bith heat shock and non-heat shock conditions even at a concentration of 3H-thymidine of as low as 37 KBq/ml. Hence the 3H labelling of radiosensitive cultured cells can lead to some deviations in cellular processes under study

  9. Syntheses of pyrimidine acyclic nucleoside phosphonates as potent inhibitors of thymidine phosphorylase (PD-ECGF) from SD-lymphoma

    Czech Academy of Sciences Publication Activity Database

    Pomeisl, Karel; Votruba, Ivan; Holý, Antonín; Pohl, Radek

    2007-01-01

    Roč. 26, 8/9 (2007), s. 1025-1028. ISSN 1525-7770. [International Roundtable /17./. Bern, 03.09.2006-07.09.2006] R&D Projects: GA MŠk 1M0508 Grant ostatní: Descartes Prize(XE) HPAW-CT-2002-9001 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * thymidine phosphorylase * pyrimidines * FPMP derivatives * fluorination Subject RIV: CC - Organic Chemistry Impact factor: 0.723, year: 2007

  10. The effect of adriamycin on dentinogenesis and 3H-thymidine incorporation into the enamel organ of the rat incisor

    International Nuclear Information System (INIS)

    The effect of adriamycin (5 mg/kg) on 3H-thymidine incorporation and on dentin formation was studied in rat incisors. Male Sprague Dawley rats received an intravenous injection of adriamycin. Some of these also received a subcutaneous injection of 3H-thymidine at a dose of 2 mCi/kg one day later. One group of control animals received an intravenous injection of a volume of physiological saline equal to that of the adriamycin dose. Another group received physiological saline, and one hour later was given an additional injection of 3H-thymidine at a similar dose as above. All the animals were killed 1 h, 1 d, 4 d, 8 d, 16 d, 28 d, and 32 d after 3H-thymidine treatment. Light microscopy revealed irregular dentin deposits between the mantle and circumpulpal layer of the labial dentin at 16 d. Within these deposits were trapped cells. The latter, through radioautographic labelling, appeared to be cells from the odontoblast layer. Also, the labelling pattern of the enamel organ in both the control and experimental groups indicated that the eruption rate of the tooth was not affected. Serial sectioning and examination of the lingual portion of the incisors at 28 d revealed a lack of dentin formation and a failure in the closure of the apical foramen. Electron microscopic observations showed an irregular and random arrangement of collagen fibers within the deposits of irregular dentin, and the presence of twisted odontoblastic processes. Examination of the lingual surface showed the presence of fibroblasts and collagen fibers bridging the gap that resulted from the failure in dentin formation. These cells, which were similar to periodontal ligament cells, appeared to have arisen from that area. (author)

  11. Influence of concomitant infusion of thymidine and inosine on methotrexate activity in normal and P388-bearing mice

    NARCIS (Netherlands)

    Uitendaal, Martin P.; Schornagel, J.H.; Leyva, A.; Pinedo, H.M.

    1984-01-01

    Combinations of thymidine and inosine (ranging from 0 to 7.5 mg/hr) were co-administered during a 72-hr continuous i.v. infusion of 3 μg/hr methotrexate in normal and P388 solid tumor-bearing DBA/2 mice. Methotrexate alone was lethal to all normal mice. Inosine at 1.0–7.5 mg/hr could reverse toxicit

  12. Sensitivity and specificity of tritiated thymidine incorporation and ELISPOT assays in identifying antigen specific T cell immune responses

    OpenAIRE

    MacLeod Beth; Slota Meredith; dela Rosa Corazon; Goodell Vivian; Disis Mary L

    2007-01-01

    Abstract Background Standardization of cell-based immunologic monitoring is becoming increasingly important as methods for measuring cellular immunity become more complex. We assessed the ability of two commonly used cell-based assays, tritiated thymidine incorporation (proliferation) and IFN-gamma ELISPOT, to predict T cell responses to HER-2/neu, tetanus toxoid (tt), and cytomegalovirus (CMV) antigens. These antigens were determined to be low (HER-2/neu), moderate (tt), and robustly (CMV) i...

  13. Labeling of thymidine analog with an organometallic complex of technetium-99m for diagnostic of cancer: radiochemical and biological evaluation

    International Nuclear Information System (INIS)

    Thymidine analogs have been labeled with different radioisotopes due to their potential in monitoring the uncontrollable cell proliferation. Considering that the radioisotopes technetium-99m still keep a privileged position as a marker due to its chemical and nuclear properties, this dissertation was constituted by the developed of a new technique of labeling of thymidine analog with 99mTc, by means of the organometallic complex. The aims of this research were: synthesis of the organometallic complex technetium-99m-carbonyl, thymidine labeling with this precursor, evaluation of stability, and radiochemical e biological evaluation with healthy and tumor-bearing animals. The preparation of the organometallic precursor, using the CO gas, was easily achieved, as well as the labeling of thymidine with this precursor, resulting itself a radiochemical pureness of ≥ 97% and ≥ 94%, respectively. Chromatography systems with good levels of trustworthiness were used, ensuring the qualification and quantification of the radiochemical samples. The result of in vitro testing of lipophilicity disclosed that the radiolabeled complex is hydrophilic, with a partition coefficient (log P) of -1.48. The precursor complex and the radiolabeled have good radiochemical stability up to 6 h in room temperature. The cysteine and histidine challenge indicated losses between 8 and 1 1 % for concentrations until 300 mM. The biodistribution assay in healthy mice revealed rapid blood clearance and low uptake by general organs with renal and hepatobiliary excretion. The tumor concentration was low with values of 0.28 and 0.18 %ID/g for lung and breast cancer, respectively. The results imply more studies in other tumor models or the modification of the structure of the organic molecule that act like ligand. (author)

  14. Somatostatin analog (SMS 201-995) inhibits the basal and angiotensin II-stimulated 3H-thymidine uptake by rat adrenal glands

    International Nuclear Information System (INIS)

    The effects of a long-acting somatostatin analog SMS 201-995 injections on the basal and angiotensin II-stimulated [3H]-thymidine uptake by the rat adrenal glands incubated in vitro were examined. It was shown that SMS 201-995 significantly inhibited the [3H]-thymidine uptake and, additionally, suppressed the stimulatory effect of a single angiotensin II injection

  15. 3H-radioactivity measurement in the rat kidney after single injection of folic acid and continuous infusion of 3H-Thymidine

    International Nuclear Information System (INIS)

    Male Sprague-Dawley rats received a single i.v. injection of folic acid. Immediately afterwards, continuous intravenous infusion of 3H-Thymidine has been started. The animals have been sacrified after 0.5 up to 10.0 days for determination of the vital weight of the kidneys, of the wet weight of kidney sections (thickness 20 μ) after removal of the paraffine coat, and of the radioactivity per dry weight unit of the kidney in the 20 μ kidney section after residue-free incineration of tissue. The radioactivity per total kidney and the percentage of 3H-Thymidine radioactivity utilized by the whole kidney in relation to the infused 3H-Thymidine radioactivity have been calculated. The various data have been compared with results obtained by autoradiographic investigations on the same model under fully identical conditions. Excellent agreement has been found between the autoradiographically obtained curves of the 3H-Thymidine labelling indices in the epithelium and in the mesenchyma of the kidney on the one hand and the curves of the 3H-Radioactivity per dry weight unit of the kidney on the other. The method of sample preparation applied largely excludes unwanted 'radioactive contamination', so that the 3H-Thymidine radioactivity measurements in agreement with the autoradiographic data can be assumed to show an incorporation of 3H-Thymidine into newly developed DNA. The percentages of 3H-Thymidine radioactivity utilized by the kidney in relation to the quantity of infused 3H-Thymidine radioactivity are almost constant during folic acid induced proliferation. The strong decline in radioactivity per dry weight unit between 3.0 and 3.5 days coincides with the occurrence of blackened, desquamated tubulus epithelia in the tubular lumen which became necrotic, and with the simultaneous occurrence of radioactively labelled, monocytic cells in the blood. These phenomena are related to the cell loss. (orig./MG)

  16. Radiation-induced binding of 2,2,6,6-tetramethyl-1,4-piperidone-N-oxyl to thymidine in oxygen-free aqueous solutions

    International Nuclear Information System (INIS)

    Steady-state γ-radiolysis of deaerated aqueous solutions of thymidine has been carried out in the presence of 2,2,6,6-tetramethyl-1,4-piperidone-N-oxyl (TAN), a well known radiosensitizing agent. The eight main radiation-induced TAN addition products to thymidine have been isolated and characterized by sup(1)H and sup(13)C nmr, cd, and fast-atom bombardment mass spectrometry measurements

  17. Elevation of radiolabelled thymidine uptake in RIF-1 fibrosarcoma and HT29 colon adenocarcinoma cells after treatment with thymidylate synthase inhibitors

    International Nuclear Information System (INIS)

    We recently showed an increase in tumour uptake of 2-[11C]thymidine in patients with gastrointestinal malignancies after thymidylate synthase (TS) inhibition. To understand the phenomenon in more detail, we investigated whether TS inhibition by different TS inhibitors leads to a dose- and time-dependent change in the uptake of radiolabelled thymidine, and whether radiotracer uptake is related to changes in cell viability resulting from treatment. RIF-1 and HT29 cells were treated with the TS inhibitors 5-fluorouracil (5-FU) and AG337 (nolatrexed dihydrochloride), as well as cisplatin as control. The cell viability and net accumulation of [3H]thymidine after a 1-h pulse was determined at different times after drug treatment. In both cell lines, [3H]thymidine uptake increased after a 2-h treatment with 5-FU, in a dose- and time-dependent manner. [3H]thymidine uptake decreased at 24 and 48 h post treatment. AG337 also produced a similar effect. In contrast to the TS inhibitors, cisplatin decreased [3H]thymidine uptake in RIF-1 and HT29 cells at all time points. Cell viability was compromised only after 24 h. Using two types of TS inhibitor, we have shown an increase in [3H]thymidine uptake, in a dose-dependent manner, a few hours after TS inhibition when the cell viability was not compromised. This effect was not seen with a non-TS inhibitor. These findings suggest that 2-[11C]thymidine positron emission tomography can be used to study TS inhibition in vivo at early time points when cell viability is not compromised and may therefore be helpful in the development of new TS inhibitors and in differentiating between patients with tumours sensitive to TS inhibitors and those unlikely to respond. (orig.)

  18. JBP1 and JBP2 are two distinct thymidine hydroxylases involved in J biosynthesis in genomic DNA of African trypanosomes.

    Science.gov (United States)

    Cliffe, Laura J; Kieft, Rudo; Southern, Timothy; Birkeland, Shanda R; Marshall, Marion; Sweeney, Kate; Sabatini, Robert

    2009-04-01

    Genomic DNA of African trypanosomes contains a hypermodified thymidine residue termed base J (beta-d-glucosyl-HOMedU). This modified base is localized primarily to repetitive DNA, namely the telomeres, and is implicated in the regulation of antigenic variation. The base is synthesized in a two-step pathway. Initially, a thymidine residue in DNA is hydroxylated by a thymidine hydroxylase (TH). This intermediate (HOMedU) is then glucosylated to form base J. Two proteins involved in J synthesis, JBP1 (J binding protein 1) and JBP2, contain a putative TH domain related to the family of Fe(2+)/2-oxoglutarate-dependent hydroxylases. We have previously shown that mutations in the TH domain of JBP1 kill its ability to stimulate J synthesis. Here we show that mutation of key residues in the TH domain of JBP2 ablate its ability to induce de novo J synthesis. While the individual JBP1 null and JBP2 null trypanosomes have reduced J levels, the deletion of both JBP1 and JBP2 generates a cell line that completely lacks base J but still contains glucosyl-transferase activity. Reintroduction of JBP2 in the J-null trypanosome stimulates HOMedU formation and site-specific synthesis of base J. We conclude that JBP2 and JBP1 are the TH enzymes involved in J biosynthesis. PMID:19136460

  19. Thymidine 5'-O-monophosphorothioate induces HeLa cell migration by activation of the P2Y6 receptor.

    Science.gov (United States)

    Gendaszewska-Darmach, Edyta; Szustak, Marcin

    2016-06-01

    ATP, ADP, UTP, and UDP acting as ligands of specific P2Y receptors activate intracellular signaling cascades to regulate a variety of cellular processes, including proliferation, migration, differentiation, and cell death. Contrary to a widely held opinion, we show here that nucleoside 5'-O-monophosphorothioate analogs, containing a sulfur atom in a place of one nonbridging oxygen atom in a phosphate group, act as ligands for selected P2Y subtypes. We pay particular attention to the unique activity of thymidine 5'-O-monophosphorothioate (TMPS) which acts as a specific partial agonist of the P2Y6 receptor (P2Y6R). We also collected evidence for the involvement of the P2Y6 receptor in human epithelial adenocarcinoma cell line (HeLa) cell migration induced by thymidine 5'-O-monophosphorothioate analog. The stimulatory effect of TMPS was abolished by siRNA-mediated P2Y6 knockdown and diisothiocyanate derivative MRS 2578, a selective antagonist of the P2Y6R. Our results indicate for the first time that increased stability of thymidine 5'-O-monophosphorothioate as well as its affinity toward the P2Y6R may be responsible for some long-term effects mediated by this receptor. PMID:26746211

  20. Radiation-induced inhibition of thymidine incorporation in vivo as a measure of the initial slope and RBEn/γ

    International Nuclear Information System (INIS)

    Radiation damage can be measured by decreased incorporation of 3H-TdR. The early effect of total body irradiation of mice, with doses up to 300-400 rad, of gamma rays or neutrons, on thymidine-3H incorporation into the DNA of murine proliferating normal and tumor cells are described. Total body irradiation with single doses up to 300 rad resulted in a steep dose-dependent depression of 3H-TdR incorporation into the DNA of the jejunal crypt, testis, spleen, fibrosarcoma (FSa), and FSa metastasis cells. The dose required to depress 3H-TdR incorporation values to 37% of control level (D37/thymidine) after γ-irradiation was calculated to be 405, 443, 72, 303, and 531 rad, for jejunal crypt, testis, spleen, FSa metastasis, and FSa tumor cells, respectively. The depression progressed during the first 3 hours after irradiation. After neutron irradiation, the D37/thymidine was calculated to be 81, 140, 35, and 155 rad for jejunal crypt, testis, spleen, and FSa metastasis cells, respectively. The RBEn/γ derived from these results were 5.00, 3.16, 2.06, and 1.95 for jejunal crypt, testis, spleen, and FSa metastasis cells, respectively. These findings show that cell survival after small doses of irradiation correlate with the effect of irradiation on the actively proliferating cells at the time of irradiation

  1. Thymidine radical formation via one-electron transfer oxidation photoinduced by pterin: Mechanism and products characterization.

    Science.gov (United States)

    Serrano, Mariana P; Vignoni, Mariana; Lorente, Carolina; Vicendo, Patricia; Oliveros, Esther; Thomas, Andrés H

    2016-07-01

    UV-A radiation (320-400nm), recognized as a class I carcinogen, induces damage to the DNA molecule and its components through different mechanisms. Pterin derivatives are involved in various biological functions, including enzymatic processes, and it has been demonstrated that oxidized pterins may act as photosensitizers. In particular, they accumulate in the skin of patients suffering from vitiligo, a chronic depigmentation disorder. We have investigated the ability of pterin (Ptr), the parent compound of oxidized pterins, to photosensitize the degradation of the pyrimidine nucleotide thymidine 5'-monophosphate (dTMP) in aqueous solutions under UV-A irradiation. Although thymine is less reactive than purine nucleobases, our results showed that Ptr is able to photoinduce the degradation of dTMP and that the process is initiated by an electron transfer from the nucleotide to the triplet excited state of Ptr. In the presence of molecular oxygen, the photochemical process leads to the oxidation of dTMP, whereas Ptr is not consumed. In the absence of oxygen, both compounds are consumed to yield a product in which the pterin moiety is covalently linked to the thymine. This compound retains some of the spectroscopic properties of Ptr, such as absorbance in the UV-A region and fluorescence properties. PMID:27154982

  2. Tritiated thymidine and deoxycytidine suicide of mouse hemopoietic colony forming cells (CFC)

    International Nuclear Information System (INIS)

    Significant enhancement of tritiated dCyd suicide occurred when unlabelled dThd was added to cultures of mouse monocytic colony-forming cells. Incorporation experiments supported the suicide experiments in that incorporation of tritiated dCyd into DNA was significantly increased. One hundred micromolar dCyd significantly reduced the radiotoxicity of 0.3 μCi of tritiated dThd; incorporation experiments indicated a dose-related reduction in the incorporation of tritiated dThd into DNA with the addition of 1-100 μM unlabelled dCyd. The addition of 1 μM aminopterin reversed the effect of 100 μM deoxycytidine; viz., incorporation of dThd into DNA was 90% of controls. Aminopterin had a similar effect on deoxyuridine reversal of tritiated dThd incorporation into DNA. Aminopterin had no effect on the reduction of tritiated dThd incorporation into DNA due to the addition of 100 μM unlabelled thymidine. Unlabelled ribonucleosides, Urd and Cyd, did not significantly affect the suicide pattern of tritiated dThd or dCyd when they were added to CFC cultures. Unlabelled deoxyribonucleosides, dThd or dCyd, did not significantly affect the suicide pattern of either tritiated Cyd or Urd when they were added to cultures containing tritiated ribonucleosides. Unlabelled Urd or Cyd was effective in reversing the suicide due to tritiated Urd or Cyd. (author)

  3. Local Treatment of Hand-Foot Syndrome with Uridine/Thymidine: In Vitro Appraisal on a Human Keratinocyte Cell Line HaCaT

    Directory of Open Access Journals (Sweden)

    J. Hartinger

    2012-01-01

    Full Text Available 5-fluorouracil (5-FU is one of the most commonly used antineoplastic drugs in the anticancer therapy. The hand-foot (HF syndrome (palmar-plantar erythrodysesthesia is an adverse effect frequently related to long-term i.v. administration of 5-FU or its orally applicable prodrug capecitabine. Its severity can even lead to interruption of the otherwise effective anticancer therapy. Tentative practice in some clinics has shown that topical application of 10% uridine ointment is beneficial for calming down the HF syndrome. This study is focused on verifying the alleged protective activity of uridine in the in vitro model of cultured human keratinocyte cell line HaCaT. We also tested the protective effects of thymidine alone or uridine-thymidine combination. The cellular viability time progression was measured in order to evaluate the effect of protective agents by three different types of cytopathogenicity tests—NTCA test (non-destructive test of cellular activity, modified MTT test and RTCA (real-time cell analyser, Roche. All three methods proved the ability of uridine and uridine-thymidine combination to protect keratinocytes against 5-FU damage in vitro. While thymidine alone did not show any remarkable effect, the thymidine-uridine combination demonstrated enhanced protective activity compared to uridine alone. Our findings provided the supporting rationale for using uridine or uridine-thymidine ointments in the HF syndrome local therapy.

  4. Incorporation of thymidine into onion root meristematic cell nuclei in presence of hydroxyurea and its role in recovery of mitotic activity

    International Nuclear Information System (INIS)

    Hydroxyurea treatment of onion roots induced mitotic block which was released by transfer of bulbs to water, and also to some extent by addition of cold or 3H-thymidine to hydroxyurea solutions. In presence of hydroxyurea there was noted very intense incorporation of 3H-thymidine into cell nuclei, giving labelling index of 40-70%. However, all the mitotic figures appearing in presence of hydroxyurea and 3H-thymidine were unlabelled. On the other hand, labelled mitotic figures were obtained when roots incubated with 3H-thymidine in presence of hydroxyurea had been transferred to water. Incorporation of 3H-uridine was unaffected by hydroxyurea. The results show that hydroxyurea arrests onion root meristematic cells, either in the S phase and the G2 phase. Enhanced incorporation of 3H-thymidine in the presence of hydroxyurea, and release by added thymidine of the mitotic block indicate that hydroxyurea induces in onion root meristematic cells a particular shortage of thymidylate. (author)

  5. Autoradiography of 3H- and 14C-Thymidine in Bone Marrow by a Double-Emulsion Technique

    International Nuclear Information System (INIS)

    In the present work a new method has been developed to study the generation time of bone- marrow cells in cultures, after double labelling with 3H- and 14C-thymidine, by means of a double-emulsion autoradiographic technique. In such an investigation it is not practicable to use a single thick layer of emulsion since this prevents satisfactory staining of the bone marrow cells with Giemsa stain. If the specimen is stained before making the autoradiograph it must be covered with a protective layer to retain the stain and this absorbs a large proportion of the 3H-electrons. The technique adopted is a modification of the method described by Baserga and Nemeroff. The bone-marrow smear is covered with a very thin layer of the fine-grain Gevaert emulsion NUC 715. After an exposure suitable for the 3H-thymidine (1-5 d)the emulsion is processed and the bone marrow can be stained adequately through the thin emulsion. It is then dipped in a 7% solution of celloidin in amyl acetate to protect the stain during subsequent procedures (celloidin in ether-alcohol mixture removes the stain). The specimen is then dipped in Kodak NTB-2 emulsion having a larger grain size than the first emulsion, and exposed to 14C-electrons (2-6 weeks). The second emulsion can then be processed without affecting the staining of the bone-marrow smear. The celloidin layer is sufficiently thick to absorb all 3H-electrons so that an increase in grain density in the second emulsion is due to 14C electrons only. Moreover the celloidin layer still permits good autoradiographic resolution in the second emulsion. Autoradiographs in the first and second emulsion are easily recognised by the difference in grain size. It is thus possible to distinguish between cells containing only 3H-thymidine and only 14C-thymidine or both 3H- and 14C-thymidine. The interpretation of the results will be discussed. (author)

  6. From Phosphosites to Kinases

    DEFF Research Database (Denmark)

    Munk, Stephanie; Refsgaard, Jan C; Olsen, Jesper V;

    2016-01-01

    Kinases play a pivotal role in propagating the phosphorylation-mediated signaling networks in living cells. With the overwhelming quantities of phosphoproteomics data being generated, the number of identified phosphorylation sites (phosphosites) is ever increasing. Often, proteomics investigations...... sequence motifs, mostly based on large scale in vivo and in vitro experiments. The context of the kinase and the phosphorylated proteins in a biological system is equally important for predicting association between the enzymes and substrates, an aspect that is also being tackled with available...

  7. Phosphatidylinositol kinase from rabbit reticulocytes

    International Nuclear Information System (INIS)

    Phosphatidylinositol (PI) kinase was isolated from the postribosomal supernatant of rabbit reticulocytes. This activity was identified by the formation of a product that comigrated with phosphatidylinositol-4-phosphate (PIP) when purified PI was phosphorylated in the presence of [32P]ATP and Mg2+. Three major peaks of PI kinase activity were resolved by chromatography on DEAE-cellulose. The first peak eluted at 50-100 mM NaCl together with several serine protein kinases, casein kinase (CK) I and protease activated kinase (PAK) I and II. The PI kinase was subsequently separated from the protein kinases by chromatography on phosphocellulose. The second peak eluted at 125-160 mM NaCl and contained another lipid kinase activity that produced a product which comigrated with phosphatidic acid on thin layer chromatography. The third peak, which eluted at 165-200 mM NaCl, partly comigrated with casein kinase (CK) II and an active protein kinase(s) which phosphorylated mixed histone and histone I. CK II and the histone kinase activities were also separated by chromatography on phosphocelluslose. The different forms of PI kinase were characterized and compared with respect to substrate and salt requirements

  8. Heterotrophic bacterial production: Relationships to biological and abiological factors in estuarine environments

    International Nuclear Information System (INIS)

    Ecotoxicological effects of creosote contamination on benthic bacterial communities in the Elizabeth River, Virginia were investigated using both structural an functional microbial parameters. Results indicated that cell specific and total heterotrophic bacterial production parameters were depressed in a dose-dependent manner with increasing sediment PAH concentrations. Toxicity effects upon production were modified by temporal trends associated with temperature as well as spatial sediment characteristics. Of the parameters employed, the tritiated thymidine production assay was found to be the most sensitive for detection of ecotoxicological effects. Bacterial abundance and production were examined during a destratification event in the lower James River, Virginia. Bacterial abundance, although significantly different between stations, did not change over the study. Bacterial production (3H-Tdr incorporation) in surface waters was significantly less during the mixed period 187 μg C·1-1· d-1 compared to the most stratified state (324μg C·1-1· d-1). Correlations between bacteria and chlorophyll were diminished during the mixed period. Total and flagellate specific grazing rates upon bacteria were reduced during the onset of destratification. Relationships between bacterial and nutrient parameters also indicated a strong influence of destratification. These results indicate that destratification changes trophic interactions within the microbial loop, which are not necessarily reflected in temporal patterns of bacterial abundance. Bacterioplankton production, and ammonium assimilation and remineralization were examined between April and August 1988 in the lower York River, Va

  9. Recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus Ankara (MVA)

    DEFF Research Database (Denmark)

    Cottingham, Matthew G; Andersen, Rikke F; Spencer, Alexandra J;

    2008-01-01

    infectious virus using a Fowlpox virus helper to supply transcriptional machinery. We apply here a similar approach to the attenuated strain Modified Vaccinia virus Ankara (MVA), now widely used as a safe non-replicating recombinant vaccine vector in mammals, including humans. Four apparently full...... using GalK counterselection to insert an antigen expression cassette lacking a tandem marker gene into the traditional thymidine kinase locus of MVA-BAC. MVA continues to feature prominently in clinical trials of recombinant vaccines against diseases such as HIV-AIDS, malaria and tuberculosis. Here we...

  10. Kinase Inhibitors from Marine Sponges

    Directory of Open Access Journals (Sweden)

    Ana Zivanovic

    2011-10-01

    Full Text Available Protein kinases play a critical role in cell regulation and their deregulation is a contributing factor in an increasing list of diseases including cancer. Marine sponges have yielded over 70 novel compounds to date that exhibit significant inhibitory activity towards a range of protein kinases. These compounds, which belong to diverse structural classes, are reviewed herein, and ordered based upon the kinase that they inhibit. Relevant synthetic studies on the marine natural product kinase inhibitors have also been included.

  11. Time-dependent labelling course of human eosinophilic granulocytes after 3H thymidine application

    International Nuclear Information System (INIS)

    After intravenous injection of 0.1 μCi/g body weight 3H-Thymidine and taking of blood samples in intervals of 6-12 hrs. on three test persons with healthy blood, the labelling course of the eosinophilic granulocytes was studied. The cells were classified in four groups, according to the relative frequency of the different degrees of labelling. The time-dependent labelling index curves showed a nawe-sheped course. Elimination of the eosinophilics from the blood is carried out according to the 'At-random'-principle. 12 hrs. p.i. already 10% of the eosinophilics in the blood were labelled with maximally 5 grains. The cell flow-in phase of 13 hrs. was succeeded by a flow-out phase of nearly the same duration, afthr the first labelling maximum of 17%. 80 hrs. p.i. the first massive in-flow of high-labelled cells containing more than 30 grains. After reaching the labelling maximum of 58%, the labelling index values decreased continuously. Until the 11th day p.i., appr. 50% of the eosinophilics were still labelled, after 17 days appr. 25%, more than 65% of which consisted of cells with only 2-4 grains. Comparison of the labelling index curves of the grain groups with each other shows at first a synchronous, then an increasingly asynchronous course, according to the desynchronization of the several eosinophilic generation cycles in the bone marrow which gets more significant in the course of time. (orig.)

