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Sample records for bacterial thymidine kinase

  1. Thymidine uptake, thymidine incorporation, and thymidine kinase activity in marine bacterium isolates

    International Nuclear Information System (INIS)

    Jeffrey, W.H.; Paul, J.H.

    1990-01-01

    One assumption made in bacterial production estimates from [ 3 H]thymidine incorporation is that all heterotrophic bacteria can incorporate exogenous thymidine into DNA. Heterotrophic marine bacterium isolates from Tampa Bay, Fla., Chesapeake Bay, Md., and a coral surface microlayer were examined for thymidine uptake (transport), thymidine incorporation, the presence of thymidine kinase genes, and thymidine kinase enzyme activity. Of the 41 isolates tested, 37 were capable of thymidine incorporation into DNA. The four organisms that could not incorporate thymidine also transported the thymidine poorly and lacked thymidine kinase activity. Attempts to detect thymidine kinase genes in the marine isolates by molecular probing with gene probes made from Escherichia coli and herpes simplex virus thymidine kinase genes proved unsuccessful. To determine if the inability to incorporate thymidine was due to the lack of thymidine kinase, one organism, Vibro sp. strain DI9, was transformed with a plasmid (pGQ3) that contained an E. coli thymidine kinase gene. Although enzyme assays indicated high levels of thymidine kinase activity in transformants, these cells still failed to incorporate exogenous thymidine into DNA or to transport thymidine into cells. These results indicate that the inability of certain marine bacteria to incorporate thymidine may not be solely due to the lack of thymidine kinase activity but may also be due to the absence of thymidine transport systems

  2. 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....... The TKs from Gram-positive bacteria are more closely related to the eukaryotic TK1 enzymes than are TKs from Gram-negative bacteria....

  3. Thymidine kinases in archaea

    DEFF Research Database (Denmark)

    Clausen, A.R.; Matakos, A.; Sandrini, Michael

    2006-01-01

    Twenty-six fully sequenced archaeal genomes were searched for genes coding for putative deoxyribonucleoside kinases (dNKs). We identified only 5 human-like thymidine kinase 1 genes (TK1s) and none for non-TK1 kinases. Four TK1s were identified in the Euryarchaea and one was found in the Crenarcha...

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

    International Nuclear Information System (INIS)

    Peterson, Kristin L.; Reid, William C.; Freeman, Alexandra F.; Holland, Steven M.; Pettigrew, Roderic I.; Gharib, Ahmed M.; Hammoud, Dima A.

    2013-01-01

    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 124 I-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 14 C-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 14 C-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

  5. Thymidine plaque autoradiography of thymidine kinase-positive and thymidine kinase-negative herpesviruses

    International Nuclear Information System (INIS)

    Tenser, R.B.; Jones, J.C.; Ressel, S.J.; Fralish, F.A.

    1983-01-01

    Plaques formed by herpes simplex virus (HSV), pseudorabies virus, and varicella-zoster virus were studied by plaque autoradiography after [ 14 C]thymidine labeling. Standard thymidine kinase-positive (TK+) viruses and TK- mutants of HSV types 1 and 2 and pseudorabies virus were studied, including cell cultured viruses and viruses isolated from animals. Autoradiography was performed with X-ray film with an exposure time of 5 days. After development of films, TK+ plaques showed dark rims due to isotope incorporation, whereas TK- plaques were minimally labeled. Plaque autoradiography of stock TK- viruses showed reversion frequencies to the TK+ phenotype of less than 10(-3). Autoradiography indicated that TK- virus retained the TK- phenotype after replication in vivo. In addition, it was shown that TK- HSV could be isolated from mouse trigeminal ganglion tissue after corneal inoculation of TK- HSV together with TK+ HSV. The plaque autoradiographic procedure was very useful to evaluate proportions of TK+ and TK- virus present in TK+-TK- virus mixtures

  6. Unchanged thymidine triphosphate pools and thymidine metabolism in two lines of thymidine kinase 2-mutated fibroblasts.

    Science.gov (United States)

    Frangini, Miriam; Rampazzo, Chiara; Franzolin, Elisa; Lara, Mari-Carmen; Vilà, Maya R; Martí, Ramon; Bianchi, Vera

    2009-02-01

    Mitochondrial thymidine kinase (TK2) catalyzes the phosphorylation of thymidine in mitochondria. Its function becomes essential for dTTP synthesis in noncycling cells, where cytosolic dTTP synthesis via R1/R2 ribonucleotide reductase and thymidine kinase 1 is turned down. Mutations in the nuclear gene for TK2 cause a fatal mtDNA depletion syndrome. Only selected cell types are affected, suggesting that the other cells compensate for the TK2 deficiency by adapting the enzyme network that regulates dTTP synthesis outside S-phase. Here we looked for such metabolic adaptation in quiescent cultures of fibroblasts from two TK2-deficient patients with a slow-progressing syndrome. In cell extracts, we measured the activities of TK2, deoxycytidine kinase, thymidine phosphorylase, deoxynucleotidases and the amounts of the three ribonucleotide reductase subunits. Patient cells contained 40% or 5% TK2 activity and unchanged activities of the other enzymes. However, their mitochondrial and cytosolic dTTP pools were unchanged, and also the overall composition of the dNTP pools was normal. TK2-dependent phosphorylation of [(3)H]thymidine in intact cells and the turnover of the dTTP pool showed that even the fibroblasts with 5% residual TK2 activity synthesized dTTP at an almost normal rate. Normal fibroblasts apparently contain more TK2 than needed to maintain dTTP during quiescence, which would explain why TK2-mutated fibroblasts do not manifest mtDNA depletion despite their reduced TK2 activity.

  7. Prodrugs of herpes simplex thymidine kinase inhibitors.

    Science.gov (United States)

    Yanachkova, Milka; Xu, Wei-Chu; Dvoskin, Sofya; Dix, Edward J; Yanachkov, Ivan B; Focher, Federico; Savi, Lida; Sanchez, M Dulfary; Foster, Timothy P; Wright, George E

    2015-04-01

    Because guanine-based herpes simplex virus thymidine kinase inhibitors are not orally available, we synthesized various 6-deoxy prodrugs of these compounds and evaluated them with regard to solubility in water, oral bioavailability, and efficacy to prevent herpes simplex virus-1 reactivation from latency in a mouse model. Organic synthesis was used to prepare compounds, High Performance Liquid Chromatography (HPLC) to analyze hydrolytic conversion, Mass Spectrometry (MS) to measure oral bioavailability, and mouse latent infection and induced reactivation to evaluate the efficacy of a specific prodrug. Aqueous solubilities of prodrugs were improved, oxidation of prodrugs by animal cytosols occurred in vitro, and oral absorption of the optimal prodrug sacrovir™ (6-deoxy-mCF3PG) in the presence of the aqueous adjuvant Soluplus® and conversion to active compound N(2)-[3-(trifluoromethyl)pheny])guanine (mCF3PG) were accomplished in mice. Treatment of herpes simplex virus-1 latent mice with sacrovir™ in 1% Soluplus in drinking water significantly suppressed herpes simplex virus-1 reactivation and viral genomic replication. Ad libitum oral delivery of sacrovir™ was effective in suppressing herpes simplex virus-1 reactivation in ocularly infected latent mice as measured by the numbers of mice shedding infectious virus at the ocular surface, numbers of trigeminal ganglia positive for infectious virus, number of corneas that had detectable infectious virus, and herpes simplex virus-1 genome copy numbers in trigeminal ganglia following reactivation. These results demonstrate the statistically significant effect of the prodrug on suppressing herpes simplex virus-1 reactivation in vivo. © The Author(s) 2015.

  8. Thymidine kinase 2 enzyme kinetics elucidate the mechanism of thymidine-induced mitochondrial DNA depletion.

    Science.gov (United States)

    Sun, Ren; Wang, Liya

    2014-10-07

    Mitochondrial thymidine kinase 2 (TK2) is a nuclear gene-encoded protein, synthesized in the cytosol and subsequently translocated into the mitochondrial matrix, where it catalyzes the phosphorylation of thymidine (dT) and deoxycytidine (dC). The kinetics of dT phosphorylation exhibits negative cooperativity, but dC phosphorylation follows hyperbolic Michaelis-Menten kinetics. The two substrates compete with each other in that dT is a competitive inhibitor of dC phosphorylation, while dC acts as a noncompetitive inhibitor of dT phosphorylation. In addition, TK2 is feedback inhibited by dTTP and dCTP. TK2 also phosphorylates a number of pyrimidine nucleoside analogues used in antiviral and anticancer therapy and thus plays an important role in mitochondrial toxicities caused by nucleoside analogues. Deficiency in TK2 activity due to genetic alterations causes devastating mitochondrial diseases, which are characterized by mitochondrial DNA (mtDNA) depletion or multiple deletions in the affected tissues. Severe TK2 deficiency is associated with early-onset fatal mitochondrial DNA depletion syndrome, while less severe deficiencies result in late-onset phenotypes. In this review, studies of the enzyme kinetic behavior of TK2 enzyme variants are used to explain the mechanism of mtDNA depletion caused by TK2 mutations, thymidine overload due to thymidine phosphorylase deficiency, and mitochondrial toxicity caused by antiviral thymidine analogues.

  9. 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 com...

  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...

  11. 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...

  12. Mutation Study of Two Thymidine Kinases 

    DEFF Research Database (Denmark)

    Skovgaard, Tine; Munch-Petersen, Birgitte; Eklund, Hans

    that phosphorylates all the natural deoxyribonucleosides and like insects, C. elegans only contains a single deoxyribonucleoside kinase-like gene. In contrast to the insects, however, the protein encoded by the elegans gene is 46 % identical to human TK1 (HuTK1) and have no homology to the insect kinase. Like HuTK1...... the C. elegans kinase (CeTK1) has thymidine as the preferred substrate, but it also displays activity with deoxyguanosine, though with high Km. A number of point mutations have been introduced in the active site of both the human and elegans TK's in order to change the substrate specificity away from...... not phosphorylate the anticancer analog 1-β-D-arabinofuranosylcytosine (AraC), however. The HuTK1 mutant has been crystallized, and azidothymidine monophosphate has been modelled into the active site....

  13. Mitochondrial deoxyribonucleoside triphosphate pools in thymidine kinase 2 deficiency.

    Science.gov (United States)

    Saada, Ann; Ben-Shalom, Efrat; Zyslin, Rivka; Miller, Chaya; Mandel, Hanna; Elpeleg, Orly

    2003-10-24

    Deficiency of mitochondrial thymidine kinase (TK2) is associated with mitochondrial DNA (mtDNA) depletion and manifests by severe skeletal myopathy in infancy. In order to elucidate the pathophysiology of this condition, mitochondrial deoxyribonucleoside triphosphate (dNTP) pools were determined in patients' fibroblasts. Despite normal mtDNA content and cytochrome c oxidase (COX) activity, mitochondrial dNTP pools were imbalanced. Specifically, deoxythymidine triphosphate (dTTP) content was markedly decreased, resulting in reduced dTTP:deoxycytidine triphosphate ratio. These findings underline the importance of balanced mitochondrial dNTP pools for mtDNA synthesis and may serve as the basis for future therapeutic interventions.

  14. 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-...

  15. Design of Thymidine Analogues Targeting Thymidilate Kinase of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Luc Calvin Owono Owono

    2013-01-01

    Full Text Available We design here new nanomolar antituberculotics, inhibitors of Mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt, by means of structure-based molecular design. 3D models of TMPKmt-inhibitor complexes have been prepared from the crystal structure of TMPKmt cocrystallized with the natural substrate deoxythymidine monophosphate (dTMP (1GSI for a training set of 15 thymidine analogues (TMDs with known activity to prepare a QSAR model of interaction establishing a correlation between the free energy of complexation and the biological activity. Subsequent validation of the predictability of the model has been performed with a 3D QSAR pharmacophore generation. The structural information derived from the model served to design new subnanomolar thymidine analogues. From molecular modeling investigations, the agreement between free energy of complexation (ΔΔGcom and Ki values explains 94% of the TMPKmt inhibition (pKi=-0.2924ΔΔGcom+3.234;R2=0.94 by variation of the computed ΔΔGcom and 92% for the pharmacophore (PH4 model (pKi=1.0206×pKipred-0.0832,  R2=0.92. The analysis of contributions from active site residues suggested substitution at the 5-position of pyrimidine ring and various groups at the 5′-position of the ribose. The best inhibitor reached a predicted Ki of 0.155 nM. The computational approach through the combined use of molecular modeling and PH4 pharmacophore is helpful in targeted drug design, providing valuable information for the synthesis and prediction of activity of novel antituberculotic agents.

  16. [3H] Thymidine incorporation to estimate growth rates of anaerobic bacterial strains

    International Nuclear Information System (INIS)

    Winding, A.

    1992-01-01

    The incorporation of [ 3 H] thymidine by axenic cultures of anaerobic bacteria was investigated as a means to measure growth. The three fermentative strains and one of the methanogenic strains tested incorporated [ 3 H] thymidine during growth. It is concluded that the [ 3 H] thymidine incorporation method underestimates bacterial growth in anaerobic environments

  17. Radioimmunoassay for herpes simplex virus (HSV) thymidine kinase

    International Nuclear Information System (INIS)

    McGuirt, P.V.; Keller, P.M.; Elion, G.B.

    1982-01-01

    A sensitive RIA for HSV-1 thymidine kinase (TK) has been developed. This assay is based on competition for the binding site of a rabbit antibody against purified HSV-1 TK, between a purified 3 H-labeled HSV-1 TK and a sample containing an unknown amount of viral TK. The assay is capable of detecting 8 ng or more of the HSV enzyme. Purified HSV-1 TK denatured to <1% of its original kinase activity is as effective in binding to the antibody as is native HSV-1 TK. Viral TK is detectable at ranges of 150-460 ng/mg protein of cell extract from infected cells or cells transformed by HSV or HSV genetic material. HSV-2 TK appears highly cross-reactive, VZV TK is slightly less so, and the vaccinia TK shows little or no cross-reactivity. This RIA may serve as a tool for monitoring the expression of the HSV TK during an active herpes virus infection, a latent ganglionic infection, or in neoplastic cells which may have arisen by viral transformation

  18. Genetic and biochemical characterization of the thymidine kinase gene from herpesvirus of turkeys

    International Nuclear Information System (INIS)

    Martin, S.L.; Aparisio, D.I.; Bandyopadhyay, P.K.

    1989-01-01

    The thymidine kinase gene encoded by herpesvirus of turkeys has been identified and characterized. A viral mutant (ATR 0 ) resistant to 1-β-D-arabinofuranosylthymine was isolated. This mutant was also resistant to 1-(2-fluoro-2-deoxy-β-D-arabinofuronosyl)-5-methyluracil and was unable to incorporate [ 125 I]deoxycytidine into DNA. The mutant phenotype was rescued by a cloned region of the turkey herpesvirus genome whose DNA sequence was found to contain an open reading frame similar to that for known thymidine kinases from other viruses. When expressed in Escherichia coli, this open reading frame complemented a thymidine kinase-deficient strain and resulted in thymidine kinase activity in extracts assayed in vitro

  19. Deoxycytidine and Deoxythymidine Treatment for Thymidine Kinase 2 Deficiency.

    Science.gov (United States)

    Lopez-Gomez, Carlos; Levy, Rebecca J; Sanchez-Quintero, Maria J; Juanola-Falgarona, Martí; Barca, Emanuele; Garcia-Diaz, Beatriz; Tadesse, Saba; Garone, Caterina; Hirano, Michio

    2017-05-01

    Thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial pyrimidine salvage pathway, is essential for mitochondrial DNA (mtDNA) maintenance. Mutations in the nuclear gene, TK2, cause TK2 deficiency, which manifests predominantly in children as myopathy with mtDNA depletion. Molecular bypass therapy with the TK2 products, deoxycytidine monophosphate (dCMP) and deoxythymidine monophosphate (dTMP), prolongs the life span of Tk2-deficient (Tk2 -/- ) mice by 2- to 3-fold. Because we observed rapid catabolism of the deoxynucleoside monophosphates to deoxythymidine (dT) and deoxycytidine (dC), we hypothesized that: (1) deoxynucleosides might be the major active agents and (2) inhibition of deoxycytidine deamination might enhance dTMP+dCMP therapy. To test these hypotheses, we assessed two therapies in Tk2 -/- mice: (1) dT+dC and (2) coadministration of the deaminase inhibitor, tetrahydrouridine (THU), with dTMP+dCMP. We observed that dC+dT delayed disease onset, prolonged life span of Tk2-deficient mice and restored mtDNA copy number as well as respiratory chain enzyme activities and levels. In contrast, dCMP+dTMP+THU therapy decreased life span of Tk2 -/- animals compared to dCMP+dTMP. Our studies demonstrate that deoxynucleoside substrate enhancement is a novel therapy, which may ameliorate TK2 deficiency in patients. Ann Neurol 2017;81:641-652. © 2017 American Neurological Association.

  20. Deoxypyrimidine monophosphate bypass therapy for thymidine kinase 2 deficiency.

    Science.gov (United States)

    Garone, Caterina; Garcia-Diaz, Beatriz; Emmanuele, Valentina; Lopez, Luis C; Tadesse, Saba; Akman, Hasan O; Tanji, Kurenai; Quinzii, Catarina M; Hirano, Michio

    2014-08-01

    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-old Tk2(-/-) mice treated with dCMP+dTMP 200 mg/kg/day each (Tk2(-/-200dCMP/) (dTMP)) demonstrated that in mutant animals, the compounds raise dTTP concentrations, increase levels of mtDNA, ameliorate defects of mitochondrial respiratory chain enzymes, and significantly prolong their lifespan (34 days with treatment versus 13 days untreated). A second trial of dCMP+dTMP each at 400 mg/kg/day showed even greater phenotypic and biochemical improvements. In conclusion, dCMP/dTMP supplementation is the first effective pharmacologic treatment for Tk2 deficiency. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

  1. Microchip Immunoaffinity Electrophoresis of Antibody-Thymidine Kinase 1 Complex

    Science.gov (United States)

    Pagaduan, Jayson V.; Ramsden, Madison; O’Neill, Kim; Woolley, Adam T.

    2015-01-01

    Thymidine kinase-1 (TK1) is an important cancer biomarker whose serum levels are elevated in early cancer development. We developed a microchip electrophoresis immunoaffinity assay to measure recombinant purified TK1 (pTK1) using an antibody that binds to human TK1. We fabricated poly(methyl methacrylate) microfluidic devices to test the feasibility of detecting antibody (Ab)-pTK1 immune complexes as a step towards TK1 analysis in clinical serum samples. We were able to separate immune complexes from unbound antibodies using 0.5X phosphate buffer saline (pH 7.4) containing 0.01% Tween-20, with 1% w/v methylcellulose that acts as a dynamic surface coating and sieving matrix. Separation of the antibody and Ab-pTK1 complex was observed within a 5 mm effective separation length. This method of detecting pTK1 is easy to perform, requires only a 10 μL sample volume, and takes just 1 minute for separation. PMID:25486911

  2. Retrospective natural history of thymidine kinase 2 deficiency.

    Science.gov (United States)

    Garone, Caterina; Taylor, Robert W; Nascimento, Andrés; Poulton, Joanna; Fratter, Carl; Domínguez-González, Cristina; Evans, Julie C; Loos, Mariana; Isohanni, Pirjo; Suomalainen, Anu; Ram, Dipak; Hughes, M Imelda; McFarland, Robert; Barca, Emanuele; Lopez Gomez, Carlos; Jayawant, Sandeep; Thomas, Neil D; Manzur, Adnan Y; Kleinsteuber, Karin; Martin, Miguel A; Kerr, Timothy; Gorman, Grainne S; Sommerville, Ewen W; Chinnery, Patrick F; Hofer, Monika; Karch, Christoph; Ralph, Jeffrey; Cámara, Yolanda; Madruga-Garrido, Marcos; Domínguez-Carral, Jana; Ortez, Carlos; Emperador, Sonia; Montoya, Julio; Chakrapani, Anupam; Kriger, Joshua F; Schoenaker, Robert; Levin, Bruce; Thompson, John L P; Long, Yuelin; Rahman, Shamima; Donati, Maria Alice; DiMauro, Salvatore; Hirano, Michio

    2018-03-30

    Thymine kinase 2 (TK2) is a mitochondrial matrix protein encoded in nuclear DNA and phosphorylates the pyrimidine nucleosides: thymidine and deoxycytidine. Autosomal recessive TK2 mutations cause a spectrum of disease from infantile onset to adult onset manifesting primarily as myopathy. To perform a retrospective natural history study of a large cohort of patients with TK2 deficiency. The study was conducted by 42 investigators across 31 academic medical centres. We identified 92 patients with genetically confirmed diagnoses of TK2 deficiency: 67 from literature review and 25 unreported cases. Based on clinical and molecular genetics findings, we recognised three phenotypes with divergent survival: (1) infantile-onset myopathy (42.4%) with severe mitochondrial DNA (mtDNA) depletion, frequent neurological involvement and rapid progression to early mortality (median post-onset survival (POS) 1.00, CI 0.58 to 2.33 years); (2) childhood-onset myopathy (40.2%) with mtDNA depletion, moderate-to-severe progression of generalised weakness and median POS at least 13 years; and (3) late-onset myopathy (17.4%) with mild limb weakness at onset and slow progression to respiratory insufficiency with median POS of 23 years. Ophthalmoparesis and facial weakness are frequent in adults. Muscle biopsies show multiple mtDNA deletions often with mtDNA depletion. In TK2 deficiency, age at onset, rate of weakness progression and POS are important variables that define three clinical subtypes. Nervous system involvement often complicates the clinical course of the infantile-onset form while extraocular muscle and facial involvement are characteristic of the late-onset form. Our observations provide essential information for planning future clinical trials in this disorder. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  3. Transgene expression of Drosophila melanogaster nucleoside kinase reverses mitochondrial thymidine kinase 2 deficiency.

    Science.gov (United States)

    Krishnan, Shuba; Zhou, Xiaoshan; Paredes, João A; Kuiper, Raoul V; Curbo, Sophie; Karlsson, Anna

    2013-02-15

    A strategy to reverse the symptoms of thymidine kinase 2 (TK2) deficiency in a mouse model was investigated. The nucleoside kinase from Drosophila melanogaster (Dm-dNK) was expressed in TK2-deficient mice that have been shown to present with a severe phenotype caused by mitochondrial DNA depletion. The Dm-dNK(+/-) transgenic mice were shown to be able to rescue the TK2-deficient mice. The Dm-dNK(+/-)TK2(-/-) mice were normal as judged by growth and behavior during the observation time of 6 months. The Dm-dNK-expressing mice showed a substantial increase in thymidine-phosphorylating activity in investigated tissues. The Dm-dNK expression also resulted in highly elevated dTTP pools. The dTTP pool alterations did not cause specific mitochondrial DNA mutations or deletions when 6-month-old mice were analyzed. The mitochondrial DNA was also detected at normal levels. In conclusion, the Dm-dNK(+/-)TK2(-/-) mouse model illustrates how dTMP synthesized in the cell nucleus can compensate for loss of intramitochondrial dTMP synthesis in differentiated tissue. The data presented open new possibilities to treat the severe symptoms of TK2 deficiency.

  4. Transgene Expression of Drosophila melanogaster Nucleoside Kinase Reverses Mitochondrial Thymidine Kinase 2 Deficiency*♦

    Science.gov (United States)

    Krishnan, Shuba; Zhou, Xiaoshan; Paredes, João A.; Kuiper, Raoul V.; Curbo, Sophie; Karlsson, Anna

    2013-01-01

    A strategy to reverse the symptoms of thymidine kinase 2 (TK2) deficiency in a mouse model was investigated. The nucleoside kinase from Drosophila melanogaster (Dm-dNK) was expressed in TK2-deficient mice that have been shown to present with a severe phenotype caused by mitochondrial DNA depletion. The Dm-dNK+/− transgenic mice were shown to be able to rescue the TK2-deficient mice. The Dm-dNK+/−TK2−/− mice were normal as judged by growth and behavior during the observation time of 6 months. The Dm-dNK-expressing mice showed a substantial increase in thymidine-phosphorylating activity in investigated tissues. The Dm-dNK expression also resulted in highly elevated dTTP pools. The dTTP pool alterations did not cause specific mitochondrial DNA mutations or deletions when 6-month-old mice were analyzed. The mitochondrial DNA was also detected at normal levels. In conclusion, the Dm-dNK+/−TK2−/− mouse model illustrates how dTMP synthesized in the cell nucleus can compensate for loss of intramitochondrial dTMP synthesis in differentiated tissue. The data presented open new possibilities to treat the severe symptoms of TK2 deficiency. PMID:23288848

  5. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten

    2010-01-01

    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 tyrosine......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...... and highlighted their importance in bacterial physiology. Having no orthologues in Eukarya, BY-kinases are receiving a growing attention from the biomedical field, since they represent a particularly promising target for anti-bacterial drug design....

  6. Bromovinyl-deoxyuridine: A selective substrate for mitochondrial thymidine kinase in cell extracts

    International Nuclear Information System (INIS)

    Franzolin, Elisa; Rampazzo, Chiara; Perez-Perez, Maria-Jesus; Hernandez, Ana-Isabel; Balzarini, Jan; Bianchi, Vera

    2006-01-01

    Cellular models of mitochondrial thymidine kinase (TK2) deficiency require a reliable method to measure TK2 activity in whole cell extracts containing two interfering deoxyribonucleoside kinases, thymidine kinase 1 (TK1) and deoxycytidine kinase. We tested the value of the thymidine analog (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) as a TK2-specific substrate. With extracts of OSTTK1 - cells containing TK2 as the only thymidine kinase and a highly specific TK2 inhibitor we established conditions to detect the low TK2 activity commonly present in cells. With extracts of TK1-proficient osteosarcoma cells and normal human fibroblasts we showed that BVDU, but not 1-(β-D-arabinofuranosyl)thymine (Ara-T), discriminates TK2 activity even in the presence of 100-fold excess TK1. A comparison with current procedures based on TK2 inhibition demonstrated the better performance of the new TK2 assay. When cultured human fibroblasts passed from proliferation to quiescence TK2 activity increased by 3-fold, stressing the importance of TK2 function in the absence of TK1

  7. Bromovinyl-deoxyuridine: A selective substrate for mitochondrial thymidine kinase in cell extracts.

    Science.gov (United States)

    Franzolin, Elisa; Rampazzo, Chiara; Pérez-Pérez, María-Jesús; Hernández, Ana-Isabel; Balzarini, Jan; Bianchi, Vera

    2006-05-26

    Cellular models of mitochondrial thymidine kinase (TK2) deficiency require a reliable method to measure TK2 activity in whole cell extracts containing two interfering deoxyribonucleoside kinases, thymidine kinase 1 (TK1) and deoxycytidine kinase. We tested the value of the thymidine analog (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) as a TK2-specific substrate. With extracts of OSTTK1- cells containing TK2 as the only thymidine kinase and a highly specific TK2 inhibitor we established conditions to detect the low TK2 activity commonly present in cells. With extracts of TK1-proficient osteosarcoma cells and normal human fibroblasts we showed that BVDU, but not 1-(beta-d-arabinofuranosyl)thymine (Ara-T), discriminates TK2 activity even in the presence of 100-fold excess TK1. A comparison with current procedures based on TK2 inhibition demonstrated the better performance of the new TK2 assay. When cultured human fibroblasts passed from proliferation to quiescence TK2 activity increased by 3-fold, stressing the importance of TK2 function in the absence of TK1.

  8. Monitoring of herpes simplex virus thymidine kinase enzyme activity using positron emission tomography

    NARCIS (Netherlands)

    Hospers, GAP; Calogero, Anna; van Waarde, A; Doze, P; Vaalburg, W; Mulder, NH; de Vries, EFJ

    2000-01-01

    9-[(1-[F-18]Fluoro-3-hydroxy-2-propoxy)methyl]guanine ([F-18]FHPG) wasevaluated as a tracer for noninvasive positron emission tomography (PET) imaging of herpes simplex virus type 1 thymidine kinase (HSV-tk) gene expression. C6 rat glioma cells with and without the HSV-tk gene were incubated with

  9. Human cytosolic thymidine kinase: purification and physical characterization of the enzyme from HeLa cells

    International Nuclear Information System (INIS)

    Sherley, J.L.; Kelly, T.J.

    1988-01-01

    The mammalian cytosolic thymidine kinase is one of a number of enzymes involved in DNA replication whose activities increase dramatically during S phase of the cell cycle. As a first step in defining the mechanisms that control the S phase induction of thymidine kinase activity, the authors have purified the human enzyme from HeLa cells and raised a specific immune serum against the purified protein. The enzyme was isolated from cells arrested in S phase by treatment with methotrexate and purified to near homogeneity by ion-exchange and affinity chromatography. Stabilization of the purified enzyme was achieved by the addition of digitonin. An electrophoretic R/sub m/ of 0.2 in nondenaturing gels characterizes the purified enzyme activity as cytosolic thymidine kinase. The enzyme has a Stoke's radius of 40 A determined by gel filtration and a sedimentation coefficient of 5.5 S determined by glycerol gradient sedimentation. Based on these hydrodynamic values, a native molecular weight of 96,000 was calculated for the purified enzyme. When electrophoresed in denaturing sodium dodecyl sulfate-polyacrylamide gels under reducing conditions, the most purified enzyme fraction was found to contain one predominant polypeptide of M/sub r/ = 24,000. Several lines of evidence indicate that this polypeptide is responsible for thymidine kinase enzymatic activity

  10. 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

    The gene encoding thymidine kinase 1 from tomato (toTK1) has in combination with azidothymidine (AZT) recently been proposed as a powerful suicide gene for anticancer gene therapy. The toTK1/AZT combination has been demonstrated to have several advantages for the treatment of glioblastomas becaus...

  11. Elimination of the truncated message from the herpes simplex virus thymidine kinase suicide gene

    NARCIS (Netherlands)

    Chalmers, D; Ferrand, C; Apperley, JF; Melo, JV; Ebeling, S; Newton, [No Value; Duperrier, A; Hagenbeek, A; Garrett, E; Tiberghien, P; Garin, M

    Introduction of the Herpes simplex virus thymidine kinase (HSV-tk) gene into target cells renders them susceptible to killing by ganciclovir (GCV). We are studying the use of HSV-tk-transduced T lymphocytes in the context of hematopoietic stem cell transplantation. We have previously shown, in vitro

  12. Retained sensitivity to cytotoxic pyrimidine nucleoside analogs in thymidine kinase 2 deficient human fibroblasts

    OpenAIRE

    Bjerke, Mia; Solaroli, Nicola; Lesko, Nicole; Balzarini, Jan; Johansson, Magnus; Karlsson, Anna

    2010-01-01

    Thymidine kinase 2 (TK2) is a mitochondrial deoxyribonucleoside kinase that phosphorylates several nucleoside analogs used in anti-viral and anti-cancer therapy. A fibroblast cell line with decreased TK2 activity was investigated in order to obtain insights in the effects of TK2 deficiency on nucleotide metabolism. The role of TK2 for the sensitivity against cytotoxic nucleoside analogs was also investigated. The TK2 deficient cells retained their sensitivity against all pyrimidine nucleoside...

  13. Contrasting ability to take up leucine and thymidine among freshwater bacterial groups: implications for bacterial production measurements

    Science.gov (United States)

    Pérez, María Teresa; Hörtnagl, Paul; Sommaruga, Ruben

    2010-01-01

    We examined the ability of different freshwater bacterial groups to take up leucine and thymidine in two lakes. Utilization of both substrates by freshwater bacteria was examined at the community level by looking at bulk incorporation rates and at the single-cell level by combining fluorescent in situ hybridization and signal amplification by catalysed reporter deposition with microautoradiography. Our results showed that leucine was taken up by 70–80% of Bacteria-positive cells, whereas only 15–43% of Bacteria-positive cells were able to take up thymidine. When a saturating substrate concentration in combination with a short incubation was used, 80–90% of Betaproteobacteria and 67–79% of Actinobacteria were positive for leucine uptake, whereas thymidine was taken up by bacterial group. Bacterial abundance was a good predictor of the relative contribution of bacterial groups to leucine uptake, whereas when thymidine was used Actinobacteria represented the large majority (> 80%) of the cells taking up this substrate. Increasing the substrate concentration to 100 nM did not affect the percentage of R-BT cells taking up leucine (> 90% even at low concentrations), but moderately increased the fraction of thymidine-positive R-BT cells to a maximum of 35% of the hybridized cells. Our results show that even at very high concentrations, thymidine is not taken up by all, otherwise active, bacterial cells. PMID:19725866

  14. Dominant negative selection of vaccinia virus using a thymidine kinase/thymidylate kinase fusion gene and the prodrug azidothymidine

    International Nuclear Information System (INIS)

    Holzer, Georg W.; Mayrhofer, Josef; Gritschenberger, Werner; Falkner, Falko G.

    2005-01-01

    The Escherichia coli thymidine kinase/thymidylate kinase (tk/tmk) fusion gene encodes an enzyme that efficiently converts the prodrug 3'-azido-2',3'-dideoxythymidine (AZT) into its toxic triphosphate derivative, a substance which stops DNA chain elongation. Integration of this marker gene into vaccinia virus that normally is not inhibited by AZT allowed the establishment of a powerful selection procedure for recombinant viruses. In contrast to the conventional vaccinia thymidine kinase (tk) selection that is performed in tk-negative cell lines, AZT selection can be performed in normal (tk-positive) cell lines. The technique is especially useful for the generation of replication-deficient vaccinia viruses and may also be used for gene knock-out studies of essential vaccinia genes

  15. Retained sensitivity to cytotoxic pyrimidine nucleoside analogs in thymidine kinase 2 deficient human fibroblasts.

    Science.gov (United States)

    Bjerke, Mia; Solaroli, Nicola; Lesko, Nicole; Balzarini, Jan; Johansson, Magnus; Karlsson, Anna

    2010-01-01

    Thymidine kinase 2 (TK2) is a mitochondrial deoxyribonucleoside kinase that phosphorylates several nucleoside analogs used in anti-viral and anti-cancer therapy. A fibroblast cell line with decreased TK2 activity was investigated in order to obtain insights in the effects of TK2 deficiency on nucleotide metabolism. The role of TK2 for the sensitivity against cytotoxic nucleoside analogs was also investigated. The TK2 deficient cells retained their sensitivity against all pyrimidine nucleoside analogs tested. This study suggests that nucleoside analog phosphorylation mediated by TK2 may be less important, compared to other deoxyribonucleoside kinases, for the cytotoxic effects of these compounds.

  16. 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...

  17. Loss of thymidine kinase 2 alters neuronal bioenergetics and leads to neurodegeneration

    OpenAIRE

    Bartesaghi, Stefano; Betts-Henderson, Joanne; Cain, Kelvin; Dinsdale, David; Zhou, Xiaoshan; Karlsson, Anna; Salomoni, Paolo; Nicotera, Pierluigi

    2010-01-01

    Mutations of thymidine kinase 2 (TK2), an essential component of the mitochondrial nucleotide salvage pathway, can give rise to mitochondrial DNA (mtDNA) depletion syndromes (MDS). These clinically heterogeneous disorders are characterized by severe reduction in mtDNA copy number in affected tissues and are associated with progressive myopathy, hepatopathy and/or encephalopathy, depending in part on the underlying nuclear genetic defect. Mutations of TK2 have previously been associated with a...

  18. Advances in study of perpes simplex virus type 1-thymidine kinase reporter gene imaging

    International Nuclear Information System (INIS)

    Liu Ying; Lan Xiaoli; Zhang Yongxue

    2007-01-01

    Radionuclide reporter gene imaging is an effect way to provide qualitative and quantitative information for gene therapy. There are three systems of reporter gene including kinase reporter gene. perpes simplex virus type 1-thymidine kinase (HSV1-tk) has perfect physical and chemical characteristic which is suit for imaging as reporter gene. It has been widely investigated and intensively researched. Two substrates of HSV1-tk are purine nucleosite derivant and acyclovir derivant, which can also be used as reporter probes of HSV1-tk. (authors)

  19. Properties of Cells Carrying the Herpes Simplex Virus Type 2 Thymidine Kinase Gene: Mechanisms of Reversion to a Thymidine Kinase-Negative Phenotype

    Science.gov (United States)

    Bastow, K. F.; Darby, G.; Wildy, P.; Minson, A. C.

    1980-01-01

    We have isolated cells with a thymidine kinase-negative (tk−) phenotype from cells which carry the herpes simplex virus type 2 tk gene by selection in 5-bromodeoxyuridine or 9-(2-hydroxyethoxymethyl)guanine. Both selection routines generated revertants with a frequency of 10−3 to 10−4, and resistance to either compound conferred simultaneous resistance to the other. tk− revertants fell into three classes: (i) cells that arose by deletion of all virus sequences, (ii) cells that had lost the virus tk gene but retained a nonselected virus-specific function and arose by deletion of part of the virus-specific sequence, and (iii) cells that retained the potential to express all of the virus-specific functions of the parental cells and retained all of the virus-specific DNA sequences. Images PMID:16789205

  20. Estimating bacterial production in marine waters from the simultaneous incorporation of thymidine and leucine.

    Science.gov (United States)

    Chin-Leo, G; Kirchman, D L

    1988-08-01

    We examined the simultaneous incorporation of [H]thymidine and [C]leucine to obtain two independent indices of bacterial production (DNA and protein syntheses) in a single incubation. Incorporation rates of leucine estimated by the dual-label method were generally higher than those obtained by the single-label method, but the differences were small (dual/single = 1.1 +/- 0.2 [mean +/- standard deviation]) and were probably due to the presence of labeled leucyl-tRNA in the cold trichloroacetic acid-insoluble fraction. There were no significant differences in thymidine incorporation between dual- and single-label incubations (dual/ single = 1.03 +/- 0.13). Addition of the two substrates in relatively large amounts (25 nM) did not apparently increase bacterial activity during short incubations (leucine incorporation rates covaried over depth profiles of the Chesapeake Bay. Estimates of bacterial production based on thymidine and leucine differed by less than 25%. Although the need for appropriate conversion factors has not been eliminated, the dual-label approach can be used to examine the variation in bacterial production while ensuring that the observed variation in incorporation rates is due to real changes in bacterial production rather than changes in conversion factors or introduction of other artifacts.

  1. Virus-specific DNA sequences present in cells which carry the herpes simplex virus thymidine kinase gene.

    Science.gov (United States)

    Minson, A C; Darby, G K; Wildy, P

    1979-11-01

    Two independently derived cell lines which carry the herpes simplex type 2 thymidine kinase gene have been examined for the presence of HSV-2-specific DNA sequences. Both cell lines contained 1 to 3 copies per cell of a sequence lying within map co-ordinates 0.2 to 0.4 of the HSV-2 genome. Revertant cells, which contained no detectable thymidine kinase, did not contain this DNA sequence. The failure of EcoR1-restricted HSV-2 DNA to act as a donor of the thymidine kinase gene in transformation experiments suggests that the gene lies close to the EcoR1 restriction site within this sequence at a map position of approx. 0.3. The HSV-2 kinase gene is therefore approximately co-linear with the HSV-1 gene.

  2. The pathogenicity of thymidine kinase-deficient mutants of herpes simplex virus in mice.

    Science.gov (United States)

    Field, H J; Wildy, P

    1978-10-01

    The pathogenicity for mice of two mutants of herpes simplex virus (type 1 and type 2), which fail to induce thymidine kinase, were compared with their respective parent strains. The mutants were much less virulent than the parents following either intracerebral or peripheral inoculation. The replication of the virus at the site of inoculation and its progression into the nervous system were studied. Following a very large inoculum in the ear, the type 1 mutant was found to establish a latent infection in the cervical dorsal root ganglia. Mice inoculated intracerebrally with small doses of the mutant viruses were solidly immune to challenge with lethal doses of the parent strain.

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

    International Nuclear Information System (INIS)

    Hlavaty, J.; Hlubinova, K.; Altanerova, V.; Liska, J.; Altaner, C.

    1997-01-01

    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 C 6 rat glioma cells with subsequent injection of HSVtk retrovirus producing cells. (author)

  4. 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

    2012-01-01

    and AtTK1b catalyze redundant reactions. The results obtained in the present study suggest a crucial role for the salvage of thymidine during early plant development. Sequence data from the present study have been deposited in the EMBL database/GenBank under accession numbers: AT3G07800.1 (AtTK1a), At5G...

  5. Preliminary validation of varicella zoster virus thymidine kinase as a novel reporter gene for PET

    International Nuclear Information System (INIS)

    Deroose, Christophe M.; Chitneni, Satish K.; Gijsbers, Rik; Vermaelen, Peter; Ibrahimi, Abdelilah; Balzarini, Jan; Baekelandt, Veerle; Verbruggen, Alfons; Nuyts, Johan; Debyser, Zeger; Bormans, Guy M.

    2012-01-01

    Introduction: Imaging of gene expression with positron emission tomography (PET) has emerged as a powerful tool for biomedical research during the last decade. The prototypical herpes simplex virus type 1 thymidine kinase (HSV1-TK) PET reporter gene (PRG) is widely used and many other PRGs have also been validated. We investigated varicella zoster virus thymidine kinase (VZV-tk) as new PRG with radiolabeled bicyclic nucleoside analogues (BCNAs) as PET tracers. Methods: The uptake and washout of four different radiolabeled BCNAs was evaluated in cells expressing VZV-tk after lentiviral vector (LV) transduction and in control cells. Metabolism of the tracers was assayed by high pressure liquid chromatography (HPLC). Mice bearing VZV-TK expressing xenografts were imaged with PET. Results: High uptake in VZV-tk expressing cells was seen for 3 of the 4 tracers tested. The uptake of the tracers could be blocked by the presence of excess thymidine in the incubation solution. Cellular retention was variable, with one tracer showing an acceptable half-life of ∼ 1 hour. The amount of intracellular tracer correlated with the titer of LV used to transduce the cells. VZV-TK dependent conversion into metabolites was shown by HPLC. No specific accumulation was observed in cells expressing a fusion protein containing an HSV1-TK moiety. VZV-tk expression in xenografts resulted in a 60% increase in uptake in vivo as measured with PET. Conclusions: We have validated the combination of VZV-tk and radiolabeled BCNAs as new PRG/PRP system. Further optimization of the PRPs and the PRG are warranted to increase the signal.

  6. 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)

    Waldman, A.S.; Milman, G.

    1984-01-01

    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 [ 3 H]thymidine into cell nuclei as measured by autoradiography

  7. 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...

  8. 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

  9. 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

    Serum thymidine kinase (S-TK) was measured weekly in 16 randomly selected patients with AIDS or AIDS-related complex (ARC; Centers for Disease Control group IV A or group IV C-2) who participated in a controlled study of the efficacy of zidovudine therapy. S-TK increased significantly (P less than...... 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...

  10. Preoperative Serum Thymidine Kinase Activity as Novel Monitoring, Prognostic, and Predictive Biomarker in Pancreatic Cancer.

    Science.gov (United States)

    Felix, Klaus; Hinz, Ulf; Dobiasch, Sophie; Hackert, Thilo; Bergmann, Frank; Neumüller, Magnus; Gronowitz, Simon; Bergqvist, Mattias; Strobel, Oliver

    2018-01-01

    The aim of the study was to investigate serum thymidine kinase 1 (S-TK) activity as a diagnostic and prognostic marker for patients with pancreatic ductal adenocarcinoma (PDAC). Using the sensitive TK activity assay DiviTum, preoperative serum samples from 404 PDAC, 28 chronic pancreatitis, and 25 autoimmune pancreatitis patients and 83 healthy volunteers were analyzed. The preoperative S-TK activities of 54 PDAC patients who received neoadjuvant therapy (nTx) were also compared with those of 258 PDAC patients who did not receive nTx. The preoperative S-TK activities of PDAC patients were significantly higher and discriminatory from autoimmune and chronic pancreatitis patients and control groups. The S-TK activity in PDAC patients was associated with overall survival. Patients with S-TK activity of less than 80 Du (DiviTum units)/L demonstrated median survival of 20.3 months with an estimated 18.0% 5-year survival rate; for S-TK activity of 80 Du/L or greater, median survival was 15.1 months with a 6.8% 5-year survival rate. For early-stage PDAC, these differences were even more pronounced. The S-TK activity in the nTx group was significantly higher than that in the group not receiving nTx. Pancreatic ductal adenocarcinomas reveal a significant increase in S-TK activity, which is associated with overall survival, especially in early tumor stages. Serum thymidine kinase 1 activity may be a useful parameter for monitoring nTx efficacy.

  11. Estimating Bacterial Production in Marine Waters from the Simultaneous Incorporation of Thymidine and Leucine

    OpenAIRE

    Chin-Leo, Gerardo; Kirchman, David L.

    1988-01-01

    We examined the simultaneous incorporation of [3H]thymidine and [14C]leucine to obtain two independent indices of bacterial production (DNA and protein syntheses) in a single incubation. Incorporation rates of leucine estimated by the dual-label method were generally higher than those obtained by the single-label method, but the differences were small (dual/single = 1.1 ± 0.2 [mean ± standard deviation]) and were probably due to the presence of labeled leucyl-tRNA in the cold trichloroacetic ...

  12. Validity of the tritiated thymidine method for estimating bacterial growth rates: measurement of isotope dilution during DNA synthesis

    International Nuclear Information System (INIS)

    Pollard, P.C.; Moriarty, D.J.W.

    1984-01-01

    The rate of tritiated thymidine incorporation into DNA was used to estimate bacterial growth rates in aquatic environments. To be accurate, the calculation of growth rates has to include a factor for the dilution of isotope before incorporation. The validity of an isotope dilution analysis to determine this factor was verified in experiments reported here with cultures of a marine bacterium growing in a chemostat. Growth rates calculated from data on chemostat dilution rates and cell density agreed well with rates calculated by tritiated thymidine incorporation into DNA and isotope dilution analysis. With sufficiently high concentrations of exogenous thymidine, de novo synthesis of deoxythymidine monophosphate was inhibited, thereby preventing the endogenous dilution of isoope. The thymidine technique was also shown to be useful for measuring growth rates of mixed suspensions of bacteria growing anaerobically. Thymidine was incorporated into the DNA of a range of marine pseudomonads that were investigated. Three species did not take up thymidine. The common marine cyanobacterium Synechococcus species did not incorporate thymidine into DNA

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

    International Nuclear Information System (INIS)

    Knight, G.B.; Gudas, J.M.; Pardee, A.B.

    1987-01-01

    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

  14. Temperature-driven adaptation of the bacterial community in peat measured by using thymidine and leucine incorporation

    OpenAIRE

    Ranneklev, Sissel Brit; Bååth, Erland

    2001-01-01

    The temperature-driven adaptation of the bacterial community in peat was studied, by altering temperature to simulate self-heating and a subsequent return to mesophilic conditions. The technique used consisted of extracting the bacterial community from peat using homogenization-centrifugation and measuring the rates of thymidine (TdR) or leucine (Leu) incorporation by the extracted bacterial community at different temperatures. Increasing the peat incubation temperature from 25°C to 35, 45, o...

  15. 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 drug discovery approach, we have identified small-molecule histidine-kinase

  16. Thymidine kinase 2 (H126N) knockin mice show the essential role of balanced deoxynucleotide pools for mitochondrial DNA maintenance

    OpenAIRE

    Akman, Hasan O.; Dorado, Beatriz; López, Luis C.; García-Cazorla, Ángeles; Vilà, Maya R.; Tanabe, Lauren M.; Dauer, William T.; Bonilla, Eduardo; Tanji, Kurenai; Hirano, Michio

    2008-01-01

    Mitochondrial DNA (mtDNA) depletion syndrome (MDS), an autosomal recessive condition, is characterized by variable organ involvement with decreased mtDNA copy number and activities of respiratory chain enzymes in affected tissues. MtDNA depletion has been associated with mutations in nine autosomal genes, including thymidine kinase (TK2), which encodes a ubiquitous mitochondrial protein. To study the pathogenesis of TK2-deficiency, we generated mice harboring an H126N Tk2 mutation. Homozygous...

  17. Thymidine Kinase 2 Deficiency-Induced mtDNA Depletion in Mouse Liver Leads to Defect beta-Oxidation

    OpenAIRE

    Zhou, Xiaoshan; Kannisto, Kristina; Curbo, Sophie; von Dobeln, Ulrika; Hultenby, Kjell; Isetun, Sindra; Gåfvels, Mats; Karlsson, Anna

    2013-01-01

    Thymidine kinase 2 (TK2) deficiency in humans causes mitochondrial DNA (mtDNA) depletion syndrome. To study the molecular mechanisms underlying the disease and search for treatment options, we previously generated and described a TK2 deficient mouse strain (TK2(-/-)) that progressively loses its mtDNA. The TK2(-/-) mouse model displays symptoms similar to humans harboring TK2 deficient infantile fatal encephalomyopathy. Here, we have studied the TK2(-/-) mouse model to clarify the pathologica...

  18. Thymidine kinase-negative herpes simplex virus mutants establish latency in mouse trigeminal ganglia but do not reactivate.

    OpenAIRE

    Coen, D M; Kosz-Vnenchak, M; Jacobson, J G; Leib, D A; Bogard, C L; Schaffer, P A; Tyler, K L; Knipe, D M

    1989-01-01

    Herpes simplex virus infection of mammalian hosts involves lytic replication at a primary site, such as the cornea, translocation by axonal transport to sensory ganglia and replication, and latent infection at a secondary site, ganglionic neurons. The virus-encoded thymidine kinase, which is a target for antiviral drugs such as acyclovir, is not essential for lytic replication yet evidently is required at the secondary site for replication and some phase of latent infection. To determine the ...

  19. Study on the measurement of serum thymidine kinase and its clinical significance in hematological neoplastic disorders

    Energy Technology Data Exchange (ETDEWEB)

    Torizumi, Kazutami; Aibata, Hirofumi; Yamada, Ryusaku; Shimizu, Eiji; Okamoto, Yukiharu; Tsujimoto, Masato; Tsuda, Tadaaki; Ota, Kiichiro

    1988-06-01

    A 'Prolifigen TK-REA' kit for measuring serum thymidine kinase (TK) was fundamentally and clinically evaluated. Laboratory findings for recovery, dilution, and reproducibility were satisfactory. There was no correlation between serum TK activity and serum lactic dehydrogenase, carcinoembryonic antigen, or ..cap alpha..-fetoprotein. The serum concentration of TK in normal volunteers ranged from 1.6 to 6.5 U/L. It was extremely high for chronic myeloid leukemia (CML) and acute myeloid leukemia (AML), as compared to the normal value. In the AML group, higher incidence of blasts in peripheral blood tended to be associated with higher serum concentration of TK. A similar tendency was seen in the case of acute lymphocytic leukemia (ALL) and myelodysplasia syndrome (MDS). A positive correlation between serum TK activity and the absolute counts of myeloblasts in peripheral blood existed in CML and AML patients. Since patients with hematological neoplastic disorders, who have abnormality in DNA metabolism, tended to have higher serum TK activity than did normal volunteers, serum TK activity may have a potential marker for abnormal DNA metabolism. (Namekawa, K.).

  20. Molecular characterization of thymidine kinase mutants of human cells induced by densely ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Kronenberg, A; Little, J B

    1989-04-01

    In order to characterize the nature of mutants induced by densely ionizing radiations at an autosomal locus, the authors have isolated a series of 99 thymidine kinase (tk) mutants of human TK6 lymphoblastoid cells iraadiated with either fast neutrons or accelerated argon ions. Individual muant clones were examined for alterations in their restriction fragment pattern after hybridization with a human cDNA probe for tk. A restriction fragment length polymorphism (RFLP) allowed identification of the active tk allele. Among the neutron-induced mutants, 34/52 exhibited loss of the previously active allele while 6/52 exhibited intragenic rearrangements. Among the argon-induced mutants 27/46 exhibited allele loses and 10/46 showed rearrangements within the tk locus. The remaining mutants had restriction patterns indistinguishable from the TK6 parent. Each of the mutant clones was further examined for structural alterations within the c-erbAl locus which has been localized to chromosome 17q11-q22, at some unknown distance from the human tk locus at chromosome 17q21-q22. A substantial proportion (54%) of tk mutants induced by densely ionizing radiation showed loss of the c-erb locus on the homologous chromosome, suggesting that the mutations involve large-scale genetic changes. (author). 51 refs.; 2 figs.; 6 tabs.

  1. Loss of thymidine kinase 2 alters neuronal bioenergetics and leads to neurodegeneration.

    Science.gov (United States)

    Bartesaghi, Stefano; Betts-Henderson, Joanne; Cain, Kelvin; Dinsdale, David; Zhou, Xiaoshan; Karlsson, Anna; Salomoni, Paolo; Nicotera, Pierluigi

    2010-05-01

    Mutations of thymidine kinase 2 (TK2), an essential component of the mitochondrial nucleotide salvage pathway, can give rise to mitochondrial DNA (mtDNA) depletion syndromes (MDS). These clinically heterogeneous disorders are characterized by severe reduction in mtDNA copy number in affected tissues and are associated with progressive myopathy, hepatopathy and/or encephalopathy, depending in part on the underlying nuclear genetic defect. Mutations of TK2 have previously been associated with an isolated myopathic form of MDS (OMIM 609560). However, more recently, neurological phenotypes have been demonstrated in patients carrying TK2 mutations, thus suggesting that loss of TK2 results in neuronal dysfunction. Here, we directly address the role of TK2 in neuronal homeostasis using a knockout mouse model. We demonstrate that in vivo loss of TK2 activity leads to a severe ataxic phenotype, accompanied by reduced mtDNA copy number and decreased steady-state levels of electron transport chain proteins in the brain. In TK2-deficient cerebellar neurons, these abnormalities are associated with impaired mitochondrial bioenergetic function, aberrant mitochondrial ultrastructure and degeneration of selected neuronal types. Overall, our findings demonstrate that TK2 deficiency leads to neuronal dysfunction in vivo, and have important implications for understanding the mechanisms of neurological impairment in MDS.

  2. Identification and nucleotide sequence of the thymidine kinase gene of Shope fibroma virus

    International Nuclear Information System (INIS)

    Upton, C.; McFadden, G.