  12. Stability of RNA and DNA in Bone Marrow Cells, Demonstrated with Tritiated Cytidine and Thymidine

    International Nuclear Information System (INIS)

    DNA and RNA metabolism was studied using tritiated thymidine (H3Th), a specific precursor for DNA, and tritiated cytidine (H3C), a common precursor for both RNA and DNA. With H3C, differential incorporation into RNA, DNA or the soluble pool was determined autoradiographically in the single cell, and/or chemically for cell populations by means of differential extraction using appropriate treatment with perchloric acid. Initial turnover studies in the Hela cell with H3C indicated the precursor role of nuclear RNA for cytoplasmic RNA. Conservation and distribution of label in the RNA fraction was consistent with major macromolecular RNA stability, and continued incorporation of label into the DNA fraction was consistent with the presence of a late precursor for DNA. Similar findings were observed in the immature bone marrow cells of the rat studied over a period of several days after intravenous administration of H3C. The amount of tritium activity in the acid-soluble' RNA and DNA fractions was followed chemically and/or autoradiographically. The three curves were found to be parallel from the first day after injection and parallel to curves for tritium label in DNA following H3Th administration. The expected rate of fall off in label, calculated from kinetics of the rat bone marrow cell populations studied separately by H3Th and autoradiography, assuming no turnover of RNA or DNA and loss of label only by loss of marrow cells by division and maturation, was in agreement with the slopes obtained. The results indicate that, once synthesized, soluble and macromolecular RNA is retained by the bone marrow cell in a manner similar to DNA. Newly formed RNA and DNA are diluted in the cells only through cell division. (author)

  13. Early assessment of therapy response in malignant lymphoma with the thymidine analogue [18F]FLT

    International Nuclear Information System (INIS)

    The aim of this study was to determine whether the thymidine analogue 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) is adequate for early evaluation of the response of malignant lymphoma to antiproliferative treatment in a mouse xenotransplant model. Immunodeficient mice bearing a follicular lymphoma xenotransplant were treated with high-dose chemotherapy (cyclophosphamide, n 10), immunotherapy (CD20 mAb, ibritumomab-tiuxetan, n = 10) or radioimmunotherapy ([90Y]CD20 mAb, Zevalin, n = 10). Forty-eight hours after treatment, antiproliferative effects were assessed with [18F]FLT. Ninety minutes after i.v. injection of 5-10 MBq [18F]FLT, mice were sacrificed and radioactivity within the tumour and normal organs was measured using a gamma counter and calculated as % ID/g. The proliferation fraction in tissue samples derived from treated and untreated tumours was evaluated by Ki-67 immunohistochemistry, which served as the reference for proliferative activity. In untreated lymphoma, the mean proliferation fraction was 83.6%. After chemotherapy, the mean proliferation fraction decreased to 39.3% (p = 0.0001), after immunotherapy to 77.6% (p = 0.0078) and after radioimmunotherapy to 78.8% (p = 0.014). In none of the animals was a significant change in tumour size observed. In untreated lymphoma, tumoural [18F]FLT uptake was 5.4% ID/g, after chemotherapy it was 1.5% (p = 0.0005), after immunotherapy, 3.9% (non-significant), and after radioimmunotherapy, 5.8% (non-significant). In a lymphoma xenotransplant model, [18F]FLT detects early antiproliferative drug activity before changes in tumour size are visible. These findings further support the use of [18F]FLT-PET for imaging early response to treatment in malignant lymphoma. (orig.)

  14. Tritiated thymidine-labeled bronchioloalveolar cells and radiation dose following inhalation of plutonium in rats

    International Nuclear Information System (INIS)

    The goal of this study is to show the relationship of inhaled Pu particle distribution and alveolar-bronchiolar target-cell response with respect to the formation of pulmonary carcinoma. The proliferation of type 2 alveolar epithelium and nonciliated bronchiolar epithelium appears critical in the induction of lung tumors associated from inhaled 239PuO2. Female, Wistar rats were either sham-exposed (40 rats) or given a single inhalation to 169Yb-239PuO2 (99 rats, ILB, 3.9 +/- 1.2 kBq) and examined at 20 time intervals from 1 day to 700 days postexposure for Pu particle distribution in airways by SEM quantitative autoradiography and for cell labeling with tritiated thymidine. Initially, deposited Pu particles were rapidly cleared from the surface of the trachea, bronchi, and bronchioles within a few days. Thereafter, about 5 times more alpha track exposure to the bronchiolar epithelium was delivered from Pu particles found in peribronchiolar alveoli than from Pu particles being cleared from bronchiolar surfaces. Exposure of bronchiolar epithelium at later times was due mostly to the formation of peribronchiolar Pu particle aggregates. A maximal increase in labeled alveolar wall cells was seen at 60 days after exposure, decreasing gradually to control levels by 400 days. Cell labeling in focal alveolar regions of Pu aggregation was about 5 fold higher. Increased bronchiolar epithelium labeling appeared in two phases. The first phase was seen 15 days after exposure, associated with initial deposition and clearance of Pu particles. The second phase slowly reached a maximum at 250 days and was associated with peribronchiolar Pu aggregate formation. The temporal-spatial dose-distribution pattern for inhaled Pu particles is an important aspect of Pu-induced pulmonary carcinogenesis

  15. Solution-state structure of the Dewar pyrimidinone photoproduct of thymidylyl-(3'→5')-thymidine

    International Nuclear Information System (INIS)

    The preparation, spectroscopic investigation, structure determination, conformational analysis, and modeling of the Dewar pyrimidinone photoproduct of thymidylyl-(3'→5')-thymidine, previously referred to as TpT3 is described. TpT3 was prepared in quantitative yield by photolysis of an aqueous solution of the (6-4) photoproduct of TpT with Pyrex-filtered medium-pressure mercury arc light. TpT3 was analyzed by FAB MS, IR, UV, and 1H, 13C, and 31P NMR spectroscopy. The spectroscopic data led to the conclusion that TpT3 results from the photoisomerization of the pyrimidinone ring of the (6-4) product of TpT to its Dewar valence isomer. Torsion angle and interproton distance information derived from coupling constants and NOE data was used to constrain ring conformation searches by utilizing the SYBYL molecular modeling program subroutine SEARCH. Sets of angles derived from the ring search procedure were then used to construct structures whose geometries were optimized by the energy-minimization subroutine MAXIMIN. A two-state model for the solution-state structure of the Dewar photoproduct was chosen which was energetically sound, fit the experimental coupling constants with an RMS deviation of 1.15 Hz, and was consistent with the NOE data. The model for the Dewar photoproduct was compared to a model for the (6-4) photoproduct and the TpT subunits of the Dickerson dodecamer structure by a least-squares fitting procedure. It was concluded that the Dewar photoproduct more closely resembles a B-form TpT unit than does the (6-4) photoproduct

  16. The partial molar heat capacity, expansion, isentropic, and isothermal compressions of thymidine in aqueous solution at T = 298.15 K

    Energy Technology Data Exchange (ETDEWEB)

    Hedwig, Gavin R., E-mail: G.Hedwig@massey.ac.nz [Institute of Fundamental Sciences-Chemistry, Massey University, Private Bag 11222, Palmerston North (New Zealand); Jameson, Geoffrey B. [Institute of Fundamental Sciences-Chemistry, Massey University, Private Bag 11222, Palmerston North (New Zealand); Hoiland, Harald [Department of Chemistry, University of Bergen, N-5020 Bergen (Norway)

    2011-12-15

    Highlights: > Solution densities and sound speeds were measured for aqueous solutions of thymidine. > Partial molar volumetric properties at infinite dilution and T = 298.15 K were derived. > The partial molar isentropic and isothermal compressions are of opposite signs. > The partial molar heat capacity for thymidine at infinite dilution was determined. - Abstract: Solution densities have been determined for aqueous solutions of thymidine at T = (288.15, 298.15, 303.15, and 313.15) K. The partial molar volumes at infinite dilution, V{sub 2}{sup 0}, obtained from the density data were used to derive the partial molar isobaric expansion at infinite dilution for thymidine at T = 298.15 K, E{sub 2}{sup 0}{l_brace}E{sub 2}{sup 0}=({partial_derivative}V{sub 2}{sup 0}/{partial_derivative}T){sub p}{r_brace}. The partial molar heat capacity at infinite dilution for thymidine, C{sub p,2}{sup 0}, at T = 298.15 K has also been determined. Sound speeds have been measured for aqueous solutions of thymidine at T = 298.15 K. The partial molar isentropic compression at infinite dilution, K{sub S,2}{sup 0}, and the partial molar isothermal compression at infinite dilution, K{sub T,2}{sup 0}{l_brace}K{sub T,2}{sup 0}=-({partial_derivative}V{sub 2}{sup 0}/{partial_derivative}P){sub T}{r_brace}, have been derived from the sound speed data. The V{sub 2}{sup 0}, E{sub 2}{sup 0}, C{sub p,2}{sup 0}, and K{sub S,2}{sup 0} results for thymidine are critically compared with those available from the literature.

  17. Biochemical and structural characterization of polyphosphate kinase 2 from the intracellular pathogen Francisella tularensis

    OpenAIRE

    Batten, Laura E.; Parnell, Alice E.; Wells, Neil J.; Amber L Murch; Oyston, Petra C. F.; Roach, Peter L.

    2016-01-01

    The metabolism of polyphosphate is important for the virulence of a wide range of pathogenic bacteria and the enzymes of polyphosphate metabolism have been proposed as an anti-bacterial target. In the intracellular pathogen Francisella tularensis, the product of the gene FTT1564 has been identified as a polyphosphate kinase from the polyphosphate kinase 2 (PPK2) family. The isogenic deletion mutant was defective for intracellular growth in macrophages and was attenuated in mice, indicating an...

  18. Listeria monocytogenes, an invasive bacterium, stimulates MAP kinase upon attachment to epithelial cells.

    OpenAIRE

    Tang, P.; Rosenshine, I.; Finlay, B B

    1994-01-01

    Protein tyrosine phosphorylation is an important regulatory mechanism for many cellular processes in eucaryotic cells. During the invasion of the gram-positive pathogen, Listeria monocytogenes, into host epithelial cells, two host proteins become tyrosine phosphorylated. We have identified these major tyrosine phosphorylated species to be two isoforms of mitogen-activated protein (MAP) kinase, the 42 and 44 kDa MAP kinases. This activation begins within 5 to 15 min of bacterial infection. The...

  19. Bacterial Nail Infection (Paronychia)

    Science.gov (United States)

    ... of nail infection is often caused by a bacterial infection but may also be caused by herpes, a ... to a type of yeast called Candida , or bacterial infection, and this may lead to abnormal nail growth. ...

  20. Tyrosine kinases in rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Kobayashi Akiko

    2011-08-01

    Full Text Available Abstract Rheumatoid arthritis (RA is an inflammatory, polyarticular joint disease. A number of cellular responses are involved in the pathogenesis of rheumatoid arthritis, including activation of inflammatory cells and cytokine expression. The cellular responses involved in each of these processes depends on the specific signaling pathways that are activated; many of which include protein tyrosine kinases. These pathways include the mitogen-activated protein kinase pathway, Janus kinases/signal transducers and activators transcription pathway, spleen tyrosine kinase signaling, and the nuclear factor κ-light-chain-enhancer of activated B cells pathway. Many drugs are in development to target tyrosine kinases for the treatment of RA. Based on the number of recently published studies, this manuscript reviews the role of tyrosine kinases in the pathogenesis of RA and the potential role of kinase inhibitors as new therapeutic strategies of RA.

  1. Prevention of bacterial adhesion

    DEFF Research Database (Denmark)

    Klemm, Per; Vejborg, Rebecca Munk; Hancock, Viktoria

    2010-01-01

    Management of bacterial infections is becoming increasingly difficult due to the emergence and increasing prevalence of bacterial pathogens that are resistant to available antibiotics. Conventional antibiotics generally kill bacteria by interfering with vital cellular functions, an approach that....... As such, adhesion represents the Achilles heel of crucial pathogenic functions. It follows that interference with adhesion can reduce bacterial virulence. Here, we illustrate this important topic with examples of techniques being developed that can inhibit bacterial adhesion. Some of these will...

  2. A study of bacterial gene regulatory mechanisms

    DEFF Research Database (Denmark)

    Hansen, Sabine

    GRNs this thesis also provided the first evidence of the sensor histidine kinase VC1831 being an additional player in the Vibrio cholerae quorum sensing (QS) GRN. Bacteria use a process of cell-cell communication called QS which enable the bacterial cells to collectively control their gene expression...... using small signaling molecules called autoinducers, thereby coordinating group behavior. At the heart of the V. cholerae QS response lie four small RNA (sRNA) molecules called the quorum regulatory RNAs (Qrr). This PhD thesis provides evidence that the sensor histidine kinase VC1831 is regulated by the...... Qrr sRNAs. It is further shown that VC1831 feeds back to activate the expression the Qrrs, presumably via phosphorylation of LuxU. Thus, VC1831, which responds to an unknown ligand, is a new player in the V. cholerae QS response. Prior to this report, the two autoinducer sensors CqsS and LuxQ were the...

  3. Inhibition of human thymidine phosphorylase by phosphonate analogues of pyrimidine nucleoside 2’(3’)-phosphates

    Czech Academy of Sciences Publication Activity Database

    Panova, Natalya; Kóšiová, Ivana; Šimák, Ondřej; Petrová, Magdalena; Liboska, Radek; Buděšínský, Miloš; Rejman, Dominik; Rosenberg, Ivan

    Lyon : Université de Lyon, 2010, s. 336-337. [International Roundtable on Nucleosides, Nucleotides and Nucleic Acids. IRT 2010. Lyon (FR), 29.08.2010-03.09.2010] R&D Projects: GA MŠk(CZ) LC06077; GA AV ČR KAN200520801; GA MŠk(CZ) LC06061 Institutional research plan: CEZ:AV0Z40550506 Keywords : human thymidine phosphorylase * phosphorylase inhibition * phosphonate nucleosides Subject RIV: CC - Organic Chemistry http://irt2010.univ-lyon1.fr

  4. Inhibition of human thymidine phosphorylase by conformationally constrained pyrimidine nucleoside phosphonic acids and their "open-structure" isosteres

    Czech Academy of Sciences Publication Activity Database

    Kóšiová, Ivana; Šimák, Ondřej; Panova, Natalya; Buděšínský, Miloš; Petrová, Magdalena; Rejman, Dominik; Liboska, Radek; Páv, Ondřej; Rosenberg, Ivan

    2014-01-01

    Roč. 74, Mar 3 (2014), s. 145-168. ISSN 0223-5234 R&D Projects: GA ČR GA203/09/0820; GA ČR GA202/09/0193; GA ČR GA13-24880S; GA ČR GA13-26526S Institutional support: RVO:61388963 Keywords : phosphonate * conformationally constrained nucleotide analog * human thymidine phosphorylase * PBMC * bi-substrate-like inhibitor * Michael addition Subject RIV: CC - Organic Chemistry Impact factor: 3.447, year: 2014

  5. Thymidine phosphorylase/platelet-derived endothelial cell growth factor expression associated with hepatic metastasis in gastric carcinoma.

    OpenAIRE

    Maeda, K; Chung, Y.S.; Ogawa, Y; Takatsuka, S.; Kang, S. M.; Ogawa, M; Sawada, T.; Onoda, N.; Kato, Y; Sowa, M

    1996-01-01

    It is known that angiogenesis plays an important role in the growth and metastasis of solid tumours. Several angiogenic factors have been identified and platelet-derived endothelial cell growth factor (PD-ECGF) is thought to be one such factor. Recently, it was reported that thymidine phosphorylase (dThdPase) is identical to PD-ECGF. Using immunohistochemical staining with an anti-dThdPase antibody, we investigated the correlation between dThdPase expression and the microvessel density in 120...

  6. Relationship between the Expression of Thymidylate Synthase,Thymidine Phosphorylase and Dihydropyrimidine Dehydrogenase and Survival in Epithelial Ovarian Cancer

    Institute of Scientific and Technical Information of China (English)

    王常玉; 翁艳洁; 王鸿雁; 石英; 马丁

    2010-01-01

    The mRNA and protein expression of thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) and their relationship with prognosis were investigated. Real-time quantitative RT-PCR (Taqman) was used to detect the mRNA expression of TS, TP and DPD in formalin-fixed and paraffin-embedded 106 samples of epithelial ovarian cancer and 29 normal ovaries. A TATA box-binding protein (TBP) was used as an endogenous reference gene. A relationship between TS, TP, DPD expression a...

  7. Lethal and mutagenic effects of tritiated water and incorporated [3H-methyl]-thymidine on extracellular phage lambda

    International Nuclear Information System (INIS)

    A study was made of lethal and mutagenic effects on extracellular phage lam mbda of β-particles from tritiated water and of [ h-methyl]-thymidine incorporated into dna. It was shown that the mean lethal dose d10 and the y yield of c-mutations per unit of the dose absorbed during external β-irradiation in 3h2o or during the decay of incorporated [3H-methyl]-th hymidine were equivalent to those obtained during 60Co-γ-irradiation in 4% nutrient broth or at a dried state respectively

  8. Oncoprotein protein kinase

    Energy Technology Data Exchange (ETDEWEB)

    Karin, Michael (San Diego, CA); Hibi, Masahiko (San Diego, CA); Lin, Anning (La Jolla, CA); Davis, Roger (Princeton, MA); Derijard, Benoit (Shrewsbury, MA)

    2003-02-04

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  9. Kinase-Catalyzed Biotinylation

    OpenAIRE

    Senevirathne, Chamara; Green, Keith D.; Pflum, Mary Kay H.

    2012-01-01

    Kinase-catalyzed protein phosphorylation plays an essential role in a variety of biological processes. Methods to detect phosphoproteins and phosphopeptides in cellular mixtures will aid in cell biological and signaling research. Our laboratory recently discovered the utility of γ-modified ATP analogues as tools for studying phosphorylation. Specifically, ATP-biotin can be used for labeling and visualizing phosphoproteins from cell lysates. Because the biotin tag is suitable for protein detec...

  10. Regulation of Autophagy by Kinases

    International Nuclear Information System (INIS)

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets

  11. Structure of 2-oxo-3-deoxygalactonate kinase from Klebsiella pneumoniae

    International Nuclear Information System (INIS)

    The crystal structure of 2-oxo-3-deoxygalactonate kinase from the De Ley–Doudoroff pathway of galactose metabolism has been determined at 2.1 Å resolution. In most organisms, efficient d-galactose utilization requires the highly conserved Leloir pathway that converts d-galactose to d-glucose 1-phosphate. However, in some bacterial and fungal species alternative routes of d-galactose assimilation have been identified. In the so-called De Ley–Doudoroff pathway, d-galactose is metabolized into pyruvate and d-glyceraldehyde 3-phosphate in five consecutive reactions carried out by specific enzymes. The penultimate step in this pathway involves the phosphorylation of 2-oxo-3-deoxygalactonate to 2-oxo-3-deoxygalactonate 6-phosphate catalyzed by 2-oxo-3-deoxygalactonate kinase, with ATP serving as a phosphoryl-group donor. Here, a crystal structure of 2-oxo-3-deoxygalactonate kinase from Klebsiella pneumoniae determined at 2.1 Å resolution is reported, the first structure of an enzyme from the De Ley–Doudoroff pathway. Structural comparison indicates that the enzyme belongs to the ASKHA (acetate and sugar kinases/hsc70/actin) family of phosphotransferases. The protein is composed of two α/β domains, each of which contains a core common to all family members. Additional elements introduced between conserved structural motifs define the unique features of 2-oxo-3-deoxygalactonate kinase and possibly determine the biological function of the protein

  12. Structure of 2-oxo-3-deoxygalactonate kinase from Klebsiella pneumoniae

    Energy Technology Data Exchange (ETDEWEB)

    Michalska, Karolina [Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory (United States); Cuff, Marianne E. [Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory (United States); Structural Biology Center, Biosciences Division, Argonne National Laboratory (United States); Tesar, Christine; Feldmann, Brian [Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory (United States); Joachimiak, Andrzej, E-mail: andrzejj@anl.gov [Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory (United States); Structural Biology Center, Biosciences Division, Argonne National Laboratory (United States); Department of Biochemistry and Molecular Biology, University of Chicago (United States)

    2011-08-01

    The crystal structure of 2-oxo-3-deoxygalactonate kinase from the De Ley–Doudoroff pathway of galactose metabolism has been determined at 2.1 Å resolution. In most organisms, efficient d-galactose utilization requires the highly conserved Leloir pathway that converts d-galactose to d-glucose 1-phosphate. However, in some bacterial and fungal species alternative routes of d-galactose assimilation have been identified. In the so-called De Ley–Doudoroff pathway, d-galactose is metabolized into pyruvate and d-glyceraldehyde 3-phosphate in five consecutive reactions carried out by specific enzymes. The penultimate step in this pathway involves the phosphorylation of 2-oxo-3-deoxygalactonate to 2-oxo-3-deoxygalactonate 6-phosphate catalyzed by 2-oxo-3-deoxygalactonate kinase, with ATP serving as a phosphoryl-group donor. Here, a crystal structure of 2-oxo-3-deoxygalactonate kinase from Klebsiella pneumoniae determined at 2.1 Å resolution is reported, the first structure of an enzyme from the De Ley–Doudoroff pathway. Structural comparison indicates that the enzyme belongs to the ASKHA (acetate and sugar kinases/hsc70/actin) family of phosphotransferases. The protein is composed of two α/β domains, each of which contains a core common to all family members. Additional elements introduced between conserved structural motifs define the unique features of 2-oxo-3-deoxygalactonate kinase and possibly determine the biological function of the protein.

  13. LTB4 stimulates growth of human pancreatic cancer cells via MAPK and PI-3 kinase pathways

    International Nuclear Information System (INIS)

    We have previously shown the importance of LTB4 in human pancreatic cancer. LTB4 receptor antagonists block growth and induce apoptosis in pancreatic cancer cells both in vitro and in vivo. Therefore, we investigated the effect of LTB4 on proliferation of human pancreatic cancer cells and the mechanisms involved. LTB4 stimulated DNA synthesis and proliferation of both PANC-1 and AsPC-1 human pancreatic cancer cells, as measured by thymidine incorporation and cell number. LTB4 stimulated rapid and transient activation of MEK and ERK1/2 kinases. The MEK inhibitors, PD98059 and U0126, blocked LTB4-stimulated ERK1/2 activation and cell proliferation. LTB4 also stimulated phosphorylation of p38 MAPK; however, the p38 MAPK inhibitor, SB203580, failed to block LTB4-stimulated growth. The activity of JNK/SAPK was not affected by LTB4 treatment. Phosphorylation of Akt was also induced by LTB4 and this effect was blocked by the PI-3 kinase inhibitor wortmannin, which also partially blocked LTB4-stimulated cell proliferation. In conclusion, LTB4 stimulates proliferation of human pancreatic cancer cells through MEK/ERK and PI-3 kinase/Akt pathways, while p38 MPAK and JNK/SAPK are not involved

  14. Measurement of short-term 11C-thymidine activity in human head and neck tumours using positron emission tomography (PET)

    International Nuclear Information System (INIS)

    Tumour uptake of 11C-thymidine labeled in the methyl position was assessed after intravenous injection in 13 patients with head and neck tumours. This activity was compared to other tracers such as C15O, 13NH3 and C15O2. In every single case a 'positive' tumour image after injection of 11C-thymidine was obtained. Time-activity curves showed the initial activity to be followed by a rapid decrease over the first 10-15 min with an apparent plateau thereafter. A similar level of uptake was found in normal salivary gland regions and myocardium, while higher activities were noted in liver and kidney parenchyma. It is suggested that both blood flow and cellular metabolism can influence 11C-thymidine imaging in this class of human tumours. (orig.)

  15. Use of metabolic inhibitors to estimate protozooplankton grazing and bacterial production in a monomictic eutrophic lake with an anaerobic hypolimnion

    International Nuclear Information System (INIS)

    Inhibitors of eucaryotes (cycloheximide and amphotericin B) and procaryotes (penicillin and chloramphenical) were used to estimate bacterivory and bacterial production in a eutrophic lake. Bacterial production appeared to be slightly greater than protozoan grazing in the aerobic waters of Lake Oglethorpe. Use of penicillin and cycloheximide yielded inconsistent results in anaerobic water and in aerobic water when bacterial production was low. Production measured by inhibiting eucaryotes with cycloheximide did not always agree with [3H]thymidine estimates or differential filtration methods. Laboratory experiments showed that several common freshwater protozoans continued to swim and ingest bacterium-size latex beads in the presence of the eucaryote inhibitor. Penicillin also affected grazing rates of some ciliates. The authors recommended that caution and a corroborating method be used when estimating ecologically important parameters with specific inhibitors

  16. Quantification of thymidine kinase (TK1) mRNA in normal and leukemic cells and investigation of structure-function relatiosnhip of recombinant TK1enzyme

    DEFF Research Database (Denmark)

    Kristensen, Tina

    lymphocytes. As the high TKI mRNA level is not translated into an active enzyme, these results indicate a defect in the regulation of TKI in CLL cells. For the studies of the structure-function relationship of TKI a recombinant TKI protein, which is expressed as a glutathione-S-transferase (GST) fusion...... protein was used. TKI protein is cleaved from the GST-part with thrombin. Two TKI mutants, TKI-l 93 and TKI-l 76, with deletions from the C-terminal were constructed by the recombinant PCR method. Deletion of 57 amino acids from the C-terminal (TKI- 176) results in an inactive enzyme. Deletion of 40 amino...

  17. Therapeutic properties of a vector carrying the HSV thymidine kinase and GM-CSF genes and delivered as a complex with a cationic copolymer

    OpenAIRE

    Alekseenko, Irina V; Snezhkov, Eugene V; Igor P Chernov; Pleshkan, Victor V.; Potapov, Victor K.; Sass, Alexander V.; Monastyrskaya, Galina S; Kopantzev, Eugene P.; Tatyana V. Vinogradova; Khramtsov, Yuri V; Ulasov, Alexey V; Rosenkranz, Andrey A.; Sobolev, Alexander S; Bezborodova, Olga A; Plyutinskaya, Anna D

    2015-01-01

    Background Gene-directed enzyme prodrug therapy (GDEPT) represents a technology to improve drug selectivity for cancer cells. It consists of delivery into tumor cells of a suicide gene responsible for in situ conversion of a prodrug into cytotoxic metabolites. Major limitations of GDEPT that hinder its clinical application include inefficient delivery into cancer cells and poor prodrug activation by suicide enzymes. We tried to overcome these constraints through a combination of suicide gene ...

  18. Effects of herpes simplex virus thymidine kinase/acyclovir system on growth of human pulmonary adenocarcinoma A549 cell line in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    HE Xiang-liang; HE Dong-hua; GUO Xian-jian; QIAN Gui-sheng; HUANG Gui-jun; CHEN Wei-zhong; LI Shu-ping

    2002-01-01

    Objective: To observe the effect of anciclovir (ACV) treatment on tumors induced by inoculation of TK gene-transfected human pulmonary adenocarcinoma A549 cells in nude mice. Methods: A recombinant plasmid containing TK gene was constructed and transfected into A549 cells by electroporation. The sensitivity of the transgenic cells (A549-TK) to ACV was examined by MTT assay in vitro and for in vivo observation, inoculation of A549-TK and A-549 cells into nude mice was separately performed to induce tumor growth, the response of which to ACV treatment was observed, and the tumor tissues were pathologically examined. Results: A recombinant plasmid containing TK gene was successfully constructed and transfected into A549 cells. The sensitivity of A549-TK cells to ACV was 43 times higher than that of A549 cells. The tumors induced by A549-TK cells showed no significant increase in size after ACV treatment (P>0. 05), and light microscopy revealed local tissue necrosis, karyoklasis, and nuclei disappearance. Conclusion: A549-TK cells acquires sensitivity to ACV both in vitro and in vivo, and ACV can inhibit the growth of tumors induced by A549-TK cell inoculation in nude mice.

  19. Sensitivity and specificity of tritiated thymidine incorporation and ELISPOT assays in identifying antigen specific T cell immune responses

    Directory of Open Access Journals (Sweden)

    MacLeod Beth

    2007-09-01

    Full Text Available Abstract Background Standardization of cell-based immunologic monitoring is becoming increasingly important as methods for measuring cellular immunity become more complex. We assessed the ability of two commonly used cell-based assays, tritiated thymidine incorporation (proliferation and IFN-gamma ELISPOT, to predict T cell responses to HER-2/neu, tetanus toxoid (tt, and cytomegalovirus (CMV antigens. These antigens were determined to be low (HER-2/neu, moderate (tt, and robustly (CMV immunogenic proteins. Samples from 27 Stage II, III, and IV HER-2/neu positive breast cancer patients, vaccinated against the HER-2/neu protein and tt, were analyzed by tritiated thymidine incorporation and IFN-gamma ELISPOT for T cell response. Results Linear regression analysis indicates that both stimulation index (SI (p = 0.011 and IFN-gamma secreting precursor frequency (p Conclusion These data underscore the importance of taking into consideration the performance characteristics of assays used to measure T cell immunity. This consideration is particularly necessary when determining which method to utilize for assessing responses to immunotherapeutic manipulations in cancer patients.

  20. Actions of exogenous histones and other proteins on [3H]-thymidine incorporation into DNA of Novikoff hepatoma cells

    International Nuclear Information System (INIS)

    The effects of exogenous proteins on the incorporation of [3H]-thymidine into DNA was studied in Novikoff hepatoma ascites cells incubated in Eagle's minimal essential medium. A liver cytosol fraction (8 mg protein/ml) caused approximately 80% inhibition of isotope incorporation. The inhibitory activity of cytosol fractions from Morris hepatomas 9618A2, 5123C and 20 were inversely related to their growth rate. Under conditions in which there appeared to be a density dependent inhibition of growth, a mean 10 to 20% stimulation of isotope incorporation was observed after addition of total calf thymus histones and individual fractions in the concentration range of 100 to 400μg/ml. In experiments with lower cell concentrations, a 60% or greater increase in [3H]-thymidine incorporation could be obtained with total calf thymus histone and with Fl and arginine-rich histones from rat liver. At concentrations of 1 to 2 mg/ml, histones inhibited DNA synthesis. Bovine serum albumin had little effect on DNA synthesis. Polylysine caused an 80 to 90% inhibition at a concentration of 1 mg/ml, but stimulatory effects were detected under certain conditions at 10μg/ml. The results suggest critical dependence on the ratio of cell and exogenous protein concentration in the action of proteins on DNA synthesis. (author)

  1. Peculiarities of the incorporation of [3H]thymidine into AT-rich regions of DNA during replicative synthesis

    International Nuclear Information System (INIS)

    The authors studied the role of the AT-rich regions in DNA replication in vivo. The authors selected cells of humans and Drosophila - organisms belonging to different types of alternation of unique and repetitive sequences - as the objects of investigation. The authors then studied the behavior of the AT-rich sequences in replication by the method of thermoelution of [3H]thymidine-labeled DNA, fragmented by ultrasound to 350 nucleotide pairs. By measuring the amount of DNA and the amount of the label in the fractions, the authors were able to construct curves of the change in the specific activity of DNA as a function of the temperature of elution from HAP and, consequently, as a function of the AT composition. The authors call them differential temperature chromatograms (DTC). Human peripheral blood lymphocytes were cultured according to the standard procedure with PHA (Difco P). A culture of D. melanogaster cells was labeled with [3H]thymidine in the logarithmic phase of growth for 1.2 and 42 h. At the end of the labeling, cell DNA was isolated from the lymphocytes and cell and nuclear DNA from a Drosophila tissue culture by the standard methods

  2. Proliferation and survival of L5178Y murine lymphoma cells exposed to tritiated water and tritiated thymidine

    International Nuclear Information System (INIS)

    Two strains of murine lymphoma cells L5178Y, inversely cross-sensitive to X-rays and UV light, were exposed to various concentrations of tritiated water and tritiated thymidine. The exposure was carried out at 370C to simulate conditions of in vivo chronic exposure. Tritiated water was used at 2, 4 and 100 μCi/ml (74 and 148 kBq/ml and 3.7 MBq/ml) and tritiated thymidine at 0.05 μCi/ml (1.85 kBq/ml). The exposure was carried out for 4, 25, 50, 100, 200 and 400 h. The strains exposed, L5178Y-R and L5178Y-S, which differ in sensitivity to acute irradiation, also differ in susceptibility to tritiated compounds. It was found that the development of the exposed cell populations proceeds according to a reproducible pattern: growth phases can be distinguished that differ both in their rate of proliferation and in their cell reproductive capacity determined after transfer into a non-radioactive medium. (author)

  3. Raman and surface-enhanced Raman spectroscopy of amino acids and nucleotide bases for target bacterial vibrational mode identification

    Science.gov (United States)

    Guicheteau, Jason; Argue, Leanne; Hyre, Aaron; Jacobson, Michele; Christesen, Steven D.

    2006-05-01

    Raman and surface-enhanced Raman spectroscopy (SERS) studies of bacteria have reported a wide range of vibrational mode assignments associated with biological material. We present Raman and SER spectra of the amino acids phenylalanine, tyrosine, tryptophan, glutamine, cysteine, alanine, proline, methionine, asparagine, threonine, valine, glycine, serine, leucine, isoleucine, aspartic acid and glutamic acid and the nucleic acid bases adenosine, guanosine, thymidine, and uridine to better characterize biological vibrational mode assignments for bacterial target identification. We also report spectra of the bacteria Bacillus globigii, Pantoea agglomerans, and Yersinia rhodei along with band assignments determined from the reference spectra obtained.

  4. Therapeutic targeting of Janus kinases

    OpenAIRE

    Pesu, Marko; Laurence, Arian; Kishore, Nandini; Zwillich, Sam; Chan, Gary; O’Shea, John J.

    2008-01-01

    Cytokines play pivotal roles in immunity and inflammation, and targeting cytokines and their receptors is an effective means of treating such disorders. Type I and II cytokine receptors associate with Janus family kinases (JAKs) to effect intracellular signaling. These structurally unique protein kinases play essential and specific roles in immune cell development and function. One JAK, JAK3, has particularly selective functions. Mutations of this kinase underlie severe combined immunodeficie...

  5. Visualizing autophosphorylation in histidine kinases

    OpenAIRE

    Casino, Patricia; Miguel-Romero, Laura; Marina, Alberto

    2014-01-01

    Reversible protein phosphorylation is the most widespread regulatory mechanism in signal transduction. Autophosphorylation in a dimeric sensor histidine kinase is the first step in two-component signalling, the predominant signal-transduction device in bacteria. Despite being the most abundant sensor kinases in nature, the molecular bases of the histidine kinase autophosphorylation mechanism are still unknown. Furthermore, it has been demonstrated that autophosphorylation can occur in two dir...

  6. Tau-tubulin kinase

    Directory of Open Access Journals (Sweden)

    Seiko Ikezu

    2014-04-01

    Full Text Available Tau-tubulin kinase (TTBK belongs to casein kinase superfamily and can phosphorylate microtubule-associated protein tau and tubulin. TTBK has two isoforms, TTBK1 and TTBK2, which contain highly homologous catalytic domains but their non-catalytic domains are distinctly different. TTBK1 is expressed specifically in the central nervous system and is involved in phosphorylation and aggregation of tau. TTBK2 is ubiquitously expressed in multiple tissues and genetically linked to spinocerebellar ataxia type 11. TTBK1 directly phosphorylates tau protein, especially at Ser422, and also activates cycline-dependent kinase 5 in a unique mechanism. TTBK1 protein expression is significantly elevated in Alzheimer’s disease brains, and genetic variations of the TTBK1 gene are associated with late-onset Alzheimer’s disease in two cohorts of Chinese and Spanish populations. TTBK1 transgenic mice harboring the entire 55-kilobase genomic sequence of human TTBK1 show progression of tau accumulation, neuroinflammation, and neurodegeneration when crossed with tau mutant mice. Our recent study shows that there is a striking switch in mononuclear phagocyte and activation phenotypes in the anterior horn of the spinal cord from alternatively activated (M2-skewed microglia to pro-inflammatory (M1-skewed infiltrating peripheral monocytes in P301L tau mutant mice by crossing with TTBK1 transgenic mice. TTBK1 is responsible for mediating M1-activated microglia-induced neurotoxicity, and its overexpression induces axonal degeneration in vitro. These studies suggest that TTBK1 is an important molecule for the inflammatory axonal degeneration, which may be relevant to the pathobiology of tauopathy including Alzheimer’s disease.

  7. Phosphatidylinositol 3-kinase in myogenesis.

    Science.gov (United States)

    Kaliman, P; Zorzano, A

    1997-08-01

    Phosphatidylinositol 3-kinase (PI 3-kinase) has been cloned and characterized in a wide range of organisms. PI 3-kinases are activated by a diversity of extracellular stimuli and are involved in multiple cell processes such as cell proliferation, protein trafficking, cell motility, differentiation, regulation of cytoskeletal structure, and apoptosis. It has recently been shown that PI 3-kinase is a crucial second messenger in the signaling of myogenesis. Two structurally unrelated highly specific inhibitors of PI 3-kinase-wortmannin and LY294002-block the morphological and biochemical differentiation program of different skeletal-muscle cell models. Moreover, L6E9 myoblasts overexpressing a dominant-negative mutant of PI 3-kinase p85 regulatory subunit (Δp85) are unable to differentiate. Furthermore, PI 3-kinase is specifically involved in the insulinlike growth factor (IGF)-dependent myogenic pathway. Indeed, the ability of IGF-I, des-1,3-IGF-I, and IGF-II to promote cell fusion and muscle-specific protein expression is impaired after treatment with PI 3-kinase inhibitors or in cells overexpressing Δp85. The identification of additional key downstream elements of the IGF/PI 3-kinase myogenic cascade is crucial to a detailed understanding of the process of muscle differentiation and may generate new tools for skeletal and cardiac muscle regeneration therapies. (Trends Cardiovasc Med 1997;7:198-202). © 1997, Elsevier Science Inc. PMID:21235885

  8. 3H-thymidine (3HTdR) incorporated into DNA; Dosimetric and radiobiological considerations

    International Nuclear Information System (INIS)

    Tritium can be selectively incorporated into DNA in vivo by administration of tritiated thymidine (3HTdR), and this labelled precursor is used widely and effectively in investigations on the mechanism of DNA synthesis, in studies on the kinetics of cell proliferation, and for the purpose of selective cell destruction. It is important, of course, to be aware of the threshold for toxicity of the isotope used in this manner. In addition, the unique dosimetric features involved present theoretical and practical problems, the solutions of which will shed light on mechanisms of radiobiology effect. The highly selective irradiation of the nucleus of those cells which incorporated 3HTdR at the time of DNA synthesis constitutes not only partial-body irradiation, but partial-cell irradiation, and homogeneity of exposure can be defined only in terms of subcellular dimensions. The toxicity of 3HTdR and the associated problems have been reviewed extensively. The object of the present paper is to: (a) evaluate from data in the literature and from data to be presented, whether observed effects from intranuclear 3H can be accounted for on the basis of calculated absorbed dose in rads to the cell nucleus, or, alternatively, whether additional factors deriving from the presence of the 3H in the DNA molecule must be invoked. In the latter case the biological effects would not be generally predictable on the basis of absorbed dose; and (b) to discuss the implications of the findings with respect to radiobiology and radiation protection. The conclusion is reached that while data are as yet inadequate for firm evaluation, biological effects of 3HTdR are those predictable on the basis of the average absorbed dose to the nucleus. The applicability of the absorbed dose concept can be evaluated satisfactorily only if adequate comparisons of the effect of incorporated 3HTdR can be made with a suitable standard external radiation such as X-rays, or possibly with the same isotope (3H

  9. Vimentin in Bacterial Infections

    DEFF Research Database (Denmark)

    Mak, Tim N; Brüggemann, Holger

    2016-01-01

    -vimentin interactions are presented in this review: the role of vimentin in pathogen-binding on the cell surface and subsequent bacterial invasion and the interaction of cytosolic vimentin and intracellular pathogens with regards to innate immune signaling. Mechanistic insight is presented involving distinct bacterial......Despite well-studied bacterial strategies to target actin to subvert the host cell cytoskeleton, thus promoting bacterial survival, replication, and dissemination, relatively little is known about the bacterial interaction with other components of the host cell cytoskeleton, including intermediate...... filaments (IFs). IFs have not only roles in maintaining the structural integrity of the cell, but they are also involved in many cellular processes including cell adhesion, immune signaling, and autophagy, processes that are important in the context of bacterial infections. Here, we summarize the knowledge...

  10. Osmotic stress-dependent serine phosphorylation of the histidine kinase homologue DokA

    Directory of Open Access Journals (Sweden)

    Oehme Felix

    2001-03-01

    Full Text Available Abstract Background Two-component systems consisting of histidine kinases and their corresponding receivers are widespread in bacterial signal transduction. In the past few years, genes coding for homologues of two-component systems were also discovered in eukaryotic organisms. DokA, a homologue of bacterial histidine kinases, is an element of the osmoregulatory pathway in the amoeba Dictyostelium. The work described here addresses the question whether DokA is phosphorylated in vivo in response to osmotic stress. Results We have endogenously overexpressed individual domains of DokA to investigate post-translational modification of the protein in response to osmotic shock in vivo. Dictyostelium cells were labeled with [32P]-orthophosphate, exposed to osmotic stress and DokA fragments were subsequently isolated by immunoprecipitation. Thus, a stress-dependent phosphorylation could be demonstrated, with the site of phosphorylation being located in the kinase domain. We demonstrate biochemically that the phosphorylated amino acid is serine, and by mutational analysis that the phosphorylation reaction is not due to an autophosphorylation of DokA. Furthermore, mutation of the conserved histidine did not affect the osmostress-dependent phosphorylation reaction. Conclusions A stimulus-dependent serine phosphorylation of a eukaryotic histidine kinase homologue was demonstrated for the first time in vivo. That implies that DokA, although showing typical structural features of a bacterial two-component system, might be part of a eukaryotic signal transduction pathway that involves serine/threonine kinases.

  11. Structural basis for the regulation mechanism of the tyrosine kinase CapB from Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Vanesa Olivares-Illana

    2008-06-01

    Full Text Available Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high-resolution structure of a bacterial tyrosine kinase reveals a 230-kDa ring-shaped octamer that dissociates upon intermolecular autophosphorylation. These observations provide a molecular basis for the regulation mechanism of the bacterial tyrosine kinases and give insights into their copolymerase function.

  12. Demonstrating Bacterial Flagella.

    Science.gov (United States)

    Porter, John R.; And Others

    1992-01-01

    Describes an effective laboratory method for demonstrating bacterial flagella that utilizes the Proteus mirabilis organism and a special harvesting technique. Includes safety considerations for the laboratory exercise. (MDH)

  13. TBK1 protects vacuolar integrity during intracellular bacterial infection.

    Directory of Open Access Journals (Sweden)

    Andrea L Radtke

    2007-03-01

    Full Text Available TANK-binding kinase-1 (TBK1 is an integral component of Type I interferon induction by microbial infection. The importance of TBK1 and Type I interferon in antiviral immunity is well established, but the function of TBK1 in bacterial infection is unclear. Upon infection of murine embryonic fibroblasts with Salmonella enterica serovar Typhimurium (Salmonella, more extensive bacterial proliferation was observed in tbk1(-/- than tbk1(+/+ cells. TBK1 kinase activity was required for restriction of bacterial infection, but interferon regulatory factor-3 or Type I interferon did not contribute to this TBK1-dependent function. In tbk1(-/-cells, Salmonella, enteropathogenic Escherichia coli, and Streptococcus pyogenes escaped from vacuoles into the cytosol where increased replication occurred, which suggests that TBK1 regulates the integrity of pathogen-containing vacuoles. Knockdown of tbk1 in macrophages and epithelial cells also resulted in increased bacterial localization in the cytosol, indicating that the role of TBK1 in maintaining vacuolar integrity is relevant in different cell types. Taken together, these data demonstrate a requirement for TBK1 in control of bacterial infection distinct from its established role in antiviral immunity.

  14. Structural basis for the regulation mechanism of the tyrosine kinase CapB from Staphylococcus aureus

    DEFF Research Database (Denmark)

    Olivares-Illana, Vanesa; Meyer, Philippe; Bechet, Emmanuelle;

    2008-01-01

    understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus...

  15. 3H thymidine an indicator of benzo(a)pyrene induced lung carcinogenesis: role of quercetin and curcumin

    International Nuclear Information System (INIS)

    Full text: Lung cancer is responsible for most of the cancer related deaths and calls for new approaches to control the menace. In the present study chemopreventive efficacy of curcumin and quercetin was investigated against benzo(a)pyrene (BP) induced lung carcinogenesis. The mice were segregated into five groups which included normal control, BP treated, BP+curcumin treated, BP+quercetin treated and BP+curcumin+quercetin treated groups. The morphological and histological analyses of tumor nodules confirmed lung carcinogenesis, after 22 weeks of single i.p. injection of BP at a dose of 100 mg/Kg body weight to mice. Tumor incidence and tumor multiplicity were observed to be 88% and 1.75, respectively in the BP treated mice. A statistically significant increase in the uptake of 3H thymidine indicative of increased DNA synthesis which in turn is the marker of uncontrolled cancer cell proliferation, was observed in the lung slices of BP treated mice. Further, BP treatment resulted in marked disruption in the histoarchitecture of lungs. Nuclei were enlarged, thickening of epithelium was seen. Structure-less masses of cells were visible all over. Nuclear pleomorphism and decreased cytoplasmic contents were also observed in BP treated mice. Squamous epithelial metaplasia, severe epithelial thickening and alveolar vocuolizations in distal airways indicative of lung carcinogensis were also observed in the BP treated mice. Supplementation with curcumin alone resulted in a significant decrease in the tumor incidence as well as tumor multicity which were observed to be 77% and 1.42 respectively. Also, quercetin significantly decreased tumor incidence and tumor multiplicity to 70% and 1.28 respectively. However, upon combined supplementation with phytochemicals, an appreciable decrease in the tumor incidence and multiplicity was observed which was found to be 60% and 1.00 respectively. Further, Supplementation with curcumin alone to BP treated mice resulted in statistically

  16. Detection and grading of soft tissue sarcomas of the extremities with F-18-3 '-fluoro-3 '-deoxy-L-thymidine

    NARCIS (Netherlands)

    Cobben, DCP; Elsinga, PH; Suurmeijer, AJH; Vaalburg, W; Maas, B; Jager, PL; Hoekstra, HJ

    2004-01-01

    Purpose: The aim of the study was to investigate the feasibility of F-18-3'-fluoro-3'-deoxy-L-thymidine positron emission tomography (FLT-PET) for the detection and grading of soft tissue sarcoma (STS). Experimental Design: Nineteen patients with 20 STSs of the extremities were scanned, using attenu

  17. Drug-resistance development differs between HIV-1-infected patients failing first-line antiretroviral therapy containing nonnucleoside reverse transcriptase inhibitors with and without thymidine analogues

    OpenAIRE

    Santoro, MM; Sabin, C.; Forbici, F; Bansi, L.; Dunn, D; Fearnhill, E.; Boumis, E; Nicastri, E.; Antinori, A; Palamara, G.; Callegaro, A.; Francisci, D; Zoncada, A.; Maggiolo, F; Zazzi, M.

    2013-01-01

    We evaluated the emergence of drug resistance in patients failing first-line regimens containing one nonnucleoside reverse transcriptase inhibitor (NNRTI) administered with zidovudine (ZDV) + lamivudine (the ZDV group) or non-thymidine analogues (non-TAs) (tenofovir or abacavir, + lamivudine or emtricitabine; the non-TA group).

  18. An automated radiosynthesis of 2-[11C]thymidine using anhydrous [11C]urea derived from [11C]phosgene

    International Nuclear Information System (INIS)

    2-[11C]Thymidine has been produced from [11C]methane via [11C]phosgene and [11C]urea. Anhydrous [11C]urea was prepared from [11C]phosgene by reaction with liquid ammonia. This novel approach avoids the problems associated with the synthesis of anhydrous [11C]urea from [11C]cyanide. A fully automated system based on a modular approach and under PLC control has been developed. The system provides 2-[11C]thymidine reliably and reproducibly for clinical PET studies. The radiosynthesis takes 45-50 min from [11C]methane and the average yield was 1.5-3.3 GBq (40-90 mCi). The specific radioactivity was typically in the range 29.6-51.8 GBq μmol-1 (0.8-1.4 Ci μmol-1) at EOS corresponding to 6-12 μg of stable thymidine. The radiochemical yield of 2-[11C]thymidine was ca. 14% from [11C]methane

  19. Activity of an attached and free-living Vibrio sp. as measured by thymidine incorporation, rho-iodonitrotetrazolium reduction, and ATP/DNA ratios

    International Nuclear Information System (INIS)

    Three independent techniques, [3H]thymidine incorporation, the reduction rate of p-iodonitrotetrazolium violet (INT) to INT formazan normalized to DNA, and the ratio of ATP to DNA, were adapted to measure the activity of attached and unattached estuarine bacteria. In experiments employing the estuarine isolate Vibrio proteolytica, nutrient concentrations were manipulated by varying the concentration of peptone-yeast extract. In the presence of exogenous nutrients, the activity of free-living cells was greater than that of attached cells as measured by [3H]thymidine incorporation and ATP/DNA ratios. In the absence of peptone-yeast extract, however, the activity of attached cells surpassed that of free-living cells as determined by [3H]thymidine incorporation and INT formazan normalized to DNA. Of the three techniques, [3H]thymidine incorporation was deemed most sensitive for detecting changes in activity resulting from slight differences in nutrient concentration. By this technique, attached cells were much less sensitive to changing nutrient concentrations than were free-living cells. Below a threshold concentration, attached cell activity remained constant, while the activity of unattached cells decreased as a function of decreasing nutrient concentration. The results suggest that loss of cell surface area available for substrate uptake due to attachment may be an important factor in determining the relative activities of attached and free-living cells

  20. MET Receptor Tyrosine Kinase

    Science.gov (United States)

    Faoro, Leonardo; Cervantes, Gustavo M.; El-Hashani, Essam; Salgia, Ravi

    2010-01-01

    MET receptor tyrosine kinase (RTK) and its ligand hepatocyte growth factor (HGF) have become important therapeutic target in oncology, especially lung cancer. MET RTK is involved in cancer cell growth/survival, motility/migration, invasion/metastasis, and in angiogenesis. MET can be overexpressed in lung cancer, sometimes mutated, and sometimes amplified. Not only can MET be overexpressed, there are subsets of lung cancer tumors that have HGF overexpression. The mutations of MET can occur in the semaphorin and/or juxtamembrane domain in a majority of times. Amplification of MET can occur de novo in primary/metastatic tumors, as well arise in the context of therapeutic inhibition. There are a number of clinical inhibitors that have been developed against MET/HGF. Small molecule inhibitors such as XL184 and PF02341066 have come to clinical fruition, as well as antibodies against MET (such as MetMAb). These inhibitors will be discussed. PMID:19861919

  1. Use of halogenated thymidine analogs as clinical radiosensitizers: rationale, current status, and future prospects: non-hypoxic cell sensitizers

    International Nuclear Information System (INIS)

    The halogenated pyrimidine analogs, bromodeoxyuridine (BUdR) and iododeoxyuridine (IUdR) have been recognized as potential clinical radiosensitizers for over two decades. In vivo and in vitro experimental studies document that radiosensitization is directly dependent on the amount of thymidine replacement in DNA by these analogs. Based on recent in vivo and clinical pharmacology studies on continuous intravenous infusions of these drugs, clinical trials are underway evaluating the potential of radiosensitization in high grade gliomass and other poorly radioresponsive tumors using the technically safer intravenous route of administration. In this paper, the authors review the basic strategy for the use of these analogs, the ongoing clinical trials and the potential areas for future experimental and clinical studies

  2. Radiotoxicity and incorporation of methyl-tritiated-thymidine on preimplantation mouse embryo. In vitro fertilization and culture

    International Nuclear Information System (INIS)

    In the present work different concentrations of methyl-3 H-thymidine was added to the culture medium micro drops containing the mouse zygotes at pro nuclear stage and the embryos were cultured in vitro at 370 C in a humidified atmosphere with 5% of CO2 for four days up to the expanded blastocyst stage, the established end point to calculate the L D50 lethal dose. The blastocyst formation rate decreased with increasing concentration of tritium in medium and a value of 2.4 X 103 Bq/ml for L D50 was obtained. The 3 H-Td R incorporation by the embryo during the preimplantation period was low at the beginning and increased quickly during the morula and the blastocyst development. (author)

  3. Fine structure of reactive cells in injured nervous tissue labeled with 3H-thymidine injected before injury

    International Nuclear Information System (INIS)

    To examine the fine structure of blood mononuclear cells in injured nervous tissue, mice were given repeated injections of 3H-thymidine. Under ether anesthesia the animals either were given a stab wound to the spinal cord or had their left hypoglossal nerve transected. Tissue sections were prepared for electron microscopic radioautography, and all labeled cells were photographed. About half the labeled cells in the injured spinal cords and almost all the labeled cells in the nuclei of the injured hypoglossal nerves had nuclei with dark staining peripheral heterochromatin, dark cytoplasm with long cisternae of granular endoplasmic reticulum, and other ultrastructural features characteristic of the cells usually identifed as microglia. The remaining labeled cells in the injured spinal cords were macrophages, fibroblasts, and vascular cells. Since uninjured nervous tissue has extremely few labeled cells, most of the labeled cells in this experiment should be derived from blood mononuclear cells

  4. Shuffling bacterial metabolomes

    OpenAIRE

    Thomason, Brendan; Read, Timothy D.