    1986-01-01

    The thymidine kinase (TK) gene of Shope fibroma virus (SFV), a tumorigenic leporipoxvirus, was localized within the viral genome with degenerate oligonucleotide probes. These probes were constructed to two regions of high sequence conservation between the vaccinia virus TK gene and those of several known eucaryotic cellular TK genes, including human, mouse, hamster, and chicken TK genes. The oligonucleotide probes initially localized the SFV TK gene 50 kilobases (kb) from the right terminus of the 160-kb SFV genome within the 9.5-kb BamHI-HindIII fragment E. Fine-mapping analysis indicated that the TK Gene was within a 1.2-kb AvaI-HaeIII fragment, and DNA sequencing of this region revealed an open reading frame capable of encoding a polypeptide of 187 amino acids possessing considerable homology to the TK genes of the vaccinia, variola, and monkeypox orthopoxviruses and also to a variety of cellular TK genes. Homology matrix analysis and homology scores suggest that the SFV TK gene has diverged significantly from its counterpart members in the orthopoxvirus genus. Nevertheless, the presence of conserved upstream open reading frames on the 5' side of all of the poxvirus TK genes indicates a similarity of functional organization between the orthopoxviruses and leporipoxviruses. These data suggest a common ancestral origin for at least some of the unique internal regions of the leporipoxviruses and orthopoxviruses as exemplified by SFV and vaccinia virus, respectively

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

    International Nuclear Information System (INIS)

    Haberkorn, Uwe; Khazaie, Khashayarsha; Morr, Iris; Altmann, Annette; Mueller, Markus; Kaick, Gerhard van

    1998-01-01

    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

  4. Elimination of proliferating cells from CNS grafts using a Ki67 promoter-driven thymidine kinase

    Directory of Open Access Journals (Sweden)

    Vannary Tieng

    2016-01-01

    Full Text Available Pluripotent stem cell (PSC-based cell therapy is an attractive concept for neurodegenerative diseases, but can lead to tumor formation. This is particularly relevant as proliferating neural precursors rather than postmitotic mature neurons need to be transplanted. Thus, safety mechanisms to eliminate proliferating cells are needed. Here, we propose a suicide gene approach, based on cell cycle-dependent promoter Ki67-driven expression of herpes simplex virus thymidine kinase (HSV-TK. We generated a PSC line expressing this construct and induced neural differentiation. In vitro, proliferating PSC and early neural precursor cells (NPC were killed by exposure to ganciclovir. In vivo, transplantation of PSC led to tumor formation, which was prevented by early ganciclovir treatment. Transplanted NPC did not lead to tumor formation and their survival and neural maturation were not affected by ganciclovir. In conclusion, the cell cycle promoter-driven suicide gene approach described in this study allows killing of proliferating undifferentiated precursor cells without expression of the suicide gene in mature neurons. This approach could also be of use for other stem cell-based therapies where the final target consists of postmitotic cells.

  5. Investigation on Cell Proliferation with a New Antibody against Thymidine Kinase 1

    Directory of Open Access Journals (Sweden)

    Naining Wang

    2001-01-01

    Full Text Available The cytosolic thymidine kinase 1 (TK1 is one of the enzymes involved in DNA replication. Based on biochemical studies, TK1 is activated at late G1 of cell cycle, and its activity correlates with the cell proliferation. We have developed a polyclonal anti‐TK1 antibody against a synthetic peptide from the C‐terminus of human TK1. Using this antibody, here we demonstrate the exclusive location of TK1 in the cytoplasm of cells. Cell cycle dependent TK1 expression was studied by simultaneous fluorescence staining for TK1 and bromodeoxyuridine, by using elutriated cells, and by quantitation of the amount TK1 in relation to the cellular DNA content. TK1, which was strongly expressed in the cells in S+G2 period, raised at late G1 and decreased during mitosis. The amount of TK1 increased three folds from late G1 to G2. TK1 positive cells were demonstrated in areas of proliferation activity of various normal and malignant tissues. The new anti‐TK1 antibody works in archival specimens and is a specific marker of cell proliferation.

  6. 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.

  7. Thymidine kinase enzyme selective imaging radiopharmaceutical. {sup 99m}Tc(CO){sub 3}-Ganciclovir

    Energy Technology Data Exchange (ETDEWEB)

    Gedik, B.; Teksoez, S.; Ichedef, C.; Kilcar, A.Y.; Medine, E.I.; Ucar, E. [Ege Univ., Bornova, Izmir (Turkey). Dept. of Nuclear Applications

    2013-03-01

    The aim of this study is to radiolabel Ganciclovir, known as having selective antiviral properties against thymidine kinase, with technetium tricarbonylcore ({sup 99m}Tc(CO){sub 3}{sup +}) and to investigate the biological behavior of this complex in vitro and in vivo. Commercially provided Ganciclovir (GCV) was radiolabeled with {sup 99m}Tc(CO){sub 3}{sup +}. Initially, optimum radiolabeling conditions were determined by analyzing factors such as temperature, pH and time. Quality control of the radiolabeled compound was performed. The radiolabeling yield was found to be 97%. The {sup 99m}Tc(CO){sub 3}-GCV complex also displayed good in vitro stability during the 24 h period. In vitro cell uptake studies showed that the {sup 99m}Tc(CO){sub 3}-GCV complex is highly uptaken in A-549, PC-3, HeLa cell lines according to the control group {sup 99m}Tc(I)-tricarbonyl core. The knowledge gained from in vivo and in vitro studies of {sup 99m}Tc(CO){sub 3}-GCV could contribute to the development of a new HSV1-tk gene imaging agent. (orig.)

  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

    1995-01-01

    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.......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 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

    International Nuclear Information System (INIS)

    Slot Christiansen, Louise; Egeblad, Louise; Munch-Petersen, Birgitte; Piškur, Jure; Knecht, Wolfgang

    2015-01-01

    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

    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.

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

    International Nuclear Information System (INIS)

    Robarts, R.D.; Wicks, R.J.; Sephton, L.M.

    1986-01-01

    The incorporation of [methyl- 3 H]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- 3 H]thymidine studies of aquatic bacterial production

  12. mtDNA depletion myopathy: elucidation of the tissue specificity in the mitochondrial thymidine kinase (TK2) deficiency.

    Science.gov (United States)

    Saada, Ann; Shaag, Avraham; Elpeleg, Orly

    2003-05-01

    Decreased mitochondrial thymidine kinase (TK2) activity is associated with mitochondrial DNA (mtDNA) depletion and respiratory chain dysfunction and is manifested by isolated, fatal skeletal myopathy. Other tissues such as liver, brain, heart, and skin remain unaffected throughout the patients' life. In order to elucidate the mechanism of tissue specificity in the disease we have investigated the expression of the mitochondrial deoxynucleotide carrier, the mtDNA content and the activity of TK2 in mitochondria of various tissues. Our results suggest that low basal TK2 activity combined with a high requirement for mitochondrial encoded proteins in muscle predispose this tissue to the devastating effect of TK2 deficiency.

  13. Synthesis of 5-radioiodoarabinosyl uridine analog for probing HSV-1 thymidine kinase gene: an unexpected chelating effect

    Energy Technology Data Exchange (ETDEWEB)

    Yu, C.-S. [Department of Nuclear Science, National Tsing-Hua University, Hsinchu 300, Taiwan (China)]. E-mail: csyu@mx.nthu.edu.tw; Chiang, L.-W. [Department of Nuclear Science, National Tsing-Hua University, Hsinchu 300, Taiwan (China); Wu, C.-H. [Department of Nuclear Science, National Tsing-Hua University, Hsinchu 300, Taiwan (China); Wang, R.-T. [Department of Nuclear Science, National Tsing-Hua University, Hsinchu 300, Taiwan (China); Chen, S.-W. [Department of Nuclear Science, National Tsing-Hua University, Hsinchu 300, Taiwan (China); Wang, H.-Y. [Department of Nuclear Science, National Tsing-Hua University, Hsinchu 300, Taiwan (China); Yeh, C.-H. [Department of Nuclear Science, National Tsing-Hua University, Hsinchu 300, Taiwan (China)

    2006-04-15

    Tumor cells transduced with herpes simplex virus thymidine kinase gene has been intensively applied to the field of positron emission tomography via imaging of its substrate. As a pilot synthesis approach, a facial preparation of 5-[{sup 125}I]iodoarabinosyl uridine starting from commercial available uridine is reported herein. Interestingly, the tin group in 5-trimethylstannyl arabinosyluridine was easily removed during purification. The destannylation through the formation of a six-ligand coordination involving 2'-hydroxyl and tin was thereby proposed.

  14. 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

    Balanced deoxynucleotide pools are known to be important for correct DNA repair, and deficiency for some of the central enzymes in deoxynucleotide metabolism can cause imbalanced pools, which in turn can lead to mutagenesis and cell death. Here we show that cells deficient for the thymidine salva...

  15. Role of the viral and cellular encoded thymidine kinase in the repair of UV-irradiated herpes simplex virus

    International Nuclear Information System (INIS)

    Rainbow, A.J.; McMaster Univ., Hamilton, ON

    1989-01-01

    A strain of herpes simplex type 1 (HSV-1:KOS) encoding a functional thymidine kinase (tk + ) gene and a thymidine kinase deficient (tk - ) mutant strain (HSC-1:PTK3B) were used as probes to examine the repair of UV-damaged viral DNA in one tk - (143) and two tk + (R970-5 and AC4) human cell lines. UV survival for each HSC-1 strain was similar for infection of both tk - and tk + cells suggesting that the repair of viral DNA was not dependent on the expression of a functional cellular tk gene. In contrast, UV survival of HSV-1:PTK3B was substantially reduced compared to HSV-1:KOS when infecting all 3 human cell lines, as well as Vero monkey kidney cells and LPM1A mouse cells. Tjese results suggest that the repair of UV-irradiated HSV-1 in lytically infected mammalian cells depends, in part at least, on the expression of the viral encoded tk. (author). 20 refs.; 1 fig

  16. Down regulation by a low-zinc diet in gene expression of rat prostatic thymidylate synthase and thymidine kinase

    Directory of Open Access Journals (Sweden)

    Sassa Shuji

    2008-05-01

    Full Text Available Abstract Background Zinc has a wide spectrum of biological activities and its deficiency is related to various abnormalities of cell metabolism. Methods Wistar male rats, at age of 4 weeks, were fed a low-zinc diet for six weeks. The levels of bromodeoxyuridine incorporated into the prostatic DNA and the mRNA expression levels of prostate thymidylate synthase and thymidine kinase were examined. Result The low-zinc diet caused a marked reduction in the body growth and organ weights, resulted in a low hematopoiesis, hypo-albuminemia and hypocholesterolemia. Although there were few differences in plasma biochemical markers, plasma levels of luteinizing hormone and testosterone were reduced by the low-zinc diet. Bromodeoxyuridine-immunoreactive (S-phase cells and mRNA expression levels of thymidylate synthase and thymidine kinase in the prostate cells were markedly affected by the low-zinc diet. Conclusion A low-zinc diet appears to reduce the body growth and organ weights including prostate, causing low plasma levels of luteinizing hormone and testosterone and reduction in prostate DNA replication in growing-rats.

  17. 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

    Thymidine kinase (TK) catalyses the ATP-dependent phosphorylation of thymidine to thymidine monophosphate, which is subsequency phosphorylated to thymidine triphosphate and utilized for DNA synthesis. Human cytosolic TK (TKI) is cell cycle regulated, e.g. the TK1 activity increases sharply at the G...... patients with chronic lymphatic leukemia (CLL). 2: Structure-function relationship of recombinant TKI. In the first part a sensitive method (competitive PCR) for quantification of TKI mRNA was established. The TKI mRNA level was quantified in quiescent lymphocytes from control donors (n = 6...... are characterized as being quiescent, the TK activity was in the same range as in quiescent lymphocytes from control donors. However, quantification of the TKI mRNA level shows that all five CLL patients had a very high level (6 to 22 x IO6 copies mg-’ protein) of TKI mRNA, corresponding to the level in dividing...

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

    International Nuclear Information System (INIS)

    Schwartz, Jeffrey L.; Tamura, Yasuko; Jordan, Robert; Grierson, John R.; Krohn, Kenneth A.

    2004-01-01

    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 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 1 activity, and FLT uptake. To accomplish this, TK 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 1 activity, and FLT uptake were essentially unchanged in the p53-normal cell line. In contrast, TK 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

  19. Targeted Transgenic Overexpression of Mitochondrial Thymidine Kinase (TK2) Alters Mitochondrial DNA (mtDNA) and Mitochondrial Polypeptide Abundance : Transgenic TK2, mtDNA, and Antiretrovirals

    OpenAIRE

    Hosseini, Seyed H.; Kohler, James J.; Haase, Chad P.; Tioleco, Nina; Stuart, Tami; Keebaugh, Erin; Ludaway, Tomika; Russ, Rodney; Green, Elgin; Long, Robert; Wang, Liya; Eriksson, Staffan; Lewis, William

    2007-01-01

    Mitochondrial toxicity limits nucleoside reverse transcriptase inhibitors (NRTIs) for acquired immune deficiency syndrome. NRTI triphosphates, the active moieties, inhibit human immunodeficiency virus reverse transcriptase and eukaryotic mitochondrial DNA polymerase pol-γ. NRTI phosphorylation seems to correlate with mitochondrial toxicity, but experimental evidence is lacking. Transgenic mice (TGs) with cardiac overexpression of thymidine kinase isoforms (mitochondrial TK2 and cytoplasmic TK...

  20. F-18-FEAU as a radiotracer for herpes simplex virus thymidine kinase gene expression : in-vitro comparison with other PET tracers

    NARCIS (Netherlands)

    Buursma, AR; Rutgers, [No Value; Hospers, GAP; Mulder, NH; Vaalburg, W; de Vries, EFJ

    Objective The herpes simplex virus thymidine kinase (HSVtk) gene has frequently been applied as a reporter gene for monitoring transgene expression in animal models. In clinical gene therapy protocols, however, extremely low expression levels of the transferred gene are generally observed.

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

    NARCIS (Netherlands)

    de Vries, EFJ; van Waarde, A; Harmsen, MC; Mulder, NH; Vaalburg, W; Hospers, GAP

    [C-11]-2'-Fluoro-5-methyl-1-beta-D-arabinofuranosyluracil ([C-11]FMAU) and [F-18]-9-[(3-fluoro-1-hydroxy-2-propoxy)methyl]guanine ([F-18]FHPG), radiolabeled representatives of two classes of antiviral agents, were evaluated as tracers for measuring herpes simplex virus thymidine kinase (HSV-tk)

  2. 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

    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...

  3. Characterization of a thymidine kinase-deficient mutant of equine herpesvirus 4 and in vitro susceptibility of the virus to antiviral agents.

    Science.gov (United States)

    Azab, Walid; Tsujimura, Koji; Kato, Kentaro; Arii, Jun; Morimoto, Tomomi; Kawaguchi, Yasushi; Tohya, Yukinobu; Matsumura, Tomio; Akashi, Hiroomi

    2010-02-01

    Equine herpesvirus 4 (EHV-4) is an important equine pathogen that causes respiratory tract disease among horses worldwide. A thymidine kinase (TK)-deletion mutant has been generated by using bacterial artificial chromosome (BAC) technology to investigate the role of TK in pathogenesis. Deletion of TK had virtually no effect on the growth characteristics of WA79DeltaTK in cell culture when compared to the parent virus. Also, virus titers and plaque formation were unaffected in the absence of the TK gene. The sensitivity of EHV-4 to inhibition by acyclovir (ACV) and ganciclovir (GCV) was studied by means of a plaque reduction assay. GCV proved to be more potent and showed a superior anti-EHV-4 activity. On the other hand, ACV showed very poor ability to inhibit EHV-4 replication. As predicted, WA79DeltaTK was insensitive to GCV. Although EHV-4 is normally insensitive to ACV, it showed >20-fold increase in sensitivity when the equine herpesvirus-1 (EHV-1) TK was supplied in trans. Furthermore, both ACV and GCV resulted in a significant reduction of plaque size induced by EHV-4 and 1. Taken together, these data provided direct evidence that GCV is a potent selective inhibitor of EHV-4 and that the virus-encoded TK is an important determinant of the virus susceptibility to nucleoside analogues. Copyright 2009 Elsevier B.V. All rights reserved.

  4. Two novel mutations in thymidine kinase-2 cause early onset fatal encephalomyopathy and severe mtDNA depletion.

    Science.gov (United States)

    Lesko, Nicole; Naess, Karin; Wibom, Rolf; Solaroli, Nicola; Nennesmo, Inger; von Döbeln, Ulrika; Karlsson, Anna; Larsson, Nils-Göran

    2010-03-01

    Deficiency of thymidine kinase-2 (TK2) has been described in children with early onset fatal skeletal myopathy. TK2 is a mitochondrial deoxyribonucleoside kinase required for the phosphorylation of deoxycytidine and deoxythymidine and hence is vital for the maintenance of a balanced mitochondrial dNTP pool in post-mitotic tissues. We describe a patient with two novel TK2 mutations, which caused disease onset shortly after birth and death at the age of three months. One mutation (219insCG) generated an early stop codon, thus preventing the synthesis of a functional protein. The second mutation (R130W) resulted in an amino acid substitution, which caused a severe reduction (TK2 enzyme activity. These two novel TK2 mutations cause an extremely severe phenotype with overwhelming central nervous system symptoms not commonly seen in patients with TK2-deficiency. We conclude that the severe clinical presentation in this patient was due to a virtual lack of mitochondrial TK2 activity. Copyright 2009 Elsevier B.V. All rights reserved.

  5. Thymidine kinases share a conserved function for nucleotide salvage and play an essential role in Arabidopsis thaliana growth and development.

    Science.gov (United States)

    Xu, Jing; Zhang, Lin; Yang, Dong-Lei; Li, Qun; He, Zuhua

    2015-12-01

    Thymidine kinases (TKs) are important components in the nucleotide salvage pathway. However, knowledge about plant TKs is quite limited. In this study, the molecular function of TKs in Arabidopsis thaliana was investigated. Two TKs were identified and named AtTK1 and AtTK2. Expression of both genes was ubiquitous, but AtTK1 was strongly expressed in high-proliferation tissues. AtTK1 was localized to the cytosol, whereas AtTK2 was localized to the mitochondria. Mutant analysis indicated that the two genes function coordinately to sustain normal plant development. Enzymatic assays showed that the two TK proteins shared similar catalytic specificity for pyrimidine nucleosides. They were able to complement an Escherichia coli strain lacking TK activity. 5'-Fluorodeoxyuridine (FdU) resistance and 5-ethynyl 2'-deoxyuridine (EdU) incorporation assays confirmed their activity in vivo. Furthermore, the tk mutant phenotype could be alleviated by nucleotide feeding, establishing that the biosynthesis of pyrimidine nucleotides was disrupted by the TK deficiency. Finally, both human and rice (Oryza sativa) TKs were able to rescue the tk mutants, demonstrating the functional conservation of TKs across organisms. Taken together, our findings clarify the specialized function of two TKs in A. thaliana and establish that the salvage pathway mediated by the kinases is essential for plant growth and development. © 2015 Institute of Plant Physiology and Ecology, SIBS, CAS New Phytologist © 2015 New Phytologist Trust.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    Sea ice is a biome of immense size and provides a range of habitats for diverse microbial communities, many of which are adapted to living at low temperatures and high salinities in brines. We measured simultaneous incorporation of thymidine (TdR) and leucine (Leu), bacterial cell abundance...... and 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...... and marine biofilm systems. Leu: TdR ratios were high (up to >300) in lowermost ice layers, and when compared to published respiration measurements, these results suggest non-specific Leu incorporation. There was evidence of polyhydroxyalkanoate (PHA)-containing bacteria in the sea ice, shown by brightly...

  7. 3'-Azido-2',3'-dideoxythymidine induced deficiency of thymidine kinases 1, 2 and deoxycytidine kinase in H9 T-lymphoid cells.

    Science.gov (United States)

    Gröschel, Bettina; Kaufmann, Andreas; Höver, Gerold; Cinatl, Jaroslav; Doerr, Hans Wilhelm; Noordhuis, Paul; Loves, Willem J P; Peters, Godefridus J; Cinatl, Jindrich

    2002-07-15

    Continuous cultivation of T-lymphoid H9 cells in the presence of 3'-azido-2',3'-dideoxythymidine (AZT) resulted in a cell variant cross-resistant to both thymidine and deoxycytidine analogs. Cytotoxic effects of AZT, 2',3'-didehydro-3'-deoxythymidine as well as different deoxycytidine analogs such as 2',3'-dideoxycytidine, 2',2'-difluoro-2'-deoxycytidine (dFdC) and 1-ss-D-arabinofuranosylcytosine (Ara-C) were strongly reduced in H9 cells continuously exposed to AZT when compared to parental cells (>8.3-, >6.6-, >9.1-, 5 x 10(4)-, 5 x 10(3)-fold, respectively). Moreover, anti-HIV-1 effects of AZT, d4T, ddC and 2',3'-dideoxy-3'-thiacytidine (3TC) were significantly diminished (>222-, >25-, >400-, >200-fold, respectively) in AZT-resistant H9 cells. Study of cellular mechanisms responsible for cross-resistance to pyrimidine analogs in AZT-resistant H9 cells revealed decreased mRNA levels of thymidine kinase 1 (TK1) and lack of deoxycytidine kinase (dCK) mRNA expression. The loss of dCK gene expression was confirmed by western blot analysis of dCK protein as well as dCK enzyme activity assay. Moreover, enzyme activity of TK1 and TK2 was reduced in AZT-resistant cells. In order to determine whether lack of dCK affected the formation of the active triphosphate of the deoxycytidine analog dFdC, dFdCTP accumulation and retention was measured in H9 parental and AZT-resistant cells after exposure to 1 and 10 microM dFdC. Parental H9 cells accumulated about 30 and 100 pmol dFdCTP/10(6) cells after 4hr, whereas in AZT-resistant cells no dFdCTP accumulation was detected. These results demonstrate that continuous treatment of H9 cells in the presence of AZT selected for a thymidine analog resistant cell variant with cross-resistance to deoxycytidine analogs, due to deficiency in TK1, TK2, and dCK.

  8. Thymidine kinase 2 and alanyl-tRNA synthetase 2 deficiencies cause lethal mitochondrial cardiomyopathy: case reports and review of the literature.

    Science.gov (United States)

    Mazurova, Stella; Magner, Martin; Kucerova-Vidrova, Vendula; Vondrackova, Alzbeta; Stranecky, Viktor; Pristoupilova, Anna; Zamecnik, Josef; Hansikova, Hana; Zeman, Jiri; Tesarova, Marketa; Honzik, Tomas

    2017-07-01

    Cardiomyopathy is a common manifestation in neonates and infants with mitochondrial disorders. In this study, we report two cases manifesting with fatal mitochondrial hypertrophic cardiomyopathy, which include the third known patient with thymidine kinase 2 deficiency and the ninth patient with alanyl-tRNA synthetase 2 deficiency. The girl with thymidine kinase 2 deficiency had hypertrophic cardiomyopathy together with regression of gross motor development at the age of 13 months. Neurological symptoms and cardiac involvement progressed into severe myopathy, psychomotor arrest, and cardiorespiratory failure at the age of 22 months. The imaging methods and autoptic studies proved that she suffered from unique findings of leucoencephalopathy, severe, mainly cerebellar neuronal degeneration, and hepatic steatosis. The girl with alanyl-tRNA synthetase 2 deficiency presented with cardiac failure and underlying hypertrophic cardiomyopathy within 12 hours of life and subsequently died at 9 weeks of age. Muscle biopsy analyses demonstrated respiratory chain complex I and IV deficiencies, and histological evaluation revealed massive mitochondrial accumulation and cytochrome c oxidase-negative fibres in both cases. Exome sequencing in the first case revealed compound heterozygozity for one novel c.209T>C and one previously published c.416C>T mutation in the TK2 gene, whereas in the second case homozygozity for the previously described mutation c.1774C>T in the AARS2 gene was determined. The thymidine kinase 2 mutations resulted in severe mitochondrial DNA depletion (to 12% of controls) in the muscle. We present, for the first time, severe leucoencephalopathy and hepatic steatosis in a patient with thymidine kinase 2 deficiency and the finding of a ragged red fibre-like image in the muscle biopsy in a patient with alanyl-tRNA synthetase 2 deficiency.

  9. Thymidine kinase 2 (H126N) knockin mice show the essential role of balanced deoxynucleotide pools for mitochondrial DNA maintenance.

    Science.gov (United States)

    Akman, Hasan O; Dorado, Beatriz; López, Luis C; García-Cazorla, Angeles; Vilà, Maya R; Tanabe, Lauren M; Dauer, William T; Bonilla, Eduardo; Tanji, Kurenai; Hirano, Michio

    2008-08-15

    Mitochondrial DNA (mtDNA) depletion syndrome (MDS), an autosomal recessive condition, is characterized by variable organ involvement with decreased mtDNA copy number and activities of respiratory chain enzymes in affected tissues. MtDNA depletion has been associated with mutations in nine autosomal genes, including thymidine kinase (TK2), which encodes a ubiquitous mitochondrial protein. To study the pathogenesis of TK2-deficiency, we generated mice harboring an H126N Tk2 mutation. Homozygous Tk2 mutant (Tk2(-/-)) mice developed rapidly progressive weakness after age 10 days and died between ages 2 and 3 weeks. Tk2(-/-) animals showed Tk2 deficiency, unbalanced dNTP pools, mtDNA depletion and defects of respiratory chain enzymes containing mtDNA-encoded subunits that were most prominent in the central nervous system. Histopathology revealed an encephalomyelopathy with prominent vacuolar changes in the anterior horn of the spinal cord. The H126N TK2 mouse is the first knock-in animal model of human MDS and demonstrates that the severity of TK2 deficiency in tissues may determine the organ-specific phenotype.

  10. A defect in the thymidine kinase 2 gene causing isolated mitochondrial myopathy without mtDNA depletion.

    Science.gov (United States)

    Leshinsky-Silver, E; Michelson, M; Cohen, S; Ginsberg, M; Sadeh, M; Barash, V; Lerman-Sagie, T; Lev, D

    2008-07-01

    Isolated mitochondrial myopathies (IMM) are either due to primary defects in mtDNA, in nuclear genes that control mtDNA abundance and structure such as thymidine kinase 2 (TK2), or due to CoQ deficiency. Defects in the TK2 gene have been found to be associated with mtDNA depletion attributed to a depleted mitochondrial dNTP pool in non-dividing cells. We report an unusual case of IMM, homozygous for the H90N mutation in the TK2 gene but unlike other cases with the same mutation, does not demonstrate mtDNA depletion. The patient's clinical course is relatively mild and a muscle biopsy showed ragged red muscle fibers with a mild decrease in complexes I and an increase in complexes IV and II activities. This report extends the phenotypic expression of TK2 defects and suggests that all patients who present with an IMM even with normal quantities of mtDNA should be screened for TK2 mutations.

  11. Targeted Transgenic Overexpression of Mitochondrial Thymidine Kinase (TK2) Alters Mitochondrial DNA (mtDNA) and Mitochondrial Polypeptide Abundance

    Science.gov (United States)

    Hosseini, Seyed H.; Kohler, James J.; Haase, Chad P.; Tioleco, Nina; Stuart, Tami; Keebaugh, Erin; Ludaway, Tomika; Russ, Rodney; Green, Elgin; Long, Robert; Wang, Liya; Eriksson, Staffan; Lewis, William

    2007-01-01

    Mitochondrial toxicity limits nucleoside reverse transcriptase inhibitors (NRTIs) for acquired immune deficiency syndrome. NRTI triphosphates, the active moieties, inhibit human immunodeficiency virus reverse transcriptase and eukaryotic mitochondrial DNA polymerase pol-γ. NRTI phosphorylation seems to correlate with mitochondrial toxicity, but experimental evidence is lacking. Transgenic mice (TGs) with cardiac overexpression of thymidine kinase isoforms (mitochondrial TK2 and cytoplasmic TK1) were used to study NRTI mitochondrial toxicity. Echocardiography and nuclear magnetic resonance imaging defined cardiac performance and structure. TK gene copy and enzyme activity, mitochondrial (mt) DNA and polypeptide abundance, succinate dehydrogenase and cytochrome oxidase histochemistry, and electron microscopy correlated with transgenesis, mitochondrial structure, and biogenesis. Antiretroviral combinations simulated therapy. Untreated hTK1 or TK2 TGs exhibited normal left ventricle mass. In TK2 TGs, cardiac TK2 gene copy doubled, activity increased 300-fold, and mtDNA abundance doubled. Abundance of the 17-kd subunit of complex I, succinate dehydrogenase histochemical activity, and cristae density increased. NRTIs increased left ventricle mass 20% in TK2 TGs. TK activity increased 3 logs in hTK1 TGs, but no cardiac phenotype resulted. NRTIs abrogated functional effects of transgenically increased TK2 activity but had no effect on TK2 mtDNA abundance. Thus, NRTI mitochondrial phosphorylation by TK2 is integral to clinical NRTI mitochondrial toxicity. PMID:17322372

  12. Regulation of proliferation and functioning of transplanted cells by using herpes simplex virus thymidine kinase gene in mice.

    Science.gov (United States)

    Tsujimura, Mari; Kusamori, Kosuke; Oda, Chihiro; Miyazaki, Airi; Katsumi, Hidemasa; Sakane, Toshiyasu; Nishikawa, Makiya; Yamamoto, Akira

    2018-04-10

    Though cell transplantation is becoming an attractive therapeutic method, uncontrolled cell proliferation or overexpression of cellular functions could cause adverse effects. These unfavorable outcomes could be avoided by regulating the proliferation or functioning of transplanted cells. In this study, we used a combination of the herpes simplex virus thymidine kinase (HSVtk) gene, a suicide gene, and ganciclovir (GCV) to control the proliferation and functioning of insulin-secreting cells after transplantation in diabetic mice. Mouse pancreatic β cell line MIN6 cells were selected as insulin-secreting cells for transfection with the HSVtk gene to obtain MIN6/HSVtk cells. Proliferation of MIN6/HSVtk cells was suppressed by GCV in a concentration-dependent manner; 0.25 μg/mL GCV maintained a constant number of MIN6/HSVtk cells for at least 16 days. MIN6 or MIN6/HSVtk cells were then transplanted to streptozotocin-induced diabetic mice. Mice transplanted with MIN6 cells exhibited hypoglycemia irrespective of GCV administration. In contrast, normal (around 150 mg/dL) blood glucose levels were maintained in mice transplanted with MIN6/HSVtk cells by a daily administration of 50 mg/kg of GCV. These results indicate that controlling the proliferation and functioning of HSVtk gene-expressing cells by GCV could greatly improve the usefulness and safety of cell-based therapy. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. Usage of adenovirus expressing thymidine kinase mediated hepatocellular damage for enabling mouse liver repopulation with allogenic or xenogenic hepatocytes.

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    Daniel Moreno

    Full Text Available It has been shown that the liver of immunodeficient mice can be efficiently repopulated with human hepatocytes when subjected to chronic hepatocellular damage. Mice with such chimeric livers represent useful reagents for medical and clinical studies. However all previously reported models of humanized livers are difficult to implement as they involve cross-breeding of immunodeficient mice with mice exhibiting genetic alterations causing sustained hepatic injury. In this paper we attempted to create chimeric livers by inducing persistent hepatocellular damage in immunodeficient Rag2(-/- γc(-/- mice using an adenovirus encoding herpes virus thymidine kinase (AdTk and two consecutive doses of ganciclovir (GCV. We found that this treatment resulted in hepatocellular damage persisting for at least 10 weeks and enabled efficient engraftment and proliferation within the liver of either human or allogenic hepatocytes. Interestingly, while the nodules generated from the transplanted mouse hepatocytes were well vascularized, the human hepatocytes experienced progressive depolarization and exhibited reduced numbers of murine endothelial cells inside the nodules. In conclusion, AdTk/GCV-induced liver damage licenses the liver of immunodeficient mice for allogenic and xenogenic hepatocyte repopulation. This approach represents a simple alternative strategy for chimeric liver generation using immunodeficient mice without additional genetic manipulation of the germ line.

  14. Long term expression of Drosophila melanogaster nucleoside kinase in thymidine kinase 2-deficient mice with no lethal effects caused by nucleotide pool imbalances.

    Science.gov (United States)

    Krishnan, Shuba; Paredes, João A; Zhou, Xiaoshan; Kuiper, Raoul V; Hultenby, Kjell; Curbo, Sophie; Karlsson, Anna

    2014-11-21

    Mitochondrial DNA depletion caused by thymidine kinase 2 (TK2) deficiency can be compensated by a nucleoside kinase from Drosophila melanogaster (Dm-dNK) in mice. We show that transgene expression of Dm-dNK in Tk2 knock-out (Tk2(-/-)) mice extended the life span of Tk2(-/-) mice from 3 weeks to at least 20 months. The Dm-dNK(+/-)Tk2(-/-) mice maintained normal mitochondrial DNA levels throughout the observation time. A significant difference in total body weight due to the reduction of subcutaneous and visceral fat in the Dm-dNK(+/-)Tk2(-/-) mice was the only visible difference compared with control mice. This indicates an effect on fat metabolism mediated through residual Tk2 deficiency because Dm-dNK expression was low in both liver and fat tissues. Dm-dNK expression led to increased dNTP pools and an increase in the catabolism of purine and pyrimidine nucleotides but these alterations did not apparently affect the mice during the 20 months of observation. In conclusion, Dm-dNK expression in the cell nucleus expanded the total dNTP pools to levels required for efficient mitochondrial DNA synthesis, thereby compensated the Tk2 deficiency, during a normal life span of the mice. The Dm-dNK(+/-) mouse serves as a model for nucleoside gene or enzyme substitutions, nucleotide imbalances, and dNTP alterations in different tissues. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. 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.

  16. Serum thymidine kinase activity of various cancer and HBV positive liver diseases

    Energy Technology Data Exchange (ETDEWEB)

    Torizumi, Kazutami; Aibata, Hirofumi; Kiji, Shigeyuki; Ohta, Kiichiro; Okamoto, Yukiharu; Ohshiro, Iwao; Hirose, Tetsuhito

    1987-03-01

    Clinical utility of determination of serum deoxythymidine kinase (TK) activity is described. It is well known that elevated TK level is observed in leukemia and other malignant diseases, or some viral infectious diseases. The TK activity was assayed on normal subjects, hepatitis B virus (HBV) positive liver diseases and various cancer by a newly developed high sensitive method, radioenzyme assay (REA) utilizing /sup 125/I-iododeoxyuridine as the substrate. Measurement of TK activity by the REA is revealed to be useful for ''the marker of DNA metabolism anomaly'' in leukemia, etc.

  17. A rapid phenotypic assay for detection of acyclovir-resistant varicella-zoster virus with mutations in the thymidine kinase open reading frame.

    Science.gov (United States)

    Sahli, R; Andrei, G; Estrade, C; Snoeck, R; Meylan, P R

    2000-04-01

    Susceptibility assays by cell culture methods are time-consuming and are particularly difficult to perform with varicella-zoster virus (VZV). To overcome this limitation, we have adapted a functional test of the viral thymidine kinase (TK) in TK-deficient (tdk mutant) bacteria to detect ACV-resistant VZV in clinical samples. After PCR amplification, the complete viral TK open reading frame (ORF) is purified from PCR primers, digested with two restriction enzymes, and ligated in an oriented fashion into a bacterial expression vector. The ligation products are then used to transform tdk mutant bacteria. After transformation, an aliquot of the bacteria is plated onto a plate with minimal medium containing (i) ampicillin to select for plasmids carrying the viral TK ORF and (ii) isopropyl beta-D-thiogalactopyranoside (IPTG) to induce its expression. An identical aliquot of bacteria is also plated onto a medium containing, in addition to the components described above, 5-fluorodeoxyuridine (FUdR). Compared to the number of transformants on FUdR-free medium, the number of colonies carrying TK derived from susceptible strains was reduced by 86%, on average, in the presence of FUdR. In contrast, the number of transformants carrying TK from resistant strains with a mutant TK were reduced by only 4%, on average, on FUdR-containing plates. We have assessed the validity of this assay with cell culture isolates and several clinical samples including two cerebrospinal fluid samples from which no virus could be isolated. This colony reduction assay allowed the correct identification of the TK phenotype of each VZV isolate tested and can be completed within 3 days of receipt of the sample.

  18. Thymidine Kinase-Negative Herpes Simplex Virus 1 Can Efficiently Establish Persistent Infection in Neural Tissues of Nude Mice.

    Science.gov (United States)

    Huang, Chih-Yu; Yao, Hui-Wen; Wang, Li-Chiu; Shen, Fang-Hsiu; Hsu, Sheng-Min; Chen, Shun-Hua

    2017-02-15

    Herpes simplex virus 1 (HSV-1) establishes latency in neural tissues of immunocompetent mice but persists in both peripheral and neural tissues of lymphocyte-deficient mice. Thymidine kinase (TK) is believed to be essential for HSV-1 to persist in neural tissues of immunocompromised mice, because infectious virus of a mutant with defects in both TK and UL24 is detected only in peripheral tissues, but not in neural tissues, of severe combined immunodeficiency mice (T. Valyi-Nagy, R. M. Gesser, B. Raengsakulrach, S. L. Deshmane, B. P. Randazzo, A. J. Dillner, and N. W. Fraser, Virology 199:484-490, 1994, https://doi.org/10.1006/viro.1994.1150). Here we find infiltration of CD4 and CD8 T cells in peripheral and neural tissues of mice infected with a TK-negative mutant. We therefore investigated the significance of viral TK and host T cells for HSV-1 to persist in neural tissues using three genetically engineered mutants with defects in only TK or in both TK and UL24 and two strains of nude mice. Surprisingly, all three mutants establish persistent infection in up to 100% of brain stems and 93% of trigeminal ganglia of adult nude mice at 28 days postinfection, as measured by the recovery of infectious virus. Thus, in mouse neural tissues, host T cells block persistent HSV-1 infection, and viral TK is dispensable for the virus to establish persistent infection. Furthermore, we found 30- to 200-fold more virus in neural tissues than in the eye and detected glycoprotein C, a true late viral antigen, in brainstem neurons of nude mice persistently infected with the TK-negative mutant, suggesting that adult mouse neurons can support the replication of TK-negative HSV-1. Acyclovir is used to treat herpes simplex virus 1 (HSV-1)-infected immunocompromised patients, but treatment is hindered by the emergence of drug-resistant viruses, mostly those with mutations in viral thymidine kinase (TK), which activates acyclovir. TK mutants are detected in brains of immunocompromised

  19. Temperature-driven adaptation of the bacterial community in peat measured by using thymidine and leucine incorporation.

    Science.gov (United States)

    Ranneklev, S B; Bååth, E

    2001-03-01

    The temperature-driven adaptation of the bacterial community in peat was studied, by altering temperature to simulate self-heating and a subsequent return to mesophilic conditions. The technique used consisted of extracting the bacterial community from peat using homogenization-centrifugation and measuring the rates of thymidine (TdR) or leucine (Leu) incorporation by the extracted bacterial community at different temperatures. Increasing the peat incubation temperature from 25 degrees C to 35, 45, or 55 degrees C resulted in a selection of bacterial communities whose optimum temperatures for activity correlated to the peat incubation temperatures. Although TdR and Leu incorporations were significantly correlated, the Leu/TdR incorporation ratios were affected by temperature. Higher Leu/TdR incorporation ratios were found at higher temperatures of incubation of the extracted bacterial community. Higher Leu/TdR incorporation ratios were also found for bacteria in peat samples incubated at higher temperatures. The reappearance of the mesophilic community and disappearance of the thermophilic community when the incubation temperature of the peat was shifted down were monitored by measuring TdR incorporation at 55 degrees C (thermophilic activity) and 25 degrees C (mesophilic activity). Shifting the peat incubation temperature from 55 to 25 degrees C resulted in a recovery of the mesophilic activity, with a subsequent disappearance of the thermophilic activity. The availability of substrate for bacterial growth varied over time and among different peat samples. To avoid confounding effects of substrate availability, a temperature adaptation index was calculated. This index consisted of the log(10) ratio of TdR incorporation at 55 and 25 degrees C. The temperature index decreased linearly with time, indicating that no thermophilic activity would be detected by the TdR technique 1 month after the temperature downshift. There were no differences between the slopes of the

  20. Epstein-Barr virus thymidine kinase is a centrosomal resident precisely localized to the periphery of centrioles.

    Science.gov (United States)

    Gill, Michael B; Kutok, Jeffery L; Fingeroth, Joyce D

    2007-06-01

    The thymidine kinase (TK) encoded by Epstein-Barr virus (EBV) differs not only from that of the alphaherpesviruses but also from that of the gamma-2 herpesvirus subfamily. Because cellular location is frequently a determinant of regulatory function, to gain insight into additional role(s) of EBV TK and to uncover how the lymphocryptovirus and rhadinovirus enzymes differ, the subcellular localizations of EBV TK and the related cercopithecine herpesvirus-15 TK were investigated. We show that in contrast to those of the other family members, the gamma-1 herpesvirus TKs localize to the centrosome and even more precisely to the periphery of the centriole, tightly encircling the tubulin-rich centrioles in a microtubule-independent fashion. Centrosomal localization is observed in diverse cell types and occurs whether the protein is expressed independently or in the context of lytic EBV infection. Surprisingly, analysis of mutants revealed that the unique N-terminal domain was not critical for targeting to the centrosome, but rather, peptide sequences located C terminal to this domain were key. This is the first herpesvirus protein documented to reside in the centrosome, or microtubule-organizing center, an amembranous organelle that regulates the structural biology of the cell cycle through control of chromosome separation and cytokinesis. More recently, proteasome-mediated degradation of cell cycle regulatory proteins, production and loading of antigenic peptides onto HLA molecules, and transient homing of diverse virion proteins required for entry and/or egress have been shown to be coordinated at the centrosome. Potential implications of centrosomal localization for EBV TK function are discussed.

  1. Thymidine kinase 2 deficiency-induced mtDNA depletion in mouse liver leads to defect β-oxidation.

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    Xiaoshan Zhou

    Full Text Available Thymidine kinase 2 (TK2 deficiency in humans causes mitochondrial DNA (mtDNA depletion syndrome. To study the molecular mechanisms underlying the disease and search for treatment options, we previously generated and described a TK2 deficient mouse strain (TK2(-/- that progressively loses its mtDNA. The TK2(-/- mouse model displays symptoms similar to humans harboring TK2 deficient infantile fatal encephalomyopathy. Here, we have studied the TK2(-/- mouse model to clarify the pathological role of progressive mtDNA depletion in liver for the severe outcome of TK2 deficiency. We observed that a gradual depletion of mtDNA in the liver of the TK2(-/- mice was accompanied by increasingly hypertrophic mitochondria and accumulation of fat vesicles in the liver cells. The levels of cholesterol and nonesterified fatty acids were elevated and there was accumulation of long chain acylcarnitines in plasma of the TK2(-/- mice. In mice with hepatic mtDNA levels below 20%, the blood sugar and the ketone levels dropped. These mice also exhibited reduced mitochondrial β-oxidation due to decreased transport of long chain acylcarnitines into the mitochondria. The gradual loss of mtDNA in the liver of the TK2(-/- mice causes impaired mitochondrial function that leads to defect β-oxidation and, as a result, insufficient production of ketone bodies and glucose. This study provides insight into the mechanism of encephalomyopathy caused by TK2 deficiency-induced mtDNA depletion that may be used to explore novel therapeutic strategies.

  2. Thymidine kinase 2 deficiency-induced mtDNA depletion in mouse liver leads to defect β-oxidation.

    Science.gov (United States)

    Zhou, Xiaoshan; Kannisto, Kristina; Curbo, Sophie; von Döbeln, Ulrika; Hultenby, Kjell; Isetun, Sindra; Gåfvels, Mats; Karlsson, Anna

    2013-01-01

    Thymidine kinase 2 (TK2) deficiency in humans causes mitochondrial DNA (mtDNA) depletion syndrome. To study the molecular mechanisms underlying the disease and search for treatment options, we previously generated and described a TK2 deficient mouse strain (TK2(-/-)) that progressively loses its mtDNA. The TK2(-/-) mouse model displays symptoms similar to humans harboring TK2 deficient infantile fatal encephalomyopathy. Here, we have studied the TK2(-/-) mouse model to clarify the pathological role of progressive mtDNA depletion in liver for the severe outcome of TK2 deficiency. We observed that a gradual depletion of mtDNA in the liver of the TK2(-/-) mice was accompanied by increasingly hypertrophic mitochondria and accumulation of fat vesicles in the liver cells. The levels of cholesterol and nonesterified fatty acids were elevated and there was accumulation of long chain acylcarnitines in plasma of the TK2(-/-) mice. In mice with hepatic mtDNA levels below 20%, the blood sugar and the ketone levels dropped. These mice also exhibited reduced mitochondrial β-oxidation due to decreased transport of long chain acylcarnitines into the mitochondria. The gradual loss of mtDNA in the liver of the TK2(-/-) mice causes impaired mitochondrial function that leads to defect β-oxidation and, as a result, insufficient production of ketone bodies and glucose. This study provides insight into the mechanism of encephalomyopathy caused by TK2 deficiency-induced mtDNA depletion that may be used to explore novel therapeutic strategies.

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

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    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.

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

    Science.gov (United States)

    Villarroya, Joan; Dorado, Beatriz; Vilà, Maya R; Garcia-Arumí, Elena; Domingo, Pere; Giralt, Marta; Hirano, Michio; Villarroya, Francesc

    2011-01-01

    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. © 2011 Villarroya et al.

  5. Identification of a thymidine kinase (RuTK1) homolog differentially expressed in blackberry (Rubus L.) prickles

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    Zhang, C.; Yang, H.; Wang, X.

    2016-01-01

    Thymidine kinase (TK) is a key enzyme in controlling DNA synthesis and plays an important role in cell proliferation. However, our understanding on the TK functions in plants is still limited. From an earlier comparative transcriptome analysis of shoot apex of blackberry cv. Boysenberry and its bud mutant cv. Ningzhi 1 with fewer and thinner prickles, we found a unigene homologous to TK, RuTK1 which was differentially expressed between them. In this study, the cDNA and genomic DNA (gDNA) sequences of RuTK1 were further analyzed. RuTK1 revealed an open reading frame (ORF) of 660 bp coding for 219 amino acid residues. The gDNA sequence, which contains four exons and three introns, is relatively conserved in most plant TK homologs. A phylogenetic analysis revealed that the TK proteins from plants were classified into three groups. In each group, TKs from the same family were relatively concentrated, and RuTK1 was classified to the dicotyledoneae class and closer to those from Rosaceae. RuTK1 was highly expressed in prickles at the early stage in Boysenberry compared to in Ningzhi1. In addition, RuTK1 expression was similarly greater in mature prickles at the late stage in both cultivars, which implies a possible involvement of RuTK1 in the cell cycle at the early stage of prickle formation. These results provide a novel foundation for the further elucidation of blackberry prickle development mechanism and the functions of TKs in plants. (author)

  6. Long Term Expression of Drosophila melanogaster Nucleoside Kinase in Thymidine Kinase 2-deficient Mice with No Lethal Effects Caused by Nucleotide Pool Imbalances*

    Science.gov (United States)

    Krishnan, Shuba; Paredes, João A.; Zhou, Xiaoshan; Kuiper, Raoul V.; Hultenby, Kjell; Curbo, Sophie; Karlsson, Anna

    2014-01-01

    Mitochondrial DNA depletion caused by thymidine kinase 2 (TK2) deficiency can be compensated by a nucleoside kinase from Drosophila melanogaster (Dm-dNK) in mice. We show that transgene expression of Dm-dNK in Tk2 knock-out (Tk2−/−) mice extended the life span of Tk2−/− mice from 3 weeks to at least 20 months. The Dm-dNK+/−Tk2−/− mice maintained normal mitochondrial DNA levels throughout the observation time. A significant difference in total body weight due to the reduction of subcutaneous and visceral fat in the Dm-dNK+/−Tk2−/− mice was the only visible difference compared with control mice. This indicates an effect on fat metabolism mediated through residual Tk2 deficiency because Dm-dNK expression was low in both liver and fat tissues. Dm-dNK expression led to increased dNTP pools and an increase in the catabolism of purine and pyrimidine nucleotides but these alterations did not apparently affect the mice during the 20 months of observation. In conclusion, Dm-dNK expression in the cell nucleus expanded the total dNTP pools to levels required for efficient mitochondrial DNA synthesis, thereby compensated the Tk2 deficiency, during a normal life span of the mice. The Dm-dNK+/− mouse serves as a model for nucleoside gene or enzyme substitutions, nucleotide imbalances, and dNTP alterations in different tissues. PMID:25296759

  7. Molecular insight into mitochondrial DNA depletion syndrome in two patients with novel mutations in the deoxyguanosine kinase and thymidine kinase 2 genes.

    Science.gov (United States)

    Wang, Liya; Limongelli, Anna; Vila, Maya R; Carrara, Franco; Zeviani, Massimo; Eriksson, Staffan

    2005-01-01

    Thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) are the two key enzymes in mitochondrial DNA (mtDNA) precursor synthesis. Deficiencies in TK2 or dGK activity, due to genetic alteration, have been shown to cause tissue-specific depletion of mtDNA. In the case of TK2 deficiency, affected individuals suffer severe myopathy and, in the case of dGK deficiency, devastating liver or multi-systemic disease. Here, we report clinical and biochemical findings from two patients with mtDNA depletion syndrome. Patient A was a compound heterozygote carrying the previously reported T77M mutation and a novel mutation (R161K) in the TK2 gene. Patient B carried a novel mutation (L250S) in the dGK gene. The clinical symptoms of patient A included muscular weakness and exercise intolerance due to a severe mitochondrial myopathy associated with a 92% reduction in mtDNA. There was minimal involvement of other organs. Patient B suffered from rapidly progressive, early onset fatal liver failure associated with profoundly decreased mtDNA levels in liver and, to a lesser extent, in skeletal muscle. Site-directed mutagenesis was used to introduce the mutations detected in patients A and B into the TK2 and dGK cDNAs, respectively. We then characterized each of these recombinant enzymes. Catalytic activities of the three mutant enzymes were reduced to about 2-4% for TK2 and 0.5% for dGK as compared to the wild-type enzymes. Altered competition between dCyd and dThd was observed for the T77M mutant. The residual activities of the two mitochondrial enzymes correlated directly with disease development.

  8. In vivo imaging of herpes simplex virus type 1 thymidine kinase gene expression: early kinetics of radiolabelled FIAU

    Energy Technology Data Exchange (ETDEWEB)

    Haubner, R.; Avril, N.; Schwaiger, M. [Technische Univ. Muenchen (Germany). Nuklearmedizinische Klinik und Poliklinik; Hantzopoulos, P.A.; Gansbacher, B. [Inst. of Experimental Oncology, Technische Univ. Muenchen (Germany)

    2000-03-01

    The aim of our study was to examine the early kinetics of I*-FIAU and the possibility of utilising iodine-123-labelled FIAU for imaging of gene expression. CMS-5 fibrosarcoma cells were transduced in vitro with the retroviral vector STK containing the HSV1-tk gene. BALB/c mice were inoculated subcutaneously with HSV1-tk(+) and tk(-) cells into both flanks. FAU (2'-fluoro-2'-deoxy-1-{beta}-d-arabinofuranosyluracil was radioiodinated ({sup 123}I, {sup 125}I)) using the iodogen method. High-performance liquid chromatography purification resulted in high specific activity and radiochemical purity for both tracers ([{sup 123}I]FIAU and [{sup 125}I]FIAU). Biodistribution studies and gamma camera imaging were performed at 0.5, 1, 2 and 4 h p.i. In addition, the genomic DNA of the tumours was isolated for measurement of the activity accumulation resulting from the [{sup 125}I]FIAU incorporation. Biodistribution studies 0.5 h p.i. showed tumour/blood and tumour/muscle ratios of 3.8 and 7.2, respectively, for the HSV1-tk(+) tumours, and 0.6 and 1.2, respectively, for negative control tumours. Fast renal elimination of the tracer from the body resulted in rapidly increasing tumour/blood and tumour/muscle ratios which reached values of 32 and 88 at 4 h p.i., respectively. Tracer clearance from blood was bi-exponential, with an initial half-life of 0.6 h followed by a half-life of 4.6 h. The tracer half-life in herpes simplex viral thymidine kinase-expressing tumours was 35.7 h. The highest activity accumulation (20.3%{+-}5.7% ID/g) in HSV1-tk(+) tumours was observed 1 h p.i. At that time, about 46% of the total activity found in HSV1-tk(+) tumours was incorporated into genomic DNA. Planar gamma camera imaging showed a distinct tracer accumulation as early as 0.5 h p.i., with an increase in contrast over time. These results suggest that sufficient tumour/background ratios for in vivo imaging of HSV1-tk expression with [{sup 123}I]FIAU are reached as early as 1 h p

  9. 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

  10. 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)

    Villarroya, Joan; Lara, Mari-Carmen; Dorado, Beatriz; Garrido, Marta; Garcia-Arumi, Elena; Meseguer, Anna; Hirano, Michio; Vila, Maya R.

    2011-01-01

    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.

  11. Identification and biosynthesis of thymidine hypermodifications in the genomic DNA of widespread bacterial viruses

    Science.gov (United States)

    Lee, Yan-Jiun; Dai, Nan; Walsh, Shannon E.; Müller, Stephanie; Fraser, Morgan E.; Kauffman, Kathryn M.; Guan, Chudi; Weigele, Peter R.

    2018-01-01

    Certain viruses of bacteria (bacteriophages) enzymatically hypermodify their DNA to protect their genetic material from host restriction endonuclease-mediated cleavage. Historically, it has been known that virion DNAs from the Delftia phage ΦW-14 and the Bacillus phage SP10 contain the hypermodified pyrimidines α-putrescinylthymidine and α-glutamylthymidine, respectively. These bases derive from the modification of 5-hydroxymethyl-2′-deoxyuridine (5-hmdU) in newly replicated phage DNA via a pyrophosphorylated intermediate. Like ΦW-14 and SP10, the Pseudomonas phage M6 and the Salmonella phage ViI encode kinase homologs predicted to phosphorylate 5-hmdU DNA but have uncharacterized nucleotide content [Iyer et al. (2013) Nucleic Acids Res 41:7635–7655]. We report here the discovery and characterization of two bases, 5-(2-aminoethoxy)methyluridine (5-NeOmdU) and 5-(2-aminoethyl)uridine (5-NedU), in the virion DNA of ViI and M6 phages, respectively. Furthermore, we show that recombinant expression of five gene products encoded by phage ViI is sufficient to reconstitute the formation of 5-NeOmdU in vitro. These findings point to an unexplored diversity of DNA modifications and the underlying biochemistry of their formation. PMID:29555775

  12. 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.

  13. Kinetic and photochemical studies and alteration of ultraviolet sensitivity of Escherichia coli thymidine kinase of halogenated allosteric regulators and substrate analogues

    International Nuclear Information System (INIS)

    Chen, M.S.; Prusoff, W.H.

    1977-01-01

    The effect of various halogenated and nonhalogenated allosteric effectors on the sensitivity of Escherichia coli thymidine kinase to ultraviolet radiations (uv,253.7 nm) was investigated. All naturally occurring dNTPs convert the monomeric form of the enzyme into the dimeric form which is less sensitive to uv inactivation. Whereas 5-iodo-2'-deoxycytidine triphosphate (IdCTP) and 5-iodo-2' deoxyuridine triphosphate (IdUTP) enhance the uv inactivation of the enzyme, 5-bromo-2'-deoxyuridine triphosphate and 5-bromo-2'-deoxycytidine triphosphate exert a protective effect similar to that produced by the corresponding naturally occurring effectors, dTTP and dCTP. The enhanced uv inactivation by IdUTP is prevented totally by dTTP, but only partially by dCTP or dThd, whereas the enhanced sensitization by IdCTP is prevented almost totally by dCTP, partially by dTTP, and not at all by dThd. The uv sensitization of thymidine kinase by IdCTP appears to be at the regulatory site since a maximum saturation effect is observed, and the concentration required to exert a 50% maximal uv sensitization is similar to its K/sub m/ for enhancement of catalytic activity. When the enzyme was irradiated in the presence of either [2- 14 C]IdUTP or [2- 14 C]IdUrd, zone sedimentation analysis in sucrose density gradients showed the sedimentation coefficient of the radioactive labeled proteins to be the same, 3.8 S. Hence uv irradiation of the effector-induced dimer resulted in not only dissociation to the monomer, but also complete loss of catalytic activity. The substitution of an azido group for the 5'-OH group of 5-iodo-, 5-bromo-, 5-chloro-, or 5-fluorodeoxyuridine greatly decreased their affinity for thymidine kinase, and in addition the kinetics of inhibition changed from a competitive to a noncompetitive pattern. The presence of the azido moiety in the 5' position of the halogenated nucleosides did not enhance the rate of uv inactivation of the enzyme

  14. Synthesis and Biological Evaluation of a New Acyclic Pyrimidine Derivative as a Probe for Imaging Herpes Simplex Virus Type 1 Thymidine Kinase Gene Expression

    Directory of Open Access Journals (Sweden)

    Simon M. Ametamey

    2013-07-01

    Full Text Available With the idea of finding a more selective radiotracer for imaging herpes simplex virus type 1 thymidine kinase (HSV1-tk gene expression by means of positron emission tomography (PET, a novel [18F]fluorine radiolabeled pyrimidine with 4-hydroxy-3-(hydroxymethylbutyl side chain at N-1 (HHB-5-[18F]FEP was prepared and evaluated as a potential PET probe. Unlabeled reference compound, HHB-5-FEP, was synthesized via a five-step reaction sequence starting from 5-(2-acetoxyethyl-4-methoxypyrimidin-2-one. The radiosynthesis of HHB-[18F]-FEP was accomplished by nucleophilic radiofluorination of a tosylate precursor using [18F]fluoride-cryptate complex in 45% ± 4 (n = 4 radiochemical yields and high purity (>99%. The biological evaluation indicated the feasibility of using HHB-5-[18F]FEP as a PET radiotracer for monitoring HSV1-tk expression in vivo.