    2006-01-01

    Horizontal gene transfer (HGT) has a far more significant role than gene duplication in bacterial evolution. This has recently been illustrated by work demonstrating the importance of HGT in the emergence of bacterial metabolic networks, with horizontally acquired genes being placed in peripheral pathways at the outer branches of the networks.

  5. Vimentin in Bacterial Infections.

    Science.gov (United States)

    Mak, Tim N; Brüggemann, Holger

    2016-01-01

    Despite well-studied bacterial strategies to target actin to subvert the host cell cytoskeleton, thus promoting bacterial survival, replication, and dissemination, relatively little is known about the bacterial interaction with other components of the host cell cytoskeleton, including intermediate filaments (IFs). IFs have not only roles in maintaining the structural integrity of the cell, but they are also involved in many cellular processes including cell adhesion, immune signaling, and autophagy, processes that are important in the context of bacterial infections. Here, we summarize the knowledge about the role of IFs in bacterial infections, focusing on the type III IF protein vimentin. Recent studies have revealed the involvement of vimentin in host cell defenses, acting as ligand for several pattern recognition receptors of the innate immune system. Two main aspects of bacteria-vimentin interactions are presented in this review: the role of vimentin in pathogen-binding on the cell surface and subsequent bacterial invasion and the interaction of cytosolic vimentin and intracellular pathogens with regards to innate immune signaling. Mechanistic insight is presented involving distinct bacterial virulence factors that target vimentin to subvert its function in order to change the host cell fate in the course of a bacterial infection. PMID:27096872

  6. Prevention of bacterial adhesion

    DEFF Research Database (Denmark)

    Klemm, Per; Vejborg, Rebecca Munk; Hancock, Viktoria

    2010-01-01

    Management of bacterial infections is becoming increasingly difficult due to the emergence and increasing prevalence of bacterial pathogens that are resistant to available antibiotics. Conventional antibiotics generally kill bacteria by interfering with vital cellular functions, an approach that ...... become valuable weapons for preventing pathogen contamination and fighting infectious diseases in the future....

  7. Vimentin in Bacterial Infections

    Directory of Open Access Journals (Sweden)

    Tim N. Mak

    2016-04-01

    Full Text Available Despite well-studied bacterial strategies to target actin to subvert the host cell cytoskeleton, thus promoting bacterial survival, replication, and dissemination, relatively little is known about the bacterial interaction with other components of the host cell cytoskeleton, including intermediate filaments (IFs. IFs have not only roles in maintaining the structural integrity of the cell, but they are also involved in many cellular processes including cell adhesion, immune signaling, and autophagy, processes that are important in the context of bacterial infections. Here, we summarize the knowledge about the role of IFs in bacterial infections, focusing on the type III IF protein vimentin. Recent studies have revealed the involvement of vimentin in host cell defenses, acting as ligand for several pattern recognition receptors of the innate immune system. Two main aspects of bacteria-vimentin interactions are presented in this review: the role of vimentin in pathogen-binding on the cell surface and subsequent bacterial invasion and the interaction of cytosolic vimentin and intracellular pathogens with regards to innate immune signaling. Mechanistic insight is presented involving distinct bacterial virulence factors that target vimentin to subvert its function in order to change the host cell fate in the course of a bacterial infection.

  8. Function, structure, and mechanism in bacterial photosensory LOV proteins

    OpenAIRE

    Herrou, Julien; Crosson, Sean

    2011-01-01

    LOV domains are protein photosensors conserved in bacteria, archaea, plants and fungi that detect blue light via a flavin cofactor. In the bacterial kingdom, LOV domains are present in both chemotrophic and phototrophic species, where they are found N-terminally of signaling and regulatory domains such as sensor histidine kinases, diguanylate cyclases/phosphodiesterases, DNA-binding domains, and σ factor regulators. In this review, we describe the current state of knowledge on the function of...

  9. Formation of cyclic 1,N2-propanodeoxyguanosine and thymidine adducts in the reaction of the mutagen 2-bromoacrolein with calf thymus DNA

    International Nuclear Information System (INIS)

    The interaction of the mutagen 2-bromoacrolein (2BA) with DNA and thymidine was studied in vitro by reaction of [3-3H]2BA with thymidine, RNA, single-stranded DNA, and double-stranded DNA in phosphate buffer (pH 7.4). After purification of the nucleic acids, they were incubated at alkaline pH to convert any (hydroxybromo)propano(deoxy)-guanosine adducts to their dihydroxy analogues. After acid or enzymatic hydrolysis, the hydrolysates were analyzed by reversed-phase high-performance liquid chromatography. At a concentration of 1.6 mM, the fraction of 2BA that became covalently bound to DNA was 2.3% of the amount added. Only 3% of the radioactivity bound to DNA after extensive purification could be accounted for as cyclic 1,N2-(6,7-dihydroxy)-propanoguanine adducts. More 2BA became covalently bound to single-stranded DNA and RNA as compared with double-stranded DNA. However, high-performance liquid chromatographic analyses showed that formation of cyclic 1,N2-(6,7-dihydroxy)propanoguanine adducts was also a minor reaction with these macromolecules. Because these data showed that other type(s) of reaction(s) are more important in the reaction of 2BA with nucleic acids, we have investigated the reaction of 2BA with other nucleosides. It was found that 2BA reacted well with thymidine in vitro, and the major product was identified by 500 MHz 1H and 75.43 MHz 13C nuclear magnetic resonance and thermospray mass spectrometry as 3-(2-bromo-3-oxopropyl)thymidine. This adduct was unstable and decomposed upon storage. After enzymatic hydrolysis of [3H]2BA-modified double-stranded DNA and subsequent analysis of the hydrolysate by high-performance liquid chromatography, 22% of the covalently bound radioactivity to DNA coeluted with decomposition products of the 3-(bromooxypropyl)thymidine adduct

  10. Neurogenesis in the vomeronasal epithelium of adult garter snakes: 3. Use of /sup 3/H-thymidine autoradiography to trace the genesis and migration of bipolar neurons

    Energy Technology Data Exchange (ETDEWEB)

    Wang, R.T.; Halpern, M.

    1988-10-01

    Use of 3H-thymidine autoradiography and unilateral vomeronasal (VN) axotomy has permitted us to demonstrate directly the existence of VN stem cells in the adult garter snake and to trace continuous bipolar neuron development and migration in the normal VN and deafferentated VN epithelium in the same animal. The vomeronasal epithelium and olfactory epithelium of adult garter snakes are both capable of incorporating 3H-thymidine. In the sensory epithelium of the vomeronasal organ, 3H-thymidine-labeled cells were initially restricted to the base of the undifferentiated cell layer in animals surviving 1 day following 3H-thymidine injection. With increasing survival time, labeled cells progressively migrated vertically within the receptor cell column toward the apex of the bipolar neuron layer. In both the normal and denervated VN epithelium, labeled cells were observed through the 56 days of postoperative survival. In the normal epithelium, labeled cells were always located within the matrix of the intact receptor cell columns. However, labeled cells of the denervated epithelium were always located at the apical front of the newly formed cell mass following depletion of the original neuronal cell population. In addition, at postoperative days 28 and 56, labeled cells of the denervated VN epithelium achieved neuronal differentiation and maturation by migrating much farther away from the base of the receptor cell column than the labeled cells on the normal, unoperated contralateral side. This study directly demonstrates that basal cells initially incorporating 3H-thymidine are indeed stem cells of the VN epithelium in adult garter snakes.

  11. Neurogenesis in the vomeronasal epithelium of adult garter snakes: 3. Use of 3H-thymidine autoradiography to trace the genesis and migration of bipolar neurons

    International Nuclear Information System (INIS)

    Use of 3H-thymidine autoradiography and unilateral vomeronasal (VN) axotomy has permitted us to demonstrate directly the existence of VN stem cells in the adult garter snake and to trace continuous bipolar neuron development and migration in the normal VN and deafferentated VN epithelium in the same animal. The vomeronasal epithelium and olfactory epithelium of adult garter snakes are both capable of incorporating 3H-thymidine. In the sensory epithelium of the vomeronasal organ, 3H-thymidine-labeled cells were initially restricted to the base of the undifferentiated cell layer in animals surviving 1 day following 3H-thymidine injection. With increasing survival time, labeled cells progressively migrated vertically within the receptor cell column toward the apex of the bipolar neuron layer. In both the normal and denervated VN epithelium, labeled cells were observed through the 56 days of postoperative survival. In the normal epithelium, labeled cells were always located within the matrix of the intact receptor cell columns. However, labeled cells of the denervated epithelium were always located at the apical front of the newly formed cell mass following depletion of the original neuronal cell population. In addition, at postoperative days 28 and 56, labeled cells of the denervated VN epithelium achieved neuronal differentiation and maturation by migrating much farther away from the base of the receptor cell column than the labeled cells on the normal, unoperated contralateral side. This study directly demonstrates that basal cells initially incorporating 3H-thymidine are indeed stem cells of the VN epithelium in adult garter snakes

  12. Bacterial tactic responses.

    Science.gov (United States)

    Armitage, J P

    1999-01-01

    Many, if not most, bacterial species swim. The synthesis and operation of the flagellum, the most complex organelle of a bacterium, takes a significant percentage of cellular energy, particularly in the nutrient limited environments in which many motile species are found. It is obvious that motility accords cells a survival advantage over non-motile mutants under normal, poorly mixed conditions and is an important determinant in the development of many associations between bacteria and other organisms, whether as pathogens or symbionts and in colonization of niches and the development of biofilms. This survival advantage is the result of sensory control of swimming behaviour. Although too small to sense a gradient along the length of the cell, and unable to swim great distances because of buffetting by Brownian motion and the curvature resulting from a rotating flagellum, bacteria can bias their random swimming direction towards a more favourable environment. The favourable environment will vary from species to species and there is now evidence that in many species this can change depending on the current physiological growth state of the cell. In general, bacteria sense changes in a range of nutrients and toxins, compounds altering electron transport, acceptors or donors into the electron transport chain, pH, temperature and even the magnetic field of the Earth. The sensory signals are balanced, and may be balanced with other sensory pathways such as quorum sensing, to identify the optimum current environment. The central sensory pathway in this process is common to most bacteria and most effectors. The environmental change is sensed by a sensory protein. In most species examined this is a transmembrane protein, sensing the external environment, but there is increasing evidence for additional cytoplasmic receptors in many species. All receptors, whether sensing sugars, amino acids or oxygen, share a cytoplasmic signalling domain that controls the activity of a

  13. Allosteric regulation of glycerol kinase by enzyme IIIglc of the phosphotransferase system in Escherichia coli and Salmonella typhimurium.

    OpenAIRE

    Novotny, M J; Frederickson, W L; Waygood, E B; Saier, M H

    1985-01-01

    The mechanism by which enzyme IIIglc of the bacterial phosphotransferase system regulates the activity of crystalline glycerol kinase from Escherichia coli has been studied, and the inhibitory effects have been compared with those produced by fructose-1,6-diphosphate. It was shown that the free, but not the phosphorylated, form of enzyme IIIglc inhibits the kinase. Mutants of Salmonella typhimurium were isolated which were resistant to inhibition by either enzyme IIIglc (glpKr mutants) or fru...

  14. MAP Kinases in Immune Responses

    Institute of Scientific and Technical Information of China (English)

    Yongliang Zhang; Chen Dong

    2005-01-01

    MAP kinases are evolutionarily conserved signaling regulators from budding yeast to mammals and play essential roles in both innate and adaptive immune responses. There are three main families of MAPKs in mammals. Each of them has its own activators, inactivators, substrates and scaffolds, which altogether form a fine signaling network in response to different extracellular or intracellular stimulation. In this review, we summarize recent advances in understanding of the regulation of MAP kinases and the roles of MAP kinases in innate and adaptive immune responses.

  15. RNA interference screen identifies Abl kinase and PDGFR signaling in Chlamydia trachomatis entry.

    Directory of Open Access Journals (Sweden)

    Cherilyn A Elwell

    2008-03-01

    Full Text Available The strain designated Chlamydia trachomatis serovar L2 that was used for experiments in this paper is Chlamydia muridarum, a species closely related to C. trachomatis (and formerly termed the Mouse Pneumonitis strain of C. trachomatis. This conclusion was verified by deep sequencing and by PCR using species-specific primers. All data presented in the results section that refer to C. trachomatis should be interpreted as referring to C. muridarum. Since C. muridarum TARP lacks the consensus tyrosine repeats present in C. trachomatis TARP, we cannot make any conclusions about the role of TARP phosphorylation and C. muridarum entry. However, the conclusion that C. trachomatis L2 TARP is a target of Abl kinase is still valid as these experiments were performed with C. trachomatis L2 TARP [corrected]. To elucidate the mechanisms involved in early events in Chlamydia trachomatis infection, we conducted a large scale unbiased RNA interference screen in Drosophila melanogaster S2 cells. This allowed identification of candidate host factors in a simple non-redundant, genetically tractable system. From a library of 7,216 double stranded RNAs (dsRNA, we identified approximately 226 host genes, including two tyrosine kinases, Abelson (Abl kinase and PDGF- and VEGF-receptor related (Pvr, a homolog of the Platelet-derived growth factor receptor (PDGFR. We further examined the role of these two kinases in C. trachomatis binding and internalization into mammalian cells. Both kinases are phosphorylated upon infection and recruited to the site of bacterial attachment, but their roles in the infectious process are distinct. We provide evidence that PDGFRbeta may function as a receptor, as inhibition of PDGFRbeta by RNA interference or by PDGFRbeta neutralizing antibodies significantly reduces bacterial binding, whereas depletion of Abl kinase has no effect on binding. Bacterial internalization can occur through activation of PDGFRbeta or through independent

  16. Insights into the structure-function relationship of Brugia malayi thymidylate kinase (BmTMK).

    Science.gov (United States)

    Doharey, Pawan Kumar; Singh, Sudhir Kumar; Verma, Pravesh; Verma, Anita; Rathaur, Sushma; Saxena, Jitendra Kumar

    2016-07-01

    Lymphatic filariasis is a debilitating disease caused by lymph dwelling nematodal parasites like Wuchereria bancrofti, Brugia malayi and Brugia timori. Thymidylate kinase of B. malayi is a key enzyme in the de novo and salvage pathways for thymidine 5'-triphosphate (dTTP) synthesis. Therefore, B. malayi thymidylate kinase (BmTMK) is an essential enzyme for DNA biosynthesis and an important drug target to rein in filariasis. In the present study, the structural and functional changes associated with recombinant BmTMK, in the presence of protein denaturant GdnHCl, urea and pH were studied. GdnHCl and urea induced unfolding of BmTMK is non-cooperative and influence the functional property of the enzyme much lower than their Cm values. The study delineate that BmTMK is more prone to ionic perturbation. The dimeric assembly of BmTMK is an absolute requirement for enzymatic acitivity and any subtle change in dimeric conformation due to denaturation leads to loss of enzymatic activity. The pH induced changes on structure and activity suggests that selective modification of active site microenvironment pertains to difference in activity profile. This study also envisages that chemical moieties which acts by modulating oligomeric assembly, could be used for better designing of inhibitors against BmTMK enzyme. PMID:27044348

  17. Isolation of chloroplastic phosphoglycerate kinase

    Energy Technology Data Exchange (ETDEWEB)

    Macioszek, J.; Anderson, L.E. (Univ. of Illinois, Chicago (USA)); Anderson, J.B. (Pennsylvania State Univ., University Park (USA))

    1990-09-01

    We report here a method for the isolation of high specific activity phosphoglycerate kinase (EC 2.7.2.3) from chloroplasts. The enzyme has been purified over 200-fold from pea (Pisum sativum L.) stromal extracts to apparent homogeneity with 23% recovery. Negative cooperativity is observed with the two enzyme phosphoglycerate kinase/glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13) couple restored from the purified enzymes when NADPH is the reducing pyridine nucleotide, consistent with earlier results obtained with crude chloroplastic extracts. Michaelis Menten kinetics are observed when 3-phosphoglycerate is held constant and phosphoglycerate kinase is varied, which suggests that phosphoglycerate kinase-bound 1,3-bisphosphoglycerate may be the preferred substrate for glyceraldehyde-3-P dehydrogenase in the chloroplast.

  18. Protein Crystals of Raf Kinase

    Science.gov (United States)

    1995-01-01

    This image shows crystals of the protein raf kinase grown on Earth (photo a) and on USML-2 (photo b). The space-grown crystals are an order of magnitude larger. Principal Investigator: Dan Carter of New Century Pharmaceuticals

  19. Heterotrophic bacterial production: Relationships to biological and abiological factors in estuarine environments

    Energy Technology Data Exchange (ETDEWEB)

    Koepfler, E.T.

    1989-01-01

    Ecotoxicological effects of creosote contamination on benthic bacterial communities in the Elizabeth River, Virginia were investigated using both structural an functional microbial parameters. Results indicated that cell specific and total heterotrophic bacterial production parameters were depressed in a dose-dependent manner with increasing sediment PAH concentrations. Toxicity effects upon production were modified by temporal trends associated with temperature as well as spatial sediment characteristics. Of the parameters employed, the tritiated thymidine production assay was found to be the most sensitive for detection of ecotoxicological effects. Bacterial abundance and production were examined during a destratification event in the lower James River, Virginia. Bacterial abundance, although significantly different between stations, did not change over the study. Bacterial production ({sup 3}H-Tdr incorporation) in surface waters was significantly less during the mixed period 187 {mu}g C{center dot}1-1{center dot} d{sup {minus}1} compared to the most stratified state (324{mu}g C{center dot}1-1{center dot} d{sup {minus}1}). Correlations between bacteria and chlorophyll were diminished during the mixed period. Total and flagellate specific grazing rates upon bacteria were reduced during the onset of destratification. Relationships between bacterial and nutrient parameters also indicated a strong influence of destratification. These results indicate that destratification changes trophic interactions within the microbial loop, which are not necessarily reflected in temporal patterns of bacterial abundance. Bacterioplankton production, and ammonium assimilation and remineralization were examined between April and August 1988 in the lower York River, Va.

  20. Restricted Distribution of the Butyrate Kinase Pathway among Butyrate-Producing Bacteria from the Human Colon

    Science.gov (United States)

    Louis, Petra; Duncan, Sylvia H.; McCrae, Sheila I.; Millar, Jacqueline; Jackson, Michelle S.; Flint, Harry J.

    2004-01-01

    The final steps in butyrate synthesis by anaerobic bacteria can occur via butyrate kinase and phosphotransbutyrylase or via butyryl-coenzyme A (CoA):acetate CoA-transferase. Degenerate PCR and enzymatic assays were used to assess the presence of butyrate kinase among 38 anaerobic butyrate-producing bacterial isolates from human feces that represent three different clostridial clusters (IV, XIVa, and XVI). Only four strains were found to possess detectable butyrate kinase activity. These were also the only strains to give PCR products (verifiable by sequencing) with degenerate primer pairs designed within the butyrate kinase gene or between the linked butyrate kinase/phosphotransbutyrylase genes. Further analysis of the butyrate kinase/phosphotransbutyrylase genes of one isolate, L2-50, revealed similar organization to that described previously from different groups of clostridia, along with differences in flanking sequences and phylogenetic relationships. Butyryl-CoA:acetate CoA-transferase activity was detected in all 38 strains examined, suggesting that it, rather than butyrate kinase, provides the dominant route for butyrate formation in the human colonic ecosystem that contains a constantly high concentration of acetate. PMID:15028695

  1. Inhibition of protein kinase B activity induces cell cycle arrest and apoptosis during early G₁ phase in CHO cells.

    Science.gov (United States)

    van Opstal, Angélique; Bijvelt, José; van Donselaar, Elly; Humbel, Bruno M; Boonstra, Johannes

    2012-04-01

    Inhibition of PKB (protein kinase B) activity using a highly selective PKB inhibitor resulted in inhibition of cell cycle progression only if cells were in early G1 phase at the time of addition of the inhibitor, as demonstrated by time-lapse cinematography. Addition of the inhibitor during mitosis up to 2 h after mitosis resulted in arrest of the cells in early G1 phase, as deduced from the expression of cyclins D and A and incorporation of thymidine. After 24 h of cell cycle arrest, cells expressed the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PKB activity in early G1 phase is required to prevent the induction of apoptosis. Using antibodies, it was demonstrated that active PKB translocates to the nucleus during early G1 phase, while an even distribution of PKB was observed through cytoplasm and nucleus during the end of G1 phase. PMID:22251027

  2. Bacterial Wound Culture

    Science.gov (United States)

    ... Home Visit Global Sites Search Help? Bacterial Wound Culture Share this page: Was this page helpful? Also known as: Aerobic Wound Culture; Anaerobic Wound Culture Formal name: Culture, wound Related ...

  3. Bacterial Meningitis in Infants

    Directory of Open Access Journals (Sweden)

    J Gordon Millichap

    2004-04-01

    Full Text Available A retrospective study of 80 infantile patients (ages 30-365 days; 47 male, 33 female with culture-proven bacterial meningitis seen over a 16 year period (1986-2001 is reported from Taiwan.

  4. Calibrating bacterial evolution

    OpenAIRE

    Ochman, Howard; Elwyn, Susannah; Moran, Nancy A

    1999-01-01

    Attempts to calibrate bacterial evolution have relied on the assumption that rates of molecular sequence divergence in bacteria are similar to those of higher eukaryotes, or to those of the few bacterial taxa for which ancestors can be reliably dated from ecological or geological evidence. Despite similarities in the substitution rates estimated for some lineages, comparisons of the relative rates of evolution at different classes of nucleotide sites indicate no basis for their universal appl...

  5. Molecular mechanisms of the synergy between cysteinyl-leukotrienes and receptor tyrosine kinase growth factors on human bronchial fibroblast proliferation

    Directory of Open Access Journals (Sweden)

    Hajime Yoshisue

    2006-12-01

    Full Text Available We have reported that cysteinyl-leukotrienes (cys-LTs synergise not only with epidermal growth factor (EGF but also with platelet-derived growth factor (PDGF and fibroblast growth factor (FGF to induce mitogenesis in human bronchial fibroblasts. We now describe the molecular mechanisms underlying this synergism. Mitogenesis was assessed by incorporation of [3H]thymidine into DNA and changes in protein phosphorylation by Western blotting. Surprisingly, no CysLT receptor antagonists (MK-571, montelukast, BAY u9773 prevented the synergistic mitogenesis. LTD4 did not cause phosphorylation of EGFR nor did it augment EGF-induced phosphorylation of EGFR, and the synergy between LTD4 and EGF was not blocked by the metalloproteinase inhibitor GM6001 or by an HB-EGF neutralising antibody. The EGFR-selective kinase inhibitor, AG1478, suppressed the synergy by LTD4 and EGF, but had no effect on the synergy with PDGF and FGF. While inhibitors of mitogen-activated protein kinase, phosphatidylinositol 3-kinase and protein kinase C (PKC prevented the synergy, these drugs also inhibited mitogenesis elicited by EGF alone. In contrast, pertussis toxin (PTX efficiently inhibited the potentiating effect of LTD4 on EGF-induced mitogenesis, as well as that provoked by PDGF or FGF, but had no effect on mitogenesis elicited by the growth factors alone. Whereas LTD4 alone did not augment phosphorylation of extracellular signal-regulated kinase (Erk-1/2 and Akt, it increased phosphorylation of PKC in a Gi-dependent manner. Addition of LTD4 prolonged the duration of EGF-induced phosphorylation of Erk-1/2 and Akt, both of which were sensitive to PTX. The effect of cys-LTs involves a PTX-sensitive and PKC-mediated intracellular pathway leading to sustained growth factor-dependent phosphorylation of Erk-1/2 and Akt.

  6. Kinetics of H3-thymidine incorporation into nuclei of cells of the regenerating liver of rats exposed to various doses of X-rays

    International Nuclear Information System (INIS)

    Action of X-radiation on the incorporation of H3-thymidine into nuclei of cells of the regenerating liver of rats has been studied. A whole-body exposure of rats to 150-300 R has been found to inhibit H3-thymidine uptake into DNA of S-cells of the regenerating liver, and this effect can be attributed to the inhibition of DNA synthesis rather than to changes in the concentration of a label in the intracellular pool of DNA precursors. In addition to the DNA synthesis decrease, inhibition of the label uptake into the pool is observed after doses of 600 to 1200 R. On the basis of the data obtained, a hypothesis is proposed that explains the mechanism of inhibition of DNA synthesis under the action of radiation

  7. Utilization of 3H from deoxyuridine and thymidine for synthesis of DNA and other macromolecules in various organs of the rat

    International Nuclear Information System (INIS)

    The reported investigation showed that liver and intestinal mucosa, but not spleen or thymus of ACI strain rats incorporated significant amounts of 3H from deoxyuridine 6-3H and thymidine- methyl-3H into a RNA fraction obtained by alkaline hydrolysis of tissue homogenates. In bone marrow, 3H for thymidine but not from deoxyuridine was incorporated into this fraction. Inhibition of DNA synthesis by 5-fluorouracils (5-FU) increased 3H in this RNA fraction for only intestinal mucosa and bone marrow, while enhancement of DNA synthesis during recovery from 5-FU toxicity was associated with an increase in 3H for the alkali-solubilized fractions of liver, intestinal mucosa, spleen and thymus but not Morris hepatoma 3924A. (U.K.)

  8. Pd-catalyzed Suzuki-Miyaura coupling reaction in the synthesis of 5-aryl-1-[2-(phosphonomethoxy)ethyl]uracils as potential multisubstrate inhibitors of thymidine phosphorylase

    Czech Academy of Sciences Publication Activity Database

    Pomeisl, Karel; Holý, Antonín; Pohl, Radek

    2007-01-01

    Roč. 48, č. 17 (2007), s. 3065-3067. ISSN 0040-4039 R&D Projects: GA MŠk 1M0508 Grant ostatní: Descartes Prize(XE) HPAW-CT-2002-9001 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * thymidine phosphorylase * Suzuki coupling * pyrimidine Subject RIV: CC - Organic Chemistry Impact factor: 2.615, year: 2007

  9. Pd-catalyzed Suzuki-Miyaura coupling reaction in the synthesis of 5-aryl substituted pyrimidine acyclic nucleoside phosphonates as potential multisubstrate inhibitors of thymidine phosphorylase

    Czech Academy of Sciences Publication Activity Database

    Pomeisl, Karel; Holý, Antonín; Pohl, Radek

    Dublin : University College Dublin, 2007. s. 329. [European Symposium on Organic Chemistry /15./. 08.07.2007-13.07.2007, Dublin] R&D Projects: GA MŠk 1M0508 Grant ostatní: Descartes Prize(XE) HPAW-CT-2002-9001 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * thymidine phosphorylase * Suzuki coupling Subject RIV: CC - Organic Chemistry

  10. Permeability changes and incorporation of labelled thymidine into DNA and whole cells of the fibroblast culture of Chinese hamsters affected by MEA and low temperature

    International Nuclear Information System (INIS)

    Action of MEA and low temperature (20degC) on the incorporation of labelled thymidine into DNA and whole cells of the fibroblast culture of chinese hamsters has been studied. It has been found that each of the above-mentioned factors equally decreases the label uptake into the cell and DNA. It is concluded that MEA and low temperature do not substantially influence the rate of DNA synthesis

  11. Impact of HIV-1 reverse transcriptase polymorphism F214L on virological response to thymidine analogue-based regimens in antiretroviral therapy (ART)-naive and ART-experienced patients

    DEFF Research Database (Denmark)

    Ceccherini-Silberstein, Francesca; Cozzi-Lepri, Alessandro; Ruiz, Lidia;

    2007-01-01

    A negative association between the polymorphism F214L and type 1 thymidine analogue (TA) mutations (TAMs) has been observed. However, the virological response to TAs according to the detection of F214L has not been evaluated.......A negative association between the polymorphism F214L and type 1 thymidine analogue (TA) mutations (TAMs) has been observed. However, the virological response to TAs according to the detection of F214L has not been evaluated....

  12. Effect of ionizing radiation on thymidine uptake, differentiation, and VEGFR2 receptor expression in endothelial cells: The role of VEGF165

    International Nuclear Information System (INIS)

    Purpose: Late thrombosis of irradiated vascular segments may be the consequence of endothelial cell (EC) dysfunction after radiation therapy. We investigated the effects of β ionizing radiation on human EC viability, thymidine uptake, and differentiation. Methods and Materials: Endothelial cells were exposed to 32P-labeled DNA oligonucleotides in incremental doses of 2, 6, and 10 Gy. The modulation of the VEGFR2 receptor expression after irradiation and the overall potential radioprotective effect of VEGF165 on these functions were assayed. Results: A dose-dependent inhibitory effect of β irradiation on ECs' thymidine uptake and differentiation was observed. EC viability, however, was not affected at levels of radiation up to 10 Gy. VEGF165 proved to have a radioprotective effect as ECs' thymidine uptake, after radiation doses of 2, 6, and 10 Gy, was increased by 1.5-, 2-, and 4-fold, respectively, in the presence of 10 ng/ml of VEGF165 (p165 also proved beneficial in maintaining cell differentiation at 16 h postirradiation when compared to controls. These biologic effects were in direct correlation with the upregulation of VEGFR2 receptor expression in irradiated ECs. Conclusions: β irradiation interacts directly with EC functions by significantly reducing their ability to differentiate and proliferate, associated with upregulation of VEGFR2. These effects can be prevented in part by pretreating cells with VEGF165, an effect potentially favored by the upregulation of VEGFR2 receptor expression after irradiation

  13. Thymidine selectively enhances growth suppressive effects of camptothecin/irinotecan in MSI+ cells and tumors containing a mutation of MRE11

    DEFF Research Database (Denmark)

    Rodriguez, Rene; Hansen, Lasse Tengbjerg; Phear, Geraldine; Scorah, Jennifer; Spang-Thomsen, Mogens; Cox, Angela; Helleday, Thomas; Meuth, Mark

    2008-01-01

    PURPOSE: DNA synthesis inhibitors and damaging agents are widely used in cancer therapy; however, sensitivity of tumors to such agents is highly variable. The response of tumor cells in culture to these agents is strongly influenced by the status of DNA damage response pathways. Here, we attempt ...... exploit the altered response of mismatch repair (MMR)-deficient colon cancer cells and tumors to camptothecin or irinotecan and thymidine by combining them to improve therapeutic response. EXPERIMENTAL DESIGN: A panel of colon cancer cell lines was assayed for response to camptothecin...... in nude mice. RESULTS: Camptothecin-thymidine combinations suppress colony formation of MMR-deficient tumor cells 10- to 3,000-fold relative to that obtained with camptothecin alone and significantly reduce the concentrations of the agents required to induce late S/G(2) arrest and senescence....... Sensitivity is not a direct result of MMR, p53, or p21 status. However MMR-deficient cell lines containing an intronic frameshift mutation of MRE11 show greatest sensitivity to these agents. Increased sensitivity to this combination is also evident in vivo as thymidine enhances irinotecan-induced growth...

  14. Cell kinetic effects of incorporated 3H-thymidine on proliferating human lymphocytes: flow cytometric analysis using the DNA/nuclear protein method

    International Nuclear Information System (INIS)

    Phytohemagglutinin-stimulated human peripheral blood lymphocytes incorporating high concentrations of 3H-thymidine accumulate in G2 and show a consequent reduction in the number of cells entering M (division delay). The simultaneous flow cytometric analysis of DNA content (propidium iodide fluorescence) and nuclear protein content (fluorescein isothiocyanate fluorescence) allows for the accurate quantitation of these events; G2 and M are separated in the bivariate distributions. A good correlation was observed between mitotic indices, quantitated by manually counting mitotic cells, and integration of the M area in DNA/nuclear protein histograms. Moreover, significant differences in G2 nuclear protein levels were found between untreated and 3H-thymidine-treated lymphocytes. In order to characterize this effect, G2 was empirically divided into low nuclear protein (G2A) and high nuclear protein (G2B) compartments. 3H-thymidine caused an initial accumulation of lymphocytes in G2A, followed within 3-6 h by a gradual movement of some cells into G2B, with a subsequent accumulation of cells in G2B. The results suggest that the distribution of cells in G2 (G2A and G2B), the average nuclear protein content of G2B cells, and the proportion of cells in M are parameters that when used in combination provide a unique description of radiobiological effects

  15. Labelling indices after 3H-thymidine incorporation during organ culture of duodenal mucosa in coeliac disease

    International Nuclear Information System (INIS)

    Incorporation of 3H-thymidine during organ culture of duodenal biopsy specimens from 34 coeliac and 10 non-coeliac patients was studied by autoradiography. High labelling indices were found in flat, coeliac mucosas. Gluten fractions, which provoked histological deterioration during culture, induced labelling of a greater proportion of crypt cells and higher migration rate than parallelly cultured specimens on gluten-free medium. No influence on clypt cell kinetics could be observed after culture with gluten fractions incapable of producing histological damage or with alpha-lactalbumin. In coeliac remission mucosas, labelling indices were at the same level as in non-coeliac biopsis, and no significant effects of gluten were observed. Autoradiography seems to be a fairly sensitive and reliable determinant of gluten toxicity by organ culture in coeliac desease and should supplement the histological appraisal of the biopsies. The increment of labelling indices provoked by gluten exposure seemed not merely to be a concequence of increased desquamation of cells from the biopsy surface but could imply a direct influence of gluten on crypt cell kinetics in coeliac disease. (Auth.)

  16. Thymidylate Synthase, Thymidine Phosphorylase and Orotate Phosphoribosyl Transferase Levels as Predictive Factors of Chemotherapy in Oral Squamous Cell Carcinoma

    International Nuclear Information System (INIS)

    We conducted a clinicopathologic study on protein and mRNA levels of thymidylate synthase (TS), thymidine phosphorylase (TP) and orotate phosphoribosyl transferase (OPRT) using biopsy tissue specimens before treatment. The mRNA levels have been measured in tumor cells microdissected from paraffin-embedded specimens (Danenberg Tumor Profile method: DTP method). We studied the mRNA and protein expression as effect predictive factors in chemotherapy. The subjects consisted of 20 cases of untreated oral squamous cell carcinoma who had undergone chemotherapy with TS-1 (16 males and 4 females, tongue in 8 cases, upper gingiva in 3 cases, lower gingiva in 3 cases, buccal mucosa in 5 cases and floor of the mouth in 1 case). TS gene expressions of the responders were lower than those for the nonresponders. Furthermore, regarding males who were less than 70 years of age, stage I and II, well differentiated type and tongue, TS mRNA expression of the responders were lower than that for the nonresponders. The mRNA expression of OPRT for the male responders was lower than that for the nonresponders. No remarkable difference was observed by immunohistochemistry. In this study, the measurement of the TS levels using the DTP method may potentially act as a predictive factor of antitumor effectiveness

  17. Incorporation of tritiated thymidine by epithelial and interstitial cells in bronchiolar-alveolar regions of asbestos-exposed rats

    International Nuclear Information System (INIS)

    Inhaled asbestos causes progressive interstitial lung disease. The authors have performed a series of studies to elucidate early pathogenetic events at sites of fiber deposition in asbestos-exposed rats. This study reports that a single 5-hour exposure to chrysotile asbestos induces significant increases in incorporation of tritiated thymidine (3HTdR) into nuclei of epithelial and interstitial cells of bronchiolar-alveolar regions. No cell populations in air-exposed or carbonyl iron-exposed control animals exhibited more than 1% labeling at any point in time. Immediately after the 5-hour asbestos exposure, incorporation was normal. By 19 hours after exposure there was a significant increase in incorporation of 3HTdR, particularly by Type II epithelial cells of the first alveolar duct bifurcations. The greatest increase in degree of incorporation (up to 18-fold) was observed 24 hours after exposure, and increased percentages of 3HTdR-labeled cells were maintained through the 48 hours postexposure period. Normal labeling was present by 8 days after exposure, and this level remained through the 1-month period studied. This apparent mitogenic response correlates with increased numbers of bronchiolar-alveolar epithelial and interstitial cells demonstrated by ultrastructural morphometry in correlative studies. The authors speculate that the incorporation of 3HTdR could be induced by the direct effects of inhaled fibers or by mitogenic factors released from macrophages attracted to the inhaled asbestos

  18. An experimental study on healing of pulp wound following pulpotomy in dogs by autoradiography with tritiated thymidine

    International Nuclear Information System (INIS)

    This experiment is an autoradiographical study of the healing process in pulp wounds following pulpotomy and direct capping with calcium hydroxide. Experimental, young adult, mongrel dogs were divided into the following two groups: A group: Pulpotomy was performed and the following intervals allowed to lapse between amputation and sacrifice. One hour before sacrifice, 1μCi/gbw 3H-thymidine was injected intravenously (flash labeling). B group: Single pulse labeling was performed either one day (1 spl) or two days (2 spl) after amputation. During the first two days after amputation, pulp stem cells under the cutting surface divided and proliferated, with the proliferative rate reaching a maximum during the second day and decreased gradually from the fourth day after pulpotomy. Cells in the first zone became columnar in shapes and assumed a regular arrangement under the necrotic layer. Though these cells were unlabeled in the A group, they were prominently labeled in the B group. These finding indicate a part of the proliferated undifferentiated mesenchymal cells migrated and regularly arranged beneath the necrotic layer, differentiated into odontoblasts and then produced a dentine bridge. The remaining undifferentiated mesenchymal cells in the second zone reverted to pulp stem cells to accomplish healing. (auth.)

  19. Estimation of cancerolytic properties of thionine from plants seeds by inclusion of C14-thymidine in tumoral cells

    International Nuclear Information System (INIS)

    Full text: It has been earlier shown that cysteine rich peptides - thionine from seeds of various plants possess expressed fungitoxic activity. It is connected to influence of thionine on cellular membranes of fungi. It was possible to assume that the substances showing cytotoxic activity will be active in relation to tumoral cells. We isolated peptide fractions from seeds bamia (Hibiscus esculentus), kenaf (Hibiscus cannabinus), abutilon (Abutilon theophrasti), euphorbia (Euphorbia virgata), palma Christi (Ricinus communis) and horse sorrel (Rumex confertus) and studied their antineoplastic and fungitoxic activity. Antiproliferative action of peptides to melanoma cells of mice was estimated in cytotoxic test by inclusion of C14-thymidine to DNA. This researches have shown that peptides from seeds of horse sorrel and palma Christi did not change a level of synthesis of DNA while peptides from euphorbia and bamia considerably reduced inclusion of labeled nucleotide to DNA and suppressed growth of tumoral cells on 14 and 39 % accordingly. Parallel tests of these peptides on fungitoxic activity in relation to virulent strains of Verticillium dahliae have shown suppression of conidial growth on 17 and 26 % accordingly. Thus, peptides from seeds of bamia and euphorbia possess the expressed property to suppress growth of tumoral cells and can be used at creation a new cancerolytic preparations for treatment of human cancer. Work is executed under the financial support of fundamental grants F - 4.19 and F-4.1.44

  20. Bacterial meningitis in children

    International Nuclear Information System (INIS)

    To demonstrate the epidemiology, clinical manifestations and bacteriological profile of bacterial meningitis in children beyond the neonatal period in our hospital. This was a retrospective descriptive study conducted at Prince Rashid Hospital in Irbid, Jordan. The medical records of 50 children with the diagnosis of bacterial meningitis during 4 years period, were reviewed. The main cause of infection was streptococcus pneumoniae, followed by Haemophilus influenza and Niesseria meningitides. Mortality was higher in infants and meningococcal infection, while complications were more encountered in cases of streptococcus pneumoniae. Cerebrospinal fluid culture was positive in 11 cases and Latex agglutination test in 39. There is a significant reduction of the numbers of bacterial meningitis caused by Haemophilus influenza type B species. (author)

  1. Diagnosis of bacterial vaginosis

    Directory of Open Access Journals (Sweden)

    Đukić Slobodanka

    2013-01-01

    Full Text Available Bacterial vaginosis is a common, complex clinical syndrome characterized by alterations in the normal vaginal flora. When symptomatic, it is associated with a malodorous vaginal discharge and on occasion vaginal burning or itching. Under normal conditions, lactobacilli constitute 95% of the bacteria in the vagina. Bacterial vaginosis is associated with severe reduction or absence of the normal H2O2­producing lactobacilli and overgrowth of anaerobic bacteria and Gardnerella vaginalis, Atopobium vaginae, Mycoplasma hominis and Mobiluncus species. Most types of infectious disease are diagnosed by culture, by isolating an antigen or RNA/DNA from the microbe, or by serodiagnosis to determine the presence of antibodies to the microbe. Therefore, demonstration of the presence of an infectious agent is often a necessary criterion for the diagnosis of the disease. This is not the case for bacterial vaginosis, since the ultimate cause of the disease is not yet known. There are a variety of methods for the diagnosis of bacterial vaginosis but no method can at present be regarded as the best. Diagnosing bacterial vaginosis has long been based on the clinical criteria of Amsel, whereby three of four defined criteria must be satisfied. Nugent’s scoring system has been further developed and includes validation of the categories of observable bacteria structures. Up­to­date molecular tests are introduced, and better understanding of vaginal microbiome, a clear definition for bacterial vaginosis, and short­term and long­term fluctuations in vaginal microflora will help to better define molecular tests within the broader clinical context.

  2. Interfering with bacterial gossip

    DEFF Research Database (Denmark)

    Bjarnsholt, Thomas; Tolker-Nielsen, Tim; Givskov, Michael

    2011-01-01

    defense. Antibiotics exhibit a rather limited effect on biofilms. Furthermore, antibiotics have an ‘inherent obsolescence’ because they select for development of resistance. Bacterial infections with origin in bacterial biofilms have become a serious threat in developed countries. Pseudomonas aeruginosa......, resistance and QS inhibition as future antimicrobial targets, in particular those that would work to minimize selection pressures for the development of resistant bacteria.......Biofilm resilience poses major challenges to the development of novel antimicrobial agents. Biofilm bacteria can be considered small groups of “Special Forces” capable of infiltrating the host and destroying important components of the cellular defense system with the aim of crippling the host...

  3. Cloning and Characterization of upp, a Gene Encoding Uracil Phosphoribosyltransferase from Lactococcus lactis

    DEFF Research Database (Denmark)

    Martinussen, Jan; Hammer, Karin

    1994-01-01

    Uracil phosphoribosyltransferase catalyzes the key reaction in the salvage of uracil in many microorganisms. The gene encoding uracil phosphoribosyltransferase (upp) was cloned from Lactococcus lactis subsp. cremoris MG1363 by complementation of an Escherichia coli mutant. The gene was sequenced......-negative bacterial strains. The phenotype of the uracil phosphoribosyltransferase-deficient strain was established. Surprisingly, the upp strain is resistant only to very low concentrations of 5-fluorouracil. Secondary mutants in thymidine phosphorylase and thymidine kinase were isolated by selection for resistance...

  4. Role(s) of IL-2 inducible T cell kinase and Bruton's tyrosine kinase in mast cell response to lipopolysaccharide.

    Science.gov (United States)

    Huang, Weishan; August, Avery

    2016-06-01

    Mast cells play critical roles during immune responses to the bacterial endotoxin lipopolysaccharide (LPS) that can lead to fatal septic hypothermia [1], [2], [3]. IL-2 inducible T cell kinase (ITK) and Bruton's tyrosine kinase (BTK) are non-receptor tyrosine kinases that act downstream of numerous receptors, and have been shown to modulate mast cell responses downstream of FcεRIα [4], however, their roles in regulating mast cell responses to endotoxic stimuli were unclear. We found that the absence of ITK and BTK alters the mast cell response to LPS, and leads to enhanced pro-inflammatory cytokine production by mast cells and more severe LPS-induced hypothermia in mice [5]. Here, we detail our investigation using microarray analysis to study the transcriptomic profiles of mast cell responses to LPS, and the roles of ITK and/or BTK expression in this process. Mouse whole genome array data of WT, Itk (-/-) , Btk (-/-) , and Itk (-/-)  Btk (-/-) bone marrow-derived mast cells (BMMCs) stimulated by PBS (control) or LPS for 1 h were used in our latest research article [5] and is available in the Gene Expression Omnibus under accession number GSE64287. PMID:27081634

  5. Bacterial extracellular lignin peroxidase

    Science.gov (United States)

    Crawford, Donald L.; Ramachandra, Muralidhara

    1993-01-01

    A newly discovered lignin peroxidase enzyme is provided. The enzyme is obtained from a bacterial source and is capable of degrading the lignin portion of lignocellulose in the presence of hydrogen peroxide. The enzyme is extracellular, oxidative, inducible by lignin, larch wood xylan, or related substrates and capable of attacking certain lignin substructure chemical bonds that are not degradable by fungal lignin peroxidases.

  6. Bacterial Skin Infections

    Science.gov (United States)

    ... or scraped, the injury should be washed with soap and water and covered with a sterile bandage. Petrolatum may be applied to open areas to keep the tissue moist and to try to prevent bacterial invasion. Doctors recommend that people do not use ...

  7. Bacterial microflora of nectarines

    Science.gov (United States)

    Microflora of fruit surfaces has been the best source of antagonists against fungi causing postharvest decays of fruit. However, there is little information on microflora colonizing surfaces of fruits other than grapes, apples, and citrus fruit. We characterized bacterial microflora on nectarine f...

  8. Protein kinase CK2 in human diseases

    DEFF Research Database (Denmark)

    Guerra, Barbara; Issinger, Olaf-Georg

    2008-01-01

    Protein kinase CK2 (formerly referred to as casein kinase II) is an evolutionary conserved, ubiquitous protein kinase. There are two paralog catalytic subunits, i.e. alpha (A1) and alpha' (A2). The alpha and alpha' subunits are linked to two beta subunits to produce a heterotetrameric structure....... The catalytic alpha subunits are distantly related to the CMGC subfamily of kinases, such as the Cdk kinases. There are some peculiarities associated with protein kinase CK2, which are not found with most other protein kinases: (i) the enzyme is constitutively active, (ii) it can use ATP and GTP and...... specifically target this protein kinase [10]. Since not all the aspects of what has been published on CK2 can be covered in this review, we would like to recommend the following reviews; (i) for general information on CK2 [11-18] and (ii) with a focus on aberrant CK2 [19-22]....

  9. Degradation of Activated Protein Kinases by Ubiquitination

    OpenAIRE

    Lu, Zhimin; Hunter, Tony

    2009-01-01

    Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular functions. Once a protein kinase is activated, its activity is subsequently downregulated through a variety of mechanisms. Accumulating evidence indicates that the activation of protein kinases commonly initiates their downregulation via the ubiquitin/proteasome pathway. Failure to regulate protein kinase activity or expression levels can cause human diseases.

  10. Determinants of homodimerization specificity in histidine kinases

    OpenAIRE

    Ashenberg, Orr; Rozen-Gagnon, Kathryn; Laub, Michael T; Keating, Amy E.

    2011-01-01

    Two-component signal transduction pathways consisting of a histidine kinase and a response regulator are used by prokaryotes to respond to diverse environmental and intracellular stimuli. Most species encode numerous paralogous histidine kinases that exhibit significant structural similarity. Yet in almost all known examples, histidine kinases are thought to function as homodimers. We investigated the molecular basis of dimerization specificity, focusing on the model histidine kinase EnvZ and...

  11. Poly-thymidine oligonucleotides mediate activation of murine glial cells primarily through TLR7, not TLR8.

    Directory of Open Access Journals (Sweden)

    Min Du

    Full Text Available The functional role of murine TLR8 in the inflammatory response of the central nervous system (CNS remains unclear. Murine TLR8 does not appear to respond to human TLR7/8 agonists, due to a five amino acid deletion in the ectodomain. However, recent studies have suggested that murine TLR8 may be stimulated by alternate ligands, which include vaccinia virus DNA, phosphothioate oligodeoxynucleotides (ODNs or the combination of phosphothioate poly-thymidine oligonucleotides (pT-ODNs with TLR7/8 agonists. In the current study, we analyzed the ability of pT-ODNs to induce activation of murine glial cells in the presence or absence of TLR7/8 agonists. We found that TLR7/8 agonists induced the expression of glial cell activation markers and induced the production of multiple proinflammatory cytokines and chemokines in mixed glial cultures. In contrast, pT-ODNs alone induced only low level expression of two cytokines, CCL2 and CXCL10. The combination of pT-ODNs along with TLR7/8 agonists induced a synergistic response with substantially higher levels of proinflammatory cytokines and chemokines compared to CL075. This enhancement was not due to cellular uptake of the agonist, indicating that the pT-ODN enhancement of cytokine responses was due to effects on an intracellular process. Interestingly, this response was also not due to synergistic stimulation of both TLR7 and TLR8, as the loss of TLR7 abolished the activation of glial cells and cytokine production. Thus, pT-ODNs act in synergy with TLR7/8 agonists to induce strong TLR7-dependent cytokine production in glial cells, suggesting that the combination of pT-ODNs with TLR7 agonists may be a useful mechanism to induce pronounced glial activation in the CNS.

  12. Uptake of 3H-thymidine by the receptor cell populations after injury of the sensory nerve fibres

    International Nuclear Information System (INIS)

    The material of the study was the skin from the beak of two-day ducklings. The investigation was carried out on the 2nd, 5th, 20th and 45th day after the crushing of the sensory nerve fibres entering the capsulated Herbst receptors. Twenty four hours before the biopsy, the ducklings were injected at 6 hours intervals with 3H-thymidine. The number of labelled index in the three cell pupulations, participating in the receptor development was determined. The cells of the subcapsular space of all control animals (with intacted suborbital nerves) have shown the highest labelled index. The index of the capsular perineural cells is about 12 times lower, while the labelled index of the Schwann receptor cells is about 10 times lower. Following the denervation, the labelled index in increasing and reaches its maximum on the 5th postoperative day. The Schwann receptor cells in comparison to the two other cell populations show the most significant deviation during the regeneration (45th day after the intervention). The investigations show that all three cell populations pass through a miotic cycle of innovation. The low labelled index of the Schwann receptors (1-2 labelled cells in 1000) is an indication of a high differentiation. One can assume that their regeneration takes place at the expense of the proper proliferation activity as well as of the differentiation of the Schwann cells from the distal section of the regenerating sensory nerve fibres. Taking into consideration the high labelled index of the other populations, it seems most probable that their regeneration takes place for the expense of their own cell populations. (A.B.)

  13. Premitotic DNA synthesis in the brain of the adult frog (Rana esculenta L.): An autoradiographic 3H-thymidine study

    International Nuclear Information System (INIS)

    Replicative synthesis of DNA in the brain of the adult frog was studied by light microscope autoradiography. Animals collected during the active period (May-June) and in hibernation (January) were used. In active frogs, 3H-thymidine labelling occurred mainly in the ependymal cells which line the ventricles. The mean labelling index (LI%) was higher in the ependyma of the lateral and fourth ventricles than in the ependyma of the lateral diencephalon and tectal parts of the mesencephalon. In the recessus infundibularis and preopticus the number of labelled cells (LCs) was several times greater than in the lateral parts of the third ventricle. LCs were seen subependymally only occasionally. The incidence of LCs in the parenchyma of the brain was much lower in most regions than in the ventricular ependyma; LCs were mainly small and, from their nuclear morphology, they were glial cells. The LI% reached the highest value in the septum hippocampi and in the nucleus entopeduncularis. In these locations, LCs were larger and closer in size to the nerve cells of these regions. From comparison with data obtained earlier in the brain of mammals, it is evident that the distribution of proliferating cells in the olfactory and limbic system is phylogenetically conservative. The occurrence of pyknotic cells in the same areas which contain LCs, suggests that cell division reflects in part the process of cell renewal observed in mammals. However, proliferating cells could also be linked to the continuous growth observed in non-mammalian vertebrates. In hibernating frogs, LCs and pyknoses were not seen or were found occasionally, which further indicates the functional significance of both processes

  14. Kinetics of [32P]orthophosphate and [3H]thymidine incorporation into newly replicating DNA in vivo

    International Nuclear Information System (INIS)

    Nuclear DNA can be empirically subdivided into three populations: (1) bulk DNA or low salt-soluble DNA (75%), (2) high salt-soluble DNA (23%), and (3) matrix DNA which remains tightly bound to the nuclear matrix (2%). Newly replicating DNA is associated with the nuclear matrix in regenerating rat liver. To study the incorporation of DNA precursors into replicating DNA via the salvage vs the de novo pathway, 100μCi [3H]thymidine (3H-Thd) and 5mCi [32P]orthophosphate (32P/sub i/) were injected into the hepatic portal vein of partially hepatectomized rats. Increasing time of 3H-Thd incorporation showed the label is chased from matrix DNA to bulk DNA. After a 10 min pulse, 13% of the total specific activity is associated with bulk DNA and 57% with matrix DNA. After 30 min, 32% and 36% of the total specific activity remain associated with bulk and matrix DNA, respectively, indicating that most of the 3H has been chased from the matrix DNA. In contrast, after injection of 32P/sub i/, the amount of label in matrix DNA increases to a maximum at 30 min and only then begins to decrease. At 10 min the specific activity/total specific activity of bulk DNA is 7% and of matrix DNA is 66% vs 8% and 82% after 30 min. The kinetic pattern of 32P/sub i/ incorporation differs dramatically from that of 3H-Thd suggesting (a) the incorporation of de novo precursors lags significantly behind that of precursors entering through the salvage pathway, or (b) there may be two distinct classes of replication forks

  15. Differences in temporal aspects of mutagenesis and cytotoxicity in Chinese hamster cells treated with methylating agents and thymidine.