  15. 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)

    Lee, TaeSup; Kim, JunYoup; Moon, ByungSeok; Kang, JooHyun; Song, Inho; Kwon, HeeChung; Kim, KyungMin; Cheon, GiJeong; Choi, ChangWoon; Lim, SangMoo

    2007-01-01

    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

  16. 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)

    Intine, R.V.; Rainbow, A.J.

    1990-01-01

    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

  17. 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.

  18. 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)

    Rainbow, A.J.

    1989-01-01

    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

  19. [{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.

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

    International Nuclear Information System (INIS)

    Iriberri, J.; Unanue, M.; Ayo, B.; Barcina, I.; Egea, L.

    1990-01-01

    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- 3 H]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 10 11 and 8.678 x 10 11 μg of C mol -1 for free-living and attached bacteria in the freshwater system, and 1.276 x 10 11 and 1.354 x 10 11 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

  1. 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)

    Kim, Jae Ho; Kim, Sang Hie; Kolozsvary, A.

    1995-01-01

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jae Ho; Kim, Sang Hie; Kolozsvary, A. [Henry Ford Hospital, Detroit, MI (United States)] [and others

    1995-11-01

    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 {mu}g/ml) preceded radiation, the sensitizer enhancement ratio (SER) for BVdU and acyclovir was 1.4 {plus_minus} 0.1 and 1.3 {plus_minus} 0.1, respectively. Exposure of cells to 10 {mu}g/ml acyclovir for two 24-h periods both pre- and postirradiation resulted in a SER of 1.6 {plus_minus} 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.

  3. 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)

    Jae, Ho Kim; Sang, Hie Kim; Kolozsvary, Andrew; Brown, Stephen L.; Ok, Bae Kim; Freytag, Svend O.

    1995-01-01

    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

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

    DEFF Research Database (Denmark)

    Christiansen, Louise Slot; Egeblad, Louise; Munch-Petersen, Birgitte

    2015-01-01

    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 t...

  5. 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.

  6. Immune responses to recombinants of the South African vaccine strain of lumpy skin disease virus generated by using thymidine kinase gene insertion.

    Science.gov (United States)

    Wallace, David B; Viljoen, Gerrit J

    2005-04-27

    The South African vaccine strain of lumpy skin disease virus (type SA-Neethling) is currently being developed as a vector for recombinant vaccines of economically important livestock diseases throughout Africa. In this study, the feasibility of using the viral thymidine kinase gene as the site of insertion was investigated and recombinant viruses were evaluated in animal trials. Two separate recombinants were generated and selected for homogeneity expressing either the structural glycoprotein gene of bovine ephemeral fever virus (BEFV) or the two structural glycoprotein genes of Rift Valley fever virus (RVFV). Both recombinants incorporate the enhanced green fluorescent protein (EGFP) as a visual marker and the Escherichia coli guanine phosphoribosyl transferase (gpt) gene for dominant positive selection. The LSDV-RVFV recombinant construct (rLSDV-RVFV) protected mice against virulent RVFV challenge. In a small-scale BEFV-challenge cattle trial the rLSDV-BEFV construct failed to fully protect the cattle against virulent challenge, although both a humoral and cellular BEFV-specific immune response was elicited.

  7. The mouse lymphoma thymidine kinase assay for the assessment and comparison of the mutagenic activity of cigarette mainstream smoke particulate phase.

    Science.gov (United States)

    Schramke, H; Meisgen, T J; Tewes, F J; Gomm, W; Roemer, E

    2006-10-29

    The mouse lymphoma thymidine kinase assay (MLA) has been optimized to quantitatively determine the in vitro mutagenicity of cigarette mainstream smoke particulate phase. To test whether the MLA is able to discriminate between different cigarette types, specially constructed cigarettes each containing a single tobacco type - Bright, Burley, or Oriental - were investigated. The mutagenic activity of the Burley cigarette was statistically significantly lower, up to approximately 40%, than that of the Bright and Oriental cigarettes. To determine the impact of two different sets of smoking conditions, American-blend cigarettes were smoked under US Federal Trade Commission/International Organisation for Standardisation conditions and under Massachusetts Department of Public Health (MDPH) conditions. Conventional cigarettes - eight from the US commercial market plus the Reference Cigarettes 1R4F and 2R4F - and an electrically heated cigarette smoking system (EHCSS) prototype were tested. There were no statistically significant differences between the two sets of smoking conditions on a per mg total particulate matter basis, although there was a consistent trend towards slightly lower mutagenic activity under MDPH conditions. The mutagenic activity of the EHCSS prototype was distinctly lower than that of the conventional cigarettes under both sets of smoking conditions. These results show that the MLA can be used to assess and compare the mutagenic activity of cigarette mainstream smoke particulate phase in the comprehensive toxicological assessment of cigarette smoke.

  8. Molecular and clinical characterization of the myopathic form of mitochondrial DNA depletion syndrome caused by mutations in the thymidine kinase (TK2) gene.

    Science.gov (United States)

    Chanprasert, Sirisak; Wang, Jing; Weng, Shao-Wen; Enns, Gregory M; Boué, Daniel R; Wong, Brenda L; Mendell, Jerry R; Perry, Deborah A; Sahenk, Zarife; Craigen, William J; Alcala, Francisco J Climent; Pascual, Juan M; Melancon, Serge; Zhang, Victor Wei; Scaglia, Fernando; Wong, Lee-Jun C

    2013-01-01

    Mitochondrial DNA (mtDNA) depletion syndromes (MDSs) are a clinically and molecularly heterogeneous group of mitochondrial cytopathies characterized by severe mtDNA copy number reduction in affected tissues. Clinically, MDSs are mainly categorized as myopathic, encephalomyopathic, hepatocerebral, or multi-systemic forms. To date, the myopathic form of MDS is mainly caused by mutations in the TK2 gene, which encodes thymidine kinase 2, the first and rate limiting step enzyme in the phosphorylation of pyrimidine nucleosides. We analyzed 9 unrelated families with 11 affected subjects exhibiting the myopathic form of MDS, by sequencing the TK2 gene. Twelve mutations including 4 novel mutations were detected in 9 families. Skeletal muscle specimens were available from 7 out of 11 subjects. Respiratory chain enzymatic activities in skeletal muscle were measured in 6 subjects, and enzymatic activities were reduced in 3 subjects. Quantitative analysis of mtDNA content in skeletal muscle was performed in 5 subjects, and marked mtDNA content reduction was observed in each. In addition, we outline the molecular and clinical characteristics of this syndrome in a total of 52 patients including those previously reported, and a total of 36 TK2 mutations are summarized. Clinically, hypotonia and proximal muscle weakness are the major phenotypes present in all subjects. In summary, our study expands the molecular and clinical spectrum associated with TK2 deficiency. © 2013.

  9. Targeted transgenic overexpression of mitochondrial thymidine kinase (TK2) alters mitochondrial DNA (mtDNA) and mitochondrial polypeptide abundance: transgenic TK2, mtDNA, and antiretrovirals.

    Science.gov (United States)

    Hosseini, Seyed H; Kohler, James J; Haase, Chad P; Tioleco, Nina; Stuart, Tami; Keebaugh, Erin; Ludaway, Tomika; Russ, Rodney; Green, Elgin; Long, Robert; Wang, Liya; Eriksson, Staffan; Lewis, William

    2007-03-01

    Mitochondrial toxicity limits nucleoside reverse transcriptase inhibitors (NRTIs) for acquired immune deficiency syndrome. NRTI triphosphates, the active moieties, inhibit human immunodeficiency virus reverse transcriptase and eukaryotic mitochondrial DNA polymerase pol-gamma. NRTI phosphorylation seems to correlate with mitochondrial toxicity, but experimental evidence is lacking. Transgenic mice (TGs) with cardiac overexpression of thymidine kinase isoforms (mitochondrial TK2 and cytoplasmic TK1) were used to study NRTI mitochondrial toxicity. Echocardiography and nuclear magnetic resonance imaging defined cardiac performance and structure. TK gene copy and enzyme activity, mitochondrial (mt) DNA and polypeptide abundance, succinate dehydrogenase and cytochrome oxidase histochemistry, and electron microscopy correlated with transgenesis, mitochondrial structure, and biogenesis. Antiretroviral combinations simulated therapy. Untreated hTK1 or TK2 TGs exhibited normal left ventricle mass. In TK2 TGs, cardiac TK2 gene copy doubled, activity increased 300-fold, and mtDNA abundance doubled. Abundance of the 17-kd subunit of complex I, succinate dehydrogenase histochemical activity, and cristae density increased. NRTIs increased left ventricle mass 20% in TK2 TGs. TK activity increased 3 logs in hTK1 TGs, but no cardiac phenotype resulted. NRTIs abrogated functional effects of transgenically increased TK2 activity but had no effect on TK2 mtDNA abundance. Thus, NRTI mitochondrial phosphorylation by TK2 is integral to clinical NRTI mitochondrial toxicity.

  10. Plasma thymidine kinase-1 activity predicts outcome in patients with hormone receptor positive and HER2 negative metastatic breast cancer treated with endocrine therapy.

    Science.gov (United States)

    Bonechi, Martina; Galardi, Francesca; Biagioni, Chiara; De Luca, Francesca; Bergqvist, Mattias; Neumüller, Magnus; Guarducci, Cristina; Boccalini, Giulia; Gabellini, Stefano; Migliaccio, Ilenia; Di Leo, Angelo; Pestrin, Marta; Malorni, Luca

    2018-03-27

    The aim of this study was to investigate if thymidine kinase-1 (TK1), a well-known proliferation marker, could represent a valid circulating biomarker to identify hormone receptor positive (HR+)/HER2 negative (HER2neg) metastatic breast cancer (MBC) patients most likely to benefit from endocrine therapy (ET). We used the DiviTum™ assay to analyze TK1 activity in cell lysates of three HR+/HER2neg BC cell lines and in plasma of 31 HR+/HER2neg MBC patients receiving ET. Blood samples were collected at treatment initiation, after one month and at disease progression. CTCs count and ESR1 / PIK3CA mutations in circulating tumor DNA were performed and correlated with TK1 activity. TK1 activity was reduced in the two endocrine-sensitive cell lines after 2 days of treatment. In patients, high baseline TK1 activity correlated with CTCs positivity (p-value=0.014). Patients with low baseline levels of TK1 activity had a significantly better PFS compared to those with high baseline TK1 activity (p-value=0.012). Patients with an early drop of TK1 activity after one month of treatment had a significantly better PFS compared to those who experienced an increase (p-value=0.0026). Our study suggests that TK1 could be a potential prognostic, predictive and monitoring marker of early ET response in HR+/HER2neg MBC patients.

  11. THE THYMIDINE KINASE-1(TK-1 AS A POTENTIAL TUMOR MARKER: SERUM LEVELS IN SERUM OF PATIENTS WITH SOLID AND SYSTEM MALIGNANCE NEOPLASMS

    Directory of Open Access Journals (Sweden)

    N. S. Sergeeva

    2017-01-01

    Full Text Available The review summarizes the results of studies of levels and/or activity in the blood serum of a metabolic marker thymidine kinase-1 (TK-1 of proliferating cells in patients with lymphoproliferative diseases (LPD and malignant neopasms (NM.Comparison of the data in the literature in some cases have been difficult due to the fundamentally different methods of detection the activity or concentration of TK-1, used by authors, even despite the presence of relatively high (but not absolute correlation between these parameters (maximum 0.8.Many clinical and laboratory studies have shown levels of correlation and/or TK-1 activity with clinical stages and different types of LPD and solid MN and can serve as a prognostic factor for overall and recurrence-free survival of patients. When solid MN shown that the activity of TK-1 accurately reflects the proliferative status of tumor.A comparison of the dynamics of TC-1 in the process of chemotherapy and its clinical efficacy, different authors have received fundamentally different results: in some cases the marker reduction was associated with treatment efficacy, and in part of publications they show that the clinically relevant effects of the treatment observed increase in the marker after the first chemotherapy.The entire set of received data demonstrates the relevance of the further development of the algorithm use of TK-1 in oncology practice.

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

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    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.

  13. A dual function fusion protein of Herpes simplex virus type 1 thymidine kinase and firefly luciferase for noninvasive in vivo imaging of gene therapy in malignant glioma.

    Science.gov (United States)

    Söling, Ariane; Theiss, Christian; Jungmichel, Stephanie; Rainov, Nikolai G

    2004-08-04

    BACKGROUND: Suicide gene therapy employing the prodrug activating system Herpes simplex virus type 1 thymidine kinase (HSV-TK)/ ganciclovir (GCV) has proven to be effective in killing experimental brain tumors. In contrast, glioma patients treated with HSV-TK/ GCV did not show significant treatment benefit, most likely due to insufficient transgene delivery to tumor cells. Therefore, this study aimed at developing a strategy for real-time noninvasive in vivo monitoring of the activity of a therapeutic gene in brain tumor cells. METHODS: The HSV-TK gene was fused to the firefly luciferase (Luc) gene and the fusion construct HSV-TK-Luc was expressed in U87MG human malignant glioma cells. Nude mice with subcutaneous gliomas stably expressing HSV-TK-Luc were subjected to GCV treatment and tumor response to therapy was monitored in vivo by serial bioluminescence imaging. Bioluminescent signals over time were compared with tumor volumes determined by caliper. RESULTS: Transient and stable expression of the HSV-TK-Luc fusion protein in U87MG glioma cells demonstrated close correlation of both enzyme activities. Serial optical imaging of tumor bearing mice detected in all cases GCV induced death of tumor cells expressing the fusion protein and proved that bioluminescence can be reliably used for repetitive and noninvasive quantification of HSV-TK/ GCV mediated cell kill in vivo. CONCLUSION: This approach may represent a valuable tool for the in vivo evaluation of gene therapy strategies for treatment of malignant disease.

  14. The Inactivation of Escherichia Coli Bacteria Labelled with Tritiated Thymidine; Inactivation de Bacteries Escherichia Coli Marquees par la Thymidine Tritiee; 0418 043d 0414 ; Inactivacion de Escherichia Coli Marcada con Timidina Tritiada

    Energy Technology Data Exchange (ETDEWEB)

    Apelgot, Sonia [Institut du Radium, Laboratoire Curie, Paris (France)

    1962-02-15

    Bacteria of the strain B{sup 3}{sub 1} thy {sup -}/Sr, which required thymine and are streptomycin-resistant, had their DNA labelled with tritiated thymidine. The radioactivity measurements were made with a liquid scintillation counting system, with two photomultipliers mounted in coincidence. Under these conditions, the efficiency of the measures was 4.5% and the background 130 counts/min. The radioactive bacteria were kept in sealed tubes either at 0 Degree-Sign C or at - 196 Degree-Sign C and their survival studied. These experiments showed that the radioactive bacteria are inactivated exponentially as a function of the number of tritium atoms disintegrated. The inactivation is temperature dependent. In both cases the killing efficiency per nuclear transmutation was determined and found as very low. The number of ion pairs generated by the {beta}-particles emitted as a consequence of the transmutation of H{sup 3} was evaluated and found quite comparable with the one found in the case of X-rays. The suicide caused by the H{sup 3} disintegrations seems to be directly linked with the ionizations produced by the {beta}-particles inside the bacterial DNA. (author) [French] Des bacteries de la souche B{sup 3}{sub 1} thy {sup -}/Sr, exigeantes en thymine et resistantes a la streptomycine, ont ete marquees dans leur ADN par la thymidine tritiee. Les mesures de radioactivite ont ete faites avec un detecteur a scintillation en milieu liquide comprenant deux photomultiplicateurs montes en coiencidence. Dans nos conditions, l'efficacite des mesures a ete de 4,5% et le mouvement propre de 130 coups/min. Les bacteries radioactives ont ete conservees en ampoules scellees soit a 0 Degree-Sign C soit a - 196 Degree-Sign C, et la survie etudiee. Ces experiences ont montre que les bacteries sont inactivees exponentiellement en fonction du nombre d'atomes de tritium desintegres. L'inactivation depend de la temperature a laquelle elles sont conservees. Le calcul montre que l

  15. A bovine herpesvirus 5 recombinant defective in the thymidine kinase (TK gene and a double mutant lacking TK and the glycoprotein E gene are fully attenuated for rabbits

    Directory of Open Access Journals (Sweden)

    S.C. Silva

    2010-02-01

    Full Text Available Bovine herpesvirus 5 (BoHV-5, the agent of herpetic meningoencephalitis in cattle, is an important pathogen of cattle in South America and several efforts have been made to produce safer and more effective vaccines. In the present study, we investigated in rabbits the virulence of three recombinant viruses constructed from a neurovirulent Brazilian BoHV-5 strain (SV507/99. The recombinants are defective in glycoprotein E (BoHV-5gEΔ, thymidine kinase (BoHV-5TKΔ and both proteins (BoHV-5gEΔTKΔ. Rabbits inoculated with the parental virus (N = 8 developed neurological disease and died or were euthanized in extremis between days 7 and 13 post-infection (pi. Infectivity was detected in several areas of their brains. Three of 8 rabbits inoculated with the recombinant BoHV-5gEΔ developed neurological signs between days 10 and 15 pi and were also euthanized. A more restricted virus distribution was detected in the brain of these animals. Rabbits inoculated with the recombinants BoHV-5TKΔ (N = 8 or BoHV-5gEΔTKΔ (N = 8 remained healthy throughout the experiment in spite of variable levels of virus replication in the nose. Dexamethasone (Dx administration to rabbits inoculated with the three recombinants at day 42 pi did not result in viral reactivation, as demonstrated by absence of virus shedding and/or increase in virus neutralizing titers. Nevertheless, viral DNA was detected in the trigeminal ganglia or olfactory bulbs of all animals at day 28 post-Dx, demonstrating they were latently infected. These results show that recombinants BoHV-5TKΔ and BoHV-5gEΔTKΔ are attenuated for rabbits and constitute potential vaccine candidates upon the confirmation of this phenotype in cattle.

  16. 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.)

  17. 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)

    Morin, Kevin W.; Duan Weili; Knaus, Edward E.; McEwan, Alexander J.B.; Wiebe, Leonard I.

    2005-01-01

    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 [ 125 I]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 [ 125 I]IVFRU was determined in cell culture, and whole body biodistribution after intravenous injection of [ 125 I]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 [ 125 I]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 [ 125 I]IVFRU. Biodistribution of [ 125 I]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

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

    Energy Technology Data Exchange (ETDEWEB)

    Morin, Kevin W. [Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, T6G 2N8 (Canada); Duan Weili [Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, T6G 2N8 (Canada); Knaus, Edward E. [Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, T6G 2N8 (Canada); McEwan, Alexander J.B. [Department of Radiology and Diagnostic Imaging, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, T6G 2N8 (Canada); Wiebe, Leonard I. [Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, T6G 2N8 (Canada)]. E-mail: leonard.wiebe@ualberta.ca

    2005-07-01

    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 [{sup 125}I]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 [{sup 125}I]IVFRU was determined in cell culture, and whole body biodistribution after intravenous injection of [{sup 125}I]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 [{sup 125}I]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 [{sup 125}I]IVFRU. Biodistribution of [{sup 125}I]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.

  19. In vitro uptakes of radiolabeled IVDU and IVFRU in herpes simplex virus type-1 thymidine kinase (HSV1-tk) gene transduced morris hepatoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Tae Sup; Choi, Tae Hyun; Ahn, Soon Hyuk; Woo, Kwang Sun; Jeong, Wee Sup; Kwon, Hee Chung; Choi, Chang Woon; Lim, Sang Moo [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Awh, Ok Doo [College of Health Sciences, Yonsei Univ., Wonju (Korea, Republic of)

    2004-02-01

    The herpes simplex virus type 1 thymidine kinase gene(HSV1-tk) is an attractive candidate as a reporter gene in noninvasive reporter gene monitoring system. The HSV1-tk gene was chosen as a reporter gene, because it has been extensively studied, and there are appropriate reporter probes, substrates of HSV1-tk gene product, to apply for HSV1-tk gene imaging. We used radiolabeled 5-iodovinyl-2'-deoxyuridine (IVDU) and 5-lodovinyl-2'-fluoro-2'-deoxyuridine (IVFRU) as reporter probes for HSV1-tk gene monitoring system. We prepared HSV1-tk gene transduced Morris hepatoma cell line using retroviral vector, MOLTEN containing HSV1-tk gene. And we confirmed the HSV1-tk gene expression by Northern blotting and Western blotting. We compared in vitro uptakes of radioiodinated IVDU and IVFRU to monitor HSV1-tk gene expression in Morris hepatoma cell line (MCA) and HSV1-tk gene tranduced MCA (MAC-tk) cells until 480 minutes. We also performed correlation analysis between percentage of HSV1-tk gene tranduced MCA cell % (MCA-tk%) and uptakes of radiolabeled IVDU or IVFRU. MCA-tk cell expressed HSV1-tk mRNA and HSV1-TK protein. Two compounds showed minimal uptake in MCA, but increased uptake was observed in MCA-tk. IVDU showed 4-fold higher accumulation than IVFRU at 480 min in MCA-tk (p<0.01). Both IVDU and IVFRU uptake were linearly correlated (R{sup 2}>0.96) with increasing MCA-tk%. The rediolabeld IVDU and IVFRU showed higher specific accumulation in retrovirally HSV1-tk gene transfected Morris hepatoma cell line. Both IVDU and IVFRU could be used as good substrates for evaluation of HSV1-tk gene expression.

  20. Production and characterisation of a novel chicken IgY antibody raised against C-terminal peptide from human thymidine kinase 1.

    Science.gov (United States)

    Wu, Chuanjing; Yang, Rongjiang; Zhou, Ji; Bao, Shing; Zou, Li; Zhang, Pinggan; Mao, Yongrong; Wu, Jianping; He, Qimin

    2003-06-01

    Egg yolk is a good source of highly specific antibodies against mammalian antigens because of the phylogenetic distance between birds and mammals. Chicken egg yolk immunoglobulins (IgY) were generated to a synthetic 31-amino acid peptide from the C-terminal of human HeLa thymidine kinase 1 (TK1) enzyme. The anti-TK1 IgY antibody was purified using affinity chromatography against the 31-amino acid peptide. The purified antibody inhibited the catalytic activity of the TK1 enzyme in the CEM TK1(+) cells and recognized the 25-kDa subunit and tetrameric form of TK1, which has a pI value of 8.3. No immunoreaction was observed in CEM TK1(-) cells. Western blot of the serum TK1 (S-TK1) also showed that only a single band was found in the serum of patients with malignancies. No band was seen in healthy serum. Furthermore, dot blots and enhanced chemiluminescence (ECL) detection of S-TK1 performed on sera of preoperative patients with gastric cancer (GC) (n=31) and healthy controls (n=62) showed that the levels of S-TK1 in the sera of cancer patients were significantly different (Pstage cancer patients (four breast carcinomas, three hepatocarcinomas and four thyroid carcinomas) indicated that a strong staining of TK1 enzyme was found in the cytoplasm of malignant cells. No staining or weak staining was seen in normal tissues. We suggest that screening for TK1 using anti-TK1 IgY may be potentially useful for serological and immunohistochemical detection of TK1 as an early prognosis and for monitoring patients undergoing treatment.

  1. Construction and growth properties of bovine herpesvirus type 5 recombinants defective in the glycoprotein E or thymidine kinase gene or both

    Directory of Open Access Journals (Sweden)

    M.C.S. Brum

    2010-02-01

    Full Text Available Bovine herpesvirus type 5 (BoHV-5 is an important pathogen of cattle in South America. We describe here the construction and characterization of deletion mutants defective in the glycoprotein E (gE or thymidine kinase (TK gene or both (gE/TK from a highly neurovirulent and well-characterized Brazilian BoHV-5 strain (SV507/99. A gE-deleted recombinant virus (BoHV-5 gE∆ was first generated in which the entire gE open reading frame was replaced with a chimeric green fluorescent protein gene. A TK-deleted recombinant virus (BoHV-5 TK∆ was then generated in which most of the TK open reading frame sequences were deleted and replaced with a chimeric β-galactosidase gene. Subsequently, using the BoHV-5 gE∆ virus as backbone, a double gene-deleted (TK plus gE BoHV-5 recombinant (BoHV-5 gE/TK∆ was generated. The deletion of the gE and TK genes was confirmed by immunoblotting and PCR, respectively. In Madin Darby bovine kidney (MDBK cells, the mutants lacking gE (BoHV-5 gE∆ and TK + gE (BoHV-5 gE/TK∆ produced small plaques while the TK-deleted BoHV-5 produced wild-type-sized plaques. The growth kinetics and virus yields in MDBK cells for all three recombinants (BoHV-5 gE∆, BoHV-5 TK∆ and BoHV-5 gE/TK∆ were similar to those of the parental virus. It is our belief that the dual gene-deleted recombinant (BoHV-5 gE/TK∆ produced on the background of a highly neurovirulent Brazilian BoHV-5 strain may have potential application in a vaccine against BoHV-5.

  2. Expression of Herpes Simplex Virus Thymidine Kinase/Ganciclovir by RNA Trans-Splicing Induces Selective Killing of HIV-Producing Cells

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    Carin K. Ingemarsdotter

    2017-06-01

    Full Text Available Antiviral strategies targeting hijacked cellular processes are less easily evaded by the virus than viral targets. If selective for viral functions, they can have a high therapeutic index. We used RNA trans-splicing to deliver the herpes simplex virus thymidine kinase-ganciclovir (HSV-tk/GCV cell suicide system into HIV-producing cells. Using an extensive in silico bioinformatics and RNA structural analysis approach, ten HIV RNA trans-splicing constructs were designed targeting eight different HIV splice donor or acceptor sites and were tested in cells expressing HIV. Trans-spliced mRNAs were identified in HIV-expressing cells using qRT-PCR with successful detection of fusion RNA transcripts between HIV RNA and the HSV-tk RNA transcripts from six of ten candidate RNA trans-splicing constructs. Conventional PCR and Sanger sequencing confirmed RNA trans-splicing junctions. Measuring cell viability in the presence or absence of GCV expression of HSV-tk by RNA trans-splicing led to selective killing of HIV-producing cells using either 3′ exon replacement or 5′ exon replacement in the presence of GCV. Five constructs targeting four HIV splice donor and acceptor sites, D4, A5, A7, and A8, involved in regulating the generation of multiple HIV RNA transcripts proved to be effective for trans-splicing mediated selective killing of HIV-infected cells, within which individual constructs targeting D4 and A8 were the most efficient.

  3. High levels of inactive thymidine kinase 1 polypeptide detected in sera from dogs with solid tumours by immunoaffinity methods: implications for in vitro diagnostics.

    Science.gov (United States)

    Kiran Kumar, J; Sharif, H; Westberg, S; von Euler, H; Eriksson, S

    2013-09-01

    Determination of serum thymidine kinase 1 (STK1) activity has been used as a proliferation marker for neoplastic diseases in both human and veterinary medicine. The purpose of this study was to determine STK1 activity and enzyme levels in different dog tumours. Serum samples from three dogs with leukaemia, five with lymphoma, 21 with solid tumours and 18 healthy dogs were analyzed for STK1 activity, using an optimized [(3)H]-deoxythymidine (dThd) phosphorylation assay, and for STK1 protein levels using an immunoaffinity/western blot assay. STK1 activity in dogs with haematological tumours was significantly higher than in the solid tumour and healthy dog groups (mean ± standard deviation [SD] = 65 ± 79, 1.1 ± 0.5, and 1.0 ± 0.4 pmol/min/mL, respectively). Serum samples were analyzed after immunoaffinity isolation by western blot and the TK1 26 kDa band intensities quantified revealing that concentrations were significantly higher in dogs with haematological tumours and solid tumours compared to healthy dogs (mean ± SD=33 ± 12, 30 ± 13, and 10 ± 5 ng/mL, respectively). Pre-incubation with the reducing agent dithioerythritol (DTE) showed a decrease in STK1 activity and protein levels in most samples, but an increase of about 20% in sera from healthy dogs and from those with haematological malignancies. Compared to animals with solid tumours, the specific STK1 activity (nmol [(3)H]-deoxythymidine monophosphate (dTMP)/min/mg of TK1 protein of 26 kDa) was 30-fold higher in haematological malignancies and 2.5-fold higher in healthy dogs, respectively. The results demonstrate that there is a large fraction of inactive TK1 protein, particularly in sera from dogs with solid tumours. The findings are important in the use of STK1 as a biomarker. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Radiochemotherapy of hepatocarcinoma via lentivirus-mediated transfer of human sodium iodide symporter gene and herpes simplex virus thymidine kinase gene

    Energy Technology Data Exchange (ETDEWEB)

    Chen Libo, E-mail: libochen888@hotmail.com [Department of Nuclear Medicine, Shanghai Sixth People' s Hospital, Shanghai Jiao Tong University, Shanghai 200233 (China); Guo Guoying [Xinyuan Institute of Medicine and Biotechnology, School of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018 (China); Liu Tianjing; Guo Lihe [Division of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Zhu Ruisen [Department of Nuclear Medicine, Shanghai Sixth People' s Hospital, Shanghai Jiao Tong University, Shanghai 200233 (China)

    2011-07-15

    Herpes simplex virus thymidine kinase (HSV-TK) gene/ganciclovir (GCV) system has been widely used as a traditional gene therapy modality, and the sodium/iodide symporter gene (NIS) has been found to be a novel therapeutic gene. Since the therapeutic effects of radioiodine therapy or prodrug chemotherapy on cancers following NIS or HSV-TK gene transfer need to be enhanced, this study was designed to investigate the feasibility of radiochemotherapy for hepatocarcinoma via coexpression of NIS gene and HSV-TK gene. Methods: HepG2 cells were stably transfected with NIS, TK and GFP gene via recombinant lentiviral vector and named HepG2/NTG. Gene expression was examined by reverse transcriptase polymerase chain reaction, fluorescence imaging and iodide uptake. The therapeutic effects were assessed by MTT assay and clonogenic assay. Results: HepG2/NTG cells concentrated {sup 125}I{sup -} up to 76-fold higher than the wild-type cells within 20 min, and the efflux happened with a T{sub 1/2eff} of less than 10 min. The iodide uptake in HepG2/NTG cells was specifically inhibited by sodium perchlorate. Dose-dependent toxicity to HepG2/NTG cells by either GCV or {sup 131}I was revealed by clonogenic assay and MTT assay, respectively. The survival rate of HepG2/NTG cells decreased to 49.7%{+-}2.5%, 43.4%{+-}2.8% and 8.6%{+-}1.2% after exposure to {sup 131}I, GCV and combined therapy, respectively. Conclusion: We demonstrate that radiochemotherapy of hepatocarcinoma via lentiviral-mediated coexpression of NIS gene and HSV-TK gene leads to stronger killing effect than single treatment, and in vivo studies are needed to verify these findings.

  5. Radiochemotherapy of hepatocarcinoma via lentivirus-mediated transfer of human sodium iodide symporter gene and herpes simplex virus thymidine kinase gene

    International Nuclear Information System (INIS)

    Chen Libo; Guo Guoying; Liu Tianjing; Guo Lihe; Zhu Ruisen

    2011-01-01

    Herpes simplex virus thymidine kinase (HSV-TK) gene/ganciclovir (GCV) system has been widely used as a traditional gene therapy modality, and the sodium/iodide symporter gene (NIS) has been found to be a novel therapeutic gene. Since the therapeutic effects of radioiodine therapy or prodrug chemotherapy on cancers following NIS or HSV-TK gene transfer need to be enhanced, this study was designed to investigate the feasibility of radiochemotherapy for hepatocarcinoma via coexpression of NIS gene and HSV-TK gene. Methods: HepG2 cells were stably transfected with NIS, TK and GFP gene via recombinant lentiviral vector and named HepG2/NTG. Gene expression was examined by reverse transcriptase polymerase chain reaction, fluorescence imaging and iodide uptake. The therapeutic effects were assessed by MTT assay and clonogenic assay. Results: HepG2/NTG cells concentrated 125 I - up to 76-fold higher than the wild-type cells within 20 min, and the efflux happened with a T 1/2eff of less than 10 min. The iodide uptake in HepG2/NTG cells was specifically inhibited by sodium perchlorate. Dose-dependent toxicity to HepG2/NTG cells by either GCV or 131 I was revealed by clonogenic assay and MTT assay, respectively. The survival rate of HepG2/NTG cells decreased to 49.7%±2.5%, 43.4%±2.8% and 8.6%±1.2% after exposure to 131 I, GCV and combined therapy, respectively. Conclusion: We demonstrate that radiochemotherapy of hepatocarcinoma via lentiviral-mediated coexpression of NIS gene and HSV-TK gene leads to stronger killing effect than single treatment, and in vivo studies are needed to verify these findings.

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

    Directory of Open Access Journals (Sweden)

    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.

  7. Hydroxyurea enhances the activity of acyclovir and cidofovir against herpes simplex virus type 1 resistant strains harboring mutations in the thymidine kinase and/or the DNA polymerase genes.

    Science.gov (United States)

    Sergerie, Yan; Boivin, Guy

    2008-01-01

    Drug-resistant herpes simplex virus type 1 (HSV-1) recombinant strains harboring mutations in the thymidine kinase and/or the DNA polymerase genes were evaluated for their susceptibility to various antivirals in the presence of 25 microg/ml of hydroxyurea (HyU). The latter compound decreased the 50% inhibitory concentrations of acyclovir by 1.5-3.8-fold and that of cidofovir by 2.7-14.4-fold. However, HyU did not affect the susceptibilities of the various recombinant mutants to foscarnet. Hydroxyurea, a ribonucleotide reductase inhibitor, can increase the activity of nucleoside/nucleotide analogues against drug-resistant viruses.

  8. Bifidobacterial recombinant thymidine kinase-ganciclovir gene therapy system induces FasL and TNFR2 mediated antitumor apoptosis in solid tumors

    International Nuclear Information System (INIS)

    Wang, Changdong; Ma, Yongping; Hu, Qiongwen; Xie, Tingting; Wu, Jiayan; Zeng, Fan; Song, Fangzhou

    2016-01-01

    Directly targeting therapeutic suicide gene to a solid tumor is a hopeful approach for cancer gene therapy. Treatment of a solid tumor by an effective vector for a suicide gene remains a challenge. Given the lack of effective treatments, we constructed a bifidobacterial recombinant thymidine kinase (BF-rTK) -ganciclovir (GCV) targeting system (BKV) to meet this requirement and to explore antitumor mechanisms. Bifidobacterium (BF) or BF-rTK was injected intratumorally with or without ganciclovir in a human colo320 intestinal xenograft tumor model. The tumor tissues were analyzed using apoptosis antibody arrays, real time PCR and western blot. The colo320 cell was analyzed by the gene silencing method. Autophagy and necroptosis were also detected in colo320 cell. Meanwhile, three human digestive system xenograft tumor models (colorectal cancer colo320, gastric cancer MKN-45 and liver cancer SSMC-7721) and a breast cancer (MDA-MB-231) model were employed to validate the universality of BF-rTK + GCV in solid tumor gene therapy. The survival rate was evaluated in three human cancer models after the BF-rTK + GCV intratumor treatment. The analysis of inflammatory markers (TNF-α) in tumor indicated that BF-rTK + GCV significantly inhibited TNF-α expression. The results suggested that BF-rTK + GCV induced tumor apoptosis without autophagy and necroptosis occurrence. The apoptosis was transduced by multiple signaling pathways mediated by FasL and TNFR2 and mainly activated the mitochondrial control of apoptosis via Bid and Bim, which was rescued by silencing Bid or/and Bim. However, BF + GCV only induced apoptosis via Fas/FasL signal pathway accompanied with increased P53 expression. We further found that BF-rTK + GCV inhibited the expression of the inflammatory maker of TNF-α. However, BF-rTK + GCV did not result in necroptosis and autophagy. BF-rTK + GCV induced tumor apoptosis mediated by FasL and TNFR2 through the mitochondrial control of apoptosis via Bid and Bim

  9. 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)

    Jiang Xinqing; Chen Liang; Wu Hongzhen; Huang Jingjun; Wei Xinhua; Mo Lei; Yang Ruimeng; Xiao Xiangsheng

    2012-01-01

    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 10 th 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) mm 3 , (754.57 ± 66.84) mm 3 , with no significant difference (t=0.488, P >0.05). The ADC values of the treatment groups were (0.98 ±0.11) × 10 -3 mm 2 /s,the ones of the control groups were (0.68 ±0.04) × 10 -3 mm 2 /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)

  10. 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

  11. Comparison of [{sup 18}F]FHPG and [{sup 124/125}I]FIAU for imaging herpes simplex virus type 1 thymidine kinase gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Brust, P.; Friedrich, A.; Scheunemann, M.; Noll, S.; Noll, B.; Johannsen, B. [Inst. of Bioinorganic and Radiopharmaceutical Chemistry, Forschungszentrum, Rossendorf (Germany); Haubner, R.; Avril, N. [Dept. of Nuclear Medicine, Technische Univ., Muenchen (Germany); Anton, M. [Inst. of Experimental Oncology, Technische Univ., Muenchen (Germany); Koufaki, O.N.; Schackert, H.K. [Dept. of Surgical Research, Technische Univ., Dresden (Germany); Hauses, M.; Schackert, G. [Dept. of Neurosurgery, Technische Univ., Dresden (Germany); Haberkorn, U. [Dept. of Oncological Diagnostics and Therapy, Deutsches Krebsforschungszentrum, Heidelberg (Germany)

    2001-06-01

    Various radiotracers based on uracil nucleosides (e.g. [{sup 124}I]2'-fluoro-2'-deoxy-5-iodo-1-{beta}-D-arabinofuranosyluracil, [{sup 124}I]FIAU) and acycloguanosine derivatives (e.g. [{sup 18}F]9-[(3-fluoro-1-hydroxy-2-propoxy)methyl]guanine, [{sup 18}F]FHPG) have been proposed for the non-invasive imaging of herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene expression. However, these radiotracers have been evaluated in different in vitro and in vivo models, precluding a direct comparison. Therefore, we directly compared [{sup 18}F]FHPG and radioiodinated FIAU to assess their potential for PET imaging of transgene expression. The uptake of [{sup 125}I]FIAU, [{sup 18}F]FHPG and [{sup 3}H]acyclovir was determined in vitro using four different HSV1-tk expressing cell lines and their respective negative controls. The in vitro tracer uptake was generally low in non-transduced parental cell lines. In HSV1-tk expressing cells, [{sup 3}H]acyclovir showed approximately a twofold higher tracer accumulation, the [{sup 18}F]FHPG uptake increased by about sixfold and the [{sup 125}I]FIAU accumulation increased by about 28-fold after 120-min incubation of T1115 human glioblastoma cells. Similar results were found in the other cell lines. In addition, biodistribution and positron emission tomography (PET) studies with [{sup 18}F]FHPG and [{sup 124/125}I]FIAU were carried out in tumour-bearing BALB/c mice. Significantly higher specific accumulation of radioactivity was found for [{sup 125}I]FIAU compared with [{sup 18}F]FHPG. The ratio of specific tracer accumulation between [{sup 125}I]FIAU and [{sup 18}F]FHPG increased from 21 (30 min p.i.) to 119 (4 h p.i.). PET imaging, using [{sup 124}I]FIAU, clearly visualised and delineated HSV1-tk expressing tumours, whereas only a negligible uptake of [{sup 18}F]FHPG was observed. This study demonstrated that in vitro and in vivo, the radioiodinated uracil nucleoside FIAU has a significantly higher specific

  12. 5-[{sup 18}F]Fluoroalkyl pyrimidine nucleosides: probes for positron emission tomography imaging of herpes simplex virus type 1 thymidine kinase gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Chacko, Ann-Marie [Institute for Environmental Medicine, Targeted Therapeutics Program, University of Pennsylvania, Philadelphia, PA 19104 (United States); Blankemeyer, Eric; Lieberman, Brian P.; Qu, Wenchao [Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104 (United States); Kung, Hank F. [Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104 (United States); Department of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104 (United States)], E-mail: kunghf@gmail.com

    2009-01-15

    Introduction: The preliminary in vivo evaluation of novel 5-[{sup 18}F]fluoroalkyl-2'-deoxyuridines ([{sup 18}F]FPrDU, [{sup 18}F]FBuDU, [{sup 18}F]FPeDU; [{sup 18}F]1a-c, respectively) and 2'-fluoro-2'-deoxy-5-[{sup 18}F]fluoroalkyl-1-{beta}-D-arabinofuranosyl uracils ([{sup 18}F]FFPrAU, [{sup 18}F]FFBuAU, [{sup 18}F]FFPeAU; [{sup 18}F]1d-f, respectively) as probes for imaging herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene expression is described. Methods: [{sup 18}F]1a-f were successfully synthesized by a rapid and efficient two-step one-pot nucleophilic fluorination reaction using 5-O-mesylate precursors and [{sup 18}F]F{sup -}. For in vivo studies, tumor xenografts were grown in nude mice by implanting RG2 cells stably expressing HSV1-tk (RG2TK+) and wild-type cells (RG2). Results: Biodistribution studies at 2 h pi revealed that the uptake of [{sup 18}F]1a-b and [{sup 18}F]1d-e in RG2TK+ tumors was not significantly different from control tumors. However, [{sup 18}F]1c and [{sup 18}F]1f had an average 1.6- and 1.7-fold higher uptake in RG2TK+ tumors than control RG2 tumors. Blood activity curves for [{sup 18}F]1c and [{sup 18}F]1f highlight rapid clearance of radioactivity in the blood. Dynamic small animal PET (A-PET) imaging studies of tumor-bearing mice with [{sup 18}F]1c and [{sup 18}F]1f showed higher initial uptake (3.5- and 1.4-fold, respectively) in RG2TK+ tumors than in control tumors, with continued washout of activity from both tumors over time. Conclusions: Biological evaluations suggest that [{sup 18}F]1c and [{sup 18}F]1f may have limited potential for imaging HSV1-tk gene expression due to fast washout of activity from the blood, thus significantly decreasing sensitivity and specificity of tracer accumulation in HSV1-tk-expressing tumors.

  13. Imaging Expression of Cytosine Deaminase-Herpes Virus Thymidine Kinase Fusion Gene (CD/TK Expression with [124I]FIAU and PET

    Directory of Open Access Journals (Sweden)

    Trevor Hackman

    2002-01-01

    Full Text Available Double prodrug activation gene therapy using the Escherichia coli cytosine deaminase (CDherpes simplex virus type 1 thymidine kinase (HSV1-tk fusion gene (CD/TK with 5-fluorocytosine (5FC, ganciclovir (GCV, and radiotherapy is currently under evaluation for treatment of different tumors. We assessed the efficacy of noninvasive imaging with [124I]FIAU (2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-iodo-uracil and positron emission tomography (PET for monitoring expression of the CD/TK fusion gene. Walker-256 tumor cells were transduced with a retroviral vector bearing the CD/TK gene (W256CD/TK cells. The activity of HSV1-TK and CD subunits of the CD/TK gene product was assessed in different single cell-derived clones of W256CD/TK cells using the FIAU radiotracer accumulation assay in cells and a CD enzyme assay in cell homogenates, respectively. A linear relationship was observed between the levels of CD and HSV1-tk subunit expression in corresponding clones in vitro over a wide range of CD/TK expression levels. Several clones of W256CD/TK cells with significantly different levels of CD/TK expression were selected and used to produce multiple subcutaneous tumors in rats. PET imaging of HSV1-TK subunit activity with [124I]FIAU was performed on these animals and demonstrated that different levels of CD/TK expression in subcutaneous W256CD/TK tumors can be imaged quantitatively. CD expression in subcutaneous tumor sample homogenates was measured using a CD enzyme assay. A comparison of CD and HSV1-TK subunit enzymatic activity of the CD/TK fusion protein in vivo showed a significant correlation. Knowing this relationship, the parametric images of CD subunit activity were generated. Imaging with [124I]FIAU and PET could provide pre- and posttreatment assessments of CD/TK-based double prodrug activation in clinical gene therapy trials.

  14. 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

  15. Profiling bacterial kinase activity using a genetic circuit

    DEFF Research Database (Denmark)

    van der Helm, Eric; Bech, Rasmus; Lehning, Christina Eva

    Phosphorylation is a post-translational modification that regulates the activity of several key proteins in bacteria and eukaryotes. Accordingly, a variety of tools has been developed to measure kinase activity. To couple phosphorylation to an in vivo fluorescent readout we used the Bacillus...... subtilis kinase PtkA, transmembrane activator TkmA and the repressor FatR to construct a genetic circuit in E. coli. By tuning the repressor and kinase expression level at the same time, we were able to show a 4.2-fold increase in signal upon kinase induction. We furthermore validated that the previously...... reported FatR Y45E mutation1 attenuates operator repression. This genetic circuit provides a starting point for computational protein design and a metagenomic library-screening tool....

  16. Evaluation of a C57BL/6J × 129S1/SvImJ Hybrid Nestin-Thymidine Kinase Transgenic Mouse Model for Studying the Functional Significance of Exercise-Induced Adult Hippocampal Neurogenesis.

    Science.gov (United States)

    Hamilton, G F; Majdak, P; Miller, D S; Bucko, P J; Merritt, J R; Krebs, C P; Rhodes, J S

    2015-01-01

    New neurons are continuously generated in the adult hippocampus but their function remains a mystery. The nestin thymidine kinase (nestin-TK) transgenic method has been used for selective and conditional reduction of neurogenesis for the purpose of testing the functional significance of new neurons in learning, memory and motor performance. Here we explored the nestin-TK model on a hybrid genetic background (to increase heterozygosity, and "hybrid vigor"). Transgenic C57BL/6J (B6) were crossed with 129S1/SvImJ (129) producing hybrid offspring (F1) with the B6 half of the genome carrying a herpes simplex virus thymidine kinase (TK) transgene regulated by a modified nestin promoter. In the presence of exogenously administered valganciclovir, new neurons expressing TK undergo apoptosis. Female B6 nestin-TK mice ( n = 80) were evaluated for neurogenesis reduction as a positive control. Male and female F1 nestin-TK mice ( n = 223) were used to determine the impact of neurogenesis reduction on the Morris water maze (MWM) and rotarod. All mice received BrdU injections to label dividing cells and either valganciclovir or control chow, with or without a running wheel for 30 days. Both the F1 and B6 background displayed approximately 50% reduction in neurogenesis, a difference that did not impair learning and memory on the MWM or rotarod performance. Running enhanced neurogenesis and performance on the rotarod but not MWM suggesting the F1 background may not be suitable for studying pro-cognitive effects of exercise on MWM. Greater reduction of neurogenesis may be required to observe behavioral impacts. Alternatively, new neurons may not play a critical role in learning, or compensatory mechanisms in pre-existing neurons could have masked the deficits. Further work using these and other models for selectively reducing neurogenesis are needed to establish the functional significance of adult hippocampal neurogenesis in behavior.

  17. X-ray structures of uridine phosphorylase from Vibrio cholerae in complexes with uridine, thymidine, uracil, thymine, and phosphate anion: Substrate specificity of bacterial uridine phosphorylases

    Energy Technology Data Exchange (ETDEWEB)

    Prokofev, I. I.; Lashkov, A. A., E-mail: alashkov83@gmail.com; Gabdulkhakov, A. G.; Balaev, V. V.; Seregina, T. A. [Russian Academy of Sciences, Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” (Russian Federation); Mironov, A. S. [State Research Institute of Genetics and Selection of Industrial Microorganisms (Russian Federation); Betzel, C. [University of Hamburg (Germany); Mikhailov, A. M. [Russian Academy of Sciences, Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” (Russian Federation)

    2016-11-15

    In many types of human tumor cells and infectious agents, the demand for pyrimidine nitrogen bases increases during the development of the disease, thus increasing the role of the enzyme uridine phosphorylase in metabolic processes. The rational use of uridine phosphorylase and its ligands in pharmaceutical and biotechnology industries requires knowledge of the structural basis for the substrate specificity of the target enzyme. This paper summarizes the results of the systematic study of the three-dimensional structure of uridine phosphorylase from the pathogenic bacterium Vibrio cholerae in complexes with substrates of enzymatic reactions—uridine, phosphate anion, thymidine, uracil, and thymine. These data, supplemented with the results of molecular modeling, were used to consider in detail the structural basis for the substrate specificity of uridine phosphorylases. It was shown for the first time that the formation of a hydrogen-bond network between the 2′-hydroxy group of uridine and atoms of the active-site residues of uridine phosphorylase leads to conformational changes of the ribose moiety of uridine, resulting in an increase in the reactivity of uridine compared to thymidine. Since the binding of thymidine to residues of uridine phosphorylase causes a smaller local strain of the β-N1-glycosidic bond in this the substrate compared to the uridine molecule, the β-N1-glycosidic bond in thymidine is more stable and less reactive than that in uridine. It was shown for the first time that the phosphate anion, which is the second substrate bound at the active site, interacts simultaneously with the residues of the β5-strand and the β1-strand through hydrogen bonding, thus securing the gate loop in a conformation.

  18. X-ray structures of uridine phosphorylase from Vibrio cholerae in complexes with uridine, thymidine, uracil, thymine, and phosphate anion: Substrate specificity of bacterial uridine phosphorylases

    Science.gov (United States)

    Prokofev, I. I.; Lashkov, A. A.; Gabdulkhakov, A. G.; Balaev, V. V.; Seregina, T. A.; Mironov, A. S.; Betzel, C.; Mikhailov, A. M.

    2016-11-01

    In many types of human tumor cells and infectious agents, the demand for pyrimidine nitrogen bases increases during the development of the disease, thus increasing the role of the enzyme uridine phosphorylase in metabolic processes. The rational use of uridine phosphorylase and its ligands in pharmaceutical and biotechnology industries requires knowledge of the structural basis for the substrate specificity of the target enzyme. This paper summarizes the results of the systematic study of the three-dimensional structure of uridine phosphorylase from the pathogenic bacterium Vibrio cholerae in complexes with substrates of enzymatic reactions—uridine, phosphate anion, thymidine, uracil, and thymine. These data, supplemented with the results of molecular modeling, were used to consider in detail the structural basis for the substrate specificity of uridine phosphorylases. It was shown for the first time that the formation of a hydrogen-bond network between the 2'-hydroxy group of uridine and atoms of the active-site residues of uridine phosphorylase leads to conformational changes of the ribose moiety of uridine, resulting in an increase in the reactivity of uridine compared to thymidine. Since the binding of thymidine to residues of uridine phosphorylase causes a smaller local strain of the β-N1-glycosidic bond in this the substrate compared to the uridine molecule, the β-N1-glycosidic bond in thymidine is more stable and less reactive than that in uridine. It was shown for the first time that the phosphate anion, which is the second substrate bound at the active site, interacts simultaneously with the residues of the β5-strand and the β1-strand through hydrogen bonding, thus securing the gate loop in a conformation

  19. The synthesis and biochemical evaluation of thymidine analogues substituted with nido carborane at the N-3 position

    International Nuclear Information System (INIS)

    Byun, Youngjoo; Yan Junhua; Al-Madhoun, A.S.; Johnsamuel, Jayaseharan; Yang Weilian; Barth, R.F.; Eriksson, Staffan; Tjarks, Werner

    2004-01-01

    Several thymidine analogues substituted with closo- and nido-carborane at the N-3 position were synthesized. The nido-carboranyl thymidine analogues were designed to be effective substrates for human thymidine kinase 1 in combination with an increased water solubility sufficient for clinical application in boron neutron capture therapy. This was done because N-3 substituted closo-carboranyl thymidine analogues previously synthesized in our laboratories were good TK1 substrates but were poorly water-soluble. Newly synthesized zwitterionic amino nido- and the corresponding neutral closo-m-carboranyl thymidine analogues exhibited excellent TK1 phosphorylation rates up to 75% relative to thymidine, indicating that these compounds were good substrates for thymidine kinase 1. Thin layer chromatographic studies were indicative of increased hydrophilicity of the synthesized nido-carboranyl thymidine analogues compared with their closo-carboranyl counterparts and previously reported closo-carboranyl thymidine analogues

  20. 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.

  1. 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

    Information on the regulation and structure-function relation of enzymes involved in DNA precursor synthesis is pivotal, as defects in several of these enzymes have been found to cause depletion or deletion of mitochondrial DNA resulting in severe diseases. Here, the effect of amino acid 106...... and 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...

  2. Bacterial Serine/Threonine Protein Kinases in Host-Pathogen Interactions*

    Science.gov (United States)

    Canova, Marc J.; Molle, Virginie

    2014-01-01

    In bacterial pathogenesis, monitoring and adapting to the dynamically changing environment in the host and an ability to disrupt host immune responses are critical. The virulence determinants of pathogenic bacteria include the sensor/signaling proteins of the serine/threonine protein kinase (STPK) family that have a dual role of sensing the environment and subverting specific host defense processes. STPKs can sense a wide range of signals and coordinate multiple cellular processes to mount an appropriate response. Here, we review some of the well studied bacterial STPKs that are essential virulence factors and that modify global host responses during infection. PMID:24554701

  3. Bacterial serine/threonine protein kinases in host-pathogen interactions.

    Science.gov (United States)

    Canova, Marc J; Molle, Virginie

    2014-04-04

    In bacterial pathogenesis, monitoring and adapting to the dynamically changing environment in the host and an ability to disrupt host immune responses are critical. The virulence determinants of pathogenic bacteria include the sensor/signaling proteins of the serine/threonine protein kinase (STPK) family that have a dual role of sensing the environment and subverting specific host defense processes. STPKs can sense a wide range of signals and coordinate multiple cellular processes to mount an appropriate response. Here, we review some of the well studied bacterial STPKs that are essential virulence factors and that modify global host responses during infection.