    Science.gov (United States)

    Peterson, A R; Peterson, H

    1982-03-01

    Equitoxic concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MeMes) produced different frequencies of 8-azaguanine-resistant mutants and different amounts of N7-methylguanine, O6-methylguanine (m6G), and N3-methyladenine in the DNA of V79 Chinese hamster cells. Thus, neither the cytotoxicities nor the mutagenicities of these methylating agents could be attributed solely to nitrogen or to oxygen methylations in the DNA. However, MNNG produced 12-fold more m6G and 5-fold more mutants than did MeMes, indicating that a substantial part of the MNNG-induced mutations resulted from m6G--thymine mispairing during DNA replication. The expression as mutants of mutagenic oxygen methylations in the DNA of cells treated with MNNG was enhanced by thymidine (dThd) and deoxycytidine (dCyd), but these nucleosides did not significantly enhance MeMes-induced mutagenesis. The cytotoxicities of MNNG and MeMes were also increased by 10 microM dThd in proportion to the amount of m6G in the DNA. These increases in cytotoxicity were abolished by dCyd, which did not greatly reduce the dThd-induced enhancements of mutagenesis. Moreover, when dThd was present only during the 2-hr treatment with MNNG, maximal cytotoxicity occurred, but MNNG-induced mutagenesis was not increased. Maximal mutagenesis occurred when the dThd was present throughout the first doubling time of the MNNG-treated cells. Thus, the expression of the cytotoxicity and the mutagenicity associated with m6G in the DNA of V79 cells occurred by quite different mechanisms. PMID:6951203

  16. Early assessment of therapy response in malignant lymphoma with the thymidine analogue [{sup 18}F]FLT

    Energy Technology Data Exchange (ETDEWEB)

    Buck, Andreas K. [University Hospital Ulm, Department of Nuclear Medicine, Ulm (Germany); Technical University Munich, Department of Nuclear Medicine, Munich (Germany); Kratochwil, Clemens; Glatting, Gerhard; Tepsic, Djurdja; Vogg, Andreas T.J.; Neumaier, Bernd; Reske, Sven N. [University Hospital Ulm, Department of Nuclear Medicine, Ulm (Germany); Juweid, Malik [University of Iowa, Department of Radiology and Holden Comprehensive Cancer Center, Iowa City, IA (United States); Bommer, Martin [University Hospital Ulm, Department of Haematology, Ulm (Germany); Mattfeldt, Torsten; Moeller, Peter [University Hospital Ulm, Institute of Pathology, Ulm (Germany)

    2007-11-15

    The aim of this study was to determine whether the thymidine analogue 3'-deoxy-3'-[{sup 18}F]fluorothymidine ([{sup 18}F]FLT) is adequate for early evaluation of the response of malignant lymphoma to antiproliferative treatment in a mouse xenotransplant model. Immunodeficient mice bearing a follicular lymphoma xenotransplant were treated with high-dose chemotherapy (cyclophosphamide, n = 10), immunotherapy (CD20 mAb, ibritumomab-tiuxetan, n = 10) or radioimmunotherapy ([{sup 90}Y]CD20 mAb, Zevalin, n = 10). Forty-eight hours after treatment, antiproliferative effects were assessed with [{sup 18}F]FLT. Ninety minutes after i.v. injection of 5-10 MBq [{sup 18}F]FLT, mice were sacrificed and radioactivity within the tumour and normal organs was measured using a gamma counter and calculated as % ID/g. The proliferation fraction in tissue samples derived from treated and untreated tumours was evaluated by Ki-67 immunohistochemistry, which served as the reference for proliferative activity. In untreated lymphoma, the mean proliferation fraction was 83.6%. After chemotherapy, the mean proliferation fraction decreased to 39.3% (p = 0.0001), after immunotherapy to 77.6% (p = 0.0078) and after radioimmunotherapy to 78.8% (p = 0.014). In none of the animals was a significant change in tumour size observed. In untreated lymphoma, tumoural [{sup 18}F]FLT uptake was 5.4% ID/g, after chemotherapy it was 1.5% (p = 0.0005), after immunotherapy, 3.9% (non-significant), and after radioimmunotherapy, 5.8% (non-significant). In a lymphoma xenotransplant model, [{sup 18}F]FLT detects early antiproliferative drug activity before changes in tumour size are visible. These findings further support the use of [{sup 18}F]FLT-PET for imaging early response to treatment in malignant lymphoma. (orig.)

  17. Preoperative Chemoradiation for Rectal Cancer Using Capecitabine and Celecoxib Correlated With Posttreatment Assessment of Thymidylate Synthase and Thymidine Phosphorylase Expression

    International Nuclear Information System (INIS)

    Purpose: Thymidylate synthase (TS) and thymidine phosphorylase (TP) expression have been shown to be predictors of response to therapy. The toxicity, efficacy, surgical morbidity, and immunohistochemical TS and TP expression were assessed in surgical resection specimens after preoperative chemoradiation. Methods and Materials: Twenty patients with clinical stage I to III rectal adenocarcinoma received preoperative chemoradiation and underwent surgical resection 6 weeks later. Results: Posttreatment tumor stages were T1 to T2 and N0 in 30% of patients; T3 to T4 and N0 in 30% of patients; and T1 to T3 and N1 to N2 in 15% of patients. Pathologic complete response (pCR) was evident in 25% and tumor regression occurred in a total of 80% of patients. Anal sphincter-sparing surgery was performed in 80% of cases. Acute and perioperative complications were minimal, with no grade 3/4 toxicity or treatment breaks. Pelvic control was obtained in 90% of patients. With a median follow-up of 65.5 months (range, 8-80 months), the 6-year actuarial survival rate was 75%. Local failure was significantly associated with nonresponse to therapy and with high TS and low TP expression (p = 0.008 and p = 0.04, respectively). Conclusions: The combination of capecitabine, celecoxib, and x-radiation therapy yields excellent response: a 25% pathologic pCR, no acute grade 3/4 toxicity, and minimal surgical morbidity. Nonresponders expressed significantly increased TS levels and decreased TP levels in posttreatment resection specimens compared to responders.

  18. Isolation and Characterization of acetate kinase and phosphotransacetylase mutants of Escherichia coli and Salmonella typhimurium.

    OpenAIRE

    Levine, S. M.; Ardeshir, F; Ames, G F

    1980-01-01

    Mutations in the ack (acetate kinase) and pta (phosphotransacetylase) genes in Salmonella typhimurium were characterized and determined to be analogous to those of previously described Escherichia coli mutants. We established that in both bacterial species these genes were cotransducible with the neighboring histidine transport operon and were distally located relative to purF. pta mutants were sensitive to the dye alizarin yellow and were unable to grow on medium containing inositol as a car...

  19. Allosteric Regulation of Histidine Kinases by Their Cognate Response Regulator Determines Cell Fate

    OpenAIRE

    Paul, Ralf; Jaeger, Tina; Abel, Sören; Wiederkehr, Irene; Folcher, Marc; Biondi, Emanuele G.; Laub, Michael T.; Jenal, Urs

    2008-01-01

    The two-component phosphorylation network is of critical importance for bacterial growth and physiology. Here, we address plasticity and interconnection of distinct signal transduction pathways within this network. In Caulobacter crescentus antagonistic activities of the PleC phosphatase and DivJ kinase localized at opposite cell poles control the phosphorylation state and subcellular localization of the cell fate determinator protein DivK. We show that DivK functions as an allosteric regulat...

  20. Heme uptake in bacterial pathogens

    OpenAIRE

    Contreras, Heidi; Chim, Nicholas; Credali, Alfredo; Goulding, Celia W.

    2014-01-01

    Iron is an essential nutrient for the survival of organisms. Bacterial pathogens possess specialized pathways to acquire heme from their human hosts. In this review, we present recent structural and biochemical data that provide mechanistic insights into several bacterial heme uptake pathways, encompassing the sequestration of heme from human hemoproteins to secreted or membrane-associated bacterial proteins, the transport of heme across bacterial membranes, and the degradation of heme within...

  1. Evolutionary transitions in bacterial symbiosis

    OpenAIRE

    Sachs, Joel L.; Skophammer, Ryan G.; Regus, John U.

    2011-01-01

    Diverse bacterial lineages form beneficial infections with eukaryotic hosts. The origins, evolution, and breakdown of these mutualisms represent important evolutionary transitions. To examine these key events, we synthesize data from diverse interactions between bacteria and eukaryote hosts. Five evolutionary transitions are investigated, including the origins of bacterial associations with eukaryotes, the origins and subsequent stable maintenance of bacterial mutualism with hosts, the captur...

  2. PKC and Ca2+Effect of Protein kinase C and Ca2+ on p42/p44 MAPK, Pyk2, and Src Activation in Rat Conjunctival Goblet Cells

    OpenAIRE

    Hodges, Robin R.; Horikawa, Yoshitaka; Rios, Jose D.; Shatos, Marie A.; Dartt, Darlene A.

    2007-01-01

    Conjunctival goblet cells synthesize and secrete mucins onto the ocular surface to lubricate it and protect it from bacterial infections. Mucin secretion is under neural control, and cholinergic agonists released from parasympathetic nerves are major stimuli of this secretion. The signal transduction pathways these agonists use to stimulate secretion involve activating protein kinase C (PKC) and increasing intracellular [Ca2+] to activate the non-receptor kinases Pyk2 and p60Src (Src) to tran...

  3. [Bacterial diseases of rape].

    Science.gov (United States)

    Zakharova, O M; Mel'nychuk, M D; Dankevych, L A; Patyka, V P

    2012-01-01

    Bacterial destruction of the culture was described and its agents identified in the spring and winter rape crops. Typical symptoms are the following: browning of stem tissue and its mucilagization, chlorosis of leaves, yellowing and beginning of soft rot in the place of leaf stalks affixion to stems, loss of pigmentation (violet). Pathogenic properties of the collection strains and morphological, cultural, physiological, and biochemical properties of the agents of rape's bacterial diseases isolated by the authors have been investigated. It was found that all the isolates selected by the authors are highly or moderately aggressive towards different varieties of rape. According to the complex of phenotypic properties 44% of the total number of isolates selected by the authors are related to representatives of the genus Pseudomonas, 37% - to Xanthomonas and 19% - to Pectobacterium. PMID:23293826

  4. Bacterial proteases and virulence

    DEFF Research Database (Denmark)

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    Bacterial pathogens rely on proteolysis for variety of purposes during the infection process. In the cytosol, the main proteolytic players are the conserved Clp and Lon proteases that directly contribute to virulence through the timely degradation of virulence regulators and indirectly by providing...... tolerance to adverse conditions such as those experienced in the host. In the membrane, HtrA performs similar functions whereas the extracellular proteases, in close contact with host components, pave the way for spreading infections by degrading host matrix components or interfering with host cell...... cell. These extracellular proteases are activated in complex cascades involving auto-processing and proteolytic maturation. Thus, proteolysis has been adopted by bacterial pathogens at multiple levels to ensure the success of the pathogen in contact with the human host....

  5. Supramolecular bacterial systems

    OpenAIRE

    Sankaran, Shrikrishnan

    2015-01-01

    For nearly over a decade, a wide variety of dynamic and responsive supramolecular architectures have been investigated and developed to address biological systems. Since the non-covalent interactions between individual molecular components in such architectures are similar to the interactions found in living systems, it was possible to integrate chemically-synthesized and naturally-occurring components to create platforms with interesting bioactive properties. Bacterial cells and recombinant ...

  6. Bacterial transformation of terpenoids

    International Nuclear Information System (INIS)

    Data on the bacterial transformation of terpenoids published in the literature in the past decade are analyzed. Possible pathways for chemo-, regio- and stereoselective modifications of terpenoids are discussed. Considerable attention is given to new technological approaches to the synthesis of terpenoid derivatives suitable for the use in the perfume and food industry and promising as drugs and chiral intermediates for fine organic synthesis. The bibliography includes 246 references

  7. DMPD: Bruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15081522 Bruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signall...ruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? PubmedID 15081522 Title Bruton...'s tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? Authors

  8. Effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin on (3H)-thymidine incorporation into rat liver deoxyribonucleic acid

    International Nuclear Information System (INIS)

    The effect of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) on (3H)-thymidine incorporation into hepatic DNA was studied in rats. In nonhepatectomized male and female animals, incorporation measured at the peak of the first round of liver DNA synthesis after TCDD treatment (10 ug/kg) was similar to that of control animals. In contrast, the first round of (3H) thymidine incorporation after a 1/3 hepatectomy was enhanced 3-fold in TCDD-treated rats. The enhanced response to 1/3 hepatectomy was produced by doses of TCDD ranging from 1 to 30 ug/kg with an apparent ED50 of 5 ug/kg. Enhanced incorporation was observed when the 1/3 hepatectomy was performed 5-10 days after an ED50 dose and it returned to the control level after 20 days. This enhanced response was not preceded by changes in food consumption or hepatic activities of ornithine decarboxylase (ODC), tyrosine aminotransferase (ITAT) or gamma glutamyl transpeptidase (GGT) when compared to respective control values. Also, the enhanced incorporation was not necessarily due to removal of 1/3 of the liver because it was also seen in TCDD-treated rats that were laparotomized. The mechanism of enhancement in laparatomized animals does not appear to involve a diminished response of the liver to the inhibitory effects of adrenal hormones on liver DNA synthesis. This was suggested by the finding that an adrenalectomy prior to the laparotomy did not block the enhanced incorporation of (3H) thymidine into hepatic DNA. The mechanism by which TCDD enhances the first round of liver DNA synthesis after a 1/3 hepatectomy or laparotomy remains to be determined. (author)

  9. Ceramide Kinase Contributes to Proliferation but not to Prostaglandin E2 Formation in Renal Mesangial Cells and Fibroblasts

    Directory of Open Access Journals (Sweden)

    Oleksandr Pastukhov

    2014-06-01

    Full Text Available Background/Aims: Ceramide kinase (CerK catalyzes the generation of the sphingolipid ceramide-1-phosphate (C1P which regulates various cellular functions including cell growth and death, and inflammation. Here, we used a novel catalytic inhibitor of CerK, NVP-231, and CerK knockout cells to investigate the contribution of CerK to proliferation and inflammation in renal mesangial cells and fibroblasts. Methods: Cells were treated with NVP-231 and [3H]-thymidine incorporation into DNA, [3H]-arachidonic acid release, prostaglandin E2 (PGE2 synthesis, cell cycle distribution, and apoptosis were determined. Results: Treatment of rat mesangial cells and mouse renal fibroblasts with NVP-231 decreased DNA synthesis, but not of agonist-stimulated arachidonic acid release or PGE2 synthesis. Similarly, proliferation but not arachidonic acid release or PGE2 synthesis was reduced in CERK knockout renal fibroblasts. The anti-proliferative effect of NVP-231 on mesangial cells was due to M phase arrest as determined using the mitosis markers phospho-histone H3, cdc2 and polo-like kinase-1, and induction of apoptosis. Moreover, loss of CerK sensitized cells towards stress-induced apoptosis. Conclusions: Our data demonstrate that CerK induces proliferation but not PGE2 formation of renal mesangial cells and fibroblasts, and suggest that targeted CerK inhibition has potential for treating mesangioproliferative kidney diseases.

  10. RIP Kinases Initiate Programmed Necrosis

    Institute of Scientific and Technical Information of China (English)

    Lorenzo Galluzzi; Oliver Kepp; Guido Kroemer

    2009-01-01

    Some lethal stimuli can induce either apoptosis or necrosis, depending on the cell type and/or experimental setting. Until recently,the molecular bases of this phenomenon were largely unknown. Now, two members of the receptor-interacting serine-threonine kinase (RIP) family, RIP1 and RIP3, have been demonstrated to control the switch between apoptotic and necrotic cell death.Some mechanistic details, however, remain controversial.

  11. Pantothenate Kinase-Associated Neurodegeneration

    OpenAIRE

    Meitinger, Thomas; Prokisch, Holger; Hartig, Monika B.; Klopstock, Thomas

    2012-01-01

    Pantothenate kinase-associated neurodegeneration (PKAN) is a hereditary progressive disorder and the most frequent form of neurodegeneration with brain iron accumulation (NBIA). PKAN patients present with a progressive movement disorder, dysarthria, cognitive impairment and retinitis pigmentosa. In magnetic resonance imaging, PKAN patients exhibit the pathognonomic "eye of the tiger" sign in the globus pallidus which corresponds to iron accumulation and gliosis as shown in neuropathological e...

  12. Oncoprotein protein kinase antibody kit

    Science.gov (United States)

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2008-12-23

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  13. Regulation and function of TPL-2,an IκB kinase-regulated MAP kinase kinase kinase

    Institute of Scientific and Technical Information of China (English)

    Thorsten Gantke; Srividya Sriskantharajah; Steven C Ley

    2011-01-01

    The IκB kinase(IKK)complex plays a well-documented role in innate and adaptive immunity.This function has been widely attributed to its role as the central activator of the NF-κB family of transcription factors.However,another important consequence of IKK activation is the regulation of TPL-2,a MEK kinase that is required for activation of ERK-1/2 MAP kinases in myeioid cells following Toll-like receptor and TNF receptor stimulation.In unstimulated cells,TPL-2 is stoichiometrically complexed with the NF-κB inhibitory protein NF-κB1 p105,which blocks TPL-2 access to its substrate MEK,and the ubiquitin-binding protein ABIN-2(A20-binding inhibitor of NF-κB 2),both of which are required to maintain TPL-2 protein stability.Following agonist stimulation,the IKK complex phosphorylates p105,triggering its K48-1inked ubiquitination and degradation by the proteasome.This releases TPL-2 from p105-mediated inhibition,facilitating activation of MEK,in addition to modulating NF-κB activation by liberating associated Rel subunits for translocation into the nucleus.IKK-induced proteolysis of 0105,therefore,can directly regulate both NF-κB and ERK MAP kinase activation via NF-κB1 p105.TPL-2 is critical for production of the proinflammatory cytokine TNF during inflammatory responses.Consequently,there has been considerable interest in the pharmaceutical industry to develop selective TPL-2 inhibitors as drugs for the treatment of TNF-dependent inflammatory,diseases,such as rheumatoid arthritis and inflammatory bowel disease.This review summarizes our current understanding of the regulation of TPL-2 signaling function,and also the complex positive and negative roles of TPL-2 in immune and inflammatory responses.

  14. Structural diversity of nucleoside phosphonic acids as a key factor in the discovery of potent inhibitors of rat T-cell lymphoma thymidine phosphorylase

    Czech Academy of Sciences Publication Activity Database

    Kočalka, Petr; Rejman, Dominik; Vaněk, Václav; Rinnová, Markéta; Tomečková, Ivana; Králíková, Šárka; Petrová, Magdalena; Páv, Ondřej; Pohl, Radek; Buděšínský, Miloš; Liboska, Radek; Točík, Zdeněk; Panova, Natalya; Votruba, Ivan; Rosenberg, Ivan

    2010-01-01

    Roč. 20, č. 3 (2010), s. 862-865. ISSN 0960-894X R&D Projects: GA ČR GA203/09/0820; GA ČR GA202/09/0193; GA MŠk(CZ) LC06061; GA MŠk(CZ) LC06077; GA MŠk 2B06065; GA AV ČR KAN200520801; GA AV ČR 1QS400550501; GA MZd NR9138 Institutional research plan: CEZ:AV0Z40550506 Keywords : phosphonate nucleotides * pyrrolidine * thymidine phosphorylase Subject RIV: CC - Organic Chemistry Impact factor: 2.661, year: 2010

  15. Dihydrothymidine and thymidine glycol triphosphates as substrates for DNA polymerases: differential recognition of thymine C5-C6 bond saturation and sequence specificity of incorporation.

    OpenAIRE

    Ide, H; Wallace, S. S.

    1988-01-01

    The ability of dihydrothymidine (DHdTTP) and thymidine glycol (dTTP-GLY) 5'-triphosphates to serve as substrates for different DNA polymerases was investigated. DHdTTP but not dTTP-GLY was used as a substrate by E. coli DNA polymerase I (Pol I). Within the detection limit of the assay used, neither T4 DNA polymerase nor avian myeloblastosis virus (AMV) reverse transcriptase used DHdTTP or dTTP-GLY as substrates. The ability of DHdTTP and dTTP-GLY to undergo enzyme-catalyzed turnover to the mo...

  16. Increased sensitivity to the prodrug 5'-deoxy-5-fluorouridine and modulation of 5-fluoro-2'-deoxyuridine sensitivity in MCF-7 cells transfected with thymidine phosphorylase.

    OpenAIRE

    Patterson, A V; Zhang, H.; Moghaddam, A; Bicknell, R.; Talbot, D. C.; Stratford, I. J.; Harris, A. L.

    1995-01-01

    Platelet-derived endothelial cell growth factor (PD-ECGF) is identical to human thymidine phosphorylase (dThdPase). The human MCF-7 breast cancer cell line was transfected with the dThdPase cDNA and expressed a 45 kDa protein that was detected with anti-dThdPase antibody. Cell lysates possessed elevated dThdPase activity and cells had up to 165-fold increased sensitivity to the prodrug 5'-deoxy-5-fluorouridine (5'-DFUR) in vitro. Sensitivity to 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridi...

  17. Effects of methylprednisolone on concanavalin A-induced human lymphocyte blastogenesis: a comparative analysis by flow cytometry, volume determination and 3H-thymidine incorporation

    International Nuclear Information System (INIS)

    The inhibition of concanavalin A-induced human peripheral blood lymphocyte blastogenesis by methylprednisolone (MP) was studied by using flow cytometry and tritiated thymidine (3H-TdR) incorporation. Flow cytometric determinations of volume, low angle forward light scatter, and nucleic acid showed MP to be a potent inhibitor of blastogenesis. The effects were concentration-dependent and correlated with 3H-TdR uptake. By using the single cell analytic capability of flow cytometry, the target stages of the cell cycle where MP affects lymphocyte activation were determined. Evidence is presented that steroids can block both early and late phases of this process

  18. A proteomic approach for comprehensively screening substrates of protein kinases such as Rho-kinase.

    Directory of Open Access Journals (Sweden)

    Mutsuki Amano

    Full Text Available BACKGROUND: Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored. METHODOLOGY/PRINCIPAL FINDINGS: We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.

  19. A role for the Tec family kinase ITK in regulating SEB induced Interleukin-2 production in vivo via c-jun phosphorylation

    OpenAIRE

    Henderson Andrew J; Hu Jianfang; Ragin Melanie J; August Avery

    2005-01-01

    Abstract Background Exposure to Staphylococcal Enterotoxin B (SEB), a bacterial superantigen secreted by the Gram-positive bacteria Staphyloccocus aureus, results in the expansion and eventual clonal deletion and anergy of Vβ8+ T cells, as well as massive cytokine release, including Interleukin-2 (IL-2). This IL-2 is rapidly secreted following exposure to SEB and may contribute to the symptoms seen following exposure to this bacterial toxin. The Tec family kinase ITK has been shown to be impo...

  20. Induction of Central Host Signaling Kinases during Pneumococcal Infection of Human THP-1 Cells.

    Science.gov (United States)

    Kohler, Thomas P; Scholz, Annemarie; Kiachludis, Delia; Hammerschmidt, Sven

    2016-01-01

    Streptococcus pneumoniae is a widespread colonizer of the mucosal epithelia of the upper respiratory tract of human. However, pneumococci are also responsible for numerous local as well as severe systemic infections, especially in children under the age of five and the elderly. Under certain conditions, pneumococci are able to conquer the epithelial barrier, which can lead to a dissemination of the bacteria into underlying tissues and the bloodstream. Here, specialized macrophages represent an essential part of the innate immune system against bacterial intruders. Recognition of the bacteria through different receptors on the surface of macrophages leads thereby to an uptake and elimination of bacteria. Accompanied cytokine release triggers the migration of leukocytes from peripheral blood to the site of infection, where monocytes differentiate into mature macrophages. The rearrangement of the actin cytoskeleton during phagocytosis, resulting in the engulfment of bacteria, is thereby tightly regulated by receptor-mediated phosphorylation cascades of different protein kinases. The molecular cellular processes including the modulation of central protein kinases are only partially solved. In this study, the human monocytic THP-1 cell line was used as a model system to examine the activation of Fcγ and complement receptor-independent signal cascades during infection with S. pneumoniae. Pneumococci cultured either in chemically defined or complex medium showed no significant differences in pneumococcal phagocytosis by phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 cells. Double immuno-fluorescence microscopy and antibiotic protection assays demonstrated a time-dependent uptake and killing of S. pneumoniae 35A inside of macrophages. Infections of THP-1 cells in the presence of specific pharmacological inhibitors revealed a crucial role of actin polymerization and importance of the phosphoinositide 3-kinase (PI3K) and Protein kinase B (Akt) as well during

  1. Induction of central host signalling kinases during pneumococcal infection of human THP-1 cells

    Directory of Open Access Journals (Sweden)

    Thomas Peter Kohler

    2016-04-01

    Full Text Available Streptococcus pneumoniae is a widespread colonizer of the mucosal epithelia of the upper respiratory tract of human. However, pneumococci are also responsible for numerous local as well as severe systemic infections, especially in children under the age of five and the elderly. Under certain conditions, pneumococci are able to conquer the epithelial barrier, which can lead to a dissemination of the bacteria into underlying tissues and the bloodstream. Here, specialized macrophages represent an essential part of the innate immune system against bacterial intruders. Recognition of the bacteria through different receptors on the surface of macrophages leads thereby to an uptake and elimination of bacteria. Accompanied cytokine release triggers the migration of leukocytes from peripheral blood to the site of infection, where monocytes differentiate into mature macrophages. The rearrangement of the actin cytoskeleton during phagocytosis, resulting in the engulfment of bacteria, is thereby tightly regulated by receptor-mediated phosphorylation cascades of different protein kinases. The molecular cellular processes including the modulation of central protein kinases are only partially solved. In this study, the human monocytic THP-1 cell line was used as a model system to examine the activation of Fcγ and complement receptor-independent signal cascades during infection with S. pneumoniae. Pneumococci cultured either in chemically defined or complex medium showed no significant differences in pneumococcal phagocytosis by phorbol 12-myristate 13-acetate (PMA differentiated THP-1 cells. Double immuno-fluorescence microscopy and antibiotic protection assays demonstrated a time-dependent uptake and killing of S. pneumoniae 35A inside of macrophages. Infections of THP-1 cells in the presence of specific pharmacological inhibitors revealed a crucial role of actin polymerization and importance of the phosphoinositide 3-kinase (PI3K and Protein kinase B (Akt

  2. Immunochemical characterization of rat brain protein kinase

    International Nuclear Information System (INIS)

    Polyclonal antibodies against rat brain protein kinase C (the Ca2+/phospholipid-dependent enzyme) were raised in goat. These antibodies can neutralize completely the kinase activity in purified enzyme preparation as well as that in the crude homogenate. Immunoblot analysis of the purified and the crude protein kinase C preparations revealed a major immunoreactive band of 80 kDa. The antibodies also recognize the same enzyme from other rat tissues. Neuronal tissues (cerebral cortex, cerebellum, hypothalamus, and retina) and lymphoid organs (thymus and spleen) were found to be enriched in protein kinase C, whereas lung, kidney, liver, heart, and skeletal muscle contained relatively low amounts of this kinase. Limited proteolysis of the purified rat brain protein kinase C with trypsin results in an initial degradation of the kinase into two major fragments of 48 and 38 kDa. Both fragments are recognized by the antibodies. However, further digestion of the 48-kDa fragment to 45 kDa and the 38-kDa fragment to 33 kDa causes a loss of the immunoreactivity. Upon incubation of the cerebellar extract with Ca2+, the 48-kDa fragment was also identified as a major proteolytic product of protein kinase C. Proteolytic degradation of protein kinase C converts the Ca2+/phospholipid-dependent kinase to an independent form without causing a large impairment of the binding of [3H]phorbol 12,13-dibutyrate. The two major proteolytic fragments were separated by ion exchange chromatography and one of them (45-48 kDa) was identified as a protein kinase and the other (33-38 kDa) as a phorbol ester-binding protein. These results demonstrate that rat brain protein kinase C is composed of two functionally distinct units, namely, a protein kinase and a Ca2+-independent/phospholipid-dependent phorbol ester-binding protein

  3. Bacterial Degradation of Pesticides

    DEFF Research Database (Denmark)

    Knudsen, Berith Elkær

    This PhD project was carried out as part of the Microbial Remediation of Contaminated Soil and Water Resources (MIRESOWA) project, funded by the Danish Council for Strategic Research (grant number 2104-08-0012). The environment is contaminated with various xenobiotic compounds e.g. pesticides......D student, to construct fungal-bacterial consortia in order to potentially stimulate pesticide degradation thereby increasing the chance of successful bioaugmentation. The results of the project are reported in three article manuscripts, included in this thesis. In manuscript I, the mineralization of 2...

  4. Bacterial mitotic machineries

    DEFF Research Database (Denmark)

    Gerdes, Kenn; Møller-Jensen, Jakob; Ebersbach, Gitte; Kruse, Torben; Nordström, Kurt

    2004-01-01

    Here, we review recent progress that yields fundamental new insight into the molecular mechanisms behind plasmid and chromosome segregation in prokaryotic cells. In particular, we describe how prokaryotic actin homologs form mitotic machineries that segregate DNA before cell division. Thus, the P......M protein of plasmid R1 forms F actin-like filaments that separate and move plasmid DNA from mid-cell to the cell poles. Evidence from three different laboratories indicate that the morphogenetic MreB protein may be involved in segregation of the bacterial chromosome....

  5. Bacterial terpene cyclases.

    Science.gov (United States)

    Dickschat, Jeroen S

    2016-01-01

    Covering: up to 2015. This review summarises the accumulated knowledge about characterised bacterial terpene cyclases. The structures of identified products and of crystallised enzymes are included, and the obtained insights into enzyme mechanisms are discussed. After a summary of mono-, sesqui- and diterpene cyclases the special cases of the geosmin and 2-methylisoborneol synthases that are both particularly widespread in bacteria will be presented. A total number of 63 enzymes that have been characterised so far is presented, with 132 cited references. PMID:26563452

  6. Comprehensive Characterization of AMP-Activated Protein Kinase Catalytic Domain by Top-Down Mass Spectrometry

    Science.gov (United States)

    Yu, Deyang; Peng, Ying; Ayaz-Guner, Serife; Gregorich, Zachery R.; Ge, Ying

    2016-02-01

    AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is essential in regulating energy metabolism in all eukaryotic cells. It is a heterotrimeric protein complex composed of a catalytic subunit (α) and two regulatory subunits (β and γ). C-terminal truncation of AMPKα at residue 312 yielded a protein that is active upon phosphorylation of Thr172 in the absence of β and γ subunits, which is refered to as the AMPK catalytic domain and commonly used to substitute for the AMPK heterotrimeric complex in in vitro kinase assays. However, a comprehensive characterization of the AMPK catalytic domain is lacking. Herein, we expressed a His-tagged human AMPK catalytic domin (denoted as AMPKΔ) in E. coli, comprehensively characterized AMPKΔ in its basal state and after in vitro phosphorylation using top-down mass spectrometry (MS), and assessed how phosphorylation of AMPKΔ affects its activity. Unexpectedly, we found that bacterially-expressed AMPKΔ was basally phosphorylated and localized the phosphorylation site to the His-tag. We found that AMPKΔ had noticeable basal activity and was capable of phosphorylating itself and its substrates without activating phosphorylation at Thr172. Moreover, our data suggested that Thr172 is the only site phosphorylated by its upstream kinase, liver kinase B1, and that this phosphorylation dramatically increases the kinase activity of AMPKΔ. Importantly, we demonstrated that top-down MS in conjunction with in vitro phosphorylation assay is a powerful approach for monitoring phosphorylation reaction and determining sequential order of phosphorylation events in kinase-substrate systems.

  7. Regulation of the interaction between protein kinase C-related protein kinase 2 (PRK2) and its upstream kinase, 3-phosphoinositide-dependent protein kinase 1 (PDK1)

    DEFF Research Database (Denmark)

    Dettori, Rosalia; Sonzogni, Silvina; Meyer, Lucas;

    2009-01-01

    The members of the AGC kinase family frequently exhibit three conserved phosphorylation sites: the activation loop, the hydrophobic motif (HM), and the zipper (Z)/turn-motif (TM) phosphorylation site. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates the activation loop of...... numerous AGC kinases, including the protein kinase C-related protein kinases (PRKs). Here we studied the docking interaction between PDK1 and PRK2 and analyzed the mechanisms that regulate this interaction. In vivo labeling of recombinant PRK2 by (32)P(i) revealed phosphorylation at two sites, the...... the mechanism that negatively regulates the docking interaction of PRK2 to the upstream kinase PDK1 is directly linked to the activation mechanism of PRK2 itself. Finally, our results indicate that the mechanisms underlying the regulation of the interaction between PRK2 and PDK1 are specific for PRK2...

  8. Bacterial contamination of enteral diets.

    OpenAIRE

    de Leeuw, I H; Vandewoude, M F

    1986-01-01

    Enteral feeding solutions can be contaminated by bacterial micro-organisms already present in the ingredients, or introduced during preparation or transport, or in the hospital ward. During jejunostomy feeding without pump or filter, ascending bacterial invasion of the feeding bag is possible. In patients with lowered immune response contaminated feedings can cause serious septic clinical problems. The progressive loss of the nutritional value of the enteral feeding solution by bacterial cont...

  9. Transport powered by bacterial turbulence

    OpenAIRE

    Kaiser, Andreas; Peshkov, Anton; Sokolov, Andrey; ten Hagen, Borge; Löwen, Hartmut; Aranson, Igor S.

    2014-01-01

    We demonstrate that collective turbulent-like motion in a bacterial bath can power and steer directed transport of mesoscopic carriers through the suspension. In our experiments and simulations, a microwedge-like "bulldozer" draws energy from a bacterial bath of varied density. We obtain that a maximal transport speed is achieved in the turbulent state of the bacterial suspension. This apparent rectification of random motion of bacteria is caused by polar ordered bacteria inside the cusp regi...

  10. Spontaneous bacterial peritonitis

    Institute of Scientific and Technical Information of China (English)

    Anastasios Koulaouzidis; Shivaram Bhat; Athar A Saeed

    2009-01-01

    Since its initial description in 1964, research has transformed spontaneous bacterial peritonitis (SBP) from a feared disease (with reported mortality of 90%) to a treatable complication of decompensated cirrhosis,albeit with steady prevalence and a high recurrence rate. Bacterial translocation, the key mechanism in the pathogenesis of SBP, is only possible because of the concurrent failure of defensive mechanisms in cirrhosis.Variants of SBP should be treated. Leucocyte esterase reagent strips have managed to shorten the 'tap-toshot' time, while future studies should look into their combined use with ascitic fluid pH. Third generation cephalosporins are the antibiotic of choice because they have a number of advantages. Renal dysfunction has been shown to be an independent predictor of mortality in patients with SBP. Albumin is felt to reduce the risk of renal impairment by improving effective intravascular volume, and by helping to bind proinflammatory molecules. Following a single episode of SBP, patients should have long-term antibiotic prophylaxis and be considered for liver transplantation.

  11. Adult bacterial meningitis

    DEFF Research Database (Denmark)

    Meyer, C N; Samuelsson, I S; Galle, M;

    2004-01-01

    Episodes of adult bacterial meningitis (ABM) at a Danish hospital in 1991-2000 were identified from the databases of the Department of Clinical Microbiology, and compared with data from the Danish National Patient Register and the Danish National Notification System. Reduced penicillin susceptibi......Episodes of adult bacterial meningitis (ABM) at a Danish hospital in 1991-2000 were identified from the databases of the Department of Clinical Microbiology, and compared with data from the Danish National Patient Register and the Danish National Notification System. Reduced penicillin...... susceptibility occurred in 21 (23%) of 92 cases of known aetiology, compared to an estimated 6% in nationally notified cases (p <0.001). Ceftriaxone plus penicillin as empirical treatment was appropriate in 97% of ABM cases in the study population, and in 99.6% of nationally notified cases. The notification rate...... was 75% for penicillin-susceptible episodes, and 24% for penicillin-non-susceptible episodes (p <0.001). Cases involving staphylococci, Pseudomonas spp. and Enterobacteriaceae were under-reported. Among 51 ABM cases with no identified risk factors, nine of 11 cases with penicillin...

  12. [Endogenous bacterial endophthalmitis].

    Science.gov (United States)

    Cornut, P-L; Chiquet, C

    2011-01-01

    Endogenous bacterial endophthalmitis, also called metastatic bacterial endophthalmitis, remains a diagnostic and therapeutic challenge. It is a rare and potentially sight-threatening ocular infection that occurs when bacteria reach the eye via the bloodstream, cross the blood-ocular barrier, and multiply within the eye. It usually affects immunocompromised patients and those suffering from diabetes mellitus, malignancy, or cardiac disease, but has also been reported after invasive procedures or in previously healthy people. In most cases, the ocular symptoms occur after the diagnosis of septicemia or systemic infection. Ocular symptoms include decreased vision, redness, discharge, pain, and floaters. The ocular inflammatory signs may be anterior and/or posterior. Bilateral involvement occurs in nearly 25% of cases. A wide range of microorganisms are involved, with differences in their frequency according to geography as well as the patient's age and past medical history, because of variations in the predisposing conditions and the source of the sepsis. The majority of patients are initially misdiagnosed, and ophthalmologists should be aware of this because prompt local and general management is required to save the eye and/or the patient's life. PMID:21145128

  13. Diacylglycerol Kinase Inhibition and Vascular Function

    OpenAIRE

    Choi, Hyehun; Allahdadi, Kyan J.; Tostes, Rita C A; Webb, R. Clinton

    2009-01-01

    Diacylglycerol kinases (DGKs), a family of lipid kinases, convert diacylglycerol (DG) to phosphatidic acid (PA). Acting as a second messenger, DG activates protein kinase C (PKC). PA, a signaling lipid, regulates diverse functions involved in physiological responses. Since DGK modulates two lipid second messengers, DG and PA, regulation of DGK could induce related cellular responses. Currently, there are 10 mammalian isoforms of DGK that are categorized into five groups based on their structu...

  14. Periodic growth of bacterial colonies

    Science.gov (United States)

    Yamazaki, Yoshihiro; Ikeda, Takemasa; Shimada, Hirotoshi; Hiramatsu, Fumiko; Kobayashi, Naoki; Wakita, Jun-ichi; Itoh, Hiroto; Kurosu, Sayuri; Nakatsuchi, Michio; Matsuyama, Tohey; Matsushita, Mitsugu

    2005-06-01

    The formation of concentric ring colonies by bacterial species Bacillus subtilis and Proteus mirabilis has been investigated experimentally, focusing our attention on the dependence of local cell density upon the bacterial motility. It has been confirmed that these concentric ring colonies reflect the periodic change of the bacterial motility between motile cell state and immotile cell state. We conclude that this periodic change is macroscopically determined neither by biological factors (i.e., biological clock) nor by chemical factors (chemotaxis as inhibitor). And our experimental results strongly suggest that the essential factor for the change of the bacterial motility during concentric ring formation is the local cell density.

  15. Allosteric small-molecule kinase inhibitors

    DEFF Research Database (Denmark)

    Wu, Peng; Clausen, Mads Hartvig; Nielsen, Thomas E.

    2015-01-01

    Small-molecule kinase inhibitors are invaluable targeted therapeutics for the treatment of various human diseases, especially cancers. While the majority of approved and developed preclinical small-molecule inhibitors are characterized as type I or type II inhibitors that target the ATP......-binding pocket of kinases, the remarkable sequential and structural similarity among ATP pockets renders the selective inhibition of kinases a daunting challenge. Therefore, targeting allosteric pockets of kinases outside the highly conversed ATP pocket has been proposed as a promising alternative to overcome...

  16. MST kinases in development and disease

    OpenAIRE

    Thompson, Barry J.; Sahai, Erik

    2015-01-01

    The mammalian MST kinase family, which is related to the Hippo kinase in Drosophila melanogaster, includes five related proteins: MST1 (also called STK4), MST2 (also called STK3), MST3 (also called STK24), MST4, and YSK1 (also called STK25 or SOK1). MST kinases are emerging as key signaling molecules that influence cell proliferation, organ size, cell migration, and cell polarity. Here we review the regulation and function of these kinases in normal physiology and pathologies, including cance...

  17. Algal and bacterial activities in acidic (pH 3) strip mine lakes

    International Nuclear Information System (INIS)

    Reservoir 29 and Lake B are extremely acid lakes (epilimnion pHs of 2.7 and 3.2, respectively), because they receive acidic discharges from coal refuse piles. They differ in that the pH of profundal sediments in Reservoir 29 increased from 2.7 to 3.8 during the period of thermal stratification, whereas permanently anoxic sediments in Lake B had a pH of 6.2. The pH rise in Reservoir 29 sediments was correlated with a temporal increase in H2S concentration in the anaerobic hypolimnion from 0 to >1 mM. The chlorophyll a levels in the epilimnion of Reservoir 29 were low, and the rate of primary production was typical of an oligotrophic system. However, there was a dense 10-cm layer of algal biomass at the bottom of the metalimnion. Production by this layer was low owing to light limitation and possibly H2S toxicity. The specific photosynthetic rates of epilimnetic algae were low, which suggests that nutrient availability is more important than pH in limiting production. The highest photosynthetic rates were obtained in water samples incubated at pH 2.7 to 4. Heterotrophic bacterial activity (measured by [14C]glucose metabolism) was greatest at the sediment/water interface. Bacterial production (assayed by thymidine incorporation) was as high in Reservoir 29 as in a nonacid mesotrophic Indiana lake

  18. Seeding Collaborations to Advance Kinase Science with the GSK Published Kinase Inhibitor Set (PKIS)

    OpenAIRE

    Drewry, David H.; Willson, Timothy M.; Zuercher, William J

    2014-01-01

    To catalyze research on historically untargeted protein kinases, we created the PKIS, an annotated set of 367 small molecule kinase inhibitors. The set has been widely distributed to academic collaborators as an open access tool. It has been used to identify chemical starting points for development of chemical probes for orphan kinases and to investigate kinase signaling in high content phenotypic assays. Access to the set comes with few restrictions other than the requirement that assay resu...

  19. Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates.

    Science.gov (United States)

    Xue, Liang; Wang, Wen-Horng; Iliuk, Anton; Hu, Lianghai; Galan, Jacob A; Yu, Shuai; Hans, Michael; Geahlen, Robert L; Tao, W Andy

    2012-04-10

    Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity. PMID:22451900

  20. Comparison of Peptide Array Substrate Phosphorylation of c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8

    NARCIS (Netherlands)

    Parikh, Kaushal; Diks, Sander H.; Tuynman, Jurriaan H. B.; Verhaar, Auke; Lowenberg, Mark; Hommes, Daan W.; Joore, Jos; Pandey, Akhilesh; Peppelenbosch, Maikel P.

    2009-01-01

    Kinases are pivotal regulators of cellular physiology. The human genome contains more than 500 putative kinases, which exert their action via the phosphorylation of specific substrates. The determinants of this specificity are still only partly understood and as a consequence it is difficult to pred

  1. [Small intestine bacterial overgrowth].

    Science.gov (United States)

    Leung Ki, E L; Roduit, J; Delarive, J; Guyot, J; Michetti, P; Dorta, G

    2010-01-27

    Small intestine bacterial overgrowth (SIBO) is a condition characterised by nutrient malabsorption and excessive bacteria in the small intestine. It typically presents with diarrhea, flatulence and a syndrome of malabsorption (steatorrhea, macrocytic anemia). However, it may be asymptomatic in the eldery. A high index of suspicion is necessary in order to differentiate SIBO from other similar presenting disorders such as coeliac disease, lactose intolerance or the irritable bowel syndrome. A search for predisposing factor is thus necessary. These factors may be anatomical (stenosis, blind loop), or functional (intestinal hypomotility, achlorydria). The hydrogen breath test is the most frequently used diagnostic test although it lacks standardisation. The treatment of SIBO consists of eliminating predisposing factors and broad-spectrum antibiotic therapy. PMID:20214190

  2. Studying bacterial multispecies biofilms

    DEFF Research Database (Denmark)

    Røder, Henriette Lyng; Sørensen, Søren Johannes; Burmølle, Mette

    2016-01-01

    The high prevalence and significance of multispecies biofilms have now been demonstrated in various bacterial habitats with medical, industrial, and ecological relevance. It is highly evident that several species of bacteria coexist and interact in biofilms, which highlights the need for evaluating...... the approaches used to study these complex communities. This review focuses on the establishment of multispecies biofilms in vitro, interspecies interactions in microhabitats, and how to select communities for evaluation. Studies have used different experimental approaches; here we evaluate the...... benefits and drawbacks of varying the degree of complexity. This review aims to facilitate multispecies biofilm research in order to expand the current limited knowledge on interspecies interactions. Recent technological advances have enabled total diversity analysis of highly complex and diverse microbial...

  3. Molecular approaches for bacterial azoreductases

    Directory of Open Access Journals (Sweden)

    Montira Leelakriangsak

    2013-12-01

    Full Text Available Azo dyes are the dominant types of synthetic dyes, widely used in textiles, foods, leather, printing, tattooing, cosmetics, and pharmaceutical industries. Many microorganisms are able to decolorize azo dyes, and there is increasing interest in biological waste treatment methods. Bacterial azoreductases can cleave azo linkages (-N=N- in azo dyes, forming aromatic amines. This review mainly focuses on employing molecular approaches, including gene manipulation and recombinant strains, to study bacterial azoreductases. The construction of the recombinant protein by cloning and the overexpression of azoreductase is described. The mechanisms and function of bacterial azoreductases can be studied by other molecular techniques discussed in this review, such as RT-PCR, southern blot analysis, western blot analysis, zymography, and muta-genesis in order to understand bacterial azoreductase properties, function and application. In addition, understanding the regulation of azoreductase gene expression will lead to the systematic use of gene manipulation in bacterial strains for new strategies in future waste remediation technologies.

  4. Induction of Macrophage Function in Human THP-1 Cells is Associated with MAPK Signaling and Activation of MAP3K7 (TAK1 Protein Kinase

    Directory of Open Access Journals (Sweden)

    Erik eRichter

    2016-03-01

    Full Text Available Macrophages represent the primary human host response to pathogen infection and link the immediate defense to the adaptive immune system. Mature tissue macrophages convert from circulating monocyte precursor cells by terminal differentiation in a process that is not fully understood. Here, we analyzed the protein kinases of the human monocytic cell line THP-1 before and after induction of macrophage differentiation by using kinomics and phosphoproteomics. When comparing the macrophage-like state with the monocytic precursor, 50% of the kinome was altered in expression and even 71% of covered kinase phosphorylation sites were affected. Kinome rearrangements are for example characterized by a shift of overrepresented cycline-dependent kinases associated with cell cycle control in monocytes to calmodulin-dependent kinases and kinases involved in proinflammatory signaling. Eventually, we show that monocyte-to-macrophage differentiation is associated with major rewiring of mitogen-activated protein kinase signaling networks and demonstrate that protein kinase MAP3K7 (TAK1 acts as the key signaling hub in bacterial killing, chemokine production and differentiation. Our study proves the fundamental role of protein kinases and cellular signaling as major drivers of macrophage differentiation and function. The finding that MAP3K7 is central to macrophage function suggests MAP3K7 and its networking partners as promising targets in host-directed therapy for macrophage-associated disease.

  5. Genetics Home Reference: pyruvate kinase deficiency

    Science.gov (United States)

    ... National (UK) Information Centre for Metabolic Diseases National Organization for Rare Disorders (NORD): Pyruvate Kinase Deficiency Genetic Testing Registry (1 link) Pyruvate kinase deficiency of red cells Scientific articles on PubMed (1 link) PubMed OMIM (1 link) ...

  6. A multisubstrate deoxyribonucleoside kinase from plants

    DEFF Research Database (Denmark)

    Clausen, Anders R.; Girandon, Lenart; Knecht, Wolfgang;

    2008-01-01

    Deoxyribonucleoside kinases catalyze the rate limiting step during the salvage of deoxyribonucleosides and convert them into the corresponding monophosphate compounds. We have identified and characterized a unique multisubstrate deoxyribonucleoside kinase from plants. The phylogenetic relationship...... suicide gene in anti-cancer therapy....

  7. On the DNA polymerase-a mutant: Immunofluorescence assay of UV-induced thymidine dimers in Aphr-4-2 cells

    International Nuclear Information System (INIS)

    Aphidicolin inhibits purified DNA polymerases-a and -d in vitro and inhibits mitosis in animal cells. The Chinese hamster V79 cell mutant, Aphr-4-2, was selected for its ability to form colonies in cultured medium supplemented with 1.0 microM aphidicolin. At this concentration, the parental wild-type V79 cells (clone 743x) have a survival rate of less than 10(-7). The mutant DNA polymerase-a is resistant to aphidicolin at concentrations that are inhibitory to the wild-type V79 DNA polymerase-a. The apparent Km for dCTP of the mutant DNA polymerase-a is consistently lower than that of the wild-type DNA polymerase-a. This mutant exhibits slow growth, mutator activity, hypersensitivity, and hypermutability to UV. We wanted to know the basis of UV hypersensitivity in this mutant. Using the antisera (UV2) raised against UV-induced thymidine dimers and a sensitive immunofluorescence assay to measure UV-induced thymidine dimers and with detection in ACAS 570 Workstation, we observed that 50% of the thymidine dimers disappeared within 5 h after irradiation and more than 80% of the dimers were removed within 24 h in both cell lines. These results indicate that the recognition, incision, and excision steps in nucleotide excision repair pathway are normal in the mutant. In order to know if there is a difference in DNA polymerase-a or -d activities in the parental V79(wt) and Aphr-4-2 cells, DNA polymerases were partially purified from the parental and the mutant cells using sequential centrifugation and column chromatographies on DEAE-cellulose (DE23 and DE52) to remove DNA polymerases-beta and -gamma. More than 90% of the enzymatic activities from both cells showed characteristics of DNA polymerase-a type on the basis of these criteria: sensitivity to butyl phenyl dGTP (1 microM) and to IgG raised against DNA polymerase-a (SJK 132-20)

  8. Influence of irradiation or thymidine (TdR) on the pattern of 3H-Tdsub(R) incorporation at each cell position in the crypts of the small intestine of the mouse

    International Nuclear Information System (INIS)

    Using autoradiography it was noted that S phase cells at the bottom of the crypts in the small intestine were the most efficient scavengers of exogenous injected thymidine. The efficiency of the incorporation of 3H-TdR (salvage pathway of DNA synthesis) by cells at the crypt base (stem cell zone) was twice as high as for the S phase cells at the top of the crypt (maturing proliferative cells). There were no such position-dependent differences in incorporation of 3H-UdR (de novo pathway of DNA synthesis). Radiation (0.75-5.0 Gy 137Cs γ-rays) inhibited incorporation of 3H-TdR very rapidly and was also cell-position dependent, the cells at the bottom of the crypt being most affected. Injection of cold thymidine before 3H-TdR changed the pattern of the incorporation of 3H-TdR along the side of the crypt in a very similar way to radiation, and the grain number was decreased predominantly in the cells at lower positions. The possibility of a regional gradient of endogenous thymidine (reutilization from intestinal sources), and the influence of irradiation on the gradient of thymidine incorporation resulting from direct and abscopal effects of whole body exposure, are discussed. (author)

  9. Protein kinases associated with the yeast phosphoproteome

    Directory of Open Access Journals (Sweden)

    Munn Alan L

    2006-01-01

    Full Text Available Abstract Background Protein phosphorylation is an extremely important mechanism of cellular regulation. A large-scale study of phosphoproteins in a whole-cell lysate of Saccharomyces cerevisiae has previously identified 383 phosphorylation sites in 216 peptide sequences. However, the protein kinases responsible for the phosphorylation of the identified proteins have not previously been assigned. Results We used Predikin in combination with other bioinformatic tools, to predict which of 116 unique protein kinases in yeast phosphorylates each experimentally determined site in the phosphoproteome. The prediction was based on the match between the phosphorylated 7-residue sequence and the predicted substrate specificity of each kinase, with the highest weight applied to the residues or positions that contribute most to the substrate specificity. We estimated the reliability of the predictions by performing a parallel prediction on phosphopeptides for which the kinase has been experimentally determined. Conclusion The results reveal that the functions of the protein kinases and their predicted phosphoprotein substrates are often correlated, for example in endocytosis, cytokinesis, transcription, replication, carbohydrate metabolism and stress response. The predictions link phosphoproteins of unknown function with protein kinases with known functions and vice versa, suggesting functions for the uncharacterized proteins. The study indicates that the phosphoproteins and the associated protein kinases represented in our dataset have housekeeping cellular roles; certain kinases are not represented because they may only be activated during specific cellular responses. Our results demonstrate the utility of our previously reported protein kinase substrate prediction approach (Predikin as a tool for establishing links between kinases and phosphoproteins that can subsequently be tested experimentally.