  4. Evaluation of F-18-labeled 5-iodocytidine ({sup 18}F-FIAC) as a new potential positron emission tomography probe for herpes simplex virus type 1 thymidine kinase imaging

    Energy Technology Data Exchange (ETDEWEB)

    Chan, Pei-Chia; Wu, Chun-Yi; Chang, Wen-Yi; Chang, Wei-Ting [Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, 11221, Taiwan (China); Alauddin, Mian [Department of Experimental Diagnostic Imaging, MD Anderson Cancer Center, University of Texas, TX, 77054 (United States); Liu, Ren-Shan [Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, 11221, Taiwan (China); Department of Nuclear Medicine, Faculty of Medicine, National Yang-Ming University, Taipei, 11221, Taiwan (China); Department of Nuclear Medicine and National PET/Cyclotron Center, Taipei Veterans General Hospital, Taipei, 11217, Taiwan (China); Lin, Wuu-Jyh [Institute of Nuclear Energy Research, Atomic Energy Council, Taoyuan, 32546, Taiwan (China); Chen, Fu-Du [College of Health and Leisure Science, TransWorld University, Yunlin, 64063, Taiwan (China); Chen, Chuan-Lin, E-mail: clchen2@ym.edu.tw [Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, 11221, Taiwan (China); Wang, Hsin-Ell, E-mail: hewang@ym.edu.tw [Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, 11221, Taiwan (China)

    2011-10-15

    Objective: Herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene in combination with radiolabeled nucleoside substrates is the most widely used reporter system. This study characterized 1-(2'-deoxy-2'-[{sup 18}F]fluoro-{beta}-D-arabinofuranosyl)-5-iodocytosine ({sup 18}F-FIAC) as a new potential positron emission tomography (PET) probe for HSV1-tk gene imaging and compared it with 2'-deoxy-2'-[{sup 18}F]fluoro-5-iodo-1-{beta}-D-arabinofuranosyluracil ({sup 18}F-FIAU) and 2'-deoxy-2'-[{sup 18}F]fluoro-5-ethyl-1-{beta}-D-arabinofuranosyluracil({sup 18}F-FEAU) (thymidine analogues) in an NG4TL4-WT/STK sarcoma-bearing mouse model. Methods: A cellular uptake assay, biodistribution study, radioactive metabolites assay and microPET imaging of NG4TL4-WT/STK tumor-bearing mice post administration of {sup 18}F-FIAC, {sup 18}F-FIAU and {sup 18}F-FEAU were conducted to characterize the biological properties of these tracers. Results: Highly specific uptake of {sup 18}F-FIAC, {sup 18}F-FIAU and {sup 18}F-FEAU in tk-transfected [tk(+)] cells was observed. The tk(+)-to-tk(-) cellular uptake ratio after a 2-h incubation was 66.6{+-}25.1, 76.3{+-}18.2 and 247.2{+-}37.2, respectively. In biodistribution studies, {sup 18}F-FIAC showed significant tk(+) tumor specificity (12.6; expressed as the tk(+)-to-tk(-) tumor uptake ratio at 2 h postinjection) comparable with {sup 18}F-FIAU (15.8) but lower than {sup 18}F-FEAU (48.0). The results of microPET imaging also revealed the highly specific accumulation of these three radioprobes in the NG4TL4-tk(+) tumor. Conclusion: Our findings suggested that the cytidine analogue {sup 18}F-FIAC is a new potential PET probe for the imaging of HSV1-tk gene expression. {sup 18}F-FIAC may be regarded as the prodrug of {sup 18}F-FIAU in vivo.

  5. Unavailability of thymidine kinase does not preclude the use of German comprehensive prognostic index: results of an external validation analysis in early chronic lymphocytic leukemia and comparison with MD Anderson Cancer Center model.

    Science.gov (United States)

    Molica, Stefano; Giannarelli, Diana; Mirabelli, Rosanna; Levato, Luciano; Russo, Antonio; Linardi, Maria; Gentile, Massimo; Morabito, Fortunato

    2016-01-01

    A comprehensive prognostic index that includes clinical (i.e., age, sex, ECOG performance status), serum (i.e., ß2-microglobulin, thymidine kinase [TK]), and molecular (i.e., IGVH mutational status, del 17p, del 11q) markers developed by the German CLL Study Group (GCLLSG) was externally validated in a prospective, community-based cohort consisting of 338 patients with early chronic lymphocytic leukemia (CLL) using as endpoint the time to first treatment (TTFT). Because serum TK was not available, a slightly modified version of the model based on seven instead of eight prognostic variables was used. By German index, 62.9% of patients were scored as having low-risk CLL (score 0-2), whereas 37.1% had intermediate-risk CLL (score 3-5). This stratification translated into a significant difference in the TTFT [HR = 4.21; 95% C.I. (2.71-6.53); P reliability [HR = 2.73; 95% C.I. (1.79-4.17); P German score. The c-statistic of the MDACC model was 0.65 (range, 0.53-0.78) a level below that of the German index [0.71 (range, 0.60-0.82)] and below the accepted 0.7 threshold necessary to have value at the individual patient level. Results of this external comparative validation analysis strongly support the German score as the benchmark for comparison of any novel prognostic scheme aimed at evaluating the TTFT in patients with early CLL even when a modified version which does not include TK is utilized. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. 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

    Energy Technology Data Exchange (ETDEWEB)

    Song, In Ho; Lee, Tae Sup; Kang, Joo Hyun; Lee, Yong Jin; Kim, Kwang Il; An, Gwang Il; Chung, Wee Sup; Cheon, Gi Jeong; Choi, Chang Woon; Lim, Sang Moo [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2009-10-15

    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.

  7. In Vitro and In Vivo Characterization of a Dual-Function Green Fluorescent Protein–HSV1-Thymidine Kinase Reporter Gene Driven by the Human Elongation Factor 1α Promoter

    Directory of Open Access Journals (Sweden)

    Gary D. Luker

    2002-04-01

    Full Text Available Toward the goal of monitoring activity of native mammalian promoters with molecular imaging techniques, we stably transfected DU145 prostate carcinoma cells with a fusion construct of enhanced green fluorescent protein (EGFP and wild-type herpes simplex virus-1 thymidine kinase (HSV1-TK as a reporter gene driven by the promoter for human elongation factor 1α (EF-1α-EGFP-TK. Using this model system, expression of EGFP was quantified by flow cytometry and fluorescence microscopy, while the HSV1-TK component of the reporter was quantified with 8-[3H]ganciclovir (8-[3H]GCV. As analyzed by flow cytometry, passage of EGFP-TK-DU145 transfected cells (ETK in vitro resulted in populations of cells with high and low expression of EGFP over time. High and low ETK cells retained 23-fold and 5-fold more GCV, respectively, than control. While differences in uptake and retention of GCV corresponded to relative expression of the reporter gene in each subpopulation of cells as determined by both flow cytometry (EGFP and quantitative RT-PCR, the correlation was not linear. Furthermore, in high ETK cells, net retention of various radiolabeled nucleoside analogues varied; the rank order was 8-[3H]GCV < 9-(4-fluoro-3-hydroxymethylbutylguanine ([18F]FHBG ≈ 8-[3H]penciclovir (8-[3H]PCV < 2′-fluoro-2′-deoxy-5-iodouracil-beta-d-arabinofuranoside (2-[14C]FIAU. Xenograft tumors of ETK cells in vivo accumulated 2.5-fold more 8-[3H]GCV per gram of tissue and showed greater fluorescence from EGFP than control DU145 cells, demonstrating that the reporter gene functioned in vivo. These data extend previous reports by showing that a human promoter can be detected in vitro and in vivo with a dual-function reporter exploiting optical and radiotracer techniques.

  8. Herpes simplex virus thymidine kinase imaging in mice with (1-(2'-deoxy-2'-[{sup 18}F]fluoro-1-{beta}-D-arabinofuranosyl)-5-iodouracil) and metabolite (1-(2'-deoxy-2'-[{sup 18}F]fluoro-1-{beta}-D-arabinofuranosyl)-5-uracil)

    Energy Technology Data Exchange (ETDEWEB)

    Nimmagadda, Sridhar; Lawhorn-Crews, Jawana M.; Shields, Anthony F. [Wayne State University, Karmanos Cancer Institute, Detroit, MI (United States); Wayne State University, Department of Medicine, Detroit, MI (United States); Mangner, Thomas J. [Wayne State University, Karmanos Cancer Institute, Detroit, MI (United States); Wayne State University, Department of Radiology, Detroit, MI (United States); Haberkorn, Uwe [University of Heidelberg, Department of Nuclear Medicine, Heidelberg (Germany)

    2009-12-15

    FIAU, (1-(2{sup '}-deoxy-2{sup '}-fluoro-1-{beta}-D-arabinofuranosyl)-5-iodouracil) has been used as a substrate for herpes simplex virus thymidine kinases (HSV-TK and HSV-tk, for protein and gene expression, respectively) and other bacterial and viral thymidine kinases for noninvasive imaging applications. Previous studies have reported the formation of a de-iodinated metabolite of {sup 18}F-FIAU. This study reports the dynamic tumor uptake, biodistribution, and metabolite contribution to the activity of {sup 18}F-FIAU seen in HSV-tk gene expressing tumors and compares the distribution properties with its de-iodinated metabolite {sup 18}F-FAU. CD-1 nu/nu mice with subcutaneous MH3924A and MH3924A-stb-tk+ xenografts on opposite flanks were used for the biodistribution and imaging studies. Mice were injected IV with either {sup 18}F-FIAU or {sup 18}F-FAU. Mice underwent dynamic imaging with each tracer for 65 min followed by additional static imaging up to 150 min post-injection for some animals. Animals were sacrificed at 60 or 150 min post-injection. Samples of blood and tissue were collected for biodistribution and metabolite analysis. Regions of interest were drawn over the images obtained from both tumors to calculate the time-activity curves. Biodistribution and imaging studies showed the highest uptake of {sup 18}F-FIAU in the MH3924A-stb-tk+ tumors. Dynamic imaging studies revealed a continuous accumulation of {sup 18}F-FIAU in HSV-TK expressing tumors over 60 min. The mean biodistribution values (SUV {+-} SE) for MH3924A-stb-tk+ were 2.07 {+-} 0.40 and 6.15 {+-} 1.58 and that of MH3924A tumors were 0.19 {+-} 0.07 and 0.47 {+-} 0.06 at 60 and 150 min, respectively. In {sup 18}F-FIAU injected mice, at 60 min nearly 63% of blood activity was present as its metabolite {sup 18}F-FAU. Imaging and biodistribution studies with {sup 18}F-FAU demonstrated no specific accumulation in MH3924A-stb-tk+ tumors and SUVs for both the tumors were similar to those

  9. JAK kinases are required for the bacterial RNA and poly I:C induced tyrosine phosphorylation of PKR

    Science.gov (United States)

    Bleiblo, Farag; Michael, Paul; Brabant, Danielle; Ramana, Chilakamarti V; Tai, TC; Saleh, Mazen; Parrillo, Joseph E; Kumar, Anand; Kumar, Aseem

    2013-01-01

    Discriminating the molecular patterns associated with RNA is central to innate immunity. The protein kinase PKR is a cytosolic sensor involved in the recognition of viral dsRNA and triggering interferon-induced signaling. Here, we identified bacterial RNA as a novel distinct pattern recognized by PKR. We show that the tyrosine phosphorylation of PKR induced by either bacterial RNA or poly I:C is impaired in mutant cells lacking TYK2, JAK1, or JAK2 kinases. PKR was found to be a direct substrate for the activated JAKs. Our results indicated that the double-stranded structures of bacterial RNA are required to fully activate PKR. These results suggest that bacterial RNA signaling is analogous in some respects to that of viral RNA and interferons and may have implications in bacterial immunity. PMID:23236554

  10. A strictly monofunctional bacterial hydroxymethylpyrimidine phosphate kinase precludes damaging errors in thiamin biosynthesis.

    Science.gov (United States)

    Thamm, Antje M; Li, Gengnan; Taja-Moreno, Marlene; Gerdes, Svetlana Y; de Crécy-Lagard, Valérie; Bruner, Steven D; Hanson, Andrew D

    2017-07-20

    The canonical kinase (ThiD) that converts the thiamin biosynthesis intermediate hydroxymethylpyrimidine (HMP) monophosphate to the diphosphate can also very efficiently convert free HMP to the monophosphate in prokaryotes, plants, and fungi. This HMP kinase activity enables salvage of HMP, but it is not substrate-specific and so allows toxic HMP analogs and damage products to infiltrate the thiamin biosynthesis pathway. Comparative analysis of bacterial genomes uncovered a gene, thiD2 , that is often fused to the thiamin synthesis gene thiE and could potentially encode a replacement for ThiD. Standalone ThiD2 proteins and ThiD2 fusion domains are small (~130-residues) and do not belong to any previously known protein family. Genetic and biochemical analyses showed that representative standalone and fused ThiD2 proteins catalyze phosphorylation of HMP monophosphate, but not of HMP or its toxic analogs and damage products such as bacimethrin and 5-(hydroxymethyl)-2-methylpyrimidin-4-ol. As strictly monofunctional HMP monophosphate kinases, ThiD2 proteins eliminate a potentially fatal vulnerability of canonical ThiD, at the cost of the ability to reclaim HMP formed by thiamin turnover. ©2017 The Author(s).

  11. 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...

  12. JAK kinases are required for the bacterial RNA and poly I:C induced tyrosine phosphorylation of PKR

    OpenAIRE

    Bleiblo, Farag; Michael, Paul; Brabant, Danielle; Ramana, Chilakamarti V; Tai, TC; Saleh, Mazen; Parrillo, Joseph E; Kumar, Anand; Kumar, Aseem

    2012-01-01

    Discriminating the molecular patterns associated with RNA is central to innate immunity. The protein kinase PKR is a cytosolic sensor involved in the recognition of viral dsRNA and triggering interferon-induced signaling. Here, we identified bacterial RNA as a novel distinct pattern recognized by PKR. We show that the tyrosine phosphorylation of PKR induced by either bacterial RNA or poly I:C is impaired in mutant cells lacking TYK2, JAK1, or JAK2 kinases. PKR was found to be a direct substra...

  13. Suppression of phytohemagglutinin-induction of thymidine uptake in guinea pig lymphocytes by methylglyoxal bis(guanylhydrazone) treatment.

    Science.gov (United States)

    Otani, S; Matsui, I; Morisawa, S

    1977-10-18

    Treatment with methylglyoxal bis(guanylhydrazone), a specific inhibitor of S-adenosylmethionine decarboxylase (EC 4.1.1.50), suppressed the phytohemagglutinin-induction of [3H]thymidine uptake by guinea pig lymphocytes. The kinetics of [3H]thymidine uptake revealed that the Km value for thymidine was not changed, but the V value was markedly lowered by the methylglyoxal bis(guanylhydrazone) treatment. The induction of ATP: thymidine 5'-phosphotransferase (EC 2.7.1.75) (thymidine kinase) activity by phytohemagglutinin was suppressed to about the same extent as the induction of thymidine uptake. These suppressions were dependent on the methylglyoxal bis(guanylhydrazone) doses and on duration of the methylglyoxal bis(guanylhydrazone) treatment. Analysis of [3H]thymidine labelled compounds of the acid-soluble fraction showed that conversion of thymidine to thymidine 5'-triphosphate was inhibited by the methylglyoxal bis(guanylhydrazone) treatment. DNA polymerase activity was less inhibited by the methylglyoxal bis(guanylhydrazone) treatment in comparison with the methylglyoxal bis(guanylhydrazone) inhibition of thymidine uptake by whole cells. These results strongly suggested that blocking of polyamine accumulation by the methylglyoxal bis(guanylhydrazone) treatment influenced phytohemagglutinin induction of thymidine phosphorylation, resulting in a decrease of thymidine incorporation into DNA.

  14. Role of glycogen synthase kinase-3 beta in the inflammatory response caused by bacterial pathogens

    Directory of Open Access Journals (Sweden)

    Cortés-Vieyra Ricarda

    2012-06-01

    Full Text Available Abstract Glycogen synthase kinase 3β (GSK3β plays a fundamental role during the inflammatory response induced by bacteria. Depending on the pathogen and its virulence factors, the type of cell and probably the context in which the interaction between host cells and bacteria takes place, GSK3β may promote or inhibit inflammation. The goal of this review is to discuss recent findings on the role of the inhibition or activation of GSK3β and its modulation of the inflammatory signaling in monocytes/macrophages and epithelial cells at the transcriptional level, mainly through the regulation of nuclear factor-kappaB (NF-κB activity. Also included is a brief overview on the importance of GSK3 in non-inflammatory processes during bacterial infection.

  15. Role of glycogen synthase kinase-3 beta in the inflammatory response caused by bacterial pathogens.

    Science.gov (United States)

    Cortés-Vieyra, Ricarda; Bravo-Patiño, Alejandro; Valdez-Alarcón, Juan J; Juárez, Marcos Cajero; Finlay, B Brett; Baizabal-Aguirre, Víctor M

    2012-06-12

    Glycogen synthase kinase 3β (GSK3β) plays a fundamental role during the inflammatory response induced by bacteria. Depending on the pathogen and its virulence factors, the type of cell and probably the context in which the interaction between host cells and bacteria takes place, GSK3β may promote or inhibit inflammation. The goal of this review is to discuss recent findings on the role of the inhibition or activation of GSK3β and its modulation of the inflammatory signaling in monocytes/macrophages and epithelial cells at the transcriptional level, mainly through the regulation of nuclear factor-kappaB (NF-κB) activity. Also included is a brief overview on the importance of GSK3 in non-inflammatory processes during bacterial infection.

  16. Thymidine secretion by hybridoma and myeloma cells

    International Nuclear Information System (INIS)

    Spilsberg, Bjorn; Rise, Frode; Petersen, Dirk; Nissen-Meyer, Jon

    2006-01-01

    Secretion of thymidine appeared to be a common property of hybridoma and myeloma cells, but not of other cell types, which were tested. Of three hybridoma cell lines tested, all secreted thymidine in amounts resulting in the accumulation of thymidine to concentrations of 10-20 μM in the culture medium. Also three of five myeloma cell lines that were analyzed secrete thymidine, but none of the other cell types that were studied. Thymidine was purified to homogeneity (4 mg purified from 3 l of culture medium) and identified as such by nuclear magnetic resonance spectroscopy. The cells that secreted thymidine showed high resistance to the growth inhibitory effect of thymidine

  17. Alkyl-fluorinated thymidine derivatives for imaging cell proliferation

    International Nuclear Information System (INIS)

    Toyohara, Jun; Hayashi, Akio; Gogami, Akie; Hamada, Masahiro; Hamashima, Yoshio; Katoh, Takahiro; Node, Manabu; Fujibayashi, Yasuhisa

    2006-01-01

    Derivatives of 2'-deoxyuridine that contain fluoroalkyl groups at the C5 position and derivatives of thymidine that contain fluoroalkyl groups at the N3 position were synthesized and examined in three in vitro assays designed to evaluate their potential as radiopharmaceuticals for imaging cellular proliferation. Three of the former nucleosides and five of the latter were synthesized. The three assays were as follows: (a) phosphoryl transfer assay, which showed that all three of the former nucleosides and four of the latter ones were phosphorylated by recombinant human thymidine kinase 1 (TK1) and that N 3 -(2-fluoroethyl)-thymidine (NFT202) was the most potent substrate of the eight nucleosides studied; (b) transport assay, which indicated that all eight nucleosides had good affinity for an 6-[(4-nitrobenzyl)thio]-9-β-D-ribofuranosylpurine-sensitive mouse erythrocyte nucleoside transporter, with inhibition constants in the range of 0.02-0.55 mM; and (c) degradation assay, which showed that all but one of the former nucleosides and none of the latter were degraded by recombinant Escherichia coli thymidine phosphorylase (an enzyme that catalyzes the glycosidic bond of thymidine and 2'-deoxyuridine derivatives). From these in vitro screening assays, we selected NFT202 as a candidate for subsequent in vivo evaluation because this compound met the three minimum requirements of the in vitro screening assays and had the most potent phosphorylation activity as a substrate for recombinant human TK1

  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)

    Yin Xiaohua; Lan Xiaoli; Wang Ruihua; Liu Ying; Zhang Yongxue

    2009-01-01

    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 131 I 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 131 I 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 131 I-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. Inhibition of phosphorylation and incorporation of thymidine in Duckweed (Lemna minor L. ) by sulfur dioxide and sulfite

    Energy Technology Data Exchange (ETDEWEB)

    Braendle, R; Stoeckli, B; Erismann, K H

    1975-05-15

    As there appears to be no thymidine kinase in duckweed (Lemna minor L.), thymidine seems to be phosphorylated by a nucleoside phosphotransferase. Phosphorylation and incorporation are inhibited by sulfur compounds such as sulfur dioxide and sulfite. The data are discussed in relation to the physiological effect of the air pollutant (SO2) on plant life. 12 references, 2 tables.

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

    Energy Technology Data Exchange (ETDEWEB)

    Jang, Su Jin; Kang, Joo Hyun; Lee, Yong Jin; Lee, Tae Sup; Kim, Kwang Il; Lee, Kyo Chul; An, Gwang II; Cheon, Gi Jeong; Lim, Sang Moo [KIRAMS, Seoul (Korea, Republic of); Lim, Sang Yong [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2011-10-15

    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 [{sup 18}F]FLT and [{sup 125}I]IVDU were measured and localized infections model in BALB/c mice was imaged with [{sup 18}F]FLT or [{sup 125}I]FIAU

  1. A bacterial E3 ubiquitin ligase targets a host protein kinase to disrupt plant immunity.

    Science.gov (United States)

    Rosebrock, Tracy R; Zeng, Lirong; Brady, Jennifer J; Abramovitch, Robert B; Xiao, Fangming; Martin, Gregory B

    2007-07-19

    Many bacterial pathogens of plants and animals use a type III secretion system to deliver diverse virulence-associated 'effector' proteins into the host cell. The mechanisms by which these effectors act are mostly unknown; however, they often promote disease by suppressing host immunity. One type III effector, AvrPtoB, expressed by the plant pathogen Pseudomonas syringae pv. tomato, has a carboxy-terminal domain that is an E3 ubiquitin ligase. Deletion of this domain allows an amino-terminal region of AvrPtoB (AvrPtoB(1-387)) to be detected by certain tomato varieties leading to immunity-associated programmed cell death. Here we show that a host kinase, Fen, physically interacts with AvrPtoB(1-387 )and is responsible for activating the plant immune response. The AvrPtoB E3 ligase specifically ubiquitinates Fen and promotes its degradation in a proteasome-dependent manner. This degradation leads to disease susceptibility in Fen-expressing tomato lines. Various wild species of tomato were found to exhibit immunity in response to AvrPtoB(1-387 )and not to full-length AvrPtoB. Thus, by acquiring an E3 ligase domain, AvrPtoB has thwarted a highly conserved host resistance mechanism.

  2. An open library of human kinase domain constructs for automated bacterial expression

    OpenAIRE

    Rodríguez-Laureano, Lucelenie; Işık, Mehtap; Chodera, John; Seeliger, Markus; Jeans, Chris; Gradia, Scott; Hanson, Sonya; Parton, Daniel; Albanese, Steven; Levinson, Nicholas; Behr, Julie

    2017-01-01

    Kinases play a critical role in many cellular signaling pathways and are dysregulated in a number of diseases, such as cancer, diabetes, and neurodegeneration. Since the FDA approval of imatinib in 2001, therapeutics targeting kinases now account for roughly 50% of current cancer drug discovery efforts. The ability to explore human kinase biochemistry, biophysics, and structural biology in the laboratory is essential to making rapid progress in understanding kinase regulation, designing selec...

  3. 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...

  4. 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

    To investigate the bactericidal activity of antiviral and anticancer nucleoside analogues against a variety of pathogenic bacteria and characterize the activating enzymes, deoxyribonucleoside kinases (dNKs). Several FDA-approved nucleoside analogue drugs were screened for their potential bacteric......-specific manner. Therefore, nucleoside analogues have a potential to be employed as antibiotics in the fight against emerging multiresistant bacteria....

  5. Calculation of cell production from [3H]Thymidine incorporation with freshwater bacteria

    International Nuclear Information System (INIS)

    Smits, J.D.; Riemann, B.

    1988-01-01

    The conversion factor for the calculation of bacterial production from rates of [ 3 H]thymidine incorporation was examined with diluted batch cultures of freshwater bacteria. Natural bacterial assemblages were grown in aged, normal, and enriched media at 10 to 20 degree C. The generation time during 101 growth cycles covered a range from 4 to >200 h. The average conversion factor was 2.15 x 10 18 cells mol -1 of thymidine incorporated into the trichloroacetic acid (TCA) precipitate, when the generation time exceeded 20 h. At generation times of 18 cells mol -1 of thymidine incorporated into TCA precipitate. The amount of radioactivity in purified DNA increased with decreasing generation time and increasing conversion factor (calculated from the TCA precipitate), corresponding to a decrease in the percentage in protein. The conversion factors calculated from purified DNA or from the TCA precipitate gave the same variability. Conversion factors did not change significantly with the medium, but were significantly higher at 20 degree C that at 15 and 10 degree C. Results suggests that incorporation of [ 3 H]thymidine into DNA is probably limited by uptake during period with generation times of 18 cells mol -1 of thymidine incorporated is used

  6. 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)

    Lindberg, C.

    1984-12-01

    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 NaHCO 3 . 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.) [de

  7. Radiosensitization of thymidine in deaerated aqueous solution

    International Nuclear Information System (INIS)

    Berger, Maurice.

    1982-09-01

    This work investigates the mode of action of various radiosensitizing agents on the radio-induced degradation of thymidine in deaerated aqueous solution. A special effort was devoted to the separation of addition products formed by one of these substances (a stable nitroxide radical: TAN) with the radio-induced neutral radicals of thymidine. The complex mixture of different diastereoisomers resulting from the covalent addition of the TAN molecule on the thymidine carbons C (5) or C (6) was resolved by HPLC. The structural determination of these adducts (absolute configuration) was achieved by various spectroscopic techniques and specific chemical syntheses. A conformational study has been undertaken [fr

  8. 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.

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

    International Nuclear Information System (INIS)

    Franzolin, Elisa; Miazzi, Cristina; Frangini, Miriam; Palumbo, Elisa; Rampazzo, Chiara; Bianchi, Vera

    2012-01-01

    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 [ 3 H]-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.

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

    DEFF Research Database (Denmark)

    Mutahir, Zeeshan; Clausen, Anders R.; Andersson, Karl-Magnus

    2013-01-01

    in higher vertebrates and was especially pronounced in mammalian and bird TK1s. We suggest that the dimer form is the original form and that the tetramer originated in the animal lineage after the split of Dictyostelium and the lineages leading to invertebrates and vertebrates. The efficient switching...

  11. Determination of thymidine in serum used for cell culture media

    International Nuclear Information System (INIS)

    Schaer, J.C.; Maurer, U.; Schindler, R.

    1978-01-01

    Thymidine concentrations in serum used for cell culture media were determined with an assay based on isotope dilution. In this assay, incorporation of (3H)-thymidine into DNA of cultured cells was measured in the presence of 5 and 20% serum as a function of the concentration of unlabeled thymidine added to the medium. Thymidine concentrations were measured using horse serum as well as fetal calf serum in the culture media. Dialysis of serum resulted in a reduction of thymidine levels by factors of at least 10

  12. 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

  13. Molecular moment similarity between several nucleoside analogs of thymidine and thymidine. sil@watson.ibm.com.

    Science.gov (United States)

    Silverman, B D; Pitman, M C; Platt, D E

    1999-06-01

    Molecular moment descriptors of the shape and charge distributions of twenty five nucleoside structures have been examined. The structures include thymidine as well as the difluorotoluene nucleoside analog which has been found to pair efficiently with adenine by polymerase catalysis. The remaining twenty three structures have been chosen to be as structurally similar to thymidine and to the difluorotoluene nucleoside analog as possible. The moment descriptors which include a description of the relationship of molecular charge to shape show the difluorotoluene nucleoside to be one of the most proximate molecules to thymidine in the space of the molecular moments. The calculations, therefore, suggest that polymerase specificity might be not only a consequence of molecular steric features alone but also of the molecular electrostatic environment and its registration with molecular shape.

  14. Photoreaction of 8-methoxypsoralen with thymidine

    International Nuclear Information System (INIS)

    Shim, S.C.; Kim, Y.Z.

    1983-01-01

    The photoreaction of 8-methoxypsoralen (8-MOP) with thymidine in solid film state yielded two 4',5'-monoadducts (a pair of diastereomers) and three 3,4-monoadducts. The stereochemistry of two 4',5'monoadducts was found to be cis-syn and trans-syn and one 3,4-monoadduct was cis-anti. In addition to these monoadducts, 3,4-, 4',5-biadducts were also formed during the reaction, but the isolation of each isomer of these adducts was not successful; however, the formation of these biadducts was confirmed by UV, IR, TLC and photosplitting experiments. (author)

  15. A procedure for the preparation of radioactive thymidine labelled with 14C

    International Nuclear Information System (INIS)

    Nejedly, Z.; Skodova, H.; Culik, K.; Filip, J.; Kolina, J.; Skoda, J.

    1990-01-01

    14 C-Labelled thymidine can be prepared by conversion of labelled or unlabelled thymine. The preparation is carried out in the presence of labelled or unlabelled 2-deoxycytidine, of a surfactant and a of reaction stimulator in a buffer at a temperature of 3 to 38 degC, under the catalytic effect of biocatalysts prepared from Escherichia coli B bacterial cells which are immobilized by embedding into an inert carrier. Sodium dodecyl sulfate can serve as the surfactant, D-glucose as the reaction-stimulating substrate, and sodium alginate as the inert cell carrier. In the procedure suggested, catalytic properties of enzymes are utilized without the need to isolate the enzymes from the bacterial cells beforehand or to purify them. The bacterial cells can be applied repeatedly in several production batches and stored in physiological solution at 5 degC. (M.D.)

  16. 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.

  17. Synthesis and evaluation of a {sup 99m}Tc-MAMA-propyl-thymidine complex as a potential probe for in vivo visualization of tumor cell proliferation with SPECT

    Energy Technology Data Exchange (ETDEWEB)

    Celen, Sofie [Laboratory for Radiopharmacy, K.U. Leuven, B-3000 Leuven (Belgium); Groot, Tjibbe de [Radiopharmacy, U.Z. Gasthuisberg K.U. Leuven, B-3000 Leuven (Belgium); Balzarini, Jan [Rega Institute for Medical Research, K.U. Leuven, B-3000 Leuven (Belgium); Vunckx, Kathleen [Nuclear Medicine, U.Z. Gasthuisberg K.U. Leuven, B-3000 Leuven (Belgium); Terwinghe, Christelle [Radiopharmacy, U.Z. Gasthuisberg K.U. Leuven, B-3000 Leuven (Belgium); Vermaelen, Peter [Nuclear Medicine, U.Z. Gasthuisberg K.U. Leuven, B-3000 Leuven (Belgium); Van Berckelaer, Lizette [Rega Institute for Medical Research, K.U. Leuven, B-3000 Leuven (Belgium); Vanbilloen, Hubert [Radiopharmacy, U.Z. Gasthuisberg K.U. Leuven, B-3000 Leuven (Belgium); Nuyts, Johan [Nuclear Medicine, U.Z. Gasthuisberg K.U. Leuven, B-3000 Leuven (Belgium); Mortelmans, Luc [Nuclear Medicine, U.Z. Gasthuisberg K.U. Leuven, B-3000 Leuven (Belgium); Verbruggen, Alfons [Laboratory for Radiopharmacy, K.U. Leuven, B-3000 Leuven (Belgium); Bormans, Guy [Laboratory for Radiopharmacy, K.U. Leuven, B-3000 Leuven (Belgium)]. E-mail: guy.bormans@pharm.kuleuven.be

    2007-04-15

    Introduction: Cytosolic thymidine kinase (TK1) catalyzes phosphorylation of thymidine to its monophosphate. TK1 activity is closely related with DNA synthesis, and thymidine analogs derivatized with bulky carboranylalkyl groups at the N-3 position were reported to be good substrates for TK1. Accordingly, we have synthesized {sup 99m}Tc-MAMA-propyl-thymidine and evaluated it as a potential tumor tracer. Methods: The bis(S-trityl)-protected MAMA-propyl-thymidine precursor (3-N-[S-trityl-2-mercaptoethyl]-N-[N'-(S-trityl-2-mercaptoethyl) amidoacetyl] -aminopropyl-thymidine) was prepared in three steps, and its structure was confirmed with {sup 1}H NMR and mass spectrometry. Deprotection of the thiols and labeling with {sup 99m}Tc were done in a two-step, one-pot procedure, yielding {sup 99m}Tc-MAMA-propyl-thymidine, which was analyzed with high-performance liquid chromatography, radio-LC-MS analysis (ESI+) and electrophoresis, and its log P was determined. The biodistribution in normal mice was evaluated, and its biodistribution in a radiation-induced fibrosarcoma (RIF) tumor mouse was compared with that of 3'-deoxy-3'-[{sup 18}F] fluorothymidine [{sup 18}F]FLT. Results: {sup 99m}Tc-MAMA-propyl-thymidine was obtained with a radiochemical yield of 70%. Electrophoresis indicated that the complex is uncharged, and its log P was 1.0. The molecular ion mass of the Tc complex was 589 Da, which is compatible with the hypothesized N{sub 2}S{sub 2}-oxotechnetium structure. Tissue distribution showed fast clearance from plasma primarily by the hepatobiliary pathway. Whole-body planar imaging after injection of {sup 99m}Tc-MAMA-propyl-thymidine in an RIF tumor-bearing mouse showed high uptake in the liver and the intestines. No uptake was observed in the tumor, in contrast to the clear uptake observed for [{sup 18}F] FLT visualized with {mu}PET. Conclusions: Although it has been reported that TK1 accepts large substituents at the N-3 position of the thymine ring

  18. Whole body autoradiography with mice using 14C-thymidine

    International Nuclear Information System (INIS)

    Hruby, R. et al

    1982-06-01

    Whole body autoradiography was performed using common histology equipment. Results were useful with some restrictions. 14C-thymidine and/or itsmetabolites were found in those tissues with high rate of mitosis. (Author)

  19. Reaction of Thymidine with Hypobromous Acid in Phosphate Buffer.

    Science.gov (United States)

    Suzuki, Toshinori; Kitabatake, Akihiko; Koide, Yuki

    2016-01-01

    When thymidine was treated with hypobromous acid (HOBr) in 100 mM phosphate buffer at pH 7.2, two major product peaks appeared in the HPLC chromatogram. The products in each peak were identified by NMR and MS as two isomers of 5-hydroxy-5,6-dihydrothymidine-6-phosphate (a novel compound) and two isomers of 5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol) with comparable yields. 5-Hydroxy-5,6-dihydrothymidine-6-phosphate was relatively stable, and decomposed with a half-life of 32 h at pH 7.2 and 37°C generating thymidine glycol. The results suggest that 5-hydroxy-5,6-dihydrothymidine-6-phosphate in addition to thymidine glycol may have importance for mutagenesis by the reaction of HOBr with thymine residues in nucleotides and DNA.

  20. Functional role of pyruvate kinase from Lactobacillus bulgaricus in acid tolerance and identification of its transcription factor by bacterial one-hybrid.

    Science.gov (United States)

    Zhai, Zhengyuan; An, Haoran; Wang, Guohong; Luo, Yunbo; Hao, Yanling

    2015-11-19

    Lactobacillus delbrueckii subsp. bulgaricus develops acid tolerance response when subjected to acid stress conditions, such as the induction of enzymes associated with carbohydrate metabolism. In this study, pyk gene encoding pyruvate kinase was over-expressed in heterologous host Lactococcus lactis NZ9000, and SDS-PAGE analysis revealed the successful expression of this gene in NZ9000. The survival rate of Pyk-overproducing strain was 45-fold higher than the control under acid stress condition (pH 4.0). In order to determine the transcription factor (TF) which regulates the expression of pyk by bacterial one-hybrid, we constructed a TF library including 65 TFs of L. bulgaricus. Western blotting indicated that TFs in this library could be successfully expressed in host strains. Subsequently, the promoter of pfk-pyk operon in L. bulgaricus was identified by 5'-RACE PCR. The bait plasmid pH3U3-p01 carrying the deletion fragment of pfk-pyk promoter captured catabolite control protein A (CcpA) which could regulate the expression of pyk by binding to a putative catabolite-responsive element (5'-TGTAAGCCCTAACA-3') upstream the -35 region. Real-time qPCR analysis revealed the transcription of pyk was positively regulated by CcpA. This is the first report about identifying the TF of pyk in L. bulgaricus, which will provide new insight into the regulatory network.

  1. Co-expression of bacterial aspartate kinase and adenylylsulfate reductase genes substantially increases sulfur amino acid levels in transgenic alfalfa (Medicago sativa L.).

    Science.gov (United States)

    Tong, Zongyong; Xie, Can; Ma, Lei; Liu, Liping; Jin, Yongsheng; Dong, Jiangli; Wang, Tao

    2014-01-01

    Alfalfa (Medicago sativa L.) is one of the most important forage crops used to feed livestock, such as cattle and sheep, and the sulfur amino acid (SAA) content of alfalfa is used as an index of its nutritional value. Aspartate kinase (AK) catalyzes the phosphorylation of aspartate to Asp-phosphate, the first step in the aspartate family biosynthesis pathway, and adenylylsulfate reductase (APR) catalyzes the conversion of activated sulfate to sulfite, providing reduced sulfur for the synthesis of cysteine, methionine, and other essential metabolites and secondary compounds. To reduce the feedback inhibition of other metabolites, we cloned bacterial AK and APR genes, modified AK, and introduced them into alfalfa. Compared to the wild-type alfalfa, the content of cysteine increased by 30% and that of methionine increased substantially by 60%. In addition, a substantial increase in the abundance of essential amino acids (EAAs), such as aspartate and lysine, was found. The results also indicated a close connection between amino acid metabolism and the tricarboxylic acid (TCA) cycle. The total amino acid content and the forage biomass tested showed no significant changes in the transgenic plants. This approach provides a new method for increasing SAAs and allows for the development of new genetically modified crops with enhanced nutritional value.

  2. Thymidine phosphorylase exerts complex effects on bone resorption and formation in myeloma.

    Science.gov (United States)

    Liu, Huan; Liu, Zhiqiang; Du, Juan; He, Jin; Lin, Pei; Amini, Behrang; Starbuck, Michael W; Novane, Nora; Shah, Jatin J; Davis, Richard E; Hou, Jian; Gagel, Robert F; Yang, Jing

    2016-08-24

    Myelomatous bone disease is characterized by the development of lytic bone lesions and a concomitant reduction in bone formation, leading to chronic bone pain and fractures. To understand the underlying mechanism, we investigated the contribution of myeloma-expressed thymidine phosphorylase (TP) to bone lesions. In osteoblast progenitors, TP up-regulated the methylation of RUNX2 and osterix, leading to decreased bone formation. In osteoclast progenitors, TP up-regulated the methylation of IRF8 and thereby enhanced expression of NFATc1 (nuclear factor of activated T cells, cytoplasmic 1 protein), leading to increased bone resorption. TP reversibly catalyzes thymidine into thymine and 2-deoxy-d-ribose (2DDR). Myeloma-secreted 2DDR bound to integrin αVβ3/α5β1 in the progenitors, activated PI3K (phosphoinositide 3-kinase)/Akt signaling, and increased DNMT3A (DNA methyltransferase 3A) expression, resulting in hypermethylation of RUNX2, osterix, and IRF8 This study elucidates an important mechanism for myeloma-induced bone lesions, suggesting that targeting TP may be a viable approach to healing resorbed bone in patients. Because TP overexpression is common in bone-metastatic tumors, our findings could have additional mechanistic implications. Copyright © 2016, American Association for the Advancement of Science.

  3. Regulatory Phosphorylation of Bacterial-Type PEP Carboxylase by the Ca2+-Dependent Protein Kinase RcCDPK1 in Developing Castor Oil Seeds.

    Science.gov (United States)

    Ying, Sheng; Hill, Allyson T; Pyc, Michal; Anderson, Erin M; Snedden, Wayne A; Mullen, Robert T; She, Yi-Min; Plaxton, William C

    2017-06-01

    Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled cytosolic enzyme situated at a crucial branch point of central plant metabolism. In developing castor oil seeds ( Ricinus communis ) a novel, allosterically desensitized 910-kD Class-2 PEPC hetero-octameric complex, arises from a tight interaction between 107-kD plant-type PEPC and 118-kD bacterial-type (BTPC) subunits. The native Ca 2+ -dependent protein kinase (CDPK) responsible for in vivo inhibitory phosphorylation of Class-2 PEPC's BTPC subunit's at Ser-451 was highly purified from COS and identified as RcCDPK1 (XP_002526815) by mass spectrometry. Heterologously expressed RcCDPK1 catalyzed Ca 2+ -dependent, inhibitory phosphorylation of BTPC at Ser-451 while exhibiting: ( i ) a pair of Ca 2+ binding sites with identical dissociation constants of 5.03 μM, ( ii ) a Ca 2+ -dependent electrophoretic mobility shift, and ( iii ) a marked Ca 2+ -independent hydrophobicity. Pull-down experiments established the Ca 2+ -dependent interaction of N-terminal GST-tagged RcCDPK1 with BTPC. RcCDPK1-Cherry localized to the cytosol and nucleus of tobacco bright yellow-2 cells, but colocalized with mitochondrial-surface associated BTPC-enhanced yellow fluorescent protein when both fusion proteins were coexpressed. Deletion analyses demonstrated that although its N-terminal variable domain plays an essential role in optimizing Ca 2+ -dependent RcCDPK1 autophosphorylation and BTPC transphosphorylation activity, it is not critical for in vitro or in vivo target recognition. Arabidopsis ( Arabidopsis thaliana ) CPK4 and soybean ( Glycine max ) CDPKβ are RcCDPK1 orthologs that effectively phosphorylated castor BTPC at Ser-451. Overall, the results highlight a potential link between cytosolic Ca 2+ signaling and the posttranslational control of respiratory CO 2 refixation and anaplerotic photosynthate partitioning in support of storage oil and protein biosynthesis in developing COS. © 2017 American Society of Plant

  4. Regulatory Phosphorylation of Bacterial-Type PEP Carboxylase by the Ca2+-Dependent Protein Kinase RcCDPK1 in Developing Castor Oil Seeds1[OPEN

    Science.gov (United States)

    Hill, Allyson T.; Anderson, Erin M.; She, Yi-Min

    2017-01-01

    Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled cytosolic enzyme situated at a crucial branch point of central plant metabolism. In developing castor oil seeds (Ricinus communis) a novel, allosterically desensitized 910-kD Class-2 PEPC hetero-octameric complex, arises from a tight interaction between 107-kD plant-type PEPC and 118-kD bacterial-type (BTPC) subunits. The native Ca2+-dependent protein kinase (CDPK) responsible for in vivo inhibitory phosphorylation of Class-2 PEPC’s BTPC subunit’s at Ser-451 was highly purified from COS and identified as RcCDPK1 (XP_002526815) by mass spectrometry. Heterologously expressed RcCDPK1 catalyzed Ca2+-dependent, inhibitory phosphorylation of BTPC at Ser-451 while exhibiting: (i) a pair of Ca2+ binding sites with identical dissociation constants of 5.03 μM, (ii) a Ca2+-dependent electrophoretic mobility shift, and (iii) a marked Ca2+-independent hydrophobicity. Pull-down experiments established the Ca2+-dependent interaction of N-terminal GST-tagged RcCDPK1 with BTPC. RcCDPK1-Cherry localized to the cytosol and nucleus of tobacco bright yellow-2 cells, but colocalized with mitochondrial-surface associated BTPC-enhanced yellow fluorescent protein when both fusion proteins were coexpressed. Deletion analyses demonstrated that although its N-terminal variable domain plays an essential role in optimizing Ca2+-dependent RcCDPK1 autophosphorylation and BTPC transphosphorylation activity, it is not critical for in vitro or in vivo target recognition. Arabidopsis (Arabidopsis thaliana) CPK4 and soybean (Glycine max) CDPKβ are RcCDPK1 orthologs that effectively phosphorylated castor BTPC at Ser-451. Overall, the results highlight a potential link between cytosolic Ca2+ signaling and the posttranslational control of respiratory CO2 refixation and anaplerotic photosynthate partitioning in support of storage oil and protein biosynthesis in developing COS. PMID:28363991

  5. Tritiated thymidine incorporation and the growth of heterotrophic bacteria in warm core rings

    International Nuclear Information System (INIS)

    Ducklow, H.W.; Hill, S.M.

    1985-01-01

    The time-course of the incorporation rate of [methyl- 3 H]thymidine ([ 3 H]TdR) was established during 6-12 h incubations of natural bacterial populations sampled from the surface layers of warm core Gulf Stream rings. Parallel estimates of changes in cell numbers were made in order to examine the relationships between TdR incorporation and population growth for oceanic bacterial populations. Their results indicate that a conversion factor of 4 x 10 18 cells produced per mole of [ 3 H]TdR incorporated yielded estimates of bacterial production which were within a factor of 2 or 3 of production estimates derived from changes in cell numbers in seawater cultures. The authors observed a significant, direct relationship between the initial rates of TdR incorporation per cell and specific growth rates and conclude that initial short term (15-45 min) assays of TdR incorporation are a valuable tool for studying bacterial production in oceanic waters. In most incubations, the rate of TdR incorporation increased more rapidly than did cell numbers. Very large conversion factor values were derived from these data. The discrepancy between growth determined from TdR incorporation rates and total bacterial numbers in seawater cultures has not been observed in previous studies of coastal, estuarine, or lacustrine bacteria, but was a consistent feature of our studies on oceanic populations

  6. Tumor imaging with PET and C-11 thymidine

    International Nuclear Information System (INIS)

    Conti, P.S.; Hilton, J.; Magee, C.A.; Anderson, J.H.

    1989-01-01

    Accurate interpretation of kinetic positron-emission tomographic (PET) data obtained following administration of C-11 thymidine requires identification of radiolabeled metabolites. The authors goal is to quantitate rapidly formed metabolites of C-11 thymidine by using reproducible high-pressure liquid chromatography (HPLC). Following coinjection of methyl-C-11 and methyl-C-14 thymidine, dogs bearing implanted glioblastoma were imaged with PET. Plasma samples were collected, and dogs were sacrificed at 60 minutes. Tissues were prepared for quantitative autoradiography and analysis of radioactivity associated with DNA, RNA protein, and acid soluble extracts. Plasma and tissue extracts were analyzed by HPLC by using C-18 reverse phase and dilute buffer mobile phase systems for the separation of catabolites and phosphorylated nucleotides

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

    International Nuclear Information System (INIS)

    Meseda, Clement A.; Schmeisser, Falko; Pedersen, Robin; Woerner, Amy; Weir, Jerry P.

    2004-01-01

    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

  8. Vertical detachment energies of anionic thymidine: Microhydration effects.

    Science.gov (United States)

    Kim, Sunghwan; Schaefer, Henry F

    2010-10-14

    Density functional theory has been employed to investigate microhydration effects on the vertical detachment energy (VDE) of the thymidine anion by considering the various structures of its monohydrates. Structures were located using a random searching procedure. Among 14 distinct structures of the anionic thymidine monohydrate, the low-energy structures, in general, have the water molecule bound to the thymine base unit. The negative charge developed on the thymine moiety increases the strength of the intermolecular hydrogen bonding between the water and base units. The computed VDE values of the thymidine monohydrate anions are predicted to range from 0.67 to 1.60 eV and the lowest-energy structure has a VDE of 1.32 eV. The VDEs of the monohydrates of the thymidine anion, where the N(1)[Single Bond]H hydrogen of thymine has been replaced by a 2(')-deoxyribose ring, are greater by ∼0.30 eV, compared to those of the monohydrates of the thymine anion. The results of the present study are in excellent agreement with the accompanying experimental results of Bowen and co-workers [J. Chem. Phys. 133, 144304 (2010)].

  9. Transforming thymidine into a magnetic resonance imaging probe for monitoring gene expression.

    Science.gov (United States)

    Bar-Shir, Amnon; Liu, Guanshu; Liang, Yajie; Yadav, Nirbhay N; McMahon, Michael T; Walczak, Piotr; Nimmagadda, Sridhar; Pomper, Martin G; Tallman, Keri A; Greenberg, Marc M; van Zijl, Peter C M; Bulte, Jeff W M; Gilad, Assaf A

    2013-01-30

    Synthetic chemistry has revolutionized the understanding of many biological systems. Small compounds that act as agonists and antagonists of proteins, and occasionally as imaging probes, have contributed tremendously to the elucidation of many biological pathways. Nevertheless, the function of thousands of proteins is still elusive, and designing new imaging probes remains a challenge. Through screening and characterization, we identified a thymidine analogue as a probe for imaging the expression of herpes simplex virus type-1 thymidine kinase (HSV1-TK). To detect the probe, we used chemical exchange saturation transfer magnetic resonance imaging (CEST-MRI), in which a dynamic exchange process between an exchangeable proton and the surrounding water protons is used to amplify the desired contrast. Initially, five pyrimidine-based molecules were recognized as putative imaging agents, since their exchangeable imino protons resonate at 5-6 ppm from the water proton frequency and their detection is therefore less affected by endogenous CEST contrast or confounded by direct water saturation. Increasing the pK(a) value of the imino proton by reduction of its 5,6-double bond results in a significant reduction of the exchange rate (k(ex)) between this proton and the water protons. This reduced k(ex) of the dihydropyrimidine nucleosides fulfills the "slow to intermediate regime" condition for generating high CEST-MRI contrast. Consequently, we identified 5-methyl-5,6-dihydrothymidine as the optimal probe and demonstrated its feasibility for in vivo imaging of HSV1-TK. In light of these findings, this new approach can be generalized for designing specific probes for the in vivo imaging of a variety of proteins and enzymes.

  10. Thymidine analogue-sparing highly active antiretroviral therapy (HAART).

    Science.gov (United States)

    Nolan, David; Mallal, Simon

    2003-02-01

    The use of alternative nucleoside reverse transcriptase inhibitors (NRTIs) to the thymidine analogues stavudine (d4T) and zidovudine(ZDV) has been advocated as a means of limiting long-term NRTI-associated toxicity, particularly the development of lipoatrophy or fat wasting. This approach reflects an increasing knowledge of the distinct toxicity profiles of NRTI drugs. However, recent clinical trials have demonstrated that the use of thymidine analogue NRTIs and newer alternative backbone NRTIs, such as tenofovir (TNF) and abacavir (ABC), is associated with comparable short-term efficacy and tolerability. Given the importance of toxicity profile differences in determining clinical management, it is important to recognise that d4T and ZDV cary significantly different risks for long-term NRTI toxicity. Recognising that all NRTIs, including thymidine analogues, have individual toxicity profiles provides a more appropriate basis for selecting optimal antiretroviral therapy. The safety and efficacy of TNF and ABC are also reviewed here, although the available data provide only limited knowledge of the long-term effects of these drugs in terms of toxicity and antiviral durability.

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

    International Nuclear Information System (INIS)

    Shaw, A.A.; Wainschel, L.A.; Shetlar, M.D.

    1992-01-01

    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)

  12. Comparison of Heterotrophic Bacterial Production-Rates in Early Spring in the Turbid Estuaries of the Scheldt and the Elbe

    NARCIS (Netherlands)

    Goosen, N.K.; Van Rijswijk, P.; Brockmann, U.

    1995-01-01

    In spring bacterial production rates were estimated by tritiated thymidine incorporation in the turbid estuaries of the rivers Scheldt and Elbe. Bacterial production rates in the Scheldt were 5 times higher than in the Elbe. In the Scheldt bacterial production rates correlated better with the DOC

  13. Formation of volatile decomposition products by self-radiolysis of tritiated thymidine

    International Nuclear Information System (INIS)

    Shiba, Kazuhiro; Mori, Hirofumi

    1997-01-01

    In order to estimate the internal exposure dose in an experiment using tritiated thymidine, the rate of volatile 3 H-decomposition of several tritiated thymidine samples was measured. The decomposition rate of (methyl- 3 H)thymidine in water was over 80% in less than one year after initial analysis. (methyl- 3 H)thymidine was decomposed into volatile and non-volatile 3 H-decomposition products. The ratio of volatile 3 H-decomposition products increased with increasing the rate of the decomposition of (methyl- 3 H) thymidine. The volatile 3 H-decomposition products consisted of two components, of which the main component was tritiated water. Internal exposure dose caused by the inhalation of such volatile 3 H-decomposition products of (methyl- 3 H) thymidine was assumed to be several μSv. (author)

  14. Using miniature osmotic infusion pumps to maintain tritiated thymidine exposure to cells in culture

    International Nuclear Information System (INIS)

    Neely, J.E.; Hake, D.A.

    1982-01-01

    To provide a constant level of tracer doses of tritiated thymidine to cultured cells during continuous infusion, miniature osmotic infusion pumps were used to provide replacement thymidine. By determining the loss of isotope from the media during nonreplacement, the rate of constant infusion replacement to maintain thymidine levels was calculated. The replacement rates were similar for the three cell lines examined and allowed a standard osmotic pump infusion

  15. Determining the specific activity of thymidine phosphorylase in leukocytes of patients with MNGIE and the plasma thymidine level by RP-HPLC

    Directory of Open Access Journals (Sweden)

    Rezaei Sh

    2011-06-01

    Full Text Available "nBackground: Thymidine phosphorylase (TP catalyses the conversion of thymidine into thymine. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE is an autosomal recessive disease which is caused by mutations in the nuclear gene encoding TP, bringing about severe impairment of TP-enzyme specific activity and accumulation of thymidine in plasma. The clinical manifestations of MNGIE are recognizable and homogenous, but not in the early stages of the disease. In patients who are suspected of having MNGIE, determination of TP-specific activity in leukocytes and thymidine levels in plasma are diagnostic. The methods that are usually used for the measurement of TP activity and plasma thymidine are not rapid or accurate enough and lack sensitivity."n "nMethods: The specific activity of TP was measured by RP-HPLC in leukocytes of both the controls and the patients exhibiting clinical features suggestive of MNGIE. Moreover, plasma thymidine was assessed by the same method."n "nResults: The patients had detectable plasma thymidine (>3 µmol/L but it was undetectable in the healthy controls. The patients' TP-specific activity decreased to less than 5% relative to the controls (14±4 nmol/h/mg vs. 525±165 nmol/h/mg, P<0.05. A diagnostic algorithm for the definitive diagnosis of MNGIE is suggestible based on the results of this study which relies on the measurement of plasma thymidine, TP-specific activity in leukocytes, or both."n "nConclusion: In this study, we set up a sensitive and rapid assay for the evaluation of TP-specific activity by using RP-HPLC in Iran. In addition, we established reference values for TP-specific activity and plasma thymidine in the Iranian patients.

  16. Characterization of a monoclonal antibody to thymidine glycol monophosphate

    International Nuclear Information System (INIS)

    Chen, B.X.; Hubbard, K.; Ide, H.; Wallace, S.S.; Erlanger, B.F.

    1990-01-01

    A monoclonal antibody specific for thymine glycol (TG) in irradiated or OsO4-treated DNA was obtained by immunizing with thymidine glycol monophosphate (TMP-glycol) conjugated to bovine serum albumin by a carbodiimide procedure. Screening by dot-immunobinding and enzyme-linked immunosorbant assay (ELISA) procedures gave eight clones that bound OsO4- treated DNA. One of them, 2.6F.6B.6C, an IgG2a kappa, was characterized further. Hapten inhibition studies with OsO4-treated DNA showed that the antibody was specific for TMP-glycol. Among the various inhibitors tested, inhibition was in the order TMP-glycol greater than 5,6-dihydrothymidine phosphate greater than TMP greater than thymidine glycol greater than TG. Inhibition by 5,6-dihydrothymidine, thymidine, thymine, AMP, and CMP was negligible. In OsO4-treated DNA, as few as 0.5 TG per 10,000 bp were detectable by direct ELISA. Inhibition assays could detect as few as 1.5 TG per 10,000 bp. The antibody was equally reactive with native or denatured DNA containing TG. Among the X-irradiated homopolymers dC, dA, dG, and dT, only dT reacted with the antibody. Using an ELISA, the antibody could detect damage in irradiated DNA at the level of 20 Gy. Thus the antibody is of potential use in assays for DNA damage caused by X rays or other agents that damage DNA by free radical interactions

  17. Preparation of tritiated thymidine of high specific activity

    International Nuclear Information System (INIS)

    Ivan'kova, E.K.; Sidorov, G.V.; Myasoedov, N.F.

    1981-01-01

    Optimum conditions for the reaction are determined; and conditions for reaction component separation on resins of Dowex-1x8 and APA-8p (HCOO - , elution with ammonium formate) are optimized. It is established that the transition from thymine preparations with the specific activity of 0.15 and 1.5 TBq/mmol to the preparation with the specific activity of 3.25 TBq/mmol brings about the reduction in the desoxyribosylation reaction rate and the decrease in the thymidine yield from 85-90 to 65% [ru

  18. Thymidine analogues to assess microperfusion in human tumors

    International Nuclear Information System (INIS)

    Janssen, Hilde L.; Ljungkvist, Anna S.; Rijken, Paul F.; Sprong, Debbie; Bussink, Jan; Kogel, Albert J. van der; Haustermans, Karin M.; Begg, Adrian C.

    2005-01-01

    Purpose: To validate the use of the thymidine analogues as local perfusion markers in human tumors (no labeling indicates no perfusion) by comparison with the well-characterized perfusion marker Hoechst 33342. Methods and Materials: Human tumor xenografts from gliomas and head-and-neck cancers were injected with iododeoxyuridine (IdUrd) or bromodeoxyuridine (BrdUrd) and the fluorescent dye Hoechst 33342. In frozen sections, each blood vessel was scored for the presence of IdUrd/BrdUrd labeling and Hoechst in surrounding cells. The percentage of analogue-negative vessels was compared with the fraction of Hoechst-negative vessels. Collocalization of the two markers was also scored. Results: We found considerable intertumor variation in the fraction of perfused vessels, measured by analogue labeling, both in the human tumor xenografts and in a series of tumor biopsies from head-and-neck cancer patients. There was a significant correlation between the Hoechst-negative and IdUrd/BrdUrd-negative vessels in the xenografts (r 85, p = 0.0004), despite some mismatches on a per-vessel basis. Conclusions: Thymidine analogues can be successfully used to rank tumors according to their fraction of perfused vessels. Whether this fraction correlates with the extent of acute hypoxia needs further confirmation

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

    International Nuclear Information System (INIS)

    Xing Yan; Zhao Jinhua

    2006-01-01

    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)

  20. Clinical Value of Thymidine Kinase in Predicting Recurrence of Breast Cancer in Postsurgical Follow up Studies

    Czech Academy of Sciences Publication Activity Database

    Karlíček, V.; Topolčan, O.; Pecen, Ladislav; Eben, Kryštof; Valenta, J.; Suvová, B.

    1995-01-01

    Roč. 10, č. 2 (1995), s. 80 ISSN 0886-3849. [International Conference on Human Tumor Markers /12./. 11.06.1995-14.06.1995, New York] R&D Projects: GA AV ČR IAA230106 Keywords : medical informatics * oncology

  1. 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

    The prognosis for malignant gliomas remains poor, and new treatments are urgently needed. Targeted suicide gene therapy exploits the enzymatic conversion of a prodrug, such as a nucleoside analog, into a cytotoxic compound. Although this therapeutic strategy has been considered a promising regimen...... suicide gene therapy system in combination with stem cell mediated gene delivery promises new treatment of malignant gliomas....

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

    DEFF Research Database (Denmark)

    Kock Jensen, Helle

    by recombinant technics to examine the relation between the TKl gene and the TKl protein. In the second part of this investigation a direct expression system for human TKl in E.coli was developed to produce a source of high amounts of TKl, to be able to examine the structure of TKl. The resulting recombinant TKl...... cells and that this modification can not be performed in E.coli....... infections. In the first part of the present investigation a sensitive test for quantitating TKl mRNA (competitive PCR) is developed and the results show that PHA stimulated lymphocytes reveal the same pattern concerning expression of TKl mRNA and TKl enzyme activity as serum-stimulated cells. This pattern...

  3. Catabolism of exogenously supplied thymidine to thymine and dihydrothymine by platelets in human peripheral blood

    International Nuclear Information System (INIS)

    Pero, R.W.; Johnson, D.; Olsson, A.

    1984-01-01

    The interference of platelets with the estimation of unscheduled DNA synthesis in human peripheral mononuclear leukocytes following genotoxic exposure was studied. A 96% reduction in the unscheduled DNA synthesis value was achieved by incubating [ 3 H]thymidine with platelet-rich plasma for 5 hr at 37 degrees. Using radioactive thymine-containing compounds, together with quantitative analyses based on thin-layer and ion-exchange chromatographies, we have shown that thymidine was converted to thymine which, in turn, was converted to dihydrothymine in platelet-rich plasma. The enzymes responsible were separated from platelet lysates by gel filtration and were identified as thymidine phosphorylase and dihydrothymine dehydrogenase. The phosphorylase reversibly catalyzed the formation of thymine from thymidine and converted bromodeoxyuridine to bromouracil. The dehydrogenase reversibly catalyzed the interconversion of thymine and dihydrothymine in a reaction dependent on NADP(H), and it was inhibited by diazouracil and by thymine. Nearly all the thymidine-catabolizing activity found in whole blood samples supplied exogenously with thymidine was accounted for by the platelets. Since most genetic toxicological tests that use blood samples do not involve removing platelets from the blood cell cultures, then it is concluded that precautions should be taken in the future to determine the influence of platelets on these test systems. This is particularly true for methods dependent on thymidine pulses such as unscheduled DNA synthesis, or those dependent on bromodeoxyuridine, such as sister chromatid exchanges, since this nucleoside is also a substrate for thymidine phosphorylase

  4. H3-THYMIDINE DERIVATIVE POOLS IN RELATION TO MACRONUCLEAR DNA SYNTHESIS IN TETRAHYMENA PYRIFORMIS

    Science.gov (United States)

    Stone, G. E.; Miller, O. L.; Prescott, D. M.

    1965-01-01

    The formation of a soluble H3-thymidine derivative pool has been examined in Tetrahymena pyriformis as a function of macronuclear DNA synthesis during the cell life cycle. An autoradiographic technique which allows the detection of water-soluble materials within a cell has shown that these cells do not take up and retain exogenous H3-thymidine during G1 or G2. Uptake of H3-thymidine is restricted to the S period of the cell cycle. Additional autoradiographic experiments show, however, that a soluble pool of H3-thymidine derivatives persists from the end of one DNA synthesis period to the beginning of the next synthesis period in the subsequent cell cycle. Since this persisting pool cannot be labeled with H3-thymidine, the pool does not turn over during non-S periods. PMID:19866660

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

    International Nuclear Information System (INIS)

    Murray, R.E.; Cooksey, K.E.; Priscu, J.C.

    1986-01-01

    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 10 4 cells cm -2 (diatoms) and 5 x 10 6 cells cm -2 (bacteria). The algal-bacterial consortia consistently exhibited higher rates of [ 3 H]thymidine incorporation than did biofilms composed solely of bacteria. The rates of [ 3 H]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 [ 3 H]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

  6. 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.

  7. Evaluation of radiation dose resulting from the ingestion of [3H]-and [14C]thymidine in the rat

    International Nuclear Information System (INIS)

    Takeda, H.; Iwakura, T.

    1987-01-01

    Average doses to rat tissues from the ingestion of 2-[ 14 C]thymidine were compared with those from methyl-[ 3 H]thymidine or 6-[ 3 H]thymidine. [ 14 C]thymidine gave the highest dose to spleen and small intestine. Doses to other tissues from [ 14 ]thymidine were almost the same or lower compared with those from [ 3 H]thymidine, irrespective of the 9 times higher β-ray energy of 14 C than that of 3 H. In the case of [ 14 C]thymidine, most of the dose was given by radioactivity incorporated into the organic tissue constituents (non-volatile radioactivity). In the case of [ 3 H]thymidine, the dose contributions by non-volatile radioactivity were very small and major contributions were rather from volatile radioactivity ( 3 HHO), formed by degradation of [ 3 H]thymidine. No significant difference in their total doses was found between the two [ 3 H]precursors, but the dose from non-volatile radioactivity alone was 2-3 times higher with methyl-[ 3 H]thymidine than with 6-[ 3 H]thymidine. Estimates of dose to cell nuclei in various tissues after ingestion of [ 3 H]thymidine were also made in order to predict more precisely possible radiation hazards. (author)

  8. Effect of pituitary gonadotrophins on tritiated thymidine uptake by rat ovary

    International Nuclear Information System (INIS)

    Nakano, Ryosuke; Mizuno, Tadayoshi; Katayama, Kazuaki; Hayashi, Kaname; Tojo, Shimpei

    1975-01-01

    The effect of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on the follicular growth in the ovary of the hypophysectomized rat was investigated using autoradiography. The numbers of DNA-synthesizing nuclei in the granulosa cell were measured by autoradiography after flashlabelling with tritiated ( 3 H) thymidine. The frequency of 3 H-thymidine labelled nuclei in the granulosa cell enhanced in the presence of FSH. In contrast, LH had no significant effect on thymidine uptake. The result suggests that FSH stimulates follicle cell division, whereas LH does not. (orig.) [de

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

    International Nuclear Information System (INIS)

    Soederberg, P.G.; Lindstroem, B.

    1990-01-01

    DNA synthesis in the lens has previously been localized by autoradiography following incorporation of 3 H-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 3 H-thymidine. The 3 H-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)

  10. Engineering of kinase-based protein interacting devices: active expression of tyrosine kinase domains

    KAUST Repository

    Diaz Galicia, Miriam Escarlet

    2018-05-01

    Protein-protein interactions modulate cellular processes in health and disease. However, tracing weak or rare associations or dissociations of proteins is not a trivial task. Kinases are often regulated through interaction partners and, at the same time, themselves regulate cellular interaction networks. The use of kinase domains for creating a synthetic sensor device that reads low concentration protein-protein interactions and amplifies them to a higher concentration interaction which is then translated into a FRET (Fluorescence Resonance Energy Transfer) signal is here proposed. To this end, DNA constructs for interaction amplification (split kinases), positive controls (intact kinase domains), scaffolding proteins and phosphopeptide - SH2-domain modules for the reading of kinase activity were assembled and expression protocols for fusion proteins containing Lyn, Src, and Fak kinase domains in bacterial and in cell-free systems were optimized. Also, two non-overlapping methods for measuring the kinase activity of these proteins were stablished and, finally, a protein-fragment complementation assay with the split-kinase constructs was tested. In conclusion, it has been demonstrated that features such as codon optimization, vector design and expression conditions have an impact on the expression yield and activity of kinase-based proteins. Furthermore, it has been found that the defined PURE cell-free system is insufficient for the active expression of catalytic kinase domains. In contrast, the bacterial co-expression with phosphatases produced active kinase fusion proteins for two out of the three tested Tyrosine kinase domains.

  11. LHRH inhibits [3H]thymidine incorporation by pituitary cells cultured IN VITRO

    International Nuclear Information System (INIS)

    Stepien, H.

    1981-01-01

    The effects of two synthetic neuropeptides, LHRH and neurotensin, on tritiated thymidine uptake by dispersed anterior pituitary cells were investigated. It was found that LHRH but not neurotensin (at concentrations between 10 -7 - 10 -11 M) inhibits incorporation of [ 3 H]thymidine into DNA of pituitary cell nuclei, in a dose-dependent manner. These results indicate that LHRH can regulate not only secretory activity of the gonadotrophic cells but also can be involved in the control of anterior pituitary cell replication

  12. The influence of metronidazole on free thymidine content of blood serum of irradiated mice

    International Nuclear Information System (INIS)

    Konov, A.V.; Ryabchenko, N.I.

    1986-01-01

    The influence of a radiosensitizer, metronidazole, on the free thymidine content of blood serum of irradiated mice was studied in aerobic and hypoxic conditions. A heated metronidazole solution (1 mg/g) was administered intraperitoneally 30 min before irradiation of animals with a dose of 3 Gy. Thymidine concentration in blood serum was determined by the radioimmunological technique. The influence of metronidazole on the level of thymidinemia was only noted in the animals exposed under hypoxic conditions

  13. Method of preparing tritium-labelled thymidine-5'-monophosphates of high specific activity

    International Nuclear Information System (INIS)

    Filip, J.; Vesely, J.; Cihak, A.

    1976-01-01

    A method is described of preparing thymidine-5'-monophosphates labelled with tritium of high specific activity based on enzyme synthesis in vitro. Phosphorylation was carried out using the catalytic effect of an enzyme contained in the supernatant fraction prepared from Yoshida ascites carcinoma in rats. The course of the enzyme reaction can be controlled by the concentration of the individual reaction mixture components. The method described allows obtaining thymidine-5'-monophosphate of radiochemical purity better than 95%. (J.B.)

  14. Blood clearance of 3H-thymidine and 3H-uridine ingrowing rats

    International Nuclear Information System (INIS)

    Carlsson, J.; Johansson, K.J.; Saefwenberg, J.O.

    1976-01-01

    The clearance of 3 H-thymidine and 3 H-uridine from the blood was studied in rats of ages 5, 15 and 30 days. The clearance curves were integrated to get measure of the total availability of the precursors. Age-dependent differences were found, especially for uridine, which showed a lower availability when the animals became older. In the case of thymidine lesser differences were found. The catabolic rate, as measured by the appearance of 3 H-water, was much increased, both in case of 3 H-thymidine and 3 H -uridine as the rats became older. It was observed that the amount of catabolic products (except 3 H-water) in the blood was much larger for uridine than for thymidine. Rats were given 160 rad on the first day after birth. Only in the case of 3 H-thymidine, in 5-days-old rats, an effect of irradiation could be seen, i.e. a somewhat lowered efficiency to catabolize thymidine. (author)

  15. 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

  16. Deoxyribonucleoside kinases in mitochondrial DNA depletion.

    Science.gov (United States)

    Saada-Reisch, Ann

    2004-10-01

    Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are a heterogeneous group of mitochondrial disorders, manifested by a decreased mtDNA copy number and respiratory chain dysfunction. Primary MDS are inherited autosomally and may affect a single organ or multiple tissues. Mutated mitochondrial deoxyribonucleoside kinases; deoxyguanosine kinase (dGK) and thymidine kinase 2 (TK2), were associated with the hepatocerebral and myopathic forms of MDS respectively. dGK and TK2 are key enzymes in the mitochondrial nucleotide salvage pathway, providing the mitochondria with deoxyribonucleotides (dNP) essential for mtDNA synthesis. Although the mitochondrial dNP pool is physically separated from the cytosolic one, dNP's may still be imported through specific transport. Non-replicating tissues, where cytosolic dNP supply is down regulated, are thus particularly vulnerable to dGK and TK2 deficiency. The overlapping substrate specificity of deoxycytidine kinase (dCK) may explain the relative sparing of muscle in dGK deficiency, while low basal TK2 activity render this tissue susceptible to TK2 deficiency. The precise pathophysiological mechanisms of mtDNA depletion due to dGK and TK2 deficiencies remain to be determined, though recent findings confirm that it is attributed to imbalanced dNTP pools.

  17. Development of canine herpesvirus based antifertility vaccines for foxes using bacterial artificial chromosomes.

    Science.gov (United States)

    Strive, Tanja; Hardy, Christopher M; French, Nigel; Wright, John D; Nagaraja, Nitin; Reubel, Gerhard H

    2006-02-13

    Using bacterial artificial chromosome (BAC) technology, a canine herpesvirus (CHV)-based recombinant vaccine vector was produced for the development of an antifertility vaccine for foxes. Infectious viruses were recovered following transfection of canid cells with a BAC plasmid carrying the complete CHV genome. In vitro growth characteristics of BAC-derived viruses were similar to that of wildtype (wt)-CHV. Two recombinant antigens, fox zona pellucida protein subunit 3 (fZPC) and enhanced green fluorescent protein (EGFP) as control antigen, were inserted into thymidine kinase (TK) locus of the CHV genome and shown to be efficiently expressed in vitro. Inoculation of foxes with transgenic CHVs induced CHV specific antibodies, but was innocuous and failed to elicit transgene-specific antibody responses. Infectious virus or viral DNA was not detected in mucosal secretions or tissues of vaccinated foxes. The CHV-BAC system proved to be a quick and reliable method to manipulate the CHV genome. It will help to readily apply changes in the vector design in order to improve virus replication in vivo.

  18. Regulation of annual variation in heterotrophic bacterial production in the Schelde estuary (SW Netherlands)

    NARCIS (Netherlands)

    Goosen, N.K.; Van Rijswijk, P.; Kromkamp, J.C.; Peene, J.

    1997-01-01

    Heterotrophic bacterioplankton production (H-3-thymidine incorporation rate) and abundance in the surface water of the Schelde estuary (The Netherlands) were studied during an annual cycle in 1991 along the salinity gradient (0.8 to 33 psu). Bacterial production and numbers increased from the lower

  19. Casein kinases

    DEFF Research Database (Denmark)

    Issinger, O G

    1993-01-01

    The present review on casein kinases focuses mainly on the possible metabolic role of CK-2, with special emphasis on its behavior in pathological tissues. From these data at least three ways to regulate CK-2 activity emerge: (i) CK-2 activity changes during embryogenesis, being high at certain...

  20. Kinases and Cancer

    OpenAIRE

    Jonas Cicenas; Egle Zalyte; Amos Bairoch; Pascale Gaudet

    2018-01-01

    Protein kinases are a large family of enzymes catalyzing protein phosphorylation. The human genome contains 518 protein kinase genes, 478 of which belong to the classical protein kinase family and 40 are atypical protein kinases [...

  1. Evaluation of storage conditions for tritiated thymidine as reference organically-bound tritium in urine

    International Nuclear Information System (INIS)

    Duong, T.; Trivedi, A.

    1997-01-01

    Interlaboratory intercomparison exercises have used tritiated thymidine as a reference material for organically-bound tritium (OBT) measurements in urine. We have examined the effects of storage conditions on the degradation behavior of tritium from OBT to tritiated water (HTO) in artificial and natural human urine samples. Tritiated thymidine decomposed less readily in artificial urine than natural urine samples. The degradation rate of tritiated thymidine in artificial urine, at -20 deg C, is about 10% for the first month. The rate of tritium conversion from OBT to HTO is the same at 4 deg C, but this storage temperature is less preferable, because of the danger of microbial contamination in the reference samples. The storage of the reference urine samples beyond three months after the preparation date is not recommended for quality control measurement data. (author)

  2. Radiation doses to the tissues of rat from tritiated thymidine administered by three different routes

    International Nuclear Information System (INIS)

    Takeda, Hiroshi; Iwakura, Tetsuo; Mabuchi, Yasuo.

    1984-01-01

    Biological behaviour of tritiated thymidine were investigated in rat over 120 days after oral, intraperitoneal or intravenous administration and the absorbed doses to different tissues were estimated. The result of present study revealed that the absorbed dose from tritiated thymidine varied with the route of administration. Among the three routes of administration, intraperitoneal injection gave the highest dose to all of the tissues examined. A significant difference due to the route of administration was found in spleen and small intestine, where the doses were, respectively, 3.3 and 4.5 times higher after intraperitoneal injection than after oral ingestion. The difference was substantially dependent on the dose value from non-volatile tritium which would be incorporated into DNA. Present observation suggests that the radiation hazards of tritiated thymidine differ depending on the route of entry into the body. (author)

  3. Deoxypyrimidine kinases of herpes simplex viruses types 1 and 2: comparison of serological and structural properties.

    Science.gov (United States)

    Thouless, M E; Wildy, P

    1975-02-01

    The kinetics of formation, the stability at 40 degrees C and the serological properties of thymidine kinase and deoxycytidine kinase activities induced by herpes simplex virus have been examined. The results are consistent with the hypothesis that both activities are carried on the same molecule-a deoxypyrimidine kinase. Mutants deficient in deoxypyrimidine kinase have been used to produce, by absorption of general antisera, deoxypyrimidine kinase-specific antisera. Using immunoprecipitation and SDS-polyacrylamide gel electrophoresis, only one size of polypeptide (mol. wt. 42400 plus or minus 200) has been found, constituting the type 2 enzyme. This is close to published values for the type i enzyme but co-electrophoresis demonstrated that the polypeptide of the type i enzyme was slightly bigger.

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

    International Nuclear Information System (INIS)

    Gay, C.V.; Leach, R.M.

    1985-01-01

    3 H-Thymidine was localized in sections of growth-plate cartilage and associated tibial dyschondroplastic lesion by autoradiography. One hour after 3 H-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

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

    International Nuclear Information System (INIS)

    Nykaenen, P.J.; Raunio, P.V.; Kankaanpaeae, P.U.

    1988-01-01

    The degradation of 3 H-thymidine under various cell culture conditions was analysed. It was found that a half of 3 H-thymidine was degraded to 3 H-thymine during 24 hours in PHA stimulation of blood lymphocytes. A control culture in which PHA was not added also caused 3 H-thymidine degradation. 3 H-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 3 H-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 3 H-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 3 H-thymidine degradation is an important phenomenon in cell cultures and that it can be prevented effectively by using 5-nitrouracil with 3 H-thymidine. (author)

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

    International Nuclear Information System (INIS)

    Higgy, N.A.

    1985-01-01

    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 3 H-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. 3 H-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

  7. 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)

    Yalowich, J.C.; Goldman, I.D.

    1984-01-01

    Uptake of 3 H after exposure of cells to [ 3 H]-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

  8. Radiation genetic injury and metabolic difference of tritiated thymidine in testis of young and adult mice

    Energy Technology Data Exchange (ETDEWEB)

    Mingyue, Lun; Shoupeng, Zhu

    1990-04-01

    The radiogenetoxicological effects on the adult testis and the metabolic difference of tritiated thymidine between the testis of young and adult BALB/C mice were studied. When 0.037 MBq/g.b.w. of tritiated thymidine was given i.v. to mice, the initial burden of tritium in the adult was larger than that of tritium in the young. But the retention of tritium in testis of the young gradually become larger than that of tritium in the adult with the passing time. Tritiated thymidine which was incorporated into DNA of the male germ cell nuclei damaged the genetic materials and caused the rising of the rates of the dominant lethal and the dominant mutation which produced skeletal abnomalities in the offspring. The relationship between the dominant lethal mutation index (Y) and the injected activity of tritiated thymidine (I, MBq/g.b.w.) is described by Y = 74.13 + 80.20 I (r = 0.95). The relationship between the incidence of the dominant skeletal mutation in the offspring (B) and the injected activity is B = 0.16 + 0.079 I ( r = 0.85).

  9. Radiation genetic injury and metabolic difference of tritiated thymidine in testis of young and adult mice

    International Nuclear Information System (INIS)

    Lun Mingyue; Zhu Shoupeng.

    1990-01-01

    The radiogenetoxicological effects on the adult testis and the metabolic difference of tritiated thymidine between the testis of young and adult BALB/C mice were studied. When 0.037 MBq/g.b.w. of tritiated thymidine was given i.v. to mice, the initial burden of tritium in the adult was larger than that of tritium in the young. But the retention of tritium in testis of the young gradually become larger than that of tritium in the adult with the passing time. Tritiated thymidine which was incorporated into DNA of the male germ cell nuclei damaged the genetic materials and caused the rising of the rates of the dominant lethal and the dominant mutation which produced skeletal abnomalities in the offspring. The relationship between the dominant lethal mutation index (Y) and the injected activity of tritiated thymidine (I, MBq/g.b.w.) is described by Y = 74.13 + 80.20 I (r = 0.95). The relationship between the incidence of the dominant skeletal mutation in the offspring (B) and the injected activity is B = 0.16 + 0.079 I ( r = 0.85)

  10. Tritiated uracil, tritiated thymidine, and bromodeoxyuridine induced mutations in eucaryotic cells

    International Nuclear Information System (INIS)

    Burki, H.J.; Moustacchi, E.; Cleaver, J.E.

    1979-02-01

    The induction of gene conversion at the ARG-4 locus in strain BZ34 of Saccharomyces cerevisiae was examined after the cells incorporated y- 3 H uracil under optimum growth conditions for 16 hours, and then received damage at 4 0 C from tritium decays at very low dose rates of 1.4 to 27.6 tritium decays per hour. The results were compared to the results of gene conversion induced by 60 Co. The induction of resistance to 6TG in Chinese hamster ovary (CHO) cells has been studied after incorporation of 3 H-methyl thymidine, 6- 3 H-thymidine, and bromodeoxyuridine under several experimental conditions. The induction of mutations by incorporated 6- 3 H-thymidine is about three times as effective as the induction of mutations by tritiated-methyl thymidine. These results suggest that the determination of the RBE for tritium decays in model eucaryotic systems like yeast and cultured Chinese hamster cells will be influenced by the precise experimental conditions employed. In particular, experiments with mammalian cells will be affected by hot times for mutagenesis in the cell cycle and hot positions within the DNA in the nucleus, and also by the position of tritium decay within the DNA-incorporated molecule

  11. Effect of various 3H-thymidine concentrations on the kinetics of chinese hamster cell division

    International Nuclear Information System (INIS)

    Yuzhakov, V.V.; Lychev, V.A.

    1985-01-01

    A study of the asynchronous culture of Chinese hamster fibroblasts by autoradiography has shown that the pulse (15 min) incorporation of 3 H-thymidine in nuclear DNA influences the kinetics of labelled cell proliferation. The results obtained suggest that one of the early biological effects of the pulse incorporation of 3 H-thymidine is a delay in the occurrence of the first mitosis. With the concentration of 3 H-thymidine 37 kBq/ml the slowing down of the movement of labelled cells in the cycle is detected by a shift and overlapping of waves of labelled and unlabelled mitotic cells. In an increase of the concentration up to 370-925 kBq/ml the pattern of the curves of labelled mitotic cells is distorted. These distortions are well interpreted by the nature of change of the index of labelled and unlabelled mitotic cells. After an increase in 3 H-thymidine concentration from 37 up to 370-925 kBq/ml the mitotic activity of cells labelled at the end of S-phase decreases from 1 to o0.6-0.1% respectively. With the concentration of 925 kBq/ml for these cells incorporating 3 H-thymidine at the end of S-phase, a delay of the entry into mitosis reaches 6-8 h. Autoradiography data with assessment of granule density suggest that mitotic activity and the period of delay in the occurrence of mitosis depend on the dose of irradiation with intranuclear tritium

  12. Bacterial meningitis

    NARCIS (Netherlands)

    Roos, Karen L.; van de Beek, Diederik

    2010-01-01

    Bacterial meningitis is a neurological emergency. Empiric antimicrobial and adjunctive therapy should be initiated as soon as a single set of blood cultures has been obtained. Clinical signs suggestive of bacterial meningitis include fever, headache, meningismus, vomiting, photophobia, and an

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

    International Nuclear Information System (INIS)

    Barth, Rolf F.; Yang, Weilian; Wu, Gong; Byun, Youngjoo; Tjarks, Werner; Eriksson, Staffan; Binns, Peter J.; Riley, Kent J.

    2006-01-01

    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. 10 B-enriched N5-2OH, solubilized in DMSO (50μg 10 B 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 mm 3 ). 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 10 3 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)

  14. An integrated chemo-enzymatic route for preparation of ß-thymidine, a key intermediate in the preparation of antiretrovirals

    CSIR Research Space (South Africa)

    Gordon, GER

    2011-01-01

    Full Text Available A chemo-enzymatic method for production of ß-thymidine, an intermediate in the synthesis of antiretrovirals, is described. Guanosine and thymine were converted by means of enzymatic transglycosylation to yield 5-methyluridine (5-MU), which...

  15. Incorporation of tritiated thymidine and uridine in normal and endopolyploid nuclei of differentiated tissue

    International Nuclear Information System (INIS)

    Bansal, Y.K.; Sen, Sumitra

    1987-01-01

    Rate of replication and transcription between normal and giant endopolyploid nuclei of differentiated tissue of Hordeum vulgare L. (2n=14) roots and Phlox drummondii Hook. (2n=14) and Zea mays L. (2n=20) endosperms were studied by labelling experiments with tritiated thymidine and uridine. The incorporation of thymidine and uridine was identical in both diploid and giant endopolyploid nuclei of the roots of H. vulgare. The endosperm cells of P. drummondii and Z. mays, however, exhibit markedly different labelling pattern in normal (i.e. triploid) and endopolyploid nuclei where both replication and transcription were rather high. The nutritive function of the endosperm is probably responsible for this high degree of activity. (author). 14 refs., 10 figs., 3 tables

  16. Deletion of the thymidine kinase gene induces complete attenuation of the Georgia isolate of African swine fever virus

    Science.gov (United States)

    African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs. There are no vaccines to control Africa swine fever (ASF). Experimental vaccines have been developed using genetically modified live attenuated ASFVs obtained by specifically de...

  17. 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

    report that MT2 includes an E2F-like binding site (GTTCGCGGGCAAA), as shown by the following evidence. (i) MT2 bound specifically to an affinity-purified fusion human E2F protein. (ii) Both MT2 and an authentic E2F site (TTTCGCGCGCTTT) bound specifically to similar or identical nuclear protein complexes...... 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...

  18. Ganciclovir nucleotides accumulate in mitochondria of rat liver cells expressing the herpes simplex virus thymidine kinase gene

    NARCIS (Netherlands)

    van der Eb, Marjolijn M.; Geutskens, Sacha B.; van Kuilenburg, André B. P.; van Lenthe, Henk; van Dierendonck, Jan-Hein; Kuppen, Peter J. K.; van Ormondt, Hans; van de Velde, Cornelis J. H.; Wanders, Ronald J. A.; van Gennip, Albert H.; Hoeben, Rob C.

    2003-01-01

    BACKGROUND: Ganciclovir exhibits broad-spectrum activity against DNA viruses such as cytomegaloviruses, herpes simplex viruses, varicella-zoster virus, Epstein-Barr virus and human herpes virus-6. Ganciclovir is widely applied for anti-herpetic treatment, cytomegalovirus prophylaxis after organ

  19. 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.

  20. 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.

    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

  1. Phylogenetic Analysis of Thymidin Kinase Gene Koi HerpesVirus (KHV in Some Freshwater Fish Aquaculture Centers in East Java

    Directory of Open Access Journals (Sweden)

    Budi Rianto Wahidi

    2014-11-01

    Full Text Available Koi Herpesvirus ( KHV is a virus that infects goldfish and koi that resulted in mass death and cause economic and social losses. The use of Polymerase Chain Reaction (PCR technique to detect KHV was done  in nila and gurame. Proof that KHV could infect nila and gurame KHV following genetic variation in each definitive host and spreading area have not been done. Based on these problems, this research was conducted to study the genetic variability and genetic relationships between KHV isolates in some freshwater fish as the definitive host koi, komet , koki, nila and gurame that exist in the area of East Java. The results showed that clinically the gills were pale and hemorrage the end of the tail fin. Electrophoresis results showed that all samples from  KHV infected  koi, koki, komet, nila and gurame  were all  positive but the sequencing results for tilapia and carp could not be further identified using the BLAST program. Genetically,  koi fish, koki and komet isolates were identical to GenBank isolates code KHV-GZ11 and Indo_0K02SS.

  2. [3H]thymidine labeling of dermal endothelial cells in scleroderma

    International Nuclear Information System (INIS)

    Fleischmajer, R.; Perlish, J.S.

    1977-01-01

    The purpose of this study was to estimate [ 3 H] thymidine labeling of endothelial cells of skin capillaries in localized scleroderma (LS) and systemic scleroderma (SS). Skin specimens from 14 patients with SS, 5 with LS, and 9 matched controls were studied by in vitro autoradiography. Capillaries from patients with SS showed a statistically significant increase in endothelial cell labeling when compared to vessels from controls

  3. In vitro incorporation of tritiated thymidine by the Sternberg-Reed cells in Hodgkin disease

    Energy Technology Data Exchange (ETDEWEB)

    Marinello, M; Tkachenko, G; Gavilondo, J; Baeza, B [National Institute of Oncology and Radiology, Havana (Cuba)

    1975-01-01

    A new DNA synthesis by the Sternberg-Reed cells in Hodgkin disease was studied using tritiated thymidine and autoradiography. The results show that after incubation pulses of 30 and 60 minutes, cells with lobulated nucleus, binucleated and trinucleated cells identifiable to the diagnostic Sternberg-Reed cells could undergo a new DNA synthesis. This points to a more dynamic interpretation of this type of cell.

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

    International Nuclear Information System (INIS)

    Schellmann, B.

    1981-01-01

    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 (+20 0 ) and of cooling (+4 0 C). 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 +4 0 C. (orig./MG) [de

  5. Synthesis, molecular docking study and thymidine phosphorylase inhibitory activity of 3-formylcoumarin derivatives.

    Science.gov (United States)

    Taha, Muhammad; Adnan Ali Shah, Syed; Afifi, Muhammad; Imran, Syahrul; Sultan, Sadia; Rahim, Fazal; Hadiani Ismail, Nor; Mohammed Khan, Khalid

    2018-03-01

    Thymidine phosphorylase (TP) over expression plays role in several pathological conditions, such as rheumatoid arthritis, chronic inflammatory diseases, psoriasis, and tumor angiogenesis. The inhibitor of this enzyme plays an important role in preventing the serious threat due to over expression of TP. In this regard, a series of seventeenanalogs of 3-formylcoumarin (1-17) were synthesized, characterized by 1 HNMR and EI-MS and screened for thymidine phosphorylaseinhibitory activity. All analogs showed a variable degree of thymidine phosphorylase inhibition with IC 50 values ranging between 0.90 ± 0.01 and 53.50 ± 1.20 μM when compared with the standard inhibitor 7-Deazaxanthine having IC 50 value 38.68 ± 1.12 μM. Among the series, fifteenanalogs such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16 and 17 showed excellent inhibition which is many folds better than the standard 7-Deazaxanthine whiletwo analogs 13 and 14 showed good inhibition. The structure activity relationship (SAR) was mainly based upon by bring about difference of substituents on phenyl ring. Molecular docking study was carried out to understand the binding interaction of the most active analogs. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. Assay for plasma somatomedin: (/sup 3/H)thymidine incorporation by isolated rabbit chondrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Ashton, I K; Francis, M J.O. [Nuffield Orthopaedic Centre, Oxford (UK)

    1977-01-01

    The incorporation of (/sup 3/H)thymidine by rabbit chondrocytes in vitro has been developed as a sensitive assay for plasma somatomedin. A concentration of normal plasma of 2.5% enhanced (/sup 3/H)thymidine incorporation by 5- to 20-fold compared with basal levels in the absence of plasma. The mean potency of plasma from normal adult men was 0.96 +-0.1 u./ml (mean +- S.D.) and from acromegalic patients 1.9 +- 0.4 u./ml. The apparent potency of hypopituitary plasma alone increased on heating which suggested the presence of heat-labile inhibitors of somatomedin activity. The potency of heated hypopituitary plasma (0.6 +- 0.09 u./ml) remained significantly lower (P < 0.01) than normal plasma. Human growth hormone (0.1 to 20 ..mu..u/ml), bovine growth hormone (0.5 to 20 ..mu..u/ml), insulin (0.5 to 5 ..mu..u/ml) and glucose (0.3 to 2 mmol/l) had no direct effect on the incorporation of (/sup 3/H)thymidine. Chondrocytes which had been previously stored frozen also showed a response to plasma somatomedin.

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

    International Nuclear Information System (INIS)

    Austin, H.K.; Findlay, S.E.G.

    1989-01-01

    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- 3 H]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- 3 H]thymidine into DNA. Despite differences in dominant vegatation and tidal regime, bacterial biomass was similar (1 x 10 3 ± 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 10 3 ± 0.05 mg C·m -2 and 0.3 ± 0.23 mg C · m -2 · hour -1 , respectively)

  8. Characterization of radiation-induced products of thymidine 3'-monophosphate and thymidylyl (3'→5') thymidine by high-performance liquid chromatography and laser-desorption fourier-transform mass spectrometry

    International Nuclear Information System (INIS)

    Yoshida, H.; Hettich, R.L.

    1994-01-01

    High-performance liquid chromatography (HPLC) and laser-desorption Fourier-transform mass spectrometry (LD FTMS) have been applied for direct measurements of radiation-induced products of nucleic acid constituents containing thymidine. Laser desorption FTMS could be used for the direct detection (neither hydrolyzed nor derivatized) of X-ray-induced decomposition products of aqueous thymidine monophosphate. After these initial experiments, a variety of hydrogenated and hydroxylated thymine standards were acquired and examined by FTMS to assist in the identification of unknown radiation-induced decomposition products of thymine-containing nucleotides and dinucleotides. To extend these studies to dinucleotides, the radiation-induced products generated by the gamma radiolysis of thymidylyl (3'→5') thymidine (TpT) were isolated by reverse-phase HPLC and identified by LD FTMS. Thymine and thymidine 3'-monophosphate were observed as the major products in this case. Several of the minor products of the HPLC profile were pooled in a single fraction and characterized simultaneously by LD FTMS. The resulting mass spectra indicated the presence of hydroxy-5,6-dihydothymidine monophosphate, 5,6-dihydrothymidine monophosphate and thymidine monophosphate, thymine glycol, hydroxy-5,6-dihydrothymine, 5-hydroxy-methyl-uracil and 5,6-dihydrothymine. The combination of HPLC purification and LD FTMS structural characterization provides a useful tool for the direct measurement of radiation-induced products of nucleotides and dinucleotides. 28 refs., 6 figs., 2 tabs

  9. Thymine utilization in Escherichia coli K12. On the role of deoxyribose 1-phosphate and thymidine phosphorylase

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank; Leer, Johan Christian; Nygaard, Per

    1973-01-01

    Exogenously supplied thymine is only poorly utilized by wild-type cells of Escherichia coli for the synthesis of their DNA. It appears that the lack of incorporation of exogenous thymine is due to a lack of endogenous deoxyribosyl groups, which are required for the synthesis of thymidine. Data...... to the external thymine concentration. The experiments in vivo led us to conclude that the incorporation of exogenous thymine occurs via thymidine, which is synthesized from thymine and deoxyribose 1-phosphate, catalyzed by thymidine phosphorylase. In accordance with this studies in vitro with purified thymidine...

  10. Bacterial prostatitis.

    Science.gov (United States)

    Gill, Bradley C; Shoskes, Daniel A

    2016-02-01

    The review provides the infectious disease community with a urologic perspective on bacterial prostatitis. Specifically, the article briefly reviews the categorization of prostatitis by type and provides a distillation of new findings published on bacterial prostatitis over the past year. It also highlights key points from the established literature. Cross-sectional prostate imaging is becoming more common and may lead to more incidental diagnoses of acute bacterial prostatitis. As drug resistance remains problematic in this condition, the reemergence of older antibiotics such as fosfomycin, has proven beneficial. With regard to chronic bacterial prostatitis, no clear clinical risk factors emerged in a large epidemiological study. However, bacterial biofilm formation has been associated with more severe cases. Surgery has a limited role in bacterial prostatitis and should be reserved for draining of a prostatic abscess or the removal of infected prostatic stones. Prostatitis remains a common and bothersome clinical condition. Antibiotic therapy remains the basis of treatment for both acute and chronic bacterial prostatitis. Further research into improving prostatitis treatment is indicated.

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

    International Nuclear Information System (INIS)

    Shabon, M.H.; El-Bedewi, A.F.

    2005-01-01

    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

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

    International Nuclear Information System (INIS)

    Saito, M.; Streffer, C.; Molls, M.

    1983-01-01

    Throughout gestation pregnant mice received drinking water which contained [methyl- 3 H]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

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

    International Nuclear Information System (INIS)

    Saito, M.; Streffer, C.; Molls, M.

    1983-01-01

    Throughout gestation pregnant mice received drinking water which contained [methyl- 3 H]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

  14. The endozepine ODN stimulates [{sup 3}H]thymidine incorporation in cultured rat astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Gandolfo, P.; Patte, C.; Thoumas, J.L.; Leprince, J.; Vaudry, H.; Tonon, M.C. [European Institute for Peptide Research (IFRMP no. 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U 413, UA CNRS, University of Rouen, 76821 Mont-Saint-Aignan (France)

    1999-05-15

    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{sup -14} to 10{sup -11} M), ODN caused a dose-dependent increase of [{sup 3}H]thymidine incorporation. At higher doses (10{sup -10} to 10{sup -5} M), the effect of ODN gradually declined. The central-type benzodiazepine receptor antagonist flumazenil (10{sup -6} M) completely suppressed the stimulatory action of ODN whereas the peripheral-type benzodiazepine receptor ligand, PK11195 (10{sup -6} M) had no effect. The ODN-induced stimulation of [{sup 3}H]thymidine incorporation was mimicked by methyl 6,7-dimethoxy-4-ethyl-{beta}-carboline-3-carboxylate (DMCM). The GABA{sub A} receptor antagonist bicuculline (10{sup -4} M) suppressed the effect of both ODN and DMCM on DNA synthesis. Exposure of cultured astrocytes to the specific GABA{sub A} agonist 3APS (10{sup -10} to 10{sup -4} M) also induced a dose-related increase of [{sup 3}H]thymidine incorporation. The present study indicates that ODN, acting through central-type benzodiazepine receptors associated with the GABA{sub A} 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.)

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

    International Nuclear Information System (INIS)

    Gandolfo, P.; Patte, C.; Thoumas, J.L.; Leprince, J.; Vaudry, H.; Tonon, M.C.

    1999-01-01

    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 [ 3 H]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 [ 3 H]thymidine incorporation was mimicked by methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM). The GABA A receptor antagonist bicuculline (10 -4 M) suppressed the effect of both ODN and DMCM on DNA synthesis. Exposure of cultured astrocytes to the specific GABA A agonist 3APS (10 -10 to 10 -4 M) also induced a dose-related increase of [ 3 H]thymidine incorporation. The present study indicates that ODN, acting through central-type benzodiazepine receptors associated with the GABA A 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.)

  16. Modifying effect of 5-fluoro-2-deoxyuridine and thymidine at G1 phase on radiation and chemically induced chromosome rearrangement

    International Nuclear Information System (INIS)

    Azatyan, R.A.; Voskanyan, A.Z.; Avakyan, V.A.; Akif'ev, A.P.

    1978-01-01

    The yield of structural chromosome mutations induced in Crepis capillaris seeds by X-rays and nitrogen mustard was studied as a function of treatment (at G 1 phase) with an inhibitor of unscheduled DNA synthesis, 5-fluoro-2-deoxyuridine (FdU), and its antagonist, thymidine. Air-dry seeds were irradiated at 10 krad and immediately placed in aqueous solutions of FdU, thymidine, or FdU + thymidine. Ionizing radiation induced only chromosome exchanges in the seeds. When EdU was used, the number of chromosome exchanges was the same although the fraction of simple and isolocus deletions was significantly greater than additive. The effect of FdU was manifested only after 10-hour incubation of the cells. Thymidine alone did not appreciably alter the frequency of radiation-induced aberrations. At the same time, the FdU + thymidine combination decreased the mutation yield i.e. was protective. Frequencies of the chromosome aberration in this experiment were the same as in the control

  17. 3H-thymidine labelling pattern of preleptotene chromosone condensation stages in the foetal sheep ovary

    International Nuclear Information System (INIS)

    Luciani, J.M.; Devictor-Vuillet, Monique; Bezard, Jacqueline; Mauleon, P.

    1979-01-01

    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

  18. Bacterial Vaginosis

    Science.gov (United States)

    ... Archive STDs Home Page Bacterial Vaginosis (BV) Chlamydia Gonorrhea Genital Herpes Hepatitis HIV/AIDS & STDs Human Papillomavirus ( ... of getting other STDs, such as chlamydia and gonorrhea . These bacteria can sometimes cause pelvic inflammatory disease ( ...

  19. Method of preparing thymidine-5'-monophosphate specifically or nonspecifically labelled with 14C or with 3H

    International Nuclear Information System (INIS)

    Nejedly, Z.; Filip, J.; Ekl, J.; Kolina, J.; Votruba, I.; Skoda, J.

    1977-01-01

    The invention claims a method for labelled thymidine-5'-monophosphate preparation by cultivating a special thymine-dependent Escherichia coli SPT - strain in the optimum synthetic culture medium containing 0.8 to 1.2 g/ml of labelled thymine. Practically the whole amount of labelled thymine is utilized for cellular deoxyribonucleic acid synthesis. The radioactive biomass obtained is processed using such chemical and enzymatic decomposition procedures as to allow separating the labelled thymidine-5'-monophosphate as the only thymine reaction product. Experiments conducted showed that the radiochemical purity of the thymidine-5'-monophosphate obtained was better than 98%. The absence of other nonactive substances was confirmed by spectrophotometric analysis. The overall product activity was 92.3% of the activity of thymine-2- 14 C introduced in the reaction. (Ha)

  20. Relationship between release of LH and incorporation of tritiated thymidine in the anterior pituitary gland of the castrated female rat.

    Science.gov (United States)

    Machiavelli, G A; Romano, M I; Burdman, J A

    1985-06-01

    Castration of female rats during diestrus increases the concentration of circulating LH from days 3 and the incorporation of 3H thymidine into pituitary DNA from day 5. Both effects are completely abolished by the administration of dihydrotestosterone. Although the i.v. injection of LHRH markedly enhances the concentration of LH in serum, it does not modify the incorporation of 3H thymidine into pituitary DNA. Castration might produce a maximal stimulation in 3H thymidine incorporation and a further stimulation of LH release with LHRH is unable to enhance the incorporation of the radioactive precursor. The results suggest a relationship between LH secretion and DNA synthesis in the pituitary gland of the rat.

  1. Positron imaging feasibility studies: characteristics of [3H]thymidine uptake in rodent and canine neoplasms

    International Nuclear Information System (INIS)

    Larson, S.M.; Weiden, P.L.; Grunbaum, J.

    1981-01-01

    Uptake [ 3 H]thymidine was studied in BALB/c mice with EMT-6 sarcoma, in Buffalo rats with Morris 7777 hepatoma, and in nine dogs with spontaneous neoplasms: four lymphomas, two osteosarcomas, two soft-tissue sarcomas, and a thyroid carcinoma. High tumor-to-tissue ratios were observed for all tumor types assayed, and absolute uptakes, when computed as percent dose per gram tumor normalized for body weight, were similar for transplanted and spontaneous tumors. In the rodent tumors, radiothymidine was retained for at least 3 hr in the tumor without appreciable loss. In canine neoplasms, although the highest uptakes were observed in cellular tumors with many mitotic figures, tumor uptake showed significant variability that did not correlate with any obvious histologic change, and thus may reflect true biologic differences in metabolism among tumors at different sites in the same animal. These studies provide additional experimental evidence that the ratios of neoplastic to normal tissue and the kinetics of thymidine uptake by tumors are suitable for positron emission tomography of neoplasms in small and large, animals, including both transplanted and spontaneous tumors

  2. The induction and repair of cyclobutane thymidine dimers in human skin

    International Nuclear Information System (INIS)

    Roza, L.; Erasmus Univ., Rotterdam; Vermeulen, W.; Schans, G.P. van der; Lohman, P.H.M.

    1987-01-01

    The most important detrimental effect of ultraviolet radiation (UV) on the living cell, so far known, is the induction of damage in the DNA. The major photoproducts induced in DNA by UV-C (200-280 nm) and UV-B (280-315 nm) are the cyclobutane-type pyrimidine dimers, which have been implicated in UV-induced mutagenesis and carcinogenesis. Dimer lesions in DNA of cells may be repaired in the dark by a multi-enzyme process (excision repair), or via a light dependent enzymatic reaction known as photoreactivation (phr) which is specific for pyrimidine dimers. Although phr has been found to occur in a wide range of organisms, studies on the presence of phr in mammalian cells have yielded conflicting results. To investigate repair of pyrimidine dimers in human skin cells irradiated in vivo, a specific and sensitive detection method was developed based on a monoclonal antibody directed against thymidine dimers. Application together with a fluorescent immunostaining permits the direct detection of thymidine dimers in human skin cells. The method is used in studies aimed at a better understanding of the role of these lesions in the process of carcinogenesis. A report is given on the isolation and characterization of the antibodies, and their application in a study on the induction of pyrimidine dimers in human skin and on photorepair in cultured cells. 10 refs.; 2 figs

  3. Bioorthogonal Metabolic DNA Labelling using Vinyl Thioether-Modified Thymidine and o-Quinolinone Quinone Methide.

    Science.gov (United States)

    Gubu, Amu; Li, Long; Ning, Yan; Zhang, Xiaoyun; Lee, Seonghyun; Feng, Mengke; Li, Qiang; Lei, Xiaoguang; Jo, Kyubong; Tang, Xinjing

    2018-04-17

    Bioorthogonal metabolic DNA labeling with fluorochromes is a powerful strategy to visualize DNA molecules and their functions. Here, we report the development of a new DNA metabolic labeling strategy enabled by the catalyst-free bioorthogonal ligation using vinyl thioether modified thymidine and o-quinolinone quinone methide. With the newly designed vinyl thioether-modified thymidine (VTdT), we added labeling tags on cellular DNA, which could further be linked to fluorochromes in cells. Therefore, we successfully visualized the DNA localization within cells as well as single DNA molecules without other staining reagents. In addition, we further characterized this bioorthogonal DNA metabolic labeling using DNase I digestion, MS characterization of VTdT as well as VTdT-oQQF conjugate in cell nuclei or mitochondria. This technique provides a powerful strategy to study DNA in cells, which paves the way to achieve future spatiotemporal deciphering of DNA synthesis and functions. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Sensitized photo-oxidation of thymidine by 2-methyl-1, 4-naphthoquinone. Characterization of the stable photoproducts

    International Nuclear Information System (INIS)

    Decarroz, C.; Cadet, J.; Murali Krishna, C.; Riesz, P.

    1986-01-01

    The near ultraviolet photolysis of an aerated aqueous solution of thymidine containing 2-methyl-1,4-naphthoquinone gives rise to two main classes of photoproducts as a result of the initial formation of a pyrimidine radical cation. These photo-oxidation products have been separated by high-performance liquid chromatography and further characterized by various spectroscopic techniques including fast atom bombardment mass spectrometry and high field 1 H and 13 C nuclear magnetic resonance analysis. This photoreaction constitutes an excellent model to study the chemical properties of the thymidine radical cation which is expected to be one of the primary consequences of the direct effects of ionizing radiation. (author)

  5. A rapid microassay for detecting antibodies against poliovirus based on [14C]thymidine uptake of treated cell cultures

    International Nuclear Information System (INIS)

    Hilfenhaus, J.; Damm, H.; Ziegelmaier, R.; Gruschkau, H.

    1977-01-01

    DNA synthesis of mammalian cells propagated in microplates can easily be measured if cell cultures incubated with [ 14 C]thymidine are harvested on to glass fibre filters by a semiautomatic harvesting technique. Soon after infection with poliovirus, [ 14 C]thymidine uptake of U cells (established, human amniotic cell line) is inhibited. This inhibition can be prevented by previous virus neutralization with antibody. Based on this effect a rapid, precise assay method was set up to determine neutralizing antibody titres against poliovirus. There was a good correlation between titres obtained by this assay and those obtained by 50% endpoint titrations in cytopathogenic effect inhibition assays

  6. Autoradiographic studies of the pancreas and adrenal cortex with 3H-thymidine, age dependence and other influencing factors

    International Nuclear Information System (INIS)

    Izbirak, C.D.

    1979-01-01

    The cell proliferation of pancreatic and corticoadrenal cells and their susceptibility towards exogenically administered kallikrein and isoprenalin was studied with the aid of 3 H-thymidine autoradiography. It can be assumed that those cells tabelled with 3 H-thymidine within the time available and which after pooling, served as labelling index also included those undergoing DNA synthesis in the mitotic phase and which therefore also join in cell proliferation. This investigation is based on material generated from 28 black mice of the strain C 57. (orig./MG) [de

  7. Bacterial abundance and activity in deep-sea sediments from the eastern North Atlantic

    Science.gov (United States)

    Eardly, D. F.; Carton, M. W.; Gallagher, J. M.; Patching, J. W.

    Results are presented from four cruises to the Porcupine Abyssal Plain (PAP site) that took place during the BENGAL project from September 1996 to March 1998, and two cruises to the PAP and an oligotrophic site (EUMELI) that took place during the DEEPSEAS project between September 1993 and March 1994. Bacterial abundances in sediment and sediment contact water were measured by epifluorescence microscopy. Bacterial activity was determined by 3H-thymidine incorporation as a measure of DNA synthesis, and by 3H-leucine incorporation as a measure of protein synthesis. Activities were measured under atmospheric and in situ pressures and temperatures. Bacterial activity was usually higher in samples incubated at in situ pressure than those incubated at atmospheric pressure indicating that a barophilic community was dominant. Inter-cruise comparisons of abundance and activity during the BENGAL project showed no firm evidence of there being a seasonal response in the benthic microbial community to any episodic phytodetritus event. This was probably because of inter-annual variations in the quality and quantity of phytodetritus deposition at the PAP site, the rapid remineralization of fresh organic material by the microbial communities and the timing of cruises to the study area. 3H-thymidine and 3H-leucine incorporation in sediments was higher during the BENGAL period than the DEEPSEAS programme. A methodological change in the 3H-thymidine incorporation technique for sediments may explain the differences in DNA synthesis observed between the two projects, whereas the lower levels of protein synthesis observed during the DEEPSEAS programme probably reflected both inter-annual variations in activity at the PAP site and the lower productivity that prevailed at surface at the EUMELI oligotrophic site. Rates of 3H-thymidine incorporation in sediment contact water were similar during both projects.

  8. Total synthesis of [2-11C]thymidine from [11C]urea: A tracer of choice for measurement of cellular proliferation using PET

    International Nuclear Information System (INIS)

    Labar, D.; Vander Borght, T.

    1990-01-01

    In preliminary studies of cellular proliferation with [methyl- 11 C]thymidine, the labelled degradative products mask the progressive incorporation of the tracer into DNA. The authors have developed a procedure for the synthesis of [2- 11 C]thymidine to circumvent this difficulty, using a [ 11 C]urea precursor

  9. Reaction products from N-methyl-N-nitrosourea and deoxyribonucleic acid containing thymidine residues. Synthesis and identification of a new methylation product, O4-methyl-thymidine

    Science.gov (United States)

    Lawley, P. D.; Orr, D. J.; Shah, S. A.; Farmer, P. B.; Jarman, M.