  10. Differential phosphorylation of ribosomal acidic proteins from yeast cell by two endogenous protein kinases: casein kinase-2 and 60S kinase

    International Nuclear Information System (INIS)

    The native 80S ribosomes isolated from ''Saccharomyces cerevisiae'' (strain W303) cells was phosphorylated by two endogenous protein kinases: multifunctional casein kinase-2 (CK-2) and specific 60S kinase. Three acidic proteins within the 60S ribosomal subunit: YP1β, YP1β' and YP2α are phosphorylated by both kinases. The other two proteins: YP1α and YP2β are predominantly phosphorylated by CK-2 but not by 60S kinase. This was confirmed in the experiment with the recombinant protein, YP2β, as a substrate, which is practically not phosphorylated by specific 60S kinase. These results together with the previous data based on the target amino-acid sequences suggest that, in addition to the multifunctional casein kinase-2 and specific 60S kinase, there exist probably other protein kinase(s) which phosphorylate the ribosomal acidic proteins in the cell. (author). 23 refs, 3 figs, 1 tab

  11. Estimation of S phase duration in goat epidermis by an in vivo intradermal double labelling technique using bromodeoxyuridine and tritiated thymidine

    International Nuclear Information System (INIS)

    When studying skin diseases resulting from alterations in the rate of epidermal cell turnover it is useful to be able to quantify parts of the epidermal cell cycle. An in vivo intradermal technique is described which uses tritiated thymidine followed by bromodeoxyuridine to label cells in the S phase of the cell cycle. Combined autoradiographic and immunocytochemical techniques were used to quantify the flux of cells into and out of S phase. These results were then used to estimate the length of S phase. The technique was found to provide clear distinctive labelling of S phase nuclei with both reagents. This avoided many of the problems encountered with double labelling techniques using two radioactive labels. S phase was calculated to be 7.7 hours for goat skin

  12. Laser flash photolysis and magnetic-field-effect studies on interaction of thymine and thymidine with menadione: role of sugar in controlling reaction pattern

    International Nuclear Information System (INIS)

    The magnetic field effect (MFE) in conjunction with laser flash photolysis has been used for the study of the interaction of one of the small drug like quinone molecules, 2-methyl, 1,4-naphthoquinone, commonly known as menadione (MQ), with one of the DNA bases, thymine (THN), and its corresponding nucleoside, thymidine (THDN), in acetonitrile (ACN) and sodium dodecylsulfate (SDS) micelles. It has been observed that THN undergoes electron transfer (ET) and hydrogen (H) abstraction with MQ, while THDN undergoes only H abstraction in both the media. However, our earlier studies showed that a purine base, adenine (ADN), and its nucleoside, 2'-deoxyadenosine (ADS), undergo ET in ACN and H abstraction in SDS. Here we have attempted to explain the differences in the reactions of these DNA bases with MQ. We also reveal the crucial role of a sugar unit in altering the behavior of purine and pyrimidine bases with respect to ET and H abstraction

  13. Crystal growth of phosphopantetheine adenylyltransferase, carboxypeptidase t, and thymidine phosphorylase on the international space station by the capillary counter-diffusion method

    International Nuclear Information System (INIS)

    Crystals of phosphopantetheine adenylyltransferase from Mycobacterium tuberculosis, thymidine phosphorylase from Escherichia coli, carboxypeptidase T from Thermoactinomyces vulgaris and its mutant forms, and crystals of complexes of these proteins with functional ligands and inhibitors were grown by the capillary counter-diffusion method in the Japanese Experimental Module Kibo on the International Space Station. The high-resolution X-ray diffraction data sets suitable for the determination of high-resolution three-dimensional structures of these proteins were collected from the grown crystals on the SPring-8 synchrotron radiation facility. The conditions of crystal growth for the proteins and the data-collection statistics are reported. The crystals grown in microgravity diffracted to a higher resolution than crystals of the same proteins grown on Earth.

  14. Laser flash photolysis and magnetic-field-effect studies on interaction of thymine and thymidine with menadione: role of sugar in controlling reaction pattern

    Directory of Open Access Journals (Sweden)

    Adity Bose, Debarati Dey and Samita Basu

    2008-01-01

    Full Text Available The magnetic field effect (MFE in conjunction with laser flash photolysis has been used for the study of the interaction of one of the small drug like quinone molecules, 2-methyl, 1,4-naphthoquinone, commonly known as menadione (MQ, with one of the DNA bases, thymine (THN, and its corresponding nucleoside, thymidine (THDN, in acetonitrile (ACN and sodium dodecylsulfate (SDS micelles. It has been observed that THN undergoes electron transfer (ET and hydrogen (H abstraction with MQ, while THDN undergoes only H abstraction in both the media. However, our earlier studies showed that a purine base, adenine (ADN, and its nucleoside, 2'-deoxyadenosine (ADS, undergo ET in ACN and H abstraction in SDS. Here we have attempted to explain the differences in the reactions of these DNA bases with MQ. We also reveal the crucial role of a sugar unit in altering the behavior of purine and pyrimidine bases with respect to ET and H abstraction.

  15. Effects of inhibitors of DNA, RNA, and protein synthesis on frequencies and types of premature chromosome condensation from x-ray induced micronuclei. [Cytosine arabinoside, azathioprine, thymidine, trenimon

    Energy Technology Data Exchange (ETDEWEB)

    Madle, S.; Nowak, J.; Obe, G.

    1976-10-28

    Cells containing x-ray induced micronuclei were treated for a few hours before fixation with inhibitors of DNA synthesis (cytosine arabinoside; azathioprine; thymidine; trenimon), of RNA synthesis (actinomycin D; ethidium bromide), and of protein synthesis (puromycin). Only the inhibitors of DNA synthesis lead to a significant suppression of the frequencies of mitoses with micronucleus derived premature chromosome condensation (PCC). We tend to interpret the result as follows: Micronuclei that are in the G1 phase of their cell cycles are accumulated at the G1/S border or in the early S phase of their cell cycles under the influence of the inhibitors of the DNA synthesis. Micronuclei blocked in this way cannot be induced to undergo PCC and seem to disappear from the cells.

  16. Asbestos and benzo[a]pyrene act synergistically to induce squamous metaplasia and incorporation of [3H]thymidine in hamster tracheal epithelium

    International Nuclear Information System (INIS)

    When exposed to either crocidolite asbestos (single 1-h exposure to 0.4 mg/ml medium) or the polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP) (less than or equal to 2.5 micrograms/ml medium, 1x weekly for 4 weeks), the epithelium of hamster tracheal explants exhibits insignificant amounts of squamous metaplasia, an atypical lesion, in comparison to amounts observed in untreated tissues. Incorporation of [3H]thymidine, an indication of DNA synthesis by epithelial cells, likewise is unchanged. However, the extent of squamous metaplasia and numbers of labeled basal and suprabasal cells are increased substantially when BaP and asbestos are added in combination. These results suggest an important mechanism of co-carcinogenesis involving chemical and physical carcinogens and support epidemiologic observations documenting an increased risk of bronchogenic carcinoma in asbestos workers who smoke

  17. Radioautographic study of the effects of colchicine in the cell cycle and in the migration of the ameloblasts in lower incisors of mice using 3H-thymidine

    International Nuclear Information System (INIS)

    The effect of colchicine in the cell cycle and in the migration of the ameloblasts throughout the enamel organ in lower incisors of mice was studied radioautographycaly using 3H-thymidine. The results showed a decrease of G(13.6h) and G1(3.7h) in the group of animals treated with colchicine. The decrease of G could be due to an increase of the number of ameloblasts in metaphase, as a consequence of the final action of the drug, which returns to the biocked cells the capacity of division. However, the decrease of G1 could be due to the decrease of G, since the duration of this time was calculated indirectly. The delay in the velocity of migration of the ameloblasts could be caused by the interference of the drug in the mechanisms of eruption and/or growth of the tooth. (Author)

  18. Phosphatase-Dependent Regulation of Epithelial Mitogen-Activated Protein Kinase Responses to Toxin-Induced Membrane Pores

    OpenAIRE

    Aguilar, Jorge L.; Kulkarni, Ritwij; Randis, Tara M.; Soman, Sandeep; Kikuchi, Alexander; Yin, Yuxin; Ratner, Adam J.

    2009-01-01

    Diverse bacterial species produce pore-forming toxins (PFT) that can puncture eukaryotic cell membranes. Host cells respond to sublytic concentrations of PFT through conserved intracellular signaling pathways, including activation of mitogen-activated protein kinases (MAPK), which are critical to cell survival. Here we demonstrate that in respiratory epithelial cells p38 and JNK MAPK were phosphorylated within 30 min of exposure to pneumolysin, the PFT from Streptococcus pneumoniae. This acti...

  19. Evolution of Bacterial Suicide

    Science.gov (United States)

    Tchernookov, Martin; Nemenman, Ilya

    2013-03-01

    While active, controlled cellular suicide (autolysis) in bacteria is commonly observed, it has been hard to argue that autolysis can be beneficial to an individual who commits it. We propose a theoretical model that predicts that bacterial autolysis is evolutionarily advantageous to an individualand would fixate in physically structured environments for stationary phase colonies. We perform spatially resolved agent-based simulations of the model, which predict that lower mixing in the environment results in fixation of a higher autolysis rate from a single mutated cell, regardless of the colony's genetic diversity. We argue that quorum sensing will fixate as well, even if initially rare, if it is coupled to controlling the autolysis rate. The model does not predict a strong additional competitive advantage for cells where autolysis is controlled by quorum sensing systems that distinguish self from nonself. These predictions are broadly supported by recent experimental results in B. subtilisand S. pneumoniae. Research partially supported by the James S McDonnell Foundation grant No. 220020321 and by HFSP grant No. RGY0084/2011.

  20. Electromagnetism of Bacterial Growth

    Science.gov (United States)

    Ainiwaer, Ailiyasi

    2011-10-01

    There has been increasing concern from the public about personal health due to the significant rise in the daily use of electrical devices such as cell phones, radios, computers, GPS, video games and television. All of these devices create electromagnetic (EM) fields, which are simply magnetic and electric fields surrounding the appliances that simultaneously affect the human bio-system. Although these can affect the human system, obstacles can easily shield or weaken the electrical fields; however, magnetic fields cannot be weakened and can pass through walls, human bodies and most other objects. The present study was conducted to examine the possible effects of bacteria when exposed to magnetic fields. The results indicate that a strong causal relationship is not clear, since different magnetic fields affect the bacteria differently, with some causing an increase in bacterial cells, and others causing a decrease in the same cells. This phenomenon has yet to be explained, but the current study attempts to offer a mathematical explanation for this occurrence. The researchers added cultures to the magnetic fields to examine any effects to ion transportation. Researchers discovered ions such as potassium and sodium are affected by the magnetic field. A formula is presented in the analysis section to explain this effect.

  1. The rare bacterial biosphere.

    Science.gov (United States)

    Pedrós-Alió, Carlos

    2012-01-01

    All communities are dominated by a few species that account for most of the biomass and carbon cycling. On the other hand, a large number of species are represented by only a few individuals. In the case of bacteria, these rare species were until recently invisible. Owing to their low numbers, conventional molecular techniques could not retrieve them. Isolation in pure culture was the only way to identify some of them, but current culturing techniques are unable to isolate most of the bacteria in nature. The recent development of fast and cheap high-throughput sequencing has begun to allow access to the rare species. In the case of bacteria, the exploration of this rare biosphere has several points of interest. First, it will eventually produce a reasonable estimate of the total number of bacterial taxa in the oceans; right now, we do not even know the right order of magnitude. Second, it will answer the question of whether "everything is everywhere." Third, it will require hypothesizing and testing the ecological mechanisms that allow subsistence of many species in low numbers. And fourth, it will open an avenue of research into the immense reserve of genes with potential applications hidden in the rare biosphere. PMID:22457983

  2. Transport Powered by Bacterial Turbulence

    Science.gov (United States)

    Kaiser, Andreas; Peshkov, Anton; Sokolov, Andrey; ten Hagen, Borge; Löwen, Hartmut; Aranson, Igor S.

    2014-04-01

    We demonstrate that collective turbulentlike motion in a bacterial bath can power and steer the directed transport of mesoscopic carriers through the suspension. In our experiments and simulations, a microwedgelike "bulldozer" draws energy from a bacterial bath of varied density. We obtain that an optimal transport speed is achieved in the turbulent state of the bacterial suspension. This apparent rectification of random motion of bacteria is caused by polar ordered bacteria inside the cusp region of the carrier, which is shielded from the outside turbulent fluctuations.

  3. Transport powered by bacterial turbulence.

    Science.gov (United States)

    Kaiser, Andreas; Peshkov, Anton; Sokolov, Andrey; ten Hagen, Borge; Löwen, Hartmut; Aranson, Igor S

    2014-04-18

    We demonstrate that collective turbulentlike motion in a bacterial bath can power and steer the directed transport of mesoscopic carriers through the suspension. In our experiments and simulations, a microwedgelike "bulldozer" draws energy from a bacterial bath of varied density. We obtain that an optimal transport speed is achieved in the turbulent state of the bacterial suspension. This apparent rectification of random motion of bacteria is caused by polar ordered bacteria inside the cusp region of the carrier, which is shielded from the outside turbulent fluctuations. PMID:24785075

  4. Crystal Structure of Pyridoxal Kinase from the Escherichia coli pdxK Gene: Implications for the Classification of Pyridoxal Kinases

    OpenAIRE

    Safo, Martin K.; Musayev, Faik N.; di Salvo, Martino L.; Hunt, Sharyn; Claude, Jean-Baptiste; Schirch, Verne

    2006-01-01

    The pdxK and pdxY genes have been found to code for pyridoxal kinases, enzymes involved in the pyridoxal phosphate salvage pathway. Two pyridoxal kinase structures have recently been published, including Escherichia coli pyridoxal kinase 2 (ePL kinase 2) and sheep pyridoxal kinase, products of the pdxY and pdxK genes, respectively. We now report the crystal structure of E. coli pyridoxal kinase 1 (ePL kinase 1), encoded by a pdxK gene, and an isoform of ePL kinase 2. The structures were deter...

  5. Physiological roles of mitogen-activated-protein-kinase-activated p38-regulated/activated protein kinase

    Institute of Scientific and Technical Information of China (English)

    Sergiy; Kostenko; Gianina; Dumitriu; Kari; Jenssen; Lgreid; Ugo; Moens

    2011-01-01

    Mitogen-activated protein kinases(MAPKs)are a family of proteins that constitute signaling pathways involved in processes that control gene expression,cell division, cell survival,apoptosis,metabolism,differentiation and motility.The MAPK pathways can be divided into conventional and atypical MAPK pathways.The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases,MAPK kinase,and MAPK.Atypical MAPK pathways are not organized into this three-tiered cascade.MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases.The latter are referred to as MAPK-activated protein kinases.This review focuses on one such MAPK-activated protein kinase,MAPK-activated protein kinase 5(MK5)or p38-regulated/activated protein kinase(PRAK).This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways.Recent findings on the regulation of the activity and subcellular localization,bona fide interaction partners and physiological roles of MK5/PRAK are discussed.

  6. Mitogen-activated protein kinases in atherosclerosis

    Directory of Open Access Journals (Sweden)

    Dorota Bryk

    2014-01-01

    Full Text Available Intracellular signalling cascades, in which MAPK (mitogen-activated protein kinases intermediate, are responsible for a biological response of a cell to an external stimulus. MAP kinases, which include ERK1/2 (extracellular signalling-regulated kinase, JNK (c-Jun N-terminal kinase and p 38 MAPK, regulate the activity of many proteins, enzymes and transcription factors and thus have a wide spectrum of biological effects. Many basic scientific studies have defined numerous details of their pathway organization and activation. There are also more and more studies suggesting that individual MAP kinases probably play an important role in the pathogenesis of atherosclerosis. They may mediate inflammatory processes, endothelial cell activation, monocyte/macrophage recruitment and activation, smooth muscle cell proliferation and T-lymphocyte differentiation, all of which represent crucial mechanisms involved in pathogenesis of atherosclerosis. The specific inhibition of an activity of the respective MAP kinases may prove a new therapeutic approach to attenuate atherosclerotic plaque formation in the future. In this paper, we review the current state of knowledge concerning MAP kinase-dependent cellular and molecular mechanisms underlying atherosclerosis.

  7. [Mitogen-activated protein kinases in atherosclerosis].

    Science.gov (United States)

    Bryk, Dorota; Olejarz, Wioletta; Zapolska-Downar, Danuta

    2014-01-01

    Intracellular signalling cascades, in which MAPK (mitogen-activated protein kinases) intermediate, are responsible for a biological response of a cell to an external stimulus. MAP kinases, which include ERK1/2 (extracellular signalling-regulated kinase), JNK (c-Jun N-terminal kinase) and p 38 MAPK, regulate the activity of many proteins, enzymes and transcription factors and thus have a wide spectrum of biological effects. Many basic scientific studies have defined numerous details of their pathway organization and activation. There are also more and more studies suggesting that individual MAP kinases probably play an important role in the pathogenesis of atherosclerosis. They may mediate inflammatory processes, endothelial cell activation, monocyte/macrophage recruitment and activation, smooth muscle cell proliferation and T-lymphocyte differentiation, all of which represent crucial mechanisms involved in pathogenesis of atherosclerosis. The specific inhibition of an activity of the respective MAP kinases may prove a new therapeutic approach to attenuate atherosclerotic plaque formation in the future. In this paper, we review the current state of knowledge concerning MAP kinase-dependent cellular and molecular mechanisms underlying atherosclerosis. PMID:24491891

  8. Mitotic regulation by NIMA-related kinases

    Directory of Open Access Journals (Sweden)

    Blot Joelle

    2007-08-01

    Full Text Available Abstract The NIMA-related kinases represent a family of serine/threonine kinases implicated in cell cycle control. The founding member of this family, the NIMA kinase of Aspergillus nidulans, as well as the fission yeast homologue Fin1, contribute to multiple aspects of mitotic progression including the timing of mitotic entry, chromatin condensation, spindle organization and cytokinesis. Mammals contain a large family of eleven NIMA-related kinases, named Nek1 to Nek11. Of these, there is now substantial evidence that Nek2, Nek6, Nek7 and Nek9 also regulate mitotic events. At least three of these kinases, as well as NIMA and Fin1, have been localized to the microtubule organizing centre of their respective species, namely the centrosome or spindle pole body. Here, they have important functions in microtubule organization and mitotic spindle assembly. Other Nek kinases have been proposed to play microtubule-dependent roles in non-dividing cells, most notably in regulating the axonemal microtubules of cilia and flagella. In this review, we discuss the evidence that NIMA-related kinases make a significant contribution to the orchestration of mitotic progression and thereby protect cells from chromosome instability. Furthermore, we highlight their potential as novel chemotherapeutic targets.

  9. Structure-based discovery of inhibitors of the YycG histidine kinase

    DEFF Research Database (Denmark)

    Qin, X.; Zhang, J.; Xu, B.;

    2006-01-01

    inhibitors of YycG histidine kinase thus are of potential value as leads for developing new antibiotics against infecting staphylococci. The structure-based virtual screening (SBVS) technology can be widely used in screening potential inhibitors of other bacterial TCSs, since it is more rapid and efficacious...... resistance to many conventional antibiotics and often results in chronic infection. It has an urgent need to design novel antibiotics against staphylococci infections, especially those can kill cells embedded in biofilm. RESULTS: In this report, a series of novel inhibitors of the histidine kinase (HK) Yyc......G protein of S. epidermidis were discovered first using structure-based virtual screening (SBVS) from a small molecular lead-compound library, followed by experimental validation. Of the 76 candidates derived by SBVS targeting of the homolog model of the YycG HATPase_c domain of S. epidermidis, seven...

  10. n-Butyrate inhibits Jun NH(2)-terminal kinase activation and cytokine transcription in mast cells

    International Nuclear Information System (INIS)

    Mast cells are well known to contribute to type I allergic conditions but only recently have been brought in association with chronic relapsing/remitting autoimmune diseases such as celiac disease and ulcerative colitis. Since the bacterial metabolite n-butyrate is considered to counteract intestinal inflammation we investigated the effects of this short chain fatty acid on mast cell activation. Using RNAse protection assays and reporter gene technology we show that n-butyrate downregulates TNF-α transcription. This correlates with an impaired activation of the Jun NH(2)-terminal kinase (JNK) but not other MAP kinases such as ERK and p38 that are largely unaffected by n-butyrate. As a consequence, we observed a decreased nuclear activity of AP-1 and NF-AT transcription factors. These results indicate that n-butyrate inhibits critical inflammatory mediators in mast cells by relatively selectively targeting the JNK signalling

  11. Cloning, functional expression and partial characterization of the glucose kinase from Renibacterium salmoninarum.

    Science.gov (United States)

    Concha, M I; León, G

    2000-05-01

    The complete glcK gene from the fish pathogen Renibacterium salmoninarum, encoding a glucose kinase, was analyzed and expressed. The partial characterization of the recombinant enzyme confirmed that it belongs to a group of glucose kinases involved in carbon catabolite repression. Multiple sequence alignments were used to deduce a new consensus sequence for this family of bacterial proteins, characterized by several conserved Cys residues. This sequence was more specific and allowed the detection of the first eukaryotic protein of this family. The recombinant enzyme was inhibited by N-ethylmaleimide and the substrates protected the enzyme from this inhibition, suggesting the presence of Cys residues in or close to the active site. PMID:10779719

  12. Bacterial flora of sturgeon fingerling

    International Nuclear Information System (INIS)

    The study on microbial populations is a suitable tool to understand and apply control methods to improve the sanitary level of production in fish breeding and rearing centers, ensure health of sturgeon fingerlings at the time of their release into the rivers and also in the conversation and restoration of these valuable stocks in the Caspian Sea, Iran. A laboratory research based on Austin methods (Austin, B., Austin, D.A. 1993) was conducted for bacterial study on 3 sturgeon species naming A. persicus, A. stellatus and A. nudiventris during different growth stages. Bacterial flora of Acinetobacter, Moraxella, Aeromonas, Vibrio, Edwardsiella, Staphylococcus, Proteus, Yersinia, Pseudomonas and Plesiomonas were determined. The factors which may induce changes in bacterial populations during different stages of fife are the followings: quality of water in rearing ponds, different conditions for growth stages, suitable time for colonization of bacterial flora in rearing pond, water temperature increase in fingerlings size and feeding condition. (author)

  13. Bacterial Communities: Interactions to Scale

    Science.gov (United States)

    Stubbendieck, Reed M.; Vargas-Bautista, Carol; Straight, Paul D.

    2016-01-01

    In the environment, bacteria live in complex multispecies communities. These communities span in scale from small, multicellular aggregates to billions or trillions of cells within the gastrointestinal tract of animals. The dynamics of bacterial communities are determined by pairwise interactions that occur between different species in the community. Though interactions occur between a few cells at a time, the outcomes of these interchanges have ramifications that ripple through many orders of magnitude, and ultimately affect the macroscopic world including the health of host organisms. In this review we cover how bacterial competition influences the structures of bacterial communities. We also emphasize methods and insights garnered from culture-dependent pairwise interaction studies, metagenomic analyses, and modeling experiments. Finally, we argue that the integration of multiple approaches will be instrumental to future understanding of the underlying dynamics of bacterial communities. PMID:27551280

  14. The mechanism of protein kinase C regulation

    Institute of Scientific and Technical Information of China (English)

    Julhash U. KAZI

    2011-01-01

    Protein kinase C (PKC) is a family ofserine/threonine protein kinases that plays a central role in transducing extracellular signals into a variety of intracellular responses ranging from cell proliferation to apoptosis.Nine PKC genes have been identified in the human genome,which encode 10 proteins.Each member of this protein kinase family displays distinct biochemical characteristics and is enriched in different cellular and subcellular locations.Activation of PKC has been implicated in the regulation of cell growth and differentiation.This review summarizes works of the past years in the field of PKC biochemistry that covers regulation and activation mechanism of different PKC isoforms.

  15. Differential AMP-activated Protein Kinase (AMPK) Recognition Mechanism of Ca2+/Calmodulin-dependent Protein Kinase Kinase Isoforms.

    Science.gov (United States)

    Fujiwara, Yuya; Kawaguchi, Yoshinori; Fujimoto, Tomohito; Kanayama, Naoki; Magari, Masaki; Tokumitsu, Hiroshi

    2016-06-24

    Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ) is a known activating kinase for AMP-activated protein kinase (AMPK). In vitro, CaMKKβ phosphorylates Thr(172) in the AMPKα subunit more efficiently than CaMKKα, with a lower Km (∼2 μm) for AMPK, whereas the CaMKIα phosphorylation efficiencies by both CaMKKs are indistinguishable. Here we found that subdomain VIII of CaMKK is involved in the discrimination of AMPK as a native substrate by measuring the activities of various CaMKKα/CaMKKβ chimera mutants. Site-directed mutagenesis analysis revealed that Leu(358) in CaMKKβ/Ile(322) in CaMKKα confer, at least in part, a distinct recognition of AMPK but not of CaMKIα. PMID:27151216

  16. The Bacterial Microflora of Fish

    OpenAIRE

    Austin, B.

    2002-01-01

    The results of numerous studies indicate that fish possess bacterial populations on or in their skin, gills, digestive tract, and light-emitting organs. In addition, the internal organs (kidney, liver, and spleen) of healthy fish may contain bacteria, but there is debate on whether or not muscle is actually sterile. The numbers and taxonomic composition of the bacterial populations often reflect those of the surrounding water. The role of the bacteria includes the ability to degrade complex m...

  17. Bacterial cellulose/boehmite composites

    Energy Technology Data Exchange (ETDEWEB)

    Salvi, Denise T.B. de; Barud, Hernane S.; Messaddeq, Younes; Ribeiro, Sidney J.L. [Universidade Estadual Paulista Julio de Mesquita Filho. UNESP. Instituto de Quimica de Araraquara, SP (Brazil); Caiut, Jose Mauricio A. [Universidade de Sao Paulo. Departamento de Quimica - FFCLRP/USP, Ribeirao Preto, SP (Brazil)

    2011-07-01

    Composites based on bacterial cellulose membranes and boehmite were obtained. SEM results indicate that the bacterial cellulose (BC) membranes are totally covered by boehmite and obtained XRD patterns suggest structural changes due to this boehmite addition. Thermal stability is accessed through TG curves and is dependent on boehmite content. Transparency is high comparing to pure BC as can be seen through UV-vis absorption spectroscopy. (author)

  18. Bacterial Culture of Neonatal Sepsis

    OpenAIRE

    AH Movahedian; R Moniri; Z Mosayebi

    2006-01-01

    Neonatal bacterial sepsis is one of the major cause of morbidity and mortality in neonates. This retrospective study was performed to determine the incidence of bacterial sepsis with focus on Gram negative organisms in neonates admitted at Beheshti Hospital in Kashan, during a 3-yr period, from September 2002 to September 2005. Blood culture was performed on all neonates with risk factors or signs of suggestive sepsis. Blood samples were cultured using brain heart infusion (BHI) broth accordi...

  19. Bacterial Alkaloids Prevent Amoebal Predation.

    Science.gov (United States)

    Klapper, Martin; Götze, Sebastian; Barnett, Robert; Willing, Karsten; Stallforth, Pierre

    2016-07-25

    Bacterial defense mechanisms have evolved to protect bacteria against predation by nematodes, predatory bacteria, or amoebae. We identified novel bacterial alkaloids (pyreudiones A-D) that protect the producer, Pseudomonas fluorescens HKI0770, against amoebal predation. Isolation, structure elucidation, total synthesis, and a proposed biosynthetic pathway for these structures are presented. The generation of P. fluorescens gene-deletion mutants unable to produce pyreudiones rendered the bacterium edible to a variety of soil-dwelling amoebae. PMID:27294402

  20. Mast cells in bacterial infections

    OpenAIRE

    Rönnberg, Elin

    2014-01-01

    Mast cells are implicated in immunity towards bacterial infection, but the molecular mechanisms by which mast cells contribute to the host response are only partially understood. Previous studies have examined how mast cells react to purified bacterial cell wall components, such as peptidoglycan and lipopolysaccharide. To investigate how mast cells react to live bacteria we co-cultured mast cells and the gram-positive bacteria Streptococcus equi (S. equi) and Staphylococcus aureus (S. aureus)...