    1973-01-01

    1. DNA was treated with N-methyl-N-nitrosourea at pH7–8, 37°C, degraded to yield 3- and 7-methylpurines and deoxyribonucleosides and the reaction products were separated by chromatography on ion-exchange resins. The following methods for identification and determination of products were used: with unlabelled N-methyl-N-nitrosourea, u.v. absorption; use of methyl-14C-labelled N-methyl-N-nitrosourea and use of [14C]thymine-labelled DNA. 2. The synthesis of O4-methylthymidine and its identification by u.v. and mass spectroscopy are reported. 3. 3-Methylthymidine and O4-methylthymidine were found as methylation products from N-methyl-N-nitrosourea with thymidine and with DNA, in relatively small yields. Unidentified products containing thymine were found in enzymic digests of N-methyl-N-nitrosourea-treated DNA, which may be phosphotriesters. 4. The possible role of formation of methylthymines in mutagenesis by N-methyl-N-nitrosourea is discussed. PMID:4798180

  10. BACTERIAL CONSORTIUM

    Directory of Open Access Journals (Sweden)

    Payel Sarkar

    2013-01-01

    Full Text Available Petroleum aromatic hydrocarbons like benzen e, toluene, ethyl benzene and xylene, together known as BTEX, has almost the same chemical structure. These aromatic hydrocarbons are released as pollutants in th e environment. This work was taken up to develop a solvent tolerant bacterial cons ortium that could degrade BTEX compounds as they all share a common chemical structure. We have isolated almost 60 different types of bacterial strains from different petroleum contaminated sites. Of these 60 bacterial strains almost 20 microorganisms were screene d on the basis of capability to tolerate high concentration of BTEX. Ten differe nt consortia were prepared and the compatibility of the bacterial strains within the consortia was checked by gram staining and BTEX tolerance level. Four successful mi crobial consortia were selected in which all the bacterial strains concomitantly grew in presence of high concentration of BTEX (10% of toluene, 10% of benzene 5% ethyl benzene and 1% xylene. Consortium #2 showed the highest growth rate in pr esence of BTEX. Degradation of BTEX by consortium #2 was monitored for 5 days by gradual decrease in the volume of the solvents. The maximum reduction observed wa s 85% in 5 days. Gas chromatography results also reveal that could completely degrade benzene and ethyl benzene within 48 hours. Almost 90% degradation of toluene and xylene in 48 hours was exhibited by consortium #2. It could also tolerate and degrade many industrial solvents such as chloroform, DMSO, acetonitrile having a wide range of log P values (0.03–3.1. Degradation of aromatic hydrocarbon like BTEX by a solvent tolerant bacterial consortium is greatly significant as it could degrade high concentration of pollutants compared to a bacterium and also reduces the time span of degradation.

  11. A screen for kinase inhibitors identifies antimicrobial imidazopyridine aminofurazans as specific inhibitors of the Listeria monocytogenes PASTA kinase PrkA.

    Science.gov (United States)

    Schaenzer, Adam J; Wlodarchak, Nathan; Drewry, David H; Zuercher, William J; Rose, Warren E; Striker, Rob; Sauer, John-Demian

    2017-10-13

    Bacterial signaling systems such as protein kinases and quorum sensing have become increasingly attractive targets for the development of novel antimicrobial agents in a time of rising antibiotic resistance. The family of bacterial P enicillin-binding-protein A nd S erine/ T hreonine kinase- A ssociated (PASTA) kinases is of particular interest due to the role of these kinases in regulating resistance to β-lactam antibiotics. As such, small-molecule kinase inhibitors that target PASTA kinases may prove beneficial as treatments adjunctive to β-lactam therapy. Despite this interest, only limited progress has been made in identifying functional inhibitors of the PASTA kinases that have both activity against the intact microbe and high kinase specificity. Here, we report the results of a small-molecule screen that identified GSK690693, an imidazopyridine aminofurazan-type kinase inhibitor that increases the sensitivity of the intracellular pathogen Listeria monocytogenes to various β-lactams by inhibiting the PASTA kinase PrkA. GSK690693 potently inhibited PrkA kinase activity biochemically and exhibited significant selectivity for PrkA relative to the Staphylococcus aureus PASTA kinase Stk1. Furthermore, other imidazopyridine aminofurazans could effectively inhibit PrkA and potentiate β-lactam antibiotic activity to varying degrees. The presence of the 2-methyl-3-butyn-2-ol (alkynol) moiety was important for both biochemical and antimicrobial activity. Finally, mutagenesis studies demonstrated residues in the back pocket of the active site are important for GSK690693 selectivity. These data suggest that targeted screens can successfully identify PASTA kinase inhibitors with both biochemical and antimicrobial specificity. Moreover, the imidazopyridine aminofurazans represent a family of PASTA kinase inhibitors that have the potential to be optimized for selective PASTA kinase inhibition.

  12. Bacterial Adhesion & Blocking Bacterial Adhesion

    DEFF Research Database (Denmark)

    Vejborg, Rebecca Munk

    2008-01-01

    , 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 significantly...... 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 parameters...

  13. Bacterial meningitis

    NARCIS (Netherlands)

    Heckenberg, Sebastiaan G. B.; Brouwer, Matthijs C.; van de Beek, Diederik

    2014-01-01

    Bacterial meningitis is a neurologic emergency. Vaccination against common pathogens has decreased the burden of disease. Early diagnosis and rapid initiation of empiric antimicrobial and adjunctive therapy are vital. Therapy should be initiated as soon as blood cultures have been obtained,

  14. Bacterial lipases

    NARCIS (Netherlands)

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

    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,

  15. Bacterial stress

    Indian Academy of Sciences (India)

    First page Back Continue Last page Graphics. Bacterial stress. Physicochemical and chemical parameters: temperature, pressure, pH, salt concentration, oxygen, irradiation. Nutritional depravation: nutrient starvation, water shortage. Toxic compounds: Antibiotics, heavy metals, toxins, mutagens. Interactions with other cells: ...

  16. Base excision repair of both uracil and oxidatively damaged bases contribute to thymidine deprivation-induced radiosensitization

    International Nuclear Information System (INIS)

    Allen, Bryan G.; Johnson, Monika; Marsh, Anne E.; Dornfeld, Kenneth J.

    2006-01-01

    Purpose: Increased cellular sensitivity to ionizing radiation due to thymidine depletion is the basis of radiosensitization with fluoropyrimidine and methotrexate. The mechanism responsible for cytotoxicity has not been fully elucidated but appears to involve both the introduction of uracil into, and its removal from, DNA. The role of base excision repair of uracil and oxidatively damaged bases in creating the increased radiosensitization during thymidine depletion is examined. Methods and Materials: Isogenic strains of S. cerevisiae differing only at loci involved in DNA repair functions were exposed to aminopterin and sulfanilamide to induce thymidine deprivation. Cultures were irradiated and survival determined by clonogenic survival assay. Results: Strains lacking uracil base excision repair (BER) activities demonstrated less radiosensitization than the parental strain. Mutant strains continued to show partial radiosensitization with aminopterin treatment. Mutants deficient in BER of both uracil and oxidatively damaged bases did not demonstrate radiosensitization. A recombination deficient rad52 mutant strain was markedly sensitive to radiation; addition of aminopterin increased radiosensitivity only slightly. Radiosensitization observed in rad52 mutants was also abolished by deletion of the APN1, NTG1, and NTG2 genes. Conclusion: These data suggest radiosensitization during thymidine depletion is the result of BER activities directed at both uracil and oxidatively damaged bases

  17. Perturbation of DNA replication and cell cycle progression by commonly used [3H]thymidine labeling protocols

    International Nuclear Information System (INIS)

    Hoy, C.A.; Lewis, E.D.; Schimke, R.T.

    1990-01-01

    The effect of tritiated thymidine incorporation on DNA replication was studied in Chinese hamster ovary cells. Rapidly eluting (small) DNA from cells labeled with 2 microCi of [ 3 H]thymidine per ml (200 microCi/mmol) for 60 min matured to a large nonelutable size within approximately 2 to 4 h, as measured by the alkaline elution technique. However, DNA from cells exposed to 10 microCi of [ 3 H]thymidine per ml (66 microCi/mmol) was more rapidly eluting initially and did not mature to a nonelutable size during subsequent incubation. Semiconservative DNA replication measured by cesium chloride gradient analysis of bromodeoxyuridine-substituted DNA was also found to be affected by the final specific activity of the [ 3 H]thymidine used in the labeling protocol. Dramatic cell cycle perturbations accompanied these effects on DNA replication, suggesting that labeling protocols commonly used to study DNA metabolism produce aberrant DNA replication and subsequent cell cycle perturbations

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

    Energy Technology Data Exchange (ETDEWEB)

    Bose, Adity [Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700 064 (India); Sarkar, Achintya K. [Department of Chemistry, Presidency College, 86/1 College street, Kolkata 700 073 (India); Basu, Samita, E-mail: samita.basu@saha.ac.i [Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700 064 (India)

    2009-10-15

    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.

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

    International Nuclear Information System (INIS)

    Bose, Adity; Sarkar, Achintya K.; Basu, Samita

    2009-01-01

    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.

  20. Evaluation of in-vitro cell labelling of mouse epithelia with tritiated thymidine

    International Nuclear Information System (INIS)

    Mackenzie, I.C.; Ettinger, R.L.

    1977-01-01

    Various factors affecting the epithelial labelling index recorded following in-vitro incubation of specimens of mouse skin and palatal mucosa with tritiated thymidine were examined. Isotope concentration, specimen size and the period of exposure of autoradiographs prior to development markedly influenced the labelling index recorded but, following standardization of such factors, a reproducible index could be obtained. Labelling indices comparable to those obtained by the standard in-vivo labelling method could be produced by adjustment of isotope concentration in incubation media. Comparison of labelling indices recorded for tissues labelled in-vitro by a standardized method appeared valid but the absolute values of indices so obtained and their comparison with indices resulting from in-vivo labelling methods were of doubtful significance. (author)

  1. Muscle phosphorylase kinase deficiency

    DEFF Research Database (Denmark)

    Preisler, N; Orngreen, M C; Echaniz-Laguna, A

    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)....

  2. 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)

    Hedwig, Gavin R.; Jameson, Geoffrey B.; Hoiland, Harald

    2011-01-01

    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 2 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 2 0 {E 2 0 =(∂V 2 0 /∂T) p }. The partial molar heat capacity at infinite dilution for thymidine, C p,2 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 S,2 0 , and the partial molar isothermal compression at infinite dilution, K T,2 0 {K T,2 0 =-(∂V 2 0 /∂P) T }, have been derived from the sound speed data. The V 2 0 , E 2 0 , C p,2 0 , and K S,2 0 results for thymidine are critically compared with those available from the literature.

  3. [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. Copyright © 2016 Elsevier España, S.L.U. All rights reserved.

  4. An active form of calcium and calmodulin dependant protein kinase ...

    African Journals Online (AJOL)

    The removal of the auto-inhibitory domain that negatively regulates the kinase activity in M. truncatula results in a constitutively-active form, inducing symbiotic responses in the absence of bacterial signals. In this study, we verified the functionality of a DMI3 variant and its ability to induce spontaneous nodules in M.

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

    OpenAIRE

    H. Habdas

    2015-01-01

    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 wh...

  6. 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.

  7. Tamoxifen enhances erlotinib-induced cytotoxicity through down-regulating AKT-mediated thymidine phosphorylase expression in human non-small-cell lung cancer cells.

    Science.gov (United States)

    Ko, Jen-Chung; Chiu, Hsien-Chun; Syu, Jhan-Jhang; Jian, Yi-Jun; Chen, Chien-Yu; Jian, Yun-Ting; Huang, Yi-Jhen; Wo, Ting-Yu; Lin, Yun-Wei

    2014-03-01

    Tamoxifen is a triphenylethylene nonsteroidal estrogen receptor (ER) antagonist used worldwide as an adjuvant hormone therapeutic agent in the treatment of breast cancer. However, the molecular mechanism of tamoxifen-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Thymidine phosphorylase (TP) is an enzyme of the pyrimidine salvage pathway which is upregulated in cancers. In this study, tamoxifen treatment inhibited cell survival in two NSCLC cells, H520 and H1975. Treatment with tamoxifen decreased TP mRNA and protein levels through AKT inactivation. Furthermore, expression of constitutively active AKT (AKT-CA) vectors significantly rescued the decreased TP protein and mRNA levels in tamoxifen-treated NSCLC cells. In contrast, combination treatment with PI3K inhibitors (LY294002 or wortmannin) and tamoxifen further decreased the TP expression and cell viability of NSCLC cells. Knocking down TP expression by transfection with small interfering RNA of TP enhanced the cytotoxicity and cell growth inhibition of tamoxifen. Erlotinib (Tarceva, OSI-774), an orally available small molecular inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, is approved for clinical treatment of NSCLC. Compared to a single agent alone, tamoxifen combined with erlotinib resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced activation of phospho-AKT and phospho-ERK1/2, and reduced TP protein levels. These findings may have implications for the rational design of future drug regimens incorporating tamoxifen and erlotinib for the treatment of NSCLC. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Bacterial mitosis

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Borch, Jonas; Dam, Mette

    2003-01-01

    Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system...... act together to generate the force required for plasmid movement during segregation. ParR protein binds cooperatively to the centromeric parC DNA region, thereby forming a complex that interacts with the filament-forming actin-like ParM protein in an ATP-dependent manner, suggesting that plasmid...

  9. Relationships between coastal bacterioplankton growth rates and biomass production: comparison of leucine and thymidine uptake with single-cell physiological characteristics.

    Science.gov (United States)

    Franco-Vidal, Leticia; Morán, Xosé Anxelu G

    2011-02-01

    Specific growth rates of heterotrophic bacterioplankton have been frequently estimated from in situ bacterial production (BP) to biomass (BB) ratios, using a series of assumptions that may result in serious discrepancies with values obtained from predator-free cultures. Here, we used both types of approaches together with a comprehensive assessment of single-cell physiological characteristics (membrane integrity, nucleic acid content, and active respiration) of coastal bacterioplankton during a complete annual cycle (February 2007-January 2008) in the southern Bay of Biscay off Xixón, Spain. Both leucine and thymidine incorporation rates were used in conjunction with empirical tracer to carbon or cells conversion factors (eCFs) to accurately derive BP. Leu and TdR incorporation rates covaried year-round, as did the corresponding eCFs at 0 and 50 m depth. eCFs peaked in autumn, with mean annual values close to the theoretical ones (3.4 kg C mol Leu(-1) and 2.0 × 10(18) cells mol TdR(-1)). Bacterial abundance (0.2-1.5 × 10(6) cells L(-1)) showed a bimodal distribution with maxima in May and October and minima in March. Live (membrane-intact) cells dominated year-round (79-97%), with high nucleic acid cells (42-88%) and actively respiring bacteria (CTC+, 1-16%) showing distinct surface maxima in April and July, respectively. BB (557-1,558 mg C m(-2)) and BP (7-139 mg C m(-2) day(-1)) presented two distinct peaks in spring and autumn, both of similar size due to a strong upwelling event observed in September. Specific growth rates (0.35-3.8 day(-1)) were one order of magnitude higher in predator-free incubations than bacterial turnover rates derived from integrated BP:BB ratios (0.01-0.16 and 0.01-0.09 day(-1), for Leu and TdR, respectively) and were not correlated, probably due to a significant contribution of low activity cells to total standing stocks. The Leu:TdR molar ratio averaged for the water column (6.6-25.5) decreased significantly with higher integrated

  10. effect of o,p'-DDD on the in vivo incorporation of 3H-thymidine into DNA

    International Nuclear Information System (INIS)

    Lund, B.-O.; Brandt, I.; Busk, L.; Hellmann, B.

    1990-01-01

    The effects of o,p'-DDD on the DNA synthesis in the C57Bl mouse lung and liver were studied. As determined by 3 H-thymidine incorporation into DNA, a selective increase in the lung DNA synthesis (+59%) was observed 2 day after a single intraperiotoneal injection of 100 mg/kg o,p'-DDD. Microautoradiography showed that incorporated 3 H-thymidine was confined to a restricted number of heavily labelled cells, presumably proliferating type II cells. At the most, a 9 times higher rate of cell proliferation was observed in the lung 4 days after an intraperitoneal injection of 500 mg/kg o,p'-DDD. Using mouse lung or liver S-9 as activating system, no mutagenic activity of o,p'-DDD was detected in the Ames test. The induced cell proliferation may indicate a tissue-selective promotor activity of o,p'-DDD in the mouse lung. (author)

  11. Pyruvate kinase blood test

    Science.gov (United States)

    ... medlineplus.gov/ency/article/003357.htm Pyruvate kinase blood test To use the sharing features on this page, ... energy when oxygen levels are low. How the Test is Performed A blood sample is needed. In the laboratory, white blood ...

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

    International Nuclear Information System (INIS)

    Santos, Rodrigo Luis Silva Ribeiro

    2007-01-01

    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 99m Tc, 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)

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

    International Nuclear Information System (INIS)

    Karim, A.C.

    1990-01-01

    The effect of adriamycin (5 mg/kg) on 3 H-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 3 H-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 3 H-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 3 H-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)

  14. 3H-thymidine labelling of DNA of radiosensitive organs of rats irradiated under alpine conditions and after adaptation to hypoxia in the altitude chamber

    International Nuclear Information System (INIS)

    Gusejnov, F.T.; Egorov, I.A.; Gladilin, K.L.; Farber, Yu.V.

    1979-01-01

    Preliminary adaptation of rats of high altitude conditions (3200 m) and training in the altitude chamber at the same imitated altitude inhibit 3 H-thymidine labelling of thymus DNA both shortly (26 h) and later (20 and 30 days) after irradiation. Whether the thymidine incorporation is activated or delayed depends on conditions of pretreatment. The data obtained are discussed from the point of view of the raioprotective effect of preadaptation of animals to high-altitude hypoxia

  15. 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...... of 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...

  16. In silico binding analysis and SAR elucidations of newly designed benzopyrazine analogs as potent inhibitors of thymidine phosphorylase.

    Science.gov (United States)

    Taha, Muhammad; Ismail, Nor Hadiani; Imran, Syahrul; Rahim, Fazal; Wadood, Abdul; Al Muqarrabun, Laode Muhammad Ramadhan; Khan, Khalid Mohammed; Ghufran, Mehreen; Ali, Muhammad

    2016-10-01

    Thymidine phosphorylase (TP) is up regulated in wide variety of solid tumors and therefore presents a remarkable target for drug discovery in cancer. A novel class of extremely potent TPase inhibitors based on benzopyrazine (1-28) has been developed and evaluated against thymidine phosphorylase enzyme. Out of these twenty-eight analogs eleven (11) compounds 1, 4, 14, 15, 16, 17, 18, 19, 20, 24 and 28 showed potent thymidine phosphorylase inhibitory potentials with IC50 values ranged between 3.20±0.30 and 37.60±1.15μM when compared with the standard 7-Deazaxanthine (IC50=38.68±4.42μM). Structure-activity relationship was established and molecular docking studies were performed to determine the binding interactions of these newly synthesized compounds. Current studies have revealed that these compounds established stronger hydrogen bonding networks with active site residues as compare to the standard compound 7DX. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Acute non-stochastic effect of very low dose whole-body exposure, a thymidine equivalent serum factor

    International Nuclear Information System (INIS)

    Feinendegen, L.E.; Muehlensiepen, H.; Porschen, W.; Booz, J.

    1982-01-01

    Whole-body irradiation of mice causes the dose-dependent appearance of a humoral factor in blood serum which inhibits incorporation of 125-IUdR into tissue culture cells. This factor appears even at doses below 0.01 Gy gamma irradiation and thus is probably not related to cell death. Data are presented relating this humoral factor to thymidine. Since at low doses the target size for this effect was calculated to be the entire cell, a cellular effect is postulated linking the site of few primary absorption events, anywhere in the cell, with the cellular membrane, thus causing changes in membrane charge, structure and/or fluidity. This may lead to blocking thymidine acceptance by the cell, and thus would cause a pile-up of thymidine in the reutilization pathway in peripheral blood and would give rise to the observed effect. The effect appears as a temporary disturbance of the physiological equilibrium and should not be related at present to any cellular damage. The acute low-dose effect described has implications for the measurement of low-dose exposure by biological dosimeters and on basic research on membrane function. (author)

  18. Early colonic dysplasia: comparison of differential mucin staining and tritiated thymidine labeling

    International Nuclear Information System (INIS)

    Chabot, J.A.; Colacchio, T.A.

    1985-01-01

    Controversy has arisen regarding the interpretation and significance of histochemical changes in the mucin produced by the globlet cells in colonic mucosa. The shift from sulfomucin to sialomucin, which is readily identified utilizing high iron diamine-alcian blue staining techniques, has been alternately interpreted as a specific, early dysplastic and premalignant change or a nonspecific generalized response to trauma and inflammation, among others. An attempt to clarify this issue was made by comparing mucin changes identified by high iron diamine-alcian blue staining techniques with increases in DNA synthetic activity identified utilizing autoradiographic analysis of tritiated thymidine uptake. Male Holtzman rats were treated with 15 weekly subcutaneous injections of dimethylhydrazine (30 mg/kg per week) (10 rats) or placebo (10 rats). The colons were prepared and fixed, sequential sections were stained with hematoxylin-eosin or high iron diamine-alcian blue, autoradiography was performed. Analyses of labeling index showed no difference in normal background crypts between the control and treatment groups nor in crypts adjacent to those displaying abnormal mucin staining. Crypts with abnormal mucin production (sialomucin dominant) had significantly higher labeling indexes when compared with those of control animals (p less than 0.005). These findings indicate that the shifts in mucin production identified with high iron diamine-alcian blue staining represent crypts with increased and abnormally distributed mitotic activity that is an early dysplastic response to the carcinogenic stimulus

  19. Proliferative activity of benign human prostate, prostatic adenocarcinoma and seminal vesicle evaluated by thymidine labeling

    International Nuclear Information System (INIS)

    Meyer, J.S.; Sufrin, G.; Martin, S.A.

    1982-01-01

    The thymidine labeling index (TLI) was measured in vitro in the epithelium and stroma of benign prostate glands and seminal vesicles and in the epithelium of prostatic adenocarcinomas. The mean epithelial TLI of normal peripheral (posterior) prostatic zone was 0.12 percent, and that of the normal central (deep) zone was 0.11 percent. Mean normal stromal TLI's were 0.08 percent and 0.06 percent, respectively. The mean TLI of epithelium in nodular hyperplasia was 0.31 percent, which differs significantly from normal epithelium (p less than 0.05), and the mean stromal TLI was also increased (0.16 percent, p less than 0.1). The mean TLI of prostatic adenocarcinomas was 0.90 percent (range 0.14 to 3.90 percent) which was significantly higher than for either normal epithelium (p less than 0.001) or epithelium of nodular hyperplasia (p less than 0.05). Trends of increasing TLI with increasing histologic grades and increasing nuclear size and numbers of nucleoli were not significant. The data support participation of both epithelial and stromal proliferation in nodular hyperplasia, and indicate a low basal proliferative rate in normal prostatic glands. The low TLI's of prostatic adenocarcinomas relative to other malignancies are consistent with their frequently slowly progressive course. The very low proliferative rate of seminal vesicular epithelium (mean TLI 0.02 percent) may account for the rarity of seminal vesicular carcinomas

  20. Proliferative activity of benign human prostate, prostatic adenocarcinoma and seminal vesicle evaluated by thymidine labeling

    International Nuclear Information System (INIS)

    Meyer, J.S.; Sufrin, G.; Martin, S.A.

    1982-01-01

    The thymidine labeling index (TLI) was measured in vitro in the epithelium and stroma of benign prostate glands and seminal vesicles and in the epithelium of prostatic adenocarcinomas. The mean epithelial TLI of normal peripheral (posterior) prostatic zone was 0.12 per cent, and that of the normal central (deep) zone was 0.11 per cent. Mean normal stromal TLI's were 0.08 per cent and 0.06 per cent, respectively. The mean TLI of epithelium in nodular hyperplasia was 0.31 per cent, which differs significantly from normal epithelium, and the mean stromal TLI was also increased. The mean TLI of prostatic adenocarcinomas was 0.90 per cent (range 0.14 to 3.90 per cent) which was significantly higher than for either normal epithelium or epithelium of nodular hyperplasia. Trends of increasing TLI with increasing histologic grades and increasing nuclear size and numbers of nucleoli were not significant. The data support participation of both epithelial and stromal proliferation in nodular hyperplasia, and indicate a low basal proliferative rate in normal prostatic glands. The low TLI's of prostatic adenocarcinomas relative to other malignancies are consistent with their frequently slowly progressive course. The very low proliferative rate of seminal vesicular epithelium may account for the rarity of seminal vesicular carcinomas

  1. Fluorescence and computational studies of thymidine phosphorylase affinity toward lipidated 5-FU derivatives

    Science.gov (United States)

    Lettieri, R.; D'Abramo, M.; Stella, L.; La Bella, A.; Leonelli, F.; Giansanti, L.; Venanzi, M.; Gatto, E.

    2018-04-01

    Thymidine phosphorylase (TP) is an enzyme that is up-regulated in a wide variety of solid tumors, including breast and colorectal cancers. It is involved in tumor growth and metastasis, for this reason it is one of the key enzyme to be inhibited, in an attempt to prevent tumor proliferation. However, it also plays an active role in cancer treatment, through its contribution in the conversion of the anti-cancer drug 5-fluorouracil (5-FU) to an irreversible inhibitor of thymidylate synthase (TS), responsible of the inhibition of the DNA synthesis. In this work, the intrinsic TP fluorescence has been investigated for the first time and exploited to study TP binding affinity for the unsubstituted 5-FU and for two 5-FU derivatives, designed to expose this molecule on liposomal membranes. These molecules were obtained by functionalizing the nitrogen atom with a chain consisting of six (1) or seven (2) units of glycol, linked to an alkyl moiety of 12 carbon atoms. Derivatives (1) and (2) exhibited an affinity for TP in the micromolar range, 10 times higher than the parent compound, irrespective of the length of the polyoxyethylenic spacer. This high affinity was maintained also when the compounds were anchored in liposomal membranes. Experimental results were supported by molecular dynamics simulations and docking calculations, supporting a feasible application of the designed supramolecular lipid structure in selective targeting of TP, to be potentially used as a drug delivery system or sensor device.

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

    International Nuclear Information System (INIS)

    Uyeki, E.M.; Wierzba, K.; Bisel, T.U.

    1981-01-01

    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. 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)

    Habdas, H.

    1977-01-01

    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 3 H-thymidine to hydroxyurea solutions. In presence of hydroxyurea there was noted very intense incorporation of 3 H-thymidine into cell nuclei, giving labelling index of 40-70%. However, all the mitotic figures appearing in presence of hydroxyurea and 3 H-thymidine were unlabelled. On the other hand, labelled mitotic figures were obtained when roots incubated with 3 H-thymidine in presence of hydroxyurea had been transferred to water. Incorporation of 3 H-uridine was unaffected by hydroxyurea. The results show that hydroxyurea arrests onion root meristematic cells, either in the S phase and the G 2 phase. Enhanced incorporation of 3 H-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)

  4. Bacterial Ecology

    DEFF Research Database (Denmark)

    Fenchel, Tom

    2011-01-01

    compounds these must first be undergo extracellular hydrolysis. Bacteria have a great diversity with respect to types of metabolism that far exceeds the metabolic repertoire of eukaryotic organisms. Bacteria play a fundamental role in the biosphere and certain key processes such as, for example......, the production and oxidation of methane, nitrate reduction and fixation of atmospheric nitrogen are exclusively carried out by different groups of bacteria. Some bacterial species – ‘extremophiles’ – thrive in extreme environments in which no eukaryotic organisms can survive with respect to temperature, salinity...... biogeochemical processes are carried exclusively by bacteria. * Bacteria play an important role in all types of habitats including some that cannot support eukaryotic life....

  5. Bacterial Actins.

    Science.gov (United States)

    Izoré, Thierry; van den Ent, Fusinita

    2017-01-01

    A diverse set of protein polymers, structurally related to actin filaments contributes to the organization of bacterial cells as cytomotive or cytoskeletal filaments. This chapter describes actin homologs encoded by bacterial chromosomes. MamK filaments, unique to magnetotactic bacteria, help establishing magnetic biological compasses by interacting with magnetosomes. Magnetosomes are intracellular membrane invaginations containing biomineralized crystals of iron oxide that are positioned by MamK along the long-axis of the cell. FtsA is widespread across bacteria and it is one of the earliest components of the divisome to arrive at midcell, where it anchors the cell division machinery to the membrane. FtsA binds directly to FtsZ filaments and to the membrane through its C-terminus. FtsA shows altered domain architecture when compared to the canonical actin fold. FtsA's subdomain 1C replaces subdomain 1B of other members of the actin family and is located on the opposite side of the molecule. Nevertheless, when FtsA assembles into protofilaments, the protofilament structure is preserved, as subdomain 1C replaces subdomain IB of the following subunit in a canonical actin filament. MreB has an essential role in shape-maintenance of most rod-shaped bacteria. Unusually, MreB filaments assemble from two protofilaments in a flat and antiparallel arrangement. This non-polar architecture implies that both MreB filament ends are structurally identical. MreB filaments bind directly to membranes where they interact with both cytosolic and membrane proteins, thereby forming a key component of the elongasome. MreB filaments in cells are short and dynamic, moving around the long axis of rod-shaped cells, sensing curvature of the membrane and being implicated in peptidoglycan synthesis.

  6. Contribution of heterotrophic bacterial production to the carbon budget of the river Seine (France).

    Science.gov (United States)

    Servais, P; Garnier, J

    1993-01-01

    Bacterial activity was measured in the river Seine by two methods, (3)H-thymidine incorporation into DNA and (3)H-leucine incorporation into proteins. Both incorporation rates are characterized by low values upstream of Paris, a large increase just downstream of the outfall of the Achères treatment plant effluents, and then decreasing values further downstream. The covariation of both activities is demonstrated by the constancy of the molar ratio (leucine to thymidine incorporation rate) in the range of 6 to 8 for all the samples, except in the perturbed area where it is higher (15 to 35). These high values of molar ratio are linked to the introduction into the river of large sized bacteria ([Symbol: see text]1 µm) with higher incorporation rates per cell or biomass unit than the small autochthonous bacteria (rates of large bacteria were on average 3.7 times higher than those of small bacteria. Bacterial production was calculated with experimentally determined conversion factors (0.5 × 10(18) cells per mole of thymidine incorporated and 900 gC per mole of leucine incorporated) and by taking into account the activity of both size classes of bacteria measured through fractionation experiments (post-incubation filtration). Production estimated in the perturbed area downstream of Ach6res was very high, up to 60 µgC liter(-1)h(-1) in the summer. Carbon consumption by bacteria in the area perturbed by the Ach6res effluents was calculated assuming a growth yield of 0.2 and compared to the load of biodegradable organic matter discharged by the treatment plant. In summer, an additional supply of organic matter is required to account for the intense bacterial activity, suggesting the importance of phytoplankton production in the carbon budget.

  7. Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

    Science.gov (United States)

    Choi, Mal-Gi; Kurnov, Vladlen; Kersting, Michael C.; Sreenivas, Avula; Carman, George M.

    2005-01-01

    The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work, we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent, and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSS25QRRHS (Vmax/Km = 17.5 mM-1 μmol min-1 mg-1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo, the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHS30LTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mM−1 μmol min−1 mg−1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PMID:15919656

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

    International Nuclear Information System (INIS)

    Walle, A.J.

    1975-01-01

    After intravenous injection of 0.1 μCi/g body weight 3 H-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.) [de

  9. Enterococcus faecalis phosphomevalonate kinase

    Science.gov (United States)

    Doun, Stephanie S.; Burgner, John W.; Briggs, Scott D.; Rodwell, Victor W.

    2005-01-01

    The six enzymes of the mevalonate pathway of isopentenyl diphosphate biosynthesis represent potential for addressing a pressing human health concern, the development of antibiotics against resistant strains of the Gram-positive streptococci. We previously characterized the first four of the mevalonate pathway enzymes of Enterococcus faecalis, and here characterize the fifth, phosphomevalonate kinase (E.C. 2.7.4.2). E. faecalis genomic DNA and the polymerase chain reaction were used to clone DNA thought to encode phosphomevalonate kinase into pET28b(+). Double-stranded DNA sequencing verified the sequence of the recombinant gene. The encoded N-terminal hexahistidine-tagged protein was expressed in Escherichia coli with induction by isopropylthiogalactoside and purified by Ni++ affinity chromatography, yield 20 mg protein per liter. Analysis of the purified protein by MALDI-TOF mass spectrometry established it as E. faecalis phosphomevalonate kinase. Analytical ultracentrifugation revealed that the kinase exists in solution primarily as a dimer. Assay for phosphomevalonate kinase activity used pyruvate kinase and lactate dehydrogenase to couple the formation of ADP to the oxidation of NADH. Optimal activity occurred at pH 8.0 and at 37°C. The activation energy was ~5.6 kcal/mol. Activity with Mn++, the preferred cation, was optimal at about 4 mM. Relative rates using different phosphoryl donors were 100 (ATP), 3.6 (GTP), 1.6 (TTP), and 0.4 (CTP). Km values were 0.17 mM for ATP and 0.19 mM for (R,S)-5-phosphomevalonate. The specific activity of the purified enzyme was 3.9 μmol substrate converted per minute per milligram protein. Applications to an immobilized enzyme bioreactor and to drug screening and design are discussed. PMID:15802646

  10. 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...

  11. Tyrosine-610 in the receptor kinase BAK1 does not play a major role in brassinosteroid signaling or innate immunity

    Science.gov (United States)

    The plasma membrane-localized BRI1-ASSOCIATED KINASE1 (BAK1) functions as a co-receptor with several receptor kinases including the brassinosteroid (BR) receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1), which is involved in growth, and the receptors for bacterial flagellin and EF-Tu, FLAGELLIN-SENSING ...

  12. Type IV pilins regulate their own expression via direct intramembrane interactions with the sensor kinase PilS

    OpenAIRE

    Kilmury, Sara L. N.; Burrows, Lori L.

    2016-01-01

    Although two-component systems are a ubiquitous means of rapid bacterial adaptation to changing environments, identification of the specific signals detected by sensor kinases can be challenging. Also, little is known about the diverse, poorly characterized family of sensor kinases that detect intramembrane signals. We show that the major type IV pilin, PilA, is an inhibitory intramembrane ligand for the PilS sensor kinase that controls pilA expression and we characterize the mechanism of sig...

  13. In Vitro Metabolism of H{sup 3} Thymidine; Metabolisme In Vitro de la Thymidine Tritiee; 041c 0435 0414 ''In Vitro''; Metabolismo In Vitro de la Timidina Tritiada

    Energy Technology Data Exchange (ETDEWEB)

    Rubini, J. R.; Keller, S.; Eisentraut, A. [Veterans Administration Hospital and Southwestern Medical School, Dallas, TX (United States); Cronkite, E. P. [Brookhaven National Laboratory, Upton, Long Island, NY (United States)

    1962-02-15

    The in vitro metabolism and fate of the DNA precursor H{sup 3} thymidine, H{sup 3}TDR, (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 H{sup 3}TDR 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 H{sup 3}TDR incorporation period. Supernatants at 1 h still contained considerable H{sup 3} activity yet failed to label fresh added cells. These ''used'' supernatants contained H{sup 3}TDR as the major H{sup 3} compound, and only small amounts of H{sup 3} thymine; H{sup 3} water was not formed. Speculations on the mechanisms involved are presented. (author) [French] Le metabolisme et le sort de la thymidine tritiee (1,9 curie/millimole), precurseur de l'ADN, ont ete etudies in vitro dans le sang leucemique humain et la moelle normale du chien. On a evalue la nouvelle synthese de l'ADN en determinant les indices de marquage et en comptant les grains d'autoradiogrammes de cellules effectues a partir de melanges au bout d'une heure. On a pu facilement reduire le marquage des cellules en ajoutant initialement a la thymidine tritiee d'infimes quantites de thymidine non marquee, ce qui met en evidence le faible volume du 'pool' de thymidine. Lorsque la thymidine est ajoutee au melange d'incubation apres 20 minutes, il n'y a pas de diminution du marquage. Cette periode de 20 minutes correspond au delai d'incorporation de la thymidine tritiee. Apres une heure, on constate que les surnageants contiennent encore du tritium tres actif, mais qui ne permet pas de marquer des cellules nouvelles. Les principal composant tritie de ces surnageants 'uses' est la thymidine tritiee; on n'y trouve que de

  14. Bacterial biomass and activity in the deep waters of the eastern Atlantic—evidence of a barophilic community

    Science.gov (United States)

    Patching, J. W.; Eardly, D.

    1997-09-01

    Bacterial biomass and activity were investigated in deep waters at two sites in the eastern Atlantic, of similar depth (4560-4800 m), but varying in their nutritional status. The Northern (N) site was eutrophic and subject to a strong seasonal input of surface derived organic matter (phytodetritus) to the sediment. The Southern (S) site was oligotrophic. Deep water at this site does not appear to receive any strong seasonal input. Bacterial numbers in the deep water column at the N site showed no significant seasonal variation but were greater than those at the S site. Deep water bacteria were typically small and free-living. From biovolume determinations, it was estimated that mean concentrations of bacterial organic carbon at depths greater than 500 m were 0.12 (0.03-0.29) μg C 1 -1 and 0.02 (0.01-0.04) μg C 1 -1 at the N and S sites, respectively. Rates of thymidine and leucine incorporation were used as indicators of bacterial activity. Bacterial communities in water in contact with the sediment (SCW; sediment contact water) at both sites (but especially at the S site) were strongly barophilic at in situ temperatures (2.5-4.1°C). The barophilic response of thymidine incorporation was enhanced when SCW samples from the N site were incubated at 11.5°C. It is proposed that this result indicated an elevating effect of pressure on cardinal temperatures and that the SCW community was obligately psychrophilic when unpressurised. Comparison of cell-specific incorporation rates determined under in situ conditions showed bacteria in the SCW to have levels of activity comparable with bacteria from a depth of 150 m. Thymidine incorporation rates were highest in SCW samples taken at the N site in May 1988 and September 1989. Thymidine incorporation by SCW samples taken immediately before (10 April 1994) the main spring-bloom-associated deposition of phytodetritus was significantly lower and comparable with that determined for the oligotrophic S site. The attributes

  15. Hemicastration causes and testosterone prevents enhanced uptake of [3H]thymidine by Sertoli cells in testes of immature rats

    International Nuclear Information System (INIS)

    Orth, J.M.; Higginbotham, C.A.; Salisbury, R.L.

    1984-01-01

    Rat pups were hemicastrated and uptake of [ 3 H]thymidine by Sertoli cells in the remaining testis was compared to that in testes of sham-operated pups at intervals of from 8 h to 21 days after surgery. Labeled thymidine was administered subcutaneously 2 h before sacrifice. Testes were processed for light microscope autoradiography and the percent of Sertoli cell nuclei that had incorporated [ 3 H]thymidine was determined by scoring nuclei in tissue sections as labeled or unlabeled. The percentage of cells labeled was increased in hemicastrates over intact controls by 8 h after surgery and testicular hypertrophy became apparent in hemicastrates by the following day. Labeling of Sertoli cells in hemicastrates remained elevated for 4 days and then returned to normal. When plasma levels of gonadotropins were measured in both groups 4 days after surgery, follicle-stimulating hormone (FSH) was found to be more than twice normal in hemicastrates while luteinizing hormone (LH) was unchanged. The effect of testosterone on the response of Sertoli cells to hemicastration was also examined. In hemicastrates, 2 days of androgen therapy depressed, and an additional 2 days abolished, the proliferative response of the Sertoli cells. Our findings suggest that increased proliferation of Sertoli cells within the remaining testis is involved in the enlargement of the testis that follows hemicastration. They also imply that prevention of compensatory hypertrophy by testosterone involves interference with this response of Sertoli cells in some way. Finally, our data implicate FSH in control of Sertoli cell proliferation in vivo in immature rats

  16. Kinetic Analysis of 2-[11C]Thymidine PET Imaging Studies of Malignant Brain Tumors: Compartmental Model Investigation and Mathematical Analysis

    Directory of Open Access Journals (Sweden)

    Joanne M. Wells

    2002-07-01

    Full Text Available 2-[11C]Thymidine (TdR, a PET tracer for cellular proliferation, may be advantageous for monitoring brain tumor progression and response to therapy. We previously described and validated a five-compartment model for thymidine incorporation into DNA in somatic tissues, but the effect of the blood–brain barrier on the transport of TdR and its metabolites necessitated further validation before it could be applied to brain tumors. Methods: We investigated the behavior of the model under conditions experienced in the normal brain and brain tumors, performed sensitivity and identifiability analysis to determine the ability of the model to estimate the model parameters, and conducted simulations to determine whether it can distinguish between thymidine transport and retention. Results: Sensitivity and identifiability analysis suggested that the non-CO2 metabolite parameters could be fixed without significantly affecting thymidine parameter estimation. Simulations showed that K1t and KTdR could be estimated accurately (r = .97 and .98 for estimated vs. true parameters with standard errors < 15%. The model was able to separate increased transport from increased retention associated with tumor proliferation. Conclusion: Our model adequately describes normal brain and brain tumor kinetics for thymidine and its metabolites, and it can provide an estimate of the rate of cellular proliferation in brain tumors.

  17. Patterns of labeling of intraspinal reactive cells in rats injected with [3H]thymidine prior to or following sciatic axotomy

    International Nuclear Information System (INIS)

    Gilmore, S.A.; Walls, R.C.

    1981-01-01

    Labeling patterns of reactive cells which occur in the spinal cord following sciatic axotomy were investigated by autoradiography following administration of [ 3 H]thymidine (2 μCi/g body weight/injection). In this investigation the labeling patterns in reactive cells were compared when [ 3 H]thymidine was injected: (1) prior to or (2) following surgery. Immature rats underwent sciatic axotomy and were killed 3 days later (20 days of age). Some of their littermates served as sham-operated controls, and others were killed on the day of surgery to evaluate intraspinal labeling at that time. In one series of animals, a single injection of [ 3 H]thymidine was administered 2 h prior to autopsy. This procedure resulted in heavy labeling of 6.2% of the cells classified as reactive cells. In the second series, 3 injections of [ 3 H]thymidine were given on the day prior to surgery. On the third post-operative day, 19.4% of the reactive cells were labeled, and the majority of these were lightly labeled, suggesting that they had undergone several cell divisions. The data from both injection protocols indicate that the magnitude of the cellular response to axotomy would be markedly underestimated, if one were to consider only labeled cells. The present investigators concluded that these particular applications of [ 3 H]thymidine autoradiography provide valuable information on reactions of the central nervous system to injury but are of little value in determining origins of the reactive cells. (Auth.)

  18. Characterization of genome-reduced Bacillus subtilis strains and their application for the production of guanosine and thymidine.

    Science.gov (United States)

    Li, Yang; Zhu, Xujun; Zhang, Xueyu; Fu, Jing; Wang, Zhiwen; Chen, Tao; Zhao, Xueming

    2016-06-03

    Genome streamlining has emerged as an effective strategy to boost the production efficiency of bio-based products. Many efforts have been made to construct desirable chassis cells by reducing the genome size of microbes. It has been reported that the genome-reduced Bacillus subtilis strain MBG874 showed clear advantages for the production of several heterologous enzymes including alkaline cellulase and protease. In addition to enzymes, B. subtilis is also used for the production of chemicals. To our best knowledge, it is still unknown whether genome reduction could be used to optimize the production of chemicals such as nucleoside products. In this study, we constructed a series of genome-reduced strains by deleting non-essential regions in the chromosome of B. subtilis 168. These strains with genome reductions ranging in size from 581.9 to 814.4 kb displayed markedly decreased growth rates, sporulation ratios, transformation efficiencies and maintenance coefficients, as well as increased cell yields. We re-engineered the genome-reduced strains to produce guanosine and thymidine, respectively. The strain BSK814G2, in which purA was knocked out, and prs, purF and guaB were co-overexpressed, produced 115.2 mg/L of guanosine, which was 4.4-fold higher compared to the control strain constructed by introducing the same gene modifications into the parental strain. We also constructed a thymidine producer by deleting the tdk gene and overexpressing the prs, ushA, thyA, dut, and ndk genes from Escherichia coli in strain BSK756, and the resulting strain BSK756T3 accumulated 151.2 mg/L thymidine, showing a 5.2-fold increase compared to the corresponding control strain. Genome-scale genetic manipulation has a variety of effects on the physiological characteristics and cell metabolism of B. subtilis. By introducing specific gene modifications related to guanosine and thymidine accumulation, respectively, we demonstrated that genome-reduced strains had greatly improved

  19. 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

  20. Suppression of leukocyte inhibitory factor (LIF) production and [3H]thymidine incorporation by concanavalin A-activated mononuclear cells

    International Nuclear Information System (INIS)

    Lomnitzer, R.; Rabson, A.R.

    1979-01-01

    The capacity of human mononuclear (MN) cells pretreated with concanavalin A (Con A) to suppress the activity of fresh phytohemagglutinin (PHA)-pulsed mononuclear cells was assessed. Con A-pretreated MN cells suppressed leukocyte inhibitory factor (LIF) activity in supernatants of PHA-pulsed cell cultures and [ 3 H]thymidine incorporation by these cells. Suppression was obtained in both allogeneic and autologous systems with mitomycin-treated, irradiated, or untreated Con A-induced cells. Lymphocytes from two patients that, following treatment with Con A, did not suppress mitogen-induced proliferative response of normal cells also did not suppress LIF production

  1. Stimulated mitogen-activated protein kinase is necessary but not sufficient for the mitogenic response to angiotensin II. A role for phospholipase D.

    Science.gov (United States)

    Wilkie, N; Morton, C; Ng, L L; Boarder, M R

    1996-12-13

    Activation of the mitogen-activated protein kinase (MAPK) cascade has been widely associated with cell proliferation; previous studies have shown that angiotensin II (AII), acting on 7-transmembrane G protein-coupled receptors, stimulates the MAPK pathway. In this report we investigate whether the MAPK pathway is required for the mitogenic response to AII stimulation of vascular smooth muscle cells derived from the hypertensive rat (SHR-VSM). AII stimulates the phosphorylation of MAPK, as determined by Western blot specific for the tyrosine 204 phosphorylated form of the protein. This MAPK phosphorylation was inhibited by the presence of the inhibitor of MAPK kinase activation, PD 098059. Using a peptide kinase assay shown to measure the p42 and p44 isoforms of MAPK, the stimulated response to AII was inhibited by PD 098059 with an IC50 of 15.6 +/- 1.6 microM. The AII stimulation of [3H]thymidine incorporation was inhibited by PD 098059 with an IC50 of 17.8 +/- 3.1 microM. PD 098059 had no effect on AII-stimulated phospholipase C or phospholipase D (PLD) activity. When the SHR-VSM cells were stimulated with phorbol ester, there was an activation of MAPK similar in size and duration to the response to AII, but there was no significant enhancement of [3H]thymidine incorporation. There was also no activation of PLD by phorbol ester, while AII produced a robust PLD response. Diversion of the product of the PLD reaction by 1-butanol caused a partial loss of the [3H]thymidine response; this did not occur with tertiary butanol, which did not interfere with the PLD reaction. These results show that in these cells the MAPK cascade is required but not sufficient for the mitogenic response to AII, and suggest that the full mitogenic response requires both MAPK in conjunction with other signaling components, one of which is PLD.

  2. A two emulsion autoradiographic technique and the discriminating of the three different types of labelling after double labelling with 3H- and 14C-thymidine

    International Nuclear Information System (INIS)

    Schultze, B.; Maurer, W.; Hagenbusch, H.

    1976-01-01

    The first part of the paper deals with a two emulsion autoradiographic technique for double labelling experiments with 3 H- and 14 C-thymidine which permits a clear discrimination of the different types of labelling. In the second part the application of this technique to cell kinetics studies is discussed. Accurate discrimination between the different types of labelling, namely purely 3 H-, purely 14 C- and double ( 3 H + 14 C) labelling, is only possible if the activity ratio of 3 H- to 14 C-thymidine is sufficiently high. This condition is necessary for a reliable distinction between those grains in the first emulsion which are due to true 3 H-labelling and spurious grains which are simultaneously produced in the same emulsion by 14 C-β- particles. Experiments are described to determine the required activity ratio of 3 H- to 14 C-thymidine. (author)

  3. 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.

  4. The Amoebal MAP Kinase Response to Legionella pneumophila Is Regulated by DupA

    OpenAIRE

    Li, Zhiru; Dugan, Aisling S.; Bloomfield, Gareth; Skelton, Jason; Ivens, Alasdair; Losick, Vicki; Isberg, Ralph R.

    2009-01-01

    The amoeba Dictyostelium discoideum can support replication of Legionella pneumophila. Here we identify the dupA gene, encoding a putative tyrosine kinase/dual-specificity phosphatase, in a screen for D. discoideum mutants altered in allowing L. pneumophila intracellular replication. Inactivation of dupA resulted in depressed L. pneumophila growth and sustained hyperphosphorylation of the amoebal MAP kinase ERK1, consistent with loss of a phosphatase activity. Bacterial challenge of wild-type...

  5. 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.

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

    International Nuclear Information System (INIS)

    Barra, R.; Beres, B.; Koch, M.R.; Lea, M.A.

    1976-01-01

    The effects of exogenous proteins on the incorporation of [ 3 H]-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 9618A 2 , 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 [ 3 H]-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)

  7. Lipofection of early passages of cell cultures derived from murine adenocarcinomas: in vitro and ex vivo testing of the thymidine kinase/ganciclovir system.

    Science.gov (United States)

    Karara, Armando L; Bumaschny, Viviana F; Fiszman, Gabriel L; Casais, Cecilia C; Glikin, Gerardo C; Finocchiaro, Liliana Me

    2002-01-01

    Early passages of cultured cells derived from four spontaneous Balb/c murine adenocarcinomas were used to explore the feasibility of a nonviral HSVtk-based suicide gene therapy system. After lipofection with pCMVtk, the transiently HSVtk expressing P07 (lung), M3, M05, and M38 (mammary gland) cells were, respectively, about 130-, 30-, 120-, and 170-fold more sensitive to ganciclovir (GCV) in vitro than their respective controls. Eighty percent of Balb/c mice subcutaneously inoculated with ex vivo pCMVtk-lipofected P07 cells, followed by intraperitoneal GCV injection for 7 days, displayed a complete inhibition of tumor growth for over 70 days. Control animals started to display tumors 13 days after inoculation. We present evidence showing that early passages of cultured tumor cells can efficiently express lipofected genes and that they are sensitive to the lipoplex-mediated HSVtk/GCV system.

  8. Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia.

    Science.gov (United States)

    Roth Flach, Rachel J; Danai, Laura V; DiStefano, Marina T; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K; Bortell, Rita; Alonso, Laura C; Czech, Michael P

    2016-07-29

    Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    International Nuclear Information System (INIS)

    Tong, W.-G.; Ding, X.-Z.; Talamonti, Mark S.; Bell, Richard H.; Adrian, Thomas E.

    2005-01-01

    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

  10. Regulation of Autophagy by Kinases

    International Nuclear Information System (INIS)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda

    2011-01-01

    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. Regulation of Autophagy by Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda, E-mail: alakananda.basu@unthsc.edu [Department of Molecular Biology and Immunology, Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2011-06-09

    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.

  12. Regulation of Autophagy by Kinases

    Science.gov (United States)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda

    2011-01-01

    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. PMID:24212825

  13. Regulation of Autophagy by Kinases

    Directory of Open Access Journals (Sweden)

    Savitha Sridharan

    2011-06-01

    Full Text Available 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 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.

  14. An improved method to unravel phosphoacceptors in Ser/Thr protein kinase-phosphorylated substrates.

    Science.gov (United States)

    Molle, Virginie; Leiba, Jade; Zanella-Cléon, Isabelle; Becchi, Michel; Kremer, Laurent

    2010-11-01

    Identification of the phosphorylated residues of bacterial Ser/Thr protein kinase (STPK) substrates still represents a challenging task. Herein, we present a new strategy allowing the rapid determination of phosphoacceptors in kinase substrates, essentially based on the dual expression of the kinase with its substrate in the surrogate E. coli, followed by MS analysis in a single-step procedure. The performance of this strategy is illustrated using two distinct proteins from Mycobacterium tuberculosis as model substrates, the GroEL2 and HspX chaperones. A comparative analysis with a standard method that includes mass spectrometry analysis of in vitro phosphorylated substrates is also addressed.

  15. Deoxyribonucleoside kinases activate nucleoside antibiotics in severely pathogenic bacteria

    DEFF Research Database (Denmark)

    Sandrini, Michael; Shannon, O.; Clausen, A.R.

    2007-01-01

    Common bacterial pathogens are becoming progressively more resistant to traditional antibiotics, representing a major public-health crisis. Therefore, there is a need for a variety of antibiotics with alternative modes of action. In our study, several nucleoside analogs were tested against pathog...... alternative for combating pathogenic bacteria.......Common bacterial pathogens are becoming progressively more resistant to traditional antibiotics, representing a major public-health crisis. Therefore, there is a need for a variety of antibiotics with alternative modes of action. In our study, several nucleoside analogs were tested against...... pathogenic staphylococci and streptococci. We show that pyrimidine-based nucleoside analogs, like 3'-azido-3'-deoxythymidine (AZT) and 2',2'-difluoro-2'deoxycytidine (gemcitabine), are specifically activated by the endogenous bacterial deoxyribonucleoside kinases, leading to cell death. Deoxyribonucleoside...

  16. 3H thymidine an indicator of benzo(a)pyrene induced lung carcinogenesis: role of quercetin and curcumin

    International Nuclear Information System (INIS)

    Nair, Parveen; Malhotra, A.; Dhawan, D.K.

    2010-01-01

    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 3 H 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

  17. 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)

    Sanders, R.W.; Porter, K.G.

    1986-01-01

    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 [ 3 H]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

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

    Directory of Open Access Journals (Sweden)

    Matthew G Cottingham

    2008-02-01

    Full Text Available The production, manipulation and rescue of a bacterial artificial chromosome clone of Vaccinia virus (VAC-BAC in order to expedite construction of expression vectors and mutagenesis of the genome has been described (Domi & Moss, 2002, PNAS99 12415-20. The genomic BAC clone was 'rescued' back to 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-length, rescuable clones were obtained, which had indistinguishable immunogenicity in mice. One clone was shotgun sequenced and found to be identical to the parent. We employed GalK recombination-mediated genetic engineering (recombineering of MVA-BAC to delete five selected viral genes. Deletion of C12L, A44L, A46R or B7R did not significantly affect CD8(+ T cell immunogenicity in BALB/c mice, but deletion of B15R enhanced specific CD8(+ T cell responses to one of two endogenous viral epitopes (from the E2 and F2 proteins, in accordance with published work (Staib et al., 2005, J. Gen. Virol.86, 1997-2006. In addition, we found a higher frequency of triple-positive IFN-gamma, TNF-alpha and IL-2 secreting E3-specific CD8+ T-cells 8 weeks after vaccination with MVA lacking B15R. Furthermore, a recombinant vaccine capable of inducing CD8(+ T cells against an epitope from Plasmodium berghei was created 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 demonstrate in proof-of-concept experiments that MVA-BAC recombineering is a viable route to more rapid and efficient generation of new candidate mutant and recombinant

  19. 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

    2008-01-01

    to 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...

  20. Long-term foscarnet therapy remodels thymidine analogue mutations and alters resistance to zidovudine and lamivudine in HIV-1

    DEFF Research Database (Denmark)

    Mathiesen, Sofie; Dam, Elisabeth; Roge, Birgit

    2007-01-01

    OBJECTIVE: To study the evolution of multi-drug-resistant HIV-1 in treatment-experienced patients receiving foscarnet (PFA) as part of salvage therapy and to investigate the virological consequences of emerging mutations. METHODS: Genotypic and phenotypic resistance tests were performed on plasma...... viruses from seven patients at baseline and during treatment with PFA. The phenotypic effects of mutations suspected to be associated with PFA resistance were evaluated by site-directed mutagenesis of wild-type or thymidine analogue mutations (TAM)-carrying pNL4-3. Reversion of single mutations...... was performed in a patient-derived recombinant clone. RESULTS: Baseline multi-drug-resistant isolates exhibited hypersusceptibility to PFA. In two patients who received > 12 months of PFA treatment, a novel mutation pattern including K70G, V75T, K219R and L228R emerged. These viruses had 3-6-fold resistance...

  1. Radiotoxicity and incorporation of methyl-tritiated-thymidine on preimplantation mouse embryo. In vitro fertilization and culture

    International Nuclear Information System (INIS)

    Kikuchi, O.K.; Ohyama, H.; Yamada, T.

    1993-04-01

    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 37 0 C in a humidified atmosphere with 5% of CO 2 for four days up to the expanded blastocyst stage, the established end point to calculate the L D 50 lethal dose. The blastocyst formation rate decreased with increasing concentration of tritium in medium and a value of 2.4 X 10 3 Bq/ml for L D 50 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)

  2. 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)

    Meerman, J.H.; Smith, T.R.; Pearson, P.G.; Meier, G.P.; Nelson, S.D.

    1989-01-01

    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

  3. 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)

    Wang, R.T.; Halpern, M.

    1988-01-01

    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

  4. 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.

  5. Receptor-interacting protein (RIP) kinase family

    OpenAIRE

    Zhang, Duanwu; Lin, Juan; Han, Jiahuai

    2010-01-01

    Receptor-interacting protein (RIP) kinases are a group of threonine/serine protein kinases with a relatively conserved kinase domain but distinct non-kinase regions. A number of different domain structures, such as death and caspase activation and recruitment domain (CARD) domains, were found in different RIP family members, and these domains should be keys in determining the specific function of each RIP kinase. It is known that RIP kinases participate in different biological processes, incl...

  6. 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...... than traditional screening technology....

  7. Isolation and characterization of recombinant human casein kinase II subunits alpha and beta from bacteria

    DEFF Research Database (Denmark)

    Grankowski, N; Boldyreff, B; Issinger, O G

    1991-01-01

    cDNA encoding the casein kinase II (CKII) subunits alpha and beta of human origin were expressed in Escherichia coli using expression vector pT7-7. Significant expression was obtained with E. coli BL21(DE3). The CKII subunits accounted for approximately 30% of the bacterial protein; however, most...

  8. Bacterial Standing Stock, Activity, and Carbon Production during Formation and Growth of Sea Ice in the Weddell Sea, Antarctica.

    Science.gov (United States)

    Grossmann, S; Dieckmann, G S

    1994-08-01

    Bacterial response to formation and growth of sea ice was investigated during autumn in the northeastern Weddell Sea. Changes in standing stock, activity, and carbon production of bacteria were determined in successive stages of ice development. During initial ice formation, concentrations of bacterial cells, in the order of 1 x 10 to 3 x 10 liter, were not enhanced within the ice matrix. This suggests that physical enrichment of bacteria by ice crystals is not effective. Due to low concentrations of phytoplankton in the water column during freezing, incorporation of bacteria into newly formed ice via attachment to algal cells or aggregates was not recorded in this study. As soon as the ice had formed, the general metabolic activity of bacterial populations was strongly suppressed. Furthermore, the ratio of [H]leucine incorporation into proteins to [H]thymidine incorporation into DNA changed during ice growth. In thick pack ice, bacterial activity recovered and growth rates up to 0.6 day indicated actively dividing populations. However, biomass-specific utilization of organic compounds remained lower than in open water. Bacterial concentrations of up to 2.8 x 10 cells liter along with considerably enlarged cell volumes accumulated within thick pack ice, suggesting reduced mortality rates of bacteria within the small brine pores. In the course of ice development, bacterial carbon production increased from about 0.01 to 0.4 mug of C liter h. In thick ice, bacterial secondary production exceeded primary production of microalgae.

  9. Measurement of the incorporation rates of four amino acids into proteins for estimating bacterial production.

    Science.gov (United States)

    Servais, P

    1995-03-01

    In aquatic ecosystems, [(3)H]thymidine incorporation into bacterial DNA and [(3)H]leucine incorporation into proteins are usually used to estimate bacterial production. The incorporation rates of four amino acids (leucine, tyrosine, lysine, alanine) into proteins of bacteria were measured in parallel on natural freshwater samples from the basin of the river Meuse (Belgium). Comparison of the incorporation into proteins and into the total macromolecular fraction showed that these different amino acids were incorporated at more than 90% into proteins. From incorporation measurements at four subsaturated concentrations (range, 2-77 nm), the maximum incorporation rates were determined. Strong correlations (r > 0.91 for all the calculated correlations) were found between the maximum incorporation rates of the different tested amino acids over a range of two orders of magnitude of bacterial activity. Bacterial production estimates were calculated using theoretical and experimental conversion factors. The productions calculated from the incorporation rates of the four amino acids were in good concordance, especially when the experimental conversion factors were used (slope range, 0.91-1.11, and r > 0.91). This study suggests that the incorporation of various amino acids into proteins can be used to estimate bacterial production.

  10. Bacterial lung abscess

    International Nuclear Information System (INIS)

    Groskin, S.A.; Panicek, D.M.; Ewing, D.K.; Rivera, F.; Math, K.; Teixeira, J.; Heitzman, E.R.

    1987-01-01

    A retrospective review of patients with bacterial lung abscess was carried out. Demographic, clinical, and radiographical features of this patient group are compared with similar data from patients with empyema and/or cavitated lung carcinoma; differential diagnostic points are stressed. The entity of radiographically occult lung abscess is discussed. Complications associated with bacterial lung abscess are discussed. Current therapeutic options and treatment philosophy for patients with bacterial lung abscess are noted

  11. Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Petranovic, Dina; Macek, B

    2006-01-01

    for phosphotyrosine-containing proteins in Streptomyces griseus by immunoaffinity chromatography identified bacterial SSBs as a novel target of bacterial tyrosine kinases. Since genes encoding protein-tyrosine kinases (PTKs) have not been recognized in streptomycetes, and SSBs from Streptomyces coelicolor (Sc......SSB) and Bacillus subtilis (BsSSB) share 38.7% identity, we used a B.subtilis protein-tyrosine kinase YwqD to phosphorylate two cognate SSBs (BsSSB and YwpH) in vitro. We demonstrate that in vivo phosphorylation of B.subtilis SSB occurs on tyrosine residue 82, and this reaction is affected antagonistically...... by kinase YwqD and phosphatase YwqE. Phosphorylation of B.subtilis SSB increased binding almost 200-fold to single-stranded DNA in vitro. Tyrosine phosphorylation of B.subtilis, S.coelicolor and Escherichia coli SSBs occured while they were expressed in E.coli, indicating that tyrosine phosphorylation...

  12. Peritonitis - spontaneous bacterial

    Science.gov (United States)

    Spontaneous bacterial peritonitis (SBP); Ascites - peritonitis; Cirrhosis - peritonitis ... who are on peritoneal dialysis for kidney failure. Peritonitis may have other causes . These include infection from ...

  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. TBK1 Protects Vacuolar Integrity during Intracellular Bacterial Infection

    Science.gov (United States)

    Radtke, Andrea L; Delbridge, Laura M; Balachandran, Siddharth; Barber, Glen N; O'Riordan, Mary X. D

    2007-01-01

    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. PMID:17335348

  15. Bacillus subtilis strain deficient for the protein-tyrosine kinase PtkA exhibits impaired DNA replication

    DEFF Research Database (Denmark)

    Petranovic, Dina; Michelsen, Ole; Zahradka, K

    2007-01-01

    Bacillus subtilis has recently come into the focus of research on bacterial protein-tyrosine phosphorylation, with several proteins kinases, phosphatases and their substrates identified in this Gram-positive model organism. B. subtilis protein-tyrosine phosphorylation system Ptk...... microscopy. B. subtilis cells lacking the kinase PtkA accumulated extra chromosome equivalents, exhibited aberrant initiation mass for DNA replication and an unusually long D period....

  16. Prevention of bacterial adhesion

    DEFF Research Database (Denmark)

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

    2010-01-01

    . 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 become...

  17. Structural analysis of Staphylococcus aureus serine/threonine kinase PknB.

    Directory of Open Access Journals (Sweden)

    Sonja Rakette

    Full Text Available Effective treatment of infections caused by the bacterium Staphylococcus aureus remains a worldwide challenge, in part due to the constant emergence of new strains that are resistant to antibiotics. The serine/threonine kinase PknB is of particular relevance to the life cycle of S. aureus as it is involved in the regulation of purine biosynthesis, autolysis, and other central metabolic processes of the bacterium. We have determined the crystal structure of the kinase domain of PknB in complex with a non-hydrolyzable analog of the substrate ATP at 3.0 Å resolution. Although the purified PknB kinase is active in solution, it crystallized in an inactive, autoinhibited state. Comparison with other bacterial kinases provides insights into the determinants of catalysis, interactions of PknB with ligands, and the pathway of activation.

  18. Chromosomal aberrations induced by caffeine, 3H-thymidine and by X-rays in two L5178Y sublines of different radiosensitivity. Part 1. Chromosomal aberrations in cells treated with 2mM caffeine and tritiated thymidine

    International Nuclear Information System (INIS)

    Bocian, E.; Bouzyk, E.; Rosiek, O.; Ziemba-Zoltowska, B.

    1982-01-01

    Chromosomal aberrations were studied in two sublines of L5178Y cells with different sensitivity to X-rays. Cells were treated with 2 mM caffeine for 12 h. Then they were examined at various time intervals from 5 to 24 h. Caffeine caused over three times more aberrations, mainly chromatid breaks and gaps, in radiation-sensitive L5178Y-S than in radiation-resistant L5178Y-R cells. The maximum frequency of chromatid breaks in both sublines was found at 8 h and that of chromatid exchanges and isochromatid breaks at 12 h after treatment. A dramatic decrease of the frequency of all types of aberrations at 24 h was observed. In the pulse labelled experiments caffeine enhanced the frequency of aberrations that were induced by 3 H-thymidine at a concentration of 1 μCi/ml. This effect of caffeine was greater in L5178Y-R than in L5178Y-S cells. (author)

  19. 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....

  20. Kinetics of /sup 3/H-thymidine incorporation into nuclei of cells of the regenerating liver of rats exposed to various doses of x rays

    Energy Technology Data Exchange (ETDEWEB)

    Aksenovich, T I; Polishchuk, A M [AN SSSR, Novosibirsk. Inst. Tsitologii i Genetiki

    1976-01-01

    Action of x radiation on the incorporation of /sup 3/H-thymidine into nuclei of cells of the regenerating liver of rats has been studied. A whole-body exposure of rats to 150 to 300 R has been found to inhibit /sup 3/H-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.

  1. 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...... subunit have been prepared and assayed for their ability to assemble with the catalytic alpha subunit to give a fully competent CK2 holoenzyme. The beta subunit contains an acidic stretch (amino acid 55-64), which is obviously responsible for a negative control of enzyme activity since mutations...

  2. 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 - others: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

  3. 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)

    Ermekova, V.M.; Kondakova, N.V.; Levitman, M.Kh.; Saugabaeva, K.M.; Ehjdus, L.Kh.

    1976-01-01

    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

  4. 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

  5. Helicobacter pylori induces cell migration and invasion through casein kinase 2 in gastric epithelial cells.

    Science.gov (United States)

    Lee, Yeo Song; Lee, Do Yeon; Yu, Da Yeon; Kim, Shin; Lee, Yong Chan

    2014-12-01

    Chronic infection with Helicobacter pylori (H. pylori) is causally linked with gastric carcinogenesis. Virulent H. pylori strains deliver bacterial CagA into gastric epithelial cells. Induction of high motility and an elongated phenotype is considered to be CagA-dependent process. Casein kinase 2 plays a critical role in carcinogenesis through signaling pathways related to the epithelial mesenchymal transition. This study was aimed to investigate the effect of H. pylori infection on the casein kinase 2-mediated migration and invasion in gastric epithelial cells. AGS or MKN28 cells as human gastric epithelial cells and H. pylori strains Hp60190 (ATCC 49503, CagA(+)) and Hp8822 (CagA(-)) were used. Cells were infected with H. pylori at multiplicity of infection of 100 : 1 for various times. We measured in vitro kinase assay to examine casein kinase 2 activity and performed immunofluorescent staining to observe E-cadherin complex. We also examined β-catenin transactivation through promoter assay and MMP7 expression by real-time PCR and ELISA. H. pylori upregulates casein kinase 2 activity and inhibition of casein kinase 2 in H. pylori-infected cells profoundly suppressed cell invasiveness and motility. We confirmed that casein kinase 2 mediates membranous α-catenin depletion through dissociation of the α-/β-catenin complex in H. pylori-infected cells. We also found that H. pylori induces β-catenin nuclear translocation and increases MMP7 expressions mediated through casein kinase 2. We show for the first time that CagA(+) H. pylori upregulates cellular invasiveness and motility through casein kinase 2. The demonstration of a mechanistic interplay between H. pylori and casein kinase 2 provides important insights into the role of CagA(+) H. pylori in the gastric cancer invasion and metastasis. © 2014 John Wiley & Sons Ltd.

  6. Tissue specificity for incorporation of [3H]thymidine by the 10- to 12-somite mouse embryo: alteration by acute exposure to hydroxyurea

    International Nuclear Information System (INIS)

    Miller, S.A.; Runner, M.N.

    1978-01-01

    Radioautograms from 10- to 12-somite mouse embryos labelled for 30 min in vitro with [ 3 H]thymidine were examined for frequency and intensity of incorporation. Results from ten tissues showed that values ranged from 82% of nuclei with a mean of 16.6 grains for visceral yolk sac to 17% of nuclei labelled with a mean of 4.4 grains for epithelium of the anterior gut tube. Labelling in the ten tissues indicated (1) a tissue-specific spectrum of incorporation of [ 3 H]thymidine, (2) close correlation between frequency and intensity of labelling within a tissue and (3) asymmetrical quantities of incorporation between right and left somatopleure. Treatment with hydroxyurea in vitro reduced the frequency of labelled nuclei by 85% to 17% of control values. Mean numbers of grains over treated nuclei, 3.3 to 4.6 grains, were well above background but were clustered below the low end of the control range. Tissues exposed to hydroxyurea showed (1) labelling of significant numbers of nuclei, (2) inhibition of labelling in selected tissues and (3) equalization of bilateral asymmetry in quantity (frequency and intensity) of incorporation in somatopleure. The selective reduction of thymidine incorporation and equalization of asymmetrical rates of proliferation may constitute mechanisms by which hydroxyurea causes abnormal morphogenesis. (author)

  7. Tritium retention in the femoral bone marrow and spleens of mice receiving single intravenous injections of tritiated water and tritiated thymidine

    International Nuclear Information System (INIS)

    Joshima, Hisamasa; Matsushita, Satoru; Fukutsu, Kumiko; Kashima, Masatoshi

    1987-01-01

    To derive parameters necessary for evaluating the possible hazards of tritium, retention of tritium in total and TCA-insoluble fractions of the femoral marrow and spleen of mice were observed after single intravenous injections of tritiated water and tritiated thymidine. Retention curves of tritium in TCA-insoluble fractions of the femoral marrow and spleen were resolved fairly well into two exponential components. After injecting tritiated thymidine, most of the activity was detected in the TCA-insoluble fraction. Tritium in this fraction decreased with half-times of 2.2 days in the femoral marrow and 3.6 days in the spleen as the first component, and 23.9 days and 30.5 days, respectively, as the second component. After tritiated water injections, the tritium incorporated into the TCA-insoluble fraction was quite small. Most of the activity was considered to be in the TCA-soluble fraction. Tritium in this fraction was estimated to decrease with half-times of 2.6 days in the femoral marrow and 2.3 days in the spleen as the first component, and 8.0 days and 8.2 days, respectively, as the second component. It is concluded that the retention curves of tritium in the bone marrow are similar to those in the spleen for tritiated water, but not for tritiated thymidine. (author)

  8. Detection of cell proliferation in adults of the water bear Hypsibius dujardini (Tardigrada) via incorporation of a thymidine analog.

    Science.gov (United States)

    Gross, V; Bährle, R; Mayer, G

    2018-04-01

    The taxon Tardigrada, commonly called "water bears", consists of microscopic, eight-legged invertebrates that are well known for their ability to tolerate extreme environmental conditions. Their miniscule body size means that tardigrades possess a small total number of cells, the number and arrangement of which may be highly conserved in some organs. Although mitoses have been observed in several organs, the rate and pattern of cell divisions in adult tardigrades has never been characterized. In this study, we incubated live tardigrades over a period of several days with a thymidine analog in order to visualize all cells that had divided during this time. We focus on the midgut, the largest part of the digestive system. Our results show that new cells in the midgut arise from the anterior and posterior ends of this organ and either migrate or divide toward its middle. These cells divide at a constant rate and all cells of the midgut epithelium are replaced in approximately one week. On the other hand, we found no cell divisions in the nervous system or any other major organs, suggesting that the cell turnover of these organs may be extremely slow or dependent on changing environmental conditions. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. Labelling indices after 3H-thymidine incorporation during organ culture of duodenal mucosa in coeliac disease

    International Nuclear Information System (INIS)

    Fluge, G.; Aksnes, L.

    1980-01-01

    Incorporation of 3 H-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.)

  10. An experimental study on healing of pulp wound following pulpotomy in dogs by autoradiography with tritiated thymidine

    International Nuclear Information System (INIS)

    Chen, Chao-How

    1978-01-01

    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 3 H-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.)

  11. Estimation of cancerolytic properties of thionine from plants seeds by inclusion of C14-thymidine in tumoral cells

    International Nuclear Information System (INIS)

    Pshenichnov, E.A.; Sultanova, E.M.; Kuznetsova, N.N.; Khashimova, Z.S.; Veshkurova, O.N.; Sadikov, A.A.; Salikhov, Sh.I.

    2004-01-01

    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 C 14 -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

  12. Roles of Aag, Alkbh2, and Alkbh3 in the Repair of Carboxymethylated and Ethylated Thymidine Lesions.

    Science.gov (United States)

    You, Changjun; Wang, Pengcheng; Nay, Stephanie L; Wang, Jianshuang; Dai, Xiaoxia; O'Connor, Timothy R; Wang, Yinsheng

    2016-05-20

    Environmental and endogenous genotoxic agents can result in a variety of alkylated and carboxymethylated DNA lesions, including N3-ethylthymidine (N3-EtdT), O(2)-EtdT, and O(4)-EtdT as well as N3-carboxymethylthymidine (N3-CMdT) and O(4)-CMdT. By using nonreplicative double-stranded vectors harboring a site-specifically incorporated DNA lesion, we assessed the potential roles of alkyladenine DNA glycosylase (Aag); alkylation repair protein B homologue 2 (Alkbh2); or Alkbh3 in modulating the effects of N3-EtdT, O(2)-EtdT, O(4)-EtdT, N3-CMdT, or O(4)-CMdT on DNA transcription in mammalian cells. We found that the depletion of Aag did not significantly change the transcriptional inhibitory or mutagenic properties of all five examined lesions, suggesting a negligible role of Aag in the repair of these DNA adducts in mammalian cells. In addition, our results revealed that N3-EtdT, but not other lesions, could be repaired by Alkbh2 and Alkbh3 in mammalian cells. Furthermore, we demonstrated the direct reversal of N3-EtdT by purified human Alkbh2 protein in vitro. These findings provided important new insights into the repair of the carboxymethylated and alkylated thymidine lesions in mammalian cells.

  13. Labelling of the thymidine and deoxyctidine bases of DNA by (2-14C) deoxycytidine in cultured L1210 cells

    International Nuclear Information System (INIS)

    Karle, J.M.; Hoeraut, R.M.; Cysyk, R.L.

    1983-01-01

    Exposure of cultured L1210 cells to (2- 14 C) deoxycytidine and analysis of radioactivity incorporated into DNA-pyrimidines revealed that 2.7-5.5-fold more radioactivity is incorporated into DNA-thymine than into cytosine bases. Thus, the pathway involving deamination of deoxycytidylate to deoxyuridylate and methylation to thymidylate is highly favoured over successive phosphorlation to dCTP. Several modified and endogenous pyrimidines altered the labelling of DNA-thymine and DNA-cytosine with (2- 14 C)-deoxycytidine. 3-deazauridine at 0.1 mM caused a 56% increase in the labelling of DNA-thymine. Both thymidine and 3-deazauridine (>=10 μM) increased the specific activity to DNA-cytosine by 4-fold. Cytosine arabinoside (ara-C) (>= 10 μM) reduced the labelling of both DNA-cytosine and DNA-thymine. Excess cytidine (0.1 mM) reduced the labelling of DNA-cytosine by 40%. Tetrahydrouridine at concentrations up to 1 mM had no effect. (author)

  14. Kinetics of the formation of a G2 block from tritiated thymidine in phytohemagglutinin-stimulated human lymphocytes

    International Nuclear Information System (INIS)

    Pollack, A.; Bagwell, C.B.; Irvin, G.L.; Jensen, J.A.

    1980-01-01

    Flow cytometry (FCM) was used to monitor the radiation effects promoted by incorporated tritiated thymidine ( 3 H-TdR) on phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes stained with propidium iodide (PI). Lymphocyte microcultures were continuously labeled or pulse-labeled for various periods of time with different 3 H-TdR concentrations. Two types of DNA histogram analyses were performed on unperturbed and 3 H]TdR perturbed lymphocytes. The data analyses consisted of statistical analyses between averaged groups of histograms (nonparametric analysis) and cell cycle analyses (parametric analysis) to determine the percentages of cells in G0 + G1, S and G2 + M. The results showed that (a) 3 H-TdR when added to proliferating lymphocytes under certain conditions (both short-term continuous and pulse-labeling) caused a highly significant increase in the proportion of tetraploid (4C) cells by FCM, (b) the increase in the proportion of 4C cells represented a block in G2 and (c) the relative increase in the percentage of 4C cells was proportional to 3 H-TdR incorporation which was proportional to labeling time and concentration. Therefore, it was concluded that short labeling times be used to minimize adverse radiation effects when 3 H-TdR is used to assay substances affecting lymphocyte proliferation or in the estimation of cell cycle time

  15. Thymidylate Synthase, Thymidine Phosphorylase and Orotate Phosphoribosyl Transferase Levels as Predictive Factors of Chemotherapy in Oral Squamous Cell Carcinoma

    International Nuclear Information System (INIS)

    Ogiuchi, Yosuke; Maruoka, Yasubumi; Ando, Tomohiro; Kobayashi, Makio; Ogiuchi, Hideki

    2008-01-01

    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

  16. 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

    Cucumber was grown in a partially sterilized sand-soil mixture with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum or left uninoculated. Fresh soil extract was places in polyvinyl chloride tubes without propagules of mycorrhizal fungi. Root tips and root segments...... and top of tubes, and of cocci with a diameter of 0.55-0.78 mum in the bulk soil in the center of tubes, were significantly reduced by VAM fungi. The extremely high bacterial biomass (1-7 mg C g-1 dry weight soil) was significant reduced by mycorrhizal colonization on root segments and in bulk soil...... biomass, and changed the spatial pattern of bacterial growth compared to non-mycorrhizal cucumbers. The [H-3]-thymidine incorporation was significantly higher on root tips in the top of tubes, and on root segments and bulk soil in the center of tubes on non-mycorrhizal plants compared to mycorrhizal...

  17. Bacterial activity in a reservoir determined by autoradiography and its relationships to phyto- and zooplankton

    International Nuclear Information System (INIS)

    Simek, K.

    1986-01-01

    In the drinking water reservoir Rimov (Southern Bohemia) bacterioplankton was studied during 1983. Special attention was given to the relationships between parameters of bacterial abundance, total and individual activity. Bacterial counts and biomass was assessed and autoradiographic determinations of the proportion of active bacteria incorporating thymidine (Th) and a mixture of amino acids (AA) and total uptake rate of AA were made over a year in the surface layer and during summer stratification from the thermocline and 15 m depth. Specific activity of metabolically active bacteria and specific activity per unit of biomass were negatively correlated with counts of metabolizing cells and with bacterial biomass, respectively. Total and individual heterotrophic activity and counts of bacteria coincided with the changes of phytoplankton biomass, whereas bacteria incorporating Th were more tightly correlated with primary production. The most significant relation of metabolically active bacteria was found to cladoceran biomass. Thus, this part of heterotrophic bacterial activity seems to be stimulated by leakage of dissolved organic matter from phytoplankton being disrupted and incompletely digested by cladocerans rather than from healthy photosynthetizing cells. (author)

  18. Postviral Complications: Bacterial Pneumonia.

    Science.gov (United States)

    Prasso, Jason E; Deng, Jane C

    2017-03-01

    Secondary bacterial pneumonia after viral respiratory infection remains a significant source of morbidity and mortality. Susceptibility is mediated by a variety of viral and bacterial factors, and complex interactions with the host immune system. Prevention and treatment strategies are limited to influenza vaccination and antibiotics/antivirals respectively. Novel approaches to identifying the individuals with influenza who are at increased risk for secondary bacterial pneumonias are urgently needed. Given the threat of further pandemics and the heightened prevalence of these viruses, more research into the immunologic mechanisms of this disease is warranted with the hope of discovering new potential therapies. Published by Elsevier Inc.

  19. 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.

  20. The molecular architecture of human N-acetylgalactosamine kinase.

    Science.gov (United States)

    Thoden, James B; Holden, Hazel M

    2005-09-23

    Galactokinase plays a key role in normal galactose metabolism by catalyzing the conversion of alpha-d-galactose to galactose 1-phosphate. Within recent years, the three-dimensional structures of human galactokinase and two bacterial forms of the enzyme have been determined. Originally, the gene encoding galactokinase in humans was mapped to chromosome 17. An additional gene, encoding a protein with sequence similarity to galactokinase, was subsequently mapped to chromosome 15. Recent reports have shown that this second gene (GALK2) encodes an enzyme with greater activity against GalNAc than galactose. This enzyme, GalNAc kinase, has been implicated in a salvage pathway for the reutilization of free GalNAc derived from the degradation of complex carbohydrates. Here we report the first structural analysis of a GalNAc kinase. The structure of the human enzyme was solved in the presence of MnAMPPNP and GalNAc or MgATP and GalNAc (which resulted in bound products in the active site). The enzyme displays a distinctly bilobal appearance with its active site wedged between the two domains. The N-terminal region is dominated by a seven-stranded mixed beta-sheet, whereas the C-terminal motif contains two layers of anti-parallel beta-sheet. The overall topology displayed by GalNAc kinase places it into the GHMP superfamily of enzymes, which generally function as small molecule kinases. From this investigation, the geometry of the GalNAc kinase active site before and after catalysis has been revealed, and the determinants of substrate specificity have been defined on a molecular level.

  1. SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

    Science.gov (United States)

    McGlade, C J; Ellis, C; Reedijk, M; Anderson, D; Mbamalu, G; Reith, A D; Panayotou, G; End, P; Bernstein, A; Kazlauskas, A

    1992-01-01

    The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors. Images PMID:1372092

  2. Bacterial vaginosis - aftercare

    Science.gov (United States)

    Bacterial vaginosis (BV) is a type of vaginal infection. The vagina normally contains both healthy bacteria and unhealthy bacteria. BV occurs when more unhealthy bacteria grow than healthy bacteria. No one knows ...

  3. Bacterial surface adaptation

    Science.gov (United States)

    Utada, Andrew

    2014-03-01

    Biofilms are structured multi-cellular communities that are fundamental to the biology and ecology of bacteria. Parasitic bacterial biofilms can cause lethal infections and biofouling, but commensal bacterial biofilms, such as those found in the gut, can break down otherwise indigestible plant polysaccharides and allow us to enjoy vegetables. The first step in biofilm formation, adaptation to life on a surface, requires a working knowledge of low Reynolds number fluid physics, and the coordination of biochemical signaling, polysaccharide production, and molecular motility motors. These crucial early stages of biofilm formation are at present poorly understood. By adapting methods from soft matter physics, we dissect bacterial social behavior at the single cell level for several prototypical bacterial species, including Pseudomonas aeruginosa and Vibrio cholerae.

  4. Bacterial intermediate filaments

    DEFF Research Database (Denmark)

    Charbon, Godefroid; Cabeen, M.; Jacobs-Wagner, C.

    2009-01-01

    Crescentin, which is the founding member of a rapidly growing family of bacterial cytoskeletal proteins, was previously proposed to resemble eukaryotic intermediate filament (IF) proteins based on structural prediction and in vitro polymerization properties. Here, we demonstrate that crescentin...

  5. Diagnosis of bacterial infection

    African Journals Online (AJOL)

    direct or indirect evidence of a compatible bacterial pathogen. Inflammation may be .... cardinal features (fever, confusion, headache and neck stiffness). .... specificity, inappropriate indications or poor sampling technique may diminish this ...

  6. CBL-interacting protein kinase 6 negatively regulates immune response to Pseudomonas syringae in Arabidopsis.

    Science.gov (United States)

    Sardar, Atish; Nandi, Ashis Kumar; Chattopadhyay, Debasis

    2017-06-15

    Cytosolic calcium ion (Ca2+) is an essential mediator of the plant innate immune response. Here, we report that a calcium-regulated protein kinase Calcineurin B-like protein (CBL)-interacting protein kinase 6 (CIPK6) functions as a negative regulator of immunity against the bacterial pathogen Pseudomonas syringae in Arabidopsis thaliana. Arabidopsis lines with compromised expression of CIPK6 exhibited enhanced disease resistance to the bacterial pathogen and to P. syringae harboring certain but not all avirulent effectors, while restoration of CIPK6 expression resulted in abolition of resistance. Plants overexpressing CIPK6 were more susceptible to P. syringae. Enhanced resistance in the absence of CIPK6 was accompanied by increased accumulation of salicylic acid and elevated expression of defense marker genes. Salicylic acid accumulation was essential for improved immunity in the absence of CIPK6. CIPK6 negatively regulated the oxidative burst associated with perception of pathogen-associated microbial patterns (PAMPs) and bacterial effectors. Accelerated and enhanced activation of the mitogen-activated protein kinase cascade in response to bacterial and fungal elicitors was observed in the absence of CIPK6. The results of this study suggested that CIPK6 negatively regulates effector-triggered and PAMP-triggered immunity in Arabidopsis. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  7. Bacterial community dynamics and activity in relation to dissolved organic matter availability during sea-ice formation in a mesocosm experiment.

    Science.gov (United States)

    Eronen-Rasimus, Eeva; Kaartokallio, Hermanni; Lyra, Christina; Autio, Riitta; Kuosa, Harri; Dieckmann, Gerhard S; Thomas, David N

    2014-02-01

    The structure of sea-ice bacterial communities is frequently different from that in seawater. Bacterial entrainment in sea ice has been studied with traditional microbiological, bacterial abundance, and bacterial production methods. However, the dynamics of the changes in bacterial communities during the transition from open water to frozen sea ice is largely unknown. Given previous evidence that the nutritional status of the parent water may affect bacterial communities during ice formation, bacterial succession was studied in under ice water and sea ice in two series of mesocosms: the first containing seawater from the North Sea and the second containing seawater enriched with algal-derived dissolved organic matter (DOM). The composition and dynamics of bacterial communities were investigated with terminal restriction fragment length polymorphism (T-RFLP), and cloning alongside bacterial production (thymidine and leucine uptake) and abundance measurements (measured by flow cytometry). Enriched and active sea-ice bacterial communities developed in ice formed in both unenriched and DOM-enriched seawater (0-6 days). γ-Proteobacteria dominated in the DOM-enriched samples, indicative of their capability for opportunistic growth in sea ice. The bacterial communities in the unenriched waters and ice consisted of the classes Flavobacteria, α- and γ-Proteobacteria, which are frequently found in natural sea ice in polar regions. Furthermore, the results indicate that seawater bacterial communities are able to adapt rapidly to sudden environmental changes when facing considerable physicochemical stress such as the changes in temperature, salinity, nutrient status, and organic matter supply during ice formation. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  8. Chitin and stress induced protein kinase activation

    DEFF Research Database (Denmark)

    Kenchappa, Chandra Shekar; Azevedo da Silva, Raquel; Bressendorff, Simon

    2017-01-01

    The assays described here are pertinent to protein kinase studies in any plant. They include an immunoblot phosphorylation/activation assay and an in-gel activity assay for MAP kinases (MPKs) using the general protein kinase substrate myelin basic protein. They also include a novel in-gel peptide...... substrate assay for Snf1-related kinase family 2 members (SnRK2s). This kinase family-specific assay overcomes some limitations of in-gel assays and permits the identification of different types of kinase activities in total protein extracts....

  9. 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.

  10. Kinetics of [32P]orthophosphate and [3H]thymidine incorporation into newly replicating DNA in vivo

    International Nuclear Information System (INIS)

    Panzeter, P.; Ringer, D.

    1986-01-01

    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 [ 3 H]thymidine ( 3 H-Thd) and 5mCi [ 32 P]orthophosphate ( 32 P/sub i/) were injected into the hepatic portal vein of partially hepatectomized rats. Increasing time of 3 H-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 3 H has been chased from the matrix DNA. In contrast, after injection of 32 P/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 32 P/sub i/ incorporation differs dramatically from that of 3 H-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

  11. Uptake of 3H-thymidine by the receptor cell populations after injury of the sensory nerve fibres

    International Nuclear Information System (INIS)

    Chuchkov, Ch.N.

    1978-01-01

    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 3 H-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.)

  12. Seasonal and spatial patterns of heterotrophic bacterial production, respiration, and biomass in the subarctic NE Pacific

    Science.gov (United States)

    Sherry, Nelson D.; Boyd, Philip W.; Sugimoto, Kugako; Harrison, Paul J.

    1999-11-01

    Heterotrophic bacterial biomass, production, and respiration rates were measured during winter, spring, and summer in the subarctic NE Pacific from September 1995 to June 1997. Sampling took place on six cruises at five hydrographic stations along the east/west line-P transect from slope waters at P4 (1200 m depth) to the open-ocean waters at Ocean Station Papa (OSP) (4250 m depth). Interannual variability was small relative to seasonal and spatial variability. Biomass, derived from cell counts (assuming 20 fg C cell -1), was ca. 12 μg C l -1 in the winter and increased to 20-35 μg C l -1 in the spring and summer all along line-P. Bacterial production from [ 3H]-thymidine and [ 14C]-leucine incorporation rates was lowest in the winter (ca. 0.5 μg C l -1 d -1) with little spatial variability. Production increased 10-fold in spring at P4 (to ca. 4.5 μg C l -1 d -1). In contrast, only a 2-fold increase in bacterial production was observed over this period at the more oceanic stations. Rates of production in late summer were highest over the annual cycle at all stations ranging from ca. 6 at P4 to ca. 2 μg C l -1 d -1 at OSP. Bacterial (rates increased >10-fold to ca. 100 μg C l -1 d -1 at P4 in the summer, but, interestingly, did not increase from spring to summer at the more oceanic stations. Thus bacterial growth efficiency, defined as production/(production+respiration), decreased in the spring westwards from the slope waters (P4) to the open-ocean (OSP), but increased westwards in the summer. Bacterial production was highly correlated with temperature at OSP ( r2=0.88) and less so at P4 ( r2=0.50). The observed temporal and spatial trends presented in this study suggest that seasonal changes in bacterial biomass were greatly affected by changes in loss processes, that bacterial biomass is regulated by different processes than bacterial production, and that bacterial production alone, without respiration measurements, is not a robust proxy for bacterial

  13. Influence of protein deposition on bacterial adhesion to contact lenses.

    Science.gov (United States)

    Subbaraman, Lakshman N; Borazjani, Roya; Zhu, Hua; Zhao, Zhenjun; Jones, Lyndon; Willcox, Mark D P

    2011-08-01

    The aim of the study is to determine the adhesion of Gram positive and Gram negative bacteria onto conventional hydrogel (CH) and silicone hydrogel (SH) contact lens materials with and without lysozyme, lactoferrin, and albumin coating. Four lens types (three SH-balafilcon A, lotrafilcon B, and senofilcon A; one CH-etafilcon A) were coated with lysozyme, lactoferrin, or albumin (uncoated lenses acted as controls) and then incubated in Staphylococcus aureus (Saur 31) or either of two strains of Pseudomonas aeruginosa (Paer 6294 and 6206) for 24 h at 37 °C. The total counts of the adhered bacteria were determined using the H-thymidine method and viable counts by counting the number of colony-forming units on agar media. All three strains adhered significantly lower to uncoated etafilcon A lenses compared with uncoated SH lenses (p 0.05). Lactoferrin coating on lenses increased binding (total and viable counts) of Saur 31 (p lenses showed significantly higher total counts (p lenses. Albumin coating of lenses increased binding (total and viable counts) of all three strains (p lenses does not possess antibacterial activity against certain bacterial strains, whereas lactoferrin possess an antibacterial effect against strains of P. aeruginosa.

  14. The regulation of bacterial production in the Chesapeake Bay

    International Nuclear Information System (INIS)

    Chin-Leo, G.

    1988-01-01

    In this study, the possibility that periods when the rates of macromolecule syntheses become uncoupled occur in natural assemblages of bacteria was examined by comparing rates of bacterial DNA and protein synthesis. A dual-label method which measures incorporation rates of [ 3 H]thymidine (TdR) into macromolecules (DNA) and of [ 14 C]leucine (Leu) into protein was developed to facilitate simultaneous estimation of these cellular activities in a single incubation. Under controlled conditions, changes in rates of Leu incorporation preceded fluctuations in TdR incorporation and the Leu:TdR ratio varied prior to shifts in growth rate indicating the uncoupling of protein and DNA synthesis which occurs during unbalanced growth. The delay between this uncoupling and a change in growth rate was always shorter than the generation time. In Chesapeake Bay, during October 1986, the Leu:TdR ratio was quite constant over a diel cycle and with depth, but during July 1987, the magnitude of this ratio and its variation through time increased with depth. Growth conditions for heterotrophic bacteria in Chesapeake Bay during summer in surface waters and throughout the water column in fall may be relatively constant leading to balanced growth. In contrast, fluctuating growth conditions in subsurface waters during summer may lead to unbalanced growth

  15. Tyr721 regulates specific binding of the CSF-1 receptor kinase insert to PI 3'-kinase SH2 domains: a model for SH2-mediated receptor-target interactions.

    Science.gov (United States)

    Reedijk, M; Liu, X; van der Geer, P; Letwin, K; Waterfield, M D; Hunter, T; Pawson, T

    1992-01-01

    Efficient binding of active phosphatidylinositol (PI) 3'-kinase to the autophosphorylated macrophage colony stimulating factor receptor (CSF-1R) requires the noncatalytic kinase insert (KI) region of the receptor. To test whether this region could function independently to bind PI 3'-kinase, the isolated CSF-1R KI was expressed in Escherichia coli, and was inducibly phosphorylated on tyrosine. The tyrosine phosphorylated form of the CSF-1R KI bound PI 3'-kinase in vitro, whereas the unphosphorylated form had no binding activity. The p85 alpha subunit of PI 3'-kinase contains two Src homology (SH)2 domains, which are implicated in the interactions of signalling proteins with activated receptors. Bacterially expressed p85 alpha SH2 domains complexed in vitro with the tyrosine phosphorylated CSF-1R KI. Binding of the CSF-1R KI to PI 3'-kinase activity, and to the p85 alpha SH2 domains, required phosphorylation of Tyr721 within the KI domain, but was independent of phosphorylation at Tyr697 and Tyr706. Tyr721 was also critical for the association of activated CSF-1R with PI 3'-kinase in mammalian cells. Complex formation between the CSF-1R and PI 3'-kinase can therefore be reconstructed in vitro in a specific interaction involving the phosphorylated receptor KI and the SH2 domains of p85 alpha. Images PMID:1314163

  16. Bacterial Cell Mechanics.

    Science.gov (United States)

    Auer, George K; Weibel, Douglas B

    2017-07-25

    Cellular mechanical properties play an integral role in bacterial survival and adaptation. Historically, the bacterial cell wall and, in particular, the layer of polymeric material called the peptidoglycan were the elements to which cell mechanics could be primarily attributed. Disrupting the biochemical machinery that assembles the peptidoglycan (e.g., using the β-lactam family of antibiotics) alters the structure of this material, leads to mechanical defects, and results in cell lysis. Decades after the discovery of peptidoglycan-synthesizing enzymes, the mechanisms that underlie their positioning and regulation are still not entirely understood. In addition, recent evidence suggests a diverse group of other biochemical elements influence bacterial cell mechanics, may be regulated by new cellular mechanisms, and may be triggered in different environmental contexts to enable cell adaptation and survival. This review summarizes the contributions that different biomolecular components of the cell wall (e.g., lipopolysaccharides, wall and lipoteichoic acids, lipid bilayers, peptidoglycan, and proteins) make to Gram-negative and Gram-positive bacterial cell mechanics. We discuss the contribution of individual proteins and macromolecular complexes in cell mechanics and the tools that make it possible to quantitatively decipher the biochemical machinery that contributes to bacterial cell mechanics. Advances in this area may provide insight into new biology and influence the development of antibacterial chemotherapies.

  17. Biodegradability of bacterial surfactants.

    Science.gov (United States)

    Lima, Tânia M S; Procópio, Lorena C; Brandão, Felipe D; Carvalho, André M X; Tótola, Marcos R; Borges, Arnaldo C

    2011-06-01

    This work aimed at evaluating the biodegradability of different bacterial surfactants in liquid medium and in soil microcosms. The biodegradability of biosurfactants by pure and mixed bacterial cultures was evaluated through CO(2) evolution. Three bacterial strains, Acinetobacter baumanni LBBMA ES11, Acinetobacter haemolyticus LBBMA 53 and Pseudomonas sp. LBBMA 101B, used the biosurfactants produced by Bacillus sp. LBBMA 111A (mixed lipopeptide), Bacillus subtilis LBBMA 155 (lipopeptide), Flavobacterium sp. LBBMA 168 (mixture of flavolipids), Dietzia Maris LBBMA 191(glycolipid) and Arthrobacter oxydans LBBMA 201(lipopeptide) as carbon sources in minimal medium. The synthetic surfactant sodium dodecyl sulfate (SDS) was also mineralized by these microorganisms, but at a lower rate. CO(2) emitted by a mixed bacterial culture in soil microcosms with biosurfactants was higher than in the microcosm containing SDS. Biosurfactant mineralization in soil was confirmed by the increase in surface tension of the soil aqueous extracts after incubation with the mixed bacterial culture. It can be concluded that, in terms of biodegradability and environmental security, these compounds are more suitable for applications in remediation technologies in comparison to synthetic surfactants. However, more information is needed on structure of biosurfactants, their interaction with soil and contaminants and scale up and cost for biosurfactant production.

  18. Non-Viral Deoxyribonucleoside Kinases

    DEFF Research Database (Denmark)

    Christiansen, Louise Slot; Munch-Petersen, Birgitte; Knecht, Wolfgang

    2015-01-01

    Deoxyribonucleoside kinases (dNKs) phosphorylate deoxyribonucleosides to their corresponding monophosphate compounds. dNks also phosphorylate deoxyribonucleoside analogues that are used in the treatment of cancer or viral infections. The study of the mammalian dNKs has therefore always been of gr...

  19. Bacterial meningitis in children

    International Nuclear Information System (INIS)

    Marji, S.

    2007-01-01

    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)

  20. Continuous 28 day iododeoxyuridine (IUdR) infusion and hyperfractionated accelerated radiotherapy (hart) for malignant glioma: a phase I clinical and thymidine replacement study

    International Nuclear Information System (INIS)

    Schulz, C.A.; Mehta, M.P.; Robins, H.I.; Badie, B.; Arzoomanian, R.; Simon, K.; Alberti, D.; Feierabend, C.; Kunugi, K.A.; Wilding, G.; Kinsella, T.J.

    1997-01-01

    Objectives: Based on preclinical studies demonstrating a direct correlation between duration of IUdR infusion and percent cells labeled as well as amount thymidine replaced by IUdR, we conducted a Phase I trial to: (1) investigate the maximum tolerated dose (MTD) and systemic toxicities of a continuous 28 day IUdR infusion; (2) analyze percent IUdR-thymidine replacement in peripheral granulocytes as a surrogate marker for IUdR incorporation into tumor cells; (3) measure steady state serum IUdR levels; and (4) assess the feasibility of continuous IUdR infusion and HART in the management of malignant glioma. Materials and Methods: Patients (pts.) were required to have a KPS ≥60% and biopsy proven malignant glioma. Pts. received 100 (level 1: 4 pts.), 200 (level 2: 3 pts.), 300 (level 3: 3 pts.), 400 (level 4: 6 pts.) or 500 (level 5:2 pts.) mg/m2/day IUdR by continuous infusion for 28 days. HART started 7 days after IUdR initiation. Total dose was 70 Gy [1.2 Gy BID x 25 days with a 10 Gy. (2.0 x 5 days - q Saturday) boost. Weekly assays were performed for % IUdR incorporation (thymidine replacement) and serum IUdR levels using standard HPLC methods. Standard Phase I toxicity methodology was used. Results: Between June 1994 and December 1996, 18 pts. with a mean age of 52 years were enrolled (16 glioblastoma multiforme and 2 anaplastic astrocytoma). All pts. completed XRT. Two pts. did not complete IUdR, one due to grade 4 IUdR-related toxicities and one due to rapid disease progression. Dose modification occurred in one pt.; drug withheld due to grade 3 AST (SGOT) elevation with re-initiation of drug at the next lowest level. There were no grade ≥3 XRT toxicities. Grade ≥ 3 IUdR toxicities, dose level at which they occurred, number of patients affected and duration of toxicity are presented in Table 1. Thymidine replacement peaks at 3 weeks and increases with dose (Figure 1). Data on steady state plasma IUdR levels will also be presented. Conclusions: Our

  1. 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...

  2. 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...

  3. Synthesis of dihydrothymidine and thymidine glycol 5'-triphosphates and their ability to serve as substrates for Escherichia coli DNA polymerase I

    International Nuclear Information System (INIS)

    Ide, H.; Melamede, R.J.; Wallace, S.S.

    1987-01-01

    5,6-Dihydrothymidine 5'-triphosphate (DHdTTP) was synthesized by catalytic hydrogenation of thymidine 5'-triphosphate (dTTP). Thymidine glycol 5'-triphosphate (dTTP-GLY) was prepared by bromination of dTTP followed by treatment with Ag 2 O. The modified nucleotides were extensively purified by anion-exchange high-performance liquid chromatography (HPLC). Alkaline phosphatase digestion of DHdTTP and dTTP-GLY gave the expected products (5,6-dihydrothymidine and cis-thymidine glycol), the identities of which were confirmed by reverse-phase HPLC using authentic markers. HPLC analysis of the alkaline phosphatase digested DHdTTP revealed that DHdTTP was a mixture of C5 diastereoisomers [(5S)- and (5R)-DHdTTP]. Despite the significant distortion of the pyrimidine ring in DHdTTP, it was incorporated in place of dTTP during primer elongation catalyzed by Escherichia coli DNA polymerase I Klenow fragment. The rate of incorporation of DHdTTP was about 10-25-fold lower than that of dTTP. On the other hand, dTTP-GLY, which also has a distorted pyrimidine ring, did not replace dTTP, and no elongation of the primer was observed. In order to study the preference of incorporation of the diastereoisomers of DHdTTP into DNA, salmon testes DNA, activated by exonuclease III, was used as a template for DNA polymerase I Klenow fragment in the presence of [ 3 H]DHdTTP (S and R mixture) and normal nucleotides. After enzymatic digestion of the DNA to nucleosides, the products were analyzed by HPLC. The result suggests that Escherichia coli DNA polymerase I uses both isomers of DHdTTP as substrates and that the overall efficiency of incorporation is primarily determined by the concentration of the isomers in the nucleotide pool

  4. Autoregulation of kinase dephosphorylation by ATP binding in AGC protein kinases.

    Science.gov (United States)

    Chan, Tung O; Pascal, John M; Armen, Roger S; Rodeck, Ulrich

    2012-02-01

    AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non- ATP-competitive kinase inhibitors that discriminate within and between protein kinase families.

  5. Autoregulation of kinase dephosphorylation by ATP binding to AGC protein kinases

    Science.gov (United States)

    Pascal, John M; Armen, Roger S

    2012-01-01

    AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non-ATP-competitive kinase inhibitors that discriminate within and between protein kinase families. PMID:22262182

  6. Src kinase regulation by phosphorylation and dephosphorylation

    International Nuclear Information System (INIS)

    Roskoski, Robert

    2005-01-01

    Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTPα, PTPε, and PTPλ. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined

  7. 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

    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...

  8. [Bacterial biofilms and infection].

    Science.gov (United States)

    Lasa, I; Del Pozo, J L; Penadés, J R; Leiva, J

    2005-01-01

    In developed countries we tend to think of heart disease and the numerous forms of cancer as the main causes of mortality, but on a global scale infectious diseases come close, or may even be ahead: 14.9 million deaths in 2002 compared to cardiovascular diseases (16.9 million deaths) and cancer (7.1 million deaths) (WHO report 2004). The infectious agents responsible for human mortality have evolved as medical techniques and hygienic measures have changed. Modern-day acute infectious diseases caused by specialized bacterial pathogens such as diphtheria, tetanus, cholera, plague, which represented the main causes of death at the beginning of XX century, have been effectively controlled with antibiotics and vaccines. In their place, more than half of the infectious diseases that affect mildly immunocompromised patients involve bacterial species that are commensal with the human body; these can produce chronic infections, are resistant to antimicrobial agents and there is no effective vaccine against them. Examples of these infections are the otitis media, native valve endocarditis, chronic urinary infections, bacterial prostatitis, osteomyelitis and all the infections related to medical devices. Direct analysis of the surface of medical devices or of tissues that have been foci of chronic infections shows the presence of large numbers of bacteria surrounded by an exopolysaccharide matrix, which has been named the "biofilm". Inside the biofilm, bacteria grow protected from the action of the antibodies, phagocytic cells and antimicrobial treatments. In this article, we describe the role of bacterial biofilms in human persistent infections.

  9. Interfering with bacterial gossip

    DEFF Research Database (Denmark)

    Bjarnsholt, Thomas; Tolker-Nielsen, Tim; Givskov, Michael

    2011-01-01

    that appropriately target bacteria in their relevant habitat with the aim of mitigating their destructive impact on patients. In this review we describe molecular mechanisms involved in “bacterial gossip” (more scientifically referred to as quorum sensing (QS) and c-di-GMP signaling), virulence, biofilm formation...

  10. Bacterial fingerprints across Europe

    NARCIS (Netherlands)

    Glasner, Corinna

    2014-01-01

    Bacterial pathogens, such as Staphylococcus aureus and carbapenemase-producing Enterobacteriaceae (CPE), impose major threats to human health worldwide. Both have a ‘Jekyll & Hyde’ character, since they can be present as human commensals, but can also become harmful invasive pathogens especially

  11. Bacterial membrane proteomics.

    Science.gov (United States)

    Poetsch, Ansgar; Wolters, Dirk

    2008-10-01

    About one quarter to one third of all bacterial genes encode proteins of the inner or outer bacterial membrane. These proteins perform essential physiological functions, such as the import or export of metabolites, the homeostasis of metal ions, the extrusion of toxic substances or antibiotics, and the generation or conversion of energy. The last years have witnessed completion of a plethora of whole-genome sequences of bacteria important for biotechnology or medicine, which is the foundation for proteome and other functional genome analyses. In this review, we discuss the challenges in membrane proteome analysis, starting from sample preparation and leading to MS-data analysis and quantification. The current state of available proteomics technologies as well as their advantages and disadvantages will be described with a focus on shotgun proteomics. Then, we will briefly introduce the most abundant proteins and protein families present in bacterial membranes before bacterial membrane proteomics studies of the last years will be presented. It will be shown how these works enlarged our knowledge about the physiological adaptations that take place in bacteria during fine chemical production, bioremediation, protein overexpression, and during infections. Furthermore, several examples from literature demonstrate the suitability of membrane proteomics for the identification of antigens and different pathogenic strains, as well as the elucidation of membrane protein structure and function.

  12. [Spontaneous bacterial peritonitis].

    Science.gov (United States)

    Velkey, Bálint; Vitális, Eszter; Vitális, Zsuzsanna

    2017-01-01

    Spontaneous bacterial peritonitis occurs most commonly in cirrhotic patients with ascites. Pathogens get into the circulation by intestinal translocation and colonize in peritoneal fluid. Diagnosis of spontaneous bacterial peritonitis is based on elevated polymorphonuclear leukocyte count in the ascites (>0,25 G/L). Ascites culture is often negative but aids to get information about antibiotic sensitivity in positive cases. Treatment in stable patient can be intravenous then orally administrated ciprofloxacin or amoxicillin/clavulanic acid, while in severe cases intravenous III. generation cephalosporin. Nosocomial spontaneous bacterial peritonitis often caused by Gram-positive bacteria and multi-resistant pathogens can also be expected thus carbapenem should be the choice of the empiric treatment. Antibiotic prophylaxis should be considered. Norfloxacin is used most commonly, but changes are expected due to increase in quinolone resistance. As a primary prophylaxis, a short-term antibiotic treatment is recommended after gastrointestinal bleeding for 5 days, while long-term prophylaxis is for patients with low ascites protein, and advanced disease (400 mg/day). Secondary prophylaxis is recommended for all patients recovered from spontaneous bacterial peritonitis. Due to increasing antibiotic use of antibiotics prophylaxis is debated to some degree. Orv. Hetil., 2017, 158(2), 50-57.

  13. Signaling network of the Btk family kinases.

    Science.gov (United States)

    Qiu, Y; Kung, H J

    2000-11-20

    The Btk family kinases represent new members of non-receptor tyrosine kinases, which include Btk/Atk, Itk/Emt/Tsk, Bmx/Etk, and Tec. They are characterized by having four structural modules: PH (pleckstrin homology) domain, SH3 (Src homology 3) domain, SH2 (Src homology 2) domain and kinase (Src homology 1) domain. Increasing evidence suggests that, like Src-family kinases, Btk family kinases play central but diverse modulatory roles in various cellular processes. They participate in signal transduction in response to virtually all types of extracellular stimuli which are transmitted by growth factor receptors, cytokine receptors, G-protein coupled receptors, antigen-receptors and integrins. They are regulated by many non-receptor tyrosine kinases such as Src, Jak, Syk and FAK family kinases. In turn, they regulate many of major signaling pathways including those of PI3K, PLCgamma and PKC. Both genetic and biochemical approaches have been used to dissect the signaling pathways and elucidate their roles in growth, differentiation and apoptosis. An emerging new role of this family of kinases is cytoskeletal reorganization and cell motility. The physiological importance of these kinases was amply demonstrated by their link to the development of immunodeficiency diseases, due to germ-line mutations. The present article attempts to review the structure and functions of Btk family kinases by summarizing our current knowledge on the interacting partners associated with the different modules of the kinases and the diverse signaling pathways in which they are involved.

  14. Corticosteroids for Bacterial Keratitis

    Science.gov (United States)

    Srinivasan, Muthiah; Mascarenhas, Jeena; Rajaraman, Revathi; Ravindran, Meenakshi; Lalitha, Prajna; Glidden, David V.; Ray, Kathryn J.; Hong, Kevin C.; Oldenburg, Catherine E.; Lee, Salena M.; Zegans, Michael E.; McLeod, Stephen D.; Lietman, Thomas M.; Acharya, Nisha R.

    2013-01-01

    Objective To determine whether there is a benefit in clinical outcomes with the use of topical corticosteroids as adjunctive therapy in the treatment of bacterial corneal ulcers. Methods Randomized, placebo-controlled, double-masked, multicenter clinical trial comparing prednisolone sodium phosphate, 1.0%, to placebo as adjunctive therapy for the treatment of bacterial corneal ulcers. Eligible patients had a culture-positive bacterial corneal ulcer and received topical moxifloxacin for at least 48 hours before randomization. Main Outcome Measures The primary outcome was best spectacle-corrected visual acuity (BSCVA) at 3 months from enrollment. Secondary outcomes included infiltrate/scar size, reepithelialization, and corneal perforation. Results Between September 1, 2006, and February 22, 2010, 1769 patients were screened for the trial and 500 patients were enrolled. No significant difference was observed in the 3-month BSCVA (−0.009 logarithm of the minimum angle of resolution [logMAR]; 95% CI, −0.085 to 0.068; P = .82), infiltrate/scar size (P = .40), time to reepithelialization (P = .44), or corneal perforation (P > .99). A significant effect of corticosteroids was observed in subgroups of baseline BSCVA (P = .03) and ulcer location (P = .04). At 3 months, patients with vision of counting fingers or worse at baseline had 0.17 logMAR better visual acuity with corticosteroids (95% CI, −0.31 to −0.02; P = .03) compared with placebo, and patients with ulcers that were completely central at baseline had 0.20 logMAR better visual acuity with corticosteroids (−0.37 to −0.04; P = .02). Conclusions We found no overall difference in 3-month BSCVA and no safety concerns with adjunctive corticosteroid therapy for bacterial corneal ulcers. Application to Clinical Practice Adjunctive topical corticosteroid use does not improve 3-month vision in patients with bacterial corneal ulcers. PMID:21987582

  15. Receptor-interacting protein (RIP) kinase family

    Science.gov (United States)

    Zhang, Duanwu; Lin, Juan; Han, Jiahuai

    2010-01-01

    Receptor-interacting protein (RIP) kinases are a group of threonine/serine protein kinases with a relatively conserved kinase domain but distinct non-kinase regions. A number of different domain structures, such as death and caspase activation and recruitment domain (CARD) domains, were found in different RIP family members, and these domains should be keys in determining the specific function of each RIP kinase. It is known that RIP kinases participate in different biological processes, including those in innate immunity, but their downstream substrates are largely unknown. This review will give an overview of the structures and functions of RIP family members, and an update of recent progress in RIP kinase research. PMID:20383176

  16. Relationship between release of LH and incorporation of tritiated thymidine in the anterior pituitary gland of the castrated male rat: effect of LHRH and its highly active analogue buserelin.

    Science.gov (United States)

    Romano, M I; Machiavelli, G A; Alonso, G E; Burdman, J A

    1986-01-01

    Castration of the adult male rat significantly (P less than 0.01) increased the concentration of LH in serum and the incorporation of (3H) thymidine into the pituitary DNA. The administration of a single dose of LHRH or its analogue buserelin stimulated the release of LH but it did not modify (3H) thymidine incorporation. When multiple doses of LHRH or buserelin were injected, there was a significant (P less than 0.01) inhibition of LH release and also the incorporation of (3H) thymidine into the DNA of the anterior pituitary gland was significantly (P less than 0.01) diminished. These observations are compatible with the idea of the close relationship between hormonal release and DNA synthesis in the anterior pituitary gland of the rat.

  17. Oncoprotein protein kinase antibody kit

    Science.gov (United States)

    Karin, Michael [San Diego, CA; Hibi, Masahiko [San Diego, CA; Lin, Anning [La Jolla, CA

    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.

  18. Bacterial infec tions in travellers

    African Journals Online (AJOL)

    namely bacterial causes of travellers' diarrhoea and skin infections, as well as .... Vaccination: protective efficacy against typhoid may be overcome by ingesting a high bacterial load. Vaccine ..... preparation such as cream sauce. Only after ...

  19. Covalent Immobilization of Candida rugosa Lipase at Alkaline pH and Their Application in the Regioselective Deprotection of Per-O-acetylated Thymidine

    Directory of Open Access Journals (Sweden)

    Cintia W. Rivero

    2016-08-01

    Full Text Available Lipase from Candida rugosa (CRL was stabilized at alkaline pH to overcome the inactivation problem and was immobilized for the first time by multipoint covalent attachment on different aldehyde-activated matrices. PEG was used as a stabilizing agent on the activity of CRL. At these conditions, CRL maintained 50% activity at pH 10 after 17 h incubation in the presence of 40% (w/v of PEG, whereas the enzyme without additive was instantaneously inactive after incubation at pH 10. Thus, this enzyme was covalently immobilized at alkaline pH on three aldehyde-activated supports: aldehyde-activated Sepharose, aldehyde-activated Lewatit105 and heterofunctional aldehyde-activated EDA-Sepharose in high overall yields. Heterogeneous stable CRL catalysts at high temperature and solvent were obtained. The aldehyde-activated Sepharose-CRL preparation maintained 70% activity at 50 °C or 30% (v/v acetonitrile after 22 h and exhibited high regioselectivity in the deprotection process of per-O-acetylated thymidine, producing the 3′-OH-5′-OAc-thymidine in 91% yield at pH 5.

  20. Impact of HIV-1 reverse transcriptase polymorphism F214L on virological response to thymidine analogue based regimens in ART-naïve and experienced patients

    DEFF Research Database (Denmark)

    Silberstein, F; Cozzi-Lepri, A; Ruiz, L

    2007-01-01

    BACKGROUND: 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. METHODS: We studied 590 patients from EuroSIDA who started ...... were observed in patients with M41L/T215Y and mixed TAM profiles detected before the initiation of cART. CONCLUSIONS: This study provides evidence that the detection of polymorphism F214L is associated with a favorable virological response to TA-based cART.......BACKGROUND: 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. METHODS: We studied 590 patients from EuroSIDA who started TA...... therapy for the first time as part of potent combination antiretroviral therapy (cART) and who were tested for genotypic resistance within the past 6 months. End points were median reduction in the week 24 viral load and time to virological failure (2 consecutive VL measurements >400 copies/mL after...

  1. Proton magnetic resonance studies of 5,6-saturated thymidine derivatives produced by ionizing radiation. Conformational analysis of 6-hydroxylated diastereoisomers

    Energy Technology Data Exchange (ETDEWEB)

    Cadet, J; Ducolomb, R [CEA Centre d' Etudes Nucleaires de Grenoble, 38 (France). Lab. de Radiobiologie; Hruska, F E [Manitoba Univ., Winnipeg (Canada). Dept. of Chemistry

    1979-06-20

    The conformational properties of ten 6-hydroxylated dihydrothymidine derivatives including the various diastereoisomers of 5,6-dihydroxy-5,6-dihydrothymidine, 6-hydroxy-5,6-dihydrothymidine and 5-bromo-6-hydroxy-5,6-dihydrothymidine have been studied by 250 MHz proton magnetic resonance in aqueous solutions. A close correlation has been established between the carbon-6 configuration and the osidic conformation. The increase in the amplitude of the puckering within the furanose ring compared to that of thymidine or 2'-deoxyuridine is more pronounced for the levortatory (6S) nucleosides than for the dextrorotatory (6R) diastereoisomers. The importance of the 2'endo conformer population decreases in the following order: (-)> (+)>thymidine. The absence of destabilizing effects on the g/sup +/ rotameric population about the C(4')-C(5') bond denotes the lack of any interaction between exocyclic hydroxymethyl group and the 6-hydroxyl function or the 2-keto group. The 5,6-saturated nucleosides adopt a preferential anti conformation. The comparison has been extended to syn nucleosides which show opposite trends in the sugar conformation and g/sup +/ distribution.

  2. 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.

  3. Kinases Involved in Both Autophagy and Mitosis.

    Science.gov (United States)

    Li, Zhiyuan; Zhang, Xin

    2017-08-31

    Both mitosis and autophagy are highly regulated dynamic cellular processes and involve various phosphorylation events catalysed by kinases, which play vital roles in almost all physiological and pathological conditions. Mitosis is a key event during the cell cycle, in which the cell divides into two daughter cells. Autophagy is a process in which the cell digests its own cellular contents. Although autophagy regulation has mainly been studied in asynchronous cells, increasing evidence indicates that autophagy is in fact tightly regulated in mitosis. Here in this review, we will discuss kinases that were originally identified to be involved in only one of either mitosis or autophagy, but were later found to participate in both processes, such as CDKs (cyclin-dependent kinases), Aurora kinases, PLK-1 (polo-like kinase 1), BUB1 (budding uninhibited by benzimidazoles 1), MAPKs (mitogen-activated protein kinases), mTORC1 (mechanistic target of rapamycin complex 1), AMPK (AMP-activated protein kinase), PI3K (phosphoinositide-3 kinase) and protein kinase B (AKT). By focusing on kinases involved in both autophagy and mitosis, we will get a more comprehensive understanding about the reciprocal regulation between the two key cellular events, which will also shed light on their related therapeutic investigations.

  4. Kinases Involved in Both Autophagy and Mitosis

    Directory of Open Access Journals (Sweden)

    Zhiyuan Li

    2017-08-01

    Full Text Available Both mitosis and autophagy are highly regulated dynamic cellular processes and involve various phosphorylation events catalysed by kinases, which play vital roles in almost all physiological and pathological conditions. Mitosis is a key event during the cell cycle, in which the cell divides into two daughter cells. Autophagy is a process in which the cell digests its own cellular contents. Although autophagy regulation has mainly been studied in asynchronous cells, increasing evidence indicates that autophagy is in fact tightly regulated in mitosis. Here in this review, we will discuss kinases that were originally identified to be involved in only one of either mitosis or autophagy, but were later found to participate in both processes, such as CDKs (cyclin-dependent kinases, Aurora kinases, PLK-1 (polo-like kinase 1, BUB1 (budding uninhibited by benzimidazoles 1, MAPKs (mitogen-activated protein kinases, mTORC1 (mechanistic target of rapamycin complex 1, AMPK (AMP-activated protein kinase, PI3K (phosphoinositide-3 kinase and protein kinase B (AKT. By focusing on kinases involved in both autophagy and mitosis, we will get a more comprehensive understanding about the reciprocal regulation between the two key cellular events, which will also shed light on their related therapeutic investigations.

  5. Structure of bacterial lipopolysaccharides.

    Science.gov (United States)

    Caroff, Martine; Karibian, Doris

    2003-11-14

    Bacterial lipopolysaccharides are the major components of the outer surface of Gram-negative bacteria They are often of interest in medicine for their immunomodulatory properties. In small amounts they can be beneficial, but in larger amounts they may cause endotoxic shock. Although they share a common architecture, their structural details exert a strong influence on their activity. These molecules comprise: a lipid moiety, called lipid A, which is considered to be the endotoxic component, a glycosidic part consisting of a core of approximately 10 monosaccharides and, in "smooth-type" lipopolysaccharides, a third region, named O-chain, consisting of repetitive subunits of one to eight monosaccharides responsible for much of the immunospecificity of the bacterial cell.

  6. Cooperative Bacterial Foraging Optimization

    Directory of Open Access Journals (Sweden)

    Hanning Chen

    2009-01-01

    Full Text Available Bacterial Foraging Optimization (BFO is a novel optimization algorithm based on the social foraging behavior of E. coli bacteria. This paper presents a variation on the original BFO algorithm, namely, the Cooperative Bacterial Foraging Optimization (CBFO, which significantly improve the original BFO in solving complex optimization problems. This significant improvement is achieved by applying two cooperative approaches to the original BFO, namely, the serial heterogeneous cooperation on the implicit space decomposition level and the serial heterogeneous cooperation on the hybrid space decomposition level. The experiments compare the performance of two CBFO variants with the original BFO, the standard PSO and a real-coded GA on four widely used benchmark functions. The new method shows a marked improvement in performance over the original BFO and appears to be comparable with the PSO and GA.

  7. Bacterial control of cyanobacteria

    CSIR Research Space (South Africa)

    Ndlela, Luyanda L

    2017-08-01

    Full Text Available of biological control appears to be direct contact. • Ndlela, L. L. et al. (2016) ‘An overview of cyanobacterial bloom occurrences and research in Africa over the last decade’, Harmful Algae, 60 • Gumbo, J.R. et al. (2010) The Isolation and identification... of Predatory Bacteria from a Microcystis algal Bloom.. African Journal of Biotechnology, 9. *Special acknowledgement goes to the National Research foundation for funding this presentation Bacterial control of cyanobacteria Luyanda...

  8. Bacterial growth kinetics

    International Nuclear Information System (INIS)

    Boonkitticharoen, V.; Ehrhardt, J.C.; Kirchner, P.T.

    1989-01-01

    Quantitative measurement of bacterial growth may be made using a radioassay technique. This method measures, by scintillation counting, the 14 CO 2 derived from the bacterial metabolism of a 14 C-labeled substrate. Mathematical growth models may serve as reliable tools for estimation of the generation rate constant (or slope of the growth curve) and provide a basis for evaluating assay performance. Two models, i.e., exponential and logistic, are proposed. Both models yielded an accurate fit to the data from radioactive measurement of bacterial growth. The exponential model yielded high precision values of the generation rate constant, with an average relative standard deviation of 1.2%. Under most conditions the assay demonstrated no changes in the slopes of growth curves when the number of bacteria per inoculation was changed. However, the radiometric assay by scintillation method had a growth-inhibiting effect on a few strains of bacteria. The source of this problem was thought to be hypersensitivity to trace amounts of toluene remaining on the detector

  9. Adaptive Bacterial Foraging Optimization

    Directory of Open Access Journals (Sweden)

    Hanning Chen

    2011-01-01

    Full Text Available Bacterial Foraging Optimization (BFO is a recently developed nature-inspired optimization algorithm, which is based on the foraging behavior of E. coli bacteria. Up to now, BFO has been applied successfully to some engineering problems due to its simplicity and ease of implementation. However, BFO possesses a poor convergence behavior over complex optimization problems as compared to other nature-inspired optimization techniques. This paper first analyzes how the run-length unit parameter of BFO controls the exploration of the whole search space and the exploitation of the promising areas. Then it presents a variation on the original BFO, called the adaptive bacterial foraging optimization (ABFO, employing the adaptive foraging strategies to improve the performance of the original BFO. This improvement is achieved by enabling the bacterial foraging algorithm to adjust the run-length unit parameter dynamically during algorithm execution in order to balance the exploration/exploitation tradeoff. The experiments compare the performance of two versions of ABFO with the original BFO, the standard particle swarm optimization (PSO and a real-coded genetic algorithm (GA on four widely-used benchmark functions. The proposed ABFO shows a marked improvement in performance over the original BFO and appears to be comparable with the PSO and GA.

  10. A dual-specificity isoform of the protein kinase inhibitor PKI produced by alternate gene splicing.

    Science.gov (United States)

    Kumar, Priyadarsini; Walsh, Donal A

    2002-03-15

    We have previously shown that the protein kinase inhibitor beta (PKIbeta) form of the cAMP-dependent protein kinase inhibitor exists in multiple isoforms, some of which are specific inhibitors of the cAMP-dependent protein kinase, whereas others also inhibit the cGMP-dependent enzyme [Kumar, Van Patten and Walsh (1997), J. Biol. Chem. 272, 20011-20020]. We have now demonstrated that the switch from a cAMP-dependent protein kinase (PKA)-specific inhibitor to one with dual specificity arises as a consequence of alternate gene splicing. We have confirmed using bacterially produced pure protein that a single inhibitor species has dual specificity for both PKA and cGMP-dependent protein kinase (PKG), inhibiting each with very high and closely similar inhibitory potencies. The gene splicing converted a protein with 70 amino acids into one of 109 amino acids, and did not change the inhibitory potency to PKA, but changed it from a protein that had no detectable PKG inhibitory activity to one that now inhibited PKG in the nanomolar range.

  11. Receptor Tyrosine Kinases in Drosophila Development

    Science.gov (United States)

    Sopko, Richelle; Perrimon, Norbert

    2013-01-01

    Tyrosine phosphorylation plays a significant role in a wide range of cellular processes. The Drosophila genome encodes more than 20 receptor tyrosine kinases and extensive studies in the past 20 years have illustrated their diverse roles and complex signaling mechanisms. Although some receptor tyrosine kinases have highly specific functions, others strikingly are used in rather ubiquitous manners. Receptor tyrosine kinases regulate a broad expanse of processes, ranging from cell survival and proliferation to differentiation and patterning. Remarkably, different receptor tyrosine kinases share many of the same effectors and their hierarchical organization is retained in disparate biological contexts. In this comprehensive review, we summarize what is known regarding each receptor tyrosine kinase during Drosophila development. Astonishingly, very little is known for approximately half of all Drosophila receptor tyrosine kinases. PMID:23732470

  12. Polymeric immunoglobulin receptor-mediated invasion of Streptococcus pneumoniae into host cells requires a coordinate signaling of SRC family of protein-tyrosine kinases, ERK, and c-Jun N-terminal kinase.

    Science.gov (United States)

    Agarwal, Vaibhav; Asmat, Tauseef M; Dierdorf, Nina I; Hauck, Christof R; Hammerschmidt, Sven

    2010-11-12

    Streptococcus pneumoniae are commensals of the human nasopharynx with the capacity to invade mucosal respiratory cells. PspC, a pneumococcal surface protein, interacts with the human polymeric immunoglobulin receptor (pIgR) to promote bacterial adherence to and invasion into epithelial cells. Internalization of pneumococci requires the coordinated action of actin cytoskeleton rearrangements and the retrograde machinery of pIgR. Here, we demonstrate the involvement of Src protein-tyrosine kinases (PTKs), focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) but not p38 mitogen-activated protein kinases (MAPK) in pneumococcal invasion via pIgR. Pharmacological inhibitors of PTKs and MAPKs and genetic interference with Src PTK and FAK functions caused a significant reduction of pIgR-mediated pneumococcal invasion but did not influence bacterial adhesion to host cells. Furthermore, pneumococcal ingestion by host cells induces activation of ERK1/2 and JNK. In agreement with activated JNK, its target molecule and DNA-binding protein c-Jun was phosphorylated. We also show that functionally active Src PTK is essential for activation of ERK1/2 upon pneumococcal infections. In conclusion, these data illustrate the importance of a coordinated signaling between Src PTKs, ERK1/2, and JNK during PspC-pIgR-mediated uptake of pneumococci by host epithelial cells.

  13. Engineering of kinase-based protein interacting devices: active expression of tyrosine kinase domains

    KAUST Repository

    Diaz Galicia, Miriam Escarlet

    2018-01-01

    is then translated into a FRET (Fluorescence Resonance Energy Transfer) signal is here proposed. To this end, DNA constructs for interaction amplification (split kinases), positive controls (intact kinase domains), scaffolding proteins and phosphopeptide - SH2-domain

  14. Measuring Kinase Activity-A Global Challenge.

    Science.gov (United States)

    Cann, Marissa L; McDonald, Ian M; East, Michael P; Johnson, Gary L; Graves, Lee M

    2017-11-01

    The kinase enzymes within a cell, known collectively as the kinome, play crucial roles in many signaling pathways, including survival, motility, differentiation, stress response, and many more. Aberrant signaling through kinase pathways is often linked to cancer, among other diseases. A major area of scientific research involves understanding the relationships between kinases, their targets, and how the kinome adapts to perturbations of the cellular system. This review will discuss many of the current and developing methods for studying kinase activity, and evaluate their applications, advantages, and disadvantages. J. Cell. Biochem. 118: 3595-3606, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  15. Reconstruction of the Chemotaxis Receptor-Kinase Assembly

    International Nuclear Information System (INIS)

    Park, S.; Borbat, P.; Gonzalez-Bonet, G.; Bhatnagar, J.; Pollard, A.; Freed, J.; Bilwes, A.; Crane, B.

    2006-01-01

    In bacterial chemotaxis, an assembly of transmembrane receptors, the CheA histidine kinase and the adaptor protein CheW processes environmental stimuli to regulate motility. The structure of a Thermotoga maritima receptor cytoplasmic domain defines CheA interaction regions and metal ion-coordinating charge centers that undergo chemical modification to tune receptor response. Dimeric CheA-CheW, defined by crystallography and pulsed ESR, positions two CheWs to form a cleft that is lined with residues important for receptor interactions and sized to clamp one receptor dimer. CheW residues involved in kinase activation map to interfaces that orient the CheW clamps. CheA regulatory domains associate in crystals through conserved hydrophobic surfaces. Such CheA self-contacts align the CheW receptor clamps for binding receptor tips. Linking layers of ternary complexes with close-packed receptors generates a lattice with reasonable component ratios, cooperative interactions among receptors and accessible sites for modification enzymes

  16. Radiology of bacterial pneumonia

    International Nuclear Information System (INIS)

    Vilar, Jose; Domingo, Maria Luisa; Soto, Cristina; Cogollos, Jonathan

    2004-01-01

    Bacterial pneumonia is commonly encountered in clinical practice. Radiology plays a prominent role in the evaluation of pneumonia. Chest radiography is the most commonly used imaging tool in pneumonias due to its availability and excellent cost benefit ratio. CT should be used in unresolved cases or when complications of pneumonia are suspected. The main applications of radiology in pneumonia are oriented to detection, characterisation and follow-up, especially regarding complications. The classical classification of pneumonias into lobar and bronchial pneumonia has been abandoned for a more clinical classification. Thus, bacterial pneumonias are typified into three main groups: Community acquired pneumonia (CAD), Aspiration pneumonia and Nosocomial pneumonia (NP).The usual pattern of CAD is that of the previously called lobar pneumonia; an air-space consolidation limited to one lobe or segment. Nevertheless, the radiographic patterns of CAD may be variable and are often related to the causative agent. Aspiration pneumonia generally involves the lower lobes with bilateral multicentric opacities. Nosocomial Pneumonia (NP) occurs in hospitalised patients. The importance of NP is related to its high mortality and, thus, the need to obtain a prompt diagnosis. The role of imaging in NP is limited but decisive. The most valuable information is when the chest radiographs are negative and rule out pneumonia. The radiographic patterns of NP are very variable, most commonly showing diffuse multifocal involvement and pleural effusion. Imaging plays also an important role in the detection and evaluation of complications of bacterial pneumonias. In many of these cases, especially in hospitalised patients, chest CT must be obtained in order to better depict these associate findings

  17. Radiology of bacterial pneumonia

    Energy Technology Data Exchange (ETDEWEB)

    Vilar, Jose E-mail: vilar_jlu@gva.es; Domingo, Maria Luisa; Soto, Cristina; Cogollos, Jonathan

    2004-08-01

    Bacterial pneumonia is commonly encountered in clinical practice. Radiology plays a prominent role in the evaluation of pneumonia. Chest radiography is the most commonly used imaging tool in pneumonias due to its availability and excellent cost benefit ratio. CT should be used in unresolved cases or when complications of pneumonia are suspected. The main applications of radiology in pneumonia are oriented to detection, characterisation and follow-up, especially regarding complications. The classical classification of pneumonias into lobar and bronchial pneumonia has been abandoned for a more clinical classification. Thus, bacterial pneumonias are typified into three main groups: Community acquired pneumonia (CAD), Aspiration pneumonia and Nosocomial pneumonia (NP).The usual pattern of CAD is that of the previously called lobar pneumonia; an air-space consolidation limited to one lobe or segment. Nevertheless, the radiographic patterns of CAD may be variable and are often related to the causative agent. Aspiration pneumonia generally involves the lower lobes with bilateral multicentric opacities. Nosocomial Pneumonia (NP) occurs in hospitalised patients. The importance of NP is related to its high mortality and, thus, the need to obtain a prompt diagnosis. The role of imaging in NP is limited but decisive. The most valuable information is when the chest radiographs are negative and rule out pneumonia. The radiographic patterns of NP are very variable, most commonly showing diffuse multifocal involvement and pleural effusion. Imaging plays also an important role in the detection and evaluation of complications of bacterial pneumonias. In many of these cases, especially in hospitalised patients, chest CT must be obtained in order to better depict these associate findings.

  18. Stimulation of Diesel Fuel Biodegradation by Indigenous Nitrogen Fixing Bacterial Consortia.

    Science.gov (United States)

    Piehler; Swistak; Pinckney; Paerl

    1999-07-01

    > Abstract Successful stimulation of N2 fixation and petroleum hydrocarbon degradation in indigenous microbial consortia may decrease exogenous N requirements and reduce environmental impacts of bioremediation following petroleum pollution. This study explored the biodegradation of petroleum pollution by indigenous N2 fixing marine microbial consortia. Particulate organic carbon (POC) in the form of ground, sterile corn-slash (post-harvest leaves and stems) was added to diesel fuel amended coastal water samples to stimulate biodegradation of petroleum hydrocarbons by native microorganisms capable of supplying a portion of their own N. It was hypothesized that addition of POC to petroleum amended water samples from N-limited coastal waters would promote the growth of N2 fixing consortia and enhance biodegradation of petroleum. Manipulative experiments were conducted using samples from coastal waters (marinas and less polluted control site) to determine the effects of POC amendment on biodegradation of petroleum pollution by native microbial consortia. Structure and function of the microbial consortia were determined by measurement of N2 fixation (acetylene reduction), hydrocarbon biodegradation (14C hexadecane mineralization), bacterial biomass (AODC), number of hydrocarbon degrading bacteria (MPN), and bacterial productivity (3H-thymidine incorporation). Throughout this study there was a consistent enhancement of petroleum hydrocarbon degradation in response to the addition of POC. Stimulation of diesel fuel biodegradation following the addition of POC was likely attributable to increases in bacterial N2 fixation, diesel fuel bioavailability, bacterial biomass, and metabolic activity. Toxicity of the bulk phase water did not appear to be a factor affecting biodegradation of diesel fuel following POC addition. These results indicate that the addition of POC to diesel-fuel-polluted systems stimulated indigenous N2 fixing microbial consortia to degrade petroleum

  19. Algal and bacterial activities in acidic (pH 3) strip mine lakes

    International Nuclear Information System (INIS)

    Gyure, R.A.; Konopka, A.; Brooks, A.; Doemel, W.

    1987-01-01

    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 H 2 S 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 H 2 S 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 [ 14 C]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

  20. 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)

    Fernandez Samperiz, M.M.; Blumen, G.

    1984-01-01

    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 3 H-thymidine. The results showed a decrease of G(13.6h) and G 1 (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 G 1 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) [pt

  1. 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.

  2. Laser flash photolysis and magnetic-field-effect studies on interaction of thymine and thymidine with menadione: role of sugar in controlling reaction pattern

    Science.gov (United States)

    Bose, Adity; Dey, Debarati; Basu, Samita

    2008-04-01

    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.

  3. 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)

    Bose, Adity; Dey, Debarati; Basu, Samita

    2008-01-01

    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

  4. Laser flash photolysis and magnetic-field-effect studies on interaction of thymine and thymidine with menadione: role of sugar in controlling reaction pattern

    Energy Technology Data Exchange (ETDEWEB)

    Bose, Adity; Dey, Debarati; Basu, Samita [Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata-700 064 (India)], E-mail: samita.basu@saha.ac.in

    2008-04-01

    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.

  5. Oncogenic Receptor Tyrosine Kinases Directly Phosphorylate Focal Adhesion Kinase (FAK) as a Resistance Mechanism to FAK-Kinase Inhibitors.

    Science.gov (United States)

    Marlowe, Timothy A; Lenzo, Felicia L; Figel, Sheila A; Grapes, Abigail T; Cance, William G

    2016-12-01

    Focal adhesion kinase (FAK) is a major drug target in cancer and current inhibitors targeted to the ATP-binding pocket of the kinase domain have entered clinical trials. However, preliminary results have shown limited single-agent efficacy in patients. Despite these unfavorable data, the molecular mechanisms that drive intrinsic and acquired resistance to FAK-kinase inhibitors are largely unknown. We have demonstrated that receptor tyrosine kinases (RTK) can directly bypass FAK-kinase inhibition in cancer cells through phosphorylation of FAK's critical tyrosine 397 (Y397). We also showed that HER2 forms a direct protein-protein interaction with the FAK-FERM-F1 lobe, promoting direct phosphorylation of Y397. In addition, FAK-kinase inhibition induced two forms of compensatory RTK reprogramming: (i) the rapid phosphorylation and activation of RTK signaling pathways in RTK High cells and (ii) the long-term acquisition of RTKs novel to the parental cell line in RTK Low cells. Finally, HER2 +: cancer cells displayed resistance to FAK-kinase inhibition in 3D growth assays using a HER2 isogenic system and HER2 + cancer cell lines. Our data indicate a novel drug resistance mechanism to FAK-kinase inhibitors whereby HER2 and other RTKs can rescue and maintain FAK activation (pY397) even in the presence of FAK-kinase inhibition. These data may have important ramifications for existing clinical trials of FAK inhibitors and suggest that individual tumor stratification by RTK expression would be important to predict patient response to FAK-kinase inhibitors. Mol Cancer Ther; 15(12); 3028-39. ©2016 AACR. ©2016 American Association for Cancer Research.

  6. Rapid accumulation of HIV-1 thymidine analogue mutations and phenotypic impact following prolonged viral failure on zidovudine-based first-line ART in sub-Saharan Africa.

    Science.gov (United States)

    Goodall, Ruth L; Dunn, David T; Nkurunziza, Peter; Mugarura, Lincoln; Pattery, Theresa; Munderi, Paula; Kityo, Cissy; Gilks, Charles; Kaleebu, Pontiano; Pillay, Deenan; Gupta, Ravindra K

    2017-05-01

    Lack of viral load monitoring of ART is known to be associated with slower switch from a failing regimen and thereby higher prevalence of MDR HIV-1. Many countries have continued to use thymidine analogue drugs despite recommendations to use tenofovir in combination with a cytosine analogue and NNRTI as first-line ART. The effect of accumulated thymidine analogue mutations (TAMs) on phenotypic resistance over time has been poorly characterized in the African setting. A retrospective analysis of individuals with ongoing viral failure between weeks 48 and 96 in the NORA (Nevirapine OR Abacavir) study was conducted. We analysed 36 genotype pairs from weeks 48 and 96 of first-line ART (14 treated with zidovudine/lamivudine/nevirapine and 22 treated with zidovudine/lamivudine/abacavir). Phenotypic drug resistance was assessed using the Antivirogram assay (v. 2.5.01, Janssen Diagnostics). At 96 weeks, extensive TAMs (≥3 mutations) were present in 50% and 73% of nevirapine- and abacavir-treated patients, respectively. The mean (SE) number of TAMs accumulating between week 48 and week 96 was 1.50 (0.37) in nevirapine-treated participants and 1.82 (0.26) in abacavir-treated participants. Overall, zidovudine susceptibility of viruses was reduced between week 48 [geometric mean fold change (FC) 1.3] and week 96 (3.4, P  =   0.01). There was a small reduction in tenofovir susceptibility (FC 0.7 and 1.0, respectively, P  =   0.18). Ongoing viral failure with zidovudine-containing first-line ART is associated with rapidly increasing drug resistance that could be mitigated with effective viral load monitoring. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.

  7. High radiochemical yield synthesis of [18F]FLT from 3'-O-nosylated thymidine and its 3-N-BOC-protected analogue

    International Nuclear Information System (INIS)

    Yoon, M. K.; Oh, S. J.; Ryu, J. S.; Moon, D. H.

    2002-01-01

    We synthesized 3'-O-nosylate and its 3-N-BOC-protected thymidine derivatives as two precursors for high radiochemical yield synthesis of [ 18 F]FLT and optimized [ 18 F]fluorination conditions. (5'-O-DMTr-2'-deoxy-3'-O-nosyl-β-D-threo-pentofuranosyl)thymidine and its s 3-N-BOD-protected analogue were prepared with 54% and 28% yield, respectively. After drying of [ 18 F]F-, 3'-O-nosylate(10-30 mg) or its 3-N-BOC-protected(11-34 mg) precursor was added to 500 μl of CH3CN, respectively. The mixtures were heated at 100-130 .deg. C for 5-30 min. For hydrolysis, 250-500 μl 1N HCl at 50 .deg. C for 5 min condition was used and 1.5ml of 2M sodium acetate was used for neutralization. Reaction mixture was purified by HPLC. The optimal [ 18 F]fluorination yield of 3'-O-nosylate precursor was 85±5.4% with 34 mg of precursor and a reaction time of 5 min at 130 .deg. C. For 3-N-BOC-protected analogue, [ 18 F]fluorination yield was 82±5.4% with 34 mg of precursor and a reaction time of 5 min at 110. deg. C. After HPLC purification, overall radiochemical yield using each precursors were 40±5.2% and 42±5.4% and radiochemical purity were 98±0.5% and 97±2.1% for two precursors, respectively. Preparation time was 60±10.5 min including HPLC purification for two precursors. From these two precursors, [ 18 F]FLT can be easily prepared with high radiochemical yield. 3-N-BOC-protected precursor required milder [ 18 F]fluorination conditions than 3'-O-nosylate precursor

  8. Characterization of clinically isolated thymidine-dependent small-colony variants of Escherichia coli producing extended-spectrum β-lactamase.

    Science.gov (United States)

    Negishi, Tatsuya; Matsumoto, Takehisa; Horiuchi, Kazuki; Kasuga, Eriko; Natori, Tatsuya; Matsuoka, Mina; Ogiwara, Naoko; Sugano, Mitsutoshi; Uehara, Takeshi; Nagano, Noriyuki; Honda, Takayuki

    2018-01-01

    Thymidine-dependent small-colony variants (TD-SCVs) are difficult to detect or test for antimicrobial susceptibility. We investigated the characteristics of clonal TD-SCVs of Escherichia coli, both with and without blaCTX-M-3, isolated from a patient. Mutation in the thyA gene was analysed by sequencing, and morphological abnormalities in the colonies and cells of the isolates were examined. Additionally, conjugational transfer experiments were performed to prove the horizontal transferability of plasmids harbouring resistance genes. The TD-SCVs contained a single nucleotide substitution in the thyA gene, c.62G>A, corresponding to p.Arg21His. Morphologically, their colonies were more translucent and flattened than those of the wild-type strain. In addition, cells of the TD-SCVs were swollen and elongated, sometimes with abnormal and incomplete divisions; a large amount of cell debris was also observed. Changing c.62G>A back to the wild-type sequence reversed these abnormalities. Conjugational transfer experiments showed that the TD-SCV of E. coli with blaCTX-M-3 failed to transfer blaCTX-M-3 to E. coli CSH2. However, the TD-SCV of E. coli without blaCTX-M-3 experimentally received the plasmid encoding blaSHV-18 from Klebsiella pneumoniae ATCC 700603 and transferred it to E. coli CSH2. Mutation in the thyA gene causes morphological abnormalities in the colonies and cells of E. coli, as well as inducing thymidine auxotrophy. In addition, TD-SCVs horizontally transmit plasmids encoding resistance genes. It is important to detect TD-SCVs based on their characteristics because they serve as reservoirs of transferable antibiotic resistance plasmids.

  9. A rice kinase-protein interaction map.

    Science.gov (United States)

    Ding, Xiaodong; Richter, Todd; Chen, Mei; Fujii, Hiroaki; Seo, Young Su; Xie, Mingtang; Zheng, Xianwu; Kanrar, Siddhartha; Stevenson, Rebecca A; Dardick, Christopher; Li, Ying; Jiang, Hao; Zhang, Yan; Yu, Fahong; Bartley, Laura E; Chern, Mawsheng; Bart, Rebecca; Chen, Xiuhua; Zhu, Lihuang; Farmerie, William G; Gribskov, Michael; Zhu, Jian-Kang; Fromm, Michael E; Ronald, Pamela C; Song, Wen-Yuan

    2009-03-01

    Plants uniquely contain large numbers of protein kinases, and for the vast majority of the 1,429 kinases predicted in the rice (Oryza sativa) genome, little is known of their functions. Genetic approaches often fail to produce observable phenotypes; thus, new strategies are needed to delineate kinase function. We previously developed a cost-effective high-throughput yeast two-hybrid system. Using this system, we have generated a protein interaction map of 116 representative rice kinases and 254 of their interacting proteins. Overall, the resulting interaction map supports a large number of known or predicted kinase-protein interactions from both plants and animals and reveals many new functional insights. Notably, we found a potential widespread role for E3 ubiquitin ligases in pathogen defense signaling mediated by receptor-like kinases, particularly by the kinases that may have evolved from recently expanded kinase subfamilies in rice. We anticipate that the data provided here will serve as a foundation for targeted functional studies in rice and other plants. The application of yeast two-hybrid and TAPtag analyses for large-scale plant protein interaction studies is also discussed.

  10. dependent/calmodulin- stimulated protein kinase from moss

    Indian Academy of Sciences (India)

    Unknown

    stimulated protein kinase; CDPK, calmodulin domain-like protein kinase; KM14, 14 amino acid synthetic peptide; .... used were obtained from Sigma Chemical Company, USA, ..... Plant chimeric Ca2+/Calmodulin-dependent protein kinase.

  11. The Protein Kinase RSK Family - Roles in Prostate Cancer

    National Research Council Canada - National Science Library

    Lannigan, Deborah

    2006-01-01

    The Ser/Thr protein kinase p90-kDa ribosomal S6 kinase (RSK) is an important downstream effector of mitogen-activated protein kinase but its roles in prostate cancer have not been previously examined...

  12. Structural coupling of SH2-kinase domains links Fes and Abl substrate recognition and kinase activation.

    Science.gov (United States)

    Filippakopoulos, Panagis; Kofler, Michael; Hantschel, Oliver; Gish, Gerald D; Grebien, Florian; Salah, Eidarus; Neudecker, Philipp; Kay, Lewis E; Turk, Benjamin E; Superti-Furga, Giulio; Pawson, Tony; Knapp, Stefan

    2008-09-05

    The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.

  13. Glycogen Synthase Kinase-3β

    DEFF Research Database (Denmark)

    Munkholm, Klaus; Lenskjold, Toke; Jacoby, Anne Sophie

    2016-01-01

    cells were quantitated using enzyme immunometric assays. The activity of GSK-3β (serine-9-phosphorylated GSK-3β/total GSK-3β) was lower at baseline compared with follow-up. No significant mean change over time was observed in levels of total GSK-3β and serine-9-phosphorylated GSK-3β. Exploratory......Evidence indicates a role for glycogen synthase kinase-3β (GSK-3β) in the pathophysiology of mood disorders and in cognitive disturbances; however, the natural variation in GSK-3β activity over time is unknown. We aimed to investigate GSK-3β activity over time and its possible correlation...... with emotional lability, subjective mood fluctuations and cognitive function in healthy individuals. Thirty-seven healthy subjects were evaluated with neuropsychological tests and blood samples at baseline and 12-week follow-up. Total GSK-3β and serine-9-phosphorylated GSK-3β in peripheral blood mononuclear...

  14. TYROSINE KINASE INHIBITORS AND PREGNANCY

    Directory of Open Access Journals (Sweden)

    Elisabetta Abruzzese

    2014-04-01

    Full Text Available The management of patients with chronic myeloid leukemia (CML during pregnancy has became recently a matter of continuous debate.  The introduction of the Tyrosine Kinase Inhibitors (TKIs in clinical practice has dramatically changed the prognosis of CML patients.  Patients diagnosed in chronic phase can reasonably expect many years of excellent disease control and good quality of life, as well as a normal life expectancy.  This fact has come the necessity to address issues relating to fertility and pregnancy. Physicians are not infrequently being asked for advice regarding the need for, and or the appropriateness of, stopping treatment in order to conceive. In this report we will review the data published in terms of fertility, conception, pregnancy, pregnancy outcome and illness control for all the approved TKIs, as well as suggest how to manage a planned and/or unplanned pregnancy.

  15. Host Immune Response to Bacterial Cyclic Diguanylic Acid (c-di-GMP)

    Science.gov (United States)

    2009-07-01

    charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work...thymidine incorporation by an automatic beta counter. Tests were per- formed in triplicates, and results were expressed as the mean cpm . Flow cytometry...thymidine was added and allowed to incubate for an additional 18 h. T cell proliferation was measured by [3H]thymidine uptake ( cpm ). , p 0.01, as

  16. Bacterial polyhydroxyalkanoates: Still fabulous?

    Science.gov (United States)

    Możejko-Ciesielska, Justyna; Kiewisz, Robert

    2016-11-01

    Bacterial polyhydroxyalkanoates (PHA) are polyesters accumulated as carbon and energy storage materials under limited growth conditions in the presence of excess carbon sources. They have been developed as biomaterials with unique properties for the past many years being considered as a potential substitute for conventional non-degradable plastics. Due to the increasing concern towards global climate change, depleting petroleum resource and problems with an utilization of a growing number of synthetic plastics, PHAs have gained much more attention from industry and research. These environmentally friendly microbial polymers have great potential in biomedical, agricultural, and industrial applications. However, their production on a large scale is still limited. This paper describes the backgrounds of PHAs and discussed the current state of knowledge on the polyhydroxyalkanoates. Ability of bacteria to convert different carbon sources to PHAs, the opportunities and challenges of their introduction to global market as valuable renewable products have been also discussed. Copyright © 2016 Elsevier GmbH. All rights reserved.

  17. Energetics of bacterial adhesion

    International Nuclear Information System (INIS)

    Loosdrecht, M.C.M. van; Zehnder, A.J.B.

    1990-01-01

    For the description of bacterial adhesion phenomena two different physico-chemical approaches are available. The first one, based on a surface Gibbs energy balance, assumes intimate contact between the interacting surfaces. The second approach, based on colloid chemical theories (DLVO theory), allows for two types of adhesion: 1) secondary minimum adhesion, which is often weak and reversible, and 2) irreversible primary minimum adhesion. In the secondary minimum adhesion a thin water film remains present between the interacting surface. The merits of both approaches are discussed in this paper. In addition, the methods available to measure the physico-chemical surface characteristics of bacteria and the influence of adsorbing (in)organic compounds, extracellular polymers and cell surface appendages on adhesion are summarized. (author) 2 figs., 1 tab., 50 refs

  18. Biosensors of bacterial cells.

    Science.gov (United States)

    Burlage, Robert S; Tillmann, Joshua

    2017-07-01

    Biosensors are devices which utilize both an electrical component (transducer) and a biological component to study an environment. They are typically used to examine biological structures, organisms and processes. The field of biosensors has now become so large and varied that the technology can often seem impenetrable. Yet the principles which underlie the technology are uncomplicated, even if the details of the mechanisms are elusive. In this review we confine our analysis to relatively current advancements in biosensors for the detection of whole bacterial cells. This includes biosensors which rely on an added labeled component and biosensors which do not have a labeled component and instead detect the binding event or bound structure on the transducer. Methods to concentrate the bacteria prior to biosensor analysis are also described. The variety of biosensor types and their actual and potential uses are described. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Bacterial mitotic machineries

    DEFF Research Database (Denmark)

    Gerdes, Kenn; Møller-Jensen, Jakob; Ebersbach, Gitte

    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.......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 Par...

  20. Exploring bacterial lignin degradation.

    Science.gov (United States)

    Brown, Margaret E; Chang, Michelle C Y

    2014-04-01

    Plant biomass represents a renewable carbon feedstock that could potentially be used to replace a significant level of petroleum-derived chemicals. One major challenge in its utilization is that the majority of this carbon is trapped in the recalcitrant structural polymers of the plant cell wall. Deconstruction of lignin is a key step in the processing of biomass to useful monomers but remains challenging. Microbial systems can provide molecular information on lignin depolymerization as they have evolved to break lignin down using metalloenzyme-dependent radical pathways. Both fungi and bacteria have been observed to metabolize lignin; however, their differential reactivity with this substrate indicates that they may utilize different chemical strategies for its breakdown. This review will discuss recent advances in studying bacterial lignin degradation as an approach to exploring greater diversity in the environment. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. 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 communities...

  2. Anaerobes in bacterial vaginosis

    Directory of Open Access Journals (Sweden)

    Aggarwal A

    2003-01-01

    Full Text Available Four hundred high vaginal swabs were taken from patients attending gynaecology and obstetrics department of Govt. medical college, Amritsar. The patients were divided into four groups i.e. women in pregnancy (Group I, in labour/post partum (Group II, with abnormal vaginal discharge or bacterial vaginosis (Group III and asymptomatic women as control (Group IV. Anaerobic culture of vaginal swabs revealed that out of 400 cases, 212(53% were culture positive. Maximum isolation of anaerobes was in group III (84% followed by group II (56%, group I (36% and control group (15%. Gram positive anaerobes (69.2% out numbered gram negatives (30.8%. Among various isolates Peptostreptococcus spp. and Bacteroides spp. were predominant.

  3. Bacterial meningitis in immunocompromised patients

    NARCIS (Netherlands)

    van Veen, K.E.B.

    2018-01-01

    Bacterial meningitis is an acute infection of the meninges, in The Netherlands most commonly caused by Streptococcus pneumoniae and Neisseria meningitides. Risk factors for acquiring bacterial meningitis include a decreased function of the immune system. The aim of this thesis was to study

  4. Bacteriële meningitis

    NARCIS (Netherlands)

    Brouwer, M. C.; van de Beek, D.

    2012-01-01

    Bacterial meningitis is a severe disease which affects 35.000 Europeans each year and has a mortality rate of about 20%. During the past 25 years the epidemiology of bacterial meningitis has changed significantly due to the implementation of vaccination against Haemophilus influenzae, Neisseria

  5. 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.

  6. The amoebal MAP kinase response to Legionella pneumophila is regulated by DupA.

    Science.gov (United States)

    Li, Zhiru; Dugan, Aisling S; Bloomfield, Gareth; Skelton, Jason; Ivens, Alasdair; Losick, Vicki; Isberg, Ralph R

    2009-09-17

    The amoeba Dictyostelium discoideum can support replication of Legionella pneumophila. Here we identify the dupA gene, encoding a putative tyrosine kinase/dual-specificity phosphatase, in a screen for D. discoideum mutants altered in allowing L. pneumophila intracellular replication. Inactivation of dupA resulted in depressed L. pneumophila growth and sustained hyperphosphorylation of the amoebal MAP kinase ERK1, consistent with loss of a phosphatase activity. Bacterial challenge of wild-type amoebae induced dupA expression and resulted in transiently increased ERK1 phosphorylation, suggesting that dupA and ERK1 are part of a response to bacteria. Indeed, over 500 of the genes misregulated in the dupA(-) mutant were regulated in response to L. pneumophila infection, including some thought to have immune-like functions. MAP kinase phosphatases are known to be highly upregulated in macrophages challenged with L. pneumophila. Thus, DupA may regulate a MAP kinase response to bacteria that is conserved from amoebae to mammals.

  7. n-Butyrate inhibits Jun NH(2)-terminal kinase activation and cytokine transcription in mast cells

    International Nuclear Information System (INIS)

    Diakos, Christos; Prieschl, Eva E.; Saeemann, Marcus D.; Boehmig, Georg A.; Csonga, Robert; Sobanov, Yury; Baumruker, Thomas; Zlabinger, Gerhard J.

    2006-01-01

    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

  8. Phosphorylation of dynamin I on Ser-795 by protein kinase C blocks its association with phospholipids

    DEFF Research Database (Denmark)

    Powell, K A; Valova, V A; Malladi, C S

    2000-01-01

    Dynamin I is phosphorylated in nerve terminals exclusively in the cytosolic compartment and in vitro by protein kinase C (PKC). Dephosphorylation is required for synaptic vesicle retrieval, suggesting that its phosphorylation affects its subcellular localization. An in vitro phospholipid binding ...... assay was established that prevents lipid vesiculation and dynamin lipid insertion into the lipid. Dynamin I bound the phospholipid in a concentration-dependent and saturable manner, with an apparent affinity of 230 +/- 51 nM. Optimal binding occurred with mixtures of phosphatidylserine...... the phosphorylation site in PKCalpha-phosphorylated dynamin I as a single site at Ser-795, located near a binding site for the SH3 domain of p85, the regulatory subunit of phosphatidylinositol 3-kinase. However, phosphorylation had no effect on dynamin binding to a bacterially expressed p85-SH3 domain. Thus...

  9. Particulate organic matter predicts bacterial productivity in a river dominated estuary

    Science.gov (United States)

    Crump, B. C.

    2015-12-01

    Estuaries act as coastal filters for organic and inorganic fluvial materials in which microbial, biogeochemical, and ecological processes combine to transform organic matter and nutrients prior to export to the coastal ocean. The function of this estuarine 'bioreactor' is linked to the residence times of those materials and to rates of microbial heterotrophic activity. Our ability to forecast the impact of global change on estuarine bioreactor function requires an understanding of the basic controls on microbial community activity and diversity. In the Columbia River estuary, the microbial community undergoes a dramatic seasonal shift in species composition during which a spring bacterioplankton community, dominated by Flavobacteriaceae and Oceanospirillales, is replaced by a summer community, dominated by Rhodobacteraceae and several common marine taxa. This annual shift occurs in July, following the spring freshet, when river flow and river chlorophyll concentration decrease and when estuarine water residence time increases. Analysis of a large dataset from 17 research cruises (1990-2014) showed that the composition of particulate organic matter in the estuary changes after the freshet with decreasing organic carbon and nitrogen content, and increasing contribution of marine and autochthonous estuarine organic matter (based on PO13C and pigment ratios). Bacterial production rates (measured as leucine or thymidine incorporation rates) in the estuary respond to this change, and correlate strongly with labile particulate nitrogen concentration and temperature during individual sampling campaigns, and with the concentration of chlorophyll in the Columbia River across all seasons. Regression models suggest that the concentration of labile particulate nitrogen and the rate of bacterial production can be predicted from sensor measurements of turbidity, salinity, and temperature in the estuary and chlorophyll in the river. These results suggest that the quality of

  10. Protein Kinase A in Cancer

    International Nuclear Information System (INIS)

    Caretta, Antonio; Mucignat-Caretta, Carla

    2011-01-01

    In the past, many chromosomal and genetic alterations have been examined as possible causes of cancer. However, some tumors do not display a clear molecular and/or genetic signature. Therefore, other cellular processes may be involved in carcinogenesis. Genetic alterations of proteins involved in signal transduction have been extensively studied, for example oncogenes, while modifications in intracellular compartmentalization of these molecules, or changes in the expression of unmodified genes have received less attention. Yet, epigenetic modulation of second messenger systems can deeply modify cellular functioning and in the end may cause instability of many processes, including cell mitosis. It is important to understand the functional meaning of modifications in second messenger intracellular pathways and unravel the role of downstream proteins in the initiation and growth of tumors. Within this framework, the cAMP system has been examined. cAMP is a second messenger involved in regulation of a variety of cellular functions. It acts mainly through its binding to cAMP-activated protein kinases (PKA), that were suggested to participate in the onset and progression of various tumors. PKA may represent a biomarker for tumor detection, identification and staging, and may be a potential target for pharmacological treatment of tumors

  11. Protein Kinase A in Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Caretta, Antonio; Mucignat-Caretta, Carla, E-mail: carla.mucignat@unipd.it [Department of Human Anatomy and Physiology, University of Padova, Via Marzolo 3, 35131 Padova (Italy)

    2011-02-28

    In the past, many chromosomal and genetic alterations have been examined as possible causes of cancer. However, some tumors do not display a clear molecular and/or genetic signature. Therefore, other cellular processes may be involved in carcinogenesis. Genetic alterations of proteins involved in signal transduction have been extensively studied, for example oncogenes, while modifications in intracellular compartmentalization of these molecules, or changes in the expression of unmodified genes have received less attention. Yet, epigenetic modulation of second messenger systems can deeply modify cellular functioning and in the end may cause instability of many processes, including cell mitosis. It is important to understand the functional meaning of modifications in second messenger intracellular pathways and unravel the role of downstream proteins in the initiation and growth of tumors. Within this framework, the cAMP system has been examined. cAMP is a second messenger involved in regulation of a variety of cellular functions. It acts mainly through its binding to cAMP-activated protein kinases (PKA), that were suggested to participate in the onset and progression of various tumors. PKA may represent a biomarker for tumor detection, identification and staging, and may be a potential target for pharmacological treatment of tumors.

  12. 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.

  13. 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.

  14. Bacterial growth on macrophyte leachate and fate of bacterial production

    International Nuclear Information System (INIS)

    Findlay, S.; Carlough, L.; Crocker, M.T.; Gill, H.K.; Meyer, J.L.; Smith, P.J.

    1986-01-01

    The role bacteria play in transferring organic carbon to other trophic levels in aquatic ecosystems depends on the efficiency with which they convert dissolved organic [ 14 C]-labelled carbon into bacterial biomass and on the ability of consumers to graze bacteria. The authors have measured the conversion efficiency for bacteria growing on macrophyte-derived dissolved organic carbon and estimated the amount of bacterial production removed by grazing. Bacteria converted this DOC into new tissue with an efficiency of 53%, substantially higher than the apparent conversion efficiency of macrophyte-derived particulate organic carbon or other types of DOC. Two estimates of grazing indicate that the decline in bacterial numbers after the bloom was probably due to grazing by flagellates. These results show the significance of the bacterial link between DOC and other trophic levels

  15. Molecular detection of human bacterial pathogens

    National Research Council Canada - National Science Library

    Liu, Dongyou

    2011-01-01

    .... Molecular Detection of Human Bacterial Pathogens addresses this issue, with international scientists in respective bacterial pathogen research and diagnosis providing expert summaries on current...

  16. 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

    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 activation loop and the Z/TM in the C-terminal extension. We provide evidence that phosphorylation of the Z/TM site of PRK2 inhibits its interaction with PDK1. Our studies further provide a mechanistic model to explain different steps in the docking interaction and regulation. Interestingly, we found...... that 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 and do...

  17. Isoprenoid biosynthesis and mevalonate kinase deficiency

    NARCIS (Netherlands)

    Henneman, L.

    2011-01-01

    Mevalonaat Kinase Deficiëntie (MKD) is een aangeboren ziekte geassocieerd met heftige koortsaanvallen die drie tot vier dagen aanhouden en gepaard gaan met koude rillingen, gewrichtsklachten, huiduitslag, hoofdpijn, duizeligheid, buikpijn, braken en diarree. De koortsaanvallen treden gemiddeld eens

  18. Expression Profiling of Tyrosine Kinase Genes

    National Research Council Canada - National Science Library

    Weier, Heinz

    2000-01-01

    ... of these genes parallels the progression of tumors to a more malignant phenotype. We developed a DNA micro-array based screening system to monitor the level of expression of tyrosine kinase (tk...

  19. MAP kinase cascades in Arabidopsis innate immunity

    DEFF Research Database (Denmark)

    Rasmussen, Magnus Wohlfahrt; Roux, Milena Edna; Petersen, Morten

    2012-01-01

    Plant mitogen-activated protein kinase (MAPK) cascades generally transduce extracellular stimuli into cellular responses. These stimuli include the perception of pathogen-associated molecular patterns (PAMPs) by host transmembrane pattern recognition receptors which trigger MAPK-dependent innate ...

  20. Protein Kinases in Human Breast Carcinoma

    National Research Council Canada - National Science Library

    Cane, William

    1998-01-01

    .... Rak is a novel nuclear tyrosine that our group has identified in breast cancer tissues and cell lines that has structural homology to the Src tyrosine kinase, with SH2 and SH3 domains at its amino terminus...